CA2453344A1

CA2453344A1 - Novel nucleic acids and secreted polypeptides

Info

Publication number: CA2453344A1
Application number: CA002453344A
Authority: CA
Inventors: Y. Tom Tang; Yonghong Yang; Gezhi Weng; Jie Zhang; Feiyan Ren; Aidong Xue; Jian-Rui Wang; Tom Wehrman; Malabika J. Ghosh; Dunrui Wang; Qing A. Zhao; Zhiwei Wang
Original assignee: Individual
Current assignee: Nuvelo Inc
Priority date: 2001-07-21
Filing date: 2002-07-19
Publication date: 2003-01-21

Abstract

The present invention provides novel nucleic acids, novel. polypeptide sequences encoded by these nucleic acids and uses thereof.

Description

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
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THAN ONE VOLUME.

NOTE: For additional volumes please contact the Canadian Patent Office.

$OSA/PC,f CA 02453344 2004-O1-21 NOVEL NUCLEIC ACIDS AND SECRETED
POLYPEPTIDES
1. CROSS REFERENCE T~ RELATED APPLICATIONS
S This application is a continuation-in-part application of U.S. Application Serial No.
09/488,725 filed January 21, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 784; U.S. Application Serial No. 09/491,404 filed January 25, 2000 entitled "Novel Contigs Obtained from Various Libraries"., Attorney Docket No.
785; U.S. Application Serial No. 09/496,914 filed February 03, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 787; U.S. Application Serial No.
09/S 15,126 filed February 28, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 798; U.S. Application Serial No. 09/S 19;705 filed March 07, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No.
789; U.S. Application Serial No. 09/540,217 filed March 31, 2000 entitled "Novel Contigs 1 S Obtained from Various Libraries", Attorney Docket No. 790; U.S.
Application Serial No.
09/5S2,929 filed April 18, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 791; U.S. Application Serial No. 09/577,408 f led May 18, entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No.
792; all of which are incorporated herein by reference in their entirety.

2. BACKGROUND OF THE INVENTION
2.1 TECHNICAL FIELD
The present invention provides novel polynucleotides and proteins encoded by such 2S polynucleotides, along with uses for these polynucleotides and proteins, far example in therapeutic, diagnostic and research methods.
2.2 BACKGROUND
Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly'° in the sense that they rely on 805AIrCT CA 02453344 2004-O1-21 information directly related to the discovered protein (i.e., partial DNA/axnino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.
Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.

3. SUMMARY OF THE INVENTION
The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA
molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBIT), and in some cases, sequences obtained from one or more public databases. The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins. These nucleic acid sequences are designated as SEQ ID NO: 1-244, or 489-706 and are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A
is adenine; C is 805A/PCT ~ 02453344 2004-O1-21 cytosine; G is guanine; T is thymine; and N is any of the four bases or unknown. In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.
The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-244, or 489-706 under stringent S hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ
ID NO: I-244, or 489-706. A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-244, or 489-706 or a degenerate variant or fragment thereof. The identifying sequence can be 100 base pairs in length.
The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-244., or 489-706.
The sequence information can be a segment of any one of SEQ ID NO: 1-244, or 489-706 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-244, or 489-706.
A collection as used in this application can be a collection of only one polynueleotide.
The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynueleotide that contains the segment. The array can be designed to detect foil-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.
This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences;
and host cells or organisms transformed with these expression vectors. Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known ~o those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA
or RNA, their chemical analogs and the like.
In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: I-244, or 706 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art. In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-244, or 489-706 or novel segments or parts of the 80St-1IPCT CA 02453344 2004-O1-21 nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-S9 (1992), as expressed sequence tags for physical mapping of the human genome.
The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ
ID NO: I-244, or 489-706; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-244, or 489-706; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: I-244, or 489-706. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide I 0 that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: I-244, or 489-706; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in SEQ ID NO: I-244, or 489-706; (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g. orthologs) of ably of the proteins recited I 5 above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.
'The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing;
or the 20 corresponding full length or mature protein. Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: I-244, or 489-706; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically active variants of any of the polypeptide sequences in the Sequence 2S Listing, and "substantial equivalents" thereof (e.g., with at least about 6S%, 70%, 7S%, 80%, 8S%, 90%, 9S%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.
30 The invention also provides compositions comprising a polypeptide of the invention.
Polypeptide compositions of the invention may further comprise an. acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

805A/PCT ~ 02453344 2004-O1-21 The invention also provides host cells transformed or transfected with a polynucleotide of the invention.
The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium S under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells. Preferred embodiments include those in which the protein produced by such processes is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., i~ situ hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.
Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.
In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.

80SA/PCT ~ 02453344 2004-O1-21 The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.
S The invention provides a method .for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected. The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.
The invention also provides kits comprising polynucleotide probes andlor 1 S monoclonal antibodies, and optionally quantitative standards, for caxrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.
The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.
Such methods can include, but are net limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention. The 2S invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the_invention is identified.
The methods of the invention also provide methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies. In addition, the invention encompasses methods for SOS~p~~ CA 02453344 2004-O1-21 treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can affect such modulation either on the level of target gene/protein expression or target protein activity.
The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in Table 2); for which they have a signature region (as set forth in Table 3); or for which they have homology to a gene family (as set forth in Table 4). If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.
4. DETAILED DESCT~IPTION OF THE INVENTION
4.1 DEFINITIONS
It must be noted that as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.
The term "active" refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.
According to the invention, the terms "biologically active" or "biological activity" refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.
Likewise "immunologically active" or "immunological activity" refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The term "activated cells" as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.
The terms "complementary" or "complementarity" refer to the natural binding of polynucleotides by base pairing. For example, the sequence 5'-AGT-3' binds to the complementary sequence 3'-TCA-5'. Complementarity between two single-stranded molecules may be "partial" such that only certain portions) of the nucleic acids bind or it may be "complete" such that total complementarity exists between the single stranded 805A/PCT ~ 02453344 2004-O1-21 molecules. The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.
The term "embryonic stem cells (ES)" refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.
The term "germ line stem cells (GSCs)" refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes. The term "primordial germ cells (PGCs)" refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells. PGCs are the source from which GSCs and ES cells are derived. The PGCs, the GSCs and the ES cells are capable of self renewal. Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.
The term "expression modulating fragment," EMF, means a series of nucleotides which modulates the expression of an operably linked ~RF or another EMF.
As used herein, a sequence is said to "modulate the expression of an operably linked sequence" when the expression of the sequence is altered by the presence of the EMF.
EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). ~ne class of EMFs are nucleic acid fragments which induce the expression of an operably linked GRF in response to a specific regulatory factor or physiological event.
The terms "nucleotide sequence" or "nucleic acid" or "polynucleotide" or "oligonucleotide" are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material. In the sequences herein A is adenine, C is cytosine, T is thymine, G
is guanine and N is A, C, G, or T (U) or unknown. It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U
(uracil).
Generally, nucleic acid segments provided by this invention rnay be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is ~~~~P~-T CA 02453344 2004-O1-21 capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
The terms "oligonucleotide fragment" or a "polynucleotide fragment", °'portion,'° or "segment°' or "probe" or "primer" are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides. The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides. Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.
Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules. A fragment or segment may uniquely identify each polynucleotide sequence of the present invention. Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-244, or 489-706.
Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Welsh et al. (Welsh, P.S. et al., 1992, PCR Methods Appl 1:241-250).
They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F.M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, both of which are incorporated herein by reference in their entirety.
The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-244, or 489-706.
The sequence information can be a segment of any one of SEQ ID NO: 1 ~-244, or 489-706 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO:
1-244, or 489-706, or those segments identified in Tables 3, 5., 6, and 8. One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is I in 300. In the human genome, there are three billion base pairs in one set of chromosomes. Because 42° possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes. Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5. When these segments are used in arrays for expression 5 studies, fifteen-mer segments can be used. The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.
Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer. The probability that the twenty-five rrrner would appear in a human 10 genome with a single mismatch is calculated by multiplying the probability for a full match (1-425) times the increased probability for mismatch at each nucleotide position (3 x 25). The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five. The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.
The term "open reading frame," ORF, means a series of nucleotide triplets coding for amino acids without any termination colons and is a sequence translatable into protein.
The terms "operably linked" or "operably associated" refer to functionally related nucleic acid sequences. For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.
While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.
The term "pluripotent" refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism. A
pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.
The terms "polypeptide" or "peptide" or "amino acid sequence" refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules. A polypeptide "fragment," "portion," or "segment" is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids. The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.

Preferably the peptide is from about 5 to about 200 amino acids. To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.
The term "naturally occurring polypeptide" refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translationa.l modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidatioxa and acylation.
The term "translated protein coding portion" means a sequence which encodes for the full-length protein which may include any leader sequence or any processing sequence.
The term "mature protein coding sequence" means a sequence which encodes a peptide or protein without a signal or leader sequence. The "mature protein portion" means that portion of the protein which does not include a signal or leader sequence. The peptide may have been produced by processing in the cell which removes any leader/signal sequence. The mature protein portion may or may not include the initial methionine residue.
The methionine residue may be removed from the protein during processing in the cell. The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.
The term "derivative" refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.
The term "variant"(or "analog") refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, a g., recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.
Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as Iigand-binding afFnities, interchain affinities, or degradation/turnover rate.
Preferably, amino acid '°substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, l, e., conservative amino acid replacements. "Conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Insertions" or "deletions" are preferably in the range of about 1 to 20 amino acids, more preferably 1 to I O amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
The terms "purified" or "substantially purified" as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).

The term "isolated" as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source. In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms '°isolated" and "purified"
do not encompass nucleic acids or polypeptides present in their natural source.
The term "recombinant," when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems. "Microbial" refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, "recombinant microbial" defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications;
polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.
The term "recombinant expression vehicle or vector" refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA
and translated into protein, and (3) appropriate transcription initiation and termination sequences.
Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
The term "recombinant expression system" means host cells which have stably integrated a recombinant transeriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.
Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed. The cells can be prokaryotic or eukaryotic.
The term "secreted" includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reti.culum. "Secreted"
proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P.A. and Young, P.R. (1992) Cytokine 4(2): 134 -143) and factors released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W.P. et. al.
(1998) Annu. Rev. Immunol. 16:27-55) Where desired, an expression vector may be designed to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell.
Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.
The term "stringent" is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in O.1X SSCI0.1% SDS at 68°C), and moderately stringent conditions (i.e., washing in 0.2X SSC/0.1% SDS at 42°C). Other exemplary hybridization conditions are described herein in the examples.
In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6X SSC/0.0~% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).
As used herein, "substantially equivalent" or "substantially similar" can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. Typically, such a substantially equivalent sequence varies from one of ~0~~~,I, CA 02453344 2004-O1-21 those listed herein by no more than about 3S% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less). Such a sequence is said to S have 6S% sequence identity to the listed sequence. In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embadiment, by no more than 2S%
(7S% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that S% {9S% sequence identity). Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 8S% sequence identity, more preferably at least 90%
sequence identity, more preferably at least 9S% sequence identity, more preferably at least 1 S 98% sequence identity, and most preferably at least 99% sequence identity.
Substantially equivalent nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.
Preferably, the nucleotide sequence has at least about 6S% identity, more preferably at least about 7S% identity, more preferably at least about 80% sequence identity, more preferably at least 8S% sequence identity, more preferably at least 90% sequence identity, more preferably at least about 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. For the purposes of 2S determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a new stop colon) should be disregarded. Sequence identity may be determined, e.g., using the Jotun Heirs method (Heirs, J. (1990) Methods Enzyrnol. 183:626-64S).
Identity between sequences can also be determined by other methods known in the art, e.g.
by varying hybridization conditions.
The term "totipotent" refers to the capability of a cell to differentiate into all of the cell types of an adult organism.
The term "transformation" means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration. The term "transfection" refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.
The term "infection" refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.
As used herein, an "uptake modulating fragment," UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.
UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below. The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.
Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.
4.2 NUCLEIC ACIDS OF THE INVENTION
Nucleotide sequences of the invention are set forth in the Sequence Listing.
The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-244, or 489-706; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 1-244, or 489-706; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polynucleotides of any one of SEQ ID NO: 1-244, or 489-706. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID
NO: 1-244, or 489-706; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing, or Table 8; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO:
1-244, or 489-706 (for example, as set forth in Tables 3, 5, 6, or 8). Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable 805ti1 PCT CA 02453344 2004-O1-21 immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.
The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., eDNA and genomic DNA, and RNA, e.g., mRNA. The polynucleotides may include entire coding region of the cDNA or may represent a portion of the coding region of the cDNA.
The present invention also provides genes corresponding to the cDNA sequences disclosed herein. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
Further 5' and 3' sequence can be obtained using methods known in the art. For example, full length cDNA or genornic DNA that corresponds to any of the polynucleotides of SEQ ID NO:
1-244, or 489-706 can be obtained by screening appropriate cDNA or genomic DNA
libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-244, or 489-706 or a portion thereof as a probe. Alternatively, the polynucleotides of SEQ ID NO:
1-244, or 489-706 may be used as the basis for suitable primers) that allow identiEcation and/or amplification of genes in appropriate genomic DNA or cDNA libraries.
The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene. The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.
The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that axe substantially equivalent to the polynucleotides recited above.
Polynucleotides according to tree invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99% sequence identity to a polynucleotide recited above.
Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide 805A/PCT ~ 02453344 2004-O1-21 sequences of SEQ ID NO: 1-244, or 489-706, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e. specifically hybridize to) any one of the polynucleotides of the invention are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes ox can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.
The sequences falling within the scope of the present invention are not limited to these I O specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-244, ox 489-706, a representative fragment thereof, or a nucleotide sequence at least 90%
identical, preferably 95% identical, to SEQ ID NO: 1-244, or 489-706 with a sequence from another isolate of the same species. Furthermore, to accommodate codon variability, the I 5 invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.
The nearest neighbor or homology results for the nucleic acids of the present invention, including SEQ ID NO: 1-244, or 489-706 can be obtained by searching a database using an 20 algorithm or a program. Preferably, a BLAST (Basic Local Alignment Search Tool) program is used to search for local sequence alignments (Altshul, S.F. J l~Iol. Evol. 36 290-300 (1993) and Altschul S.F. et al. J. Mol. Biol. 21:403-410 (1990)). Alternatively a FASTA
version 3 search against Genpept, using FASTX~' algorithm may be performed.
Species homologs (or orthologs) of the disclosed polynucleotides and proteins are 25 also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which 30 also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.
The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may be 8O5 A mCT CA 02453344 2004-O1-21 prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation. Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.
These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous. Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.
Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.
In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis. This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983). A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template DNA are used as starting material, primers) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. PCR
amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA

g~s~~Z CA 02453344 2004-O1-21 fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.
A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:31 S (1985); and other mutagenesis techniques S well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those 10 which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.
Polynucleotides encoding preferred polypeptide truncations of the invention could be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.
1 S The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides 20 of the desired sequence identities.
In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ 1D NO: 1-244, or 489-706, or functional equivalents thereof, may be used to generate recombinant DNA
molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate 2S host cells. Also included are the cDNA inserts of any of the clones identified herein.
A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
In general, the vector contains an origin of replication functional in at least one organism, convenient ~~~~~,I, CA 02453344 2004-O1-21 restriction endonuclease sites, and a selectable marker for the host cell.
Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-244, or 489-706 or a fragment thereof or any other polynucleotides of the invention. In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-244, or 489-706 or a fragment thereof is inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.
The following vectors are provided by way of example: Bacterial: pBs, phagescript, PsiXl74, pBluescript SK, pBs KS, pNHBa, pNHl6a, pNHlBa, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia); Eukaryotic: p~VLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
Many suitable expression control sequences axe known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R.
Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein "operably linked"
means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse ~O~~~T CA 02453344 2004-O1-21 metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. cola and S. cerevisiae TRP1 gene, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.
Such promoters can be derived from operons encoding glycolytic enzymes such as phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiatian and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E coli, Pacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

0o« mCT CA 02453344 2004-O1-21 2'~
Polynucleotides of the invention can also be used to induce immune responses.
For example, as described in Fan et al., Nat. Biotech 17, 870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably infra-muscular injection of the DNA.
The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.
4.3 ANTISENSE
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-244, or 489-706, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense'° nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 1-244, or 489-706 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-244, or are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence of the invention. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence of the invention. The term "noncoding region" refers to 5' and 3' sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-244, or 489-706, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more g05til r CT CA 02453344 2004-O1-21 2~
preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of an mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of an mRNA. An antisense oligonucleotide can be, for example, about S, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: S-fluorouracil, 5-bromouracil, 5-chlorouracil, .5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, S-(carboxyhydroxylmethyl) uracil, 5-I S carboxymethylaminomethyl-2-thiouridine, S-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, S-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, S-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, S'-methoxycarboxymethyluracil, S-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, S-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated i~ situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the 805A/PCT ~ 02453344 2004-O1-21 case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target 5 selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve 10 sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II
or pol III
promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific 15 double-stranded hybrids with complementary RNA in which, contrary to the usual a-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15:
6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (moue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (moue et al. ( 1987) FEBS Lett 215: 327-330).
4.4 RIBOZYMES AND PNA MOIETIES
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of an mRNA. A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-244, or 489-706). F'or example, a derivative of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
Alternatively, mRNA of the invention can be used to select a catalytic RNA having a specific ribonuclease 80SA/PCT ~ 02453344 2004-O1-21 activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Sciehce 261:1411-1418.
Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple S helical structures that prevent transcription of the gene in target cells.
See generally, Helene.
{1991) Ahticahcer Drug Des. 6: S69-84; Helene. et al. {1992) Ahn. N. Y. Acad.
Sci.
660:27-36; and Maher (1992) Bioassays 14: 807-1 S.
In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. Far example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: S-23). As used herein, the terms "peptide nucleic acids"
or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural 1 S nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. {1996) above; Perry-O'Keefe et al.
(1996) PATAS 93:
14670-675.
PNAs of the invention can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e. g. , PNA directed PCR clamping; as artificial restriction enzymes 2S when used in combination with other enzymes, e.g., S1 nucleases {Hyrup B.
(1996) above);
or as probes or primers for DNA sequence and hybridization (Hyrup et al.
(1996), above;
Perry-O'Keefe (1996), above).
In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA. Svch chimeras allow DNA
recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA

g~S~~~T CA 02453344 2004-O1-21 portion while the PNA portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thyrnidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
(1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al.
( 1996) above). Alternatively, chimerie molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. U.SA.
86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.
W089/10134).
In addition, oligonucleotides cm be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechhiques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
4.5 HOSTS
The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention. For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides axe in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.

