CA2504679A1

CA2504679A1 - Compositions and methods for the treatment of tumor

Info

Publication number: CA2504679A1
Application number: CA002504679A
Authority: CA
Inventors: David Botstein; Audrey Goddard; Austin L. Gurney; Victoria Smith; Colin K. Watanabe; William I. Wood
Original assignee: Individual
Current assignee: Genentech Inc
Priority date: 1999-06-09
Filing date: 2000-05-15
Publication date: 2000-12-14
Also published as: CA2501815A1

Abstract

The invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans. The invention is based upon the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product as compared to normal cells of the same tissue type and contribute to tumorigenesis.
Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain cancers, and may act as predictors of the prognosis of tumor treatment. 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

COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR
Field of the Invention The present invention relates to compositions and methods for the diagnosis and treatment of tumor.
Background of the Invention Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring etal., CA Cancel J. Clin.. 43:7 [1993]).
Cancer is characterized by an increase in the numberof abnormal, or neoplastic cells derived from a normal I S 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.
Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differentiation which are a common feature of all cancers. The genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes. which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth. Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype (Hunter. Cell, 6:1:1129 [ 1991 ] and Bishop, Cell. 64:235-248 [1991]).
A well known mechanism of gene (e.g., oncogene) overexpression in cancer cells is gene amplification.
This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced.
The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome (Alitalo et al., Adv. Cancer Res.. 47:235-281 [ 1986]). It is believed that the overexpression of the gene parallels gene amplification, i.e., is proportionate to the number of copies made.
Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer. For example, it has been found that the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor (p 185"r"''; HER2) related to the epidermal growth factor receptor EGhR), is overexpressed in about 25% to 300 of human breast cancer lSlamon et al., Science. 235:177-182 [1987]: Slamon et al., Science. 244:707-712 [ 1989]).
It has been reported that gene amplification of a proto-oncogene is an event typically involved in the more malignant forms of cancer, and could act as a predictor of clinical outcome (Schwab et al., Genes Chromosomes Cancer. 1:181-193 [1990]; Alitalo etal., supra). Thus, erbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al., [ 1987] and [ 1989], supra; Ravdin and Chamness, Gene, 159:19-27 [ 1995]; and Hynes and Stern, Biochim. Biophys.
Acta, 1198:165-184 (1994]), and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines (Baselga et al., Oncology, 11 (3 Suppl 1):43-48 [1997]). However, despite the association of erbB2 overexpression with poor prognosis, the odds of HER2-positive patients responding clinically to treatment with taxanes were greater than three times those of HER2-negative patients (Ibis. A recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or HerceptinT"') has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baselga et al.,1. Clin. Oncol., 14:737-744 [1996]).
In light of the above, there is obvious interest in identifying novel methods and compositions which are useful for diagnosing and treating tumors which are associated with gene amplification.
Surnmary of the Invention A. Embodiments The present invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans. The present invention is based on the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product and contribute to tumorigenesis.
Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain cancers, and may act as predictors of the prognosis of tumor treatment.
In one embodiment, the present invention concerns an isolated antibody which binds to a polypeptide designated herein as a PRO polypeptide_ In one aspect, the isolated antibody specifically binds to a PRO
polypeptide. In another aspect, the antibody induces the death of a cell which expresses a PRO polypeptide. Often, the cell that expresses the PRO polypeptide is a tumor cell that overexpresses the polypeptide as compared to a normal cell of the same tissue type. In yet another aspect, the antibody is a monoclonal antibody, which preferably has non-human complementarily determining region (CDR) residues and human framework region (FR) residues.
The antibody may be labeled and may be immobilized on a solid support. In yet another aspect, the antibody is an antibody fragment. a single-chain antibody, or a humanized antibody which binds, preferably specifically, to a PRO
polypeptide.
In another embodiment, the invention concerns a composition of matter which comprises an antibody which binds, preferably specifically, to a PRO polypeptide in admixture with a pharmaceutically acceptable carrier.
In one aspect, the composition of matter comprises a therapeutically effective amount of the antibody. In another aspect, the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent. Preferably. the composition is sterile.
In a further embodiment, the invention concerns isolated nucleic acid molecules which encode anti-PRO
antibodies, and vectors and recombinant host cells comprising such nucleic acid molecules.
In a still further embodiment, the invention concerns a method for producing an anti-PRO antibody, wherein the method comprises culturing a host cell transformed with a nucleic acid molecule which encodes the antibody under conditions sufficient to allow expression of the antibody, and recovering the antibody from the cell culture.
The invention further concerns antagonists of a PRO polypeptide that inhibit one or more of the biological and/or immunological functions or activities of a PRO polypeptide.
In a further embodiment, the invention concerns an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a PRO polypeptide or the complement thereof.
The isolated nucleic acid molecule is preferably DNA. and hybridization preferably occurs under stringent hybridization and wash conditions. Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the transcription and/or uanslation of the respective amplified genes, or as antisense primers in amplification reactions. Furthermore, such sequences can be used as pan of a ribozyme and/or a triple helix sequence which, in turn, may be used in regulation of the amplified genes.
In another embodiment, the invention provides a method for determining the presence of a PRO
polypeptide in a sample suspected of containing a PRO polypeptide, wherein the method comprises exposing the sample to an anti-PRO antibody and determining binding of the antibody to a PRO polypeptide in the sample. In another embodiment, the invention provides a method for determining the presence of a PRO polypeptide in a cell, wherein the method comprises exposing the cell to an anti-PRO antibody and determining binding of the antibody to the cell.
In yet another embodiment, the present invention concerns a method of diagnosing tumor in a rnamntal, comprising detecting the level of expression of a gene encoding a PRO
polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test tissue cells were obtained.
In another embodiment, the present invention concerns a method of diagnosing tumor in a mammal, comprising (a) contacting an anti-PRO antibody with a test saritple of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-PRO antibody and a PRO polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal. The detection may be qualitative or quantitative, and rnay be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type. A larger quantity of complexes formed in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained. The antibody preferably carries a detectable label. Complex formation can be monitored, for example. by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art.
The test sample is usually obtained from an individual suspected to have neoplastic cell growth or proliferation (e.g. cancerous cells).

1n another embodiment, the present invention concerns a cancer diagnostic kit comprising an anti-PRO
antibody and a carrier (e.g.. a buffer) in suitable packaging. The kit preferably contains instructions for using the antibody to detect the presence of a PKO polypeptide in a sample suspected of containing the same.
In yet another embodiment, the invention concerns a method for inhibiting the growth of tumor cells comprising exposing tumorcells which express a PRO polypeptide to an effective amount of an agent which inhibits a biological and/or immunolo~ical activity and/or the expression of a PRO
polypeptide, wherein growth of the tumor cells is thereby inhibited. The a_ent preferably is an anti-PRO antibody, a small organic and inor_anic molecule, peptide, phosphopeptide, antisense or ribozyme molecule, or a triple helix molecule. In a specific aspect, the agent, e.g., the anti-PRO antibody, induces cell death. In a further aspect, the tumor cells are further exposed to radiation treatment and/or a cytotoxic or chemotherapeutic anent.
In a further embodiment, the invention concerns an article of manufacture, comprising:
a container;
a label on the container; and a composition comprising an active agent contained within the container;
wherein the composition is effective for inhibiting the growth of tumor cells and the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO
polypeptide as compared to a normal cell of the same tissue type. In particular aspects, the active agent in the composition is an agent which inhibits an activity and/or the expression of a PRO polypeptide. In preferred aspects, the active agent is an anti-PRO antibody or an antisense oligonucleotide.
The invention also provides a method for identifying a compound that inhibits an activity of a PRO
polypeptide, comprising contacting a candidate compound with a PRO polypeptide under conditions and for a time sufficient to allow these two components to interact and deterntining whether a biological and/or immunological activity of the PRO polypeptide is inhibited. In a specific aspect, either the candidate compound or the PRO
polypeptide is immobilized on a solid suppon. In another aspect, the non-immobilized component carries a detectable label. In a preferred aspect, this method comprises the steps of (a) contacting cells and a candidate compound to be screened in the presence of the PRO polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide and (b) deterrnining the induction of said cellular response to deterntine if the test compound is an effective antagonist.
In another embodiment, the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that express the polypeptide, wherein the method comprises contacting the cells with a candidate compound and determining whether the expression of the PRO polypeptide is inhibited.
In a preferred aspect. this method comprises the steps of (a) contacting cells and a candidate compound to be screened under conditions suitable for allowing expression of the PRO
polypeptide and (b) determining the inhibition of expression of said polypeptide.
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 PRO polypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87%
nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94%
nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97%
nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82%a nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87%
nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94%
nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97%
nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO polypeptide cDNA
as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide.
with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA
molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity. alternatively at least about 83%
nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity. alternatively at least about 86%
nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93%
nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97%
nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a).
Another aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO 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 PRO polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO
antibody or as antisense oligonucleotide probes. Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence fragments) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Aiso contemplated are the PRO
polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody.
In another embodiment, the invention provides an isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a cenain aspect, the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83%
amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92%o amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94%
amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide. as disclosed herein or any other specifically defined fra<oment of the ful l-length amino acid sequence as disclosed herein.
In a further aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity. alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85%
amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96%
amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99%
amino acid sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein.
In a futther aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence scoring at least about 80% positives, alternatively at least about 81 %
positives, alternatively at least about 82%
positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least _7_ about 85% positives, alternatively at least about 86% positives, alternatively at least about 87% positives.
alternatively at least about 88% positives, alternatively at least about 89%
positives. alternatively at least about 90%
positives, alternatively at least about 91 % positives. alternatively at least about 92% positives, alternatively at least about 93% positives, alternatively at least about 9d% positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97%
positives. alternatively at least about 98%
positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO
polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide without the N-tetirtinal 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 underconditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
Another aspect of the present invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are also herein described. wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
In yet another embodiment, the invention concerns antagonists of a native PRO
polypeptide as defined herein. In a particular embodiment, the antagonist is an anti-PRO antibody or a small molecule.
In a furtherembodirnent, the invention concerns a method of identifying antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.
In a still further embodiment, the invention concerns a composition of matter comprising a PRO
polypeptide, or an antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
Another embodiment of the present invention is directed to the use of a PRO
polypeptide, or an antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an antagonist thereof or an anti-PRO antibody.
In additional embodiments of the present invention, the invention provides vectors comprising DNA
encoding any of the herein described polypeptides. Host cells 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.
_g_ CA 02504ti79 2005-06-28 In other embodiments, the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterolo~ous polypeptide or amino acid sequence.
Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin.
In yet another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fraement or sinele-chain antibody.
In yet other embodiments, the invention provides oligonucleotide 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 Figures Figure 1 shows the nucleotide sequence (SEQ ID NO:1 ) of a cDNA containing a nucleotide sequence encoding native sequence PR05800, wherein the nucleotide sequence (SEQ ID NO:
I ) is a clone designated herein as DNA 108912-2680. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 2 shows the amino acid sequence (SEQ ID N0:2) of a native sequence PR05800 polypeptide as derived from the coding sequence of SEQ ID NO:1 shown in Figure 1.
Figure 3 shows the nucleotide sequence (SEQ ID N0:3) of a cDNA containing a nucleotide sequence encoding native sequence PR06000, wherein the nucleotide sequence (SEQ ID
N0:3) is a clone designated herein as DNA102880-2689. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 4 shows the amino acid sequence (SEQ ID N0:4) of a native sequence PR06000 polypeptide as derived from the coding sequence of SEQ ID N0:3 shown in Figure 3.
Figure 5 shows the nucleotide sequence (SEQ ID N0:5) of a cDNA containing a nucleotide sequence encoding native sequence PR06016, wherein the nucleotide sequence (SEQ ID
N0:5) is a clone designated herein as DNA96881-2699. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 6 shows the amino acid sequence (SEQ ID N0:6) of a native sequence PR06016 polypeptide as derived from the coding sequence of SEQ ID N0:5 shown in Figure 5.
Figure 7 shows the nucleotide sequence (SEQ ID N0:7) of a cDNA containing a nucleotide sequence encoding native sequence PR06018, wherein the nucleotide sequence (SEQ ID
N0:7) is a clone designated herein as DNA98565-2701. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 8 shows the amino acid sequence (SEQ ID N0:8) of a native sequence PR06018 polypeptide as derived from the coding sequence of SEQ ID N0:7 shown in Figure 7.
Figure 9 shows the nucleotide sequence (SEQ ID N0:9) of a cDNA containing a nucleotide sequence encoding native sequence PR06496. wherein the nucleotide sequence (SEQ ID
N0:9) is a clone designated herein as DNA 119302-2737. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 10 shows the amino acid sequence (SEQ ID NO:10) of a native sequence PR06496 polypeptide as derived from the coding sequence of SEQ ID N0:9 shown in Figure 9.
Figure 11 shows the nucleotide sequence (SEQ ID NO:1 1 ) of a cDNA containing a nucleotide sequence encoding native sequence PR07154, wherein the nucleotide sequence ISEQ ID NO:
I I ) is a clone deli gnated herein as DNA 108760-2740. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 12 shows the amino acid sequence (SEQ ID N0:12) of a native sequence PR07154 polypeptide as derived from the coding sequence of SEQ ID NO:11 shown in Figure I I.
Figure l3 shows the nucleotide sequence (SEQ ID N0:13) of a cDNA containing a nucleotide sequence encoding native sequence PR07170, wherein the nucleotide sequence (SEQ ID
N0:13 ) is a clone designated herein as DNA 108722-2743. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 14 shows the amino acid sequence (SEQ ID N0:14) of a native sequence PR07170 polypeptide as derived from the coding sequence of SEQ ID N0:13 shown in Figure 13.
Figure 1 S shows the nucleotide sequence (SEQ ID N0:15) of a cDNA containing a nucleotide sequence encoding native sequence PR07422, wherein the nucleotide sequence (SEQ ID
N0:15) is a clone designated herein as DNAI 19536-2752. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 16 shows the anuno acid sequence (SEQ ID N0:16) of a native sequence PR07422 polypeptide as derived from the coding sequence of SEQ ID N0:15 shown in Figure 15.
Figure 17 shows the nucleotide sequence (SEQ ID N0:17) of a cDNA containing a nucleotide sequence encoding native sequence PR07431, wherein the nucleotide sequence (SEQ ID
N0:17) is a clone designated herein as DNA 119542-2754. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 18 shows the amino acid sequence (SEQ ID N0:18) of a native sequence PR07431 polypeptide as derived from the coding sequence of SEQ ID N0:17 shown in Figure 17.
Figure 19 shows the nucleotide sequence (SEQ ID N0:19) of a cDNA containing a nucleotide sequence encoding native sequence PR07476. wherein the nucleotide sequence (SEQ ID
N0:19) is a clone designated herein as DNA115253-2757. Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 20 shows the amino acid sequence (SEQ ID N0:20) of a native sequence PR07476 polypeptide as derived from the coding sequence of SEQ ID N0:19 shown in Figure 19.
Detailed Description of the Invention Definitions The phrases "gene amplification" and "gene duplication" are used interchangeably and refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The duplicated region (a stretch of amplified DNA) is often referred to as "amplicon."
Usually, the amount of the messenger RNA
(mRNA) produced. i.e., the level of gene expression, also increases in the proportion of the number of copies made of the particular gene expressed.
"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, gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladdercancer, hepatoma, colorectal cancer, endometriat carcinoma, salivary gland carcinoma, kidney cancer, liver 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 ueatment 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.
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, spons, or pet animals, such as dogs, horses, cats. cattle, pigs, sheep, etc.
Preferably, the mammal is human.
"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 pI-I 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) polypeptides; proteins, such as serum albumin. gelatin, or immunoglobulins: hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose.
mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol: salt-forming counterions such as sodium: and/or nonionic surfactants such as TWEENT".
polyethylene glycol (PEG), and PLURONICST"
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I'''. I'=5, Yy° and Re'"'~), 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 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 Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere Rhone-Poulenc Rorer..~ntony, Rnace), toxotere. methotrexate.
cisplatin, rnelphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone. vincrisiine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin. mitomycins, esperamicins (see, U.S. Pat. No. 4,675,187), 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan, and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit homtone 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 cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo. Thus.
the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes 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 G1 arrest and M-phase arrest.
Classical M-phase blockers include the vincas (vineristine and vinblasdne), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest GI also spill over into S-phase arrest, for example, DNA
alkyladng agents such as tamozifen, prednisone, dacarbazine, mochlorethamine, cispladn, ttxthotrexate, 5-fluorouracil, and an-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, ids., Chapter ! , endued "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami etal., (WB
Saunders: Philadelphia, 1995), especially p. 13.
"Doxorubicin" is an anthracycline andbiodc. The full chemical name of doxorubicin is (8S-cis~l0-[(3 amino-2,3,frtrideoxy-a-L-lyxo-hexapyranosyl)oxyJ-7,8,9,10-tetrahydro-6.8,11-trihydroxy-8-(hydroxyacetyl~l methoxy-5:12-naphthacenedione.
The urm "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 lymphokirtes, tnonokines, and traditional polvpeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-mechionvl 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;
prolactin; placental lactogen; tumor necrosis factor-a and-p; mullerian-inhibiting substance; mouse gonadotropin-associated peptide: inhibin: activin;
vascular endothelial growth factor, integrin: thrombopoietin ('IPO); 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-I and -II;
erythropoietin (EPO); osteoinducdve factors: interferons such as interferon -a. -Vii, and -y; colony stimulating factors (CSFs) such as macrophage-CSF lM-CSF): granulocyte-macrophage-CSF lGM-CSF);
and granulocvte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL- 1a, IL-2. IL-3, IL-4, IL-5. IL-6.
IL-7, IL-8. IL-9. IL-11. IL-12: a tumor necrosis factor such as TNF-a or TNF-Q: and otherpolypeptide factors including LIF and kit ligand lKLI. As used *-trademark 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, 14:375-382, 615th Meeting, Belfast ( 1986), and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery', Directed Drug Delivery. Borchardt et al., (ed.), pp. 147-267, Humana Press ( 1985). The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate~ontaining prodrugs, peptide-containingprodrugs, D-amino acid-modified prodrugs, glysocylated prodrugs, B-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
An "effective amount" of a polypeptide disclosed herein or an antagonist thereof, in reference to inhibition of neoplastic cell growth, tumor growth or cancer 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 and/or cytotoxic effect and/or apoptosis of the target cells. An "effective amount"
of a PRO polypeptide antagonist for purposes of inhibiting neoplastic cell growth, tumor growth or cancer 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 PRO polypeptide antagonist for purposes of treatment of tumor may be determined empirically and in a routine manner.
A "growth inhibitory amount ' of a PRO antagonist 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 PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
A "cytotoxic amount'' of a PRO antagonist is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cel l, either in vitro or in vivo. A "cytotoxic amount" of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers to specific polypeptide sequences as described herein. The terms "PRO/number polypeptide."
and "PRO/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence PRO polypeptide"
specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring atlelic variants of the polypeptide. In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop colons are shown in bold font and underlined in the figures. However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position l in the figures may be employed as the starting amino acid residue for the PRO
polypeptides.
'The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein. Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are contemplated by the present invention.
The approximate location of the "signal peptides" of the various PRO
polypeptides disclosed herein are shown in the present specification and/or the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence elertient (e.g., Nielsen et al., Prot. EnQ., 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.
"PRO potypeptide variant" means an active PRO polypeptide as defined above or below having at least about 8096 amino acid sequence identity with a full-length native sequence PRO
polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide. with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO
polypeptide sequence as disclosed herein. Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a PRO polypeptide variant will have at least about 80~%c amino acid sequence identity, alternatively at least about 81 cJc amino acid sequence identity, alternatively at least about 82010 amino acid sequence identity, alternatively at least about 8390 amino acid sequence identity, alternatively at least about 849 amino acid sequence identity, alternatively at least about 85cIo amino acid sequence identity. alternatively at least about 86CC amino acid sequence identity, alternatively at least about 8790 amino acid sequence identity, alternatively at least about 889c amino acid sequence identity, alternatively at least about 89% amino acid sequence identity.
alternatively at least about 9090 amino acid sequence identity, alternatively at least about 9190 amino acid sequence identity. alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95cIc amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 9890 amino acid sequence identity and l5 alternatively at least about 99cic amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, PRO
variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or store.
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO 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, ~/o 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, lnc., and the source code shown in Table 1 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 is 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 plc 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 9c amino acid sequence identity of A to B will not equal the ~Yo amino acid sequence identity of B
I S to A. As examples of 9o 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 otherwise, all ~Yo amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computerprogram.
However, R6 amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Alcschul et ttl.. Nucleic Acids Res.. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from National Center for Biotechnology Information. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, tutmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for mufti-pass = 25, dropoff for final gapped alignment = 25 and ~o~g matrix = BLOSL1M62.
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the St; 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 96 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 S6 amino acid sequence identity of A to B will not equal the 96 amino acid sequence identity of B
to A.
In addition. 96 amino acid sequence identity may also be determined using the WLJ-BLAST-2 computer program (Altschul et al.. Methods in Enzvmolow, 266:460-480 11996)). Most of the WLJ-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 = I, overlap traction = 0.125, word threshold (T) = I I, and scorin~ 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 polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide,of interest is being compared which may be a PRO
variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO
polypeptide of interest. For example, in the statement "a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B". the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein. a full-length native sequence PRO polypeptide sequence tacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein.
Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity. alternatively at least about 93%
nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97%
nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence PRO polypeptidesequence as disclosed herein. a full-length native sequence PRO
polypeptide sequence lacking the signal peptide as disclosed herein. an extracellular domain of a PRO
polypeptide. with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO
polypeptide sequence as disclosed herein.
Variants do not encompass the native nucleotide sequence.
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length. alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length.
alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length.
alternatively at least about 600 _17_ nucleotides in length, alternatively at least about 900 nucleotides in length, or more.
"Percent (q6) nucleic acid sequence identity" with respect to the PRO
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 PRO 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, Ine., and the source code shown in Table 1 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.
IS TXUS10087. 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 I. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UMX V4.OD. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the °!o 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 ~fo nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 tirnes the fraction W/Z
where W is the number of nucleotides scot~ed 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 ~k nucleic acid sequence identity of C to D will not equal the ~ nucleic acid soquence identity of D to C. As examples of 9fc nucleic acid sequence identity calculations, Tables 2C-2D demonstrate how to calculate the ~k nucleic acid sequence idencityof the nucleic acid sequence designated"Cort>parison DNA" to the nucleic acid sequence designated "PRO-DNA".
Unless specifically stated otherwise, all 9E nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program.
However, 9e nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et at., ~iucleic Acids Res., 25:3389-3402 (1997)).
NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occucrettces =10, minimum tow complexity lenettt =15/5, mold-pass e-value =
0.01, constant for multi-pass = 25, -18_ dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the %
nucleic acid sequence identity of a given nucleic acid sequence C to, with. or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
In addition, %r 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 = 1, overlap fraction = 0.125, word threshold (T) = 1 I, 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
polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide-encoding nucleic acid molecule of interest. For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules that encode an active PRO
polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encodins the full-length PRO polypeptide shown in the accompanying figures. PRO
variant polypeptides may be those that are encoded by a PRO variant polynucleotide.
The tetzrt "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.
For 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 9c 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 of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within recombinant cells.
since at least one component of the PRO
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 PRO polypeptide or an "isolated" nucleic acid encoding an anti-PRO 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 of the PRO-encoding nucleic acid or the anti-PRO-encoding nucleic acid. Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated. An isolated PRO-encoding nucleic acid molecule or an anti-PRO-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 PRO-encoding nucleic acid molecule or the anti-PRO-encoding nucleic acid molecule as it exists in natural cells. However. an isolated nucleic acid molecule encoding a PRO polypeptide or an isolated nucleic acid molecule encoding an anti-PRO
antibody includes PRO-nucleic acid molecules or anti-PRO-nucleic acid molecules contained in cells that ordinarily express PRO polypeptides or express anti-PRO antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide: a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome bindins 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 secretorv 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 olisonucleotide adaptors or linkers are used in accordance with conventional practice.
The term "antibody" is used in the broadest sense and specitically covers, for example, single anti-PRO
S monoclonal antibodies (includinc antasonist, and neutralising antibodies), anti-PRO antibody compositions with polyepitopic specificity, single chain anti-PRO antibodies, and fragments of anti-PRO antibodies (see below). The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially 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 Bioloev, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that:
( 1 ) employ low ionic strength and high temperature fer washing, for example 0.015 M sodium chloride/0.0015 M
sodium citratel0.196 sodium dodecyl sulfate at 50°C: (2) employ during hybridization a denaturing agent, such as fortnamide, for example, 5096 (vlv) forntamide with 0.196 bovine serum albuminJ0.19o Ficoll0.196 polyvinylpyrrolidond50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ SOS6 formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.196 sodium pyrophosphate, 5 x Denhardt's solution.
sonicated salmon sperm DNA (50 ~cg/ml), 0.19 SDS, and 1096 dextran sulfate at 42"C. with washes at 42"C in 0.2 x SSC (sodium chloride/sodium citrate) and 5096 formamide at 55"C. followed by a high-stringency wash consisting of 0.1 x SSC containing FrDTA
at 55°C.
-"~Nloderately stringent conditions" may be identified as described by Sambrook er vl., Molecular Clonins:
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 96 SDS) less stringent than those described above.
An example of moderately stringent conditions is overnight incubation at 37"C
in a solution comprising: 209c fotmatrtide. 5 x SSC ( 150 mM NaCI, 15 mM trisodium citrate). 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution,109E dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 35°C-50"C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accornmodate factors such as probe Icngth and the tike.
The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a PRO
polypeptide fused to a "tag polypeptide". The tag polypeptide has tnough residues to provide an epitope against which an antibody can be made, yet is shoe enough such that it does not interfere with activity of the polypeptide *-trademark to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
"Active" or "activity" for the purposes herein refers to forms) of PRO
polypeptides which retain a biological and/or an immunological activitylproperty of a native or naturally-occurring PRO polypeptide, wherein "biological" activity refers to a function (either inhibitory or stimulatory) caused by a native or naturally-occurring PRO polypeptide other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO polypeptide and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO polypeptide.
"Biological activity'' in the context of an antibody or another antagonist molecule 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 bind or complex with the polypeptides encoded by the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins or I S otherwise interfere with the transcription or translation of a PRO
polypeptide. A preferred biological activity is growth inhibition of a target tumor cell. Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell.
The term "biological activity" in the context of a PRO polypeptide means the ability of a PRO polypeptide to induce neoplastic cell growth or uncontrolled cell growth.
The phrase "immunological activity" means immunological cross-reactivity with at least one epitope of a PRO polypeptide.
"Immunological cross-reactivity" as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO
polypeptide having this activity with polyclonal antisera raised against the known active PRO 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,followedbyboosterintraperitonealorsubcutaneousinjectioninincompleteFre unds. Theimmunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologicallycross-reactive molecule (e.g., antibody) identified, to the corresponding PRO polypeptide is significantly higher (preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 8-times, most preferably at least about 10-times higher) than the binding affinity of that molecule to any other known native polypeptide.
The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO polypeptide disclosed herein or the transcription or translation thereof. Suitable antagonist molecules specifically include antagonist antibodies or antibodyfragrnents, fragments, peptides, small organic molecules, anti-sense nucleic acids. etc.
Included are methods for identifying antagonists of a PRO polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO polypeptide.
A "small molecule" is defined herein to have a molecular weight below about 500 Daltons.
"Antibodies" (Abs) and "immunoglobulins" (Igs) are olycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoslobulins include both antibodies and other antibody-like molecules which lack antigen specificity.
Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. The term "antibody"
is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
"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 of different immunoglobulin isotypes. Each heavy and li ght 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 complementarity-determining 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 (FR) regions. The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a (3-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the p-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 (L1 ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H 1 ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Seguences of Proteins of Immunolosical 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 (LI ), 50-52 lL2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1). 53-55 (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')=, and Fv fragments;
diabodies; linearantibodies (Zapataetal., Protein Ena. , 8( 10):1057-1062 [
1995]); single-chain antibody molecules;
and multispeciftc antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fraQrnents, each with a single antigen-binding site, and a residual "Fc" fragment, whose name retlects its ability to crystallize readily. Pepsin treatment yields an F(ab'), fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site.
This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-V~ dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) 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 1 ) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain 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')= antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (tc) 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, irrununoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG l, IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, b, e, y, and lt, respectively. The subunit structures and three-dimensional configurations of different classes of irnmuno~lobulins are well known.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantial ly homogeneous antibodies, i. e.. the individual antibodies comprising the population are identical except for possible natural ly occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to. conventional (polyclonal) antibody preparations which typically include different antibodies directed against differentdeterminants(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 monoclonal 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], or tray 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, 352:624-628 [ 1991 ] and Marks et al.. J. Mol. Biol.. 222:581-597 ( 1991 ), for example.

