JP2006517094A - Novel nucleic acids and polypeptides - Google Patents

Abstract

The composition of the present invention comprises a novel isolated polypeptide, a novel isolated polynucleotide encoding such a peptide, including as a recombinant DNA molecule, a cloned gene or Their modified variants, in particular 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 that produce such antibodies.

Description

1. CROSS REFERENCE TO RELATED APPLICATIONS This application is a US Provisional Patent Application No. 60 / 416,186, filed Oct. 2, 2002, entitled “Novel Nucleic Acids and Polypeptides,” which includes materials previously disclosed in the following applications. Alleged priority benefit: Attorney Docket No. 21272-502 US Provisional Patent Application No. 10 / 084,643, filed Feb. 26, 2002 entitled "Novel Nucleic Acids and Polypeptides"; PCT Application No. PCT / US00 / 35017 filed Dec. 22, 2000, entitled "Novel Contiguouss From Various Libraries"; Attorney Docket No. 785CIP3 / PCT PCT Application No. PCT / US01 / 02623, filed January 25, 2001, entitled "Novel Congs Obtained from Various Libraries"; Attorney Docket No. 787CIP3 / PCT PCT Application No. PCT / US01 / 03800 filed February 5, PCT Application No. PCT / US01 / filed February 26, 2001 entitled Attorney Docket No. 788CIP3 / PCT "Novel Contigs Obtained from Various Libraries"04927; Attorney Docket No. 789 CIP3 / PCT “Novel Contigs Obtained from Var PCT Application No. PCT / US01 / 04941, filed March 5, 2001, entitled “Ous Libraries”; Attorney Docket No. 790 CIP3 / PCT, entitled “Novel Contiguouss From Various Libraries”, March 30, 2001 PCT Application No. PCT / US01 / 08631; Attorney Docket No. 791 CIP3 / PCT PCT Application No. PCT / US01 / 08656 filed April 18, 2001 entitled “Novel Contigs Obtained from Various Libraries” All of which are hereby incorporated by reference in their entirety).

2. BACKGROUND OF THE INVENTION 2.1 TECHNICAL FIELD The present invention relates to novel polynucleotides and proteins encoded by such polynucleotides, as well as uses of such polynucleotides and proteins, eg, in therapeutic, diagnostic and research methods. provide.

2.2 Background Techniques aimed at the discovery of protein factors, including cytokines such as lymphokines, interferons, circulating soluble factors, chemokines and interleukins, have matured rapidly over the past decade. Currently conventional hybridization cloning and expression cloning techniques allow new polynucleotides to be directly related to the protein in which they were discovered (ie, partial DNA / protein amino acid sequences in the case of hybridization cloning; Cloning “directly” in the sense that it depends on the activity of the protein in the case of expression cloning. More recently, “indirect” cloning techniques, such as signal sequence cloning (which includes the presence of current well-known secretory leader sequence motifs, as well as various PCR-based or low stringency hybridization-based cloning techniques. The DNA sequence on the basis of the In the case of PCR-based technology, the status of this technology has been advanced by their cellular or tissue source or thanks to structural similarities to other genes of known biological activity.

  Identified polynucleotide and polypeptide sequences have many applications in, for example, diagnostics, forensics, and genetic mapping; to identify mutations that carry genetic disorders or other traits, to assess biological diversity And to generate many other types of data and products that depend on DNA and amino acid sequences.

3. SUMMARY OF THE INVENTION The composition of the present invention comprises a novel isolated polypeptide, a novel isolated polynucleotide encoding such a peptide, including as a recombinant DNA molecule, cloned. Specifically recognize naturally occurring variants, particularly naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and one or more epitopes present on such polypeptides As well as hybridomas that produce such antibodies.

  The composition of the present invention further comprises a vector, such as an expression vector, comprising a polynucleotide of the present invention, a cell genetically engineered to contain such a polynucleotide, and a genetic engineer to express such a polynucleotide. Cells.

  The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from an expressed sequence tag (EST) primarily isolated by sequencing by hybridization (SBH), and in some cases one or more Relating to sequences obtained from published databases. The invention also relates to the proteins encoded by such polynucleotides and the therapeutic, diagnostic and research utility of these polynucleotides and proteins. These nucleic acid sequences are named SEQ ID NO: 1-684 or 1369-1966 and are provided in the sequence listing. In the nucleic acids provided in this sequence listing, A is adenine, C is cytosine; G is guanine; T is thymine; and N is any of the four bases or unknown. In the amino acids shown in this sequence listing, an asterisk (*) corresponds to a stop codon.

  The nucleic acid sequences of the present invention can also be a nucleic acid sequence that hybridizes under stringent hybridization conditions to the complement of SEQ ID NOs: 1-684 or SEQ ID NOs: 1369-1966; Nucleic acid sequences that are species homologs, or nucleic acid sequences that encode peptides containing specific domains or truncations of the peptides encoded by SEQ ID NOs: 1-684 or 1369-1966. A polynucleotide comprising a nucleotide sequence having at least 90% identity to the same sequence of SEQ ID NOs: 1-684 or 1369-1966 or a modified variant or fragment thereof. The sequence to be identified may be 100 base pairs long.

  The nucleic acid sequences of the present invention also include sequence information derived from the nucleic acid sequences of SEQ ID NOs: 1-684 or 1369-1966. This sequence information is a segment of any one of SEQ ID NO: 1-684 or SEQ ID NO: 1369-1966 that uniquely identifies or corresponds to the sequence information of SEQ ID NO: 1-684 or 1369-1966 Also good.

  A collection as used in this application may be a collection of only one polynucleotide. A collection of sequence information or identification information for each sequence may be provided on the array of nucleic acids. In one embodiment, a segment of sequence information is provided on an array of nucleic acids to detect a polynucleotide containing that segment. The array may be designed to detect perfect matches or mismatches to the polynucleotide containing the segment. This collection may be provided in a computer readable format.

  The present invention also provides a reverse or direct complement of any nucleic acid sequence as described above; a cloning or expression vector comprising this nucleic acid sequence; and a host cell or organism transformed with these expression vectors. Including. Nucleic acid sequences according to the present invention (or their reverse or direct complements) can be used in a number of applications in various techniques known to those skilled in molecular biology, such as hybridization probes, PCR primers, etc. Applications in arrays, applications in computer readable media, applications for sequencing full-length genes, applications in chromosome and gene mapping, applications in recombinant production of proteins, and antisense DNA or RNA, their chemistry It is applied to the use in the generation of dynamic analog.

  In a preferred embodiment, the nucleic acid sequence of SEQ ID NOs: 1-684 or 1369-1966, or a novel segment or portion of this nucleic acid, is used as a primer in expression assays well known in the art. In certain preferred embodiments, the nucleic acid sequence of SEQ ID NOs: 1-684 or 1369-1966 or a novel segment or portion of this nucleic acid provided herein, as is well known in the art, and in Volllath et al., Used as an expressed sequence tag for physical mapping of the human genome in diagnostics to identify expressed genes, as exemplified in Science 258: 52-59 (1992).

  As the isolated polynucleotide of the present invention, a polynucleotide comprising any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 684 or 1369 to 1966; encoding the sequence of SEQ ID NOs: 1 to 684 or 1369 to 1966 A polynucleotide comprising any of the full-length proteins; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NOs: 1-684 or 1369-1966. The polynucleotide of the present invention also includes (a) the complement of any one of the nucleotide sequences represented by SEQ ID NOs: 1 to 684 or 1369 to 1966; (b) the amino acids represented by SEQ ID NOs: 1 to 684 or 1369 to 1966 A nucleotide sequence encoding any one of the sequences; (c) a polynucleotide which is an allelic variant of any of the above polynucleotides; (d) a species homologue (eg, an ortholog) of any of the above proteins; A polynucleotide encoding; or (e) comprising any particular domain or truncation of a polypeptide comprising SEQ ID NO: 65-1368 or 1967-2564, or a polypeptide comprising the amino acid sequence set forth in Tables 3A, 3B, 5, 7 or 8 Against the polynucleotide encoding the polypeptide. Although polynucleotides include that hybridizes under stringent hybridization conditions are not limited thereto.

  Isolated polypeptides of the present invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences shown in the sequence listing; or the corresponding full-length protein or mature protein. The polypeptide of the present invention also comprises (a) any polynucleotide having the nucleotide sequence set forth in SEQ ID NOs: 1-684 or 1369-1966; or (b) the polynucleotide of (a) under stringent hybridization conditions And a biologically active polypeptide encoded by a polynucleotide that hybridizes to the complement of. Biologically active variants of any of the polypeptide sequences in the sequence listing, and preferably “substantial equivalents” thereof that retain biological activity (eg, at least about 65%, 70% , 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity). The polypeptides of the present invention can be chemically synthesized in whole or in part, but are preferably produced by recombinant methods using the genetically engineered cells (eg, host cells) of the present invention.

  The present invention also provides a composition comprising the polypeptide of the present invention. The polypeptide composition of the present invention may further comprise an acceptable carrier, eg, a hydrophilic one, eg, a pharmaceutically acceptable carrier.

  The invention also provides a host cell transformed or transfected with a polynucleotide of the invention.

  The invention also relates to a method of producing a polypeptide of the invention, which method allows expression of the desired polypeptide and under conditions that allow purification of the polypeptide from culture or from host cells. Growing a culture of the host cells of the invention in a suitable culture medium. Preferred embodiments include those in which the protein produced by such a process is the mature form of the protein.

  The polynucleotides of the present invention have many applications in various techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, use for PCR, chromosome and gene mapping, use in recombinant production of proteins, and antisense DNA or RNA chemistry Applications in the generation of dynamic analogs. For example, where mRNA expression is largely limited to a particular cell or tissue type, the polynucleotides of the invention can be used to detect the presence of a particular cell or tissue mRNA in a sample, eg, using in situ hybridization. It can be used as a hybridization probe.

  In other exemplary embodiments, the polynucleotide is used in diagnosis as an expressed sequence tag to identify an expressed gene, or as is well known in the art and as described in Volllath et al., Science 258: 52-59 ( 1992), used as an expressed sequence tag for physical mapping of the human genome.

  The polypeptides according to the present invention can be used in various conventional procedures and methods currently applied to other proteins. For example, the polypeptides of the present invention can be used to generate antibodies that specifically bind to the polypeptides. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantifying polypeptides in tissues. The polypeptides of the present invention may also be used as molecular weight markers and as food supplements.

  Also for prevention, treatment, or amelioration in medical conditions, comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a peptide of the invention and a pharmaceutically acceptable carrier. A method is also provided.

  In particular, the polypeptides and polynucleotides of the present invention can be utilized, for example, in methods for the prevention and / or treatment of disorders involving abnormal protein expression or biological activity.

  The invention further relates to a method for detecting the presence of a polynucleotide or polypeptide of the invention in a sample. Such methods can be utilized, for example, as part of the prediction and diagnostic evaluation of disorders as mentioned herein, and for the identification of subjects who are predisposed to such conditions. The present invention provides a method for detecting a polynucleotide of the present invention in a sample, which comprises a compound that binds to the polynucleotide of interest and forms a complex with the polynucleotide. And a step for contacting the substrate for a time sufficient to form the complex and under conditions sufficient to form the complex, and detecting the complex, and as a result, the complex is detected. And the step of detecting the polynucleotide of interest. The method also provides a method for detecting a polypeptide of the invention in a sample comprising the sample and a compound that binds to and forms a complex with the polynucleotide. In a condition and for a time sufficient to form a complex, and a step of detecting the formation of the complex and, if the complex is formed, detecting the polypeptide. Is included.

  The invention also provides kits with polynucleotide probes and / or monoclonal antibodies, and optionally quantitative criteria, for performing the methods of the invention. In addition, the present invention provides a method for assessing drug efficacy and monitoring patient progression, which is involved in clinical trials for the treatment of disorders as described above.

  The invention also provides methods for the identification of compounds that modulate (ie, increase or decrease) the expression or activity of the polynucleotides and / or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate the symptoms of a disorder as described herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact (eg, bind) with the polypeptides of the invention. The present invention provides a method for identifying a compound that binds to a polypeptide of the present invention, wherein the method comprises the step of combining the compound with the present invention in a cell for a time sufficient to form a polypeptide / compound complex. Contacting the polypeptide, wherein the complex drives expression of the reporter gene sequence in the cell; detecting the complex by detecting the reporter gene sequence expression, and thereby When the expression of the reporter gene is detected, the method includes the step of identifying a compound that binds to the polypeptide of the present invention.

  The methods of the present invention also provide a method for treatment comprising administration of a polynucleotide or polypeptide of the present invention to an individual who exhibits symptoms or propensity. Furthermore, the present invention encompasses a method for treating a disease or disorder as recited herein, the method comprising administering a compound and other substances that modulate the overall activity of the target gene product. Is included. Compounds and other substances may exert such modulation on either the target gene / protein expression or the level of target protein activity.

  The polypeptides of the present invention and the polynucleotides encoding them also have homology (shown in Tables 2A and 2B), have signature regions (shown in Tables 3A and 3B); or a gene family Useful for polypeptides and polynucleotides having the same homology (shown in Tables 4A and 4B) and the same functions known to those skilled in the art. If no homology is shown for the sequences, the polypeptides and polynucleotides of the invention are useful in a variety of applications as described herein, including use in arrays for detection.

4). Detailed Description of the Invention 4.1 Definitions In this specification and the appended claims, this single ("a", "an" and "the"), which is singular, is clearly stated in particular in text. Note that it includes more than one case unless otherwise indicated.

  The term “activity” refers to a form of a polypeptide that retains the biological and / or immune activity of any naturally occurring polypeptide. According to the present invention, the terms “biologically active” or “biological activity” have the structural, regulatory or biochemical function of a naturally occurring molecule. Refers to a protein or peptide. Similarly, “immunologically active” or “immunological activity” induces a specific immune response in an appropriate animal or cell and binds to a specific antibody. Refers to the ability of a natural, recombinant or synthetic polypeptide.

  The term “activated cell” as used in this application includes the export of secreted molecular cells or enzyme molecules as part of normal or disease processes, extracellular or intracellular membranes. Cells involved in the transport of

  The term “complementary” or “complementarity” refers to the natural binding of polynucleotides by base pairing. For example, the sequence 5'-AGT-3 'binds to the complementary sequence 3'-TCA-5'. Complementarity between two single stranded molecules may be “partial” complementarity where only a small portion of the nucleic acid binds, or there may be total complementarity between the single stranded molecules. It may be “complete” complementarity as it exists. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

  The term “embryonic stem cells (ES)” refers to cells capable of developing many differentiated cell types, including germ cells, in the embryo or adult. The term “germ line stem cells (GSC)” refers to stem cells derived from primordial stem cells that stably and continuously supply embryonic cells for the production of gametes. “The term primordial germ cells (PGC) is used during embryogenesis from other cell lines, particularly yolk sac, mesentery, or gonads, with the ability to differentiate into germ cells and other cells. PGC is a source from which GSC and ES cells are derived, PGC, GSC and ES cells have a self-renewal function, so these cells proliferate the germ line In addition to generating a plurality of terminally differentiated cells that form adult special organs, they can also regenerate themselves.

  The term “expression modulation fragment”, EMF, means a series of nucleotides that regulate the expression of an operably linked ORF or another EMF.

  As used herein, a sequence is said to “modulate the expression of an optionally linked sequence” if the expression of the sequence is altered by the presence of EMF. Is called. EMF includes, but is not limited to, promoters and promoter regulatory sequences (inducible elements). One class of EMF is a nucleic acid fragment that induces expression of an ORF operably linked in response to a specific modulator or physiological event.

  The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably, which means nucleotides or these nucleotides Refers to a heteromultimer of sequence. These phrases also refer to genomic or synthetically derived DNA or RNA, which may be single stranded or double stranded, and may be a sense strand or an antisense strand, Refers to peptide nucleic acid (PNA) or refers to any DNA-like or RNA-like substance. In the sequences herein, A is adenine, C is cytosine, T is thymine, G is guanine, N is A, C, G or T (U) or an unknown substance It is. When the polynucleotide is RNA, it is believed that T (thymine) in the sequences provided herein is replaced with U (uracil). In general, the nucleic acid segments provided by the present invention can be assembled from genomic fragments and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides to obtain a synthetic nucleic acid, Nucleic acids can be expressed in recombinant transcription units containing regulatory elements derived from microbial or viral operons, or eukaryotic genes.

  The terms “oligonucleotide fragment” or “polynucleotide fragment”, “portion” or “segment” or “probe” or “primer” Used interchangeably and at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, even more preferably at least about 11 nucleotides, and most preferably Refers to a sequence of nucleotide residues that is at least about 17 nucleotides. A 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 50 nucleotides, and most preferably less than about 30 nucleotides. It is a nucleotide. Preferably, the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from 17 to 30 nucleotides, and most preferably from about 20 to 25 nucleotides. Of nucleotides. Preferably, the fragments can be used to identify or amplify identical or related portions of mRNA or DNA molecules in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures. A fragment or segment can unambiguously identify each polynucleotide sequence of the invention. Preferably, the fragment comprises a sequence substantially similar to any one of SEQ ID NOs: 1-684 or 1369-1966.

  Probes can be used, for example, to determine whether a particular mRNA molecule is present in a cell or tissue, or to isolate a similar nucleic acid sequence from chromosomal DNA described by Walsh et al. ( Walsh, PS et al., 1992, PCR Methods Appl 1: 241-250). They can be labeled by methods such as nick translation, Klenow fill-in reaction, PCR, etc. well known to those skilled in the art. The probes of the present invention, their preparation and / or labeling are described in Sambrook, J. et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. et al. M.M. 1989, Current Protocols in Molecular Biology, John Willy & Sons, New York, NY, both of which are hereby incorporated by reference in their entirety.

The nucleic acid sequences of the present invention also include sequence information from the nucleic acid sequences of SEQ ID NOs: 1-684 or 1369-1966. This sequence information uniquely identifies the sequence information of the sequence of SEQ ID NO: 1-684 or 1369-1966 or corresponds to any one segment of SEQ ID NO: 1-684 or 1369-1966, or Table 3A, It may be a segment identified as 3B, 5, 7 or 8. One such segment may be a 20-mer nucleic acid sequence. This is because the probability of a perfect match of 20 mers in the human genome is 1/300. In the human genome, there are 3 billion base pairs in a set of chromosomes. 4 Since ²⁰ 20-mers can exist, there will be 300-times 20-mers of the number of base pairs present in a set of human chromosomes. Using the same analysis, the probability that a 17mer is a perfect match in the human genome is approximately one fifth. If these segments are used as an array for expression studies, 15-mer segments may be used. The probability of a perfect match in the expressed sequence of the 15 mer is also almost 1/5 since the expressed sequence is composed of less than about 5% of the total genomic sequence.

Similarly, when using sequence information to detect a single mismatch, a segment may be a 25mer. The probability of a 25-mer representing only one mismatch in the human genome is calculated by multiplying the probability of a perfect match (1 ÷ 4 ²⁵ ) by the increased probability of mismatch at each nucleotide position (3 × 25). The probability that 18-mers with one mismatch can be detected in one array for expression studies is about one-fifth. The probability that a 20mer with one mismatch can be detected in the human genome is about one-fifth.

  The term “open reading frame”, ORF, means a series of nucleotide triplets that encode an amino acid without a stop codon and is a sequence that can be transcribed into a protein.

  The term “operably linked” or “operably associated” refers to a nucleic acid sequence that is functionally related. For example, if a promoter controls transcription of a coding sequence, the promoter is operably associated with or linked to the coding sequence. When an operably linked nucleic acid sequence is contiguous and within the same reading frame, a particular genetic element, such as a repressor gene, is still present even when not ligated adjacent to the code sequence. Transcription / translation of the coding sequence can be controlled.

  The term “pluripotent” refers to the ability of a cell to differentiate into a number of differentiated cell types present in an adult organ. A pluripotent cell has a limited ability to differentiate as compared to a totipotent cell.

  The term “polypeptide” or “peptide” or “amino acid sequence” refers to a sequence of oligopeptides, peptides, polypeptides or proteins, or fragments thereof, occurring in nature Refers to a molecule or a synthetic molecule. A “fragment”, “portion”, or “segment” of a polypeptide has at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids. Or most preferably a stretch of a series of amino acid residues of at least about 17 or more amino acids. The peptide is preferably no more than about 200 amino acids, more preferably less than 150 amino acids, and most preferably less than 100 amino acids. Preferably, the peptide is about 5 to 200 amino acids. In order to be active, the polypeptide must have a sufficient length to exhibit biological and / or immunological activity.

  The term “naturally occurring polypeptide” refers to a polypeptide produced by a cell that has not been genetically engineered, in particular, acetylation, carboxylation, glycosylation, phosphate esterification. Specifically means various polypeptides resulting from post-translational modifications of polypeptides including, but not limited to, lipidation, and acylation.

  The term “translated protein coding portion” refers to a sequence that encodes a full-length protein and may include any leader sequence or any processing sequence.

  The term “mature protein coding sequence” means a sequence that encodes a peptide or protein without a signal or leader sequence. By “mature protein portion” is meant a portion of a protein that contains neither a signal sequence nor a leader sequence. This peptide may be generated by intracellular processing that removes the leader / signal sequence. This mature protein may or may not contain the first methionine residue. This methionine residue can be removed from the protein during processing in the cell. The peptide may be produced synthetically or the protein may be produced using a polynucleotide that encodes only the mature protein coding sequence.

  The term “derivative” refers to ubiquitin binding, labeling (eg with respect to radionuclides or various enzymes), covalent polymer addition, eg pegylation (induction by polyethylene glycol) and ornithine. A polypeptide that has been chemically modified by techniques such as chemical amino acid insertion or substitution, and that does not occur naturally in human proteins.

  The term “variant” (or “analog”) is a polypeptide that differs from naturally-occurring polypeptides by amino acid insertions, deletions, and substitutions, eg, using recombinant DNA technology. It refers to the polypeptide produced using. A guide for determining which amino acid residues may be substituted, added or deleted without negating the desired activity is to compare the sequence of a particular polypeptide with the sequence of homologous peptides; And by minimizing the number of amino acid sequence changes that occur in regions of high homology (conserved regions) or by substituting amino acids with consensus sequences.

  Alternatively, recombinant sudden variants that encode these same or similar polypeptides may be synthesized or selected by taking advantage of the “redundancy” of the genetic code. Various codon substitutions, such as silent changes that create various restriction sites, can be introduced into plasmids or viral vectors or expression in specific prokaryotic or eukaryotic systems to optimize cloning. Mutations in the polynucleotide sequence are reflected in the domain of the polypeptide or other peptides appended to the polypeptide to modify the properties of any part of the polypeptide, such as ligand binding affinity, interchain affinity or Properties such as degradation / turnover rate are changed.

  Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, ie, conservative amino acid substitutions. “Conservative” amino acid substitutions can be made based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine Positively charged (basic) amino acids include argin, 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 1-20 amino acids, more preferably 1-10 amino acids. Permissible variation is experimentally determined by systematically making amino acid insertions, deletions, or substitutions in the polypeptide molecule using recombinant DNA technology, and assaying the resulting recombinant variants for activity. Can be determined.

  Alternatively, if a change in function is desired, modified polypeptides can be engineered by insertion, deletion or non-conservative changes. Such alterations can, for example, alter one or more biological functions or biochemical properties of the polypeptides of the invention. For example, such changes can alter polypeptide properties such as ligand binding affinity, interchain affinity, or degradation / turnover rate. Further, such alterations can be selected to generate polypeptides that are more suitable for expression, scale up, etc. in the host cell selected for expression. For example, a cysteine residue may be removed or replaced with another amino acid residue to remove a difluide bridge.

  The terms “purified” or “substantially purified” are used herein to designate a designated nucleic acid or polynucleotide as another biological macromolecule, such as a polynucleotide, It means to exist in the substantial absence of protein or the like. In one embodiment, the polynucleotide or polypeptide is purified to be at least 95%, more preferably at least 99% by weight of the designated biological macromolecule present (provided that water, buffer, And other small molecules, especially molecules having a molecular weight of less than 1000 daltons may be present).

  The term “isolated” as used herein is from at least one other component (eg, nucleic acid or polypeptide) that is present together when the nucleic acid or polypeptide is in its natural source. It means an isolated nucleic acid or polypeptide. In one example, the nucleic acid or polypeptide is found where only the solvent, buffer, ion, or other component normally present in the solution is present (if present). The term “isolated” or “purified” does not include nucleic acids or polypeptides present in natural sources.

  The term “recombinant” as used herein in reference to a polypeptide or protein is a polypeptide or protein derived from a recombinant (eg, microbial, insect, or mammalian) expression system. Means that. “Microbial” refers to a recombinant polypeptide or protein produced in a bacterial or fungal (eg, yeast) expression system. As a product, a “recombinant microbial” defines a polypeptide or protein that is essentially free of naturally occurring endogenous substances and without the associated natural glycosylation. Most bacterial cultures such as E. coli. Polypeptides or proteins expressed in E. coli do not have glycosylation modifications; polypeptides or proteins expressed in yeast have glycosylation patterns that are generally different from those expressed in mammalian cells.

  The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector for expressing a polypeptide from a DAN (RNA) sequence. An expression vehicle is (1) a genetic element or elements that have a regulatory role in gene expression, such as a promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, And (3) a transcription unit comprising an assembly of suitable transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence that allows extracellular secretion of the protein translated by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may contain an amino terminal methionine residue. Residues may or may not be subsequently cleaved from the expressed recombinant protein to obtain the final product.

  The term “recombinant expression system” means a host cell that stably incorporates a recombinant transcription unit into chromosomal DNA or carries the recombinant transcription unit extrachromosomally. A recombinant expression system as defined herein expresses a heterologous polypeptide or protein upon introduction of a regulatory element linked to the DNA segment or synthetic gene to be expressed. The term also refers to a host cell that stably incorporates recombinant genetic element or elements that have a regulatory role in gene expression, such as a promoter or enhancer. A recombinant expression system as defined herein expresses a polypeptide or protein that is endogenous to the cell upon introduction of a regulatory element linked to the endogenous DNA segment or gene to be expressed. It is. The cell may be prokaryotic or eukaryotic.

  The term “secreted” is a protein that is transported across or through a membrane, which includes a signal sequence in its amino acid sequence when it is expressed in an appropriate host cell. The resulting transport is mentioned. A “secreted” protein includes, but is not limited to, a protein that is completely (eg, soluble protein) or partially (eg, receptor) secreted from the cell in which it is expressed. "Secreted" proteins also include, but are not limited to, proteins that are transported across the endoplasmic reticulum membrane. “Secreted” proteins are also considered to encompass proteins containing atypical signal sequences (eg, interleukin-1β, Krasney, PA, and Young, PR (1992)). Cytokine 4 (2): 134-143) and factors released from disrupted cells (eg, interleukin-1 receptor antagonist, Arend, WP et al. (1998) Annu. Rev. Immunol. 16). : See 27-55).

