CA2401505A1 - Novel nucleic acids and polypeptides - Google Patents

Abstract

Published without an Abstract

Description

NOVEL NUCLEIC ACIDS AND POLYPEPTIDES
1. TECIrINICAL FIELD
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.

2. BACKGROUND
Technology aimed at the discovery of protein factors (including e.g., cytolcines, such as lymphokines, interferons, CSFs, 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 information directly related to the discovered protein (i.e., partial DNA/amino 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 ntunerous 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~rnvention 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 (SBH), 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: 1-172, or 345-S 16. The polypeptides sequences are designated SEQ ID NO: 17, 3-344, or 517-688. The nucleic acids and polypeptides are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A
is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases. 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: 1-172, or 345-516 under stringent 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: 1-172, or 345-516. A
polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-172, or 345-516 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: 1-172, or 345-516. The sequence information can be a segment of any one of SEQ ID NO: 1-172, or 345-516 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-172, or 345-516.
A collection as used in this application can be a collection of only one polynucleotide. 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 is provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readableformat.
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 to 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-readablemedia, 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: 1-172, or 345-516 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: 1-172, or 345-516 or novel segments or parts of the 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 258:52-59 (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: 1-172, or 345-516 ; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID
NO: 1-172, or 345-516 ; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-172, or 345-516. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-172, or 345-516; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing; (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 any of the proteins recited 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 SEQ ID NO: 173-344, or 517-688; or the 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: 1-172, or 345-516; or (b) polynucleotidesthat hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and "substantial equivalents"thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 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.

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.
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 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 process 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., in 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.

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. 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 monoclonal antibodies, and optionally quantitative standards, for carrying 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 not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention. The 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 the 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 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 effect 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 DESCRIPTION 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 axe 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 some of the nucleic acids bind or it may be "complete" such that total complementarity exists between the single stranded 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 ORF 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). One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.
The terms "nucleotide sequence" or "nucleic acid" or "polynucleotide" or "oligonculeotide" 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). It is contemplated that where the polynucleotide is RNA, the T (tlrymine) in the sequences provided herein is substituted with U (uracil). Generally, nucleic acid segments provided by this invention may 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 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 NOs:l-20.
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 Walsh et al. (Walsh, 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: 1-172, or 345-516.
The sequence information can be a segment of any one of SEQ ID NO: 1-172, or 345-516 that uniquely identifies or represents the sequence information of that sequence of SEQ ID
NO: 1-172, or 345-516. 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 1 in 300. In the hmnan 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 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 3 0 be a twenty-five mer. The probability that the twenty-five mer would appear in a human 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 3 5 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 codons 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 500 amino acids, more preferably less than 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-translational modifications of the'polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation 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 ligand-binding affinities, 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, i.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 10 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 ox 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 transcriptional, 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 eulcaryotic.
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 reticulum. "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 SSC/0.1% SDS at 68°C), and moderately stringent conditions (i.e., washing in 0.2X SSClO.l % 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/0.05% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligos), 55°C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).
As used herein, "substantially equivalent" 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 those listed herein by no more than about 35% (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 have 65% 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 embodiment, by no more than 25% (75% 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 rio more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% 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 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98%
identity, and most preferably at least 99% identity. Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code. Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 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 determining equivalence, tnmcation of the matuxe sequence (e.g., via a mutation which creates a spurious stop codon) should be' disregarded. Sequence identity may be determined, e.g., using the Jotun Hein method (Rein, J.

(1990) Methods Enzymol. 183:626-645). 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 axe set forth in the Sequence Listing.
The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-172, or 345-516; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 173-344, or 517-688; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 173-344, or 517-688. 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: 1-172, or 345-516; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing as SEQ ID NO: 173-344, or 517-688; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of airy 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: 173-344, or 517-688.
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 imrnunoglobulin-like proteins include the variable 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., cDNA and genomic DNA, and RNA, e.g., mRNA. The polynucleotides may include all of the 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 genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-172, or 345-516 can be obtained by screening appropriate cDNA or genornic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-172, or 345-516 or a portion thereof as a probe.
Alternatively,the polynucleotidesof SEQ ID NO: 1-172, or 345-516 may be used as the basis for suitable primers) that allow identification 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 are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the 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 sequences of SEQ ID NO: 1-172, or 345-516, or complements thereof, which fragment is greater than about 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 5 differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or 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 specific sequences, but also include allelic and species variations thereof.
Allelic and species variations can be routinely determined by comparing the sequence provided SEQ
ID NO: 1-172, or 345-516, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-172, or 345-516 with a sequence from another isolate of the same species. Furthermore, to accommodate codon variability, the 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 result for the nucleic acids of the present invention, including SEQ ID NO: 1-172, or 345-516, can be obtained by searching a database using an algorithm or a program. Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S.F. J Mol. 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 Fastxy algorithm.
.Species homologs (or orthologs) of the disclosed polynucleotides and proteins are 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 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 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 diffexent 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 adj acent 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 vaxiants 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 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., Gehe 34:315 (1985); and other mutagenesis techniques 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 which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.
Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.
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 of the desired sequence identities.
In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-172, or 345-516, 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 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, N~. 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 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: 1-172, or 345-516or 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: 1-172, or 345-516 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, PsiX174, pBluescript SK, pBs KS, pNHBa, pNHl6a, pNHlBa, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia). Eukaryotic: pWLneo, 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 Kaufinan et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods ira Ehzymology 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 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 arnpicillin resistance gene of E. coli and S cef~evisiae 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 3-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 initiation 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, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Str~eptomyces, 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.
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 intramuscular injection of the DNA. The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of nalced DNA.
4.3 ANTISENSE NUCLEIC ACIDS
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: 1-172, or 345-516, 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: 173-344, or 517-688 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-172, or 345-516 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 which 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: 1-172, or 345-516), 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 preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.
For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA. An antisense oligonucleotide can be, for example, about 5, 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: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-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 ih 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 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 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 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 double-stranded hybrids with complementary RNA in which, contrary to the usual (3-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 a mRNA transcripts to thereby inhibit translation of a 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: 1-172, or 345-516). For example, a derivative of a 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 SECX-encoding 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, SECX
mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter andlor enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N. Y. Acad. Sci.
660:27-36; and Maher (1992) Bioassays 14: 807-15.
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. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.
(1996) Bioorg Med Cher?z 4: 5-23). 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 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) PNAS 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 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. Such chimeras allow DNA
recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA 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-thymidine 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, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioo~g Med Chena Lett 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors ih vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.
U.S.A. 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 can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pha~m. 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 are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.
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, PGT International Publication No. W092/20808, and PCT
International Publication No. W091/09955. 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, aspaxtate 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 construct into the host cell can be effected by calcium phosphate transfection, DEAF-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 heterologous 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, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 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 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 expxess 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 cells, 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 Saccha~omyces cerevisiae, Schizosacchas~onayces pombe, Kluyve~omyces strains, Caudida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimu~ium, 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.
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 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, 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 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
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 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 natL~rally 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 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 I S 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 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 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.
PCTlUS90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.
4.6 POLYPEPTIDES OF THE INVENTION
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: 173-344, or 517-688 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ
ID NO: I-172, or 345-516 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: 1-172, or 345-516 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 173-344, or 517-688 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: 173-344, or 517-688 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: 173-344, or S 17-688.
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 may 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 immunoglobulins for many purposes, including increasing the valency of protein binding sites.
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 mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell. 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.
Protein compositions of the present invention may further comprise an acceptable carxier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The present invention ftu-ther 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 biolbgical 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 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. For example, the methods of the invention include a process for producing a polypeptide in which a host cell 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, Protein Purification.' Principles aid Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A
Laboratory Manual; Ausubel et al., Current Protocols in 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 in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules 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 than 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: 173-344, or 517-688.
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 immunological methodologies may also be easily made by those skilled in the azrt 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 employing an insect expression system. Materials and methods for baculoviruslinsect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Ca'lif., 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 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 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.
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 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 (R1'-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant 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 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.
4.6.1 DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE IDENTITY
AND SIMILARITY
Preferred identity andlor similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity axe 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., Nucleic 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). The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST
Manual, Altschul, S., et al. NCB NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol.
Biol. 215:403-410 (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.
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 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, ifz situ, or i~ vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical 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: 1275-1281 (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 ifa 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 PublicationNo. WO 94112650, PCT International PublicationNo. WO 92/20808, and PCT
InternationalPublicationNo. WO 91/09955. 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 coding sequence, amplification of the marker DNA by standard selection methods results in co-amplificationof 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 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-attachmentregions, 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 polyadenylationsignals, 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 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 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 axe 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 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 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 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.; InternationalApplicationNo.
PCTlUS92/09627 (W093/09222)by Selden et al.; and InternationalApplicationNo. PCT/US90/06436 (W091 /06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.
4.9 TRANSGENIC 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 Iine of animals using homologous recombination [Capecchi, Science 244:1288-1292 (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 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 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 ih 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 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 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.
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 subj ect in need of treatment.
Thus, "therapeutic compositions of the invention" include compositions comprising isolated 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 PGR 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 used 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.
3s 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 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. Kimmel 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 administered 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.
4.10.3 CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION
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. 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 dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DAlG, T10, B9, B9/1 l, BaF3, MC9lG, 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 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); Talcai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol.
145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 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 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 1 pp. 6.8.I-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 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. 173:1205-121 l, 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 Immunology. J. E.
Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc.
Natl. Aced. Sci.
U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11--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; 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 Tlitro 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 GROWTH FACTOR 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 fox 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), 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 andlor 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 stem 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 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 (~Vobus et al., Differentiation, 48: 173-182, (1991); Klug et aL, J. Clin. Invest., 98(I): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Py~i~ciples of Tissue E~gihee~ihg 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 and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
Ih 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: 2316-2321 (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 cytolcines, 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 granulocytes and monocyteslmacrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes 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 conjunction 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: Johansson 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 89:5907-591 l, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Cultl~re 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 Hematopoietic 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, 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 TISSUE GROWTH 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 iu healing of burns, incisions and ulcers.
A polypeptide of the present invention which induces cartilage and/or bone growth in 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 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 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 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 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 tendonlligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament 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 ex 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.
A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, 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/I6035 (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. I. 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 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, urticaria, 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: 501-9), and marine 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 (GVHD). 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, marine models of GVHD (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 promote 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 marine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and marine 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 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 in~rnune 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.

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 S patient. Another method of enhancing anti-viral immune responses would be to 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.
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) of an 1 S 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 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 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. Kmisbeek, 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
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol. 13S:1S64-1572, 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., 3S 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 for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, 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 Inununology, 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; 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, 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 at al., Cell 66:233-243, 1991; Zacharchuk, Journal ofImmunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 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:I I1-117, 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/INIiIBIN ACTIVITY
so 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 fertility 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 I O ~ 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.
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, 1985; Forage et al., Proc. Natl. Acad. Sci.
USA 83:3091-3095, 1986.
4.10.9 CHEMOTACTICICHEMOKINETIC ACTIVITY
A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, fox example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial andlor 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, monocytes 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 or 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 for 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. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta 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:
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 polynucleotidelpolypeptide 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) andlor prohibiting metastasis by reducing tumor cell motility or invasiveness.
Therapeutic compositions of the invention may be effective in adult and pediatric oncology 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 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 (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, squamous cell carcinoma, basal 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 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 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: 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), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTV, Mitomycin, Mitoxantrone HCI, Octreotide, Plicarnycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleulun-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.
Ih 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 RECEPTOR/LIGAND 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 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 ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptorlligand 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), Talcai 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 solution, afFxed 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 appxopriate 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, Cur-f-. Opih.
Biotechv~ol. 8:701-707 (1997). For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol. Biotech~ol, 9(3):205-23 (1998); Hruby et al., Cu~~ 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 i~ 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. ' 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 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 not. The response 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 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 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
5~

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 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, 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 1 S limited to, sepsis, acute pancreatitis, endotoxin shock, cytolcine 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 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, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) 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 thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or 5s 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 inj cries;
(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 nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immlmodeficiency 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, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;
(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) increased production of a neuron-associated molecule in culti.~re or ih 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 Pestronk 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 selectively affect neurons such as amyotrophic 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 Hereditary 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;
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 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 all 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 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 mglkg. The control consists of administering PBS only.
The procedure for testing the effects of the test compound would consist of 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 p.g/kg to 100 mglkg of body weight, with the preferred dose being about 0.1 ~,g/kg to 10 mg/lcg of patient body 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, Ringer'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-l, 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, TNFI, 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 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 Hyl, 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 first 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, lymphokines 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 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 hematopoietic 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
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-malting, 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, 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 barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For 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, 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 sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). 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 suitable coatings. For this purpose, concentrated sugar solutions rrxay 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.
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 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 formulated in conventional manner.
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 rnay 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 compomds 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 caxboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may 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.
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:SV~ consists of VPD diluted 1:l with a 5% 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 dimethylsulfoxide 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, 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 may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ~.g to about 100 mg (preferably about 0.1 ~.g to about 10 mg, more preferably about 0.1 ~.g to about 1 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. When 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 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, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the 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 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, 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 defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised 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 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 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 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 derived growth factor (PDGF), transforming growth factors (TGF-cc 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. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various 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 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 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 DOSAGE
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 EDSO (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 ED;o. Compounds which exhibit high therapeutic 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 EDso with little or no toxicity. The 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 i~ vit~~o 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 SO-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 p.g/kg to 100 mglkg of body weight daily, with the preferred dose being about 0.1 q,gllcg 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 PACKAGING
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 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')2 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 IgGI, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference 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 inununogens. 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:
173, 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 15 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 region of -related protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the humaxi 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 I~yte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, P~oc. 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.
Various procedures known within the art may be used for the production of 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 manlrnal) may be immunized by one or more injections with the 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 imtnunogenic proteins include but are not limited to keyhole limpet hemocyanin, 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 which 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.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immwized 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 (Goding, Monoclonal Antibodies:
Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
Usually, rat or mouse myeloma cell lines axe employed. The hybridoma 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 hybiidomas 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 rnyeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al:, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.
51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against 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, for example, be determined by the Scatchard analysis of Munson and Pollaxd, 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 hybridoma cells can be grown in vivo as ascites 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 procedL~res (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of marine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA 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 marine sequences (U.8. Patent No. 4,816,567;
Morrison, Nature 365, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe r coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
4.13.3 HUMANIZED ANTIBODIES
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 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 GDR 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 which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the 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 ANTIBODIES
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 I~ozbor, 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: 2026-2030) or by transforming human B-cells with Epstein Barr Virus 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);
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. 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/Technolo~y 10, 779-783 (1992));
Lonberg et al.
(Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnolo~y 14, 845-51 (1996)); Neuberger (Nature Biotechnolo~y 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which 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 W094102602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immuno~globulins 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 the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM
as disclosed in PCT publications WO 96133735 and WO 96/34096. This animal produces B cells which 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 antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the 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 J 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. 5,916,771. 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 irnmunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 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 libxaries (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')z fragment; (iii) an 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 are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the so 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 bispecif c antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similax procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO 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 paxt 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 Enzymolo~y, 121:210 (1986).
According to another approach described in WO 96/2701 l, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, owe or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.
tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing laxge 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 s1 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 malting and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jim 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 heterodirners. This method can also be utilized for the production of antibody homodimers. The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) 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 FcyRIII (CD16) 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 HETEROCONJUGATE 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 921200373;
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.
4,676,980.
4.13.8 EFFECTOR FUNCTION ENGINEERING
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. For example, cysteine residues) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus 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:
1191-1195 (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: 2560-2565 (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
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 immwoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, croon, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of radionuclides axe available for the production of radioconjugated antibodies.
Examples include 2iaBi~ i3lh l3iln~ 90Y~ ~d ia6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as I5 bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (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 (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
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 WordPerfect 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: 1-172, or 345-516 or a representative fragment thereof; or a nucleotide sequence at least 95%
identical to any of the nucleotide sequences of SEQ ID NO: 1-172, or 345-516 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer 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. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag 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 may 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 s5 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 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 for 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 seaxches 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 152-41:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense - Olmno, J.
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.
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 su~cient 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. One 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 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 axe 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 are well known in the art.
4.17 MEDICAL IMAGING
ss 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 ih vivo at the target site.
4.18 SCREENING ASSAYS
Using the isolated proteins and polynucleotides 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:
1-172, or 345-516, 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 polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene 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 ant 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 and 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 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 rnRNA itself (antisense - Okano, J.
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 subj ect invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-172, or 345-516. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-172, or 345-516 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.
Any suitable liybridization 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.
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 i~ vitr~o by means of the addition of the appropriate RNA polymerase as T7 or 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 al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY.
Fluorescent ih 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 (265:1981f). 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 PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES
Oligonucleotides, i.e., small nucleic acid segments, may be readily preparedby, 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 standar d synthesizers.
Immobilization can be achievedusing 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.
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 biotinylatedprobes, 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 3 0 surface termed Covalink NH. CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads 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 phosphoramidatebond 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 phosphoramidatebond 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 arias. 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/ul) 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-MeIm7), is then added to a final concentration of 10 mM 1-MeIm~. A
ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.
Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 u1 added per well. The strips are incubated for 5 hours at 50°C. After incubationthe strips are washed using, e.g., Nunc-Immuno 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).
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 8c Maskos), incorporatedherein by reference. This method of preparing an oligonucleotidebound to a support involves attaching a nucleoside 3'-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried 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 photodeprotectionmay 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 linked to Teflon using the method of Duncan & Cavalier (1988) Anal. Biochem.
169(1) 104-8; all references being specificallyincorporatedherein.

To link an oligonucleotideto a nylon support, as describedby Van Ness et al.
(1991), requires activation of the nylon surface via alkylation and selective activation of the 5'-amine of oligonucleotideswith cyanuric chloride.
One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91 (11 ) 5022-6, incorporated herein by reference). These authors used current photolithographictechniques to generate arrays of immobilized oligonucleotideprobes (DNA chips). These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5'-protectedN acyl-deoxynucleosidephosphoramidites, 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 NUCLEIC 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 ~3).
DNA fragments may be prepared as clones in M 13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplificationmethods. 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
fragmentationmethods.
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 for 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 pUC 19 (2688 base pairs). Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI* * digest of pUC 19 that was size fractionatedby a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M 13 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 agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); 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. 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 DNA 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 DNA 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 subarray 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 fixed 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 those skilled in the art upon considerationof 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 incorporatedby reference in their entirety.
S. EXAMPLES
S.Ir EXAIYI(P:1JE t Novel Nucleic Acid Seduences Obtained 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 oligonucleotideprobes (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 subj ected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencerto obtain the novel nucleic acid sequences. In some cases RACE
(Random Amplification of cDNA Ends) was performed to further extend the sequence in the 5' direction.

