CA2348157A1

CA2348157A1 - Methods and compositions for inhibiting neoplastic cell growth

Info

Publication number: CA2348157A1
Application number: CA002348157A
Authority: CA
Inventors: Jian Chen; Audrey Goddard; Austin L. Gurney; Kenneth Hillan; Mary Napier; William I. Wood
Original assignee: Genentech, Inc.; Jian Chen; Audrey Goddard; Austin L. Gurney; Kenneth Hillan; Mary Napier; William I. Wood
Current assignee: Genentech Inc
Priority date: 1998-12-01
Filing date: 1999-11-30
Publication date: 2000-06-08
Also published as: KR20010086072A; WO2000032778A3; WO2000032778A2; JP2002531092A; EP1135491A2; AU3107000A

Abstract

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

Description

METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL
GROWTH
FIELD OF THE INVENTION
The present invention concerns methods and compositions for inhibiting neoplastic cell growth. In particular, the present invention concerns antitumor compositions and methods for the treatment of tumors. The invention further concerns screening methods for identifying growth inhibitory, e.g., antitumor compounds.
BACKGROUND OF THE INVENTION
Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et ul., CA Cancel J. Clin.. 43:7 ( 1993)).
Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or Lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
Despite recent advances in cancer therapy, there is a great need for new therapeutic agents capable of inhibiting neoplastic cell growth. Accordingly, it is the objective of the present invention to identify compounds capable of inhibiting the growth of neoplastic cells, such as cancer cells.
SUMMARY OF THE INVENTION
A. Embodiments The present invention relates to methods and compositions for inhibiting neoplastic cell growth. More particularly, the invention concerns methods and compositions for the treatment of tumors, including cancers, such as breast, prostate. colon, lung, ovarian, renal and CNS cancers, leukemia, melanoma, etc., in mammalian patients, preferably humans.
In one aspect, the present invention concerns compositions of matter useful for the inhibition of neoplastic cell growth comprising an effective amount of a PR065~, PR0364 or PR0344 polypeptide as herein defined, or 2S an agonist thereof, in admixture with a pharmaceutically acceptable carrier. In a preferred embodiment, the composition of matter comprises a growth inhibitory amount of a PR0655, PR0364 or PR0344 polypeptide, or an agonist thereof. In another preferred embodiment, the composition comprises a cytotoxic amount of a PR0655, PR0364 or PR0344 polypeptide, or an agonist thereof. Optionally. the compositions of matter may contain one or more additional ;rowth inhibitory and/or cytotoxic and/or other chemotherapeutic agents.
In a further aspect, the present invention concerns compositions of matter useful for the treatment of a tumor in a mammal comprising a therapeutically effective amount of a PR0655.
PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof. The tumor is preferably a cancer.
In another aspect, the invention concerns a method for inhibiting the Qrowth of a tumor cell comprising exposing the cell to an effective amount of a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof. !n a particular embodiment, the aaonist is an anti-PR0655, anti-PR0364 or anti-PR0344 agonist antibody. In another embodiment, the agonist is a small molecule that mimics the biological activity of a PR0655, PR0364 or PR0344 polypeptide. The method may be performed in vilro or in vivo.
In a still further embodiment, the invention concerns an article of manufacture comprising:
(a) a container;
(b) a composition comprising an active agent contained within the container:
wherein the composition is effective for inhibiting the neoplastic cell growth. e. ~..
growth of tumor cells, and the active agent in the composition is a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said PR0655, PR0364 or PR0344 polypeptide or agonist thereof, for the inhibition of neoplastic cell growth, wherein the agonist may be an antibody which binds to the PR0655, PR0364 or PR0344 polypeptide.
!n a particular embodiment, the agonist is an anti-PR0655. anti-PR0364 or anti-PR0344 agonist antibody. In another embodiment, the agonist is a small molecule that mimics the biological activity of a PR0655, PR0364 or PR0344 polypeptide. Similar articles of manufacture comprising a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof in an amount that is therapeutical ly effective for the treatment oftumor are also within the scope of the present invention. Also within the scope of the invention are articles of manufacture comprising a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an agonist thereof, and a further growth inhibitory agent, cytotoxic agent or chemotherapeutic went.
B. Additional Embodiments In other embodiments of the present invention, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PR0655, PR036.1 or PR0344 polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82%
sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at feast about 84%
sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86%
sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88%
sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90%
sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92%
sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94%

sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96%
sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98%
sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule encoding a PR0655, PR0364 or PR0344 polypeptide having a ful I-length am ino 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% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82%
sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84%
sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86%
sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88%
sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90%
sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92%
sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94%
sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96%
sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98%
sequence identity and yet more preferably at least about 99% sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PR0655, PR0364 or PR034.1 polypeptide cDNA as disclosed herein, the coding sequence ofa PR0655, PR0364 or PR0344 polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PR0655, PR0364 or PR0344 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).
!n a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% 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).
Anotheraspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PR065~. PR0364 or PR0344 polypeptide which is either transmembrane 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 PR0655, PR0364 or PR0344 polypeptides are contemplated.
Another embodiment is directed to fragments of a PR065~, PR0364 or PR0344 polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PR0655, PR0364 or PR0344 polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PR0655, anti-PR0364 or anti-PR0344 antibody or as antisense oligonucleotide probes.
Such nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about I 10 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length, yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleotides in length, yet more preferably at least about 180 nucleotides in length, yet more preferably at least about 190 nucleotides in length, yet more preferably at least about 200 nucleotides in length, yet more preferably at least about 250 nucleotides in length, yet more preferably at least about 300 nucleotides in length, yet more preferably at least about 350 nucleotides in length, yet more preferably at least about 400 nucleotides in length, yet more preferably at least about 450 nucleotides in length, yet more preferably at least about 500 nucleotides in length, yet more preferably at least about 600 nucleotides in length, yet more preferably at least about 700 nucleotides in length, yet more preferably at least about 800 nucleotides in length, yet more preferably at least about 900 nucleotides in length and yet more preferably 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 PR0655, PR0364 or PR0344 polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PR0655, PR0364 or PR0344 potypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number ofweil known sequence alignment programs and determining which PR065.i, PR0364 or PR0344 polypeptide-encoding nucleotide sequence fragments) are novel. All of such PR0655, PR0364 or PR0344 polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PR0655, PR0364 or PR0344 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PR0655, PR0364 or PR0344 polypeptide fragments that comprise a binding site for an anti-PR065~, anti-PR0364 or anti-PR0344 antibody.
!n another embodiment, the invention provides isolated PR0655, PR0364 or PR0344 polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PR0655, PR0364 or PR0344 polypeptide, comprising an amino acid sequence havin~_ at least about 80% sequence identity, preferably at least about 81%
sequence identity. more preferably at least about 82% sequence identity, yet more preferably at least about 83%
sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85%
sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87%
sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89%
sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93%
sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95%
sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97%
sequence identity. yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to a PR0655. PR0364 or PR0344 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 ofa 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 PR0655, PR0364 or PR0344 polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81%
sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83%
sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85%
sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87%
sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89%
sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93%
sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95%
sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97%
sequence identity. yet more preferably at least about 98% sequence identity and yet more preferably at least about 99% sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein.
In a further aspect, the invention concerns an isolated PR0655, PR0364 or PR0344 polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81 % positives, more preferably at least about 82% positives, yet more preferably at least about 83% positives, yet more preferably at least about 84% positives, yet more preferably at least about 85%
positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives, yet more preferably at least about 90% positives, yet more preferably at least about 91% positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95%
positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives and yet more preferably at least about 99% positives when compared with the amino acid sequence of a PRO65~. PR0364 or PR034-I 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 ofatransmembrane 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 specific aspect, the invention provides an isolated PR06». PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide and recovering the PR065~, PR0364 or PR03.~4 polypeptide from the cell culture.
Another aspect of the invention provides an isolated PR0655. PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide and recovering the PR0655. PR0364 or PR0344 polypeptide from the cell culture.
In yet another embodiment, the invention concerns agonists of a native PR0655, PR0364 or PR0344 polypeptide as defined herein. In a particular embodiment, the agonist is an anti-PR065.i, anti-PR0364 or anti-PR0344 agonist antibody or a small molecule.
In a further embodiment, the invention concerns a method of identifying agonists to a PR0655, PR0364 or PR0344 polypeptide which comprise contacting the PR0655, PR0364 or PR0344 polypeptide with a candidate molecule and monitoring a biological activity mediated by said PR0655, PR0364 or PR0344 polypeptide.
Preferably, the PR0655, PR0364 or PR0344 polypeptide is a native PR0655, PR0364 or PR0344 polypeptide.
In a still further embodiment, the invention concerns a composition of matter comprising a PROb55, PR0364 or PR0344 polypeptide, or an agonist of a PR0655, PR0364 or PR0344 polypeptide as herein described, or an anti-PR0655, anti-PR0364 or anti-PR0344 agonist 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 PR0655. PR0364 or PR0344 polypeptide, or an agonist thereof as hereinbefore described, or an anti-PR0655, anti-PR0364 or anti-PR0344 agonist antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PR0655, PR0364 or PR0344 polypeptide, an agonist thereof or an anti-PR065~, anti-PR0364 or anti-PR0344 agonist 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, yeast, or Baculovirus-infected insect cells. 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 ofthe 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 ta;
sequence or a Fc region of an immunoglobulin_ In another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides. Optional ly, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucieotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID NO: I ) of a native sequence PR065~ cDNA, wherein SEQ
ID NO:1 is a clone designated herein as "DNA50960-1224".
Figure 2 shows the amino acid sequence (SEQ ID N0:2) derived from the coding sequence of SEQ ID
NO:I shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ I D N0:6) of a native sequence PR0364 cDNA, wherein SEQ
ID N0:6 is a clone designated herein as "DNA47365-1206".
Figure 4 shows the amino acid sequence (SEQ 1D N0:7) derived from the coding sequence of SEQ ID
N0;6 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ lD N0:16) of a native sequence PR0344 cDNA, wherein SEQ ID N0:16 is a clone designated herein as "DNA40592-1242".
Figure 6 shows the amino acid sequence (SEQ ID N0:17) derived from the coding sequence of SEQ ID
N0:16 shown in Figure 5.
DETAILED DESCRIPTION OF THE INVENTION
The terms "PR0655", "PR0364" or"PR0344" polypeptide or protein when used herein encompass native sequence PR0655, PR0364 or PR0344 and PR0655, PR0364 or PR0344 variants (which are further defined herein). The PR0655, PR0364 or PR0344 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods.
A "native sequence PR0655", ''native sequence PR0364" or "native sequence PR0344" comprises a polypeptide having the same amino acid sequence as the PR0655. PR0364 or PR0344 polypeptide as derived from nature. Such native sequence PR0655, PR0364 or PR0344 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means. The term "native sequence"
PR0655. PR0364 or PR0344 specifically encompasses naturally-occurring truncated or secreted forms (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the PR0655. PR0364 and PR0344 polypeptides. In one embodiment of the invention, the native sequence PR0655, PR0364 or PR0344 polypeptide is a mature or full-length native sequence PR0655, PR0364 or PR0344 polypeptide as shown in Figure 2 (SEQ ID N0:2), Figure 4 (SEQ ID N0:7), or Figure 6 (SEQ ID NO:I7), respectively. Also, while the PR0655, PR0364. and PR0344 polypeptides disclosed in Figure 2 (SEQ ID N0:2), Figure 4 (SEQ ID N0:7), or Figure 6 (SEQ ID N0:17), respectively, are shown to begin with the methionine residue designated therein as amino acid position I, it is conceivable and possible that another methionine residue located either upstream or downstream from amino acid position I in Figure 2 (SEQ ID N0:2), Figure 4 (SEQ !D
N0:7), or Figure 6 (SEQ ID N0:17), respectively, may be employed as the starting amino acid residue for the PR0655, PR0364 or PR0344 polypeptide.
IO The "extracellular domain" or "ECD" ofa polypeptide disclosed herein refers to a form ofthe polypeptide which is essentially free ofthe transmembrane and cytoplasmic domains.
Ordinarily, a polypeptide ECD will have less than about I% of such transmembrane and/or cytoplasmic domains and preferably, will have less than about 0.5% of such domains. It will be understood that any transmembrane domains) identified for the polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of IS 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 and as shown in the appended figures. As such, in one embodiment of the present invention, the extracel lular domain of a polypeptide ofthe present invention comprises amino acids I to X ofthe mature amino acid sequence, wherein X is any amino acid within 5 amino acids on either side of the extracellular domain/transmembrane domain boundary.
20 The approximate location of the "signal peptides" of the various PRO
polypeptides disclosed herein are shown in 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 ofthe 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 e~ al., 2S Prot. Ene., 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 peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
30 "PR0655 variant polypeptide" means an active PR0655 polypeptide (other than a native sequence PR0655 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues I or about 22 to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), (b) X to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X is any amino acid residue from 17 to 26 of Figure 2 (SEQ 1D N0:2), or (c) another specifically derived fragment of the amino acid sequence 3S shown in Figure 2 (SEQ ID N0:2).
"PR0364 variant polypeptide" means an active PR0364 polypeptide (other than a native sequence PR0364 polypeptide) as defined below. having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues I or about 26 to 241 of the PR0364 polypeptide shown in Figure 4 (SEQ ID N0:7), (b) X to 24 I of the PR0364 polypeptide shown in Figure 4 (SEQ ID N0:7), wherein X is any amino acid residue from 21 to 30 of Figure 4 (SEQ ID N0:7), (c) 1 or about 26 to X of Figure 4 (SEQ ID N0:7), wherein X is any amino acid from amino acid 158 to amino acid 167 of Figure 4 (SEQ ID N0:7) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID N0:7).
"PR0344 variant polypeptide" means an active PR0344 polypeptide (other than a native sequence PR0344 polypeptide) as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues I or about 16 to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17), (b) X to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17), wherein X is any amino acid residue from 11 to 20 of Figure 6 (SEQ ID NO: ! 7), or (c) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID N0:17).
Such PR0655, PR0364 and PR0344 variants include, for instance, PR0655, PR0364 and PR0344 polypeptides wherein one or more amino acid residues are added. or deleted, at the N- or C-terminus, as well as within one or more internal domains of the native sequence.
Ordinarily, a PR0655 variant will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity. more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at feast about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 22 to 208 of the PR0655 poiypeptide shown in Figure 2 (SEQ ID
N0:2), (b) X to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X is any amino acid residue from 17 to 26 of Figure 2 (SEQ ID N0:2), or (c) another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID N0:2).
Ordinarily. a PR0364 variant will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identiy. more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity. more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 26 to 241 of the PR0364 polypeptide shown in Figure 4 (SEQ ID
N0:7), (b) X to 241 of the PR0364 polypeptide shown in Figure 4 (SEQ ID N0:7), wherein X is any amino acid residue from 21 to 30 of Figure 4 (SEQ ID N0:7), (c) 1 or about 26 to X of Figure 4 (SEQ ID N0:7), wherein X
is any amino acid from amino acid 158 to amino acid 167 of Figure 4 (SEQ ID
N0:7) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID N0:7).
Ordinarily, a PR0344 variant will have at least about 80% amino acid sequence identity, more preferably at least about 81 % amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at Least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 16 to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID
N0:17), (b) X to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID
N0:17), wherein X is any amino acid residue from 11 to 20 of Figure 6 (SEQ ID N0:17), or (c) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID N0:17).
Ordinarily, PR0655, PR0364 and PR0344 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about ~0 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about l00 amino acids in length, more often at least about I50 amino acids in length, more often at least about 200 amino acids in length, more often at least about 250 amino acids in length, more often at least about 300 amino acids in length, or more.
As shown below, Table I provides the complete source code for the ALIGN-3 sequence comparison computer program. This source code may be routinely compiled for use on a UNIX
operating system to provide the ALIGN-2 sequence comparison computer program.
In addition, Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-3B) and °% nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO"
represents the amino acid sequence of a hypothetical PR0655, PR0364 or PR0344 polypeptide of interest.
"Comparison Protein" representstheamino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, "PRO-DNA"
represents a hypothetical PR0655-, PR0364-or PR0344-encoding nucleic acid sequence of interest,"Comparison S DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, "X", "Y", and "Z" each represent different hypothetical amino acid residues and "N", "L" and "V" each represent different hypothetical nucleotides.