  If desired, the expression vector may be designed to include a “signal or leader sequence” that directs the polypeptide through the cell membrane. Such sequences may be naturally occurring in the polypeptides of the invention, or may be provided from heterologous protein sources by recombinant DNA techniques.

The term “stringent” refers to conditions that are stringent as commonly understood by one of ordinary skill in the art. Stringent conditions include highly stringent conditions (ie, 0.5M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS) on filter-fixed DNA, hybridization in 1 mM EDTA at 65 ° C., and 68 ° C. And washing with 0.1 × SSC / 0.1% SDS) and moderately stringent conditions (ie, washing with 0.2 × SSC / 0.1% SDS at 42 ° C.). . Other exemplary hybridization conditions are described in the Examples herein.

  For deoxyoligonucleotide hybridization, additional exemplary stringent hybridization conditions include 37 ° C. (for 14 base oligonucleotides), 48 ° C. (for 17 base oligonucleotides), 55 ° C. Washing in 6 × SSC / 0.05% sodium pyrophosphate at 60 ° C. (for a 23 base oligonucleotide) and 60 ° C. (for a 23 base oligonucleotide).

  As used herein, “substantially equivalent” or “substantially similar” refers to both nucleotide and amino acid sequences, eg, one or more from a reference sequence. It may refer to a sequence of an abrupt variant that is altered by substitution, deletion or addition and whose net effect does not result in a deleterious functional difference between this reference sequence and the test sequence. Typically, such substantially equivalent sequences will vary by about 35% from one of the sequences listed herein (ie, substantially equivalent when compared to the corresponding reference sequence). The number of individual residue substitutions, additions, and / or deletions in a given sequence divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less). Such a sequence is said to have 65% sequence identity to the listed sequence. In one embodiment, a substantially equivalent, eg, abrupt variant sequence of the invention, differs by only 30% (70% sequence identity) from the listed sequence; for certain mutations of this embodiment, 25 % Changes (75% sequence identity); and further mutations of this embodiment only change 20% (80% sequence identity); and further mutations of this embodiment only change 10% (90% sequence identity), and further mutations of this embodiment change only 5% (95% sequence identity). A substantially equivalent, eg, suddenly variant, amino acid sequence according to the present invention preferably has at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least at least the listed amino acid sequence. It has 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity. A substantially equivalent nucleotide sequence of the present invention may have a lower percent sequence identity, for example, taking into account the redundancy or degeneracy of the genetic code. Preferably, the nucleotide sequence is at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least 85% sequence identity, Preferably it has at least 90% sequence identity, more preferably at least about 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. For purposes of determining equivalence, truncation of the mature sequence (eg, via abrupt variants that generate a new stop codon) should be excluded. Sequence identity can be determined, for example, 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, eg, by changing hybridization conditions.

  The term “totipotent” refers to the ability of a cell to differentiate into all cell types of an adult organ.

  The term “transformation” refers to the process of introducing DNA into a suitable host cell so that the DNA can be reproduced either as an extrachromosomal element or by chromosomal integration. The term “transfection” means that the expression vector is taken up by a suitable host cell, regardless of whether the coding sequence is actually expressed. The term “infection” refers to the introduction of a nucleic acid into a suitable host cell by use of a virus or viral vector.

  As used herein, “uptake modulating fragment” UMF means a series of nucleotides that mediate the incorporation of linked DNA fragments into cells. UMF can be easily identified using known UMF as a target sequence or target motif in conjunction with a computer-based system as described below. The presence and activity of UMF can be confirmed by binding suspect UMF to the marker sequence. The resulting nucleic acid molecule is then incubated with appropriate host cells under appropriate conditions to determine the uptake of the marker sequence. As mentioned above, UMF increases the frequency of incorporation of linked marker sequences.

  Each of the above terms is meant to encompass all meanings set forth for each, unless the context indicates otherwise.

4.2 Nucleic acids of the invention The nucleotide sequences of the invention are shown in the sequence listing.

  The isolated polynucleotide of the present invention includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1-684 or 1369-1966; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 1-684 or 1369-1966 And a polynucleotide comprising a nucleotide sequence that encodes the mature protein coding sequence of any one of the polynucleotides of SEQ ID NOs: 1-684 or 1369-1966. Polynucleotides of the invention also include, but are not limited to, polynucleotides that hybridize under stringent conditions to: (a) any nucleotide sequence of SEQ ID NOs: 1-684 or 1369-1966 (B) a nucleotide sequence encoding any one of the amino acid sequences shown in the Sequence Listing or Table 7; (c) a polynucleotide which is an allelic variant of any of the polynucleotides described above; (d) A polynucleotide encoding a species homologue of any of the proteins described above; or A polynucleotide encoding a polypeptide containing a domain or truncation of Plastid. The domain of interest can depend on the nature of the encoded polypeptide; for example, the domain of a receptor-like polypeptide includes a ligand binding, extracellular, transmembrane, or cytoplasmic domain, or a combination thereof; an immunoglobulin-like Protein domains include variable immunoglobulin-like domains; enzyme-like polypeptide domains include catalytic and substrate binding domains; and ligand polypeptide domains include receptor binding domains.

  The polynucleotides of the present invention include naturally occurring DNA, or fully or partially synthesized DNA, such as cDNA and genomic DNA, and RNA, such as mRNA. The polynucleotide may comprise the entire coding region of the cDNA or may be part of the coding region of the cDNA.

  The present invention also provides genes corresponding to the cDNA sequences disclosed herein. This corresponding gene can be isolated according to known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identifying and / or amplifying genes in appropriate genomic libraries, or other sources of genomic material. Additional 5 'and 3' sequences can be obtained using methods known to those skilled in the art. For example, a full-length cDNA or genomic DNA corresponding to any of the polynucleotides of SEQ ID NOs: 1 to 684 or 1369 to 1966 is suitable using any of the polynucleotides of SEQ ID NOs: 1 to 684 or 1369 to 1966 or a part thereof as a probe. Can be obtained by screening a library of appropriate cDNA or genomic DNA under suitable hybridization conditions. Alternatively, the polynucleotides of SEQ ID NOs: 1-684 or 1369-1966 are used as the basis for appropriate primer (s) that allow identification and / or amplification of genes in appropriate genomic DNA or cDNA libraries. Can do.

  The nucleic acid sequences of the present invention may be assembled from one or more public databases, such as ESTs and sequences (including cDNA and genomic sequences) obtained from dbEST, gbppri and UniGene. The EST sequence can provide sequence information identification, representative fragment or segment information, or new segment information for the full-length gene.

  The polynucleotide of the present invention also provides a polynucleotide comprising a nucleotide sequence that is substantially equivalent to the polynucleotide described above. The polynucleotide according to the present invention is, for example, at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, Typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically It may have at least about 95%, 96%, 97%, 98%, 99% sequence identity.

  What is included in the range of the nucleic acid sequence in the present invention is a nucleic acid sequence fragment that hybridizes under stringent conditions to any one of the nucleotide sequences of SEQ ID NOs: 1 to 684 or 1369 to 1966, or a complement thereof. And this fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides, and most preferably greater than 17 nucleotides. For example, contemplate a fragment of 15, 17 or 20 or more nucleotides that is selective for (ie, specifically hybridizes to) any one of the polynucleotides of the invention. Yes. A probe capable of specifically hybridizing to a polynucleotide can distinguish a polynucleotide sequence of the invention from other polynucleotide sequences in the same family of genes, or can distinguish human genes from other types of genes, and preferably Based on unique nucleotide sequence.

  Sequences falling within the scope of the invention are not limited to these specific sequences, but also include allelic variations and species variations. Allelic and species variations are at least 90% identical, preferably 95% identical to the sequences provided in SEQ ID NOs: 1-684 or 1369-1966, representative fragments thereof, or SEQ ID NOs: 1-684 or 1369-1966 The nucleotide sequence of can be routinely determined by comparing it to a sequence from another isolate of the same species. Furthermore, in line with codon variability, the present invention includes nucleic acid molecules that encode the same amino acid sequence as the specific ORFs disclosed herein. In other words, in the coding region of one ORF, it is expressly intended to replace one codon with another codon encoding the same amino acid.

  The closest approximation or homology result of the nucleic acids of the invention, including SEQ ID NOs: 1-684 or 1369-1966, can be obtained by searching a database using an algorithm or program. Preferably, a BLAST (Basic Local Alignment Search Tool) program is used to search for alignment of localized sequences (Altshu, SFJ Mol. Evol. 36 290-300). (1993) and Altschul SF et al., J. Mol. Biol. 21: 403-410 (1990)). Alternatively, a FASTXY algorithm may be used to perform a FASTA version 3 search for Genpept.

  Homologs (or orthologs) of the disclosed polynucleotide and protein species are also provided by the present invention. Species homologs can be isolated and identified by creating appropriate probes or primers from the sequences provided herein and screening appropriate nucleic acid sources from the desired species.

  The present invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring, alternative forms of isolated polynucleotides, and those encoded by the polynucleotides. Those that encode the same, homologous or related proteins.

  The nucleic acid sequences of the present invention further relate to sequences that encode variants of the described nucleic acids. These amino acid sequence variants can be prepared by methods known to those skilled in the art by introducing appropriate nucleotide changes into the native or variant polynucleotide. There are two variables in the composition of amino acid sequence variants: the location of the mutation and the nature of the mutation. Nucleic acids encoding amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode non-naturally occurring amino acid sequences. These nucleic acid modifications may be performed at sites where the nucleic acid differs from different species (variable positions) or at highly conserved regions (constant regions). A site at such a position can be identified, for example, by substitution with a conservative selection first (eg, a hydrophobic amino acid to a different hydrophobic amino acid) and further substitution at a further distance (eg, charging a hydrophobic amino acid). Amino acids), typically corrected sequentially, followed by deletions or insertions at the target site. Amino acid sequence deletions generally range from approximately 1 to 30 residues, preferably approximately 1 to 10 residues, and are typically contiguous. Amino acid insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from 1 to 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may generally range from about 1 to 10 amino acid residues, preferably 1-5 residues. Examples of terminal insertions include heterologous signal sequences necessary for secretion or intracellular targeting in different host cells, and sequences such as FLAG, or polyhistidine sequences that aid in purification of the expressed protein.

  In a preferred method, the polynucleotide encoding the novel amino acid sequence is altered via site-directed mutagenesis. This method uses an oligonucleotide sequence to change the polynucleotide, encodes the desired amino acid variant, and sufficient adjacent nucleotides on either side of the amino acid being changed, on either side of the site to be converted. A stable duplex is formed. In general, the technique of site-directed mutagenesis is well known to those skilled in the art, and this technique is exemplified by publications such as Edelman et al., DNA 2: 183 (1983). A diverse and efficient method for creating site-specific changes in polynucleotide sequences is described by Zoller and Smith, Nucleic Acids Res. 10: 6487-6500 (1982). PCR can also be used to create amino acid sequence variants of novel nucleic acids. When a small amount of template DNA is used as a starting material, the desired amino acid variant can be created with primer (s) slightly different in sequence from the corresponding region in the template DNA. PCR amplification results in a population of DNA fragment products that differ from the polynucleotide template that encodes the polypeptide at the location specified by the primer. The product of this DNA fragment replaces the corresponding region in the plasmid, thereby yielding a polynucleotide encoding the desired amino acid variant.

  Additional techniques for generating amino acid variants include cassette mutagenesis techniques described in Wells et al., Gene 34: 315 (1985); and, for example, the technique of Sambrook et al. (Supra) and Current Protocols. Other mutagenesis techniques well known in the art, such as in Molecular Biology (Ausubel et al.). Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can be used for the cloning and expression of these novel nucleic acids. You may use for implementation. Such a DNA sequence includes a sequence capable of hybridizing to an appropriate novel nucleic acid sequence under stringent conditions.

  A polynucleotide encoding a cleavage portion of a preferred polypeptide of the present invention can be used to generate a chimeric or fusion protein comprising one or more domains of the present invention, and a polynucleotide encoding a heterologous protein sequence. .

  The polynucleotide of the present invention further includes the complement of any of the above polynucleotides. This polynucleotide may be DNA (genome, cDNA, amplified or synthesized) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and include, for example, determining hybridization conditions that can conventionally isolate a polynucleotide having the same desired sequence. The method of doing can be mentioned.

  In accordance with the present invention, a polynucleotide sequence comprising a mature protein coding sequence corresponding to any one of SEQ ID NOs: 1-684 or 1369-1966, or a functional equivalent thereof, is transformed into the nucleic acid in a suitable host cell. It can be used to generate recombinant DNA molecules that direct expression, or functional equivalents thereof. Also included are cDNA inserts of any of the clones identified herein.

  A polynucleotide according to the present invention can be conjugated to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). See Useful nucleotide sequences that bind to polynucleotides include various vectors well known in the art, such as plasmids, cosmids, derivatives of lambda phage, phagemids, and the like. Accordingly, the present invention also provides a vector comprising the polynucleotide of the present invention and a host cell comprising the polynucleotide. In general, the vector contains an origin of replication that functions in at least one organism, a convenient restriction endonuclease site, and a host cell selectable marker. Vectors according to the present invention include expression vectors, replication vectors, probe generation vectors, and sequence vectors. A host cell according to the present invention may be a prokaryotic or eukaryotic cell, and may be a unicellular organism or part of a multicellular organism.

  The present invention further provides a recombinant construct comprising any of the nucleotide sequences of SEQ ID NOs: 1-684 or 1369-1966, a nucleic acid having any of its fragments, or other polynucleotides of the present invention. In one embodiment, a recombinant construct of the present invention comprises a vector such as a plasmid or virus, in which a nucleic acid having either the nucleotide sequence of SEQ ID NOs: 1-684 or 1369-1966, or a fragment thereof. Inserted forward or backward. In the case of a vector of the invention or comprising one of the ORFs, the vector may further comprise regulatory sequences, which include, for example, a promoter operably linked to the ORF. Numerous suitable vectors and promoters are known to those of skill in the art and are commercially available for producing the recombinant products of the present invention. Examples include the following vectors: bacteria: pBs, phage script, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3pR5p Eukaryotes: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

  The isolated polynucleotides of the present invention can be prepared using the methods of Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), and can be operably linked to expression control sequences such as pMT2 or pED expression vectors. A number of suitable expression control sequences are known in the art. General expression methods for recombinant proteins are also known. Illustrated in Kaufman, “Methods in Enzymology” 185, 537-566 (1990). As defined herein, “operably linked” means that the isolated polynucleotide and expression control sequences of the invention are placed in a vector or cell and the protein is Means that the ligated polynucleotide / expression control sequence is expressed by a host cell that has been transformed (transfected).

  The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vector containing a selectable marker. Two suitable vectors are pKK232-8 and pCM7. Bacterial promoters with specific names include lacI, lacZ, T3, T7, gpt, λPR, and trc. Eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTR, and mouse metallothionein-I. The selection of appropriate vectors and promoters is well within the level of ordinary skill in the art. In general, vectors for recombinant expression include origins of replication and selectable markers that allow transformation of the host cell, eg, E. coli. E. coli ampicillin resistance gene and S. coli. cerevisiae TRP1 gene, as well as promoters derived from highly expressed genes that direct transcription of downstream structural sequences. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock proteins, among others. The heterologous sequence is assembled with translation initiation and termination sequences at the appropriate stage, and preferably with a leader sequence that can direct secretion of the translated protein into the periplasmic space or extracellular medium. If desired, the heterologous sequence can encode a fusion protein comprising an amino terminal identification peptide that confers desired properties, eg, stabilization or simple purification of the expressed recombinant product. Useful expression vectors for use in bacteria are by inserting a structural DNA sequence encoding the desired protein, together with appropriate translation start and stop signals, at the stage of an operable read with a functional promoter. Built. The vector contains one or more phenotypic selectable markers and an origin of replication to ensure the maintenance of the vector and, if desired, amplify it in the host. Prokaryotic hosts suitable for transformation include E. coli. There are various species of E. coli, Bacillus subtilis, Salmonella typhimurium and Pseudomonas, Streptomyces and Staphylococcus, but other hosts can also be used as selectable objects.

  As a representative but non-limiting example, expression vectors useful for use in bacteria include bacterial selectable markers and origins of replication from commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). But you can. Examples of such commercially available vectors include pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, WI, USA). These “backbone” portions of pBR322 are combined with a suitable promoter and the structural sequence to be expressed. After transformation of the appropriate host strain and growth of the host strain to the appropriate cell density, the selected promoter is induced or repressed by appropriate means (eg, temperature shift or chemical induction) to further Incubate for a period of time. Cells are typically collected by centrifugation, disrupted by physical or chemical methods, and the resulting crude extract is stored for further purification.

  The polynucleotides of the present invention can also be used to induce an immune response. See, for example, Fan et al., Nat. Biotech. 17: 870-872 (1999), the nucleic acid sequence encoding the polypeptide can be obtained after local administration or after injection of naked plasmid DNA, and preferably after intramuscular injection of DNA. It can be used to generate antibodies against the encoded polypeptide. The nucleic acid sequence is preferably inserted into a recombinant expression vector and may be in the form of naked DNA.

4.3 Antisense Another aspect of the invention is hybridizable to or complementary to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-684 or 1369-1966, or a fragment, analog or derivative thereof. Involved in isolated antisense nucleic acid molecules. An “antisense” nucleic acid is complementary to a “sense” nucleic acid encoding a protein, eg, complementary to the coding sequence of a double-stranded cDNA molecule or complementary to an mRNA sequence. A nucleotide sequence that is In certain aspects, an antisense nucleic acid molecule comprises an antisense that comprises a sequence that is complementary to at least about 10, 25, 50, 100, 250, or 500 nucleotides, or the entire coding strand, or a portion thereof. Nucleic acid molecules are provided. A nucleic acid molecule encoding a fragment, homologue, derivative and analog of the protein of SEQ ID NO: 1-684 or 1369-1966, or an antisense nucleic acid complementary to the nucleic acid sequence of SEQ ID NO: 1-684 or 1369-1966 Further provided.

  In one embodiment, the antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention. The term “coding region” refers to a region of a nucleotide sequence that includes codons translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of the nucleotide sequence of the invention. The term “noncoding region” refers to 5 ′ and 3 ′ sequences flanking the coding region that are not translated into amino acids (ie, also referred to as 5 ′ and 3 ′ untranslated regions).

  When considering the sequences of the coding strands encoding the nucleic acids disclosed herein (eg, SEQ ID NOs: 1-684 or 1369-1966, the antisense nucleic acids of the present invention are Watson and Crick, or Hoogsteen bases An antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferred is an oligonucleotide that is antisense only to a portion of the coding or non-coding region of the mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the mRNA, eg, about 5, 10, 15, 20, 25, 30, 35 , 40, 45 or 50 nucleotides in length. Sense nucleic acids may be constructed using chemical synthesis, or enzyme-linked reactions using procedures known in the art, for example, antisense nucleic acids (eg, antisense oligonucleotides) are naturally occurring nucleotides, or Chemical synthesis using a variety of modified nucleotides designed to improve the biostability of a molecule or to improve the physical stability of a duplex formed between an antisense and a sense nucleic acid For example, phosphorothioate derivatives and acridine substituted nucleotides can be used.

  Examples of modified nucleotides that can be used to generate antisense nucleic acids include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- Acetylcytosine, 5- (carboxylhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcuocin, inosine, N6-isopentenyladenine 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminome Luuracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylcuocin, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid ( v), wivetoxosin, pseudouracil, cuocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (V), 5-methyl-2-thiouracil, 3 (3-amino-3-N-2-carboxylpropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, antisense nucleic acids can be biologically generated using expression vectors in which the nucleic acid is subcloned in the antisense orientation (ie, RNA transcribed from the inserted nucleic acid is detailed in the subsections below). In the antisense direction with respect to the target nucleic acid of interest).

  An antisense nucleic acid molecule of the invention is typically administered to a subject or generated in situ so that it hybridizes with cellular mRNA and / or genomic DNA encoding a protein of the invention or Bind, thereby inhibiting, for example, transcription and / or translation to inhibit protein expression. Hybridization may be by complementation of conventional nucleotides to form the appropriate duplex, or, for example, in the case of antisense nucleic acid molecules that bind to DNA duplexes, within the large groove of the duplex. It may be through a specific interaction. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules may be modified to target selected cells and then administered systemically. For example, for systemic administration, it specifically binds to an antigen expressed on the surface of the receptor or selected cells, eg, by linking an antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. As such, antisense molecules may be modified. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. In order to achieve sufficient intracellular concentrations of the antisense molecule, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

  In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms a specific double-stranded hybrid with the complementary RNA, where the strands are parallel to each other, unlike the normal α unit (Gaultier et al. (1987) Nucleic Acids Res. 15: 6625-6641). Antisense nucleic acid molecules can also be 2′-o-methyl ribonucleotides (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or chimeric RNA-DNA analogs (Inoue et al. (1987) FEBS Lett 215: 327- 330).

4.4 Ribozymes and PNA moieties In yet another embodiment, the antisense nucleic acid of the invention is a ribozyme. A ribozyme is a catalytic RNA molecule having ribonuclease activity and can cleave a single-stranded nucleic acid such as mRNA having a region complementary thereto. Thus, ribozymes (eg, Hammerhead ribozymes; described in Haselhoff and Gerlach (1988) Nature 334: 585-591) are utilized to catalytically cleave mRNA transcripts and thereby inhibit translation of mRNA. it can. Ribozymes having specificity for the nucleic acids of the invention can be designed based on the nucleotide sequences of the DNA disclosed herein (ie, SEQ ID NOs: 1-684 or 1369-1966). For example, a derivative of Tetrahymena L-19 IVS RNA may be constructed such that the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the mRNA. See, for example, Cech et al., US Pat. No. 4,987,071, and Cech et al., US Pat. No. 5,116,742. Alternatively, catalytic RNA having specific ribonuclease activity may be selected from a pool of RNA molecules using the mRNA of the present invention. See, for example, Bartel et al. (1993) Science 261: 1411-1418.

  Alternatively, gene expression is inhibited by targeting a nucleotide sequence complementary to a regulatory region (eg, a promoter and / or enhancer) to form a triple helix structure that prevents transcription of the gene in the target cell. obtain. See generally, Helene (1991) Anticancer Drug Des. 6: 569-84; Helene et al. (1992) Ann. N. Y. Acad. Sci. 660: 27-36; and Maher (1992) Bioassays 14: 807-15.

  In various embodiments, the nucleic acids of the invention may be modified with a base moiety, sugar moiety, or phosphate backbone, for example, to improve molecular stability, hybridization, or solubility. For example, the deoxyribose phosphate backbone of the nucleic acid may be modified to produce peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the term “peptide nucleic acids” or “PNA” refers to nucleic acid mimetics, eg, DNA mimetics, where the deoxyribose phosphate backbone is replaced by the pseudopeptide backbone. Only four natural nucleobases are retained. The neutral backbone of PNA has been shown to be able to specifically hybridize 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) (supra); Perry-O'Keefe et al. (1996) PNAS 93: 14670-675. .

  The PNA of the present invention can be used in therapeutic and diagnostic applications. For example, PNA can be used as an antisense or antigene factor in the sequence-specific regulation of gene expression by inducing transcription or translational arrest, or inhibiting replication. The PNAs of the present invention are used, for example, in the analysis of single base pair mutations in genes by PNA-directed PCR clamping; when used in combination with other enzymes, such as S1 nuclease (Hyrup B. ( 1996), supra); or as DNA sequences and hybridization probes or primers (Hyrup et al. (1996) supra; Perry-O'Keefe (1996) supra).

  In another embodiment, the PNAs of the invention can be produced by, for example, adding lipophilic or other auxiliary groups to the PNA, by forming a PNA-DNA chimera, or by improving the stability or cellular uptake, or of liposomes. It can be modified by use or use of other techniques of drug delivery known in the art. For example, PNA-DNA chimeras that can combine the advantageous properties of PNA and DNA may be generated. Such chimeras allow DNA recognition enzymes, such as RNase H and DNA polymerase, to interact with the DNA moiety, while at the same time the PNA moiety provides high binding affinity and specificity. PNA-DNA chimeras may be linked using linkers of appropriate length selected taking into account base stacking, the number of bonds between base nucleic acids, and orientation (Hyrup (1996) supra). Synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) supra and in Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, the DNA strand may be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs such as 5 ′-(4-methyoxytrityl) amino-5′-deoxy- Thymidine phosphoramidites can be used between PNA and the 5 ′ end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). The PNA monomers are then ligated in a stepwise fashion to produce a chimeric molecule having a 5 'PNA segment and a 3' DNA segment (Finn et al. (1996) supra). Alternatively, the chimeric molecule may be synthesized using a 5 'DNA segment and a 3' PNA segment. See Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

  In other embodiments, oligonucleotides include other adducts such as peptides (eg, to target host cell receptors in vivo), or cell membranes (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86: 6553-6556; Lemaire et al., 1987, Proc. Natl.Acad.Sci.84: 648-652; see PCT Publication No. W088 / 09810) or the blood brain barrier (eg, , See PCT Publication No. W089 / 10134). In addition, oligonucleotides can be hybridization-induced cleavage factors (see, eg, Krol et al., 1988, BioTechniques 6: 958-976) or intercalating agents (eg, Zon, 1988, Pharm. Res. 5: 539). ˜549)). For this purpose, the oligonucleotide may be conjugated with another molecule, such as a peptide, a hybridization-inducing cross-linking agent, a transport factor, a hybridization-inducing cleavage factor, and the like.

4.5 Host The present invention further provides a host cell that has been genetically engineered to include a polynucleotide of the present invention. For example, such a host cell may contain a nucleic acid of the invention introduced into the host cell using known transformation, transfection or infection methods. The invention further provides a host cell that has been genetically engineered to express a polynucleotide of the invention, wherein such a polynucleotide is against a host cell that drives expression of the polynucleotide in the cell. It is operatively associated with a heterologous regulatory sequence.

  With knowledge of the nucleic acid sequence, cells can be modified to allow or increase expression of the endogenous polypeptide. The cell may express the polypeptide by replacing all or part of the naturally occurring promoter with all or part of a heterologous promoter so that the cell expresses the polypeptide at a higher level. Modifications can be made to increase (eg, by homologous recombination). The heterologous promoter is inserted in such a way as to be operably linked to the coding sequence. See, for example, PCT International Publication No. WO94 / 12650, PCT International Publication No. WO92 / 20808, and PCT International Publication No. WO91 / 09955. In addition to heterologous promoter DNA, amplifiable marker DNA (for example, multifunctional CAD gene encoding ada, dhfr, and carbamyl phosphate synthase, aspartate transcarbamylase and dihydroorotase) and / or intron It is contemplated that DNA may be inserted with heterologous promoter DNA. If linked to a coding sequence, amplification of the marker DNA by standard selection methods results in simultaneous amplification of the desired protein coding sequence in the cell.

