WO2001087917A1 - Nouveaux acides nucleiques et polypeptides - Google Patents

Nouveaux acides nucleiques et polypeptides Download PDF

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Publication number
WO2001087917A1
WO2001087917A1 PCT/US2001/014826 US0114826W WO0187917A1 WO 2001087917 A1 WO2001087917 A1 WO 2001087917A1 US 0114826 W US0114826 W US 0114826W WO 0187917 A1 WO0187917 A1 WO 0187917A1
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Prior art keywords
ofthe
polypeptide
polynucleotide
protein
cells
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PCT/US2001/014826
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English (en)
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WO2001087917A8 (fr
Inventor
Y. Tom Tang
Vinod Asundi
Ping Zhou
Aidong J. Xue
Feiyan Ren
Jie Zhang
Jian-Rui Wang
Yonghong Yang
Qing A. Zhao
Ryle W. Goodrich
Chenghua Liu
Radoje T. Drmanac
Tom Wehrman
Rui-Hong Chen
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Hyseq, Inc.
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Priority to AU2001263005A priority Critical patent/AU2001263005A1/en
Priority to EP01937252A priority patent/EP1282635A4/fr
Priority to CA002409164A priority patent/CA2409164A1/fr
Publication of WO2001087917A1 publication Critical patent/WO2001087917A1/fr
Priority to US10/294,433 priority patent/US20070049743A1/en
Publication of WO2001087917A8 publication Critical patent/WO2001087917A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.
  • compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
  • the compositions ofthe present invention additionally include vectors, including expression vectors, containing the polynucleotides ofthe 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: 1-216, or 433-648.
  • the polypeptides sequences are designated SEQ ID NO: 217-432, or 649-864.
  • the nucleic acids and polypeptides are provided in the Sequence Listing.
  • A is adenosine
  • C is cytosine
  • G is guanine
  • T is thymine
  • N is unknown or any ofthe four bases.
  • * corresponds to the stop codon.
  • the nucleic acid sequences ofthe present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-216, or 433-648 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any ofthe nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation ofthe peptides encoded by SEQ ID NO: 1-216, or 433-648.
  • a polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-216, or 433-648 or a degenerate variant or fragment thereof.
  • the identifying sequence can be 100 base pairs in length.
  • the nucleic acid sequences ofthe present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-216, or 433-648.
  • the sequence information can be a segment of any one of SEQ ID NO: 1-216, or 433-648 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-216, or 433-648.
  • 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.
  • segments of sequence information are 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-readable format.
  • This invention also includes the reverse or direct complement of any ofthe 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-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.
  • nucleic acid sequences of SEQ ID NO: 1-216, or 433-648 or novel segments or parts ofthe nucleic acids ofthe invention are used as primers in expression assays that are well known in the art.
  • nucleic acid sequences of SEQ ID NO: 1-216, or 433-648 or novel segments or parts ofthe 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 ofthe human genome.
  • the isolated polynucleotides ofthe invention include, but are not limited to, a polynucleotide comprising any one ofthe nucleotide sequences set forth in SEQ ID NO: 1-216, or 433-648; a polynucleotide comprising any ofthe full length protein coding sequences of SEQ ID NO: 1-216, or 433-648; and a polynucleotide comprising any ofthe nucleotide sequences ofthe mature protein coding sequences of SEQ ID NO: 1-216, or 433-648.
  • the polynucleotides ofthe present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one ofthe nucleotide sequences set forth in SEQ ID NO: 1-216, or 433-648; (b) a nucleotide sequence encoding any one ofthe 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.
  • the isolated polypeptides ofthe invention include, but are not limited to, a polypeptide comprising any ofthe amino acid sequences set forth in SEQ ID NO: 217-432, or 649-864; or the corresponding full length or mature protein.
  • Polypeptides ofthe invention also include polypeptides with biological activity that are encoded by (a) any ofthe polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-216, or 433-648; or (b) polynucleotides that hybridize to the complement ofthe polynucleotides of (a) under stringent hybridization conditions.
  • Biologically or immunologically active variants of any ofthe polypeptide sequences in the Sequence Listing, and * "substantial equivalents" thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%o or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.
  • the polypeptides ofthe invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) ofthe invention.
  • compositions comprising a polypeptide ofthe invention.
  • Polypeptide compositions ofthe 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 ofthe invention comprising growing a culture ofthe host cells ofthe invention in a suitable culture medium under conditions permitting expression ofthe 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 ofthe 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 ofthe invention can be used as hybridization probes to detect the presence ofthe particular cell or tissue mRNA in a sample ' using, e.g., in situ hybridization.
  • 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 ofthe 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.
  • a polypeptide ofthe 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 ofthe 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 ofthe present invention and a pharmaceutically acceptable carrier.
  • a composition comprising a polypeptide ofthe present invention and a pharmaceutically acceptable carrier.
  • the polypeptides and polynucleotides ofthe 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 ofthe polynucleotides or polypeptides ofthe 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 ofthe 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 ofthe 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 ofthe complex such that if a complex is formed, the polypeptide is detected.
  • kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods ofthe 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 ofthe polynucleotides and/or polypeptides ofthe 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 ofthe invention.
  • the invention provides a method for identifying a compound that binds to the polypeptides ofthe invention comprising contacting the compound with a polypeptide ofthe 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 ofthe reporter gene is detected the compound the binds to a polypeptide ofthe invention is identified.
  • the methods ofthe invention also provide methods for treatment which involve the administration ofthe polynucleotides or polypeptides ofthe invention to individuals exhibiting symptoms or tendencies.
  • the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity ofthe target gene products. Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.
  • polypeptides ofthe 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 ofthe present invention are useful for a variety of applications, as described herein, including use in arrays for detection.
  • active refers to those forms ofthe polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.
  • biologically active or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.
  • immunologically active or “immunological activity” refers to the capability ofthe natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • activated cells are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.
  • ES embryonic stem cells
  • GSCs germ line stem cells
  • primordial stem cells primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.
  • primordial germ cells PGCs
  • PGCs primordial germ cells
  • PGCs primordial germ cells
  • PGCs primordial germ cells
  • 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.
  • EMF expression modulating fragment
  • 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.
  • 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.
  • PNA peptide nucleic acid
  • A is adenine
  • C cytosine
  • T thymine
  • G guanine
  • N A, C, G or T (U).
  • nucleic acid segments provided by this invention may be assembled from fragments ofthe 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.
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • a fragment or segment may uniquely identify each polynucleotide sequence ofthe present invention.
  • the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-216, or 433-648.
  • 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 ofthe 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 ofthe present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-216, or 433-648.
  • the sequence information can be a segment of any one of SEQ ID NO: 1-216, or 433-648 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-216, or 433- 648.
  • 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 human genome, there are three billion base pairs in one set of chromosomes. Because 4 20 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.
  • the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.
  • 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% ofthe entire genome sequence.
  • a segment when using sequence information for detecting a single mismatch, a segment can 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 ⁇ 4 25 ) times the increased probability for mismatch at each nucleotide position (3 x 25).
  • the probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.
  • the probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.
  • ORF open reading frame
  • operably linked refers to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription ofthe coding sequence.
  • 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 ofthe coding sequence.
  • 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.
  • 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.
  • any polypeptide must have sufficient length to display biological and/or immunological activity.
  • 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 ofthe polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • translated protein coding portion means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.
  • 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 ofthe 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 an 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.
  • 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.
