CA2425827A1 - Novel nucleic acids and polypeptides - Google PatentsNovel nucleic acids and polypeptides
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- CA2425827A1 CA2425827A1 CA 2425827 CA2425827A CA2425827A1 CA 2425827 A1 CA2425827 A1 CA 2425827A1 CA 2425827 CA2425827 CA 2425827 CA 2425827 A CA2425827 A CA 2425827A CA 2425827 A1 CA2425827 A1 CA 2425827A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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NOVEL NUCLEIC ACIDS AND POLYPEPTIDES
1. TECHNICAL FIELD
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.
Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of .
expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNAlamino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.
Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.
3. SUMMARI' OF THE INVENTION
The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA
molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBIi), and in some cases, sequences obtained from one or more public databases.
The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins. These nucleic acid sequences are designated as SEQ m NO: 1-446. The polypeptides sequences are designated SEQ ID NO: 447-892. The nucleic acids and polypeptides are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is unknown or any of the four bases.
The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-446 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID
NO: I-446. A
polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-446 or a degenerate variant or fragment thereof. The identifying sequence can be 100 base pairs in length.
The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: I-446. The sequence information can be a segment of any one of SEQ ID NO: 1-446 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-446.
A collection as used in this application can be a collection of only one polynucleotide.
The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.
This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences;
and host cells or organisms transformed with these expression vectors. Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA
or RNA, their chemical analogs and the like.
In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-446 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art. In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-446 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome. .
The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ
ID NO: 1-446;
a polynucleotide comprising.any of the full length protein coding sequences of SEQ ID NO: 1-446; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-446. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID
NO: 1-446; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.
orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in SEQ ID NO: 447-892; or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-446; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and "substantial equivalents" thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) ofthe invention.
The invention also provides compositions comprising a polypeptide of the invention.
Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The invention also provides host cells transformed or transfected with a polynucleotide of the invention.
The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.
Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.
In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.
The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.
The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected. The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.
The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.
Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.
The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.
Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention. The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide%ompound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound the binds to a polypeptide of the invention is identified.
The methods of the invention also provide methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies. In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.
The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in Table 2); for which they have a signature region (as set forth in Table 3); or for which they have homology to a gene family (as set forth in Table 4). If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.
4. DETAILED DESCRIPTION OF THE INVENTION
It must be noted that as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.
The term "active" refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring poIypeptide.
According to the invention, the terms "biologically active" or "biological activity" refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.
Likewise "immunologically active" or "immunological activity" refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The term "activated cells" as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.
The terms "complementary" or "complementarity" refer to the natural binding of polynucleotides by base pairing. For example, the sequence 5'-AGT-3' binds to the complementary sequence 3'-TCA-5'. Complementarity between two single-stranded molecules may be "partial" such that only some of the nucleic acids bind or it may be "complete" such that total complementarity exists between the single stranded molecules:
The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.
The term "embryonic stem cells (ES)" refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.
The term "germ line stem cells (GSCs)" refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes. The term "primordial germ cells (PGCs)" refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells. PGCs are the source from which GSCs and ES cells are derived. The PGCs, the GSCs and the ES cells are capable of self renewal. Thus these cells not only populate~the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.
The term "expression modulating fragment," EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.
As used herein, a sequence is said to "modulate the expression of an operably linked sequence" when the expression of the sequence is altered by the presence of the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.
The terms "nucleotide sequence" or "nucleic acid" or "polynucleotide" or "oligonculeotide" are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material. In the sequences herein A is adenine, C is cytosine, T is thymine, G
is guanine and N is A, C, G or T (U). It is contemplated that where the polynucleotide is RNA, the T
(thymine) in the sequences provided herein is substituted with U (uracil).
Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
The terms "oligonucleotide fragment" or a "polynucleotide fragment", "portion," or "segment" or "probe" or "primer" are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides. The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides. Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides. Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules. A fragment or segment may uniquely identify each polynucleotide sequence of the present invention. Preferably the fragment comprises a sequence substantially similar to any one of SEQ lD NO: 1-446.
Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA
as described by Walsh et al. (Walsh, P.S. et al., 1992, PCR Methods Appl 1:241-250). They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art. Probes ofthe present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F.M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, both of which are incorporated herein by reference in their entirety.
The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-446. The sequence information can be a segment of any one of SEQ ID NO: 1-446 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-446. One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one set of chromosomes. Because 42° possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.
Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5. When these segments are used in arrays for expression studies, fifteen-mer segments can be used: The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise Iess than approximately 5% of the entire genome sequence.
Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer. The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1=4~) times the increased probability for mismatch at each nucleotide position (3 x 25). The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five. The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.
The term "open reading frame," ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.
The terms "operably linked" or "operably associated" refer to functionally related nucleic acid sequences. For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.
While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.
The term "pluripotent" refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism. A
pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.
The terms "polypeptide" or "peptide" or "amino acid sequence" refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules. A polypeptide "fragment," "portion," or "segment" is a stretch of amino acid residues of at least about S amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids. The peptide preferably is not greater than about 500 amino acids, more preferably less than 200 amino acids more preferably less than I SO amino acids and most preferably less than 100 amino acids. Preferably the peptide is from about 5 to about 200 amino acids. To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.
The term "naturally occurring polypeptide" refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
The term "translated protein coding portion" means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.
The term "mature protein coding sequence" means a sequence which encodes a peptide or protein without a signal or leader sequence. The "mature protein portion" means that portion of the protein which does not include a signal or leader sequence. °The peptide may have been produced by processing in the cell which removes any leader/signal sequence.
The mature protein portion may or may not include an initial methionine residue. The methionine residue may be removed from the protein during processing in the cell. The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.
The term "derivative" refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.
The term "variant"(or "analog") refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, a g., recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.
Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as Iigand-binding amities, interchain affinities, or degradation/turnover rate.
Preferably, amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. "Conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Insertions" or "deletions" are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells m chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
The terms "purified" or "substantially purified" as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).
The term "isolated" as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source. In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms "isolated" and "purified" do not encompass nucleic acids or polypeptides present in their natural source.
The term "recombinant," when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems. "Microbial" refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, "recombinant microbial" defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.
The term "recombinant expression vehicle or vector" refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
The term "recombinant expression system" means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.
Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed. The cells can be prokaryotic or eukaryotic.
The term "secreted" includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum. "Secreted"
proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P.A. and Young, P.R. (1992) Cytokine 4(2): 134 -143) and factors released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W.P. et. al.
(1998) Annu. Rev. Immunol. 16:27-55) Where desired, an expression vector may be designed to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell.
Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.
The term "stringent" is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in O.1X SSC/0.1% SDS at 68°C), and moderately stringent conditions (i.e., washing in 0.2X SSC/0.1% SDS at 42°C). Other exemplary hybridization conditions are described herein in the examples.
In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6X SSC10.05% sodium pyrophosphate at 37°C
(for 14-base oligonucleotides), 48°C (for 17-base oligos), 55°C
(for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).
. As used herein, "substantially equivalent" can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.
Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less). Such a sequence is said to have 65% sequence identity to the listed sequence. In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).
Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85%
sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99%
identity. Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code. Preferably, nucleotide sequence has at least about 65%
identity, more preferably at least about 75% identity, more preferably at least about 80%
sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90%
sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded. Sequence identity may be determined, e.g., using the Jotun Hein method (Rein, J. (1990) Methods Enzymol. 183:626-645). Identity between sequences can also be determined by other methods known in the art, e.g. by varying hybridization conditions.
The term "totipotent" refers to the capability of a cell to differentiate into all of the cell types of an adult organism.
The term "transformation" means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.
The term "transfection" refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed. The term "infection" refers to the introduction of nucleic acids into a suitable host cell 6y use of a virus or viral vector.
As used herein, an "uptake modulating fragment," UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below. The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.
Each ofthe above terms is meant to encompass all that is described for each, unless the context dictates otherwise.
4.2 NUCLEIC ACIDS OF THE INVENTION
Nucleotide sequences of the invention are set forth in the Sequence Listing.
The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-446; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 447-892; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 447-892. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ lD NO: 1-446; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing as SEQ
ID NO: 447-892; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) is a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 447-892. Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof;
domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains;
domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.
The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.
The present invention also provides genes corresponding to the cDNA sequences disclosed herein. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
Further S' and 3' sequence can be obtained using methods known in the art. For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO:
1-446 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-446 or a portion thereof as a probe. Alternatively, the polynucleotides of SEQ ID NO: 1-446 may be used as the basis for suitable primers) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.
The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene. The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.
The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.
Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.
Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-446, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e: specifically hybridize to) any one of the polynucleotides of the invention are contemplated'. Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.
The sequences falling within the scope ofthe present invention are not limited to these specific sequences, but also include allelic and species variations thereof.
Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-446, a representative fragment thereof, or a nucleotide sequence at least 90%
identical, preferably 95% identical, to SEQ ID NO: 1-446 with a sequence from another isolate of the same species. Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific OI2Fs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.
The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-446, can be obtained by searching a database using an algorithm or a program. Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S.F. J MoI. Evol. 36 290-300 (1993) and Altschul S.F. et al. J. Mol. Biol. 21:403-410 (1990)). Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.
Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.
The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation. Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.
These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous. Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to~l0 amino residues, preferably from 1 to S residues.
Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.
In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis. This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983). A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, 1s Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template DNA are used as starting material, primers) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. PCR
amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA
fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.
A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.
Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.
°The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.
In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-446, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.
Also included are the cDNA inserts of any of the clones identified herein.
A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.
Vectors accordingto the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ >D NO: 1-446 or a fragment thereof or any other polynucleotides of the invention. In one embodiment, the recombinant constructs of the present invention comprise a.vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-446 or a fragment thereof is inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNHl6a, pNHl8a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufinan et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R.
Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotidelexpression control sequence.
ao Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E.
coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Polynucleotides of the invention can also be used to induce immune responses.
For example, as described in Fan et al., Nat. Biotech. 17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA. The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.
4.3 ANTISENSE NUCLEIC ACIDS
Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ lD NO: 1-446, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense"
nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ lD NO: 447-892 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-446 are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence of the invention. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence of the invention. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ >D NO: 1-446), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, S-carboxymethylaminomethyluracil, dihydrouracil, beta-D=galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil; uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
To achieve su~cient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II
or pol III
promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641).
The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (moue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (moue et al.
(1987) FEBSLett 215: 327-330).
4.4 RIBOZYMES AND PNA MOIETIES
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoffand Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA. A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-446). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID
NO: 1-446 (see, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.
Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene.
(1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N. Y. Acad.
and Maher (1992) Bioassays 14: 807-15.
In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.
(1996) Bioorg Med Chem 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA
under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
PNAs of the invention can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S 1 nucleases (Hyrup B.
or as probes or primers for DNA sequence and hybridization (Hyrup et al.
Perry-O'Keefe (1996), above).
In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, e.g., RNase H and DNA polyrnerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).
The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., S'-(4-methoxytrityl)amino-S'-deoxy-thymidine phosphoramidite, can be used between the PNA and the S' end of DNA (Mag et al.
(1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a S' PNA segment and a 3' DNA segment (Finn et al.
(1996) above). Alternatively, chimeric molecules can be synthesized with a S' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g , for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
86:6553-6SS6; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.
W089/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents.
(See, e.g., Zon, 1988, Pharm. Res. S: 539-S49). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention. For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.
Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurnng promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences. See, for example, PCT
International Publication No. W094/12650, PCT International Publication No.
W092/20808, and PCT International Publication No. W091/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.
The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAF-dextran mediated transfection, or electroporation (Davis, L. et al., Basie Methods in Molecular Biology (1986)). The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an OI2F) or can be used to produce a heterologous protein under the control of the EMF.
Any host/vector system can be used to express one or more of the ORFs of the present invention. 'These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and S~ cells, as well as prokaryotic host such as E. coli and B.
subtilis. The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.
Mature proteins can be expressed in mammalian cells, yeast; bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N,ew York (1989), the disclosure of which is hereby incorporated by reference.
Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981). Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Co1o205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U93?, HaK or Jurkat cells. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding site's, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. I~NA
sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharo~nyces pombe, Kluyveromyces strains, C'andida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhirnurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the 2s control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic .
engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.
The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element;
for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occun-ing elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S.
Patent No. 5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (W093/09222) by Selden et al.; and International Application No.
PCT/US90/06436 (W091/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.
4.6 POLYPEPTIDES OF THE INVENTION
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ
1D NO: 447-892 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO:
1-446 or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO:
1-446 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ
ID NO: 447-892 or (c) polynucleofides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.
The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ 1D NO: 447-892 or the corresponding full length or mature protein; and "substantial equivalents" thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.
McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding 30~ sites.
The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.
The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences. The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell. The sequence ofthe mature form of the protein is also determinable from the amino acid sequence of the full-length form.
Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided. In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.
Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, earner.
The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By "degenerate variant" is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical 1 S polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.
A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. This technique is particularly useful in producing small peptides and fragments of larger polypeptides.
Fragments are useful, for example, in generating antibodies against the native polypeptide.
Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level. One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.
The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown. For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.
The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified. Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.
In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Protein.
Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in lllolecular Biology.
Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.
The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.
In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity ofthe binding molecule for SEQ ID NO: 447-892.
The protein of the invention may also be expressed as a product of transgenic animals, e.g , as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein. a The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications, in the peptide or DNA
sequence, can be made by those skilled in the art using known techniques.
Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S.
Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein. Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity. This type of analysis determines the importance of the substituted amino acids) in biological activity. Regions of the protein that are important for protein function may be determined by the eMATRIX program.
~ther fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are encompassed by the present invention.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBatTM kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseTM; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoa~nity chromatography.
Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRH, or as a His tag. Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("FLAG~") is commercially available from Kodak (New Haven, Conn.).
Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present f invention as an "isolated protein."
The polypeptides of the invention include analogs (variants). This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted. Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such analogs may exhibit improved properties such as activity and/or stability.
Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells. Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids. Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.
4.6.1 DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE IDENTITY
Preferred identity and/or similarity are designed to give the largest match between the sequences tested. Methods fo determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP
(Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, WI7, BLASTP, BLASTN, BLASTX, FASTA
(Altschul, S.F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S.F. et al., Nucleic Acids Res. vol. 25, pp. 3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp. Biol., Vol. 6, pp. 219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol. 4, pp. 202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol. 26(I), pp. 320-322 (1998), herein incorporated by reference), the GeneAtlas software (Molecular Simulations Inc. (MSI), San Diego, CA) (Sanchez and Sali (1998) Proc. Natl. Acad. Sci., 95, 13597-13602; Kitson DH et al, (2000) "Remote homology detection using structural modeling - an evaluation" Submitted; Fischer and Eisenberg (1996) Protein Sci. 5, 947-955), Neural Network SignaIP V 1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark), and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference). The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al. NCB
Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).
4.7 CHIMERIC AND FUSION PROTEINS
The invention also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" comprises a polypeptide of the invention operatively linked to another polypeptide. Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention. In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention. In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention. Within the fusion protein, the term "operatively linked" is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other. The polypeptide can be fused to the N-terminus or C-terminus.
For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.
In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.
In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprise one or more domains fused to sequences derived from a member of the immunoglobulin protein family.
The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo. The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand. Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.
A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT
PROTOCOLS n1 MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.
4.8 GENE THERAPY
Mutations in the polynucleotides of the invention may result in loss of normal function of the encoded protein. The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention. Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989);
Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).
Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex viva in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in viva for therapeutic purposes. Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.
Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art. Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.
The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.
Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.
Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.
See, for example, PCT International Publication No. WO 94/12650, PCT
International Publication No. WO 92/20808, and PCT International Publication No. WO
91/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.
In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA
The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Patent No. 5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International Application No.
PCT/CTS92/09627 (W093/09222) by Selden et al.; and International Application No.
PCT/US90/06436 (W091/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.
4.9 TRANSGEhTIC ANIMALS
In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Patent No. 5,557,032, incorporated herein by reference.
Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S.
Patent No 5,489,743 and PCT Publication No. W094/28122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.
The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.
Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.
In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Patent No. 5,557,032, incorporated herein by reference.
Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S.
Patent No 5,489,743 and PCT Publication No. W094/28122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.
4.10 USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins ofthe present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.
Thus, "therapeutic compositions of the invention" include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity. Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.
The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.
4.10.1 RESEARCH USES AND UTILITIES
The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA
sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA
antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F.
Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. I~immel eds., 1987.
4.10.2 NUTRITIONAL USES
Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the polypeptide ox polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
4.10.3 CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION
A polypeptide of the present invention may exhibit activity relating to cytokine, cell ,proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. A
polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RBS, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the invention can be used in the following:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M.
Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;
Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. ImmunoI. 149:3778-3783, 1992;
Bowman et al., I.
Immunol. 152:1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T
cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E.
e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, 'Toronto. 1994; and Measurement of mouse and human interleukin-y, Schreiber, R. D. In Current Protocols in Immunology. J. E.
e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E.
In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938,1983;
Measurement of mouse and human interleukin 6--Nordan, R. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991;
Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11--Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;
Measurement of mouse and human Interleukin 9--Ciarletta, A:, Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp.
6.13.1, John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M.
Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.
Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 1 I :405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-S 12, 1988.
4.10.4 STEM CELL GROWTH FACTOR ACTIVITY
A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells. Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.
The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.
It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LlF), Flt-3 ligand (Flt-3L), any ofthe interleukins, recombinant soluble IL-6 receptor fused to 1L-6, macrophage inflammatory protein 1-alpha (M1P-I-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).
Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.
Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations. This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.
Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo. Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see il.S. Patent No. 5,690,926).
Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types. These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mItNA to create cDNA libraries and templates for polymerase chain reaction experiments. These studies . would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.
Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions. For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders. The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue. In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.
Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types. A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker. The selectable marker allows only cells of the desired type to survive. For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.
Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In:
Principles of Tissue Engineering eds. Lanza et al., Academic Press (1997)). Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity. Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al. Proc. Natl. Acad.
Sci, U.S.A., 92:
7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines. The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g. as described by Bernstein et al., Blood, 77: 2316-2321 (1991).
4.10.5 HEMATOPOIESIS REGULATING ACTIVITY
A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or Lymphoid cell disorders.
Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; andlor in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.
Therapeutic compositions of the invention can be used in the following:
Suitable assays for proliferation and differentiation of various hematopoietic Lines are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, I 993.
Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in:
Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.
R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.
1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemachei, R. E. In Culture of Hematopoietic Cells. R. I.
Freshney, et al.
eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.
4.10.6 TISSUE GROWTH ACTIVITY
A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament andlor nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.
A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation ofartificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or orlcologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of 4~
bone-forming cells. Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes may also be possible using the composition of the invention. ' Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendonlligament formation. Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth oftendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.
Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate. A polypeptide of the present invention may also exhibit angiogenic activity.
A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A composition of the present invention may also be useful for promoting or inhibiting differentiation oftissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
Therapeutic compositions of the invention can be used in the following:
Assays for tissue generation activity include, without limitation, those described in:
International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal); International Patent Publication No.
W091/07491 (skin, endothelium).
Assays for wound healing activity include, without limitation, those described in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Dermatol 71:382-84 (1978).
4.10.7 IMMUNE STIMULATING OR SUPPRESSING ACTIVITY
A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A polynucleotide of the invention can encode a polypeptide exhibiting such activities. A protein may be useful in the treatment of various immune deficiencies and so disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein (or antagonists thereof, including antibodies) ofthe present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.
Other conditions,, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention. The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol.
73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.
Environ. Health 53:563-79).
Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of s~
an immune response. The functions of activated T cells may be inhibited by suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression of T
cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or energy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.
Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or.more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T
cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, a lack of costimulation may also be sufficient to energize the T cells, thereby inducing tolerance in a subject.
Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, 'it may also be necessary to block the function of a combination of B lymphocyte antigens.
The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Aced.
Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.
Examples include marine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T
cells in vivo.
A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and ~i2 microglobulin protein or an MHC
class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC
class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II
MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.
Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA
encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T
cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
I S The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.
M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;
Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad.
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986;
Takai et al., J. Immunol. 140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998;
Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/'Th2 profiles) include, without limitation, those described in: Maliszewski, J.
Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a.
Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. I 994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.
M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;
Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995;
Nair et al., Journal of Virology 67:4062-4069,1993; Huang et aL, Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989;
Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without (imitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et aL, Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et aL, Cytometry 14:891-897, 1993;
Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et aL, Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
4.10.8 ACTIVIN/INIiIBIN ACTIVITY
A polypeptide of the present invention may also exhibit activin- or inhibin-related activities. A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of su~cient amounts of other inhibins can induce infertility in these mammals. Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example, U.S. Pat. No. 4,798,885. A polypeptide of the invention may also be useful for advancement of the onset.of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.
The activity of a polypeptide of the invention may, among other means, be measured by the following methods.
Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986;
Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl.
Acad. Sci. USA 83:3091-3095, 1986.
4.10.9 CHEMOTACTIC/CHEMOKINETIC ACTIVITY
A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A
polynucleotide of the invention can encode a polypeptide exhibiting such attributes.
Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic compositions (e.g.
proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
Therapeutic compositions of the invention can be used in the following:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995;
Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.
4.10.10 HEMOSTATIC AND THROMBOLYTIC ACTIVITY
A polypeptide of the invention may also be involved in hemostasis or thrombolysis or thrombosis. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A
composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
Therapeutic compositions of the invention can be used in the following:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
4.10.11 CANCER DIAGNOSIS AND THERAPY
Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis. Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.
For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition. Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.
Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.
Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.
Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer. Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without s8 necessarily eradicating the cancer.
The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer treatment is routine. Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include:
Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCI
(Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCI, Doxorubicin HCI, Estramustine phosphate sodium, Etoposide (V 16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCI (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.
In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer. There are hereditary conditions and/or environmental situations (e.g.
exposure to carcinogens) known in the art that predispose an individual to developing cancers. Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.
In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment. These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY
Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J.
Natl. Can. Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl. J.
Dev. Biol., 40: 1189-97 (1999) and Li et al., Clin. Exp. Metastasis,17:423-9 (1999), respectively.
Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.
4.10.12 RECEPTOR/LIGAND ACTIVITY
A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions. A
polynucleotide of the invention can encode a polypeptide exhibiting such characteristics. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses. Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
The activity of a polypeptide of the invention may, among other means, be measured by the following methods:
Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
Margulies, E, M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp.
Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989;
Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).
Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.
Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.
The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods. ("Guide to Protein Purification" Murray P. Deutscher (ed) Methods in Enzymology Yol.
182 (1990) Academic Press, Iric. San Diego). Examples of radioisotopes include, but are not limited to, tritium and carbon-14 . Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.
Examples of toxins include, but are not limited, to ricin.
4.10.13 DRUG SCREE1~TING
This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof. Drugs are screened against such transformed cells in competitive binding assays.
Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.
Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.
Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as "hits" or "leads" via natural product screening.
The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves. Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998).
Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews and examples'of peptidomimetic libraries, see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997); Dorner et al., BioorgMed Chem, 4(5):709-15 (1996) (alkylated dipeptides).
Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or "lead") to optimize the capacity of the "hit" to bind a polypeptide of the invention. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.
The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention. Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.
4.10.14 ASSAY FOR RECEPTOR ACTIVITY
The invention also provides methods to detect specific binding of a polypeptide e.g. a ligand or a receptor. The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention. For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention. There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention. Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands,,or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not. The response of the two cell populations to the addition of ligands(s) are then compared.
Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s). As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined. For example, a chimeric protein in.which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell. The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor. Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.
Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.
4.10.15 ANTI-INFLAn~VIATORY ACTIVITY
Compositions of the present invention may also exhibit anti-inflammatory activity.
The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
Compositions ofthis invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.
Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides andlor polypeptides of the invention. Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, .chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).
4.10.17 NERVOUS SYSTEM DISORDERS
Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:
(i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;
(ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
(iii) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;
(iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;
(v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
(vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;
(vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention:
(i) increased survival time of neurons in culture;
(ii) increased sprouting of neurons in culture or in vivo;
(iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.
Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol.
70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron.
dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor' neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
4.T0.18 OTHER ACTIVITIES
A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites;
effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s);
effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects;
promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act ~as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
4.10.19 IDENTIFICATION OF POLYMORPHISMS
The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment. Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.
For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.
Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced. Alternatively, the DNA
may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides). In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA
depending on the presence or absence of the polymorphism) may be performed.
Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms. The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention. In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.
Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.
4.10.20 ARTHRITIS AND INh'LANIMATION
The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis is determined in an experimental animal model system. The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J.
Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.
Arch. Allergy Appl. Immunol., 23:129. Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA). The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture. The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. The control consists of administering PBS only.
The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24. At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.
4.11 THERAPEUTIC METHODS
The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods. Examples of therapeutic applications include, but axe not limited to, those exemplified herein.
One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.
While the mode of administration is not particularly important, parenteral administration is preferred. An exemplary mode of administration is to deliver an intravenous bolus. The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician. It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient. Typically, the amount of polypeptide administered per dose will be in the range of about O.OI p.g/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 p,g/kg to 10 mg/kg of patient body weight. For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.
The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.
4.12 PHARMACEUTICAL FORMULATIONS AND ROUTES OF
A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-g, IL-9, IL-10, IL-11, IL-12, II,-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-a and TGF-/3), insulin-like growth factor (IGF), as well as cytokines described herein.
The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects. Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side erects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-lRa, IL-1 Hyl,1L-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents). A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).
Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.
When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated. Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co- administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
4.12.1 ROUTES OF ADNIn~TISTRATION
Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation. In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes will be targeted to and taken up selectively by the afflicted tissue.
The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action. The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art. Preferably for wound treatment, one administers the therapeutic compound directly to the site. Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.
Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90%
by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.
When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing andlor dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system.
VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD
co-solvent system (VPD:SV~ consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers fox hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions. Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.
The pharmaceutical composition of the invention may be in the form of a complex of the proteins) or other active ingredients) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigens) to T lymphocytes. The antigen components could also be supplied as purified IVIHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T
cells can be combined with the pharmaceutical composition of the invention.
The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.
Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.
The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.
Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response. Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ~,g to about 100 mg (preferably about 0.1 ~g to about 10 mg, more preferably about 0.1 p,g to about 1 mg) of protein or other active ingredient of the present invention per kg body weight. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
Topical administration may be suitable for wound healing and tissue repair:
Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation.
Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging'from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and polyvinyl alcohol).
The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone andlor cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-oc and TGF-(3), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary applications.
Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
For example, the addition of other known growth factors, such as IGF I
(insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
4.12.3 EFFECTIVE DOSAGE
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the ICso as determined in cell culture (i.e., the concentration of the test compound which achieves a half maximal inhibition of the protein's biological activity). Such information can be used to more accurately determine useful doses in humans.
A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDSO (the dose lethal to 50% of the population) and the EDSO (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LDso and EDso. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch.
1 p.1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays uan be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 p.g/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 p,glkg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions fox administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
Also included in the invention are antibodies to proteins, or fragments of proteins of the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab. and F~ab')a fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
Certain classes have subclasses as well, such as IgG~, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as the amino acid sequences shown in SEQ ID NO:
447-892, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of -related protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for g1 targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of . polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies:
A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
4.13.1 POLYCLONAL ANTIBODIES
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
Additional examples of adjuvants which can be employed include MPL-TDM
adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, por an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoa~nity chromatography. Purification of immunoglobulins is discussed, for example, by D.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
4.13.2 MONOCLONAL ANTIBODIES
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse effciently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia.
Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);
Brodeur et al., Monoclonal Antibody Production Techniques and Applications Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be deternnined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA
also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
4.13.3 HUMANIZED ANTIBODIES
The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')Z
or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature. 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S.
Patent No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. 0_p. Struct. Biol.
4.13.4 HUMAN ANTIBODIES
Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV
hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol.. 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.
(Bio/Technolo~y 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnolo~y 4 845-51 (1996)); Neuberger (Nature BiotechnoloQV 4 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 ( 1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT
publication W094/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides alt the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse~ as disclosed in PCT publications WO
and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in s7 culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT
publication WO 99/53049.
4.13.5 Fab FRAGMENTS AND SINGLE CHAIN ANTIBODIES
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule;
(ii) an Fab fragment generated by reducing the disulfide bridges of an F~ab~)a fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F~ fragments.
4.13.6 BISPECIFIC ANTIBODIES
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by amity chromatography steps. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in WO 96/2701 l, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers 1 S which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments.
These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lyric activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.
See, Gruber et al., J. hnmunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one ofwhich originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as Fc~yRI (CD64), FcyRII (CD32) and FcyRIII
(CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
4.13.7 HETEROCONJUGATE ANTIBODIES
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360;
92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
For example, immunotoxins can be constructed using a disulfide exchange reaction .or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No. 4,676,980.
4.13.8 EFFECTOR FUNCTION ENGINEERING
It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.
For example, cysteine residues) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176:
(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A
chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of radionuclides are available for the production of radioconjugated antibodies.
Examples include 2i2Bi,13~h 131~~ soy and ~$~Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
4.14 COMPUTER READABLE SEQUENCES
In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, "computer readable media"
refers to any medium which can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM;
electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention. As used herein, "recorded" refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.
A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available sofl:ware such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.
By providing any of the nucleotide sequences SEQ ID NO: 1-446 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-446 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer sofl:ware is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE
(Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.
Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.
As used herein, "a computer-based system" refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, "data storage means" refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.
As used herein, "search means" refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.
Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA
(NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems. As used herein, a "target sequence"
can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues. However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.
As used herein, "a target structural motif," or "target motif," refers to any rationally selected sequence or combination of sequences in which the sequences) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.
There are a variety of target motifs known in the art. Protein target motifs include, but are S not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
4.15 TRIPLE HELIX FORMATION
In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.
Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense -Olmno, J. Neurochem: 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA
hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonueleotide.
4.16 DIAGNOSTIC ASSAYS AND HITS
The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.
In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample. Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.
In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.
In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.
Conditions for incubating a nucleic acid probe or antibody with a test sample vary.
Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.
One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G.R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.
Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.
In detail, a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or probe. Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.
4.17 MEDICAL IMAGING
°The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection). See, e.g., Kunkel et al., U.S. Pat. NO. 5,413,778. Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.
4.18 SCREENING ASSAYS
Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID
NO: 1-446, or bind to a specific domain of the polypeptide encoded by the nucleic acid. In detail, said method comprises the steps of (a) contacting an agent with an isolated protein encoded by an OItF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.
In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention.can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, arid detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.
Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.
Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.
Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound). Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound). Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.
The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.
For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be "rationally selected or designed"
when the agent is chosen based on the configuration of the particular protein.
For example, one skilled in the art can readily adapt currently available procedures to generate peptides, - pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides," In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.
In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above such agents can be randomly screened or rationally designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control. One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.
Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense -Okano, J.
Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.
Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.
4.19 USE OF NUCLEIC ACIDS AS PROBES
Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences. The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-446. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from any of the nucleotide sequences SEQ ID NO: 1-446 can be used as an indicator of the presence of RNA
of cell type of such a tissue in a sample.
Any suitable hybridization technique can be employed, such as, for example, in situ hybridization. PCR as described in US Patents Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences. Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.
The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.
Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA
probes. Such , vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences. The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY.
loo Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.
Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f). Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.
4.20 PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES
Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon. One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.
Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin. Microbiol.
using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey & Collins, (1989) Mol. Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989);
all references being specifically incorporated herein.
Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker. For example, Broude et al. (1994) Proc. Natl. Acad.
Sci. USA 91(8) 3072-6, describe the use of biotinylated probes, although these axe duplex probes, that are immobilized on streptavidin-coated magnetic beads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Of course, this same linking chemistry is applicable to coating any surface with streptavidin. Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, CA).
Nunc Laboratories (Naperville, IL) is also selling suitable material that could be used.
Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH. CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.
CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules may be bound ORFs of the present invention can to CovaLink exclusively at the 5'-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal. Biochem. 198(1) 138-42).
The use of CovaLink NH strips for covalent binding of DNA molecules at the 5'-end has been described (Rasmussen et al., (1991). In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res. 11(8) 6513-29). This is beneficial as immobilization using only a single covalent bond is preferred. The phosphoramidate bond joins the DNA to the CovaLinle NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm. To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5'-end phosphate group. It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.
More specifically, the linkage method includes dissolving DNA in water (7.5 ng/pl) and denaturing for 10 min. at 95°C and cooling on ice for 10 min. Ice-cold 0.1 M
1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM
1-MeIm7. The single-stranded DNA solution is then dispensed into CovaLink NH
strips (75 p,l/well) standing on ice.
Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm~, is made fresh and 25 p.1 added per well. The strips are incubated for 5 hours at 50°C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50°C).
It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern ~ Maskos), incorporated herein by reference. This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3'-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support. The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.
Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.
An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed. For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al. (1991) Science 251(4995) 767-73, incorporated herein by reference. Probes may also be immobilized on nylon supports as described by Van Ness et al. (1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.
Biochem. 169(1) 104-8; all references being specifically incorporated herein.
To link an oligonucleotide to a nylon support, as described by Van Ness et al.
(1991), requires activation of the nylon surface via alkylation and selective activation of the S'-amine of oligonucleotides with cyanuric chloride.
One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by reference). These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips). These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5'-protected N acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies. A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.
15~ 4.21 PREPARATION OF NUCLEIC ACID FRAGMENTS
The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC
inserts, and RNA, including mRNA without any amplification steps. For example, Sambrook et al.
(1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).
DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.
Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA samples may be prepared in 2-S00 ml of final volume.
The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.
Low pressure shearing is also appropriate, as described by Schriefer et al.
(1990) Nucleic Acids Res. 18(24) 7455-6, incorporated herein by reference). In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures. A
lever device allows controlled application of low to intermediate pressures to the cell. The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.
One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.
(1992) Nucleic Acids Res. 20(14) 3753-62. These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.
The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA
fragments form the small molecule pUCl9 (2688 base pairs). Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a Cvi3I** digest of pUCl9 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z
minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI**
restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.
As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 pg instead of 2-5 p,g); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.
This is achieved by incubating the DNA solution for 2-5 minutes at 80-90°C. °The solution is then cooled quickly to 2°C to prevent renaturation of the DNA fragments before they are contacted with the chip. Phosphate groups must also be removed from genomic DNA by methods known in the art.
4.22 PREPARATION OF DNA ARRAYS
Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane. Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 n1 of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density of the wells is achieved. One to 25 dots may be accommodated in 1 mm2, .depending on the type of label used. By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed. Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones. Each of the subarrays may represent replica spotting of the same samples. In one example, a selected gene segment may be amplified from 64 patients. For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).
A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples may be spotted on one 8 x 12 cm membrane. Subarrays may contain 64 samples, one from each patient.
Where the 96 subarrays are identical, the dot span may be 1 mma and there may be a 1 mm space between subarrays.
Another approach is to use membranes or plates (available from NUNC, Naperville, Illinois) which may be partitioned by physical spacers e.g. a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips. A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.
The present invention is illustrated in the following examples. Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects ofthe present invention not be limited to the disclosure ofthe following examples. The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention. Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.