/_ Table 1 * C-C increased from 12 to 15 * Z is average of EQ
* B is average of ND
* match with stop is M; stop-stop = 0; 1 (joker) match = 0 *%
A'defne M -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 */
/* A */ { 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0, M. 1. 0,-2, 1, 1, 0. 0,-6, 0,-3, 0}, /* B */ { 0. 3,-4, 3, 2.-5, 0, 1,-2, 0, 0,-3,-2. 2, M,-1, 1, 0, 0. 0, 0,-2,-5, 0,-3, 1}, /* C *I {-2.-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4, M,-3,-5.-4. 0.-2, 0,-2,-8, 0, 0,-5}, /* D */ { 0, 3,-5, 4, 3,-6. 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2.-1, 0, 0, 0,-2,-1, 0,-4, 2}, /* E */ { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3.-2, l,_M,-I. 2.-1, 0, 0, 0,-Z,-7, 0,-4, 3}, /* F */ {-4,-5,-4,-6,-5. 9,-5,-2, 1, 0,-5, 2, 0,-4, M,-5,-S,-4,-3,-3, 0,-1, 0, 0, 7,-5}.
1* G */ { 1. 0,-3, l, 0,-5, 5,-2,-3, 0,-2,-4,-3, O, M,-1,-1,-3. I, 0, 0,-1,-7, 0,-5, 0}.
/* H *1 {-1. 1,-3. 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0, 3, 2.-1,-l, 0,-2,-3.
0, 0, 2}, /* I */ {-1,-2,-2.-2,-2, 1,-3,-2, 5, 0,-2, 2. 2,-2, M,-2,-2.-2,-1, 0. 0, 4.-5, 0.-1,-2}, /* J */ { 0, 0, 0, 0, 0, 0, 0. 0, 0, 0. 0. 0, 0, 0, M, 0. 0, 0. 0, 0, 0. 0, 0, 0, 0, 0}, /* K */ {-1. 0,-5. 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1, M.-1. 1. 3, 0. 0. 0,-2,-3, 0,-4, 0}.
I* L */ {-2.-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3.-3,-I, 0, 2.-2, 0,-1,-2}, /* M */ {-1.-2,-5,-3,-2, 0,-3,-2, 2, 0. 0, 4, 6,-2, M,-2.-1. 0,-2,-1. 0, 2,-0, 0,-2,-1}, /* N */ { 0, 2,-4. 2. I,-4, 0. 2,-2, 0, 1,-3,-2, 2, M,-1, 1, 0, 1, 0, 0.-2,-4, 0.-2. 1}, /* O */ { M,_M,_M. M, M, M._M. M, M,_M,_M. M._M,_M, O, M, M, M, M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,-1,-3,-l,-1,-5,-1, 0,-2, 0,-1,-3,-2,-1, M, 6, 0, 0, 1, 0, 0,-1.-6, 0,-5, 0}, I* Q *1 { 0, 1,-5. 2, 2,-S,-1, 3,-2, 0. 1,-2,-1, 1, M, 0, 4, 1,-1,-1, 0,-2,-5, 0,-4, 3}, /* R *1 {-2. 0,-4,-l,-l.-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0. 1, 6, 0.-1, 0,-2, 2, 0,-4, 0}, /* S */ { 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1, M, l,-1, 0. 2. 1, 0,-1,-2, 0,-3, 0}, /* T */ { 1, 0,-2. 0, 0,-3, 0,-l, 0, 0, 0,-1,-1, 0, M. 0,-1,-1, 1, 3, 0, 0,-5, 0,-3, 0}, /* U */ { 0, 0, 0, 0, 0, 0, 0, 0, 0. 0, 0, 0, 0, O, M, 0, 0, 0, 0, 0, 0. 0. 0.
0, 0, 0}, /* V */ { 0,-2,-2,-2,-2,-1,-I,-2, 4, 0,-2, 2, 2,-2, M,-I,-2,-2,-1. 0, 0, 4,-6, 0,-2,-2}, /* W */ {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4,_M,-6,-5, 2,-2,-5. 0,-6,17, 0, 0,-6}.
/* X */ { 0, 0, 0, 0, 0, 0, 0, 0, 0. 0, 0, 0, 0. O, M, 0. 0. 0. 0, 0, 0. 0, 0, 0, 0, 0}, 1* Y *I {-3,-3, 0,-4,-4, 7,-5, 0,-I, 0,-4,-I,-2,-2, M,-5,-4,-4,-3,-3, 0.-2. 0, 0,10,-4}, !* Z */ { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-I, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4}
};
Page 1 of day.h /*

*I

#include< >
stdio.h #include<
ctype.h >

#defineMAXJMP 16 l* max jumps in a diag *I

#defineMAXGAP l* don't continue to penalize 24 gaps larger than this *I

#defineJMPS 1024 /* max jmps in an path */

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

#defineDMAT 3 /* value of matching bases */

#detineDMIS 0 /* penalty for mismatched bases */

#de6neDINSO8 /* penalty for a gap *1 #defineDINSI1 !* penalty per base */

#defmePINSO8 /* penalty for a gap */

#definePINS14 /* penalty per residue */

struct jmp {

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

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

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

struct diag {

int score; 1* score at last jmp */

long offset; /* offset of prev block */

shortijmp; l* current jmp index */

strnct l* list of jmps */
jmp jp;

struct path {

int spc; /* number of leading spaces */

shortn[JMPS]; /* size of jmp (gap) */

int x[JMPS]; l* loc of jmp (last elem before gap) *I

}:

char *ofile; /* output file name */

char *namex(2];/* seq names: getseqsp *1 char *prog; I* prog name for err msgs *I

char *seqx(2]; /* seqs: getseqsQ *1 int dmax: /* best diag: nwp */

int dmax0; /* final diag */

int dna; /* set if dna: main() *1 int endgaps; I* set if penalizing end gaps *I

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

int IenO, lent;l* seq lens */

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

int smax; /' max score: nwp */

int *xbm; /* bitmap for matching */

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

structdiag *dx; /* holds diagonals *1 structpath pp[2]; /* holds path for seas */

char *callocQ, Q, *indexQ, *strcpyQ;
*mailoc char *getseqQ, *g callocQ;

Page 1 of nw.h /* Needleman-Wunsch alignment program * usage: props filet filet * where filet and filet are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity * Any lines beginning with ';', ' > ' or ' < ' are ignored * Max file length is 65535 (limited by 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 tmp file in Itmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 */
~ittclude "nw.h"
#inciude "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 static _pbval[26] _ {
1, 2~(1 < <('D'-'A'))~(1 < <('N'-'A')), 4, 8. 16. 32, 64.
128, 256. OxFFFFFFF, 1 < < 10, 1 < < I 1, 1 < < 12, 1 < < 13, I < < 14, l«15, 1«l6, 1«17, 1«18, 1«19, 1«20, 1«21, 1«22, 1«23. 1«24, 1«25(1«('E'-'A'))~(1«('Q'-'A')) main(ac, av) I>t181I1 int ac;
sitar *avp;
prop = av(0];
if (ac ! = 3) {
fprintf(stderr,"usage: %s filet filet\n", prop);
fprintf(stderr,"where filet and filet are two dna or two protein sequences.ln");
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 \"align.out\"1n");
exit( I );
namex[0] = av[1];
namex[I] = av(2];
seqx[0] = getseq(namex[0], &len0):
seqx[1] = getseq(namex[1], &lenl);
xbm = (dna)? dbval : pbval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "align.out"; /* output file */
nwQ; l* fill in the matrix, get the possible jmps */
readjmpsQ; /* get the actual jmps */
print(); /* print stars, aligtunent */
cleanup(0); I* unlink any tmp files *1 Page 1 of nw.c I* do the alignment, return best score: main() * 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.
*/
nwQ IIW

{

char *px, *py; /* seqs and ptrs *!

int *ndely, I* keep track of dely *I
*dely;

int ndelx, delx;/* keep track of delx */

int *tmp; /* for swapping row0, cowl */

int mis; /* score for each type */

int ins0, insl;/* insertion penalties */

register id; I* diagonal index *I

register ij; /* jmp index */

register *col0, *coll;/* score for curt, last row */

register xx, yy; /* index into seqs *1 dx = (struct diag *)g calloc("to get diags", IenO+lenl+I, 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 calloc("to get col0", lenl + 1, sizeof(int));
coi l = (int *)g calloc("to get coi l ", lenl + 1, sizeof(int));
ins0 = (dna)? DINSO : PINSO;
insl = (dna)? DINS1 : PINS1;
smax = -10000;
if (endgaps) {
for (col0[0] = dely[O) _ -ins0, yy = 1; yy < = lenl; yy++) {
col0[yy] = dely(yy] = col0[yy-1] - insl;
ndely[yYl = YY:
col0[0] = 0; /* Waterman Bull Math Biol 84 */
else for (yy = 1; yy < = lent; yy++) dely(yy] _ -ins0;
/* fill in match matrix */
for (px = seqx(0], xx = 1; xx < = len0; px++, xx++) {
/* initialize tirst entry in col *I
if (endgaps) {
if (xx = = I ) coil[0] = delx = -(ins0+insl);
else coil[0] = delx = col0[0] - insl;
ndelx = xx;
else {
col l (0] = 0;
delx = -ins0;
ndelx = 0;
Page 2 of nw.c ...nw for (py = seqx[1], yy = 1: yy < = lent: py++, yy++) {
mis = col0[yy-1];
if (dna) mss + _ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else mis += day[*px-'A'][*py-'A'j;
/* update penalty for del in x seq;
* favor new del over ongong del * ignore MAXGAP if weighting endgaps */
if (endgaps ~ ~ ndely[yy] < MAXGAP) {
if (col0[yy] - ins0 > = dely[yy]) {
dely[yy] = col0(yy] - (ins0+insl);
ndely[yy] = 1;
} else {
dely[yy] -= insl;
ndely[yy] + +;
}
} else {
if (col0[yy] - (ins0+insl) > = dely[yy]) {
dely[yy] = col0[yy] - (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 > = delx) {
delx = coll[yy-1] - (ins0+insl);
ndelx = 1;
} else {
deix -= insl;
ndelx+ +;
}
} else {
if (coil[yy-1] - (ins0+insl) > = delx) {
deli = colt[yy-1] - (ins0+insl);
ndelx = 1;
} eLse ndelx++;
}
/* pick the maximum score; we're favoring * mis over any del and delx over dely */
Page 3 of nw.c id = xx - yy + lent - l ;
if (mis > = delx && mis > = dely[yy]) ...nw cot l [yyJ = mis;
else if (delx > = dely[yy]) {
colt[yy] = deli;
ij = dx[id].ijmp;
if (dx[id].jp.n(O] && (!dna ~ ~ (ndelx > = MAXJMP
&& xx > dx[id].jp.x[ijJ+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx(idJ.ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
dx[id].jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
else {
colt[yY1 = dely(YY];
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndely[yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
dx[id].jp.n[ij] _ -ndely[yYl;
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
if (xx = = IenO && yy < ten 1 ) {
/* last cot x~
if (endgaps) coll(yy] -= ins0+insl*(lenl-yy);
if (cot 1 [yy] > smax) {
smax = coll(yyJ;
dmax = id;
if (endgaps && xx < IenO) coll[yy-I] -= ins0+insl*(len0-xx);
if (coll[yy-1] > smax) {
smax = coll(yy-1];
dmax = id;
tmp = col0; col0 = cot 1; cot l = tmp;
(void) free((char *)ndely);
(void) free((c6ar *)dely);
(void) free((char *)col0);
(void) free((char *)coll);
Page 4 of nw.c !*
* print() -- only routine visible outside this module * static:
* getmat() -- trace back best path, count matches: print() * pr align() -- print alignment of described in array p[]: print() * dumpblockQ -- dump a block of lines with numbers, stars: pr align() * numsp -- put out a number line: dumpblockp * putlineQ -- put out a line (name, [num], seq, [num]): dumpblockQ
* starsp - -put a line of stars: dumpblockp * stripnameQ -- strip any path and prefix from a seqname */
A~include "nw.h"
define SPC 3 Atdefine P LINE 256 /* maximum output line *1 A~define P SPC 3 !* space between name or num and seq */
extern day[26][26];
int oleo; I* set output line length *I
FILE *fx; /* output file *1 primp p)rlIlt {
int lx, ly, firstgap, lastgap; I* overlap *I
if ((fx = fopen(ofile, "w")) _ = 0) {
fprintf(stderr,"%s: can't write %s\n", prog, ofile);
cleanup( 1 );
fprintf(fx, " < first sequence: %s (length = % d)\n", namex[0], IenO);
fprintf(fx. "<second sequence: %s (length = %d)\n", namex[1], ienl);
oleo = 60;
Ix = IenO:
ly = lenl;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x */
pp[O].spc = firstgap = lenl - dmax - 1;
ly __ ~[Ol,spc:
else if (dmax > lenl - I) { /* leading gap in y */
pp[i].spc = firstgap = dmax - (lenl - 1);
Ix -= PP[ll.sPc:
if (dmax0 < IenO - I) { /* trailing gap in x */
lastgap = IenO - dmax0 -I;
Ix -= lastgap;
else if (dmax0 > len0 - 1) { 1* trailing gap in y */
lastgap = dmax0 - (IenO - 1 );
ly _= lastgap;
getmat(lx, ly, firstgap, lastgap);
pr alignQ;
Page 1 of nwprint.c /*
* trace back the best path, count matches */
static getmat(Ix, ly, firstgap, lastgap) g8ltlIlat int Ix, ly; /* "core" (minus endgaps) */
int firstgap, lastgap; /* leading trailing overlap */
{
int tun, i0, il, siz0, sizl;
char outx[32];
double pct;
register n0, nl;
register char *p0, *pl;
I* get total matches, score */
i0 = il = siz0 = sizl = 0;
p0 = seqx[0] + pp[lJ.spc;
pl = seqx[1] + pp[0].spc;
n0 = pp[1].spc + 1;
nl = pp[0].spc + l;
nm = 0;
while ( *p0 8t& *pl ) {
if (siz0) {
pl++;
nl++;
siz0--;
else if (sizl) {
p0++;
n0++;
siz 1--;
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[ilJ) sizf = pp[1].n[il++];
p0++;
pl++;
I* pct homology:
* if penalizing endgaps, base is the shorter seq * else, knock off overhangs and take shorter core i/
d (eAdga~) Ix = (len0 < lenl)? IenO : lenl;
else Ix = (Ix < ly)? !x : ly;
pct = 100.*(double)tun/(double)Ix;
fprimf(fx, "1n");
fprintf(fx, "< 96d match%s in an overlap of %d: %.2f percent similarityln", tun, (tun == 1)? "" . "es". lx, pct):
Page 2 of nwprint.c fprintf(fx, " < gaps in first sequence: %d", gapx); ...gCtIllat if (gapx) {
(void) sprintf(outx. " (%d %s%s)", ngapx, (dna)'? "base":"residue", (ngapx == 1)' ""~"s");
fprintf(fx,"%s", outx):
fprintf(fx, ", gaps in second sequence: %d", gapy);
if (gapY) {
(void) sprintf(outx, " (%d %s%s)", ngapy,(dna)? "base":"residue", (ngapy = = 1)? "":"s");
tprintf(fx,"%s", outx);
if (dna) fprintf(fx, "\n < score: %d (match = %d, mismatch = % d, gap penalty = %d + % d per base)\n", smax, DMAT, DMIS, DINSO, DINSI);
else fprintf(fx.
"\n < score: %d (Dayhoff PAM 250 matrix, gap penalty = % d + %d per residue)\n", stnax, PINSO, PINSI);
if (endgaps) fprintf(fx.
"<endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n", firstgap, (dna)? "base" : "residue", (firstgap == 1)? "" . "s", lastgap, (dna)? "base" : "residue", (lastgap = = I)? ~" . "s~);
else fprintf(fx, " < endgaps not penalized\n");
sGttic nm; l* matches in core -- for checking */

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

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

static nc[2]; 1* number at start of current line */

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

static siz[2];

static char *ps[2]: /* ptr to current element *1 static char *po[2]; /* ptr to next output char slot */

static char out[2][P I* output line *I
LINE];

static char star[P LINE];/* set by stars() *!