  • variant refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e 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 ofthe 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.
  • recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use ofthe "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 ofthe polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.
  • 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 replacements may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature ofthe residues involved.
  • 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.
  • insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.
  • Such alterations can, for example, alter one or more ofthe biological functions or biochemical characteristics of the polypeptides ofthe invention.
  • such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.
  • 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.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
  • purified or substantially purified 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.
  • the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, ofthe 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).
  • isolated 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.
  • 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 ofthe same.
  • isolated and purified do not encompass nucleic acids or polypeptides present in their natural source.
  • recombinant when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.
  • Microbial refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.
  • 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.
  • 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 component thereof.
  • 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.
  • recombinant protein 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.
  • 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 ofthe 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 ofthe regulatory elements linked to the endogenous DNA segment or gene to be expressed.
  • the cells can be prokaryotic or eukaryotic.
  • 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 ofthe 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.
  • an expression vector may be designed to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell.
  • a signal or leader sequence may be naturally present on the polypeptides ofthe present invention or provided from heterologous protein sources by recombinant DNA techniques.
  • 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 NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.1X SSC/0.1% SDS at 68°C), and moderately stringent conditions (i.e., washing in 0.2X SSC/0.1% SDS at 42°C).
  • highly stringent conditions i.e., hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.1X SSC/0.1% SDS at 68°C
  • moderately stringent conditions i.e., washing in 0.2X SSC/0.1% SDS at 42°C.
  • Other exemplary hybridization conditions are described herein in the examples.
  • 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).
  • 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.
  • 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.
  • a substantially equivalent, e.g., mutant, sequence ofthe 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 no 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 ofthe invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy ofthe genetic code.
  • 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.
  • sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.
  • truncation of the mature sequence e.g. , via a mutation which creates a spurious stop codon
  • Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, 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.
  • totipotent refers to the capability of a cell to differentiate into all ofthe cell types of an adult organism.
  • transformation means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.
  • 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.
  • infection refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.
  • 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 ofthe marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.
  • the isolated polynucleotides ofthe invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-216, or 433-648; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 217-432, or 649-864; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence ofthe polypeptides of any one of SEQ ID NO: 217-432, or 649-864.
  • the polynucleotides ofthe present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any ofthe nucleotides sequences of SEQ ID NO: 1-216, or 433-648; (b) nucleotide sequences encoding any one ofthe amino acid sequences set forth in the Sequence Listing as SEQ ID NO: 217-432, or 649-864; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any ofthe proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation ofthe polypeptides of SEQ ID NO: 217-432, or 649-864.
  • Domains of interest may depend on the nature ofthe encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable 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 ofthe 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 ofthe coding region ofthe cDNA or may represent a portion ofthe 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.
  • full length cDNA or genomic DNA that corresponds to any ofthe polynucleotides of SEQ ID NO: 1-216, or 433-648 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any ofthe polynucleotides of SEQ ID NO: 1-216, or 433-648 or a portion thereof as a probe.
  • the polynucleotides of SEQ ID NO: 1-216, or 433-648 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.
  • the nucleic acid sequences ofthe 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.
  • polynucleotides ofthe 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.
  • nucleic acid sequence fragments that hybridize under stringent conditions to any ofthe nucleotide sequences of SEQ ID NO: 1-216, or 433-648, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e. specifically hybridize to) any one ofthe polynucleotides ofthe invention are contemplated.
  • Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences ofthe 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.
  • sequences falling within the scope ofthe 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 in SEQ ID NO: 1-216, or 433-648, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-216, or 433-648 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 ofthe present invention can be obtained by searching a database using an algorithm or a program.
  • 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)).
  • a FASTA version 3 search against Genpept using Fastxy algorithm.
  • Species homologs (or orthologs) ofthe 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 ofthe disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms ofthe isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.
  • nucleic acid sequences ofthe invention are further directed to sequences which encode variants ofthe 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 ofthe mutation and the nature ofthe mutation. Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature. These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).
  • Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.
  • Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.
  • Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.
  • 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.
  • polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.
  • This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides ofthe changed amino acid to form a stable duplex on either side ofthe site of being changed.
  • site-directed mutagenesis is 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.
  • PCR may also be used to create amino acid sequence variants ofthe novel nucleic acids.
  • primer(s) 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., Gene 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 ofthe genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice ofthe 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 ofthe invention and heterologous protein sequences.
  • the polynucleotides ofthe invention additionally include the complement of any ofthe 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 ofthe desired sequence identities.
  • polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-216, or 433-648, 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 ofthe clones identified herein.
  • a polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
  • Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide ofthe invention and a host cell containing the polynucleotide.
  • 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 ofthe nucleotide sequences of SEQ ID NO: 1-216, or 433-648 or a fragment thereof or any other polynucleotides ofthe invention.
  • the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any ofthe nucleotide sequences of SEQ ID NO: 1-216, or 433-648 or a fragment thereof is inserted, in a forward or reverse orientation.
  • the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.
  • suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs ofthe present invention. The following vectors are provided by way of example.
  • Bacterial pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); P Trc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLneo, ⁇ SV2cat, pOG44, PXTI, pSG (Stratagene) ⁇ SVK3, pBPV, pMSG, pSVL (Pharmacia).
  • the isolated polynucleotide ofthe invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al.,
  • operably linked means that the isolated polynucleotide ofthe 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 lad, 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.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation ofthe host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • 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.
  • PGK 3-phosphoglycerate kinase
  • 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.
  • the heterologous sequence can encode a fusion protein mcluding 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 ofthe 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, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements ofthe well known cloning vector pBR322 (ATCC 37017).
  • 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.
  • 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.
  • appropriate means e.g., temperature shift or chemical induction
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Polynucleotides ofthe invention can also be used to induce immune responses.
  • 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 ofthe DNA.
  • the nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.
  • Another aspect ofthe 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: 1-216, or 433-648, 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.
  • antisense nucleic acid molecules 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: 217-432, or 649-864 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-216, or 433-648 are additionally provided.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence ofthe invention.
  • the term “coding region” refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region” ofthe coding strand of a nucleotide sequence ofthe invention.
  • 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).
  • antisense nucleic acids ofthe 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 ofthe coding or noncoding region of a mRNA.
  • 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 ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fiuorouracil, 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,
  • 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 ofthe invention are typically administered to a subject or generated in 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 ofthe 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 ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • 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.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -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 (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBSLett 215: 327-330).
  • an antisense nucleic acid ofthe 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.
  • 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 ofthe invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1- 216, or 433-648).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-216, or 433-648 (see, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).
  • polynucleotides ofthe invention 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.
  • gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N Y. Acad. Sci.
  • the nucleic acids ofthe invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23).
  • 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 ofthe invention can be used in therapeutic and diagnostic applications.
  • 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 ofthe 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., SI 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).
  • PNAs ofthe 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.
  • 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.
  • 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) Bioorg Med Chem Lett 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl Acad. Sci. U.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).
  • peptides e.g., for targeting host cell receptors in vivo
  • 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.
  • 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, Pharm. Res. 5: 539-549).
  • 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.
  • the present invention further provides host cells genetically engineered to contain the polynucleotides ofthe invention.
  • host cells may contain nucleic acids ofthe 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 ofthe invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression ofthe 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. WO94/12650, PCT International Publication No. WO92/20808, and PCT International Publication No. WO91/09955.
  • amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted along with the heterologous promoter DNA. If linked to the coding sequence, amplification ofthe marker DNA by standard selection methods results in co- amplification ofthe 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 ofthe recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).
  • the host cells containing one ofthe polynucleotides ofthe 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 ofthe EMF.
  • Any host/vector system can be used to express one or more ofthe ORFs ofthe 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 ofthe present invention.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • 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 Colo205 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 r flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 viral genome for example, S V40 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 ofthe 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.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • yeast strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, 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 ofthe appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides ofthe invention under the control of inducible regulatory elements, in which case the regulatory sequences ofthe endogenous gene may be replaced by homologous recombination.
  • 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.
  • sequences which affect the structure or stability ofthe 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 ofthe 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 ofthe regulatory sequence, placing the gene under the control ofthe new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.
  • the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
  • 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.
  • the naturally occurring sequences are deleted and new sequences are added.
  • the identification ofthe 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 ofthe 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 ofthe 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 isolated polypeptides ofthe invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 217-432, or 649-864 or an amino acid sequence encoded by any one ofthe nucleotide sequences SEQ ID NO: 1-216, or 433-648 or the corresponding full length or mature protein.
  • Polypeptides ofthe invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one ofthe nucleotide sequences set forth in SEQ ID NO: 1-216, or 433-648 or (b) polynucleotides encoding any one ofthe amino acid sequences set forth as SEQ ID NO: 217-432, or 649-864 or (c) polynucleotides that hybridize to the complement ofthe polynucleotides of either (a) or (b) under stringent hybridization conditions.
  • the invention also provides biologically active or immunologically active variants of any ofthe amino acid sequences set forth as SEQ ID NO: 217-432, or 649-864 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: 217-432, or 649-864.
  • Fragments ofthe proteins ofthe present invention which are capable of exhibiting biological activity are also encompassed by the present invention.
  • Fragments ofthe 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) ofthe disclosed proteins.
  • the protein coding sequence is identified in the sequence listing by translation ofthe 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 ofthe mature form ofthe protein is also determinable from the amino acid sequence ofthe full-length form.
  • proteins ofthe present invention are membrane bound, soluble forms ofthe proteins are also provided. In such forms, part or all ofthe 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 ofthe present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
  • the present invention further provides isolated polypeptides encoded by the nucleic acid fragments ofthe present invention or by degenerate variants ofthe nucleic acid fragments ofthe present invention.
  • degenerate variant is intended nucleotide fragments which differ from a nucleic acid fragment ofthe present invention (e.g. , an ORF) by nucleotide sequence but, due to the degeneracy ofthe genetic code, encode an identical polypeptide sequence.
  • Preferred nucleic acid fragments ofthe present invention are the ORFs that encode proteins.
  • the amino acid sequence can be synthesized using commercially available peptide synthesizers.
  • the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. This technique is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
  • polypeptides and proteins ofthe present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.
  • 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 ofthe polypeptides or proteins ofthe present invention.
  • the invention also relates to methods for producing a polypeptide comprising growing a culture of host cells ofthe invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.
  • the methods ofthe invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide ofthe invention is cultured under conditions that allow expression ofthe 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 ofthe protein.
  • 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 ofthe isolated polypeptides or proteins ofthe present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affmity chromatography. See, e.g., Scopes, Protein Purification: Principles and Practice, Springer- Veriag (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 then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival ofthe animal/cells.
  • the peptides ofthe 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.
  • toxins e.g., ricin or cholera
  • the toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity ofthe binding molecule for SEQ ID NO: 217-432, or 649-864.
  • the protein ofthe invention may also be expressed as a product of transgenic animals, e.g., as a component ofthe 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.
  • one or more ofthe cysteine residues may be deleted or replaced with another amino acid to alter the conformation ofthe 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).
  • such alteration, substitution, replacement, insertion or deletion retains the desired activity ofthe protein.
  • Regions ofthe protein that are important for the protein function can be determined by various methods known in the art mcluding 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.
  • the protein may also be produced by operably linking the isolated polynucleotide ofthe invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • kits Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif, U.S.A. (the MaxBatTM kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.
  • an insect cell capable of expressing a polynucleotide ofthe present invention is "transformed.”
  • the protein ofthe 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 (/. e. , from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification ofthe 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 immunoaffinity chromatography.
  • affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseTM
  • hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether
  • immunoaffinity chromatography immunoaffinity chromatography
  • the protein ofthe invention may also be expressed in a form which will facilitate purification.
  • 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.
  • FLAG® is commercially available from Kodak (New Haven, Conn.).
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all ofthe 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.”
  • polypeptides ofthe 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 ofthe polypeptides ofthe invention embrace fusions ofthe polypeptides or modifications ofthe polypeptides ofthe 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.
  • 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.
  • 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.
  • polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.
  • identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S.F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S.F.
  • 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
  • a "chimeric protein" or “fusion protein” comprises a polypeptide ofthe invention operatively linked to another polypeptide.
  • the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.
  • a fusion protein comprises at least one biologically active portion of a protein according to the invention.
  • a fusion protein comprises at least two biologically active portions of a protein according to the invention.
  • 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.
  • a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.
  • the fusion protein is a GST-fusion protein in which the polypeptide sequences ofthe invention are fused to the C-terminus ofthe GST (i.e., glutathione S-transferase) sequences.
  • 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 ofthe immunoglobulin protein family.
  • the immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein ofthe 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 ofthe 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.
  • the immunoglobulin fusion proteins ofthe 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 ofthe invention with a ligand.
  • a chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques.
  • 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.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • 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).
  • 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
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a nucleic acid encoding a polypeptide ofthe invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein ofthe invention.
  • Mutations in the polynucleotides ofthe invention may result in loss of normal function of the encoded protein.
  • the invention thus provides gene therapy to restore normal activity ofthe polypeptides ofthe invention; or to treat disease states involving polypeptides ofthe invention.
  • Delivery of a functional gene encoding polypeptides ofthe invention to appropriate cells is effected ex vivo, in situ, ox in 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.
  • polypeptides ofthe invention in other human disease states, preventing the expression of or inhibitmg the activity of polypeptides ofthe 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 ofthe invention.
  • Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids ofthe present invention, their complements, or their translated RNA sequences, by methods known in the art. Further, the polypeptides ofthe present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.
  • the present invention still further provides cells genetically engineered in vivo to express the polynucleotides ofthe invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression ofthe polynucleotides in the cell. These methods can be used to increase or decrease the expression ofthe polynucleotides of the present invention.
  • DNA sequences allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.
  • Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.
  • the heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.
  • amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding sequence, amplification ofthe marker DNA by standard selection methods results in co-amplification ofthe desired protein coding sequences in the cells.
  • cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides ofthe invention under the control of inducible regulatory elements, in which case the regulatory sequences ofthe endogenous gene may be replaced by homologous recombination.
  • 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.
  • sequences which affect the structure or stability ofthe RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties ofthe 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 ofthe regulatory sequence, placing the gene under the control ofthe new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.
  • the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
  • 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.
  • the naturally occurring sequences are deleted and new sequences are added.
  • the identification ofthe 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 ofthe 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 ofthe negatively selectable marker.
  • Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xantbine-guanine phosphoribosyl-transferase (gpt) gene.
  • 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.
  • Knockout 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 ofthe 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. WO94/28122, incorporated herein by reference.