All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.
5.1 EXAMPLE 1 Novel Nucleic Acid Seguences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques. The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts. Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences. The clones were clustered into groups of similar or identical sequences.
Representative clones were selected for sequencing.
In some cases, the 5' sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol. PCR products were purified and subjected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences 5.2 EXAMPLE 2 Assemblage of Novel Nucleic Acids The nucleic acids of the present invention, designated as SEQ ID NO: 1-446 were assembled using an EST sequence as a seed. Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST, gb pri, UniGene, and exons from public domain genomic sequences predicated by GenScan) that belong to this assemblage. The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage. Further, inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.
Using PHRAP (Univ. of Washington) or CAP4 (Paracel), full-length gene sequences and their corresponding protein sequences were generated from the assemblage.
Any frame shifts and incorrect stop codons were corrected by hand editing. During editing, the sequence was checked using FASTXY algorithm against Genbank (i.e., dbEST, gb pri, UniGene, and Genpept). Other computer programs which may have been used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and gc-zip-2 (Hyseq, Inc.). The full-length nucleotide sequences are shown in the Sequence Listing as SEQ ID NO:
1-446. The corresponding polypeptide sequences are SEQ ID NO: 447-892.
Table 1 shows the various tissue sources of SEQ ID NO: 1-446.
The nearest neighbor results for polypeptides encoded by SEQ ID NO: 1-446 (i.e.
SEQ 1D NO: 447-892) were obtained by a BLASTP (version 2.0a1 19MP-WashU) search against Genpept, Geneseq and SwissProt databases using BLAST algorithm. The nearest neighbor result showed the closest homologue with functional annotation for SEQ ID NO: 1-446. The translated amino acid sequences for which the nucleic acid sequence encodes are shown in the Sequence Listing. The homologues with identifiable functions for SEQ ID NO:
1-446 are shown in Table 2 below.
Using eMatrix software package (Stanford University, Stanford, CA) (Wu et al., J.
Comp. Biol., Vol. 6 pp. 219-235 (1999) herein incorporated by reference), polypeptides encoded by SEQ ID NO: 1-446 (i.e. SEQ ID NO: 447-892) were examined to determine whether they had identifiable signature regions. Table 3 shows the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-values) and the positions) of the signature within the polypeptide sequence.
Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res., Vol.
26(1) pp. 320-322 (1998) herein incorporated by reference) polypeptides encoded by SEQ 1D NO:
1-446 (i.e. SEQ ID NO: 447-892) were examined for domains with homology to certain peptide domains. Table 4 shows the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.
The GeneAtlas'2' software package (Molecular Simulations Inc. (MSI), San Diego, CA) was used to predict the three-dimensional structure models for the polypeptides encoded by SEQ ID NO: 1-446 (i.e. SEQ ID NO: 447-892). Models were generated by (1) PSI-BLAST which is a multiple alignment sequence profile-based searching developed by Altschul et al, (Nucl. Acids. Res. 25, 3389-3408 (1997)), (2) High Throughput Modeling (HTM) (Molecular Simulations Inc. (MSI) San Diego, CA,) which is an automated sequence and structure searching procedure (http://www.msi.comn, and (3) SeqFold~ which is a fold recognition method described by Fischer and Eisenberg (J. Mol. Biol. 209, 779-791 (1998)).
This analysis was carried out, in part, by comparing the polypeptides of the invention with the known NMR (nuclear magnetic resonance) and x-ray crystal three-dimensional structures as templates. Table 5 shows, "PDB ID", the Protein DataBase (PDB) identifier given to template structure; "Chain ID", identifier of the subcomponent of the PDB
"Compound Information", information of the PDB template structure and/or its subcomponents; "PDB Function Annotation" gives function of the PDB template as annotated by the PDB files (http:/www.rcsb.or DBE; start and end amino acid position of the protein sequence aligned; PSI-BLAST score, the verify score, the SeqFold score, and the Pgtential(s) of Mean Force (PMF). The verify score is produced by GeneAtlas'~
software (MSI), is based on Dr. Eisenberg's Profile-3D threading program developed in Dr. David Eisenberg's laboratory (US patent no. 5,436,850 and Luthy, Bowie, and Eisenberg, Nature, 356:83-85 (1992)) and a publication by R. Sanchez and A. Sali, Proc. Natl.
Acad. Sci. USA, 95:13597-12502. The verify score produced by GeneAtlas normalizes the verify score for proteins with different lengths so that a unified cutoff can be used to select good models as follows:
IO Verify score (normalized) _ (raw score- 112 high score)/(1/2 high score) The PFM score, produced by GeneAtlas'~ software (MSI), is a composite scoring function that depends in part on the compactness of the model, sequence identity in the alignment used to build the model, pairwise and surface mean force potentials (MFP). As 15 given in Table 5, a verify score between 0 to 1.0, with 1 being the best, represents a good model. Similarly, a PMF score between 0 to 1.0, with 1 being the best, represents a good model. A SeqFold'h' score of more than 50 is considered significant. A good model may also be determined by one of skill in the art based all the information in Table 5 taken in totality.
The nucleotide sequence within the sequences that codes for signal peptide sequences 20 and their cleavage sites can be determined from using Neural Network SignalP V 1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark). The process for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication " Identification of prokaryotic and eukaryotic signal peptides and prediction of 25 their cleavage sites" Protein Engineering, Vol. 10, no. l, pp. I-6 (1997), incorporated herein by reference. A maximum S score and a mean S score, as described in the Nielson et al, as reference, were obtained for the polypeptide sequences. Table 6 shows the position of the last amino acid of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.
30 Table 7 correlates each of SEQ ID NO: 1-446 to a specific chromosomal location.
Table 8 is a correlation table of the novel polynucleotide sequences SEQ ID
NO: 1-446, and their corresponding priority nucleotide sequences in the priority application USSN
09/687,527, herein incorporated by reference in its entirety.
Tissue RNA/'TissueLibrarySEQ ID NO:
Ori in Source Name adult GIBCO AB3001 31 35-36 52-53 57-59 63-64 69 brain 73-74 86-87 102 109 138 adult GIBCO ABD003 2 6-8 10 13 20-22 35-36 38-39 brain 43 45 51-54 58 60 64 68-adult Clontech ABR001 34 40 93 97 130 155 160 190 276 brain 307 341 436 adult Clontech ABR006 15 30 61 65 68 70 74 88 95 99 brain 106 109 113 129 134 138 adult Clontech ABR008 1 3-5 8-10 22 26 28-29 33-34 brain 37 42 46 51 55-56 58 62-adult Clontech ABR011 52 155 160 315 brain adult BioChain ABR012 64 67 164 284 brain adult BioChain ABR013 356 brain adult InvitrogenABR014 58 122 128 174 212 231 248 260 brain adult InvitrogenABR015 6-7 58 63 72 80 122-123 269 brain adult Invitro ABR016 20-21 36 58 93 131 167 217 285 brain en adult InvitrogenABT004 13 33 36 58 63 75 93 95 99 102 brain 107 120-121 123 127 cultured StratageneADP001 34-37 55 60 67 80 86-87 106 109 preadi 270 280 414 ocytes adrenal Clontech ADR002 8 19-21 25 36 42 44-45 47 55 gland 59 62 68 72-73 84 87-88 adult GIBCO AHR001 6-9 15 19-21 30 33-36 39 43 45 heart 49-51 53-55 57-59 61-64 adult GIBCO AKD001 6-8 10 12 15 20-22 25-26 28-30 kidney 33-34 36-37 39 4345 48 adult InvitrogenAKT002 3-4 6-9 13 23 28-29 34 36 61-63 kidney 68 70 76 95 97-99 I 15 Tissue 12NA/TissueLibrary. SEQ ID NO:
Ori in Source Name adult GIBCO ALG001 6-8 34 40 53 58-59 64 67-68 73 lung 76 109 112 118 129 134 lymph Clontech ALN001 37 39 56 62-63 67 99 104 149 node 152 163-164 174 196 217 young GIBCO ALV001 20-21 33 54-55 59-61 72 76 88 liver 95 100 115 121 123 125 adult InvitrogenALV002 30 36 39 51-52 69 75 84 88 119-121 liver 123 127 145 185 adult Clontech ALV003 26 80 liver adult InvitrogenAOV001 2-4 6-10 12 15 19-23 25-26 28-30 ovary 32-34 36-39 42-43 47 adult Invitro APL001 43 59 77 181 209 lacenta en lacenta InvitrogenAPL002 10 22 24 34 36 73 77 121 285 adult GIBCO ASP001 6-7 10 12 16-17 20-23 30 35 48 spleen 51 55 59 62-64 67 72 76 adult GIBCO ATS001 5 9 13 19-2I 34 39 49-50 59 62 testis 64 69 72 90 94-95 102 bladder InvitrogenBLD001 38 42 51 67 73 93 95 98 107 127 bone marrowClontech BMD001 2 6-7 9 1 I-12 19-21 23 33-34 bone marrowClontech BMD002 8-10 20-25 27 36-37 39-40 45 bone marrowClontech BMD004 40 64 279 adult InvitrogenCLN001 27 48 58 100 122 128 157 179 colon 185 212 246-247 317 355 mixture various GTL016 103-104 323 of 16 tissues! vendors*
mIZNA*s mixture various CTL021 64 179 260 323 445 of 16 tissues! vendors*
ml2NA*s adult BioChain CVX001 3-4 6-7 9-10 12-13 20-23 25-26 cervix 30 36-37 39-40 43 45 47 Tissue RNA/TissueLibrary SEQ ID NO:
Ori in Source Name dia hra BioChain DIA002 215 endothelialStratageneEDT001 2 5-13 19-23 28-30 32-37 39 cells 68-69 73 76 80-81 84 86-88 91-92 eso hagusBioChain ES0002 64 196 279 fetal Clontech FBR001 55 85 395 brain fetal Clontech FBR004 91-92 199-200 316 brain fetal Clontech FBR006 5 12 14 28-29 31 33-34 37 43 brain 46 58 61-63 65 68 73-74 fetal Clontech FBRS03 112 brain fetal InvitrogenFBT002 5 10 22 31 33-34 42 52 55 58 brain 64 66 73 75 84 98 107 109 fetal InvitrogenFHR001 64 67-69 86-87 90 202 206 213-215 heart 217 225 245 272-fetal Clontech FKD001 30 57 62 64 88 163 171 198 200 kidney 238 261 437-438 fetal Clontech FKD002 146 156 176 255 kidney fetal InvitrogenFKD007 122 316 kidney fetal Clontech FLG001 37 78 90 112 269 354 lun fetal InvitrogenFLG003 5 12 48 51 69 104 120 128 137 lung 177 I 94 202 212 216 250 fetal Clontech FLG004 63 76 126 lung fetal Columbia FLS001 1-15 18-50 52-58 60-113 115 liver- 118-120 122-124 126-128 spleen University 131-132134136142144-145149153158-159162-165 245-24? 255 259 261 272-fetal Columbia FLS002 5 9-12 15 20-26 28-31 34-35 liver- 38-41 44 47 49-50 53-55 64 spleen University 67-69 71-75 78-79 81 85-89 91-92 fetal Columbia FLS003 1 12 36 61 74 78 88 111 125 liver- 174 221 291 378 433 spleen University fetal InvitrogenFLV001 10 13 22 31 33 36 41 60 69 84 liver 114 I20-121 126 164 219 fetal Clontech FLV002 261313 liver Tissue RNA/TissueLibrary~~ SEQ ID NO:
Ori in Source _ Name fetal Clontech FLV004 16-17 36 53 68 80 86-87 132 171 liver 179 183 204 221 272-fetal InvitrogenFMS001 28-29 31 36 45 48 62 67 74 102 muscle 107 122 181 196 208 fetal InvitrogenFMS002 5 23 38 51 61 85 90 102 108 151 muscle 174 183 187 189 204 fetal InvitrogenFSK001 8 11 23 30 36 45 48 51 53 58 skin 60.64 67 70 73 81 84 86-87 fetal InvitrogenFSK002 8 31 39 67 79 86-87 90 97 118 skin 168 174 181 203 207 216 fetal BioChain FSP001 67 203 238 s Teen umbilicalBioChain FUC001 15 20-21 33 36 38-39 51 54 59-60 cord 97-98 103-104 109 117-118 120 fetal GIBCO HFB001 3-4 8-10 12 15 18-22 30-31 33-34 brain 36 43 45 47 52 55 57-infant Columbia IB2002 5 8-9 13 15-17 20-21 25-26 28-29 brain 31 33 36 43 51-56 59 University 67-70 73 80 84 86-88 90 93 95 infant Columbia IB2003 5 10-11 22 40 42 46 51-52 54 brain 56 62 65 70 93 97-98 102 University 117 121 123 128 134-135 140 151 infant Columbia IBM002 93 95 123 140 181 brain University infant Columbia IBS001 54 73 93 123 176 188 220 255-256 brain 331 University lung, StratageneLFB001 6-7 32 35 55 60 64 71 103-104 ~ 109-110 118 123 128 137 fibroblast 140 145 161 163 175 187 193 217 lung tumorInvitrogenLGT002 3-4 6-7 10-12 14-15 20-22' 27 34 36 38-39 42 48 5.1-52 LymphocytesATCC LPC001 6-7 9 16-17 25 28-29 33 36 53 Tissue )ttNA/TissueLibrarySEQ ID NO:
Ori 'n Source Name LeukocyteGIBCO LUC001 6-8 12 16-17 19-21 23 25 28-30 LeukocyteClontech LUC003 51 62 68 70 73 80 95 97 117 163 melanoma Clontech MEL004 9 15 20-21 34 51-52 61 64 68 from cell 124 163 172 186-187 196 223-224 line 226-227 258 262 291 ATCC #CRL 302 341 396 415 mammary InvitrogenMMG001 8 10 13 15 22 28-29 33-34 36-37 gland 63 72-73 84-85 88 90 95 98 102 induced StratageneNTD001 9 36 45 68 73 76 97 106 112 I
neuron 179 264 306 341 376 401 cells retinoic StratageneNTR001 36 118 134 221 261 401 418 acid-induced neuronal cells neuronal StratageneNTU001 33 36 46 68 72 81 91-92 98 102 cells 112 160 182 190-191 pituitaryClontech PIT004 20-21 36 55 65 68 137-138 148 land 356 430 placenta Clontech PLA003 12 30 67 194 302 417 436 prostate Clontech PRT001 9-10 22-23 29 36 38 43 112 118 rectum InvitrogenREC001 27 42 60 69-70 98 103-104 123 salivary Clontech SAL001 6-7 9 33 48 53 62 157 164 170 gland 177 190 194 257 268 287 skin fibroblastATCC SFB001 63 112 skin fibroblastATCC SFB003 I 12 small Clontech SIN001 9-10 12 22 30 33 36 40 45 52 intestine 119 123-124 127 129 134 136 149-I
skeletal Clontech SKM001 3-4 6-7 20-21 64 103-104 120 muscle 215 278-279 330 386 skeletal Clontech SKMS04 42 muscle spinal Clontech SPC001 9 12 33-34 36 38-39 45 53 56 cord 58 61 64 66 78 86-87 90 adult Clontech SPLc01 25 125 136 138 168 171 176 275 s Teen 416 stomach Clontech STO001 69 73 94 97 100 141 177 231 233 thalamus Clontech THA002 58 72 78 93 127 133 138 160 184 Tissue ItNA/TissueLibrarySEQ ID NO:
Ori in Source Name thymus Clontech THM001 6-7 9 1216-17 19 33 42 59 61 222 231 237 239 243 272-274'299 thymus Clontech THMc02 6-7 9 12 14 16-17 19 28-29 37-38 thyroid Clontech THR001 3-4 6-7 9-10 12 20-22 25-26 30 gland 36 39-40 42 47 53-S4 59-trachea Clontech TRC001 6-7 36 S9 78 127 152 190 240 uterus Clontech UTR001 3-4 59 118 123 137 177 217 219 * The 16 tissuelmRNAs and their vendor sources are as follows: 1) Normal adult brain mRNA (Invitrogen), 2) Normal adult kidney mRNA (Invitrogen), 3) Normal fetal brain mRNA (Invitrogen), 4) Normal adult liver mRNA (Invitrogen), 5) Normal fetal kidney mRNA (Invitrogen), 6) Normal fetal liver mRNA (Invitrogen), 7) normal fetal skin mRNA
(Invitrogen), 8) human adrenal gland mRNA (Clontech), 9) Human bone marrow mRNA
(Clontech), 10) Human leukemia lymphoblastic mRNA (Clontech), 11) Human thymus mRNA (Clontech), 12) human lymph node mRNA (Clontech), 13) human so\spinal cord mRNA (Clontech), 14) human thyroid mRNA (Clontech), 15) human esophagus mRNA
(BioChain), 16) human conceptional umbilical cord mRNA (BioChain).
SEQ AccessionSpecies Description Score ID
NO: No. Identi 447 AAB87354Homo Sapiens22-MAY-2001 31-AUG-2000731 100 Human gene 13 encoded secreted protein HFVJP07, SEQ ID N0:95.
448 X97490 Mus musculusPNG rotein 739 96 449 AK001950Homo SapiensFLJ11088 fis, clone 1157 100 PLACE1005287, weakly similar to INNER
450 AK001950Homo SapiensFLJ11088 fis, clone 790 73 PLACE1005287, weakly similar to INNER
451 AB044385Homo SapiensmRNA for transmembrane 4492 99 molecule with thrombospondin module, complete cds.
452 BC005361Homo Sapiensproteasome (prosome, 1334 100 macropain) subunit, alpha type, 4, clone MGC:12467 IMAGE:3685931, mRNA, com lete cds.