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulinsl 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 of the 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 et al., Proc.
Natl. Acad. Sci. USA. 81:6851-6855 [ 1984] ).
"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. For the most pan, 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 IS 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, vat7able 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 irrununoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al.. Nature, 321:522-525 ( 1986); Reichmann et al.. Nature, 332:323-329 [1988]; and Presta, Curr. On. Struct. Biol., '?:593-596 (1992).
The humanized antibody includes a PRIMATIZED"" antibody wherein the antigen-binding region of the antibody is derived froman antibody produced by immunizing macaque monkeys with the antigen of interest. ' "Single-chain Fv" or "sFv" antibody fragments comprise the V" and VL domains of antibody. wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V" and V~ domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see, Pluckthun in The Pharmacoloov of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V~) in the same polypeptide chain (VH - V~). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP -104,097; WO 93/1 1161; and Hollinger et al.. Proc. Natl. Acad. Sci. USA, 90:6444-64:18 (1993).
An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments. the antibody will be puritied ( 1 ) to greater than 959c 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 I S residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in sire 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 (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. Radionuclides that can serve as detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. 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 of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heteroiogous 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 pan 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 rnay be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG~ subtypes, IgA (including IgA-I and IgA-2), IgE, IgD or IgM.
As shown below, Table 1 provides the complete source code for the ALIGN-2 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-2B) 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 PRO polypeptide of interest. "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, "PRO-DNA" represents a hypothetical PRO-encoding nucleic acid sequence of interest, "Comparison DNA"
represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, "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; ) (joker) match = 0 */
Ndefine M -8 J* 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 0 P Q R S T U V W X Y Z */
I* A */ { 2, 0,-2, 0, 0,-4, l,-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. ?. M,-1, 1. 0, 0, 0, 0,-2,-5, 0,-3, 1 }, /* C */ {-2,-4,15.-5,-5,-4,-3.-3,-2. 0,-S,-6,-S,-4, M,-3,-5.-4, 0,-2, 0,-2,-8, 0, 0,-5}, /* D */ { 0, 3,-S, 4, 3.-6, 1, 1,-2, 0, 0,-4.-3. 2, M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2}, /* E */ { 0, 2,-S, 3, 4.-5, 0, 1,-2, 0, 0,-3,-2. 1, M,-1. 2.-1, 0, 0, 0,-2,-7, 0,-4, 3}, /* F */ {-4,-S,-4,-6.-5, 9,-S,-2, I, 0,-5, 2, 0.-4._M,-5,-S,-4,-3,-3, 0,-1, 0, 0, 7,-5}, /* G *l { 1. 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3. 0,_M.-1,-1,-3, 1, 0, 0,-1,-7, 0,-S, 0}, /* H */ {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2. M. 0, 3, 2,-I,-1, 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}, I*J*I {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 */ {-I, 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}, /* L */ {-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4.-3,_M,-3,-2,-3,-3,-1, 0. 2,-2, 0,-1,-2}, /* M */ {-I,-2,-5,-3,-2, 0,-3.-2, 2. 0, 0, 4, 6.-2, M,-2,-1, 0,-2,-1, 0, 2,-4, 0,-2,-1}, /* N */ { 0, 2,-4, 2, 1,-4. 0, 2,-2, 0, 1,-3,-2. 3. M,-1, 1, 0, 1, 0, 0.-2,-4, 0,-2, 1}, /* O */ { M, M, M, M, M, M, M, M, M. M,_M._M. M, M, 0, M,_M. M,_M, M, M.-M, M, M. M, M}, /* P */ { 1,-1.-3; 1,-1,-5,-1, 0,-2, 0,-1,-3,-2,-I,_M, 6, 0. 0. 1, 0, 0,-1,-6, 0,-5, 0}, /* Q */ { 0. 1,-S, 2, 2,-S,-1, 3,-2, 0, 1,-2,-1, I, M, 0, 4, I,-1,-1, 0,-2,-5, 0,-4, 3}, /* R */ {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3. 0, 0, M, 0, I, 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. I,-1, 0, 2, 1, 0,-1.-2, 0,-3, 0}, /* T */ { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, O, 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. 0, M, 0, 0, 0, 0; 0, 0, 0, 0, 0, 0, 0}, /* V */ { 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2. M,-1,-2,-2,-1, 0, 0, 4,-6, 0,-2,-2}, /* W */ {-6,-S.-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*/ {0,0,0,0,0,0,0,0Ø0,0,0,0,0, M,0,0,0,0,0,0.0,0,0,0,0}, /* Y */ {-3,-3, 0.-4,-4, 7,-5, 0,-1, 0,-4,-1,-2.-2, M,-5,-4,-4,-3,-3, 0,-2, 0, 0,10,-4}, /* Z */ { 0, I.-S, 2, 3,-5, 0, 2,-2. 0. 0,-2,-1, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4}
};

Table 1 (cony) /*

*/

liinclude< >
stdio.
h Ninclude< h >
ctype.

JldefineMAXJMP /* max jumps in a diag 16 */

kdefineMAXGAP /* don't continue to penalize 24 gaps larger than this */

JldefineIMPS 1024 /* max jmps in an path */

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

#defineDMAT 3 1* value of matching bases */

/IdefineDMIS 0 /* penalty for mismatched bases */

NdefineDINSO8 /* penalty for a gap */

JldetineDINS11 /* penalty per base *1 !!definePINSO8 /* penalty for a gap */

NdefinePINS14 /* penalty per residue */

structp jm {

shortn[MAXJ MP]; /* size of jmp (neg for dely) */

tmsigned MP]; /* base no. of jmp short in seq x */
x(MAXJ

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

structag di {

int score; /* score at last jmp */

long offset; /* offset of prey block */

shortijmp; /* current jmp index */

struct /* list of jmps */
}: jmp jp;

struct path {

int spc; /* number of leading spaces */

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

int x[IMPS]; jmp (last elem before gap) /* loc */
of };

char *ofile; /* output file name */

char *namex[2];I* seq names: getseqsQ
*/

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

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

int dmax; /* best diag: nwQ */

int dmax0; I* final diag *I

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

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

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

int IenO, /* seq lens */
lenl;

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

int smax; /* max score: nw() */

int *xbm; /* bitmap for matching */

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

structdiag *dx; /* holds diagonals */

structpath pp(2]; l* holds path for seqs *I

char *callocQ,, *index(), *strcpyQ;
*mallocQ

char *getseqQ.
*g_calloc();

Table 1 (cony) /* 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 /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 */
Xinclude "nw.h"
#include "day.h"
static _dbval[26] _ {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10.0 static _pbval[26] _ {
1, 2~(1 < <('D'-'A'))~(1 < <('N'-'A')), 4, 8, 16, 32, 64, 128, 256, OxFFFFFFF, 1 < < 10, 1 < < 11, 1 < < 12, 1 < < 13, 1 < < 14, 1«15. 1«16, 1«17, 1«l8, 1«19, 1«20, 1«21. 1«22, 1 < < 23, 1 < < 24, 1 < < 25 ~ ( 1 < < (' E'-' A' )) ~ ( 1 < < (' Q'-' A' )) main(ac, av) Ink int ac;
char *av~;
prop = av[O];
if (ac ! = 3) {
fprintf(stderr,"usage: qbs filet file2ln", prop);
fprintf(stderr,"where filet and filet are two dna or two protein sequences.\n");
fprintf(stderr,"The sequences can be in upper- or lower-case\n");
fprintf(stderr,"Any lines beginning with ';' or ' <' are ignored\n");
fprintf(stderr,"Output is in the file \"align.out\"\n");
exit(1);
namex(O] = av[1];
namex(I] = av[2];
seqx(0] = getseq(namex[0], &IenO);
seqx[l] = getseq(namex[1], &lenl);
xbm = (dna)? dbval : pbval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "align.out"; /* output file */
nwQ; /* fill in the matrix, get the possible jmps */
readjmpsQ; /* get the actual jmps */
printQ; /* print stars, alignment */
cleanup(0); l* unlink any tmp tiles */

Table 1 (cony) /* 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.
*/
nw() nW

{

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

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

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

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

int mis; /* score for each type */

int ins0, insl;/* insertion penalties */

register id; /* diagonal index */

register ij; /* jmp index */

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

register xx, yy; /* index into seqs */

dx = (struct diag *)g calloc("to get diags", IenO+lenl +I, sizeoflstruct 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+I, sizeof(int));
col l = (int *)g calloc("to get col l ", lenl + I, sizeof(int));
ins0 = (dna)? DINSO : PINSO;
insl = (dna)? DINS1 : PINS1;
smax = -10000;
if (endgaps) {
for (col0[0] = dely[0] _ -ins0, yy = 1; yy < = lenl; yy++) {
col0[yy] = dety[yyJ = col0[yy-1] - insl;
ndely[yy] = YY:
col0[0] = 0; /* Waterman Bull Math Biol 84 *I
else for (yy = 1; yy < _ lent; yy++) dely[yy] _ -ins0;
/* fill in match matrix */
for (px = seqx[0], xx = I; xx < = len0; px++, xx++) {
/* initialize first entry in col */
if (endgaps) {
if (xx = = 1 ) coil[0] = delx = -(ins0+insl);
else coll[0] = delx = col0[OJ - insl;
ndelx = xx;
else {
col l [0] = 0:
delx = -ins0;
ndelx = 0;

Table 1 (cony) for(py = seqx[1], yy = 1: yy < = lenl; py++, yy++) {
mis = col0[yy-(];
if (dna) mis +_ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else mis += day[*px-'A'][*py-'A'];
/* 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 {
deiy[yy] -= insl;
ndely[yy) + +;
}
} else {
if (col0[yy] - (ins0+insl ) > = dely[yY)) {
deiy[yy] = col0(yy) - (ins0+insl);
ndely[yy] = 1:
} else ndely[yy)++;
/* update penalty for del in y seq;
* favor new dei over ongong del */
if (endgaps J J ndelx < MAXGAP) {
if (coll[yy-1) - ins0 > = delx) {
delx = coll(yy-1] - (ins0+insl);
ndclx = 1;
} else {
delx -= insl;
ndelx++;
}
}else{
if (coll[yy-1] - (ins0+insl l > = delx) {
delx = coll[yy-1] - (irts0+insl);
ndelx = 1:
} else ndelx++;
}
/* pick the maximum score; we're favoring * mis over any del and delx over delY
*1 ...nw Table 1 (cony) id=xx-yy+lenl-1;
if (mis > = delx && mis > = dely[yy]) col 1 [yy] = mis;
else if (delx > = dely[yy]) {
colt[yy] = delx;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndelx > = 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] = ndelx;
dx[id].jp.x(ij] = xx;
dx[id].score = delx;
else {
coll(yy] = 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(yy];
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
if ixx _= IenO && yy < lenl) {
/* last col */
if (endgaps) coil[yy] -= ins0+insl*(lenl-yy);
if (coll[yy] > smax) {
smax = coll(yy];
dtnax = id;
if (endgaps && xx < IenO) coil[yy-1] -= ins0+insl*(IenO-xxj;
if (col l [yy- I ] > smax) {
smax = coi l [yy-1 ];
dmax = id;
tmp = col0: col0 = coll; coil = tmp;
(void) free((char *)ndely);
(voidi free((char *)dely);
(voidi free((char *)col0);
(voidi free((char *)coll);
...nw Table 1 (cony) i*
* 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_alien() * numsQ -- put out a number line: dumpblock() * putline() -- put out a line (name, [num], seq, [numb): dumpblockQ
* stars() - -put a line of stars: dumpblockQ
* stripttameQ -- strip any path and pretix from a seqname */
Ninclude "nw.h lldefine SPC 3 lldefine P LINE 256 1* maximum output line */
Hdefine PSPC 3 /* space between name or num and seq */
extern day[26][26];
int oleo; /* set output line length */
FILE *fx; I* output file *I
print() pTlnt {
int lx, ly, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) _ = 0) {
fpcintf(stderr," ~s: can't write 9~s\n", prog, ofile);
cleanup(1);
fprintf(fx, "<first sequence: %s (length = ~d)\n", namex[0], IenO);
fprintf(fx, "<second sequence: ~s (length = hod)\n", namex(1], lenl);
oleo = 60;
lx = IenO;
'ly = lent;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x */
pp[0]-spc = firstgap = lenl - dmax - l;
ly _= pP[01~~:
else if (dtnax > lenl - l) { /* leading gap in y */
pp[1]-spc = firstgap = dmax - (lenl - 1);
lx -= PP[1]~sPc;
if (dmax0 < IettO- I) { /~' trailing gap in x *1 lastgap = IenO - dmax0 -1;
Ix -= lastgap;
else if (dmax0 > IenO - 1 ) { /* trailing gap in y */
lastgap = dmax0 - (IenO - 1);
ly _= lastgap;
getmat(Ix, ly, firstgap, lastgapl;
pr align();

/*
* trace back the best path, count matches */
static Tabte 1 (cony) getmat(Ix, ly, firstgap, lastgap) getnlat int Ix, ly; /* "core" (minus endgaps) *!
int firstgap, lastgap; /* leading trailine_ overlap *!
int nm, i0, il, siz0, sizl;
char outx[32];
double pct;
register n0, nl;
register char *p0, *pl;
/* get total matches, score */
i0 = il = siz0 = sizl = 0;
p0 = seqx(0] + pp[1].spc;
P1 = ~qx(1] + PP[Ol.sPc:
n0 = pp[lJ.spc + 1;
nl = pp(O].spc + 1;
tun = 0;
white ( *p0 && *pl if (siz0) {
pl++;
nl++;
siz0--;
else if (sizl) {
p0++;
n0++;
siz l--;
else {
if (xbm[*p0-'A']&xbm[*pl-'A' nm++;
if (n0++ _= pp[OJ.x[i0J) siz0 = pp[0].n[i0++I;
if (nl++ _= pp(1].x[ilJ) sizl = pp[1].n[il++];
p0++;
pl++;
/* pct homology:
* if penalizing endgaps, base is the shorter seq * else, knock off overhangs and take shorter core */
if (endgaps) Ix = (IenO < lenl)? IenO : lenl;
else Ix = (Ix < ly)'? Ix : ly;
pct = 100.*(doublelrun/(double)Ix;
fprintflfx, "\n");
fprintf(fx, "< 5~d match's in an overlap of 5(d: 9.2f percent similarity\n", tun, (nm == 1)? ",. . ,.es,., Ix, pct);

Table 1 (cont'1 tprintf(fx. " < gaps in first sequence: %d", gapx); ...getltlat if (gapxj {
(void) sprintf(outx, " (%d ~s%s)", ngapx, (dna)? "base":"residue", (ngapx == 1f' "":"s");
tprintf(fx," l s", outx);
fprintf(fx, ", gaps in second sequence: '~d", gapy);
if (gapY) {
(void) sprintf(outx, " (7od 56s%s)", ngapy, (dna)? "base":"residue", (ngapy == 1)? "":"s");
tprintf(fx,"'ks", outxj;
if (dna) fprintf(fx, "\n< score: %d (match = ld, 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 + god per residue)\n", stnax, PINSO, PINSI);
if (endgaps) tprintf(fx, "<endgaps penalized. left endgap: %d %s%s, right endgap: ~d %s%s\n", firstgap, (dna)? "base" : "residue",(firstgap = = I)? "' . "s", lastgap. (dna)? "base" : "residue", (lastgap == I)? .., ~ "s");
else fprintf(fx, " < endgaps not penalized\n");
staticnm; /* matches in core -- for checking *!

statichnax; /* lengths of stripped file names */

staticij[2]; /* jmp index for a path */

staticnc[2]; /* number at start of current line */

staticni[Z]; /* current elem number -- for gapping *I

staticsiz[2];

static*ps(2]; /* ptr to current char element */

static*po[2]; /* ptr to next output char char slot */

staticout[2][P /* output line */
char LINE];

staticstar[P LINE]:i* set by stars() char */

/*
* print alignment of described in struct path pp[]
*1 static pr_augnp pr_align int nn: /* char count */
int more; -register i:
for ii = 0, lmax = 0: i < Z: i++) {
nn = stripname(namex[i]);
if (nn > Imax) Imax = nn;
nc[i] = I:
ni[i] = I:
siz[i] = ij[i] = 0:
ps[ij = seqx(i];
po[i] = out[i]:

Table 1 (cony) for (nn = nm = 0, more = 1; more; ) { ...pr_align for (i = more = 0; i < 2; i++) {
I*
* do we have more of this sequence'?
*/
if (!*ps[i]) continue:
more+ +;
if (pp[i].spc) { /* leading space *1 *po[i]++ _ .
PP(i].sPc-:
else if (siz[i]) { l* in a gap */
*po[i]++ _ , siz[i]__;
else { /* we're putting a seq element */
*Po(il = *Ps(i1;
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(il.x(i.Jfi]D {
/*
* we need to merge all gaps * at this location */
siz[i] = pp[i].n[ij(i]++]:
while (ni(i] _= pp[i].x(ij[i]]) siz[i] += pp[i].n[ij[i]++]:
ni[i]++;
if (++nn == olen ~ ~ !more && nn) {
dumpblockQ;
for (i = 0; i < 2; i++) po[i] = out(i];
nn = 0;
/*
* dump a block of lines. including numbers, stars: pr align() */
static dumpblockp dumpblock register for (i = 0; i < 2; i++) *po[;]__ _ ';0':

Table I (cony) (void) putt('\n', fx):
for (i = 0; i < 2: i++) {
if (*out[i] && t*out[i[ ~ _ , . I I *(po[i]) ! _ ' ')) {
if (i = = 0) nums(i):
if (i == 0 && *out[1]) starsQ:
purl inet i):
if (i == 0 && *out[1]) fprintf(fx, star);
if (i == 1) nums(i);
/*
* put out a number line: dumpblock() */
static ...dumpblock nums(ix) hums int ix; I* index in out[] holding seq line *I
{
char mine[P-LINE];
register i, j;
register char *pn, *px, *py;
for (pn = nline, i = 0; i < Imax+P SPC; i++, pn++) *pn = , for (i = nc[ix], py = out[ix[; *py; py++, pn++) {
if (*pY =- ~ I I *PY =- ~-~) *Pn =
else {
if (ilol0 == 0 I I (i == 1 && nc[ix] != 1)) {
j = (i < 0)? -i : i;
for (px = pn; j; j /= 10, px--) *px = j910 + '0';
if (i < 0) *p -else *pn = .
i++;
*Pn = .\0.;
nc[ix] = i;
for (pn = mine; *pn; pn++) (void) putt(*pn, fx);
(voidj putt('\n', fx);
/*
* put out a line (name. [num], seq, [num]): dumpblock() */
Static pudine(ix) putline int ix; {

Table 1 (cony) int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px ! _ ':'; px++, i++) (void) pate(*px. fx);
for (; i < Imax+P SPC; i++) (void) pate(' ', fx);
/* these count from 1:
* ni[] is current element (from 1) * txQ is number at start of current line *I
for (px = out[ixJ; *px; px++) (void) pate(*px&Ox7F, fx);
(void) putt('\n', fx);
...putline /*
* put a line of stars (seqs always in out[0], out[1[): dumpbiockQ
*/
static stars() stars {
int i:
register char *p0, *pl, cx, *px;
if (!*out[OJ II (*out[0] _ _ ' &8c *(po[Oj) _ - ' ') II
!*out[1] I I (*out[1] __ ' && *(Po[Il) _- ' 7) return;
px = star;
for (i = Imax+P SPC: i; i--) *px++ _ ' , for (p0 = out[OJ, pl = out[1); *p0 && *pl; p0++, pl ++) {
if (isalpha(*p0) 8r.8c isalpha(*p1)) {
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++ _ '\n';
*Px = '\0';
cx = , Table 1 (cont'1 /*
* 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 = 0:
for (px = pn; *px; px++) if (*px =_ '/') py=px+1;
if (py) (void) strcpy(pn, py);
return(strlen(pn));
-:l0-Table 1 (cony) /*
* cleanup() -- cleanup any tmp tile * getseqQ -- read in seq, set dna, len, maxlen * g callocQ -- calloc() with error checkin * readjmpsp -- eet the good jmps, from tmp file if necessary * writejmps() -- write a filled array of jmps to a tmp file: nwQ
*l kinclude "nw.h"
Ninclude <sys/file.h>
char *jname = "hmp/homgXXXXXX"; /* tmp tile for jmps */
FILE *fj;
int cleanup(); /* cleanup tmp file */
long (seek();
/*
* remove any tmp file if we blow */
cleanup(i) Cleanup int i:
{
if (fj) (void) unlinkQname);
exit(i);
*
* read, return ptr to seq, set dna, len, maxlen * skip lines staving with ';', ' <', or ' > ' * seq in upper or tower case */
char gecseq(file, len) getseq char *file; /* file name */
int *len; /* seq ten */
{
char line(1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp;
if ((fp = fopen(file, "r")) _ = 0) {
fprintf(stderr."Sbs: can't read 9bs\n", prog, file);
exit(1);
tlen = natgc = 0;
while (fgets(Iine, 1024, fp)) {
if (*line =_ .' ~ ~ *line =_ ' <' ~ ~ *line =_ ' >') continue;
for (px = line; *px !_ '\n'; px++) tf (isupper(*px) ~ ~ islower(*px)) tlen++;
if ((pseq = malloc((unsigned)(tlen+6))) _ = 0) {
fprintf(stderr."~s: malloc() failed to get ~kd bytes for ~ks\n", prog, tlen+6, file):
exit(1);
Ps~1(Ol = P~9(17 = Pse9I21 = P~I(31 = '\0';
~ll-Table 1 (cony) py = pseq + 4;
*len = tlen:
rewind(fp);
while (fgets(line, 1024, fp)) {
if (*line =- .' ~ ~ *line =- ' <' ~ ~ *line =- ' >') continue;
for (px = line; *px ! _ '\n'; px++) {
' if (isupper(*px)) *py++ _ *px;
else if (islower(*px)) *py++ = toupper(*px);
if (index("ATGCU",*(py-1))) natgc+ +;
*py + + _ '\0';
*py = '\0';
(void) fclose(fp);
dna = natgc > (tlen/3);
return(pseq+4);
..getseq char g calloc(msg, nx, sz) g_calloc char *msg; /* program, calling routine *!
int nx, sz; /* number and size of elements */
{
char *px, *calloc();
if ((px = calhx((unsigned)nx, (unsigned)sz)) _= 0) {
if (*msg) {
fprintflstderr, "%s: g callocQ failed %s (n= 5~d, sz= 5bd)\n", prog, msg, nx, sz);
exit( 1 );
return(px);
/*
* get tinal jmps from dx[) or tmp tile, sec pp[j, reset dmax: main() *I
readjmpsp readjmps {
int fd = -I;
int siz, i0, il;
register i, j, xx;
if (fj) {
(void) fclose(fj);
if ((fd = open(jname, O_RDONLY, 0)) < 0) {
tprintf(stderr, 9s: can't open() SEs\n", prog, jname);
cleanup( 1 );
for (i = i0 = il = 0, dmaxU = dmax, xx = IenO; ; i++) {
while (1) {
for (j = dx[dmaxj.ijmp; j > = 0 && dx[dtnax].jp.x[j] > = xx; j--) Table 1 (cony) ...readjmps if (j < 0 && dx[dmax].offset && fj) {
(void) Iseek(fd, dx[dmax].offset, 0);
(voidl read(fd, char *)&dx[dmax].jp, sizeof(struct jmpj);
(void) read(fd. (char *)&dx[dmax].offset, sizeof(dx[dmax].offsetj);
dx(dmax].ijmp = MAX1MP-I;
else break:
if (i > = JMPS) {
fprintf(stderr, "'~s: too many gaps in alignmentln", prog);
cleanup(1);
ifQ >=0){
siz = dx[dmax].jp.n[j];
xx = dx(dmaxJ.jp,x[j];
dmax + = siz;
if (siz < 0) { /* gap in second seq */
pp[lJ.n(il] _ -siz;
xx + = siz;
/*id=xx-vv+lenl-1 */ .-pp(1].x[il] = xx-dmax + lenl - 1;
gapy++;
ngapy -= siz:
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP ~ ~ endgaps)'? -siz : MAXGAP;
il++;
else if (siz > 0) { /* gap in first seq */
PP[Ol.n(i0] = siz:
pp[Oj.x[i0J = xx;
gapx+ +;
ngapx + = siz:
I* ignore MAXGAP when doing endgaps *I
siz = (siz < MAXGAP ~ ~ endgaps)? siz : MAXGAP;
i0++;
) ) else break;
) I* reverse the order of jmps */
for (j = 0, i0--; j < i0; j++, i0--) {
i = PP[Ol.n~]; PP(0]~n~l = PP[0].n[iOJ; PP[OJ.n[i0] = i:
i = PP[0].xUJ; PP[OI.xGJ = PP[Ol~x[i0]: PP[O].x[i0] = i;
for (j = 0, il--; j < il; j++, il--) {
i = PP[1]-n~J; PP[Il~n~l = PP[1].n[il]: PP[1]~n[ilJ = i:
i = PPfI]~xGl; PP(ll.x~J = PP[ll.x[il]; PP[1].x(il] = i:
) if (fd > = 0) (void) close(fd);
if (fj) {
(void) unlink(jname);
fj = 0;
offset = 0:
--t3-Table 1 (cony) i*
* write a filled jmp struct offset of the prey one (if any): nw() *~
writejmps(ix) WTItejIllpS
int ix;
char *mktempQ;
if (!tj) {
if (mktemp(jname) < 0) {
fprintf(stderr, "'~s: can't mktempp '~s\n", prog, jname);
cleanup( 1 );
if ((fj = fopen(jname, '.w'~)) _= 0) {
fprintf(stderr, ":Es: can't write 5bs\n", prog, jname);
exit(1 );
(void) fwrite((char *)Bcdx[ix].jp, sizeof(struM jmp), 1, fj);
(void) fwrite((char *)&dx[ix).offset, sizeof(dx[ix].offset), 1, fj);
_44_ 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
polypeptide) _ divided by 15 = 33.3 Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids) Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids) % amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _ divided by 10 = 50%
-.~6-Table 2C
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides j '7o 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%
=17-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 %

II. Compositions and Methods of the Invention A. Full-length PRO Polyuentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides. In particular.
cDNA encoding PRO polypeptides has been identified and isolated, as disclosed in further detail in the Examples below. It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ
number is unique for any given DNA and the encoded protein, and will not be changed. However, for sake of simplicity, in the present specification the proteins encoded by the herein disclosed nucleic acid sequences as well as all further native homologues and variants included in the foregoing definition of PRO
polypeptides will be referred to as "PRO"
regardless of their origin or mode of preparation.
As disclosed in the Examples below, cDNA clones have been deposited with the ATCC. The actual nucleotide sequence of the clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequences can be determined from the nucleotide sequences using routine skill. For the PRO polypeptides and encoding nucleic acid described herein, Applicants have identified what are believed to be the reading frames best identifiable with the sequence information available at the time.
B. PRO Polvnentide Variants In addition to the full-length native sequence PRO polypeptides described herein, it is contemplated that PRO polypeptide variants can be prepared. PRO polypeptide variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA and/or by synthesis of the desired PRO
polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-uanslational processes of the PRO polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PRO polypeptide or in various domains of the PRO
polypeptidedescribed herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No.

