EP1054961A2 - Proteine de la famille des recepteurs a chaine courte humains du tnf - Google Patents

Proteine de la famille des recepteurs a chaine courte humains du tnf

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Publication number
EP1054961A2
EP1054961A2 EP99905816A EP99905816A EP1054961A2 EP 1054961 A2 EP1054961 A2 EP 1054961A2 EP 99905816 A EP99905816 A EP 99905816A EP 99905816 A EP99905816 A EP 99905816A EP 1054961 A2 EP1054961 A2 EP 1054961A2
Authority
EP
European Patent Office
Prior art keywords
sctr
polynucleotide
sequence
seq
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99905816A
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German (de)
English (en)
Inventor
Y. Tom Tang
Neil C. Corley
Preeti Lal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Incyte Corp
Original Assignee
Incyte Pharmaceuticals Inc
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Filing date
Publication date
Application filed by Incyte Pharmaceuticals Inc filed Critical Incyte Pharmaceuticals Inc
Publication of EP1054961A2 publication Critical patent/EP1054961A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TNF -receptor family protein and to the use of these sequences in the diagnosis, treatment, and prevention of developmental, reproductive, neoplastic. and immunological disorders.
  • MPs have been associated with tissue growth, cell proliferation, tissue or cell differentiation, and cell death.
  • MPs also act as important mediators and regulators of the inflammatory response.
  • Tumor necrosis factor is a pleiotropic cytokine that a mediates immune regulation and inflammatory responses.
  • TNF-related cytokines generate partially overlapping cellular responses, including differentiation, proliferation, NF- ⁇ B activation, and cell death, by triggering the aggregation of receptor monomers.
  • the cellular responses triggered by TNF are initiated through its interaction with distinct cell surface receptors.
  • the tumor necrosis factor receptor (TNFR) superfamily includes the 60 kDa and 80 kDa forms of TNFR (TNFRI and TNFRII).
  • nerve growth factor receptor (NGFR) CD40, CD27. CD30, OX-40, 4- 1 BB, Fas, WSL-S 1 , GITR. TRICK-2A, and TRAIL receptor.
  • NGFR nerve growth factor receptor
  • CD40 CD27. CD30, OX-40
  • 4- 1 BB Fas
  • WSL-S 1 GITR. TRICK-2A
  • TRAIL receptor TRAIL receptor
  • CD40, 5 CD27, 4- IBB, OX-40, WSL-S1, and GITR are generally less than 300 amino acids in length (short chain), whereas TNFRI, TNFRII, CD30, Fas, TRICK-2A, and TRAIL receptor are more than 300 residues in length (long chain).
  • WSL-S1/LARD-3 appears to be a soluble isoform of the transmembrane protein WSL-l /LARD-1, truncated before the transmembrane domain.
  • binding of the signaling TNFR ligand to TNFR superfamily proteins initiates a second messenger cascade which often leads to programmed cell death, also known as apoptosis.
  • a sequence motif termed the death domain is present in some TNFR proteins and is a critical structural element involved in signal transduction which leads to apoptosis.
  • the death domain averaging 70-80 amino acids, is always localized near one end of the protein. This motif is also found in TNFR-binding proteins. (Hofman, K. and Tschopp, J. (1995) FEBS Lett. 371 :321-323.)
  • TNFR superfamily proteins antagonize the apoptosis-inducing second messenger cascade, either by interfering with the ligand-receptor interaction or by
  • TNFR and NGFR bind more than one type of ligand. and therefore members of the TNFR superfamily may interact with more than one type of ligand.
  • Defective expression of TNFR genes can prevent signal transduction and induction of apoptosis.
  • the present invention is based on the discovery of a new short-chain tumor necrosis factor receptor family protein (SCTR), the polynucleotides encoding SCTR, and the use of these compositions for the diagnosis, treatment, or prevention of developmental, reproductive, neoplastic, and immunological disorders.
  • SCTR tumor necrosis factor receptor family protein
  • the invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.
  • the invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.
  • the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO:l .
  • the invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:l or a fragment of SEQ ID NO:l.
  • the invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:l or a fragment of SEQ ID NO:l, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.
  • the invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
  • the invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
  • the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.
  • the expression vector is contained within a host cell.
  • the invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 , the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO:l in conjunction with a suitable pharmaceutical carrier.
  • the invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:l or a fragment of SEQ ID NO:l. as well as a purified agonist and a purified antagonist of the polypeptide.
  • the invention also provides a method for treating or preventing a developmental disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide having the amino acid sequence of SEQ ID NO:l or a fragment of SEQ ID NO:l.
  • the invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide having the amino acid sequence of SEQ ID NO:l or a fragment of SEQ ID NO:l.