/*
* print aligtunent of described instruct path pp[]
*/
static pr alignQ
pr align ~t nn: /* char count */
int more;
register i;
for (i = 0, Imax = 0; i < 2; i++) {
nn = stripname(namex[i]);
if (nn > Imax) lmax = nn;
~fi] = I;
ni[i] = 1;
siz[i] = ij[i] = 0;
ps(i] = seqx[i];
po[i] = aut[i];
Page 3 of nwprint.c for (nn = nm = 0, more = 1; more: ) { ...pT align for (i = more = 0; i < 2; i++) {
/*
* do we have more of this sequence'?
*/
if (!*ps[il) continue;
more++;
if (pp[i].spc) { /* leading space */
*po[i]++ _ , PP[i].spc__;
else if (siz[i]) { I* in a gap *I
*po[i] + + _ siz[i]__;
else { l* we're puuing a seq element */
*Pofil = *Ps[i];
if (islower(*ps[i])) *ps[i] = toupper(*ps[i]);
po[i]++;
ps[i]++;
/*
* are we at next gap for this seq?
*/
if (ni[i] _= pp[i].x[ij[i]]) {
l*
* we need to merge all gaps * at this location */
siz[i] = pp[i].n[ij[i]++];
white (ni[i] _- pp[i].x[ijfilD
siz(i] += pp[i].n[ij[i]++];
ni[i]++;
if (++nn == olen ~ ~ !more 8r.8c nn) {
dumpblockQ;
for (i = 0; i < Z; i++) po[i] = out[i];
nn = 0;
/*
* dump a block of lines, including numbers, stars: pr align() */
static dttmpblackp dumpblock {
renter i;
for (i = 0; i < 2; i++) *po[i]__ ~ '10':
Page 4 of nwprint. c (void) putc('\n', fx);
for(i=O:i<2;i++){
if (*out[i] && (*out[i] ! _ ' ' I I *(po(i]) ! _ ' ')) {
if (i == 0) nums(i);
if (i == 0 && *out[1]) starsQ;
putline(i);
if (i = = 0 && *out[ 1 ]) fprintf(fx, star);
if (i == 1) nums(i);
...dumpblock /*
* pttt out a number line: dumpblockQ
*I
static nums(ix) nUlriS
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 < Imax+P SPC: i++, pn++) *pn = , for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
~(*PY =- ' I I *PY =- '-7 *Pn = , else {
if (i%10 == 0 I I (i == 1 && nc[ix] != I)) {
j = (i < 0)? -i : i;
for (px = pn; j; j /= 10, px--) *px = j% 10 + '0';
if (i < 0) *px = , else *Pn = , i++;
*pn = '\0';
ttc[ix] = i;
for (pn = nline; *pn; pn++) (void) putc(*pn, fx);
(void) putc('\n', fx);
/*
* put out a line (name, [num], seq, [num]): dumpblockQ
*I
static P~u~(~) putline iot ix;
{
Page 5 of nwprint.c ...putline int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px ! _ ':'; px++, i++) (void) putc(*px, fx);
for (; i < Imax+P SPC; i++) (void) putc(' ', fx);
/* these count from I:
* niQ 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);
/*
* put a line of stars (seqs always in out[O], out[1]): dumpblockQ
*/
static stars() Stll'S
{
int i;
register char *p0, *pl, cx, *px;
if (!*out(0] ~ ~ (*out[0] _- ' && *(po[O]) _- ' ') ~ ~
!*out(1] ~ ~ (*out[I] _ _ ' && *(Poll]) _- ' ')) return;
px = star;
for (i = Imax+P SPC; i; i--) *px++ _ , for (p0 = out[O], 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 else cx = , *px++ = cx;
*px++ _ '1n';
*Px = '\0~:
cx = , Page 6 of nwprint.c /*
* strip path or prefix from pn, return len: pr align() */
static stripname(pn) stripname char *pn; /* file name (may be path) */
register char *px, *py;
PY = ~:
for (px = pn; *px; px++) if (*px = _ '/') py=px+ 1;
(PY) (void) strcpy(pn, py);
return(strlen(pn));
Page 7 of nwprint.c /*
* cleanup() -- cleanup any tmp tile * getseqQ -- read in seq, set dna, len. maxlen * g callocQ -- callocp with error checkin * readjmpsQ -- get the good jmps, from tmp fife if necessary * writejmpsQ -- write a f-filled array of jmps to a tmp file: nwp */
Ninclude "nw.h"
~linclude < sys/file.h >
char *jname = "/tmp/homgXXXXXX"; !* tmp file for jmps */
FILE *tj;
int cleanupQ; /* cleanup tmp file */
long IseekQ:
/*
* remove any tmp file if we blow */
cleanup(i) cleanup int i:
{
if (tj) (void) unlink(jname);
exit(i);
/*
* read. return ptr to seq, set dna, len, maxlen * skip lines starting with ': , ' <', or ' >' * seq in upper or lower case */
char *
getseq(file, len) geltSeq char *file: I* file name */
int *len; l* seq fen */
{
char line[1024], *pseq;
register char *px, *py:
int natgc, tlen;
FILE *tp;
if ((fp = fopen(file."r")) _= 0) {
fprintf(stderr,"%s: can't read %s\n", grog, file):
exit(1);
tlen = natgc = 0:
while (fgets(line. 1024, fp)) {
if (*line =- .' ~ ~ *line =- ' <' ~ ~ *line =_ ' >') continue;
for (px = line; *px !_ '1n': px++) if (isupper(*px) ~ ~ islower(*px)) tlen++;
if ((pseq = malloc((unsigned)(tlen+6))) _= 0) {
fprintf(stderr,"%s: mallocQ failed to get %d bytes for %s\n", prog, tlen+6, file);
exit(1);
P~qI01 = P~qll] = Pseq[21 = Pseq[3] _ '\0':
Page 1 of nwsubr.c ...getseq py = pseq + 4:
*len = tlen;
rewind(fp):
while (fgets(line. 1024, fp)) {
if (*line =- .' ~ ~ *line = _ ' <' ( ~ *line =- ' >') continue:
for (px = line; *px ! _ '1n'; px++) {
if (isupper(*px)) *py + + _ *px;
else if (islower(*px)) *py++ = toupper(*px);
if (index("ATGCU".*(py-1))) natgc + + ;
*py++ _ '\0';
*py = '\0':
(void) fclose(fp):
dna = natgc > (tlen/3):
return(pseq+4);
char g calloc(msg, nx, sz) char *msg; /* program, calling routine */
int nx, sz; /* number and size of elements *l {
char *px, *callocp;
if ((px = calloc((unsigned)nx. (unsigned}sz)) _ = 0) {
if (*msg) {
fprintf(stderr. "%as: g callocQ failed %s (n=%d, sz=%d)1n", prog, msg, nx, sz);
exit(1);
return(px);
l*
* get final jmps from dx[] or tmp file, set pp[], reset dmax: main() */
readjmpsQ readjmps {
int fd = -I:
int siz, i0, i 1;
register i. j, xx;
if (fj) {
(void) fclose(fj);
if ((fd = open(jname. O_RDONLY, 0)) < 0) {
fprintf(stderr. %s: can't open() %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--) Page 2 of nwsubr.c ...readjmps if (j < 0 && dx[dmax].offset && tj) {
(void) Iseek(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, "9os: too many gaps in alignment\n", prog):
cleanup( I );
if (j > = 0) {
siz = dx[dmax].jp.n[j];
xx = dx[dmax).jp.x(j];
dmax += siz;
if (siz < 0) { /* gap in second seq */
PP[1].n[il] _ -siz;
xx + = siz:
/* id = xx - yy + lenl - 1 */
pp[1].x[il] = xx - dmax + lent - l;
gapy + + ;
ngapy -= siz;
I* ignore MAXGAP when doing endgaps *I
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;
I* ignore MAXGAP when doing endgaps *I
siz = (siz < MAXGAP ~ ~ endgaps)'? siz : MAXGAP:
i0++;
else break;
/* reverse the order of jmps */
for (j = 0, i0--: j < i0; j + + , i0--) {
i = PP[0l.nGl; PP[Ol.nU1 = PP[0].n[i0]; PP[Ol.n[i0] = i:
i = PP[OI~xG]; PP[OI.xG] = PP[0].x[i0); PP[0].x[i0] = i;
for (j = 0. il--; j < il; j++, il--) {
i = PP[1].nU]; PP[ll.nLl] = PP[ll.nlil]; PP[1].n[il] = i:
i = PP[1].x[j]; PP[ll.xLJ] = PP[11.x[il]: PP[ll.x[il] = i;
if (fd > = 0) (void) close(fd);
if (fj) {
(void) unlink(jname);
fj = 0;
offset = 0;
Page 3 of nwsubr.c /*
* write a tilled jmp struct offset of the prev one (if any): nwp */
writejmps(ix) writejmps int ix;
char *mktempQ;
if (!fj) {
if (mktemp(jname) < 0) {
fprintf(stderr, "%s: can't mktemp() %s\n", prog, jname);
cleanup(/);
if ((fj = fopen(jname, "w")) _ = 0) {
fprintf(stderr, " % s: can't write % stn", prog, jname);
exit( 1 );
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset. sizeof(dx[ixJ.offset), l, fj);
Page 4 of nwsubr.c Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparison Protein XXXXXYYYYYYY (Length = 12 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
poiypeptide) _ divided by 15 = 33.3 %

Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids) Comparison Protein XXXXXYYYYYYZZYZ (Length = IS 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) _ divided by LO = 50%

Table 2C
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 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (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 "Percent (°'°) amino acid sequence identity" with respect to the PR06S5. PR0364 and PR034.1 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO6SS, PR0364 or PR0344 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. ALIGN-2 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 obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-

2 program is provided in Table 1. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table I 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. 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.
For purposes herein, 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, Tables 2A-2B
demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein'' to the amino acid sequence designated "PRO".
Unless specifically stated othenvise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program.
However, % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res., 25:3389-3402 ( 1997)). The NCB/-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCB/-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values includin=, for example. unmask = yes, strand = all, expected occurrences = 10.
minimum low complexity length = I S!S, multi-pass e-value = 0.01, constant for multi-pass = 2S. 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 % amino acid sequence identity of A to B will not equal the %
amino acid sequence identity of B
to A.
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzvmoloav, 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 = O.I25, word threshold (T) = 11, and scoring matrix =
BLOSUM62. For purposes herein, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO poiypeptide 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 am ino 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 iriterest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
"PR0655 variant polynucleotide" or "PR06S5 variant nucleic acid sequence"
means a nucleic acid molecule which encodes an active PR06SS polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues I or about 22 to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), (b) a nucleic acid sequence which encodes amino acids X to 208 ofthe PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X
is any amino acid residue from 17 to 26 of Figure 2 (SEQ ID N0:2), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID N0:2).
Ordinarily, a PR0655 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at feast about 84°ro nucleic acid sequence identir<~, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues I or about 22 to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), (b) a nucleic acid sequence which encodes amino acids X to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X is any amino acid residue from 17 to 26 of Figure 2 (SEQ ID N0:2), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID N0:2). PR0655 polynucleotide variants do not encompass the native PR0655 nucleotide sequence.
"PR0364 variant polynucleotide" or "PR0364 variant nucleic acid sequence"
means a nucleic acid molecule which encodes an active PR0364 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues I or about 26 to 241 of the PR0364 polypeptide shown in Figure 4 (SEQ ID N0:7), (b) a nucleic acid sequence which encodes amino acids X to 241 ofthe PR0364 poiypeptide shown in Figure 4 (SEQ ID N0:7), wherein X
is any amino acid residue from 21 to 30 of Figure 4 {SEQ ID N0:7), (c) a nucleic acid sequence which encodes amino acids I or about 26 to X
of Figure 4 (SEQ ID N0:7), wherein X is any amino acid from amino acid 158 to amino acid I67 of Figure 4 (SEQ
ID N0:7) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ 1D N0:7). Ordinarily, a PR0364 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity' more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues I or about 26 to 2:1 I of the PR0364 polypeptide shown in Figure 4 (SEQ
ID N0:7), (b) a nucleic acid sequence which encodes amino acids X to 241 of the PR0364 polypeptide shown in Figure 4 (SEQ ID N0:7), wherein X is any amino acid residue from 21 to 30 of Figure 4 (SEQ ID N0:7), (c) a nucleic acid sequence which encodes amino acids 1 or about 26 to X of Figure 4 (SEQ ID N0:7), wherein X is any amino acid from amino acid 158 to amino acid 167 of Figure 4 (SEQ ID N0:7) or (d) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID N0:7).
PR0364 polynucleotide variants do not encompass the native PR0364 nucleotide sequence.
"PR0344 variant polynucleotide" or "PR0344 variant nucleic acid sequence"
means a nucleic acid molecule which encodes an active PR0344 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues I or about 16 to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17), (b) a nucleic acid sequence which encodes amino acids X to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17), wherein X
is any amino acid residue from 11 to 20 of Figure 6 (SEQ ID N0:17),or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID N0:17).
Ordinarily, a PR0344 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 16 to 243 of the PR0344 poiypeptide shown in Figure 6 (SEQ ID N0:17), (b) a nucleic acid sequence which encodes amino acids X to 243 of the PR0344 poiypeptide shown in Figure 6 (SEQ ID N0:17), wherein X is any amino acid residue from I 1 to 20 of Figure 6 (SEQ ID N0:17),or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID N0:17). PR0344 polynucleotide variants do not encompass the native PR0344 nucleotide sequence.
Ordinarily, PR0655, PR0364 and PR0344 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in ien~th. more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length. or more.
"Percent (%) nucleic acid sequence identity" with respect to the PR065~, PR0364 and PR0344 polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PR0655, PR0364 or PR0344 polypeptide-encoding nucleic acid sequence, 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, ALIGN-2 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, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table I
IS 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. TXUSi0087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided inTable I.' 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.
For purposes herein, 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 nucleicacidsequenceidentitycalculations,Tables 2C-2D demonstrate how to calculate the%nucleicacid sequence identity ofthe nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO-DNA".
Unless specifically stated otherwise. all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program.
However, % 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)). The NCBI-BLAST2 sequence comparison program may be downloaded from http:/iwww.ncbi.nlm.nih.gov. NCB/-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 NCB/-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 % nucleic acid sequence identity of D to C.
In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymolosy, 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 = l, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein, 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
pofynucleotide) 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 molecute of interest.
In other embodiments, PR0655, PR0364 and PR0344 variant polynucleotides are nucleic acid molecules that encode an active PR0655, PR0364 or PR0344 polypeptide, respectively, and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PR065~ polypeptide shown in Figure 2 (SEQ ID N0:2), to nucleotide sequences encoding the full-length PR0364 poiypeptide shown in Figure 4 (SEQ ID N0:7), to nucleotide sequences encoding the full-length PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17), respectively. PR0655, PR0364 and PR0344 variant polypeptides may be those that are encoded by a PR0655, PR0364 or PR0344 variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity comparisons performed as described above. includes amino acid residues in the sequences compared that are not only identical. but also those that have similar properties. Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest.
Por purposes herein, the % value of positives 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 % positives 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 scoring a positive value as defined above 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 % positives of A to B will not equal the % positives of B to A.
"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. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. 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 15 residues ofN-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 sire within recombinant cells, since at least one component of the PR0655, PR0364 or PR0344 natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
An "isolated" nucleic acid molecule encoding a PR0655, PR0364 or PR0344polypeptide or an "isolated"
nucleic acid molecule encoding an anti-PR0655, anti-PR0364 or anti-PR0344 antibody 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 ofthe PR0655-, PR0364- or PR0344-encoding nucleic acid or the anti-PR0655-, anti-PR0364- or anti-PR0344-encoding nucleic acid. Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated. An isolated PR0655-, PR0364- or PR0344-encoding nucleic acid molecule or an isolated anti-PR065 ~-, anti-PR0364- or anti-PR0344-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the PR0655-, PR0364- or PR0344-encoding nucleic acid molecule or from the anti-PR0655-, anti-PR0364- or anti-PR0344-encoding nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule encoding a PR0655, PR0364 or PR0344 polypeptide or an isolated nucleic acid molecule encodingan anti-PR0655, anti-PR0364 or anti-PR0344 antibody includes PR0655-,PR0364-orPR0344-nucleic acid molecules or anti-PR0655-, anti-PR0364- or anti-PR0344-nucleic acid molecules contained in cells that ordinarily express PR065S, PR0364 or PR0344 polypeptides or anti-PR06S5, anti-PR0364 or anti-PR0344 antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that ofnatural 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 known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably finked" 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 ligation 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-PR06SS, anti-PR0364 and anti-PR0344 monoclonal antibodies (including agonistantibodies),anti-PR0655,anti-PR0364 and anti-PR0344 antibody compositions with polyepitopic specificity, single chain anti-PR0655, anti-PR0364 and anti-PR0344 antibodies, and fragments of anti-PR06S5, anti-PR0364 and anti-PR0344 antibodies (see below). The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially 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 v Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that:
( l ) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chioride/0.001 S M
sodium citrate/0.1% sodium dodecyl sulfate at SO°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/O.I% Ficoll/0.1%