  • Transgenic animals can be prepared wherein all or part of a promoter ofthe polynucleotides ofthe invention is either activated or inactivated to alter the level of expression ofthe polypeptides ofthe 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.
  • polynucleotides ofthe present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides ofthe 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 ofthe polypeptides ofthe invention.
  • 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.
  • Knockout 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 ofthe 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. WO94/28122, incorporated herein by reference.
  • Transgenic animals can be prepared wherein all or part ofthe polynucleotides ofthe invention promoter is either activated or inactivated to alter the level of expression ofthe polypeptides ofthe 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.
  • polynucleotides and proteins ofthe present invention are expected to exhibit one or more ofthe uses or biological activities (including those associated with assays cited herein) identified herein.
  • Uses or activities described for proteins ofthe 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 ofthe invention, the polynucleotides ofthe invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.
  • compositions ofthe invention include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides ofthe invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity ofthe target gene products, either at the level of target gene/protein expression or target protein activity.
  • 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 ofthe 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 ofthe polypeptides ofthe invention.
  • polypeptides ofthe present invention may likewise be involved in cellular activation or in one ofthe other physiological pathways described herein.
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and
  • 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.
  • 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 ofthe 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 ofthe binding interaction.
  • Polynucleotides and polypeptides ofthe 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.
  • the polypeptide or polynucleotide ofthe 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.
  • the polypeptide or polynucleotide ofthe invention can be added to the medium in or on which the microorganism is cultured.
  • a polypeptide ofthe 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 ofthe 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.
  • compositions ofthe 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, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DAI, 123, Tl 165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.
  • Therapeutic compositions ofthe 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.
  • 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- ⁇ , Schreiber, R. D. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomry, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al, J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
  • a polypeptide ofthe 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 ofthe polypeptide ofthe invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.
  • the ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.
  • 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
  • organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.
  • exogenous growth factors and or cytokines may be administered in combination with the polypeptide ofthe invention to achieve the desired effect, including any ofthe 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 ofthe 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).
  • SCF stem cell factor
  • LIF leukemia inhibitory factor
  • Flt-3 ligand Flt-3 ligand
  • MIP-1 -alpha macrophage inflammatory protein 1-alpha
  • G-CSF G-CSF
  • GM-CSF GM-CSF
  • stroma cells transfected with a polynucleotide that encodes for the polypeptide ofthe 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 ofthe invention to induce autocrine expression ofthe polypeptide ofthe 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.
  • polypeptides ofthe 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 ofthe 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.
  • 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 ofthe polypeptide ofthe invention and its effect on stem cells can also be manipulated to achieve controlled differentiation ofthe 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 ofthe desired type to survive.
  • stem cells can be induced to differentiate into cardiomyocytes (Wobus et al, Differentiation, 48: 173-182, (1991); Klug et al., J. Clin. Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.
  • 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 ofthe polypeptide ofthe invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
  • a differentiation factor such as retinoic acid and an antagonist ofthe polypeptide ofthe invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
  • stem cells In vitro cultures of stem cells can be used to determine if the polypeptide ofthe 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 ofthe polypeptide ofthe invention alone or in combination with other growth factors or cytokines.
  • the ability ofthe polypeptide ofthe 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).
  • a polypeptide ofthe 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.
  • erythroid progenitor cells in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (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 ofthe above-mentioned hematopoietic cells and therefore find therapeutic utility in various
  • compositions ofthe invention can be used in the following:
  • 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 include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G.
  • a polypeptide ofthe present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.
  • a polypeptide ofthe 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 ofthe 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 ofthe invention.
  • tissue regeneration activity that may involve the polypeptide ofthe 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.
  • compositions ofthe 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 ofthe 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 in vivo to effect tissue repair.
  • the compositions ofthe 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.
  • compositions ofthe 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 ofthe 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 cerebro vascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition ofthe invention.
  • diseases ofthe 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, Hunt
  • compositions ofthe 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 ofthe 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.
  • organs including, for example, pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • Part ofthe desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.
  • a polypeptide ofthe present invention may also exhibit angiogenic activity.
  • a composition ofthe 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 ofthe 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.
  • compositions ofthe invention can be used in the following:
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (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).
  • a polypeptide ofthe present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a polynucleotide ofthe 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 (SOD)), 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.
  • SOD severe combined immunodeficiency
  • 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.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein ofthe present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • proteins ofthe 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 ofthe 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) ofthe 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.
  • allergic reactions and conditions e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema,
  • a protein (or antagonists thereof) ofthe present invention may also be treatable using a protein (or antagonists thereof) ofthe present invention.
  • the therapeutic effects ofthe 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 (Vohr et al., Arch. Toxocol. 73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).
  • 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 ofthe 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 ofthe 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).
  • B lymphocyte antigen functions such as, for example, B7
  • GVHD graft- versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection ofthe 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 ofthe invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • 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).
  • murine 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 ofthe 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 ofthe diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/Ipr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.
  • an antigen function e.g., a B lymphocyte antigen function
  • Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.
  • enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.
  • 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 ofthe present invention or together with a stimulatory form of a soluble peptide ofthe present invention and reintroducing the in vitro activated T cells into the 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 ofthe present invention as described herein such that the cells express all or a portion ofthe 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.
  • 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 MHC class I alpha chain protein and ⁇ 2 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.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and ⁇ 2 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.
  • 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.
  • 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 ofthe invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al, J. Immunol.
  • 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.
  • MLR Mixed lymphocyte reaction
  • 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.
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al, Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al, 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 84:111-117, 1994; Fine et al, Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
  • a polypeptide ofthe present invention may also exhibit activin- or inhibin-related activities.
  • a polynucleotide ofthe 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).
  • FSH follicle stimulating hormone
  • a polypeptide ofthe present invention alone or in heterodimers with a member ofthe 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.
  • the polypeptide ofthe invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells ofthe anterior pituitary. See, for example, U.S. Pat. No. 4,798,885.
  • a polypeptide ofthe invention may also be useful for advancement ofthe 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 ofthe 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.
  • a polypeptide ofthe present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • a polynucleotide ofthe 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 ofthe invention
  • 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.
  • the protein or peptide has the ability to directly stimulate directed movement of cells.
  • Therapeutic compositions ofthe 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
  • a polypeptide ofthe invention may also be involved in hemostasis or thrombolysis or thrombosis.
  • a polynucleotide ofthe 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 ofthe 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).
  • compositions ofthe 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.
  • Polypeptides ofthe invention may be involved in cancer cell generation, proliferation or metastasis. Detection ofthe presence or amount of polynucleotides or polypeptides ofthe invention may be useful for the diagnosis and/or prognosis of one or more types of cancer. For example, the presence or increased expression of a polynucleotide/polypeptide ofthe invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence ofthe 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.
  • compositions ofthe 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 ofthe female genital tract including ovarian
  • Polypeptides, polynucleotides, or modulators of polypeptides ofthe 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.
  • composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.
  • An anti-cancer cocktail is a mixture ofthe polypeptide or modulator ofthe 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 ofthe invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis- DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (VI 6-213), 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
  • Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.
  • therapeutic compositions of the invention may be used for prophylactic treatment of cancer. There are hereditary conditions and/or environmental situations (e.g.
  • in vitro models can be used to determine the effective doses ofthe polypeptide ofthe 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.
  • Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.
  • a polypeptide ofthe present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.
  • a polynucleotide ofthe invention can encode a polypeptide exhibiting such characteristics.