  • the invention also provides a method for treating or preventing a neoplastic disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide having the amino acid sequence of SEQ ID NO:l or a fragment of SEQ ID NO:l.
  • the invention also provides a method for treating or preventing an immunological disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.
  • the invention also provides a method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:l or a fragment of SEQ ID NO:l in the biological sample.
  • the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.
  • Figures 1A, IB, 1C, ID, and IE show the amino acid sequence (SEQ ID NO:l) and nucleic acid sequence (SEQ ID NO:2) of SCTR.
  • the alignment was produced using MacDNASIS PROTM software (Hitachi Software Engineering Co. Ltd., San Bruno, CA).
  • Figures 2A, 2B, and 2C show the amino acid sequence alignments among SCTR (1321844; SEQ ID NO:l), human 4- IBB (GI 728738; SEQ ID NO:3), human WSL- Sl/LARD-3 (GI 1669692; SEQ ID NO:4). and human apoptosis-inducing protein TRICK- 2A (GI 2407651 : SEQ ID NO:5), produced using the multisequence alignment program of DNASTARTM software (DNASTAR Inc. Madison WI).
  • a includes a plurality of such host cells
  • an antibody is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
  • SCTR refers to the amino acid sequences of substantially purified SCTR obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which, when bound to SCTR, increases or prolongs the duration of the effect of SCTR.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of SCTR.
  • alleles are an alternative form of the gene encoding SCTR. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding SCTR include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same SCTR or a polypeptide with at least one functional characteristic of SCTR. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding SCTR, and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding SCTR.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent SCTR.
  • Deliberate 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, as long as the biological or immunological activity of SCTR is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
  • amino acid or amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules.
  • fragments refer to fragments of SCTR which are preferably about 5 to about 15 amino acids in length and which retain some biological activity or immunological activity of SCTR.
  • amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art. (See, e.g., Dieffenbach, C.W. and G.S. Dveksler (1995) PCR Primer, a Laboratory Manual. Cold Spring Harbor Press, Plainview, NY, pp.1-5.)
  • PCR polymerase chain reaction
  • antagonist refers to a molecule which, when bound to SCTR, decreases the amount or the duration of the effect of the biological or immunological activity of SCTR.
  • Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of SCTR.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fa, F(ab') 2 , and Fv fragments, which are capable of binding the epitopic determinant.
  • Antibodies that bind SCTR polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired.
  • a carrier protein e.g., bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • antigenic determinant refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition containing a nucleic acid sequence which is complementary to a specific nucleic acid sequence.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
  • Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant. or synthetic SCTR, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base pairing.
  • sequence A-G-T
  • complementary sequence T-C-A
  • 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 nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • composition comprising a given polynucleotide sequence or a “composition comprising a given amino acid sequence,” as these terms are used herein, refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation, an aqueous solution, or a sterile composition.
  • Compositions comprising polynucleotide sequences encoding SCTR or fragments of SCTR may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCRTM (Perkin Elmer. Norwalk, CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GEL VIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence .
  • XL-PCRTM Perkin Elmer. Norwalk, CT
  • GEL VIEWTM Fragment Assembly system GEL VIEWTM Fragment Assembly system
  • the term "correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding SCTR, by northern analysis is indicative of the presence of nucleic acids encoding SCTR in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding SCTR.
  • a “deletion,” as the term is used herein, refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to the chemical modification of SCTR, of a polynucleotide sequence encoding SCTR, or of a polynucleotide sequence complementary to a polynucleotide sequence encoding SCTR.
  • Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially homologous.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency.
  • a substantially homologous sequence or hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of reduced stringency.
  • Percent identity refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MegAlign program (DNASTAR, Inc.. Madison WI). The MegAlign program can create alignments between two or more sequences according to different methods, e.g., the Clustal method. (See, e.g., Higgins, D.G. and P. M. Sharp (1988) Gene 73:237-244.) The Clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
  • the percentage similarity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance. (See, e.g., Harrington, J.J. et al. (1997) Nat Genet. 15:345-355.)
  • humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R ⁇ ,t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • insertion or “addition,” as used herein, refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines. and other signaling molecules, which may affect cellular and systemic defense systems.
  • microarray refers to an arrangement of distinct polynucleotides or oligonucleotides arrayed on a substrate, e.g., paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • modulate refers to a change in the activity of SCTR. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of SCTR.
  • nucleic acid or “nucleic acid sequence,” as used herein, refer to an oligonucleotide. nucleotide, polynucleotide, or any fragment thereof, 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.
  • fragments refers to those nucleic acid sequences which are greater than about 60 nucleotides in length, and most preferably are at least about 100 nucleotides, at least about 1000 nucleotides, or at least about 10,000 nucleotides in length.