5.2 EXAIV~II'I~E 2 Assemblage of Novel Nucleic Acids The contigs or nucleic acids of the present invention, designated as SEQ ID
NO: 345-516 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 version 114, gb pri 114, and UniGene version 1 O l ) 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%.
Table 6 sets forth the novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 517-688) of the present invention, and their corresponding nucleotide locations to each of SEQ ID NO: 517-688. Table 6 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:/lfasta.bioch.virginia,edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W.R.
Pearson, Methods in Enzymology, l 83 :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/compositionalproperlies (C. Burge and S. Karlin, J. Mol. Biol., 268:78-94 (1997), incorporatedhereinby 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 EXAlVIPLE 3 Novel Nucleic Acids Using PHRAP (Univ. of Washington) or CAP4 (Paracel), full-length gene cDNA
sequences and their corresponding protein sequences were generated from the assemblage.
Any frame shifts and incorrect stop colons were corrected by hand editing. During editing, the sequence was checked using FASTY and/or BLAST against Genebank. Other computer programs which may have been used in the editing process were phredPhrap and Consed ((University of Washington) and ed-ready, ed-ext and gc-zip-2 (Hyseq, Inc.)). The full-length nucleotide sequences are shown in the Sequence Listing as SEQ ID NO: 1-23. The amino acids are SEQ ID NO: 173-195.
Table 1 shows the various tissue sources of SEQ ID NO: 1-23.

The nearest neighbor results for SEQ ID NO: 1-23 were obtained by a BLASTP
(version 2.0a1 19MP-WashU) search against Genpept release 121 and Geneseq release 200101 (Derwent), using BLAST algorithm. The nearest neighbor result showed the closest homologue for SEQ ID
NO: 1-23 from Genpept . The translated amino acid sequences for which the nucleic acid sequence encodes are shown in the Sequence Listing. The homologs with identifiable functions for SEQ ID NO: 1-23 are shown in Table 2 below.
Using eMatrix software package (Stanford University, Stanford, CA) (Wu et al., J. Comp.
Biol., Vol. 6 pp. 219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions. Table 3 shows the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-values) and the positions) of the signature within the polypeptide 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 domain found, the description, the p-value and the pFam score for the identified domain within the sequence.
The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determine from using Neural Network SignalP V l .l 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 HenrikNielson, 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), incorporatedherein by reference. A maximum S score and a mean S score, as described in the Nielson et as reference, was obtained for the polypeptide sequences. Table 7 shows the position of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.
5.4 EXAMPLE 4 Novel Nucleic Acids 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 sequence was checked using FASTY and/or BLAST against Genbank (i.e. dbEST version 117, gb pri 117, UniGene version 117, Genpept release 117). Other computer programs which may have been used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and gc-zip-2 (Hyseq, Inc.). The full-length nucleotide, including splice variants resulting from these procedures are shown in the Sequence Listing as SEQ ID NOS: 24-137. The corresponding amino acids are SEQ ID NO: 196-309.
Table 1 shows the various tissue sources of SEQ ID NO: 24-137.
The nearest neighbor results for SEQ ID NO: 24-137 were obtained by a BLASTP
version 2.0a1 19MP-WashU search against Genpept release 121 and Geneseq release 200101 (Derwent), using BLAST algorithm. The nearest neighbor result showed the closest homologue for SEQ ID NO: 24-137 from Genpept . The translated amino acid sequences for which the nucleic acid sequence encodes are shown in the Sequence Listing. The homologs with identifiable functions for SEQ ID NO: 24-137 are shown in Table 2 below.
Using eMatrix software package (Stanford University, Stanford, CA) (Wu et al., J. Comp.
Biol., Vol. 6 pp. 219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions. Table 3 shows the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-values) and the positions) of the signature within the polypeptide sequence.
Using the pFam software program (Sonnhanuner 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 domain found, the description, the p-value and the pFam score for the identified domain within the sequence.
The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determine from using Neural Networlc 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 as reference, was obtained for the polypeptide sequences. Table 7 shows the position of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.
5.5 EXAMPLE 5) Novel Nucleic Acids 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 sequence was checked using FASTY and/or BLAST against Genbank (i.e., dbEST version 1 I 8, gb pri 118, UniGene version I 18, Genpept release 118). Other computer programs which may have been used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and gc-zip-2 (Hyseq, Inc.). The full-length nucleotide, including splice variants resulting from these procedures are shown in the Sequence Listing as SEQ ID NOS: 138-172. The corresponding amino acid sequences are SEQ ID NO: 310-344.
Table 1 shoves the various tissue sources of SEQ ID NO: 138-172.
The homology results for SEQ ID NO: 138-172 were obtained by a BLASTP version 2.0a1 19MP-WashU search against Genpept release 121 and Geneseq release 200101 (Derwent), using BLAST algorithm. The nearest neighbor result showed the homologs for SEQ
ID NO:
138-172 from Genpept. The translated amino acid sequences for which the nucleic acid sequence encodes are shown in the Sequence Listing. The homologues with identifiable functions for SEQ ID NO: 138-172 axe shown in Table 2 below.
Using eMatrix software package (Stanford University, Stanford, CA) (Wu et al., J. Comp.
Biol., Vol. 6 pp. 219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions. Table 3 shows the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-values) and the positions) of the signature within the polypeptide 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 domain found, the description, the p-value and the pFam score for the identified domain within the sequence.
The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determine 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 eon 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 as reference, was obtained for the polypeptide sequences. Table 7 shows the position of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.
Table 5 is a correlation table of all of the sequences and SEQ ID Nos.

Tissue RNA SourceLibrary SEQ ID NOS:

Origin Name fibroblast StrategeneLFB001 37 62-63 69 87-89 108 124 126-127 NULL NULL CGSd001 25 54 74 NULL NULL CGSd004 26 80 NULL NULL CGSd005 11 91 166 NULL NULL CGSd006 11 25-26 34 39 60 139 143 160 NULL NULL CGSd009 13 45 NULL NULL CGd007 34 NULL NULL CGd008 34 39 76 adipocytes StratageneADP001 6 8 38 91 95 97 104 108 110 144,-adrenal Clontech ADR002 6 9 14 16 22 27 38-39 41 45 gland 62-63 74 79 81 90 107 111 123 adult BioChain ABR012 38 126 brain adult BioChain ABR013 35 53 brain adult Clontech ABR001 15 17 22 35 72 84 126 brain adult Clontech ABR006 11 22-23 31-32 39 43 52 54-55 brain 81 84 102 110 139 149 168 adult Clontech ABR008 1-4 7-12 l8 22-24 27-28 31-33 brain 40 44-45 50 52-53 55 57-60 62 ' 74 81 84 87-89 93-96 98-99 102 123 130-131 136 142-145 l47-148 adult Clontech ABR011 23 38 brain adult GIBCO AB3001 6 17 19 22 38 52 54-55 62 74 brain 104 122 127-128 131 164 adult GIBCO ABD003 6 10 18 22-23 32 35 38 40 52-53 brain ~ 55 58 62 71-72 74 79-81 94-95 adult InvitrogenABR014 57 62 76 78 96 120-121 161 brain adult InvitrogenABR015 6 37 76 91 99 brain adult InvitrogenABR016 57 63 brain adult Invitrogen.ABT004 10 23 32 40 43 55 58 60 74 84 brain 112 120-121 148 167-168 Tissue RNA SourceLibrary SEQ ID NOS:

Origin Name adult GIBCO AHR001 2 6 14 18-19 22 30 38-39 45 heart 57 60 62-63 66-67 74 76 79 81 adult GIBCO AKD001 2 6-10 14 17-19 22 25 28-29 kidney 38-39 42-45 52 54-55 58 60-63 adult InvitrogenAKT002 1-2 6 20 34 38 41 43 52 62 76 kidney 87-90 94-95 105 138 152-154 adult Clontech ALV003 38 57 76 95 149 liver adult InvitrogenALV002 9 14 22 26 41-42 45 55 62 65 8'7-liver 89 97 111 120-121 141 149 160 adult lung GIBCO ALG001 38 41 54-55 57 62 76 79 81 86 adult lung InvitrogenLGT002 1-3 5 8 10 14 17 19 21-23 25 adult Clontech SPLc01 9 16 52 55 62 81 143 150 153-154 spleen adult GIBCO ASP001 1-2 6 10 18 34 38 40 42 51 57 spleen 72 74-76 78 80-81 86 90-91 95 bladder InvitrogenBLD001 1 15 18 34 51 87-89 95 97 132 bone Clonetech BMD007 57 76 138 marrow bone Clontech BMD001 1-3 6 8-11 13-14 19 21 31 36 marrow 41 50 53-54 57 60-62 69 74 76 bone Clontech BMD004 38 57 76 91 98 marrow bone GF BMD002 9 12 16 19 23 25 27 39 41-44 marrow 59 62 73-74 76 78 81 84 91 94-99 cervix BioChain CVX001 2 6-7 10 17 19-20 23-24 27 34 Tissue RNA SourceLibrary SEQ ID NOS:

Origin Name colon InvitrogenCLN001 23-24 55 65 72 95 120-123 142 diaphragm BioChain DIA002 62 126 153-154 endothelia StrategeneEDT001 1 6-7 10-11 14 16-19 22 24 27 1 cells 37-40 42-43 47 49 52 58 60 62-63 107-l08 120-124 126-133 135-136 esophagus BioChain ES0002 23 fetal Clontech FBR001 60 91 brain fetal Clontech FBR004 1 3 ll 22 32 74 brain fetal Clontech FBR006 1 9-12 14 16 22 29 32-33 36 brain 45 57 59-60 72 80-81 84 91 94 fetal GIBCO HFB001 1-2 6-7 10 13 22-23 27-28 32 brain 39 42 44 48 51-52 54 57-58 60 fetal InvitrogenFBT002 1 3 8 22-23 40 42 62 74 78 80 brain 91 95 98 100-101 108 122 124 fetal InvitrogenFHR001 57 160 heart fetal Clontech FKD001 7 38 40 60 62 85 87-89 95 147 kidney fetal Clontech FKD002 42 95 kidney fetal InvitrogenFKD007 57 62 78 91 95 kidney fetal Clontech FLV004 41 44 57 62 69 72 95 111 149 liver fetal InvitrogenFLV001 3 18 22 27 34 52 55 57-58 76 liver 87-89 91 111 120-121 123 131 fetal Snares FLS001 1-3 5-6 10-11 13-14 16-19 23-24 liver- 26-27 29 31 34-35 38-44 46 50-52 spleen 54 57-65 67-70 72 74 76 78-81 l11-112 117 l20-124 126-128 130-133 l55-156 160-162 165 167-172 fetal Snares FLS002 3 5-6 8-10 13 16-19 23-24 26-27 liver- 34-35 37 41 44-45 50-51 53-55 spleen 58 62-64 68-70 74-75 79 81 84 89 91 93-94 96 98-99 103-l05 Tissue RNA Source Library SEQ ID NOS:

Origin Name fetal Soares FLS003 26 50 53 57 62 76 78 96 149-150 liver-spleen fetal lungClontech FLG001 94-95 115 131 149 168 fetal lungInvitrogen FLG003 1 24 40 57 62 73 76 78 80-81 fetal Invitrogen FMS001 24 27 30 54,-58 90 94 99 119 muscle 131 140 142 147 164 168 fetal Invitrogen FMS002 42 122 155 muscle fetal skinInvitrogen FSK001 1 6 18 22-24 27 43 45 52 55 fetal skinInvitrogen FSK002 1 29 40-41 54-55 62 69 94 96 fetal BioChain FSP001 91 95 spleen infant NULL IBM002 3 11 23 32 128 brain infant NULL IBS001 44 59 120-121 170 brain infant Soares IB2002 3 10-13 18-19 22-23 25 27 32 brain 40 43 54-55 57 59-63 77-78 84 infant Soares IB2003 1 3 23 27 37 40 51 60 62 72 brain 104 108 111 120-123 128 136 leukocytesClontech LUC003 1-2 38 62 76 84 94 97 128 136 leukocytesGIBCO LUC001 2 6-8 11 13-14 16 18-19 22-24 lung 37 62-63 lung Strategene LFB001 1-2 6-7 10 14 19 lymph nodeClontech ALN001 6 19 34 39 62 80 90 93 95 112-114 lymphocyteATCC LPC001 37-38 43 51 55 57 62 70 72 76 macrophageInvitrogen HMP001 113-114 mammary Invitrogen MMG001 1-2 8 13-14 18 22-23 27 33 40 gland 52 55 58-60 70 74 84 86-90 93 Tissue RNA SourceLibrary SEQ ID NOS:

Origin Name melanoma Clontech MEL004 2 6 40 42 51 60 62-63 67 74 mix NULL SUP005 26 40 52 57 62 78 95 149 153-154 mix NULL SUP008 1 57 78 84 120-121 mix NULL SUP009 26 40 44 57 76 78 144-145 153-154 mixed EST CGd010 34 mixed NULL CGd0l2 11 25-26 31 33-34 39 7S 147 mixed NULL CGd013 26 mixed NULL CGd015 25-26 39 44 47 52 148 153-154 mixed NULL CGd016 147 neuron StrategeneNTD001 6 23 47 49 60 74 81 94-95 113-114 neuron StrategeneNTR001 6 59 91 95 128 136 148 neuronal StrategeneNTU001 14 23 55 59 95 100-101 108 111-cells 112 122 132-133 136 151 168 ovary InvitrogenAOV001 1-3 6-9 11 14 16-19 21-23 27-29 100-101 103 105 107-108 l11-115 119-124 126-128 130-132 l35 pituitary Clontech PIT004 2 34 57 62 74 78 gland placenta InvitrogenAPL001 42 59 95 148 placenta InvitrogenAPL002 24 33-34 62 76 87-90 95 99 122 prostate Clontech PRT001 6 17 37-38 54 62-63 65-66 72 rectum InvitrogenREC001 22 24 34 54 95 97 122 168 salivary Clontech SAL001 6 59 87-89 91 107-108 131 138 gland 144-145 150 160 skeletal Clonetech SKMS03 153-154 muscle skeletal Clontech SKM001 1 56-57 81 86 90-91 94 123 153-muscle 155 skeletal NULL S1CMS04 38 muscle skin ATCC SFB001 72 95 fibroblast skin ATCC SFB003 62 95-96 fibroblast -small Clontech SIN001 2 25 33 37-40 54 62 65 70 74 intestine 81 84 86 90 136 143 153-155 spinal Clontech SPC001 3 6 12 37-39 55 57-58 70 74 cord 91 94-95 98 104 113-114 126 stomach Clontech ST0001 1 25 33 35 39 43 74 95 132 testis GIBCO ATS001 1 14 17-18 38 44 46 60 74 76 (thalamus Clontech THA002 5 18 22 24 41 43 52 65 84 99 Tissue RNA SourceLibrary SEQ ID NOS:

Origin Name thymus Clonetech THM001 6 9 18-19 34 38 41-42 45 52-54 thymus Clontech THMc02 1 3 7 9 16 23 27 35 40 44 51-53 thyroid Clontech THR001 1 3 6 16-19 22 29 35 38 42 45 gland 54 56 58 60-63 74 76 81 84 87-89 trachea Clontech TRC001 5 69 76 80 90-91 93 113-114 umbilical BioChain FUC001 6-7 10-11 14 22 29 41 43 51 cord 74 76 78 80-81 91 94 97 105 uterus Clontech UTR001 37 52 67 76 81 95 108 112 118 young GIBCO ALV001 2 26 29 38 52 54 63 72 74 95-97 liver 99 104 108 111 120-121 123 149 SEQ ACCESSION SPECIES DESCRIPTION SMITH- % IDENTITY

ID NUMBER WATERMAN

NO: SCORE

1 W80293 Homo SapiensHuman translocation1016 96 associated protein designated Gp25L-H.

2 M74089 Homo sapiensTB 1 2226 99 3 D88827 Homo Sapienszinc finger protein447 46 4 234818 Homo Sapiensvoltage-dependent1363 96 L-type Ca channel alpha 1 subunit U60269 Homo Sapiensputative envelope370 98 protein;

orf similar to env of Type A

and Type B retroviruses and to class II HERVs 6 247087 Homo SapiensRNA polymerase 865 100 II

elongation factor-like protein 7 AL078461 Homo sapiensdJ90108.1 (continues782 97 in dJ1121G12 (AL109965)) 8 X93357 Mus musculushomolog of human 870 53 SYT

9 AF227156 Homo SapiensRRN3 1679 98 AC004908 Homo Sapienssimilar to ribosomal377 94 protein L23a; similar to P29316 (PID:g132848) 11 U10556 SaccharomycesYhr085wp 175 31 ' cerevisiae 12 M29581 Homo sapienszinc finger protein2949 99 8 (ZFP8) 13 M64788 Homo SapiensGTPase activating1235 55 protein 14 AK026016 Homo sapiensunnamed protein 1846 100 product 600637 Homo sapiensHuman secreted 166 64 protein, SEQ ID NO: 4718.

16 AF201941 Homo SapiensDC8 1226 100 17 283826 Homo SapiensdJ473B4.1 (novel 1083 100 protein similar to predicted human and worm genes) 18 AB031052 Homo Sapiensvaccinia related 2521 100 kinase 3 19 AL050091 Homo Sapienshypothetical protein1011 100 AF090931 Homo SapiensPR00483 137 65 21 U39320 Rattus norvegicuscysteine string 747 68 protein 22 Y66718 Homo sapiensMembrane-bound 1554 70 protein PR01106.

24 AE003612 Drosophila CG12393 gene product244 36 melanogaster V00662 Homo SapiensATPase 6 232 100 ' 26 T12920_cdlHomo Sapiens11-AUG-1994 Human731 100 serum albumin gene with restriction site between domains 2 and 3.