5,364.934. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO
polypeptide that results in a change in the amino acid sequence of the PRO polypeptide as compared with the native sequence PRO polypeptide.
Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO polypeptide. 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 PRO polypeptide 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 replacins 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 ranee of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

WU UU/75317 rw mu~uumss~u PRO 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 PRO polypeptide.
PRO polypeptide fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO polypeptide fragments by enzymatic digestion. e.g., by treatin_ 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 enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR).
Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide.
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 20Arg (R) lys; gln; asn IYs Asn (N) gln; his; lys; arg gln Asp (D) flu glu Cys (C) ser ser Gln (Q) asn asn 25Glu (E) asp asp Gly (G) pro; ala ala His (H) asn: gln; lys; arg arg Ile (I) leu; val; met: ala;
phe;

norleucine leu 30Leu (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; leu tyr 35Pro (P) ala ala Ser (S) thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe 40Val (V) ile; leu; met; phe;

ala; norleucine leu Substantial modifications in function or immunological identity of the polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution. for example, as a sheet or helical conformation. (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro: and (6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
Such substituted residues also may be inuoduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as oligonuclebtide-mediated (site-directedl mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.
. Acids Res., 13:4331 ( 1986); Zoller et al., Nucl. Acids Res.. 10:6487 ( 1987)], cassette mutagenesis [Wells et al..
Gene, 3:315 ( 1985)], restriction selection mutagenesis [Wells etal., Philos.
Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science. 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant. an isoteric amino acid can be used.
C. Modifications of PRO Polvnentides Covalent modifications of the PRO polypeptide are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a PRO polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO
polypeptide. Derivatization with bifunctional agents is useful, for instance.
for crosslinking the PRO polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies. and vice-versa. Commonly used crosslinking agents include. e.g., 1.1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde. N-hydroxysuccinimideesters, for example, esters with 4-azidosaiicylic acid.
homobifunctional imidoesters. including disuccinimidyl esters such as 3.3'-dithiobis(succinimidylpropionate).
bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding ~lutamyl and aspartyl residues. respectively, hydroxylation of proline and lysine. phosphorylation of hydroxyl groups of seryi or threonyl residues, methyiation of the a-amino groups of lysine. arginine. and histidine side chains [T.E. Creiohton. Proteins: Structure and Molecular Properties, W.H. Freeman &
Co., San Francisco, pp. 79-86 ( 1983)], acetylation of the N-terminal amine. and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the PRO polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
"Altering the native ~lycosylation pattern"
is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO
polypeptides (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO polypeptide. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO polypeptide (for O-linked glycosylation sites). The PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PRO
polypeptide is by chetnical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO
87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 ( 1981 ).
Rernoval of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzyrrtatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, er al., Arch. Biochem. Bionhys., 259:52 ( 1987) and by Edge et al., Anal. Biochem.. 118:131 ( 1981 ).
Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al.. Meth. Enzvmol., I 38:350 (1987).
Another type of covalent modification of PRO polypeptides comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene slycol, 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 1.179,337.
The PRO polypeptides of the present invention may also be modified in a way to form a chimeric molecule comprising the PRO polypeptide fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO
polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the PRO polypeptide.
The presence of such epitope-tagged forms of the PRO polypeptide can be detected using an antibody against the tai polypeptide. Also. provision of the epitope tag enables the PRO 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 et al., Mol. Cell. Biol., 8:2159-2165 ( 1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular BioloUv, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky etal., Protein En~ineerin~, 30:547-553 ( 1990)]. Other tag polypeptides include the Flab peptide [Hopp etal., BioTechnolow, 6:1204-1210 ( 1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 ( 1992)];
an a-tubulin epitope peptide [Skinner et al., J. Biol. Chem.. 266:15163-15 I
66 ( 1991 )]; and the T7 gene 10 protein peptide tag [Lutz-Freyetmuth etal., Proc. Natl. Acad. Sci. USA. 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule tray comprise a fusion of the PRO polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent fotlrt of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The I~ fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) fomn of a PRO
polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunogiobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH 1, CH2 and CH3 regions of an IgG 1 molecule. For the production of immunoglobulin fusions see also, U.S. Patent No. 5,428,130 issued June 27,1995.
D. Preparation of PRO Polweptides The description below relates primarily to production of PRO polypeptides by culturing cells transformed or transfected with a vector containing the PRO nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PRO polypeptides.
For instance, the PRO 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)]. !n vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosysterns Peptide Synthesizer (Foster City, CA) using manufacturer's instructions. Various portions of the PRO polypeptide may be chemjcally synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO polypeptide.
a. Isolation of DNA Encodine a PRO Polyneptide DNA encoding the PRO polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level. Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The PRO-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.
Libraries can be screened with probes (such as antibodies to the PRO
polypeptide, or oligonucleotides of at feast about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or oenornic library with the selected probe may be conducted using standard procedures. such as described in Sambrook et al.. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding the PRO polypeptide is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory YYV VV//JJ1/ rt.1/VJVV/1JJJ0 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 orenzyme 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 an and as described herein.
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein for PRO
polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transfotmants, 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 Biotechnolow: a Practical Approach, 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, CaCh. CaPO,, liposome-mediated and electroporation. Depending on the host cell used. transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., srlpra, or electroporation is generally used for prokaryotes. Infection with Agrobacrerium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene. ?3:315 ( 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. V iroloev, 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 -etal., J. Bact., 130:946 ( 1977j and Hsiao etal., Proc. Natl. Acad.
Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection. electroporation, bacterial protoplast fusion with intact cells. or polycations, e.g., polybrene.
polyornithine, may also be used. For various techniques for transforming mammalian cells, see, Keown etal., Methods in Enzvmolow, 185:527-53711990) and Mansour et al.. Nature, 336:348-35211988).

wv uur tasi t YC1-/USUU/13358 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. colt.
Various E. colt strains are publicly available, such as E. colt K12 strain MM294 (ATCC 31.-146); E. colt X 1776 (ATCC 31,537); E. colt strain W31 10 (ATCC 27,325) and E colt strain K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. colt, Ernerobacter, Emvirria.
Klebsiella, Proteus. Salmonella, e.g., Salmonella yphirnurinm, Serratia, e.g., Serraria nrorcescans, and Shigella, as well as Bacilli such as B. snbtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudonronas such as P. aernginosa, and Streptomrces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. colt W31 10 strain 1A2, which has the complete genotype ronA ; E. colt W3110 strain 9E4, which has the complete genotype ronA ptr3; E. colt W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype torvl ptr3 phoA EIS (argF-lac)l69 degP ompT kan'; E. colt W3110 strain 37D6. which has the complete genotype torrA ptr3 phoA EIS (argF-lac)l fig degP ompT rbs7 IIvG
kan '; E. colt W31 10 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation;
and an E. colt strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946.783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes; eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [ 1981 ]: EP
139,383 published 2 May 1985); Kluyveromyres hosts (U.S. Patent No. 4,943,529;
Fleer et al.. Bio!>'echnoloov, 9: 968-975 (1991 )) such as, e.g., K. lacris 1MW98-8C, CBS683, CBS4574;
Louvencourt er al., J. Bacteriol.. 7:7 [1983]), K. fragilis (ATCC 12,~i24), K. bulgaricus (ATCC 16,045). K.
wickeramii (ATCC _'-1.1781. K. waltii (ATCC 56,500), K. drosophilarnm (ATCC 36.906; Vanden Berg et al., Bioll'echnoloey, 8:135 ( 1990)), K.
thermotolerans, and K. marxianns: yarrowia (EP402,226); Pichia pastoris (EP
183,070; Sreekrishna eral., J. Basic Microbiol., 28:265-278 [ 1988]); Candida; Trichoderma reesia f EP 244,234);
Nenrospora crassa (Case eral., Proc.
Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomcces such as Schwqnnionyres occidenralis (EP 394.538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora.
Penicilliunr. Tolopocladiunr (WO
91/00357 published 10 January 1991 ), and Aspergillus hosts such as A.
nidularu (Ballance et al.. Biochem.
Biophvs. Res. Commun., 112:284-289 [ 1983]; Tilburn et al., Gene, 26:205-221 [
1983]; Yelton er al.. Proc. Natl.
Acad. Sci. USA, 81:1470-147:1 [1984]) and A. niger (Kelly and Hynes. EMBO 1.. -1:.J75-479 [1985]).
Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of Urowth on methanol selected from the genera consisting of Hansenula, Candida. Kloeckera. Pichia.
Saccharomyces. Tornlopsu, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methvlotrophs, 269 (1982).
Suitable host cells for the expression of glycosylated PRO polypeptides 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) and COS cells.
More specific examples include monkey kidney CV 1 line transformed by S V40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham er al.. J. Gen. Virol., 36:59 ( 1977)); Chinese hamster ovary cells/-DHFR (CHO), Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 ( 1980)): mouse sertoli cells (TM4, Mather, Biol. Reprod.. 23:243-25 I
( 1980)); human lung cells (W 138, ATCC CCL 75); human liver cells (Hep G2, HB 8065j; 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.
c. Selection and Use of a Reolicable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding the PRO polypeptide 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 termination sequence.
Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
The PRO polypeptide tray be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the PRO-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, 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 Saccharonrcces and Klucveromyces 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, SUCK 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 2~r plasmid origin is suitable for yeast. andwarious 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 wv vur rim r YC1YU5UU/13358 not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR
activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 ( 1980). A
suitable selection gene for use in yeast is the np I 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 ap I
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-I [Jones, Genetics. 85: l2 ( 1977)].
Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.
Promoters suitable foruse with prokaryotic hosts include the (3-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 ( 1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer etal., Proc. Natl. Acad. Sci. USA, 80:21-25 ( 1983)].
Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding the PRO polypeptide.
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 Rep., 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.
PRO polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (S V40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
Transcription of a DNA encoding the PRO polypeptide 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 rnamrnalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication on gin (bp 100-270), wv uutiasi7 PCTliJS00/13358 the cytomegalovirus early promoter enhancer. the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the PRO coding sequence. but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect. plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the ntRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs orcDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the PRO polypeptide.
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO polypeptides in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 ( 1981 ); Mantei et al., Nature. 281:40-06 ( 1979); EP 117,060; and EP 117,058.
d. Detecting Gene Amplificatiott/Exnression 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 rnRNA
[Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies rnay be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
The antibodies in tum may be labeled and the assay tray 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 irnmunological methods, such as ittuttunohistochemical 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 imcnunohistochemical staining and/or assay of sample fluids logy be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against am exogenous sequence fused to PRO
DNA and encoding a specific antibody epitope.
e. Purification of Polvoeutide Forms of PRO polypeptides tnay be recovered from culture medium or from host cell iysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in expression of the PRO polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
It rnay be desired to purify the PRO polypeptide 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 IQG;
and metal chelating columns *-trademark -58-to bind epitope-tagged forms of the PRO polypeptide. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzvmolooy, 182 ( 1990): Scopes, Protein Purification: Principles and Practice. Springer-Verlag. New York ( 1982). The purification steps) selected will depend, for example, on the nature of the production process used and the particular PRO
polypeptide produced.
E. Amplification of Genes Encoding PRO Polvpeptides in Tumor Tissues and Cell Lines The present invention is based on the identification and characterization of genes that are amplified in certain cancer cells.
The genome of prokaryotic and eukaryotic organisms is subjected to two seemingly conflicting requirements. One is the preservation and propagation of DNA as the genetic information in its original form, to guarantee stable inheritance through multiple generations. On the other hand.
cells or organisms must be able to adapt to lasting environmental changes. The adaptive mechanisms can include qualitative or quantitative modifications of the genetic material. Qualitative modifications include DNA
mutations, in which coding sequences are altered resulting in a structurally and/or functionally different protein.
Gene amplitication is a quantitative modification, whereby the actual number of complete coding sequence, i.e.. a gene, increases, leading to an increased number of available templates for transcription, an increased number of translatable transcripts, and, ultimately, to an increased abundance of the protein encoded by the amplified gene.
The phenomenon of gene amplification and its underlying mechanisms have been investigated in vitro in several prokaryotic and eukaryotic culture systems. The best-characterized example of gene amplification involves the culture of eukaryotic cells in medium containing variable concentrations of the cytotoxic drug methotrexate (MTX). MTX is a folic acid analogue and interferes with DNA synthesis by blocking the enzyme dihydrofolate reductase (DHFR). During the initial exposure to low concentrations of MTX
most cells (>99.9°!0) will die. A
small number of cells survive, and are capable of growing in increasing concentrations of MTX by producing large amounts of DHFR-RNA and protein. The basis of this overproduction is the amplification of the single DHFR
gene. The additional copies of the gene are found as extrachromosomal copies in the form of small, supernumerary chromosomes (double minutes) or as integrated chromosomal copies.
Gene amplification is most commonly encountered in the development of resistance to cytotoxic drugs (antibiotics for bacteria and chemotherapeutic agents for eukaryotic cells) and neoplastic transformation.
Transformation of a eukaryotic cell as a spontaneous event or due to a viral or chemical/environmental insult is typically associated with changes in the genetic material of that cell. One of the most common genetic chances observed in human malignancies are mutations of the p53 protein. p53 controls the transition of cells from the stationary (G1 ) to the replicative lS) phase and prevents this transition in the presence of DNA damage. In other words, one of the main consequences of disabling p53 mutations is the accumulation and propagation of DNA
damage, i.e., genetic changes. Common types of genetic changes in neoplastic cells are, in addition to point mutations, amplifications and Gross. structural alterations, such as translocations.
The amplification of DNA sequences may indicate a specific functional requirement as illustrated in the DHFR experimental system. Therefore, the amplification of certain oncogenes in malignancies points toward a causative role of these genes in the process of malignant transtotmation and maintenance of the transformed phenotype. This hypothesis has gained support in recent studies. For example, the bcl-2 protein was found to be amplified in certain types of non-Hodgkin's lymphoma. This protein inhibits apoptosis and leads to the progressive accumulation of neoplastic cells. Members of the gene family of growth factor receptors have been found to be amplified in various types of cancers suggesting that overexpression of these receptors may make neoplastic cells less susceptible to limiting amounts of available growth factor. Examples include the amplification of the androgen receptor in recurrent prostate cancer during androgen deprivation therapy and the amplification of the groH~th factor receptor homologue ERB2 in breast cancer. Lastly, genes involved in intracellular signaling and control of cell cycle progression can undergo amplification during malignant transformation.
This is illustrated by the amplification of the bcl-I and ras genes in various epithelial and lymphoid neoplasms.
These earlier studies illustrate the feasibility of identifying amplified DNA
sequences in neoplasms, because this approach can identify genes important for malignant transformation. The case of ERB2 also demonstrates the feasibility from a therapeutic standpoint, since transforming proteins may represem novel and specific targets for tumor therapy.
Several different techniques can be used to demonstrate amplified genomic sequences. Classical cytogenetic analysis of chromosome spreads prepared from cancer cells is adequate to identify gross structural alterations. such as translocations, deletions and inversions. Amplified genomic regions can only be visualized, if they involve large regions with high copy numbers or are present as extrachromosomal material. While cytogenetics was the first technique to demonstrate the consistent association of specific chromosomal changes with particular neoplasms, it is inadequate for the identification and isolation of manageable DNA sequences. The more recently developed technique of comparative genomic hybridization (CGH) has illustrated the widespread phenomenon of genomic amplification in neoplasms. Tumor and normal DNA are hybridized simultaneously onto metaphases of normal cells and the entire genome can be screened by image analysis for DNA
sequences that are present in the tumor at an increased frequency. (WO 93/18,186; Gray et al., Radiation Res., 137:275-289 [ 1994]). As a screening method, this type of analysis has revealed a large number of recurring arriplicons (a stretch of amplified DNA) in a variety of human neoplasrns. Although CGH is more sensitive than classical cytogenetic analysis in identifying amplified stretches of DNA, it does not allow a rapid identification and isolation of coding sequences within the amplicon by standard molecular genetic techniques.
The mostsensitive methods to detect gene amplification are polymerase chain reaction (PCR)-based assays.
These assays utilize very small amount of tumor DNA as starting material, are exquisitely sensitive, provide DNA
that is amenable to further analysis, such as sequencing and are suitable for high-volume throughput analysis.
The above-mentioned assays are not mutually exclusive, but are frequently used in combination to identify amplifications in neoplasms. While cytogenetic analysis and CGH represent screening methods to survey the entire aenome for amplified regions, PCR-based assays are most suitable for the final identitication of coding sequences, i.e., genes in amplified regions.
According to the present invention, such genes have been identified by quantitative PCR (S. Gelmini et al., Clin. Chem.. 43:752 [1997)), by comparing DNA from a variety of primary tumors, including breast. lung, colon, prostate. brain. liver: kidney, pancreas, spleen. thymus, testis, ovary, uterus, etc., tumor. or tumor cell lines.
_6p_ with pooled DNA from healthy donors. Quantitative PCR was performed using a TaqManT" instrument (ABI).
Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs.
Human lung carcinoma cell lines include A549 (SRCC768), Calu-1 (SRCC769), Calu-6 (SRCC770), H157 (SRCC771). H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774), SW900 ISRCC775), (SRCC832),and H810(SRCC833),allavailablefromATCC. Primary human lung tumor cells usually derive from adenocarcinomas, squamous cell carcinomas, large cell carcinomas. non-small cell carcinomas, small cell carcinomas, and broncho alveolar carcinomas, and include, for example, SRCC724 (adenocarcinoma. abbreviated as "AdenoCa")(LTI), SRCC725 (squamous cell carcinoma, abbreviated as "SqCCa)(LTIa), SRCC726 (adenocarcinoma)(LT2), SRCC727 (adenocarcinoma)(LT3), SRCC728 (adenocarcinoma)(LT4), SRCC729 (squamous cell carcinoma)(LT6), SRCC730 (adeno/squamous cell carcinoma)(LT7), (adenocarcinoma)(LT9), SRCC732 (squamous cell carcinoma)(LT10), SRCC733 (squamous cell carcinoma)(LTII), SRCC734 (adenocarcinoma)(LT12), SRCC735 (adeno/squamous cell carcinoma)(LT13), SRCC736 (squamous cell carcinoma)(LT15), SRCC737 (squamous cell carcinorna)(LT16), SRCC738 (squamous cell carcinoma)(LT17), SRCC739 (squamous cell carcinoma)(LT18), SRCC740 (squamous cell carcinoma)(LT19), SRCC741 (lung cell carcinoma, abbreviated as "LCCa")(LT21 ), SRCC811 (adenocarcinoma)(LT22), SRCC825 (adenocarcinoma)(LT8), SRCC886 (adenocarcinoma)(LT25), SRCC887 (squamous cell carcinoma) (LT26), SRCC888 (adeno-BAC carcinoma) (LT27), SRCC889 (squamous cell carcinoma) (LT28), SRCC890 (squamous cell carcinoma) (LT29), SRCC891 (adenocarcinoma) (LT30), SRCC892 (squamous cell carcinoma) (LT31), SRCC894 (adenocarcinoma) (LT33). Also included are human lung tumors designated SRCC I I 25 [HF-000631 ], SRCC1127 [HF-000641], SRCC1129 [HF-000643], SRCC1133 [HF-000840], SRCC1135 (HF-000842], SRCC1227 [HF-001291], SRCC1229 (HF-001293], SRCC1230 [HF-001294], SRCC1231 (HF-001295], SRCC1232 [HF-001296], SRCC1233 (HF-001297], SRCC1235 (HF-001299], SRCC1236 [HF-001300], SRCC1296[HF-001640],SRCC1299[HF-001643],SRCC1300[HF001644],SRCC1301 [HF-001645],SRCC1302 [HF-001646], SRCC1303 [HF-001647], and SRCC1304 [HF-001648].
Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), Co1o320 (carcinoma, SRCC778), HT29 (adenocarcinoma. SRCC779), HM7 (a high mucin producing variant of ATCC colon adenocarcinoma cell line, SRCC780, obtained from Dr. Robert Warren, UCSF), CaWiDr (adenocarcinotna, SRCC781 ), HCT116 (carcinoma, SRCC782), SKCO1 (adenocarcinoma, SRCC783), SW403 (adenocarcinoma, SRCC784), LS
174T (carcinoma, SRCC785), Coto205 (carcinoma, SRCC828), HCT15 (carcinoma, SRCC829), HCC2998 (carcinoma, SRCC830), and KM12 (carcinoma, SRCC831). Primary colon tumors include colon adenocarcinomas designated CT2 (SRCC742), CT3 (SRCC743) ,CTB (SRCC744), CTIO (SRCC745), CT12 ISRCC746), CT14 (SRCC747), CT15 (SRCC748), CT16 (SRCC7491, CT17 (SRCC750), CT1 (SRCC751), CT4 (SRCC752), CTS
(SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CTl 1 (SRCC757), CT18 (SRCC758), CT19 (adenocarcinoma;
SRCC906), CT20 (adenocarcinoma. SRCC907), CT21 (adenocarcinoma, SRCC908), CT22 (adenocarcinorna, SRCC909), CT23 (adenocarcinoma, SRCC910), CT24 (adenocarcinorria. SRCC91 l), CT25 (adenocarcinoma, SRCC912), CT26 (adenocarcinoma, SRCC913), CT27 (adenocarcinoma, SRCC914),CT28 (adenocarcinoma, SRCC915), CT29 (adenocarcinoma, SRCC916), CT30 (adenocarcinoma, SRCC917), CT31 (adenocarcinoma, SRCC918), CT32 (adenocarcinoma. SRCC919), CT33 (adenocarcinoma, SRCC920), CT35 (adenocarcinoma.
SRCC921 ), and CT36 (adenoc:arcinoma. SRCC922). Also included are human colon tumor centers designated SRCC1051 [HF-000499]. SRCC1052 (HF-000539), SRCC1053 [HF-000575], SRCC1054 (HF-000698], SRCC1059 [HF-000755], SRCC1060 [HF-000756], SRCC1142 (HF-000762), SRCC1144 [HF-000789], SRCCI 146 [HF-000795) and SRCC1148[HF-000811].
Human breast carcinoma cell lines include, for example. HBL 100 (SRCC759), MB435s (SRCC7601, T47D
(SRCC761 ). MB468(SRCC76?). MB 175 (SRCC763), MB361 (SRCC764>, BT20 (SRCC765), MCF7 (SRCC766), and SKBR3 (SRCC767), and human breast tumor center designated SRCC 1057 (HF-000545]. Also included are human breast tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC1100, SRCC1101, and human breast-met-lung-NS tumor designated SRCC893 [LT
32].
Human rectum tumors include SRCC981 [HF-000550] and SRCC982 [HF-000551].
Human kidney tumor centers include SRCC989 [HF-000611 ) and SRCC1014 (HF-000613].
Human testis tumor center include SRCCl001 [HF-000733] and testis tumor margin SRCC999 [HF-000716].
Human parathyroid tumors include SRCC1002 (HF-000831 ] and SRCC1003 [HF-000832).
Human lymph node tumors include SRCC1004 [HF-000854], SRCC1005 [HF-000855], and SRCC1006 [HF-000856].
F. Tissue Distribution The results of the gene amplification assays herein can be verified by further studies, such as, by determining mRNA expression in various human tissues.
As noted before, gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.
Acad. Sci. USA, 77:5201-5205 [ 1980] ), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively.
antibodies may be employed that can recognize specific duplexes. including DNA duplexes, RNA duplexes. and DNA-RNA
hybrid duplexes or DNA-protein duplexes.
Gene expression in various tissues, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, io quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal.
Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to sequence PRO DNA and encoding a specific antibody epitope. General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided hereinbelow.
G. Chromosome Mapping If the amplification of a given gene is functionally relevant. then that gene should be amplified more than neighboring genomic regions which are not important for tumor survival. To test this, the gene can be mapped to a particular chromosome, e.g., by radiation-hybrid analysis. The amplification level is then determined at the location identified. and at the nei_hboring ~enomic region. Selective or preferential amplification at the genomic region to which the gene has been mapped is consistent with the possibility that the gene amplification observed promotes tumor growth or survival. Chromosome mapping includes both framework and epicenter mapping. For further details see, e.g., Stewart et al., Genome Research. 7:422-433 ( 1997).
H. Antibody Binding Studies The results of the gene amplification study can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inhibit the expression of PRO
polypeptides on tumor (cancer) cells is tested.
IO Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific.
and heteroconjugate antibodies, the preparation of which will be described hereinbelow.
Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays. and immunoprecipitation assays.
Zola, Monoclonal Antibodies: A
Manual of Technioues, pp.147-158 (CRC Press, Inc., 1987).
Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyze for binding with a limited amount of antibody. The amount of target protein (encoded by a gene amplified in a tumor cell) in the test sample is inversely proponional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubilized before or after the competition, so that the standard and analyze that are bound to the antibodies may conveniently be separated from the standard and analyze which remain unbound.
Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyze is bound by a first antibody which is ittunobilized on a solid support, and thereafter a second antibody binds to the analyze, thus forming an insoluble three-part complex. See, e.g., U.S. Patent No. 4,376,110.
The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable trbiety (indirect sandwich assay).
Foc example. one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
For immunohistochemistry, the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as fotmalin, for example.
I. Cell-Based Tumor Assays Cell-based assays and animal models for tumors (e.g., cancers) can be used to verify the findings of the gene amplification assay, and further understand the relationship between the genes identified herein and the development and pathogenesis of neoplastic cell growth. The role of gene products identified herein in the development and pathology of tumor or cancer can be tested by using primary tumor cells or cells lines that have been identified to amplify the genes herein. Such cells include, for example, the breast, colon and lung cancer cells and cell lines listed above.

WO 00/75317 . PCT/US00/13358 In a different approach, cells of a cell type known to be involved in a particular tumor are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth is analyzed. Suitable cells include, for example, stable tumor cell lines such as, the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and rns-transfected NIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorigenic growth. Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumorigenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells. or by mediating antibody-dependent cellular cytotoxicity (ADCC). Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of cancer.
In addition, 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]).
Animal Models A variety of well known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other antagonists of the native polypeptides, including small molecule antagonists. 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 nu gene has been introduced into a very large numberof distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c. B lO.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, 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 Oncology Research. E. Boven and B. Winograd, eds., CRC Press, Inc..
1991.
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 B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu 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 conditions, involving freezing and storing in liquid nitrogen lKartnali et al., Br. J.
Cancer, :18:689-696 (1983]).

Tumor_ cells can be inuoduced into animals, such as nude mice. by a variety of procedures. The subcutaneous (s.c.) space in mice is very suitable for tumor implantation.
Tumors can be uansplanted s.c. as solid blocks. as needle biopsies by use of a trochar, or as cell suspensions. For solid block or trochar implantation, tumor tissue fragments of suitable size are inuoduced 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 neuroblastomacells (from which the neu oncogen was initially isolated), or neu-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al., PNAS USA, 83:9129-9133 (1986).
Similarly, animal models of colon 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 ntice has been described, for example, by Wang et al., Cancer Research, 54:4726728 ( 1994) and Too er af., 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 vitro 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 (DeL.eo et al., J. Exo. Med., 146:720 [1977]), which provide a highly conuoliable model system for studying the anti-tumor activities of various agents (Palladino et al., J. Immunol., 138:4023-4032 [ 1987]). 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 l Ox 10' to I
Ox 10' cells/ml. The animals are then infected subcutaneously with 10 to 100 E,d 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 beneficial effects in the ueatment of human patients diagnosed with small cell carcinoma of the lung (SCCL). This tumor can be inuoduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi er al., Br. J. Cancer, 41 auppl. 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 drus 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 Spank Thomsen, Proc. 6th Int. Worksh~ on Immune-Det7cient Animals. Wu and Sheng eds., 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 of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals.
Animals that can serve as a target for transgenic 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 et al., Cell, 56:313-321 [1989)); electroporation of embryos (Lo, Mol. Celt Biol., 3:1803-1814 [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, uansgenic animals include those that carry the uansgene only in pan 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 et al., Proc. Natl. Acad. Sci. USA, 89:6232-6236 ( 1992).
The expression of the transgene 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.
Alternatively, "knock out" animals can be constructed which have a defective or altered gene encoding a PRO polypeptide identified herein. as a result of homologous recombination between theendogenous gene encoding the polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal. For example, cDNA encoding a PRO polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques. A portion of the genomic DNA encoding a particular PRO
polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see, e.g., Thomas and Capecchi, Cell, 51:503 ( 1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see, e.g., Li et al.. C~ 69:915 ( 1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see. e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford. 1987), pp. 1 13-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
Progeny harboring the homologously recombined DNA in their germ cells can he identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, by their ability to defend a_ainst certain pathological conditions and by their development of pathological conditions due to absence of the PRO
polypeptide.
Theefticacy of antibodies specifically binding the polypeptides identified herein and otherdrug 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 of cats, accounting for over 60~/c 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 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 squamous 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. After 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 tumor in animals.
K. Screening Assays for Drug Candidates Screening assays for drug candidates are designed to identify compounds that bind or complex with the polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
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 pert~ormed in a variety of formats, including protein-protein binding assays.
biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
All assays are common in that they call for contacting the drug candidate with a polypeptide encoded by a nucleic acid identified herein under conditions and for a tune sufficient to allow these two components to interact.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment. the polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiterplate, 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 solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
If the candidate compound interacts with but does not bind to a particular PRO
polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, cross-linking, co-irnmunoprecipitation, 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 Sono, Nature, 340:245-246 (1989); Chien er 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 tnodular 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 of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALL -lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL-l activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for p-Qalactosidase. 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.
Compounds that interfere with the interaction of a PRO-encoding gene identified herein and other intra-or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the amplified gene and the infra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a test compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture. to serve as positive control. The binding (complex formation) between the test compound and the infra-or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reactions) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
To assay for antagonists, the PRO polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO
polypeptide. Alternatively, antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay.
The PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun., 112: Chapter 5 ( 1991 ). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA
library created from this RNA is divided into pools and used to transfect COS
cells or other cells that are not responsive to the PRO polypeptide. Transfected cells that are grown on glass slides are exposed to labeled PRO
polypeptide. The PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled PRO
polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
In another assay for antagonists. mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with the PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO
polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide.
Another potential PRO polypeptide antagonist is an antisense RNA or DNA
construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein uanslation.
Antisense technology can be used to wo oons3m Pcr/usoon33ss control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. Forexample, the S' coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptide herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see. Lee et al., Nucl_ Acids Res., 6:3073 ( 1979); Cooney et al., Science, 241: 456 ( 1988); Dervan et al.. Science, 251:1360 ( 1991 )), thereby preventing transcription and the production of the PRO polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA
in viva and blocks translation of the mRNA molecule into the PRO polypeptide (antisense - Okano, Neurochem., 56:560 (1991);
Olieodeoxvnucleotides as Antisense Inhibitors of Gene Expression (CRC Press:
Boca Raton, FL, 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed ut viva to inhibit production of the PRO polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in lenD h, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length. about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, Or more.
Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes Z5 act by sequence-specific hybridization to the complementary target RNA.
followed by endonucleolytic cleavage.
Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Bioloov, 4:469-471 (1994), and PCT
publication No. WO 97/33551 (pubiished September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT
publication No. WO 97/33551, supra.
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
L. Compositions and Methods for the Treatment of Tumors The compositions useful in the treatment of tumors associated with the amplification of the genes identified herein include, without limitation. antibodies, small organic and inorganic molecules, peptides, phosphopeptides.

antisense and ribozyme molecules. triple helix molecules. etc., that inhibit the expression and/or activity of the target gene product.
For example, antisense RNA and RNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage.
Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biolow, 4:469-471 ( 1994), and PCT
publication No. WO 97/33551 (published September 18, 1997).
Nucleic acid molecules in triple helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT
publication No. WO 97/33551. supra.
These molecules can be identified by any or any combination of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
M. Antibodies Some of the mostpromising drug candidates according to the present invention are antibodies and antibody fragments which tray inhibit the production or the gene product of the amplified genes identified herein and/or reduce the activity of the gene products.
1. Polyclonal Antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan.
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and. if desired, an adjuvant.
Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PRO
polypeptide or a fusion protein thereof.
It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue expenmentatton.
2. Monoclonal Antibodies The anti-PRO antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein. Nature, 256:495 ( 1975). In _71_ a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the PRO polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies:
Principles and Practice, Academic Press, ( 1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the 1 S 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 :r..~!; ine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection (ATCC), Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol.,133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Technigues and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO polypeptides. 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 orascites fluid by conventional immunoglobulin purification procedures such as, forexample, 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 ~=enes encodin;_ the heavy and light chains of murine 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 ovan (CHO) cells, or myelonta cells that do not otherwise produce immuno~rlobulin protein. to obtain the synthesis of montx;lonal 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 rnurine sequences [U.S.
Patent No. 4,816,567; Morrison et nl., supra] or by covalently joining to the immuno~~lobulin codins sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable l~ 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 immunonlobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc re~~ion so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or 1 5 are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce fragments thereof, particularly, Fab fi~agments, can be accomplished using routine techniques known in the art.
Human and Humanized Antibodies The anti-PRO antibodies may further comprise humanized antibodies or human antibodies. Humanized 2~ 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) 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 25 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 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 30 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 immunoalobulin [Jones et al., Nature, 321:522-525 ( 1986): Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized 35 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 pertbrmed foliowin~ the method of Winter and co-workers [Jones et crl., Nature. 321:522-525 (1986); Riechmann et al., Nature, 332:323-~~7 (1988): Verhoeyen et al., Science, 239:1534-1536 ( 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 ip 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 phase 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 Therapy, Alan R.
Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes 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/Technology, 10:779-783 (1992); Lonberg etal., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-13 ( 1994); Fishwild et al., Nature Biotechnolow, 14:845-51 ( 1996); Neuberger, Nature Biotechnology, 14:826 ( 1996); Lonberg and Huszar, Intern.
Rev. Immunol., 13:65-93 (1995).
4. Antibody Dependent Enzyme Mediated Prodrue Theranv (ADEPT) The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 /01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U. S. Patent No. 4,975,278.
The enzyme componentoftheimmunoconjugateusefulforADEPTincludesanyenzymecapableofacting on a prodrug in such as way so as to convert it into its more active, cytotoxic form.
Enzymes that are useful in the method of this invention include, but are not limited to,=lycosidase, glucose oxidase, human lysozyme, human glucuronidase, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs: cytosine deaminase useful forconverting non-toxic 5-fluorocytosine into the anti-cancer drug 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase A) and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents;
carbohydrate-cleaving enzymes such as ~3-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; ~i-lactamase useful for convening drugs derivatized with p-lactams into free drugs; and penicillin amidases, such as penicillin Vamidase or penicillin G amidase, useful for convening drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively;
_7.f_ antibodies with enzymatic activity. also known in the art as~'abzymes" ran be used to convert the prodrugs of the invention into tree active drugs (see. e.,~., Massey. Nature. 328:457-458 ( 1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
The enzymes of this invention can be covalently bound to the anti-PRO
antibodies by techniques well known in the art such as the use of the heterobifunctional cross-linking a_ents discussed above. Alternatively, fusion proteins comprising at least the anti~_en binding region of the antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed usin~_ recombinant DNA techniques well known in the art (see. e.g.. Neuberger et al., Nature. 312:604-608 ( 1984)).
5. Bi~ecitic Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different anti~_ens. In the present case, one of the binding specificities is for the PRO
polypeptide 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 of bispecific antibodies is based on the co-expression of two immunoglobuiin 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 lquadromas) 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.. 10:3655-3659 f 1991 ).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant re_ion ICH 1 ) containin_ the site necessary for li_ht-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired. the immuno~lobulin light chain, are inserted into separate expression vectors. and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh etnl., Methods in Enzvmolow, I ? 1:210 ( 1986).
According to another approach described in WO 96/27011. 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 part of the CH3 region of an antibody constant domain.
In this method. one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larder side chains te. ~., tyrosine or tryptophan 1.
Compensatory "cavities" of identical or similar size to the lar~le side chainls) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones ie. ~.. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanti:d end-products such as homodimers.
Bispecific antibodies can be prepared as full len!~th antibodies or antibody fragments (e.g.. F(ab'):
bispecific antibodies). Techniques for generating bispecitic antibodies from antibody fragments have been described in the literature. For example, bispecitic antibodies can be prepared using chemical linkage. Brennan er al.. Science, 229:81 ( 1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab'), fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to thionitrobenzoate ITNB ) 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 bispecit3c 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 bispecitic antibodies. . Shalaby er al.. J. Exn. Med., ! 75:217-225 ( 1992) describe the production of a fully humanized bispecific antibody F(ab')= molecule. Each Fab' fragment was separately secreted from E. call and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecitic antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lyric activity of human cytotoxic lymphocytes a_ainst human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecit3c antibodies have been produced using leucine zippers.
Kostelny et oL, J. Immunol., 148(5):1547-1553 ( 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody"
technology described by Hollinger er ol., 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 (V") connected to a light-chain variable domain (V~) by a linker which is too short to allow pairing between the two domains on the same chain.
Accordingly, the 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. lmmunol..
I 52:5368 ( 1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol., 147:60 ( I 991 ).
Exemplary bispecific antibodies may bind to two different epitopes on a _iven polypeptide herein.
Alternatively. an anti-polypeptide arm may be combined with an arm which binds to a trigsering 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), FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular polypeptide. Bispecitic antibodies may also be used to localize cytotoxic a«ents to cells 3$ which express a particular polypeptide. These antibodies possess a polypeptide-binding arm and an arm which binds a cytotoxic went or a radionuclide chelator. such as EOTUBE, DPTA. DOTA.
or TETA. Another bispecitis antibody of interest binds the polypeptide and further binds tissue factor (TF).