  • operably associated refers to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the encoded polypeptide but still bind to operator sequences that control expression of the polypeptide.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
  • oligonucleotide is substantially equivalent to the terms “amplimer,” “primer,” “oligomer,” and “probe,” as these terms are commonly defined in the art.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
  • PNAs preferentially bind complementary single stranded DNA and RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)
  • sample as used herein, is used in its broadest sense.
  • a biological sample suspected of containing nucleic acids encoding SCTR, or fragments thereof, or SCTR itself may comprise a bodily fluid: an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solid support; a tissue; a tissue print; etc.
  • the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • stringent conditions refers to conditions which permit hybridization between polynucleotide sequences and the claimed polynucleotide sequences.
  • stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide. or raising the hybridization temperature.
  • hybridization under high stringency conditions could occur in about 50% formamide at about 37°C to 42°C.
  • Hybridization could occur under reduced stringency conditions in about 35% to 25% formamide at about 30°C to 35°C.
  • hybridization could occur under high stringency conditions at 42°C in 50% formamide.
  • Hybridization could occur under reduced stringency conditions as described above, but in 35% formamide at a reduced temperature of 35°C.
  • the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90%) free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • Transformation describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a “variant" of SCTR refers to an amino acid sequence that is altered by one or more amino acids.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have
  • nonconservative changes e.g., replacement of glycine with tryptophan.
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.
  • the invention is based on the discovery of a new human short-chain TNF -receptor family protein (SCTR), the polynucleotides encoding SCTR, and the use of these compositions for the diagnosis, treatment, or prevention of developmental, reproductive, neoplastic. and immunological disorders. Nucleic acids encoding the SCTR of the present invention were first identified in
  • Incyte Clone 1321844 from the bladder cDNA library (BLADNOT04) using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:2 was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 1321884 (BLADNOT04), 1919431 (BRSTTUT01), 1450213 and 1450646 (PENITUT01). 3189157 (THYMNON04), 1208771 (BRSTNOT02), 1219551 (NEUTGMT01), and 1358156 (LUNGNOT09).
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:l, as shown in Figures 1A, IB, 1C, ID. and IE.
  • SCTR is 291 amino acids in length and has one potential cAMP and cGMP-dependant phosphorylation site at residue T-200; two potential casein kinase II phosphorylation sites at residues S-l 13 and S-206; four potential protein kinase C phosphorylation sites at residues S-l 1, S-24, S-25, and S-228, and a potential TNFR superfamily cysteine-rich region signature from about residue C-l 12 to about residue C-210.
  • SCTR has chemical and structural homology with human 4- IBB (GI 728738; SEQ ID NO:3), human WSL-Sl/ LARD-3 (GI 1669692; SEQ ID NO:4), and human apoptosis-inducing protein TRICK-2A (GI 2407651 : SEQ ID NO:5).
  • SCTR shares 13 % identity with human 4-1BB and human WSL-Sl /LARD-3.
  • the internal region of SCTR from about residue 112 to about 235 containing the cysteine-rich region signature shares 26 % and 36 % identity, respectively with the corresponding regions in human 4- IBB and human WSL-Sl / LARD-3.
  • SCTR human 4-1BB, and human WSL-S1/LARD-3
  • SCTR human 4-1BB, and human WSL-S1/LARD-3
  • the fragment of SEQ ID NO:2 from about nucleotide 463 to about nucleotide 495 is useful for designing oligonucleotides or for use as a hybridization probe.
  • Northern analysis shows the expression of this sequence in various libraries, at least 53 % of which are immortalized or cancerous and at least 23 % of which involve immune response.
  • SCTR in reproductive, neuronal, gastrointestinal, cardiovascular, and developmental tissues.
  • the invention also encompasses SCTR variants.
  • a preferred SCTR variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the SCTR amino acid sequence, and which contains at least one functional or structural characteristic of SCTR.
  • the invention also encompasses polynucleotides which encode SCTR.
  • the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO:2, which encodes a SCTR.
  • the invention also encompasses a variant of a polynucleotide sequence encoding SCTR.
  • a variant polynucleotide sequence will have at least about 80%, more preferably at least about 90%. and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding SCTR.
  • a particular aspect of the invention encompasses a variant of SEQ ID NO:2 which has at least about 80%. more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:2.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of SCTR.
  • nucleotide sequences which encode SCTR and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring SCTR under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding SCTR or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode
  • SCTR and SCTR derivatives, or fragments thereof, entirely by synthetic chemistry After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding SCTR or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:2 or a fragment of SEQ ID NO:2 under various conditions of stringency.
  • SEQ ID NO:2 or a fragment of SEQ ID NO:2 under various conditions of stringency.
  • Methods for DNA sequencing are well known and generally available in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp., Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE Amplification System (GlBCO/BRL, Gaithersburg, MD).
  • the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • the nucleic acid sequences encoding SCTR may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • various methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus.