  The host cell may 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 such as a bacterial cell. Prokaryotic cells may also be used. Introduction of recombinant constructs into host cells can be performed by calcium phosphate transfection, DEAE, dextran-mediated transfection, or electroporation (Davis, L, et al., Basic Methods in Molecular Biology (1986)). Host cells having one of the polynucleotides of the invention can be used in a conventional manner to produce the gene product encoded by the isolated fragment (in the case of an ORF), or using this It is possible to produce heterologous proteins under the control of EMF.

  Any host / vector system can be used to express one or more ORFs of the invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cells, COS cells, 293 cells and Sf9 cells, and prokaryotic hosts such as E. coli. E. coli and B.I. subtilis. The most preferred cells are usually cells that do not express a particular polypeptide or protein, or cells that express a polypeptide or protein at a natural low level. The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of a suitable promoter. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, New York (1989), The disclosure of which is hereby incorporated by reference.

  Recombinant proteins can also be expressed using various mammalian cell culture systems. Examples of mammalian cell expression systems include the monkey kidney fibroblast COS-7 strain described in Gluzman, Cell 23: 175 (1981). Those capable of expressing compatible vectors in other cell lines include, for example, C127, monkey COS cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human epidermis A431 cells, human Colo 205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from primary tissue in vitro, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Mammalian cell expression vectors contain an origin of replication, a suitable promoter and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and non-transcribed sequences flanking 5 '. The DNA sequence from the viral genome of SV40, such as the SV40 origin, early promoter, enhancer, splice, and polyadenylation site, can be used to provide the necessary non-transcribed genetic elements. Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from the cell pellet, followed by one or more salting out, aqueous ion exchange, or size exclusion chromatography steps. If necessary, a step of refolding the protein may be used to complete the conformation of the mature protein. Finally, high performance liquid chromatography (HPLC) may be used as the final purification step. Microbial cells used for protein expression may be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cytolytic agents.

  Alternatively, it may be possible to produce proteins in lower eukaryotes such as yeast or insects, or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing a heterologous protein. Possible suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing a heterologous protein. If the protein is made in yeast or bacteria, in order to obtain a functional protein it is necessary to modify the protein produced there, for example by phosphorylation or glycosylation at the appropriate site There is a case. Such covalent bond addition can be accomplished using known chemical or enzymatic methods.

  In another embodiment of the invention, cells and tissues can be designed to express an endogenous gene comprising a polynucleotide of the invention under the control of an inducible regulatory element, in which case the endogenous gene These regulatory sequences can be replaced by homologous recombination. As described herein, gene targeting can be used to replace an existing regulatory region of a gene with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Can be replaced. Such regulatory sequences may be composed of promoters, enhancers, scaffold attachment regions, negative regulatory elements, transcription initiation sites, and regulatory protein binding sites, or combinations of such sequences. Alternatively, sequences that affect the structure or stability of the generated RNA or protein may be substituted, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences to enhance or modify protein transport or secretion properties, or alter or improve the function or stability of a protein or RNA molecule. , Other sequences.

  This target event may be simply the insertion of a regulatory sequence, placing the gene under the control of a new regulatory sequence, eg, inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the target event may be a simple deletion of a regulatory element, for example a deletion of a tissue specific negative regulatory element. Alternatively, the target event may replace an existing element; for example, a tissue-specific enhancer may be replaced with an enhancer that has a broader or different cell type specificity than a naturally occurring element. Also good. Here, naturally occurring sequences are deleted and new sequences are added. In all cases, identification of target events is facilitated by the use of one or more selectable marker genes flanked by the target DNA, which allows selection of cells that have integrated exogenous DNA into the host cell genome. become. Identification of the target event is also the correct homology with the sequence in the host cell genome such that the negative selectable marker is adjacent to the exogenous DNA, but this negative selectable marker is constructed adjacent to the target sequence. It can also be facilitated by the use of one or more marker genes that exhibit negative selection properties such that recombination does not result in stable integration of this negative selectable marker. Useful markers for this purpose include the herpes simplex virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyltransferase (gpt) gene.

  Gene targeting or gene activation techniques that can be used in accordance with this aspect of the invention are described in further detail below: US Pat. No. 5,272,071 to Chapel; US Pat. No. 5,578, Sherwin et al. International Application No. PCT / US92 / 09627 (WO93 / 09222) by Selden et al .; and International Application No. PCT / US90 / 06436 (WO91 / 06667) by Skoultchi et al. (Each of which is herein incorporated by reference in its entirety). ).

4.6 Polypeptides of the Present Invention Isolated polypeptides of the present invention include, but are not limited to, polypeptides including: shown as any one of SEQ ID NOs: 685-1368 or 1967-2564 An amino acid sequence, or an amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NOs: 1-684 or 1369-1966, or the corresponding full-length or mature protein. Polypeptides of the present invention also include polypeptides preferably having biological or immunological activity encoded by: (a) nucleotides set forth in SEQ ID NOs: 1-684 or 1369-1966 A polynucleotide having any one of the sequences, or (b) a polynucleotide encoding any one of the amino acid sequences set forth in SEQ ID NOs: 685-1368 or 1967-2564, or (c) stringent hybridization A polynucleotide that hybridizes to the complement of the polynucleotide of either (a) or (b) under conditions. The invention also provides a biological or immune activity variant of any of the amino acid sequences set forth as SEQ ID NOs: 685-1368 or 1967-2564, or the corresponding full-length or mature protein; and retains biological activity. Their “substantial equivalents” (eg, 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 Having at least about 99% amino acid identity). A polypeptide encoded by an allelic variant may be similar, higher, or lower in activity as compared to a polypeptide comprising SEQ ID NOs: 685-1368 or 1967-2564.

  Fragments of the proteins of the present invention that are capable of exhibiting biological activity are also encompassed by the present invention. The protein fragment may be in a linear form or, for example, both of which are hereby incorporated by reference. U. Saragovi et al., Bio / Technology 10,773-778 (1992) and R.A. S. McDowell et al. Amer. Chem. Soc. 114, 9245-9253 (1992), they may be circularized using known methods. Such fragments may be fused with a carrier molecule such as an immunoglobulin for many purposes, such as increasing the valency of the binding site of the protein. Fragments are also identified in Table 3A, 3B, 5, 7 or 8.

  The present invention also provides both full-length and mature forms (eg, no signal or precursor sequences) of the disclosed proteins. Protein coding sequences are identified in the sequence listing by translation of the disclosed nucleotide sequences. The putative signal sequence is shown in Table 5. The mature form of such a protein can be obtained and confirmed by expressing the full length polynucleotide in a suitable mammalian cell, or other host cell, and sequencing the cleaved product. One skilled in the art will recognize that the actual cleavage site may be different from the cleavage site estimated in Table 5. The mature form of the protein can also be determined from the full-length amino acid sequence. Where the protein of the invention is associated with a membrane, a soluble form of this protein is also provided. In such a form, part or all of the region that binds the protein to the membrane is deleted such that the protein is completely secreted from the cell in which it is expressed (eg, incorporated herein by reference). Sakal et al., Prep. Biochem. Biotechnol. (2000), 30 (2), pages 107-23).

  The protein composition of the present invention may further comprise an acceptable carrier, such as a pharmaceutically acceptable hydrophilic carrier.

  The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. A “degenerate variant” is a nucleotide sequence that differs from a nucleic acid fragment of the invention (eg, ORF), but encodes the same polynucleotide sequence due to the degeneracy of the genetic code. Nucleotide fragment. A preferred nucleic acid fragment of the invention is an ORF encoding a protein.

  Various methodologies known in the art can be utilized to obtain any of the isolated polypeptides or proteins of the present invention. At the simplest level, amino acid sequences can be synthesized using commercially available peptide synthesizers. Syntheticly constructed protein sequences share primary, secondary, or tertiary and / or higher-order structural features with the protein and therefore share biological properties, including protein activity, in common There is. This technique is particularly useful for generating fragments of small peptides and large polypeptides. Fragments are useful, for example, for generating antibodies against natural polypeptides. Thus, they can be used as biologically active or immunological substitutes for naturally purified proteins, in screening therapeutic compounds, and in the immunological process of antibody development. .

  The polypeptides and proteins of the invention may alternatively be purified from cells that have been altered to express the desired polypeptide or protein. As used herein, if genetic engineering results in the production of a polypeptide or protein that the cell does not normally produce, or that is usually produced only at low levels, then the cell has the desired polypeptide or protein. It has been changed to express. Those skilled in the art will readily adjust procedures to introduce and express either recombinant or synthetic sequences into eukaryotic or prokaryotic cells to generate cells that produce one of the polypeptides or proteins of the invention. can do.

  The present invention also relates to a method of producing a polypeptide, the method comprising growing a culture of a host cell of the invention in a suitable culture medium and the protein from the culture in which the cell or cell is grown. A step of purification. For example, the method of the present invention comprises a process for producing a polypeptide comprising culturing host cells having an appropriate expression vector comprising a polynucleotide of the present invention under conditions that allow expression of the encoded polypeptide. Is included. The polypeptide may be recovered and further purified from the culture, conveniently from the culture medium, or from lysates prepared from the host cells. Preferred embodiments include those in which the protein produced by such a process is the full-length or mature form of the protein.

  In another method, the polypeptide or protein is purified from bacterial cells that naturally produce the polypeptide or protein. One skilled in the art can readily utilize known methods for separation of polypeptides and proteins to obtain one of the isolated polypeptides or proteins of the invention. These include, but are not limited to, immunochromatography, HPLC, size exclusion chromatography, ion exchange chromatography, and immunoaffinity chromatography. See, for example, Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook et al., Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocol. Polypeptide fragments that retain biological / immunological activity include fragments comprised of greater than about 100 amino acids, or greater than about 200 amino acids, and fragments encoding specific protein domains.

  The purified polypeptide can be used in in vitro binding assays well known in the art to identify molecules that bind to the polypeptide. These molecules include, but are not limited to, small molecules, molecules from combinatorial libraries, antibodies or other proteins. 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 short, molecules are titrated into multiple cell cultures or animals and then tested for either cell / animal death or animal / cell long-term survival.

  Furthermore, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins such as ricin or cholera, or other compounds that are toxic to cells. The toxin binding molecule complex can then be targeted to tumors or other cells by the specificity of the binding molecule for SEQ ID NOs: 685-1368 or 1967-2564.

  The protein of the present invention may also be used as a component of somatic cells containing a nucleotide sequence encoding the protein, or a product of a transgenic animal characterized by germ cells, such as transgenic bovine, goat, pig or sheep milk. It may be expressed.

  Proteins provided herein also include proteins characterized by an amino acid sequence that is similar to the amino acid sequence of the purified protein, but is naturally modified or artificially designed. For example, modification with a peptide or DNA sequence can be performed by one skilled in the art using known techniques. Desired modifications in the protein sequence can include alterations, substitutions, substitutions, insertions or deletions of selected amino acid residues in the coding sequence. For example, one or more cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Such techniques of alteration, substitution, replacement, insertion or deletion are known to those skilled in the art (see, eg, US Pat. No. 4,518,584). Preferably, such alterations, substitutions, substitutions, insertions or deletions retain the desired activity of the protein. Protein regions important for protein function can be determined by various methods well known in the art, including the alanine scanning method. The alanine scanning method involved the step of systematically replacing a single or contiguous amino acid with alanine and testing the resulting variant containing alanine for biological activity. This type of analysis determines the importance of the substituted amino acid (s) in biological activity. Protein regions important for protein function may be determined by the eMATRIX program.

  Other fragments and derivatives of protein sequences that are expected to retain all or part of the activity of the protein and are useful in screening or other immunological methods are also disclosed herein. It can be easily made by those skilled in the art in consideration. Such modifications are encompassed by the present invention.

Proteins can also be produced by operably linking the isolated polynucleotides of the present invention to appropriate control sequences in one or more insect expression vectors and using insect expression systems. Materials and methods for baculovirus / insect cell expression systems are described, for example, in Invitrogen, San Diego, Calif. , U. S. A. Kit (the ^MaxBat TM kit) is commercially available as such methods, Summers and Smith are incorporated by reference herein, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) is well known in the art. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed”.

The proteins of the present invention can be prepared by culturing transformed host cells under culture conditions suitable for expressing the recombinant protein. The resulting protein can then be purified from such cultures (ie, from culture media or cell extracts) using known purification processes such as gel filtration and ion exchange chromatography. Examples of protein purification include the following substances. One or more column steps on an affinity resin such as concanavalin A-agarose, heparin-toyopearl ^™ or Cibacrom blue 3GA Sepharose ^™ ; One or more steps, including hydrophobic interaction chromatography using resins such as ether, butyl ether, or propyl ether; or immunoaffinity chromatography.

  Alternatively, the proteins of the invention may also be expressed in a form that facilitates purification. For example, it may be expressed as a fusion protein such as maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or a HIS tag, 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, NJ) and Invitrogen, respectively. The protein may also be tagged with epitopes and subsequently purified by using antibodies specific for such epitopes. One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).

  Finally, one or more reverse phase high performance liquid chromatography (RP-HPLQ) steps (using hydrophobic RP-HPCL media such as silica gel with pendant methyl or other aliphatic groups) are used to further Can be purified. By using some or all of the above purification steps in various combinations, isolated homologous proteins that are substantially homologous can also be obtained. Proteins purified in this way are substantially free of other mammalian proteins and are defined in the present invention as "isolated proteins".

  The polypeptides of the present invention include analogs (variants). This includes fragments as well as peptides in which one or more amino acids have been deleted, inserted or substituted. Also, an analog of a polypeptide of the invention encompasses fusion of the polypeptide, or modification of a polypeptide of the invention, where the polypeptide or analog is another part (s), eg, a target Fused to a partial or another therapeutic agent. Such analogs may exhibit improved properties such as activity and / or stability. Examples of moieties that can be fused to a polypeptide or analog include, for example, target moieties that deliver the polypeptide to pancreatic cells, such as antibodies to pancreatic cells, immune cells such as T cells, monocytes, dendritic cells , Antibodies against granulocytes and the like, and receptors and ligands expressed in the pancreas or immune cells. Other moieties that can be fused to the polypeptide include therapeutic agents used for therapy, such as immunosuppressive agents such as cyclosporine, SK506, azathioprine, CD3 antibodies and steroids. The polypeptide may also be fused to immune modulators and other cytokines such as alpha interferon or beta interferon.

4.6.1 Determining Identity and Similarity of Polypeptides and Polynucleotides Preferred identities and / or similarities are designed to obtain the greatest match between the sequences tested. Methods for determining identity and similarity are organized in computer programs, including but not limited to computer programs, including: GCG program packages 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, SF et al., J. Molec. Biol. -BLAST (Altschul SF et al., Nucleic Acids Res. 25th, incorporated herein by reference. Vol., 3389-3402), eMatrix software (incorporated herein by reference, Wu et al., J. Comp. Biol. Vol. 6, 219-235 (1999)), eMotif software (referenced herein). Nevil-Manning et al., ISMB-97, Vol. 4, pp. 202-209), Pfam software (Sonhammer et al., Nucleic Acids Res. Vol. 26 (1) 320, incorporated herein by reference. 322 (1998)), and Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol Biol, 157, 105-31 (1982)), GeneAtlas software (Molecular Simulations Inc. (MSI), San Diego, CA) (Sanchez and Sali (1998) Proc. Natl. Acad. Sci., 95, 13597-13602, incorporated herein by reference; Kitson DH et al. (2000) “Remote”. homology detection using structural modeling-an evaluation "submitted content; Fischer and Eisenberg (1996) Protein Sci.5,947~955), Neural Network SignalP V1.1 program (Center for Biological Sequence Analysis, The Technical University of Denmark)). Polypeptide sequences are examined by the proprietary algorithm SeqLoc, which divides the protein into three sets of positions (intracellular, membrane, or secretion). This prediction is based on three characteristics of each polypeptide, including the percentage of cysteine residues, the Kyte-Doolittle score of the first 20 amino acids of each protein, and the longest hydrophobic stretch of the protein Including the Kyte-Doolittle score. The estimated protein values are compared against values from a set of 592 proteins of known cellular localization from the Swisssport database (http://www.expasy.ch/sprot). The prediction is based on maximum likelihood estimation.

  The presence of the transmembrane region (s) was detected using the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html).

  The BLAST program is available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S. et al. NCBI NLM NIH Bethesda, MD 20894; Altschul, S et al., J. Mol. 1990)).

4.7 Chimeric proteins and fusion proteins The present invention also provides chimeric or fusion proteins. As used herein, a “chimeric protein” or “fusion protein” includes a polypeptide of the invention operably linked to another polypeptide. Within the fusion protein, the polypeptide of the invention may correspond to all or part of the protein according to the invention. In one embodiment, the fusion protein comprises at least one biologically active part of a protein according to the invention. In another embodiment, the fusion protein comprises at least two bioactive portions of the protein according to the invention. Within the fusion protein, the term “operably linked” is intended to indicate that the polypeptide of the invention and another polypeptide are fused together in a frame. The polypeptide can be fused to the N-terminal, C-terminal, or middle.

  For example, in one embodiment, the fusion protein comprises a polypeptide of the invention operably linked to the extracellular domain of a second protein.

  In another embodiment, the fusion protein is a GST-fusion protein and the polypeptide sequence of the invention is fused to the C-terminus of the GST (ie glutathione S-transferase) sequence.

  In another embodiment, the fusion protein is an immunoglobulin fusion protein, wherein a polypeptide sequence according to the present invention comprises one or more domains fused to a sequence derived from a member of an immunoglobulin protein family. The immunoglobulin fusion protein of the invention is incorporated into a pharmaceutical composition and administered to a subject to inhibit the interaction between the ligand on the surface of the cell and the protein of the invention, thereby in vivo signal. Transmission can be suppressed. Immunoglobulin fusion proteins can be used to affect the bioavailability of cognate ligands. Inhibition of ligand / protein interactions may be useful therapeutically both in the treatment of proliferation and differentiation disorders, such as cancer, and in the regulation (eg, promotion or suppression) of cell survival. In addition, the immunoglobulin fusion proteins of the invention can be used as immunogens to generate antibodies in a subject, purify ligands, and molecules that inhibit the interaction between the polypeptides of the invention and ligands in screening assays. Identify

  The chimeric or fusion proteins of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences may be used in accordance with conventional techniques, for example, to use blunt ends or staggered ends for ligation, to provide suitable ends. Ligated together in-frame by restriction enzyme digestion, optionally sticky end filling, alkaline phosphatase treatment to avoid undesired binding, and enzyme ligation. In another embodiment, the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of a gene fragment can be performed using an anchor primer that produces a complementary overhang between two consecutive gene fragments that are substantially annealed and re-amplified. Thus, chimeric gene sequences can be generated (see, eg, Ausubel et al. (Ed.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). In addition, many expression vectors are commercially available that already encode a fusion moiety (eg, a GST polypeptide). Nucleic acids encoding the polypeptides of the invention may be cloned into such expression vectors such that the fusion moiety is linked in-frame to the protein of the invention.

4.8 Gene Therapy Mutations in the polynucleotide of the gene of the invention can result in a loss of normal function of the encoded protein. Thus, the present invention provides gene therapy to restore normal activity of the polypeptides of the present invention; or to treat disease states associated with the polypeptides of the present invention. Delivery of a functional gene encoding a polypeptide of the invention to an appropriate cell may be accomplished ex vivo, in situ, or in vivo by use of a vector, more particularly a viral vector (eg, adenovirus, adeno-associated virus or retrovirus). Or by use of physical DNA transfer methods (eg, liposomes or chemotherapy). For example, Anderson, Nature. Addendum of Volume 392, no. 6679, pages 25-20 (1998). For additional reviews of gene therapy techniques, see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). That. Introduction of any one of the genes encoding the nucleotides of the invention or the polypeptides of the invention can also be achieved using extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). In addition, cells can be cultured ex vivo in the presence of the protein of the present invention in order to proliferate the cells or to give such cells the desired effect or activity. The treated cells can then be introduced in vivo for therapeutic purposes. Alternatively, in other human disease states, preventing expression of the polypeptide of the invention or inhibiting its activity would be useful for treating this disease state. It is also contemplated to apply antisense therapy or gene therapy to negatively regulate the expression of the polypeptides of the invention.

  Other methods of inhibiting protein expression include introducing antisense molecules, their complements, or translated RNA sequences to the nucleic acids of the invention by methods known in the art. Furthermore, the polypeptides of the invention can be inhibited using targeted deletion methods or by insertion of negative regulatory elements such as tissue-specific silencers.

  The present invention further provides cells that have been engineered in vivo to express the polynucleotides of the present invention, where such polynucleotides drive the expression of the polynucleotide in the cells. It is operably associated with regulatory sequences that are heterologous to the host cell. These methods can be used to increase or decrease the expression of a polypeptide of the invention.

  With the knowledge of the DNA sequences provided by the present invention, cell modifications can allow, increase or decrease the expression of endogenous polypeptides. Modify the cell (eg, by homologous recombination) to replace all or part of the naturally occurring promoter with all or part of the heterologous promoter, so that the cell expresses the protein at a higher level In addition, the expression of the polypeptide can be increased. This heterologous promoter is inserted in such a way as to be operably linked to the desired protein encoding sequence. See, for example, PCT International Publication No. WO94 / 12650, PCT International Publication No. WO92 / 20808, and PCT International Publication No. WO91 / 09955. In addition to heterologous promoter DNA, an amplifiable marker DNA (eg, ada, dhfr and multifunctional CAD genes encoding carbamyl phosphate synthase, aspartate transcarbamylase and dihydroorotase) and / or intron DNA It is also contemplated that it can be inserted with heterologous promoter DNA. When linked to the coding sequence of the desired protein, amplification of the marker DNA by standard selection methods results in simultaneous amplification of the desired protein coding sequence in the cell.

  In another embodiment of the invention, cells and tissues may be designed to express an endogenous gene comprising a polynucleotide of the invention under the control of inducible regulatory elements, in which case the endogenous The regulatory sequence of the gene can be replaced by homologous recombination. As described herein, gene targeting can be used to replace an existing regulatory region of a gene with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. May be substituted. Such regulatory sequences may be composed of promoters, enhancers, scaffold attachment regions, negative regulatory elements, transcription initiation sites, regulatory protein binding sites, or combinations of such sequences. Alternatively, sequences that affect the structure or stability of the generated RNA or protein may be replaced, removed, added or modified by targeting methods. These sequences may include polyadenylation signals, mRNA stability elements, splice sites, leader sequences to improve or modify protein transport or secretion properties, or other that alter or improve the function or stability of a protein or RNA molecule. Contains an array.

  The target event may be simply the insertion of a regulatory sequence, placing the gene under the control of a new regulatory sequence, eg, inserting a new promoter or enhancer or both upstream of the gene. Alternatively, the target event may be a simple deletion of a regulatory element, eg a deletion of a tissue specific negative regulatory element. Alternatively, the target event may replace an existing element; for example, a tissue specific enhancer may be replaced with an enhancer that has a broader or different cell type specificity than the naturally occurring element. Here, naturally occurring sequences are deleted and new sequences are added. In either case, identification of the target event can be facilitated by the use of one or more selectable marker genes that are flanked by the target DNA, which allows selection of cells that have exogenous DNA integrated into the cell genome. . Identification of the target event is also a sequence in the host cell genome such that the negative selectable marker is linked to exogenous DNA, but this negative selectable marker is configured to flank the target sequence. Correct homologous recombination with can be facilitated by the use of one or more marker genes that exhibit negative selection properties that do not result in stable integration of this negative selectable marker. Useful markers for this purpose include the herpes simplex virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyltransferase (gpt) gene.

  Gene targeting or gene activation techniques that can be used in accordance with this aspect of the invention are described in Chapel, US Pat. No. 5,272,071, each of which is hereby incorporated by reference in its entirety; Sherwin et al. U.S. Pat. No. 5,578,461; Selden et al. International Application No. PCT / US92 / 09627 (WO 93/09222); and Skoultchi et al. International Application No. PCT / US90 / 06436 (WO 91/06667). Is done.

4.9 Transgenic Animals In a preferred method of determining the biological function of a polypeptide of the invention in vivo, one or more genes provided by the invention are overexpressed in the animal's reproductive system using homologous recombination. Either inactive or inactivated [Capechi, Science 244: 1288-1292 (1989)]. Animals in which a gene is overexpressed under the regulatory control of an exogenous or endogenous promoter element are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. The knockout animal is preferably a non-human mammal, which can be prepared as described in US Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful for determining the role that the polypeptides of the invention play in biological processes, preferably in disease states. Transgenic animals are useful as model systems for identifying compounds that modulate lipid metabolism. Transgenic animals are preferably non-human mammals, but are made using the methods described in US Pat. No. 5,489,743 and PCT Publication No. WO 94/28122, incorporated herein by reference. The

  If all or part of the promoter of the polynucleotide of the present invention is activated or inactivated to change the expression level of the polypeptide of the present invention, a transgenic animal can be produced. Inactivation can be performed using the homologous recombination method described above. Activation can be achieved by supplementing or replacing homologous promoters to increase protein expression. Homologous promoters can be supplemented by the insertion of one or more heterologous enhancer elements known to confer promoter activity to specific tissues.

  The polynucleotides of the invention can also be developed by, for example, homologous recombination or knockout strategies of animals that cannot express the polypeptides of the invention or that express various polypeptides. Such animals are useful as models for studying the in vivo activity of polypeptides and for studying modulators of polypeptides of the invention.

  In a preferred method for determining the biological function of a polypeptide of the invention in vivo, one or more genes provided by the invention are overexpressed in the germline of an animal using homologous recombination or Inactivated [Capecchi, Science 244: 1288-1292 (1989)]. Animals in which genes are overexpressed under the regulatory control of exogenous or endogenous promoter elements are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. The knockout animal is preferably a non-human mammal, which can be prepared as described in US Pat. No. 5,557,032, which is incorporated herein by reference. Transgenic animals are useful in determining the role that a polypeptide of the invention plays in a biological process, preferably a disease state. Transgenic animals are useful as model systems for identifying compounds that modulate lipid metabolism. The transgenic animal is preferably a non-human mammal but is created using the methods described in US Pat. No. 5,489,743 and PCT Publication No. WO 94/28122, incorporated herein by reference. .