805A1i'CT CA 02453344 2004-O1-21 Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences. See, for example, PCT International Publication No. W094/12650, PCT International Publication No. W092/20808, and PCT International Publication No. WO91/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA
IO (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA
may be inserted along with the heterologous promoter DNA. If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.
The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant canstruct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L, et al., Basic Methods in Molecular Biology (1986)). The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a hetervlogous protein under the control of the EMF.
Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eul~aryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and S~3 cells, as well as prokaryotic host such as E. coli and B. subtilis. The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A
Laboratory 805A/L'CT CA 02453344 2004-O1-21 Manual, Second Edition, Cold Spring Harbor, New York (1989), the disclosure of which is hereby incorporated by reference.
Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981). Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Co1o205 cells, 3T3 cells, CV-1 Bells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Cahdida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typlzimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

80SA/PCT ~ 02453344 2004-O1-21 In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene 5 targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, and regulatory protein binding sites or combinations of said sequences.
10 Alternatively, sequences which affect the structure or stability of the RNA
or protein produced may be replaced, removed, added, or otherwise modified by targeting.
These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA
15 molecules.
The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory 20 element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are 25 contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but conf gured such that the negatively selectable marker flanks the targeting 30 sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

805A/PCT ~ 02453344 2004-O1-21 The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S.
Patent No.
5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.;
International Application No. PCTIUS92/09627 (W093/09222) by Selden et al.; and International Application No. PCT/US90/06436 (W091/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.
4.6 PoLYPEPTIDES OF TILE IN~~ENTIDN
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ
ID NO: 245-488, or 707-924 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-244, or 489-706 or the corresponding full length or mature protein.
Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-244, or 489-706 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SIEQ ID NO: 245-488, or 707-924 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions. The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 245-488, or 707-924 or the corresponding full length or mature protein; and "substantial equivalents" thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at Least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96°/~, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 245-488, or 707-924.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they rnay be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.
McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as 805A/PCT ~ 02453344 2004-O1-21 immunoglobulins for many purposes, including increasing the valency of protein binding sites. Fragments are also identified in Tables 3, 5, 6, and 8.
The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.
The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences. The predicted signal sequence is set forth in Table 6.
The mature form of such protein may be obtained and confirmed by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell and sequencing of the cleaved product. One of skill in the art will recognize that the actual cleavage site may be different than that predicted in Table 6. The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form. Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided. In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.
1 S Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By "degenerate variant" is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.
A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. This technique is particularly useful in producing small peptides and fragments of larger polypeptides.
Fragments are useful, for example, in generating antibodies against the native polypeptide.
Thus, they may be employed as biologically active or immunological substitutes for natural, purified g05tilPCT CA 02453344 2004-O1-21 proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level. One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.
The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown. Far example, the methods of the invention include a process for producing a polypeptide in which a host cell I S containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.
The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified. Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.
In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Proteih Purification: Principles ahd Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubei et al., Current Protocols iu Molecular Biology. Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.
The purified polypeptides can be used in iu vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules ~~~A~~.I. CA 02453344 2004-O1-21 include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.
In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 245-488, or 707-924.
The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications, in the peptide or DNA
sequence, can be made by those skilled in the art using known techniques.
Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S.
Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein. Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity. This type of analysis determines the importance of the substituted amino acids) in biological activity. Regions of the protein that are important for protein function may be determined by the eMATRIX program.
Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other gOS~~.I. CA 02453344 2004-O1-21 immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are encompassed by the present invention.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and 5 employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBatTM kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a 10 polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i. e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange 15 chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA
SepharoseTM;
one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffmity chromatography.
20 Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag. Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and 25 Invitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("FLAG~") is commercially available from Kodak (New Haven, Conn.).
Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant 30 methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is 805ti/PCT CA 02453344 2004-O1-21 substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein."
The polypeptides of the invention include analogs (variants). This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted. Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such analogs may exhibit improved properties such as activity and/or stability.
Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells. Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids. Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.
4.6.i DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE
IDENTITY AND SIMILARITY
Preferred identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP
(Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, BLASTX, FASTA
(Altschul, S.F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S.F. et al., Nucleic Acids Res. vol. 25, pp. 3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp. Biol., Vol. 6, pp. 219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol. 4, pp. 202-209, herein incorporated by reference), Pfam software (Sonnhammer et al.,1\~ucleic Acids Res., Vol. 26(1), pp. 320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
polypeptide sequences were examined by a proprietary algorithm, SeqLoc that separates the proteins into three sets of locales: intracellular, membrane, or secreted.
This prediction is g~SA/PC~I~ CA 02453344 2004-O1-21 based upon three characteristics of each polypeptide, including percentage of cysteine residues, Kyte-Doolittle scores for the first 20 amino acids of each protein, and Kyte-Doolittle scores to calculate the longest hydrophobic stretch of the said protein. Values of predicted proteins are compared against the values from a set of 592 proteins of known cellular localization from the Swissprot database (http:/lwww.expas .~prot).
Predictions are based upon the maximum likelihood estimation.
The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.
NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., :1. Mol. Biol. 215:403-(1990).
4.7 CHIMERIC AND FUSION PROTEINS
The invention also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" comprises a polypeptide of the invention operatively linked to another polypeptide. Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention. In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention. In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention. Within the fusion protein, the term "operatively linked" is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other. The polypeptide can be fused to the N-terminus or C-terminus, or to the middle.
For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.
In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST
(i.e., glutathione S-transferase) sequences.
In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprise one or more domains fused to sequences derived from a member of the immunoglobulin protein family.
The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo. The immunoglobulin fusion proteins can be used to affect the bioavailability of a 805A/PCT ~ 02453344 2004-O1-21 cognate ligand. Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e.g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.
A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley ~ Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.
4.8 GENE THERAPY
Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein. The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention. Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, ih situ, or in viva by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use ofphysical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998).
For additional reviews of gene therapy technology see Friedmann, Science, 244:

go5~'- T CA 02453344 2004-O1-21 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).
Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes. Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.
Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art. Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.
The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.
Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.
Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.
See, for example, PCT International Publication No. WO 94/12650, PCT
International Publication No. WO 92/20808, and PCT International Publication No. WO
91109955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein codiilg sequence, ~~SA~CT CA 02453344 2004-O1-21 amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.
In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control 5 of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.
Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment 10 regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA
stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion 15 properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.
The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e,g., in.serting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple 20 deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element;
for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may 25 be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA
has integrated into the cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the 30 negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the 805A/PCT ~ 02453344 2004-O1-21 Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Patent No. 5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International Application No.
PCT/LJS92/09627 (W093/09222) by Selden et al.; and International Application No.
PCT/LTS90/06436 (W091/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.
4.9 TIgANSGENIC ANIMALS
In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory 1 S control of exogenous or endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals. Knockout animals, preferably non-human mammals, can be prepared as described in IJ.S. Patent No. 5,557,032, incorporated herein by reference.
Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S.
Patent No 5,489,743 and PCT Publication No. W094/28122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.
The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.
Such animals are 805A/PCT ~ 02453344 2004-O1-21 useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.
In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:2288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Patent No. 5,557,032, incorporated herein by reference.
Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S.
Patent No 5,489,743 and PCT Publication No. W094/28122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the Ievel of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.
4.10 USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.
Thus, "therapeutic compositions of the invention" include compositions comprising isolated ~~SA~CT CA 02453344 2004-O1-21 polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity. Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.
The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.
4.10.1 RESEARCH USES AND UTILITIES
The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA
sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out'° known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA
antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be ~zsed in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

805A/PCT ~ 02453344 2004-O1-21 The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or S its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F.
Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Bergen S. L. and A. R. Kirnmel eds., 1987.
4.10.2 NUTRITIONAL USES
Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be adminstered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
4.10. CYTOKINE AND CELL PROLIFERATION/D:IFFERENTIATION
ACTIVITY
A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.

805A/PCT ~ 02453344 2004-O1-21 Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor 5 dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RBS, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the invention can be used in the following:
Assays for T-cell or thymocyte proliferation include without limitation those 10 described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M.
Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19;
Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 15 133:327-341, 1991; Bertagnolli, et al., I. Immunol. 149:3778-3783, 1992;
Bowman et al., I.
Immunol. 152:1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T
cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E.
e.a. Coligan 20 eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin-y, Schreiber, R. D. In Current Protocols in Immunology. J. E.
e.a. Coligan eds. Vol I pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of° Human and Murine 25 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P.
E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173 :1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938, 1983; Measurement of mouse and human interleukin 6--Nordan, R. In Current Protocols in 30 Immunology. J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. /991;
Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin I I--Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;

805A/rCT CA 02453344 2004-O1-21 Measurement of mouse and human Interleukin 9--Ciarletta, A., Giannotti, J., Clark, S. C.
and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In l~it~o assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.
Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988.
4.10.4 STEM CELL GR~WTH FACT~R ACTIVITY
A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells. Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.
The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and Lung.
It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), ~~S~CT CA 02453344 2004-O1-21 Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).
Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.
Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations. This can be accomplished by direct administration of the polypeptide of the invention to the culture medium. Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo. Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Patent No. 5,690,926).
Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types. These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments. These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.
Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions. For example, polypeptides of the present invention may be used to manipulate stern cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders. The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue. In addition, the expanded stem cell populations can also be 805A/PCT ~ 02453344 2004-O1-21 genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.
Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types. A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker. The selectable marker allows only cells of the desired type to survive. For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.
Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In:
Principles of Tissue Engineering eds. Lanza et al., Academic Press (1997)). Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid aaad an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity. Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al. Proc. Natl. Acad.
Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines. The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g. as described by Bernstein et al., Blood, 77: 231-2321 (1991).
4.10.5 HEMATOPOIESIS REGULATING ACTIVITY
A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.
Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as 805tiI PCT CA 02453344 2004-O1-21 granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent rnyelo-suppression; in supporting the growth and proliferation of rnegakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in <;onjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.
Therapeutic compositions of the invention can be used in the following:
Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: .Iohansson et al. Cellular Biology 15:141-151, 1995;
Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in:
Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.
R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.
1994;
Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994;
Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of I-Iematopoietic Cells.
R. I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y.
1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, goSA/PC,I, CA 02453344 2004-O1-21 Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. 'Vol pp. 139-162, ~Wiley-Liss, Inc., New York, N.Y. 1994.
4.10.6 'fISSiJE GR~i~VTH ACTIVITY' A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.
A polypeptide of the present invention which induces cartilage and/or bone growth in 10 circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent 15 contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells. Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or 20 periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes may also be possible using the composition of the invention.
Another category of tissue regeneration activity that may involve the polypeptide of 25 the present invention is tendon/ligament formation. Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing 30 damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament ~~SA/PCT CA 02453344 2004-O1-21 defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors e~ vivo for return i~ vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.
Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate. A polypeptide of the present invention may also exhibit angiogenic activity.

805A/PCT ~ 02453344 2004-O1-21 A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fabrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
Therapeutic compositions of the invention can be used in the following:
Assays for tissue generation activity include, without limitation, those described in:
International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal); International Patent Publication No.
W091/07491 (skin, endothelium).
Assays for wound healing activity include, without limitation, those described in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. l, and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest.
Dermatol 71:382-84 (1978).
4.10.7 IMMUNE STIMULATING OR SUPPRESSING ACTIVITY'' A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A polynucleotide of the invention can encode a polypeptide exhibiting such activities. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus 805A/PCT ~ 02453344 2004-O1-21 erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, w-ticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention. The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Voter et al., Arch. Toxocol. 73: SOl-9), and murine local lymph node assay {Kimber et al., J. Toxicol. Environ. Health 53: 563-79).
Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression of T
cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.
Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (G"VHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
Moreover, a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, murine models of CaVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which proanote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.
Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen B~SA~CT CA 02453344 2004-O1-21 arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.
840-856).
Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a 5 means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.
10 Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T
cells into the patient. Another method of enhancing anti-viral immune responses would be to 15 isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T
cells in vivo.
20 A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.
In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) 25 of an MHC class I alpha chain protein and (32 microglobulin protein or an MHC class II
alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I
or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II
MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor 30 cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC
class II associated protein, such as the invariant chain, can also be cotransfected with a DNA
encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T

80511/ r CT CA 02453344 2004-O1-21 cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.
M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;
Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad.
Sci. USA
IO 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986;
Takai et al., J. Immunol. 140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998;
Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in:
Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays fox B cell function:
In vitro antibody production, Mond, J. J. and Brunswicl~, M. In Current Protocols in Immunology. J.
E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.
M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Gxeene Publishing Associates and Wiley-Interscience {Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;
Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 805A/rCT CA 02453344 2004-O1-21 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytornetry 14:891-897, 1993;
Gorczyca et al., International Journal of ~ncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995;
Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
4.10.8 ACTIVIN/INHIBIN ACTIVITY
A polypeptide of the present invention may also exhibit activin- or inhibin-related activities. A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone {FSH). Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fextility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example, U.S. Pat. No. 4,798,885. A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.

805A/PCT ~ 02453344 2004-O1-21 The activity of a polypeptide of the invention may, among other means, be measured by the following methods.
Assays for activin/inhibin activity include, without limitation, those described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1.985; Forage et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
4.10.9 CHEMOTACTIC/CHEMOKINETIC ACTIVITY
A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A
polynucleotide of the invention can encode a polypeptide exhibiting such attributes.
Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic compositions (e.g. proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, rnonocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation ox movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity fox a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
Therapeutic compositions of the invention can be used in the following:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A. M. Kxuisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.
Greene Publishing Associates and Wiley-Tnterscience (Chapter 6.12, Measurement of alpha and beta ,,;. ,.. :; : .. _ .. .. ~, ,."". ., _. .. ,.,. .. ..

805A/PCT ~ 02453344 2004-O1-21 Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995;
Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.
4,10.10 HEMOSTATIC AND THROMBOLYTIC ACTIVITY
A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A
composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
Therapeutic compositions of the invention can be used in the following:
I S Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
4.10.11 CANCER DIAGNOSIS AND THERAPY
Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis. Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.
For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition. Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.
Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.
Therapeutic compositions of the invention may be effective in adult and pediatric oncology gO~~~T CA 02453344 2004-O1-21 including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including 5 small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine 10 (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squarnous cell carcinoma, basal 15 cell carcinoma, hemangiopericytoma and Karposi's sarcoma.
Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer. Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant 20 cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.
The composition can also be administered in therapeutically effective amounts as a 25 portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer treatment is routine. Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include:
30 Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCI, Doxorubicin HCI, Estramustine phosphate sodium, Etoposide (V 16-213), Floxuridine, 5-Fluorouracil (5-Fu), S~~~p~T, CA 02453344 2004-O1-21 Flutamide, Hydroxyurea (hydroxycarbamide), Lfosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.
In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer. There are hereditary conditions and/or environmental situations (e.g.
exposure to carcinogens) known in the art that predispose an individual to developing cancers. Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.
Ira vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment. These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY
Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J.
Natl. Can. Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl. J. Dev.
Biol., 40: 1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999), respectively.
Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.
4.10.12 RECEPTOIt/LIGAN1) ACTIVITY
A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions. A
polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.
Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular 805ti1PCT CA 02453344 2004-O1-21 adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses. Receptors and Iigands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptorlligand interaction. A
protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
The activity of a polypeptide of the invention may, among other means, be measured by the following methods:
Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1- 7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987;
Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989;
Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).
Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.
Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.
The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods.
("Guide to Protein Purification" Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990) Academic Press, Inc. San Diego). Examples of radioisotopes include, but are not limited to, tritium and carbon-14 . Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules. Examples of toxins include, but are not limited, to ricin.
4.10.13 DRUG SCREENING
This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The polypeptides or fragments employed in such a test may either be free in 805A/PCT ~ 02453344 2004-O1-21 solution, axed to a solid support, borne on a cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof. Drugs are screened against such transformed cells in competitive binding assays.
Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.
Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include ( 1 ) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.
Chemical Libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as "hits" or "Leads" via natural product screening.
The sources of natural product Libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves. Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998).
Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews and examples of peptidomirnetic libraries, see Al-Obeidi et al., Mol. Biotechnol, 9{3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997); Dorner et al., BioorgMed Chem, 4(5):709-15 (1996) (alkylated dipeptides).
Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or "lead") to optimize the capacity of the "hit"

to bind a polypeptide of the invention. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.
S The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention. Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.
4.10.14 ASSAY FOR RECEPTOR ACTIVITY
The invention also provides methods to detect specific binding of a polypeptide e.g. a ligand or a receptor. The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention. For 1 S example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention. There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i. e., increase or decrease) biological activity of a polypeptide of the invention. Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does 2S not. The responses of the two cell populations to the addition of ligands(s) are then compared. Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s). As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined. For example, a chimeric protein in ~~S~P~Z CA 02453344 2004-O1-21 which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell. The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor. Known downstream proteins 5 involved in intracellular signaling can then be assayed for expected modifications i.e.
phosphorylation. Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.
4.10.15 ANTI-INFLAMMATORY ACTIVITY
10 Compositions of the present invention may also exhibit anti-inflammatory activity.
The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or 15 suppressing production of other factors which more directly inhibit or promote an inflammatory response. Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, 20 complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, 25 acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to 30 intrauterine infections.
4.10.16 LEUKEMIAS

Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention. Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, S promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytie) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).
4.10.17 NERVOUS SYSTEM DISORDERS
Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thLis observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:
(i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;
(ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
(iii)infectious lesions, in which a portion of the n ervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;
(iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not Limited to degeneration associated with Parkinson's disease, Alzheirner9s disease, Huntington's chorea, or amyotrophic lateral sclerosis;

805t11hCT CA 02453344 2004-O1-21 (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B 12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
(vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;
(vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention:
(i) increased survival time of neurons in culture;
(ii) increased sprouting of neurons in culture or in vivo;
(iii)increas ed production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.
Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in hestronlc et al. (1980, Exp. Neurol.
70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured;
and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that S selectively affect neurons such as arnyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and FIereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
4.10.18 OTHER ACTIVITIES
A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites;
1 S effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms;
effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s);
effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects;
promoting 2S differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
4.10.19 IDENTIFICATION OF POLYMORPHISMS

The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment. Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.
For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.
Polymorphisms can be identified in a variety of ways known in the art which alI
generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced. Alternatively, the DNA
may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides). In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA
depending on the presence or absence of the polymorphism) may be performed.
Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms. The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention. In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.
Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.
4.10.20 ARTHRITIS AND INFLAMMATION
The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis is determined in an experimental animal model system. The 805A/PCT ~ 02453344 2004-O1-21 experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int. Arch.
Allergy Appl. Immunol., 23:129. Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA). The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture. The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. The control consists of administering PB S only.
The procedure for testing the effects of the test compound would consist of IO intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24. At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.
4.11 THERAPEUTIC METHODS
The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods. Examples of therapeutic applications include, but are not limited to, those exemplified herein.
4.11.1 EXAMPLE
One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.
While the mode of administration is not particularly important, parenteral administration is preferred. An exemplary mode of administration is to deliver an intravenous bolus. The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician. It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient. Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 ~,g/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 ~g/kg to I 0 mg/kg of patient body g~SA/PCT CA 02453344 2004-O1-21 weight. For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art and examples include water, saline, lRnger's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.
The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.
4.12 PHARMACEUTICAL FORMULATIONS AND ROUTES OF
ADMINISTRATION
A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-a and TGF-(3), insulin-like growth factor (IGF), as well as cytokines described herein.
The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active 005AJrCT CA 02453344 2004-O1-21 ingredient of the invention, or to minimize side effects. Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti- inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-lRa, IL-1 HyI, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents). A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the f rst protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).
Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated. Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, ly~nphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or ~oSA~C,h CA 02453344 2004-O1-21 anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hernatopoietic factor(s), thrombolytic or anti-thrombotic factors.
4.12.1 ROUTES OF ADMINISTRATION
Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation. In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes will be targeted to and taken up selectively by the afflicted tissue.
The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action. The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art. Preferably for wound treatment, one administers the therapeutic compound directly to the site. Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.
Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.
4.12.2 COMPOSITIONS/FORMULATIONS

~OS~CT CA 02453344 2004-O1-21 Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90%
by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.
When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The pharmaceutical composition of the present invention may also contain stabilizers, 805AIPCT ~ 02453344 2004-O1-21 preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the 5 barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Fox oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, 10 capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or 15 sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, andlor polyvinylpyrrolidone (PVl'). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with 20 suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, andlor titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
25 Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved 30 or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges forn~ulated in conventional manner.