polyvinylpyrrolidone/~OmM 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, ~ x Denhardt's solution, sonicated salmon sperm DNA (50 pg/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 50% formamide at 55 °C, followed by a high-stringency wash consisting of0.1 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 ( 150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 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 PR0655, PR0364 or PR0344 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 am ino 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 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 lgM.
"Active" or "activity" for the purposes herein refers to forms) of PR0655, PR0364 or PR0344 which retain a biological andlor an immunological activity of native or naturally-occurring PR0655, PR0364 or PR0344, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring PR0655, PR0364 or PR0344 other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344.
"Biological activity" in the context of an antibody or another agonist that can be identified by the screening assays disclosed herein (e.g., an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to invoke one or more of the effects listed herein in connection with the definition of a "therapeuticallyeffective amount." In a specific embodiment, "biological activity" is the ability to inhibit neoplastic cell growth or proliferation. A preferred biological activity is inhibition, including slowing or complete stopping, of the growth of a target tumor (e.g., cancer) cell. Another preferred biological activity is cytotoxic activity resulting in the death ofthe target tumor (e.g., cancer) cell. Yet another preferred biological activity is the induction of apoptosis of a target tumor (e.g., cancer) cell.
The phrase "immunologicai activity" means immunological cross-reactivity with at least one epitope of a PR0655, PR0364 or PR0344 polypeptide.
"Immunological cross-reactivity" as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PR0655, PR0364 or PR0344 polypeptide having this activity with polyclonal antisera raised against the known active PR0655, PR0364 or PR0344 polypeptide.
Such antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds. The immunological cross-reactivity preferably is "specific". which means that the bindingaffinity ofthe immunologically cross-reactive molecule (e. g., antibody) identified, to the corresponding PR0655, PR0364 or PR0344 polypeptide is significantly higher(preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 6-times, most preferably at least about 8-times higher) than the binding affinity of that molecule to any other known native polypeptide.
"Tumor", as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
The "pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response. etc.
An "effective amount" of a polypeptide disclosed herein or an agonist thereof, in reference to inhibition of neoplastic cell growth, is an amount capable of inhibiting, to some extent, the growth of target cells. The term includes an amount capable of invoking a growth inhibitory, cytostatic andlor cytotoxic effect and/or apoptosis of the target cells. An "effective amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
A "therapeutically effective amount", in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects: ( 1 ) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest: (2) reduction in the number of tumor cells; (3) reduction in tumor size;
(4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs;
(5) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; and/or (7) relief, to some extent, of one or more symptoms associated with the disorder.
A "therapeutically effective amount"
of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes of treatment of tumor may be 1S determined empirically and in a routine manner.
A "growth inhibitory amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A
"growth inhibitory amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
A "cytotoxic amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A
"cytotoxic amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
The term "cytotoxic anent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I"', I'=', Y9° and Re'e°), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of tumor, e.g., cancer.
Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers SquibbOncology, Princeton, NJ), and doxetaxel (Taxotere, Rhdne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, meiphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (see, U.S. Patent No. 4,675,187), melphalan and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.
A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially tumor, e.g., cancer cell. either in vitro or in viva.
Thus, the growth inhibitory agent is one which significantly reduces the percentage of the target cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G 1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II
inhibitors such as doxorubicin, epirubicin. daunorubicin, etoposide, and bleomycin. Those agents that arrest G 1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, S-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds., Chapter I, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al., (WB Saunders: Philadelphia, 1995), especially p. 13.
The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin;
proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor;
proiactin; placental lactogen; tumor necrosis factor-a and -p; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as NGF-(i; platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and TGF-p; insulin-like growth factor-1 and -fI; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-a, -Vii, and -y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-S, IL-6, IL-7, IL-8, IL-9, IL-11, IL-I2; a tumor necrosis factor such as TNF-a or TNF-Vii; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions, 14, pp. 375-382, 6lSth Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery,"
Directed Drug Delivery. Borchardt et al., (ed.), pp. 247-267, Humana Press ( 1985). The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, glycosylated prodrugs or optionally substituted phenylacetamide-containing prodrugs, S-fluorocytosine and other 5-fluorouridine prodrugs which can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
The term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native PR06SS, PR0364 or PR0344 poiypeptide disclosed herein.
Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PR0655, PR0364 or PR0344 polypeptides, peptides, small organic molecules, etc.
Methods for identifying agonists of a PR0655, PR0364 or PR0344 polypeptide may comprise contacting a tumor cell with a candidate agonist molecule and measuring the inhibition of tumor cell growth.
"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, hut rather is cyclic in nature.
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and fatrrt 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;
low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpytroiidone; 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 PLURONICSTM
"Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (V,,) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarily-detetmtining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a (i-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the (3-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et al., NIH Publ. No.91-3242, Vol. I, pages 647-669 (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarily determining region" or "CDR" (i.e., residues 24-34 (L 1 ), SO-S6 (L2) and 89-97 (L3) in the light chain variable domain and 31-3S (H1), SO-65 (H2) and 9S-102 (H3) in the heavy chain variable domain; Kabat et al., Seauences of Proteins of Immunolo~,ical Interest, Sth Ed. Public Health Service, National Institute of Health, Bethesda, MD. [ 1991 j) and/or those residues from a "hypervariable loop" (i.
e., residues 26-32 (L 1 ), SO-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H 1 ), 53-SS (H2) and 96-101 (H3) in the heavy chain variable domain; Clothia and Lesk, J. Mol. Biol., 196:901-917 [1987]).
"Framework" or"FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
"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 Ens;., 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')Z 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 ofone heavy- and one tight-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 (CH I ) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH 1 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')z antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Outer chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one oftwo 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, lgE, IgG, and IgM, and several ofthese may be further divided into subclasses (isotypes), e.g., IgG I, IgG2, IgG3, IgG4, IgA, and IgA2.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially 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. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthetzttore, in contrast to conventional (polyclonal) antibody preparationswh ichtypically includedifferent antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. in addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monocional antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature. 256:495 [ 1975], ormay be made by recombinant DNA methods (see, e.g., U.S. Patent No.
4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, x:624-628 [1991] and Marks et al., J. Mol. Biol.. 222:581-597 (1991), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder ofthe chains) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison er al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 [ 1984J).
"Humanized" fotrns of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a 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 FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, 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 sequence. The humanized antibody optimally also will comprise at least a portion of an immunoelobulin constant region (Fc), typically that ofa human immunoglobulin. For further details, see, Jones et al., Nature. X21:522-525 (1986); Reichmann et al., Nature.
X32:323-329 [1988]; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanized antibody includes a PR1MATIZEDTMantibodywhereinthe antigen-binding region ofthe antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
"Single-chain Fv" or "sFv" antibody fragments comprise the V" and V~ 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 Pharmacoloay of Monoclonal Antibodies, Vol. 113. Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V") 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 andlor 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 enrymes, 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-tenminal 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.
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 (eg., 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. The label may also be a non-detectable entity such as a toxin.
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 ofvarious types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PR0655, PR0364 or PR0344 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.
S A "small molecule" is defined herein to have a molecular weight below about 500 Daltons.
II. Comt~ositions and Methods of the Invention A. Full-len th h PR0655. PR0364 and PR0344 Polvpeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0655, PR0364 and PR0344. In particular, cDNAs encoding PR0655, PR0364 and PR0344 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below.
As disclosed in the Examples below, cDNA clones encoding PR0655, PR0364 and PR0344 polypeptides have been deposited with the ATCC. The actual nucleotide sequences of the clones can readily be determined by the skilled artisan by sequencing of the deposited clones using routine methods in the art. The predicted amino acid sequences can be determined from the nucleotide sequences using routine skill.
For the PR0655, PR0364 and PR0344 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 lime.
B. PR0655. PR0364 and PR0344 Variants In addition to the full-length native sequence PR0655, PR0364 and PR0344 polypeptides described herein, it is contemplated that PR0655, PR0364 and PR0344 variants can be prepared. PR0655, PR0364 and PR0344 variants can be prepared by introducing appropriate nucleotide changes into the PR0655, PR0364 or PR0344 DNA, and/or by synthesis of the desired PR0655, PR0364 or PR0344 polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PR0655, PR0364 or PR0344 polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PR0655, PR0364 or PR0344 or in various domains of the PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 that results in a change in the amino acid sequence of the PR0655, PR0364 or PR0344 as compared with the native sequence PR0655, PR0364 or PR0344. 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 PR0655, PR0364 or PR0344. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PR0655, PR0364 or PR0344 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 I 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.
PR0655, PR0364 and PR0344 polypeptide 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 biological activity of the PR0655, PR0364 or PR0344 polypeptide.
PR0655, PR0364 and PR0344 fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PR0655, PR0364 and PR0344 fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enrymes 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 S' and 3' primers in the PCR. Preferably, PR0655, PR0364 and PR0344 polypeptide fragments share at least one biological and/or immunological activity with the native PR06SS, PR0364 or PR0344 polypeptide shown in Figure 2 (SEQ ID
N0:2), Figure 4 (SEQ ID N0:7), and Figure 6 (SEQ 1D N0:17), respectively.
In particular embodiments, conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened.

Table 3 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his: lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn 10Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg lle (I) leu; val: met; ala; phe;

norleucine leu 15Leu (L) norleucine; ile; val;

met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu 20Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe 25Val (V) iie; ieu; met; phe;

ala; norleucine leu Substantial modifications in function or immunological identity of the PR0655, PR0364 or PR0344 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 30 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;
35 (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 40 the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as ofigonucleotide-mediated (site-directed) mutagenesis, alanine scanning; and PCR mutaoenesis. Site-directed mutagenesis [Carter et al., Nucl.
Acids Res.. 13:4331 ( 1986); Zoller et al., Nucl. Acids Res.. 10:6487 ( 1987)], cassette mutagenesis [Wells er al., Gene. 34:315 ( 1985)], restriction selection mutagenesis [Wells et a/., Philos. Traps. R. Soc. London SerA _317:415 ( 1986)] or other known techniques can be performed on the cloned DNA to produce the PR0655, PR0364 or PR0344 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 [Cunnineham 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 PR0655 PR0364 and PR0344 Covalent modifications of PR0655, PR0364 and PR0344 are included within the scope of this invention.
One type of covalent modification includes reacting targeted amino acid residues of a PR0655, PR0364 or PR0344 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PR0655, PR0364 or PR0344. Derivatization with bifunctional agents is useful, for instance, for crosslinking PR0655, PR0364 or PR0344 to a water-insoluble support matrix or surface for use in the method for purifying anti-PR0655, anti-PR0364 or anti-PR0344 antibodies, and vice-versa. Commonly used crosslinkingagents include, e.g., I,1-bis(diazoacetyl)-2-phenylethaneglutaraldehyde,N-hydroxysuccinimide esters, forexampie, esters with 4-azidosalicylicacid, homobifunctional imidoesters, inciuding 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 aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups ofseryl orthreonyl 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 PR0655, PR0364 or PR0344 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native giycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PR0655, PR0364 or PR0344 (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 PR0655, PR0364 or PR0344. 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 PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 (for O-linked glycosylation sites). The PR0655, PR0364 or PR0344 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide is by chemical or enzymatic coupling of glycosides to the pofypeptide. Such methods are described in the art, e.g., in WO 87/05330 published I 1 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem..
pp. 259-306 ( 1981 ).
Removal of carbohydrate moieties present on the PR0655, PR0364 or PR0344 polypeptide may be accompl fished chem ically or enzymatically or by mutational substitution of codons encoding for am ino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance. by Hakimuddin, et al., Arch. Biochem. Biophvs., 259:52 ( 1987) and by Edge et al., Anal. Biochem..
I 18:131 ( 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 al., Meth.
Enzvmol.. 138:350 (1987).
Another type of covalent modification of PR0655, PR0364 or PR0344 comprises linking the PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide of the present invention may also be modified in a way to form a chimeric molecule comprising PRO655, PR0364 or PR0344 fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PR0655, PR0364 or PR0344 polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
The epitope tag is generaliy placed at the amino- or carboxyl- terminus of the PR0655, PR0364 or PR0344 polypeptide. The presence of such epitope-tagged forms of the PR0655, PR0364 or PR0344 polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PR0655, PR0364 or PR0344 polypeptide 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 er al., Mol. Cell.
Biol., 8:2159-2165 ( 1988)]; the c-myc tag and the SF9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Bioloev.