  A transgenic animal can be prepared in which all or part of the polynucleotide of the promoter of the present invention is activated or inactivated to change the level of expression of the polypeptide of the present invention. Inactivation can be performed using the homologous recombination method described above. Activation can be achieved by recruiting or replacing homologous promoters to increase protein expression. Homologous promoters can be supplemented by inserting one or more heterologous enhancer elements known to confer promoter activity to specific tissues.

4.10 Applications and Bioactivity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the applications or bioactivities identified herein, including those related to the assays recited in the present invention. Is done. The uses or activities described for the proteins of the invention can be provided by the administration or use of such proteins, or polynucleotides encoding such proteins (eg, gene therapy or vectors suitable for the introduction of DNA, etc. ). The mechanism underlying the particular condition or pathology is whether the polypeptide of the invention, the polynucleotide of the invention or their modulator (activator or inhibitor) is beneficial to a subject in need of treatment. Decide what. Thus, "therapeutic compositions of the invention" includes compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and denatured variants thereof), or The overall activity of the target gene product, either in the polypeptides of the invention (including full-length proteins, mature proteins and their truncations or domains), or the level of target gene / protein expression or target protein activity Include compounds and other substances that modulate. Such modulators include polypeptides, analogs (variants), including fragments and fusion proteins, antibodies and other binding proteins; directly or indirectly activating or inhibiting the polypeptides of the invention Compounds (such as those identified herein, eg, identified through drug screening assays); antisense polynucleotides and polynucleotides suitable for triple helix formation; and, in particular, polypeptides of the invention Antibodies or other binding partners that specifically recognize one or more of the epitopes.

  The polypeptides of the present invention may be involved in cell activation as well, or may be involved in one of the other physiological pathways described herein.

4.10.1 Research Uses and Usefulness The polynucleotides obtained by the present invention can be used for various purposes by research organizations. This polynucleotide can be used for the following purposes: to express a recombinant protein for analysis, characterization, or therapeutic use; as a marker for tissues in which the corresponding protein is preferentially expressed (structurally or As specific molecular weight markers on gels; as chromosomal markers or tags (if labeled) to identify chromosomes or create map maps of relevant gene locations; To compare with the patient's endogenous DNA sequence to identify potential genetic disorders; as a probe to hybridize to and discover new relevant DNA sequences; PCR primers for gene fingerprinting As a source of information; other novel polynucleotides As a probe to “subtract-out” known sequences in the process of discovery; for attachment to a “gene chip” or other support, including for testing expression patterns To screen and create oligomers; to elicit anti-protein antibodies using DNA immunization techniques; and as an antigen to elicit anti-DNA antibodies or elicit another immune response. If a polynucleotide encodes a protein that binds or is likely to bind to another protein (eg, as in a receptor-ligand interaction), the polynucleotide can be used in an interaction trap assay (eg, , Gyuris et al., Cell 75: 791-803 (1993)), polynucleotides encoding other proteins of the binding partner can be identified, or inhibitors of binding interactions can be identified.

  Polypeptides provided by the present invention can also be used in assays to determine biological activity, including: in a multi-protein panel for high-throughput screening; for raising antibodies or other immune responses To induce; as a reagent (including a labeling reagent) in an assay designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid; the corresponding polypeptide is preferentially expressed As a marker of the tissue being done (structurally or at a particular stage in tissue differentiation, development or disease state); and, of course, to isolate correlated receptors or ligands. Proteins involved in these binding interactions can also be used to screen for inhibitors or agonists of binding interaction peptides or small molecules.

  Any or all of these research utilities can be developed in the form of commercially available reagent grades or kits as research products.

  Methods for implementing the applications as described above are well known to those skilled in the art. References disclosing such methods include, but are not limited to, “Molecular Cloning: A Laboratory Manual” 2nd Edition, Cold Spring Harbor Laboratory Press, Sambrook, J. MoI. , E.C. F. Fritsch and T.W. Maniatis, 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S .; L. And A. R. Kimmel ed., 1987.

4.10.2 Nutritional use The polynucleotides and polypeptides of the present invention can also be used as a nutrient source or supplement. Such uses include, but are not limited to, use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source, and use as a carbohydrate. In that case, the polypeptide or polynucleotide of the invention may be added to the feed of a particular organism, or as a separate solid or liquid preparation, eg in the form of a powder, tablet, solution, suspension or capsule. It may be administered in the form. In the case of a microorganism, the polypeptide or polynucleotide of the present invention may be added to a medium in which the microorganism is cultured.

4.1.3 Cytokines and cell proliferation / differentiation activity Polypeptides of the invention exhibit activity associated with cytokines, cell proliferation (either induction or inhibition), or cell differentiation (either induction or inhibition). Or may induce the production of other cytokines in a particular cell population. The polynucleotide of the present invention can encode a polypeptide exhibiting such characteristics. Many protein factors discovered so far, including all known cytokines, have shown activity in one or more factor-dependent cell proliferation assays, which makes this assay a convenient method for confirming cytokine activity. is there. The activity of the therapeutic composition of the present invention is not limited to 32D, DA2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (preB M +), 2E8, RB5, DA1, 123, T1165. , HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco, as demonstrated by any one of a number of conventional factor-dependent cell proliferation assays. The therapeutic composition of the present invention can be used in the following cases:
T-cell or thymocyte proliferation assays, including but not limited to those described below: Current Protocols in Immunology, edited by J. Am. E. Coligan, A.M. M.M. Kruisbeek, D.M. H. Margulies, E .; M.M. Shevach, W.M. Strober, Pub. Green Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro Assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunological Studies in H.). Immunol. 137: 3494-3500, 1986; Bertagnolli et al., J. MoI. Immunol. 145: 1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli et al., I. et al. Immunol. 149: 3778-3783, 1992; Bowman et al. Immunol. 152: 1756-1761, 1994.

  Assays for cytokine production and / or proliferation of pancreatic cells, lymphoid cells or thymocytes, including but not limited to those described below: Polyclonal T cell simulation, Kruisbeek, A. et al. M.M. And Shevach, E .; M.M. In Currnet Protocols in Immunology, J. MoI. E. e. a. Edited by Coligan. Volume I, 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin-γ, Schreiber, R .; D. In Current Protocols in Immunology. J. et al. E. e. a. Coligan ed., Vol. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

  Assays for proliferation and differentiation of hematopoietic cells and lymphocyte producing cells, including but not limited to those described below: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K. et al. Davis, L .; S. And Lipsky P.M. E. In Current Protocols in Immunology J. et al. E. e. a. Edited by Coligan. Volume 1. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; de Vries et al., J. MoI. Exp. Med. 173: 1205-1211, 1991; Moreau et al., Nature 336: 690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U. S. A. 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R.M. In Current Protocols in Immunology. J. et al. E. Edited by Coligan. Volume 1 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U. S. A. 83: 1857-1861, 1986; Measurement of human Interleukin 11-Bennett, F .; , Giannotti, J .; Clark, S .; C. And Turner, K .; J. et al. In Current Protocols in Immunology. J. et al. E. Edited by Coligan. Volume 6.1 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9-Cialletta, A .; , Giannotti, J .; Clark, S .; C. And Turner, K .; J. et al. In Current Protocols in Immunology. J. et al. E. Edited by Coligan. Volume 6.1, 6.13.1, John Wiley and Sons, Toronto. 1991.

  In an assay of the response of a T-cell clone to an antigen, which specifically identifies proteins that affect APC-T cell interactions and direct T cell effects by measuring proliferation and cytokine production. Including, but not limited to, those described below: Current Protocols in Immunology, edited by J. Am. E. Coligan, A.M. M.M. Kruisbeek, D.M. H. Margulies, E .; M.M. Shevach, W.M. Strober, Pub. Green Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro Assays for Mouse Lymphocyte Function, Chapter 7, Cytokines and therial cell; Natl. Acad. Sci. USA 77: 6091-6095, 1980; Weinberger et al., Eur. J. et al. Immun. 11: 405-411, 1981; Takai et al., J. MoI. Immunol. 137: 3494-3500, 1986; Takai et al., J. Biol. , Immunol. 140: 508-512, 1988.

4.10.4 Stem Cell Growth Factor Activity The polypeptides of the present invention may exhibit stem cell growth factor activity and are pluripotent and totipotent, including primordial germ cells, embryonic stem cells, hematopoietic stem cells, and / or germ line stem cells Stem cell proliferation, differentiation and survival. Administration of the polypeptides of the present invention to stem cells in vivo or ex vivo is expected to maintain and proliferate cell populations in a totipotent or pluripotent state, which may result in the regeneration of damaged or diseased tissue. Useful for construction, transplantation, biopharmaceutical manufacturing, and biosensor development. The ability to produce large amounts of human cells has important applications in the following areas: production of human proteins that must be taken from non-human sources or donors, Parkinson's disease, Alzheimer's disease and Transplantation of cells to treat diseases such as other neurodegenerative diseases; transplanted tissues such as bone marrow, skin, cartilage, tendons, bones, muscles (including myocardium), blood vessels, retina, nerve cells, gastrointestinal Cells; and organs for transplantation, such as kidney, liver, pancreas (including islet cells), heart and lung.

  Any growth factor listed herein, including other stem cell maintenance factors, and in particular stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any interleukin Recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-α (MIP-1-α), G-CSF, GM-CSF, thrombopoietin (TPO), platelet fourth factor (PF-4) ), A plurality of different growth factors and / or cytokines, including platelet derived growth factor (PDGF), nerve growth factor, and basic fibroblast growth factor (bFGF), to achieve the desired effect. It is contemplated that it may be administered in combination with a polypeptide.

  Totipotent stem cells can give rise to virtually any mature cell type, and growing these cells in culture can promote the production of large amounts of mature cells. Stem cell culture techniques are known in the art and the polypeptides of the invention are expected to be administered with other growth factors and / or cytokines as needed to improve the survival and proliferation of the stem cell population. . This can be achieved by administering the polypeptide of the present invention directly to the culture medium. Alternatively, stromal cells transfected with a polynucleotide encoding a polypeptide of the invention can be used as a support cell layer for a culture medium or an in vivo stem cell population. The stromal feeder cells of the feeder cell layer may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (US Pat. No. 5,690,926). checking).

  Stem cells themselves can be transfected with the polynucleotides of the invention to induce autocrine expression of the polypeptides of the invention. This allows the generation of undifferentiated totipotent / pluripotent stem cell lines that are useful as they are and can then be differentiated into the desired mature cell type. These stable cell lines can also be used to generate cDNA libraries and templates for polymerase chain reaction experiments as a source of undifferentiated totipotent / pluripotent mRNAs. These studies allow the isolation and identification of genes that are differentially expressed in stem cell populations that regulate stem cell proliferation and / or maintenance.

  The growth and maintenance of totipotent stem cell populations is useful for the treatment of many pathological conditions. For example, the polypeptides of the present invention manipulate stem cells in culture to produce neuroepithelial cells that are supplemented with cells damaged by disease, autoimmune disease, accidental injury, or genetic disorder, or Can be used to replace. The polypeptides of the present invention may be useful for inducing proliferation of nerve cells and for regeneration of nerve and brain tissue, i.e. diseases and disorders of the central and peripheral nervous system, and nerve cells or It may be useful for the treatment of mechanical and traumatic disorders involving neural tissue degeneration, death or injury. In addition, the expanded stem cell population can be genetically altered for gene therapy and to reduce host rejection of the replacement tissue after grafting or transplantation.

  The expression of the polypeptide of the present invention and its effect on stem cells can also manipulate the controlled differentiation of stem cells into more differentiated cell types. A widely applicable method for obtaining a pure population of a particular differentiated cell type from an undifferentiated stem cell population involves the use of a cell type specific promoter that drives a selectable marker. The selectable marker can only survive the desired type of cell. For example, stem cells may be 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 LW, Principles of Tissue Engineering, edited by Lanza et al., Academic Press (1997)). Alternatively, the stem cells were oriented by culturing the stem cells in the presence of an antagonist of the polypeptide of the present invention that inhibits the effects of the activity of differentiation factors such as retinoic acid and endogenous stem cell factor to promote differentiation. Differentiation can be achieved.

  In vitro cultures of stem cells can be used to determine whether the polypeptides of the present invention exhibit stem cell growth factor activity. Stem cells are isolated from any one of a variety of cell sources (including hematopoietic stem cells and embryonic stem cells) and in the presence of the polypeptide alone or in combination with other growth factors or cytokines, Thompson et al., Proc. Natl. Acad. Sci, U.I. S. A. , 92: 7844-7848 (1995). The ability of the polypeptides of the invention to induce stem cell proliferation is determined by colony formation on a semi-solid support, as described, for example, in Bernstein et al., Blood, 77: 2316-2321 (1991). .

4.10.5 Hematopoietic modulating activity The polypeptides of the present invention may be involved in the regulation of hematopoiesis and thus in the treatment of bone marrow or lymphoid disorders. Any biological activity in supporting colony-forming cells or in supporting factor-dependent cell lines indicates involvement in the regulation of hematopoiesis, for example in supporting growth and proliferation by erythroid progenitor cells alone or with other cytokines. This shows utility in the following cases: for example in various anemia treatments or for use in combination with irradiation / chemotherapy to stimulate the production of erythrocyte precursors and / or erythrocytes; In support of the growth and proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity) useful in combination with chemotherapy for the prevention or treatment of subsequent myelosuppression; Megakaryocytes and subsequent platelet growth and in combination with chemotherapy to prevent or treat myelosuppression In support of growth and thereby enabling the prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use instead of or as an adjunct to platelet transfusions; and / or In support of hematopoietic stem cell growth and proliferation that allows maturation into any or all of the above-mentioned hematopoietic cells, and thus various stem cell disorders (usually but not limited to aplastic anemia and paroxysmal nocturnal hemoglobinuria) Post-radiation / chemotherapy, either in vivo or in vitro (ie, in conjunction with bone marrow transplantation or peripheral progenitor cell transplantation (homologous or xenogeneic)), etc. Repopulating the stem cell fraction as normal cells or as genetically engineered cells for gene therapy. Find therapeutic utility Te.

The therapeutic composition of the present invention can be used in the following cases:
Suitable assays for the growth and differentiation of various hematopoietic cell lines are cited above.

  As an assay for embryonic stem cell differentiation (identifying proteins that affect embryonic differentiation hematopoiesis elsewhere), see Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al., Molecular and Cellular Biology 13: 473-486, 1993; McClanahan et al.

  Assays for stem cell survival and differentiation (specifically identifying proteins that regulate lympho-hematopoiesis) include the methylcellulose colony forming assays, Freshney, M. et al. G. Culture of Hematopoietic Cells. R. I. Freshney, et al., Vol. 265-268, Wiley-Liss, Inc. , New York, N .; Y. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89: 5907-5911, 1992; Primitive herponicity colony forming cells with high proliferative potential, McNice, I. K. and Bridgedell, R.A. A. Culture of Hematopoietic Cells,. R. I. Freshney et al., Ed., Vol 23-39, Wiley-Liss, Inc New York, N .; Y. 1994; Neben et al., Experimental Hematology 22: 353-359, 1994; E. Culture of Hematopoietic Cells,. R. I. Freshney et al., Ed., Vol. 1-21, Wiley-Liss, Inc. , New York, N .; Y. 1994; Long term bone marlow cultures in the presence of stromal cells, Sponcer, E .; Dexter, M .; And Allen, T .; Culture of Hematotopic Cells. . R. I. Freshney et al., Vol 163-179, Wiley-Liss, Inc. , New York, N .; Y. 1994; Long term culture initiating cell assay, Sutherland, H .; J. et al. In Culture of Hematopoietic Cells. . R. I. Freshney, et al., Vol 139-162, Wiley-Liss, Inc. , New York, N .; Y. Examples include, but are not limited to, the assays described in 1994.

4.10.6 Tissue proliferative activity Polypeptides of the present invention are also used in bone or cartilage, tendon, ligament and / or nerve tissue growth or regeneration, as well as in wound healing and repair and replacement, and in burns, incisions and ulcers. Can also be involved in healing.

  The polypeptides of the present invention that induce cartilage and / or bone growth when bone is not normally formed are applied in healing fractures and ligament injuries or defects in humans and other animals. The polypeptides, antibodies, binding partners or other modulator compositions of the invention can be used prophylactically in the reduction of subcutaneous and open fractures, and can also be used in improving prosthetic fixation. Novel bone formation induced by osteogenic substances contributes to the repair of craniofacial defects by congenital, trauma-induced or tumor resection, and is also useful in cosmetic surgery.

  The polypeptides of the present invention may also be involved in attracting osteogenic cells, stimulating osteogenic cell growth, or inducing differentiation of osteogenic cell precursors. Osteoporosis, deformity, such as methods by blocking bone and / or cartilage repair or by blocking inflammation or tissue destruction processes mediated by inflammatory processes (collagenase activation, osteoclast activation, etc.) Treatment of osteoarthritis, bone degenerative disorders, or periodontal disease may also be possible using the compositions of the present invention.

  Another category of tissue regeneration activity that may involve the polypeptides of the invention is tendon / ligament formation. Induction of tendon / ligament-like tissue or other tissue formation is the result of tendon or ligament damage, malformations and other tendons or ligaments in humans and other animals if such tissue is not properly formed It is applied in the healing of deficiencies. Such a preparation using a tendon / ligament-like tissue-inducing protein is a prophylactic application in preventing damage to tendon or ligament tissue, as well as improved fixation of tendon or ligament to bone or other tissue And in repairing tendon or ligament tissue defects. Novel tendon / ligament-like tissue formation induced by the compositions of the present invention contributes to the repair of other tendons or ligament defects that are congenital, traumatic, or other causes, and also tendons or ligaments It is also useful for cosmetic surgery for bonding or repair. The composition of the present invention attracts tendon or ligament-forming cells, stimulates the growth of tendon or ligament-forming cells, induces differentiation of tendon or ligament-forming cell precursors, or acts on tissue repair in vivo. An environment may be provided that induces ex vivo tendon / ligament cell or progenitor growth to revert. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The composition may also include suitable substrates and / or sequestering substances such as carriers that are well known in the art.

  The compositions of the present invention may also be useful for nerve cell growth and nerve and brain tissue regeneration, ie, for the treatment of diseases and neurological disorders of the central and peripheral nervous system, as well as nerve cells or nerve tissue. It may also be useful for mechanical and traumatic disorders, including degeneration, death or trauma. More particularly, diseases of the peripheral nervous system, such as peripheral nerve injury, peripheral neuropathy and localized neuropathy, and diseases of the central nervous system, such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis The composition can also be used for the treatment of Sjogren's disease and Shy Dräger syndrome. In addition, conditions that can be treated by the present invention include mechanical and traumatic disorders such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathy resulting from chemotherapy or other medical treatment may also be treatable using the compositions of the present invention.

  The compositions of the present invention may also be used to promote better or faster closure of non-healing wounds, including but not limited to pressure ulcers, ulcers associated with vascular dysfunction, surgical wounds, and traumatic wounds. May also be useful.

  The compositions of the present invention can also be used for tissues of organs (including pancreas, liver, intestine, kidney, skin, endothelium), muscles (smooth muscle, skeletal muscle or myocardium) and blood vessels (including vascular endothelium). It can also be involved in the generation or regeneration of other tissues or in promoting the growth of cells containing such tissues. Some of the desired effects may be due to inhibition or modulation of fibrous scars that may allow normal tissue regeneration. The polypeptides of the present invention may also exhibit angiogenic effects.

  The compositions of the present invention may also be useful for conditions resulting from intestinal protection or regeneration and treatment of lung or liver fibrosis, various tissue reperfusion injury, and systemic cytokine damage.

  The compositions of the invention may also be useful for promoting or inhibiting the differentiation of the tissue from precursor tissues or cells; or for inhibiting the growth of the tissue.

The therapeutic composition of the present invention can be used in the following cases:
Assays for tissue production activity include International Patent Publication No. WO95 / 16035 (bone, cartilage, tendon); International Patent Publication No. WO95 / 058446 (nerve, nerve cell); International Patent Publication No. WO91107491 (skin, endothelium). Examples include, but are not limited to, the described assays.

  As an assay for wound healing activity, Winter, Epidemial Wound Healing, pages 71-112 (Maibach, HI and Loveee, DT, ed.), Year Book Medical Publishers, Inc. Improved by Eaglstein, Chicago, and Mertz, J .; Invest. Dermatol 71: 382-84 (1978) includes, but is not limited to, the assays described in Dermatol 71: 382-84 (1978).

4.10.7 Immunostimulatory or Suppressive Activity Polypeptides of the invention may also exhibit immunostimulatory or immunosuppressive activity, including but not limited to activity according to the assays described herein. The polynucleotide of the present invention can encode a polypeptide exhibiting such activity. Proteins are used in the treatment of various immunodeficiencies and disorders (such as severe combined immunodeficiency (SCID)), for example in the regulation (upper or lower) of T and / or B lymphocyte growth and proliferation. As well as in the effect on the cytolytic activity of NK cells and other cell populations. These immunodeficiencies may be genetic or may be caused by viral (eg, HIV) and bacterial or fungal infections, or may result from autoimmune disorders. More particularly, some infectious diseases caused by viruses, bacteria, fungi or other infectious diseases can be treated using the proteins of the present invention, including HIV, hepatitis virus, herpes virus. , Infections due to various fungal infections such as actinomycetes, Leishmania spp (genus Leishmania), malaria species, and candita. Of course, in this regard, the proteins of the invention may also be useful where boosting to the immune system may generally be desired, ie in the treatment of cancer.

  Examples of autoimmune diseases that can be treated using the protein of the present invention include connective tissue diseases (collagen disease), multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pneumonia, Guillain-Barre syndrome, autoimmunity Thyroiditis, insulin-dependent diabetes, myasthenia gravis, graft-versus-host disease, and autoimmune inflammatory eye disease. Such proteins of the invention (or their antagonists including antibodies) also have allergic reactions and conditions (eg anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergy, mastocytosis, allergic rhinitis, hypersensitivity) Pneumonia, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, polymorphic erythema, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, vaginal keratoconjunctivitis, giant papillae Conjunctivitis and contact allergy), for example asthma (especially allergic asthma) or other respiratory disorders may also be useful. Other conditions where immunosuppression is desired (eg, including organ transplantation) may also be treated using the protein (or antagonist thereof) of the present invention. The therapeutic effect of the polypeptide or its antagonist on the allergic reaction is described by the cumulative contact sensitization test (Lastbom et al., Toxicology 125: 59-66, 1998), the 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 mouse local lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53: 563). ~ 79) can be evaluated by in vivo animal models.

  When using the protein of the present invention, it is also possible to modulate the immune response in a number of ways. Down-regulation may be performed in a manner that inhibits or blocks an immune response that is already in progress, or may be involved in preventing induction of an immune response. The function of activated T cells can be inhibited by suppression of T cell responses, by induction of specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active non-antigen specific process that requires sustained exposure of T cells to suppressors. Tolerance associated with induction of non-responsiveness or anergy (no response) in T cells is immunosuppressive in that it is generally antigen-specific and persists after exposure to the tolerizing agent is discontinued. Be identifiable. Operationally, tolerance can be demonstrated by the absence of a T cell response upon re-exposure to a specific antigen in the absence of a tolerizing agent.

  Down-regulation or prevention of one or more antigen functions, including but not limited to B lymphocyte antigen functions (eg, B7 etc.), eg prevention of high levels of lymphokine synthesis by activated T cells is Useful in the state of organ transplantation, as well as in graft-versus-host disease (GVHD). For example, blocking T cell function should reduce tissue destruction in tissue transplantation. Typically, in tissue transplantation, transplant rejection is initiated by T cells recognizing it as foreign, followed by an immune response that destroys the graft. Administration of the therapeutic composition of the present invention prevents cytokine synthesis by immune cells such as T cells, thereby acting as an immunosuppressant. Furthermore, the absence of co-stimulation is sufficient for the T cells to be anergized (deresponsive), which may induce tolerance in the subject. Induction of long-term tolerance by the B lymphocyte antigen blocking reagent can avoid the need for repeated administration of these blocking agents. To obtain sufficient immunosuppression or tolerance in a subject, it may be necessary to block the function of the B lymphocyte antigen combination.

  The efficacy of a particular therapeutic composition in preventing organ transplant rejection or GVHD can be assessed using animal models that predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart grafts in rats and cross-species islet cell grafts in mice, all of which are described in Lenschow et al., Science 257: 789-792 (1992). ) And Turka et al., Proc. Natl. Acad. Sci USA, 89: 11102-11105 (1992) and has been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo. In addition, the mouse model of GVHD (see Paul, Fundamental Immunology, Raven Press, New York, 1989, pages 846-847) measures the effect of the therapeutic composition of the present invention during the progression of this disease. Can be used for

  Blocking antigen function may also be useful therapeutically for the treatment of autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive to self tissue and promote the production of cytokines and autoantibodies involved in the pathology of the disease. Prevention of activation of autoreactive T cells can reduce or eliminate the pathology. Administration of reagents that block T cell stimulation can inhibit T cell activation and prevent the production of autoantibodies or T cell-derived cytokines that may be involved in the course of the disease. In addition, blocking reagents may induce antigen-specific tolerance of autoreactive T cells, which can lead to long-term remission from the disease. The efficacy of blocking reagents in preventing or ameliorating autoimmune disorders can be measured using a number of well-characterized animal models of human autoimmune diseases. Examples include mouse experimental autoimmune encephalitis, systemic lupus erythematosus in MEL / lpr / lpr mice or NZB hybrid mice, mouse autoimmune collagen arthritis, diabetes in NOD mice and BB rats, and mouse experimental myasthenia gravis (Ref. Paul, Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

  Upregulation of antigen function (eg, B lymphocyte antigen function) as a means of upregulating the immune response may also be useful in therapy. Up-regulation of the immune response can be done in a manner that enhances an existing immune response or elicits an initial immune response. For example, an enhanced immune response may be useful in the case of viral infections including systemic viral diseases such as influenza, colds, and encephalitis.

  Alternatively, the antiviral immune response may involve removing T cells from a patient in an infected patient, expressing a peptide of the invention, or a viral antigen together with a stimulating form of the soluble peptide of the invention. It can be enhanced by co-stimulating T cells in vitro with a purified APC and reintroducing the in vitro activated T cells into the patient. Another method of enhancing the antiviral immune response is to isolate an infected cell from a patient and use the protein of the invention described herein such that the cell expresses all or part of the protein on its surface. Transfecting the cells with the encoding nucleic acid and reintroducing the transfected cells into the patient. Here, the infected cells can transmit a costimulatory signal, thereby activating T cells in vivo.