27 AL109804 Homo sapiensdJ1009E24.3 (A 920 100 novel protein) 28 AL021918 Homo Sapiensb34I8.1 (Kruppel 649 54 related Zinc Finger protein 184) 29 601131 Homo SapiensHuman secreted 666 99 protein, SEQ ID NO: 5212.

Y73373 Homo SapiensHTRM clone 9218031225 100 protein sequence.

31 AJ293573 Homo Sapienszinc finger protein2243 100 Cezanne SEQ ACCESSION SPECIES DESCRIPTION SMITFI- % IDENTITY
ID NUNISER WATERMAN
NO: SCORE

32 AF157634 Homo sapienscollapsin response2943 99 mediator protein-5 33 J00287 Homo Sapienspepsinogen 2032 100 34 AF264014 Homo Sapiensscavenger receptor4523 99 cysteine-rich type 1 protein precursor 35 AK024433 Homo SapiensFLJ00023 protein 918 100 36 AF119855 Homo SapiensPR01847 253 71 37 AF176667 Xenopus F-box protein 426 80 laevis 28 38 M85148 Macaca mulattacytochrome oxidase178 68 subunit III

39 883119 Homo sapiensHuman cisplatin 396 98 resistance protein.

40 X58521 Homo Sapiensnucleoporin p62 2068 99 41 Y86211 Homo SapiensNuclear transport1432 87 protein clone hfb066 protein sequence.

42 AL035587 Homo SapiensdJ475N16.3 (novel1243 99 protein similar to RPL7A
(60S
ribosomal protein L7A)) 43 AF233522 Homo Sapiensgamma-adaptin 3196 100 related protein, GGA2 44 AF151066 Homo sapiensHSPC232 1513 93 45 AF113539 Homo sapienshypothalamus protein1647 99 46 AF090901 Homo sapiensPR00195 120 60 47 J02459 bacteriophageD (head-DNA 561 99 lambda stabilization;110) 48 AF068294 Homo SapiensHDCMB45P 234 72 49 J02459 bacteriophageR (cell lysis;l58)834 100 lambda 50 U71363 Homo Sapienszinc finger protein396 47 zfp6 51 AL022393 Homo Sapiensp373c6.1 2751 99 52 AF257330 Homo SapiensCOBW-like protein1188 99 53 Y07010 Homo SapiensBreast cancer 767 98 associated antigen precursorsequence.

54 AL158056 Schizosaccharomsimilar to yeast 813 25 yces pombe Ecm29 cell wall stucture/biosynthesis protein 55 Y44559 Homo SapiensHuman Rhotekin 2721 100 protein.

56 D86639 Mus musculusstac 360 38 57 V00488 Homo Sapiensalpha globin 382 98 58 AL033378 Homo SapiensdJ323M4.1 (KIAA07906030 100 protein) 59 AB026111 Homo SapiensXAB2 4470 99 60 AB043550 Mus musculusneural activity-related3852 99 ring forger protein 61 D38112 Homo SapiensATPase subunit 260 98 62 X15187 Homo Sapiensprecursor polypeptide4112 100 (AA -21 to 782) 63 D50369 Homo Sapienslow molecular 383 98 mass ubiquinone-binding protein 64 AF161356 Homo sapiensHSPC093 126 58 65 211502 Homo Sapiensintestine-specific1426 99 annexin 66 J03941 Mus musculusferritin heavy 962 100 chain 67 AB034206 Homo SapiensMCT-1 962 100 68 AF118086 Homo SapiensPR01992 107 58 69 AF182416 Homo SapiensMDS015 1739 97 70 603240 Homo sapiensHuman secreted 108 56 ~ protein, SEQ ACCESSION SPECIES DESCRIPTION SMITH- % IDENTITY

ID NUMBER WATERMAN

NO: SCORE

SEQ ID NO: 7321.

71 600397 Homo SapiensHuman secreted 161 62 protein, SEQ ID NO: 4478.
' 72 X76538 Homo sapienshMpvl7 569 100 73 AC005626 Homo sapiensR29124_l 1199 97 74 601332 Homo sapiensHuman secreted 407 87 protein, SEQ ID NO: 5413.

75 Y53883 Homo SapiensA suppressor of 1250 100 cytokine signalling protein designated HSCOP-3.

76 M34059 Homo Sapiensbeta-globin 962 100 78 V00488 Homo Sapiensalpha globin 733 100 79 Z11907_cdlHomo sapiens19-JAN-1999 Human1282 100 potassium channel I~+Hnov28 cDNA
(5' splice variant 1).

80 U47924 Homo SapiensC10 627 100 81 AF134894 Homo SapiensSWAP-70 homolog 3019 100 82 AB042027 Mus musculusGolgi-associated 4806 93 band 4.1-like protein 83 603793 Homo SapiensHuman secreted 209 71 protein, SEQ ID NO: 7874.

84 X83301 Homo SapiensSMAS 754 97 85 V00662 Homo sapiensURF 2 (NADH 211 91 dehydrogenase subunit) 86 W88627 Homo SapiensSecreted protein 230 69 encoded by gene 94 clone HPMBQ32.

87 Y27607 Homo SapiensHuman secreted 1461 100 protein encoded by gene No. 41.

88 Y27607 Homo SapiensHuman secreted 1340 94 protein encoded by gene No. 41.

89 Y94295 Homo SapiensHuman coenzyme 1068 100 A-utilising enzyme CoAEN-3.

90 AL080169 Homo Sapienshypothetical protein973 98 91 D14531 Homo Sapiens'human homologue 974 100 of rat ribosomal protein L9' 93 AC006465 Homo Sapienssupported by mouse437 100 EST

AA277724 (NID:g1917632) and Genscan 94 AL137798 Homo SapiensdJl 182A14.5.1 2075 94 (novel gene (isoform 1)) 95 M77232 Homo Sapiensribosomal protein706 97 96 AF261717 Homo SapiensSARI 1032 100 97 X06256 Homo Sapiensintegrin alpha 5541 100 5 subunit precursor 98 AC004472 Homo SapiensP1.11659_5 542 98 99 W85459 Homo SapiensSecreted protein 1650 98 encoded by clone dh1135_9.

100 AF201333 Homo Sapiensdelta-tubulin 2380 100 101 AF201333 Homo Sapiensdelta-tubulin 1345 100 102 AB036836 Homo SapiensCarbonic anhydrase-related1740 100 protein 10 104 W80748 Homo SapiensHuman mitochondrial1102 100 chaperone protein (Hmt-GrpE).

105 600376 Homo sapiensHuman secreted 130 73 protein, SEQ ID NO: 4457.

106 Y99444 Homo sapiensHuman PR01575 1241 94 (LTNQ781) amino acid SEQ ACCESSION SPECIES DESCRIPTION SMITH- % IDENTITY
ID NUMBER WATERMAN
NO: SCORE

sequence SEQ ID
N0:358.

107 603787 Homo sapiensHuman secreted 232 74 protein, SEQ ID NO: 7868.

108 AJ237734 Homo Sapiensribophorin II 2619 100 110 Y99349 Homo SapiensHuman PRO 1110 1683 100 (UNQ553) amino acid sequence SEQ ID
N0:31.

111 AF258341 Homo SapiensNADPH-cytochrome 3540 99 reductase I AB044547 Homo Sapiensheterogeneous 3178 99 12 nuclear ribonucleoprotein L

113 AF067804 Homo SapiensHDCMC04P 2124 94 114 AF067804 Homo SapiensHDCMC04P 2219 97 115 U73778 Homo Sapienscollagen type 16043 99 XII alpha-1 116 602922 Homo SapiensHuman secreted 120 69 protein, SEQ ID NO: 7003.

118 603133 Homo SapiensHuman secreted 175 59 protein, SEQ ID NO: 7214.

119 AFI 15345 Homo sapienscalcium-regulated282 100 heat stable protein 120 AF265228 Homo sapiensDNMT1 associated 2422 100 protein-121 AF265228 Homo sapiensDNMT1 associated 1320 85 protein-122 AB018189 Xenopus N027 907 76 laevis 123 AB033603 Homo sapienscop9 complex subunit1364 100 7a 124 AL121805 Drosophila BACN4L24.f 200 32 melanogaster 125 Y03000 Homo SapiensFragment of human153 72 secreted protein encoded by gene 121.

126 D38112 Homo SapiensNADH dehydrogenase448 90 subunit 4L

127 AL050091 Homo sapienshypothetical protein1011 100 128 AK023702 Homo Sapiensunnamed protein 2774 100 product 129 702459 bacteriophageM (tail component;109)578 100 lambda 130 AF116272 Homo SapiensT-cell activation532 85 protein 131 W74836 Homo SapiensHuman secreted 655 100 protein encoded by gene 108 clone HEBEK93.

132 AF146793 Mus musculusPDCL2 705 58 133 702459 bacteriophageW (head-tail joining;68)336 100 lambda 134 AB011539 Homo SapiensMEGF6 131 58 135 600827 Homo SapiensHuman secreted 523 98 protein, SEQ ID NO: 4908.

136 Y35969 Homo SapiensExtended human 1777 99 secreted protein sequence, SEQ ID
NO. 218.

137 AB007861 Homo sapiensKIAA0401 1856 100 138 AK000529 Homo sapiensunnamed protein 1027 91 product 139 A17783 unidentifiedNC28 583 100 140 Y73373 Homo SapiensHTRM clone 921803561 100 protein sequence.

141 D00762 Homo Sapiensproteasome subunit686 93 142 X76091 Homo SapiensDNA binding protein3747 99 143 AK000004 Homo SapiensFLJ00004 protein 3106 96 144 Y73377 Homo SapiensHTRM clone 1645941852 41 SEQ ACCESSION SPECIES DESCRIPTION SMITH- % H)ENTITY

H) NUMBER WATERMAN

NO: SCORE

protein sequence.

145 Y73377 Homo SapiensHTRM clone 1645941591 45 protein sequence.

146 U 12329 Cricetulus mutant sterol 373 94 griseus regulatory element binding protein-2 147 U80735 Homo sapiensCAGF28 3779 97 148 Y91577 Homo SapiensHuman secreted 155 , 96 protein sequence encoded by gene 2 SEQ ID N0:250.

149 X00129 Homo Sapiensprecursor RBP 1005 96 150 X97675 Homo Sapiensplakophilin 2a 4288 99 151 J02459 bacteriophageI (tail component;223)1111 99 lambda 152 Y94702 Homo SapiensHuman androgen 839 100 shutoff gene 3 (AS3) protein sequence.

153 X95325 Homo SapiensDNA-binding protein1975 100 154 AF 171061 Canis familiarisY-box protein 1426 89 ZONAB-A

155 X99584 Homo sapiensSMT3A protein 522 98 156 Y99397 Homo sapiensHuman PR01298 1554 100 (UNQ666) amino acid sequence SEQ ID
N0:210.

157 X78926 Homo Sapienszinc finger protein426 50 158 J02459 bacteriophageE (capsid component;341)1754 100 lambda 159 602922 Homo SapiensHuman secreted 160 48 protein, SEQ ID NO: 7003.

160 AF157317 Homo SapiensAD-015 protein 869 100 161 Y36495 Homo sapiensFragment of humanI I 1 67 secreted protein encoded by gene 27.

162 Y94870 Homo SapiensHuman protein 1707 100 clone HP02545.

163 AF204172 Homo Sapienspopeye protein 1872 9,8 164 B01391 Homo SapiensNeuron-associated376 58 protein.

165 299259 Schizosaccharomputative phosphotransferase396 43 yces pombe 166 U28831 Homo Sapiensprotein that is 518 55 immuno-reactive with anti-PTH

polyclonal antibodies 167 AF225419 Homo SapiensHSCARG 1546 100 168 X04412 Homo Sapiensplasma gelsolin 4101 100 169 X13482 Homo SapiensU2 snRNP-specific1284 99 A' protein (AA 1-255) 170 X59618 Homo Sapienssmall subunit 1389 100 ribonucleotide reductase 171 AC004882 Homo Sapienssimilar to cytochrome326 100 Bcl J

chain; similar to 1BGY

(PID:g4139401) 172 AL034374 Homo SapiensdJ483K16.1.1 (novel1651 100 protein (isoform 1)) SEQ ACCESSION DESCRIPTION RESULTS
ID

NO: NUMBER

1 PFOl 105 emp24/gp25L/p24 PF01105B 25.12 1.257e-39170-222 family. .

2 BL00215 Mitochondria) BL00215B 10:44 1.900e-09 energy 481-494 transfer proteins.

3 PD00066 PROTEIN ZINC-FINGERPD00066 13.92 6.400e-16 427-440 METAL-BINDI. PD00066 13.92 7.923e-15 371-384 PD00066 13.92 4.600e-14 343-356 PD00066 13.92 3.500e-13 399-412 4 PR00167 CALCIUM CHANNEL PR00167D 13.86 3.400e-34 SIGNATURE PR00167E 20.54 2.800e-27 PR00167F 16.75 4.000e-20 PR00167G 10.89 5.050e-16 PR00167D 13.86 3.778e-12 6 BL00517 Ribonuclease III BL00517C 7.29 9.386e-10 98-112 family proteins.

8 PR00209 ALPHA/BETA GLIADINPR00209B 4.88 5.500e-09 325-344 FAMILY SIGNATURE

PR00925 NONHISTONE PR00925B 3.73 3.089e-10 11-24 CHROMOSOMAL

FAMILY SIGNATURE

12 PD01066 PROTEIN ZINC FINGERPD01066 19.43 8.953e-31 27-66 ZINC-FINGER METAL-BINDING NU.

14 PF00521 DNA PF00521G 18.47 7.904e-09 gyrase/topoisomerase IV, subunit A.

18 BL00107 Protein kinases BL00107A 18.39 7.207e-11 binding region proteins.

21 PR00625 DNAJ PROTEIN PR00625A 12.84 2.750e-19 FAMILY SIGNATURE PR00625B 13.48 4.490e-15 22 PR00927 ADENINE PR00927B 14.66 6.262e-11 NUCLEOTIDE PR00927E 14.93 3.160e-09 SIGNATURE

26 PR00802 SERUM ALBUMIN PR00802C 12.28 2.370e-11 FAMILY SIGNATURE PR00802G 14.57 8.734e-09 28 PR00048 C2H2-TYPE ZINC PR00048A 10.52 5.500e-13 FINGER SIGNATURE PR00048A 10.52 7.429e-13 PR00048A 10.52 1.000e-1135-49 PR00048B 6.02 6.538e-11 137-147 PR00048A 10.52 6.684e-11 PR00048B 6.02 2.421 e-09 30 PR00988 URIDINE KINASE PR00988A 6.39 8.905e-09 2-20 SIGNATURE

31 BL00315 Dehydrins proteins.BL00315A 9.35 8.119e-09 41-69 33 PR00792 PEPSIN (A1) ASPARTICPR00792A 11.54 6.250e-23 PROTEASE FAMILY PR00792D 12.74 5.200e-18 SIGNATURE PR00792B 12.78 7.750e-14 PR00792C 9.10 1.000e-12 274-286 34 BL00420 Speract receptor BL00420B 22.67 3.089e-38 repeat 408-463 proteins domain BL00420B 22.67 8.875e-38 proteins. 618-673 BL00420B 22.67 6.143e-24 BL00420B 22.67 7.692e-23 SEQ ACCESSION DESCRIPTION RESULTS
ID

NO: NUMBER

BL00420B 22.67 3.778e-21 BL00420B 22.67 7.253e-15 BL00420C 11.90 1.900e-12 BL00420C 11.90 3.700e-12 BL00420C 11.90 9.250e-12 39 BL00667 Respiratory-chain BL00667A 20.80 1.000e-40 dehydrogenase subunit proteins.

41 BLOT 160 Kinesin light chainBL01160B 19.54 8.703e-10 repeat 407-461 proteins. BL01160B 19.54 2.373e-09 42 BL00634 Ribosomal protein BL00634 34.38 7.796e-20 proteins.

50 PD01066 PROTEIN ZINC FINGERPD01066 19.43 9.842e-2416-55 ZINC-FINGER METAL-BINDING NU.

51 PD00066 PROTEIN ZINC-FINGERPD00066 13.92 8.200e-16 METAL-BINDI. PD00066 13.92 4.462e-15 PD00066 13.92 8.615e-15 PD00066 13.92 5.200e-14 PD00066 13.92 3.000e-13 53 BLOI 160 I~inesin light BLOT I60B 19.54 8.093e-09 chain repeat 586-640 proteins.

54 PF00956 Nuclesosome assemblyPF00956B 23.14 2.534e-09 protein (NAP).

56 BL00479 Phorbol esters BL00479A 19.86 6.4DOe-13 diacylglycerol BL00479B 12.57 5.860e-10 binding 116-132 domain proteins.

57 BL01033 Globins profile. BL01033A 16.94 3.250e-20 58 PR00910 LUTEOVIRUS ORF6 PR00910A 2.51 4.321 e-09 PROTEIN SIGNATURE

60 BL00518 Zinc finger, C3HC4BL00518 12.23 9.143e-10 type 65-74 (RING finger), proteins.

61 BL00449 ATP synthase a BL00449C 13.17 3.813e-21 subunit 32-55 proteins.

62 BL00298 Heat shock hsp90 BL00298A 10.97 1.000e-40 proteins 74-119 family proteins. BL00298E' 27.30 1.000e-40 BL00298F 11.21 1.000e-40 BL00298H 20.50 1.000e-40 BL00298C 16.40 2.286e-40 BL00298B 15.64 1.290e-39 BL00298G 24.57 5.345e-39 BL00298I 30.07 7.818e-34 BL00298D 17.97 6.226e-33 65 BL00223 Annexins repeat BL00223C 24.79 l .000e-40 proteins 208-263 domain proteins. BL00223B 28.47 9.679e-39 BL00223A 15.59 6.000e-27 BL00223C 24.79 1.000e-16 BL00223A 15.59 2.306e-16 66 BL00540 Ferritin iron-bindingBL00540A 15.06 1.000e-40 regions proteins. BL00540B 18.82 1.000e-40 BL00540C 13.00 7.500e-15 73 DM00372 CARCINOEMBRYONIC DM00372A 19.18 2.800e-22 ANTIGEN PRECURSOR DM00372B 20.31 2.523e-19 AMINO-TERMINAL

DOMAIN.