6. Heteroconiu~ate Antibodies Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S.
Patent No. 4.676.980]. and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinkin~
agents. For example, immunotoxins may be constructed usins a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example. in U.S. Patent No.
1,676,980.
7. Effector function en~ineerin~
It may be desirable to modify the antibody of the invention with respect to effector function. so as to enhance the effectiveness of the antibody in treating cancer, for example. For example, cysteine residues) may be introduced in 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. Exp. Med.. 176:1191-I 195 ( 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 which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See. Stevenson et al., Anti-Cancer Drug Desien, 3:219-230 ( 1989).

8. Immunoconiusates The invention also pertains to irnmunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fra_ments thereof. or a small rnolecule toxin), or a radioactive isotope (i.e.. a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.
Enzymaticallyactive protein toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from Pseadomonas aerrrginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, AleurireS fordii proteins. dianthin proteins, Phmolaca americana proteins (PAPI, PAPA, and PAP-S), momordica charantia inhibitor, curcin, croon, sapaonaria officinalis inhibitor, relonin. saporin. mitogellin, restrictocin.
phenomycin, enomycin and the tricothecenes. Small molecule toxins include. for example. calicheamicins, maytansinoids, palytoxin and CC1065.
A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include='=Bi.
'3'L'''In, ~"Y and'"~Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol ) propionate ISPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives _77_ (such as bis-ip-diazoniumbenzoyll-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate). ;end bis-active tluorine compounds (such as 1.5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta er aL. Science. 238:1098 (1987). Carbon-14-labeled 1-isothiocvanatoExnzvl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, W094/I 1026.
In another embodiment, the antibody may be conjugated to a "receptor" {such as streptavidin) for utilization in tumor pretargetin~ wherein the antibody-receptor conjugate is administered to the patient. followed by removal of unbound conjugate from the circulation using a clearin_ agent and then administration of a "Iigand"
(e.g., avidin) which is conjugated to a cytotoxic went (e.g., a radionucleotide).

9. Immunoliposomes 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. Sei. USA, 82:3688 ( 1985); Hwang et al.. Proc. Natl. Acad. Sci. USA. 77:4030 ( 1980):
and U.S. Patent Nos. 1,485,035 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 of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem.. 257:286-288 (1982) via a disulfide interchange reaction. A
chemotherapeutic agent !such as Doxorubicin) is optionally contained within the liposome. See. Gabizon etal..
J. National CancerInst.. 81 ( 19):1484 ( 1989).
N. Pharmaceutical COmDOSItIOfIS
Antibodies specifically binding the product of an amplified gene identit7ed herein, as welt as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions.
If the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitors.
internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody.
or an antibody fragment, into cells. Where 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 e~ al.. Proc. Natl. Acad. Sci. USA. 90:7889-7893 [ 1993[).
Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers lReminoton's Pharmaceutical Sciences, 16th edition. Osoh 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 _78-wu uui75317 PCT/US00113358 employed. and include buffers such as phosphate. citrate, and other organic acids: antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl 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 nr-cresol); low molecular weight (less than about 10 residues) polypeptides: proteins, such as serum albumin, gelatin, or immunoglobulins: hydrophilic polymers such as polyvinylpyrrolidone: amino acids such as glycine, glutamine.
asparagine, 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 TWEENr"', PLURONICST"" or polyethylene glycol (PEG).
Non-antibody compounds identified by the screening assays of the present invention can be formulated in an analogous manner. using standard techniques well known in the art.
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, hydroxymethylcel lulose or gelatin-inicrocapsu les 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 Remin~ton'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.
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.R., tilms or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides t Lt.S. Pat. No.
3,773,919), copolymers of L-~_=lutamic acid and 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 biolo~Tical activity and possible changes in immunogenicity. Rational strate~Ties can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions. controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

O. Methods of Treatment It is contemplated that the antibodies and other anti-tumor compounds of the present invention may be used to treat various conditions. including those characterized by overexpression and/or activation of the amplified genes identified herein. Exemplary conditions or disorders to be treated with such antibodies and other compounds, including, but not limited to, small organic and inorganic molecules, peptides, antisense molecules, etc., include benign or malignant tumors (e.~., renal, liver, kidney. bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, tune, vulva(, thyroid, hepatic carcinomas: sarcomas:
glioblastomas: and various head and neck tumors):
leukemias and lymphoid malignancies: other disorders such as neuronal, filial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders: and inflammatory, angiogenic and immunoloEic disorders.
The anti-tumor agents of the present invention, e.g., antibodies. 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, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, infra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.
Other therapeutic regimens may be combined with the administration of the anti-cancer agents, e.g., antibodies 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, achemotherapeutic 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 Chemotheraoy Service Ed., M.C. Petry, Williams & Wilkins. Baltimore, MD
(1992). The chemotherapeutic a;ent may precede, or follow administration of the anti-tumor went, e.g., antibody, or may be given simultaneously therewith. The antibody 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 other tumor associated antigens. such as antibodies ~5 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 antigens disclosed herein may be co administered to the patient. Sometimes. it may be benet7cial to also administer one or more cytokines to the patient.
In a preferred embodiment, the antibodies herein are co-administered with a growth inhibitory went. For example, the growth inhibitory a=ent may be administered first. followed by an antibody of the present invention. However, simultaneous administration or administration of the antibody of the present invention first is also contemplated.
Suitable dosages for the growth inhibitory a_ent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory went and the antibody herein.
For the prevention or treatment of disease, the appropriate dosa~_=e of an anti-tumor agent, e./; ., an antibody 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 a_ent is suitably administered to the patient at one time or over a series of treatments.
For example, depending on the type and severity of the disease, about 1 pg/kg to I 5 mg/kg (e. e., 0..1-20 mg/kg ~ of antibody 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 mi;ht range from about 1 Ecglk to 100 mJk~ or more. depending on the factors mentioned above. For repeated administrations over several days or lon~~er. depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
P. 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 hypodetTrtic injection needle). The active agent in the composition is usually an anti-tumor agent capable of interfering with the activity of a gene product identified herein, e.g., an antibody. 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 pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint. including other buffers, diluents, filters. needles, syringes, and package inserts with instructions for use.
Q. Diagnosis and Prognosis of Tumors While cell surface proteins, such as growth receptors overexpressed in certain tumors are excellent targets for drug candidates or tumor (e.g., cancer) treatment, the same proteins along with secreted proteins encoded by the genes amplified in tumor cells trod additional use in the diagnosis and prognosis of tumors. For example.
antibodies directed against the protein products of genes amplified in tumor cells can be used as tumor diagnostics or prognostics.
For example, antibodies. including antibody fragments, can be used to qualitatively or quantitativel v detect the expression of proteins encoded by the amplified genes ("marker gene products"). The antibody preferably is equipped with a detectable, e.~., fluorescent label, and binding can be monitored by light microscopy, flow cytomet~~. tluorimetry, or other techniques known in the art. These techniques are particularly suitable. if the amplified gene encodes a cell surface protein, e.g., a growth factor. Such binding assays are performed essentially as described in section 5 above.
!u si~u detection of antibody binding to the marker gene products can be performed, for example. by immunot7uorescence or immunoelectron microscopy. For this purpose, a histolo~ical specimen is removed from the patient. and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample.
This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent for those skilled in the an that a wide variety of histological methods are readily available for in -AI-situ detection.
The following examples are offered for illustrative purposes only. and are not intended to limit the scope of the present invention in any way.
EXAMPLES
Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification. by ATCC accession numbers is the American Type Culture Collection. 10801 University Blvd.. Manassas, VA 20110-2209. All original deposits referred to in the present application 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 deposit will be made available by ATCC
under the terms of the Budapest Treaty, and subject to an agreement between Genentech. Inc., and ATCC.
20 Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks:
Gait, Oli>ronucleotide Synthesis. IRL Press. Oxford.1984: R.1. Freshney, Animal Cell Culturo. 1987; Coligan er al., Current Protocols in ImmunoloQV, 1991.
EXAMPLE I
elation of cDNA Clones Encoding a Human PR05800 The extracellular domain (ECD) sequences finctuding 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). The search was performed using the computer program BLAST or BLAST2 [Altschul et ctl.. Methods in Enzvrnoloav, x: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 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 vvV vUrraal r rt.lruDUUriaa70 above. This consensus sequence is herein desi=oared DNA 102836. In some cases.
the consensus sequence derives from an intermediate consensus DNA sequence which was extended usin_ repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as tar as possible using the sources of EST sequences discussed above.
S Based on the DNA 102836 consensus sequence, oligonucleotides were synthesized: I ) to identity 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 PR05800. Forward and reverse PCR primers ~~enerally range from 20 to 30 nucleotides and are often designed to =ive a PCR product of about 100-1000 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-l.5kbp. In order to screen several libraries for a full-len~_th clone. DNA from the libraries was screened by PCR amplification. as per Ausubel et 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.
PCR primers (forward and reverse) were synthesized:
forward PCR primer 1:
5'-CAGCGAACCGGGTGCCGGGTC-3' (SEQ ID N0:21 ) forwaM PCR primer 2:
5'-GAGCGACGAGCGCGCAGCGAAC-3' (SEQ ID N0:22) forward PCR primer 3:
ZO 5'-ATACTGCGATCGCTAAACCACCATGCGCCGCCGCCTGTGGCTG-3' (SEQ ID N0:23) reverse PCR primer l:
5'-GCCGGCCTCTCAGGGCCTCAG-3' (SEQ ID N0:24) reverse PCR primer 2:
5'-CCCACGTGTACAGAGCGGATCTC-3' (SEQ ID N0:25) reverse PCR primer 3:
5'-GAGACCAGGACGGGCAGGAAGTG-3' (SEQ ID N0:26) reverse PCR primer 4:
5'-CAGGCACCTTGGGGAGCCGCC-3' (SEQ ID N0:27) reverse PCR primer 5:
5'-CCCACGTGTACAGAGCGGATCTC-3' (SEQ ID N0:28) reverse PCR primer 6:
5'-GAGACCAGGACGGGCAGGAAGTG-3' (SEQ ID N0:29) Additionally, a synthetic olieonucleotide hybridization probe was constructed from the consensus DNA 102836 sequence which had the following nucleotide sequence: .
hybridization probe:
5'-CTCTACGGGTACTGCAGGTTCCGGGAGCGCATCGAAGAGAACGG-3' (SEQ ID N0:30) . RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. The cDNA
libraries used to isolate the eDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen. San Diego. CA. The cDNA was primed with olio dT containing a NotI
site, linked with blunt to SaII hemikinased adaptors, cleaved with NotI. sized appropriately by gel electrophoresis.
and cloned in a detined orientation into a suitable clonins vector (such as pRKB or pRKD: pRKSB is a precursor of pRKSD that does not contain the SfiI site; gee. Holmes et al.. Science.
?53:1 ?78-1280 ( I 991 )) in the unique XhoI
$ and Notl sites.
DNA sequencing of the clones isolated as described above ~=ave the full-length DNA sequence for a full-length PROS800 polypeptide (designated herein as DNA108912-2680 (Figure 1. SEQ ID NO: 1 ]) and the derived protein sequence for that PROS800 polypeptide.
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 7-9 and a stop signal at nucleotide positions 517-519 (Figure 1. SEQ ID NO:1 ).
The predicted polypeptide precursor is 170 amino acids long, has a calculated molecular weight of approximately 19.663 daltons and an estimated pI of approximately I 1.81. Analysis of the full-length PROS800 sequence shown in Figure 2 (SEQ ID N0:2) evidences the presence of a variety of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above.
1S Clone DNA 108912-2680 has been deposited with ATCC on May 25. 1999 and is assigned ATCC deposit no. PTA-124.
An analysis of the Dayhoff database (version 3S.4S SwissProt 3S), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 2 (SEQ ID N0:2), evidenced sequence identity between the PROS800 _ _amino acid sequence and the following Dayhoff sequences: P W52S9S, P WS7313. FGFA HUMAN, P_WS7264, FGFA_RAT, P_WS2597, MMU94S17_l, FGFA MOUSE, P_W57306 and D86333_1.

Isolation of cDNA Clones Encodine a Human PR06000 A cDNA clone (DNA 102880-2689) encoding a native human PR06000 polypeptide was identified using a yeast screen, in a human uterine cDNA library that preferentially represents the S' ends of the primary cDNA
clones.
Clone DNA 102880-2689 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 28-30 and ending at the stop codon at nucleotide positions 580-S82 (Figure 3: SEQ ID
N0:3). The predicted polypeptide precursor is 184 amino acids long (Figure :~:
SEQ ID N0:4). The full-length PR06000 protein shown in Figure 4 has an estimated molecular weight of about 21.OS2 daltons and a pI of about 5.01. Analysis of the full-length PR06000 sequence shown in Figure 4 (SEQ ID
NO: 4) evidences the presence of a variety of important polypeptide domains as shown in Figure 4, wherein the locations given for those important polypeptide domains are approximate as described above. Clone DNA 102880-2689 has been deposited with ATCC
on July 20. 1999 and is assigned ATCC Deposit No. PTA-383.
An analysis of the Davhoff database i version 3S.4S SwissProt 3S), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 4 (SEQ ID NO: 4 ).
evidenced sequence identity between the PR06000 amino acid sequence and the following Dayhoff sequences: SPS-VICFA, ADU8S448_I. 664635.
AE001 S 16_3. P_W20328, P W20747, and SPS SPIOL.

Isolation of cDNA Clones Encodins a Human PR06016 DNA96881-2699 was identitied by applying a proprietary signal sequence finding algorithm developed by Genentech. Inc., (South San Francisco. CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g.. Genbank) and/or private (LIFESEQ'', Incvte Pharmaceuticals, Inc., Palo Alto, CA) databases. The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surcounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence frasment under consideration. The nucleotides following the first ATG
must code for at least 35 unambiguous amino acids without any stop codons. If the tirst ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals.
Use of the above described signal sequence algorithm allowed identification of an EST sequence from the LIFESEQ'~ database. Incyte Pharmaceuticals. Palo Alto, CA, designated herein as 3035248H I . This EST sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e.g., Genbank) and a proprietary EST DNA database (LIFESEQ°, Incyte PhatTrtaceuticals, Palo Alto, CA) to identify existing homologies. The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al., Methods in Enzymolow, 266:460-480 ( 1996)). Those comparisons resulting in a BLAST
score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into aconsensus DNA sequence with the program "phrap" (Phil Green, University of Washington, Seattle, Washington).
The consensus sequence obtained therefrom is herein designated DNA82389.
In tight of an observed sequence homology between the DNA82389 sequence and an EST sequence encompassed within clone no. 3035248H 1 from the LIFESEQ=' database, Incyte Pharmaceuticals, Palo Alto, CA, clone no. 3035248H1 was purchased and the cDNA insert was obtained and sequenced. It was found herein that that cDNA insert encoded a full-length protein. The sequence of this cDNA
insert is shown in Figure ~ and is herein desisnated as DNA96881-2699.
Clone DNA96881-2699 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 1005-1007 (Figure 5: SEQ ID
NO:S). The predicted polypeptide precursor is 315 amino acids long (Figure 6:
SEQ ID N0:6). The full-length PR06016 protein shown in Figure 6 has an estimated molecular weight of about 35,963 daltons and a pI of about 5.38. Analysis of the full-length PR06016 sequence shown in Figure 6 (SEQ ID
NO: 6) evidences the presence of a variety of important polypeptide domains as shown in Figure 6. wherein the locations given for those important polypeptide domains are approximate as described above. Clone DNA9688 I -2699 has been deposited with ATCC
on August 17. 1999 and is assigned ATCC Deposit No. PTA-553, An analysis of the Davhoff database (version 35.45 SwissProt 35), using the ALIGN-? sequence alignment analysis of the full-length sequence shown in Figure 6 (SEQ ID NO: 6).
evidenced sequence identity between the PRO6016 _ -amino acid sequence and the following Dayhoff sequences: P W88499:
HGS RE347; P_W88647;

Y087 CREEL: S~-1095; P W03626; P_W03627: IE68_HSVSA; PN0009: RNU51583_I.
EXAMPLE d Isolation of eDNA Clones Encoding a Human PR06018 DNA98565-2701 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech. Inc.. (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g., Genbank) and/or private (LIFESEQ'', Incyte Pharmaceuticals, Inc., Palo Alto. CA) databases. The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG
must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids. the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion sienals.
Use of the above described signal sequence algorithm allowed identification of an EST sequence from the LIFESEQ~ (Incyte Pharmaceuticals, Palo Alto, CA) database, designated herein as 745575H I . This EST sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e.g., Genbank) and a proprietary EST DNA database (LIFESEQ'~. Incyte Pharmaceuticals, Palo Alto. CA) to identify existing homologies. The homology search was performed using the computer program BLAST or BLAST2 (Altshul eral., Methods in Enzymoloay, 266:460-480 ( 1996)). Those comparisons resulting in a BLAST
score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into aconsensus DNA sequence with iheprogram "phrap" (Phil Green, Universityof Washington, Seattle, Washington).
The consensus sequence obtained therefrom is herein designated DNA82411.
In light of an observed sequence homology between the DNA82411 sequence and an EST sequence encompassed within clone no. 745575H 1 from the LIFESEQ'° (Incyte Pharmaceuticals. Palo Alto. CA 1 database, clone no. 745575H1 was purchased and the cDNA insert was obtained and sequenced. It was found herein that that eDNA insert encoded a full-length protein. The sequence of this cDNA
insert is shown in Figure 7 and is herein desienated as DNA98565-2701.
Clone DNA98565-2701 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 352-357 and ending at the stop codon at nucleotide positions 3085-3087 (Figure 7: SEQ ID
N0:7). The predicted polypeptide precursor is 911 amino acids long (Figure 8:
SEQ ID N0:8). The full-length PR06018 protein shown in Figure 8 has an estimated molecular weight of about 99.117 daltons and a p1 of about 4.62. Analysis of the full-length PR06018 sequence shown in Figure 8 (SEQ ID
NO: 8) evidences the presence of a variety of important polypeptide domains as shown in Figure 8. wherein the locations Viven for those important polypeptide domains are approximate as described above. Clone DNA98565-2701 has been deposited with ATCC
on August 3. 1999 and is assigned ATCC Deposit No. PTA-481.
An analysis of the Dayhoff database ( version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 8 (SEQ ID NO: 8), evidenced sequence identity between the PR06018 _amino acid sequence and the followimt Dayhoff sequences: PGCB_BOVIN;
P 885442; P_R77034;
P_R12609: AC0031 10_2; PGCV-HUMAN; AFI 16856_1: P W75099: HGS-A 176;
A098460_1.

Isolation of cDNA Clones Encoding a Human PR06496 The extracellular domain IECD) sequences ~includina 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 a proprietary EST database (LIFESEQ'~, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et ol., Methods in Enzvmoloey, 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 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 DNA43048. In some cases, the DNA43048 consensus sequence derives from an intermediate consensus DNA sequence which was extended usins 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 DNA43048 consensus sequence, and in light of an observed sequence homology between the DNA43048 sequence and an EST sequence encompassed within clone no. 1636952 from the LIFESEQ°
database, Incyte Pharmaceuticals. Palo Alto, CA, clone no. 1636952 was purchased and the cDNA insert was obtained and sequenced. It was found herein that that cDNA insert encoded a full-length protein. The sequence of this cDNA insert is shown in Figure 9 and is herein designated as DNA119302-2737.
The full length clone identitied above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 63-65 and a stop signal at nucleotide positions 2328-2330 (Figure 9. SEQ ID
NO: 9). The predicted polypeptide precursor is 755 amino acids long. has a calculated molecular weight of approximately 82.785 daltons and an estimated p1 of approximately 8.71.
Analysis of the full-length PR06496 sequence shown in Figure 10 (SEQ ID NO: 10) evidences the presence of a variety of important polypeptide domains as shown in Figure 10. wherein the locations given for those important polypeptide domains are approximate as described above. Clone DNA 1 19302-2737 has been deposited with ATCC on August 10, I 999 and is assigned ATCC Deposit No. PTA-520.
An analysis of the Dayhoff database ( version 35.45 SwissProt 35). using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 10 (SEQ ID NO: 10), evidenced sequence identity between the PR06496 amino acid sequence and the following Dayhoff sequences: P W81365:
NEC 3 MOUSE;
I~fUSPRCONI.I_I; FURI_HUMAN: P_R775.10; S71340: P W73932; DROFURlISO-I;
GEN12660: and P_R59784.
_g7_ Isolation of cDNA Clones Encodine a Human PR0715=1 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 (1) public EST databases m.g., Merck/Washington University). and (2) a proprietary EST
database (LIFESEQ'~, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et ul.. Methods in Enzymolow, 266:460-.180 ( 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 cases, 90) or ~~reater 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 DNA38237. In some cases, the DNA38237 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of 1$ BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST
sequences discussed above.
Based on the DNA38237 consensus sequence, and in light of an observed sequence homology between the DNA38237 sequence and an EST sequence encompassed within clone no. 1855755 from the LIFESEQ°
database, Incyte Pharmaceuticals, Palo Alto. CA, clone no. 1855755 was purchased and the cDNA insert was obtained and sequenced. It was found herein that that eDNA insert encoded a full-length protein. The sequence of this cDNA insert is shown in Figure 11 and is herein designated as DNA
108760-2740.
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 102-104 and a stop signal at nucleotide positions 1083-1085 (Figure 11, SEQ
ID NO: 11 ). The predicted polypeptide precursor is 327 amino acids long, has a calculated molecular weight of approximately 34.348 daltons and an estimated pI of approximately 7.88.
Analysis of the full-length PR07154 sequence shown in Figure 12 (SEQ ID NO: I?) evidences the presence of a variety of important polypeptide domains as shown in Figure 12, wherein the locations given for those important polypeptide domains are approximate as described above. Clone DNA 108760-2740 has been deposited with ATCC on August 17,1999 and is assigned ATCC Deposit No. PTA-548.
An analysis of the Dayhoff database t version 35.45 SwissProt 35), using the ALIGN-2 sequence alisnment analysis of the futl-length sequence shown in Figure 12 (SEQ ID NO: 12), evidenced sequence identity between the PR07154 amino acid sequence and the following Dayhoff sequences:
AF061022_I; AF061024_l;
HS889N I S_ 1; GGY 14064_ I : GGY I 4063_ ! : AF061023_ 1; XLU43330_ I ; GEN
1:1531; MMCARH_ 1: and MMU90715 1.

Isolation of cDNA Clones Encaline a Human PR07170 DNA 108722-2743 was identified by applying a proprietary signal sequence finding algorithm developed _88_ VYV VU//JJ1/ rl.I/UJVV/1JJJ8 by Genentech. Inc.. (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g.. Genbank) and/or private (LIFESEQ"'. Incvte Pharmaceuticals.
Inc.. Palo Alto, CA) databases. The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the tirst and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG
must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals.
Use of the above described signal sequence algorithm allowed identification of an EST cluster sequence from the LIFESEQ'~ database, Incyte Pharmaceuticals. Palo Alto, designated herein as CLU57836. This EST
cluster sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e.g., Genbank) and a proprietary EST DNA database (LIFESEQ°, Incyte Pharmaceuticals. Palo Alto. CA) to identify existing homologies. The homology search was performed using the computer program BLAST or BLAST2 (Altshul et at., Methods in Enzvmoloov, 266:460-480 ( 1996)).
Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into a consensus DNA sequence with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). The consensus sequence obtained therefrom is herein designated DNA58756.
In light of an observed sequence homology between the DNA58756 sequence and an EST sequence encompassed within clone no. 2251462 from the LIFESEQ~ database. Incyte Pharmaceuticals, Palo Alto, CA, clone no. 2251462 was purchased and the cDNA insert was obtained and sequenced. It was found herein that that cDNA
insert encoded a full-length protein. The sequence of this cDNA insert is shown in Figure 13 and is herein desienated as DNA108722-2743.
Clone DNA108722-2743 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 1506-1508 (Figure ! 3: SEQ ID
N0:13). The predicted polypeptide precursor is 482 amino acids long (Figure 14: SEQ ID NO:1-1). The full-length PR07170 protein shown in Figure 14 has an estimated molecular weight of about 49,060 daltons and a pI of about 4.74. Analysis of the full-length PR07170 sequence shown in Figure 14 (SEQ ID
NO: 14) evidences the presence of a variety of important polypeptide domains as shown in Figure 14, wherein the locations given for those important polypeptide domains are approximate as described above. Clone DNA108722-2743 has been deposited with ATCC on August 17, 1999 and is assigned ATCC Deposit No. PTA-552.
An analysis of the Davhoff database (version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 14 (SEQ ID NO: 1-I), evidenced sequence identity between the PR07170 amino acid sequence and the following Dayhoff sequences: P_Y12291, I-17141. D88733_l, DMC56G7-1, P_Y I 1606. HWP I CANAL. HSMUCSBEX_ 1, HSU78550-1, HSU70136-1, and SGS3_DROME.

w~ uui75317 PCT/US00/13358 Isolation of cDNA Clones Encodin<_ a Human PR07-122 DNA 1 19536-2752 was identified by applying a proprietary siUnal sequence finding algorithm developed by Genentech, Inc., (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from S public (e.g., Gen$ank) and/or private (LIFESEQ", Incyte Pharmaceuticals.
Inc.. Palo Alto, CA) databases. The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the tirst ATG
must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids. the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion sienals.
Use of the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database, designated herein as 81575. This EST cluster sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e:g., GenBank) and a proprietary EST DNA database (LIFESEQ~, Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies. The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al.. Methods in Enzvmolow, 266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into a consensus DNA sequence with the program "phrap" (Phil Green, University of Washington, Seattle, Washineton). The consensus sequence obtained therefrom is herein designated DNA104391.
In light of an observed sequence homology between the DNA104391 sequence and an EST sequence encompassed within clone no. 1922888 from the Incyte database, clone no.
1922888 was purchased and the cDNA
insert was obtained and sequenced. It was found herein that that cDNA insert encoded a full-length protein. The sequence of this cDNA insert is shown in Figure 15 and is herein designated as DNA I 19536-2752.
Clone DNA 119536-2752 contains a single open reading frame with an apparent translational initiation site at nucleotide positions :17-49 and ending at the stop codon at nucleotide positions 311-313 (Figure 15; SEQ ID
N0:15). The predicted polypeptide precursor is 88 amino acids long (Figure 16). The full-length PR07422 protein shown in Figure 16 has an estimated molecular weight of about 9.645 daltons and a pI of about 5.45. Analysis of the full-length PR07-122 sequence shown in Figure 16 (SEQ ID N0:16) evidences the presence of a variety of important polypeptide domains as shown in Figure 16, wherein the locations given for those important polypeptide domains are approximate as described above. Clone DNA 1 I 9536-2752 has been deposited with ATCC on August 17, 1999 and is assigned ATCC deposit no. PTA-551.