The polypeptides of the present invention can provide the T cells with the necessary stimulatory signals to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells lacking MHC class I or MHC class II molecules or unable to reexpress sufficient amounts of MHC class I or MHC class II molecules are treated with MHC class I α chain protein and β ₂ microglobulin protein or MHC class II α. Transfects with nucleic acids encoding all or part of the chain protein and MHC class II β chain protein (eg, cytoplasmic domain cleavage portion), thereby expressing MHC class I or MHC class II proteins on the cell surface be able to. T cell mediated against transfected tumor cells by expressing appropriate class I or class II MHC with peptides having the activity of B lymphocyte antigens (eg B7-1, B7-2, B7-3) An immune response is induced. Optionally, a gene encoding an antisense construct that blocks expression of an MHC class II binding protein such as an invariant chain can also be co-transfected with DNA encoding a peptide having B lymphocyte antigen activity, Can promote the presentation of tumor-associated antigens and induce tumor-specific immunity. Thus, induction of a T cell mediated immune response in a human subject can sufficiently overcome tumor specific tolerance in the subject.

  The activity of the protein of the present invention can be measured by the following method, although there are other methods.

  Suitable assays for thymic or splenocyte cytotoxicity include, but are not limited to, Current Protocols in Immunology, J. MoI. E. Coligan, A.M. M.M. Kruisbeek, D.M. H. Margulies, E .; M.M. Shevach, W.M. Published by Strober, Green Publishing Associates and Wiley-Interscience (Chapter 3, In vitro assignments for Mouse Lymphocyte Function 3.1. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Biol. Immunol. 128: 1968-1974, 1982; Immunol. 135: 1564-1572, 1985; Takai et al. Immunol. 137: 3494-3500, 1986; Takai et al., J. Biol. Immunol. 140: 508-512, 1988; Bowman et al., J. MoI. Virology 61: 1992-1998; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Brown et al., J. Biol. Immunol. 153: 3079-3092, 1994.

  Assays for T cell-dependent immunoglobulin responses and isotype switches (modulating T cell-dependent antibody responses and distinguishing proteins that affect the Thl / Th2 profile from others) include, but are not limited to, Mariszewski, J. et al. Immunol. 144: 3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond. J. et al. And Brunswick, M .; In Current Protocols in Immunology. J. et al. E. e. a. Edited by Coligan, Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. The assay described in 1994 is mentioned.

  Mixed lymphocyte reaction (MLR) assays (which primarily distinguish proteins that produce Th1 and CTL responses from others) include, but are not limited to, Current Protocols in Immunology, J. MoI. E. Coligan, A.M. M.M. Kruisbeek, D.M. H. Margulies, E .; M.M. Shevach, W.M. Edited by Strober, published by Green Publishing Associates and Wiley-Interscience (Chapter 3, In vitro assignments for Mouse Lymphocyte Function, 3.1-3.19; Chapter 7, Immunologics. Immunol. 137: 3494-3500, 1986; Takai et al., J. Biol. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. MoI. Immunol. 149: 3778-3783, 1992.

  Dendritic cell-dependent assays (which distinguishes proteins expressed by dendritic cells that activate naive T cells from others) include, but are not limited to, Guery et al. Imimmol. 134: 536-544, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 1991; Mactonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgado et al. 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; l of Clinical Investigation 94: 797~807,1994; and Inaba et al., Journal of Experimental Medicine 172: 631~640,1990 include assays described.

  Assays for lymphocyte survival / apoptosis (which distinguishes proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, but are not limited to, Darzynewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al., Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1955-1951, 1993; Itoh et al., Cell 66: 233-243, 1991; 1990; Zamai et al., Cytometry 14: 891-897, 1993; Gorczyca et al., In ernational Journal of Oncology 1: 639~648,1992 include assays described.

  Protein assays that affect the early stages of T cell orientation and development include, but are not limited to, Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 155: 111-122, 1994; Blood 85: 2770-2778, 1995; Toki, et al., Proc. Nat. Acad Sci. USA 88: 7548-7551,1991.

4.10.8 Activin / Inhibin Activity Polypeptides of the invention may also exhibit activin or inhibin-related activity. The polynucleotide of the present invention can encode a polypeptide exhibiting such properties. Inhibin is characterized by its ability to inhibit the release of follicle stimulating hormone (FSH), while activin is characterized by its ability to stimulate the release of follicle stimulating hormone (FSH). Thus, the polypeptide of the present invention alone or as a heterodimer (heterodimer) with a member of the inhibin family reduces fertility of female mammals with inhibin, and spermatogenesis in male mammals. It may be useful as a contraceptive based on the ability to reduce Infertility can be induced in these mammals by administering sufficient amounts of other inhibins. Alternatively, the polypeptides of the invention are based on the ability of activin molecules in stimulating FSH (follicle stimulating hormone) release from anterior pituitary cells as homodimers or as heterodimers with other protein subunits of the inhibin group. Thus, it may be useful as a fertility induction therapeutic agent. See, for example, U.S. Pat. No. 4,798,885. The polypeptides of the present invention are also useful for accelerating the onset of fertilization in sexually immature mammals so as to improve the lifetime fertility of such livestock, not limited to cattle, sheep and pigs. possible.

  The activity of the polypeptide of the present invention can be measured by the following method, although there are other methods.

  Assays for activin / inhibin activity include, but are not limited to, Vale et al., Endocrinology 91: 562-572, 1972; Ling et al., Nature 321: 779-782, 1986; Vale et al., Nature 321: 776-779, 1986; Mason et al., Nature 318: 659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83: 3091-3095, 1986.

4.10.9 Chemotaxis / chemomotor activity Polypeptides of the present invention include, for example, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells. It may be involved in the chemotactic or chemokinetic activity of the mammalian cells it contains. The polynucleotide of the present invention can encode a polypeptide exhibiting such attributes. The activity of chemotactic and chemokinetic receptors can be used to mobilize or attract a desired population of cells to the desired site of action. Chemotaxis or chemokinetic compositions (eg, proteins, antibodies, binding partners, or modulators of the invention) are of particular benefit in the treatment of wounds and other trauma to tissues and in the treatment of local infections. give. For example, attraction of lymphocytes, monocytes or neutrophils to a tumor or site of infection can improve the immune response to the tumor or infectious agent.

  A protein or peptide has chemotactic activity against such a cell population if it can directly or indirectly stimulate the direction or movement of a particular cell population. Preferably, the protein or peptide has a function of directly stimulating directed movement of cells. Whether a particular protein has chemotactic activity against a cell population can be readily determined by using such proteins or peptides in any known cell chemotaxis assay.

The therapeutic composition of the present invention can be used in the following cases:
Assays for chemotactic activity (identifying proteins that induce or prevent chemotaxis) are the ability of proteins to induce cell migration within the membrane, as well as the ability of proteins to migrate from one cell population to another. Consists of an assay that also measures the ability to induce adhesion. Suitable assays for migration and attachment include, but are not limited to, Current Protocols in Immunology, J. MoI. E. Coligan, A.M. M.M. Kruisbeek, D.M. H. Marguiles, E .; M.M. Shevach, W.M. Edited by Strober, published by Green Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al., J. 76. Int. Lind et al., APMIS 103: 140-146, 1995; Muller et al. Eur. J. Immunol.25: 1744-1748; Gruber et al., J. of Immunol.152: 5860-5867, 1994; of Immunol.153: 1762-1768,1994.

4.10.10 Hemostasis and Thrombolytic Activity The polypeptides of the present invention may also be involved in hemostasis or thrombolysis or thrombosis. The polynucleotide of the present invention may encode a polypeptide exhibiting such attributes. The composition is useful for enhancing coagulation and other hemostasis in the treatment of various coagulation disorders (including genetic disorders such as hemophilia) or in the treatment of wounds resulting from trauma, surgery or other causes. possible. The compositions of the present invention are also for the treatment and prevention of thrombus dissolution or formation and conditions resulting therefrom (eg, heart and central nervous system vascular infarctions (eg, stroke)). Can be useful.

The therapeutic composition of the present invention can be used in the following cases:
Hemostatic and thrombolytic activity assays include, but are not limited to, Linet et al. Clin. Pharmacol. 26: 131-140, 1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostaglandins 35: 467-474, 1988.

4.1.11 Cancer Diagnosis and Treatment The polypeptides of the present invention may also be involved in the production, growth or metastasis of cancer cells. Detection of the presence or amount of a polynucleotide or polypeptide of the invention can be useful for the diagnosis and / or prediction of one or more cancers. For example, increased presence or expression of a polynucleotide / polypeptide of the invention may indicate a genetic risk of cancer, a precancerous condition, or an advanced malignancy. Conversely, a genetic defect, or absence of this polypeptide, can be associated with a cancerous condition. Identification of single nucleotide gene polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prediction.

  The treatment of cancer involves the inhibition of tumor cell proliferation, inhibition of angiogenesis (new blood vessel growth required to support tumor growth) and / or reducing tumor cell motility or invasiveness to prevent metastasis. Promote regression. The therapeutic composition of the present invention may be effective in adult and pediatric tumors, including solid tumors / malignant tumors, locally advanced tumors, human soft tissue sarcomas, metastatic cancers such as lymphatic metastases Blood cell malignancies such as multiple myeloma, acute and chronic leukemia, and lymphoma, head and neck cancer including mouth cancer, laryngeal cancer and thyroid cancer, lung cancer including small cell cancer and non-small cell cancer, Urinary organs including breast cancer, including small cell and ductal cancer, esophageal cancer, gastric cancer, colorectal cancer, gastrointestinal cancer, including polyps associated with colorectal and colorectal neoplasms, pancreatic cancer, liver cancer, bladder cancer and prostate cancer Cancer, ovarian cancer, uterine (including endometrial) cancer and female reproductive organ malignancies including solid follicular tumor, renal cancer including renal cell carcinoma, endogenous brain tumor, neuroblastoma, astrocytes (astrocytic) Cell) brain tumor, nerve , Brain tumor including metastatic tumor cell invasion in the central nervous system, bone cancer including osteoma, malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangioblastoma and Skin cancer including Kaposi's sarcoma.

  The polypeptides, polynucleotides or polypeptide modulators of the present invention (including inhibitors and stimulators of the biological activity of the polypeptides of the present invention) may be administered for the treatment of cancer. The therapeutic composition can be administered alone or in a therapeutically effective dose in conjunction with cancer adjunct therapy such as surgery, chemotherapy, radiation therapy, hyperthermia and laser therapy, and Although it is not necessary to eradicate cancer, it may provide beneficial effects such as reducing tumor size, reducing tumor growth rate, inhibiting metastasis, or improving overall clinical status .

  The composition may be administered in a therapeutically effective amount as part of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of a polypeptide or modulator of the present invention and one or more anti-cancer agents in addition to a pharmaceutically acceptable carrier for delivery. It is conventional to use anti-cancer cocktails as a treatment for cancer. Well known anticancer agents in the art that can be used therapeutically in combination with the polypeptides or modulators of the present invention include actinomycin D, aminoglutethimide, asparaginase, bleomycin, busulfan, carboplatin, carmustine, chlorambutyl, cisplatin (cis-DDP). , Cyclophosphamide, cytarabine hydrochloride (cytosine / arabinoside), dacarbazine, dactinomycin, daunorubicin hydrochloride, doxorubicin hydrochloride, estramustine phosphate sodium, etoposide (V16-213), floxuridine, 5-fluorouracil (5 -Fu), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, interferon α-2a, interferon α-2b, leuprolide acetate (L HRH-free factor analog), lomustine, mechlorethamine hydrochloride (nitrogen mustard), melphalan, mercaptopurine, mesna, methotrexate (MTX), mitomycin, mitoxantrone hydrochloride, octreotide, pricamycin, procarbazine hydrochloride, streptozocin, quencher Examples include acid tamoxifen, thioguanine, thiotepa, vinblastine sulfate, vincristine sulfate, amsacrine, azacitidine, hexamethylmelamine, interleukin-2, mitoguazone, pentostatin, semustine, teniposide, and vindesine sulfate.

  Furthermore, the therapeutic composition of the present invention can be used for preventive treatment of cancer. There are genetic and / or environmental conditions (eg, exposure to carcinogens) known in the art that the individual is predisposed to developing cancer. Under these circumstances, it may be advantageous to treat these individuals with a therapeutically effective amount of a polypeptide of the present invention to reduce the risk of developing cancer.

  In vitro models can be used to determine an effective amount of a polypeptide of the invention as a potential cancer treatment. These in vitro models include cultured tumor cell growth assays, cultured tumor cell growth in soft agar (Freshney, (1987) Culture of Animal Cells: A manual of Basic Technique, Wily-Liss, New York, NY Chapter 18 And Chapter 21), Giovanella et al., J. MoI. Natl. Can. Inst, 52: 921-30 (1974), a tumor system in nude mice, Pilkington et al., Anticancer Res. , 17: 4107-9 (1997), and the potential for tumor cell mobility and invasiveness in the Boyden Chamber assay, and Ribatta et al., Intl. Dev. Biol. , 40: 1189-97 (1999) and Li et al., Clin. Exp. Examples include angiogenesis assays such as induction of angiogenesis of chicken chorioallantoic membranes or induction of vascular endothelial cell migration, each described in Metastasis, 17: 423-9 (1999). Suitable tumor cell lines are available, for example, from the catalog of the American Type Tissue Culture Collection.

4.1.10. Receptor / Ligand Activity Polypeptides of the invention may also exhibit activity as receptors, receptor ligands, or inhibitors or agonists of receptor / ligand interactions. The polynucleotide of the present invention may encode a polypeptide exhibiting such properties. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (such as selectins, integrins and their ligands). Including, but not limited to, cell adhesion molecules), and receptor / ligand pairs involved in antigen presentation, antigen recognition and the development of cellular and humoral immune responses. Receptors and ligands are also useful for screening for peptides or small molecule inhibitors with potential related receptor / ligand interactions. The proteins of the present invention (including but not limited to receptor fragments and ligands) may themselves be useful as inhibitors of receptor / ligand interactions.

  The activity of the polypeptide of the present invention can be measured by the following method, although there are other methods.

  Suitable assays for receptor ligand activity include, but are not limited to, Current Protocols in Immunology, J. MoI. E. Coligan, A.M. M.M. Kruisbeek, D.M. H. Margulies, E .; M.M. Shevach, W.M. Edited by Strober, published by Green Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.2.1-2.c. Natl. Acad. Sci. USA 84: 6864-6868, 1987: Bierer et al., J. Biol. Exp. Med. 168: 1145-1156, 1988; Rosenstein et al., J. MoI. Exp. Med. 169: 149-160 1989; Stoltenburg et al., J. MoI. Immunol. Methods 175: 59-68, 1994; Stitt et al., Cell 80: 661-670, 1995.

  For example, the polypeptides of the present invention may be used as receptors for ligand (s), thereby transmitting the biological activity of the ligand (s). Ligands can be identified by binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays or other methods known in the art.

  Studies characterizing whether a drug or protein is an agonist, antagonist, partial agonist, or partial antagonist requires the use of other proteins as competitive ligands. The polypeptide of the invention or its ligand (s) may be conjugated and labeled with radioisotopes, colorimetric molecules or toxin molecules by conventional methods ("Guide to Protein Purification" Murray P. Deutscher). (Eds.) Methods in Enzymology Vol. 182 (1990) Academic Press, Inc. San Diego). Examples of radioactive isotopes include, but are not limited to, tritium and carbon-14. Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules. Examples of toxins include, but are not limited to, ricin.

4.1.13 Drug Screening The present invention is particularly useful for screening compounds by using novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The polypeptide or fragment used in such a test may be released in solution, immobilized on a solid support, generated on the cell surface, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells that are stably transformed with a recombinant nucleic acid expressing the polypeptide or fragment thereof. Drugs are screened against such transformed cells in binding protein competition assays. Viable or fixed forms of such cells can be used in standard binding assays. For example, measuring the formation of a complex between the polypeptide or fragment of the invention and the agent being tested, or in forming a complex between the novel polypeptide and an appropriate cell line well known in the art. You can examine the decrease.

  Sources of test compounds that can be screened for their ability to bind to the polypeptides of the invention or to modulate (ie, increase or decrease) their activity include (1) libraries of inorganic and organic compounds, (2) natural products Libraries and (3) combinatorial libraries consisting of random or mimetic peptides, oligonucleotides, or organic molecules.

  Chemical libraries can be readily synthesized or purchased from a number of commercial sources, including “hit” or “leads” via structural analogs of known compounds, or natural product screening. ) "Can be mentioned.

  Sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation or marine organisms, and mixed libraries for screening are (1) from soil, plant or marine microorganisms Broth fermentation and extraction, or (2) extraction from the microorganism itself. Natural product libraries include polyketides, non-ribosomal peptides and their (non-naturally occurring) variants. For a review, see Science 282: 63-68 (1998).

  Combinatorial libraries consist of a large number of peptides, oligonucleotides or organic compounds and can be easily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular importance is a combinatorial library of peptides and oligonucleotides. Other interesting libraries include peptides, proteins, peptidomimetics, multi-parallel synthetic collections, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8: 701-707 (1997). For reviews and examples of libraries of peptidomimetics, see Al-Obeidi et al., Mol. Biotechnol, 9 (3): 205-23 (1998); Hruby et al., Curr Opin Chem Boy, 1 (1): 114-19 (1997): Dorner et al., Bioorg Med Chem, 4 (5): 709-15 ( 1996) (alkylated dipeptides).

  Candidate “hits” for optimizing the ability of “hits” to bind to the polypeptides of the invention by identifying modulators using the various libraries described herein. "(Or" lead ") can be modified. The molecules identified in the binding capacity assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models well known in the art. In short, the molecule is titrated into a number of cell cultures or animals and then tested for cell / animal death or animal / cell survival extension.

  The binding molecules identified in this way may be complexed with toxins, such as ricin or cholera, or complexed with other compounds that are toxic to the cell, such as radioisotopes. Also good. The toxin binding molecule complex then targets a tumor or other cell with the specificity of the binding molecule for the polypeptide of the invention. Alternatively, the binding molecule may be complexed with an imaging agent for targeting and imaging purposes.

4.10.14 Receptor Activity Assay The present invention also provides a method for detecting specific binding of a polypeptide, eg, a ligand or receptor. This technique provides a number of assays that are particularly useful for identifying previously unknown binding partners of receptor polypeptides of the invention. For example, expression cloning using mammalian or bacterial cells or a die hybrid screening assay can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography using a suitably immobilized polypeptide of the present invention can be used to isolate a polypeptide that recognizes and binds to the polypeptide of the present invention. There are many different libraries that are used to identify compounds, particularly small molecules, that modulate (ie, increase or decrease) the biological activity of the polypeptides of the invention. A ligand for a receptor polypeptide of the invention can also be identified by adding an exogenous ligand or a cocktail of ligands to two genetically identical cell populations except for expression of the receptor of the invention: one cell The population expresses the receptor of the present invention, but not other cell populations. The response of the two cell populations to the addition of ligand (s) is then compared. Alternatively, the expression library can be co-expressed with the polypeptides of the invention in a cell and assayed for an autocrine response to identify potential ligand (s). Furthermore, as another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including (1) organic and inorganic chemistry Libraries, (2) natural product libraries, and (3) combinatorial libraries consisting of random peptides, oligonucleotides or organic molecules.

  The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptides of the invention can be determined. For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of the protein for which the ligand has been identified is produced in the host cell. The cells are then incubated with a ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor. The known downstream proteins involved in intracellular signaling can then be assayed for the expected modification, ie phosphorylation. Other methods known in the art can also be used to identify signaling molecules involved in receptor activity.

4.1.15 Anti-inflammatory activity The compositions of the present invention also exhibit anti-inflammatory activity. Anti-inflammatory activity stimulates cells involved in the inflammatory response, inhibits or promotes cell-cell interactions (eg, cell adhesion), or inhibits or promotes the chemotaxis of cells involved in the inflammatory process Or by inhibiting or promoting the extravasation of cells or stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Compositions having such activity are inflammatory state infections including chronic or acute conditions, including but not limited to infections (eg septic shock, sepsis or systemic inflammatory response syndrome (SIRS)) , Ischemic reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, or cytokines such as TNF or IL-1 It can be used to treat those caused by overproduction. The compositions of the present invention may also be useful for the treatment of anaphylaxis or hypersensitivity to an antigenic substance or antigenic material. Compositions of the present invention include, but are not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine-induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage due to type 1 diabetes, graft-versus-host disease, inflammatory bowel disease To prevent or treat conditions such as inflammation associated with lung disease, other autoimmune diseases or inflammatory diseases, as an antiproliferative agent for acute, chronic myelogenous leukemia, etc. or in uterine infection Can be used in the prevention of secondary preterm birth.

4.10.16 Leukemia Leukemia and related disorders may be treated or prevented by administering a therapeutic agent that promotes or inhibits the function of the polynucleotides and / or polypeptides of the present invention. Such leukemias and related disorders include acute leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic Myelocyte (granulocytic) leukemia and chronic lymphocytic leukemia (for review of these diseases, see Fishman et al., 1985, Medicine, 2nd edition, JB Lippincott Co., Philadelphia). However, it is not limited to these.

4.1.17 Nervous System Disorders Involves cell types that can test the efficacy of interventions with compounds that modulate the activity of the polynucleotides and / or polypeptides of the present invention and can therefore be treated while observing indicators of therapeutic utility Nervous system disorders include, but are not limited to, nervous system disorders and diseases or disorders that result in either axonal disconnection, neuronal regression or degeneration, or demyelination. Nervous system lesions that can be treated in patients (including humans and non-human mammals) according to the present invention include any of the following central (including spinal cord, brain) or peripheral nervous system lesions: Not limited to:
(I) lesions caused by physical injury or associated with surgery, such as lesions that cut off part of the nervous system, or traumatic lesions including compression injury (ii) cerebral infarction or cerebral ischemia, or spinal cord infarction or Ischemic lesions resulting in neuronal damage or death resulting from oxygen deprivation in parts of the nervous system, including ischemia (iii) As a result of infection, eg no infection by human immunodeficiency virus, herpes zoster, herpes simplex virus Infectious lesions that are destroyed or damaged with or with them, or with Lyme disease, tuberculosis, syphilis;
(Iv) Degeneration in which part of the nervous system is destroyed or damaged as a result of a degenerative process including, but not limited to, Parkinson's disease, Alzheimer's disease, Huntington's disease or amyotrophic lateral sclerosis (V) a nutritional disorder, or a lesion associated with a nutritional disorder, in which part of the nervous system is destroyed or damaged by a nutritional or metabolic disorder, including vitamin B12 deficiency, folate deficiency, Wernicke Disease, tobacco-alcoholic amblyopia, Marcaphava Vinyami disease (primary degeneration of the corpus callosum) and alcoholic cerebellar degeneration.

  (Vi) Neurological lesions associated with systemic disease, including but not limited to diabetes (diabetic neuropathy, facial paralysis), systemic lupus erythematosus, cancer or sarcoma.

(Vii) lesions caused by toxic substances including alcohol, lead or certain neurotoxins; and (viii) demyelinating lesions in which part of the nervous system is destroyed or damaged by demyelinating disease, which is multiple sclerosis Demyelinating lesions including, but not limited to, neuropathy, human immunodeficiency virus-associated myelopathy, transverse myelopathy, or various etiology, progressive multifocal leukoencephalopathy, and central pontine myelin lysis.

  Useful therapeutic agents for the treatment of nervous system disorders according to the present invention can be selected by examining biological activity in promoting neuronal survival or differentiation. For example, without limitation, therapeutic agents that induce any of the following effects may be useful according to the present invention.

  (I) Extension of neuronal survival time in culture.

  (Ii) Increased neuronal sprouting in culture or in vivo.

(Iii) increased production of neuronal cell-related molecules, such as choline acetyltransferase or acetylcholinesterase associated with motor neurons, in culture or in vivo; or (iv) reduction of symptoms of neuronal dysfunction in vivo.

  Such an effect can be measured by any method known in the art. Preferably, not limited to embodiments, the increase in neuronal survival can be measured by the method shown in Arakawa et al. (1990, J. Neurosci. 10: 3507-3515); Detectable by the method shown in Pestron et al., (1980, Exp. Neurol. 70: 65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4: 17-42); Can be measured by bioassays, enzymatic assays, antibody binding, Northern blot assays, etc., depending on the molecule to be measured; and motor neuron dysfunction is a motor neuron disorder such as weakness Motor neuron transmission speed or functional disability It can be measured by the evaluation of physical symptoms.

  In certain embodiments, motor neuron disorders that can be treated according to the present invention can affect infarcts, infections, exposure to toxins, trauma, surgical damage, degenerative diseases or other components of the motor neuron and nervous system. Disorders such as malignant tumors and disorders that selectively affect neurons such as amyotrophic lateral sclerosis include, but are not limited to, progressive spinal muscular atrophy, progressive Ball paralysis, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis in early childhood (Fatio-Londe syndrome), gray leukitis and post-polio syndrome, and hereditary motor sensory neuropathy (Charcot-Marie -Tooth disease), but is not limited to these.

4.1.18 Other Activities The polypeptides of the present invention may also further exhibit one or more of the following additional activities or actions: bacteria, viruses, fungi and other parasites, including but not limited to Inhibiting or killing the growth, infection or function of infectious agents including, but not limited to, height, weight, hair color, eye color, skin, body fat ratio or other tissue pigmentation, or organ Or act on physical characteristics (such as increase or decrease in breast size, or changes in bone morphology and shape) such as body size or shape (suppression or enhancement); affect biorhythms or circadian rhythms or rhythms Affect fertility in male or female subjects; dietary fat, lipids, proteins, carbohydrates, vitamins, minerals, Affecting the metabolism, catabolism, assimilation, processing, utilization, storage or excretion of a factor or other nutritional factor or ingredient (s); including but not limited to appetite, libido, stress, cognition (including cognitive impairment) Act on behavioral characteristics including depression (including depressive disorder) and ferociousness; provide analgesic or other pain relief; promote differentiation and growth of embryonic stem cells in non-hematopoietic systems Hormonal or endocrine activity; in the case of enzymes, correcting enzyme deficiencies and treating deficiency-related diseases; treating hyperproliferative disorders (eg, psoriasis); immunoglobulin-like activities (eg, antigen or Such as the ability to bind complement); as well as acting as an antigen in the vaccine component to cross-react with such proteins or such proteins The ability to promote an immune response against another substance or entity.

4.1.19 Definition of polymorphism Proving polymorphism allows the identification of such polymorphisms in human subjects, and this pharmacogenetic information can be used for diagnosis and treatment . Such polymorphism can be associated with, for example, differential predisposition or susceptibility to various disease states (such as diseases involving inflammation or immune response), or differential response to drug administration, and this genetic information Can be used to tailor prophylaxis or treatment appropriately. For example, if there is a polymorphism associated with a predisposition to inflammation or autoimmune disease, this condition in humans can be diagnosed by identifying the presence of this polymorphism.