  • genomic DNA is first amplified in the presence of a primer which is complementary to a linker sequence within the vector and a primer specific to a region of the nucleotide sequence.
  • the amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
  • the primers may be designed using commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, MN) or another appropriate program to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to 72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations may be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g., GenotyperTM and Sequence NavigatorTM, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode SCTR may be used in recombinant DNA molecules to direct expression of SCTR, or fragments or functional equivalents thereof, in appropriate host cells. 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 produced, and these sequences may be used to clone and express SCTR.
  • SCTR-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • the nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter SCTR-encoding sequences for a variety of reasons including, but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • nucleic acid sequences encoding SCTR may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence For example, to screen peptide libraries for inhibitors of SCTR activity, it may be useful to encode a chimeric SCTR protein that can be recognized by a commercially available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the SCTR encoding sequence and the heterologous protein sequence, so that SCTR may be cleaved and purified away from the heterologous moiety.
  • sequences encoding SCTR may be synthesized, in whole or in part, using chemical methods well known in the art.
  • the protein itself may be produced using chemical methods to synthesize the amino acid sequence of SCTR, or a fragment thereof.
  • peptide synthesis can be performed using various solid-phase techniques.
  • Automated synthesis may be achieved using the ABI 431 A Peptide Synthesizer (Perkin Elmer).
  • the amino acid sequence of SCTR, or any part thereof may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol.
  • composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1983) Proteins. Structures and Molecular Properties. WH Freeman and Co., New York, NY.)
  • nucleotide sequences encoding SCTR or derivatives thereof may be inserted into appropriate expression vector, i.e.. a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding SCTR. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid. or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • the invention is not limited by the host cell employed.
  • control elements are those non-translated regions, e.g., enhancers, promoters, and 5' and 3' untranslated regions, of the vector and polynucleotide sequences encoding SCTR which interact with host cellular proteins to carry out transcription and translation.
  • Such elements may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters e.g., hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, La Jolla, CA) or pSportlTM plasmid (GlBCO/BRL
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding SCTR. vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • a number of expression vectors may be selected depending upon the use intended for SCTR.
  • vectors which direct high level expression of fusion proteins that are readily purified may be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the sequence encoding SCTR may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced, and pIN vectors.
  • Bluescript® Stratagene
  • pIN vectors See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH, may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH
  • the expression of sequences encoding SCTR may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
  • These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews. (See, e.g., Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, NY; pp. 191-196.)
  • An insect system may also be used to express SCTR.
  • SCTR Autographa californica nuclear polyhedrosis virus
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the sequences encoding SCTR may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of sequences encoding SCTR will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which SCTR may be expressed.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding SCTR may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing SCTR in infected host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding SCTR. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding SCTR and its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular cell system used. (See, e.g., Scharf. D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation. carboxylation, glycosylation, phosphorylation, lipidation. and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding, and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda. MD) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • cell lines capable of stably expressing SCTR can be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase genes and adenine phosphoribosyltransferase genes, which can be employed in tk ⁇ or apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11 :223-232; and Lowy, I. et al.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • npt confers resistance to the aminoglycosides neomycin and G-418
  • als o ⁇ pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine.
  • Visible markers e.g., anthocyanins, ⁇ glucuronidase and its substrate GUS, luciferase and its substrate luciferin may be used.
  • Green fluorescent proteins (GFP) (Clontech, Palo Alto, CA) can also be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. et al. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding SCTR is inserted within a marker gene sequence, transformed cells containing sequences encoding SCTR can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding SCTR under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • GFP Green fluorescent proteins
  • host cells which contain the nucleic acid sequence encoding SCTR and express SCTR may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
  • polynucleotide sequences encoding SCTR can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding SCTR.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding SCTR to detect transformants containing DNA or RNA encoding SCTR.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding SCTR include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding SCTR, or any fragments thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • RNA polymerase such as T7, T3, or SP6
  • Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent. or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding SCTR may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode SCTR may be designed to contain signal sequences which direct secretion of SCTR through a prokaryotic or eukaryotic cell membrane.
  • Other constructions may be used to join sequences encoding SCTR to nucleotide sequences encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp., Seattle, WA.
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA)
  • One such expression vector provides for expression of a fusion protein containing SCTR and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on immobilized metal ion affinity chromatography (IMAC).
  • IMAC immobilized metal ion affinity chromatography
  • the enterokinase cleavage site provides a means for purifying SCTR from the fusion protein.
  • Fragments of SCTR may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques.
  • Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • Various fragments of SCTR may be synthesized separately and then combined to produce the full length molecule.
  • SCTR Chemical and structural homology exists among SCTR and human 4- IBB (GI 728738), human WSL-S1/LARD-3 (GI 1669692), and human apoptosis-inducing protein TRICK-2A (GI 2407651).