Isolation of cDNA Clones Encoding Human PR07-i31 DNA I 19542-2754 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc.. (South San Francisco. CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g., GenBank) and/or private (LIFESEQ", Incyte Pharmaceuticals, Inc..
Palo Alto. CA) databases. The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG
must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals.
Use of the above described signal sequence algorithm allowed identification of an EST sequence from the LIFESEQ~, Incyte Pharmaceuticals, Inc., Palo Alto, CA database, designated herein as clone No. 2201182. This EST sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQJ, Incyte Pharmaceuticals, Palo Alto, CA) to identity existing homologies. The homology search was performed using the computer program BLAST or BLAST2 (Altshul er al., Methods in Enzymoloey, 266:460-480 ( 1996)).
Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into a consensus DNA sequence with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). The consensus sequence obtained therefrom is herein designated DNA 104392.
In light of an obsen~ed sequence homology between the DNA 104392 sequence and an EST sequence encompassed within clone no. ??01 I 82 from the Incyte database, clone no:
2201 l 82 was purchased and the cDNA
insert was obtained and sequenced. It was found herein that that cDNA insert encoded a full-length protein. The sequence of this cDNA insert is herein designated as DNA I 19542-2754.
Clone DNA 119542-2754 contains a single open readin _ frame with an apparent translational initiation site at nucleotide positions 2-17-2~t9 and ending at the stop codon at nucleotide positions 838-8d0 (Fieure 17: SEQ ID
N0:17). The predicted polypeptide precursor is 197 amino acids long (Figure 181. The full-length PR07431 protein shown in Figure 18 has an estimated molecular weight of about 21,992 daltons and a pI of about 12.18.
Analysis of the full-length PR07-131 sequence shown in Figure 18 (SEQ ID
NO:18) evidences the presence of a variety of important polypeptide domains as shown in Figure 18, wherein the locations given for those important polypeptidedornains are approximate as described above. Clone DNA 119542-2754 has been deposited with ATCC
on August 31, 1999 and is assigned ATCC deposit no. PTA-619.
An analysis of the Davhoff database (version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 18 ISEQ ID NO: 18), evidenced sequence identity between the PR07431 amino acid sequence and the following Dayhoff sequences:
AF061943_l; RNU08136-I;
MAV011838_1: HXAA_HLt~IAN: Y653_HUMAN: P_R51263; P_R74041; AF101057_1;
AF101058;

_yl_ EXAh'IPLE 10 Isolation of cDNA Clones Encodine a Human PR07476 A search was performed using the computer program BLAST or BLAST2 (Altschul et al.. Methods in Enzvmolow, 266:460-48U ( 1996)] for cytokine/~rowth factor homoloUs. A 94.5 KB
piece was found to contain exons encoding growth factor homologs, however. this piece was broken up by large introns. The introns were removed by a computer algorithm. Based on the DNA102863 consensus sequence.
oligonucleotides were 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 PR07476. 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 lens=th. In some cases, additional oli~onucleotides are synthesized when the consensus sequence is greater than about 1-l.5kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel el al., Current Protocols in Molecular Bioloay, 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.
PCR primers (forward and reverse) were synthesized:
forward PCR primer:
5'-ATGCAGCTCCCACTGGCCCTG-3' (SEQ ID NO: 31 ) reverse PCR primer:
5'-CTAGTAGGCGTTCTCCAGCTCGGCCTG-3' (SEQ ID N0:32) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA 102863 sequence which had the following nucleotide sequence:
hybridization probe:
5'-CTTCCGCTGCATCCCCGACCGCTACCGCGCGCAGCGCGTG-3' (SEQ ID NO: 33) DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR07476 polypeptide (desi~=nated herein as DNA1 15253-2757 [Figure 19. SEQ ID NO: 19]) and the derived protein sequence for that PR07476 polypeptide.
The full length clone identit3ed above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 62-64 and a stop signal at nucleotide positions 701-703 (Figure 19, SEQ ID
NO: 19). The predicted polypeptide precursor is 213 amino acids long, has a calculated molecular weight of approximately 24.031 daltons and an estimated pI of approximately 9.59.
Analysis of the full-length PR07476 sequence shown in Figure 20 (SEQ ID NO: 20) evidences the presence of a variety of important polypeptide domains as shown in Figure 20, wherein the locations given for those important polypeptide domains are approximate as described above. Clone DNA 1 15253-2757 has been deposited with ATCC on August 31. 1999 and is assigned ATCC Deposit No. PTA-612.
An analysis of the Davhoff database t version 35.45 SwissProt 35 ). using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 20 (SEQ ID NO: 2U), evidenced sequence identity between the PR07476 - -amino acid sequence and the followingT Dayhoff sequences:
P_W58704: P W9S71 l; P W09408:

~~V VV//JJ1/ rt.I/VJUV/iJJJO
P Y 12009: T08710; P-W44090: P_W27654: P-1'03'_''_'5: LSHB_MELGA: AB011 U30_ 1.

Gene Amolitication This example shows that the PR05800-. PR06000-. PR06016-. PR06018-. PR06496-.
PR07154-, PR07170-. PR07422-, PR07431-or PR07476-encoding genes are amplified in the genome of certain human lung, colon and/or breast cancers and/or cell lines. Amplification is associated with overexpression of the _ene product, indicating that the polypeptides are useful tar~~ets for therapeutic intervention in certain cancers such as colon, lung, breast and other cancers. Therapeutic agents may take the form of antagonists of PR05800, PR06000. PR06016, PR06018. PR06496, PR07154. PR07170. PR07422. PR07431 or PR07476 polypeptides, for example. murine-human chimeric, humanized or human antibodies against a PR05800. PR06000.
PR06016, PR06018. PR06496, PR07154. PR07170. PR07422. PR07431 or PR07476 polypeptide.
The starting material for the screen was genomic DNA isolated from a variety of cancers. The DNA is quantitated precisely, e.g., tluorometrically. As a negative control, DNA was isolated from the cells of ten normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (not shown). The 5' nuclease assay (for example, TaqManT") and real-time quantitative PCR (for example, ABI Prizm 7700 Sequence Detection System'"' (Perkin Elmer, Applied Biosystetns Division, Foster City, CA)), were used to find genes potentially amplified in certain cancers. The results were used to determine whether the DNA
encoding PR05800, PR06000, PR06016. PR06018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 is over-represented in any of the primary lung or colon cancers or cancer cell lines or breast cancer cell lines that were screened. The primary lung cancers were obtained from individuals with tumors of the type and stage as indicated in Table 4. An explanation of the abbreviations used for the designation of the primary tumors listed in Table 4 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore.
The results of the TaqMan~"' are reported in delta i01 Ct units. One unit corresponds to 1 PCR cycle or approximately a 2-fold amplitication relative to normal. two units corresponds to 4-told. 3 units to 8-fold amplification and so on. Quantitation was obtained using primers and a TaqManT" fluorescent probe derived from the PR05800-. PR06000-, PR06016-, PR06018-, PR06496-, PR07154-, PR07170-, PR07422-, PR07431-or PR07476-encoding gene. Regions of PR05800, PR06000. PR06016. PR060 I 8.
PR06496, PR07154. PR07170, PR07422, PR07431 or PR0747b which are most likely to contain unique nucleic acid sequences and which are least likely to have spliced out introns are preferred for the primer and probe derivation, e.g.. 3'-untranslated regions.
The sequences for the primers and probes (forward, reverse and probe) used for the PR05800, PR06000, PR06016, PR06U18. PRO(~i96, PR07154, PR07170, PR07-122. PR07431 or PR07476 gene amplification analysis were as follows:
PR05800 f DNA 108912-2b801:
108912.tm.f 1:
5'-GCGTCGTGGTCATCAAAG-3' (SEQ ID N0:34i _93_ I 08912.tm.r i 5'-TGCAGTCCACGGTGTAGAG-3'(SEQ ID
N0:35) 108912.tm.p I

5'-CTTCTACGTGGCCATGAACCGC-3'(SEQ ID
N0:36) I 08912.tm.f2:

5'-CCTGGAGATCCGCTCTGTA-3'(SEQ ID
N0:37) 108912.tm.p2:

5'-CTTTGATGACCACGACGCCCA-3'(SEQ ID
N0:38) 108912.tm.r2:

5'-ACGTAGAAGCCTGAGGACACT-3'(SEQ ID
N0:39) PR06000 fDNA102880-2689):
102880.tm, f t 5'-GATGCTCCAGCTGAAATCC-3' (SEQ ID N0:40) 102880.tm.r 1:
5'-CACATGGCTGGAAATGATG-3' (SEQ ID N0:41 ) 102880.tm.p 1:
5'-AAGCTAAGCTCCCAACTGACAGCCA-3' (SEQ ID N0:42) PR06016 (DNA96881-2699):
96881.tm.fl:
5'-TGGCCTACATGTGTCTTCATC-3' (SEQ ID N0:43) 96881.tm.r 1:
5'-CACAACTTTCTGGTCATATTCCAT-3' (SEQ ID N0:44) 96881.tm.p 1:
5'-CCTGCCCCAAGACGGCATTAG-3' (SEQ ID N0:45) PR06018 (DNA98565-2701 ):
98565.tm.f 1:
5'-CCTGGGCACCAGATCTTC-3' ISEQ ID N0:46) 98565.tm.r I
5'-AGGGCAGTTGAGGCACTT-3' (SEQ ID N0:47) 98565.tm.pl:
5'-CATCAGGGCCGGAGTAAATCCCT-3' (SEQ ID N0:48) PR06496 (DNA119302-2737):
119302.tm.f I
5'-TCCATGGACCTCCCACTATAC-3' (SEQ ID N0:49) _94_ I 19302_tm.r l S'-GCTGACAACTTCAGGTTCCA-3' (SEQ ID N0:50) 1 19302.tm.p I
S'-ACCCCCACCAAACCCCAGGT-3' (SEQ IDNO:S I ) PR071 S4 IDNA 108760-2740):
108760.tm.f 1:
S'-GATCTCTGAGCACACTTGTATGAG-3' (SEQ ID N0:52) 108760.tm.r 1:
S'-GGCAGACGAGGGTCTTTC-3' (SEQ ID N0:53) I 08760.tm.p 1:
5'-CAGGAACCCCTTGCTAGAATCAGCC-3' (SEQ ID N0:54) PR07170 (DNA108722 ?743):
108722.tm.f 1:
S'-CCCAGAAGGTTCCCATGA-3' (SEQ ID NO:SS) 108722.tm.r 1:
S'-GGGTCCTGTTGCCACATC-3' (SEQ ID N0:56) I 08722.tm.p 1:
S'-CAGCATGTCCAAGCCCCTAACCC-3' (SEQ ID N0:57) PR07-l22 (DNAI 19536-2752):
119536.tmf I
S'-TCTCCCCGATTCTCATCTG-3' (SEQ ID N0:58) I 19536.tm.r 1:
S'-CCCTGAGAGTCCTGCACAT-3' (SEQ ID N0:59) 119536.tm.pl:
5'-CCCATAATCATGGACACAGCCCC-3' (SEQ ID N0:60) PR07-431 (DNA 1195:12-27541:
119542.tm.f 1:
S'-AGTGAAGTTTCTCCAGTCCCTAGT-3' (SEQ ID N0:61 ) 119542.tm.r I
S'-CCTGGGGTAAGTGAGCAAA-3' (SEQ ID N0:62) 1195=4?.tm.p I
S'-CCTCTCTTTTCACCCACCTTCCTCAG-3' (SEQ ID N0:63) _9;_ PR07476 IDNA 1 15253-2757):
115253.tm.f1:
5'-GGGACTGGTTAAGAAAGTTGGAT-3' (SEQ ID N0:64) 115253.tm.rl 5'-CGCCTCAGGCTTTCTGAT-3' (SEQ ID N0:65) l 15253.tm.p 1:
5'-AGATTCCCCCTTGCACCTCGC-3' (SEQ ID N0:66) The 5' nuclease assav reaction is a tluorescent PCR-based technique which makes use of the S' exonuclease activity of Taq DNA polymerise enzyme to monitor amplification in real time.
Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerise enzyme, and is labeled with a reporter fluorescent dye and a quencher tiuorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerise enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is tree from the quenching effect of the second f7uorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
The 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 7700TM
Sequence Detection. The system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer. The systemamplifies samples in a 96-well format on a thermocycler.
During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.
5' Nuclease assay data are initially expressed as Ct. or the threshold cycle.
This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence. The ~Ct values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer DNA results to normal human DNA results.
Table 4 describes the sta_e, T stage and N stage of various primary tumors which were used to screen the PR05800, PR06000, PR06016, PR06018. PR06496, PR0715=l, PR07170, PR07~22, PR07=l31 or PR07:f76 compounds of the invention.

WV UU!'/5317 Y~.1/UJUU/153~5 Table d Primary Lung and Colon Tumor Profiles Primary Tumor StageOther Stage Dukes Stage T Stage N Staee Human lung tumor AdenoCa IIA T1 N 1 (SRCC724) [LT1 ]

Human lung tumor SqCCa (SRCC725)IIB T3 NO
[LTIa) Human lung tumor AdenoCa IB T2 NO
(SRCC726) [LT2]

Human lung tumor AdenoCa IIIAT1 N2 (SRCC727) [LT3J

Human lung tumor AdenoCa IB T2 NO
(SRCC72$) (LT-(]

Human lung tumor SqCCa (SRCC729)IB T2 NO
[LT6]

Human tune tumor Aden/SqCCa IA T1 NO
(SRCC7301 [LT7]

Human Lung tumor AdenoCa IB T2 NO
(SRCC731 ) [LT9]

Human lung tumor SqCCa (SRCC732)IIB T2 NI
[LT10J

Human lung tumor SyCCa (SRCC733)IIA Tl N 1 [LT11 ]

Human lung tumor AdenoCa IV T2 NO
(SRCC734) [LT12J

Human lung tumor AdenoSqCCa T2 NO
(SRCC735)[LT13] IB

Human lung tumor SqCCa (SRCC736)IB T2 NO
(LT15]

Human lung tumor SqCCa (SRCC737)IB T2 NO
[LT16]

Human lung tumor SqCCa (SRCC738)IIB T2 N I
[LT17]

Human lung tumor SqCCa (SRCC739)IB T2 NO
[LT18]

Human lunv tumor SqCCa (SRCC740)IB T2 NO
(LT19J

Human tuns tumor LCCa (SRCC741IIB T3 N I
) [LT21 J

Human lung AdenoCa (SRCC811 lA T1 NO
) [LT22]

Human colon AdenoCa (SRCC742) M 1 D pT4 NO
[CT2]

Human colon AdenoCa (SRCC743) B pT3 NO
(CT3]

Human colon AdenoCa (SRCC B T3 NO
744) [CT8J

Human colon AdenoCa (SRCC745) A pT2 NO
[CT10J

Human colon AdenoCa (SRCC746) MO, R1 B T3 NO
[CT12]

Human colon AdenoCa (SRCC747) pMO, RO B pT3 pN0 (CT14J

Human colon AdenoCa (SRCC748) M1, R2 D T4 N2 [CT15]

Human colon AdenoCa (SRCC749) pM0 B pT3 pN0 [CT16]

Human colon AdenoCa (SRCC750) C1 pT3 pNl [CT17]

Human colon AdenoCa (SRCC751 MO, R 1 B pT3 NO
) [CTl ]

Human colon AdenoCa (SRCC752) B pT3 MO
[CT4]

Human colon AdenoCa (SRCC753) G2 C1 pT3 pN0 [CTS]

Human colon AdenoCa (SRCC754) pMO, RO B pT3 pN0 [CT6]

Human colon AdenoCa (SRCC755) G1 A pT2 pN0 [CT7]

Human colon AdenoCa (SRCC756) G3 D pT-I pN2 [CT9]

Human colon AdenoCa (SRCC7S7) B T3 NO
[CTI 1 ]

Human colon AdenoCa (SRCC758) MO, RO B pT3 pN0 [CT18]

DNA Preparation:
DNA was prepared from cultured cell (fines, primary tumors, and normal human blood. The isolation was performed using purification kit, buffer set and protease and all from Qiagen.
according to the manufacturer's instructions and the description below.
Cell culture lvsis:
Cells were washed and trypsinized at a concentration of 7.5 x 10' per tip and pelleted by centrifuging at 1000 rpm for 5 minutes at 4"C. followed by washing a~~ain with 1/2 volume of PBS and recentrifugation. The pellets were washed a third time. the suspended cells collected and washed 2x with PBS. The cells were then suspended into 10 ml PBS. Buffer C1 was equilibrated at 4"C. Qiagen protease #19155 was diluted into 6.25 mi cold ddH,O to a final concentration of 20 mg/ml and equilibrated at 4"C. 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse A stock ( 100 mg/ml ) to a final concentration of 200 ug/ml.
Buffer C1 ( 10 ml, 4"C) and ddH20-(:IO ml. 4"C) were then added to the 10 ml of cell suspension. mixed by inverting and incubated on ice for 10 minutes. The cell nuclei were pelleted by centrifuging in a Beckman swinging bucket rotor at 2500 rpm at 9"C for 15 minutes. The supernatant was discarded and the nuclei were S suspended with a vortex into 2 ml Buffer CI (at.f"C) and 6 mI ddH,O, followed by a second 4"C centrifugation at 2500 rpm for I 5 minutes. The nuclei were then resuspended into the residual buffer using 200 gel per tip. G2 buffer ( 10 ml) was added to the suspended nuclei while gentle vortexing was applied.
Upon completion of buffer addition.
vigorous vortexing was applied for 30 seconds. Qiagen protease (200 ~cl, prepared as indicated above) was added and incubated at 50"C for 60 minutes. The incubation and centrifugation were repeated until the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min., ~"C).
Solid lmmart nunor sample preparation and lysis:
Tumor samples were weighed and placed into 50 ml conical tubes and held on ice. Processing was limited to no more than ?50 mg tissue per preparation ( 1 tip/preparation). The protease solution was freshly prepared by diluting into 6.25 ml cold ddH=O to a final concentration of 20 mg/ml and stored at 4"C. G2 buffer (20 ml) was 1S prepared by diluting DNAse A to a final concentration of 200 mg/ml (from 100 mJml stock). The tumor tissue was homogenated in 19 mhG2 buffer for 60 seconds using the large tip of the polytron in a laminar-flow TC hood in order to avoid inhalation of aerosols, and held at room temperature.
Between samples, the.polytron was cleaned by spinning at ? x 30 seconds each in 2L ddH,O, followed by G2 buffer (50 ml).
If tissue was stilt present on the generator tip, the apparatus was disassembled and cleaned.
Qiagen protease (prepared as indicated above, l.0 ml) was added. followed by vortexing and incubation at 50°C for 3 hours. Ttte incubation and centrifugation were repeated until the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for I O tttin., 4"C).
Human blood preparation and lysis:
Blood was drawn from healthy volunteers using standard infectious agent protocols and citrated into 10 ml samples per tip. Qiagen protease was freshly prepared by dilution into 6.25 ml cold ddH,O to a final concentration of 20 mJml and stored at 4"C. G2 buffer was prepared by diluting RNAse A to a final concentration of 200 ~~ml from 100 mJml stock. The blood ( 10 ml) was placed into a 50 ml conical tube and 10 ml C 1 buffer and 30 ml ddH.O (both previously equilibrated to 4"C) were added. and the components mixed by inverting and held on ice for 10 minutes. The nuclei were pelleted with a Beckman swinging bucket rotor at 2500 rpm, -t"C for 15 minutes and the supernatant discarded. With a v ortex, the nuclei were suspended into 2 ml Cl buffer (.i"C) and 6 ml ddH,O (-t"C). Vonexing was repeated until the pellet was white. The nuclei were then suspended into the residual buffer using a 200 ~.I tip. G2 butter ( 10 ml) was added to the suspended nuclei while gently vortexing, followed by vigorous vortexing for 30 seconds. Qiagen protease was added (200 ul) and incubated at 50"C for 60 rtunutes. The incubation and centrifu_ation were repeated until the lysates were clear (e.g.. incubating additional 3S 30-60 minutes. pelletin~ at 3000'x g for 10 min., 4"C).
Purincarion of cleared h~.rates:
( 1 ) Isolation of aenomic DNA:
Genomic DNA was equilibrated ( 1 sample per maxi tip preparation) with 10 ml QBT butter. QF elution WO 00!?531T PCT/US00/13358 buffer was equilibrated at SO"C. The samples were vortexed for 30 seconds.
then loaded onto equilibrated tips and drained by cnavity. The tips were washed with 2 x I S ml QC buffer. The DNA
was eluted into 30 ml silanized.
autoclaved 30 ml Corex tubes with I S ml QF butter (50"C). Isopropanol l 10.5 ml ) was added to each sample, the tubes covered with paratin and mixed by repeated inversion until the DNA
precipitated. Samples were pelleted by centrifugation in the SS-34 rotor at 15,000 rpm for lU minutes at 4"C. The pellet location was marked, the supernatant discarded. and 10 ml 709c ethanol (4"C1 was added. Samples were pelleted again by centrifugation on the SS-34 rotor at 10,000 rpm for 10 minutes at 4"C. The pellet location was marked and the supernatant discarded.
The tubes were then placed on their side in a drying rack and dried 10 minutes at 37"C. taking care not to overdry the samples.
Afterdrying, the pellets were dissolved into 1.0 ml TE (pH 8.5) and placed at 50°C for I -2 hours. Samples were held overnisht at 4"C as dissolution continued. The DNA solution was then transferred to 1.5 ml tubes with a 26 gauge needle on a tuberculin syringe. The transfer was repeated Sx in order to shear the DNA. Samples were then placed at 50°C for l-2 hours.
(2) Quantitation of oenomic DNA and nreoaration for gene amplification assay:
The DNA levels in each tube were quantified by standard A;MJ Ate"
spectrophotornetry on a 1:20 dilution (5 ul DNA +95 ul ddH,O) using the 0.1 ml quartz cuvettes in the Beckman DU640 spectrophotometer. A,",/A~, ratios were in the range of 1.8-1.9. Each DNA sample was then diluted further to approximately 200 nglml in TE
(pH 8.5). If the original material was highly concentrated (about 700 ng/~cl), the material was placed at 50°C for several hours until resuspended.
Fluoromctric DNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidelines as modified below. This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometer to warm-up for about 15 minutes. The Hoechst dye working solution (#H33258.10 ul. Prepared within 12 hours of use) was diluted into 100 ml 1 x THE buffer. A 2 ml cuvette was filled with the fluorometer solution, placed into the machine, and the machine was zer oed. pGEM 3Zf(+) (2 ul, lot #360851026) was added to 2 ml of fluorometer solution and calibrated at 200 units. An additional 2 ul of pGEM
3Zff+) DNA was then tested and the reading confirnxd at 400+/-10 units. Each sample was then read at least in triplicate. When 3 samples were found to tie within 10'Xc of each other. their average was taken and this value was used as the quantification value.
The fluorortxtricly determined concentration was then used to dilute each sample to 10 ng/ul in ddH;O.
This was done simultaneously on all template samples for a single TaqManr"
plate assay, and with enough material to run 500-1000 assays. The samples were tested in triplicate with Taqtnanr"
primers and probe both B-actin and GAPDH on a single plate with normal human DNA and no-template controls. The diluted samples were used provided that the CT value of normal human DNA subtracted from test DNA was +l-I Ct. The diluted, lot-qualified genomic DNA was stored in 1.U ml aliquots at -80"C. Aliquots which were subsequently to be used in the~gene amplification assay wrre stored at 4"C. Each I ml aliquot is enough for 8-9 plates or 64 tests.
Gene ampfificariorr assay:
The PR05800, PR06000. PR06016. PR06018. PR06496. PR07154. PR07170. PR07422.

or PR07476 compounds of the invention were screened in the following prirnary tumors and the resulting ~Ct values arc reported in Table 5.
*-trademark -99-r V N V'.

r ~ N cr;
O - ~i N

_ G

M
'T V.
' OC V
.

a N

N
~f a r O

a %:

r :J r x O

a , r r c~ r C

O

E' d T

a -- ~O r N r!
U

a J o c ;

:u , O

a .!

a j v: r = N

O ~- m f ~i .J

4 a O

z y oc a r x O c-i a a .r.- ~ ~i~ a - d w , a --rr J

f~.M r 'T'7 'y', C70--a a a r '7 V
, n ~ y _:x x r r_ ~ ~J ~JN ~!.c ,c ~ X X x X

_ ~ X X

C L ;_ L L :L L
~

T T

DISCUSSION AND CONCLUSION:
PR05800 (DNA 108912-2680):
The Act values for DNA108912=_'680 in a variety of tumors are reported in Table 5. A ~lCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that siUniticant amplification of nucleic acid DNA 108912-2680 encoding PR05800 occurred in primary tune tumors: HF-001644 and HF-001647.
Because amplification of DNA 108912-2680 occurs in various lung tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA 108912-2680 (PR05800) would be expected to have utility in cancer therapy.
PR06000 (DNA 102880-2689):
The Act values for DNA102880-2689 in a variety of tumors are reported in Table 5. A OCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA 102880-2689 encoding PR06000 occurred in primary tuns tumor HF-001295.
Becauseamplification of DNA 102880-2689 occurs in lung tumor, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA102880-2689 (PR06000) would be expected to have utility in cancer therapy.
PR06016 (DNA96881-2699):
The ~Ct values for DNA96881-2699 in a variety of tumors are reported in Table 5. A Act of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA96881-2699 encoding PR06016 occurred: (1 ) in primary lung tumor HF-000641; and (2) in primary colon tumor centers: HF-000762, HF-000789 and HF-000811.
Because amplification of DNA96881-2699 occurs in various tumors. it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA96881-2699 (PR06016) would be expected to have utility in cancer therapy.
PR06018 (DNA98565-2701 ):
The GICt values for DNA98565-270 i in a variety of tumors are reported in Table 5. A Act of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA98565-2701 encoding PR06018 occurred: ( 1 ) in pritttary lung tumor HF-0008=l0: and (2) in primary colon tumor center HF-00081 1.
Because amplification of DNA98565-2701 occurs in various tumors. it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists t e.g., antibodies) directed against the protein encoded by DNA98565-2701 (PR06018) would be expected to have utility in cancer therapy.

PR06496 (DNAI 19302-2737):
The ACt values for DNA 119302-2737 in a variety of tumors are reported in Table 5. A OCt of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table indicates that significant amplification of nucleic acid DNA 119302-2737 encoding PR06496 occurred in primary 5 lung tumors: HF-000840, HF-000842, HF-001294 and HF-001296.
Because amplification of DNA 119302-2737 occurs in various lung tumors, it is highly probable to play a significant role in tumor formation or growth. As a result. antagonists (e.g., antibodies) directed against the protein encoded by DNA I 19302-2737 (PR06496) would be expected to have utility in cancer therapy.
PR07154 (DNA108760-2740):
The ~Ct values for DNA108760-2740 in a variety of tumors are reported in Table 5. A OCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA 108760-2740 encoding PR07154 occurred in primary lung tumors: HF-001296 and HF-001299.
Because amplification of DNA108760-2740 occurs in various lung tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA108760-2740 (PR07154) would be expected to have utility in cancer therapy.
PR07170 (DNA 108722-2743):
The ~Ct values for DNA108722-2743 in a variety of tumors are reported in Table 5. A OCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA 108722-2743 encoding PR07170 occurred in primary lung tumor HF-001296.
Because amplification of DNA 108722-2743 occurs in lung tumor. it is highly probable to play a si gnifieant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA108733-'743 (PR07170) would be expected to have utility in cancer therapy.
PR07422 (DNA119536-2752):
The ~Ct values for DNA1 19536-2752 in a variety of tumors are reported in Table 5. A ~Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplification of nucleic acid DNA 119536-2752 encodin=PR07-122 occurred in primary tune tumor HF-001647.
Because amplification of DNAI 19536-2752 occurs in lung tumor, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA119536-2752 (PR07422) would be expected to have utility in cancer therapy.
PR07431 (DNA 119542-2754):
The ACt values for DNA I I 9542-2754 in a variety of tumors are reported in Table S. A ACt of > 1 was typically used as the threshold value for amplification scorinn, as this represents a doubling of gene copy. Table indicates that significant amplitication of nucleic acid DNAI 19542-2754 encoding PR07431 occurred: (1) in testis tumor center HF-000733: ( 21 in primary colon tumor center: HF-0005 39:
and (3) in primary lung tumors: HF-000842 and HF-001296.
5 Because amplification of DNA119542-2754 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.S..
antibodies) directed against the protein encoded by DNA119542-2754 (PR07431 ) would be expected to have utility in cancer therapy.
PR07476 (DNA I 15253-27571:
The ~Ct values for DNA I 15253-2757 in a variety of tumors are reported in Table 5. A ~Ct of > 1 was typically used as the threshold value for amplit3cation scoring, as this represents a doubling of gene copy. Table 5 indicates that significant amplitication of nucleic acid DNA 115253 ?757 encoding PR07476 occurred: (I ) in testis tumor center HF-000733: (?) in primary colon tumor centers: HF-000539 and HF-000575; and (31 in primary lung turnors HF-001294 and HF-001296.
Because amplification of DNA115253-2757 occurs in various tumors. it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA115253-2757 (PR07476) would be expected to have utility in cancer therapy.