  Polymorphisms can be identified in a variety of ways known in the art, all of which generally involve obtaining a sample from a patient, analyzing the DNA from this sample, as needed. And including the step of isolating or amplifying the DNA and identifying the presence of polymorphisms in the DNA. For example, PCR can be used to amplify a suitable genomic DNA fragment, which can then be sequenced. Alternatively, the DNA is subjected to allele-specific oligonucleotide hybridization (where appropriate oligonucleotides are hybridized to DNA under conditions that allow detection of single base mismatches) or single nucleotide extension assays (polymorphisms) The oligonucleotide that hybridizes directly next to the position of 1 is extended with one or more labeled nucleotides). In addition, traditional restriction enzyme fragment length polymorphism analysis (using restriction enzymes that result in differential digestion of genomic DNA depending on the presence or absence of polymorphisms) may be performed. The array of nucleotide sequences of the present invention can be used for polymorphism detection. This array may contain the modified nucleotide sequences of the present invention to detect the nucleotide sequences of the present invention. Alternatively, any one of the nucleotide sequences of the present invention can be replaced on the array to detect changes in these sequences.

  Alternatively, polymorphisms that result in changes in the amino acid sequence can also be detected by detecting a corresponding change in the amino acid sequence of the protein, eg, by an antibody specific for the mutant sequence.

4.10.20 Arthritis and inflammation The immunosuppressive effect of the composition of the invention on rheumatoid arthritis is determined in an experimental animal model system. The experimental model system is adjuvant-induced arthritis in rats. The protocol is described by Horositz et al., 1983, Science, 219: 56 or Waksman et al., 1963, Int. Arch. Allergy Appl. Immunol. 23: 129. Induction of this disease can be accomplished by a single administration, typically by intradermal injection, of a killed tuberculosis suspension in complete Freund's adjuvant (CFA). The route of injection can vary, but in rats, a mixture of adjuvants may be injected into the base of the tail. The polypeptide is administered in phosphate buffer (PBS) at a dose of about 1-5 mg / kg. Control consists of administration of PBS only.

  The procedure for testing the effect of a test compound consists of an intradermal injection of killed M. tuberculosis contained in CFA, followed by administration of the test compound immediately followed by administration every other day up to 24 days. On days 14, 15, 18, 20, 22, and 24 after injection of mycobacterial CFA, the above-mentioned J. An overall score for arthritis is obtained as described by Holoshitz. The results of data analysis reveal that the test compound has a dramatic effect on joint swelling by measuring the reduction in arthritic score.

4.11 Therapeutic Methods Compositions of the present invention (including polypeptide fragments, analogs, variants and antibodies, or other binding partners or modulators such as antisense polynucleotides) have many applications in various therapeutic methods. . Examples of therapeutic applications include, but are not limited to, those exemplified herein.

4.11.1 Examples One example of the present invention is an effective amount of a polypeptide or other composition of the present invention for an individual suffering from a disease or disorder that can be modulated by modulating a peptide of the present invention. Administration. The mode of administration is not particularly important, but parenteral administration is preferred. An exemplary dosage form is to deliver an intravenous bolus. The dosage of the polypeptide or other composition of the invention is usually determined by the prescribing physician. Dosages are expected to vary with the patient's age, weight, condition and response. Typically, the amount of polypeptide administered per dose ranges from about 0.01 μg / kg to 100 mg / kg per kg patient body weight, with doses of about 0.1 μg / kg to 10 mg / kg. Preferably there is. For parenteral administration, the polypeptides of the invention are formulated in an injectable form in combination with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art, examples of which include water, saline, Ringer's solution, dextrose solution, and a solution consisting of a small amount of human serum albumin. The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such a solution belongs to the category of the field.

4.12 Pharmaceutical Formulations and Routes of Administration Proteins or other compositions of the invention, including but not limited to recombinant and non-recombinant sources, including but not limited to Pharmaceutical composition (including peptide antibodies and other binding partners) mixed with suitable carrier or excipient (s) on their own or in doses necessary to treat or ameliorate various disorders It may be administered to the patient in need thereof. Such compositions may be used as needed (in addition to proteins or other active ingredients and carriers), diluents, extenders, salts, buffers, stabilizers, solubilizers, and others well known in the art. May also be included. The term “pharmacologically acceptable” means a non-toxic substance that does not interfere with the effectiveness of the bioactivity of the active ingredient (s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the present invention also comprises 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, TNF-1, TNF- 2, G-CSF, Meg-CSF, etc., thrombopoietin, stem cell factor, and erythropoietin may be included. In further compositions, the proteins of the invention may be combined with other factors that are advantageous for the treatment of the disease or disorder in question. These factors include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factor (TGF-α and TGF-β), insulin-like growth factor (IGF). And the cytokines described herein.

  The pharmaceutical composition may further comprise other factors that enhance the activity of the protein or other active ingredient or that complement the activity or use in therapy. Such additional elements and / or factors may be included in the pharmaceutical composition to produce a synergistic effect with the protein or other active ingredient of the invention or to minimize side effects. Conversely, coagulation factors, cytokines, lymphokines, other hematopoietic factors, thrombolytic factors, or antithrombotic factors, or anti-inflammatory drugs (eg, IL-1Ra, IL-1Hy1, IL-1Hy2, anti-TNF, corticosteroids, immunity) In order to minimize the side effects of the inhibitor (suppressor), the protein of the present invention or other active ingredient is added to a specific coagulation factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic factor or antithrombotic factor, or anti-inflammatory agent. It may be included in the formulation. The proteins of the present invention may be active in multimers (eg, heterodimers or homodimers), or may be active on their own or in complex with other proteins. As a result, the pharmaceutical composition of the present invention may contain the protein of the present invention in the form of a multimer or a complex.

  Instead of being included in a pharmaceutical composition of the invention comprising a first protein, a second protein or therapeutic agent can also be administered with the first protein (eg, at the same time or the therapeutic concentration of the combined substances at the treatment site). At different times in the conditions realized). The compound formulation and administration techniques for immediate application are described in “Remington's Pharmaceutical Sciences” Mack Publishing Co. , Easton, PA, final version. Furthermore, a therapeutically effective dose is a recovery of symptoms, eg, treatment, cure, prevention or remission in a related medical situation, or an increase in the rate of treatment, cure, prevention or remission of such a condition. The amount of the compound sufficient to provide When an individual active ingredient is administered alone, a therapeutically effective dose refers to that active ingredient alone. When used in combination, a therapeutically effective dose means the total amount of active ingredient that provides a therapeutic effect, whether administration is in combination, in succession, or at the same time.

  In practicing the method of treatment or use of the present invention, a therapeutically effective amount of the protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated. The protein or other active ingredient of the present invention may be administered alone according to the method of the present invention or administered in combination with other therapeutic methods such as treatment with cytokines, lymphokines or other hematopoietic factors. Also good. When co-administered in combination with one or more of cytokines, lymphokines, other hematopoietic factors, the protein or other active ingredient of the present invention includes cytokine (s), lymphokine (s), other hematopoiesis The agent (s), thrombolytic factor, or antithrombotic factor may be administered simultaneously or sequentially. When administered continuously, the protein or other active ingredient of the present invention is cytokine (s), lymphokine (s), other hematopoietic factor (s), thrombolytic factor, or antithrombotic factor The appropriate order will be determined by the attending physician.

4.12.1 Routes of administration Suitable routes of administration include, for example, oral, rectal, transmucosal, or enteral administration; parenteral delivery including intramuscular, subcutaneous, intramedullary injection, and subarachnoid, direct heart There may be mentioned indoor injection, intravenous injection, intraperitoneal injection, intranasal injection or intraocular injection. Administration of the protein or other active ingredient of the invention used in a pharmaceutical composition, or practice of the method of the invention can be accomplished by a variety of conventional methods such as oral ingestion, inhalation, topical application or application of the skin, subcutaneous, intraperitoneal, It can be done by parenteral injection or intravenous injection. Intravenous administration to the patient is preferred.

  Alternatively, the compound can be administered locally rather than systemically. For example, via direct injection of the compound into joints or fibrous tissue suffering from arthritis, in which case many are depot or sustained release formulations. It is also possible to administer the compounds locally, for example as eye drops, in order to prevent scarring, which tends to occur as a complication of glaucoma surgery. In addition, the drug can be administered using a drug-targeted delivery system, eg, targeting arthritis or fibrous tissue, coated with a specific antibody, eg, a liposome. Liposomes target affected tissues and are selectively absorbed.

  The polypeptides of the present invention are administered by any route that delivers an effective amount to the desired site of action. Determination of the appropriate route of administration and effective dosage for a particular indication is within the level of the art. Preferably for wound treatment, the therapeutic compound is administered directly to the site. Suitable dosage ranges for the polypeptides of the present invention can be extrapolated from these dosages or from similar studies with appropriate animal models. Then, in order to obtain the maximum therapeutic effect, the dose may be adjusted by the clinician as necessary.

4.12.2 Composition / formulation (formulation)
Accordingly, a pharmaceutical composition for use according to the present invention comprises one or more physiologically acceptable excipients that contain excipients and adjuvants that help shape the active compound into a pharmaceutically available preparation. It can be formulated by conventional methods using carriers. These pharmaceutical compositions are known per se, such as conventional methods such as mixing, dissolving, granulating, dragee-making, micronizing, emulsifying, encapsulating, entrapping or lyophilizing processes. Can be manufactured. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of a protein or active ingredient of the present invention is administered orally, the protein or active ingredient of the present invention is in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the pharmaceutical composition of the invention may further comprise a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 5% to 95% of the protein or other active ingredient of the present invention, preferably about 25% to 95% of the protein or active ingredient of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, animal or vegetable oils such as peanut oil, mineral oil, soybean oil or sesame oil, or synthetic oil may be added. Liquid pharmaceutical compositions may further comprise physiological saline, glucose or other saccharide solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition comprises about 0.5 to 90% by weight of the protein or other active ingredient of the present invention, preferably about 1 to 50% by weight of the protein or other active ingredient of the present invention. Including.

  When a therapeutically effective amount of a protein or other active ingredient of the present invention is administered by intravenous, dermal, or subcutaneous injection, the protein or other active ingredient of the present invention does not contain a pyrogen and is administered parenterally. It is in the form of an acceptable aqueous solution. It is within the field of the art to prepare solutions of such parenterally acceptable proteins or other active ingredients in consideration of pH, isotonicity, stability and the like. Preferred pharmaceutical compositions for intravenous, dermal or subcutaneous injection include, in addition to the protein of the invention or other active ingredient, isotonic vehicles such as sodium chloride injection, Ringer's injection, glucose injection, Glucose and sodium chloride injections, lactated Ringer's injections, or other vehicles known in the art should also be included. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. For injection, the substances of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

  For oral administration, the compounds can be readily formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds of the present invention to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for ingestion by the patient to be treated. become. Pharmaceutical preparations for oral use, if necessary, to obtain tablets or dragee cores, after adding the appropriate adjuvants, if necessary, grind the resulting mixture, granulate mixture It can be processed and obtained from solid excipients. Suitable excipients are in particular sugars including bulking agents such as lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum , Methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If necessary, disintegrating agents such as bridged polyvinylpyrrolidone, agar or alginic acid, or a salt thereof such as sodium alginate may be added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, among which gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or A solvent mixture may be included as required. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

  Pharmaceutical preparations that 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 glycol or sorbitol. Push-fit capsules may contain the active ingredient in admixture with a bulking agent such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. . In the case of soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Further stabilizers may be added. All formulations for oral administration must be in dosages suitable for such administration. For buccal administration, the composition may take the form of tablets or lozenges formulated in conventional manner.

  For administration by inhalation, the compounds for use according to the invention are in the form of aerosol spray preparations from pressurized packs or nebulizers, suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, Conveniently delivered using carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a fixed amount. Capsules and cartridges such as gelatin for use in an inhaler or insufflator may be formulated to contain a powder mixture of the compound with a suitable powder base such as lactose or starch. The compound may be formulated for parenteral administration, eg, by bolus injection or injection by continuous infusion. The injectable formulation may be in unit dosage form, for example, in an ampoule or multi-dose container with the addition of a preservative. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulations such as suspending, stabilizing, and / or dispersing agents. Good.

  Pharmaceutical formulations for parenteral administration include aqueous solutions containing the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Liquid injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. If desired, the suspension may also contain suitable stabilizers or factors that increase the solubility of the compounds, allowing the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form and combined with a suitable vehicle, such as sterile pyrogen-free water, before use.

  The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulation methods described above, the compounds may be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, formulating the compound as a suitable polymeric or hydrophobic material (eg as an acceptable emulsion in oil), or as an ion exchange resin, or a slightly soluble derivative, eg as a slightly soluble salt it can.

  A pharmaceutical carrier for the hydrophobic compounds of the present invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The auxiliary solvent system may be a VDP auxiliary solvent system. VPD is a solution of 3% w / v benzyl alcohol, 8% w / v nonpolar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300, made in absolute ethanol. is there. The VPD co-solvent system (VPD: 5W) consists of VPD diluted 1: 1 with a 5% aqueous glucose solution. This co-solvent system dissolves hydrophobic compounds well and itself produces less toxicity when administered systemically. Of course, the proportion of the co-solvent system can vary considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may vary: for example, other low toxicity nonpolar surfactants may be used in place of polysorbate 80; the fraction size of polyethylene glycol may vary Other biocompatible polymers may be replaced with polyethylene glycols such as polyvinylpyrrolidone; and other sugars or polysaccharides may be substituted for glucose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be used. Well known examples of delivery vehicles or carriers for hydrophobic drugs are liposomes and emulsions. Certain organic solvents such as dimethyl sulfoxide can be used, but usually have a high cost of toxicity. In addition, sustained release systems such as solid hydrophobic polymer semipermeable substrates containing therapeutic agents may be used to deliver the compounds. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained release capsules release the compound over 2-3 weeks to 100 days, depending on its chemical nature. Depending on the chemical nature of the therapeutic reagent and the stability of the organism, additional strategies may be used for the stabilization of proteins or other active ingredients.

  The pharmaceutical composition may also comprise a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol. Many of the active ingredients of the present invention may be provided as salts with pharmaceutically compatible counterions. Such pharmaceutically acceptable base addition salts are those that retain biological effectiveness and free acid properties, such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamines, dialkylamines. It can be obtained by reaction with an inorganic or organic base such as monoalkylamine, dibasic amino acid, sodium acetate, potassium benzoate, triethanolamine and the like.

  The pharmaceutical composition of the present invention may be in the form of a complex of the protein (s) or other active ingredient (s) of the present invention and a protein or peptide antigen. Protein and / or peptide antigens carry a stimulatory signal to both B and T lymphocytes. B lymphocytes respond to antigens through immunoglobulin receptors on their surface. T lymphocytes respond to antigen through the T cell receptor (TCR) after presentation of the antigen by MHC proteins. MHC and structurally related proteins, including those encoded by class I and class II MHC genes on host cells, serve to present peptide antigen (s) to T lymphocytes. The antigen component can also be supplied with purified MHC peptide complex alone or with a costimulatory molecule capable of transmitting a signal directly to T cells. Alternatively, antibodies capable of binding to surface immunoglobulins and other molecules on B cells, and antibodies capable of binding to TCR and other molecules on T cells may be combined with the pharmaceutical compositions of the invention.

  The pharmaceutical composition of the present invention may be in the form of liposomes, wherein the protein of the present invention is combined with an amphipathic substance such as a lipid in addition to other pharmaceutically acceptable carriers. The lipid may be present in aggregated form as a layered phase in micelles, insoluble monolayers, liquid crystals or aqueous solutions. Suitable lipids for liposome formulations include but are not limited to monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids and the like. The preparation of such liposome formulations is described, for example, in US Pat. Nos. 4,235,871; 4,501,728; 4,837,028, all incorporated herein by reference. And within the state of the art as disclosed in US Pat. No. 4,737,323.

  The amount of the protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend on the nature of the condition being treated and its severity, and the nature of the prior treatment received by the patient. Ultimately, in treating each individual patient, the attending physician will decide the amount of protein or other active ingredient of the present invention. The attending physician will initially administer small amounts of the protein or other active ingredient of the present invention and observe the response in the patient. Until the highest therapeutic effect is obtained for the patient, the dose of the protein or other active ingredient of the present invention is increased and administered, at which point the dosage is no longer increased. Various pharmaceutical compositions used to practice the methods of the present invention comprise from about 0.01 μg to about 100 mg (preferably from about 0.1 μg to about 10 mg, preferably from about 0.1 μg to about 10 mg of protein or other active ingredient of the present invention per kg of body weight. Preferably, it should contain from 0.1 μg to about 1 mg). For compositions of the invention that are useful for bone, cartilage, tendon, or ligament regeneration, the method of treatment includes administering the composition locally, systemically, or locally as an implant or device. . Upon administration, the therapeutic composition for use in the present invention will, of course, be in a physiologically acceptable form that does not contain pyrogens. Further, the composition may desirably be encapsulated in a viscous form or injected for delivery to a site of bone, cartilage, or tissue injury. Local administration may be appropriate for wound healing and tissue repair. Substances useful for treatment other than the protein or other active ingredient of the present invention (which may optionally be included in the above composition) instead of or in addition to the composition in the method of the present invention, Administration may be simultaneous or sequential. Preferably, for bone and / or cartilage formation, it is possible to send a composition containing proteins or other active ingredients to the site of bone and / or cartilage injury, for the development of bone and cartilage The composition comprises a substrate that can comprise the following structure, and preferably can be reabsorbed by the body. Such a substrate may be formed of materials currently used in medical applications for other implants.

  The choice of substrate material is based on biocompatibility, biodegradability, mechanical properties, appearance and interactive properties. The specific application of this composition will determine the appropriate formulation. Possible substrates for this composition may be biodegradable and chemically defined synthetic calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. . Other possible substances are biodegradable and biologically distinct such as bone or skin collagen. Other substrates are composed of pure protein or extracellular matrix components. Other possible substrates are non-biodegradable and chemically distinct, for example sintered hydroxyapatite, bioglass, aluminate, or other ceramics. The substrate may be composed of any combination of the above types of materials, for example, polylactic acid and hydroxyapatite, or collagen and tricalcium phosphate. Bioceramics can also be treated with varying compositions such as calcium-aluminate-phosphate to change pore size, particle size, particle shape and biodegradability. Presently preferred is a 50:50 (molecular weight) copolymer of lactic acid and glycolic acid in the form of porous particles having a diameter of 150-800 microns. For some applications, it is useful to utilize a sequestering agent such as carboxymethylcellulose or autoclots to prevent the protein composition from dissociating from the substrate.

  A preferred family of sequestering agents are cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose. Most preferred are the cation salts of carboxymethylcellulose (CMC). 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% by weight of the total formulation, preferably 1-10% by weight, which prevents the detachment of the protein from the polymer substrate. And the amount necessary for proper handling of the composition, but not so much as to prevent the progenitor cells from entering the matrix, which is the amount that the protein helps the osteogenic activity of the progenitor cells . In further compositions, the proteins or other active ingredients of the invention may be combined with other substances useful for the treatment of bone and / or cartilage defects, wounds, or problematic tissues. Such substances include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factor (TGF-α and TGF-β), and insulin-like growth factor (IGF). ).

  Therapeutic compositions are also currently beneficial for veterinary applications. Such treatment with the protein or other active ingredient of the present invention is in addition to humans, especially patients where livestock and thoroughbred horses are desired. The dosage regimen of the pharmaceutical composition containing the protein used for tissue regeneration may vary depending on various factors that alter the action of the protein, such as the desired tissue weight to be formed, the damaged portion, the condition of the damaged tissue, the wound Determined by the attending physician in view of size, type of tissue damaged (eg, bone), patient age, gender and diet, severity of infection, administration time, and other clinical factors. The dosage may vary depending on the type of substrate used for the regeneration and other proteins contained 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 affect the dosage. Tissue / bone growth and / or repair can be monitored for progress by periodic assessment, eg, with X-rays, histomorphometry determination methods, and tetracycline labeling methods.

  The polynucleotides of the present invention can also be used in gene therapy. Such polynucleotides can be introduced into cells either in vivo or ex vivo for expression in a mammalian subject. The polynucleotides of the invention may also be administered by other known methods for introducing nucleic acids into cells or organisms, including but not limited to viral vectors or naked DNA forms. Cells can also be cultured ex vivo in the presence of the protein of the present invention to grow or to obtain the desired effect or activity on such cells. The treated cells can then be introduced in vivo for therapeutic purposes.

4.12.3 Effective Dosage A pharmaceutical composition suitable for use in the present invention includes a composition comprising an active ingredient in an amount effective to achieve its purpose. More particularly, a therapeutically effective amount means an amount effective to prevent or alleviate the progression of current symptoms in the subject being treated. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from suitable in vitro assays. For example, using an animal model, a dose is formulated to determine a range of circulating concentrations that can be used to more accurately determine useful doses in humans. For example, an animal model may be used to formulate a dose to determine the range of circulating concentrations in which the IC ₅₀ (ie, the concentration of the test compound that obtains half-maximal inhibition of protein bioactivity) determined in cell culture is determined. included. Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compound that achieves recovery of symptoms or prolongation of patient survival. The toxicity and therapeutic efficacy of such compounds can be determined, for example, by cell cultures to determine the LD ₅₀ (dose that kills 50% of the population) and ED ₅₀ (the therapeutically useful dose in 50% of the population) or It can be determined by standard pharmaceutical procedures in laboratory animals. 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 ₅₀ . Compounds that exhibit high therapeutic indices are preferred. Data obtained from this cell culture assay and animal studies can be used to formulate a range of dosage for use in humans. The dosage of such compounds lies preferably within the range of circulating concentrations that include the ED ₅₀ and has no or little toxicity. The dosage may vary within this range depending upon the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, for example, Chapter 1, page 1 of Fingl et al., 1975, “The Pharmacological Basis of Therapeutics”. Dosage amount and interval can be adjusted individually so that plasma levels of the active moiety are sufficient to maintain the desired effects or to maintain a minimum effective concentration (MEC). The MEC will vary for each compound but can be estimated based on in vitro data. The dosage required to achieve MEC depends on the individual characteristics and route of administration. However, plasma concentrations can be determined using HPLC assays or bioassays.

  Dosage intervals can also be determined using the MEC value. The compounds should be administered using a regimen that maintains plasma levels above the MEC at 10-90%, preferably 30-90%, most preferably 50-90% of the time. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

  Exemplary dosage regimens for the polypeptides or other compositions of the invention vary between adults and children, and range from about 0.01 μg / kg to 100 mg / kg body weight daily, with preferred doses being daily. It is in the range of about 0.1 μg / kg to 25 mg / kg per kg patient body weight. Administration may be once a day or the delivery interval may be shortened or lengthened as long as the dose is equal.

  Of course, the amount of the composition will depend on the subject being treated, the age and weight of the subject, the severity of the symptoms, the manner of administration and the diagnosis of the prescribing physician.

4.12.4 Packaging
The composition can also be administered in packs or dispenser devices that can contain one or more dosage forms containing the active ingredients therein, if desired. The pack may include, for example, a metal or plastic foil, such as a blister pack. The pack or dispenser device can be accomplished by a construct for administration. A composition comprising a compound of the invention may be formulated in a pharmaceutically compatible carrier, which may be prepared, placed in a suitable container and labeled for treatment of the indication.

4.13 Antibody The present invention also includes an antibody against the protein of the present invention or a fragment of the protein. As used herein, the term “antibody” specifically binds (immunoreacts with) an immunologically active portion of an immunoglobulin molecule and an immunoglobulin (Ig) molecule, ie, an antigen. Refers to a molecule having an antigen binding site. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, F _ab , F _{ab ′} and F _{(ab ′) 2} fragments, and F _ab expression libraries. Generally, antibody molecules obtained from humans are related to any class of IgG, IgM, IgA, IgE, and IgD, but these molecules differ from each other due to the nature of the heavy chain present in the molecule. ing. Certain classes have subclasses such as IgG ₁ , IgG ₂ and the like. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies encompasses all such classes, subclasses, and types of human antibody species.

  It is also conceivable to use an isolated protein related to the protein of the invention as an antigen, or part or fragment thereof, and use it further as an immunogen to standard techniques for polyclonal and monoclonal antibody preparation Can also be used to generate antibodies that immunospecifically bind to an antigen. A full-length protein can be used, or the present invention provides an antigenic peptide fragment of an antigen used as an immunogen. The antigenic peptide fragment is an amino acid sequence of a full-length protein, for example, at least 6 amino acid residues of the amino acid sequences shown in column numbers 685 to 1368 or 1967 to 2564 or Tables 3A, 3B, 5, 7 or 8. And surrounds the epitope so that antibodies raised against this peptide form a specific immune complex with the full-length protein or with any fragment containing the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. A preferred epitope surrounded by an antigenic peptide is a region of the protein located on its surface; this is usually a hydrophilic region.

  In certain embodiments of the invention, at least one of the epitopes surrounded by the antigenic peptide is a surface region of the protein, eg, a hydrophilic region. Hydrophobic analysis of related human protein sequences indicates which regions of the related protein are particularly hydrophilic and are therefore likely to encode useful surface residues for targeting antibody production. As a method for targeting antibody production, a hydrophobic hydrophilicity indicator plot showing hydrophilic and hydrophobic regions can be generated by any method well known in the art, including, for example, Kyte Doolitel or Hopp The Woods method (with or without Fourier transform) may be mentioned. See, for example, Hopp and Woods, 1981, Proc., Each of which is incorporated herein by reference in its entirety. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. MoI. Mol. Biol. 157: 105-142. Also provided herein are antibodies specific for one or more domains in an antigenic protein, or derivative, fragment, analog or homologue thereof.

  The protein of the present invention, or a derivative, fragment, analog, homologue or orthologue thereof can be used as an immunizing antigen in the production of antibodies that immunospecifically bind to these protein components.

  The term “specific for” means that the variable regions of the antibodies of the invention recognize and bind exclusively to the polypeptides of the invention (ie, sequence identity, homology found in the family of polypeptides). The polypeptides of the invention can be distinguished from other similar polypeptides despite similarity) but also with sequences outside the variable region of the antibody, in particular with sequences in the constant region of the molecule. Indicates that the interaction may also interact with other proteins (eg, S. aureus protein A or other antibodies in ELISA technology). Screening assays for determining the binding specificity of the antibodies of the invention are well known in the art and routinely performed. For a comprehensive discussion of such assays, see Harlow et al., (Eds.), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. As mentioned above, antibodies that recognize and bind to fragments of the polypeptide of the invention are also contemplated, provided that the antibody is first specific for the full-length polypeptide of the invention. Similar to antibodies specific for the full-length polypeptides of the invention, antibodies of the invention that recognize fragments are of the same family, despite the inherent sequence identity, homology, and similarity found in the family of proteins. An antibody that can distinguish a polypeptide from a polypeptide.