  • SCTR is expressed in neoplastic, immunological, reproductive, neuronal, gastrointestinal, cardiovascular, and developmental tissues. Therefore, SCTR appears to play a role in developmental, reproductive, neoplastic, and immunological disorders.
  • SCTR or a fragment or derivative thereof may be administered to a subject to treat or prevent a developmental disorder.
  • developmental disorder refers to any disorder associated with development or function of a tissue, organ, or system of a subject (such as the brain, adrenal gland, kidney, skeletal or reproductive system).
  • Such developmental disorders can include, but are not limited to, renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome, Smith- Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spinal bifida, and congenital glaucoma, cataract, or sensorineural hearing loss.
  • a vector capable of expressing SCTR or a fragment or derivative thereof may be administered to a subject to treat or prevent a developmental disorder including, but not limited to, those described above.
  • a pharmaceutical composition comprising a substantially purified SCTR in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a developmental disorder including, but not limited to, those provided above.
  • an agonist which modulates the activity of SCTR may be administered to a subject to treat or prevent a developmental disorder including, but not limited to, those listed above.
  • SCTR or a fragment or derivative thereof may be administered to a subject to treat or prevent a reproductive disorder.
  • reproductive disorders can include, but are not limited to, disorders of prolactin production; infertility, including tubal disease, ovulatory defects, and endometriosis; disruptions of the estrous cycle, disruptions of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, autoimmune disorders, ectopic pregnancy, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea: disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis. cancer of the prostate, benign prostatic hyperplasia, and prostatitis, carcinoma of the male breast and gynecomastia.
  • a vector capable of expressing SCTR or a fragment or derivative thereof may be administered to a subject to treat or prevent a reproductive disorder including, but not limited to, those described above.
  • a pharmaceutical composition comprising a substantially purified SCTR in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a reproductive disorder including, but not limited to, those provided above.
  • an agonist which modulates the activity of SCTR may be administered to a subject to treat or prevent a reproductive disorder including, but not limited to, those listed above.
  • SCTR or a fragment or derivative thereof may be administered to a subject to treat or prevent a neoplastic disorder.
  • a neoplastic disorder may include, but is not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
  • a vector capable of expressing SCTR or a fragment or derivative thereof may be administered to a subject to treat or prevent a neoplastic disorder including, but not limited to, those described above.
  • a pharmaceutical composition comprising a substantially purified SCTR in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a neoplastic disorder including, but not limited to, those provided above.
  • an agonist which modulates the activity of SCTR may be administered to a subject to treat or prevent a neoplastic disorder including, but not limited to, those listed above.
  • an antagonist of SCTR may be administered to a subject to treat or prevent an immunological disorder.
  • immunological disorders can include, but are not limited to, AIDS, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis. bronchitis, cholecystitis, contact dermatitis. Crohn's disease, atopic dermatitis, dermatomyositis.
  • diabetes mellitus emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease. Hashimoto's thyroiditis, hypereosinophilia.
  • an immunological disorder may include, but is not limited to, those discussed above.
  • an antibody which specifically binds SCTR may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express SCTR.
  • a vector expressing the complement of the polynucleotide encoding SCTR may be administered to a subject to treat or prevent an immunological disorder including, but not limited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art. according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of SCTR may be produced using methods which are generally known in the art. In particular, purified SCTR may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind SCTR.
  • Antibodies to SCTR may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with SCTR or with any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions. KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corvnebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to SCTR have an amino acid sequence consisting of at least about 5 amino acids, and. more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of SCTR amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to SCTR may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.
  • the hybridoma technique the human B-cell hybridoma technique
  • EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
  • chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • techniques developed for the production of “chimeric antibodies” such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used.
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce SCTR-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)
  • Antibody fragments which contain specific binding sites for SCTR may also be generated.
  • fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between SCTR and its specific antibody.
  • the polynucleotides encoding SCTR. or any fragment or complement thereof may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding SCTR may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding SCTR.
  • complementary molecules or fragments may be used to modulate SCTR activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding SCTR.
  • Expression vectors derived from retroviruses, adeno viruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequences complementary to the polynucleotides of the gene encoding SCTR. (See. e.g., Sambrook, supra: and Ausubel, supra.)
  • Genes encoding SCTR can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding SCTR. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA. such vectors may continue to transcribe RNA molecules until they are disabled by,,endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions of the gene encoding SCTR.
  • Oligonucleotides derived from the transcription initiation site e.g., between about positions -10 and +10 from the start site, are preferred.
  • inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases. transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber. B.E.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding SCTR.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU. and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding SCTR. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine. and wybutosine, as well as acetyl-, methyl-, thio-. and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-466.)