  • 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 ofthe relevant receptor/ligand interaction.
  • a protein ofthe present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
  • the activity of a polypeptide ofthe invention may, among other means, be measured by the following methods:
  • Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1- 7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
  • polypeptides ofthe 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.
  • polypeptides ofthe present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods.
  • radioisotopes include, but are not limited to, tritium and carbon- 14 .
  • colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.
  • toxins include, but are not limited, to ricin.
  • 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, affixed to a solid support, borne on a cell surface or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the 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.
  • Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides ofthe 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 ofthe organisms themselves.
  • Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof. For a review, see Science 282:63-6% (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.
  • peptide and oligonucleotide combinatorial libraries are peptide and oligonucleotide combinatorial libraries.
  • Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
  • combinatorial chemistry and libraries created therefrom see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).
  • peptidomimetic libraries see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol,
  • 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.
  • toxins e.g., ricin or cholera
  • the toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity ofthe binding molecule for a polypeptide ofthe invention.
  • the binding molecules may be complexed with imaging agents for targeting and imaging purposes.
  • the invention also provides methods to detect specific binding of a polypeptide e.g. a ligand or a receptor.
  • 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 ofthe 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 ofthe invention can be used to isolate polypeptides that recognize and bind polypeptides ofthe invention.
  • Ligands for receptor polypeptides ofthe 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 ofthe receptor ofthe invention: one cell population expresses the receptor ofthe invention whereas the other does not. The response ofthe two cell populations to the addition of ligands(s) are then compared.
  • an expression library can be co-expressed with the polypeptide ofthe invention in cells and assayed for an autocrine response to identify potential ligand(s).
  • BIAcore assays 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.
  • downstream intracellular signaling molecules in the signaling cascade ofthe polypeptide ofthe invention can be determined.
  • a chimeric protein in which the cytoplasmic domain ofthe polypeptide ofthe 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 ofthe 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.
  • compositions ofthe 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 ofthe invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.
  • conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegen
  • Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function ofthe polynucleotides and/or polypeptides ofthe invention.
  • 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).
  • Nervous system disorders involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity ofthe polynucleotides and/or polypeptides ofthe 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 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 ofthe nervous system, or compression injuries;
  • ischemic lesions in which a lack of oxygen in a portion ofthe nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
  • infectious lesions in which a portion ofthe nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;
  • degenerative lesions in which a portion ofthe 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;
  • neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;
  • demyelinated lesions in which a portion ofthe 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.
  • therapeutics which elicit any ofthe following effects may be useful according to the invention:
  • a neuron-associated molecule in culture or in vivo, e.g. , choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or
  • Such effects may be measured by any method known in the art.
  • 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.
  • 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.
  • 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 ofthe 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).
  • 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 ofthe 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
  • a polypeptide ofthe invention may also exhibit one or more ofthe 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
  • 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.
  • 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 ofthe 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 ofthe DNA, and identifying the presence ofthe polymorphism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.
  • 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 ofthe polymorphism is extended with one or more labeled nucleotides).
  • allele-specific oligonucleotide hybridization in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch
  • a single nucleotide extension assay in which an oligonucleotide that hybridizes immediately adjacent to the position ofthe polymorphism is extended with one or more labeled nucleotides.
  • traditional restriction fragment length polymorphism analysis using restriction enzymes that provide differential digestion ofthe genomic DNA depending on the presence or absence ofthe polymorphism
  • the array can comprise modified nucleotide sequences ofthe present invention in order to detect the nucleotide sequences ofthe present invention.
  • any one ofthe nucleotide sequences ofthe present invention can be placed on the array to detect changes from those sequences.
  • polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence ofthe protein, e.g., by an antibody specific to the variant sequence.
  • the immunosuppressive effects ofthe compositions ofthe 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 ofthe disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • the route of injection can vary, but rats may be injected at the base ofthe tail with an adjuvant mixture.
  • the polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.
  • the control consists of administering PBS only.
  • the procedure for testing the effects ofthe 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.
  • an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis ofthe data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease ofthe arthritis score.
  • compositions including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides
  • therapeutic applications include, but are not limited to, those exemplified herein.
  • One embodiment ofthe invention is the administration of an effective amount ofthe polypeptides or other composition ofthe invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides ofthe 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 ofthe polypeptides or other composition ofthe 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 ofthe individual patient. Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 ⁇ g/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 ⁇ g/kg to 10 mg/kg of patient body weight.
  • polypeptides ofthe invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts ofthe human serum albumin.
  • the vehicle may contain minor amounts of additives that maintain the isotonicity and stability ofthe polypeptide or other active ingredient. The preparation of such solutions is within the skill ofthe art.
  • a protein or other composition ofthe present 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.
  • 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.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredient(s).
  • the pharmaceutical composition ofthe invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • proteins ofthe invention may be combined with other agents beneficial to the treatment ofthe disease or disorder in question.
  • agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF- and TGF- ⁇ ), insulin-like growth factor (IGF), as well as cytokines described herein.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • TGF- and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • the pharmaceutical composition may further contain other agents which either enhance the activity ofthe 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 ofthe invention, or to minimize side effects.
  • protein or other active ingredient ofthe present invention may be included in formulations ofthe particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti- inflammatory agent to minimize side effects ofthe 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 ofthe present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • pharmaceutical compositions ofthe invention may comprise a protein ofthe invention in such multimeric or complexed form.
  • 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 ofthe combination of agents is achieved at the treatment site).
  • a therapeutically effective dose further refers to that amount ofthe compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration ofthe relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts ofthe active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • a therapeutically effective amount of protein or other active ingredient ofthe present invention is administered to a mammal having a condition to be treated.
  • Protein or other active ingredient ofthe present invention may be administered in accordance with the method ofthe invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.
  • protein or other active ingredient ofthe present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.
  • the attending physician will decide on the appropriate sequence of administering protein or other active ingredient ofthe present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • 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 ofthe present invention used in the pharmaceutical composition or to practice the method ofthe 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.
  • the compounds may be administered topically, for example, as eye drops.
  • 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 ofthe invention are administered by any route that delivers an effective dosage to the desired site of action.
  • a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.
  • Suitable dosage ranges for the polypeptides ofthe 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.
  • 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 ofthe active compounds into preparations which can be used pharmaceutically.
  • physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing ofthe active compounds into preparations which can be used pharmaceutically.
  • These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
  • protein or other active ingredient ofthe present invention When a therapeutically effective amount of protein or other active ingredient ofthe present invention is administered orally, protein or other active ingredient ofthe present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition ofthe 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 ofthe present invention, and preferably from about 25 to 90% protein or other active ingredient ofthe present invention.
  • 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 ofthe pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition When administered in liquid form, contains from about 0.5 to 90%) by weight of protein or other active ingredient ofthe present invention, and preferably from about 1 to 50% protein or other active ingredient ofthe present invention.
  • protein or other active ingredient ofthe present invention When a therapeutically effective amount of protein or other active ingredient ofthe present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient ofthe present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • 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 ofthe 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 ofthe present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the agents ofthe invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • 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 ofthe 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).
  • 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.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or 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.
  • compositions 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.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix ofthe 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 multi-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.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions ofthe active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentrated solutions.
  • 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.
  • 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.
  • 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 ofthe 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 ofthe nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.
  • This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity ofthe 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.
  • 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.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • 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.
  • 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.