  The antibodies of the invention are useful, for example, for therapeutic purposes (by modulating the activity of the polypeptides of the invention), for diagnostic purposes to detect or quantify the polypeptides of the invention, and for purification of the polypeptides of the invention. It is. Also included are kits comprising the antibodies of the invention for any of the purposes described herein. In general, the kit of the invention also includes a control antigen in which the antibody is immunospecific. Furthermore, the present invention provides a hybridoma producing the antibody according to the present invention. The antibody of the present invention is useful for the detection and / or purification of the polypeptide of the present invention.

  Monoclonal antibodies that bind to the proteins of the present invention may be useful diagnostic agents for protein immunodetection. Neutralizing monoclonal antibodies that bind to proteins may also be useful therapeutic agents for protein-related conditions as well as for the treatment of some forms of cancer involving abnormal expression of the protein. In the case of cancer cells or leukemia cells, neutralizing monoclonal antibodies against the protein may be useful for detecting and preventing metastatic spread of cancer cells that may be mediated by the protein.

  The labeled antibodies of the invention can be used in in vitro, in vivo and in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed. This antibody can also be used directly in therapy or other diagnostics. Furthermore, the present invention provides the antibody described above immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose®, acrylic resins and eg polyacrylamide and latex beads. Techniques for conjugating antibodies to such solid supports are well known to those skilled in the art (Weir, DM, et al., “Handbook of Experimental Immunology” 4th Edition, Blackwell Scientific Publications, Oxford, Chapter 10). (1986); Jacobi, WD et al., Meth. Enzym. 34 Academic Press, NY (1974)). The immobilized antibodies of the invention can be used for in vitro, in vivo, in situ assays, as well as for immunoaffinity purification of the proteins of the invention.

  Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies against a protein of the invention, or agonist derivatives, fragments, analog homologs or orthologs thereof (eg, incorporated herein by reference). (See, Antibodies: A Laboratory Manual, Harlow E. and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.) Some of these antibodies are discussed below.

4.13.1 Polyclonal antibodies For the production of polyclonal antibodies, various suitable host animals (eg, rabbits, goats, etc.) are injected by one or more injections with natural proteins, synthetic variants thereof, or derivatives thereof. Mice or other mammals) may be immunized. Suitable immunogen preparations can include, for example, naturally occurring immunogenic proteins, chemically synthesized polypeptides representative of immunogenic proteins, or recombinantly expressed immunogenic proteins. In addition, the protein may be conjugated to a second protein known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation may further include an adjuvant. Various adjuvants used to enhance immunological response include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels (eg, aluminum hydroxide), surfactants (eg, lysolecithin, pluronic polyols) , Polyanions, peptides, oil emulsions, dinitrophenols, etc.), adjuvants useful in humans, such as Bacillus Calmette-Guerin and Corynebacterium parvum or similar immunostimulants. Further examples of adjuvants that can be used include MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolic acid).

  Polyclonal antibody molecules against an immunogenic protein can be isolated from a mammal (eg, from blood) and further purified by well-known techniques such as affinity chromatography using protein A or protein G, thereby Obtain mainly IgG fraction of immune serum. Thereafter, or alternatively, it is also possible to immobilize on the column the specific antigen or epitope thereof that is the sought-after immunoglobulin target and purify the immunospecific antibody by immunoaffinity chromatography. Immunoglobulin purification is described, for example, in D.C. Considered by Wilkinson (The Scientific, published by The Scientist, Inc., Philadelphia PA, Vol. 14, Issue 8 (April 17, 2000) pp. 25-28).

4.13.2 Monoclonal Antibody As used herein, the terms “monoclonal antibody” (MAb) or “monoclonal antibody composition” refer to the unique light chain gene product and the unique light chain gene product. It refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a heavy chain gene product. In particular, the complementarity determining regions (CDRs) of monoclonal antibodies are the same in all molecules of the population. Thus, MAbs contain an antigen binding site that can immunoreact with a particular epitope of that antigen, characterized by the specific binding affinity of that antigen.

  Monoclonal antibodies can be prepared using hybridoma methods such as those described in Kohler and Milstein, Nature, 256: 495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to raise lymphocytes that produce or can produce antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

  The immunizing agent typically includes a protein antigen, a fragment thereof or a fusion protein thereof. Generally, peripheral blood lymphocytes are used when human-derived cells are desired, or spleen cells or lymph node cells are used when non-human mammalian sources are desired. Lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 59-103. ). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells from rodents, cattle and humans. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth and survival of unfused immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine / guanine / phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma is typically a substance that prevents the growth of HGPRT-deficient cells. Contains hypoxanthine, aminopterin, and thymidine ("HAT medium").

  Preferred immortal cell lines are those that fuse efficiently, support antibody expression by selected antibody-producing cells at a stable high level, and are sensitive to a medium such as HAT medium. A further preferred immortal cell line is the murine myeloma cell line, which can be obtained from, for example, the Salk Institute Cell Distribution Center, San Diego, California, and American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Production Techniques and Applications). Marcel Dekker, Inc., New York (1987) 51-63).

  The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation reactions or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be determined according to, for example, Scatchard analysis of Munson and Polard, Anal. Anal Biochem. 107: 220 (1980). Preferably, antibodies with a high degree of specificity and high binding affinity for the target antigen are isolated.

  After the desired hybridoma cells are identified, this clone may be subcloned by limiting dilution and propagated by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

  Monoclonal antibodies secreted by subclones are isolated or purified from 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. can do.

  Monoclonal antibodies can also be made by recombinant DNA methods such as those described in US Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (eg, specific for genes encoding the heavy and light chains of murine antibodies). By using an oligonucleotide probe capable of binding to the target). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, this DNA is loaded onto an expression vector, which is then host cells that normally do not produce immunoglobulin proteins, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. To synthesize monoclonal antibodies in recombinant host cells. This DNA can, for example, replace the coding sequence of the human heavy and light chain constant domains in place of the homologous mouse sequence (US Pat. No. 4,816,567; Morrison, Nature 368, 812-12 (1994). )) Or by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be used in place of the constant domain of the antibody of the present invention or in place of the variable domain of the antigen-binding site of the antibody of the present invention to create a chimeric bivalent antibody. .

4.13.3 Humanized antibodies Antibodies to protein antigens of the present invention may further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without causing an immune response in humans even when immunoglobulins are administered. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, antibody Fv, Fab, Fab ′, F (ab ′) ₂ , or other antigen binding sequences) that are predominantly Consisting of human immunoglobulin sequences and minimally containing sequences derived from non-human immunoglobulin. Humanization of antibodies is performed by the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534. ˜1536 (1988)), the corresponding sequence of a human antibody can be used instead of the rodent CDR sequence. (See also US Pat. No. 5,225,539). In some cases, human immunoglobulin Fv framework residues are replaced with corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient's antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody comprises substantially at least one and typically all two variable domains, wherein all or substantially all of the CDR regions are non-human immunoglobulin. And all or substantially all of the framework region is that of a human immunoglobulin consensus sequence. Humanized antibodies preferably 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. Stgruc.Biol.2: 593-596 (1992)).

4.13.4 Human antibodies Fully humanized antibodies are related to antibody molecules in which the substantially entire sequence of both the light and heavy chains, including the CDRs, is of human gene origin. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies”. Human monoclonal antibodies are prepared by trioma technology; human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72), and EBV hybridoma technology to produce human monoclonal antibodies (Cole et al. , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96). Human monoclonal antibodies are available in the practice of the present invention, and by using human hybridoma cells (see Cote et al., 1983, Proc Natl Acad Sci USA 80: 2026-230) or human B cells can be produced by transformation in vitro with Epstein-Barr virus (see Cole et al., 1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96).

  In addition, human antibodies also include phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991)). Can also be produced. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, eg, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, we observed human antibody production, which closely resembles that seen in the human body in all respects, including gene rearrangement, assembly, and antibody repertoire. This is described, for example, in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 633,425; 5,661,016, and Marks et al. (Bio / Technology 10, 779-783 (992); Lonberg et al. (Nature 368, 856-859 (1994))); Morrison (Nature 368, 812-13 (1994); Fishwild et al. (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Im. unol.13, 65~93 (1995)).

Human antibodies can also be produced using transgenic non-human animals that have been modified to produce fully humanized antibodies rather than the animal's endogenous antibodies in response to challenge with the antigen ( PCT publication WO 94/02602). Disables endogenous genes encoding immunoglobulin heavy and light chains in non-human hosts and inserts active loci encoding heavy and light immunoglobulins into the host genome . The human gene is introduced using, for example, a yeast artificial chromosome containing a necessary part of a human DNA segment. Then, by interbreeding an intermediate transgenic animal that does not completely contain the modified portion, an animal that provides all the desired modified portions is obtained as a progeny. A preferred embodiment of such a non-human animal is the mouse, referred to as Xenomous ^™ as described in PCT Publications WO 96/33735 and WO 96/34096. This animal produces B cells that secrete fully humanized immunoglobulins. The antibody may be obtained directly from this animal after immunization with the target immunogen, eg, in preparation of a polyclonal antibody, or from an immortalized B cell derived from an animal such as a hybridoma producing a monoclonal antibody. Is also possible. Further, a gene encoding an immunoglobulin having a human variable region may be recovered and expressed to directly obtain an antibody, or may be further modified, for example, an antibody analog such as a single chain Fv molecule You can also get

  An example of a method for producing a non-human host, such as a mouse, that lacks expression of an endogenous immunoglobulin heavy chain is disclosed in US Pat. No. 5,939,598. It comprises the step of deleting the J segment gene from at least one endogenous heavy chain locus in the embryonic stem cell, preventing rearrangement of this locus and rearranging the immunoglobulin heavy chain locus The deletion is carried out by a target vector containing a gene encoding a selectable marker; a transgenic containing a gene encoding a selectable marker in somatic cells and germ cells And producing a mouse from embryonic stem cells.

  Methods for producing antibodies of interest such as human antibodies are disclosed in US Pat. No. 5,916,771. It introduces an expression vector comprising a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture and introduces an expression vector comprising a nucleotide sequence encoding a light chain into another mammalian host cell. And a step of fusing the two cells to form a hybrid cell. Hybrid cells express antibodies comprising heavy and light chains.

  In a further refinement in this procedure, a method for identifying clinically relevant epitopes of an immunogen, and a related method for selecting antibodies that immunospecifically bind to the relevant epitope with high affinity are described in PCT publication. It is disclosed in WO99 / 53049.

4.13.5 FAB fragments and single chain antibodies The technology according to the present invention is adaptable for the production of single chain antibodies specific for the antigenic proteins of the present invention (see eg US Pat. No. 4,946,778). thing). In addition, the method is adapted to construct _Fab expression libraries to allow rapid and effective identification of monoclonal _Fab fragments with the desired specificity for proteins, or derivatives, fragments, analogs, homologs thereof. (See, for example, Huse et al., 1989 Science 246: 1275-1281). Antibody fragments containing idiotypes against a protein antigen can be produced by techniques known in the art, including but not limited to: (i) produced by pepsin digestion of antibody molecules. produced by treating the antibody molecule with (iii) papain and a reducing agent; _{F (ab ')} that _{2 fragments; (ii) F (ab'} ) Fab fragment generated by reducing the disulfide bridges of the ₂ fragments _Fab fragments, and (iv) _Fv fragments.

4.13.6 Bispecific antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the case of the present invention, one of the binding specificities is the antigenic protein of the present invention. The second binding target is any other antigen, advantageously a cell surface protein or a receptor or receptor subunit.

  Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, where the two heavy chains have different specificities. (Milstein and Cuello, Nature, 305: 537-539 (1983)). Because immunoglobulin heavy and light chains are randomly combined, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which is the correct bispecific It has a structure. Purification of the correct molecule is usually performed by an affinity chromatography step. A similar procedure was published on May 13, 1993, WO 93/088829 and Traunecker et al., 1991 EMBO J. et al. 10: 3655-3659.

  Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) may be fused to immunoglobulin constant domain sequences. Fusion with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions is preferred. It is preferred to have a first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. The immunoglobulin heavy chain fusion and, if desired, DNA encoding the immunoglobulin light chain is inserted into another expression vector and cotransfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

  According to another approach described in WO 96/27011 it is also possible to manipulate the interface between a pair of antibody molecules so as to maximize the proportion of heterodimers recovered from the recombinant cell culture. . A preferred interface comprises at least a portion of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). Compensating “cavities of the same or similar size to the large side chain (s) by replacing the large amino acid side chain with a smaller amino acid side chain (eg, alanine or threonine) Is made on the junction of the second antibody molecule. This provides a mechanism for increasing the production of heterodimers over undesirable end products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) ₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229, 81 (1985) describe a procedure for proteolytic cleavage of intact antibodies to generate F (ab ′) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and inhibit intermolecular disulfide bond formation. The resulting Fab ′ fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab′-TNB derivatives is then reduced with mercaptoethylamine and converted back to Fab′-thiol and mixed with an equimolar amount of other Fab′-TNB derivatives to form bispecific antibodies. The produced bispecific antibody can be used as an enzyme selective immobilization agent.

In addition, Fab ′ fragments were It is recovered directly from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al. Exp. Med. 175: 217-225 (1992) describes the production of _two molecules of the fully humanized bispecific antibody F (ab ′). Each Fab ′ fragment is E. coli. It is secreted separately from E. coli and subjected to direct chemical binding in vitro to form bispecific antibodies. The bispecific antibody thus formed is capable of binding to cells overexpressing ErbB2 receptor and normal human T cells, while lysing human cytotoxic lymphocytes against human breast cancer targets. Induces activity.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab ′ portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. Hollinger et al., Proc. Nati. Acad. Sci. USA, 90: 6444-6448 (1993), the “bispecific antibody” technology provides an alternative mechanism for making fragments of bispecific antibodies. This fragment contains a heavy chain variable region (VH) and a light chain variable region (VL) joined together by a linker that is too short to pair between two domains on the same chain. Accordingly, the V _H and V _L domains of one fragment, by complementary V _L and V _H domains pair of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by use of single chain Fv (sFv) dimers has also been reported. Gruber et al. Immunol. 152: 5368 (1994).

  Trivalent or higher antibodies are also conceivable. For example, trispecific antibodies can be prepared. Tutt et al. Immunol. 147: 50 (991).

  A typical bispecific antibody can bind to two different epitopes, at least one of which is derived from a protein antigen of the invention. Alternatively, the anti-antigenic arm of an immunoglobulin molecule is linked to a triggering molecule on a leukocyte, eg, a T cell receptor molecule (eg, CD2, CD3, CD28 or B7), or an IgG Fc receptor (FcγR), eg, FcγRI (CD64). ), FcγRII (CD32), and an arm that binds to FcγRIII (CD16), thereby concentrating the cellular defense mechanisms to cells that express a particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells that express a particular antigen. These antibodies possess an antigen-binding arm and an arm that binds a cytotoxic agent or radionuclide chelator, such as EOTUBE, DPATA, DOTA, or TETA. Another bispecific antibody of interest binds to the protein antigen described herein and also binds to tissue factor (TF).

4.13.7 Heteroconjugate antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently bound antibodies. Such antibodies are, for example, for attacking unwanted cells with immune system cells (US Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360; WO 92/003733; EP 03089). Has been proposed. This antibody is believed to be prepared in vitro using methods known in synthetic protein chemistry, including those involving cross-linking 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 the reagents disclosed, for example, in US Pat. No. 4,676,980.

4.13.8 Effector Functional Engineering It may be desirable to modify the antibodies of the present invention with respect to effector function, thereby enhancing, for example, the effectiveness of the antibody in the treatment of cancer. For example, cysteine residue (s) are introduced into the Fc region, for example, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can improve internalization ability and / or increase complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Caron et al. Exp. Med. 176: 1911-1195 (1992); and Shopes, J. et al. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff et al., Cancer Research, 53: 2560-2565 (1993). . Alternatively, an antibody with a double Fc region can be engineered to improve complement lysis and ADCC capacity. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1980).

4.13.9 Immunoconjugates The invention also provides cytotoxic agents, such as chemotherapeutic agents, toxins (eg, enzymatically active toxins from bacteria, fungi, plants, or animals, or fragments thereof), Or it is associated with an immune complex comprising an antibody conjugated to a radioisotope (ie, a radioconjugate).

Chemotherapeutic agents useful for the generation of such immune complexes are described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modesin A chain, α-sarcin, Aleurites fordiii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), mordicica charantia inhibitor, crucin, clotine, saponiaria gentinolithin, inhibitor Mention may be made of phenomycin, enomycin and trichothecene. Various radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

  The binding of the antibody to the cytotoxic substance can be achieved by the dual action of various bifunctional protein binders such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolene (IT), and imidoester. Derivatives (eg, dimethyl adipimidate HCL), active esters (eg, disuccinimidyl suberate), aldehydes (eg, glutaraldehyde), bisazide compounds (eg, bis (P-azidobenzoyl) hexanediamine ), Bis-diazonium derivatives (eg, bis- (P-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg, triene 2,6-diisocyanate), and bis-active fluorine compounds (eg, 1,5-difluoro) -2,4-dinitrobenzene) That. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a representative chelator for the binding of radionuclides to antibodies. See WO94 / 11026.

  In another embodiment, an antibody can be conjugated to a “receptor” (such as streptavidin) for use in tumor pre-targeting, but here antibody-receptor binding The body is administered to the patient, after which the unbound conjugate is removed from the circulatory system with a clarifying agent, followed by administration of a “ligand” (eg, avidin), which is then a cytotoxic agent. To join.

4.14 Computer-readable sequences In one application of this embodiment, the nucleotide sequences of the present invention can be recorded on a computer-readable medium. As used herein, “computer readable media” refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as CD-ROM; RAM and Electronic storage media such as ROM; as well as hybrids of these categories such as magnetic / optical storage media. Those skilled in the art will readily understand how any of the currently known computer readable media can be used to produce a product containing a computer readable medium recording the nucleotide sequences of the present invention. As used herein, “recorded” refers to the process of storing information on a computer-readable medium. One of ordinary skill in the art can readily produce a product containing the nucleotide sequence information of the present invention using any of the currently known methods for recording information on computer readable media.

  A variety of data storage constructs are available to those skilled in the art to create computer readable media recording the nucleotide sequences of the present invention. The selection of the data storage structure is generally based on the selected means of accessing the recorded information. In addition, the nucleotide sequence information of the present invention can be recorded on computer readable media using a variety of data processing programs and formats. This sequence information can be represented in a word processor text file formatted in commercial software such as WordPerfect and Microsoft Word, or in the form of an ASCII file stored in a database application such as DB2, Sybase, Oracle, etc. It can also be expressed as One skilled in the art can readily adapt any number of data processor structuring formats (eg, text files or databases) to obtain a computer readable medium recording the nucleotide sequence information of the present invention. Is possible.

  Any of the nucleotide sequences of SEQ ID NOs: 1-684 or 1369-1966, or a representative fragment thereof; or any nucleotide sequence that is at least 95% identical to any of the nucleotide sequences of SEQ ID NOs: 1-684 or 1369-1966 Is provided in a computer readable form so that one skilled in the art can routinely access the sequence information for various purposes. Computer software that makes sequence information provided as computer-readable media accessible to those skilled in the art is publicly available. The BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17: 203-207 (1993)) search algorithms on the Sybase system by the following example A method of identifying an open reading frame (ORF) within a nucleic acid sequence using software that performs is shown. Such ORFs may be protein-encoded fragments and are useful for the production of commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites. There may be.

  As used herein, “a computer-based system” refers to the hardware, software, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware required for the computer-based system of the present invention comprises a central processing unit (CPU), an input device, an output device, and a data storage device. One skilled in the art can readily appreciate which of the currently available computer-based systems are suitable for use with the present invention. As described above, the computer-based system of the present invention comprises a data storage device recording the nucleotide sequence of the present invention, and hardware and software means necessary to support and execute the search means. . As used herein, “data storage means” refers to a storage device (memory) that can record the nucleotide sequence information of the present invention or a product that records the nucleotide sequence information of the present invention. Any memory access device that can be accessed.

  As used herein, “search means” is performed on a computer-based system to compare a target sequence or target structural motif with sequence information recorded in a data storage device. One or more programs. Search means are used to identify fragments or regions of known sequence that match a particular target sequence or target motif. Various known algorithms have been publicly disclosed, and various commercially available software can be used in the computer-based system of the present invention to execute the search means. Examples of such software include, but are not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN, and BLASTA (NPOLYPEPTIDEIA). One skilled in the art can readily understand which of the algorithms or execution software packages available to perform homology searches can be adapted for use in the computer-based system of the present invention. As used herein, the term “target sequence” may be any nucleic acid sequence or amino acid sequence of 6 or more nucleotides or 2 or more amino acids. One skilled in the art can readily appreciate that the longer the target sequence, the less likely it will occur randomly in the database. The most preferred sequence length of the target sequence is about 10-300 amino acids, more preferably about 30-100 nucleotide residues. However, as is already well known, searches for commercially important fragments such as sequence fragments involved in gene expression and protein processing may be performed in even shorter lengths.

  As used herein, “a target structural motif” or “target motif” refers to any reasonably selected sequence or combination of sequences. The singular (s) is selected based on the three-dimensional structure formed upon folding of the target motif. Various target motifs are known in the art. Protein target motifs include, but are not limited to, active sites and signal sequences of enzymes. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures, and inducible expression elements (protein binding sequences).

4.15 Triple helix formation Furthermore, the fragments of the present invention can be used in a broad sense to control gene expression through triple helix formation or antisense DNA or RNA. Or based on binding to RNA. Polynucleotides suitable for these methods are preferably 20-40 bases in length, and regions of genes involved in transcription (triple helix-Lee et al., Nucl. Acids Res. 6: 3073 (1979); See Cooney et al., Science, 15241: 456 (1988); and Dervan et al., Science, 251: 1360 (1991)), or mRNA itself (Antisense-Olmno, J. Neurochem. 56: 560 ( 1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix formation preferably blocks RNA transcription from DNA at optimal conditions, but antisense RNA hybridization blocks translation of mRNA molecules into polypeptides. Both technologies have proven effective in model systems. Information contained in the sequences of the present invention is necessary for the design of antisense or triple helix oligonucleotides.

4.16 Diagnostic Assays and Kits The present invention further uses one nucleic acid probe or antibody of the present invention (optionally conjugated or associated with an appropriate label) to produce one ORF or Also provided is a method of identifying the presence or expression of the homolog in a test sample.

  The method of detecting a polynucleotide of the present invention generally comprises contacting a compound that binds to a polynucleotide to form a complex with a sample for a time sufficient to form the complex, detecting the complex, As a result, if a complex is detected, the polynucleotide of the present invention may be detected in the sample. Such a method also includes contacting the sample with a nucleic acid primer that anneals to the polynucleotide of the invention under stringent hybridization conditions, and amplifying the annealed polynucleotide, so that the polynucleotide Can be included, the polynucleotide of the present invention will be detected in the sample.

  Methods for detecting a polypeptide of the invention generally involve contacting a compound that binds to a polypeptide to form a complex with a sample for a time sufficient to form the complex, detecting the complex, As a result, if a complex is detected, it may include a step in which the polypeptide of the invention will be detected in the sample.

  Specifically, such a method comprises incubating a test sample with one or more of the antibodies of the invention, or one or more of the nucleic acid probes, and binding of the nucleic acid probe or antibody to a component within the test sample. Assaying.

  Conditions for incubating the nucleic acid probe or antibody with the test sample vary. Incubation conditions depend on the format used in the assay, the detection method used, the type and nature of the nucleic acid probe or antibody used in the assay. Those skilled in the art will recognize that any one of the commonly available hybridization, amplification, or immunological assay formats can be readily adapted to use the nucleic acid probes or antibodies of the present invention. Examples of such assays are described in Chard, T .; , An Introduction to Radioimmunoassay and Related Technologies, Elsevier Science Publishers, Asterdam, The Netherlands (1986); R. , Techniques in Immunocytochemistry, Academic Press, Orlando, FL, Volume 1 (1982), Volume 2 (1983), Volume 3 (1985); Tijssen, P. et al. , Practice and Theory of Immunoassays: Lumpry Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterham, 198 Test samples of the present invention include cells, cellular protein or membrane extracts, or biological fluids (eg sputum, blood, serum, plasma, or urine). The test sample used in the above-described method depends on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample to be assayed. Methods for preparing cellular protein extracts or membrane extracts are well known in the art and can be readily adapted to obtain a sample suitable for the system utilized.

  In another embodiment of the invention, a kit is provided that contains the reagents necessary to perform an assay of the invention. Specifically, the present invention provides a compartment kit that closes and receives one or more containers, the kit including: (a) one of the probes or antibodies of the present invention. A first container comprising; and (2) one or more other containers comprising one or more of the following: a reagent capable of detecting the presence of a washing reagent, bound probe or antibody.

  Specifically, compartment kits include any kit that contains reagents in a separate container. Such containers include small glass containers, plastic containers, or pieces of plastic or paper. Such containers allow for efficient transfer of reagents from one compartment to another, so that the sample and reagent are not cross-contaminated and the substance or solution in each container is quantitatively reduced by 1 It is also possible to add from one compartment to another. Such containers include containers for test samples, containers for antibodies used in the assay, containers for wash reagents (eg, phosphate buffered saline, Tris buffer, etc.), bound antibodies or probes. A container containing a reagent used for detection can be mentioned. The type of detection reagent includes a labeled nucleic acid probe, a labeled secondary antibody, or an enzyme capable of reacting with the labeled antibody or an antibody binding reagent when the primary antibody is labeled. Those of skill in the art will readily appreciate that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats well known in the art.

4.17 Medical Images The novel polypeptides and binding partners of the present invention are useful for medical images of sites expressing molecules of the present invention (eg, inflammation or infection if the polypeptide of the present invention is involved in an immune response). Useful for the image part of the part). See, for example, Kunkel et al., US Pat. No. 5,413,778. Such methods include chemical coupling of a labeling agent or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging of the labeled polypeptide at a target site in vivo.

4.18 Screening Assay By using the isolated proteins and polynucleotides of the present invention, the present invention is encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NOs: 1-684 or 1369-1966. Further provided are methods of obtaining and identifying a substance that binds to a polypeptide that is bound to, or a specific domain of a polypeptide encoded by the nucleic acid. Specifically, the method includes the following steps:
(A) contacting the ORF of the present invention or an isolated protein encoded by the nucleic acid of the present invention with a substance;
(B) A step of determining whether or not the substance binds to the protein or the nucleic acid.