Use of PR05800. PR06000. PR06016, PR06018, PR06496. PR07154. PR07170. PR07422, PRO?431 or PR07476 as a hybridization probe The following method describes use of a nucleotide sequence encoding a PR05800. PR06000, PR06016, PR06018, PR06496, PR07154. PR07170, PR07422, PR07431 or PR07476 polypeptide as a hybridization probe.
DNA comprising the coding sequence of a full-length or mature "PR05800", "PR06000", "PR06016", "PR06018", "PR06496", "PR07154", "PR07170", "PR07422". "PR07431 " or "PR07476"
polypeptide as disclosed herein and/or fragments thereof may be employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurrins variants of PR05800, PR06000, PR06016, PR06018. PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476) in human tissue cDNA libraries or human tissue genomic libraries.
Hybridization and washingoffilterscontainingeitherlibraryDNAs isperfortnedunderthefollowinghigh stringency conditions. Hybridization of radiolabeled PR05800-, PR06000-, PR06016-, PR06018-. PR06496-, PR07154-, PR07170-, PR07422-, PR07431- or PR07476-derived probe to the Yilters is performed in a solution of SOclc fotmamide, 5x SSC, 0.19c SDS, 0.1 % sodium pyrophosphate. 50 mM
sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42"C for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1 x SSC and 0.1 % SDS at 42"C.
DNAs having a desired sequence identity with the DNA encoding full-length native sequence PR05800, PR06000, PR06016, PR06018. PR06496. PR07154. PR07170, PR07-122, PR0743 I or PR07476 can then be identified using standard techniques known in the art.

Expression of PROS800. PR06000. PR06016. PR06018. PR06496. PR07154. PR07170.
PR074?2.
PR074 31 or PR07476 Polvpeptides in E. coli.
This example illustrates preparation of an unglycosylated form of PR05800.
PR06000, PR06016, PR06018. PR06496, PR07154. PR07170. PR07422. PR07431 or PR07476 by recombinant expression in E.
coli.
The DNA sequence encoding the PRO polypeptide of interest is initially amplified using selected PCR
primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli: see Bolivar et al., Gene. 2:95 ( I 977)) 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 ST'II codons, poly-His sequence, and enterokinase cleavage site), the PR05800, PR06000, PR06016, PR06018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 coding region, lambda transcriptional terminator.
and an arQU 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 brown 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 cel l pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PR05800. PR06000. PR06016. PR06018. PR06496. PR07154, PR07170. PR07422.
PR07431 or PR07476 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
PR05800, PR06000. PR06016, PR06018, PR06496, PR07154, PR07170. PR07422.
PR07431 or PR07476 may be expressed in E. toll in a poly-His tagged form using the following procedure. The DNA encoding PR05800, PR06000, PR06016, PR06018. PR06496. PR07154, PR07170. PR07422, PR07431 or PR07476 is initially amplified using selected PCR primers. The primers 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-amplitied, poly-His tagged sequences are then ligated into an expression vector. which is used to transform an E. toll host based on strain 52 (W31 10 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(IacIq).
Transfortnants are tirst grown in LB containing 50 mg/ml carbenicillin at 30°C with shaking until an O.D. of 3-5 at 600 nm is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 (NH,)=SO" 0.71 g sodium citrate~2H,0, 1.07 g KCI. 5.36 g Difco yeast extract, 5.368 Sheffield hycase SF in 500 ml water, as well as 110 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 ~ pellets) is resuspended in 10 volumes (w/v) in 7 M
suanidine, 20 mM Tris, pH 8 buffer. Solid sodium sultite and sodium tetrathionate are 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 .10.000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiaaen Ni =*-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The proteins are 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 protein is refolded by diluting sample slowly into freshly prepared refolding buffer consisting of:
20 mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20 tnM glycine and 1 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 R1/H reversed phase column using a mobile buffer of 0:1 % TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A=H"
absorbance are analyzed on SDS
polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
Fractions concainins the desired folded PR05800, PR06000, PR06016. PR06018.
PR06496, PR07154, PR07170, PR07422. PR0743 I or PR07476 protein are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM
Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.

Expression of PR05800. PR06000. PR06016. PR06018. PR06496. PR07154. PR07170.
PR07422.
PR07431 or PR07476 in mammalian cells This example illustrates preparation of a potentially glycosyiated form of PR05800. PR06000, PR06016, PR06018. PR06496. PR0715=1. PR07170, PR07422, PR07-131 or PR07476 by recombinant expression in mammalian cells.
-IOS-The vector. pRKS (see EP 307,2.17, published March 15. 1989), is employed as the expression vector.
Optionally, the PR05800, PR06000. PR06016, PR06018. PR06496, PR07154. PR07170, PR07-122. PR07431 or PR07476 DNA is ligated into pRKS with selected restriction enzymes to allow insertion of the PR05800, PR06000, PR06016, PR06018, PR06496. PR07154. PR07170, PR07422, PR0743 I or PR07476 DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRKS-(PR05800, PR06000, PR06016, PR06018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476].
In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to contluence in tissue culture plates in medium such as DMEM
supplemented with fetal calt serum and optionally, nutrient components and/or antibiotics. About 10 ~g pRKS-[PR05800, PR06000, PR06016, PR06018, PR06496, PR07154, PR07170, PR07422. PR07431 or PR07476] DNA is mixed with about 1 pg DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 ( 1982)] and dissol ved in 500 pl of 1 mM Tris-HCI, 0.1 mM EDTA, 0.227 M CaCI=. To this mixture is added, dropwise, 500 ul of 50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25"C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37"C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ~Ci/ml'SS-cysteine and 200 ~Ci/ml'SS-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a l5ola SDS
gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the PR05800, PR06000, PR06016, PR06018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 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; PR05800, PR06000, PR06016, PR06018, PR06496, PR07154. PR07170, PR07422, PR07431 or PR07476 DNA may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et af.. Proc. Natl. Acad. Sci., 12:7575 ( 1981 ). 393 cells are grown to maximal density in a spinner flask and 700 ~g pRKS-[PR05800, PR06000, PR06016.
PR06018. PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476] 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 ug/ml bovine insulin and 0.1 ug/ml bovine transfetrin. After about four days, the conditioned media is centrifuged and tittered to remove cells and debris.
The sample containing expressed PR05800, PR06000, PR06016, PR06018. PR06496, PR07154. PR07170.
PR07422, PR07431 or PR07476 can then be concentrated and purified by any selected method. such as dialysis and/or column chromatography.
In another embodiment PR058U0. PR06000, PR06016. PR06018. PR06496. PR07154.
PR07170, PR07422, PR07431 or PR07476 can be expressed in CHO cells. The pRKS-[PR05800.
PR06000. PR06016, PR06018, PR06496, PR07154, PR07170, PR07422. PR07431 or PR07476] vector can be transfected into CHO

cells using known rea~_ents such as CaPO, or DEAF-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containin~_ a radiolabel such as'SS-methionine. After determining the presence of the PR05800. PR06000, PR06U16.
PR06018. PR06496, PR07154. PR07170, PR07-1?2. PR07431 or PR07476 polypeptide. the culture medium may be replaced with serum tree medium. Preferably, the cultures are incubated for about 6 days.
and then the conditioned medium is harvested. The rnediumcontaining the expressed PR05800. PR06000. PR06016.
PR060 i 8, PR06496. PR07154, PR07170, PR07422, PR07431 or PR07476 can then be concentrated and puritied by any selected method.
Epitope-tagged PR05800. PR06()D0. PR06016. PR060! 8. PR06496. PR07154, PR07170. PR07422, PR07431 or PR07476 may also be expressed in host CHO cells. The PR05800.
PR06000, PR06016. PR06018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 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 Bacutovirus expression vector. The poly-His tagged PR05800, PR06000, PR06016.
PR06018, PR06496, PR07154, PR07170. PR0742'?. PR07431 or PR07476 insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones.
Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tasged PR05800, PR06000, PR06016, PR06018. PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 can then be concentrated and purified by any selected method, such as by Ni='-chelate affinity chromato~
aphy. Expression in CHO and/or COS
cells may also be accomplished by a transient expression procedure.
PR05800. PR06000. PR06016, PR06018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 may be expressed in CHO cells by a transient procedure. Stable expression in CHO cells can be 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 IQG1 constant region sequence containing the hinge, CH2 and CH2 domains and/or in a poly-His tagged form.
Following PCR amplitication. the respective DNAs are subcloned in a CHO
expression vector using standard techniques as described in Ausubel er al.. Current Protocols of Molecular Bioio_v, Unit 3.16. John Wiley and Sons ( 1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA
of interest to allow the convenient shuttling of cDNA's. The vector used for 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 are introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect'°
(Qiagen). Dosper~' or Fugene" (Boehringer Mannheim). The cells are Grown as described in Lucas er al., .supra.
Approximately 3 x 1 U' cells are frozen in an ampule for further growth and production as described below.
The ampuies containin~_ the plasmid DNA are thawed by placement into a water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing J O 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 tittered PS20 with Sclc 0.2 um diatiltered fetal bovine serum). The cells are then aliquoted into a I UO ml spinner containing 90 ml of selective media. After I-2 days, the cells are transferred into a 250 ml spinner filled with 150 ml selective growth medium and incubated at 37"C. After another 2-3 days. 250 ml, 500 ml and 2000 ml spinners are seeded v,~ith 3 x 10' cells/ml. The cell media is exchanged with fresh media by centrifugation and resuspension S 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 I(,. 1992 is actually used. 3L
production spinner is seeded at 1.2 x 10"
cells/ml. On day 0. the cell number and pH are determined. On day 1, the spinner is sampled and spar~_in~ with filtered air is commenced. On day 2, the spinner is sampled, the temperature shitted to 33"C, and 30 ml of 500 JL
~~lucose and 0.6 ml of 10% antifoam (e.g., 35~c polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) added. Throughout the production. the pH is adjusted as necessary to keep at around 7.2. After 10 days.
or until viability drops below 70nc, the cell culture is harvested by centrifugation and filtered through a 0.23 .gym 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 (Qiasen). 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 hishly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCI and 49c mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which 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 ~.I of I 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.
EXAMPLE I S
Expression of PR05800. PR06000. PR06016. PR06018. PR06496. PR07154. PR07170.
PR07422.
PR07431 or PR07476 in Yeast The following method describes recombinant expression of PR05800, PR06000, PR06016. PR06018.
PR06496, PR07154, PR07170. PR07422, PR07431 or PR07476 in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PR05800.
PR06000. PR06016. PR0601 S. PR06496, PR07154, PR07170. PR07-122. PR07431 or PR07476 from the ADH2/GAPDH promoter. DNA encodin~_ PR05800. PR06000. PR06U16. PR06018.
PR06496. PR07154, PR07170. PR07422. PR07-is I or PR07476 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR05800. PR06000.
PR06016. PR06018. PR06-t96.
PR07154. PRO7170, PR07422. PR07431 or PR07476_ For secretion. DNA encodinn PR05800, PR06000.

PR06016, PR06018. PR06496. PR07154. PR071 70. PR07-122. PR07431 or PR07-t76 can be cloned into the selected plasmid, together with DNA encodin~= the ADH2/GAPDH promoter, a native PR05800. PR06000, PR06016. PR06018. PR06496. PR07154. PR07170. PR07422, PR07431 or PR07476 signal peptide or other mammalian si~=nal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence,. and linker sequences ( if needed) for expression of PR05800, PR06000. PR06016, PR06018, PR06=196. PR07154.
PR07170. PR07422. PR07431 or PR07476.
Yeast cells, such as yeast strain AB I 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 l0~lo trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
Recombinant PR05800, PR06000. PR06016, PR06018, PR06496. PR07154, PR07170, PR07422, PR07431 or PR07476 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing PR05800, PR06000, PR06016. PR06018, PR06496. PR07154, PR07170, PR07422. PR07431 or PR07476 may further be purified using selected column chromatography resins.

Exnressian of PR05800 PR06000. PR06016. PR06018. PR06496. PR07154. PR07170.
PR07422.
PR07431 or PR07476 in Baculovirus-infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells.
The sequence coding for PR05800. PR06000, PR06016, PR06018, PR06496, PR07154, PR07I70, PR07422, PR07431 or PR07476 is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-His tags and immuno~lobulin tags (like Fc regions of IgG). A variety of plasmids may be employed. including plasmids derived from commercially available plasmids such as pVL1393 (Novagenl. Briefly. the sequence encoding PR05800. PR06(>D0. PR06016. PR06018.
PR06496. PR07154.
PR0717U, PR07422. PR0743 I or PR07476 or the desired portion of the coding sequence of PR05800. PR06000, PR06016. PR06018, PR06496, PR07154. PR07170. PR07422, PR07431 or PR07476 [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 tlanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldT" virus DNA
l Pharmingen i into Spodoprera fiwgiperda ("Sf9" ) cel Is (ATCC CRL 171 1 ) using I ipofectin ( commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C. the released viruses are harvested and used for turther 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 ( 199=1).
Expressed poly-His tagged PR05800. PR06000. PR06016. PR06018. PR06496.
PR0715=1, PR07170.
PR07422. PR07431 or PR07476 can then be purified. for example, by Ni='-chelate aftinity chromatography as WO 00/75317 Y(:1 /UJUU11335ti follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert er al.. Mature.
362:175-179 ( 1993). Brietly, St~9 cells are washed. resuspended in sonication buffer (25 ml Hepes. pH 7.9: 12.5 rnM MgCI=; U.1 mM EDTA; 10% glycerol: 0.1 % NP-40; O.d M KCl), and sonicated twice for 20 seconds on ice, The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer i s0 mM
phosphate. 300 mM NaCI. 10% glycerol, pH 7.8) and filtered through a 0.45 arm filter. A Ni='-NTA agarose column tcommercially 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.~ ml per minute. The column is washed to baseline A..~, with loading buffer, at which point fraction collection is started.
Next, the column is washed with a secondary wash buffer (50 rnM phosphate; 300 rnM NaCI, 10% glycerol, pH
6.0), which elutes nonspecifically bound protein. After reaching A_H"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 tQiagen).
Fractions containing the eluted His"; tagged PR05800, PR06000, PR06016, PR06018, PR06496. PR07154, PR07170, PR07422, PR07431 or PR07476, respectively, are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PR05800, PR06000.
PR06016. PR06018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
While expression is actually performed in a 0.5-2 L scale, it can be readily scaled up for larger t e.g., 8 L) preparations. The proteins are expressed as an IgG construct (immunoadhesin), in which the protein extracellular region is fused to an IgG I constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms.
Following PCR amplification, the respectiveeoding 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~
baculovirus DNA (Pharmingen) are co-transfected into 105 Spodoptera frugiperda ("Sfl7") cells (ATCC CRL
1711 ), using Lipofectin (Gibco BRL). pb.PH.IQG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (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 ~. Czlls 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 of Ni ='-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A
Sepharose CL--~B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The tirst viral amplification supernatant is used to infect a spinner culture (500 ml I of Sf9 cells crown 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 are repeated. as necessary, until expression of the spinner culture is cont3tTned.

WO 00/75317 PCT/fJS00/13358 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 titters. For the poly-His tagged constructs, the protein construct is purified using a Ni ='-NTA column (Qiagen). Before purification, imidazolt is added to the conditioned media to a concentration of 5 tnM. The conditioned media is pumped onto a 6 ml Ni ='-NTA column equilibrated in 20 rnM
Hepes. pH 7.-i, 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 mM Hepes. 0.14 M NaCI and -l~'c mannitol, pH 6.8, with a 25 ml G25 Supe~ne (Pharmacies) column and stored at -80°C.
10 Irnmunoadhesin (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 (Pharmacial 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 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 proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edtnan degradation.
Alternatively, a modified baculovitus 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'-of) of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly His tags and imtnunoglobulin tags (like Fc regions of IgG). A variety of plasmids tray be employed, including plasmids derived from commercially available plasmids such as pIEI-1 (Novagen). The pIEI-1 and pIEI-2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus ie 1 promoter in stably-transforrrxd insect cells. The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for ie 1-mediated gene expression in uninfected insect cells as well as the hr5 enhancer element, pIEI-1 and pIEI-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 exvacellular domain of a transmembrane protein) is amplified by PCR with pritncrs complementary to the 5' and 3' regions. The 5' pritner may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subc)oned into the expression vector. For example, derivatives of pIEI-I can include the Fc region of human IgG (pb.PH.IgG) oran 8 histidine (pb.PH.His> tag downstream (3'-of) the desired sequence. Preferably, the vector conswct is sequenced for confirmation.
High 5 cells are grown to a contluency of 5096 under the conditions of 27 °C, no CO:, NO pen/strep. For each I 50 mm plate, 30 ~sg of pIE based vector containing the sequence is mixed with 1 ml Ex-Cel l medium (Media:
Ex-Cell 4U1 + I/100 LGIu JRH Biosciences #14401-78P (note: this tnedia is light sensitive)), and in a separate tube.100 ~I of CeIIFectin (CeIIFECTIN (GibcoBRL #10362-010) (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 i 5 minutes. 8 ml of Ex-Cell modia is addod to the 2 ml of DNA/CeIIFECIIN mix and this is layered on high 5 cells that have been washed once with Ex-Cell rnedia. The plate is then incubated in darkness for 1 hour at room temperature. The *-trademark -III-DNA/CeIIFECTIN mix is then aspirated. and the cells are washed once with Ex-Cell to remove excess CelIFECTIN, 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28"C. The supernatant is harvested and the expression of the sequence in the baculovirus expression vector is determined by batch binding of ( ml of supernatant to 25 ml of Ni ='-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for I~_G 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 tagged constructs, the protein comprising the sequence is puritied 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, butter containing 0.3 M NaCI and 5 mM imidazole at a flow rate of 4-5 mllmin. at 48°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 4~1c mannitol, pH 6.8. with a 25 ml G25 Superfine (Phatmtacia) 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 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 ml of I 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.
PR06018, PR07154. PR07170 and PR07476 were successfully expressed by the above modified baculovirus procedure incorporating high 5 cells.

Preparation of Antibodies that Bind PR05800. PR06000. PR06016, PR06018.
PR06496. PR07154, PR07170. PR07422. PR07431 or PR07476 This example illustrates preparation of monoclonal antibodies which can specifically bind PR05800, PR06000, PR06016, PR06018. PR06496, PR07154. PR07170, PR07422, PR07431 or PR07476.
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 PR05800, PR06000. PR06016. PR06018, PR06496. PR07154, PR07170. PR07422, PR07431 or PR07476 fusion proteins containing PR05800.
PR06000, PR06016, PR06018. PR06496, PR07154, PR07170. PR07=122. PR07431 or PR07476 and cells expressing recombinant PROS800. PR06000, PR06016. PR06018, PR06496, PR07154.
PR07170. PR07422, PR07431 or PR07476 on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
_I1~_ wV UU/75317 PCT/US00/13358 Mice, such as Balb/c, are immunized with the PR05800. PR06000, PR06016.
PR06018. PR06496.
PR07154. PR07170, PR07422, PR07431 or PR07476 immunogen emulsitied in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from l-100 micrograms. Alternatively. the immunogen is emulsitied in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind toot 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 ELISA assays to detect anti-PR05800, anti-PR06000. anti-PR06016, anti-PR06018, anti-PR06496, anti-PR07154, anti-PR07170, anti-PR07422, anti-PR07431 or anti-PR07476 antibodies.
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a t7nal intravenous injection of PR05800, PR06000, PR06016. PR06018, PR06496.
PR07154, PR07170, PR07422, PR07431 or PR07476. 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 PR05800, PR06000, PR06016, PR06018, PR06496, PR07I54, PR07170, PR07422, PR07431 or PR07476. Determination of "positive"
hybridoma cells secreting the desired monoclonal antibodies against PR05800, PR06000. PR06016, PR06018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 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-PR05800, anti-PRO6000, anti-PR06016, anti-PR06018, anti-PR06496, anti-PR07154, anti-PR07170. anti-PR07422, anti-PR07431 or anti-PR07476 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.
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 Deposit Deposit Date No.:

DNA108912-2680 PTA-124 May 25, 1999 DNA102880-2689 PTA-383 July 20, 1999 DNA96881-2699 PTA-553 August 17.

DNA98565-2701PTA-481 August 3, DNA119302-2737 PTA-520 August 10, DNA 108760-27J0PTA-5:18 .4ugust 17.

DNA 108723-27~J3PTA-SS? August 17. 1999 DNA I 19536-2752PTA-551 August 17. 1999 DNA I 19542-'_'75:JPTA-619 August 31. 1999 S DNA11S2S3-2757PTA-612 Au~ust31.1999 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 the maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc., and the ATCC, 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 conswed 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 considued to be sufficient to enable one slut led 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 illusuations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to chose skilled in the art from the foregoing description and fall within the scope of the appended claims.