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of SCTR, antibodies to SCTR, and mimetics. agonists, antagonists, or inhibitors of SCTR.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal. subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules
  • auxiliaries can be added, if desired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, 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 product identification or to characterize the quantity of active compound, i.e., dosage.
  • 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 coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate. and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution. Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • 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, triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example SCTR or fragments thereof, antibodies of SCTR. and agonists, antagonists or inhibitors of SCTR, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED50 (the dose therapeutically effective in 50% of the population) or LD50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the ED50/LD50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • antibodies which specifically bind SCTR may be used for the diagnosis of disorders characterized by expression of SCTR, or in assays to monitor patients being treated with SCTR or agonists, antagonists, or inhibitors of SCTR.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for SCTR include methods which utilize the antibody and a label to detect SCTR in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • SCTR A variety of protocols for measuring SCTR, including ELISAs. RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of SCTR expression.
  • Normal or standard values for SCTR expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to SCTR under conditions suitable for complex formation The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of SCTR expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding SCTR may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of SCTR may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of SCTR. and to monitor regulation of SCTR levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding SCTR or closely related molecules may be used to identify nucleic acid sequences which encode SCTR.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding SCTR, alleles, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the SCTR encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:2, or from genomic sequences including promoters, enhancers, and introns of the SCTR gene.
  • Means for producing specific hybridization probes for DNAs encoding SCTR include the cloning of polynucleotide sequences encoding SCTR or SCTR derivatives into vectors for the production of mRNA probes.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding SCTR may be used for the diagnosis of a disorder associated with expression of SCTR.
  • a disorder associated with expression of SCTR include, but are not limited to, a developmental disorder such as, renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome, Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spinal bifida, and congenital glaucoma, cataract, or sensorineural hearing loss; a reproductive disorder such as, disorders of prolactin production; infertility, including tubal disease,
  • a neoplastic disorder such as, adenocarcinoma. leukemia, lymphoma. melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; and an immunological disorder such as, AIDS, Addison ' s disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis.
  • an immunological disorder such as, AIDS, Addison ' s disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis.
  • the polynucleotide sequences encoding SCTR may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patients to detect altered SCTR expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding SCTR may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding SCTR may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding SCTR in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding SCTR. under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • oligonucleotides designed from the sequences encoding SCTR may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding SCTR, or a fragment of a polynucleotide complementary to the polynucleotide encoding SCTR, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of SCTR include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
  • radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymo ⁇ hisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • nucleic acid sequences encoding SCTR may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs). bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries.
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding SCTR on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
  • the nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals. In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • SCTR in another embodiment, SCTR, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the formation of binding complexes between SCTR and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT application WO84/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • test compounds are reacted with SCTR, or fragments thereof, and washed. Bound SCTR is then detected by methods well known in the art. Purified SCTR can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • nucleotide sequences which encode SCTR may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • the BLADNOT04 cDNA library was constructed from microscopically normal bladder tissue obtained from a 28-year-old Caucasian male who died from a self-inflicted gun shot wound.
  • the frozen tissue was homogenized and lysed using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, NJ) in guanidinium isothiocyanate solution.
  • the lysate was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 ⁇ m at ambient temperature.
  • the RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water, and treated with DNase at 37°C RNA extraction and precipitation were repeated as before.
  • the mRNA was then isolated using the Qiagen Oligotex kit (QIAGEN, Inc.; Chatsworth, CA) and used to construct the cDNA library.
  • mRNA was handled according to the recommended protocols in the Superscript plasmid system (Catalog #18248-013, G co-BRL).
  • cDNA synthesis was initiated with a Notl-oligo d(T) primer.
  • Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with Notl, fractionated on a Sepharose CL4B column (Catalog #275105-01; Pharmacia), and those cDNAs exceeding 400 bp were ligated into the Notl and EcoRI sites of the pINCY 1 vector (Incyte).
  • the plasmid pINCY 1 was subsequently transformed into DH5 ⁇ TM competent cells (Catalog #18258-012; GiBCO-BRL).
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Catalog #26173; QIAGEN, Inc.). The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog #22711, GiBCO-BRL) with carbenicillin at 25 mg/1 and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours and at the end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4° C
  • the cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol. Biol. 94:44 If), using a Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with Peltier Thermal Cyclers (PTC200 from MJ Research, Watertown, MA) and Applied Biosystems 377 DNA Sequencing Systems, and reading frame was determined.
  • nucleotide sequences and/or amino acid sequences of the Sequence Listing were used to query sequences in the GenBank, SwissProt, BLOCKS, and Pima II databases. These databases, which contain previously identified and annotated sequences, were searched for regions of homology using BLAST (Basic Local Alignment Search Tool). (See, e.g., Altschul, S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol. Biol. 215:403-410.) BLAST produced alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments.