453 BC005361Homo Sapiensproteasome (prosome, 1098 100 macropain) subunit, alpha type, 4, clone MGC:12467 IMAGE:3685931, mRNA, com lete cds.
454 AK001930Homo sapiensFLJ11068 fis, clone 1742 100 PLACE1004918, weakly similar to L-LACTATE
DEHYDROGENASE M CHAIN
455 AF151042Homo SapiensHSPC208 740 100 456 AL365512Homo Sapienshuman gene mapping to 251 99 chomosome 22. I
457 AF279307Homo Sapiensfunction IB (ASF1B) 1075 99 mRNA, complete cds.
458 AF212243Homo sa mRNA, complete cds. 1104 100 iens 459 AAY13360Homo sapienS25-JUN-1999 16-SEP-19982350 100 Amino acid sequence of protein PR0269.
460 AAB65692Homo Sapiens27-MAR-2001 26-MAY-20002758 96 Novel protein kinase, SEQ
ID NO: 220.
461 AK001061Homo SapiensFLJ10199 fis, clone 1305 100 HEMBA1004850.
462 AF042380Homo Sapiensadaptor protein (Grf40)1785 100 mRNA, complete cds.
463 AF042380Homo Sapiensadaptor protein (Grf40)809 100 mRNA, complete cds.
464 AL137422Homo SapienscDNA DKFZp761A1623 (from410 98 clone DKFZp761A1623); partial cds.
465 AF220193Homo Sapienshypothalamus protein 1039 100 HT007 mRNA, complete cds.
466 AAB60505Homo sapiens24-APR-2001 21-JUL-20003419 100 Human cell cycle and proliferation protein CCYPR-53, SEQ ID N0:53.
467 AAB69556Homo Sapiens27-APR-2001 10-MAR-20003315 99 Human Repro-EN-1.0 protein.
468 AL365512Homo Sapienshuman gene mapping to 2294 99 chomosome 22.
469 AAB90821Homo Sapiens15-JUN-2001 02-OCT-20003011 99 Human shear stress-response protein SEQ ID
470 AF195821Homo Sapiens(TNG2) mRNA, com lete 562 100 cds.
471 AK000399Homo sa FLJ20392 fis, clone 2281 99 iens KAIA4653.
472 AK001240Homo SapiensFLJ10378 fis, clone 1654 100 NT2RM2002004, weakly similar to LA
SEQ AccessionSpecies Description Score ID No. Identi NO:
473 Y11339 Homo Sapiensfor GaINAc alpha-2, 3182 100 6-sialyltransferase I, long form.
474 AAY90296Homo Sapiens24-OCT-2000 I1-JAN-2000936 100 Human a tidase, HPEP-13 rotein se uence.
475 AAY90296Homo Sapiens24-OCT-2000 1 I-JAN-20002087 99 Human a tidase, HPEP-13 protein se uence.
476 AJ250193Mus musculusmuscle rotein 637 730 72 477 AK001706Homo sa FLJ10844 fis, clone 959 100 iens NT2RP4001353.
478 AAB56847Homo Sapiens13-MAR-2001 08-MAR-2000749 100 Human prostate cancer antigen protein sequence SEQ ID N0:1425.
479 AB049952Homo SapiensmRNA for mitochondria) 1074 100 ribosomal protein S 18a, com lete cds.
480 AK011757Mus musculusutative 589 100 481 BC012167Homo sapiensSimilar to RIKEN cDNA 899 99 gene, clone MGC:20409 IMAGE:4637888, mRNA, complete cds.
482 AF038129Ovis ariesolyubi uitin ~ 771 100 483 AK012782Mus musculusutative 2562 92 484 AK001214Homo sa FLJ10352 fis, clone 2770 100 iens NT2RM2001152.
485 AK021681Homo sapiensFLJ11619 fis, clone 2337 100 HEMBA1004131, moderately similar to HOMOLOG.
486 AJ252060Homo Sapiensfor TRABID protein (TRABID3796 100 gene).
487 AL137301Homo SapienscDNA DKFZp434N1429 (from261 60 clone DKFZp434N1429); artial cds.
488 BC007588Homo SapiensSimilar to RIKEN cDNA 1328 92 gene, clone IMAGE:3342825, mRNA, partial cds.
489 AB015335Homo SapiensmRNA, partial cds. 617 100 490 AAY66765Homo Sapiens05-APR-2000 02-JUN-19991251 99 Membrane-bound rotein PR01384.
491 AC005154Homo Sapiensclone RP4-777023 from 994 100 7p14-p15, com fete sequence.
492 AF155140Homo Sapienstesticular RNA helicase1902 99 mRNA, complete cds.
493 AK001760Homo SapiensFLJ10898 fis, clone 2575 99 NT2RP5003492.
494 BC007396Homo Sapiensclone IMAGE:3834655, 1428 100 mRNA, partial cds.
495 AK001374Homo sa FLJ10512 fis, clone 2604 100 iens NT2RP2000658.
496 AK001374Homo sa FLJ10512 fis, clone 1902 97 iens NT2RP2000658.
497 AK000507Homo SapiensFLJ20500 fis, clone 1189 100 KAT09159.
498 AK000650Homo SapiensFLJ20643 fis, clone 1490 99 KAT02633.
499 AK001766Homo SapiensFLJ10904 fis, clone 2403 100 OVARC1000013, weakly similar to APOPTOTIC
500 AF233224Homo Sapiensprotein FBS (FBS) mRNA,1698 100 - complete cds.
501 BC005030Homo Sapiensclone MGC:12628 IMAGE:3690254,1853 100 mRNA, complete cds.
502 AK001449Homo sa FLJ10587 fis, clone 3440 100 iens NT2RP2004042.
503 AF326206Homo Sapienstranscription factor 2149 99 mRNA, complete cds.
504 AF220191Homo Sapienshypothalamus protein 1099 100 HSMNPI mRNA, com lete cds.
505 AF155096Homo Sapiensantigen mRNA, partial 2008 98 cds.
506 BC008250Homo SapiensSimilar to RIKEN cDNA 1332 100 SEQ AccessionSpecies Description Score ID
NO: No. Identi gene, clone MGC:9282 IMAGE:3872059, mRNA, complete cds.
507 AAY90287Homo Sapiens24-OCT-2000 11-JAN-20002514 100 Human a tidase, HPEP-4 protein se uence.
508 BC000802Homo SapiensSimilar to ribosomal 976 99 protein S9, clone MGC:5482 IMAGE:3452221, mRNA, com fete cds.
509 BC010011Homo SapiensSimilar to RIKEN cDNA 1691 97 gene, clone MGC:15407 IMAGE:4309613, mRNA, com lete cds.
510 AK010605Mus musculusputative 1023 99 511 AK020026Mus musculusputative 799 97 512 AB051853Homo Sapiensgene for rho-GTPase 1119 100 activating protein, com fete cds.
513 J04695 Mus musculusalpha-2 type IV collagen4444 87 514 AL512733Homo SapienscDNA DKFZp762P093 (from1380 100 clone DKFZ 762P093 .
515 AF284752Homo sa GK004) mRNA, com lete 654 100 iens cds.
516 AK001055Homo sa FLJ10193 fis, clone 767 100 iens HEMBA1004763.
518 AB037669Homo SapiensmRNA for L-type amino 2790 100 acid transporter 2, com lete cds.
519 AC007059Homo sa 19, cosmid 826549, com 4163 100 iens lete sequence.
520 AF119863Homo sa PRO2160 483 100 iens 521 AF151063Homo SapiensHSPC229 984 100 522 BC012123Homo Sapiensgolgi phosphoprotein 1528 100 3, clone MGC:20187 IMAGE:4558305, mRNA, complete cds.
523 BC002717Homo SapiensSimilar to chorionic 1128 100 somatomammotropin hormone (placental lactogen), clone MGC:3714 IMAGE:3631916, mRNA, com lete cds.
524 AAB36522Homo Sapiens07-MAR-2001 13-APR-20003431 99 Human CLASP related protein sequence SEQ ID
525 AAB08763Homo Sapiens02-JAN-2001 29-FEB-2000608 99 A human leukocyte and blood related protein (LBAP).
526 BC001810Homo Sapiensenolase 1, (alpha), 2206 99 clone MGC:2414 IMAGE:2906988, mRNA, complete cds.
527 AAY87267Homo Sapiens11-MAY-2000 25-JUN-19991790 100 Human signal peptide containing protein HSPP-44 SEQ ID N0:44.
528 AK002043Homo SapiensFLJ11181 fis, clone 682 100 PLACE1007460.
529 AKOOI795Homo sa FLJI0933 fis, clone 891 100 ienS OVARC1000605.
530 AF182412Homo Sapiens(MDS025) mRNA, complete1104 98 cds.
531 AF345564Homo sa FKSG76 1327 99 iens 532 AK008759Mus musculus, putative 1314 96 533 BC010543Homo Sapiensclone MGC:17544 IMAGE:3462146,1093 100 mRNA, com lete cds.
534 AK023510Homo SapiensFLJ13448 fis, clone 1257 100 PLACE1002993.
535 AL110245Homo SapienscDNA DKFZp434F011 (from301 91 clone DKFZ 434F011); artial cds.
536 U04520 Homo SapiensIV collagen alpha 5 3630 100 chain (COL4A5) gene, exon 51 and complete cds, alternatively spliced.
537 AK000516Homo SapiensFLJ20509 fis, clone 1088 100 KAT09623.
SEQ AccessionSpecies ~ Description Score ID
NO: No. Identi 538 AK000516Homo sa FLJ20509 fis, clone 788 100 iens KAT09623.
539 AC004865Homo sa clone RP4-728D4, com 3759 100 iens lete se uence.
540 AF286162Homo Sapiens4-phosphate Adaptor _ 99 Protein-1 mRNA, 1570 com lete cds.
541 AL591714Homo sa human gene ma ing to 821 100 iens chomosome 20.
542 AX179297Homo Sapiens21615 ADH 1243 100 543 AL136844Homo sapienscDNA DKFZp434G1730 (from_ 100 clone 1583 DKFZp434G1730); com lete cds.
544 AK001371Homo sa FLJ10509 fis, clone 3677 100 iens NT2RP2000617.
545 AK000213Homo sa FLJ20206 fis, clone 2343 100 iens COLF1582.
546 AK008020Mus musculusputative 1919 71 547 AF119870Homo SapiensPR02266 616 100 548 AK000763Homo sa FLJ20756 fis, clone 3152 100 iens HEP01556.
549 AK023550Homo SapiensFLJ13488 fis, clone 1215 99 PLACE1003915, weakly similar to PROBABLE
ARGINYL-TRNA SYNTHETASE, CYTOPLASMIC (EC 126.96.36.199 .
550 AK023550Homo SapiensFLJ13488 fis, clone 2069 99 PLACE1003915, weakly similar to PROBABLE
ARGINYL-TRNA SYNTHETASE, CYTOPLASMIC (EC 188.8.131.52).
551 AC002126Homo Sapiensfrom chromosome 19-cosmids449 100 R30102:R29350:R27740 containing MEF2B, genomic sequence, complete sequence.
552 BC012182Homo Sapiensclone MGC:20469 IMAGE:4554554,1582 99 mRNA, complete cds.
553 AL136528Homo sapiensDNA sequence from clone271 100 1092A11 on chromosome 1p36.2-36.33 Contains the gene for KIAA0495 protein, the TP73 (tumor protein p73) gene, a gene containing a WD
repeat domain, ESTs, STSs, GSSs and CpG Islands, com lete se uence.
554 AF334161Homo Sapiensfinger protein mRNA, 1561 98 complete cds.
555 AJ277587Homo SapiensmRNA for Spir-1 protein3012 99 (Spir-1 gene).
556 AY014283Homo SapiensmRNA, com lete cds. 1066 100 557 AF090938Homo sapiensHQ0628 PR00628 mRNA, 278 100 complete cds.
558 AF161511Homo SapiensHSPC162 480 100 559 AF039942Homo Sapienstranscription factor 1382 100 Zhangfei (ZF) mRNA, com fete cds.
560 AF271782Homo SapiensmRNA, complete cds. 1280 100 561 AF107495Homo Sapiensand putative FWP002 783 100 mRNA, complete cds.
562 AK015086Mus musculusputative 183 70 563 AL353936Homo SapienscDNA DKFZp761K1423 (from533 100 clone DKFZp761 K I 423).
564 X87241 Homo sa mRNA for hFat protein. 1997199 iens 565 BC004896Homo SapiensSimilar to ribosomal 1494 100 protein, mitochondrial, L5, clone MGC:3400 IMAGE:3529006, mRNA, complete cds.
566 AB062594Bos taurusutative 704 87 567 AL136683Homo SapienscDNA DKFZp564D0478 (from1034 100 clone DKFZp564D0478); complete cds.
~ 568 AAY87355Homo sapiens11-MAY-2000 25-JUN-1999952 100 ~ ~ ~ Human ~ ~
SEQ AccessionSpecies Description Score ID
NO: No. Identi signal peptide containing protein HSPP-132 SEQ ID N0:132.
569 BC008967 Homo Sapiensclone IMAGE:3010666, 1024100 mRNA, partial cds.
570 AK022754 Homo SapiensFLJ12692 fis, clone NT2RM4002623,242599 weakly similar to ASPARTYL-TRNA
SYNTHETASE (EC 184.108.40.206).
571 AF083106 Homo Sapienstype 1 (SIRTI) mRNA, 3929100 complete cds.
572 AK000017 Homo SapiensFLJ20010 fis, clone ADKA03268.611 100 573 AF308801 Homo sapiensprotein sorting protein 254199 16 (VPS16) mRNA, com lete cds.
574 BC001686 Homo Sapiensmethionine adenosyltransferase131598 II, alpha, clone MGC:2907 IMAGE:3010820, mRNA, com lete cds.
575 AK000675 Homo sapiensFLJ20668 fis, clone KAIA585.1474100 576 X68242 Homo sapiensmRNA for Hin-1. 757 100 577 BC001245 Homo sapiensSimilar to uncharacterized150499 bone marrow protein BM036, clone MGC:4957 IMAGE:3460193, mRNA, complete cds.
578 BC009782 Homo Sapienshypothetical protein 432 98 dJ12208.2, clone MGC:13493 IMAGE:4092710, mRNA, complete cds.
579 AL133109 Homo SapienscDNA DKFZp566N1047 (from341699 clone DKFZ 566N1047); artial ' cds.
580 AF161494 Homo sa HSPC145 1562100 iens 581 AAY22465 Homo Sapiens29-SEP-1999 17-DEC-1998 216 23 Human hippocam al sel-10 protein sequence.
582 AF312864 Homo sa mRNA, com lete cds. 627 100 iens 583 AAY70236 Homo Sapiens06-JUN-2000 20-AUG-1999 2310100 Human RNA-associated protein-17 (RNAAP-17).
584 AF240769 Macaca VAMP-2 ~ 584 100 mulatta 585 AAB98084 Homo Sapiens16-AUG-2001 26-OCT-2000 248299 Human rotein sequence SEQ ID
586 AK002058 Homo SapiensFLJ11196 fis, clone PLACE1007688,255199 weakly similar to LA
587 AK000500 Homo SapiensFLJ20493 fis, clone KAT08512.834 100 588 AF251296 Homo SapiensmRNA, com lete cds. 1299100 589 AF169149 Homo Sapiens(CABPI) mRNA, complete I 99 cds. 172 590 M96859 Homo Sapiensdipeptidyl aminopeptidase224652 like protein mRNA, com lete cds.
591 AAB88489 Homo Sapiens23-MAY-2001 07-JUL-2000 967 100 Human membrane or secretory protein clone PSEC0265.
592 AB063495 Mus musculusSpred-1 220592 593 AF155661 Homo sapiensdehydrogenase (PDH) mRNA,3050100 complete cds.
594 AAY90962 Homo Sapiens05-SEP-2000 12-OCT-1999 140399 Human 6713 protein sequence SEQ ID N0:5.
595 AF315378 Rattus suppressor of profilinlp4l197598 of actin-norvegicusrelated complex 2/3 596 AB036693 Homo Sapiensfor RAB9-like protein, 1067100 com lete cds.
597 AF359284 Homo SapiensmRNA, complete cds. 500499 598 AK001877 Homo SapiensFLJ11015 fis, clone PLACE1003302,274699 SEQ AccessionSpecies Description Score ID
NO: No. Identi highly similar to ZINC
599 AAB71913Homo Sapiens09-MAY-2001 16-AUG-20001516 100 Human ISOM-5.
600 L27867 Rattus neurexophilin 1448 98 norvegicus 601 AC004991Homo Sapiensclone RPS-1186C1 from 311 100 7q21.2-q31.1, complete sequence.
602 AF057019Dictyosteliuminteraptin 146 26 discoideum 603 AF247177Mus musculussphingosine-1-phosphate523 36 phosphohydrolase 604 BC007704Homo Sapiensclone MGC:10277 IMAGE:3952366,746 100 mRNA, complete cds.
605 UI8920 Homo Sapienschromosome 17q12-21 455 81 mRNA, clone pOV-3, partial cds.
606 U48363. Mus musculusal ha-NAC, muscle-s 810 30 ecific form p220 607 AF014008Bos taurusmyocardial vascular 490 100 inhibition factor 608 AL136604Homo SapienscDNA DKFZp564F2122 (from2716 96 clone ' DKFZp564F2122); com lete cds.
609 AK007689Mus musculusutative 289 100 610 AAB57020Homo Sapiens13-MAR-2001 08-MAR-2000384 100 Human prostate cancer antigen protein sequence SEQ ID N0:1598.