11 436CA Sequence Sequence Listing <110> Genentech, Inc.
Botstein,David A.
Goddard,AUdrey Gurney, Austin L.
Smith, Victoria watanabe,Colin K.
Wood,William I.
<120> Compositions and Methods for the Treatment of Tumor <130> P3031R1PCT
<140> PCT/u500/13358 <141> 2000105P15 <150> US 60/138,385 <151> 1999106P09 <150> US 60/144,790 <151> 1999107P20 <150> US 60/146,843 <151> 1999108P03 <150> US 60/148,188 <151> 1999108P10 <150> US 60/149,320 <151> 1999108P17 <150> US 60/149,327 <151> 1999108P17 <150> US 60/149,396 <151> 1999108P17 <150> US 60/150,114 <151> 1999108P20 <150> US 60/151,700 <151> 1999108P31 <150> US 60/151,734 <151> 1999108P31 <160> 66 <210> 1 <211> 520 <212> DNA
<213> Homo Sapien <400> 1 cgggtcatgc gccgccgcct gtggctgggc ctggcctggc tgctgctggc 50 gcgggcgccg gacgccgcgg gaaccccgag cgcgtcgcgg ggaccgcgca 100 gctacccgca cctggagggc gacgtgcgct ggcggcgcct cttctcctcc 150 actcacttct tcctgcgcgt ggatcccggc ggccgcgtgc agggcacccg 200 11 436CA Sequence ctggcgccac ggccaggaca gcatcctgga gatccgctct gtacacgtgg 250 gcgtcgtggt catcaaagca gtgtcctcag gcttctacgt ggccatgaac 300 cgccggggcc gcctctacgg gtcgcgactc tacaccgtgg actgcaggtt 350 ccgggagcgc atcgaagaga acggccacaa cacctacgcc tcacagcgct 400 ggcgccgccg cggccagccc atgttcctgg cgctggacag gagggggggg 450 ccccggccag gcggccggac gcggcggtac cacctgtccg cccacttcct 500 gcccgtcctg gtctcctgag 520 <210> 2 <211> 170 <212> PRT
<213> Homo Sapien <400> 2 Met Arg Arg Arg Leu Trp Leu Gly Leu Ala Trp Leu Leu Leu Ala Arg Ala Pro Asp Ala Ala Gly Thr Pro Ser Ala Ser Arg Gly Pro Arg Ser Tyr Pro His Leu Glu Gly Asp Val Arg Trp Arg Arg Leu Phe Ser Ser Thr His Phe Phe Leu Arg val Asp Pro Gly Gly Arg Val Gln Gly Thr Arg Trp Arg His Gly Gln Asp Ser Ile Leu Glu Ile Arg Ser val His val Gly val val val Ile Lys Ala Val Ser Ser Gly Phe Tyr Val Ala Met Asn Arg Arg Gly Arg Leu Tyr Gly Ser Arg Leu Tyr Thr Val Asp Cys Arg Phe Arg Glu Arg Ile Glu Glu Asn Gly His Asn Thr Tyr Ala Ser Gln Arg Trp Arg Arg Arg Gly Gln Pro Met Phe Leu Ala Leu Asp Arg Arg Gly Gly Pro Arg Pro Gly Gly Arg Thr Arg Arg Tyr His Leu Ser Ala His Phe Leu Pro Val Leu Val Ser <210> 3 <211> 841 <212> DNA
<Z13> Homo Sapien <400> 3 11 436CA Sequence ggatgggcga gcagtctgaa tgccagaatg gataaccgtt ttgctacagc 50 atttgtaatt gcttgtgtgc ttagcctcat ttccaccatc tacatggcag 100 cctccattgg cacagacttc tggtatgaat atcgaagtcc agttcaagaa 150 aattccagtg atttgaataa aagcatctgg gatgaattca ttagtgatga 200 ggcagatgaa aagacttata atgatgcact ttttcgatac aatggcacag 250 tgggattgtg gagacggtgt atcaccatac ccaaaaacat gcattggtat 300 agcccaccag aaaggacaga gtcatttgat gtggtcacaa aatgtgtgag 350 tttcacacta actgagcagt tcatggagaa atttgttgat cccggaaacc 400 acaatagcgg gattgatctc cttaggacct atctttggcg ttgccagttc 450 cttttacctt ttgtgagttt aggtttgatg tgctttgggg ctttgatcgg 500 actttgtgct tgcatttgcc gaagcttata tcccaccatt gccacgggca 550 ttctccatct ccttgcagat accatgctgt gaagtccagg ccacatggag 600 gtgtcctgtg tagatgctcc agctgaaatc ccaagctaag ctcccaactg 650 acagccaaca tcatttccag ccatgtgtgg gagccatcct ggatgtccag 700 ccttaacaag ccttcagagg acttcagcca cagctattat cttactacat 750 ccttgtgaga ctctaataaa gaaccaacta gctgagccca atcaacctat 800 ggaactgata gaaataaaat gaattgttgt tttgtgccgt t 841 <210> 4 <211> 184 <212> PRT
<213> Homo Sapien <400> 4 Met Asp ASn Arg Phe Ala Thr Ala Phe Val Ile Ala Cys Val Leu Ser Leu Ile Ser Thr Ile Tyr Met Ala Ala Ser Ile Gly Thr Asp Phe Trp Tyr Glu Tyr Arg Ser Pro val Gln Glu Asn Ser Ser asp Leu Asn Lys Ser Ile Trp Asp Glu Phe Ile Ser Asp Glu Ala Asp Glu Lys Thr Tyr Asn Asp Ala Leu Phe Arg Tyr Asn Gly Thr Val Gly Leu Trp Arg Arg Cys Ile Thr Ile Pro Lys Asn Met His Trp Tyr Ser Pro Pro Glu Arg Thr Glu Ser Phe Asp Val Val Thr Lys Cys Val Ser Phe Thr Leu Thr Glu Gln Phe Met Glu Lys Phe Val 11 436CA Sequence Asp Pro Gly Asn His Asn Ser Gly Ile Asp Leu Leu Arg Thr Tyr Leu Trp Arg Cys Gln Phe Leu Leu Pro Phe Val Ser Leu Gly Leu Met Cys Phe Gly Ala Leu Ile Gly Leu Cys Ala Cys Ile Cys Arg Ser Leu Tyr Pro Thr Ile Ala Thr Gly Ile Leu His Leu Leu Ala Asp Thr Met Leu <210> 5 <211> 1130 <212> DNA
<213> Homo Sapien <400> 5 gggcctggcg atccggatcc cgcaggcgcg ctggctgcgc tgcccggctg 50 tctgtcgtca tggtggggcc ctgggtgtat ctggtggcgg cagttttgct 100 catcggcctg atcctcttcc tgactcgcag ccggggtcgg gcggcagcag 150 ctgacggaga accactgcac aatgaggaag agagggcagg agcaggccag 200 gtaggccgct ctttgcccca ggagtctgaa gaacagagaa ctggaagcag 250 accccggcgt cggagggact tgggcagccg tctacaggcc cagcgtcgag 300 cccagcgagt ggcctgggaa gacggggatg agaatgtggg tcaaactgtt 350 attccagccc aggaggaaga aggcattgag aagccagcag aagttcaccc 400 aacagggaaa attggagcca agaaactacg gaagctagag gaaaaacagg 450 ctcgaaaggc tcagcgagag gcagaggagg ctgaacgtga agaacggaaa 500 cgcctagagt cccaacgtga ggccgaatgg aagaaggaag aggaacggct 550 tcgcctgaag gaagaacaga aggaggagga agagaggaag gctcaggagg 600 agcaggcccg gcgggatcac gaggagtacc tgaaactgaa ggaggccttc 650 gtggtagaag aagaaggtgt tagcgaaacc atgactgagg agcagtctca 700 cagcttcctg acagaattca tcaattacat caagaagtcc aaggttgtgc 750 ttttggaaga tctggctttc cagatgggcc taaggactca ggacgccata 800 aaccgcatcc aggacctgct gacggagggg actctaacag gtgtgattga 850 cgaccggggc aagtttatct acataacccc agaggaactg gctgccgtgg 900 ccaatttcat ccgacagcgg ggccgggtgt ccatcacaga gcttgcccag 950 gccagcaact ccctcatctc ctggggccag gacctccctg cccaggcttc 1000 11 436CA Sequence agcctgactc cagtccttcc ttgagtgtat cctgtggcct acatgtgtct 1050 tcatccttcc ctaatgccgt cttggggcag ggatggaata tgaccagaaa 1100 gttgtggatt aaaggcctgt gaatactgaa 1130 <210> 6 <211> 315 <212> PRT
<213> Homo Sapien <400> 6 Met Val Gly Pro Trp Val Tyr Leu Val Ala Ala Val Leu Leu Ile Gly Leu Ile Leu Phe Leu Thr Arg Ser Arg Gly Arg Ala Ala Ala Ala Asp Gly Glu Pro Leu His Asn Glu Glu Glu Arg Ala Gly Ala Gly Gln Val Gly Arg Ser Leu Pro Gln Glu Ser Glu Glu Gln Arg Thr Gly Ser Arg Pro Arg Arg Arg Arg Asp Leu Gly Ser Arg Leu Gln Ala Gln Arg Arg Ala Gln Arg Val Ala Trp Glu Asp Gly Asp Glu Asn Val Gly Gln Thr Val Ile Pro Ala Gln Glu Glu Glu Gly Ile Glu Lys Pro Ala Glu Val His Pro Thr Gly Lys Ile Gly Ala Lys Lys Leu Arg Lys Leu Glu Glu Lys Gln Ala Arg Lys Ala Gln Arg Glu Ala Glu Glu Ala Glu Arg Glu Glu Arg Lys Arg Leu Glu Ser Gln Arg Glu Ala Glu Trp Lys Lys Glu Glu Glu Arg Leu Arg Leu Lys Glu Glu Gln Lys Glu Glu Glu Glu Arg Lys Ala Gln Glu Glu Gln Ala Arg Arg Asp His Glu Glu Tyr Leu Lys Leu Lys Glu Ala Phe Val Val Glu Glu Glu Gly Val Ser Glu Thr Met Thr Glu Glu Gln Ser His Ser Phe Leu Th'r Glu Phe Ile Asn Tyr Ile Lys Lys Ser Lys Val Val Leu Leu Glu Asp Leu Ala Phe Gln Met Gly Leu Arg Thr Gln Asp Ala Ile Asn Arg Ile Gln Asp Leu Leu Thr 11 436cA Sequence Glu Gly Thr Leu Thr Gly val Ile Asp Asp Arg Gly Lys Phe Ile Tyr Ile Thr Pro Glu Glu Leu Ala Ala Val Ala Asn Phe Ile Arg Gln Arg Gly Arg Val Ser Ile Thr Glu Leu Ala Gln Ala Ser Asn Ser Leu Ile Ser Trp Gly Gln Asp Leu Pro Ala Gln Ala Ser Ala <210> 7 <211> 3476 <212> DNA
<213> Homo Sapien <400> 7 gctctatgcc gcctaccttg ctctcgccgc tgctgccgga gccgaagcag 50 agaaggcagc gggtcccgtg accgtcccga gagccccgcg ctcccgacca 100 gggggcgggg gcggccccgg ggagggcggg gcaggggcgg ggggaagaaa 150 gggggttttg tgctgcgccg ggagggccgg cgccctcttc cgaatgtcct 200 gcggccccag cctctcctca cgctcgcgca gtctccgccg cagtctcagc 250 tgcagctgca ggactgagcc gtgcacccgg aggagacccc cggaggaggc 300 gacaaacttc gcagtgccgc gacccaaccc cagccctggg tagcctgcag 350 catggcccag ctgttcctgc ccctgctggc agccctggtc ctggcccagg 400 ctcctgcagc tttagcagat gttctggaag gagacagctc agaggaccgc 450 gcttttcgcg tgcgcatcgc gggcgacgcg ccactgcagg gcgtgctcgg 500 cggcgccctc accatccctt gccacgtcca ctacctgcgg ccaccgccga 550 gccgccgggc tgtgctgggc tctccgcggg tcaagtggac tttcctgtcc 600 cggggccggg aggcagaggt gctggtggcg cggggagtgc gcgtcaaggt 650 gaacgaggcc taccggttcc gcgtggcact gcctgcgtac ccagcgtcgc 700 tcaccgacgt ctccctggcg ctgagcgagc tgcgccccaa cgactcaggt 750 atctatcgct gtgaggtcca gcacggcatc gatgacagca gcgacgctgt 800 ggaggtcaag gtcaaagggg tcgtctttct ctaccgagag ggctctgccc 850 gctatgcttt ctccttttct ggggcccagg aggcctgtgc ccgcattgga 900 gcccacatcg ccaccccgga gcagctctat gccgcctacc ttgggggcta 950 tgagcaatgt gatgctggct ggctgtcgga tcagaccgtg aggtatccca 1000 tccagacccc acgagaggcc tgttacggag acatggatgg cttccccggg 1050 gtccggaact atggtgtggt ggacccggat gacctctatg atgtgtactg 1100 11 436CA sequence ttatgctgaa gacctaaatg gagaactgtt cctgggtgac cctccagaga 1150 agctgacatt ggaggaagca cgggcgtact gccaggagcg gggtgcagag 1200 attgccacca cgggccaact gtatgcagcc tgggatggtg gcctggacca 1250 ctgcagccca gggtggctag ctgatggcag tgtgcgctac cccatcgtca 1300 cacccagcca gcgctgtggt gggggcttgc ctggtgtcaa gactctcttc 1350 ctcttcccca accagactgg cttccccaat aagcacagcc gcttcaacgt 1400 ctactgcttc cgagactcgg cccagccttc tgccatccct gaggcctcca 1450 acccagcctc caacccagcc tctgatggac tagaggctat cgtcacagtg 1500 acagagaccc tggaggaact gcagctgcct caggaagcca cagagagtga 1550 atcccgtggg gccatctact ccatccccat catggaggac ggaggaggtg 1600 gaagctccac tccagaagac ccagcagagg cccctaggac gctcctagaa 1650 tttgaaacac aatccatggt accgcccacg gggttctcag aagaggaagg 1700 taaggcattg gaggaagaag agaaatatga agatgaagaa gagaaagagg 1750 aggaagaaga agaggaggag gtggaggatg aggctctgtg ggcatggccc 1800 agcgagctca gcagcccggg ccctgaggcc tctctcccca ctgagccagc 1850 agcccaggag aagtcactct cccaggcgcc agcaagggca gtcctgcagc 1900 ctggtgcatc accacttcct gatggagagt cagaagcttc caggcctcca 1950 agggtccatg gaccacctac tgagactctg cccactccca gggagaggaa 2000 cctagcatcc ccatcacctt ccactctggt tgaggcaaga gaggtggggg 2050 aggcaactgg tggtcctgag ctatctgggg tccctcgagg agagagcgag 2100 gagacaggaa gctccgaggg tgccccttcc ctgcttccag ccacacgggc 2150 ccctgagggt accagggagc tggaggcccc ctctgaagat aattctggaa 2200 gaactgcccc agcagggacc tcagtgcagg cccagccagt gctgcccact 2250 gacagcgcca gccgaggtgg agtggccgtg gtccccgcat caggtgactg 2300 tgtccccagc ccctgccaca atggtgggac atgcttggag gaggaggaag 2350 gggtccgctg cctatgtctg cctggctatg ggggggacct gtgcgatgtt 2400 ggcctccgct tctgcaaccc cggctgggac gccttccagg gcgcctgcta 2450 caagcacttt tccacacgaa ggagctggga ggaggcagag acccagtgcc 2500 ggatgtacgg cgcgcatctg gccagcatca gcacacccga ggaacaggac 2550 ttcatcaaca accggtaccg ggagtaccag tggatcggac tcaacgacag 2600 gaccatcgaa ggcgacttct tgtggtcgga tggcgtcccc ctgctctatg 2650 agaactggaa ccctgggcag cctgacagct acttcctgtc tggagagaac 2700 11 436CA Sequence tgcgtggtca tggtgtggca tgatcaggga caatggagtg acgtgccctg 2750 caactaccac ctgtcctaca cctgcaagat ggggctggtg tcctgtgggc 2800 cgccaccgga gctgcccctg gctcaagtgt tcggccgccc acggctgcgc 2850 tatgaggtgg acactgtgct tcgctaccgg tgccgggaag gactggccca 2900 gcgcaatctg ccgctgatcc gatgccaaga gaacggtcgt tgggaggccc 2950 cccagatctc ctgtgtgccc agaagacctg cccgagctct gcacccagag 3000 gaggacccag aaggacgtca ggggaggcta ctgggacgct ggaaggcgct 3050 gttgatcccc ccttccagcc ccatgccagg tccctagggg gcaaggcctt 3100 gaacactgcc ggccacagca ctgccctgtc acccaaattt tccctcacac 3150 cttgcgctcc cgccaccaca ggaagtgaca acatgacgag gggtggtgct 3200 ggagtccagg tgacagttcc tgaaggggct tctgggaaat acctaggagg 3250 ctccagccca gcccaggccc tctcccccta ccctgggcac cagatcttcc 3300 atcagggccg gagtaaatcc ctaagtgcct caactgccct ctccctggca 3350 gccatcttgt cccctctatt cctctaggga gcactgtgcc cactctttct 3400 gggttttcca agggaatggg cttgcaggat ggagtgtctg taaaatcaac 3450 aggaaataaa actgtgtatg agccca 3476 <210> 8 <211> 911 <212> PRT
<213> Homo Sapien <400> 8 Met Ala Gln Leu Phe Leu Pro Leu Leu Ala Ala Leu Val Leu Ala Gln Ala Pro Ala Ala Leu Ala Asp Val Leu Glu Gly Asp Ser Ser Glu Asp Arg Ala Phe Arg Val Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val Leu Gly Giy Ala Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro Pro Pro Ser Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr Phe Leu Ser Arg Gly Arg Glu Ala Glu val Leu Val Ala Arg Gly Val Arg Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro Ala Tyr Pro Ala Ser Leu Thr Asp Val 11 436CA Sequence Ser Leu Ala Leu Ser Glu Leu Arg Pro Asn Asp Ser Gly Ile Tyr Arg Cys Glu Val Gln His Gly Ile Asp Asp Ser Ser asp Ala Val Glu Val Lys Val Lys Gly Val Val Phe Leu Tyr Arg Glu Gly Ser Ala Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln Glu Ala Cys Ala Arg Ile Gly Ala His Ile Ala Thr Pro Glu Gln Leu Tyr Ala Ala Tyr Leu Gly Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu Ser Asp Gln Thr Val Arg Tyr Pro Ile Gln Thr Pro Arg Glu Ala Cys Tyr Gly Asp Met Asp Gly Phe Pro Gly Val Arg Asn Tyr Gly Val Val Asp Pro Asp Asp Leu Tyr Asp Val Tyr Cys Tyr Ala Glu Asp Leu Asn Gly Glu Leu Phe Leu Gly Asp Pro Pro Glu Lys Leu Thr Leu Glu Glu Ala Arg Ala Tyr Cys Gln Glu Arg Gly Ala Glu Ile Ala Thr Thr Gly Gln Leu Tyr Ala Ala Trp Asp Gly Gly Leu Asp His Cys Ser Pro Gly Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln Arg Cys Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro Asn Gln Thr Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val Tyr Cys Phe Arg Asp Ser Ala Gln Pro Ser Ala Ile Pro Glu Ala Ser Asn Pro Ala Ser Asn Pro Ala Ser Asp Gly Leu Glu Ala Ile Val Thr Val Thr Glu Thr Leu Glu Glu Leu Gln Leu Pro Gln Glu Ala Thr Glu Ser Glu Ser Arg Gly Ala Ile Tyr Ser Ile Pro Ile Met Glu Asp Gly Gly Gly Gly Ser Ser Thr Pro Glu Asp Pro Ala Glu Ala Pro Arg Thr Leu Leu Glu Phe Glu 11 436CA Sequence Thr Gln Ser Met Val Pro Pro Thr Gly Phe Ser Glu Glu Glu Gly Lys Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu Glu Glu Lys Glu Glu Glu Glu Glu Glu Glu Glu Val Glu Asp Glu Ala Leu Trp Ala Trp Pro Ser Glu Leu Ser Ser Pro Gly Pro Glu Ala Ser Leu Pro Thr Glu Pro Ala Ala Gln Glu Lys Ser Leu Ser Gln Ala Pro Ala Arg Ala Val Leu Gln Pro Gly Ala Ser Pro Leu Pro Asp Gly Glu Ser Glu Ala Ser Arg Pro Pro Arg Val His Gly Pro Pro Thr Glu Thr Leu Pro Thr Pro Arg Glu Arg Asn Leu Ala Ser Pro Ser Pro Ser Thr Leu Val Glu Ala Arg Glu Val Gly Glu Ala Thr Gly Gly Pro Glu Leu Ser Gly Val Pro Arg Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu Gly Ala Pro Ser Leu Leu Pro Ala Thr Arg Ala Pro Glu Gly Thr Arg Glu Leu Glu Ala Pro Ser Glu Asp Asn Ser Gly Arg Thr Ala Pro Ala Gly Thr Ser Val Gln Ala Gln Pro Val Leu Pro Thr Asp Ser Ala Ser Arg Gly Gly Val Ala Val Val Pro Ala Ser Gly Asp Cys Val Pro Ser Pro Cys His Asn Gly Gly Thr Cys Leu Glu Glu Glu Glu Gly Val Arg Cys Leu Cys Leu Pro Gly Tyr Gly Gly Asp Leu Cys Asp Val Gly Leu Arg Phe Cys Asn Pro Gly Trp Asp Ala Phe Gln Gly Ala Cys Tyr Lys His Phe Ser Thr Arg Arg Ser Trp Glu Glu Ala Glu Thr Gln Cys Arg Met Tyr Gly Ala His Leu Ala Ser Ile Ser Thr Pro Glu Glu Gln Asp Phe Ile Asn Asn Arg Tyr Arg Glu Tyr Gln Trp Ile Gly Leu Asn Asp Arg 11 436CA Sequence Thr Ile Glu Gly Asp Phe Leu Trp Ser Asp Gly Val Pro Leu Leu Tyr Glu Asn Trp Asn Pro Gly Gln Pro Asp Ser Tyr Phe Leu Ser Gly Glu Asn Cys Val Val Met Val Trp His Asp Gln Gly Gln Trp Ser Asp Val Pro Cys Asn Tyr His Leu Ser Tyr Thr Cys Lys Met Gly Leu Val Ser Cys Gly Pro Pro Pro Glu Leu Pro Leu Ala Gln Val Phe Gly Arg Pro Arg Leu Arg Tyr Glu Val Asp Thr Val Leu Arg Tyr Arg Cys Arg Glu Gly Leu Ala Gln Arg Asn Leu Pro Leu Ile Arg Cys Gln Glu Asn Gly Arg Trp Glu Ala Pro Gln Ile Ser Cys Val Pro Arg Arg Pro Ala Arg Ala Leu His Pro Glu Glu Asp Pro Glu Gly Arg Gln Gly Arg Leu Leu Gly Arg Trp Lys Ala Leu Leu Ile Pro Pro Ser Ser Pro Met Pro Gly Pro <210> 9 <211> 2663 <212> DNA
<213> Homo Sapien <400> 9 cccactcggc ggtttggcgg gagggagggg ctttgcgcag gccccgctcc 50 cgccccgcct ccatgcggcc cgccccgatt gcgctgtggc tgcgcctggt 100 cttggccctg gcccttgtcc gcccccgggc tgtggggtgg gccccggtcc 150 gagcccccat ctatgtcagc agctgggccg tccaggtgtc ccagggtaac 200 cgggaggtcg agcgcctggc acgcaaattc ggcttcgtca acctggggcc 250 gatcttctct gacgggcagt actttcacct gcggcaccgg ggcgtggtcc 300 agcagtccct gaccccgcac tggggccacc gcctgcacct gaagaaaaac 350 cccaaggtgc agtggttcca gcagcagacg ctgcagcggc gggtgaaacg 400 ctctgtcgtg gtgcccacgg acccctggtt ctccaagcag tggtacatga 450 acagcgaggc ccaaccagac ctgagcatcc tgcaggcctg gagtcagggg 500 ctgtcaggcc agggcatcgt ggtctctgtg ctggacgatg gcatcgagaa 550 11 436CA Sequence ggaccacccg gacctctggg ccaactacga ccccctggcc agctatgact 600 tcaatgacta cgacccggac ccccagcccc gctacacccc cagcaaagag 650 aaccggcacg ggacccgctg tgctggggag gtggccgcga tggccaacaa 700 tggcttctgt ggtgtggggg tcgctttcaa cgcccgaatc ggaggcgtac 750 ggatgctgga cggtaccatc accgatgtca tcgaggccca gtcgctgagc 800 ctgcagccgc agcacatcca catttacagc gccagctggg gtcccgagga 850 cgacggccgc acggtggacg gccccggcat cctcacccgc gaggccttcc 900 ggcgtggtgt gaccaagggc cgcggcgggc tgggcacgct cttcatctgg 950 gcctcgggca acggcggcct gcactacgac aactgcaact gcgacggcta 1000 caccaacagc atccacacgc tttccgtggg cagcaccacc cagcagggcc 1050 gcgtgccctg gtacagcgaa gcctgcgcct ccaccctcac caccacctac 1100 agcagcggcg tggccaccga cccccagatc gtcaccacgg acctgcatca 1150 cgggtgcaca gaccagcaca cgggcacctc ggcctcagcc ccactggcgg 1200 ccggcatgat cgccctagcg ctggaggcca acccgttcct gacgtggaga 1250 gacatgcagc acctggtggt ccgcgcgtcc aagccggcgc acctgcaggc 1300 cgaggactgg aggaccaacg gcgtggggcg ccaagtgagc catcactacg 1350 gatacgggct gctggacgcc gggctgctgg tggacaccgc ccgcacctgg 1400 ctgcccaccc agccgcagag gaagtgcgcc gtccgggtcc agagccgccc 1450 cacccccatc ctgccgctga tctacatcag ggaaaacgta tcggcctgcg 1500 ccggcctcca caactccatc cgctcgctgg agcacgtgca ggcgcagctg 1550 acgctgtcct acagccggcg cggagacctg gagatctcgc tcaccagccc 1600 catgggcacg cgctccacac tcgtggccat acgacccttg gacgtcagca 1650 ctgaaggcta caacaactgg gtcttcatgt ccacccactt ctgggatgag 1700 aacccacagg gcgtgtggac cctgggccta gagaacaagg gctactattt 1750 caacacgggg acgttgtacc gctacacgct gctgctctat gggacggccg 1800 aggacatgac agcgcggcct acaggccccc aggtgaccag cagcgcgtgt 1850 gtgcagcggg acacagaggg gctgtgccag gcgtgtgacg gccccgccta 1900 catcctggga cagctctgcc tggcctactg ccccccgcgg ttcttcaacc 1950 acacaaggct ggtgaccgct gggcctgggc acacggcggc gcccgcgctg 2000 agggtctgct ccagctgcca tgcctcctgc tacacctgcc gcggcggctc 2050 cccgagggac tgcacctcct gtcccccatc ctccacgctg gaccagcagc 2100 11 436CA Sequence agggctcctg catgggaccc accacccccg acagccgccc ccggcttaga 2150 gctgccgcct gtccccacca ccgctgccca gcctcggcca tggtgctgag 2200 cctcctggcc gtgaccctcg gaggccccgt cctctgcggc atgtccatgg 2250 acctcccact atacgcctgg ctctcccgtg ccagggccac ccccaccaaa 2300 ccccaggtct ggctgccagc tggaacctga agttgtcagc tcagaaagcg 2350 accttgcccc cgcctgggtc cctgacaggc actgctgcca tgctgcctcc 2400 ccaggctggc cccagaggag cgagcaccag cacccgacgc ctggcctgcc 2450 agggatgggc cccgtggaac cccgaagcct ggcgggagag agagagagag 2500 aagtctcctc tgcattttgg gtttgggcag gagtgggctg gggggagagg 2550 ctggagcacc ccaaaagcca ggggaaagtg gagggagaga aacgtgacac 2600 tgtccgtctc gggcaccgcg tccaacctca gagtttgcaa ataaaggttg 2650 cttagaaggt gaa 2663 <210> 10 <211> 755 <212> PRT
<213> Homo Sapien <400> 10 Met Arg Pro Ala Pro Ile Ala Leu Trp Leu Arg Leu Val Leu Ala Leu Ala Leu Val Arg Pro Arg Ala Val Gly Trp Ala Pro Val Arg Ala Pro Ile Tyr Val Ser Ser Trp Ala Val Gln Val Ser Gln Gly Asn Arg Glu Val Glu Arg Leu Ala Arg Lys Phe Gly Phe Val Asn Leu Gly Pro Ile Phe Ser Asp Gly Gln Tyr Phe His Leu Arg His Arg Gly Val Val Gln Gln Ser Leu Thr Pro His Trp Gly His Arg Leu His Leu Lys Lys Asn Pro Lys val Gln Trp Phe Gln Gln Gln Thr Leu Gln Arg Arg val Lys Arg Ser val Val Val Pro Thr Asp Pro Trp Phe Ser Lys Gln Trp Tyr Met Asn Ser Glu Ala Gln Pro Asp Leu Ser Ile Leu Gln Ala Trp Ser Gln Gly Leu Ser Gly Gln Gly Ile Val Val Ser Val Leu Asp Asp Gly Ile Glu Lys Asp His 11 Sequence ProAspLeu TrpAlaAsn TyrAspPro LeuAla TyrAsp Phe Ser AsnAspTyr AspProAsp ProGlnPro ArgTyr ProSer Lys Thr GluAsnArg HisGlyThr ArgCysAla GlyGlu AlaAla Met Val AlaAsnAsn GlyPheCys GlyValGly ValAla AsnAla Arg Phe Ile Gly Gly Val Arg Met Leu Asp Gly Thr Ile Thr Asp Val Ile Glu Ala Gln Ser Leu Ser Leu Gln Pro Gln His Ile His Ile Tyr Ser Ala Ser Trp Gly Pro Glu Asp Asp Gly Arg Thr Val Asp Gly Pro Gly Ile Leu Thr Arg Glu Ala Phe Arg Arg Gly Val Thr Lys Gly Arg Gly Gly Leu Gly Thr Leu Phe Ile Trp Ala Ser Gly Asn Gly Gly Leu His Tyr Asp Asn Cys Asn Cys Asp Gly Tyr Thr Asn Ser Ile His Thr Leu Ser Val Gly Ser Thr Thr Gln Gln Gly Arg Val Pro Trp Tyr Ser Glu Ala Cys Ala Ser Thr Leu Thr Thr Thr Tyr Ser Ser Gly Val Ala Thr Asp Pro Gln Ile Val Thr Thr Asp Leu His His Gly Cys Thr Asp Gln His Thr Gly Thr Ser Ala Ser Ala Pro Leu Ala Ala Gly Met Ile Ala Leu Ala Leu Glu Ala Asn Pro Phe Leu Thr Trp Arg Asp Met Gln His Leu Val Val Arg Ala Ser Lys Pro Ala His Leu Gln Ala Glu Asp Trp Arg Thr Asn Gly Val Gly Arg Gln Val Ser His His Tyr Gly Tyr Gly Leu Leu Asp Ala Gly Leu Leu Val Asp Thr Ala Arg Thr Trp Leu Pro Thr Gln Pro Gln Arg Lys Cys Ala Val Arg Val Gln Ser Arg Pro Thr Pro Ile Leu Pro Leu Ile Tyr Ile Arg Glu Asn Val Ser Ala Cys Ala 11 436CA Sequence Gly Leu His Asn Ser Ile Arg Ser Leu Glu His Val Gln Ala Gln Leu Thr Leu Ser Tyr Ser Arg Arg Gly Asp Leu Glu Ile Ser Leu Thr Ser Pro Met Gly Thr Arg Ser Thr Leu Val Ala Ile Arg Pro Leu Asp Val Ser Thr Glu Gly Tyr Asn Asn Trp Val Phe Met Ser Thr His Phe Trp Asp Glu Asn Pro Gln Gly Val Trp Thr Leu Gly Leu Glu Asn Lys Gly Tyr Tyr Phe Asn Thr Gly Thr Leu Tyr Arg Tyr Thr Leu Leu Leu Tyr Gly Thr Ala Glu Asp Met Thr Ala Arg Pro Thr Gly Pro Gln Val Thr Ser Ser Ala Cys Val Gln Arg Asp Thr Glu Gly Leu Cys Gln Ala Cys Asp Gly Pro Ala Tyr Ile Leu Gly Gln Leu Cys Leu Ala Tyr Cys Pro Pro Arg Phe Phe Asn His Thr Arg Leu Val Thr Ala Gly Pro Gly His Thr Ala Ala Pro Ala Leu Arg Val Cys Ser Ser Cys His Ala Ser Cys Tyr Thr Cys Arg Gly Gly Ser Pro Arg Asp Cys Thr Ser Cys Pro Pro Ser Ser Thr Leu Asp Gln Gln Gln Gly Ser Cys Met Gly Pro Thr Thr Pro Asp Ser Arg Pro Arg Leu Arg Ala Ala Ala Cys Pro His His Arg Cys Pro Ala Ser Ala Met Val Leu Ser Leu Leu Ala Val Thr Leu Gly Gly Pro Val Leu Cys Gly Met Ser Met Asp Leu Pro Leu Tyr Ala Trp Leu Ser Arg Ala Arg Ala Thr Pro Thr Lys Pro Gln Val Trp Leu Pro Ala Gly Thr <210> 11 <211> 1161 <212> DNA
<213> Homo Sapien 11 436CA Sequence <220>
<221> unsure <222> 1149 <223> unknown base <400> 11 gcccgggcgg ctgcccttgg gtgctccctt ccctgcccga cacccagacc 50 gaccttgacc gcccacctgg caggagcagg acaggacggc cggacgcggc 100 catggccgag ctcccggggc cctttctctg cggggccctg ctaggcttcc 150 tgtgcctgag tgggctggcc gtggaggtga aggtacccac agagccgctg 200 agcacgcccc tggggaagac agccgagctg acctgcacct acagcacgtc 250 ggtgggagac agcttcgccc tggagtggag ctttgtgcag cctgggaaac 300 ccatctctga gtcccatcca atcctgtact tcaccaatgg ccatctgtat 350 ccaactggtt ctaagtcaaa gcgggtcagc ctgcttcaga acccccccac 400 agtgggggtg gccacactga aactgactga cgtccacccc tcagatactg 450 gaacctacct ctgccaagtc aacaacccac cagatttcta caccaatggg 500 ttggggctaa tcaaccttac tgtgctggtt ccccccagta atcccttatg 550 cagtcagagt ggacaaacct ctgtgggagg ctctactgca ctgagatgca 600 gctcttccga gggggctcct aagccagtgt acaactgggt gcgtcttgga 650 acttttccta caccttctcc tggcagcatg gttcaagatg aggtgtctgg 700 ccagctcatt ctcaccaacc tctccctgac ctcctcgggc acctaccgct 750 gtgtggccac caaccagatg ggcagtgcat cctgtgagct gaccctctct 800 gtgaccgaac cctcccaagg ccgagtggcc ggagctctga ttggggtgct 850 cctgggcgtg ctgttgctgt cagttgctgc gttctgcctg gtcaggttcc 900 agaaagagag ggggaagaag cccaaggaga catatggggg tagtgacctt 950 cgggaggatg ccatcgctcc tgggatctct gagcacactt gtatgagggc 1000 tgattctagc aaggggttcc tggaaagacc ctcgtctgcc agcaccgtga 1050 cgaccaccaa gtccaagctc cctatggtcg tgtgacttct cccgatccct 1100 gagggcggtg agggggaata tcaataatta aagtctgtgg gtacccttna 1150 aaaaaaaaaa a 1161 <210> 12 <211> 327 <212> PRT
<213> Homo Sapien <400> 12 Met Ala Glu Leu Pro Gly Pro Phe Leu Cys Gly Ala Leu Leu Gly 11 436CA Sequence Phe Leu Cys Leu Ser Gly Leu Ala Val Glu Val Lys Val Pro Thr Glu Pro Leu Ser Thr Pro Leu Gly Lys Thr Ala Glu Leu Thr Cys Thr Tyr Ser Thr Ser Val Gly Asp Ser Phe Ala Leu Glu Trp Ser Phe Val Gln Pro Gly Lys Pro Ile Ser Glu Ser His Pro Ile Leu Tyr Phe Thr Asn Gly His Leu Tyr Pro Thr Gly Ser Lys Ser Lys Arg Val Ser Leu Leu Gln Asn Pro Pro Thr Val Gly Val Ala Thr Leu Lys Leu Thr Asp Val His Pro Ser Asp Thr Gly Thr Tyr Leu Cys Gln Val Asn Asn Pro Pro Asp Phe Tyr Thr Asn Gly Leu Gly Leu Ile Asn Leu Thr Val Leu Val Pro Pro Ser Asn Pro Leu Cys Ser Gln Ser Gly Gln Thr Ser Val Gly Gly Ser Thr Ala Leu Arg Cys Ser Ser Ser Glu Gly Ala Pro Lys Pro Val Tyr Asn Trp Val Arg Leu Gly Thr Phe Pro Thr Pro Ser Pro Gly Ser Met Val Gln Asp Glu Val Ser Gly Gln Leu Ile Leu Thr Asn Leu Ser Leu Thr Ser Ser Gly Thr Tyr Arg Cys Val Ala Thr Asn Gln Met Gly Ser Ala Ser Cys Glu Leu Thr Leu Ser Val Thr Glu Pro Ser Gln Gly Arg Val Ala Gly Ala Leu Ile Gly Val Leu Leu Gly Val Leu Leu Leu Ser Val Ala Ala Phe Cys Leu Val Arg Phe Gln Lys Glu Arg Gly Lys Lys Pro Lys Glu Thr Tyr Gly Gly Ser Asp Leu Arg Glu Asp Ala Ile Ala Pro Gly Ile Ser Glu His Thr Cys Met Arg Ala Asp Ser Ser Lys Gly Phe Leu Glu Arg Pro Ser Ser Ala Ser Thr Val Thr Thr Thr Lys Ser Lys Leu Pro Met Val Val 11 436CA sequence <210> 13 <211> 2015 <212> DNA
<213> Homo Sapien <400> 13 ggaaaaggta cccgcgagag acagccagca gttctgtgga gcagcggtgg 50 ccggctagga tgggctgtct ctggggtctg gctctgcccc ttttcttctt 100 ctgctgggag gttggggtct ctgggagctc tgcaggcccc agcacccgca 150 gagcagacac tgcgatgaca acggacgaca cagaagtgcc cgctatgact 200 ctagcaccgg gccacgccgc tctggaaact caaacgctga gcgctgagac 250 ctcttctagg gcctcaaccc cagccggccc cattccagaa gcagagacca 300 ggggagccaa gagaatttcc cctgcaagag agaccaggag tttcacaaaa 350 acatctccca acttcatggt gctgatcgcc acctccgtgg agacatcagc 400 cgccagtggc agccccgagg gagctggaat gaccacagtt cagaccatca 450 caggcagtga tcccgaggaa gccatctttg acaccctttg caccgatgac 500 agctctgaag aggcaaagac actcacaatg gacatattga cattggctca 550 cacctccaca gaagctaagg gcctgtcctc agagagcagt gcctcttccg 600 acggccccca tccagtcatc accccgtcac gggcctcaga gagcagcgcc 650 tcttccgacg gcccccatcc agtcatcacc ccgtcacggg cctcagagag 700 cagcgcctct tccgacggcc cccatccagt catcaccccg tcatggtccc 750 cgggatctga tgtcactctc ctcgctgaag ccctggtgac tgtcacaaac 800 atcgaggtta ttaattgcag catcacagaa atagaaacaa caacttccag 850 catccctggg gcctcagaca tagatctcat ccccacggaa ggggtgaagg 900 cctcgtccac ctccgatcca ccagctctgc ctgactccac tgaagcaaaa 950 ccacacatca ctgaggtcac agcctctgcc gagaccctgt ccacagccgg 1000 caccacagag tcagctgcac ctcatgccac ggttgggacc ccactcccca 1050 ctaacagcgc cacagaaaga gaagtgacag cacccggggc cacgaccctc 1100 agtggagctc tggtcacagt tagcaggaat cccctggaag aaacctcagc 1150 cctctctgtt gagacaccaa gttacgtcaa agtctcagga gcagctccgg 1200 tctccataga ggctgggtca gcagtgggca aaacaacttc ctttgctggg 1250 agctctgctt cctcctacag cccctcggaa gccgccctca agaacttcac 1300 cccttcagag acaccgacca tggacatcgc aaccaagggg cccttcccca 1350 ccagtaggga ccctcttcct tctgtccctc cgactacaac caacagcagc 1400 11 436CA Sequence cgagggacga acagcacctt agccaagatc acaacctcag cgaagaccac 1450 gatgaagccc caacagccac gcccacgact gcccggacga ggccgaccac 1500 agacgtgagt gcaggtgaaa atggaggttt cctcctcctg cggctgagtg 1550 tggcttcccc ggaagacctc actgacccca gagtggcaga aaggctgatg 1600 cagcagctcc accgggaact ccacgcccac gcgcctcact tccaggtctc 1650 cttactgcgt gtcaggagag gctaacggac atcagctgca gccaggcatg 1700 tcccgtatgc caaaagaggg tgctgcccct agcctgggcc cccaccgaca 1750 gactgcagct gcgttactgt gctgagaggt acccagaagg ttcccatgaa 1800 gggcagcatg tccaagcccc taaccccaga tgtggcaaca ggaccctcgc 1850 tcacatccac cggagtgtat gtatggggag gggcttcacc tgttcccaga 1900 ggtgtccttg gactcacctt ggcacatgtt ctgtgtttca gtaaagagag 1950 acctgatcac ccatctgtgt gcttccatcc tgcattaaaa ttcactcagt 2000 gtggcccaaa aaaaa 2015 <210> 14 <211> 482 <212> PRT
<213> Homo Sapien <400> 14 Met Gly Cys Leu Trp Gly Leu Ala Leu Pro Leu Phe Phe Phe Cys Trp Glu Val Gly val Ser Gly Ser Ser Ala Gly Pro Ser Thr Arg Arg Ala Asp Thr Ala Met Thr Thr Asp Asp Thr Glu val Pro Ala Met Thr Leu Ala Pro Gly His Ala Ala Leu Glu Thr Gln Thr Leu Ser Ala Glu Thr Ser Ser Arg Ala Ser Thr Pro Ala Gly Pro Ile Pro Glu Ala Glu Thr Arg Gly Ala Lys Arg Ile Ser Pro Ala Arg Glu Thr Arg Ser Phe Thr Lys Thr Ser Pro Asn Phe Met val Leu Ile Ala Thr Ser Val Glu Thr Ser Ala Ala Ser Gly Ser Pro Glu Gly Ala Gly Met Thr Thr Val Gln Thr Ile Thr Gly Ser Asp Pro Glu Glu Ala Ile Phe Asp Thr Leu Cys Thr Asp Asp Ser Ser Glu Glu Ala Lys Thr Leu Thr Met Asp Ile Leu Thr Leu Ala His Thr 11 436CA Sequence Ser Thr Glu Ala Lys Gly Leu Ser Ser Glu Ser Ser Ala S.er Ser Asp Gly Pro His Pro Val Ile Thr Pro Ser Arg Ala Ser Glu Ser Ser Ala Ser Ser Asp Gly Pro His Pro Val Ile Thr Pro Ser Arg Ala Ser Glu Ser Ser Ala Ser Ser Asp Gly Pro His Pro Val Ile Thr Pro Ser Trp Ser Pro Gly Ser Asp Val Thr Leu Leu Ala Glu Ala Leu Val Thr Val Thr Asn Ile Glu Val Ile Asn Cys Ser Ile Thr Glu Ile Glu Thr Thr Thr Ser Ser Ile Pro Gly Ala Ser Asp Ile Asp Leu Ile Pro Thr Glu Gly Val Lys Ala Ser Ser Thr Ser Asp Pro Pro Ala Leu Pro Asp Ser Thr Glu Ala Lys Pro His Ile Thr Glu val Thr Ala Ser Ala Glu Thr Leu Ser Thr Ala Gly Thr Thr Glu Ser Ala Ala Pro His Ala Thr Val Gly Thr Pro Leu Pro Thr Asn Ser Ala Thr Glu Arg Glu Val Thr Ala Pro Gly Ala Thr Thr Leu Ser Gly Ala Leu Val Thr Val Ser Arg Asn Pro Leu Glu Glu Thr Ser Ala Leu Ser val Glu Thr Pro Ser Tyr val Lys val Ser Gly Ala Ala Pro Val Ser Ile Glu Ala Gly Ser Ala Val Gly Lys Thr Thr Ser Phe Ala Gly Ser Ser Ala Ser Ser Tyr Ser Pro Ser Glu Ala Ala Leu Lys Asn Phe Thr Pro Ser Glu Thr Pro Thr Met Asp Ile Ala Thr Lys Gly Pro Phe Pro Thr Ser Arg Asp Pro Leu Pro Ser Val Pro Pro Thr Thr Thr Asn Ser Ser Arg Gly Thr Asn Ser Thr Leu Ala Lys Ile Thr Thr Ser Ala Lys Thr Thr Met 11 436CA Sequence Lys Pro Gln Gln Pro Arg Pro Arg Leu Pro Gly Arg Gly Arg Pro Gln Thr <210> 15 <211> 332 <212> DNA
<213> Homo Sapien <400> 15 atgaggaagc tccagggcag gatggtttac ctgcctggac agcaagatga 50 tggctacact agcccccatt ctctgggcgc ctggatttgc ccaccagatc 100 tcctcacctc ttgcccttca cctcctgctg tacctacaag gtctccccga 150 ttctcatctg cccataatca tggacacagc cccaggatgt gcaggactct 200 cagggaccat ctggagttcc agctggaatc tgggcctggt ggagtgggag 250 tggggcaggg gcctgcattg ggctgactta gagagcacag ttattccatc 300 catatggaaa taaacatttt ggattcctga tc 332 <210> 16 <211> 88 <212> PRT
<213> Homo Sapien <400> 16 Met Met Ala Thr Leu Ala Pro Ile Leu Trp Ala Pro Gly Phe Ala His Gln Ile Ser Ser Pro Leu Ala Leu His Leu Leu Leu Tyr Leu Gln Gly Leu Pro ASp Ser His Leu Pro Ile Ile Met Asp Thr Ala Pro Gly Cys Ala Gly Leu Ser Gly Thr Ile Trp Ser Ser Ser Trp Asn Leu Gly Leu Val Glu Trp Glu Trp Gly Arg Gly Leu His Trp Ala Asp Leu Glu Ser Thr Val Ile Pro Ser Ile Trp Lys <210> 17 <211> 1302 <zlz> DNA
<213> Homo Sapien <220>
<221> unsure <222> 121801253 <223> unknown base <400> 17 tttgcagtgg ggtcctcctc tggcctcctg cccctcctgc tgctgctgct 50 11 436cA Sequence gcttccattg ctggcagccc agggtggggg tggcctgcag gcagcgctgc 100 tggcccttga ggtggggctg gtgggtctgg gggcctccta cctgctcctt 150 tgtacagccc tgcacctgcc ctccagtctt ttcctactcc tggcccaggg 200 taccgcactg ggggccgtcc tgggcctgag ctggcgccga ggcctcatgg 250 gtgttcccct gggccttgga gctgcctggc tcttagcttg gccaggccta 300 gctctacctc tggtggctat ggcagcgggg ggcagatggg tgcggcagca 350 gggcccccgg gtgcgccggg gcatatctcg actctggttg cgggttctgc 400 tgcgcctgtc acccatggcc ttccgggccc tgcagggctg tggggctgtg 450 ggggaccggg gtctgtttgc actgtacccc aaaaccaaca aggatggctt 500 ccgcagccgc ctgcccgtcc ctgggccccg gcggcgtaat ccccgcacca 550 cccaacaccc attagctctg ttggcaaggg tctgggtcct gtgcaagggc 600 tggaactggc gtctggcacg ggccagccag ggtttagcat cccacttgcc 650 cccgtgggcc atccacacac tggccagctg gggcctgctt cggggtgaac 700 ggcccacccg aatcccccgg ctactaccac gcagccagcg ccagctaggg 750 ccccctgcct cccgccagcc actgccaggg actctagccg ggcggaggtc 800 acgcacccgc cagtcccggg ccctgccccc ctggaggtag ctgactccag 850 cccttccagc ccaaatctag agcattgagc actttatctc ccacgactca 900 gtgaagtttc tccagtccct agtcctctct tttcacccac cttcctcagt 950 ttgctcactt accccaggcc cagcccttcg gacctctaga caggcagcct 1000 cctcagctgt ggagtccagc agtcactctg tgttctcctg gcgctcctcc 1050 cctaagttat tgctgttcgc ccgctgtgtg tgctcatcct caccctcatt 1100 gactcaggcc tggggccagg ggtggtggag ggtgggaaga gtcatgtttt 1150 ttttctcctc tttgattttg tttttctgtc tcccttccaa cctgtcccct 1200 tccccccacc aaaaaaannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1250 nnnaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1300 as 1302 <210> 18 <211> 197 <212> PRT
<213> Homo Sapien <400> 18 Met Gly Val Pro Leu Gly Leu Gly Ala Ala Trp Leu Leu Ala Trp Pro Gly Leu Ala Leu Pro Leu Val Ala Met Ala Ala Gly Gly Arg 11 436CA Sequence Trp Val Arg Gln Gln Gly Pro Arg Val Arg Arg Gly Ile Ser Arg Leu Trp Leu Arg Val Leu Leu Arg Leu Ser Pro Met Ala Phe Arg Ala Leu Gln Gly Cys Gly Ala Val Gly Asp Arg Gly Leu Phe Ala Leu Tyr Pro Lys Thr Asn Lys Asp Gly Phe Arg Ser Arg Leu Pro Val Pro Gly Pro Arg Arg Arg Asn Pro Arg Thr Thr Gln His Pro Leu Ala Leu Leu Ala Arg Val Trp Val Leu Cys Lys Gly Trp Asn Trp Arg Leu Ala Arg Ala Ser Gln Gly Leu Ala Ser His Leu Pro Pro Trp Ala Ile His Thr Leu Ala Ser Trp Gly Leu Leu Arg Gly Glu Arg Pro Thr Arg Ile Pro Arg Leu Leu Pro Arg Ser Gln Arg Gln Leu Gly Pro Pro Ala Ser Arg Gln Pro Leu Pro Gly Thr Leu Ala Gly Arg Arg Ser Arg Thr Arg Gln Ser Arg Ala Leu Pro Pro Trp Arg <210> 19 <211> 2329 <212> DNA
<213> Homo Sapien <400> 19 atccctcgac ctcgacccac gcgtccgctg gaaggtggcg tgccctcctc 50 tggctggtac catgcagctc ccactggccc tgtgtctcgt ctgcctgctg 100 gtacacacag ccttccgtgt agtggagggc caggggtggc aggcgttcaa 150 gaatgatgcc acggaaatca tccccgagct cggagagtac cccgagcctc 200 caccggagct ggagaacaac aagaccatga accgggcgga gaacggaggg 250 cggcctcccc accacccctt tgagaccaaa gacgtgtccg agtacagctg 300 ccgcgagctg cacttcaccc gctacgtgac cgatgggccg tgccgcagcg 350 ccaagccggt caccgagctg gtgtgctccg gccagtgcgg cccggcgcgc 400 ctgctgccca acgccatcgg ccgcggcaag tggtggcgac ctagtgggcc 450 11 436CA Sequence cgacttccgc tgcatccccg accgctaccg cgcgcagcgc gtgcagctgc 500 tgtgtcccgg tggtgaggcg ccgcgcgcgc gcaaggtgcg cctggtggcc 550 tcgtgcaagt gcaagcgcct cacccgcttc cacaaccagt cggagctcaa 600 ggacttcggg accgaggccg ctcggccgca gaagggccgg aagccgcggc 650 cccgcgcccg gagcgccaaa gccaaccagg ccgagctgga gaacgcctac 700 tagagcccgc ccgcgcccct ccccaccggc gggcgccccg gccctgaacc 750 cgcgccccac atttctgtcc tctgcgcgtg gtttgattgt ttatatttca 800 ttgtaaatgc ctgcaaccca gggcaggggg ctgagacctt ccaggccctg 850 aggaatcccg ggcgccggca aggcccccct cagcccgcca gctgaggggt 900 cccacggggc aggggaggga attgagagtc acagacactg agccacgcag 950 ccccgcctct ggggccgcct acctttgctg gtcccacttc agaggaggca 1000 gaaatggaag cattttcacc gccctggggt tttaagggag cggtgtggga 1050 gtgggaaagt ccagggactg gttaagaaag ttggataaga ttcccccttg 1100 cacctcgctg cccatcagaa agcctgaggc gtgcccagag cacaagactg 1150 ggggcaactg tagatgtggt ttctagtcct ggctctgcca ctaacttcct 1200 gtgtaacctt gaactacaca attctccttc gggacctcaa tttccacttt 1250 gtaaaatgag ggtggaggtg ggaataggat ctcgaggaga ctattggcat 1300 atgattccaa ggactccagt gccttttgaa tgggcagagg tgagagagag 1350 agagagaaag agagagaatg aatgcagttg cattgattca gtgccaaggt 1400 cacttccaga attcagagtt gtgatgctct cttctgacag ccaaagatga 1450 aaaacaaaca gaaaaaaaaa agtaaagagt ctatttatgg ctgacatatt 1500 tacggctgac aaactcctgg aagaagctat gctgcttccc agcctggctt 1550 ccccggatgt ttggctacct ccacccctcc atctcaaaga aataacatca 1600 tccattgggg tagaaaagga gagggtccga gggtggtggg agggatagaa 1650 atcacatccg ccccaacttc ccaaagagca gcatccctcc cccgacccat 1700 agccatgttt taaagtcacc ttccgaagag aagtgaaagg ttcaaggaca 1750 ctggccttgc aggcccgagg gagcagccat cacaaactca cagaccagca 1800 catccctttt gagacaccgc cttctgccca ccactcacgg acacatttct 1850 gcctagaaaa cagcttctta ctgctcttac atgtgatggc atatcttaca 1900 ctaaaagaat attattgggg gaaaaactac aagtgctgta catatgctga 1950 gaaactgcag agcataatag ctgccaccca aaaatctttt tgaaaatcat 2000 11 436CA Sequence ttccagacaa cctcttactt tctgtgtagt ttttaattgt taaaaaaaaa 2050 aagttttaaa cagaagcaca tgacatatga aagcctgcag gactggtcgt 2100 ttttttggca attcttccac gtgggacttg tccacaagaa tgaaagtagt 2150 ggtttttaaa gagttaagtt acatatttat tttctcactt aagttattta 2200 tgcaaaagtt tttcttgtag agaatgacaa tgttaatatt gctttatgaa 2250 ttaacagtct gttcttccag agtccagaga cattgttaat aaagacaatg 2300 aatcatgaaa aaaaaaaaaa aaaaaaaaa 2329 <210> 20 <211> 213 <212> PRT
<213> Homo Sapien <400> 20 Met Gln Leu Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Thr Ala Phe Arg Val Val Glu Gly Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr 11 436CA Sequence <210>21 <211>21 <212>DNA