  • BLAST was especially useful in determining exact matches or in identifying homologs which may be of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin.
  • Other algorithms could have been used when dealing with primary sequence patterns and secondary structure gap penalties.
  • the sequences disclosed in this application have lengths of at least 49 nucleotides and have no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
  • BLAST searched for matches between a query sequence and a database sequence.
  • BLAST evaluated the statistical significance of any matches found, and reported only those matches that satisfy the user-selected threshold of significance.
  • threshold was set at 10 "25 for nucleotides and 10 "8 for peptides.
  • Incyte nucleotide sequences were searched against the GenBank databases for primate (pri), rodent (rod), and other mammalian sequences (mam), and deduced amino acid sequences from the same clones were then searched against GenBank functional protein databases, mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp), for homology.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; and Ausubel, F.M. et al. supra, ch. 4 and 16.)
  • Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQTM database (Incyte Pharmaceuticals). This analysis is much faster than multiple membrane- based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
  • the basis of the search is the product score, which is defined as:
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40. the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • the results of northern analysis are reported as a list of libraries in which the transcript encoding SCTR occurs. Abundance and percent abundance are also reported. Abundance directly reflects the number of times a particular transcript is represented in a cDNA library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library.
  • the nucleic acid sequence of Incyte Clone 1321844 was used to design oligonucleotide primers for extending a partial nucleotide sequence to full length.
  • One primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide.
  • Primers were used to facilitate the extension of the known sequence "outward" generating amplicons containing new unknown nucleotide sequence for the region of interest.
  • the initial primers were designed from the cDNA using OLIGO 4.06 (National Biosciences, Madison, MN), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations was avoided.
  • High fidelity amplification was obtained by following the instructions for the XL- PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. PCR was performed using the Peltier Thermal Cycler (PTC200; M.J. Research, Watertown, MA), beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, with the following parameters:
  • Step 1 94° C for 1 min (initial denaturation)
  • Step 2 65° C for 1 min
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 6 68° C for 7 min Step 7 Repeat steps 4 through 6 for an additional 15 cycles
  • Step 8 94° C for 15 sec
  • Step 11 Repeat steps 8 through 10 for an additional 12 cycles Step 12 72° C for 8 min
  • coli mixture was plated on Luria Bertani (LB) agar (See, e.g., Sambrook, supra. Appendix A, p. 1) containing 2x Carb. The following day, several colonies were randomly picked from each plate and cultured in 150 ⁇ l of liquid LB/2x Carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 ⁇ l of each overnight culture was transferred into a non-sterile 96-well plate and. after dilution 1 :10 with water, 5 ⁇ l from each sample was transferred into a PCR array.
  • LB Luria Bertani
  • PCR amplification For PCR amplification, 18 ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions:
  • Step 2 94° C for 20 sec
  • Step 3 55° C for 30 sec
  • Step 5 Repeat steps 2 through 4 for an additional 29 cycles
  • Hybridization probes derived from SEQ ID NO:2 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham, Chicago, IL), and T4 polynucleotide kinase (DuPont NEN ® , Boston, MA).
  • the labeled oligonucleotides are substantially purified using a Sephadex G-25 superfine resin column (Pharmacia & Upjohn, Kalamazoo, MI). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II (DuPont NEN, Boston, MA).
  • the DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus. Schleicher & Schuell, Durham. NH). Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester, NY) is exposed to the blots to film for several hours, hybridization patterns are compared visually.
  • oligonucleotides for a microarray one of the nucleotide sequences of the present invention is examined using a computer algorithm which starts at the 3' end of the nucleotide sequence. For each, the algorithm identifies oligomers of defined length that are unique to the nucleic acid sequence, have a GC content within a range suitable for hybridization, and lack secondary structure that would interfere with hybridization. The algorithm identifies approximately 20 oligonucleotides corresponding to each nucleic acid sequence. For each sequence-specific oligonucleotide.
  • a pair of oligonucleotides is synthesized in which the first oligonucleotides differs from the second oligonucleotide by one nucleotide in the center of the sequence.
  • the oligonucleotide pairs can be arranged on a substrate, e.g. a silicon chip, using a light-directed chemical process. (See, e.g., Chee, supra.)
  • Probe sequences may be selected by screening a large number of clones from a variety of cDNA libraries in order to find sequences with conserved protein motifs common to genes coding for signal sequence containing polypeptides.
  • sequences identified from cDNA libraries are analyzed to identify those gene sequences with conserved protein motifs using an appropriate analysis program, e.g., the Block 2 Bioanalysis Program (Incyte, Palo Alto, CA).
  • This motif analysis program based on sequence information contained in the Swiss-Prot Database and PROSITE, is a method of determining the function of uncharacterized proteins translated from genomic or cDNA sequences. (See, e.g., Bairoch, A. et al. (1997) Nucleic Acids Res.