611 AAB20328Homo Sapiens29-MAY-2001 14-SEP-2000798 100 Human protein hosphatase and kinase protein-7.
612 BC007618Homo Sapiensclone MGC:15730 IMAGE:3355289,2163 100 mRNA, com lete cds.
613 AAZ94941Homo SapiensO1-AUG-2000 29-SEP-1999654 100 Human carbohydrate-associated protein CRBAP-1 cDNA.
614 M91669 Homo SapiensBullous pemphigoid autoantigen8016 99 gene, 3' end.
615 AF116649Homo sa PR00566 248 100 iens 616 AK001837Homo SapiensFLJ10975 fis, clone 2198 100 PLACE1001383, weakly similar to ZINC-FINGER
617 AF116672Homo sa PR01905 553 99 iens 618 BC011707Homo sapiensnuclear receptor binding1471 100 factor-2, clone MGC:19778 IMAGE:3687848, mRNA, complete cds.
619 AL133606Homo SapienscDNA DKFZp434A2017 (from5012 100 clone DKFZp434A2017); partial cds.
620 AAY58618Homo SapiensI I-APR-2000 I 1-JUN-19991778 100 Protein regulating gene expression PRGE-11.
621 AF276707Homo sapienscarcinoma susceptibility121 100 protein I
(HCCA3) mRNA, complete cds.
622 AF161554Homo sa HSPC069 3072 98 iens 623 AAY73327Homo Sapiens24-FEB-2000 04-MAY-19991668 100 HTRM
clone 0 52927 protein sequence.
624 AAP60958Homo sapiens_ 564 100 Sequence of human endogenous benzodiazepineoid(EBZD) polypeptide.
625 AK010262Mus musculusputative 1767 94 626 AK001317Homo sa FLJI0455 fis, clone 2539 99 iens NT2RP1001014.
627 M80899 Homo Sapiensnovel protein AHNAK 6618 99 mRNA, artial SEQ AccessionSpecies Description ~ ~ Score ID
NO: No. Identi se uence.
628 AAB21018Homo Sapiens19-DEC-2000 28-JAN-2000 262999 Human nucleic acid-bindin protein, NuABP-22.
629 LI9183 Homo sa MAC30 mRNA, 3' end. 901 97 iens 630 BCOOOS40Homo SapiensDKFZP434H132 protein, 739 100 clone MGC:3034 IMAGE:3163610, mRNA, com lete cds.
631 BC002857Homo Sapiensclone MGC:3442 IMAGE:3636594,1033100 mRNA, complete cds.
632 AAW78188Homo Sapiens13-APR-1999 1 I-JUN-19981300100 Human secreted protein encoded by gene 63 clone HPMCC16.
633 AKOOOS87Homo sa FLJ20580 fis, clone REC00516.848 100 iens 634 AF116637Homo sa PR01489 266 100 iens 635 BCOI Mus musculusRIKEN cDNA 1110060018 124289 1495 ene 636 AL157473Homo SapienscDNA DKFZp761L0424 (from216099 clone DKFZp761 L0424).
637 AFOOS067Homo sa mRNA, complete cds. 14156S
iens 638 AL117532Homo SapienscDNA DKFZp434E192 (from 3706100 clone DKFZp434E 192); partial cds.
639 AF2S1441Homo Sapiensmotif and leucine zipper4234100 containing kinase AZK mRNA, com lete cds.
640 BC010493Homo Sapiensclone MGC:16982 IMAGE:3048997,249699 mRNA, complete cds.
641 AK017S31Mus musculusputative 79S SO
642 BC000204Homo Sapiensribosomal protein S3A, 1367100 clone MGC:3109 IMAGE:3350750, mRNA, complete cds.
643 AF226076Homo sa (CHRACIS) mRNA, complete651 100 iens cds.
644 AK023267Homo SapiensFLJ1320S fis, clone NT2RP3004534,412999 highly similar to Mouse oncogene (ect2) mRNA.
645 AE006465Homo sapienSsequence section 4 of 1605100 8.
646 AF272973Homo sa mRNA, complete cds. 1411100 iens 647 AF237982Homo sapiens7alpha-hydroxylase (CYP39A1)2478100 mRNA, complete cds.
648 AK023139Homo sapiensFLJ13077 fis, clone NT2RP3001944,1754100 moderately similar to HYPOTHETICAL
47.6 KD PROTEIN C 16C
649 AF315591Homo sa 2 PUMH2 mRNA, com lete 298594 iens cds.
650 AF302691Mus musculusmyelin ex ression factor-3-like943 77 rotein 651 AL136S92Homo SapienscDNA DKFZp761I172 (from 139399 clone DKFZp761I172); complete cds.
6S2 AABS8279Homo Sapiens14-MAR-2001 08-MAR-2000 678 100 Lung cancer associated polypeptide sequence SEQ ID 617.
653 AF104927Homo Sapiensligase (TTLLI) mRNA, 2260100 complete cds.
654 AL163792Arabidopsisputative protein 587 49 . thaliana 6SS AF233395Homo Sapiensprotein type 7 (SIRT7) 2086100 mRNA, complete cds.
656 AF233223Homo Sapiensprotein FBG2 (FBG2) mRNA,1602100 complete cds.
657 AF317549Homo Sapiensfinger protein 268 (ZNF268)288599 mRNA, complete cdS.
658 AK02S426Homo SapiensFLJ21773 fis, clone COLF7849.1172100 SEQ AccessionSpecies Description Score ID
NO: No. Identi 659 AL049548Homo SapiensDNA sequence from clone771 100 39863 on chromosome 6q25.1-25.3.
Contains the 3'part of the gene for the ortholog of rat CPG2, part of a novel gene, ESTs, STSs and GSSs, com lete se uence.
660 AK000947Homo SapiensFLJ10085 fis, clone 931 100 HEMBA1002161, moderately similar to MYOSIN HEAVY
CHAIN, CARDIAC MUSCLE
661 AAY48487Homo Sapiens08-DEC-1999 20-MAR-1998432 36 Human breast tumour-associated rotein 32.
662 AE003564DrosophilaCG13295 gene product 377 29 melanogaster 663 AB049955Homo sapiensmRNA for mitochondria) 313 100 ribosomal rotein 521, complete cds.
664 BC005357Homo SapiensSimilar to RIKEN cDNA 418 100 gene, clone MGC:12458 IMAGE:3511019, mRNA, com lete cds.
665 L08240 Homo sa MG81 mRNA, artial cds. 3398 99 iens 666 AK022732Homo SapiensFLJI2670 fis, clone 1551 99 NT2RM4002301.
667 AAY57900Homo Sapiens23-MAR-2000 28-MAY-1999996 100 Human transmembrane rotein HTMPN-24.
668 BC005805Homo Sapiensclone MGC:1003 IMAGE:2988344,862 100 mRNA, complete cds.
669 AF151073Homo sa HSPC239 1535 100 iens 670 AF151073Homo SapiensHSPC239 1209 100 671 AK000197Homo sa FLJ20190 fis, clone 1754 100 iens COLF0714.
672 AJ010071Homo sa TOM)-like rotein. 2444 99 iens 673 AJ010071Homo sapiensTOM1-like protein. 1236 97 674 BC004395Homo SapiensSimilar to apolipoprotein1700 100 L, clone MGC:10978 IMAGE:3636011, mRNA, complete cds.
675 AC016526Homo Sapiens14 clone RP11-361H10 2554 99 map 14q24.3, com lete sequence.
676 AJ279246Homo SapiensNPHS2 gene for podocin,1939 100 exon 1 and joined CDS.
677 AL136628Homo SapienscDNA DKFZp564C182 (from732 100 clone DKFZ 564C182 ; complete cds.
678 AF116636Homo sa PR01488 362 100 iens 679 AF116694Homo SapiensPR02219 414 100 680 AK001867Homo SapiensFLJ11005 fis, clone 859 100 PLACE1002996.
681 AK027746Homo sa FLJ14840 fis, clone 1531 99 iens OVARC1001916.
682 AK026486Homo sa FLJ22833 fis, clone 623 100 iens KAIA4266.
683 AK001421Homo sapiensFLJ10559 fis, clone 1650 100 NT2RP2002618, weakly similar to PROTEIN
684 AK000521Homo SapiensFLJ20514 fis, clone 1313 100 KAT09756.
685 X59869 Homo SapiensTCF-1 mRNA for T cell 1375 99 factor 1 (splice form A).
686 AK002135Homo sapiensFLJ11273 fis, clone 1419 100 PLACE1009338.
687 AAY57896Homo Sapiens23-MAR-2000 28-MAY-1999733 100 Human transmembrane rotein HTMPN-20.
688 AF189692Homo sapiensCdc42 effector protein 452 100 SPEC2 mRNA, complete cds.
689 AJ401269Bos tauruspolyadenylate-binding 2439 99 protein 1 SEQ AccessionSpecies Description Score ID
NO: No. Identi 690 AK016776Mus musculusutative 1801 69 691 AB038523Homo sa for MBIP, complete cds.1772 100 iens 692 AK000241Homo sapiensFLJ20234 fiS, clone 2398 100 COLF5673.
693 AJ245719Homo sa for brk kinase substrate2154 100 iens (BKS gene).
694 AAB97378Homo sapiens17-AUG-2001 08-NOV-20001533 100 Human kringle domain containing protein 1.
695 AB038523Homo sapiensfor MBIP, complete cds.1552 99 696 AK026105Homo SapiensFLJ22452 fis, clone 2419 100 HRC09667.
697 AAB00187Homo Sapiens08-FEB-2001 15-MAR-2000635 45 Breast cancer rotein BCNI.
698 AAY99425Homo sapiens08-AUG-2000 Ol-SEP-19991343 100 Human PR01558 (UNQ766) amino acid se uence SEQ ID N0:306.
699 AK009886Mus musculusputative 1329 75 700 AK016154Mus musculusputative 1166 79 701 AF151072Homo SapiensHSPC238 843 100 702 AB045180Homo SapiensmRNA for toll-like receptor5466 100 9, complete cds.
703 AAB58961Homo Sapiens27-MAR-2001 08-MAR-2000460 98 Breast and ovarian cancer associated antigen protein sequence SEQ ID 669.
704 BC007556Homo SapiensSimilar to TEA domain 2365 100 family member 2, clone MGC:15481 IMAGE:2967735, mRNA, complete cds.
705 AAB74726Homo sapiens12-JUN-2001 14-AUG-20001601 48 Human membrane associated protein MEMAP-32.
706 AF217413Homo Sapiens3 isoform gene, complete4450 100 cds, alternatively spliced.
707 AAG01129Homo Sapiens06-OCT-2000 21-FEB-2000230 77 Human secreted protein, SEQ
ID NO: 5210.
708 AK024066Homo sapiensFLJ14004 fis, clone 1791 100 Y79AA1002351.
709 AF259799Homo Sapiensacid transporter system2560 100 A2 (ATA2) mRNA, corn lete cds.
710 AJ250839Homo Sapiensfor serine/threonine 2227 100 protein kinase.
711 AK027057Homo SapiensFLJ23404 fis, clone 410 91 HEP18862.
712 AF116652Homo SapiensPR00813 1023 100 713 AF208845Homo sa BM-003 861 65 iens 714 AK001123Homo sa FLJ10261 fis, clone 3127 100 iens HEMBB1000975.
715 BC002571Homo Sapiensclone MGC:2481 IMAGE:3143135,1419 99 mRNA, complete cds.
716 BC004169Homo SapiensSimilar to RIKEN cDNA 1266 100 gene, clone MGC:2714 IMAGE:2821548, mRNA, complete cds.
717 AK026147Homo SapiensFLJ22494 fis, clone 1152 99 HRC11131.
718 AJ400877Homo sapienSgene, CEGPI gene, CI 1094 99 lorfl4 gene, Cl IorflS gene, CI lorfl6 gene and C 1 l orfl 7 gene.
719 AJ400877Homo Sapiensgene, CEGPI gene, Cllorfl4575 100 gene, C 1 I orfl 5 gene, C
11 orfl6 gene and C 11 orfl7 gene.
720 AJ400877Homo Sapiensgene, CEGP1 gene, Cllorfl4379 97 gene, C 11 orfl 5 gene, C
11 orfl 6 gene and C I I orf 17 gene.
721 AK021919Homo SapiensFLJ11857 fis, clone 1851 99 HEMBA1006807, moderately similar to Homo Sapiens SEQ AccessionSpecies Description Score ID
NO: No. Identi mRNA for SPOP.
722 AAB93876Homo Sapiens26-JUN-2001 28-JUL-2000552 39 Human rotein se uence SEQ
723 BC005827Homo SapiensH2B histone family, 392 93 member Q, clone MGC:1729 IMAGE:2989788, mRNA, complete cds.
724 BC010929Homo Sapiensclone MGC:13522 IMAGE:4291498,932 99 mRNA, complete cds.
725 BC010929Homo Sapiensclone MGC:13522 IMAGE:4291498,1446 100 mRNA, complete cds.
726 AAU00875Homo Sapiens04-JUL-2001 30-AUG-20002015 100 Human cancer related protein 10.
727 AK004371Mus musculusutative 1096 89 728 AK000846Homo sa FLJ20839 fis, clone 1379 100 iens ADKA02346.
729 AF090939Homo SapiensHQ0641 PRO0641 mRNA, 275 100 complete cds.
730 AF201950Homo sapiensprotein mRNA, complete 399 100 cds.
731 AF129756Homo Sapiensgene, partial cds; and 691 99 CLIC1, DDAH, G6b, G6c, GSb, G6d, G6e, G6f, BATS, GSb, CSK2B, BAT4, G4, Apo M, BATS, BAT2, AIF-I, 1C7, LST-I, LTB, TNF, and LTA genes, complete cds.
732 226593 Homo Sapiensrearranged mRNA for 573 97 T-cell receptor alpha chain.
733 BC004366Homo Sapiensclone MGC:10334 IMAGE:3641657,1219 100 mRNA, complete cds.
734 AK001243Homo sa FLJ10381 fis, clone 2326 100 iens NT2RM2002055.
735 BC008947Homo SapiensSimilar to RIKEN cDNA 2319 99 gene, clone MGC:3422 IMAGE:3028566, mRNA, com lete cds.
736 AK000702Homo sa FLJ20695 fis, clone 1554 100 iens KAIA2502.
737 AF090937Homo SapiensHQ0618 PR00618 mRNA, 492 100 complete cds.
738 BC005357Homo sapiensSimilar to RIKEN cDNA 597 99 gene, clone MGC:12458 IMAGE:3511019, mRNA, complete cds.
739 BC005357Homo SapiensSimilar to RIKEN cDNA 603 99 gene, clone MGC:12458 IMAGE:3511019, mRNA, complete cds.
740 AL390216Homo SapienscDNA DKFZp762K222 (from1120 100 clone DKFZ 762K222).
741 AK006724Mus musculusputative 1077 80 742 AK000615Homo SapiensFLJ20608 fis, clone 1038 98 KAT05987.
743 AK000615Homo SapiensFLJ20608 fis, clone 778 98 KAT05987.
744 AAY99669Homo Sapiens03-NOV-2000 23-NOV-19991013 99 Human GTPase associated rotein-20.
745 AL137516Homo SapienscDNA DKFZp564M2178 (from3506 99 clone DKFZ 564M2178); artial cds.
746 BC006006Homo SapiensSimilar to RIKEN cDNA 597 100 gene, clone MGC:14832 IMAGE:4283597, mRNA, complete cds.
747 AB002819Perilla actin 142 96 frutescens 748 AJ271290Homo Sapiensfor facilitative glucose866 99 transporter GLUTll (SLC2A11 ene).
749 AK000157Homo SapiensFLJ20150 fis, clone 1559 99 COL08263.
SEQ AccessionSpecies Description Score ID No. Identi NO:
750 AF231410Homo Sapienssperm protein ropporin 205 87 mRNA, complete cds.
751 AB044755Homo SapiensmRNA for basic-helix-loop-helix1723 100 rotein, com lete cds.
752 AK001610Homo SapiensFLJ10748 fis, cloneNT2RP3001819,1852 100 weakly similar to RING
753 AF208864Homo SapiensARF 688 99 754 AF209931Homo Sapiensprotein mRNA, partial 1222 96 cds.
755 AF064604Homo Sapiensprotein mRNA, artial 1350 93 cds.
756 AK000042Homo SapiensFLJ20035 fis, clone 2029 100 COL00213.
757 AF208864Homo SapiensARF 688 99 758 AK016624Mus musculusutative 847 84 759 AF332890Homo Sapienszinc finger FEZL 1561 99 760 AK000602Homo SapiensFLJ20595 fis, clone 764 100 KAT08558.
761 AAB73227Homo Sapiens11-MAY-2001 11-AUG-20002733 99 Human hos hatase NP 060232 h.
762 AF016903Homo sa precursor mRNA, partial8478 100 iens cds.
763 BC002912Homo Sapiensclone MGC:11279 IMAGE:3944940,1512 98 mRNA, com lete cds.
764 AB035179Homo Sapiensfor HES6, com lete cds.1143 98 765 AK000506Homo SapiensFLJ20499 fis, clone 3811 99 KAT09034.
766 AAG71494Homo Sapiens31-JUL-2001 06-OCT-2000613 98 Human olfactory receptor polypeptide, SEQ ID
767 BC001005Homo Sapienscytochrome c oxidase 329 100 subunit VIII, clone MGC:8432IMAGE:2821167, mRNA, com lete cds.
768 AF104260Homo SapiensmRNA, partial cds. 1327 51 769 AF116709Homo SapiensPRO2605 642 100 770 AF176330Homo sapiens(PCBP4) mRNA, com lete 2041 100 cds.