SEQ ACCESSION DESCRIPTION RESULTS
ID

NO: NUMBER

74 PDOl 168 SYNTHETASE LIGASEPD01168L 9.47 4.500e-09 369-384 PROTEIN ALANYL.

76 BL01033 Globins profile. BL01033A 16.94 7.923e-18 BL01033B 13.81 1.000e-15 78 BL01033 Globins profile. BL01033A 16.94 3.250e-20 BL01033B 13.81 7.000e-14 79 PR00169 POTASSIUM CHANNELPR00169A 16.77 2.075e-11 SIGNATURE

81 PF00992 Troponin. PF00992A 16.67 6.921e-09417-452 82 PR00935 BAND 4.1 PROTEIN PR00935D 10.20 7.833e-09 FAMILY SIGNATURE

87 BL00166 Enoyl-CoA BL00166D 22.87 7.250e-20 hydratase/isomeraseBL00166C 18.93 2.537e-19 proteins. BL00166B 16.92 5.500e-09 88 BL00166 Enoyl-CoA BL00166D 22.87 7.250e-20 hydratase/isomeraseBL00166C 18.93 2.537e-19 proteins. BL00166B 16.92 5.500e-09 89 BL00166 Enoyl-CoA BL00166D 22.87 7.250e-20 hydratase/isomeraseBL00166B 16.92 5.500e-09 proteins.

91 BL00700 Ribosomal proteinBL00700B 18.26 1.000e-40162-201 proteins 2. BL00700D 21.51 1.000e-40 BL00700A 19.34 9.308e-33 BL00700C 11.76 4.000e-13 95 BL00578 Ribosomal proteinBL00578B 15.15 1.000e-40 S6e 39-90 proteins. BL00578A 14.24 2.800e-37 96 BL01020 SAR1 family proteins.BL01020C 15.35 1.000e-40 BL01020D 19.09 8.071e-40 BL01020B 11.70 6.750e-38 BL01020A 11.87 1.400e-28 97 BL00242 Integrins alpha BL00242C 16.86 4.176e-30 chain 319-349 proteins. BL00242E 9.03 3.864e-27 1000-1029 BL00242D 13.57 4.706e-26 BL00242D 13.57 7.407e-13 BL00242A 13.80 2.500e-11 BL00242B 8.13 4.913e-11 222-232 BL00242D 13.57 3.700e-10 100 BL00227 Tubulin subunits BL00227C 25.48 2.525e-23 alpha, 114-166 beta, and gamma BL00227F 21.16 5.610e-12 proteins. 400-454 101 BL00227 Tubulin subunits BL00227C 25.48 2.525e-23 alpha, 114-166 beta, and gamma BL00227F 21.16 5.610e-12 proteins. 298-352 102 BL00162 Eukaryotic-type BL00162C 17.78 1.000e-19126-163 carbonic anhydrases proteins.BL00162E 14.93 5.922e-16 BL00162D 15.06 7.864e-12 BL00162F 22.68 7.750e-11268-302 BL00162A 22.92 3.282e-10 104 BL01071 grpE protein. BL01071A 24.88 2.674e-23 BL01071B 18.21 6.447e-16191-215 106 PR00002 BONE MATRIX GLA PR00002A 11.56 1.750e-16 DOMAIN SIGNATURE PR00002B 8.36 7.632e-13 500-511 111 PR00369 FLAVODOXIN PR00369D 20.20 7.261e-16 SIGNATURE PR00369A 11.29 7.600e-14 PR00369C 11.27 8.313e-12 PR00369B 13.29 1.600e-11 112 PD02784 PROTEIN NUCLEAR PD02784B 26.46 4.713e-16 SEQ ACCESSION DESCRIPTION RESULTS
ID

NO: NUMBER

RIBONUCLEOPROTE1N.

113 PF00856 SET domain proteins.PF00856B 16.42 3.302e-17 PF00856A 26.14 3.714e-15 114 PF00856 SET domain proteins.PF00856B 16.42 3.302e-17 PF00856A 26.14 3.714e-15 115 PR00453 VON WILLEBRAND PR00453A 12.79 5.737e-17 FACTOR TYPE A PR00453A 12.79 3.571 e-16 DOMAIN SIGNATURE PR00453A 12.79 5.125e-15 PR00453A 12.79 1.563e-13 PR00453B 14.65 7.667e-13 PR00453B 14.65 7.818e-12 PR00453B 14.65 9.455e-12 PR00453B 14.65 1.243e-11478-493 PR00453C 12.26 4.462e-10 PR00453C 12.26 9.308e-10 122 DM00668 ZEIN. DM00668C 8.89 8.794e-09 24-48 126 PF00420 NADH- PF00420B 16.51 4.150e-31 ubiquinone/plastoquinonePF00420A 16.63 3.571 e-24 oxidoreductase chain 4L.

128 BL00405 43 Kd postsynapticBL00405G 7.78 9.919e-10 285-322 protein.

134 PR000I TYPE III EGF-LIKEPROOOl 1D 14.03 2.108e-10 SIGNATURE PROOO11B 13.08 6.055e-09 PROOO11A 14.06 6.178e-09 139 BL00472 Small cytokines BL00472C 20.76 6.850e-34 (intercrine/chemokine)C-BL00472BI4.678.579e-1544-62 C subfamily signatur.BL00472A 7.45 2.895e-11 11-23 140 PR00988 URIDINE KINASE PR00988A 6.39 8.905e-09 2-20 SIGNATURE

141 BL00388 Proteasome A-typeBL00388A 23.14 5.500e-39 subunits proteins.BL00388B 31.38 1.3 10e-27 BL00388C 18.79 3.842e-10121-143 142 PD02699 PROTEIN DNA- PD02699C 24.84 1.000e-40 BINDING BINDING PD02699A 8.91 3.250e-35 235-264 DNA. PD02699B 18.28 6.571e-21 143 DM01970 0 kw ZK632.12 DMOI970B 8.60 2.612e-13 551-564 ENDOSOMAL III.

144 PR00624 HISTONE HS PR00624G 4.08 6.900e-09 180-200 SIGNATURE

146 BL00038 Myc-type, 'helix-loop-BL00038B 16.97 9.027e-10 helix' dimerization domain proteins.

147 PR00209 ALPHA/BETA GLIADINPR00209B 4.88 4.494e-12 393-412 FAMILY SIGNATURE

149 PR00179 LIPOCALIN PR00179B 9.56 2.895e-13 128-141 SIGNATURE PROOI79A 13.78 3.250e-1140-53 PR00179C 19.02 6.040e-11 152 PR00929 AT-HOOK-LIKE PR00929C 5.26 8.759e-09 55-66 DOMAIN SIGNATURE

153 BL00352 'Cold-shock' DNA-bindingBL00352B 23.66 5.886e-25 domain proteins. BL00352A 12.19 1.429e-17 154 BL00352 'Cold-shock' DNA-bindingBL00352B 23.66 5.886e-25 domain proteins. BL00352A 12.19 1.429e-17 155 BL00299 Ubiquitin domain BL00299 28.84 2.250e-25 32-84 I ~ proteins.

SEQ ACCESSION DESCRIPTION RESULTS
ID

NO: NUMBER

156 PF00534 Glycosyl transferasesPF00534B 14.47 8.364e-15 group 1.

157 BL00028 Zinc finger, C2H2BL00028 16.07 8.615e-11 46-63 type, domain proteins.

164 BL00880 Acyl-CoA-binding BL00880 17.52 8.875e-34 46-96 protein.

168 BL00740 MAM domain proteins.BL00740B 19.76 3.813e-09 ~ 690-711 170 BL00368 Ribonucleotide BL00368A 36.98 1.000e-40 reductase 84-139 small subunit BL00368B 22.06 5.846e-26 proteins. 161-187 172 BL01188 GNS1/SUR4 family BL01188C 22.65 6.667e-10 proteins.

SEQ PFAM NAME DESCRIPTION p-value PFAM
ID SCORE
NO:

1 EMP24_GP25Lemp24/gp25L/p24 family 6.4e-90 312.2 2 mito_carr Mitochondria) carrier 1.1 e-093 6.2 proteins 3 SCAN SCAN domain 1e-56 201.9 4 ion_trans Ion transport protein S.Se-260877.1 6 Skpl Skpl family 2.2e-64 227.3 12 zf C2H2 Zinc finger, C2H2 type 3.2e-53 190.2 13 Rap GAP Rap/ran-GAP 5.4e-117402.1 17 MSP_domain MSP (Major sperm protein)2.9e-10 47.5 domain 18 pkinase Eukaryotic protein kinase0.022 12.5 domain 21 DnaJ DnaJ domain 2.4e-34 127.5 22 mito_carr Mitochondria) carrier 1.1 e-85296.7 proteins 24 PH PH domain 2.4e-14 56.3 26 transport_protSerum albumin family 2.4e-06 11.0 28 zf C2H2 Zinc finger, C2H2 type 2.7e-22 87.5 31 zf A20 A20-like zinc finger 0.0056 23.3 32 DihydroorataseDihydroorotase-like 7.6e-88 305.3 33 asp Eukaryotic aspartyl protease5.7e-211714.3 34 SRCR Scavenger receptor cysteine-rich4.3e-187634.9 domain 37 F-box F-box domain. 0.089 19.3 39 NADHdh NADH dehydrogenases 1.3e-39 145.0 42 Ribosomal_L30Ribosomal protein L30p/L7e1.2e-10 40.0 43 G_Adapt Gamma-adaptin, C-terminus1.7e-67 237.6 CT

50 KRAB KRAB box 7.4e-12 52.8 51 SCAN SCAN domain 5.4e-67 236.0 55 PH PH domain 1.1e-06 28.9 56 SH3 SH3 domain 1.8e-14 61.5 57 globin Globin 2.1e-26 96.5 60 NHL NHL repeat 1.7e-46 167.9 61 ATP-synt ATP synthase A chain 1.3e-10 48.7 A

62 HSP90 Hsp90 protein 0 1548.4 -65 annexin Annexin 1.9e-77 270.7 66 ferritin Ferritins 4.1e-114386.1 67 PUA PUA domain 1.7e-16 68.2 69 DUF34 Domain of unknown fimction1.2e-33 125.2 73 ig Immunoglobulin domain 0.0015 16.5 75 ExonucleaseExonuclease 0.076 -29.3 76 globin Globin 5.9e-53 189.1 78 globin Globin 4.6e-57 203.0 79 I~_tetra K+ channel tetramerisation5.6e-22 86.4 domain 81 PH PH domain 2.2e-21 81.4 82 Band_41 FERM domain (Band 4.1 1.1e-22 73.0 family) 85 oxidored_qlNADH-Ubiquinone/plastoquinone3.6e-06 25.3 (complex I) 87 ECH Enoyl-CoA hydratase/isomerase4.1e-52 186.6 family 88 ECH Enoyl-CoA hydratase/isomerase4.1 e-52186.6 family 89 ECH Enoyl-CoA hydratase/isomerase1.3e-20 81.9 family 91 Ribosomal_L6Ribosomal protein L6 9e-99 341.6 95 Ribosomal Ribosomal protein S6e 1.8e-90 314:0 S6e 96 arf ADP-ribosylation factor 5.4e-98 339.0 family 97 FG-GAP FG-GAP repeat 1.4e-77 271.2 SEQ PFAM NAME DESCRIPTION p-value PFAM
LD SCORE
NO:

100 tubulin Tubulin/FtsZ family 1.8e-49 177.8 101 t~.ibulin Tubulin/FtsZ family 1.8e-49 177.8 102 carb_anhydraseEukaryotic-type carbonic 2.3e-48 174.1 anhydrase 104 GrpE GrpE 5.9e-70 245.8 111 FAD_bindingFAD binding domain 7.2e-122418.3 112 rrm RNA recognition motif. 9.7e-12 52.4 113 SET SET domain 3.3e-34 127.1 114 SET SET domain 3.3e-34 127.1 115 fn3 Fibronectin type III domain2.6e-292984.5 126 oxidored_q2NADH-ubiquinone/plastoquinone1.8e-44 161.2 oxidoreduct 132 Phosducin Phosducin 0.097 9.7 139 IL8 Small cytokines (intecrine/chemokine),3.6e-38 131.6 inter 141 proteasome Proteasome A-type and 6.1 e-39142.8 B-type 142 RFX_DNA RFX DNA-binding domain 4.1e-43 156.7 bin ding 143 RhoGEF RhoGEF domain 8.1e-64 225.4 146 HLH Helix-loop-helix DNA-binding1.4e-05 31.9 domain 147 BRCT BRCAl C Terminus (BRCT) 1.2e-28 108.6 domain 149 lipocalin Lipocalin / cytosolic 2.5e-41 146.4 fatty-acid binding pr 150 Armadillo Armadillo/beta-catenin-like7.8e-10 46.1 seg repeats 153 CSD 'Cold-shock' DNA-binding 1.1e-33 124.6 domain 154 CSD 'Cold-shock' DNA-binding 1.1e-33 124.6 domain 156 Glycos_transfGlycosyl transferases 6.7e-39 142.7 1 group 1 157 zf C2H2 Zinc finger, C2H2 type 0.00041 27.1 162 ig Immunoglobulin domain 1 e-06 26.7 164 ACBP Acyl CoA binding protein 1.1e-39 145.2 165 DUF60 Domain of unknown function2.2e-36 134.3 168 Gelsolin Gelsolin repeat. 9.3e-109374.7 169 LRR Leucine Rich Repeat 0.00025 27.8 170 ribonuc_redRibonucleotide reductases1.5e-122358.6 172 GNS 1 SUR4 GNS llSUR4 family 2.7e-09 -26.6 SEQ ID SEQ ID SEQ ID SEQ ID Priority docketSEQ ID NO:
NO: of NO: NO: NO: number_correspondingin full- of full-lengthof contigof contigSEQ ID NO: USSN 091515,126 length peptide nucleotidepeptide in priority nucleotidesequence sequence sequence application sequence 1 173 345 517 788CIP2 3 788_9798 2 174 346 518 788CIP2 5 788_10429 3 175 347 519 788CIP2 6 788_10672 4 176 348 520 788CIP2 7 788_10828 177 349 521 788CIP2 8 788_10879 6 178 350 522 . 788CIP2 9 788 11026 ' _11045 8 180 352 524 788CIP2 11 788_11381 9 181 353 525 788CIP2 12 788_11481 182 354 526 788CIP2 13 788_11927 ' 11 183 355 527 788CIP2 788_12700 12 184 356 528 788CIP2 15 788_12706 13 185 357 529 788CIP2 16 788_12741 14 186 358 530 788CIP2 17 788_12744 187 359 531 788CIP2 18 788_13017 16 188 360 532 788CIP2 20 788_13633 17 189 361 533 788CIP2 21 788_13637 18 190 362 534 788CIP2 22 788_13645 19 191 363 535 788CIP2 23 788_13695 192 364 536 788CIP2 24 788_13756 21 193 365 537 788CIP2 25 788_13858 22 194 366 538 788CIP2 26 788_13886 23 195 367 539 788CIP2 27 788_14039 24 196 368 540 788CIP2B 2 788_31 197 369 541 788CIP2B 3 788_54 26 198 370 542 788CIP2B 4 788_58 27 199 371 543 788CIP2B 5 788_768 28 200 372 544 788CIP2B 6 788_1718 29 201 373 545 788CIP2B 7 788_1748 202 374 546 788CIP2B 8 788_1751 31 203 375 547 788CIP2B 9 788_1850 32 204 376 548 788CIP2B 10 788_1946 33 205 377 549 788CIP2B 11 788_1967 34 206 378 550 788CIP2B 12 788_1994 207 379 551 788CIP2B 13 788_2331 36 208 380 552 788CIP2B 14 788_2725 37 209 381 553 788CIP2B_15 788_2735 38 210 382 554 788CIP2B 16 788_2961 39 211 383 555 788CIP2B 18 788_3039 212 384 556 788CIP2B 19 788_3287 41 213 385 557 788CIP2B 20 788_4305 42 214 386 558 788CIP2B 21 788_4356 43 215 387 559 788CIP2B 22 788_4389 44 216 388 560 788CIP2B 24 788_4458 217 389 561 788CIP2B 25 788_4685 46 218 390 562 788CIP2B 26 788_4825 47 219 391 563 788CIP2B 27 788_4829 ~

SEQ ID SEQ ID SEQ ID SEQ ID Priority docketSEQ ID NO:
NO: of NO: NO: NO: number correspondingin full- of full-lengthof contigof contigSEQ ID NO: USSN 09/515,126 length peptide nucleotidepeptide in priority nucleotidesequence sequence sequence application sequence 49 221 393 565 788CIP2B 29 788_4917 ' _4943 52 224 396 568 788CIP2B 32 788_5175 53 225 397 569 788CIP2B 33 788_5254 54 226 398 570 788CIP2B 34 788_5566 55 227 399 571 788CIP2B 35 788_5725 56 228 400 572 788CIP2B 36 788_5794 ' _5841 59 231 403 575 788CIP2B 39 788_5922 60 232 404 576 788CIP2B 40 788_5942 61 233 405 577 788CIP2B 41 788_5980 62 234 406 578 788CIP2B 42 788_6025 63 235 407 579 788CIP2B 43 788_6151 64 236 408 580 788CIP2B 44 788_6849 65 237 409 581 788CIP2B 45 788_7191 66 238 410 582 788CIP2B 46 788_7281 67 239 411 583 788CIP2B 47 788_8402 68 240 412 584 788CIP2B 48 788_9636 69 241 413 585 788CIP2B 49 788_9645 70 242 414 586 788CIP2B 50 788_9784 ' _10223 73 245 417 589 788CIP2B 53 788_10369 74 246 418 590 788CIP2B 54 788_10413 75 247 419 591 788CIP2B 55 788_10441 76 248 420 592 788CIP2B 56 788_10607 77 249 421 593 788CIP2B 57 788_10625 78 250 422 594 788CIP2B 58 788_10857 79 251 423 595 788CIP2B 59 788_10868 80 252 424 596 788CIP2B 60 788_10962 81 253 425 597 788CIP2B 61 788_11218 82 254 426 598 788CIP2B 62 788_11224 83 255 427 599 788CIP2B 63 788_11251 84 256 428 600 788CIP2B 64 788_11262 85 257 429 601 788CIP2B 65 788_11597 86 258 430 602 788CIP2B 66 788_11708 87 259 431 603 788CIP2B 67 788_11719 88 260 432 604 788CIP2B 68 788_11719 89 261 433 605 788CIP2B 69 788_11719 90 262 434 606 788CIP2B 70 788_11823 91 263 435 607 788CIP2B 71 788_11865 92 264 436 608 788CIP2B 72 788_11939 93 265 437 609 788CIP2B 73 788_11961 94 266 438 610 788CIP2B 74 788_12119 95 267 439 611 788CIP2B 75 788_12128 96 268 440 612 788CIP2B 76 788_12190 97 269 441 613 788CIP2B 77 788_12212 98 270 442 614 788CIP2B 78 788_12233 SEQ ID SEQ ID SEQ ID SEQ ID Priority docketSEQ ID NO:
NO: of NO: NO: NO: number correspondingin full- of full-lengthof contigof contigSEQ ID NO: USSN 09/515,126 length peptide nucleotidepeptide in priority nucleotidesequence sequence sequence application sequence 100 272 444 616 788CIP2B 80 788_12518 101 273 445 617 788CIP2B 81 788_12518 102 274 446 618 788CIP2B 82 788_12525 103 275 447 619 788CIP2B 83 788_12594 104 276 448 620 788CIP2B 84 788_12737 105 277 449 621 788CIP2B 85 788_12818 .