5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky e~ al., Protein EnQineerinQ. 'y6 :547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp er al., Bio'TechnoloQV. 6: I 204-1210 ( 1988)]; the KT3 epitope peptide [Martin et al., Science. 255: I 92-194 ( 1992)]; an WO 00/32778 PC'T/US99/28409 a-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266: I 5 I 63-1 S I
66 ( 1991 )]; and the T7 gene ! 0 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 PR0655, PR0364 or PR0344 polypeptide 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 Lhe Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PR0655, PR0364 or PR0344 polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH I, CH2 and CH3 regions of an (gG I molecule. For the production of immunoglobulin fusions see also, US Patent No. 5,428,130 issued June 27, 1995.
D. Preuaration of PR0655, PR0364 and PR0344 The description below relates primarily to production of PR0655, PR0364 or PR0344 by culturing cells transformed or transfected with a vector containing PR0655, PR0364 or PR0344 nucleic acid. it is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PR0655, PR0364 or PR0344. For instance, the PR0655, PR0364 or PR0344 polypeptide 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 PR0655, PR0364 or PR0344 polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PR0655, PR0364 or PR0344 polypeptide.
Isolation of DNA Encoding PR0655, PR0364 or PR0344 DNA encoding PR0655, PR0364 or PR0344 may be obtained from a cDNA library prepared from tissue believed to possess the PRO655, PR0364 or PR0344 m RNA and to express it at a detectable level. Accordingly, human PR0655, human PR0364 or human PR0344 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The PR0655-, PR0364- or PR0344-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 PR0655, PR0364 or PR0344 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 Clonine: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press. 1989). An alternative means to isolate the gene encoding PR0655, PR0364 or PR0344 is to use PCRmethodology [Sambrook er al., supra; Dieffenbach etal.. 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 it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like'=P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within defined regions of the 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 e~ al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
2. Selection and Transformation of Host Cells Host cells are uansfected or transformed with expression or cloning vectors described herein for PR0655, PR0364 or PR0344 production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnoloev: a Practical Apuroach, M. Butler, ed. (IRL Press, 1991 ) and Sambrook et al., supra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCI=, 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 er al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tnmefaciens is used for transformation of certain plant cells, as described by Shaw er al., Gene. 23:315 ( 1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Viroloev, 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 ( I 977) 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 3S protoplast fusion with intact cells, or polycations, e.g., polybrene, polyomithine, may also be used. For various techniques for transforming mammalian cells, see, Keown et al., Methods in Enzvmolow, I 85:527-537 ( I 990) and 5$

Mansour et al., Nature. 336:348-352 ( I 988).
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. toll strains are publicly available, such as E. toll K 12 strain MM294 (ATCC 31,446); E. toll X 1776 (ATCC 31..537); E. toll strain W3110 (ATCC 27,325) and KS 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. toll, Enterobacter, Erwinia, Klebsiella, Proteus.
Salmonella, e.g.. Salmonellatvphimuritrm, Serratia, e.g., Serratia nrarcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD ?66,710 published 12 April 1989), Pseudomonas such as P. aertrginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W31 I 0 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.
toll W31 10 strain I A2, which has the complete genotype tonA ; E. toll W31 10 strain 9E4, which has the complete genotype tonA ptr3; E. toll W31 t0 strain 27C7 (ATCC 55,244). which has the complete genotype tonAptr3 phoA
EI S (argF lac) 169 degP ompT karf; E. toll W3110 strain 37D6, wh ich has the complete genotype tonA ptr3 phoA
EIS (argF lac)169 degP ompT rbs7 ilvG karr ; E. toll W3110 strain 4084, which is strain 37D6 with a non kanamycin resistant degP deletion mutation; and an E. toll 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 polymerise reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PR0655-, PR0364- or PR0344-encoding vectors.
Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schcosaccharomyces pombe (Beach and Nurse, Nature. 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluymeromyces hosts (U.S. Patent No. 4,943,529;
Fleer et al., Bio/Technolow, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574;
Louvencourt et al., J. Bacteriol.. 737 [ 1983]), K. fragilis (ATCC 12,424), K.
bulgaricrrs (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilurum (ATCC
36,906; Van den Berg et al., Bio/Technolosy. 8: I 35 ( 1990)), K. thermotolerans, and K. marxiantrs;
yarrowia (EP 402.236); 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. Acid. Sci. USA, 76:259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991 ), and. Aspergillus hosts such as A. nidnlans (Ballance et al., Biochem. Biophvs. Res. Commun., I 12:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]: Yelton et al., Proc. Natl. Acid. Sci. USA 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J.. ~t:47S-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. Sacclurronrrces, Torrrlopsis, 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 PR0655, PR0364 or PR0344 are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO}
S and COS cells. More specific examples include monkey kidney CV I 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, Masher. Biol.
Renrod., 23:243-251 ( 1980)); human lung cells (W 138, ATCC CCL ?5); 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.
Selection and Use of a Reolicable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding PR0655, PR0364 or PR0344 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 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 term ination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
The PR0655, PR0364 or PR0344 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 PR0655-. PR036d- or PR0344-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, ipp, or heat-stable enterotoxin II
leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomvces and Kluyveromvces 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 polypeptides of the same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2u 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, c.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 PR0655-, PR0364- or PR0344-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 trpl 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 trpl 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 ( t 977)].
Expression and cloning vectors usually contain a promoter operably finked to the PR0655-, PR0364- or PR0344-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety ofpotential 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, 28 I :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.
Sci. USA. 80:21-35 ( t 983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding PR0655, PR0364 or PR0344.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem.. 255:2073 ! 1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Red 7:149 ( 1968); Holland, Biochemistry, 17:4900 ( 1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase. pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
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.
PR0655. PR0364 or PR0344 transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowipox virus (UK 2,211,504 published S July 1989), adenovirus (such as Adenovirus 3), 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 PR0655, PR0364 or PR0344 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, onewill use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer 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 S' or 3' to the PR0655, PR0364 or PR0344 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 oftranscription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslatedregionsofeukaryoticorviraIDNAsorcDNAs.
Thesere~ionscontainnucleotidesegmentstranscribed I 5 as polyadenylated fragments in the untranslated portion of the mRNA
encoding PRO655, PR0364 or PR0344.
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PR0655, PR0364 or PR0344 in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981);
Mantel et al., Nature, 281:40-46 ( 1979); EP 117,060; and EP I 17,058.
4. Detecting Gene Am~ificationlExoression 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 siW 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 immunohistochemical 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 PR0655, PR0364 or PR0344 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PR0655, PR0364 or PR0344 DNA and encoding a specific antibody epitope.
5. Purification of Polvpeptide Forms of PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 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 canon-exchange resin such as DEAE; 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 PR0655, PR0364 or PR0344. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzvmoloev, I 82 ( 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 PR06S5, PR0364 or PR0344 produced.
E. Antibodies Some drug candidates for use in the compositions and methods of the present invention are antibodies and antibody fragments which mimic the biological activity of a PR065S, PR0364 or PR0344 polypeptide.
Polvclonal 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 PR06S5, PR0364 or PR0344 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 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 PR06~5. PR0364 or PR0344 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 [coding, Monoclonal Antibodies: Principles and Practice. Academic Press, ( t 986) pp. 59-103]. Immortalized cell lines are usually transformed mammaliancel(s, particularly myelomacells ofrodent, 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 enryme 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 Die_o, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described forthe production of human monoclonal antibodies [Kozbor, J. Immunoi., 133:3001 ( 1984); Brodeur et al., Monoclonal Antibody Production Techniaues and Applications, Marcel Dekker. Inc., New York, (1987) pp. S1-63].
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PR0655, PR036.1 or PR0344. 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, supra]. 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 can be 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 murine sequences [U.S.
Patent No. 4,816,567: Morrison et al., supra) or by covalently joinin; to the immunoglobulin coding sequence all or pan 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 known 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 crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion ofantibodiesto produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
Human and Humanized Antibodies The antibodies of the invention may further comprise humanized antibodies or human antibodies.
Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) ofthe 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 immunoglobulin 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 typical ly two, variable domains.
in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and al!
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 ul., Nature, 321:522-~2~ ( 1986);
Riechmann e~ al., Nature, 332:323-329 ( 1988); and Presta, Curr. Op. Struct. Biol.. ?:593-596 ( 1993)].
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 referred 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, 331:522-525 (1986}: Riechmann er al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 339: I 534-1536 ( 1988)), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No.
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): Marks 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 Theranv. Alan R.
Liss, p. 77 ( 1985) and Boerner et al., J. lmmunol., 147 l :86-95 (1991)]. Similarly, human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes 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/Technolow, 10: 779-783 ( 1992); Lonberg et al., Nature. 368: 856-859 ( 1994); Morrison. Nature, 368:
812-13 ( I 994); Fishwild et al., Nature Biotechnoloev, 14:845-51 ( 1996); Neuberger, Nature Biotechnoloav. 14: 826 ( 1996); Lonberg and Huszar, Intern.
Rev. Immunol., I 3 :6S-93 ( 1995).
4. Bisnecific Antibodies Bispecific 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 specificities is for the PR065S, PR0364 or PR0344, 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 known in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of two immuno~lobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 ( 1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., I 0:3655-3659 ( t 991 ).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge. CH?, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH 1 ) 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 detailsofgeneratingbispecificantibodiessee, for example, Suresh erul., Methods in Enzvmoloev 121:210(1986).
According to another approach described in WO 96/2701 1. the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a pan 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 ofthe 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'), bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229:81 ( I 985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')= fragments. These fragments are reduced in the presence of the dithio) 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. toll and chemically coupled to form bispecific antibodies. Shalaby e~ al., J. Ex~. Med., 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')_ molecule. Each Fab' fragment was separately secreted from E. toll and subjected to directed chemical coupling in virro 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 tar_ets.
Various techniques 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 e~ 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 wdiabody"
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 pairin; between the two domains on the same chain.
Accordingly, the V" and V~ domains of one fragment are forced to pair with the complementary V~ and V" 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..
I X2:5368 ( 1994 ).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et aL, J. Immunol., Id7:60 ( 1991 ).
Exemplarv_ bispecific antibodies may bind to two different epitopes on a given PR0655, PR0364 or PR0344 polypeptide herein. Alternatively, an anti-PR06~.i. anti-PR0364 or anti-PR0344 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 (FcyR), such as FcyRI
(CD64), FcyRl1 (CD32) and FcyRl l l {CD 16) so as to focus cellular defense mechanisms to the cel I
expressing the particular PR0655, PR0364 or PR0344 polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PR0655, PR0364 or PR0344 polypeptide. These antibodies possess a PR0655-, PR0364- or PR0344-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 PR0655, PR0364 or PR0344 polypeptide and further binds tissue factor (TF).
1$ 5. Heteroconiugate 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. 1,676,980], 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 involvingcrosslinkingagents. 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-d-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 cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See, Caron et al., J. Ex~. Med., 176:
I 191-1195 ( 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. Sc~e, Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 ( 1989).
6s Immunoconiugates The invention also pertains to immunoconjugates comprising an antibody conjugated toacytotoxic 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.
Enrymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonus aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phyrolaca americarra proteins (PAPI, PAPI1, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ='=Bi. "'l, "'In, ~°Y, and'B6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate(SPDP).
iminothiolane(IT),bifunctionalderivatives of imidoesters (such as dimethyladipimidate 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 I,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-isothiocyanatobenryl-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 as 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. Immunolinosomes The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein er aL, Proc. Natl. Acad. Sci. USA
82: 3688 (1985); Hwang e~ aL, Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. 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 ofdefined 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., J. Biol. Chem., 257: 386-288 (1982) via a disulfide-interchange reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. .See, Gabizon e~ al, J. National Cancer Inst.. 81 ( 19):
1484 ( 1989).