~0~~~~T CA 02453344 2004-O1-21 For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in mufti-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. optionally, the suspension rnay also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

g05A~LT, CA 02453344 2004-O1-21 A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system.
VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD
co-solvent system (VPD:SW) consists of VPD diluted 1:1 with a S% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dirnethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions. Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, 805A/PCT ~ 02453344 2004-O1-21 ., 78 trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.
The pharmaceutical composition of the invention may be in the form of a complex of the proteins) or other active ingredients) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T
cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigens) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR
and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.
Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.
The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.
Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response. Larger doses of protein or other active ingredient of the present invention ~zay be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not ~~~~~.,,I, CA 02453344 2004-O1-21 increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.0I ~.g to about 100 mg (preferably about 0.1 ~g to about IO mg, more preferably about 0.1 ~g to about I mg) of protein or other active ingredient of the present invention per kg body weight. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. iUhen administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or I O injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
Topical administration may be suitable for wound healing and tissue repair.
Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, rnay alternatively or additionally, be administered simultaneously or sequentially with the composition in the I 5 methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted 20 medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation.
Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, 25 tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defned, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised 30 of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole 805AIPCT ~ 02453344 2004-O1-21 weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents 10 include hyaluronic acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and polyvinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the 15 progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.
These agents include various growth factors such as epidermal growth factor (EGF), platelet 20 derived growth factor (PDGF), transforming growth factors (TGF-a and TGF-(3), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary applications.
Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.
25 The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering vaxious factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of 30 administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
For example, the addition of other known growth factors, such as IGF I
(insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by 805t1/i-CT CA 02453344 2004-O1-21 periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
4.12.3 EFFECTIVE DGSAGE
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the ICSO as determined in cell culture (i. e. , the concentration of the test compound which achieves a half maximal inhibition of the protein's biological activity). Such information can be used to more accurately determine useful doses in humans.
A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDSO (the dose lethal to 50%
of the population) and the FDso (the dose therapeutically effective in 50% of the population).
The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LDSO and EDso. Compounds which exhibit high therapeutic goSA/PL-T CA 02453344 2004-O1-21 $2 indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. T'he dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in "The Pharmacological basis of Therapeutics", Ch. 1 p.1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from ivc vitro data. Dosages necessary to achieve the MEC
will depend on individual characteristics and route of administration. However, HPLC
assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 pg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 ~g/kg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
4.12.4 PACKAGIllTG
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be 8OStI/PCT CA 02453344 2004-O1-21 prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
4.13 ANTIBODIES
Also included in the invention are antibodies to proteins, or fragments of proteins of the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab° and F~ab~~z fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
Certain classes have subclasses as well, such as IgGr, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. I~.eference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO: 1-244, or 489-706, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 1 S amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface;
commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a surface region of the protein, e.g., a hydrophilic region. A
hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J.
Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety.
Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
The term "specific for" indicates that the variable regions of the antibodies of the invention recognize and bind polypeptides of the invention exclusively (i.e., able to distinguish the polypeptide of the invention from other similar polypeptides despite sequence I 5 identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA
techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al.
(Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY
(1988), Chapter 6. Antibodies that recognize and bind fragments of the polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific far, as defined above, full-length polypeptides of the invention. As with antibodies that are specific for full length polypeptides of the invention, antibodies of the invention that recognize fragments are those which can distinguish polypeptides from the same family of polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.
Antibodies of the invention are useful for, for example, therapeutic purposes (by modulating activity of a polypeptide of the invention), diagnostic purposes to detect or quantitate a polypeptide of the invention, as well as purification of a polypeptide of the invention. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control 805A/PCT ~ 02453344 2004-O1-21 antigen for which the antibody is immunospecific. The invention further provides a hybridoma that produces an antibody according to the invention. Antibodies of the invention are useful for detection and/or purification of the polypeptides of the invention.
Monoclonal antibodies binding to the protein of the invention may be useful 5 diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and 10 preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
The labeled antibodies of the present invention can be used for i~c vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed. The antibodies may also be used directly in therapies or other diagnostics. The 15 present invention further provides the above-described antibodies immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose~, acrylic resins and such as polyacrylamide and latex beads. Techniques fox coupling antibodies to such solid supports are well known in the art (Weir, D.M. et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell 20 Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W.D.
et al., Meth.
Enzym. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vavo, and in situ assays as well as for immuno-affinity purification of the proteins of the present invention.
Various procedures known within the art may be used for the production of 25 polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies:
A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
4.13.1 POLYCLONAL ANTIBODIES
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the 805A/PCT ~ 02453344 2004-O1-21 native protein, a synthetic variant thereof, or a derivative of the foregoing.
An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hernocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants that can be employed include MPL-TDM
adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
4.13.2 MONOCLONAL ANTIBODIES
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

805A/PCT ~ 02453344 2004-O1-21 Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256, 495 (1970. In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (coding, Monoclonal Antibodies: Principles and Practice, Academic Press, (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 hybridorna cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are marine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia.
Human myelorna and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);
Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
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, far example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107, 220 (1980).
Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridama cells can be grown in vivo as aseites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA
also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 {1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a ehimeric bivalent antibody.
4.13.3 HUMANIZED ANTIBODIES

805A/PCT ~ 02453344 2004-O1-21 The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric S immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321, 522-525 (1986); Riechmann et al., Nature, 332, 323-327 (1988); Verhoeyen et al., Science, 239, 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct.
Biol., 2, 593-596 (1992)).
4.13.4 HUMAN ANTII~ODIES
Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies"
herein. Human monoclonal antibodies can be prepared by the trioma technique;
the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV
hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80, ~O~~P~.I, CA 02453344 2004-O1-21 2026-2030) or by transforming human B-cells with Epstein Barr Wirus in vitro (see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227, 381 (1991);
5 Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing 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.
10 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 Marks et al.
(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368, 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol.
13, 65-93 15 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals that are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT
publication W094/02602). The endogenous genes encoding the heavy and light immunoglobulin chains 20 in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than 25 the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the ~enomouseTM as disclosed in PCT
publications WO
96/33735 and WO 96/34096. This animal produces B cells that secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal 30 antibody, or alternatively from immortalized B cells derived from the animal, such as hybridornas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the pn'r A IPCT CA 02453344 2004-O1-21 antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S.
Patent No. 5,939,598. It can be obtained by a method including deleting the S
segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,7X. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT
publication W~ 99/53049.
4.13.5 FAB FRAGMENTS AND SINGLE CHAIN ANTIBODIES
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246, 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F~ab')2 fragment produced by pepsin digestion of an antibody molecule;
(ii) an Fab fragment generated by reducing the disulfide bridges of an F~ab~~2 fragment; (iii) an 805A/PCT ~ 02453344 2004-O1-21 Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F~ fragments.
4.13.6 BISPECIFIC ANTIBODIES
Bispecific antibodies a.re monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is 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 immunoglobulin heavy-chainllight-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305, 537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecue is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., 1991 EM~O J., 10, 3655-3659.
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 region (CHl) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121, 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 that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine ar tryptophan). Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229, 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E, coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med, 175, 217-225 (1992) describe the production of a fully humanized bispecific antibody F{ab')Z
molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecitic antibody fragments directly from recombinant cell culture have also been described. For. example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5), (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
I1SA 90, 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a 805ti/PCT CA 02453344 2004-O1-21 ' 94 light-chain variable domain (V L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.
See, Gruber et al., J. Immunol. 152, 5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147, 60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyIZIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
4.13.7 HETEROCON.IUGATE ANTIBODIES
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360;
WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No. 4,6?6,980.
4.13.8 EFFECTOR FUNCTION ENGINEERING

805tiIPCT CA 02453344 2004-O1-21 It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. Fox example, cysteine residues) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus 5 generated can 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, 1191-1 I95 (1992) and Shopes, J. Immunol., 148, 2918-2922 (1992).
Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53, 2560-10 2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3, 219-230 (1989).
4.13.9 IMMUNOCONJUGATES
15 The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have 20 been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin .A chain, modeccin A
chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins {PAPI, PAPA, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria 25 officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, i3'In, 9oY, and is6Re.
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 30 (SPDP), iminothiolane (IT), bifunetional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds {such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates ~~~~~T CA 02453344 2004-O1-21 (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (M%-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094i11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
4.14 COMPUTER READABLE SEQUENCES
In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, "computer readable media"
refers to any medium which can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM;
electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention. As used herein, "recorded" refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.
A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as V~ordPerfect 805A/PCT ~ 02453344 2004-O1-21 and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e. g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.
By providing any of the nucleotide sequences SEQ ID NO: I-244, or 489-706 or a representative fragment thereof; or a nucleotide sequence at least 95%
identical to any of the nucleotide sequences of SEQ ID NO: 1-244, or 489-706 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer I O software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. i~Iol. Biol. 215:403-410 (1990)) and BLAZE (Bnxtlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence. Such ORFs may be protein-encoding fragments and rnay be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.
As used herein, "a computer-based system" refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, "data storage means" refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.
As used herein, '°search means" refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.
Search means are 805A/PCT ~ 02453344 2004-O1-21 used to identify fragments or regions of a known sequence which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages far conducting homology searches can be adapted for use in the present computer-based systems. As used herein, a "target sequence" can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.
However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.
As used herein, "a target structural motif," or "target motif," refers to any rationally selected sequence or combination of sequences in which the sequences) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.
There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
4.15 TRIPLE HELIX FORMATION
In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.
Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are 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 15241, 456 (1988); and Dervan et al., Science 251, 1360 (1991)) or to the mRNA itself (antisense-Olmno, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides a.s Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA
hybridization blocks translation of an mRNA molecule into polypeptide. Botr~ techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.
4.16 DIAGNOSTIC ASSAYS AND KITS
The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.
In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.
Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.
In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.
In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components 'within the test sample.
Conditions for incubating a nucleic acid probe or antibody with a test sample vary.
Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.
~ne skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science 805A/PCT ~ 02453344 2004-O1-21 Publishers, Amsterdam, The Netherlands (1986); Bullock, G.R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.
Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b} one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.
In detail, a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or probe. Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. One skilled in the art will readily recognize that the disclosed probes and.
antibodies of the present invention can be readily incorporated into one of the established kit formats which axe well known in the art.

805t-1/PCT CA 02453344 2004-O1-21 4.17 MEDICAL IMAGING
The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection). See, e.g., Kunkel et al., U.S. Pat. NO. 5,413,778. Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.
4.18 SCREENING ASSAYS
Using the isolated proteins and poiynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-244, or 489-706, or bind to a specific domain of the polypeptide encoded by the nucleic acid. In detail, said method comprises the steps of:
(a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.
In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.
Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.
Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptidelcompound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene ~~~~~T CA 02453344 2004-O1-21 sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.
Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound). Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound). Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.
The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected a.nd screened at random or rationally selected or designed using protein modeling techniques.
For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be "rationally selected or designed"
when the agent is chosen based on the configuration of the particular protein.
For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides," In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.
In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control. One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic 805A/PCT ~ 02453344 2004-O1-21 phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.
Agents suitable for use in these methods preferably contain 20 to 40 bases and are 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); and Dervan et al., Science 251, 1360 (1991)) or to the mRNA itself (antisense-Okano, 3.
Neurochem. 56, 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.
Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.
4.19 USE OF NUCLEIC ACIDS AS PROBES
Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences. The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-244, or 489-706. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from any of the nucleotide sequences SEQ ID NO: 1-244, or 489-706 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.
Any suitable hybridization technique can be employed, such as, for example, in situ hybridization. PCR as described in US Patents Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences. Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.
The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.