106 278 450 622 788CIP2B 86 788_12825 107 279 451 623 788CIP2B 87 788_12868 108 280 452 624 788CIP2B 88 788_13029 109 281 453 625 788CIP2B 89 788_13031 110 282 454 626 788CIP2B 90 788_13037 I11 283 455 627 788CIP2B 91 788_13051 112 2$4 456 628 788CIP2B 92 788_13061 113 285 457 629 788CIP2B 93 788_13105 114 286 458 630 788CIP2B 94 788_13105 115 287 459 631 788CIP2B 95 788_13131 116 288 460 632 788CIP2B 96 788_13281 117 289 461 633 788CIP2B 97 788_13325 ' 118 290 462 634 788CIP2B 788_13453 119 291 463 635 788CIP2B 99 788_13557 120 292 464 636 788CIP2B 100 788_13638 121 293 465 637 788CIP2B 101 788_13638 122 294 466 638 788CIP2B 102 788_13655 123 295 467 639 788CIP2B 103 788_13656 124 296 468 640 788CIP2B 104 788_13668 125 297 469 641 788CIP2B 105 788_13681 126 298 470 642 788CIP2B 106 788_13725 127 299 471 643 788CIP2B 107 788_13796 128 300 472 644 788CIP2B 108 788_13802 129 301 473 645 788CIP2B 109 788_13814 130 302 474 646 788CIP2B 110 788_13816 131 303 475 647 788CIP2B 111 788_13836 132 304 476 648 788CIP2B 112 788_13852 133 305 477 649 788CIP2B 113 788_13855 ' 134 306 478 650 788CIP2B 788_13990 135 307 479 651 788CIP2B 115 788_14056 136 308' 480 652 788CIP2B 116 788_14066 137 309 481 653 788CIP2B 117 788_14070 138 310 482 654 788CIP2C 1 788_360 139 311 483 655 788CIP2C 2 788_1175 140 312 484 656 788CIP2C 3 788_3964 141 313 485 657 788CIP2C 4 788_5522 142 314 486 r 658 788CIP2C 5 788_5537 143 315 487 659 788CIP2C 6 788_5573 144 316 488 660 788CIP2C 7 788_5868 145 317 489 661 788CIP2C 8 788_5868 146 318 490 662 788CIP2C 9 788_6265 147 319 491 663 788CIP2C 10 788_7013 148 320 492 664 788CIP2C 11 788_7017 149 321 493 665 788CIP2C 12 788_7019 SEQ ID SEQ ID SEQ ID SEQ ID Priority docketSEQ ID NO:
I NO: NO: NO: NO: number correspondingin of full-of full-lengthof contigof contigSEQ ID NO: USSN 091515,126 I lengthpeptide nucleotidepeptide in priority nucleotidesequence sequence sequence application 1i sequence ' _8816 153 325 497 669 788CIP2C 16 788_8916 154 326 498 670 788CIP2C 17 788_8916 ' _9067 157 329 501 673 788CIP2C 20 788_9254 158 330 502 674 788CIP2C 21 788_9285 159 331 503 675 788CIP2C 22 788_10049 160 332 504 676 788CIP2C 23 788_10858 161 333 505 677 788CIP2C 24 788_11054 162 334 506 678 788CIP2C 25 788_11872 1163 335 507 679 788CIP2C 26 788_12230 164 336 508 680 788CIP2C 27 788_12369 j 165 337 509 681 788CIP2C 28 788_12464 1166 338 510 682 788CIP2C 29 788_12708 167 339 511 683 788CIP2C 30 788_13024 1168 340 512 684 788CIP2C 31 788_13199 1169 341 513 685 788CIP2C 32 788_13601 170 342 514 686 788CIP2C 33 788_13666 I 171 343 515 687 788CIP2C 34 788_13749 SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, f=Stop peptide sequence codon, l=possible nucleotide deletion, \=possible sequence nucleotide insertion 517 A 40 359 LKIPMQFLHSGFWFSFFVFFGF*KFGFGP

QGGRQGG\NKTKGEKLPPGKKKIPGPNP

QENREKKGPPKTLKKFGNLKKKGGKTR

GPRGEKNSDPKGTPGQNPGNKKK

DGFDGLGYRGGARDEQGFGGAFPARSFI

TGSDLGHWVTTLTDI\PGRRN\LHWGVK

SPPYGVPTNCTPYEGPTEDPFDSGSG

GWLAAPSCFGFSCHFLKKGFLGPGTVAH

SCNPSTLGGRGGWI/T/RGQEIKTILANTV

KP

KKLPGTASPEKKQE\LVDEPAVGESKEE

KIELKSITADGESPPASKINMDALQPNEN

EDKSPYPTPESTGEEDEEEPEMPVGPRPR

PLSELHLKEKTVPMPEASAC

521 A 1 , 451 KINTELQTEVAMLKSMVLWLGEQVQSL

QLQQQLRHHFNHIHICVTNSEYNQSEYP

WDLVKAHLQGAFTSNITFDTGEFQNTIID

STK*PHDL*ASLDDCS*I*DGLSILASVLM

LAAPVLALQTPSWMLFMALPVSLSRLK

AYFLVSVLS

522 A 354 549 IFWVGKNALHEDE*NTERRTDDIPVWDH

EFLKVDQGTLFELILAANYLYIRGWLDV

TCKTGANMI

523 A 448 489 YARSAIGLLVFQSLMLVAAGDQFLFRP*

GE*RQPR* *LKMTKHPPNRRGISFEVGAQ

LEARDRLKNWYPAHIEDIDYEEGKVLIH

FKRWNHRYDEWFCWDSPYLRPLEKIQL

RKEGLHEEDGSSVSNKSGKFCSMQGQPL

GCLCFNL

524 A 35 402 KLFLGFPILHGSSSPP*LKVPSLTHKVSAL

VSSNSF*VMYLVIFHMNMKVAWRRGSL

LHSFQLGFYKSQHFGRPRWADRLRLGV

RDQPGQHGETPSLLV QKKI

*MQLLRRLRWEDQLSSGGRGCSGP*LHH

CTVAWATEQD

VMVISVSSRNALIDMPRNPCLTSCVCLSI

QSSRRLRLTIRIFMPDSWLKSCLTSYVCL

SIQSSRCLRLTIRIFMPDSWLKSCLTSYVC

LSIQSSRRLQLTIRIFMPDSWLKSCLTSYV

CLSIQSSRCLQLTIRiFMPDSWLKSCLTSY

VCLLIQSSRCLKLTVRIFMPLVPRLA

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, irLeucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, *=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion LQNATPTNFKTKTIHLPEQLKEDGTLPTN

NRKLNIKDLLSQMHHYNAGVKQRALLG

LKDLLSQYPFIIDAHLLNILSEVTAVFTD

KDANVRLAAVQLLQFLAPKIRAEQISPFF

PLV SAHLS SAMTHITEGIQED SLKVLDIL

LEQWALITGRSSILLKNFVELISHQQLSK

GLDK

FTQEEWGQLDPTQRILYRDVMLETFDHL

LSIGPELTKPEDISQLEQGTDLWAETLT

PPPAS/RLPCIEARGGQ*SLPSLILQPGGA

GWGILLPNSVLPPAPCTPQSHVSLPSAGP

CAPLGGRRQAPLGPRAAEPCRVRGCGA

AALSCALPALGRGTFGWKTGI

APVGEWLPPNGP*SDDELPLSQQCKCRT

KSLAPPSSA*GS*HRT*LPGTGH*CGLARI

ESSAGVLEEASAAK*GSGEAPGSKQHSS

SPAATSAPSAGSWHPGTAAAHPPG/SSAS

FHTFAAFTVSLSASELGS**QSRVKHFFL

SCSSTSCCSRALIHTKSSESGMSSRRSAFS

SPSSGGISLSWLGPKSRISSVTSSRCSRVS

*NFLSSEVTFVS\VSSHSSPQATLE*LCVK

SQTSTMLSYAVELPSMPEPAPLLG*GLSH

SHPHAVRDSGEAC*SMGSVTGPLYSGYK

EEVVCCTLVEVFPSLLQVSRNPRMPFDF

LGILICTPWGS

531 A 12 245 CAKNECQGGQVESGSF*AIYL*TVI\YLC

WLSVCLSIYLSWLSIYLSIYLSIYLSIYP

SIHPSIQLASI*SIFLWG

ALVSATPREDFRVRCTSKRAVTEMLQLC

GRFVQKLGDALPEEIREPALRDAQWTFE

SAVQENISINGQAWQEASDNCFMDSDIK

VLEDQFDEIIVDIATKRKQYPRKILECVIK

TIKAKQEILKQYHPWHPLDLKYDPDPA

PHMENLKCRGETVAKEISEAMKSLPALI

EQGEGFSQVLRMQPVIHLQRIHQEVFSS

CHRKPDAKPENFITQIETTPTETASRKTS

DWLKRKQTKDCPQRKWLRPKKINL

DT

NLPVFWPTELIFYADDQSTHKQVLTLY

NPYEFALKFKVLCTTPNKYV WDAAGA

VKPQCCVDIVIRHRDVRSCHYGVIDKFR

LQVSEQSQRKALGRKEWATLLPSAKEQ

QKEEEEKRLKEHLTESLFFEQSFQPENRA

VSSGPSLLTVFLGWCIAALMLPTLGDV

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K--Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion ESLVPLYLHL,SVNQKLVAAYILGLITMAI

LRT

EEHVGSQTFVNPHVSSFQGSKRGLNSSF

ETSPKKVKWSSTVTSPRLSLFSDGDSSES

EDTLSSSERSKGSGSRPPTPKSSPQKTRK

SPQVTRGSPQKTSCSPQKTRQSPQTLKRS

RVTTSLEALPTGTVLTDKSGRQWKLKSF

QTRDNQGILYEAAPTSTLTCDSGPQKQK

FSLKLDAKDGRLFNEQNFFQRAAKPLQV

NKWKKLYSTPLLAIPTCMGFGVHQDKY

RFLVLPSLGRSLQSALDVSPKHVLSERSV

LQVACRLLDALEFLHENEYVHGNVTAE

NIFVDPEDQSQVTLAGYGFAFRYCPSGK

HVAYVEGSRSPHEGDLEFISMDLHKGCG

PSRRSDLQSLGYCMLKWLYGFLPWTNC

LPNTEDIMKQKQKFVDKPGPFVGPCGH

WIRPSETLQKYLKVVMALTYEEKPPYA

MLRNNLEALLQDLRVSPYDPIGLPMVP

EVDVGTDKAQNSDPILDTSSLVPGCSSV

DNIKSSQTSQNQGLGRPTLEGDEETSEVE

YTVNKGPASSNRDRVPPSSEASEHHPRH

RVS SQAEDTS SSFDNLFIDRLQRITIADQ

GEQQSEENASTKNLTGLSSGTEKKPHYM

EVLEMRAKNPVPQLRKFKTNVLPFRQN

DSSSHCQKSGSPISSEERRRRDKQHLDDI

TAARLLPLHHMPTQLLSIEESLALQKQQ

KQNYEEMQAKLAAQKLAERLNIKMRSY

NPEGESSGRYREVRDEDDDWSSDEF

ARWLVPVIPALWEAEVGRSFEARSSRTA

WATYGGSLFYKK

KGASNEEIKKTYRKLALKHHPDKNPDDP

AATEKFKEITDAHAILTDISKRSIYDKYG

SLGLYVAEQFGDENVNTYFMLSSWWA

KALFVIVGLLTGCYFCCCLCCCCNCCCG

HCRPESSVPEEDFYVSPEDLEEQIKSDME

KDVDFPVFLQPTNANEKTQLIKEGSRSY

CTDS

CRASPPPRAGAMRGSPGDAERRQRWGR

LFEELDSNKDGRVDVHELRQGLARLGG

GNPDPGAQQGISSEGDADPDGGLDLEEF

SRYLQEREQRLLLMFHSLDRNQDGHIDV

SEIQQSFRALGISISLEQAEKILHSMDRDG

TMTIDWQEWRDHFLLHSLENVEDVLYF

WKHSTVLDIGECLTVPDEFSKQEKLTGM

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, amino Q=Glutamine, to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion WWKQLVAGAVAGAVSRTGTAPLDRLK

VFMQVHASKTNRLNILGGLRSMVLEGGI

RSLWRGNGINVLKIAPESAIKFMAYEQIK

RAILGQQETLHVQERFVAGSLAGATAQT

IIYPMEVLKTRLTLRRTGQYKGLLDCAR

RILEREGPRAFYRGYLPNVLGIIPYAGIDL

AVYETLKNWWLQQYSHDSADPGILVLL

ACGTISSTCGQIASYPLALVRTRMQAQA

SIEGGPQLSMLGLLRHILSQEGMRGLYR

GIAPNFMKVIPAV SISYWYENMKQALG

VTSRSFC

539 A 474 1098 ~ RRRCFQGFLKKKKIIQPKGEGTGRGRGV

VPFIHNIVNIHGLYYMCFSLVFACLSVRL

SVWGLDLRSRAPLLPQPPPPCPTLGTELE

QVWPPNHRWEPAGSAWATASTLAPESC

VCKCVCVYACLSMCMYVHGEPVCKYV

QRETVTVTEAGGRWGCWRRGLPGADSS

QVDPPQVSGRCDRVALRAELLGTQGPQ

PALLAVHVPTLDGR

SRTWRMPK*QEGENPCPLPKRNLGNA*T

FNS*KRTGPG/SPDSKGPDLGPI/SVPHILS

GSPGPSS~PTLPQLHDLLAIPVMQGREAG

GLRGHRALPLA*EEAGRALQLPLSQPPP

CRPAAPFFNQ/GPQGCCPWRGSGHSPGP

QLGG*EPGAYSGWQQTSRMSGVAKPGS

GVRPATGSPTRVPPGACSQSLPGTAPSPR

PPGPPALLRPAGVGALQGHQSRWQRQT

PRAREPRPVPLCSLPAPPGSVGPGPRDSQ

RRIAGCP*GQTSIFGQCHWGSVGSLGPPR

CAGSPCGPLGSR*VHSGLHSSRSASWLP

REQQGS/PGGGQGPLV WPHSSALRPPG/V

PGNLPPPIAPKPKRAIPARDPAPPPGLQH

E

541 A 1 438 , MLAIFKYLINNRLITT*Q/WTEKLTS*QMI

TIHNTKGLT* SLILLSLIIFIATTNLLGLLP

YSFTPTTQLSINLSMAIPL*TVAEFIGFRS

KIKNALSHFLPQGTPTPLIPILVIIETISLLI

QPILLDVLLTANITAGHLLIHLN

542 A 13 577 LNINKKYNYIKR*DYYKIII~NKKMNII*IIIV

K*YNKE*YK*NMKKIIK\Y*IYYSQV
STPT

IVKVSRNLGKVGSKCCKHPEAKRMI'CA

EDYLYVVLNQL*VLHEKTPVNDRVTKC

*KESLVNRRPCFSTLEVDKTYVTKKFNA

ETFTFHADICTLSEKERQIKKQTALDELV

KHKPKATKEQLKAVMDD

D*GTQRGGSGRGGRGRGR/DGSTNEGRG

ERRGQG

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, IrLeucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutarnine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, Y=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion LGQEIATCTKIPSRKRDIEFNEFVKNFTV

RSILVAEQIDPMEENCHKYGTC*KMLKQ

NSDLIIQ\ESMMEKKKPCKYSECGRTFRG

HITLVQHQITHCGERPCI

KEELEPILLNLFQKIEKEEILSNSFRETSIA

LIPKPGKDITKEVNHRPI SLMNRDAKVL

NKMLSN*IQ

PPKKKKFPPPPGVYFFFFLRPPPPLFFFFF

FFFFFFFF*EGGGEKPSPP/IKKGFFKPKKF

FSPPPGGKKKPPFLKKKICKKICDVPQSSN

LESSLSFPIHKTGTIIPASLNV

ASGPPCSS*ACCPRFCQSCPVC*PQNLLS

SCPSPDLALTSPSAGRPRCWWNCCQLTP

CLSLQTQEWTGAHLQLRRWGTQAGPEE

PVHQRTVGSTSHSCSYA

548 A 419 2 RWSARSRITGEEGSESAVQER*LTYHTH

GGIANRDRTHCPVSLVN\VSSMSAGDVIT

AGKMQGKV VLAEPTAALCHGN\GLHYY

RQDWSHAAAYVTVPPVRVDTNTSTYLM

SLLANDTLNIVASDRRPFTTKQKAMGNV

549 A 201 550 GCLVSLGVSHAWQRVGTWEIFVG*MNA

G*MSA*TRGTEISRIGQWMGGEGMSGLT

SWFLLGEMNP*GLRELNLLLTLSITMNG

ETIACAEGCQAICDTGTSLLT/GPTSPIAQ

HQSDI

LTQPLFPCLANV SDPYLSAVPEGSALICL

EDKRLRLVDGDSRCAGRVEIYHDGFWG

TICDDGWDLSDAHVVCQKLSCGVAFNA

TV SAHFG/V GAGPI W/LDDL/NCTGMESH

LRQ/CPF

FXF~~~XXFFFFFXGXXXXFXXFXXXX

XFFFFFFFFFFFFFFFFFFFXCY

552 A 255 338 EVMHFCLFVF*DRVLLCHPGWSAVAQS

Q

PP**GALLKPSPGPRFNTAWPGKPAPRES

LKRAPSLGGGPKRPNPPPEVKASGADKK

L

554 A 3 365 YHIVQPSP*PLAGALSALLMTSGLAMRS

HFHSITLLILGLLSSTLTIYQ*WRDVTRES

TYQGHHTPPVRKGLRYGIILFITSEVIFFA

GLF*AFYHSSLAPTPQLGGHWPPTGITPL

NTLE

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation 1=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K--Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, r=Stop peptide sequence codon, l=possible nucleotide deletion, \=possible sequence nucleotide insertion S55 A 1 125 LMNSNSIPPIPLRPTHTPPMKKL*IRTAYP