771 AF169226Homo Sapiensconserved domain protein972 100 1 (ACDPI) mRNA, complete cds.
772 283851 Homo SapiensDNA sequence from clone474 100 989H11 on chromosome 22q13.1-13.2.
Contains part of a novel gene, ESTs, GSSs and four putative CpG islands, complete sequence.
773 AK000130Homo SapiensFLJ20123 fis, clone 998 100 COL06041.
774 AC004908Homo Sapiensclone RPS-855D21, complete171 91 sequence.
775 X68879 Homo sa EMXl mRNA. 819 100 iens 776 AAR78692Homo sapiens15-MAR-1996 24-DEC-1993832 100 Human skeletal muscle stress protein, p20.
777 AF042831Homo Sapienstranscription factor 615 100 (FKHL18) mRNA, partial cds.
778 AK001050Homo SapiensFLJ10188 fis, clone 1312 100 HEMBA1004693.
779 AL096817Homo SapiensDNA sequence from clone320 100 on chromosome 6q15-16.1.
Contains an HSP60 (TCP-1/cpn60 chaperonin family) pseudogene, three novel genes, ESTs, STSs and GSSs, com lete sequence.
781 AF202922Homo Sapiens(LRP16) mRNA, complete 1511 95 cdS.
782 AL365514Homo sapienshuman gene mapping to 2255 100 chomosome 22.
783 AF023859Papio cyclophilin A 538 95 hamadryas 784 X03491 Mus musculus57 kd keratin (aa I-524)2099 80 SEQ AccessionSpecies Description Score ID No. Identi NO:
785 AAB27242Homo Sapiens27-MAR-2001 10-MAY-20002327 98 Human EXMAD-20 SEQ ID NO:
786 AAY99414Homo Sapiens08-AUG-2000 OI-SEP-19992270 100 Human PR01461 (UNQ742) arr~ino acid sequence SEQ ID N0:269.
787 AF043350Homo Sapiensprotein 1 (LSP1) gene, 361 100 LSP1-5 allele, artial cds.
788 BCO11551Homo Sapiensclone MGC:19971 IMAGE:4561164,1606 88 mRNA, complete cds.
789 AL050256Homo sa human gene ma ing to 881 100 iens chomosome 22.
790 AY014302Homo Sapiensene, exon 2 and complete1409 100 cds.
791 AK000314Homo SapiensFLJ20307 fis, clone 5380 99 HEP07254.
792 AK023886Homo SapiensFLJ13824 fis, clone 1377 100 THYRO1000505.
793 AK019547Mus musculusputative 265 96 794 AK005789Mus musculusutative 475 97 795 AK001783Homo sa FLJ10921 fis, clone 1246 100 iens OVARC1000411.
796 AK027598Homo sapiensFLJ14692 fis, clone 3134 99 NT2RP2005344, weakly similar to PROBABLE
5 (EC 220.127.116.11).
797 U60269 Homo sapiensendogenous retrovirus 381 100 HERV-K(HML6) proviral clone HML6.17 putative polymerase and envelope genes, partial cds, and 3'LTR.
798 AL137651Homo SapienscDNA DKFZp434O0213 (from1366 100 clone DKFZ 43400213); artial cds.
799 AK000061Homo SapiensFLJ20054 fis, clone 1751 99 COL00849.
800 AF233588Homo sa (RIS) mRNA, com lete 1353 100 iens cds.
801 576838 Mus s . Dbs 1469 49 802 AB033168Mus musculusnuclear protein ZAP 1946 89 803 AB049591Homo sa related with psoriasis,647 100 iens com lete cds.
804 AF093249Homo Sapiensisoform 4 (PHRETl) mRNA,1046 100 alternatively spliced, complete cds.
805 AL049679Homo Sapiensgene from PAC 97K10, 1527 100 chromosome X, similar to heparan-sulphate sulfotransferase.
806 AB015329Homo sapiensmRNA, artial cds. 1055 97 807 AF077034Homo sa HSPCO10 163 96 iens 808 AF241833Mus musculussecretory carrier membrane1256 98 protein 5 809 AK001352Homo SapiensFLJ10490 fis, clone 697 100 NT2RP2000233.
810 AF138860Homo sa PR00843 649 100 iens 811 272496 Homo sa MUCSB gene (partial). 18275100 iens 812 AK000361Homo sa FLJ20354 fis, clone 3585 99 iens HEP15013.
813 AK001072Homo SapiensFLJ10210 fis, clone 2372 100 HEMBA1006344, weakly similar to RADIXIN.
814 AK001707Homo SapiensFLJ10845 fis, clone 2161 100 NT2RP4001372, weakly similar to IRREGULAR
CHIASM C-ROUGHEST PROTEIN
815 S79854 Homo Sapiens3 iodothyronine deiodinase774 100 mRNA, complete cds.
816 AB036704Homo SapiensmRNA for phosphodiesterase2541 100 1 1A, complete cds.
817 BC010181Homo sapiensclone MGC:20197 IMAGE:4543414,387 89 mRNA, complete cds.
818 AAB68074Homo sa 09-JUL-2001 10-NOV-20001960 99 iens Amino acid SEQ AccessionSpecies Description Score ID No. Identi NO:
sequence of a human chordin-like homologue splice variant.
819 AF227516Homo sa mRNA, com lete cds. 1444 97 iens 820 AF077202Homo sa HSPC016 100 100 iens 821 AK002945Mus musculusputative 615 94 822 AK000047Homo SapiensFLJ20040 fis, clone 193 97 COL00417.
823 AF119878Homo sapiensPR02353 401 100 824 BC005827Homo sapiensH2B histone family, 385 100 member Q, clone MGC:1729 IMAGE:2989788, mRNA, complete cds.
825 AAB73230Homo Sapiens11-MAY-2001 11-AUG-2000423 97 Human phos hatase AA493915 h.
826 AK000513Homo sa FLJ20506 fis, clone 707 100 iens KAT09493.
827 AK001021Homo sapiensFLJ10159 fis, clone 1471 100 HEMBA1003528.
828 AJ002535Homo Sapiensfor obscurin (OBSCN 466 100 gene).
829 AJ243662Homo sa for NICE-1 protein. 566 100 iens 830 AK000268Homo sa FLJ20261 fis, clone 2659 100 iens COLF7630.
831 AC005396Arabidopsisputative proline-rich 113 32 thaliana protein 832 AJ249977Homo Sapiensfor AMP-activated protein2518 99 kinase gamma 3 subunit (AMPK gamma 3 gene).
833 AAY44985Homo sapiens23-MAY-2000 27-JUL-1999616 88 Human epidermal protein-2.
834 AJ006692Homo sa KerB ene. 923 76 iens 835 M88166 Sus scrofasmall roline-rich rotein190 59 836 AK000139Homo sapiensFLJ20132 fis, clone 2479 100 COL06441.
837 AK000520Homo sapiensFLJ20513 fis, clone 805 99 KAT09741.
838 AC004744Homo sapiensclone GS1-465N13 from 293 98 7p15-p21, complete se uence.
839 AJ002535Homo sa for obscurin (OBSCN 466 100 iens ene).
840 AAY87354Homo Sapiens1 I-MAY-2000 25-JUN-19991546 100 Human signal peptide containing protein HSPP-131 SEQ ID N0:131.
841 AK000054Homo sapiensFLJ20047 fis, clone 4964 100 COL00577.
842 AAB47129Homo sapiens04-JUN-2001 14-SEP-2000672 100 CDIFF-7, Incyte ID No. 2027937CD1.
843 281024 Homo sa mRNA for TCR al ha (TCRAV).604 90 iens 844 _ Homo sa FLJ10858 fis, clone 3226 99 AK001720ienS NT2RP4001555.
845 AE000660Homo sapiensreceptor alpha delta 561 99 locus from bases 501613 to 752736 (section 3 of 5) of the Com lete Nucleotide Se uence.
846 U61084 Homo Sapiensprotein mRNA, complete 1281 97 cdS.
847 AF161550Homo SapiensHSPC065 954 99 848 AK001002Homo sapiensFLJI0140 fis, clone 896 99 HEMBA1003179, moderately similar to PROBABLE
YLTRANSFERASE (EC 18.104.22.168).
849 AJ406946Homo Sapiensfor keratin associated 1079 95 protein 9.2 KRTAP9.2 ene .
850 AF339106Mus musculusforkhead-related transcri1480 99 tion factor 2 851 AF081797Mus musculushigh cysteine keratin-associated469 58 protein 12.1 852 AF071081Mycobacteriumproline-rich mucin homolog121 36 tuberculosis SEQ AccessionSpecies Description Score ID No. Identi NO:
853 AF116686Homo sa PR02116 192 100 iens 854 AF070655Homo sa F1F0-ty a ATP synthase 443 89 iens subunit g 855 AAY41710Homo Sapiens07-DEC-1999 08-MAR-19994232 98 Human PR0618 rotein se uence.
856 AAB47276Homo sa 06-AUG-2001 12-JUL-2000887 98 iens hOATS.
857 AF113013Homo SapiensPR00806 345 100 858 X60661 Rattus potential ligand-binding344 74 rattus protein 859 AF119902Homo SapiensPR02832 406 100 860 AK009462Mus musculusputative 1723 100 861 AAB95296Homo Sapiens26-JUN-2001 28-JUL-20004692 99 Human protein sequence SEQ
862 AB017927Homo SapiensmRNA for p53DINPlb, 878 100 complete cds.
863 AAB83845Homo Sapiens23-JUL-2001 30-OCT-20001346 54 Amino acid sequence of a human protein expressed in tumour cells.
864 AX149579Homo SapiensDNA encoding a transmembrane562 98 serine rotease (Endotheliase 2-S) protein 865 BC012048Homo Sapiensclone IMAGE:3502817, 1225 99 mRNA, partial cds.
866 AK000575Homo sa FLJ20568 fis, clone 664 99 iens REC00775.
867 X76383 Homo sa mRNA for HE3(al ha . 807 100 iens 868 AF286598Homo SapiensmRNA, complete cds. 2381 100 869 AK022643Homo SapiensFLJ12581 fis, clone 721 92 NT2RM4001140, weakly similar to HOMEOBOX
870 AF119891Homo SapiensPR02706 363 100 871 AK009258Mus musculuSputative 1246 80 872 U66412 Mus musculuSadenomatous pol osis 133 88 coli 873 AK001162Homo SapiensFLJI0300 fis, clone 184 100 NT2RM2000030.
874 AL033518Homo sapienSDNA sequence from clone199 100 on chromosome 6p21.1-21.31.
Contains part of the gene for a novel protein similar to C. elegans C05C8.6 (Tr:016313), STSs and GSSs, complete sequence.
875 AF116601Homo sa PR00128 446 100 iens 876 AF156889Homo Sapienshomeobox protein 3 isoform2148 100 b (LHX3) mRNA, complete cds.
877 AK026671Homo sa FLJ23018 fis, clone 385 100 iens LNG00903.
878 AAY92515Homo Sapiens10-AUG-2000 06-OCT-19992523 99 Human OXRE-12.
879 AL136818Homo SapienscDNA DKFZp434F1726 (from1736 99 clone DKFZp434F 1726).
880 AB05531 Homo Sapiensfor RanBPM, complete 2172 67 I cds.
881 AF006465Mus musculusB cell antigen receptor1286 61 Ig beta associated protein 1 882 AF143956Mus musculuscoronin-2 1020 72 883 AK008237Mus musculusputative 653 84 884 AK008237Mus musculusputative 653 84 885 AF221846Homo Sapiensgastric protein ZG12P 182 100 mRNA, complete cds.
886 BC001005Homo Sapienscytochrome c oxidase 304 93 subunit VIII, clone MGC:8432 IMAGE:2821167, mRNA, com lete cds.
887 X01715 Homo Sapiensgene fragment for the 2543 100 ~ acetylcholine SEQ AccessionSpecies Description Score ID No. Identi NO:
receptor gamma subunit precursor (exons 1 and 2).
888 AK001974Homo sa FLJ11112 fis, clone 955 100 iens PLACE1005925.
889 AF212016Homo sa rece for 9 IL1R9 mRNA, 3607 100 iens com lete cds.
890 D88437 Homo Sapiensfor G-protein coupled 2455 100 receptor SALPR, complete cds.
891 AK002298Mus musculusutative 833 97 892 X96389 Bos taurusprocollagen I N-proteinase440 34 SEQ ID AccessionDescription Results*
451 PD01719 PRECURSOR GLYCOPROTEINPD01719A 12.89 8.200e-17 SIGNAL RE. 371 452 BL00388 Proteasome A-type BL00388A 23.14 5.875e-40 subunits 5-51 proteins. BL00388B 31.38 6.538e-29 106 BL00388D 20.71 1.391e-26 18.79 2.000e-453 BL00388 Proteasome A-type BL00388B 31.38 6.538e-29 subunits 33-75 proteins. BL00388D 20.71 1.391e-26 147 BL00388C 18.79 2.000e-22 454 BL00064 L-lactate dehydrogenaseBL00064C 17.28 8.442e-22 proteins. 293-338 BL00064A 21.16 5.574e-12 459 BL01187 Calcium-binding EGF-likeBL01187B 12.04 1.257e-10 domain 218-proteins attern proteins.234 460 BL00107 Protein kinases ATP-bindingBL00107B 13.31 9.100e-15 region roteins. 215 462 PR00678 PI3 KINASE P85 REGULATORYPR00678H 9.13 1.529e-1 SUBUNIT SIGNATURE
463 PR00678 PI3 KINASE P85 REGULATORYPR00678H 9.13 1.529e-1 . 1 64-87 SUBUNIT SIGNATURE
469 PD00930 PROTEIN GTPASE DOMAINPD00930B 33.72 6.250e-17 ACTIVATION. 487 PD00930A 25.62 2.841e-13 472 PR00302 LUPUS LA PROTEIN PR00302A 11.32 3.318e-14 473 PF00777 Sialyltransferase PF00777C 18.60 9.416e-26 family. 363-418 PF00777D 22.05 3.681e-11 476 BL00360 Ribosomal protein BL00360B 20.22 5.705e-19 S9 proteins. 317-353 BL00360C 17.65 4.857e-18 479 BL00057 Ribosomal protein BL00057 24.94 8.800e-14 S18 roteins. 81-129 482 BL00299 Ubiquitin domain BL00299 28.84 1.000e-40 proteins. 16-68 BL00299 28.84 I.OOOe-40 483 BL00039 DEAD-box subfamily BL00039D 21.67 9.000e-37 dependent helicases 367 BL00039A 18.44 proteins. 3.893e-24 28-67 BL00039C 15.63 8.269e-17 19.19 4.818e-485 PR00828 FORM1N SIGNATURE PR00828B 5.23 8.218e-10 489 PR00581 PROSTANOID EP2 RECEPTOR1'R00581E 3.48 9.875e-10 SIGNATURE
490 BL00615 C-type lectin domainBL00615A 16.68 8.200e-11 proteins. 113-492 BL00039 DEAD-box subfamily BL00039D 21.67 4.176e-23 dependent helicases 437 BL00039A 18.44 proteins. 7.065e-16 19.19 5.395e-15.63 9.820e-11 24 493 BL00479 Phorbol esters / _ diacylglycerol BL00479B 12.57 9.518e-09 binding domain proteins.488 SEQ ID AccessionDescription Results*
494 PR00929 AT-HOOK-LIKE DOMAIN PR00929B 4.38 I.OOOe-10 SIGNATURE
499 BL00678 Trp-Asp (WD) repeat BL00678 9.67 4.000e-10 proteins 339-350 roteins.
S00 PF00646 F-box domain proteins.PF00646A 14.37 4.375e-09 503 PD01066 PROTEIN ZINC FINGER PD01066 19.43 3.800e-30 FINGER METAL-BINDING
504 DM00191 w SPAC8A4.04C RESISTANCEDM00191A 8.16 4.360e-09 SPAC8A4.OSC 223 DAUNORUBICIN.
506 PR00060 RIBOSOMAL PROTEIN PR00060A 10.94 6.023e-09 507 PF00646 F-box domain roteins.PF00646A 14.37 9.036e-10 508 BL00632 Ribosomal rotein BL00632 23.79 2.821e-12 S4 roteins. 104-147 510 BL01191 Ribosomal protein BL01191A 15.57 I.OOOe-40 S3Ae proteins. 13-64 BL01191B 13.33 I.OOOe-40 512 PD00930 PROTEIN GTPASE DOMAINPD00930B 33.72 6.063e-25 ACTIVATION. 203 PD00930A 25.62 8.297e-15 513 BL00604 Synaptophysin l synaptoporinBL00604F 5.96 7.718e-10 567-.612 proteins.