805A/PCT ~ 02453344 2004-O1-21 Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA
probes. Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes an vatro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences. The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well-known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et aI (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY.
Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.
Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981fj. Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.
4.20 PREPARATIOhl OF SUPPORT BOUND OLIGONUCLEOTIDES
Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon. One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.
Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.
Microbiol. 28(6), 1469-72); using UV light (Nagata et al., 1985; Dahlen et al , 1987; Morrissey &
Collins, (1989) Mol.
Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988;
1989); all references being specifically incorporated herein.

~~~~~T CA 02453344 2004-O1-21 Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker. For example, Broude et al. (1994) Proc. Natl. Acad.
Sci. USA 91(8), 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Of course, this same linking chemistry is applicable to coating any surface with streptavidin. Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, CA).
Nunc Laboratories (Naperville, IL) is also selling suitable material that could be used.
Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH. CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridgeheads for further covalent coupling.
CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules may be bound to CovaLink exclusively at the 5'-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal. Biochem. 198(1) 138-42).
The use of CovaLink NH strips for covalent binding of DNA molecules at the 5'-end has been described (Rasmussen et al., (1991). In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res. 11(8) 6513-29). This is beneficial as immobilization using only a single covalent bond is preferred. The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm. To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5'-end phosphate group. It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.
More specifically, the linkage method includes dissolving DNA in water (7.5 ng/~.1) and denaturing for 10 min. at 95°C and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm~), is then added to a final concentration of 10 mM 1-MeIm~.
A ss DNA solution is then dispensed into CovaLink NH strips (75 ~.1/well) standing on ice.
Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIrn~, is made fresh and 25 N,l added per well. The strips are incubated for 5 hours at SO°C. After incubation the strips are washed using, e.g., Nunc-Irnmuno Wash;
first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS
heated to 50°C).

805A/PCT ~ 02453344 2004-O1-21 It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference. This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3'-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups earned by the support. The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.
Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.
An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed. For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al. (1991) Science 251 (4995), 767-73, incorporated herein by reference. Probes may also be immobilized on nylon supports as described by Van Ness et al.
(1991) Nucleic Acids Res., 19(12) 3345-50; or Iinked to Tetlon using the method of Dunean &
Cavalier (1988) Anal. Biochem. 169(1), 104-8; all references being specifically incorporated herein.
To link an oligonucleotide to a nylon support, as described by Van Ness et al.
(1991), requires activation of the nylon surface via alkylation and selective activation of the 5'-amine of oligonucleotides with cyanotic chloride.
One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) Proc. Nat'1. Acad.
Sci., USA 91(11), 5022-6, incorporated herein by reference). These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA
chips). These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5'-protected N acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies. A
matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.
4.21 PREPARATION OF hIITCLEIC ACID FRAGMENTS
The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC
inserts, and RNA, including mRNA without any amplification steps. For example, Sambrook et al. (1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).

805A/PCT ~ 02453344 2004-O1-21 DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.
Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA
samples may be prepared in 2-500 ml of final volume.
The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.
Low pressure shearing is also appropriate, as described by Schriefer et al.
(1990) Nucleic Acids Res. 18(24), 7455-6, incorporated herein by reference). In this method, DNA
samples are passed through a small French pressure cell at a variety of low to intermediate pressures. A lever device allows controlled application of low to intermediate pressures to the cell. The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.
One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.
(1992) Nucleic Acids Res. 20(14) 3753-62. These authors described an approach far the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.
The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions, which alter the specificity of this enzyme (CviJI* *), yield a quasi-random distribution of DNA fragments form the small molecule pUCl9 (2688 base pairs). Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUCl9 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z
minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI**
restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.
As reported in the literature, advantages of this approach compared to sonication and agaxose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ~,g instead of 2-5 ~.g); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed).
Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.

805A/PCT ~ 02453344 2004-O1-21 This is achieved by incubating the DNA solution for 2-5 minutes at 80-90°C. The solution is then cooled quickly to 2°C to prevent renaturation of the D1~TA
fragments before they are contacted with the chip. Phosphate groups must also be removed from genomic DNA by methods known in the art.
4.22 PREPARATION OF L1NA ARRAYS
Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane. Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 n1 of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density of the wells is achieved. One to 25 dots may be accommodated in 1 mm2, depending on the type of label used. By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed. Samples in one suba.rray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones. Each of the subarrays may represent replica spotting of the same samples. In one example, a selected gene segment may be amplified from 64 patients. For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).
A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples may be spotted on one 8 x 12 cm membrane. Subarrays may contain 64 samples, one from each patient.
Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.
Another approach is to use membranes or plates (available from NUNC, Naperville, Illinois) which may be partitioned by physical spacers e.g. a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips. A f red physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.
The present invention is illustrated in the following examples Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples. The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention. Indeed, numerous modifications and variations in the practice of the invention are expected to occur to 805AlPCT ~ 02453344 2004-O1-21 those skilled in the art upon consideration of the present preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.
All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.
5.0 EXAMPLES
5.1 EXAMPLE 1 Novel Nucleic Acid Seauences ~btained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques. The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts. Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences. The clones were clustered into groups of similar or identical sequences.
Representative clones were selected for sequencing.
In some cases, the 5' sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol. PCR products were purified and subjected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.
5.2 EXAMPLE 2 Assemblage of Novel Nucleic Acids The contigs or nucleic acids of the present invention, designated as SEQ ID
NO: 489-706 were assembled using an EST sequence as a seed. Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST, gb pri, and UniGene, and exons from public domain genomic sequences predicated by GenScan) that belong to this assemblage. The algorithm terminated when there were no additional sequences from the above databases that would extend the assemblage. Further, inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.

Table 8 sets forth the novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 489-706) of the present invention, and their corresponding translation start and stop nucleotide locations to each of SEQ
ID N~: 489-706.
Table 8 also indicates the method by which the polypeptide was predicted.
Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fasta.bioch.vir~inia edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W.R. Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference). Method B
refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C.
Burge and S. Karlin, J. Mol. Biol., 268:78-94 (1997), incorporated herein by reference).
Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.
5.3 EXAMPLE 3 Novel Nucleic Acids The novel nucleic acids of the present invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases. The nucleic acids were assembled using an EST sequence as a seed. Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (Hyseq's database containing EST sequences, dbEST, gb pri, and UniGene) that belong to this assemblage. The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage. Inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST
score greater than 300 and percent identity greater than 95%.
Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length gene cDNA
sequence and its corresponding protein sequence were generated from the assemblage. Any frame shifts and incorrect stop codons were corrected by hand editing. During editing, the sequences were checked using PASTY and/or BLAST against Genebank (i.e., dbEST, gb pri, UniGene, and Genpept) and the Geneseq (Derwent). Other computer programs which may 805A/PCT ~ 02453344 2004-O1-21 T ~ 111 have been used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and cg-zip-2 (Hyseq, Inc.). The full-length nucleotide and amino acid sequences, including splice variants resulting from these procedures are shown in the Sequence Listing as SEQ ID NO: 1-488.
S SEQ ID NO: 1-132 were classified as secreted according to their predicted cellular localization using the Pfam software program (Sonnhammer et al., Nucleic Acids Res., Vol.
26(1) pp. 320-322 (1998), and http:/lpfam.wustl.edu/, herein incorporated by reference).
SEQ ID NO: 133-197 were determined to contain signal peptide sequences and their cleavage sites using Neural Network SignalP V 1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark). The process for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication " Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites" Protein Engineering, Vol. 10, no. l, pp. 1-6 (1997), incorporated herein by reference.
A maximum S score and a mean S score, as described in the Nielson et al reference, was obtained for the polypeptide sequences.
SEQ ID NO: 198-244 were determined to be secreted polypeptides using a proprietary algorithm, SeqLocTM (Ilyseq Inc.). SeqLocTM classifies the proteins into three sets of locales: intracellular, membrane, or secreted. This prediction is calculated using maximum likelihood estimation of three characteristics of each polypeptide, 1 ) percentage of cysteine residues, 2) Kyte-Doolittle scores for the first 20 amino acids of each protein (J.
Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference), a.nd 3) Kyte-Doolittle scores to calculate the longest hydrophobic stretch (LHS) of the said protein (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference). The LHS is calculated by finding the stretch of 20 amino acid residues in the protein that have the highest sum of Kyte-Doolittle hydrophobicity values.
Table 1 shows the various tissue sources of SEQ ID NO: 1-244.
The nearest neighbor results for polypeptides SEQ ID NO: 245-488, that correspond to nucleotide sequences SEQ ID NO: 1-244 were obtained by a BLASTP (version 2.0a1 19MP-WashU) searches against Genpept release 124 and the Geneseq release (Derwent) using BLAST algorithm. The nearest neighbor results showed the closest homologue with functional annotation for SEQ ID NO: 245-488 from Genpept 124 and Geneseq. The translated amino acids sequences for which the nucleic acid sequence encodes 805tilrCT CA 02453344 2004-O1-21 are shown in the Sequence Listing. The homologues with identifiable functions for SEQ ID
NO: 245-488 are shown in Table 2.
Using eMatrix software package (Stanford University, Stanford, CA) (Wu et al., J.
Comp. Biol., Vol. 6, 219-235 (1999), http://motif.stanford.edyernatrix-search/
herein incorporated by reference), all the polypeptide sequences were examined to determine whether they had identifiable signature regions. Scoring matrices of the eMatrix software package are derived from the BLOCKS, PRINTS, PFAM, PRODOM, and DOMO
databases. Table 3 shows the accession number of the homologous eMatrix signature found in the indicated polypeptide sequence, its description, and the results obtained which include accession number subtype; raw score; p-value; and the position of signature in amino acid sequence.
Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res., Vol.
26(1) pp. 320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains. Table 4 shows the name of the Pfam model found, the description, the e-value, the Pfam score for the identified model within the sequence, number of similar domains found, and the position of the domain in the SEQ ID NO: being interrogated. Further description of the Pfam models can be found at http://pfam.wustl.eduJ.
The GeneAtlas~ software package (Molecular Simulations Inc. (MSI), San Diego, CA) was used to predict the three-dimensional structure models for the polypeptides encoded by SEQ ID NO 1-244 (i.e. SEQ ID NO: 245-488). Models were generated by (1) PSI-BLAST which is a multiple alignment sequence profile-based searching developed by Altschul et al, (Nucl. Acids Res. 25, 3389-3408 (1997)), (2) High Throughput Modeling (HTM) (Molecular Simulations Inc. (MSI) San Diego, CA,) which is an automated sequence and structure searching procedure (http://www.msi.com/), and (3) SeqFoldrM
which is a fold recognition method described by Fischer and Eisenberg (J. Mol. Biol. 209, 779-791 (1998)).
This analysis was carried out, in part, by comparing the polypeptides of the invention with the known NMR (nuclear magnetic resonance) and x-ray crystal three-dimensional structures as templates. Table 5 shows: "PDB ID°°, the Protein DataBase (PDB) identifier given to template structure; "Chain ID", identifier of the subcomponent of the PDB
template structure; "Compound Information", information of the PDB template structure and/or its subcomponents; "PDB Function Annotation" gives function of the PDB template as annotated by the PDB files (http:/www.rcsb.or~lPDB/); start and end amino acid position of gO~~~.I. CA 02453344 2004-O1-21 the protein sequence aligned; PSI-BLAST score, the verify score, the SeqFold score, and the Potentials) of Mean Force (PMF). The verify score is produced by GeneAtlasTM
software (MSI), is based on Dr. Eisenberg's Profile-3D threading program developed in Dr. David Eisenberg's laboratory (US patent no. 5,436,850 and Luthy, Bowie, and Eisenberg, Nature, 356:83-8S (1992)) and a publication by R. Sanchez and A. Sali, Proc. Natl.
Acad. Sci. USA, 9S:13S97-12502. The verify score produced by GeneAtlas normalizes the verify score for proteins with different lengths so that a unified cutoff can be used to select good models as follows:
Verify score (normalized) _ (raw score - 1 /2 high score)/( 1 /2 high score) The PFM score, produced by GeneAtlas~ software (MSI), is a composite scoring function that depends in part on the compactness of the model, sequence identity in the alignment used to build the model, pairwise and surface mean force potentials (MFP). As 1 S given in table S, a verify score between 0 to 1.0, with 1 being the best, represents a good model. Similarly, a PMF score between 0 to 1.0, with 1 being the best, represents a good model. A SeqFold~ score of more than SO is considered significant. A good model may also be determined by one of skill in the art based all the information in Table S taken in totality.
The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determined from using Neural Network SignalP V
1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark). The process for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication " Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites" Protein Engineering, Vol. 10, no. l, pp. 1-6 ( 1997), incorporated herein by reference. A maximum S score and a mean S
score, as described in the Nielson et al reference, was obtained for the polypeptide sequences. Table 6 shows the position of the last amino acid of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.
Table 7 correlates SEQ ID NO: 1-244 to a specific chromosomal location.
Table 8 is a correlation table of the novel polynucleotide sequences SEQ ID
NO: 1-244, their corresponding polypeptide sequences SEQ ID NO: 24S-488, their corresponding ~~~~P~.I. CA 02453344 2004-O1-21 priority contig nucleotide sequences SEQ ID NO: 489-706, their corresponding priority contig polypeptide sequences SEQ ID NO: 707-924, and the US serial number of the priority application in which the contig sequence was filed.

805tiIrCT CA 02453344 2004-O1-21 IAS
Tahla 1 Tissue RNA source Librar Name SEQ ID NO:
on in adrenal Clontech ADR002 4 7 9 13 24-26 31 33 42-43 gland 56 60 71 84-adult bladderInvitrogen BLD001 8 12-I3 25 71 122-123 adult brainBioChain ABR013 183 adult brainClontech ABR001 13 22 64 66 102 112 182 _ adult brainClontech ABR006 1-5 18 22 24 36-39 47 adult brainClontech ABR008 1-2 4 7-8 I 1 13-14 22 adult brainGIBCO AB3001 _ adult brainGIBCO ABD003 1 8 11 13 43 55 62 64 adult brainInvitro ABR014 140 176 189 en adult brainInvitro ABR015 133 136 208 241 en adult brainInvitro ABR416 8 13 186 en _ adult brainInvitro ABT004 11 42 48 60 85 129 133 en 141-142 147 149 adult cervixBioChain CVX001 1 7 12 14 21 24 26-27 adult colonInvitrogen CLN001 13 71 75 93 122 129 134 adult heartGIBCO A~IR001 1 4 6-8 13 21 23-24 42-43 adult kidneyGIBCO AKD001 4 6 8 12-13 20 23-26 34 adult kidneyInvitrogen AKT002 6 12-13 24 39 42-43 51 adult liverClontech ALV003 36-38 51 73 135-136 165 adult liverInvitrogen ALV002 4-5 12-13 32 39 42 48 adult lun GIBCO ALG001 25-26 41 48 60 74 81 146 adult ovaryInvitrogen AOV001 4 7 9 12-14 20-21 23-26 Tahla t Tissue RNA source Library Name SE ID NO:
on in adult lacentaClontech APLOO x 43 73 17_8 adult spleenClontech SPLc01 7-8 14 36-38 53-54 59 71 125 129 133 140-1.42 162 adult spleenGIBCO ASP001 4 8 13 23-24 27 41 48 64 adult testisGIBCO ATS001 10 13 34 43 46 60 81 102 bone marrowClontech BMD001 _ bone marrowClontech BMD007 11 bone marrowGF BMD002 1-2 11-12 14 x9 28-29 36-38 21_2 225 240-241 243 cultured Stratagene ADP001 10 25 42-43 48 75 81 91 readi oc 144 163 178 186 205 241 tes endothelialStratagene EDT001 1 4 7-8 10-11 13 19-20 cells 62 64 66 75 78 81 90-91 fetal brainClontech FBR001 35 53-54 129 182 fetal brainClontech FBR004 36-38 70 94 126 171 187 23 x 238 fetal brainClontech FBR006 1-2 5 7 13 15 24 32 35-39 212 2x8 220 222 228 230-231 fetal brainGIBCO HFB001 1 4 11-13 15 24 26 30 32 81 90 94 1 x2 I25 130 133 fetal brainInvitrogen FBT002 9 34 36-38 81 102 127 147 fetal heartInvitrogen FHR001 4 7-8 10 13-14 21 23 27 fetal kidneClontech FKD001 6 23 66 81 146 fetal kidneyClontech FKD002 19 26 42 60 78-79 92 102 fetal kidneInvitro FKD007 122 189 en fetal liverClontech FLV002 2 11 42 133 173-175 180 fetal liverClontech FLV004 2 11 3S-38 40 48 98 118 x27 133 136 186 189 x96-fetal liverInvitrogen FLV001 23 31 42 70 75 122 133 fetal liver-Columbia FLS041 1-13 17 20-21 23 25 30 spleen ~ University~ 63-64 75-76 79 85 90 95 11'7 TahlP l Tissue RNA source Librar Name ~ SE ID N~:
on in fetal liver-Columbia FLS002 1 4-5 8 11 13 17-18 20-25 spleen University 48 51 56 63-64 79 90-91 fetal liver-Columbia _ 1 3 9 13 21 43 50 61 66 spleen University 122 130 136 173-175 187-188 fetal lun Clontech FLG001 6 8 32 35 62 122 129 197 fetal lungInvitrogen FLG003 10 39-40 69 83 98 102 fetal muscleInvitrogen FMS001 4 8 10 2127 33 49 102 fetal muscleInvitrogen FMS002 7-8 10 13 23 26 33 42 fetal skinInvitrogen FSK001 1 4 9-10 12-13 27 36-38 fetal skinInvitrogen FSK002 10 14 22-23 25 39 48 88-91 fibroblastStratagene LFB001 1 4 8 12-13 30 66 81 117 Genomic Research BAC002 80 DNA-from- Genetics (CITB

BAC-393I6 BAC libr ) Genomic Genomic BAC003 80 DNA

DNA-from- from Genetic BAC-393I6 Research induced Stratagene NTD001 1 22 30 32 42 84 117 125 neuron-cells infant Columbia IB2002 4 9-10 15 22-23 33 43 brain 48-49 55 63 67 73 University 75 81 85 90 99 102 120 infant Columbia IB2003 7 10 12 22 47 49 53-54 brain 61 75 84-85 90 University 94-95 102 122 133 135 141-i42 147 176 infant Columbia IBM002 12 157-158 224 I
brain Universi infant Columbia IBS001 6 10 33 108 135 233 brain Universi leukoc Clontech LUC003 14 27 81 146 197 20I 204 to 242 leukocyte GIBCO LUC001 1-2 4 7-8 12-14 17 21-22 OQStiITCT CA 02453344 2004-O1-21 11~
Table 1 Tissue RNAsource Library Name SEQ ID NO:
on in lung tumorInvitrogen LGT002 1 3-4 12 15 39 42-43 48 I m h nodeClontech ALN001 8 13 18 25 35-38 lymphocytesATCC LPC001 4 14 21 25-27 35 46 48 macro ha Invitro HMP001 1 12 25 140 144 181 194 a en 197 mammary Invitrogen MMG001 4 6 9 12-13 25 27 31 33-34 gland 51 53-56 60-62 70 72 75 144 14.6 156 163 172 176 melanoma Clontech MEL004 4 52 81 130 133 143 146 from-cell- 200 212 218 line-ATCC-#CRL-1424 mix of various CTL016 71 122 207 16 vendors tissues-mRNAs mix of various CTL021 189 16 vendors tissues-mRNAs mix B/I/C SUP005 1136173-175181185 mix I B/I/C SUP008 48 55 122 130 173-176 mix B/I/C SUP009 1 140 173-175 189 mixed EST clones CGd010 7 20 31 116-I 19 132 150 neuronal Stratagene NTU001 3-4 15 60 63 75 120 122 cells 133 140 171 pituitary Clontech PIT004 13 20 33 43 66 74 90 123 land placenta Clontech PLA003 4-5 7 25 36-39 56 93 100-101 lacenta Invitro APL002 41 75 224 en prostate Clontech PRT001 20 26 34 62 72 81 143 rectum Invitrogen REC001 3 25 33 51 74 88-89 122 retinoic Stratagene NTR001 1 3 34 104 124 129 140 acid- 225 241 induced-neuronal-cells saliva Clontech SALs03 179 land saliva Clontech SAL001 1 18 34 69 71 120 179 land 204 214 235-236 skeletal Clontech SKM001 7 42 49 73 75 102 130 muscle small Clontech SIN001 1 4 7-8 10 12 20-22 26 intestine 51 61 68 71 75 86 91-92 805A/PCT ~ 02453344 2004-O1-21 Table 1 Tissue RNA source Librar Name SEA II;i NO:
on in spinal Clontech SPC001 2 8 13 24 26 35 43 63-64 cord 127-128 130 stomach Clontech STO001 32 99 143 161 172 189 thalamus Clontech THA002 7 10-11 60 79 98 127 131 thymus Clontech THM001 1 14 26 30 46 50 74 79 thymus Clontech THMc02 I-2 4 10 13 24-25 30 32 thyroid Clontech THR001 1 4-5 7-8 20-21 24 26 gland 31 43 49 53-54 64 trachea Clontech TRC001 4-5 40 48 62 111 144 146 umbilical BioChain FUC001 6 12 18 32 36-38 40 61 cord 95 98-99 118 122 127 140 uterus Clontech UTR001 10 12-13 21 71 130 134 young liverGIBCO ALV001 1 24 27 48 73 85 136 173-175 *The 16 tissue-mRNAs and their vendor source are ass follows: 1) Normal adult brain mRNA (Invitrogen), 2) normal kidney mRNA (Invitrogen), 3) normal adult liver mRNA
(Invitrogen), 4) normal fetal brain mRNA (Invitrogen), 5) normal fetal kidney mRNA
(Invitrogen), 6) normal fetal liver mRNA (Invitrogen), 7) normal fetal skin mRIVA
(Invitrogen), 8) human adrenal gland mRNA (Clontech), 9) human bone marrow mRNA
(Clontech), 10) human leukemia lymphoblastic mRNA (Clontech), 11) human thymus mRNA (Clontech), 12) human lymph node mRNA (Clontech), 13) human spinal cord mRNA (Clontech), 14) human thyroid mRNA (Clontech), 15) human esophagus mRNA
(BioChain), 16) human conceptional umbilical cord mRNA (BioChain).

805A/PCT ~ 02453344 2004-O1-21 Table 2 SEQ AccessionSpecies Description Score ID

NO: Number identit 245 AAW78199 Homo SapiensHUMA- Human secreted 294899 protein encoded b Qene 74 clone HGBAC 11.

245 114915787Mus musculusWAC 291294 245 g111139753Homo SapiensbA48B24.1 (A novel protein280599 containing a formin binding protein (FBP28) domain 246 AAM43622 Homo sapiensHUMA- Human polypeptide 890 99 SEQ ID

NO 300.

246 g118766553Caenorhabditisubiquitin c-terminal 202 25 hydrolase ele ans 246 AAM71031 Homo SapiensMOLE- Human bone marrow 199 100 expressed probe encoded protein SEQ ID NO:

31337.

247 AAM93769 Homo sapiensHELI- Human polypeptide,341299 SEQ ID

NO: 3772.

247 g113559026Homo SapiensbG174L6.2 (MSF: megakaryocyte221 21 stimulatin factor 247 AAB29773 Homo SapiensRHOD- Human megakaryocyte220 21 stimulating factor (MSF), SEQ ID

NO:1.

248 111967711Homo sa Tsa24 rotein 10136 99 iens 248 1642252 Mus musculusis 24 943792 248 AAB95540 Homo SapiensHELI- Hurnan protein 743999 sequence SEQ ID

N0:18147.

249 115823648Homo sa ALS2CR9 296991 fens 249 118605620Mus museulussimilar to roline-rich 118843 protein 48 249 AAG74705 Homo sapiensHUMA- Human colon cancer929 96 antigen rotein SEQ ID N0:5469.

250 AAU74823 Homo sa INCY- Human REPTR 6 rotein.378510 iens 250 g113623799Homo sapiensseven-transmembrane receptor378510 Frizzled-250 g110334640Homo sapiensbA425A6.1 (frizzled (Drosophila)378510 homolo 8 251 AAB95372 Homo SapiensHELI- Human protein sequence338999 SEQ ID

N0:17692.

251 g110176983ArabidopsisGTP-binding membrane 183858 protein LepA

thaliana homolog 251 g120515955ThermoanaeroMembrane GTPase LepA 168052 bacter ten con ensis 252 AAB58241 Homo SapiensROSE/ Lung cancer associated825 90 of a tide se uence SEQ
ID 579.

252 11780755 Homo sa DJ-1 rotein 825 90 iens 252 116751471Homo sa DJ-1 825 90 iens 253 g121428404DrosophilaLD05365p 227 27 melanoaaster 253 g17190399Chlamydia phospholipase D family 180 31 protein muridarum 253 g12313422Helicobactermembrane bound endonuclease171 30 (nuc) Lori 26695 254 11869810 Homo sa SH3-containin Grb-2-like186695 iens 1 254 16120106 Homo sa SH3-containing rotein 186695 iens EEN

254 1 12654853Homo sa SH3-domain GRB2-like 186695 iens 1 255 AAY51529 Homo sa INNO- Human al ha-s nuclein579 86 iens rotein.

255 AAW88131 Homo sa USSH Human al ha s nucleic579 86 iens rotein.

Table 2 SEQ AccessionSpecies Description Score ID

NO: Number identi 255 AAY07277 Homo Sa UYSA- Human al ha-s nuclein.579 86 iens 256 Qi14587851Homo sa Graft 327899 iens 256 113310137Mus musculusPSGAP-m 293089 256 113310135Mus musculusPSGAP-s 293089 257 AAB58241 Homo SapiensROSE/ Lung cancer associated728 82 oly a tide se uence SEQ
I_D 579.

257 g116751469CercopithecusDJ-1 728 82 aethio s 257 12460318 Homo sa RNA-bindin rotein re 728 82 iens ulator subunit 258 AAM50136 Homo sapiensMILL- Human GTPase activating214510 molecule GAP-5.

258 15020264 Mus musculusCdcA.2 GTPase-activatin 919 48 rotein 258 AAE13842 Homo SapiensCORI- Human lung tumour-specific774 50 rotein 20129.

259 112005724Homo sa mixed linea a kinase 561810 iens MLK1 259 AAE11775 Homo Sa INCY- Human kinase (PKIN)-9530392 iens rotein.

259 g121410177Mus musculusSimilar to mitogen-activated360790 protein kinase kinase kinase 260 AAR85092 Homo SapiensAMGE- EPH-like receptor 489393 protein tyrosine kinase HEK11.

260 g1551608 Homo sa rece for rotein-tyrosine489393 iens kinase 260 g1755568 Rattus Ehk-3, full length form 482492 norve icus 261 AAQ13290_Homo SapiensUYSF- Leukocyte derived 618 96 growth aal factor ene.

261 AAB15804 Homo SapiensNEOR- Human chemokine 618 96 NO: 46.

261 AAW96716 Homo Sa UNMI A latelet basic 618 96 iens rotein (PBP).

262 AAM00862 Homo SapiensNYSE- Human bone marrow 971 93 protein, SEQ ID NO: 225.

262 AAM00975 Homo SapiensNYSE- Human bone marrow 827 100 protein, SEQ ID NO: 451.

262 AAU77835 Homo SapiensMERE Human N-terminal 815 91 acetyl transferase HUTUDO1).

263 AAG01995 Homo SapiensGEST Human secreted protein,523 100 SEQ ID

NO: 6076.

263 AAU19731 Homo SapiensHUMA- Human novel extracellular439 89 matrix rotein, Se ID
No 381.

263 AAU19744 Homo SapiensHUMA- Human novel extracellular296 95 matrix rotein, Se ID
No 394.

264 AAY44988 Homo sa INCY- Human a idermal 162194 iens rotein-5.

264 114249975Homo sa Similar to Sh3 domain 162194 iens YSC-like 1 264 11944389 Mus musculusSh3 I1 151187 265 g1 19916191Methanosarcinkinesin light chain 508 36 a acetivorans~

str. C2A]

[Methanosarci na acetivorans 265 g120907522Methanosarcintetratricopeptide repeat479 36 family protein a mazei Goel 265 g12645229Plectonema kinesin light chain 445 37 bo anum 266 AAB94232 Homo SapiensHELI- Human protein sequence307599 SEQ ID

N0:14606.

Table 2 SEQ AccessionSpecies Description Score ID

NO: Number identit 266 AAY83079 Homo sa UYNY F-box rotein FBP-11.661 99 iens 266 16164741 Homo sa F-box rotein Fbxl 1 661 99 iens 267 118676718Homo sa FLJ002S8 rotein 393398 iens 267 1 10441465Homo sa actin filament associated152143 iens rotein 2 113129531Gallus actin filament-associated148642 67 anus rotein _ AAB93991 Homo SapiensHELI- Human protein sequence212099 N0:14091.

268 AAU 17462Homo SapiensHUMA- Novel signal transduction211999 athwa rotein, Se ID 1027.

268 AAU17440 Homo SapiensHUMA- Novel signal transduction204099 athwa rotein, Se ID 1005.

269 ABB50205 Homo sapiensINCY- Human transcription124610 factor TRFX-56.

269 g117862100DrosophilaLD08718p 723 52 melanoaaster 269 AAM00911 Homo SapiensNYSE- Human bone marrow 472 54 protein, SEQ ID NO: 387.

270 AAG80184 Homo SapiensWIRT/ Human MEK kinase 698399 rotein fra ent.

270 AAB60291 Homo sa ISIS- Human MEKK1. 698399 iens 270 12815888 Homo sa MEK kinase 1 698399 iens 271 13002590 Homo sa interleukin-1 rece tor-associated350489 iens kinase 271 AAW14306 Homo SapiensT'LTLA- Interleukin-1 349089 receptor-associated rotein kinase.

271 11220313 Homo sa interleukin-1 rece tor-associated349089 iens kinase 272 AAY97638 Homo sa UNMI A of 1 WD rotein 577788 iens se uence.

272 g15051670Homo Sapiensapoptotic protease activating577788 factor-1 lon isoform APAF-1L

272 AAY97641 Homo SapiensUNMI Apaf 1XL-LlOA protein575587 se uence.

273 AAY97638 Homo sa UNMI A of 1 WD rotein 584788 iens se uence.

273 g15051670Homo Sapiensapoptotic protease activating584788 factor-1 long isoform APAF-1L
_ 273 AAY97636 Homo sa UNMI A of 1XL rotein 582588 iens se uence.

274 AAY52186 Homo sapiensCURA- Human enhancer 433 82 of rudimentary gene (ERH) amino acid se uence.

274 AAG03943 Homo SapiensGEST Human secreted protein,433 82 SEQ ID

NO: 8024.

274 g11374695Homo Sapienshuman protein homologous433 82 to DROER

rotein 275 11657835 Mus musculusRho- uanine nucleotide 253180 exchan a factor 275 ABB44551 Homo sapiensSWIT- Human wound healing928 42 related of a tide SEQ ID NO 8.

275 g15199316Homo Sapiensnon-ocogenic Rho GTPase-specific690 35 GTP exchan a factor 276 g18388704Leishmaniaprobable CG14353 protein678 43 !

ma' or 276 AAG03317 Homo sapiensGEST Human secreted protein,285 98 SEQ ID

_ NO: 7398.

2?6 g115291701DrosophilaLD24014p 115 24 melano aster 277 AAH78272_Homo SapiensMILL- Coding sequence 252910 of human aal GTPase activator rotein ~
26651.

Table 2 SEQ AccessionSpecies Description Score ID

NO: Number _ identi 277 AAG67550 Homo SapiensMILL- Amino acid sequence252910 of human GTPase activator rotein 26651.

277 AAH78271_Homo SapiensMILL- Nucleotide sequence252910 of human gal GTPase activator protein 26651.

278 114488252_ ras-like roteinlVTS58635 108210 Homo sa iens 278 ABB06136 Homo SapiensCOMP- Human NS protein 681 99 sequence SEQ ID N0:228.

278 11666073 Homo sa RRP22 rotein 501 51 iens 279 119387193Homo sa StAR-related 1i id transfer111110 iens rotein 4 279 119387189Mus musculusStAR-related 1i id transfer983 86 rotein 4 279 g113542895Mus musculusSimilar to RIKEN cDNA 979 86 ene 280 AAB94355 Homo SapiensHELI- Human protein sequence426399 SEQ ID

N0:14877, 280 120372683Homo sa euchromatic histone meth 416399 iens ltransferase 1 280 114211561Homo sa GLP1 237810 iens 281 AAB94355 Homo sapiensHELI- Human protein Sequence393694 SEQ ID

N0:14877.

281 120372683Homo sa euchromatic histone meth 383694 iens Itransferase 1 281 114211561Homo SapiensGLP1 204489 282 AAB94355 Homo SapiensHELI- Human protein sequence410496 SEQ ID

N0:14877.

282 120372683Homo sa euchromatic histone meth 400496 iens Itransferase 1 282 114211561Homo sa GLP1 221994 iens 283 AAB64405 Homo sapiensINCY- Arnino acid sequence204399 of human intracellular signalling molecule INTRA37.

283 g115530218Homo sapiensSimilar to RIKEN cDNA 204399 ene 283 115930031Mus musculusRIKEN cDNA 1300006M19 176785 ene 284 AAW88399 Homo sapiensGEMY Human testis secreted260599 protein dx290 1.

284 g15281051Arabidopsisstress-induced protein 162 28 stil-like protein thaliana 284 1872116 Gl cine sti (stress inducible 159 26 max rotein) 285 17363368 Mus musculusinhibito ada ter molecule178777 285 16492338 Mus musculusada for rotein; DOKL 178777 285 13043919 Homo sa docking rotein 501 37 iens 286 AAM78693 Homo SapiensHYSE- Human protein SEQ 246297 ID NO

1355.

286 i36619Ho rno sa serine/threonine rotein 246297 iens kinase 286 115990456Homo sa PCTAIRE rotein kinase 246297 iens 1 287 AAB64420 Homo SapiensINCY- Amino acid sequence225510 of human intracellular signalling molecule INTRA52.

287 a121411454Mus musculusRIKEN cDNA 4833427E09 19 gene 1685 287 g112484136Rattus SMHS2 _ norve ices 288 AAG67823 Homo SapiensSHAN- Human guanine-nucleotide909 58 releasin factor 52 protein.

288 ABB04984 Homo SapiensMERE Human new ras guanine-908 58 nucleotide-exchange factor N0:2.

288 g118490322Homo sapiensSimilar to RIKEN cDNA 681 50 ene ODStI/rCT CA 02453344 2004-O1-21 Table 2 SEQ AccessionSpecies Description Score ID

N~: Number identit 289 ai10801596Mus musculusDoc2 aroma 109878 289 AAW25032 Homo SapiensSHIO Human Doc2-beta colon601 44 cancer rotein.

289 gi1235722Homo sa Doc2 beta 601 44 iens 290 ABB04984 Homo sapiensMERE Human new ras guanine-170110 nucleotide-exchange factor N0:2.

290 AAG67823 Homo SapiensSHAN- Human guanine-nucleotide168799 releasin factor 52 rotein.

290 gi18490322Homo SapiensSimilar to RIKEN cDNA 111964 ene 291 AAM40118 Homo SapiensHYSE- Human polypeptide 171070 SEQ ID NO

3263.

291 AAM41904 Homo SapiensNYSE- Human polypeptide 100180 SEQ ID NO

6835.

291 gi4099012Dictyosteliumdrainin 722 39 discoideum 292 AAB34844 Homo SapiensHUMA- Human secreted protein843 84 sequence encoded by gene 44 S:EQ ID

N0:132.

292 1550060 Homo sa GTP-bindin rotein 843 84 iens 292 16969622 Mus musculussmall GTP-bindin rotein 843 84 293 AAH78263~Homo sapiensMILL- Nucleotide sequence310610 of human aal kinase 14760.

293 1 13194657Homo sa skeletal m osin 1i t chain310610 iens kinase 293 g118073328Homo Sapiensskeletal muscle-specific 310610 myosin light chain kinase 294 13599940 Mus musculusfacio enital dys lasia 254582 rotein 2 294 g13342246Rattus actin-filament binding 153352 protein Frabin norve ices 294 115705415Mus musculusactin-bindin rotein frabin-al152654 ha 295 a13851202Homo sa ZO-3 482998 iens 295 g13033501Canis ZO-3 385783 familiaris 295 115214772Mus musculusSimilar to ti ht 'unction368180 rotein 3 296 AAB95184 Homo SapiensHELI- Human protein sequence285499 SEQ ID

N0:17254.

296 ABB03717 Homo SapiensHUMA- Human rnusculoskeletal422 98 system related polypeptide SEQ ID NO

1664.

296 g13108057Mus musculuschannel interactin PDZ 138 27 domain rotein 297 AAB48307 Homo sa UYYA Human ZF26 rotein. 227479 iens 297 AAY83085 Homo sa UYNY F-box rotein FBP-17.227479 iens 297 110764488Homo sa dactylin 221710 iens 298 AAB48307 Homo sa UYYA Human ZF26 ratein. 211475 iens 298 AAY83085 Homo sa UYNY F-box rotein FBP-17.211475 iens 298 g110764488Homo Sapiensd 200092 actylin 299 AAY97293 Homo Sapiens_ 227578 INCY- Lipid associated protein (LIPAP 3335404CD1.

299 15670328 Homo sa co ine III 131749 iens 299 AAM39997 Homo sapiensHYSE- Human polypeptide _ 3142.

300 AAE09604 Homo sapiensHUMA- Human gene 12 encoded423 97 novel rotein HE9RA75, SEQ ID
N0:40.

300 110178646H dra vuI dishevelled 193 48 aris 805A/PCT ~ 02453344 2004-O1-21 Table 2 SEQ AccessionSpecies Description Score ID

NO: Number _ identit 300 AAB68347 Homo Sapiens_ 190 41 REGC Amino acid sequence of dishevelled (dsh) rotein.

301 AAM41632 Homo sapiensHYSE- Human polypeptide 235610 SEQ ID NO

6563.

301 AAM39846 Homo SapiensHYSE- Human polypeptide 227110 SEQ ID NO

2991.

301 AAW90868 Homo sa GEHO Human d sferlin 113 33 iens roteiii.

302 i51144Musmusculus h2-cal onin 102576 302 117391137Mus musculuscal onin 2 102576 302 a11962 Sus scrofah2-cal onin 102476 303 g1 15082283Homo sapiensSimilar to small glutamine-rich153910 tetratricopeptide repeat (TPR)-containin 303 g12909372Homo Sapienssmall glutamine-rich 854 58 tetratricopeptide (SGT) 303 g14539082Homo Sapienssmall glutamine-rich 854 58 tetratricopeptide re eat containin rotein 304 111345052Homo sa SM-20 231410 iens 304 114547146Homo sa EGLN1 rotein 231410 iens 304 114547239Mus musculusEGLN1 rotein 156378 305 AAU16939 Homo sapiensHUMA- Human novel secreted161099 protein, SEQ ID 180.