  • suitable solid or gel phase 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 ofthe active ingredients ofthe 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 ofthe 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 ofthe invention may be in the form of a complex ofthe protein(s) or other active ingredient(s) 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 ofthe antigen by MHC proteins.
  • TCR T cell receptor
  • 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 antigen(s) 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.
  • the pharmaceutical composition ofthe invention may be in the form of a liposome in which protein ofthe 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.
  • 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.
  • liposomal formulations are 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 ofthe present invention in the pharmaceutical composition ofthe 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.
  • the attending physician will decide the amount of protein or other active ingredient ofthe present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein or other active ingredient ofthe present invention and observe the patient's response.
  • the various pharmaceutical compositions used to practice the method ofthe 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 ofthe present invention per kg body weight.
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • 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 ofthe 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 ofthe invention.
  • 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.
  • compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • 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 ofthe 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.
  • 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.
  • 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
  • CMC carboxymethylcellulose
  • Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl 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 ofthe protein from the polymer matrix and to provide appropriate handling ofthe 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 ofthe progenitor cells.
  • proteins or other active ingredients ofthe invention may be combined with other agents beneficial to the treatment ofthe bone and/or cartilage defect, wound, or tissue in question.
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • 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 ofthe proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition ofthe 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.
  • IGF I insulin like growth factor I
  • Polynucleotides ofthe 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 ofthe 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 ofthe 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.
  • 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 ofthe subject being treated. Determination ofthe effective amount is well within the capability of those skilled in the art, especially in light ofthe detailed disclosure provided herein.
  • 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 IC 50 as determined in cell culture (i.e. , the concentration of the test compound which achieves a half-maximal inhibition ofthe 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 ofthe 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 LD 50 (the dose lethal to 50% ofthe population) and the ED 50 (the dose therapeutically effective in 50% ofthe population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 50 and ED 0 .
  • 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 ED 50 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 ofthe patient's condition. See, e.g., Fingl et al., 1975, in "The
  • Dosage amount and interval may be adjusted individually to provide plasma levels ofthe active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
  • Dosage intervals can also be determined using MEC value.
  • Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% ofthe time, preferably between 30-90% and most preferably between 50-90%.
  • the effective local concentration ofthe drug may not be related to plasma concentration.
  • An exemplary dosage regimen for polypeptides or other compositions ofthe invention will be in the range of about 0.01 ⁇ g/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 ⁇ g/kg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity ofthe affliction, the manner of administration and the judgment ofthe prescribing physician.
  • 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 ofthe invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • antibodies to proteins, or fragments of proteins ofthe invention.
  • the term "antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (lg) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (lg) 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, F a b, F ab ' and F (ab')2 fragments, and an F ab expression library.
  • an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG], IgG 2 , 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 ofthe 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 ofthe antigen for use as immunogens.
  • An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein, such as the amino acid sequences shown in SEQ ID NO: 217-432, or 649-864, 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.
  • 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 ofthe protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of -related protein that is located on the surface ofthe protein, e.g., a hydrophilic region.
  • a hydrophilic region e.g., a hydrophilic region.
  • a hydrophobicity analysis ofthe human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte
  • a protein ofthe invention may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • 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.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
  • immunogenic 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.
  • 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 ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of i munoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope ofthe 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).
  • 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.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • 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 are 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 ofthe unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • 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 murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • 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 ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • 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 ofthe invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells ofthe invention serve as a preferred source of such DNA.
  • 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.
  • 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 ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens ofthe 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') or other antigen- binding subsequences of antibodies) that are principally comprised ofthe 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.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe 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)).
  • Fc immunoglobulin constant region
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice ofthe 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).
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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 immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications.
  • nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 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.
  • 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.
  • U.S. Patent No. 5,939,598 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 ofthe 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.
  • F ab FRAGMENTS AND SINGLE CHAIN ANTIBODIES techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F a b 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( a b') 2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F ab')2 fragment; (iii) an F a fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one ofthe binding specificities is for an antigenic protein ofthe invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature. 305:537-539 (1983)).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions.
  • CHI first heavy-chain constant region
  • 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 ofthe CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface ofthe 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 chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab' fragments 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') molecule.
  • Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
  • This method can also be utilized for the production of antibody homodimers.
  • the "diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.
  • the fragments comprise a " heavy-chain variable domain (VH) connected to a light-chain variable domain (V ) by a linker - which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and V domains of one fragment are forced to pair with the complementary VL and V H domains of another fragment, thereby forming two antigen-binding sites.
  • VH and V domains of one fragment are forced to pair with the complementary VL and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • 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.
  • bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen ofthe invention.
  • 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 (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.
  • 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.
  • 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).
  • Heteroconjugate antibodies are also within the scope ofthe present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • the antibody ofthe invention can be desirable to modify the antibody ofthe invention with respect to effector function, so as to enhance, e.g., the effectiveness ofthe antibody in treating cancer.
  • cysteine residue(s) 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 antitumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.
  • 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).
  • 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).
  • 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).
  • 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, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 L 131 In, 90 Y, and 186 Re.
  • Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the antibody in another embodiment, can be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
  • a "receptor” such as streptavidin
  • a nucleotide sequence ofthe present invention can be recorded on computer readable media.
  • 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.
  • 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
  • hybrids of these categories such as magnetic/optical storage media.
  • recorded refers to a process for storing information on computer readable medium.
  • a skilled artisan can readily adopt any ofthe presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information ofthe 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 ofthe present invention.
  • the choice ofthe data storage structure will generally be based on the means chosen to access the stored information.
  • a variety of data processor programs and formats can be used to store the nucleotide sequence information ofthe 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 ofthe present invention.
  • nucleotide sequences SEQ ID NO: 1-216, or 433-648 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any ofthe nucleotide sequences of SEQ ID NO: 1-216, or 433-648 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.
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information ofthe present invention.
  • the minimum hardware means ofthe computer-based systems ofthe present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • the computer-based systems ofthe present invention comprise a data storage means having stored therein a nucleotide sequence ofthe present invention and the necessary hardware means and software means for supporting and implementing a search means.
  • data storage means refers to memory which can store nucleotide sequence information ofthe present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information ofthe present invention.
  • 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 ofthe present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA).
  • EMBL Smith-Waterman
  • BLASTN BLASTN
  • BLASTA NPOLYPEPTIDEIA
  • 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.
  • searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing may be of shorter length.
  • a target structural motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding ofthe target motif.
  • 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).
  • fragments ofthe present invention 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 ofthe gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res.
  • the present invention further provides methods to identify the presence or expression of one ofthe ORFs ofthe present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention, optionally conjugated or otherwise associated with a suitable label.
  • methods for detecting a polynucleotide ofthe 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 ofthe 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 ofthe invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide ofthe invention is detected in the sample.
  • methods for detecting a polypeptide ofthe invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide ofthe invention is detected in the sample.
  • methods comprise incubating a test sample with one or more ofthe antibodies or one or more ofthe nucleic acid probes ofthe present invention and assaying for binding ofthe 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 ofthe nucleic acid probe or antibody used in the assay.
  • One skilled in the art will recognize that any one ofthe commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies ofthe 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.
  • test samples ofthe 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 ofthe detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.
  • kits which contain the necessary reagents to carry out the assays ofthe present invention.
  • the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one ofthe probes or antibodies ofthe present invention; and (b) one or more other containers comprising one or more ofthe following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.
  • 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.
  • 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.