515 BL01152 Hypothetical hesB/yadR/yfhFBL01152C 25.93 1.900e-29 family proteins. 128 BLOI 152B 20.12 6.121e-11 516 PR00049 WILM'S TUMOUR PROTEINPR00049D 0.00 1.000e-09 SIGNATURE
518 BL00218 Amino acid permeasesBL00218D 21.49 3.797e-1 proteins. I 243-288 BL00218B 21.44 1.621e-10 75-107 BL00218E 23.30 3.520e-523 PR00836 SOMATOTROPIN HORMONEPR00836B 16.59 2.895e-16 FAMILY SIGNATURE 120 PR00836D 13.05 1.621e-13 195-210 PR00836A 14.40 2.800e-11.95 4.913e-13179-196 526 BL00164 Enolase proteins. BL00164B 16.22 I .000e-40 141 BL00164C 15.66 I.OOOe-40 144-194 BL00164G 12.13 I.OOOe-10.48 3.813e-39 313-349 21.97 2.588e-38 220-263 BL00164A 11.58 1.529e-27 BL00164E 8.80 9.100e-20 529 BL00790 Receptor tyrosine BL00790R 16.20 3.516e-09 kinase class V 21-6S
530 PR00288 PUROTHIONIN SIGNATUREPR00288B 13,09 9.870e-09 536 DM00250 kw ANNEXIN ANTIGEN DM00250B 13.84 8.541 e-09 426-PROLINE TUMOR. 450 540 BL00495 Apple domain proteins.BL00495G 12.47 8.920e-09 542 PR00080 ALCOHOL DEHYDROGENASEPR00080C 17.16 4.750e-12 I 551 PR00926 MITOCHONDRIAL CARRIERPR00926F 17.75 1.964e-20 PROTEIN SIGNATURE
SEQ ID AccessionDescription Results*
552 BL00795 Involucrin proteins.BL00795C 17.06 2.286e-12 148 BL00795C 17.06 5.208e-12 102-147 BL00795C 17.06 8.953e-17.06 1.000e-09114-159 BL00795C
17.06 1.400e-09 97-142 BL00795C 17.06 3.200e-09 149 BL00795C 17.06 4.I OOe-09 101-146 BL00795C 17.06 4.800e-556 PF00628 PHD-finger. PF00628 15.84 6.806e-09 559 PR00041 CAMP RESPONSE ELEMENTPR00041E 7.20 7.072e-12 BINDING (CREB) PROTEIN
564 BL01119 Copper-fist domain BL01119B 18.30 2.385e-09 proteins. 3818-568 BL00814 Adrenodoxin family, BL00814B 23.55 9.372e-22 iron-sulfur 127-binding region proteins.165 BL00814A 15.33 3.769e-15 570 PF00152 tRNA synthetases PF00152D 21.30 4.774e-29 class II. 434-473 PF00152C 28.03 7.107e-25 571 PR00608 CLASS II CYTOCHROME PR00608A 13.74 7.000e-09 574 BL00376 S-adenosylmethionineBL00376A 10.62 LOOOe-40 synthetase 19-74 proteins. BL00376D 18.36 I.OOOe-40 201 BL00376C 11.943.571e-38 122-157 BL00376B 14.91 3.500e-' 19 99-I 16 579 BL00415 Synapsins proteins. BL00415N 4.29 6.058e-12 580 BL00475 Ribosomal protein BL00475B 8.20 6.769e-09 L15 proteins. 46-56 BL00475D 16.25 9.578e-09 581 BL00678 Trp-Asp (WD) repeat BL00678 9.67 4.000e-13 proteins 241-252 roteins.
583 PD02784 PROTEIN NUCLEAR PD02784B 26.46 3.629e-13 RIBONUCLEOPROTE1N. 139 PD02784C 20.76 6.894e-09 584 BL00417 Synaptobrevin proteins.BL00417B 18.48 I.OOOe-40 113 BL00417A 7.74 3.700e-34 585 BL01013 Oxysterol-binding BL01013D 26.81 9.578e-17 protein family 267-proteins. 311 BL01013C 9.97 6.308e-13 I.33 3.717e-586 PR00302 LUPUS LA PROTEIN PR00302A 11.32 3.647e-13 589 BLOOOI8 EF-hand calcium-bindingBL00018 7.41 I.OOOe-12 domain 209-222 roteins.
590 BL00708 Prolyl endopeptidaseBL00708B 24.91 2.235e-15 family serine 619-proteins. 650 593 BLOI032 Protein phosphatase BL01032H 11.25 I.OOOe-10 2C proteins. 446-459 BL01032C 6.14 4.474e-09 ~ 596 BLO1 I GTP-binding nuclear BL01115A 10.22 3.600e-16 ~ 15 ~ protein ran 8-52 SEQ ID AccessionDescription Results*
S97 BL00226 Intermediate filamentsBL00226D 19.10 4.450e-18 proteins. 113-S98 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 2.800e-14 METAL-BINDI. PD00066 13.92 2.800e-14 PD00066 13.92 5.200e-14167-180 PD00066 13.92 5.500e-13 PD00066 13.92 1.857e-12 PD00066 13.92 2.714e-12 PD00066 13.92 9.143e-12 PD00066 13.92 9.143e-12 PD00066 13.92 4.913e-1 PD00066 13.92 1.346e-10 PD00066 13.92 2.200e-09 599 BL00194 Thioredoxin family BL00194 12.16 5.500e-14176-189 proteins.
BL00194 12.16 4.913e-13 604 PD00289 PROTEIN SH3 DOMAIN PD00289 9.97 9.550e-11 REPEAT PRESYNA.
607 BL00960 BTGl family proteins.BL00960C 12.68 3.647e-26 609 PR00366 ENDOTHELIN RECEPTOR PR00366A 14.10 4.222e-09 SIGNATURE
611 BL00383 Tyrosine specific BL00383E 10.35 6.368e-09 protein 93-phosphatases proteins.104 612 BL00290 Immunoglobulins and BL00290B 13.17 8.773e-10 major 266-histocompatibility 284 complex proteins.
614 BL00415 Synapsins proteins. BL00415C 7.09 3.182e-09 616 PF00628 PHD-finger. PF00628 15.84 5.125e-1 619 BL00322 Histone H3 proteins.BL00322B 13.68 8.514e-10 622 PD02411 PROTEIN TRANSCRIPTIONPD02411 21.89 4.214e-15 REGULATION NUCLEAR.
624 BL00880 Acyl-CoA-bindin rotein.BL00880 17.52 1.000e-40 628 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 7.300e-17 METAL-BINDI. PD00066 13.92 3.400e-14 PD00066 13.92 4.000e-14 PD00066 13.92 8.000e-13 PD00066 13.92 9.500e-13 631 BL00226 Intermediate filamentsBL00226D 19.10 4.667e-11 proteins. 121-632 PD01613 RIBOSOME FACTOR PROTEINPD01613 23.39 6.121e-17169-215 RECYCLIN.
636 PR00049 WILM'S TUMOUR PROTEINPR00049D 0.00 9.500e-10 637 PF00855 PWWP domain proteins.PF00855 13.75 3.872e-17 638 PR00671 INHIBIN BETA B CHAINPR00671C 4.18 9.671e-10 639 BL00240 Receptor tyrosine BL00240F 17.74 7.645e-1 kinase class III I 157-proteins. 205 BL00240G 28.45 1.818e-10 642 BL01191 Ribosomal protein BL01191A 15.57 1.000e-40 S3Ae proteins. 13-64 BL01191B 13.33 1.000e-40 140 BL01191C 16.50 1.000e-40 SEQ ID AccessionDescription Results*
643 PR00950 FLAGELLAR BIOSYNTHETICPR00950B 14.12 6.571e-09 644 BL00741 Guanine-nucleotide BL00741B 14.27 7.808e-09 dissociation 558-stimulators CDC24 581 family si n.
646 PD02059 CORE POLYPROTEIN PD02059B 24.48 7.211e-09 PROTEIN GAG CONTAINS:160 P.
647 BL00086 Cytochrome P450 cysteineBL00086 20.87 7.395e-13 heme- 404-436 iron ligand proteins.
649 PF00806 Pumilio-family RNA PF00806B 11.32 4.176e-12 binding 766-domain proteins (aka776 PF00806C 7.81 PUM-HD, 5.263e-11 Pumilio homol. 838-847 PF00806C 7.81 7.632e-650 PR00221 CAULIMOVIRUS COAT PR00221H 12.82 7.614e-09 651 PF00023 Ank repeat proteins.PF00023A 16.03 9.571e-11 654 BL01279 Protein-L-isoaspartate(D-aspartate)BL01279A 24.27 6.967e-10 O-methyltransferase 138 signa.
657 BL00028 Zinc finger, C2H2 BL00028 16.07 1.000e-14 type, domain 351-368 proteins. BL00028 16.07 4.706e-14 BL00028 16.07 7.882e-14 BL00028 16.07 S.SOOe-13 BL00028 16.07 5.950e-13 BL00028 16.07 2.174e-12 BL00028 16.07 2.957e-12 BL00028 16.07 8.043e-12 BL00028 16.07 9.217e-12 BL00028 16.07 2.038e-11 BL00028 16.07 3.769e-11 BL00028 16.07 4.115e-11 BL00028 16.07 8.615e-11 BL00028 16.07 8.962e-11 BL00028 16.07 5.200e-10 BL00028 16.07 9.100e-10 BL00028 16.07 9.100e-10 658 PF00850 Histone deacetylase PF00850E 8.88 4.750e-12 family. 52-78 PF00850D 14.76 8.696e-11 PF00850G 22.75 5.382e-10 660 PR00193 MYOSIN HEAVY CHAIN PR00193A 15.41 6.294e-22 661 BL00478 LIM domain proteins.BL00478B 14.79 5.500e-13 665 PF00566 Probable rabGAP domainPF00566B 11.92 6.100e-09 proteins. 330-676 BL01270 Band 7 protein familyBL01270D 20.87 1.509e-21 proteins. 232-270 BL01270B 18.74 4.136e-16 164-203 BL01270A 9.40 8.953e-13.03 8.500e-12 270-299 685 PD02448 TRANSCRIPTION PROTEINPD02448A 9.37 3.927e-09 DNA-BINDIN. 198 686 PR00625 DNAJ PROTEIN FAMILY PR00625D 11.93 7.828e-10 SIGNATURE
689 PF00658 Poly-adenylate bindingPF00658B 28.57 1.000e-40 protein, 105-unique domain proteins.152 PF00658C 16.33 8.500e-36 696 PF00566 . Probable rabGAP PF00566A 12.64 1.409e-1 domain proteins. I 210-SEQ ID AccessionDescription ~ Results*
698 BLO1100 NNMT/PNMT/TEMT familyBLO1100E 12.25 9.277e-09 of 171-methyltransferases 215 roteins.
699 BL00569 Myelin basic protein.BL00569A 16.70 3.632e-09 702 PR00019 LEUCINE-RICH REPEAT PR00019A 11.19 7.261e-10 SIGNATURE 693 PR00019B 11.36 7.300e-10 676-690 PR00019B 11.36 8.650e-11.36 4.240e-09 122-136 11.36 4.240e-09 307-321 PR00019A 11.19 4.333e-09 431 PR00019A 11.19 8.000e-09 703 BL00025 P-t e'Trefoif domainBL00025 17.17 9.217e-21 roteins. 53-74 704 BL00554 TEA domain proteins.BL00554A 11.66 1.000e-40 107 BL00554C 12.10 1.000e-40 326-379 BL00554D 12.30 1.000e-10.31 8.875e-39 262-303 706 PR00878 CHOLINESTERASE PR00878F 5.37 4.780e-13 SIGNATURE
709 BL00594 Aromatic amino acidsBL00594A 16.75 5.688e-10 permeases 76-roteins. 120 ?10 BL00107 Protein kinases ATP-bindingBL00107A 18.39 3.647e-20 region proteins. 167 BL00107B 13.31 6.727e-13 711 PD01066 PROTEIN ZINC FINGER PD01066 19.43 3.250e-35 FINGER METAL-BINDING
713 BL00028 Zinc finger, C2H2 BL00028 16.07 4.857e-09 type, domain 6-23 proteins.
715 PR0011 ALPHABETA HYDROLASE PROOI I IA 11.49 4.200e-11 721 PF00651 BTB (also known as PF00651 15.00 2.895e-1 BR-C/Ttk) I 213-226 domain proteins.
722 BL00069 Glucose-6-phosphate BL00069C 16.11 7.723e-09 dehydrogenase proteins.
723 PR00621 HISTONE H2B SIGNATUREPR00621A 12.25 8.714e-23 PR00621B 4.91 5.034e-21 724 BL00919 Deoxyribonuclease BL00919F 14.41 9.010e-09 I proteins. 108-725 BL00919 Deoxyribonuclease BL00919F 14.41 9.0I
I proteins. Oe-09 108-726 BL00790 Receptor tyrosine BL00790I 20.01 2.375e-12 kinase class V 192-proteins. 223 727 BL01115 GTP-binding nuclear BLOI I 15A 10.22 3.089e-10 protein ran 23-67 proteins.
731 BL00983 Ly-6 / u-PAR domain BL00983C 12.69 4.981e-09 proteins. 83-99 732 DM00031 IMMUNOGLOBULIN V DM00031B 15.41 1.797e-09 REGION. 113 740 PF00078 Reverse transcriptasePF00078A 8.82 9.438e-09 (RNA- 803-811 de endent DNA olymerase).
744 BL01020 SARI family proteins.BL01020C 15.35 7.038e-20 745 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 3.000e-13 METAL-BINDI. PD00066 13.92 I.OOOe-12 SEQ ID AccessionDescription Results*
PD00066 13.92 5.286e-12 PD00066 13.92 6.143e-12 PD00066 13.92 9.571e-12 747 PR00190 ACTIN SIGNATURE PR00190F 7.80 7.506e-09 748 BL00216 Sugar transport proteins.BL00216B 27.64 4.512e-16 749 PF00622 Domain in SPIa and PF00622B 21.00 9.795e-09 the 166-RYanodine Receptor. 188 750 DM0I513 CAMP-DEPENDENT PROTEINDM01513A 13.61 1.491e-0910-51 KINASE REGULATORY
751 BL00038 Myc-type, 'helix-loop-helix'BL00038B 16.97 4.750e-14 dimerization domain 105 BL00038A 13.61 proteins. 4.750e-11 753 BL01019 ADP-ribosylation BL01019A 13.20 4.882e-2447-87 factors family proteins.
754 PD00930 PROTEIN GTPASE DOMAINPD00930B 33.72 7.000e-17 ACTIVATION.
757 BLOIOI9 ADP-ribosylation BLOl0I9A 13.20 4.882e-24 factors family 47-87 roteins.
759 BL00028 Zinc finger, C2H2 BL00028 16.07 6.400e-13 type, domain 279-296 roteins.
761 DM01724 kw ALLERGEN POLLEN DM01724 8.14 9.526e-09192-212 HOL-LI. ' 762 DM00758 AGRIN. DM00758 13.12 8.250e-14 763 PR00421 THIORED03~IN FAMILY PR00421B 11.40 7.400e-09 SIGNATURE
764 BL00038 Myc-type, 'helix-loop-helix'BL00038A 13.61 5.667e-10 dimerization domain roteins.
766 PR00245 OLFACTORY RECEPTOR PR00245A 18.03 2.373e-1326-48 SIGNATURE ' 770 PF00013 KH domain proteins PF00013 5.78 7.300e-09 family of 32-44 RNA binding roteins.
775 BL00027 'Homeobox' domain BL00027 26.43 1.600e-29 proteins. 85-128 776 BL01031 Heat shock hsp20 BL01031C 17.68 7.000e-13 proteins family 100-profile. 125 BL01031B 15.78 4.300e-11 777 BL00657 Fork head domain BL00657B 22.27 4.789e-37 proteins. 63-106 BL00657A 19.39 1.600e-32 782 BL0049I Aminopeptidase P BL00491C 12.15 8.800e-18 and proline 363-dipeptidase proteins.378 BL00491D 8.33 2.946e-12 5.42 5.320e-783 BL00170 Cyclophilin-type BL00170C 18.49 3.571e-32 peptidyl-prolyl 35-80 cis-trans isomerase signatur.
784 BL00226 Intermediate filamentsBL00226D 19.10 6.143e-40 proteins. 418-465 BL00226B 23.86 5.696e-35 13.23 2.174e-12.77 3.57Ie-12150-165 23.86 1.113e-10 202-250 BL00226B 23.86 5.395e-09 427 BL00226B 23.86 9.163e-09 SEQ ID AccessionDescription Results*
_ 785 PF00624 Flocculin re eat PF00624I 9.10 8.875e-10 roteins. 96-126 786 BL00021 Kringle domain proteins.BL00021D 24.56 3.942e-22 418 BL00021B 13.33 4.214e-14 790 BL00027 'Homeobox' domain BL00027 26.43 7.750e-34 proteins. 207-250 791 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 7.000e-14 METAL-BINDI. PD00066 13.92 1.500e-13 PD00066 13.92 S.OOOe-13 PD00066 13.92 4.429e-12 PD00066 13.92 3.400e-09 796 BL01228 Hypothetical cof BL01228D 17.44 7.150e-11 family proteins. 232-800 PR00449 TRANSFORMING PROTEINPR00449A 13.20 7.577e-10 RAS SIGNATURE
801 BL00741 Guanine-nucleotide BL00741B 14.27 5.250e-10 dissociation 748-stimulators CDC24 771 family si n.
802 PR00918 CALICIVIRUS NON- PR00918A 13.76 2.500e-11 FAMILY SIGNATURE
811 BL01185 C-terminal cystine BL01185D 23.45 8.043e-19 knot proteins. 4238-4291 BL01185C 15.86 9.852e-i5 813 BL00660 Band 4.1 family domainBL00660C 23.36 4.774e-17 proteins. 217-261 BL00660A 31.50 2.091e-16 45-98 BL00660B 17.33 1.396e-09 814 DM00179 w KINASE ALPHA ADHESIONDM00179 13.97 8.435e-09 T-CELL.