305 ABB10234 Homo SapiensHUMA- Human cDNA SEQ 161099 ID NO:

542.

305 111527997Homo sa NOTCH2 rotein 1483 iens 99 306 AAM48953 Homo SapiensCHIR Human colon cancer __ 100 related 776 rotein SEQ ID NO: 4.

306 AAG02355 Homo SapiensGEST Human secreted protein,597 96 SEQ ID

NO: 6436.

306 AAU20658 Homo SapiensHUMA- Human secreted 184 43 protein, Seq ID No 650.

307 AAM78475 Homo sapiensHYSE- Human protein SEQ 589895 ID NO

1137.

307 AAM70260 Homo sapiensMOLE- Human bone marrow 369299 expressed probe encoded protein SEQ ID NO:

30566.

307 AAM57843 Homo SapiensMOLE- Human brain expressed369299 single exon probe encoded protein SEQ ID

NO: 29948.

308 AAB93852 Homo sapiensHELI- Human protein sequence205399 SEQ ID

NO:1 3705.

308 AAM39549 Homo sapiens_ 205399 HYSE- Human polypeptide SEQ ID NO

2694.

308 AAG64490 Homo SapiensSHAN- Human lissencephaly204399 protein 43.

309 AAT61456_aHomo SapiensUYJE- C-proteinase clone383699 pCP-1.

al 309 ABB90755 Homo SapiensUYJO Human Tumour Endothelial383699 Marker of a tide SEQ
ID NO 242.

309 AAW13670 Homo SapiensUYJE- C-proteinase encoded383699 by clone CP-2.

310 AAB93117 Homo SapiensHELI- Human protein sequence197899 SEQ ID

N0:11991.

310 AAB94167 Homo sapiensHELI- Human protein sequence913 100 SEQ ID

N0:14468.

pnG p mCT CA 02453344 2004-O1-21 1z6 Table 2 SEQ Accession Species Description Score ID

NO: Number identit 310 g16650370 Dictyosteliumrac serine/threonine 113 29 kinase homolog discoideum 311 AAB65650 Homo SapiensSUGE- Novel protein kinase,201692 SEQ ID

NO: 177.

311 15139689 Homo sa MOK rotein kinase 201692 iens 311 15139691 Mus musculusMOK rotein kinase 166077 312 AAE04546 Homo sapiensINCY- Human G-protein 457599 coupled rece tor-2 GCREC-2) rotein.

312 g119387136Homo SapiensPYRIN-containing APAFI-like456399 protein 312 AAU00023 Homo SapiensBIOJ Human activated 416591 T-lymphocyte associated se uence 2, ATLAS-2.

313 AAE18955 Homo sapiensINCY- Human cell cyc3e 155899 ' protein and mitosis-associated molecule (CCPMAM-3).

313 AAB95737 Homo SapiensHELI- Human protein sequence654 100 SEQ ID

N0:18627.

313 g115722097Homo SapiensbA690P14.1 (novel cyclin463 39 (contains FLJ10895) 314 AAG67393 Homo SapiensSUGE- Amino acid sequence357599 of human rotein kinase SGK223.

314 AAG67394 Homo SapiensSUGE- Amino acid sequence104940 of human rotein kinase SGK269.

314 AAM25743 Homo sapiensHYSE- Human protein sequence100199 SEQ

ID N0:1258.

315 g13327808 Homo Sapienslatent transforming growth804593 factor-beta bindin rotein 4S

315 12190402 Homo sa latent TGf'-beta bindin 777489 iens rotein-4 315 g13327814 Homo Sapienslatent transforming growth387199 factor-beta bindin rotein 4 316 16624055 Homo sa similar to rin motif 683 100 iens ; note:

316 11655418 Homo sa an rin motif 551 98 iens 316 AAB92646 Homo SapiensHELI- Human protein sequence497 50 SEQ ID

N0:10979.

317 AAM40636 Homo sapiensHYSE- Human polypeptide 231999 SEQ ID NO

5567.

317 g114307916Mus musculusmyosin phosphatase targeting221782 subunit 3 317 AAM38850 Homo SapiensHYSE- Human polypeptide 215910 SEQ ID NO

1995.

318 1 19343951Homo sa Similar to GTP-rho bindin347099 iens rotein 1 318 119697913 Homo sa rho hilin-1 343796 iens 318 11176422 Mus musculusrho hilin 234171 319 117389232 Homo sa block of roliferation 397410 iens 1 319 113938293 Homo sa Similar to block of roliferation390910 iens 1 319 116416407 Homo sa KM-PA-2 rotein 338910 iens 320 AAU78329 Homo sapiensMILL- Tetratricopeptide 207299 repeat containin meth ltransferase TPR

320 AAB56481 Homo sapiensROSE/ Human prostate 769 97 cancer antigen rotein se uence SEQ ID
N0:1059.

_ 320 ABB89399 Homo SapiensHUMA- Human polypeptide 473 95 SEQ ID

NO 1775.

321 g113279080Homo SapiensSimilar to protein interacting206710 with uanine nucleotide exchan a factor ~05~CT CA 02453344 2004-O1-21 s ~ 127 Table 2 SEQ AccessionSpecies Description Score ID

NO: Number __ identit 321 g12459833Rattus Maxpl 169186 norve 'cus 321 117386088Homo sa ras effector-like rotein 968 98 iens 322 115529066Homo sa sortin nexin 27 129597 iens 322 AAB45187 Homo sapiensHUMA- Human secreted protein874 100 sequence encoded by gene N0:128.

322 AAM41948 Homo sapiensHYSE- Human polypeptide 534 94 SEQ ID NO

6879.

323 AAG67293 Homo sapiensLEXI- Amino acid sequence583510 of a human rotein.

323 g110764778Homo Sapiensphosphoinositol 3-phosphate-binding583510 rotein-2 323 AAB47871 Homo sa INCY- IS1G1'-I. 312210 iens 324 ABB11747 Homo sapiensHYSE- Human IL-1 delta 922 100 homologue, SEQ ID N0:2117.

324 AAB 19923Horno sapiensHYSE- Human interleukin-1906 100 Hy2 (extended form, artial se uence).

324 AAB19924 Homo SapiensHYSE- Human interleukin-1868 100 Hy2 (long version .

325 AAU20396 Homo sapiensHUMA- Human secreted protein,916 100 Seq ID No 388.

325 AAU20594 Homo sapiensHUMA- Human secreted protein,669 99 Seq ID No 586.

325 ABB03519 Homo SapiensHUMA- Human musculoskeletal669 99 system related polypeptide SEQ ID NO

1466.

326 AAB65609 Homo SapiensSUGE- Novel protein kinase,147610 SEQ ID

NO: 135.

326 AAU17266 Horno SapiensHUMA- Novel signal transduction976 98 athwa rotein, Se ID 831.

326 g120901962CaenorhabditisC. elegans CEH-20 protein485 43 ele ans comes ondin se uence F31E3.2d) 327 111596127Homo sa d nein axonemal intermediate318399 iens chain 327 111493148Homo sa intermediate d ein chain 317999 iens 327 g114189848Cynops dynein intermediate chain229069 rrho aster 328 ABB07495 Homo SapiensINCY- Human lipid metabolism901 80 molecule (LMM) polypeptide (ID:

2970737CD 1 .

' 328 12565396 Mus m_usculusschwannoma-associated 355 39 r otein 328 AAB90716 Homo sapiens_ 347 42 GEMY Human CI1480 9 protein se uence SE ID 128.

329 AAB95777 Homo SapiensHELI- Human protein sequence233510 SEQ ID

N0:18722.

329 g11772658Rattus Srgl 207695 norve ices 329 115991284Mus musculuss na tota min XII 205895 330 g115779080Homo Sapiens_ 191110 Similar to RIKEN cDNA

ene 330 AAM95146 Homo SapiensHUMA- Human reproductive 445 83 system related anti enSE ID NO:
3804..

330 AAG03274 Homo SapiensGEST Human secreted protein,424 100 SEQ ID

NO: 7355.

Table 2 SEQ Accession Species Description Score ID

NO: Number identit 331 gi21309836Trichinellaglutamic acid-rich protein272 22 cNBL1700 s iralis 331 gi9837385 Takifugu retinitis pigmentosa 249 21 GTPase regulator-rubri es like rotein 331 gi16611639Encephalitozospore wall protein 2 247 21 precursor on intestinalis 332 AAB65684 Homo sapiensSUGE- Novel protein kinase,113899 SEQ ID

NO: 212.

332 AAI66821 Homo SapiensMILL- Human protein kinase102698 a al of a tide 13295 codin se uence.

332 AAG65760 Homo SapiensMILL- Human protein kinase102698 of a tide 13295.

333 AAI66821 Homo SapiensMILL- Human protein kinase133210 a al of a tide 13295 codin se uence.

333 AAG65760 Homo sapiensMILL- Human protein kinase133210 of a tide 13295.

333 AAI66820_aHomo sapiensMILL- Human protein kinase133210 al of a tide 13295 encodin cDNA.

334 AAB65663 Homo SapiensSUGE- Novel protein kinase,711399 SEQ ID

NO: 191.

334 AAG67800 Homo SapiensMILL- Amino acid sequence711399 of human rotein kinase 14790.

334 AAH78649- Homo sapiensMILL- Nucleotide sequence710399 of human aal rotein kinase 14790.

335 gi2352277 Homo sa MAP kinase kinase kinase809896 iens 335 gi1504010 Homo SapiensSimilar to Mouse TFIIi-associated779710 transactivator factor pl7(GB RO:MMU11548):
Containing ratein kinase motif 335 11932805 Mus musculusMEK kinase 4b 723489 336 118307483 Homo sa hos hoinositide-bindin 232310 iens roteins 336 g118700711Mus musculusdual-specificity Rho- 206590 and Arf GTPase activatin rotein 1 336 ABB07500 Homo SapiensINCY- Human GTP-binding 847 40 protein GTPB) (ID: 1299273CD1).

337 AAB65607 Homo SapiensSUGE- Novel protein kinase,390998 SEQ ID

NO: 133.

337 16088096 Homo sa rotein kinase PKNbeta 390998 iens 337 1914100 Homo sa rotein kinase PRK2 200650 iens 338 AAM25715 Homo SapiensNYSE- Human protein sequence358999 SEQ

ID N0:1230.

338 g16448792 Rattus activator of G-protein 151891 signaling 3 norve icus 338 118204662 Mus musculusSimilar to LGN rotein 574 43 339 AAB93093 Homo sapiensHELI- Human protein sequence258510 SEQ ID

N0:11941.

339 118307483 Homo sa hos hoinositide-bindin 258510 iens roteins 339 g118700711Mus musculusdual-specificity Rho- 227088 and Arf=GTPase activatin rotein 1 340 AAG74346 Homo SapiensHUMA- Human colon cancer712 96 antigen rotein SEQ ID NO:5110.

340 AAB38475 Homo SapiensHUMA- Fragment of human 504 36 secreted protein encoded by gene 33 clone HACBZ59.

340 112005908 Homo sa AD037 499 37 iens 805A/PCT ~ 02453344 2004-O1-21 Table 2 SEQ Accession Species Description Score ID

NO: Number identit 341 g120071809Mus musculusSimilar to expressed 112687 sequence 341 g113881165MycobacteriuLAO/AO transport system 783 50 kinase m tuberculosis 341 g121324296CorynebacteriPutative periplasmic 735 45 protein kinase um ArgK and related GTPases of G3E

glutamicumfamily 342 120339623 Homo sa KRIT1 isoform 387210 iens 342 19998950 Homo sa an in re eat-containin 387210 iens rotein 342 20 Homo sa krev interaction tra 386499 44278 iens ed 1 343 _ Homo sapiensmembrane-associated guanylate471399 _ kinase-g112003994 related MAGI-3 343 g110945428Homo Sapiensmembrane-associated guanylate465696 kinase 343 g112003992Mus musculusmembrane-associated guanylate445494 kinase-related MAGI-3 344 AAB93844 Homo SapiensHELI- Human protein sequence102376 SEQ ID

NO;13683.

344 g117980216Drosophilarolling pebbles isoform 916 45 melano aster 344 g117980214Drosophilarolling pebbles isoform 916 45 melanogaster 345 g117980216Drosophilarolling pebbles isoform 143247 melano aster 345 g117980214Drosophilarolling pebbles isoform 143247 melano aster 345 g116974692Drosophilarolling pebbles isoform 143247 melano aster 346 g113436428Homo SapiensSimilar to feminization 287285 1 a homolog (C.

ele ans) 346 g13930525 Mus musculussex-determination protein258677 homolog Fem 1 a 346 114318743 Mus musculusfeminization 1 a homolo 258677 (C. elegans 347 g112274842Homo sa bA157P1.1.1 laminin al 20092 100 iens ha 5) 347 120147503 Homo sa laminin al has chain 20032 99 iens recursor 347 a12599232 Mus musculuslaminin al ha 5 chain 15805 79 348 120269788 Homo sa PH domain containin rotein255310 iens 348 AAU 17064 Homo SapiensHUMA- Novel signal tra.nsduction251098 athwa rotein, Se ID 629.

348 AAU17496 Homo SapiensHUMA- Novel signal transduction897 99 athwa rotein, Se ID 1061.

349 ABA96187- Homo SapiensMERE Human hTSSK3 encoding141510 aal cDNA SE ID NO 1.

349 AAM47999 Homo sa MERE Human hTSSK3 SEQ 141510 iens ID NO 2.

349 AAE19154 Homo sapiensTHOR/ Human kinase polypeptide141510 (PKIN-12 .

350 AAB94849 Homo SapiensHELI- Human protein sequence183099 SEQ ID

N0:16030.

350 AAG01248 Homo SapiensGEST Human secreted protein,288 100 SEQ ID

NO: 5329.

350 AAG68345 Homo sapiensBODA- Human integrin 127 92 85 protein SEQ

ID N0:2.

Table 2 SEQ AccessionSpecies Description Score ID

NO: Number identit 351 gi21309836Trichinellaglutamic acid-rich protein272 22 cNBL1700 s iralis 351 gi9837385Takifugu retinitis pigmentosa 249 21 GTPase regulator-rubri es like rotein 351 gi21309834Trichineilaglutamic acid-rich protein242 23 cNBL1500 s iralis 352 117940756Homo sa cask-interactin rotein 631110 iens 2 352 117940760Mus musculuscask-interactin rotein 559690 352 11794 Homo sa cask-interactin rotein 228342 0758 iens 1 353 _ DrosophilaLD29485p 113254 g116769404 melano aster 353 19502080 Mus musculustubb su er-famil rotein 364 29 353 19502082 Homo sa tubb su er-famil rotein 363 28 iens 354 AAB65684 Homo SapiensSUGE- Novel protein kinase,151198 SEQ ID

NO: 212.

354 AAI66821 Homo sapiensMILL- Human protein kinase141198 a al pol peptide 13295 codin se uence.

354 AAG65760 Homo sapiensMILL- Human protein kinase141198 of a tide 13295.

355 AAB85425 Homo SapiensLEXI- Novel human membrane132110 protein (NHP).

355 AAW05732 Homo SapiensUSSH Human metastasis 304 28 tumour su ressor ene KAI1 roduct.

355 1258295 Homo sa C33 antigen 304 28 iens 356 g14689229Rattus b-tomosyn isoform 401963 norveaicus 356 g13790389Rattus m-tomosyn 399163 norve icus 356 g14689231Rattus s-tomosyn isoform 390862 norve icus 357 g119354084Mus musculusSimilar to myosin X 870 43 357 g11755049Bos taurusmyosin X 869 43 357 i6996SS8 Mus musculusm osin X 867 43 358 AAE17499 Homo sapiensINCY- Human secretion 312010 and trafficking rotein-8 (SAT-8).

358 ABB05693 Homo SapiensGEHU- Human cell 312010 signaling/communication protein clone am 2 2013.

358 g114210270Rattus synaptotagmin 3 296595 norve ieus 359 AAM78959 Homo SapiensNYSE- Human protein SEQ 11167 99 ID NO

1621.

3S9 AAM79943 Homo SapiensNYSE- Human protein SEQ 11161 99 ID NO

3589.

359 AAM49177 Homo SapiensHELI- Human MAST205 431862 (hMAST205).

360 121070344Homo sa GAS2-related rotein isoform147598 iens beta 360 121070342Homo sa GAS2-related rotein isoform984 94 iens al ha 360 11666071 Homo sa GAR22 rotein 791 55 iens 361 AAB6S663 Homo sapiensSUGE- Novel protein kinase,854410 SEQ ID

NO: 191.

361 AAG67800 Homo sapiensMILL- Amino acid sequence854410 of human rotein kinase 14790.

361 AAH78649-Homo SapiensMILL- Nucleotide sequence853499 of human aal rotein kinase 14790.

805A/PCT ~ 02453344 2004-O1-21 Table 2 SEQ AccessionSpecies Description Score /~
ID

NO: Number identit 362 g120379563Homo SapiensSimilar to neural precursor314599 cell expressed, developmentally down-reaulated ene 1 362 1286103 Mus musculusnedd-1 rotein 268984 362 AAG74568 Homo SapiensHUMA- Human colon cancer201798 antigen rotein SEQ ID N0:5332.

363 117426439Homo sa bA445O16.1 (DXF34 123310 lens 363 114317890Gallus s indlin 117783 anus 363 AAU11882 Homo SapiensMDSP- Human Ndr-interacting110084 protein, S indlin.

364 AAB56854 Homo SapiensROSEI Human prostate 981 99 cancer antigen rotein se uence SE ID
N0:1432.

364 ABB90390 Homo SapiensHUMA- Human potypeptide 902 98 SEQ ID

NO 2766.

364 g12330663Schizosaccharcoronin-like protein 593 28 om ces ombe 365 AAU76874 Homo sa MERE Human E hA full 253283 lens len th kinase.

365 118694546Homo sa Full len th kinase 253283 lens 365 AAE19158 Homo SapiensTHOR/Human kinase polypeptide247x80 PKIN-16).

366 g113603394Homo sapienstype VI collagen alpha 450699 2 chain recursor 366 i49809Musmusculus al ha-2 colla en 421291 366 i62882Gallus callus a VI colla en subunit 326174 al hat 367 AAE14257 Homo sapiensLEXI- Human calcium-calmodulin243210 de endent rotein kinase.

367 AAEI6267 Homo sa INCY- Human kinase PKIN-13243210 lens rotein.

367 112830367Homo sa serine/threonine kinase 243210 lens 33 368 g117529995Homo Sapiensoxysterol-binding protein-Like491599 protein 368 112382787Homo sa OSBP-related rotein 6; 491599 lens ORP6 368 AAB95255 Homo sapiensHELI- Human protein sequence379099 SEQ ID

N0:17425.

369 AAB64404 Homo sapiensINCY- Amino acid sequence447410 of human intracellular signalling molecule INTRA36.

369 14539084 Homo sa GRIP1 rotein 436410 lens 369 ABB11493 Homo SapiensHYSE- Human GRIP1 protein427498 homolo ue, SEQ ID NO:1863.

370 g117980216Drosophilarolling pebbles isoform 216942 melano aster 370 g117980214Drosophilarolling pebbles isoform 216942 melanogaster 370 g116974692Drosophilarolling pebbles isoform 216942 melanoaaster 371 113097174Homo sa Similar to CGI-130 rotein104710 lens 371 14929729 Homo sa CGI-130 rotein 815 82 lens 371 ABB89176 Homo SapiensHUMA- Human polypeptide 471 100 SEQ ID

NO 1552.

372 116945899Homo sa cortactin-bindin rotein 862710 lens 2 372 g117488611Takifugu Brain ankyrin 2 282740 rubri es 372 AAM54754 Homo SapiensMOLE- Human brain expressed282210 single exon probe encoded protein SEQ ID

NO: 26859.

805t-llrCT CA 02453344 2004-O1-21 Table 2 SEQ AccessionSpecies Description Score ID

NO: Number identit 373 112382789Homo sa OSBP-related rotein 7; 447310 iens ORP7 373 g117529991Homo Sapiensoxysterol-binding protein-like447099 protein 373 g117529995Homo sapiensoxysterol-binding protein-like216849 protein 374 117483737Homo sa ZFP106 981899 iens 374 111493840Homo sa zinc fin er rotein 106 981899 iens 374 13372657 Mus musculuszinc fin er rotein 106 732977 375 AAB99783 Homo SapiensBIOW- Human SHC protein 123410 43 protein SEQ ID N0:2.

375 AAU17199 Homo sapiensHUMA- Novel signal transduction121898 athwa rotein, Se ID 764.

375 ABB16299 Homo SapiensHUMA- Human nervous system506 96 related of a tide SEQ ID NO 4956.

376 AAE02777 Homo sapiensCURA- Human PRO-C-MG.72 473 49 protein encoded by DNA-C-MG.72-1776 cDNA clone.

376 AAB90821 Homo SapiensNOJI/ Human shear stress-response467 48 rotein SEQ ID NO: 150.

376 114625021Homo sa MacGAP 467 48 iens 377 AAY14555 Homo SapiensINCY- Human NADH dehydrogenase229894 subunit 1 rotein.

377 g13337443Homo SapiensNADH-ubiquinone oxidoreductase229894 NDUFS2 subunit 377 g114250796Homo SapiensNADH dehydrogenase (ubiGuinone)229894 Fe-S protein 2 (49kD) (NADH-coenzyme Q reductase 378 g1550013 Homo sa ribosomal rotein L5 653 72 iens 378 g111640568Homo sa MSTP030 653 72 iens 378 g121483852E uus caballusribosomal protein L5 647 71 379 AAB94219 Homo SapiensHELI- Human protein sequence146510 SEQ ID

N0:14579.

379 AAG67148 Homo sapiensINCY- Amino acid sequence117310 of a human en me.

379 g12853081ArabidopsisATP binding protein-like778 59 thaliana 380 AAP90342 Homo sa KAGA Human realbumin. 641 87 iens 380 138b999 Homo sa realbumin recursor 641 87 iens 380 Qi189584 Homo sa realbumin 641 87 iens 381 AAG932S6 Homo sa NISC- Human rotein HP10416.105099 iens 381 AAG89214 Homo SapiensGEST Human secreted protein,103498 SEQ ID

NO: 334.

381 AAM40797 Homo sapiensHYSE- Human polypeptide 377 44 SEQ ID NO

5728.

382 AAG93256 Homo sa NISC- Human rotein HP10416.828 83 iens 382 AAG89214 Homo SapiensGEST Human secreted protein,812 82 SEQ ID

NO: 334.

382 AAM40797 Homo SapiensHYSE- Human polypeptide 208 44 SEQ ID NO

5728.

383 AAG93256 Homo sa NISC- Human rotein HP10416.631 99 iens 383 AAG89214 Homo SapiensGEST Human secreted protein,615 98 SEQ ID

NO: 334.

383 115030010Mus musculusRIKEN cDNA 0610033H09 315 59 ene 384 112803105Homo sa nucleobindin 1 196486 iens 384 11144316 Homo sa nucleobindin 194285 iens Table 2 EQ ID AccessionSpecies Description Score NO: Number identit 384 AAR49667 Homo sa YOSH- Human nucleobindin.191184 iens 385 120987450Homo sa LOC146433 127499 iens 385 AAB44867 Homo SapiensHUMA- Human secreted 130 96 protein encoded by gene 38.

385 11517914 Homo sa monocytic leukaemia zinc97 43 iens fm er protein 386 g120987450Homo sa LOC146433 111910 iens 386 AAB44867 Homo SapiensHUMA- Human secreted 130 96 protein encoded b ene 38.

386 g1643447 Malus x S3-RNase precursor 74 24 domestica 387 g113661132Homo Sapiensnon-biotin containing 292310 ~ subunit of 3-meth Icroton 1 CoA carbo lace 387 g110934059Homo Sapiensnon-biotin containing 2923100 subunit of 3-meth lcrotonyl-CoA carbox lase 387 g113925684Homo sapiens3-methylcrotonyl-CoA 2923100 carboxylase subunit MCCB