  • the disclosed probes and antibodies ofthe present invention can be readily incorporated into one ofthe established kit formats which are well known in the art.
  • novel polypeptides and binding partners ofthe invention are useful in medical imaging of sites expressing the molecules ofthe invention (e.g., where the polypeptide ofthe 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 ofthe labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.
  • the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any ofthe nucleotide sequences set forth in SEQ ID NO: 1-216, or 433-648, or bind to a specific domain of the polypeptide encoded by the nucleic acid.
  • said method comprises the steps of:
  • such methods for identifying compounds that bind to a polynucleotide ofthe 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 ofthe invention is identified.
  • such methods for identifying compounds that bind to a polypeptide ofthe invention can comprise contacting a compound with a polypeptide ofthe 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 ofthe invention is identified.
  • Methods for identifying compounds that bind to a polypeptide ofthe invention can also comprise contacting a compound with a polypeptide ofthe 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 ofthe invention is identified.
  • Compounds identified via such methods can include compounds which modulate the activity of a polypeptide ofthe invention (that is, increase or decrease its activity, relative to activity observed in the absence ofthe compound).
  • compounds identified via such methods can include compounds which modulate the expression of a polynucleotide ofthe invention (that is, increase or decrease expression relative to expression levels observed in the absence ofthe compound).
  • Compounds, such as compounds identified via the methods ofthe invention can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.
  • the agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.
  • the agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.
  • 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 ofthe present invention.
  • agents may be rationally selected or designed.
  • an agent is said to be "rationally selected or designed" when the agent is chosen based on the configuration ofthe particular protein.
  • 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.
  • one class of agents ofthe present invention can be used to control gene expression through binding to one ofthe ORFs or EMFs ofthe 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 ofthe gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 3:173 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251 :1360 (1991)) or to the mRNA itself (antisense - Okano, J. Neurochem. 56:560
  • Agents which bind to a protein encoded by one ofthe ORFs ofthe present invention can be used as a diagnostic agent. Agents which bind to a protein encoded by one ofthe ORFs ofthe present invention can be formulated using known techniques to generate a pharmaceutical composition.
  • Another aspect ofthe subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.
  • the hybridization probes ofthe subject invention may be derived from any ofthe nucleotide sequences SEQ ID NO: 1-216, or 433-648. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from any ofthe nucleotide sequences SEQ ID NO: 1-216, or 433-648 can be used as an indicator ofthe presence of RNA of cell type of such a tissue in a sample.
  • any suitable hybridization technique can be employed, such as, for example, in situ hybridization.
  • PCR as described in US Patents Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.
  • 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.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.
  • the nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.
  • the nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.
  • Oligonucleotides i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • Support bound oligonucleotides may be prepared by any ofthe methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.
  • One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers. Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin. Microbiol. 28(6) 1469-72); using UV light (Nagata et al, 1985; Dahlen et al, 1987; Morrissey & Collins, (1989) Mol. Cell
  • Another strategy that may be employed is the use ofthe strong biotin-streptavidin interaction as a linker.
  • biotinylated probes although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.
  • Streptavidin-coated beads may be purchased from Dynal, Oslo.
  • Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies
  • 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 ⁇ /., (1991) Anal. Biochem. 198(1) 138-42).
  • CovaLink NH strips for covalent binding of DNA molecules at the 5'-end has been described (Rasmussen et al., (1991). In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res. 11 (8) 6513-29). This is beneficial as immobilization using only a single covalent bond is preferred.
  • the phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.
  • 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.
  • the linkage method includes dissolving DNA in water (7.5 ng/ ⁇ l) 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-Melm 7 ), is then added to a final concentration of 10 mM 1-Melm 7 . The single-stranded DNA solution is then dispensed into CovaLink NH strips (75 ⁇ l/well) standing on ice.
  • EDC l-ethyl-3-(3-dimethylaminopropyl)-carbodiirnide
  • a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.
  • This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3 '-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups 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.
  • addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al. (1991) Science 251(4995) 767-73, incorporated herein by reference.
  • Probes may also be immobilized on nylon supports as described by Van Ness et al. (1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal. Biochem. 169(1) 104-8; all references being specifically incorporated herein.
  • the nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA
  • DNA DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.
  • Sambrook et al. (1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p.
  • DNA fragments may be prepared as clones in Ml 3, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods. Samples may be prepared or dispensed in multiwell plates. About 100- 1000 ng of DNA samples may be prepared in 2-500 ml of final volume.
  • nucleic acids would then be fragmented by any ofthe 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
  • the restriction endonuclease CviJl 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 (Cvz ' JI* *), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).
  • Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a Cvz ' JI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus Ml 3 cloning vector. Sequence analysis of 76 clones showed that Cvz ' JI* * restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.
  • advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ⁇ g instead of 2-5 ⁇ g); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed
  • 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 nl of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density ofthe wells is achieved. One to 25 dots may be accommodated in 1 mm 2 , 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 ofthe subarrays may represent replica spotting ofthe same samples.
  • a selected gene segment may be amplified from 64 patients.
  • the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample). A plate for each ofthe 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 mm 2 and there may be a 1 mm space between subarrays.
  • membranes or plates available from NUNC, Naperville, Illinois
  • 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.
  • 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 ofthe library were amplified with PCR using primers specific for the vector sequences which flank the inserts.
  • Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.
  • the clones were clustered into groups of similar or identical sequences. Representative clones were selected for sequencing.
  • the 5' sequence ofthe amplified inserts was then deduced using a typical Sanger sequencing protocol.
  • PCR products were purified and subjected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.
  • SEQ ID NO: 433-648 The contigs or nucleic acids ofthe present invention, designated as SEQ ID NO: 433-648 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 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicated by GenScan) that belong to this assemblage. The algorithm terminated when there was no additional sequences from the above databases that' ould extend the assemblage. Further, inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.
  • Table 6 sets forth the novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 433-648) ofthe present invention, and their corresponding translation start and stop nucleotide locations to each of SEQ ID NO: 433-648. 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://fasta.bioch.virginia.edu) which selects a polypeptide based on a comparison ofthe translated novel polynucleotide to known polynucleotides (W.R. Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).
  • Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol. Biol., 268:78-94 (1997), incorporated herein by reference).
  • Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.
  • the full-length nucleotide sequences are shown in the Sequence Listing as SEQ ID NO: 1-216.
  • the corresponding polypeptide sequences are SEQ ID NO: 217-432.
  • Table 1 shows the various tissue sources of SEQ ID NO: 1-216.
  • the nearest neighbor results for polypeptides encoded by SEQ ID NO: 1-216 i.e. SEQ ID NO: 217-432 were obtained by a BLASTP (version 2.0al 19MP-WashU) search against Genpept release 123 and Genseq release 200110 (Derwent), using BLAST algorithm.
  • the nearest neighbor result showed the closest homologue with functional annotation for SEQ ID NO: 1-216 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: 1-216 are shown in Table 2 below.
  • polypeptides encoded by SEQ ID NO: 1-216 were examined to determine whether they had identifiable signature regions.
  • Table 3 shows the signature region found in the indicated polypeptide sequences, the description ofthe signature, the eMatrix p-value(s) and the position(s) ofthe signature within the polypeptide sequence.
  • polypeptides encoded by SEQ ID NO: 1- 216 were examined for domains with homology to certain peptide domains.
  • Table 4 shows the name ofthe domain found, the description, the p-value and the pFam score for the identified domain within the sequence.
  • nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determined from using Neural Network SignalP VI .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. 1, pp. 1-6 (1997), incorporated herein by reference.
  • the GeneAtlasTM software package (Molecular Simulations Inc. (MSI), San Diego, CA) was used to predict the three-dimensional structure models for the polypeptides encoded by SEQ ID NO 1-216 (i.e. SEQ ID NO: 217-432).
  • Models were generated by (1) PSI-BLAST which is a multiple alignment sequence profile-based searching developed by Altschul et al, (Nucl. Acids. Res. 25, 3389-3408 (1997)), (2) High Throughput Modeling (HTM) (Molecular Simulations Inc. (MSI) San Diego, CA,) which is an automated sequence and stracture searching procedure (http://www.msi.com/). and (3) SeqFoldTM which is a fold recognition method described by Fischer and Eisenberg (J. Mol.
  • the verify score is produced by GeneAtlasTM software (MSI), is based on Dr. Eisenberg's Profile-3D threading program developed in Dr. David Eisenberg's laboratory (US patent no. 5,436,850 and Luthy, Bowie, and Eisenberg, Nature, 356:83-85 (1992)) and a publication by R. Sanchez and A. Sali, Proc. Natl. Acad. Sci. USA, 95:13597-12502.
  • the verify score produced by GeneAtlas normalizes the verify score for proteins with different lengths so that a unified cutoff can be used to select good models as follows:
  • Verify score (normalized) (raw score - 1/2 high score)/(l/2 high score)
  • the PFM score produced by GeneAtlasTM software (MSI), is a composite scoring function that depends in part on the compactness ofthe model, sequence identity in the alignment used to build the model, pairwise and surface mean force potentials (MFP). As given in table 8, a verify score between 0 to 1.0, with 1 being the best, represents a good model. Similarly, a PMF score between 0 to 1.0, with 1 being the best, represents a good model. A SeqFoldTM score of more than 50 is considered significant. A good model may also be determined by one of skill in the art based all the information in Table 8 taken in totality. Table 5 is a correlation table of all ofthe sequences and SEQ ID numbers.
  • the 16 tissue-mRNAs and their vendor source are as follows: 1) Normal adult brain mRNA (Invitrogen), 2) normal adult kidney mRNA (Invitrogen), 3) normal adult liver mRNA (Invitrogen), 4) normal fetal brain mRNA (Invitrogen), 5) normal fetal kidney mRNA (Invitrogen), 6) normal fetal liver mRNA (Invitrogen), 7) normal fetal skin mRNA (Invitrogen), 8) human adrenal gland mRNA (Clontech), 9) human bone marrow mRNA (Clontech), 10) human leukemia lymphablastic mRNA (Clontech), 11) human thymus mRNA (Clontech), 12) human lymph node mRNA (Clontech), 13) human spinal cord mRNA (Clontech), 14) human thyroid mRNA (Clontech), 15) human esophagus mRNA (BioChain), 16) human conceptional umbilical cord m

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Abstract

La présente invention concerne de nouveaux acides nucléiques, de nouvelles séquences polypeptidiques codées par lesdits acides nucléiques et leurs utilisations.
PCT/US2001/014826 2000-05-18 2001-05-16 Nouveaux acides nucleiques et polypeptides WO2001087917A1 (fr)

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AU2001263005A AU2001263005A1 (en) 2000-05-18 2001-05-16 Novel nucleic acids and polypeptides
EP01937252A EP1282635A4 (fr) 2000-05-18 2001-05-16 Nouveaux acides nucleiques et polypeptides
CA002409164A CA2409164A1 (fr) 2000-05-18 2001-05-16 Nouveaux acides nucleiques et polypeptides
US10/294,433 US20070049743A1 (en) 2000-05-18 2002-11-13 Novel nucleic acids and polypeptides

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US57740800A 2000-05-18 2000-05-18
US09/577,408 2000-05-18
US66729800A 2000-09-22 2000-09-22
US09/667,298 2000-09-22
US69578100A 2000-10-24 2000-10-24
US09/695,781 2000-10-24
US71586900A 2000-11-17 2000-11-17
US09/715,869 2000-11-17
US77533001A 2001-02-01 2001-02-01
US09/775,330 2001-02-01

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WO2001083524A2 (fr) * 2000-04-28 2001-11-08 Incyte Genomics, Inc. Proteines du metabolisme de l'arn
WO2006034278A2 (fr) * 2004-09-21 2006-03-30 Matritech, Inc. Procedes et compositions de detection du cancer au moyen de composants de la particule spliceosomale u2
EP1434783A4 (fr) * 2001-03-16 2006-06-07 Lilly Co Eli Proteines de mammiferes lp et reactifs associes
US7846737B2 (en) 2001-06-13 2010-12-07 Millennium Pharmaceuticals, Inc. Genes, compositions, kits, and methods for identification, assessment, prevention and therapy of cervical cancer

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DE19811193A1 (de) * 1998-03-10 1999-09-16 Metagen Gesellschaft Fuer Genomforschung Mbh Menschliche Nukleinsäuresequenzen aus Prostatatumorgewebe
WO2000009552A1 (fr) * 1998-08-14 2000-02-24 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO2000056891A2 (fr) * 1999-03-22 2000-09-28 Incyte Pharmaceuticals, Inc. Proteines transmembranaires humaines

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DATABASE GENEMBL [online] 7 September 2000 (2000-09-07), SCHMITT ET AL., XP002947927, Database accession no. AX018076 *
DATABASE GENESEQ [online] 16 June 2000 (2000-06-16), JACOBS ET AL., XP002947926, Database accession no. AAA16650 *
DATABASE GENESEQ [online] 8 December 1999 (1999-12-08), SPECHT ET AL., XP002947924, Database accession no. AAZ33446 *
DATABASE GENESEQ [online] 8 February 2001 (2001-02-08), YUE ET AL., XP002947925, Database accession no. AAA96498 *
See also references of EP1282635A4 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001083524A2 (fr) * 2000-04-28 2001-11-08 Incyte Genomics, Inc. Proteines du metabolisme de l'arn
WO2001083524A3 (fr) * 2000-04-28 2002-06-20 Incyte Genomics Inc Proteines du metabolisme de l'arn
EP1434783A4 (fr) * 2001-03-16 2006-06-07 Lilly Co Eli Proteines de mammiferes lp et reactifs associes
US7846737B2 (en) 2001-06-13 2010-12-07 Millennium Pharmaceuticals, Inc. Genes, compositions, kits, and methods for identification, assessment, prevention and therapy of cervical cancer
US8062892B2 (en) 2001-06-13 2011-11-22 Millennium Pharmaceuticals, Inc. Genes, compositions, kits, and methods for identification, assessment, prevention and therapy of cervical cancer
WO2006034278A2 (fr) * 2004-09-21 2006-03-30 Matritech, Inc. Procedes et compositions de detection du cancer au moyen de composants de la particule spliceosomale u2
WO2006034278A3 (fr) * 2004-09-21 2006-08-31 Matritech Inc Procedes et compositions de detection du cancer au moyen de composants de la particule spliceosomale u2

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EP1282635A1 (fr) 2003-02-12
EP1282635A4 (fr) 2005-05-11
AU2001263005A1 (en) 2001-11-26
AU2001263005A8 (en) 2007-08-30
WO2001087917A8 (fr) 2007-07-19
CA2409164A1 (fr) 2001-11-22

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