  Thus, in general, such a method for identifying a compound that binds to a polynucleotide of the invention comprises contacting the polynucleotide of the invention with the compound 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 the polynucleotide of the invention is identified.

  Thus, in general, such a method for identifying a compound that binds to a polypeptide of the invention is sufficient to form a polypeptide / compound complex between the polypeptide of the invention and the compound. The step of contacting with time may include the step of detecting the complex and, consequently, if a polypeptide / compound complex is detected, identifying the compound that binds to the polynucleotide of the invention.

  A method for identifying a compound that binds to a polypeptide of the present invention also comprises contacting the compound of the present invention with a compound for a time sufficient to form a polypeptide / compound complex in the cell. Wherein the complex drives the receptor gene sequence expression in the cell and detects the complex by detecting the expression of the reporter gene sequence, so that if the polypeptide / compound complex When a body is detected, it can include the step of identifying compounds that bind to the polypeptides of the invention.

  Compounds identified through such methods modulate the activity of the polypeptide of the invention (ie, increase or decrease its activity relative to that observed in the absence of the compound). A compound can be mentioned. Alternatively, a compound identified by such a method includes a compound that modulates the expression of a polynucleotide of the invention (ie, increases or decreases expression relative to the expression level observed in the absence of the compound). Can be mentioned. Compounds such as those identified via the methods of the invention can be tested for ability to modulate activity / expression using standard assays well known to those skilled in the art.

  Factors screened in the above assays can include, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical factors. Such factors may be randomly selected or screened, or may be rationally screened or designed using protein modeling techniques.

  In the case of random screening, factors such as peptides, carbohydrates, pharmaceutical substances, etc. are randomly selected and assayed for the ability to bind to the protein encoded by the ORF of the invention. Alternatively, the substance may be rationally screened or designed. As used herein, a factor is said to be “rationally selected or designed” if it is selected based on the structure of a particular protein. For example, one of ordinary skill in the art can readily apply currently available procedures to generate peptides, pharmaceutical agents, etc. that can bind to specific peptide sequences to produce rationally designed anti-peptide peptides (eg, Hurby et al., Application of Synthetic Peptides: Antisense Peptides,) Synthetic Peptides, A User's Guide, W., et al. H. Freeman, NY (1992) 289-307, and Kaspczak et al., Biochemistry 28: 9230-8 (1989)), or pharmaceutical factors, etc. can be generated.

  In addition to the foregoing, a class of factors of the present invention can be used in a broad sense to control gene expression through binding to one of the ORFs or EMFs of the present invention. As noted above, such factors may be randomly screened or rationally designed / selected. By targeting ORFs or EMFs, one of ordinary skill in the art can design sequence-specific or element-specific factors, thereby allowing single ORFs or multiple ORFs (both depending on the same EMF for expression control). Either expression can be adjusted. One class of DNA binding agents are those that contain residues of bases that hybridize to or form triple helices by binding to DNA or RNA. Such materials may be based on classical phosphodiester, ribonucleic acid backbones, or may be various sulfhydryl derivatives or polymer derivatives having base attachment ability.

  Factors suitable for use in these methods preferably contain 20-40 bases and regions of the gene involved in transcription (triple helix-Lee et al., Nuel. Acids. Res. 6: 3073 (1979); Cooney et al. , Science, 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991)), or mRNA itself (Antisense-Okano, J. Neurochem. 56: 560 (1991); It is designed to be complementary to Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix formation has the best results in blocking RNA transcription from DNA, while antisense RNA hybridization blocks translation of mRNA molecules into polypeptides. Both technologies have proven effective in model systems. Information contained in the sequences of the present invention is necessary for the design of antisense or triple helix oligonucleotides and other DNA binding agents.

  A factor that binds to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent. An agent that binds to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques for producing pharmaceutical compositions.

4.19 Use of Nucleic Acids as Probes Another aspect of the present invention is to provide polypeptide-specific nucleic acid hybridization probes that can hybridize to naturally occurring nucleotide sequences. The hybridization probe of the present invention can be derived from any of the nucleotide sequences SEQ ID NO: 1-684 or 1369-1966. Since the corresponding genes are expressed only in a limited number of tissues, hybridization probes derived from any of the nucleotide sequences SEQ ID NOs: 1-684 or 1369-1966 will be used for cell types of such tissues in the sample. It can be used as an index indicating the presence of RNA.

  For example, any suitable hybridization technique such as in situ hybridization may be used. The PCR described in US Pat. Nos. 4,683,195 and 4,965,188 provides another use of oligonucleotides based on nucleotide sequences. Such probes used in PCR may be derived from recombination, chemically synthesized, or a mixture of both. This probe contains different nucleotide sequences for the detection of the same sequence, or a degenerate pool of potential sequences for the identification of closely related genomic sequences.

  Another method of producing a hybridization probe specific for a nucleic acid involves cloning the nucleic acid sequence into a vector for the production of mRNA probes. Such vectors are known in the art and are commercially available for synthesizing RNA probes in vitro by adding an appropriate RNA polymerase or SP6 RNA polymerase as T7 and an appropriate radiolabeled nucleotide. Can be used for This nucleotide sequence can be used to construct a hybridization probe for mapping each genomic sequence. The nucleotide sequences provided herein can be mapped to a particular region of a chromosome or chromosome using well-known gene and / or chromosome mapping techniques. These techniques include in situ hybridization, linkage analysis to known chromosomal markers, hybridization screening using known chromosome-specific libraries or chromosomal preparations subjected to flow classification, and the like. There are other techniques for fluorescence in situ hybridization of chromosome distributions, such as Verma et al. (1988) Human Chromosomes: A Manual of Basic Technologies, Pergamon Press, New York NY. It is described in.

  Fluorescence in situ hybridization of chromosomal preparations and other physical chromosomal mapping techniques can be correlated with additional genetic map data. An example of genetic map data can be found in 1994 Genome Issue of Science (265: 1981f). The correlation between the location of the nucleic acid on the physical chromosomal map and a particular disease (or predisposition to a particular disease) will help to limit the region of DNA associated with that genetic disease. The nucleotide sequences of the present invention can be used to detect genetic sequence differences between normals, carriers or affected individuals.

4.20 Preparation of oligonucleotides immobilized on a support Oligonucleotides, ie small nucleic acid segments, can be synthesized by chemical means, as is commonly done, for example, using automated oligonucleotide synthesizers. It can be easily prepared by direct synthesis.

  Support-immobilized oligonucleotides can be prepared by any method known to those of skill in the art using any suitable support such as glass, polyethylene or Teflon. One strategy is to accurately spot oligonucleotides synthesized by standard synthesizers. Immobilization uses passive adsorption (Inouye and Honda, 1990 J. Clin Microbiol 28 (6) 1469-72); ultraviolet light (Nagata et al., 1985; Dahlen et al., 1987, Morrissey and Collins (1989) Mol. Cell Probes. 1989 3 (2) 189-207) or by covalent attachment of base-modified DNA (Keller et al., 1988; 1989); all references are incorporated in detail herein. Yes.

  Another strategy that can be used is to use a strong biotin-streptavidin interaction as a linker. See, for example, Broude et al. (1994) Proc. Natl. Acad. Sci USA 91 (8) 3072-6 describes the use of biotinylated probes, but they are dual probes and are immobilized on streptavidin-coated magnetic beads. Streptavidin-coated beads can be purchased from Dynal, Oslo. Of course, this same conjugation chemistry can be applied to any surface coating with streptavidin. Biotinylated probes can be purchased from a variety of vendors such as, for example, Operan Technologies (Alameda, Calif.).

  Appropriate materials are also available from Nunc Laboratories (Nepperville, IL). Nunc Laboratories has developed a method that allows DNA to be covalently bound to a microwell surface called Covalent NH. CovaLink NH is a polystyrene surface grafted with secondary amino groups (> HN) that serve as bridgeheads for further covalent bonding. CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules can be linked only to the 5 ′ end of CovaLink by phosphoramidite linkage, which allows the immobilization of DNA above 1 pmol (Rasmussen et al. (1991) Anal Biocher 198 (l) 138-42). ).

  The use of CovaLink NH strips for the covalent attachment of DNA molecules to 5 'has been published (Rasmussen et al., 1991). In this technique, phosphoramidite linkages are used (Chu et al. (1983) Nucleic Acids 11 (18) 6513-29). This has the advantage that it is preferable to fix using only one covalent bond. Phosphoramidate linkage binds DNA via a 2 nm long spacer arm to the CovaLink NH secondary amino group located at the end of the spacer arm that is covalently grafted to the polystyrene surface. . In order to link an oligonucleotide to CovaLink NH via a phosphoramidate bond, the oligonucleotide end must have a 5'-end phosphate group. Perhaps biotin can be covalently bound to CovaLink and then bound to the probe using streptavidin.

More specifically, this ligation method includes a step of dissolving DNA in water (7.5 ng / μl), a step of denaturing at 95 ° C. for 10 minutes, and a step of cooling with ice for 10 minutes. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm ₇ ) is then added to a final concentration of 10 mM I-MeIm ₇ . The ss DNA solution is then fed to a CovaLink NH strip (75 μl / well) placed on ice.

Carbodiimide 0.2M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) dissolved in 10 mM 1-MeIm ₇ was newly prepared and 25 μl was added per well. The strip was allowed to incubate at 50 ° C. for 5 hours. After incubation, the strip was washed using, for example, a Nunc-Immuno Wash. First, the wells are washed three times, then immersed in a washing solution for 5 minutes, and finally washed three times (the washing solution is 0.4N NaOH, 0.25% SDS heated to 50 ° C.).

  A more suitable method for the present invention is believed to be that described in PCT patent application WO 90/03382 (Southern & Maskos), incorporated herein by reference. This method of preparing an oligonucleotide immobilized on a support adds a nucleoside 3'-reagent via a phosphate group via a covalent phosphodiester linkage to an aliphatic hydroxyl group carried by the support. Process. This oligonucleotide is then synthesized on a supported nucleoside to remove the protecting group from the synthetic oligonucleotide strand under standard conditions that do not cleave the oligonucleotide from the support. Suitable reagents include nucleoside phosphoramidites and nucleoside hydrogen phosphorates.

  For the preparation of the DNA probe array, operations on the chip for DNA probe preparation can be performed. For example, addressable laser-initiated photodeprotection can be performed directly on the glass surface as described in Fodor et al. (1991) Science 251 (4995) 767-73, incorporated herein by reference. Can be used for synthesis. Probes are also described in Van Ness et al. (1991) Nucleic Acids Res. 19 (12) 3345-50 may be immobilized on a nylon support; the probe may also be attached to Duncan and Cavalier (1988) Anal. Biochem. 169 (l) 104-8 may be used to link to Teflon; all references are incorporated in detail herein.

  To link an oligonucleotide to a nylon support, it is necessary to activate the nylon surface by alkylation and selectively activate the 5′-amine of the oligonucleotide with cyanuric chloride as described in Van Ness et al. (1991). There is.

  One particular method for preparing oligonucleotides immobilized on a support is described by Pease et al. (1994) Proc. Nat'l. Acad. Sci USA 91 (11) 5022-6 is to use photogenerated synthesis. These authors use current photolithographic techniques to generate an array of immobilized oligonucleotide probes (DNA chips). These methods of synthesizing oligonucleotide probes using light in a high-density miniaturized array include photolabile 5'-protected N-acyl-deoxynucleoside phosphoramidite surface linker chemistry and diverse combinatorial synthesis Use strategies. By this method, a matrix of oligonucleotide probes spatially divided into 256 can be created.

4.21 Preparation of Nucleic Acid Fragments Nucleic acid is obtained from any suitable source, for example, RNA, including cDNA, genomic DNA, chromosomal DNA, microcleaved chromosomal bands, cosmid or YAC inserts, and mRNA without amplification steps. Can do. For example, Sambrook et al. (1989) describe three protocols for isolating high molecular weight DNA from mammalian cells (p. 9.14-9.23).

  DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and / or directly from genomic DNA or cDNA by PCR or other amplification methods. Samples may be prepared or arranged in multiwell plates. About 100-1000 ng of DNA sample may be prepared in a final volume of 2-500 ml.

  The nucleic acid is then fragmented by any method known to those skilled in the art including, for example, using the restriction enzymes described in Sambrook et al. (1989) 9.24-9.28, ultrasonic shearing and NaOH treatment. .

  The low pressure shear method is also described by Schriefer et al. (1990) Nucleic Acids Res. 18 (24) 7455-6 is appropriate. In this method, a DNA sample is passed through a small French press cell under various pressures ranging from low pressure to medium pressure. The low to medium pressure regulation on the cells can be controlled with a lever device. The results of these studies indicate that low pressure shear is useful as an alternative to sonic and enzymatic DNA fragmentation methods.

  One particular method that is suitable for fragmenting DNA is the Fitzgerald et al. (1992) Nucleic Acids Res. 20 (14) 3753-62, a method using the two-base discrimination endonuclease CviJI is considered. These authors describe an approach for rapid fragmentation and fractionation of DNA to a specific size that they deemed appropriate for shotgun cloning and sequencing.

  The restriction endonuclease CviJI usually cleaves the recognition sequence PuGCPy between G and C, leaving a blunt end. Due to special reaction conditions that change the specificity of this enzyme (CviJI **), semi-randomly distributed DNA fragments are generated from the small molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992) directly linked the randomization of this fragment strategy to the lacZ minus M13 cloning vector using CviJI ** digests of pUC19 size fractionated by rapid gel filtration without end repair. To evaluate quantitatively. Sequence analysis of 76 clones showed that CviJI ** restricts pyGCPy and PuGCPu in addition to the PuGCPy site, and that new sequence data accumulates at a rate consistent with random fragmentation. .

  As reported in the literature, the advantages of this approach include the following compared to sonication and agarose gel fractionation: less DNA required (0.2-0.5 μg instead of 2-5 μg) ); And fewer processing steps (no need for pre-ligation, end repair, chemical extraction, or agarose gel electrophoresis and elution, etc.).

  Regardless of how the nucleic acid fragment is obtained or prepared, it is important to denature the DNA to obtain a single strand that can be used for hybridization. This is accomplished by incubating the DNA solution at 80-90 ° C. for 2-5 minutes. The solution is then quenched to 2 ° C. to prevent the DNA fragment from regenerating before contacting the chip. Phosphate groups must also be removed from genomic DNA by methods known to those skilled in the art.

4.22 Preparation of DNA Arrays Arrays can be prepared by spotting DNA samples on a support such as a nylon membrane. Spotting can be performed by repeatedly transferring approximately 20 nl of DNA solution to a nylon membrane using an array of metal pins (the location corresponds to an array of wells in a microtiter plate). By offset printing, a dot density higher than that of the well can be obtained. Depending on the type of marker used, 1-25 dots per mm ² can be provided. By avoiding spotting in several preselected numbers of rows and columns, another subset (subarray) can be formed. The samples in one subarray may be the same genomic segment (or the same gene) of DNA from different individuals, or may be different overlapping genomic clones. Each subarray may correspond to replica spotting of the same sample. In one example, the selected gene segment may be amplified from 64 patients. For each patient, the amplified gene segments may be placed in one 96 well plate (all 96 wells contain the same sample). Prepare one plate for each of the 64 patients. By using a 96 pin device, all samples may be spotted on a single 8 × 12 cm film. The subarray may include 64 samples from each patient. If 96 subarrays are identical, the dot span may be 1 mm ² and there may be a 1 mm spacing between the subarrays.

  Another approach is to use a physical spacer, for example a plastic lattice molded on the membrane, a lattice similar to the classification of membranes provided at the bottom of a multiwell plate, or a membrane that can be separated by a hydrophobic strip or Plate (available from NUNC, Naperville Illinois). A fixed physical spacer is not preferred for imaging by exposing a flat phosphorus storage screen or x-ray film.

  The invention is illustrated by the following examples. In view of this disclosure, it will be apparent to those skilled in the art that many other embodiments and variations may be made within the scope of the invention. Accordingly, it is contemplated that the broad aspects of the invention are not limited to the disclosure of the following examples. The invention should not be limited by the exemplary embodiments intended to illustrate only one aspect of the invention, and functionally equivalent compositions and methods are within the scope of the invention. In fact, many modifications and variations will occur to those skilled in the art when considering the preferred embodiments of the present invention. Accordingly, the only limitations in the scope of the invention are set forth in the appended claims.

  All references cited in this specification are hereby incorporated by reference in their entirety.

5.0 Example 5.1 Example 1
Novel nucleic acid sequences obtained from different libraries Multiple novel nucleic acids were obtained from cDNA libraries prepared from different human tissues and in some cases standard PCR, SBH sequence characterization and Sanger sequencing Isolated from a genomic library derived from human chromosomes using technology. The library insert was amplified by PCR using primers specific for the vector sequence adjacent to the insert. Clones from the cDNA library were spotted on nylon membrane filters and screened with oligonucleotide probes (eg, 7-mer) to obtain characteristic sequences. The clones were classified into groups with similar or identical sequences. A representative clone was selected for sequencing.

  In some cases, the 5 'sequence of the amplified insert was then deduced using a typical Sanger sequencing protocol. The PCR product was purified and subjected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was performed using a 377 Applied Biosystems (ABI) sequencer to generate new nucleic acid sequences.

5.2 Example 2
Novel nucleic acid assembly The contigs or nucleic acids of the invention named SEQ ID NO: 1369-1966 were assembled using EST sequences as seeds. A circular algorithm is then used to extend the seed EST into the extension assemblage, where different databases belonging to this assemblage (ie, databases containing Hyseq EST sequences, dbEST, gb pri, and UniGene, and estimated by GenScan) Was performed by deriving additional sequences from the exons from the public domain genomic sequences. The algorithm stopped when no additional sequences were available from the above database to extend the assemblage. In addition, incorporation of component sequences into assemblages was based on BLASTN hits against elongating assemblages having a BLAST score greater than 300 and a percent identity greater than 95%.

  Table 7 shows each of the novel predicted polypeptides (including proteins), SEQ ID NOs: 1967-2564 (encoded by the novel polynucleotides of the invention (SEQ ID NOs: 1369-1966)), and SEQ ID NOs: 1369-1966 The positions of their corresponding translation start and stop nucleotides relative to are indicated. Table 7 also shows how the polypeptide was predicted. Method A uses a software program called FASTY (available from http: //fasta.bioch.virginia, edu) that selects polypeptides based on a comparison of translated novel polynucleotides to known polynucleotides. (W. R. Pearson, Methods in Enzymology, 183: 63-98 (1990), incorporated herein by reference). Method B provides a software program called GenScan (available from Stanford University, Office of Technology Licensing) for human / vertebrate sequences that predicts polypeptides based on a probabilistic model of genetic structure / constitutive properties Refers to the polypeptide obtained by use (C. Burge and S. Karlin, J. Mol. Biol., 268: 78-94 (1997), incorporated herein by reference). Method C is Hyseq's proprietary software that translates a novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and selects the polypeptide with the longest open reading frame. It refers to the polypeptide obtained by using the program.

5.3 Example 3
Novel nucleic acids The novel nucleic acids of the invention are assembled from sequences obtained from a cDNA library by the method described in Example 1 above, and in some cases from sequences obtained from one or more public databases. did. Nucleic acids were assembled using EST sequences as seeds. A circular algorithm is then used to extend the seed EST into the extension assembly, where additional sequences are derived from different databases belonging to this assembly (databases containing Hyseq EST sequences, dbEST, gb pri, and UniGene). Was done by. The algorithm stopped when no more sequences were available to extend the assemblage from the above database. Incorporation of component sequences into assemblages was based on BLASTN hits for elongating assemblages with a BLAST score greater than 300 and a percent identity greater than 95%.

  Using PHRAP (University of Washington) or CAP4 (Paracel), the full-length gene cDNA sequence and its corresponding protein sequence were generated from this assemblage. Any frameshifts and incorrect stop codons were corrected by manual editing. During editing, sequences were checked using FASTY and / or BLAST against Genebank (ie dbEST, gb pri, and UniGene and Genpept) and Geneseq (Derwent). Other computer programs that could be used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and cg-zip-2 (Hyseq, Inc.). The full length nucleotide and amino acid sequences including the splicing variants obtained from these procedures are shown in the sequence listing as SEQ ID NOs: 1-1368.

  The TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html) was used to confirm that the nucleic acid sequences of the present invention have at least one transmembrane domain. One skilled in the art will recognize that the proteins of the invention can be utilized as either membrane-bound targets or soluble proteins.

  Table 1 shows various tissue sources of SEQ ID NOs: 1-684.

  The homologues of the polypeptide SEQ ID NO: 685 to 1368 corresponding to the nucleotide sequence SEQ ID NO: 1 to 684 were obtained by BLAST version 2.0a1 19MP-WashU search against Genept and Geneseq (Derwent) using the BLAST algorithm. . The results showing the homologues of SEQ ID NOs: 685 to 1368 are shown in Tables 2A and 2B.

  eMatrix software package (Stanford University, Stanford, CA) (Wu et al., J. Comp. Biol., Vol. 219-235 (1999), http: //motif.stanford. All polypeptide sequences were tested using edu / ematrix-search /) to determine if they had identifiable sign regions. The eMatrix software package scoring matrix is derived from the BLOCKS, PRINTS, PFAM, PRODOM and DOMO databases. Tables 3A and 3B show the accession number of the homologous eMatrix signature found in the polypeptide sequence shown, its description and obtained results including accession number subtype; raw score; p value; and amino acid sequence The position of the sign at.

  Using the Pfam software program (Sonhammer et al., Nucleic Acids Res., 26 (1), pages 320-322 (1998), incorporated herein by reference), homology to specific peptide domains All polypeptide sequences were tested for having domains. Tables 4A and 4B show the names, descriptions, e-values, and Pfam scores for the identified models in the sequence, of the found Pfam models. Further description of the Pfam model can be found at http: // pfam. Wustl. edu / can be seen.

  Table 5 shows the position of the signal peptide in each polypeptide and its score using the Neural Network SignalP V1.1 program (Center for Biological Sequence Analysis, The Technical University of Denmark) Show. Processes for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also described in Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication, which are incorporated herein by reference. "Identification of prokaryotic and eukaryotic signal peptides and prediction of the cleavage sites," Protein Engineering, Vol. 10, No. 1, pages 1-6 (1997). Maximum and average S-scores were obtained for polypeptide sequences as described in Nielson et al.

  Table 6 relates to the nucleotide sequences of the present invention for specific chromosomal locations, if assignable.

  Table 8 shows the number of transmembrane regions for each of SEQ ID NOs: 651 to 1368 with a TMPred score of 500 or greater using the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html). , Indicate its position (s) and the resulting TMPred score.

  Table 9 shows the novel polynucleotide sequence SEQ ID NO: 1-684, its corresponding polypeptide sequence SEQ ID NO: 65-1368, its corresponding preferred contiguous nucleotide sequence SEQ ID NO: 1369-1966, its corresponding preference. Contig polypeptide sequence SEQ ID NO: 1967-2564, as well as US application number correlation table of priority applications (all of which are hereby incorporated by reference in their entirety) in which the contig sequences are filed.

Claims (26)

An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-684. An isolated polynucleotide encoding a biologically active polypeptide, which hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions. An isolated polynucleotide encoding a polypeptide having biological activity, wherein the polynucleotide has greater than about 99% sequence identity with the polynucleotide of claim 1. The polynucleotide of claim 1, wherein the polynucleotide is DNA. 2. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises a complementary sequence. A vector comprising the polynucleotide of claim 1. An expression vector comprising the polynucleotide according to claim 1. A host cell genetically engineered to contain the polynucleotide of claim 1. A host cell genetically engineered to contain a polynucleotide of claim 1 operatively associated with a regulatory sequence that regulates expression of the polynucleotide in the host cell. An isolated polypeptide comprising:
Encoded by (a) a polypeptide encoded by any one of the polynucleotides of claim 1 and (b) a polynucleotide that hybridizes under stringent conditions with any one of SEQ ID NOs: 1-684. The polypeptide to be
A polypeptide selected from the group consisting of: A composition comprising the polypeptide of claim 10 and a carrier. An antibody against the polypeptide according to claim 10. A method for detecting the polynucleotide of claim 1 in a sample comprising:
a) contacting the sample with a compound that binds to the polynucleotide of claim 1 to form a complex with the polynucleotide for a time sufficient to form a complex;
b) detecting the complex and, as a result, detecting a complex, detecting the polynucleotide of claim 1;
Including the method. A method for detecting the polynucleotide of claim 1 in a sample comprising:
a) contacting the sample under stringent hybridization conditions with a nucleic acid primer that anneals to the polynucleotide of claim 1 under such conditions;
b) amplifying a product comprising at least a portion of the polynucleotide of claim 1;
c) detecting the product and thereby detecting the polynucleotide of claim 1 in the sample;
Including the method. 15. The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing the annealed RNA molecule to a cDNA polynucleotide. A method for detecting a polypeptide according to claim 10 in a sample comprising:
a) contacting the sample with a compound that binds to the polypeptide to form a complex with the polypeptide under conditions and for a time sufficient to form the complex;
b) detecting the formation of the complex and, as a result, detecting the formation of a complex, the step of detecting the polypeptide of claim 10;
Including the method. A method for identifying a compound that binds to the polypeptide of claim 10 comprising:
a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide / compound complex;
b) detecting the complex, so that if the polypeptide / compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified; and
Including the method. A method for identifying a compound that binds to the polypeptide of claim 10 comprising:
a) contacting the compound with the polypeptide of claim 10 in a cell under conditions sufficient to form a polypeptide / compound complex, wherein the complex comprises a reporter gene sequence in the cell; Driving expression;
b) detecting the complex by detecting reporter gene sequence expression so that when the polypeptide / compound complex is detected, a compound binding to the polypeptide of claim 10 is identified. Process,
Including the method. A method for producing the polypeptide of claim 10, comprising:
a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of any of the polynucleotides of claims 1-684 under conditions sufficient to express the polypeptide in the cell Process and;
b) isolating the polypeptide from the cell culture or cells of step (a);
Including the method. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of any one of polypeptide SEQ ID NOs: 685 to 1368. 21. The polypeptide of claim 20, wherein the polypeptide is provided in a polypeptide array. A collection of polynucleotides consisting of at least one of SEQ ID NOs: 1-684. 23. The collection of claim 22, wherein the collection is provided on a nucleic acid array. 24. The collection of claim 23, wherein the array detects a perfect match against any one of the polynucleotides in the collection. 24. The collection of claim 23, wherein the array detects a mismatch to any one of the polynucleotides in the collection. 23. The collection of claim 22, wherein the collection is provided in a computer readable format.