388 AAY48524 Homo SapiensMETA- Human breast tumour-257 100 associated rotein 69.

389 AAG74912 Homo SapiensHUMA- Human colon cancer223 85 antigen rotein SE ID N0:5676.

390 AAM79919 Homo SapiensHYSE- Human protein S.EQ177510 ID NO

3565.

390 AAM78935 Homo sapiensNYSE- Human protein SEQ 177510 ID NO

1597.

390 AAB50287 Homo SapiensUYNE- Human schizophrenia174910 related rotein SE ID NO: 21.

391 AAB94276 Homo sapiensHELI- Human protein sequence107010 SEQ ID

N0:14703.

391 g13171934Mus musculusneuronal-STOP rotein 184 34 391 g11370291Rattus STOP protein 183 34 norve icus 392 120073109Mus musculusRIKEN cDNA 1110035L05 350 37 ene 392 AAG74306 Homo sapiensHUMA- Human colon cancer139 35 antigen rotein SEQ ID N0:5070.

392 g19957242Canis progesterone receptor 120 30 familiaris 393 111386113Homo sa FKSG25 266410 iens 393 113548673Homo sa SCOT-t 265899 iens 393 120988313Homo sa 3-oxoacid CoA transferase264410 iens 2 394 ABB84989 Homo SapiensGETH Human PR09821 protein965 100 se uence SE ID N0:346.

394 AAY27573 Homo SapiensHUMA- Human secreted 331 46 protein encoded by ene No. 7.

394 AAY31830 Homo SapiensGEMY Ltuman adult brain 328 54 secreted rotein nh899 8.

395 ABB 14720Homo sapiensHUMA- Human nervous system811 94 related of a tide SEQ ID NO 3377.

395 AAM64934 Homo sapiensMOLE- Human brain expressed177 100 single exon probe encoded protein SEQ ID

NO: 37039.

395 AAU74618 Homo sapiensUYCA- Oestrogen-regulated77 23 famil rotein AX083511 Hs.

396 AAM74834 Homo sapiensMOLE- Human bone marrow 500 93 expressed probe encoded protein SEQ ID NO:

35140.

~~S~CT CA 02453344 2004-O1-21 Table 2 SEQ AccessionSpecies Description Score ID

NO: Number identit 396 AAM62032 Homo sapiensMOLE- Human brain expressed500 93 single exon probe encoded protein SEQ ID

NO: 34137.

396 AAB90685 Homo SapiensI GEMY Human BR595 4 492 95 protein se uence SEQ ID 51.

397 111094311Homo sa brain link rotein-I 186910 iens 397 AABI2304 Homo sapiensHUMA- Human secreted 185699 protein encoded b ene 4 clone HFXHC41.

397 g111094297Rattus brain link protein-1 170591 nerve icus 398 AAB94977 Homo SapiensHELI- Human protein sequence886 100 SEQ ID

N0:16558.

398 118558591Mus musculusvomeronasal rece for 82 22 398 gi~20822105~Mus musculussimilar to vomeronasal 82 22 1 receptor, C3 ref~XP_1447 04.1 399 AAE03765 Homo SapiensHUMA- Human gene 2 encoded408110 secreted protein HCE3C63, SEQ ID

N0:35.

399 g120988899Mus musculussimilar to deleted in 400997 bladder cancer chromosome re ion candidate 399 AAV83819_Homo sapiensCURI- Tumour suppresser 213953 gene aal IB3089A (also known as DBCCRl).

400 AAG03S76 Homo SapiensGEST Human secreted protein,152 81 SEQ ID

NO: 7657.

400 AA006224 Homo SapiensNYSE- Human polypeptide 97 42 SEQ ID NO

20116.

400 AAW76734 Homo SapiensKIRI Human mDia Rho targeting92 51 rotein.

401 AAB74696 Homo SapiensINCY- Human membrane 240510 associated rotein MEMAP-2.

401 1 15420832Homo sa NOE3-3 240510 iens 401 118490927Homo sa olfactomedin 3 239299 iens 402 AAU12240 Homo sapiensGETH Human PR04399 polypeptide245996 se uence.

402 115420828Homo sa NOE3-1 245996 iens 402 119386930Mus musculuso timedin form B 244396 403 ABB 10359Homo sapiensHUMA- Human cDNA SEQ 122188 I:D NO:

667.

403 AAU18038 Homo SapiensHUMA- Human immunoglobulin116085 of a tide SEQ ID No 183.

403 AAU18085 Homo sapiensHUMA- Human immunogIobulin987 86 of a tide SE ID No 230.

404 119071209Homosa CD109 295196 iens 404 AAB 12127Homo sapiensPROT- Hydrophobic domain294596 protein isolated from HT-1080 cells.

404 g120070080Mus musculusGPI-anchored alpha-2 209967 macroglobulin-related rotein 405 AAB94142 Homo sapiensHELI- Human protein sequence328910 SEQ ID

N0:14414.

405 118676648Homo sa FLJ00223 rotein 253999 iens 405 gi590I808DrosophilaBcDNA.GH03694 111641 melano aster 406 114039834Homo sa elon ation factor G1 136610 iens 406 114285174Homo sa elon ation factor G 136610 iens 13.5 Tahle 2 SEQ AccessionSpecies Description Score ID

N~: Number identit 406 gi310102 Rattus elongation factor G 121589 norve ices 407 AAM25864 Homo sapiensHYSE- Human protein sequence146299 SEQ

ID NO:1379.

407 AAB88448 Homo sapiensHELI- Human membrane 142194 or secretory rotein clone PSEC0233.

407 AAY76143 Homo SapiensHUMA- Human secreted 138098 protein encoded b ene 20.

408 110716074Mus musculusM83 rotein 309 33 408 Qi13543090Mus musculusSimilar to transmembrane309 33 rotein 6 408 ABB89854 Homo SapiensHUMA- Human polypeptide 290 31 SEQ ID

NO 2230.

409 g116877172Homo sapienscytochrome P450, subfamily281099 IVF, oly epode 11 409 AAB85779 Homo sapiensINCY- Human drug metabolizing280599 en me (IDNo.6825202CD1).

409 110303605Homo sa CYP4Fl I 280499 iens 410 AAB27230 Homo sapiensINCY- Human EXMAD-8 SEQ 132683 ID NO:

8.

410 g118496364Oncorhynchusotolin-1 581 40 keta 410 118676606Homo sa FLJ0020I rotein 535 41 iens 411 AAB27230 Homo sapiensINCY- Human EXMAD-8 SEQ 149510 ID NO:

8.

411 g118496364Oncorhynchusotolin-1 518 42 keta 41 I g1687606 Lepomis saccular collagen 476 40 macrochirus 412 AAB27230 Homo sapiensdNCY- Human EXMAD-8 SEQ 158890 ID NO:

8.

412 g118496364Oncorhynchusotolin-1 725 46 keta 412 118676606Homo sa FLJ00201 rotein 613 41 iens 413 AAB27230 Homo SapiensINCY- Human EXMAD-8 SEQ 162698 ID NO:

8.

413 g118496364Oncorhynchusotolin-1 583 42 keta 413 118676606Homo sa FLJ00201 rotein 490 37 iens 414 AAU77493 Homo SapiensINCY- Human lipid metabolism213598 enzyme, LMM-1.

414 1505053 Homo sa I sosomal acid 1i ase 115053 iens 414 g1460143 Homo Sapienslysosomal acid lipase/cholesteryl114853 ester h drolase 415 AAG68347 Homo SapiensBODA- Human zinc finger 283910 protein 59 SEQ ID N0:2.

415 AAB08899 Homo SapiensHUMA- :Human secreted 260299 protein sequence encoded by gene N0:56.

415 120146520Homo sa SLTP003 955 100 iens 416 AAB61188 Homo SapiensMILL- Human INTERCEPT 189286 rotein.

416 AAB61190 Homo SapiensMILL- Mature human INTERCEPT178885 217 protein.

416 AAB61191 Homo sapiensMILL- Human INTERCEPT 178093 extracellular domain.

Table 2 SEQ Accession Species Description ~ Score ID

NO: Number _ __ identit 417 AAB30550 Homo SapiensPICO- Amino acid sequence183995 of an al ha-2HS- Tyco rotein precursor.

417 AAY56991 Homo sa FARB Human fetuin oly 183995 iens epode.

417 AAW61492 Homo SapiensPICO- Human fetuin glycoprotein183995 type 2.

418 AAB30550 Homo SapiensPICO- Amino acid sequence185095 of an al ha-2HS- 1 co rotein recursor.

418 AAY56991 Homo sa FARB Human fetuin of 1$5095 iens a tide.

418 AAW61492 Homo sapiensPICO- Human fetuin glycoprotein185095 type 2.

419 AAB30550 Homo sapiensPICO- Amino acid sequence183194 of an al ha-2HS- I co rotein recursor.

419 AAY56991 Homo sa FARB Human fetuin of 183194 iens a tide.

419 AAW61492 Homo SapiensPICO- Human fetuin glycoprotein183194 type 2.

420 AAB94839 Homo SapiensHELL- Human protein sequence315310 SEQ ID

N0:16010.

420 AAB73691 Homo SapiensINCY- Human oxidoreductase315310 protein ORP-24.

420 g118028283Homo Sapiensvery-long-chain acyl-CoA314999 deh dro enase VLCAD

421 g111125672Homo SapiensdJ591C20.1 (novel protein251110 similar to mouse NG26) 421 AAB94489 Homo sapiensHELI- Human protein sequence135057 SEQ ID

NO:15176.

421 AAY91669 Homo SapiensHUMA- Human secreted 135057 protein sequence encoded by gene N0:342.

422 AAD02606- Homo SapiensHYSE- Human angiopoietin,190590 aal CG007a1t1, cDNA.

422 AAB53070 Homo SapiensGETH Human angiogenesis-associated190590 rotein PR0197, SEQ ID
N0:31.

422 AAY72621 Homo SapiensNYSE- Human angiopoietin190590 protein, CG007a1t1. _ 423 1467671 Homo sa ZN-al ha-2- I co rotein 127383 iens 423 i38026Ho rno sa Zn-al hat- 1 co rotein 126483 iens 423 1340442 Homo sa Zn-al ha-2- I co rotein 125983 iens 424 AAE07119 Homo sapiensHUMA- I-3uman gene 12 241198 encoded secreted protein fragment, SEQ ID

N0:136.

424 AAE07062 Homo SapiensHUMA- Human gene 12 encoded24I

secreted protein HE8FD93, SEQ ID

N0:79.

424 AAM99932 Homo SapiensHUMA- Human polypeptide 143198 SEQ ID

NO 48.

425 AAI67788 Homo SapiensUYHA- Human lysyl oxidase-like228699 a al L OXL3) rotein encodin cDNA.

425 AAD24786- Homo sapiensINCY- Human secreted 228699 protein-3 aal (SECP) cDNA.

425 AAA47799_ Homo SapiensMILL- Human lysyl oxidase228699 related aal rotein (Lor)-2 cDNA (CDS).

426 AAB88385 Homo SapiensHELI- Human membrane 157099 or secretory rotein clone PSEC0128.

426 AAD24790- Homo SapiensINCY- Human secreted 154910 protein-7 aal (SECP) cDNA.

Table 2 SEQ AccessionSpecies Description Score /~
ID

NO: Number identit 426 AAA96354 Homo SapiensGETH cDNA encoding a 154910 novel _ oly a tide desi nated aal PR06030.

427 AAY71471 Homo SapiensZYMO Human prostaglandin558 95 s nthase (PD2 s nthase).

427 ai189772 Homo sa rosta landin D2 s nthase558 95 iens 427 112963879Homo sa rosta landin D s nthase 558 95 iens 428 AAB24476 Homo SapiensHUMA- Human secreted 129877 protein sequence encoded by gene NO:101.

428 g112018147Chlamydomonvegetative cell wall 115 46 protein gpl as reinhardtii 428 g17715585StreptococcusPspA 114 31 pneumoniae 429 AAB95763 Homo sapiensHELI- Human protein sequence134110 SEQ ID

N0:18691.

429 AAM40777 Homo sapiensNYSE- Human polypeptide 77 34 SEQ ID NO

5708.

429 gi56625Rattus microtubule associated 76 28 protein 2 norve icus 430 AAE04221 Homo SapiensHUMA- Human gene 5 encoded614 97 secreted protein HUVFB80, SEQ ID

N0:76.

430 AAE04203 Homo sapiensHUMA- Human gene 5 encoded612 97 secreted protein HUVFB80, SEQ ID

N0:57.

430 AAM90448 Homo SapiensHUMA- Human 256 77 immune/haematopoietic antigen SEQ

ID N0:18041.

431 gi~20853599~Mus musculussimilar to dJ287G14.1 144781 (exon of a yet reflXP~1369 unidentified gene, or part of a 24.1 ~ pseudogene?; similar to parts of BMP

and Tolloid proteins) 431 gi~4826463~eHomo sapiensdJ287G14.1 (exon of a 620 99 yet unidentified mb~CAB428 gene, or part of a pseudogene?;
similar 99.1 to arts of BMP and Tolloid roteins) 431 gi~4557503~reHomo sapienscubilin precursor; cubilin;211 36 intrinsic fINP 001072 factor-cobalamin receptor;
intrinsic .l factor B 12-rece for 432 g113774338Homo sapienscytochrome P450 subfamily262499 IIIA

polype tide 43 432 112642642Homo sa cytochrome P450 CYP3A43 262499 iens 432 g111225240Homo sapienscytochrome P450 subfamily261299 IIIA

of a tide 43 433 AAV60292-Homo SapiensVEDA DNA sequence encoding210793 death aal associated protein (DAP)-7 (cathepsin D).

433 AAQ89844-Homo sapiensVEDA Human death associated210793 protein aa1 DAP-7, also called cathe sin D.

433 AAA46901-Homo SapiensGETH cDNA encoding novel210793 aal of a tide PR0292.

434 AAG01648 Homo SapiensGEST Human secreted protein,281 100 SEQ ID

NO: 5729.

434 AAU81958 Homo SapiensGETH Human PR0346. 254 31 434 AAB80266 Homo sa GETH Human PR0346 rotein.254 31 iens Tabh 2 SEQ ID AccessionSpecies Description Score NO: Number identit 435 g13168604Homo Sapiensproline and glutamic 510798 acid rich nuclear rotein isoform 435 AAW31186 Homo sa DAND Human 160 pol eptide472398 iens 160.2.

435 AAW31185 Homo sa DAND Human 160 0l a tide313980 iens 160.1.

436 AAM25961 Homo SapiensHYSE- Human protein sequence832 100 SEQ

ID N0:1476.

436 ABB84895 Homo sapiensGETH Human PR01190 protein650 99 se uence SEQ ID N0:158.

436 AAY99357 Homo SapiensGETH Human PR01190 (L1NQ604)650 99 amino acid se uence SEQ
ID N0:58.

437 110799172Homo sa uterine-derived 14 kDa 742 100 iens rotein 437 119550451Homo sa o us 2 81 42 iens 437 16957462 Homo sa J159A19.3 novel rotein) 75 35 iens d 438 ABB11737 Homo Sapiens_ 598 100 HYSE- Human secreted protein homolo e, SEQ ID N0:2107.

438 AAY02692 Homo SapiensHUMA- Human secreted 459 98 protein encoded b ene 43 clone HTADX17.

438 116356681Homo SapiensCD2 family 10 459 98 439 AAY08326 Homo SapiensSTRD Human granulysin 454 96 P522 active fra went.

439 AAW59874 Homo SapiensHUMA- Amino acid sequence454 96 of the cDNA clone CAT-1 (HTXET53).

439 AAU84278 Homo sapiensBGHM Human endometrial 269 66 cancer related rotein, GNLY.

440 119912826Ciona savilarval mesench me s ecifc130449 n 1 rotein 440 16572165 Homo sa d31119A7.5 novel rotein 546 99 iens (isoform 2)) 440 AAM68752 Homo SapiensMOLE- Human bone marrow 444 100 expressed probe encoded protein SEQ ID NO:

29058.

441 AAB84327 Homo SapiensINCY- Amino acid sequence220299 of a human lyase and associated protein HLYAP-2.

441 AAM93587 Homo SapiensHELI- Human polypeptide,220299 SEQ ID

NO: 3385.

441 AAU83711 Homo SapiensGETH Human PRO protein, 216298 Seq ID No 240.

442 AAU82017 Homo sapiensINCY- Human secreted 101646 protein SECP43.

442 118676716Homo sa FLJ00257 rotein 100345 iens 442 _ Homo SapiensINCY- CDIFF-15, Incyte 764 44 AAB47134 ID No.

3478571 CD 1.

443 14235144 Homo sa BC39498 1 145265 iens 443 g121265141Homo SapiensSimilar to zinc forger 143365 protein 91 (HPF7, HTF 10) 443 19802037 Homo sa zinc fm er rotein SBZ:F3140161 iens 444 111493481Homo sa PR02474 433 100 iens 444 ABB06613 Homo sapiensCUBA- G protein-coupled 68 28 receptor GPCRBa rotein SEQ ID
N0:36.

444 g11171608Plasmodiumrps7 67 38 falci arum 446 112751092Homo sa PNAS-x23 346 100 iens 447 gi65030Xenops ~ transcription factor 94 25 IIF subunit laevis 447 g121428364DrosophilaGM14375p 94 30 melano aster 805A/PCT ~ 02453344 2004-O1-21 Table 2 SEQ Accession Species Description Score ID

NO: Number identit 447 g121430981Xenopus RAP74 subunit of transcription93 25 factor laevis IIF

448 g116265514OdontobutisNADH dehydrogenase subunit?4 36 obscura 448 AAW87504 Homo SapiensSIBI- Human N-methyl-D-aspartate72 30 receptor subunit encoded by clone NMDA24.

448 AAW87503 Homo SapiensSIBI- Human N-methyl-D-aspartate72 30 receptor subunit encoded by clone NMDA22.

449 ABB89065 Homo sapiensHUMA- Human polypeptide 116199 SEQ ID

NO 1441.

449 AAY19743 Homo SapiensHUMA- SEQ ID NO 461 from972 99 W09922243.

449 AAY19541 Homo SapiensHUMA- Amino acid sequence265 100 of a human secreted rotein.

450 AAB47433 Homo sa BIOD- Human zinc-fm er 223510 iens rotein 43.

450 115929737 Mus musculusSimilar to zinc fm er 182954 rotein 347 450 113752754 Homo sa zinc fin er 1111 178850 iens 451 AAB58357 Homo SapiensROSE/ Lung cancer associated465 98 oly a tide se uence SEQ
ID 695.

451 g113569765Giardia variable surface protein73 53 14f intestinalis 451 g113569785Giardia variable surface protein71 53 42d intestinaiis 452 AAG02783 Homo SapiensGEST Human secreted protein,344 100 SEQ ID

NO: 6864.

452 AA013848 Homo SapiensHYSE- Human polypeptide 80 35 SEQ ID NO

27740.

452 1469232 Bos taurusvacuolar H+ATPase subunit70 43 453 1l 1493502Homo sa PR03102 545 100 iens 453 16822268 Mus musculusCIP7 78 36 453 a119070521Homo sa metallothionein 1M 77 29 iens 454 AAG02139 Homo SapiensGEST Human secreted protein,287 98 SEQ ID

NO: 6220.

454 AAM96469 Homo SapiensHUMA- Human reproductive94 95 system related antigen SEQ m NO: 5127.

454 g120151347DrosophilaGH06335p 75 26 melano aster 455 AAU16313 Homo SapiensHUMA- Human novel secreted121598 protein, Se ID 1266.

455 ABB05662 Homo sapiensGEHU- Human signal transduction980 100 rotein clone am 2 10h17.

455 g121040537Homo SapiensSimilar to RIKEN cDNA 137 40 ene 456 1881564 Homo sa ZNF157 104947 iens 456 g1 15787774Homo sapiensbB479F 17.1 (zinc finger104347 protein 157 HZF22 ) 456 116797860 Homo sa ZNF317-2 rotein 104150 iens 457 AAG00579 Homo sapiensLEST Human secreted protein,254 94 SEQ ID

NO: 4660.

458 1202219 Mus musculusal ha-tubulin iso a M-a1ha-6405 100 458 g1213862 Oncorhynchusalpha-tubulin 405 100 m kiss 458 g12843123 Homo Sapiensalpha tubulin ~ 405 100 ~ ~ ~

$O5 A mC.,T CA 02453344 2004-O1-21 Table 2 SEQ Accession Species Description Score ID

N~: Number identit 459 1532688 Horno sa thrombos ondin- 50 292 97 iens 459 1553801 Homo sa Thrombos ondin 139 100 iens 459 1567240 Mus musculusthrombos ~ondin 1 129 57 460 19885325 Homo sa RAGE-1 796 100 iens 460 AAU85524 Homo sa CORI- L552S lun tumour 663 99 iens rotein.

460 AAB76869 Homo SapiensCORI- Human lung tumour 663 99 protein related protein sequence SEQ ID

N0:791.

461 AAU81995 Homo sapierisINCY- Human secreted 171299 protein SECP21.

461 AAU10030 Homo SapiensUYJO Human elongation 171299 of fatty acids (ELF) rotein.

461 g114594722Homo Sapienselongation of very long 171299 chain fatty acids rotein 462 g1159725 Octopus alpha tubulin 274 77 dofleini 462 g12098753 Gecarcinusalpha-2-tubulin 271 75 lateralis 462 g19994 Paracentrotusalpha-tubulin (AA 1-452)271 75 lividus 463 g112314165Homo SapiensbA526D8.4 (novel KRAB 453710 box containing C2H2 type zinc finger rotein 463 AAM79958 Homo SapiensNYSE- Human protein SEQ 235061 ID NO

3604.

463 AAM78974 Homo sapiensNYSE- Human protein SEQ 235061 ID NO

1636.

464 AAB92490 Homo SapiensHELI- Human protein sequence517 60 SEQ ID

N0:10585.

464 AAB92452 Homo sapiensHELI- Human protein sequence129 38 SEQ ID

N0:10484.

464 AAM95329 Homo sapiensHUMA- Human reproductive129 38 system related anti en SEQ ID
NO: 3987.

465 AAE02058 Homo sapiensHUMA- Human four disulfide536 43 core domain (FDCD)-containin rotein.

465 112655452 Homo sa keratin associated rotein495 44 iens 4.7 465 112655456 Homo sa keratin associated rotein471 41 iens 4.9 466 AAM93935 Homo SapiensHELI- Human polypeptide,215610 SEQ ID

NO: 4112.

466 AAG64944 Homo sa SHAN- Human zinc-finger 184 iens protein 44. 010 466 AAM93807 Homo SapiensHELI- Human polypeptide,_ NO: 3849.

467 112314284 Homo sa dJ353C17.1 (novel rotein)628 99 iens 467 g118892440PyrococcusATP-dependentprotease _ 29 LA (lon) 79 furiosus DSM

467 1558671 Homo sa re ulato artner for cdk572 33 iens kinase 468 AAM25660 Homo SapiensNYSE- Human protein sequence711 100 SEQ

ID N0:1175.

468 111493560 Homo sa PR02730 711 100 iens 468 ABB89111 Homo sapiensHUMA- Lluman polypeptide634 100 SEQ ID

NO 1487.

469 AAE12784 Homo SapiensINCY- Human delta 1-pyrroline-5-572 100 carboxylate reductase homologue (PSCRH).

Table 2 SEQ AccessionSpecies Description Score ID

NO: Number identit 469 g115928817Homo SapiensSimilar to pyrroline 572 100 5-carboxylate reductase isoform 469 AAB74779 Homo sapiensUYFU- Human Py-CR protein543 97 SEQ ID

N0:4.

470 Qi2689443Homo sa 828830 2 307510 iens 470 AAM68615 Homo SapiensMOLE- Human bone marrow 297010 expressed probe encoded protein SEQ ID NO:

28921.

470 AAM56237 Homo SapiensMOLE- Human brain expressed297010 single exon probe encoded protein SEQ ID

NO: 28342.

471 g11167849Homo sapiensNAD (H)-specific isocitrate199297 dehydrogenase gamma subunit recursor 471 g1 1673432Homo sapiensNAD(H)-specific isocitrate199297 dehydrogenase gamma-subunit recursor 471 g14096803Homo sapiensNAD+specific isocitrate 199297 dehydrogenase amma subunit recursor 472 17459861 Homo sa Zinc fin er rotein ZNF45961 36 iens 472 11160977 Homo sa zinc fm er rotein 957 36 iens 472 AAB21003 Homo SapiensINCY- Human nucleic acid-binding942 35 rotein, NuABP-7.

473 AAB94397 Homo SapiensHELL- Human protein sequence912 100 SEQ ID

N0:14966.

473 AAR13520 Homo SapiensUYSF- Leukocyte derived 72 34 growth factor analo ue.