  • PROSITE may be used to identify functional or structural domains that cannot be detected using conserved motifs due to extreme sequence divergence. The method is based on weight matrices. Motifs identified by this method are then calibrated against the SWISS-PROT database in order to obtain a measure of the chance distribution of the matches.
  • HMMs Hidden Markov models
  • HMMs were initially developed to examine speech recognition patterns, but are now being used in a biological context to analyze protein and nucleic acid sequences as well as to model protein structure.
  • HMMs have a formal probabilistic basis and use position-specific scores for amino acids or nucleotides. The algorithm continues to inco ⁇ orate information from newly identified sequences to increase its motif analysis capabilities.
  • a chemical coupling procedure and an ink jet device can be used to synthesize oligomers on the surface of a substrate.
  • An array analogous to a dot or slot blot may also be used to arrange and link fragments or oligonucleotides to the surface of a substrate using or thermal, UV, mechanical, or chemical bonding procedures, or a vacuum system.
  • a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
  • nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray may be assessed through analysis of the scanned images.
  • full-length cDNAs or Expressed Sequence Tags comprise the elements of the microarray.
  • Full-length cDNAs or ESTs corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention are arranged on an appropriate substrate, e.g., a glass slide.
  • the cDNA is fixed to the slide using, e.g., U.V. cross-linking followed, by thermal and chemical and subsequent drying.
  • U.V. cross-linking followed, by thermal and chemical and subsequent drying.
  • Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
  • Sequences complementary to the SCTR-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring SCTR.
  • oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
  • Appropriate oligonucleotides are designed using Oligo 4.06 software and the coding sequence of SCTR.
  • a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oligonucleotide is designed to prevent ribosomal binding to the SCTR-encoding transcript.
  • SCTR is accomplished by subcloning the cDNA into an appropriate vector and transforming the vector into host cells.
  • This vector contains an appropriate promoter, e.g., ⁇ -galactosidase upstream of the cloning site, operably associated with the cDNA of interest.
  • an appropriate promoter e.g., ⁇ -galactosidase upstream of the cloning site, operably associated with the cDNA of interest.
  • IPTG isopropyl beta-D- thiogalactopyranoside
  • SCTR short-chain TNF-receptor family protein
  • the assay for short-chain TNF-receptor family protein measures the prevention or induction of apoptosis in a transfected cell line.
  • SCTR can be expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with an eukaryotic expression vector encoding SCTR.
  • Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art.
  • the cells with and without the SCTR expression vector are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression of SCTR. Phase microscopy is subsequently used to compare the mitotic index of transformed versus control cells. An increase in the mitotic index where SCTR stimulates cell proliferation by blocking apoptosis indicates SCTR activity. Likewise, a decrease in cell numbers where SCTR stimulates apoptosis indicates SCTR activity.
  • SCTR substantially purified using PAGE electrophoresis (see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
  • the SCTR amino acid sequence is analyzed using DNASTAR software (DNASTAR Inc) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C -terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel et al. supra, ch.
  • the oligopeptides are 15 residues in length, and are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to KLH (Sigma, St. Louis, MO) by reaction with N-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • MBS N-maleimidobenzoyl-N- hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 1% BSA. reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • XII Purification of Naturally Occurring SCTR Using Specific Antibodies
  • Naturally occurring or recombinant SCTR is substantially purified by immunoaffinity chromatography using antibodies specific for SCTR.
  • An immunoaffinity column is constructed by covalently coupling anti-SCTR antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • Media containing SCTR are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of SCTR (e.g., high ionic strength buffers in the presence of detergent).
  • SCTR preferential absorbance of SCTR
  • the column is eluted under conditions that disrupt antibody/SCTR binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and SCTR is collected.
  • SCTR SCTR, or biologically active fragments thereof, are labeled with I25 I Bolton-Hunter reagent.
  • I25 I Bolton-Hunter reagent See, e.g., Bolton et al. (1973) Biochem. J. 133:529.
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled SCTR, washed, and any wells with labeled SCTR complex are assayed. Data obtained using different concentrations of SCTR are used to calculate values for the number, affinity, and association of SCTR with the candidate molecules.

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Abstract

L'invention porte sur une protéine de la famille des récepteurs à chaîne courte humains du TNF (SCTR) et sur des polynucléotides identifiant le SCTR et codant pour lui. L'invention porte également sur des vecteurs d'expression, des cellules hôtes, des agonistes et des antagonistes ainsi que sur des méthodes de traitement ou prévention de troubles associés à l'expression du SCTR.
EP99905816A 1998-02-17 1999-02-05 Proteine de la famille des recepteurs a chaine courte humains du tnf Withdrawn EP1054961A2 (fr)

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