815 BL01205 Iodothyronine deiodinasesBL01205A 28.90 1.581e-25 proteins.
816 BL00126 3'S'-cyclic nucleotideBL00126C 22.07 LOOOe-28 phosphodiesterases 286 BL00126E 35.22 proteins. 6.878e-22 372-427 BL00126D 25.50 1.857e-27.56 4.545e-18 179-216 15.20 2.385e-14 219-231 818 BL01208 VWFC domain proteins.BL01208B 15.83 5.667e-11 BL01208B 15.83 7.750e-10 821 BL01107 Ribosomal protein BL01107B 16.28 1.000e-40 L27e proteins. 46-90 BLO1 I 07A 12.03 7.529e-34 824 PR00621 HISTONE H2B SIGNATUREPR00621A 12.25 8.714e-23 PR00621B 4.91 7.207e-21 827 PR00211 GLUTELIN SIGNATURE PR00211B 0.86 8.083e-09102-829 PR00049 WILM'S TUMOUR PROTEINPR00049D 0.00 9.924e-1 SIGNATURE
830 BL00226 Intermediate filamentsBL00226B 23.86 4.600e-33 proteins. 244-292 BL00226D 19.10 8.054e-29 410-457 BL00226C 13.23 8.125e-12.77 4.960e-14 139-154 833 PR00021 SMALL PROLINE-RICH PR00021A 4.31 2.440e-10 PROTEIN SIGNATURE PR00021 B 7.29 3.647e-09 834 PR00876 NEMATODE PR00876B 7.66 5.014e-09143-SEQ ID AccessionDescription Results*
835 PR00021 SMALL PROLINE-RICH PR00021A 4.31 7.366e-16 PROTEIN SIGNATURE PR00021A 4.31 8.291e-09 836 PF01062 Putative membrane PF01062F 17.08 I.OOOe-40 protein. 277-331 PF01062E 16.81 8.603e-26 214-258 PF01062D 18.73 8.636e-16.52 6.339e-22 20-60 PF01062B
15.58 6.906e-18 62-92 PF01062C
15.18 5.135e-12 92-123 841 BL00232 Cadherins extracellularBL00232B 32.79 5.579e-22 repeat 18-66 proteins domain proteins.BL00232B 32.79 9.169e-18 284 BL00232B 32.79 6.803e-14 340-388 BL00232C 10.65 8.500e-32.79 - 2.098e-12120-168 BL00232C
10.65 3.415e-12 16-34 32.79 9.451e-12 451-499 842 PR00021 SMALL PROLINE-RICH PR00021A 4.31 5.333e-15 PROTEIN SIGNATURE
843 DM00031 IMMUNOGLOBULIN V DM0003IB 15.41 6.108e-IO
REGION. 125 844 BL01242 Formamidopyrimidine-DNABL01242F 17.92 7.722e-14 glycosylase proteins.211 BL01242G 25.36 3.084e-10 846 BL00903 Cytidine and deoxycytidylateBL00903 12.93 5.821e-09 deaminases zinc-binding region s.
848 BL00564 Argininosuccinate BL00564A 19.93 6.114e-09 synthase 7-44 proteins.
849 BL00273 Heat-stable enterotoxinsBL00273 12.24 7.638e-10 proteins. 140-153 BL00273 12.24 8.875e-10 850 BL00657 Fork head domain BL00657A 19.39 9.438e-21 proteins. 74-855 BL00021 Kringle domain proteins.BL00021B 13.33 3.143e-18 604 BL00021D 24.56 3.613e-17 860 BL00798 Aldo/keto reductase BL00798F 23.30 LOOOe-40 family 238-proteins. 287 BL00798E 20.32 8.759e-31 177-215 BL00798B 16.01 3.172e-7.65 1.375e-14.97 2.565e-15 8-23 BL00798C
11.15 2.800e-15 70-83 861 DM01117 2 kw TRANSPOSASE DM01117B 13.11 8.333e-09 TRANSPOSITION VASOTOCIN.530 862 PR00930 HIGH MOBILITY GROUP PR00930E 5.98 6.143e-09 PROTEIN (HMGY) SIGNATURE
863 PD00919 CALCIUM-BINDING PD00919B 9.47 4.822e-09 R.
864 BL00021 Kringle domain proteins.BL00021D 24.56 3.647e-33 865 PR00910 LUTEOVIRUS ORF6 PROTEINPR00910A 2.51 4.889e-10 SIGNATURE
868 PR00833 POLLEN ALLERGEN POA PR00833H 2.30 8.500e-10 SIGNATURE 297 PR00833H 2.30 6.769e-09 SEQ ID AccessionDescription Results*
_ 869 BL00032 'Homeobox' antennapedia-typeBL00032B 10.83 1.281e-11 protein. 138 876 BL00027 'Homeobox' domain BL00027 26.43 3.500e-25 proteins. 177-220 877 PR00049 WILM'S TUMOUR PROTEINPR00049D 0.00 9.557e-13 SIGNATURE 149 PR00049D 0.00 2.500e-12 0.00 2.500e-0.00 4.000e-12138-153 0.00 4.000e-12 139-154 PR00049D 0.00 4.000e-12 155 PR00049D 0.00 4.000e-12 0.00 4.000e-0.00 4.000e-12 143-158 0.00 4.000e-12 144-159 PR00049D 0.00 4.000e-12 160 PR00049D 0.00 4.000e-12 0.00 4.000e-0.00 4.000e-12148-163 0.00 7.126e-11 132-147 PR00049D 0.00 9.244e-11 164 PR00049D 0.00 1.643e-10 0.00 7.643e-0.00 8.714e-10133-148 0.00 2.831 e-09 130-145 PR00049D 0.00 5.576e-09150-880 PF00624 Flocculin repeat PF00624I 9.10 9.646e-09 proteins. 409-439 881 PF00624 Flocculin repeat PF00624I 9.10 9.646e-09 proteins. 448-478 882 PR00320 G-PROTEIN BETA WD-40PR00320B 12.19 3.571e-10 REPEAT SIGNATURE 1136 PR00320A 16.74 9.206e-10 13.01 1.000e-091121-1136 16.74 I.878e-09 1121-1136 PR00320C 13.01 3.700e-09 1186 PR00320B 12.19 5.950e-09 883 BL00904 Protein prenyltransferasesBL00904D 1.47 6.945e-10 alpha 197-subunit repeat proteins238 roteins.
884 BL00904 Protein prenyltransferasesBL00904D 1.47 6.945e-10 alpha 179-subunit re eat roteins220 roteins.
887 PR00254 NICOTINIC ACETYLCHOLINEPR00254D 15.50 1.857e-18 RECEPTOR SIGNATURE 116 PR00254A 11.23 2.588e-14 1.36 3.045e-13 79-92 PR00254B 12.97 5.179e-13 889 PD02870 RECEPTOR INTERLEUKIN-1PD02870B 18.83 7.571e-19 PRECURSOR. 134 PD02870C 24.41 4.643e-10 890 BL00237 G-protein coupled BL00237A 27.68 9.500e-25 receptors 148-proteins. 188 BL00237D 11.23 5.235e-15 13.19 1.360e-5.28 8.875e-1 I 276-288 SEQ ID AccessionDescription Results*
892 PD01719 PRECURSOR GLYCOPROTEINPD01719A 12.89 8.132e-18 SIGNAL RE.
* Results include: Accession number, sub type, Ematrix p-value, and the position of signature sequence.
SEQ Pfam Model Description E-valueScore ID
451 is 1 Thrombos ondin ty a 1 4.9e-1356.8 domain 452 proteasome Proteasome A-t a and 2.1e-49177.6 B-ty a 453 proteasome Proteasome A-type and 1.5e-39144.8 B-type 454 ldh lactate/malate dehydrogenase,1.4e-2080.1 NAD binding do 536 Colla en Collagen tri 1e helix 1.4e-71251.2 repeat (20 copies .
540 PH PH domain 7.9e-1454.2 542 adh_short short chain dehydrogenase1.1e-70248.3 545 UPF0066 Uncharacterised protein 8.1e-38139.1 family UPF0066 546 Peptidase_M48Pe tidase family M48 0.013 -49.3 550 tRNA-synt tRNA synthetases class 1.3e-1117.9 Id I R
551 mito carr Mitochondrial Garner 1.3e-2081.9 protein 554 zf CCCH Zinc finger C-x8-C-x5-C-x3-H1.2e-0945.5 ty a 559 bZIP bZIP transcription factor6e-05 22.9 564 cadherin Cadherin domain 0 1932.1 565 TGS TGS domain 0.071 5.1 568 fer2 2Fe-2S iron-sulfur cluster2.3e-0634.6 binding domain 570 tRNA-synt tRNA synthetases class 3.6e-33123.6 2 II D, K and N) 571 SIR2 Sir2 family 1e-97 338.1 574 S-AdoMet S-adenosylmethionine 1.5e-98340.9 syntD2 synthetase, cent 576 OTU OTU-like cysteine protease0.006 13.2 579 R3H R3H domain 5.5e-1459.9 580 Ribosomal Ribosomal rotein L15 4.3e-1356.9 L15 amino terminal re 581 WD40 WD domain, G-beta repeat1.2e-2082.1 583 rrm RNA recognition motif. 5.3e-0530.1 584 synaptobrevinSynaptobrevin Se-36 133.1 585 Oxysterol Oxysterol-binding protein7.5e-34125.9 BP
589 efhand EF hand 3.4e-26100.5 590 DPPIV N termDi a tidyl peptidase 3.5e-173588.7 IV (DPP IV) N-termi 592 WHI WH1 domain 0.0045 7.1 593 PP2C Protein phosphatase 2C 1.3e-74261.3 595 WD40 WD domain, G-beta re 3.1e-1667.4 eat 596 ras Ras family 2.6e-86300.1 597 filament Intermediate filament 1.5e-0628.4 protein 598 zf C2H2 Zinc finger, C2H2 type 1.4e-106367.4 599 thiored Thioredoxin 8.9e-46156.1 603 PAP2 PAP2 superfamily 0.0057 10.0 604 PDZ , PDZ domain (Also known 3.4e-2390.5 as DHR or GLGF) 606 NAC NAC domain 1.6e-26101.5 607 Anti-proliferatBTGI family 5.2e-2286.5 609 Ribosomal Ribosomal protein S27 8.4e-30112.4 S27e 611 DSPc Dual specificity phosphatase,2.4e-0623.2 catalytic doma 612 ig Immunoglobulin domain 9e-10 36.5 613 Gal-bind_lectinGalactoside-binding lectin1.9e-0720.0 614 Collagen Collagen triple helix 9e-41 148.9 repeat (20 copies) 616 PHD PHD-fin er 4.9e-2080.0 618 bZIP bZIP transcription factor0.0062 15.8 620 AP endonucleaslAP endonuclease family 0.021 10.3 I
622 SET SET domain 2e-54 194.2 623 zf C3HC4 Zinc finger, C3HC4 type 1.3e-1038.7 (RING finger) 624 ACBP ~ Acyl CoA binding protein4.4e-57203.1 ~
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~ ono ono ono ono ono ono ono ono ono Z ~
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
~~ TTENANT LES PAGES 1 A 261 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
1-446, an active domain coding portion of SEQ ID NO: 1-446, and complementary sequences thereof.
(a) a polypeptide encoded by any one of the polynucleotides of claim 1;
and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-446.
a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.
a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions;
b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.
a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.
a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.
a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.
Priority Applications (3)
|Application Number||Priority Date||Filing Date||Title|
|PCT/US2001/027760 WO2002031111A3 (en)||2000-10-12||2001-10-11||Novel nucleic acids and polypeptides|
|Publication Number||Publication Date|
|CA2425827A1 true true CA2425827A1 (en)||2002-04-18|
Family Applications (1)
|Application Number||Title||Priority Date||Filing Date|
|CA 2425827 Abandoned CA2425827A1 (en)||2000-10-12||2001-10-11||Novel nucleic acids and polypeptides|
Country Status (3)
|EP (1)||EP1325120A4 (en)|
|CA (1)||CA2425827A1 (en)|
|WO (1)||WO2002031111A3 (en)|
Families Citing this family (46)
|Publication number||Priority date||Publication date||Assignee||Title|
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|US6515109B1 (en) *||2000-10-12||2003-02-04||Exelixis, Inc.||Human ECT2 polypeptide|
|EP1338649A4 (en) *||2000-10-30||2004-10-13||Takeda Chemical Industries Ltd||Novel gene overexpressed in heart and skeletal muscle and use tehreof|
|US7153678B2 (en)||2000-12-20||2006-12-26||Bristol-Myers Squibb||Polynucleotides encoding the novel human phosphatase, RET31, and variants thereof|
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|EP1790662A1 (en) *||2001-04-10||2007-05-30||Agensys, Inc.||Nucleic acid and corresponding protein entitled 121P2A3 useful in treatment and detection of cancer|
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|US7705120B2 (en)||2001-06-21||2010-04-27||Millennium Pharmaceuticals, Inc.||Compositions, kits, and methods for identification, assessment, prevention, and therapy of breast cancer|
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|JP4301541B2 (en) *||2002-09-05||2009-07-22||公立大学法人横浜市立大学||Smg-1 binding proteins and screening method for a substance which controls its activity|
|US20060199181A1 (en) *||2002-09-11||2006-09-07||Genentch, Inc.||Compositions and methods for the treatment of immune related diseases|
|EP1604033A2 (en) *||2002-11-08||2005-12-14||Bayer HealthCare AG||Diagnostics and therapeutics for diseases associated with human phosphodiesterase 11a3 (pde11a3)|
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|US20050026194A1 (en) *||2003-06-20||2005-02-03||Tularik Inc.||Gene amplification and overexpression in cancer|
|WO2005014817A3 (en) *||2003-08-07||2005-04-21||Compugen Ltd||Chordin-like homologs|
|US20070275872A1 (en) *||2003-10-28||2007-11-29||Cooper Garth J||Peptides with Anti-Obesity Activity and Other Related Uses|
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|CN101123994B (en)||2004-08-16||2012-11-14||夸克医药公司||Therapeutic uses of inhibitors of rtp801|
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|EP2292796A1 (en) *||2005-02-10||2011-03-09||Oncotherapy Science, Inc.||Method of diagnosing bladder cancer|
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|NL2000439C2 (en)||2006-01-20||2009-03-16||Quark Biotech||Therapeutic uses of inhibitors of RTP801.|
|US7723052B2 (en)||2006-05-11||2010-05-25||Quark Pharmaceuticals, Inc.||Screening systems utilizing RTP801|
|WO2007141796A3 (en)||2006-06-09||2010-12-16||Quark Pharmaceuticals, Inc.||Therapeutic uses of inhibitors of rtp801l|
|DK2091965T3 (en)||2006-10-17||2013-08-05||Oncotherapy Science Inc||Peptide vaccines against cancers that express MPHOSPH1- or polypeptides DEPDC1|
|EP2137205A2 (en)||2007-02-26||2009-12-30||Quark Pharmaceuticals, Inc.||Inhibitors of rtp801 and their use in disease treatment|
|US8614311B2 (en)||2007-12-12||2013-12-24||Quark Pharmaceuticals, Inc.||RTP801L siRNA compounds and methods of use thereof|
|CA2732142A1 (en) *||2008-07-28||2010-02-04||Idera Pharmaceuticals, Inc.||Modulation of toll-like receptor 9 expression by antisense oligonucleotides|
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|US8815779B2 (en)||2009-09-16||2014-08-26||SwitchGear Genomics, Inc.||Transcription biomarkers of biological responses and methods|
|GB201009654D0 (en) *||2010-06-09||2010-07-21||Cancer Res Technology||Nucleic acid methyltransferase|
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|WO2016086088A3 (en) *||2014-11-25||2016-08-18||Northwestern University||Wound healing through sirt1 overexpression|
Family Cites Families (2)
|Publication number||Priority date||Publication date||Assignee||Title|
|EP1212343A4 (en) *||1999-09-03||2004-11-03||Human Genome Sciences Inc||52 human secreted proteins|
|CA2405525A1 (en) *||2000-04-12||2001-10-25||Principia Pharmaceutical Corporation||Albumin fusion proteins|
Also Published As
|Publication number||Publication date||Type|
|WO2001057182A2 (en)||Nucleic acids, proteins, and antibodies|
|WO2001036432A2 (en)||18 human secreted proteins|
|WO2001054708A1 (en)||22 human secreted proteins|
|WO1999051744A2 (en)||A interleukin-1 receptor antagonist and uses thereof|
|WO2002077013A2 (en)||Human secreted proteins|
|WO2001032910A2 (en)||27 human secreted proteins|
|US6824973B2 (en)||Method of promoting stem cell proliferation or survival by contacting a cell with a stem cell factor-like polypeptide|
|US6569662B1 (en)||Nucleic acids and polypeptides|
|WO2001021658A1 (en)||32 human secreted proteins|
|US20040219521A1 (en)||Novel nucleic acids and polypeptides|
|US20050192215A1 (en)||Methods and materials relating to novel polypeptides and polynucleotides|
|US6783969B1 (en)||Cathepsin V-like polypeptides|
|US20030165921A1 (en)||Novel nucleic acids and polypeptides|
|US6743619B1 (en)||Nucleic acids and polypeptides|
|US20040048249A1 (en)||Novel nucleic acids and secreted polypeptides|
|US6436703B1 (en)||Nucleic acids and polypeptides|
|US20020197679A1 (en)||Novel nucleic acids and polypeptides|
|US7411052B2 (en)||Methods and materials relating to stem cell growth factor-like polypeptides and polynucleotides|
|US20040053245A1 (en)||Novel nucleic acids and polypeptides|
|US20020146692A1 (en)||Methods and materials relating to G protein-coupled receptor-like polypeptides and polynucleotides|
|US6541623B1 (en)||Interleukin—1 receptor antagonist and uses thereof|
|US20030232054A1 (en)||Novel nucleic acids and polypeptides|
|WO2001055317A2 (en)||Nucleic acids, proteins, and antibodies|
|US6673904B2 (en)||Stem cell growth factor-like polypeptides|
|US20030194708A1 (en)||Human homolog of crossveinless materials and methods|