Identification of Proteins Capable of Inhibiting Neoplastic Cell Growth or Proliferation The proteins disclosed in the present application have been assayed in a panel of 60 tumor cell lines currently used in the investigational, disease-oriented, in vi~ro drug-discovery screen of the National Cancer Institute (NCI). The purpose of this screen is to identify molecules that have cytotoxic andlor cytostatic activity $ against different types of tunl,ors. NCI screens more than 10,000 new molecules per year (Monks el al., J. Natl.
Cancer Inst., 83:757-766 ( 1991 ): Boyd, Cancer: Princ. Pract. Oncol. Update, 3 10 : I-12 ([ 1989]). The tumor cell lines employed in this study have been described in Monks et al.. supra. The cell lines the growth of which has been significantly inhibited by the proteins of the present application are specified in the Examples.
The results have shown that the proteins tested show cytostatic and, in some instances and concentrations, cytotoxic activities in a variety of cancer cell lines, and therefore are useful candidates for tumor therapy.
Other cel I-based assays and animal models for tumors (e.g., cancers) can also be used to verify the findings of the NCI cancer screen, and to further understand the relationship between the protein identified herein and the development and pathogenesis of neoplastic cell growth. For example, primary cultures derived from tumors in transgenic animals (as described below) can be used in the cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e.g., Small et al., Mol. Cell. Biol.. 5_:642-648 [ 1985]).
G. Animal Models A variety of well known animal models can be used to further understand the role of the molecules identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other agonists ofthe native polypeptides, including small molecule agonists. The in vivo nature of such models makes them particularly predictive of responses in human patients. Animal models of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g.. colon cancer cells implanted in colonic tissue.
(See, e.g., PCT publication No. WO 97/33551, published September 18, 1997).
Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice. The observation that the nude mouse with hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose. The autosomal recessive ntr gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD. I/st, NC, NFR, NFS, NFSM, NZB, NZC, NZW, P, RIII and SJL. In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts. For further details see, e.g., The Nude Mouse in Oncoloev Research, E. Boven and B. WinoVrad, eds., CRC
Press. lnc., 199(.
The cells introduced into such animals can be derived from known tumor/cancer cell lines. such as, any of the above-listed tumor cell lines, and, for example. the 8104- I-1 cell line (stable NIH-3T3 cell line transfected with the ne:r protooncogene); ras-transfected NIH-3T3 cells: Caco-2 (ATCC HTB-37): a moderately well-differentiated grade II human colon adenocarcinoma cell line. HT-29 (ATCC HTB-38), or from tumors and cancers. Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard S conditions, involving freezing and storing in liquid nitrogen (Karmali et al., Br. J. Cancer, 48:689-696 [1983)).
Tumor cells can be introduced into animals. such as nude mice, by a variety of procedures. 'Fhe subcutaneous (s.c.) space in mice is very suitable for tumor implantation.
Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions. For sol id block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s.c. space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells can also be injected as subdermal implants. In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s.c. tissue. Boven and Winograd ( 1991 ), supra. Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or neu-transformed NIH-3T3 cells into nude mice. essentially as described by Drebin et al.. Proc. Natl. Acad. Sci. USA.
83:9129-9133 ( 1986).
Similarly, animal models ofcolon cancer can be generated by passaging colon cancer cells in animals, e.g., nude mice, leading to the appearance of tumors in these animals. An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al., Cancer Research. 54:4726-4728 ( 1994) and Too et al., Cancer Research. 55:681-684 ( 1995). This model is based on the so-called "METAMOUSE" sold by Anticancer, Inc., (San Diego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from the in virro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines.
For example, Meth A, CMS4, CMSS, CMS21, and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et aL, J. Exo. Med., 146:720 [ 1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Palladino et al., J. Immunol.. 138:4023-4032 [ t 987]). Briefly, tumor cells are propagated in vitro in cell culture. Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about 1 Ox 10°
to I Ox 10' cells/ml. The animals are then infected subcutaneously with 10 to I 00 ,ul of the cell suspension, allowing one to three weeks for a tumor to appear.
In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model. Efficacy in this tumor model has been correlated with benefcial effects in the treatment ofhuman patients diagnosed with small cell carcinoma ofthe lung (SCCL). This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al., Br. J. Cancer. 41, suppl.
4:309 [ 1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive. For further information about this tumor model see, Zacharski, Haemostasis, 16:300-320 [1986]).
One way of evaluating the efficacy of a test compound in an animal model on an implanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions. The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually convened into the corresponding volume by using a mathematical formula. However, the measurement of tumor size is very inaccurate. The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay. Another important variable in the description of tumor growth is the tumor volume doubling time. Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals Wu and Sheng gds., Basel, 1989, 301.
It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially. Therefore, these changes need to be carefully monitored, by a combination of a morphometric method and flow cytometric analysis.
Recombinant (transgenic) animal models can be engineered by introducing the coding portion ofthe genes identified herein into thegenomeofanimalsofinterest,usingstandardtechniquesforproducingtransgenicanima ls.
Animals that can serve as a target for transeenic manipulation include.
without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys. Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U.S.
Patent No. 4,873,191 ); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al., Proc. Natl.
Acad. Sci. USA, 82:6148-615 [1985]); gene targeting in embryonic stem cells (Thompson etal., C~ 56:313-321 [1989]); electroporation of embryos (Lo, Mol. Cell. Biol.. 3_:1803-18t4 (1983]); sperm-mediated gene transfer (Lavitrano et al., Cell, 57:717-73 [ 1989]). For review, see, for example, U.S. Patent No. 4,736,866.
For the purpose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals"). The transgene can be integrated either as a single transgene, or in concatamers, e.g., head-to-head or head-to-tail tandems. Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko er al., Proc. Natl. Acad. Sci. USA. _89:6232-636 ( 1992).
The expression of the trans~ene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry. The animals are further examined for signs of tumor or cancer development.
The efficacy of antibodies specifically binding the polypeptides identified herein and other drug candidates, can be tested also in the treatment of spontaneous animal tumors.
A suitable target for such studies is the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy ofcats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short WO 00/32778 PC'f/US99/28409 survival times for cats with this tumor. These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor. Prior to entry into the study, each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT).
Cats diagnosed with sublingual oral squarnous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor. the animals may not be able to feed themselves. Each cat is treated repeatedly, over a longer period of time.
Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck. A fter treatment. each cat undergoes another CT
scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter.
The data are evaluated for differences in survival, response and toxicity as compared to control groups.
Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested. Of these mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans. However, the use of this model is limited by the rare occurrence of this type of rumor in animals.
H. Screenine Assavs for Drue Candidates Screening assays for drug candidates are designed to identify compounds that competitively bind or complex with the receptors) of the polypeptides identified herein, or otherwise signal through such receptor(s).
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, including peptides, preferably soluble peptides, (poly)peptide-immunoglobulin fusions, 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. 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.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, a receptor of a 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 polypeptide and drying. Alternatively, an immobilized antibody. e.g., a monoclonal antibody, specific for the 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 sol id 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.
Ifthe candidate compound interacts with but does not bind to a particular receptor, its interaction with that polypeptide can be assayed by methods wel I known for detectin_ protein-protein interactions. Such assays include traditional approaches, such as. 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 Sone. Nature (London), 340:245-246 ( 1989); Chien et al., Proc. 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, while 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 ofGAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL t-lacZ
reporter gene under control of a GAL4-activated promoter depends on reconstitution of GALA activity via protein-protein interaction. Colonies 1 S containing interacting poiypeptides are detected with a chromogenic substrate for [i-galactosidase. A complete kit (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.
I. Pharmaceutical Compositions The potypeptides of the present invention, agonist antibodies specifically binding proteins identified herein, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions.
Where antibody fragments are used, the smallest inhibitory fragment which 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 which 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 er al., Proc. Natl. Acad. Sci. USA 90:7889-7893 [1993]).
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or _rowth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
Therapeutic formulations ofthe polypeptides identified herein, oragonists thereof are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Renrinmnn's PHarmaceurica! 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 andmethionine:preservatives(suchasoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride: benzalkonium chloride, benzethonium chloride; phenol. butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben: catechol;
resorcinol; cyclohexanol; 3-pentanol;
and m-cresol); low molecular weight (less than about 10 residues) polvpeptides: proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparasine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose. mannose, or dextrins: chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol: salt-forming counter-ions such as sodium: metal complexes (e.g.~ Zn-protein complexes);
and/or non-ionic surfactants such as TWEENT", PLURONICST" or polyethylene glycol (PEG).
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 a cytotoxic agent, cytokine 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 ReminQton's Pharmaceutical Sciences, 16th edition, Osol, A.
ed. ( 1980).
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.
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 DEPOTT"
(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, controllin~z moisture content. using appropriate additives, and developing specific polymer matrix compositions.
Methods of Treatment It is contemplated that the polypeptides of the present invention and their agonists, including antibodies, peptides, and small molecule agonists, may be used to treat various tumors, e.g., cancers. Exemplary conditions or disorders to be treated include benign or malignant tumors (e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal. prostate, pancreatic, lung> vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; and various head and neck tumors); leukemias and lymphoid malignancies; other disorders such as neuronal, glial, astrocytai, hypothalamic and other glandular. macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders. The anti-tumor agents of the present invention (including the polypeptides disclosed herein and agonists which mimic their activity, e.g., antibodies, peptides and small organic molecules), are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intracerobrospinal, intraocular.
intraarterial.intralesional,subcutaneous,intraarticular,intrasynovial>
intrathecal, oral, topical, or inhalation routes.
Other therapeutic regimens may be combined with the administration of the anti-cancer agents of the instant invention. For example, the patient to be treated with such anti-cancer agents may also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotheraov Service, ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may precede, or follow administration of the anti-tumor agent of the present invention, or may be given simultaneously therewith. The anti-cancer agents of the present invention may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
It may be desirable to also administer antibodies against tumor associated antigens, such as antibodies which bind to the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies binding the same or.two or more different cancer-associated antigens may be co-administered to the patient. Sometimes, it may be beneficial to also administer one or more cytokines to the patient.
In a preferred embodiment, the anti-cancer agents herein are co-administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by the administration of an anti-cancer agent of the present invention. However, simultaneous administration or administration of the anti-cancer agent of the present invention first is also contemplated. Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the antibody herein.
For the prevention or treatment of disease, the appropriate dosage of an anti-tumor agent herein will depend on the type of disease to be treated. as defined above. the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling ofeffective 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 Drus Development, Yacobi et al.. eds., Pergamon Press, New York 1989, pp. 42-96.
For example. depending on the type and severity of the disease, about I ,ug/kg to (5 mg/kg (e.g., 0.1-20 mg/kg) of an antitumor agent is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about I ~cg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be useful. The progress ofthis therapy is easily monitored by conventional techniques and assays. 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.
K. Articles of Manufacture In another embodiment of the invention, an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic.
The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is an anti-tumor agent of the present invention. The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice. The article of manufacture may further comprise a second container comprising a phatntaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, f Iters, needles, syringes, and package inserts with instructions for use.
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 CultureCollection.Manassas, VA.

Isolation of cDNA clones Encoding PR065~. PR0364 and PR0344 (A) PR0655 An expressed sequence tag (EST) DNA database (Ll FESEQh . ! ncyte Phamaceuticals, Palo Alto. CA) was searched and an EST was identified which showed homology to interferon.
Possible homology was noted between Incyte EST 3728969 (subsequently renamed as DNA49668) and mammalian alpha interferons. The homology was confirmed by inspection.
RNA for construction of cDNA libraries was then isolated from human small intestine (LIB 99). The cDNA libraries used to isolate the cDNA clones encoding human PR0655 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 Notl site, linked with blunt to Sall hemikinased adaptors, cleaved with Noti, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain the SfiI
site; see, Holmes ei al., Science, 253:1278-1280 ( 1991 )) in the unique Xhol and Notl.
Oligonucleotides probes based upon the above described EST sequence were then synthesized: 1 } to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO655. 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 40-55 by in length. 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 Protocols in Molecular Bioloey, supra, 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 oligonucleotide probes employed were as follows:
reverse PCR primer 1:
5'-TCTCTGCTTCCAGTCCCATGAGTGC-3' (SEQ ID N0:3) reverse PCR primer 2:
5'-GCTTCCAGTCCCATGAGTGCTTCTAGG-3' (SEQ 1D N0:4) hvbridization probe 5'-GGCCATTCTCCATGAGATGCTTCAGCAGATCTTCAGCCTCTTCAGGGCAA-3' (SEQ ID NO:S) A full length clone was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 621-623 and a stop signal at nucleotide positions 1245-1247 (Figure I, SEQ
ID NO:1 ). The predicted polypeptide precursor is 208 amino acids long, and has a calculated molecular weight of approximately 24,414 daltons and an estimated p1 of approximately 8.92.
Analysis of the full-length PR0655 sequence shown in Figure 2 (SEQ ID N0:2) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0655 sequence (Figure 2; SEQ ID N0:2) evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 31: N-glycosylation sites from about amino acid 95 to about amino acid 99, and from about am ino acid I 04 to about amino acid I 08: a casein kinase 11 phosphorylation site from about amino acid l81 to about amino acid I 85; an N-myristoylation site from about amino acid 133 to about amino acid 139; and an interferon alpha, beta and delta family signature from about amino acid 147 to about amino acid 166. Clone DNA50960-1224 has been deposited with ATCC on December 3, 1997 and is assigned ATCC deposit no.209509.
An analysis ofthe Dayhoffdatabase (version35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis ofthe full-length sequence shown in Figure 2 (SEQ ID N0:2), evidenced about 35-40% sequence identity between the PR0655 amino acid sequence and various human IFN-a species. The homology is highest within the 22-189 amino acid region of the sequence shown in Figure 2 (SEQ ID N0:2). At the nucleotide level, the homology with the coding sequence of IFN-a is about 60%.
(B) PR0364 An expressed sequence tag (EST) DNA database and a proprietary EST database (LIFESEQ~, Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST (Incyte ESTno.
3003460) was identified which showed homology to members of the tumor necrosis factor receptor (TNFR) family of polypeptides.
A consensus DNA sequence was then assembled relative to the Incyte EST no.
3003460 and other EST
sequences using repeated cycles of BLAST (Altshul et al., Methods in Enzvmoloev, 266:460-480 (1996)) and "phrap" (Phil Green, University of Washington, Seattle, Washington). This consensus sequence is herein designated DNA44825.
Based upon the DNA44825 consensus sequence, oligonucleotides probes were then synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0364. 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 40-55 by in length. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel e~ al.. Current Protocols in Molecular Biolosv, supra, 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 oligonucleotide probes employed were as follows:
forward PCR urimer 1:
5'-CACAGCACGGGGCGATGGG-3' (SEQ ID N0:8) forward PCR primer 2:
5'-GCTCTGCGTTCTGCTCTG-3' (SEQ ID N0:9) forward PCR primer 3:
5'-GGCACAGCACGGGGCGATGGGCGCGTTT-3' (SEQ ID NO:10) reverse PCR primer 1:
5'-CTGGTCACTGCCACCTTCCTGCAC-3' (SEQ ID NO: I t ) reverse PCR primer 2:
5'-CGCTGACCCAGGCTGAG-3' (SEQ ID N0:12) reverse PCR primer 3:
5'-GAAGGTCCCCGAGGCACAGTCGATACA-3' (SEQ ID N0:13) hybridization probe I
5'-GAGGAGTGCTGTTCCGAGTGGGACTGCATGTGTGTCCAGC-3' (SEQ ID NO: I4) hybridization arobe 2:
5'-AGCCTGGGTCAGCGCCCCACCGGGGGTCCCGGGTGCGGCC-3' (SEQ ID NO: l5) RNA for construction ofcDNA libraries was then isolated from human small intestine tissue. The cDNA
libraries used to isolate the cDNA clones encoding human PR0364 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 Nott site, linked with blunt to Sall hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD;
pRKSB is a precursor of pRKSD that does not contain the SfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique Xhol and Notl.
A full length clone for DNA47365-1206 was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 121-123 and a stop signal at nucleotide positions 844-846 (Figure 3, SEQ ID N0:6). The predicted polypeptide precursor is 241 amino acids long, and has a calculated molecular weight of approximately 26,000 daltons and an estimated pl of approximately 6.34.
Analysis of the full-length PR0364 sequence shown in Figure 4 (SEQ ID N0:7) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0364 sequence evidenced the following: a signal peptide from about amino acid 1 to about amino acid 25; a potential transmembrane domain from about amino acid 163 to about amino acid 183: an N-glycosylation site from about amino acid 146 to about amino acid 1 S0; N-myristoylation sites from about amino acid ~ to about amino acid I I, from about amino acid 8 to about amino acid 14, from about amino acid 25 to about amino acid 31, from about amino acid 30 to about amino acid 36, from about amino acid 33 to about amino acid 39, from about amino acid 118 to about amino acid 124, from about amino acid 122 to about amino acid 128, and from about amino acid 156 to about amino acid 162; a prokaryotic membrane lipoprotein lipid attachment site from about amino acid 166 to about amino acid 177; and a leucine zipper pattern from about amino acid 171 to about amino acid 193..
Clone DNA47365-1206 has been deposited with ATCC on November 7, 1997 and is assigned ATCC
deposit no. 209436.
An analysisofthe Dayhoff database (version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure d (SEQ ID N0:7), evidenced sequence identity between the PR0364 amino acid sequence and members of the tumor necrosis factor receptor family, thereby indicating that PR0364 may be a novel member of the tumor necrosis factor receptor family.
A detailed review of the amino acid sequence of the full-length native PR0364 polypeptide and the nucleotide sequence that encodes that amino acid sequence evidences sequence homology with the mouse GITR
protein reported by Nocentini et al., Proc. Natl. Acad. Sci. USA, 91:6216-6221 (1997). It is possible, therefore, that PR0364 represents the human counterpart to the mouse GITR protein reported by Nocentini et al.
(C) PR0344 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 EST databases (e.g., GenBank), and a proprietary EST
database (LIFESEQ~, Incyte Pharmaceuticals, Palo Alto, CA}. The search was performed using the computer program BLAST or BLAST2 [Altschul et aL, Methods in Enzvmolopyy, 266:460-480 ( 1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some 1S 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, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA34398. tn some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.
Based on the DNA34398 consensus sequence oligonucleotides were synthesized: I
) to identify by PCR
a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0344. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about I 00-1000 by in length. The probe sequences are typically 40-55 by in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. 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 Protocols in Molecular Bioloev s:rpra, 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.
PCR primers (forward and reverse) were synthesized:
forward PCR primer 1:
5'-TACAGGCCCAGTCAGGACCAGGGG-3' (SEQ ID N0:18) forward PCR primer 2:
5'-AGCCAGCCTCGCTCTCGG-3' (SEQ ID N0:19) forward PCR primer 3:

5'-GTCTGCGATCAGGTCTGG-3' (SEQ ID N0:20) reverse PCR primer I
5'-GAAAGAGGCAATGGATTCGC-3' (SEQ ID N0:21 ) reverse PCR primer 2:
S 5'-GACTTACACTTGCCAGCACAGCAC-3' (SEQ ID N0:22) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA34398 sequence which had the following nucleotide sequence:
hybridization probe:
5'-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3' (SEQ ID N0:23) RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. 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 Notl site, linked with blunt to SaII hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain the Sfil site; see, Holmes et al.. Science, 253:1278- t 280 ( I 991 )) in the unique Xhol and Notl sites.
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0344 polypeptide (designated herein as DNA40592-1242 [Figure S, SEQ ID NO: 16)) and the derived protein sequence for that PR0344 polypeptide.
The full length clone identifced above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 227-229 and a stop signal at nucleotide positions 956-958 (Figure 5, SEQ ID
N0:16). The predicted polypeptide precursor is 243 amino acids long, and has a calculated molecular weight of approximately 25,298 daltons and a pI ofabout 6.44. Analysis ofthe full-length PR0344 sequence shown in Figure 6 (SEQ 1D N0:17) evidences the presence of a variety of important polypeptide domains, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0344 sequence evidenced the following: a signal peptide from about amino acid 1 to about amino acid 15; N-myristoylation sites from about amino acid I f to about amino acid 17, from about amino acid 68 to about amino acid 74, and from about amino acid 216 to about amino acid 223: and a cell attachment sequence from about amino acid 77 to about amino acid 80.
Clone DNA40592-1242 has been deposited with ATCC on November 2l, 1997 and is assigned ATCC
deposit no. 209492.