RFRYDQLIHLL*KNFLPLTLALLI*HVKK
KKKK

FPPPPKGFFFPNPPRGFFFPPP*KKKFFFPP

PPFFCPPPVFFFCPPPLFFFFFFFFFFFFFFF

FF

DCSKLRRPLKRNRITEGIYGSTFLYLKFL

VVWALVLLADFVLEFRFEYLWPFWLFIR

SVYDSFRYQGLVCLVIHVFHVWGPFSCL

E*MLLAILR*LC

LELPDKC SLAFV VRIKRIDGGSLLVQRTI

ARLCLKKIFSGVFLKMLRIVEPYVT*GFP

NLKSVWEFILKHGQAKVKNKTIGQTQW

LTPVIPALWEAKDGWIMRSGDRDHPGQ

ALQVVYERCEKLRPTLFRLASDTTDDDD

ALSEILQANDLLTQGVLLYKQVMEGRV

TFGNRVTSSLGDIPVSRVFQNPAGCMKT

CPLIDLEVDNGPAQMGTVVPSLLHQDLA

ALGNSDAPVTGMV

EGRYEYERIPRERAPPRSHPSDESGYRW

TRDDHSASRQPEYRDMRDGFRRKSFYSS

HYARERSPYKRDNTFFRESPVGRKDSPH

SRSGSSVSSRSYSPERSKSYSFHQSQHRK

SVRPGASYKRQNEGNPERDKERPVQSLK

TSRDTSPSSGSAVSSSKVLDKPSRLTEKE

LAEAASKWAAEKLEKSDESNLPEISEYE

AGSTAPLFTDQPEEPESNTTHGIELFEDS

QLTTRSKAIASKTKEIEQVYRQDCETFG

MVVKMLIEKDPSLEKSIQFALRQNLHEI

GERCVEELKHFIAEYDTSTQDFGEPF

MTLANHWNCPVLSSDSDFCIFDLKTGFC

PLNSFQWRNMNTIKGTQNYIPAKCFSLD

AFCHHFSNMNKALLPLFAGLCGNDHVN

LPIMETFLSKARLPLGATS SKGRRHTIRIL

GLLNWLSHFANPTEALDNVLKYLPKKD

RENVKELLCCSMEEYQQSQVKLQDFFQ

CGTYVCPDALNLGLPEW VLVALAKGQL

SPFISDALVLRRTILPTQVENMQQPNAHR

ISQPIRQIIYGLLLNASPHLDKTSWNALPP

DLSRLTELSLRRRQMLLLETLKVKQTILE

PIPTSLKLPIAVSCYWLQHTETKAKLHHL

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K--Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, *=Stop peptide sequence codon, l=possible nucleotide deletion, \=possible sequence nucleotide insertion KMLYAEFQRVKAQTRLGTRLDLDTAHI

FCQWQSCLQMGDVSQPAAVHSSPRARP

NSTVQWKPGARTMPATASIDLCRKSPEH

MS

CVCVCVCVCVCVCVYSSAFKKTRRMHR

WIR

PGYVKPKHEVNPQMTLRRLPDEDPQNL

ADPAYRRRRIIMQNMRDEELAIAQVEEM

QAVSAVLKGKYTMTG

KEKIF WKEF SILDANKNTDVS WKEIKIAT

LTGVWKKLILTFETVSYKSQNQH

STGAGRYQFLSRWWDAYRKQLGLKDFS

PKSQAAVALQQIKERGALPMIDRGDIRQ

AIDRCSNMWASLPGAGYGQFEHKADSLI

A

LGCWYGAKDETPSKQTLSIQQESPLRTH

WTGGNDVTLKLRLEYTGMVIPHCGLKL

LGLK

SLGKTETLICLKISWHSLRIRTYWAVRSC

WRYSRGLRISLWFETESHCVPQVGVRW

CDLGSLKPLPPGFRRFS

LERYLADLPTLEDHQKQSQQLKDSELKS

TELQEKVTELESLLEETQAICREKEIQLES

LRQRETEFSSAGHSLQDKQSVEETSGEG

PEVEMESWQKRYDSLQKIVEKQQQKMD

QLRSQVQSLEQEVAQEEGTSQALREEAQ

RRDSALQQLRTAVKELSVQNQDLIEKNL

TLQEHLRQAQPGSPPSPDTAQLALELHQ

ELASCLQDLQAVCSIVTQRAQGHDPNLS

L

KMCDPAKGAAGQRTIAALLPCLLDKGM

MSTVTEVRALSINTLVKISKSAGAMLKP

HAPKLIPALLESLSVLEPQVLNYLSLRAT

THREGARLETPPPWLAMFTDQPALPNPC

SPASVGP

PLSQVNNKFGLRCKNCKTNIHEHCQSYV

EMQRCFGKIPPGFHRAYS SPLYSNQQYA

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, l=possible nucleotide deletion, \=possible sequence nucleotide insertion CVKDLSAANRNDPVFETLRTGVIMANK

ERKKGQADKKNPVAAMMEEEPESARPE

EGKPQDGNPEGDKKAEKKTPDDKHKQP

GFQQSHYFVALYRFKALEKDDLDFPPGE

KITVIDDSNEEWGRGKIGEKVGFFPSNFII

WGPA

ALERMFLSFPTTCI

574 A 384 1 ~ RTDSSTTNRESVNSEDGDAKEPPYRGPF

CGRDRVHTDFTPSPYDTDSLKLKKGDII

DIISKPPMGTWMGLLNNKVGTFKFIYVD

VLSEDEEKPKRPTRRRRKGRPPQPKSVE

DLLDRINIKEHMPTCI

DNGQLDDARVILEKATKVNFKQVDDLA

SVWCQCGELELRHENYDEALRLLRKAT

ALPARRAEYFDGSEPVQNRVYKSLKVW

SMLADLEESLGTFQSTKAVYDRILDLRIA

TPHVFGRSEDQASTR

QVQLDAVNKRLPEIDSALQFISEIIHQLT

NQKASIVDDIHSTFDELQKTLNVRKSVL

LMELEVNYGLKHKVLQSQLDTLLQGQE

SIKSCSNFTAQTLNHGTKA

577 A 1 45 LLYTYVLLASGVWVA*AYHRLIENNRN

QIIQALLITILLGLYFTLLQASE\HLQSPFTI

SDGIYGSTFFVATGFHRLHVIIGSTLLTIC

FIPQLIFDFTSKHHFGIQAAV*YWHLVDG

V*LFMHGSI*WVA

SPASDYLELDTIKNLVKKYSQFINFPIYV

WSSKTETVEEPMEEEEAAKEEKEESDDE

AAVEEEEEEKKPKTKKVEKTV WDWEL

MNDIKPIWQRPSKEVEEDEYKAFYKSFS

KESDDPMAYIHFTAEGEVTFKSILFVPTS

APRGLFDEYGSKKSDYIKLYVRRVFITD

DFHDMMPKYLNFVKGWDSDDLPLNVS

RETLQQHKLLKVIRKKLVRKTLDMIKKI

ADDKYNDTFWKEFGTNIKLGVIEDHSNR

TRLAKLLRFQSSHHPTDITSLDQYVERM

KEKQDKIYFMAGSSRKEAESSPFVERLL

KKGYEVIYLTEPVDEYCIQALPEFDGKR

FQNVAKEGVKFDESEKTKESREAVEKEF

EPLLNWMKDKALKDKIEKAVVSQRLTE

SPWLLWVGQPSTDWSGNMRDFMKAQA

YQPGK\DILPN\YYASQK\KTFEIN1PRPPL\

IRDMLRRIK\EDE\DDKTVLDLA\V
VLV *N

RQRLGSGYLFTQTLKAYGDW/RLERML

RLSFEHLTPDAKGGRRAPKEEP*KRQQK

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K--Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino , to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion DTSRKTQSRDEE/MQEMGW WETDEEEE

TSQRNPTA

FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

FFFFFF*FYILKIFFFFFILEILCYLSNYGVG

VTY/IRMSYYK*MLESYFFFTINIFFIGNN

YLISLIYNIYAVKRQL

LPRLVLSSWPQVIFLPWPPKFLGLRT

581 A 1 272 ESSCDWLNAYLTLVRCAQD*ADYFAER

LYKSMKGAGTDEETLIRIIVTRAEVDLH

GIKAKFQEKYQKSLSDMVRSDTFGDFRK

LLVALLH

582 A 4.01 1 LYIFSPSRLIFFRGFSFFFFPPKKIFFLKIFP

NDFFFPSHFFKIPSLGFFFYTFFIRKIFFFFP

IIYFSPPGFFF*SPPFFFFFFFFFFFFFFFFFF

FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFG

WYKQRPSQARV WTASLDFTL

KFDEKENVSNCIQLKTSVIKGIKNQLIEQ

FPGIEPWLNQIMPKKDPVKIVRCHEHIEI

LTVNGELLFFRQREGPFYPTLRLLHKYPF

ILPHQQVDKGAIKFVLSGANIMCPGLTSP

GAKLYPAAVDTIVAIMAEGKQHALCVG

VMKMSAEDIEKVNKGIGIENIHYLNDGL

WHMKTYK

584 A 1 343 FFFFEDKVSLCHPRWECSDDITAH\CSLN

LGFK* S SHL\SLPK*LCLLGTQHYAPANF

LYFVFFVETGFRHVGQAGLEHLGSTDPP

ASA/FPKCWDYRCEPPCSTRFYYLS*LNF

G

AFMDWRALLSSLNDFASLSFAESWDNV

GLLVEPSPPHTVNTLFLTNDLTDEVMDE

VLLKKAHLILSYHPSIFRPMKRITWNTW

KELLVIRALENRVGIYSPYTT*EA\APQG

VNNWVA

SASRVAGITGMRHHAQLIFVFLVETGFH

HVGQAGLELLTSGDPPASASQSAGITGM

SHHAWPDVNYLKNFQVILMGQG\WNPC

SGPIRAPGCHKRMHALTPVGLGGFGNVL

LLF*DG/CLSVAHPGAQWHDHSSLQPRP

PGLR\YPPASAS * V SASGFHDDLGPRHTT

LVLFRGQSCTRSPSATAPPRSTDMQS*A

ALFPRGSCVGFLGPSVD/YRTPAEDLLG

MPHCLCFPSSLNTQSGLAMMPLCLLASP

PLEQPSWHATLLAAGHCAFCLYRSAVM

LPK*QQQQQQQRLGTWLMPVFPAVRDQ

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K--Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, amino Q=Glutamine, to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown,''=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion PGQHGETLSL

587 A 236 3 VDHLRSEV*DQPGQHIVKPRLY*KIQKL

AGHGGACL*SPLLGKLRQENRLNPG\AR

SCSEPRSHYCTPAWGTE*NSVS

KVQVLTADFWGGRQHCWHASSYTCRP

RPPGLKD/IASCSSLQEATSCPSAFCA*NS

KATKQLVERRGLQEHQRGRTLTMVS

KCIP/WQGLLLTAS\LLAL*EAPTTAWLFI

ASAPYEVAEGENVHLSVWLRENLYSY

GWKGKTVEPNQLIAAYVIDTHVRTPGP

AYSGRETISPSGDLHFQNVTLEDTGYYN

LQVTYRNSQIEQASHHLRVYESVAQPSI

QASSCI

VLGWC/AELRVLCKLEENVQATNSPSEA

EEFQLEVSGLLGEMNCPYLSLTSGDVTK

RLLIQKNCLLLLTYLISELEAARMLCW

APPKKS

RFS\CLSLPS

VDEVGGEALVKLIGAYPWTQRFFESFGD

LSTPDAVMGNPNGKAHGAKVLGAFSDG

LAHLDNLKGTFATLSELHCDKLHVDAE

NlFMLLGNVLVCELA

SQVLRGLRHKNCLNPGDGGCSEPRWH

CTPAWTEEDPV SLSKKREMDGNNTTF

TPSQL

594 A 1 418 PQTQREPTMGLSAAD*TNV*AGWGKVG

AHAGEYGAEALERMFLSFPTTKTYFPHF

DLSHGYAQDKGHGMKVADALTNAVAH

VDDMPNALYALSDLHAHKL*VDPVNFK

LLNHCLWTLACHLLAEVTPGVHAFLD

KFPG

DPVTLNVGGHLYTTILTTLTRYPDSMLG

AMNGGDFPTA*DPQGNYFIDLDGPLFRY

VLNFLRTSELTLPLDFKEFDLLLK*SDFY

QIEPLIQCIN

596 A 83 341 . SFRRPMASASTQPAALSAEQAKASIH*FL

ALSAGPAVCSPTPTRDASLGCFWRAVVL

AEVIQAFSAPENAVRMDEARDNACNDM

GK

597 A 102 1 EHRPGWAHACNPSTSGGQGEWIT*GQE

FKTSLG

598 ~ A ~ 180 - 3 KKKKKKKKRGEIGKFYSLLEELTARSWF~
- ~

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, r=Stop peptide sequence colon, l=possible nucleotide deletion, \=possible sequence nucleotide insertion SCFFIF*DKQGLPWWLGISYKGSGQYDIQ

A

LAARKQSPFFLAVFGIYHLLITIHF*RQK

KKIYLSIYLSIYLSIYLSIYLNDLTEENC

600 A 364 219 NFCTTAS*GRGCSEPR*RHCTPAWVTDY

DSISNKN*TGGDFIKNIMRNLQDAIIGSV

- YFHVERVNRLN

601 A 61 284 EDHHAGTLITALSSH*FFT*VGLEINMLA

FIPVLTKKKKKKRGGPFKGTKFKARGGG

RKNFFKGAPKLNSGAGV

DIEREIGAILQNAGTVILELSKEKTNERLL

DRQAAAFTASVQHVEAELSAQIRYLTQV

ATGQPHEGSSYSSRKDCQMALKRVDYA

RLKLSDVARTCEQMLEN

MNRPSARNALGNVFVSELLETLAQLRED

RQVRVLLFRSGVKGVFCAGADLLDREH

MS

MNRP SARNALGNVF V SELLETLAQLRED

RQVRVLLFRSGVKGVFCAGADLLDREH

MS

MNRPSARNALGNVF V SELLETLAQLRED

RQVRVLLFRSGVKGVFCAGADLLDREH

MS

CFVLRRESCSVARAGVQWRDLGSLQAP

PPGFTPF S WKGEDAV SLNRSIAPQPERQS

ETLGSNRLRRWRKNYKARRGGHMPVL

QVLS

607 A 1 ' 268 RMKTILSNQTVDIPENVDITLKGRTGIVK

GPRGTLRRDFNHINVELSLLGKKKKRL,R

VDKWRGNTKELSTDRTMCIHCHNMIMV

VIHVW

ETHASA

TDEQRMTARGKTSDIEANQPLETNKENS

S SVTV SDPEMENKAGQTLENS SLMAELL

SDVPVTLAPHVLAG

FHTCSLKSSGALWCMIFRGRFHSLVCFS

RLSGVLMEVEEHEVLQDSLDRCYSTPPM

FFDIPD

611 A 2 363 NSAPEAGSCWKMKVIVISFPATGCQI~LIE

VDDERKLRTFYDNRMAPEVAVDALVEE

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, l=possible nucleotide deletion, \=possible sequence nucleotide insertion WKGYWRIIGGNDKQGFPMKHGDLTHG

RVRLLLSKGHSCYRPRRTGERKRKSLRG

CIVDANLSVL

KLVFLGLDNAGKTTLLHMLKDDRLGQH

VPTLHPTSEELTIAGMTFTTFDLGGHEQA

RRV WKNYLPAINGIVFLVDCADHSRLVE

SKVELNA

NKSDPVPPLTRKKKKKKIKKKKKKKKKK

KKKKKICKKKRGAL

RTLLGNFEESLLRGRFAPSGHIDGYTAEI

GASGSYCPQHVT

VGSEQAEATMEQCACVERELDKVLQKF

LTYGQHCERSLEELLHYVGQLRAELASA

ALQGTPLSATLSLVMSQCCRKIKDTVQK

LASDHKDIHSSVSRVGKAIDRNFDSEICG

VVSDAVWDAREQQQQILQMAIVEHLYQ

QGMLSVAEELCQESTLNVDLDFKQPFLE

LNRILEALHEQDLGPALEWAVSHRQRLL

ELNSSLEFKLHRLHFIRLLAGGPAKQLEA

LSYARHFQPFARLHQREIQVMMGSLVY

LRLGLEKSPYCHLLDSSHWAEICETFTR

DACSLLGLSVESPLSVSFASGCVALPVL

MNIKAVIEQRQCTGVWNHKDELPIEIEL

GMKCWYHSVFACPILY

616 A 220 627 VGPKF'EPDRSAGRRLAVVFRGNTPSLWL

SMSIVTVQLGQCGNQIGFEVFDALLSDS

HSSQGLCSMRENEAYQASCKERFFSEEE

NGVPIARAVLVDMEPKVINQTLSKAAQS

GQWKYGQHACFCQKQGSGNNWAYG

SMSIVTVQLGQCGNQIGFEVFDALLSDS

HSSQGLCSMRENEAYQASCKERFFSEEE

NGVPIARAVLVDMEPKVINQTLSKAAQS

GQWKYGQHACFCQKQGSGNNWAYG

AQQNSPKIHEGWWAYKEWQGSFVPVP

SFWGLVNSAWNLCSVGK

QCNLCLLSPSDSPP

SIPVLALSPGLLCSARS\TKQKNSCQQLK

EDVD\QTKWGTDTPSMD*ILMEEVKLEE

QLKEWEEYKLALSDTEGLQQSIQKLVD

EANMCSIQGFCMDSLEVADVLENGTQC

VS*QKNSCQQLKEDVDPNQMGDRYSLY

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, ~ M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, #=Stop peptide sequence codon, !=possible nucleotide deletion,1=possible sequence nucleotide insertion GLDTHGRSQVRRATEGGCGRI