473 gi~20823620~Mus musculussimilar to gene 11-1 97 26 protein precursor -reflXP-1442 malaria parasite (Plasmodium 24.1 falci arum) (fra menu 474 19802037 Homo sa zinc fin er rotein SBZF3257699 iens 474 14235144 Homo sa BC39498 1 146161 iens 474 AAM93961 Homo SapiensHELL- Human polypeptide,142759 SEQ ID

NO: 4169.

475 AAB21040 Homo Sapiens1NCY- Human nucleic acid-binding269792 rotein, NuABP-44.

475 13294544 Homo sa C2H2- a zinc fm er rotein129945 iens 475 15757625 Homo sa C2H2 zinc fm er rotein 129945 iens 476 AAM70492 Homo SapiensMOLE- Human bone marrow 611 56 expressed probe encoded protein SEQ ID NO:

30798.

476 AAM58051 Homo SapiensMOLE- Human brain expressed611 56 single exon probe encoded protein SEQ ID

NO: 30156.

476 AAE02058 Homo SapiensHUMA- Human four disulfide563 41 core domain (FDCD)-containin rotein.

477 ABB04717 Homo SapiensSHAN- Human PP1030 protein127089 SEQ ID

NO:S.

477 AAY86431 Homo SapiensHUMA- Human gene 35-encoded230 94 rotein fra ent, SEQ ID
NO:346.

477 AAY86430 Homo SapiensHUMA- Human gene 35-encoded154 91 rotein fra ent, SEQ ID
NO:345.

_ 478 AAY14426 Homo SapiensHUMA-Human secreted protein222 100 encoded b ene 16 clone HSAVP17.

805AlPCT ~ 02453344 2004-O1-21 Table 2 SEQ AccessionSpecies Description Score ID

NO: Number identit 478 AAY14481 Homo SapiensHUMA- Fragment of human 79 100 secreted rotein encoded b ene 16.

478 gi14026136MesorhizobiuABC transporter sugar 66 28 permease m loti 479 112407395Homo sa tri artite motif rotein 116910 iens TRIM7 479 a115150298Homo sa I co enin-interactin 108898 iens rotein 1 479 1 12407397Mus musculustri artite motif rotein 913 84 480 AAB73600 Homo sa SHAN- Zinc fin er rotein290699 iens 57.

480 g16467206Homo Sapiensgonadotropin inducible 146549 transcription re ressor-4 480 AAY58627 Homo sapiensINCY- Protein regulating141948 gene ex ression PRGE-20.

481 AAB94433 Homo sapiensHELI- Human protein sequence373899 SEQ ID

N0:15052.

481 a114764499Homo sa zinc fin er rotein 138044 iens 481 1 1504006Homo sa similarto human ZFY rotein.117243 iens 482 AAG03930 Homo sapiensGEST Human secreted protein,90 50 SEQ ID

NO: 8011.

482 g13116064Squalus s-sgkl 89 44 acanthias 482 g13116066Squalus s-sgk2 83 42 acanthus 483 AAB 12318Homo SapiensHUMA- Human secreted 494 96 protein encoded b ene 18 clone HE2FL70.

483 gi~2827286~gHomo sapiensnovel antagonist of FGF 68 32 signaling b~AAC39567 .1 484 AAY08325 Homo SapiensSTRD Human granulysin 327 76 P520 active fra ent.

484 AAW59874 Homo SapiensHUMA- Amino acid sequence327 76 of the cDNA clone CAT-1 HTXET53).

484 AAR23732 Homo SapiensMINU Gene 519 cDNA derived327 76 a tide.

485 AAG89277 Homo SapiensGEST Human secreted protein,883 100 SEQ ID

NO: 397.

485 a120147667Homo sa ADP-ribos lation factor-like201 31 iens rotein 1 485 113937801Homo sa ADP-ribos lation factor-like201 31 iens 1 486 118151760Homo sa Offenes Leseraster TB7 867 100 iens 486 AAU16453 Homo SapiensHUMA- Human novel secreted650 97 protein, Seq ID 1406.

486 AAU16018 Homo sapiensHUMA- Human novel secreted552 94 protein, Se ID 971.

487 AAE10184 Homo SapiensNYSE- Human bone marrow 332510 derived rotein, SEQ ID NO: 28.

487 118676608Homo sa FLJ00203 rotein 275 22 iens 487 AAB95523 Homo SapiensHELI- Human protein sequence261 21 SEQ ID

N0:18106.

488 AAB93782 Homo SapiensHELI- Human protein sequence252510 SEQ ID

N0:13516.

488 g120809447Homo sapienssimilar to zinc forger 103543 protein 14 (KOX

6); GIOT-4 for gonadotropin inducible transcri tion re ressor-4 488 AAY58627 Homo SapiensINCY- Protein regulating988 44 gene ex ression PRGE-20.

gOS~PCT CA 02453344 2004-O1-21 Table 3 SEQ AccessionDescription Results*
ID

NO: Number 245 BL01159 WW/rs S/WWP domain BL01159 13.85 3.755e-10 roteins. 101-115 245 PR00403 WW DOMAIN SIGNATURE PR00403B 12.19 1.305e-09 245 DM00215 PROLINE-RICH PROTEIN DM00215 19.43 5.881e-09 3. 403-435 246 PR00450 RECOVERIN FAMILY PR00450C 12.22 1.818e-12 SIGNATURE

247 PR00659 CHROMOGRANIN SIGNATUREPR00659B 13.09 9.746e-09 248 BL00115 Eukaryotic RNA polymeriseBLOOI 15Z 3.12 4.176e-09 he to a tide re eat roteins.

249 BL00904 Protein prenyltransferasesBL00904A 8.30 1.574e-09 alpha 628-677 subunit re eat roteins roteins.

249 PD02059 CORE POLYPROTEIN PROTEINPD02059B 24.48 7.136e-10 GAG CONTAINS: P. PD02059B 24.48 5.817e-09 249 PR00049 WILM'S TUMOUR PROTEIN PR00049D 0.00 9.557e-13 SIGNATURE PR00049D 0.00 4.000e-12 PR00049D 0.00 4.000e-12 PR00049D 0.00 4.000e-12 PR00049D 0.00 4.000e-12 PR00049D 0.00 4.000e-12 PR00049D 0.00 4.000e-12 PR00049D 0.00 4.000e-12 PR00049D 0.00 5.125e-12 PR00049D 0.00 5.125e-12 PR00049D 0.00 6.899e-11 PR00049D 0.00 7.126e-11 PR00049D 0.00 9.168e-11 PR00049D 0.00 1.214e-10 PR00049D 0.00 2.071e-10 PR00049D 0.00 2.071e-10 PR00049D 0.00 2.071e-10 PR00049D 0.00 7.857e-10 PR00049D 0.00 1.153e-09 PR00049D 0.00 1.458e-09 PR00049D 0.00 1.610e-09 PR00049D 0.00 5.576e-09 PR00049D 0.00 5.729e-09 PR00049D 0.00 6.797e-09 PR00049D 0.00 6.949e-09 PR00049D 0.00 7.102e-09 249 PR00910 LUTEOVIRUS ORF6 PROTEINPR00910A 2.51 7.429e-09 SIGNATURE PR00910A 2.51 7.536e-09 249 PR00211 GLUTELIN SIGNATURE PR00211B 0.86 8.269e-10 PR00211B 0.86 1.833e-09 PR00211B 0.86 6.250e-09 PR00211B 0.86 7.500e-09 PR00211B 0.86 7.750e-09 249 PR00806 VINCULIN SIGNATURE PR00806A 6.63 8.767e-09 249 DM00215 PROL1NE-RICH PROTEIN DM00215 19.43 6.559e-18 3. 615-647 DM00215 19.43 8.875e-15 DM00215 19.43 2.957e-13 DM00215 19.43 3.739e-13 DM00215 19.43 5.891e-13 DM00215 19.43 9.413e-13 DM00215 19.43 7.563e-12 DM00215 19.43 9.250e-12 I DM00215 19.43 1.353e-11 DM00215 19.43 2.412e-11 805A/PCT ~ 02453344 2004-O1-21 Table 3 SEQ ID AccessionDescription Results*

NO: Number DM00215 19.43 4.353e-11 DM00215 19.43 4.706e-11 DM00215 19.43 5.235e-11 DM00215 19.43 5.588e-11 DM00215 19.43 9.294e-11 DM00215 19.43 9.647e-1 l 607-639 DM00215 19.43 1.321e-10 DM00215 19.43 2.929e-10 DM00215 19.43 5.500e-10 DM00215 19.43 1.153e-09 DM00215 19.43 1.915e-09 DM00215 19.43 2.068e-09 DM00215 19.43 2.831e-09 DM00215 19.43 3.288e-09 DM00215 19.43 3,746e-09 DM00215 19.43 3.898e-09 DM00215 19.43 4.051 e-09 DM00215 19.43 4.203e-09 DM00215 19.43 5,119e-09 DM00215 19.43 5.881 e-09 DM00215 19.43 6.339e-09 DM0021 S 19.43 6.492e-09 DM00215 19.43 6.797e-09 DM00215 19.43 7.254e-09 DM0021 S 19.43 7.559e-09 DM00215 19.43 8.017e-09 DM00215 19.43 9.237e-09 DM00215 19.43 9.237e-09 DM00215 19.43 9,695e-09 250 PR00489 FRIZZLED PROTEIN SIGNATUREPR00489C 9.29 2.250e-28 PR00489E 9.95 4.808e-25 PR00489G 8.99 6.478e-25 PR00489B 13.69 4.273e-24 PR00489A 11.81 7.353e-24 PR00489D 15.68 2,703e-22 PR00489F 14.55 1.675e-21 250 PR00341 PRION PROTEIN SIGNATUREPR00341D 0.26 5.442e-10 PR00341C 0.07 8.043e-10_641-656 250 BL00604 Synaptophysin / synaptoporinBL00604F 5.96 5.014e-09625-669 roteins.

250 DM01724 kw ALLERGEN POLLEN CIM1DM01724 8.14 4.553e-09 HOL-Ll. DM01724 8.14 7.987e-09 250 PR00527 GASTRIN RECEPTOR PR00527I 5.36 8.412e-09 SIGNATURE

250 PR00124 ATP SYNTHASE C SUBUNIT PR00124A 8.81 9.069e-09 SIGNATURE

250 BL00291 Prion protein. BL00291A 4.49 1.867e-10 BL00291A 4.49 4.931e-09 BL00291A 4.49 9.379e-09 250 BL00180 Glutamine synthetase BL00180D 13.26 9.746e-09 roteins. 182-203 251 PR00315 GTP-BINDING ELONGATION PR00315A 11.81 B.OOOe-14 FACTOR SIGNATURE PR00315C 13.85 3.250e-12 251 BL00301 GTP-binding elongation BL00301B 20.09 2.080e-24 factors 139-170 roteins. BL00301A 12.41 5.125e-12 251 BL01176 Initiation factor 2 BL01176B 8.74 7.153e-11 roteins. 136-173 251 PR00449 TRANSFORMING PROTEIN PR00449E 13.50 7.214e-09 805A/PCT ~ 02453344 2004-O1-21 Table 3 SE[ AccessionDescription Itesults*
ID

NO: Number RAS SIGNATURE

253 PF00614 Phospholipase D. ActivePF00614B 14.45 3.294e-09 site proteins 200-219 motifs.

254 BL50002 Src homology 3 (SH3) BL50002A 14.19 4.7SOe-12 domain 332-350 roteins rofile.

254 PR00452 SH3 DOMAIN SIGNATURE PR004S2B 11.65 5.500e-09 256 BL50002 Src homology 3 (SH3) BL500028 15.18 5.200e-10 domain 693-706 roteins rofile.

256 PF00620 GTPase-activator proteinPF00620B 14.20 6.000e-10 for Rho- 372-388 like GTPases.

256 PD00930 PROTEIN GTPASE DOMAIN PD00930B 33.72 4.000e-18 ACTIVATION. PD00930A 25.62 6.684e-10 256 PR00452 SH3 DOMAIN SIGNATURE PR00452D 17.02 2.385e-09 258 PD00930 PROTEIN GTPASE DOMAIN PD00930B 33.72 2.098e-20 ACTIVATION.

258 PF00620 GTPase-activator proteinPF00620B 14.20 4.913e-13 for Rho- 86-102 like GTPases.

259 BL00107 Protein kinases ATP-bindingBL00107B 13.31 1.000e-14 region 329-344 roteins.

259 BL00239 Receptor tyrosine kinaseBL002398 25.15 7.286e-18 class II 191-238 proteins. BL00239E 17.14 5.655e-14 BL00239F 28.15 1.122e-12 259 BL00790 Receptor tyrosine kinaseBL007900 7.68 5.747e-13 class V 308-340 roteins. BL00790Q 15.61 6.400e-11 259 BL00240 Receptor tyrosine kinaseBL00240F 17.74 8.953e-18 class III 300-347 roteins. BL00240G 28.45 8.011e-11 259 PR00452 SH3 DOMAIN SIGNATURE PR00452D 17.02 7.188e-12 PR00452B 11.65 2.000e-09 259 PR00109 TYROSINE KINASE CATALYTICPR00109E 14.414.176e-14 DOMAIN SIGNATURE PR00109D 17.04 9.471e-12 PR00109C 12.85 3.250e-09 2S9 PR00761 BINDIN PRECURSOR PR00761E 14.32 1.758e-09 4.32 4.600e-09 24-42 259 BL50001 Src homology 2 (SH2) _ domain BL50001D 11.00 6.250e-09 roteins rofile.

259 BL50002 Src homology 3 (SH3) BL50002B 15.18 3.769e-11 domain 100-113 roteins rofile. BL50002A 14.19 7.750e-09 260 BL00790 Receptor tyrosine kinaseBL00790B 21.59 1.000e-40 class V 62-113 proteins. BL00790C 16.65 1.000e-40 BL00790E 29.58 1.000e-40 BL00790G 22.06 1.000e-40 BL00790J 14.21 1.000e-40 BL00790K 9.30 1.000e-40 BL007900 7.68 1.000e-40 BL00790R 16.20 1.000e-40 BL00790N 13.25 7.618e-33 BL00790I 20.01 4.094e-28 BL00790D 12.41 2.125e-27 BL00790H 13.42 2.957e-27 BL00790M 8.74 3.483e-27 BL00790L 11.16 2.350e-25 BL00790F 15.90 6.143e-25 BL00790A 19.74 2.688e-18 BL00790P 12.33 1.261e-16 260 BL00240 Rece for rosine kinaseBL00240F 17.74 8.640e-18 class III 787-834 Table 3 SEQ~ AccessionDescription I~esults*
ID

NO: Number roteins. BL00240E 11.56 3.778e-16 260 BL00107 Protein kinases ATP-bindingBL00107A 18.39 6.063e-21 region 748-778 roteins. BL00107B 13.31 1.000e-13 260 PR00109 TYROSINE KINASE CATALYTICPR00109D 17.04 7.158e-23 DOMAIN SIGNATURE PR00109B 12.27 4.706e-20 PR00109C 12.85 5.765e-12 PR00109A 15.00 8.269e-11 260 PR00014 FIBRONECTIN TYPE III PR00014D 12.04 7.545e-13 SIGNATURE PR00014B 14.77 5.154e-11 PR00014C 15.44 8.500e-11 260 BL00239 Receptor tyrosine kinaseBL00239E 17.14 3.813e-26 class II 788-837 proteins. BL00239B 25.15 1.655e-22 BL00239C 18.75 8.263e-13 BL00239D 16.81 8.627e-11 260 BL50001 Src homology 2 (SH2) BL50001B 17.40 8.875e-14 domain 745-765 proteins profile, BL50001C 10.17 2.200e-09 BL50001D 11.00 6.250e-09 260 BL00243 Integrins beta chain BL00243I 31.77 6.704e-09 cysteine-rich 273-315 domain proteins.

260 PD02520 RECEPTOR PRECURSOR PD02520C 10.48 7.266e-09 TRANSMEMBRANE.

261 BL00471 Small cytokines BL00471 23.92 1.000e-40 (intercrine/chemokine) C-x-C

subfamil si nat.

261 PR00437 SMALL CXC CYTOKINE PR00437B 14.81 2.421e-22 FAMILY SIGNATURE PR00437C 14.85 8.579e-19 PR00437A 9.50 3.813e-11 261 PR00436 INTERLEUKIN-8 SIGNATUREPR00436C 10.51 6.382e-09 262 PD00126 PROTEIN REPEAT DOMAIN PD00126A 22.53 3.483e-09 NUCLEA.

263 PR00360 C2 DOMAIN SIGNATURE PR00360A 14.59 8.839e-10 PR00360B 13.61 3.455e-09 264 PR00499 NEUTROPHIL CYTOSOL PR00499D 10.18 1.875e-12 264 BL50002 Src homology 3 (SH3) BL50002A 14.19 3.077e-11 domain 271-289 roteins rofile. BL50002B 15.18 5.800e-10 264 PR00452 SH3 DOMAIN SIGNATURE PR00452B 11.65 5.645e-10 PR00452D 17.02 8.773e-10 265 BL01160 Kinesin light chain BL01160F 9.68 8.161 e-21 repeat proteins. 399-439 BL01160F 9.68 6.243e-17 BL01160E 8.74 6.938e-17 BL01160E 8.74 5.140e-16 BLOT 160E 8.74 7.300e-16 BL01160E 8.74 3.972e-14 BL01160E 8.74 5.075e-I4 BL01160F 9.68 2.017e-13 BL01160F 9.68 4.913e-13 BL01160F 9.68 6.009e-13 BL01160E 8.74 7.300e-13 BL01160C 2.94 1.354e-12 BL01160G 13.67 2.948e-12 BLOI 160F 9.68 6.067e-12 BL01160F 9.68 6.748e-12 BL01160G 13.67 1.089e-1 BL01160G 13.67 4.653e-1 g~~~P~T CA 02453344 2004-O1-21 Table 3 SEQ AccessionDescription Results*
ID

NO: Number BL01160C 2.94 7.614e-11 BL01160E 8.74 9.773e-11 BL01160G 13.67 4.600e-10 BL01160C 2.94 4.971e-10 BL01160I 12.96 7.165e-10 BL01160I 12.96 9.575e-10 BL01160C 2.94 1.503e-09 BL01160G 13.67 4.436e-09 BL01160G 13.67 5.909e-09 BL01160I 12.96 8.241e-09 BL01160I 12.96 9.797e-09 265 PR00381 KINESIN LIGHT CHAIN PR00381D 13.94 1.318e-10 SIGNATURE PR00381E 8.75 7.364e-10 PR00381D 13.94 8.380e-09 PR00381F 9.13 9.010e-09 PR00381E 8.75 1.000e-08 266 PF00646 F-box domain roteins. PF00646A 14.37 3.893e-10 269 PF00642 Zinc finger C-x8-C-x5-C-x3-HPF00642 11.59 4.673e-10 type 312-322 and similar).

270 PR00109 TYROSINE KINASE CATALYTICPR00109B 12.27 5.059e-12 DOMAIN SIGNATURE

270 BL00107 Protein kinases ATP-bindingBL00107A 18.39 1.818e-15 region 1198-1228 roteins. BL00107B 13.31 1.643e-11 270 BL00239 Receptor tyrosine kinaseBL00239B 25.15 5.792e-13 class II 1133-1180 roteins. BL00239E 17.14 2,528e-09 270 PR00578 LATERAL EYE OPSIN PR00578E 9.62 4.447e-09 SIGNATURE

270 BL00240 Receptor tyrosine kinaseBL00240E 11.56 5.286e-09 class III 1184-1221 roteins.

271 BL00107 Protein kinases ATP-bindingBL00107A 18.39 8,650e-17 region 356-386 roteins.

271 BL00239 Receptor tyrosine kinaseBL00239B 25.15 7.545e-13 class II 296-343 roteins.

272 BL00678 Trp-Asp (WD) repeat BL00678 9.67 8.615e-1 proteins 1 1038-1048 proteins. BL00678 9.67 9.400e-10 BL00678 9.67 1.474e-09 BL00678 9.67 3.842e-09 BL00678 9.67 6.684e-09 272 PR00368 FAD-DEPENDENT PYRIDINE PR00368B 12.10 6.760e-09 NUCLEOTIDE REDUCTASE

SIGNATURE

272 PR00320 G-PROTEIN BETA WD-40 PR00320A 16.74 7.353e-14 REPEAT SIGNATURE PRU0320C 13.01 3.000e-12 PR00320B 12.19 1.000e-11 PR00320A 16.74 2.862e-11 PR00320C 13.01 5.304e-11 PR00320A 16.74 6.586e-11 PR00320B 12.19 4.086e-10 PR003208 12.19 6.657e-10 PR00320A 16.74 6.824e-10 PR00320A 16.74 1.000e-09 PR00320C 13.01 1.000e-09 PR00320C 13.01 3.700e-09 PR00320B 12.19 4.600e-09 PR00320B 12.19 5.500e-09 805A/PCT ~ 02453344 2004-O1-21 Table 3 SEQ AccessionDescription Results*
ID

NO: Number PR00320A 16.74 6.707e-09 PR00320C 13.01 7.300e-09 PR00320C 13.01 7.300e-09 13?8-1392 273 BL00678 Trp-Asp (WD) repeat BL00678 9.67 8.615e-11 proteins 711-721 proteins. 8L00678 9.67 9.400e-10 BL00678 9.67 1.474e-09 BL00678 9.67 3.842e-09 BL00678 9,67 6.684e-09 273 PR00368 FAD-DEPENDENT PYRIDINE PR00368B 12.10 6.760e-09 NUCLEOTIDE REDUCTASE

SIGNATURE

273 PR00320 G-PROTEIN BETA VJD-40 PR00320A 16.74 7.353e-14 REPEAT SIGNATURE PR00320C 13.01 3.000e-12 PR00320B 12.19 1.000e-11 PR00320A 16.74 2.862e-11 PR00320C 13.01 5.304e-11 PR00320A 16.74 6.586e-11 PR00320B 12.19 4.086e-10 PR00320B 12.19 6.657e-10 PR00320A 16.74 6.824e-10 PR00320A 16.74 1.000e-09 PR00320C 13.01 1.000e-09 PR00320C 13.01 3.700e-09 PR00320B 12.19 4.600e-09 PR00320B 12.19 S.SOOe-09 PR00320A 16.74 6.707e-09 PR00320C 13.01 7.300e-09 PR00320C 13.01 7.300e-09 274 BL01290 Enhancer of rudimentaryBL01290B 17.01 4.231e-39 proteins. 39-78 BL01290A 11.13 6.226e-19 276 BL00678 Trp-Asp (WD) repeat BL00678 9,67 3.769e-11 proteins 134-144 roteins.

276 PR00320 G-PROTEIN BETA WD-40 PR00320B 12.19 S.SOOe-15 REPEAT SIGNATURE PR00320A 16.74 4.600e-12 PR00320C 13.01 8.435e-11 276 PR00319 BETA G-PROTEIN PR00319B 11.47 8.143e-09 (TRANSDUCIN) SIGNATURE

278 PR00449 TRANSFORMING PROTEIN PR00449A 13.20 9.206e-14 RAS SIGNATURE PR00449D 10.79 6.276e-10 280 PF00791 Domain present in ZO-i PF00791B 28.49 9.053e-12 and UncS- 821-875 like netrin rece tors.

280 PF00023 Ank re eat roteins. PF00023A 16.03 7.750e-10 280 PD02329 KINASE ACETYLGLUTA~\iIATEPD02329B 16.24 4.838e-09 NAG DEHYD.

281 PF00791 Domain present in ZO-I PF00791B 28.49 9.053e-12 and UncS- 773-827 like netrin rece tors.

281 PF00023 Ank re eat roteins. PF00023A 16.03 7.750e-10 281 PD02329 KINASE ACETYLGLUTAMATE PD02329B 16.24 4.838e-09 NAG DEHYD.

282 PF00791 Domain present in ZO-I PF00791B 28.49 9.053e-12 and UncS- 796-850 like netrin rece torn, 282 PF00023 Ank re eat roteins. PF00023A 16.03 7.750e-10 282 PD02329 KINASE ACETYLGLUTAMATE PD02329B 16.24 4.838e-09 NAG DEHYD.

286 BL00107 Protein kinases ATP-bindinBL00107A 18.39 I.OOOe-23 re ion 262-292 DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME l~ DE -NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.

NOTE: For additional volumes please contact the Canadian Patent Office.

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-244.

2. An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.

3. An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 99% sequence identity with the polynucleotide of claim 1.

4. The polynucleotide of claim 1 wherein said polynucleotide is DNA.

5. An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.

6. A vector comprising the polynucleotide of claim 1.

7. An expression vector comprising the polynucleotide of claim 1.

8. A host cell genetically engineered to comprise the polynucleotide of claim 1.

9. A host cell genetically engineered to comprise the polynucleotide of claim operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.

10. An isolated polypeptide, wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide encoded by any one of the polynucleotides of claim 1;
and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-244.

11. A composition comprising the polypeptide of claim 10 and a carrier.

12. An antibody directed against the polypeptide of claim 10.

13. A method for detecting the polynucleotide of claim 1 in a sample, comprising:
a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.

14. A method for detecting the polynucleotide of claim 1 in a sample, comprising:

a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions;
b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.

15. The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA
polynucleotide.

16. A method for detecting the polypeptide of claim 10 in a sample, comprising:

a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.

17. A method for identifying a compound that binds to the polypeptide of claim 10, comprising:

a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.

18. A method for identifying a compound that binds to the polypeptide of claim 10, comprising:

a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.

19. A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of any of the polynucleotides from SEQ ID NO: 1-244, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).

20. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of any one of the polypeptides SEQ ID NO: 245-488.

21. The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.

22. A collection of polynucleotides, wherein the collection comprising of at least one of SEQ ID NO: 1-244.

23. The collection of claim 22, wherein the collection is provided on a nucleic acid array.

24. The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.

25. The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.

26. The collection of claim 22, wherein the collection is provided in a computer-readable format.