In sitzr Hybridization In suu hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription. identify and localize viral infection, follow changes in specific mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision. I: 169-176 (1994), using PCR-generated '3P-labeled riboprobes.
Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for IS minutes at 37°C, and further processed for in sinr hybridization as described by Lu and Gillett, srrpra. A ("-P)UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2T"' nuclear track emulsion and exposed for 4 weeks.
"P-Ribonrobe svnthesis 6.0 ul (125 mCi) of "P-UTP (Amersham BF 1002. SA<2000 Ci/mmol) were speed-vacuum dried. To each tube containing dried "P-UTP, the following ingredients were added:
2.0Ic1 Sx transcription buffer 1.0 ~I DTT (100 mM) 2.0 ,ul NTP mix (2.5 mM: 10 tcl each of 10 mM GTP, CTP & ATP + 10 ~1 H=O) t.0 ul UTP (50 ~cM) 1.0 ~cl RNAsin I.0 ~cl DNA template ( 1 fig) 1.0 ~I H,O
1.0 ~cl RNA polymerise (for PCR products T3 = AS, T7 = S, usually) The tubes were incubated at 37°C for one hour. A total of 1.0 ul RQ1 DNase was added, followed by incubation at 37°C for 15 minutes. A total of 90 ~cl TE ( 10 mM Tris pH

7.6/1 mM EDTA pH 8.0) was added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a MICROCON-SOT"' ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, a total of 100 ~I TE was added, then t ~I of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR IIT"' The probe was run on a TBE/urea gel. A total of 1-3 ul of the probe or 5 ul of RNA Mrk III was added to 3 ~l of loading buffer. After heating on a 95°C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes. The gel was wrapped in plastic wrap (SARANT"' brand) and exposed to XAR film with an intensifying screen in a -70°C freezer one hour to overnight.
"P-Hybridization A. Pretreatment of frozen sections The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes. The trays were placed in a 55 °C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (2S ml 20 x SSC + 975 ml SQ H=O). After deproteination in 0.5 ~g/ml proteinase K for 10 minutes at 37°C (12.5 ul of 10 mglml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, and 100%

8 PCT/US99/28409 ethanol, 2 minutes each.
B. Pretreatment oJparajfrn-embedded sections The slides were deparaffinized, placed in SQ H,O, and rinsed twice in 2 x SSC
at room temperature, for minutes each time. The sections were deproteinated in 20 ~g/ml proteinase K
(500 ul of l0 mg/ml in 250 ml 5 RNase-free RNase buffer; 37°C, 15 minutes) for human embryo tissue.
or 8 x proteinase K ( l00 ul in 250 ml Rnase buffer, 37°C, 30 minutes) for formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
C. Prehybridi=anon The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50%
formamide) - saturated filter paper. The tissue was covered with 50 ~1 of hybridization buffer (3.75 g dextran sulfate+6 ml SQ H,O), vortexed, and heated in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.75 ml fotrnamide, 3.75 ml 20 x SSC, and 9 ml SQ H=O were added, and the tissue was vortexed well and incubated at 42°C for 1-4 hours.
D. Hybridisation 1.0 x 10° cpm probe and I .0 ul tRNA (50 mg/ml stock) per slide were heated at 95 °C for 3 minutes. The slides were cooled on ice, and 48 ~cl hybridization buffer was added per slide. After vortexing, 50 ul "P mix was added to 50 ul prehybridization on the slide. The slides were incubated overnight at 55°C.
E. Washes Washing was done for 2x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0.25 M EDTA, Vr4L), followed by RNAseA treatment at 37°C for 30 minutes (500 ~I of 10 mg/ml in 250 ml Rnase buffer=20 ~cg/ml), The slides were washed 2 x 10 minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55 °C, 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, V f=4L).
F. Oligon:~cleotides In situ analysis was performed on one of the DNA sequences disclosed herein.
The oligonucleotides employed for this analysis are as follows:
( 1 ) DNA47365-1206 (PR0364) pl:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC AAC CCG AGC ATG GCA CAG CAC-3' (SEQ 1D
N0:24) p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA TCT CCC AGC CGC CCC TTC TC-3' (SEQ ID
N0:25) (G) Results !n situ analysis was performed on the above DNA sequence disclosed herein. The results from this analysis is as follows:
(I) DNA47365-1206 (PR0364) (novel TNF-receptor Homoloe) In the fetus, expression was observed in the fascia lining the anterior surface of the vertebral body. In addition, expression was seen over the fetal retina. Low level expression was seen over fetal neurones. All other tissues were negative.

Use of PR0655, PR0364 or PR0344 as a Hybridization Probe The following method describes use ofa nucleotide sequence encoding PR0655, PR0364 or PR0344 as a hybridization probe.
DNA comprising the coding sequence of full-length or mature PR0655, PR0364 or PR0344 (as shown in Figure I, 3, and 5, respectively, SEQ ID NOS: I, 6, and 16, respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PR0655, PR0364 or PR0344) 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 radioiabeled probe derived from the gene encoding a PR0655, PR0364 or PR0344 polypeptide to the filters is performed in a solution of 50%
formamide, Sx SSC, 0.1% SDS, 0. I % 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 O. l x SSC and 0.1% SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art.

Expression of PR0655, PR0364 or PR0344 in E. coli This example illustrates preparation of an unglycosylated form of PR0655, PR0364 or PR0344 by recombinant expression in E. toll.
The DNA sequence encoding PR0655, PR0364 or PR0344 is initially amplified using selected PCR
primers. The primers should contain restriction enzyme sites which correspond to the restriction enryme 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. toll; 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 trp promoter, a poly-His leader (including the first six STII codons, poly-H is sequence, and enterokinase cleavage site), the PR0655, PR0364 or PR0344 coding region, lambda transcriptionalterminator, and an argU gene.
The ligation mixture is then used to transform a selected E. toll 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 known in the art, and the solubilized PR0655, PR0364 or PR0344 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
PR0655, PR0364 or PR0344 may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding PR0655, PR0364 or PR0344 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 (W31 l0 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(laclq).
Transfotmants are first grown in LB containing 50 mg/ml carbenicillin at 30°C with shaking until an OD6~ of 3-5 is reached. Cultures are then diluted 50-I00 fold into CRAP media (prepared by mixing 3.57 g (NH,,),SO,, 0.71 g sodium citrate~2H20, 1.07 g KCI, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 1 10 mM
MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO,) 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 sulfite and sodium tetrathionate is added to make final concentrations of 0.1 M 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 0.33 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 SO 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. ?0 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 R i /H reversed phase column using a mobile buffer of 0. I % TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A=a° absorbance are analyzed on SDS
polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the property 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 higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removes endotoxin from the samples.
Fractions containing the desired folded PR0655, PR0364 or PR0344 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 Supe~ne (Pharmacia) resins equilibrated in the formulation buffer and sterile ftltered.
PR0655 and PR0364 were successfully expressed in E. coli in a poly-His tagged form by the above procedure.

Expression of PR0655, PR0364 or PR0344 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PR0655, PR0364 or PR0344 by recombinant expression in mammalian cells.
The vector, pRKS (see EP 307,247, published March I5, 1989), is employed as the expression vector.
Optionally, the PR0655, PR0364 or PR0344 DNA is ligated into pRKS with selected restriction enzymes to allow insertion of the PR0655, PR0364 or PR0344 DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRKS-PR0655, pRKS-PR0364 or pRKS-PR0344.
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 componentsand/orantibiotics. About I 0 ~g pRKS-PR0655, pRKS-PR0364 or pRKS-PR0344 DNA is mixed with about I beg DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 ul of 1 mM Tris-HC1, 0. l mM EDTA, 0.227 M CaCI=. To this mixture is added, dropwise, 500 ~cl of 50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaPO,, 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 ~eCi/ml "S-cysteine and 200 ~cCi/ml "S-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 the PR0655, PR0364 or PR0344 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, PR065~. PR0364 or PR0344 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac er al.. Proc. Natl.
Acad. Sci.. 12:7575 ( 1981 ). 293 cells are grown to maximal density in a spinner flask and 700 ~g pRKS-PR0655, pRKS-PR0364 or pRKS-PR0344 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. I ug/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PR0655, PR0364 or PR0344 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
In another embodiment, PR065~. PR0364 or PR034a can be expressed in CHO cells.
The pRKS-PR0655, pRKS-PR0364 or pRKS-PR0344 can be transfected into CHO cells using known reagents such as CaP04 or DEAF-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 ''S-methionine. After determining the presence of a PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide can then be concentrated and purified by any selected method.
Epitope-tagged PR0655, PR0364 or PR0344 may also be expressed in host CHO
cells. The PR0655, PR0364 or PR0344 may be subcloned out of the pRKS vector. 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 PR0655, PR0364 or PR0344 insert can then be subcloned into a SV~FO 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 PR0655, PR0364 or PR0344 can then be concentrated and purified by any selected method, such as by Ni='-chelate affinity chromatosraphy.
PR0655, PR0364 or PR0344 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 IgG 1 constant region sequence containing the hinge, CH2 and CH2 domains and/or as a poly-His tagged fotitt.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using standard techniques as described in Ausubel w al., Current Protocols of Molecular Biolow, Unit 3.16, John Wiley and Sons ( I 997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA
of interest to allow the convenient shuttl in, of cDNA's. The vector used in 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~ (Quiagen), Dosper'~' or Fugene~ (8oehringer Mannheim). The cells are grown as described in Lucas er 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 a water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing I 0 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 um filtered PS20 with 5% 0.2 ~cm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 ml spinner containing 90 ml of selective media. After I-2 days, the cells are transferred into a 250 ml spinner filled with I50 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 10° cells/ml. On day 0, the cell number and pH is determined. On day I, 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 drops below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 gem filter. The filtrate is 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 NaCI and 4%
mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharcnacia) 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 S mi Protein A column (Phatmacia) which has been equilibrated in 20 mM
Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with i 00 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ~cl 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.
PR0364 was stably expressed in CHO cells by the above described method. In addition, PR0364 was expressed in CHO cells by the transient expression procedure.

Expression of PR065~. PR036-l or PR03d4 in Yeast The following method describes recombinant expression of PR0655. PR0364 or PR0344 in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PR0655, PR0364 or PR0344 from the ADH2/GAPDH promoter. DNA encoding PR0655. PR0364 or PR0344 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR0655, PR0364 or PR0344. For secretion, DNA encoding PR065~, PR0364 or PR0344 can be cloned into the selected plasmid, together with DNA encoding the ADH2!GAPDH promoter, a native PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344.
Yeast cells, such as yeast strain ABl 10, 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 PR0655, PR0364 or PR0344 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected carnidge filters. The concentrate containing PR0655, PR0364 or PR0344 may further be purified using selected column chromatography resins.

Expression of PR0655, PR0364 or PR0344 in Baculovirus-Infected Insect Cells The fotlowing method describes recombinant expression in Baculovirus-infected insect cells.
The sequence coding for PR0655, PR0364 or PR0344 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 IQG). A variety of plasmids may be employed, including plasmids derived from commercially available 2S plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PR0655, PR0364 or PR0344 or the desired portion of the coding sequence of PR0655, PR0364 or PR0344 (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 5' 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 plasmidand BaculoGoIdT" virus DNA
(Pharmingen)intoSpodopterafr:rgiperda("Sf~9")cells(ATCCCRL
1711)usinglipofectin(commerciallyavailable from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications. 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 PR0655, PR0364 or PR0344 can then be purified, for example, by Ni='-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Std cells as described by Rupert et al., Nature, 362: i 75-179 ( 1993). Briefly. Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCf,; O.l mM EDTA; 10°.%
glycerol; 0.1% NP-40; 0.4 M KC1), 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 mm filter. A Ni='-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 A,s°
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 A:e° 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 Ni='-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted H is,o tagged PR0655, PR0364 or PR0344, respectively, are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PR0655, PR0364 or PR0344 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
FollowingPCRamplification,the respective coding sequences are subcloned into a baculovirus expression vector(pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the vector and Baculogold~
baculovirvs DNA (Pharmingen) are co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL
l7l 1), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL 1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences. The cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells are incubated for 5 days at 28°C. The supernatant is harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells are incubated for 3 days at 28°C. The supernatant is harvested and the expression of the constructs in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml ofNi '-'-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B
beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The first vita! amplification supernatant is used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells are incubated for 3 days at 28 °C. The supernatant is harvested and filtered. Batch binding and SDS-PAGE analysis is repeated, as necessary, until expression of the spinner culture is confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein construct is 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 ~ 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 NaCI and 4% mannitol, pH 6.8, with a 25 ml G2~ Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows.
The conditioned media is pumped onto a ~ ml Protein A column (Pharmacia) which has 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 mi 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 of the proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
PR0344 was expressed in baculovirus infected Sf9 insect cells.
Alternatively, a modified baculovirus procedure may be used incorporating high-5 cells. In this procedure, the DNA encoding the desired sequence is amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-00 of an epitope tag contained with 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 pIE 1-1 (Novagen). The pIE 1-1 and pIE 1-2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus ie I promoter in stably-transformed insect cells ( I ). The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for iel-mediated gene expression in uninfected insect cells as well as the hr5 enhancer element. pIE 1-1 and pIE 1-2 include the translation initiation site and can be used to produce fusion proteins. Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector. For example, derivatives of plEl-I can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3'-of) the desired sequence. Preferably, the vector construct is sequenced for confirmation.
High-5 cells are grown to a confluency of 50% under the conditions of, 27°C, no CO=, NO pen/strep. For each 150 mm plate, 30 ~cg of pIE based vector containing the sequence is mixed with I ml Ex-Cell medium (Media:
Ex-Cell 401 + 1/100 L-Glu JRH Biosciences # 14401-78P (note: this media is light sensitive)), and in a separate tube, 100 ~I ofCeIIFectin (CeIIFECTIN (GibcoBRL # t 0362-O I 0) (vortexed to mix)) is mixed with I ml of Ex-Cell medium. The two solutions are combined and allowed to incubate at room temperature for 15 minutes. 8 ml of 3S Ex-Cell media is added to the 2 ml of DNA/CeIIFECTIN mix and this is layered on high-~ cells that have been washed once with Ex-Cell media. The plate is then incubated in darkness for 1 hour at room temperature. The DNA/CeIIFECTIN mix is then aspirated. and the cells are washed once with Ex-Cell to remove excess CeIIFECT1N, 30 m I of fresh Ex-Cel I media is added and the cel Is are incubated for 3 days at 28°C. The supernatant is harvested and the expression ofthe sequence in the baculovirus expression vector is determined by batch binding of 1 ml ofsupernatent to 25 ml ofNi ='-NTA beads (QIAGEN) forhistidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IeG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The conditioned media from the transfected cells (0.5 to 3 L ) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His ta~~ed constructs, the protein comprising the sequence is 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 of4-5 mUmin. at48°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 then subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows.
The conditioned media 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 ! 00 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml 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 of the sequence is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary.
PR0364 and PR0344 were expressed using the above baculovirus procedure employing high-5 cells.