621 A 648 937 RKGQQQTCLS\LRVSWLYRKVTFSIFFFF

LIL*K*RWGLTMVTQAGLKLLGTSNPPA

FALPKCWDYRCKSLPPAQKLTFSKATTK

KTPQICQSMKVS

EVEKSSDGPGAAQEPTWLTDVPAAMEFI

AATEDLEIPAVPILHSMVQKFPGVSFGIS

TDSEVLTHYNITGNTICLFRLVDNEQLDL

EDEDIESIDATKLSRFIEINSLHMVTEYNP

VTVIGLFNSVIQIHLLLINE/PRPPQKYEE

NMHRYQKAAKLFQGKIPPLFLVGQL\DE

RKMGKVISFFKLKESQLPAL\QFTRL*DD

EWDTLPTAEVSVEHVQNFCDGFLSGKLL

KENRESEGKTPKVEL

/RFSCLSLS\GSWDYRRPPPRPANFFVFLV

ETGFHCGLDLLTS*SARLGLPKCWDYKR

EPPHPA

QPAARTPAGPCIGGMAPPGFKHVSSMLA

\LTIIAST\WALTPTHYLTKHDVERLKASL

DRPFTNLESAFYS\IVGLSSLG\AQVP\DA

KKACTYI\RSNLDP\SNV\DSLFYGWPRAS

Q\ALSGM*RSLFSNE\TKDLAFGQLFS*GT

SSVYPRSYHAS/VAALKWALGLPLASQE

ALSALTARLSKEETVLATVQALQTASHL

SQQADLRSIVEEIEDLVARLDELGGVYL

Q\FEEGLETTAL\FVAATYKA/L,MDH\VG

TE\PSIKE\DQVIQLMN\AIF\SKKNFE\SLS

EAFSV\ASAAAVLSHNRYHVPVWVPEG

SASDTHEQAILRLQVTNVLSQPLTQATV

KLEHAKSVAS\RATVLQKTSFTP\VGIVFE

LNFMN\VKFS\SGY\YDFLGRKLKGDNRY

IS\NTVELRVQDPPTEVGITNVDLSTV1DK

DQSIAP\QTTRVTYP\AKAKGTFIADSHQ

NFALFFQL\VGVNTGAELTPHQTFVRLH

NQKTGQ\EVVFVAEPDNKNVYKFELDTS

ERKGLNLTSRSGTYTFY\LIIGDATLKNPI

LWNVADVVIKFPEEEAPSTVLSQNLFTP

KQEIQHLFREPEKRPPTWSNTFTALILSP

LLLLFALWIRIGANVSNFTFAPSTIIFHLG

HAAMLGLMYVYWTQLNMFQTLKYLAI

LGSVTFLAGNRMLAQQAVKRTAH

GTLI*ASEVRK/SG*HQCQR*LWEGDL\L

HC**QCELMEPTWKMAWHQLLEKKTHS

PL/DLPVPHQGLDMAVM/CYQGA/CARN

I LYSQILTIAPNWNQPTYPSGREWMLKNA

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, amino Q=Glutamine, to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion RIAIG

TTTTSSSGLGSPMIVGSPRALTQPLGLLR

LLQLVSTCVAFSLVASVGAWTGSMGNW

SMFTWCFCFSVTLIILIVEL\SGLQARFPL

SWRNFPITFACYAALFCLSASIIYPTTYV

QFLSHGRSRDHAIAATFFSCIACVAYATE

VAWTRARPGEITGYMATVPGLLKVLET

FVACIIFAFISDPNLYQHQPALEWCVAVY

AICFILAAIAILLNLGECT'NVLPIPFPSFLS

GLA\FCLSSSMPPPLFSGPSTSSMRSMAA

SLGAREM*AAAAAMPTTCVPGTADWL

WPS

VSLFSMTDMILFSLIVGLLTYWFLFRKK

KEEVPEFTKIQTLTSSVRESSFVEKMKKT

GRNIIVFYGSQTGTAEEFANRLSKDAHR

YGMRGMSADPEEYDLADLSSLPEIDNAL

VVFCMATYGEGDPTDNAQDFYDWLQE

TDVDLSGVKFAVFGLGNKTYEHFNAMG

KYVDKRLEQLGAQRIFELGLGDDDGNL

EEDFITWREQFWPAVCEHFGVEATGEES

SIRQYELV VHTDIDAAKVYMGEMGRLK

SYENQKPPFDAKNPFLAAVTTNRKLNQ

GTERHLMHLELDISDSKIRYESGDHV\AV

YPANDSALVNQLGKILGADLDV VMSLN

NLDEESNKKHPFPCPTSYRTALTYYLDIT

NPPRTNVLYELAQYASEPSEQELLRKMA

S S S GEGKELYLS W WEARRHILAILQDC

PSLRPPIDHLCELLPRLQARYYSIAS
SSKV

HPNS VHICAV V VEYETKAGRINKGVATN

WLRAKEPVGENGGRALVPMFVRKSQFR

LPFKATTPVIMVGPGTGVAPFIGFIQERA

WLRQQGKEVGETLLYYGCRRSDEDYLY

REELAQFI~DGALTQLNVAFSREQSHK

VYVQHLLKQDREHLWKLIEGGAHIYVC

GDARNMARDVQNTFYDIVAELGAMEH

AQAVDYIKKLMTKGRYSLDVWS

RSGAMVKMLAAAGGGGGGGRYYGGGSE

GGRAPKRLKTDNAGDQHGGGGGGGGG

AGAAGGGGGGENYDDPHKTPASPV
VHI

RGLIDGV VEADLVEALQEFGPISYV
V VM

PKKRQALVEFEDVLGACNAVNYAADN

QIYIAGHPAFVNYSTSQKISRPGDSDDSR

SVNSVLLFTILNPIYSITTDVLYTICNPCG

PVQRIVIFRKNGVQAMVEFDSVQSAQRA

KASLNGADIYSGCCTLKIEYAKPTRLNV

FKNDQDTWDYTNPNLSGQGDPGSNPNK

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end CLCysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, amino Q=Glutamine, to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of , X=Unknown,''~=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion RQRQPPLLGDHPAEYGGPHGGYHSHYH

DEGYGPPPPHYEGRRMGPPVGGHRRGP

SRYGPQYGHPPPP\PPPPEYGPHADSPVL

MVYGLDQSKMNCDRVFNVFCLYGNVE

KVKFMKSKPGAAMVEMADGYAVDRAI

THLNNNFMFGQKLNVCVSKQPAIMPGQ

SYGLEDGSCSYKDFSESRNNRFSTPEQA

AKNRIQHPSNVLHFFNAPLEVTEENFFEI

CDELGVKRPSSVKVFSGKSERSSSGLLE

WESKSDALETLGFLNHYQMKNPNGPYP

YTLKLCFSTAQHAS

AGSEPESVEASPVVVEKSNSYPHQLYTS

S SHHSHSYNGFAPMRTHNRVLVPPPNTP

APPPP\SVLISKNEVGHIYPLPNFDETSQC

LPTISTS\EDGSYGTDVT\RCICGFTHDDG

YMICCDKCSVWQHIDCMGIDRQHIPDTY

LCERCQPRNLDKERAVLLQRRKRENMS

DGDTSATESGDEVPVELYTAFQHTPTSIT

LTASRV SKVNDKRRKKSGEKEQHISKCK

KGSAPEIDPSSDGSNFGWETKIKAWMDR

YEEANNNQYSEGVQREAQRIALRLGNG

NDKKEMNKSDLNTNNLLFKPPVESHIQK

NK\KiLKSAK\DWPP\DALIIEYRGKFM\L

R\EQFEANGYFF\KRP\YPFVLF\YSKF\HG

LEMCVDARTFGNEARFIRRSCTPNAEVR

HEIQDGTIHLYIYSIHSIPKGTEITIAFDFD

YGNCKYKVDCACLKENPECPVLKRSSES

MENINSGYETRRKKGKKDKDISKEKDT

QNQNITLDCEGTTNKMKSPETKQRKLSP

LRLSV SNNQEPDFIDDIEEKTPISNEVEM

ESEEQIAERKRKMNWTVDGCGSWFWFR

AEASVKALGLTLNVLFG

AGSEPESVEASPVVVEKSNSYPHQLYTS

SSHHSHSYNGFAPMRTHNRVLVPPPNTP

APPPP\SVLISKNEVGHIYPLPNFDETSQC

LPTISTS\EDGSYGTDVT\RCICGFTHDDG

YMICCDKCSV WQHIDCMGIDRQHIPDTY

LCERCQPRNLDKERAVLLQRRKRENMS

DGDTSATESGDEVPVELYTAFQHTPTSIT

LTASRVSKVNDKRRKKSGEKEQHISKCK

KGSAPEIDPSSDGSNFGWETKIKAWMDR

YEEANNNQYSEGVQREAQRIALRLGNG

NDKKEMNKSDLNTNNLLFKPPVESHIQK

NK\KILKSAK\DWPP1DALIIEYRGKFM\L

R\EQFEANGYFF\KRP\YPFVLF\YSKF\HG

LEMCVDARTFGNEARFIRRSCTPNAEVR

HEIQDGTIHLYIYSIHSIPKGTEITIAFDFD

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, *=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion YGNCKYKVDCACLKENPECPVLKRSSES

MENINSGYETRRKKGKKDKDISKEKDT

QNQNITLDCEGTTNKMKSPETKQRKLSP

LRLSVSNNQEPDFIDDIEEKTPISNEVEM

ESEEQIAERKRI~MNWTVDGCGSWFWFR

AEASVKALGLTLNVLFG

VCVCVCLPNHAIIVMLNRTEPYVPEGGA

YLPEREPFIVPVEPERTAEYEDYGADEPA

EEPPEPHRRWR\RALPHGPGQ

KVDKSLKMRKKQRKKAENSKNQNASSP

PKEHNSSPAKEQN*TENEFDELTEVGFR

M

LSS*DYRACI

634 A 88 328 RHKKRCLTYLTIK*MQIKTKMRYHFTHS

RMFIVKKDSKCWQICGEIEILTHCWWKC

TMVQSLWKIV*PFLKMMEKMRSEF

PQPPTHQASVGLLDTPWSRERSPSPLRG

NVVPSPLPTRRTRTFSA*VTGSSGLGSTL

NLQGAFLVSHPQPTAQPQVCLFGSPGHR

PKNGQRHSESLASGKRSI

ARAQVAEGAVRNIQAEGHRQIPDLTWT

TLLLLARPFNSPPLRSGLAPPPDLSLVLL

QALTLDLRWLPHLSGAMATGADVRDIL

ELGGPEGDAASGTI SKKDIINPDKKKSKK

SSETLTFKRPEGMHREVYALLYSDKKDA

PPLLPSDTGQGYRTVKAKLGSKKVRPW

KWMPFT'NPARKDGAMFFHWRRAAEEG

KDYPFARFNKTVQVPVYSEQEYQLYLH

DDAWTKAETDHLFDLSRRFDLRFVVIHD

RYDHQQFKKRSVEDLKERYYHICAKLA

NVRAVPGTDLKIPVFDAGHERRRKEQLE

RLYNRTPEQVAEEEYLLQELRKIEARKK

EREKRSQDLQKLITAADTTAEQRRTERK

APKKKLPQKKEAEKPAVPETAGIKFPDF

KSAGVTLRSQRMKLPSSVGQKKIKALEQ

MLLELGVELSPTPTEELVHMFNELRSDL

VLLYELKQACANCEYELQMLRHRI~AL

ARAGVLGGPATPASGPGPASAEPAVTEP

GLGPDPKDTIIDWGAPLTPNSRKRRESA

SSSSSVKKAKKP

ARAQVAEGAVRNIQAEGHRQIPDLTWT

TLLLLARPFNSPPLRSGLAPPPDLSLVLL

QALTLDLRWLPHLSGAMATGADVRDIL

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C'=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, ~=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion ELGGPEGDAASGTISKKDIINPDKKKSKK

S SETLTFKRPEGMHREVYALLYSDKKDA

PPLLPSDTGQGYRTVKAKLGSKKVRPW

KWMPFTNPARKDGAMFFHWRRAAEEG

KDYPFARFNKTVQVPVYSEQEYQLYLH

DDAWTKAETDHLFDLSRRFDLRFV VIHD

RYDHQQFKKRSVEDLKERWHICAKLA

NVRAVPGTDLKIPVFDAGHERRRKEQLE

RLYNRTPEQVAEEEYLLQELRKIEARKK

EREKRSQDLQKLITAADTTAEQRRTERK

APKKKLPQKKEAEKPAVPETAGIKFPDF

KSAGVTLRSQRMKLPSSVGQKKIKALEQ

MLLELGVELSPTPTEELVHMFNELRSDL

VLLYELKQACANCEYELQMLRHRHEAL

ARAGVLGGPATPASGPGPASAEPAVTEP

GLGPDPKDTIIDWGAPLTPNSRKRRESA

SSSSSVKKAKKP

ALASVSFDSRDSKMAAQSAPKVVLKSTT

KMSLNERFTNMLKNKQPTPVNIRASMQ

QQQQLASARNRRLAQQMENRPSVQAAL

KLKQSLKQRLGKSNIQARLGRPIGALAR

GAIGGRGLPIIQRGLPRGGLRGGRATRTL

LRGGMSLRGQNLLRGGRAVAPRMGLRR

GGVRGRGGPGRGGLGRGAMGRGGIGG

RGRGMIGRGRGGFGGRGRGRGRGRGAL

ARPVLTKEQLDNQLDAYMSKTKGHLDA

ELDAYMAQTDPETND

VTGQNQEQFLLLAKSAKGAALATLIHQ

VLEAPGW VFGELLDMPNVRELAESDF

ASTFRLLTVFAYGTYADYLAEARNLPPL

TEAQKNKLRHLSVVTLAAKVKCIPYAV

LLEALALRNVRQLEDLVIEAVYADVLRG

SLDQRNQRLEVDYSIGRDIQRQDLSAIAR

TLQEWCVGCEWLSGIEEQVSRANQHK

EQQLGLKQQIESEVANLKKTIKVTTAAA

AAATSQDPEQHLTELREPAPGTNQRQPS

KKASKGKGLRGSAKIWSKSN

HNGKTIFAYFTGSKDAGGKSWCPDCVQ

AEPWREGLKHISEGCVFIYCQVGEKPY

WKDPNNDFRKNLKVTAVPTLLKYGTPQ

KLVESECLQANLVEMLFSED

LLPRLECSGAVMARCSLNLLGSGDPPAS

ASQVAGTTGACHHTSLIFPFVLYVDSTG

LTLKT

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=AsparticAcid,E=GlutamicAcid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K--Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Onknown, f=Stop peptide sequence codon, I=possible nucleotide deletion, \=possible sequence nucleotide insertion LGGTISLLGRLWRSHLISSLLCLEGIILSL

FIIATLITLNTHSLLTNIGPIAILVFAACEA

AGGLSLLGSISNTYGLNWHNLNLLQC

EVDVGTDKAQNSDPILDTSSLVPGCSSV

DNIKSSQTSQNQGLGRPTLEGDEETSEVE

YTVNKGPASSNRDRVPPSSEASEHHPRH

RVSSQAEDTSSSFDNLFIDRLQRITIADQ

GEQQSEENASTKNLTGLSSGTEKKPHYM

EVLEMRAKNPVPQLRKFKTNVLPFRQN

DSSSHCQKSGSPISSEERRRRDKQHLDDI

TAARLLPLHf-INIPTQLLSIEESLALQKQQ

KQNYEEMQAKLAAQKLAERLNIKMRSY

NPEGESSGRYREVRDEDDDWSSDEF

GLLGVEQSDKTDQFLVTDSGRTVILYKV

SDQKPLGSWSVKQGQIITCPAVCNFQTG

EYV WHDNKVLRI WNNEDVNLDKV
FK

ATLSAEVYRILSVQGTEPLVLFKEGAVR

GLEALLADPQQKIETVISDEEVIKWTKFF

WFRHPVLIFITEKHGNYFAYVQMFNSRI

LTKYTLLLGQDENSVIKSFTASVDRKFIS

LMSLS SDGCIYETLIPIRPADPEKNQSLV

KSLLLKAWSGNARNGVALTALDQDHV

AVLGSPLAASKECLSVWIKFQTLQTSK

ELPQGTSGQLWWGEHLFMLHGKSLTVI

PYKCEVSSLAGALGKLKHSQDPGTHVV

SHFVNWETPQGCGLGFQNSEQSRRILRR

RKIEVSLQPEVPPSKQLLSTIMKDSEKHIE

VEVRKFLALKQTPDFHTVIGDTVTGLLE

RCKAEPSFYPRNCLMQLIQTHVLSYSLC

PDLMEIALKKKDVQLLQLCLQQFPDIPES

VTCACLKIFLSIGDDSLQETDVNMESVF

DYSINS VHDEKMEEQTEILQNGFNPEED

KCNNCDQELNKKPQDETKESTSCPWQ

KRAALLNAILHSAYSETFLLPHLKDIPAQ

HITLFLKYLYFLYLKCSENATMTLPGIHP

PTLNQIMDWICLLLDANFTVWMMPEA

KRLLINLYKLVKSQISWSELNKIEVSFR

ELQKLNQEKNNRGLYSIEVLELF

645 A 118 473 WWPVLRRWTMKT'F'RWKVKPGMDVAS

VPSVRKVRFGDGYSQRAPAGLNANLKT

YSVTLSVPREEATVLESFLEEHGGWKSF

LWTPPYEWRQIKVTCAKWSSRVSMLRV

EFSAEFEQWN

TAVLRKRQEEMRPLDIDEVEAPEEVEVL

EPEEDFEQFLLPVINEMREDIASLIREHGR

AYLRTRSKLWEMDNMLIQIKTQVEASEE

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, amino Q=Glutamine, to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, I=possible nucleotide deletion, \=possible sequence nucleotide insertion SALNHVQHPSGEADERVSELCEKAEEKA