<213>ArtificialSequence <220>

<223>syntheticoligonucleotideprobe <400>21 cagcgaaccg ccgggt c 21 ggtg <210>22 <211>22 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>22 gagcgacgag cagcga ac 22 cgcg <210>23 <211>43 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>23 atactgcgat aaacca ccatgcgccg 43 cgct ccgcctgtgg ctg <210>24 <211>21 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>24 gccggcctct gcctca g 21 cagg <210>25 <211>23 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>25 cccacgtgta 23 cagagcggat ctc <210>26 <211>23 <212>DNA

<213>ArtificialSequence 11 436cA Sequence <220>

<223> Synthetic oligonucleotideprobe <400> 26 gagaccagga cgggcaggaa gtg 23 <210> 27 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic oligonucleotideprobe <400> 27 caggcacctt ggggagccgc c 21 <210> 28 <211> 23 <212> DNA

<213> Artificiail Sequence <220>

<221> Artificial Sequence <222> full <223> Synthetic oligonucleotideprobe <400> 28 cccacgtgta cagagcggat ctc 23 <210> 29 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic oligonucleotideprobe <400> 29 gagaccagga cgggcaggaa gtg 23 <210> 30 <211> 44 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic oligonucleotideprobe <400> 30 ctctacgggt actgcaggtt ccgggagcgc atcgaagaga acgg44 <210> 31 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic oligonucleotideprobe <400> 31 atgcagctcc cactggccct g 21 11 436CA Sequence <210>32 <211>27 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>32 ctagtaggcg ccagct cggcctg 27 ttct <210>33 <211>40 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>33 cttccgctgc ccgacc gctaccgcgc 40 atcc gcagcgcgtg <210>34 <211>18 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>34 gcgtcgtggt aaag 18 catc <210>35 <211>19 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>35 tgcagtccac tagag 19 ggtg <210>36 <211>22 <212>DNA

<213>ArtificialSequence <220>

<223>Syntheticoligonucleotideprobe <400>36 cttctacgtg 22 gccatgaacc gc <210>37 <211>19 <212>DNA

<213>ArtificialSequence <220>

11 436CA Sequence <223> Synthetic oligonucleotide probe <400> 37 cctggagatc cgctctgta 19 <210> 38 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 38 ctttgatgac cacgacgccc a 21 <210> 39 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 39 acgtagaagc ctgaggacac t 21 <210> 40 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 40 gatgctccag ctgaaatcc 19 <210> 41 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 41 cacatggctg gaaatgatg 19 <210> 42 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 42 aagctaagct cccaactgac agcca 25 <210> 43 <211> 21 11 436cA Sequence <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 43 tggcctacat gtgtcttcat c 21 <210> 44 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 44 cacaactttc tggtcatatt ccat 24 <210> 45 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 45 cctgccccaa gacggcatta g 21 <210> 46 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> synthetic oligonucleotide probe <400> 46 cctgggcacc agatcttc 18 <210> 47 <211> 18 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic oligonucleotide probe <400> 47 agggcagttg aggcactt 18 <210> 48 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 48 11 436CA Sequence catcagggcc ggagtaaatc cct 23 <210> 49 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 49 tccatggacc tcccactata c 21 <210> 50 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 50 gctgacaact tcaggttcca 20 <210> 51 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 51 acccccacca aaccccaggt 20 <210> 52 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> synthetic oligonucleotide probe <400> 52 gatctctgag cacacttgta tgag 24 <210> 53 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 53 ggcagacgag ggtctttc 18 <210> 54 <211> 25 <212> DNA
<213> Artificial Sequence 11 436CA sequence <220>

<223> Synthetic oligonucleotideprobe <400> 54 caggaacccc ttgctagaat cagcc 25 <210> 55 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> Synthetic oligonucleotideprobe <400> 55 cccagaaggt tcccatga 18 <210> 56 <211> 18 <212> DNA

<213> Artificial sequence <220>

<223> synthetic oligonucleotideprobe <400> 56 gggtcctgtt gccacatc 18 <210> 57 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> Synthetic oligonucleotideprobe <400> 57 cagcatgtcc aagcccctaa ccc 23 <220> 58 <211> Z9 <212> DNA

<213> Artificial sequence <220>

<223> Synthetic oligonucleotideprobe <400> 58 tctccccgat tctcatctg 19 <210> 59 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> Synthetic oligonucleotideprobe <400> 59 ccctgagagt cctgcacat lg <210> 60 11 436CA Sequence <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> Synthetic oligonucleotideprobe <400> 60 cccataatca tggacacagc ccc 23 <210> 61 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic oligonucleotideprobe <400> 61 agtgaagttt ctccagtccc tags 24 <210> 62 <211> 19 <212> DNA

<213> Artificial sequence <220>

<223> synthetic oligonucleotideprobe <400> 62 cctggggtaa gtgagcaaa lg <210> 63 <211> 26 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic oligonucleotideprobe <400> 63 cctctctttt cacccacctt cctcag 26 <Z10> 64 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> Synthetic oligonucleotideprobe <400> 64 gggactggtt aagaaagttg gat 23 <210> 65 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic oligonucleotideprobe <400> 65 11 436CA Sequence cgcctcaggc tttctgat 18 <210> 66 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic oligonucleotide probe <400> 66 agattccccc ttgcacctcg c 21

Claims

1. An isolated antibody that binds to a PRO6018 (SEQ ID NO:8) polypeptide.

2. The antibody of Claim 1 which induces the death of a cell that expresses said polypeptide.

3. The antibody of Claim 2, wherein said cell is a cancer cell that overexpresses said polypeptide as compared to a normal cell of the same tissue type.

4. The antibody of Claim 1 which is a monoclonal antibody.

5. The antibody of Claim 4 which comprises a non-human complementarity determining region (CDR) or a human framework region (FR).

6. The antibody of Claim 1 which is labeled.

7. The antibody of Claim 1 which is an antibody fragment or a single-chain antibody.

8. A composition of matter which comprises an antibody of Claim 1 in admixture with a pharmaceutically acceptable carrier.

9. The composition of matter of Claim 8 which comprises a therapeutically effective amount of said antibody.

10. The composition of matter of Claim 8 which further comprises a cytotoxic or a chemotherapeutic agent.

11. An isolated nucleic acid molecule that encodes the antibody of Claim 1.

12. A vector comprising the nucleic acid molecule of Claim 11.

13. A host cell comprising the vector of Claim 12.

14. A method for producing an antibody that binds to a PRO6018 (SEQ ID NO:8) polypeptide, said method comprising culturing the host cell of Claim 13 under conditions sufficient to allow expression of said antibody and recovering said antibody from the cell culture.

15. An antagonist of a PRO6018 (SEQ ID NO:8) polypeptide.

16. The antagonist of Claim 15, wherein said antagonist inhibits tumor cell growth.

17. An isolated nucleic acid molecule that hybridizes to a nucleic acid sequence that encodes a PRO6018 (SEQ ID NO:8) polypeptide, or the complement thereof.

18. The isolated nucleic acid molecule of Claim 17, wherein said hybridization is under stringent hybridization and wash conditions.

19. A method for determining the presence of a PRO6018 (SEQ ID NO:8) polypeptide in a sample suspected of containing said polypeptide, said method comprising exposing the sample to an anti-PRO6018 antibody and determining binding of said antibody to said polypeptide in said sample.

20. The method of Claim 19, wherein said sample comprises a cell suspected of containing a PRO6018 (SEQ ID NO:8) polypeptide.

21. The method of Claim 20, wherein said cell is a cancer cell.

22. A method of diagnosing tumor in a mammal, said method comprising detecting the level of expression of a gene encoding a PRO6018 (SEQ ID NO:8) polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test tissue cells were obtained.

23. A method of diagnosing tumor in a mammal, said method comprising (a) contacting an antiPRO6018 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between said antibody and a PRO6018 (SEQ ID NO:8) polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.

24. The method of Claim 23, wherein said antibody is detectably labeled.

25. The method of Claim 23, wherein said test sample of tissue cells is obtained from an individual suspected of having neoplastic cell growth or proliferation.

26. A cancer diagnostic kit comprising an anti-PRO6018 antibody and a carrier in suitable packaging.

27. The kit of Claim 26 which further comprises instructions for using said antibody to detect the presence of a PRO6018 (SEQ ID NO:8) polypeptide in a sample suspected of containing the same.

28. The use of an effective amount of an agent that inhibits a biological activity of a PRO6018 (SEQ ID NO:8) polypeptide for inhibiting the growth of tumor cells that express said polypeptide.

29. The use of Claim 28, wherein said tumor cells overexpress said polypeptide as compared to normal cells of the same tissue type.

30. The use of Claim 28, wherein said agent is an anti-PRO6018 antibody.

31. The use of Claim 30, wherein said anti-PRO6018 antibody induces cell death.

32. The use of Claim 28, wherein said tumor cells are further exposed to radiation treatment, a cytotoxic agent or a chemotherapeutic agent.

33. The use of an effective amount of an agent that inhibits the expression of a PRO6018 (SEQ ID NO:8) polypeptide for inhibiting the growth of tumor cells that express said polypeptide.

34. The use of Claim 33, wherein said tumor cells overexpress said polypeptide as compared to normal cells of the same tissue type.

35. The use of Claim 33, wherein said agent is an antisense oligonucleotide that hybridizes to a nucleic acid which encodes the PRO6018 (SEQ ID NO:8) polypeptide or the complement thereof.

36. The use of Claim 35, wherein said tumor cells are further exposed to radiation treatment, a cytotoxic agent or a chemotherapeutic agent.

37. An article of manufacture, comprising: a container; a label on the container; and a composition comprising an active agent contained within the container, wherein the composition is effective for inhibiting the growth of tumor cells and wherein the label on the container indicates that the composition is effective for treating conditions characterized by overexpression of a PRO6018 (SEQ ID NO:8) polypeptide in said tumor cells as compared to in normal cells of the same tissue type.

38. The article of manufacture of Claim 37, wherein said active agent inhibits a biological activity of and/or the expression of said PRO6018 (SEQ ID NO:8) polypeptide.

39. The article of manufacture of Claim 38, wherein said active agent is an anti-PRO6018 antibody.

40. The article of manufacture of Claim 38, wherein said active agent is an antisense oligonucleotide.

41. A method of identifying a compound that inhibits a biological or immunological activity of a PRO6018 (SEQ ID NO:8) polypeptide, said method comprising contacting a candidate compound with said polypeptide under conditions and for a time sufficient to allow the two components to interact and determining whether a biological or immunological activity of said polypeptide is inhibited.

42. The method of Claim 41, wherein said candidate compound is an antiPRO6018 antibody.

43. The method of Claim 41, wherein said candidate compound or said PRO6018 (SEQ ID NO:8) polypeptide is immobilized on a solid support.

44. The method of Claim 43, wherein the non-immobilized component is detestably labeled.

45. A method of identifying a compound that inhibits an activity of a PRO6018 (SEQ
ID NO:8) polypeptide, said method comprising the steps of (a) contacting cells and a candidate compound to be screened in the presence of said polypeptide under conditions suitable for the induction of a cellular response normally induced by said polypeptide and (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist, wherein the lack of induction of said cellular response is indicative of said compound being an effective antagonist.

46. A method for identifying a compound that inhibits the expression of a (SEQ ID NO:8) polypeptide in cells that express said polypeptide, wherein said method comprises contacting said cells with a candidate compound and determining whether expression of said polypeptide is inhibited.

47. The method of Claim 46 wherein said candidate compound is an antisense oligonucleotide.

48. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an amino acid sequence shown in Figure 8 (SEQ
ID
NO:8).

49. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence shown in Figure 7 (SEQ ID NO:7).

50. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a full-length coding sequence of the nucleotide sequence shown in Figure 7 (SEQ ID
NO:7).

51. A vector comprising the nucleic acid of any one of Claims 48 to 50.

52. The vector of Claim 51 operably linked to control sequences recognized by a host cell transformed with the vector.

53. A host cell comprising the vector of Claim 51.

54. The host cell of Claim 53, wherein said cell is a CHO cell.

55. The host cell of Claim 53, wherein said cell is an E coli.

56. The host cell of Claim 53, wherein said cell is a yeast cell.

57. The host cell of Claim 53, wherein said cell is a Baculovirus-infected insect cell.

58. A process for producing a PRO6018 (SEQ ID NO:8) polypeptide comprising culturing the host cell of Claim 53 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.

59. An isolated polypeptide having at least 80% amino acid sequence identity to the amino acid sequence shown in Figure 8 (SEQ ID NO:8).

60. A chimeric molecule comprising a polypeptide according to Claim 58 or 59 fused to a heterologous amino acid sequence.

61. The chimeric molecule of Claim 60, wherein said heterologous amino acid sequence is an epitope tag sequence.

62. The chimeric molecule of Claim 60, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.

63. An antibody which specifically binds to a polypeptide according to Claim 58 or 59.

64. The antibody of Claim 63, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.

65. Isolated nucleic acid having at least 80% nucleic acid sequence identity to:
(a) a nucleotide sequence encoding the polypeptide shown in Figure 8 (SEQ ID
NO:8), lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 8 (SEQ ID NO:8), with its associated signal peptide; or (c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 8 (SEQ ID NO:8), lacking its associated signal peptide.

66. An isolated polypeptide having at least 80% amino acid sequence identify to:
(a) the polypeptide shown in Figure 8 (SEQ ID NO:8), lacking its associated signal peptide;
(b) an extracellular domain of the polypeptide shown in Figure 8 (SEQ ID
NO:8), with its associated signal peptide; or (c) an extracellular domain of the polypeptide shown in Figure 8 (SEQ ID
NO:8).
lacking its associated signal peptide.