Preparation of Antibodies that Bind PR0655 PR0364 or PR0344 This example illustrates preparation of monoclonal antibodies which can specifically bind PR0655, PR0364 or PR0344.
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 PR0655, PR0364 or PR0344, fusion proteins containing PR0655, PR03b4 or PR0344, and cellsexpressingrecombinant PR0655. PR0364 or PR0344 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 PR0655, PR0364 or PR0344 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 EL1SA assays to detect anti-PR065~, anti-PR0364 or anti-PR0344 antibodies.
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PR0655, PR0364 or PR0344. 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 murine myeloma cell line such as P3X63AgU.1, 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 PR0655, PR0364 or PR0344.
Determination of"positive" hybridoma cells secreting the desired monoclonal antibodies against PR0655, PR0364 or PR0344 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-PR0655, anti-PR0364 or anti-PR03~1-t 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.

Purification of PR0655, PR0364 or PR0344 Polvoentides Using, Specific Antibodies Native or recombinant PR0655, PR0364 or PR0344 polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-PR0655, pro-PR0364 or pro-PR0344 polypeptide, mature PR0655, mature PR0364 or mature PR034.1 polypeptide, or pre-PR0655, pre-PR0364 or pre-PR0344 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PR0655, PR0364 or PR0344 polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-PR0655, anti-PR0364 or anti-PR0344 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 antibodiesare prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalentiy 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 immunoaffinity column is utilized in the purification of the PR0655, PR0364 or PR0344 polypeptide by preparing a fraction from cel Is containing the PR06>j, PR0364 or PR0344 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 PR0655. PR0364 or PR0344 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
A soluble PR0655, PR0364 or PR0344 polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of the PR0655, PR0364 or PR0344 polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PR06», antibody/PR0364 or antibody/PR0344 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 the PR0655, PR0364 or PR0344 polypeptide is collected.

Drua Screenine This invention is particularly useful for screening compounds by using PR065~.
PR0364 or PR0344 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques. The PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 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 a PR0655, PR0364 or PR0344 polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide-associated disease or disorder. These methods comprise contacting such an agent with a PR0655, PR0364 or PR0344 polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the PR0655, PR0364 or PR0344 polypeptide or fragment, or (ii) for the presence of a complex between the PR0655, PR0364 or PR0344 polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays, the PR0655, PR0364 or PR0344 polypeptide or fragment is typically labeled. After suitable incubation, the free PR0655, PR0364 or PR0344 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 the PR065~, PR0364 or PR0344 polypeptide or to interfere with the PR0655, PR0364 or PR0344 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 l3, 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 PR0655. PR0364 or PR0344 polypeptide, the peptide test compounds are reacted with the PR0655, PR0364 or PR0344 polypeptide and washed. Bound PR0655, PR0364 WO 00/32778 PC'f/US99/28409 or PR0344 polypeptide is detected by methods well known in the art. Purified PR0655. PR0364 or PR0344 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 a PR0655, PR0364 or PR0344 polypeptide specifically compete with a test compound for binding to the PR0655, PR0364 or PR0344 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 a PR0655, PR0364 or PR0344 polypeptide.

Rational Drug Desien The goal of rational drug design is to produce structural analogs of a biologically active polypeptide of interest (i.e., a PR0655, PR0364 or PR0344 poiypeptide) 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 PR0655, PR0364 or PR0344 polypeptide or which enhance or interfere with the function of the PR0655, PR0364 or PR0344 polypeptide in vivo (cf., Hodason.
Bio/Technoloey, 9: 19-21 ( (991 )).
In one approach, the three-dimensional structure of the PR0655, PR0364 or PR0344 polypeptide, or of a PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous PR0655, PR0364 or PR0344 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. 3 I :7796-7801 ( 1992) or which act as inhibitors.
agonists, or antagonists of native peptides as shown by Athauda et ul., J.
Biochem.. 1 13:742-746 ( 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 minor 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 PR0655, PR0364 or PR0344 polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge ofthe PR0655, PR0364 or PR0344 polypeptide amino acid sequence provided herein wilt provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.

In G'irro Antitumor Assav The antiproliferative activity of the PR0655, PR0364 and PR0344 polypeptides was determined in the investigational, disease-oriented in vitro anti-cancer drub discovery, assay of the National Cancer Institute (NCI), using a sulforhodam ine B (SRB) dye binding assay essentially as described by Skehan et al., J. Natl. Cancer Inst., 82:1107-I 112 ( 1990). The 60 tumor cell lines employed in this study ("the NCI panel"), as well as conditions for their maintenance and culture in vitro have been described by Monks et al., J.
Natl. Cancer lnst.. 83:757-766 ( 1991 ). The purpose of this screen is to initially evaluate the cytotoxic and/or cytostatic activity of the test compounds against different types of tumors (Monks et al., supra; Boyd, Cancer: Princ. Pract. Oncol Update ~:1-12 [1989]).
Cells from approximately 60 human tumor cell lines were harvested with trypsin/EDTA (Gibco), washed once, resuspended in IMEM and their viability was determined. The cell suspensions were added by pipet (100 ~cl volume) into separate 96-well microtiter plates. The cell density for the 6-day incubation was less than for the 2-day incubation to prevent overgrowth. Inoculates were allowed a preincubation period of24 hours at 37°C for stabilization. Ditutions at twice the intended test concentration were added at time zero in 100 ~I aliquots to the microtiter plate wells ( 1:2 dilution). Test compounds were evaluated at five half log dilutions ( 1000 to 100,000-fold). Incubations took place for two days and six days in a 5% CO, atmosphere and 100% humidity.
After incubation, the medium was removed and the cells were fixed in 0. t ml of 10% trichloroacetic acid at 40°C. The plates were rinsed five times with deionized water, dried, stained for 30 minutes with 0.1 ml of 0.4%
sulforhodamine B dye (Sigma) dissolved in I% acetic acid, rinsed four times with 1% acetic acid to remove unbound dye, dried, and the stain was extracted for five minutes with 0.1 ml of 10 mM Tris base [tris(hydroxymethyl)aminomethane],pH 10.5.
Theabsorbance(OD)ofsulforhodamineBat492nmwasmeasured using a computer-interfaced, 96-well microtiter plate reader.
A test sample is considered positive if it shows at least 40% growth inhibitory effect at one or more concentrations. The results are shown in the following Table 4, where the tumor cell type abbreviations are as follows:
NSCL = non-small cell lung carcinoma; CNS = central nervous system Table 4 CompoundConcentrationDays Tumor Cell Tvpe Desi nQ anon PR0655 22.2 nM 2 Ovarian OVCAR-4 PR0655 22.2 nM 6 Colon HCT-15 PR0655 22.2 nM 6 Melanoma UACC-257 PR0655 18.0 nM 6 Melanoma LOX IMVI

PR0655 18.0 nM 6 Colon KM-12 PR0364 27.23 nM 6 NSCL HOP62 PR0364 27.33 nM 6 Colon KM-12 PR0364 27.23 nM 6 CNS SF295 PR0364 27.23 nM 6 Melanoma LOX 1MVI

PR0364 27.23 nM 6 Renal UO-31 PR0364 135.00 nM 2 Colon KM-12 PR0364 135.00 nM 6 NSCL HOP62 PR0364 135.00 nM 6 Melanoma LOX IMVI

PR0364 135.00 nM 6 Colon HT-29 PR0364 135.00 nM 6 Ovarian IGROVI

PR0364 135.00 nM 6 Breast MDA-MB 435 PR0344 1.2 nM 2 Leukemia HL-60 (TB) PR0344 1.2 nM 6 Renal UO-31 and PR0344 14.9 nM 2 Colon KM-12 PR0344 14.9 nM 2 CNS SF-268 PR0344 14.9 nM 2 Ovarian OVCAR-4 PR0344 14.9 nM 2 Renal CAKI-1 PR0344 14.9 nM 2 Breast MDA-MB-435 PR0344 14.9 nM 6 Leukemia HL-60 (TB) PR0344 14.9 nM 6 Colon KM-12 PR0344 14.9 nM 6 CNS SF-295 PR0344 14.9 nM 6 NSCL HOP62 Deposit of Material The following materials have been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas. VA 20110-2209, USA (ATCC):
Material ATCC Dep. No. Deposit Date_ DNA50960-1224 209509 December 3, 1997 DNA47365-1206 209436 November 7, 1997 DNA40592-1242 209492 November 21, 1997 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 U.S.C. ~
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.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
I. A composition of matter useful for the inhibition of neoplastic cell growth, said composition comprising an effective amount of a PRO655. PRO364 or PRO344 polypeptide, or an monist thereof. in admixture with a pharmaceutically acceptable carrier.
2. The composition of matter of Claim 1 comprising a growth inhibitory amount of a PRO655.
PRO364 or PRO344 polypeptide, or an agonist thereof.
3. The composition of matter of Claim I comprising a cytotoxic amount of a PRO655, PRO364 or PRO344 polypeptide, or an agonist thereof.
4. The composition of matter of Claim 1 additionally comprising a further growth inhibitory agent, cytotoxic agent or chemotherapeutic agent.
5. A composition of matter useful for the treatment of a tumor in a mammal.
said composition comprising a therapeutically effective amount of a PRO655. PRO364 or PRO344 polypeptide, or an agonist thereof.
6. The composition of matter of Claim 5, wherein said tumor is a cancer.
7. The composition of matter of Claim 6, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, renal cancer, colorectal cancer, uterine cancer, prostate cancer, lung cancer, bladder cancer, central nervous system cancer, melanoma and leukemia.
8. A method for inhibiting the growth of a tumor cell comprising exposing said tumor cell to an effective amount of a PRO655, PRO364 or PRO344 polypeptide, or an agonist thereof.
9. The method of Claim 8, wherein said agonist is an anti-PRO655, anti-PRO364 or anti-PRO344 agonist antibody.
10. The method of Claim 8, wherein said agonist is a small molecule mimicking the biological activity of a PRO655, PRO364 or PRO344 polypeptide.
l I . The method of Claim 8, wherein said step of exposing occurs in vitro.
12. The method of Claim 8, wherein said step of exposing occurs in vivo.
13. An article of manufacture comprising:

a container; and a composition comprising an active agent contained within the container;
wherein said active agent in the composition is a PRO655, PRO364 or PRO344 polypeptide, or an monist therof.
14. The article of manufacture of Claim 13, further comprising a label affixed to said container, or a package insert included in said container, referring to the use of said composition for the inhibition of neoplastic cell growth.
15. The article of manufacture of Claim 13, wherein said agonist is an anti-PRO655, anti-PRO364 or anti-PRO344 agonist antibody.
16. The article of manufacture of Claim 13, wherein said agonist is a small molecule mimicking the biological activity of a PRO655, PRO364 or PRO344 polypeptide.
17. The article of manufacture of Claim 13, wherein said active agent is present in an amount that is effective for the treatment of tumor in a mammal.
18. The article of manufacture of Claim 3l,wherein said composition additionaliycomprisesa further growth inhibitory agent, cytotoxic agent or chemotherapeutic agent.
19. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), and Figure 6 (SEQ ID NO:17).
20. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in Figure 1 (SEQ ID NO:1 ), Figure 3 (SEQ
ID NO:6), and Figure 5 (SEQ ID NO:16).
21. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the full-length coding sequence of the nucleotide sequence shown in Figure 1 (SEQ ID NO:1), Figure 3 (SEQ ID NO:6), and Figure S (SEQ ID NO:16).
22. 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 209509, 209436 or 209492.
23. A vector comprising the nucleic acid of any one of Claims 19 to 22.
24. The vector of Claim 23 operably linked to control sequences recognized by a host cell transformed with the vector.
25. A host cell comprising the vector of Claim 23.
26. The host cell of Claim 25, wherein said cell is a CHO cell.
27. The host cell of Claim 25, wherein said cell is an E. coli.
28. The host cell of Claim 25, wherein said cell is a yeast cell.
29. The host cell of Claim 25, wherein said cell is a Baculovirus-infected insect cell.
30. A process for producing a PRO655, PRO364 or PRO344 polypeptide comprising culturing the host cell of Claim 25 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
31. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), and Figure 6 (SEQ ID NO:17).
32. An isolated polypeptide scoring at least 80% positives when compared to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ
ID NO:7), and Figure 6 (SEQ ID NO:17).
33. 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 209509, 209436 or 209492.
34. A chimeric molecule comprising a polypeptide according to any one of Claims 31 to 33 fused to a heterologous amino acid sequence.
35. The chimeric molecule of Claim 34, wherein said heterologous amino acid sequence is an epitope tag sequence.
36. The chimeric molecule of Claim 34, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.
37. An antibody which specifically binds to a polypeptide according to any one of Claims 31 to 33.
38. The antibody of Claim 37, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.
39. Isolated nucleic acid having at least 80% nucleic acid sequence identity to:
(a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID
NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO:17), lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO:17), with its associated signal peptide; or (c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO:17), lacking its associated signal peptide.
40. An isolated poiypeptide having at least 80% amino acid sequence identity to:
(a) the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ 1D NO:7), or Figure 6 (SEQ
ID NO:17), lacking its associated signal peptide;
(b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID
NO:2), Figure 4 (SEQ ID
NO:7), or Figure 6 (SEQ ID NO:17), with its associated signal peptide; or (c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID
NO:2), Figure 4 (SEQ ID
NO:7), or Figure 6 (SEQ ID NO:17), lacking its associated signal peptide.