KEIAKMAEMLVELVWRIERSESS

NSSREPGWETAMAKDILGEAGLHFDEL

NKLRVLDPEVTQQTIELKEECKDFVDKI

GQFQKIVGGLIELVDQLAKEAENEKMK

AIGARNLLKSIAKQREAQQQQLQALIAE

KKMQLERYRVEYEALCKVEAEQNEFID

QFIFQK

ESLKELEEEAEEEQRILQQSVVKTYEDM

TLEELEDHEDEFNEEDERAIEMYRRRRL

AEWKATKLKNKFGEVLEISGKDYVQEV

TKAGEGLW VILHLYKQGIPLCALINQHL

SGLARKFPDVKFIKAISTTCIPNYPDRNLP

TIFVYLEGDIKAQFIGPLVFGGMNLTRDE

LEWKLSESGAIMTDLEENPKKPIEDVLLS

SVRRSVLMKRDSDSEGD

QKDGRRVEFTATSVSDLKKYIAELEVQT

GMTQRRTGPAGFYV

PKTEKCLCPRGRTGARCDAG

TSGQMEDTIKEKQYILNEARTLFRKNKN

LTDTDLIKQCIDECTARIEIGLHYKIPYPR

PIHLPPMGLTPLRGRGLRSQEKLRKLSKP

VYLRSHDEVS

SAELVAKGTVGKRKWGCAGASSSGSAL

LPPCRELLMGHRFLRGLLTLLLPPPPLYT

RI~RMLGPES VPPPKRSRSKLMAPPRIGT

HNGTFHCDEALACALLRLLPEYRDAEIV

RTRDPEKLASCDIWDVGGEYDPRRHRY

DHHQRSFTETMSSLSPGKPWQTKLSSAG

LIYLHFGHKLLAQLLGTSEEDSMVGTLY

DKMYENFVEEVDAVDNGISQWAEGEPR

YALTTTLSARVARLNPTWNHPDQDTEA

GFKRAMDLVQEEFLQRLDFYQHS WLPA

RALVEEALAQRFQVDPSGEIVELAKGAC

PWKEHLYHLESGLSPPVAIFFVIYTDQAG

QWRIQCVPKEPHSFQSRLPLPEPWRGLR

DEALDQV SGIPGCIFVHASGFIGGHRTRE

GALSMARATLAQRSYLPQIS

SCRQRQLEARKAAAEKKKIQKEKLSTPE

KIKQEALELAGITSDPGLSLKGGLSQQGL

KPSLKVEPQNHFSSFKYSGNAWESYSV

LGNCRPSDPYSMNSVYSYHSYYAQPSLT

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, I=possible nucleotide deletion, \=possible sequence nucleotide insertion SVNGFHSKYALPSFSYYGFPSSNPVFPSQ

a FLGPGAWGHSGSSGSFEKKPDLHALHNS

LSPAYGGAEFAELPSQA VPTDAHHPTPH

HQQPAYPGPKEYLLPKAPLLHSVSRDPS

PFAQSSNCYNRSIKQEPVDPLTQAEPVPR

DAGKMGK'TPLSEVSQNGGPSHLWGQYS

GGPSMSPKRTNGVGGSWGVFSSGESPAI

VPDKLSSFGASCLAPSHFTDGQWGLFPG

EGQQAASHSGGRLRGKPWSPCKFGNST

SALAGPSLTEKPWALGAGDFNSALKGSP

GFQDKLWNPMKGEEGRIPAAGASQLVF

YQHKNLNQPNHGLALWEAKMKQLAER

ARARQEEAARLGLGQQEAKLYGKKRK

WGGTVVAEPQQKEKKGVVPTRQALAV

PTDSAVTVSSYAYTKVTGPYSRWI

654 A 653 2 KYRWGFTVLARMVSIS*SRDLPVSASQS

AGIIGVSHRARPRFYFLISLWHLLYFLIFF

QS*Q*MKNLCLKKLPDDSNYCSCTTLFK/

TGNLSCLPDILCTKS*YSVS/CKSFANSYI

QKKHDKM*KENGVR*GIINNLRLSIILILI

LIS*LSYFNDFH*NSGTSLIVS*HYYLNKH

DLHPNYFILLFCFSF/VRQGLRSVAQAGV

QWCDLGSLEPPPPRF

FPP*NFFFSPRGLIFGGGFFQFFPPPKKRFF

SKIPEAFLKVPPKRKKIVFATAPVIYFGPP

REFFKGPPP

PPPFFIPPPRIFFLGPLKKKKTPPPPGGKNF

FF*RAPPSP

YTNGAMCVLLPRATPGTLSLTPAFATW

GGIKQIPLPYAFGDVFTKYC

PSSSVAGENFPCEEGLEAGPSPTVLGAH

AEMALDSQVPKVTPQEEADSDVGEEPD

SENTPQKADKDAGLAQHSGPQKLLHIA

QELLHTEETYVKRLHLLDQVFCTRLTDA

GIPPEVIMGIFSNISSIHRFHGQFLLPELKT

RITEEWDTNPRLGDILQKLAPFLKMYGE

YVKNFDRAVGLVSTWTQRSPLFKDVVH

SIQVRRRRG

KAARF SCDIEQLKAQIMLCGEIPHPKNN

YSSRTPCSSLLPLLNAHAATSGKQSNFSR

KS STHNKPS EGKAANPKMV S SLPS
TARP

SHQTMTANKQNGE

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=AsparticAcid,E=GlufamicAcid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR--Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence colon, /=possible nucleotide deletion, l=possible sequence nucleotide insertion KAARFSCDIEQLKAQIMLCGEIPHPKNN

YSSRTPCSSLLPLLNAHAATSGKQSNFSR

KSSTHNKPSEGKAANPKMVSSLPSTADP

SHQTMTANKQNGE

GFREALVGAVNWSLVEDWFMAAVQN

PALTALTTPIQTAALQVPVRAAFSPTLLP

PPLFLEVLVFRDVKQVPGGVKQLEPPKE

GERRTT\HNIIEKRIRSS\INDKIIELKDLV

MGTDAKVGAKQNCVLCSTISPLPVFASG

A*RSPRLKVSEYKTELEVLFY*SILVSCQ

DYKILKNIRDW *GLIRQ*PAETFDSFGYA

LQLMGTPLSEAEKDACHSVWDTGLCW

WSQLMA

SFRKLMEHKQNSVGRVESASESVIALYH

IQRSVTEKLIFFSL\SLSEIILISCENDLHLC

REYFARGIGTFPRLPV

664 A 558 175 ASLSCGGLHPVRASWLLCL'TKQAWAME

GAPPPASLPPCSLISDCCASNQRDSVGVG

PSEPCAGWLWRRFLSRSEKRNIRVGV

TRFSRCV/LSPLSLTQKGNSLTPCASQAR

QCLALLRLAHGARTH

EFKMKYWGVTSFLQKGNDDHWIVDTD

YDTYAGQYSCRLLNLDGTCADSYSFVFS

RDPNGLPPEAQKIVRQRQEELCLARQYR

LIVHNGYCDGRSKRNLL

ARPTLVF

DGGQVWIGR

ESTQSTPQKGRGRPSKTPSPSQPKKWR

VGRSKQAATKENDSSEEVNVFQGSSPVD

DIPQEETEEEEVSTVNVRRRSAKRERR

PAPPTQQSSAE

PAPPTQQSSAE

LSKLMKAYCERQGLSMRHITLRFDGQSI

NETDTPAQLEMEDEDTIDVFQQQTESVP

DSILAGHSF

FLRSFSDKQKISLLHSCTCVLYTPSNEHF

GIVPLEAMYMQCPVIAWSGGPLESIDH

SVTGFLCEPDPVHFSEAIEKFIREPSLKAT

MGLAGRARVKEKFSPEAFTEQLYRWT

144 .

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K--Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, X=Stop peptide sequence codon, !=possible nucleotide deletion, \=possible sequence nucleotide insertion KLAG

PRGGHQCGSALSCDEGVPAAQGASSEKP

HECTKCGKALCCRSDLRVHHGVHAGEK

SSACSERGSGFREKLCPDKQGTHTKEKP

ARDSRSGKTIFRKTRLCVPGTVHAGAKF

YKC WECEKTSHKSRLIEHLRSHTGEKPC

GCRECGKAFFQKSHLILRQRTHTGEKPC

GCAECGKAAPRTFAS

TTGDPAREFTMIHSAPLMLLADPDEFES

VQLAQSWPLGAIVSLWRSPCRKMN

KSMKMRKS*CKKAENSKNQNGSSPAKD

YNSSPAREQNWMENWLDKLTEVGFIRW

IITNSFKLKEHVLTQCKELRS/FEKKLEEL

LTKIISLEKTISV*GEG*IQILDRSL*RVCV

GYLRRSC

LHTTEGSLLATYGWIVFSCTLLYVVFQ

KLSARLRALRHRQLDRAAAAVEPDVDV

KLQEALAAARLKMQEELNAQVEKHKE

KLKQLE*EKRRQTIEMW SMQDGKSY

677 A 588 3 GGWLAFPLACPGRCHQPPAAl'ASAALL

NLGRPEAPAHPQTAGSVRPAAVPPGC*G

RTGGAGSPAGHSATGPAAPPPRGSTSSA

PPARQAEAGLESSSAQVIRPPRSSKVLGL

QA*ATAPGLPQLSEGDPGFPSGRVTWIG

QSPASEDSWRPSAVSSLTYSPVSPARGC

RSPS*KSGKT*KSRQPKRENPPGTAP

NF SLESHNISLTEHS SMPVENNITLERP
SN

VNLTCQFTTSGD

YIYIHIYTYTHTYNFFFLERELHS

LLTPPNWSGPPGLPPAASQPIILPGPGPSP

NLLKSSTSSERSRGAK

REQDRDVQLSKALSYALRHGALKI~GLP

MGADGFVPLGTLLQLPQFRGFSAEDVQ

RWDTNRKQRFALQLGDPSTGLLIRANQ

GHSLQVPKLELMPLETPQALPPMLVHGT

FWKHWPSILLKGLSCQGRTHIHLAPGLP

GDPGIISGMRSRCEIAWIDGPLALADGIP

FFRSANGVILTPGNTDGFLLPKYFKEALQ

LRPTRKPLSLAGDEETECQSSPKHSSRER

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C~Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S=Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, x=Stop peptide sequence codon, I=possible nucleotide deletion, \=possible sequence nucleotide insertion KTRRV SSRKKPALKATSDEKDSFSNITR

GKKDGEISRKVSSQKPPTLKGTSDEEDS

VLGIARENKDGEKSRTVSSEKPPGLKAS

SDEKENVLNIARVYCVRCRGS

KRRRVPRDPGGANCAGGGVSADTSGLA

AAARGPPTLGFRERRSARSLDRIRPILVL

MVDKKLVWFGGTGAQGGSVARTLLE

DGTFKVRWTRNPRKKAAKELRLQGAE

VVQGDQDDQVIMELALNGAYATFIVTN

YWESCSQEQEVKQGKLLADLARRLGLH

YVWSGLENIKKLTAGRLAAAHFDGKG

EVEEYFRDIGVPMTSVRLPCYFENLLSHF

LPQKAPDGKSYLLSLPTGDVPMDGMSV

SDLGPWLSLLKMPEKWGQNIGLSTCR

HTAEEYAALLTKH~PARSCTMPR*LLRTT

KSLAFPVPGTW/LNMFRFYALRPDRDIEL

TLRLNPKALTLDQWLEQHKGDFNLL

DLVPVPTNLYGDFFTGDAWILKWQLR

NGNLQYDLHYWLGNECSQDESGAAAIF

TVQLDDYLNGRAVQHREVQGFESATFL

GYFKSGLKYKKGGVASGFKHWPNEW

VQRLFQVKGRRVVRATEVPVSWESFNN

GDCFILDLGNNIHQWCGSNSNRYERLKA

TQVSKGIRDNERSGRARVHVSEEGTEPE

AMLQVLGPKPALPAGTEDTAKEDAANR

KLAKLYKVSNGAGTMSVSLVADENPFA

QGALKSEDCFILDHGKDGKIFV WKGKQ

ANTEERKAALKTASDFITKMDYPKQTQ

VSVLPEGGETPLFKQFFKNWRDPDQTDG

LGLSYLS SHIANVERVPFDAATLHTSTA

MAAQHGMDDDGTGQKQIWRIEGSNKV

PVDPATYGQFYGGDSYIILYNYRHGGRQ

GQIIYNWQGAQSTQDEVAASAILTAQLD

EELGGTPVQSRVVQGKEPAHLMSLFGG

KPMIIYKGGTSREGGQTAPASTRLFQVR

ANSAGATRAVEVLPKAGALNSNDAFVL

KTPSAAYLWGTGASEAEKTGAQELLR

VLRAQPVQVAEGSEPDGFWEALGGKAA

YRTSPRLKDKKMDAHPPRLFACSNKIGR

FVIEEVPGELMQEDLATDDVMLLDTWD

QVFVWGKDSQEEEKTEALTSAKRYIET

DPANRDRRTPITWKQGFEPPSFVGWFL

GWDDDY\WSVGPLGTGPMAGAGLP

685 A 361 570 NCFLVFSPARSTVGEFASMSSEECI*MHV

VFLFPLTLCWSL*ERQEAEKMFKGKRG

AQLAKDIARRSKT

686 A ~ 2484 1122 QAQPMGRVGGMAQPMGRAGAPKPMG
~

SEQ MethodPredicted Predicted Amino acid sequence (A=Alanine end C=Cysteine, ID beginning nucleotideD=Aspartic Acid, E=Glutamic Acid, NO: nucleotidelocation F=Phenylalanine, G=Glycine, H=Histidine, location correspondingI=Isoleucine, K=Lysine, L=Leucine, M=Methionine, correspondingto last N=Asparagine, P=Proline, Q=Glutamine, amino to first acid residueR=Arginine, S--Serine, T=Threonine, amino of V=Valine, acid residuepeptide W=Tryptophan, Y=Tyrosine, of X=Unknown, Y=Stop peptide sequence codon, /=possible nucleotide deletion, \=possible sequence nucleotide insertion RAGSARRGRFKGCWSEGSPVHPVPAVL

SWLLALLRCASTMLSLRVPLAPITDPQQ

LQLSPLKGLSLVDKENTPPALSGTRVLA

SKTARRIFQEPTEPKTKAAAPGVEDEPLL

RENPRRFVIFPIEYHDIWQMYKKAEASF

WTAEEVDLSKDIQHWESLKPEERYFISH

VLAFFAASDGIVNENLVERFSQEVQITEA

RCFYGFQIAMENIHSEMYSLLIDTYIKDP

KEREFLFNAIETMPCVKKKADWALRWI

GDKEATYGERVVAFAAVEGIFFSGSFASI

FWLKKRGLMPGLTFSNELISRDEGLHCD

FACLMFKHLVHKPSEERVREIIINAVRIE

QEFLTEALPVKLIGMNCTLMKQYIEFVA

DRLMLELGFSKVFRVENPFDFMENISLE

GKTNFFEKRVGEYQRMGVMS SPTENSF

TLDADF

GVMFFERAFDQGADAIYDHVNEGKLW

KHIKHKYENK

CCCSKLLPPLGLKGFQMEHFDASLSTYF

KALLGPRDTRVKGWFLLDNYIPTFICSVI

YLLIVWLGPKYMRNKQPFSCRGILVVYN

LGLTLLSLYMFCELVTGV WEGKYNFFC

QGTRTAGESDMKIIRVLWWYYFSKLIEF

MDTFFFILRKNNHQITVLHVYHHASMLN

IWWFVMNWVPCGHSYFGATLNSFIHVL

MYSYYGLSSVPSMRPYLWWKKYITQGQ

LLQFVLTIIQTSCGVIWPCTFPLGWLYFQI

GYMISLIALFTNFYIQTYNKKGASRRKD

HLKDHQNGSMAAVNGHTNSFSPLENNV

KPRKLRKD

SEQ Position of end MaxS (maximum Means (mean ID of signal score) score) NO: in amino acid sequence 1 29 0.962 0.801 S 17 0.989 0.926 15 33 0.933 0.780 20 39 0.899 0.608 23 18 0.992 0.867 25 32 0.928 0.768 26 37 0.979 0.808 27 48 0.977 0.661 28 18 0.965 0.805 29 16 0.972 0.860 30 21 0.948 0.785 31 23 0.954 0.824 32 18 0.966 0.896 33 15 0.989 0.956 39 16 0.988 0.971 61 37 0.979 0.808 62 21 0.990 0.901 73 48 0.977 0.661 86 18 0.965 0.805 87 16 0.949 0.749 88 16 0.949 0.749 89 16 0.949 0.749 91 16 0.972 0.860 102 21 0.948 0.785 103 19 0.893 0.604 104 28 0.945 0.556 106 17 0.965 0.869 107 16 0.980 0.957 108 22 0.969 0.917 11 S 23 0.954 0.824 126 18 0.966 0.896 138 38 0.901 0.607 139 33 0.976 0.754 140 27 0.966 0.909 147 38 0.901 0.607 149 22 0.929 0.83 8 162 32 0.949 0.704 168 27 0.966 0.909 171 42 0.895 ~ 0.609

Claims (28)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-172, or 345-516, a mature protein coding portion of SEQ ID NO: 1-172, or 345-516, an active domain coding portion of SEQ ID NO: 1-172, or 345-516, and complementary sequences thereof.

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 90% 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 1 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: 1-172, or 345-516.

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 SEQ ID NO: 1-172, or 345-516, a mature protein coding portion of SEQ ID NO: 1-172, or 345-516, an active domain coding portion of SEQ ID NO: 1-172, or 345-516, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ
ID NO: 1-172, or 345-516, 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: 173-344, or 517-688, the mature protein portion thereof, or the active domain thereof.

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

22. A collection of polynucleotides, wherein the collection comprising the sequence information of at least one of SEQ ID NO: 1-172, or 345-516.

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.

27. A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim or 20 and a pharmaceutically acceptable carrier.

28. A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.