[go: up one dir, main page]

EP1015588A1 - Regulateur de la croissance cellulaire - Google Patents

Regulateur de la croissance cellulaire

Info

Publication number
EP1015588A1
EP1015588A1 EP98953187A EP98953187A EP1015588A1 EP 1015588 A1 EP1015588 A1 EP 1015588A1 EP 98953187 A EP98953187 A EP 98953187A EP 98953187 A EP98953187 A EP 98953187A EP 1015588 A1 EP1015588 A1 EP 1015588A1
Authority
EP
European Patent Office
Prior art keywords
celr
sequence
sequences
polynucleotide sequence
fragments
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Withdrawn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
EP98953187A
Other languages
German (de)
English (en)
Inventor
Preeti Lal
Neil C. Corley
Purvi Shah
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
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Incyte Pharmaceuticals Inc filed Critical Incyte Pharmaceuticals Inc
Publication of EP1015588A1 publication Critical patent/EP1015588A1/fr
Withdrawn legal-status Critical Current

Links

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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TECHNICAL FIELD This invention relates to nucleic acid and amino acid sequences of a new human cell growth regulator and to the use of these sequences in the diagnosis, prevention, and treatment of inflammation and disorders associated with cell proliferation and apoptosis
  • the p53 protein is a tumor suppressor and remains the most frequently mutated gene in human cancers p53 functions primarily m transc ⁇ ptional regulation of its down-stream effector genes The regulation is achieved either through direct interaction of the p53 protein with transcription factors or via sequence-specific binding of the transc ⁇ ptional activation domain of p53 to DNA
  • growth-responsive genes transc ⁇ ptionally regulated by p53 include p22 F ' /aPI , MDM2, CGR11, and CGR19 p2j WAFi/ c ipi QQRH anc j CGR19 have been characterized as giowth suppressing genes while MDM2 is associated with promoting cell growth
  • p2J WAH/ap ' encodes a kinase inhibitor which suppresses cell growth by interacting with cychn-dependent kinases (Harper, J W et al (1993) Cell 75 805-816) Decrease in p53 regulatory activity by MDM2 leads to continuous cell
  • CGR19 is a zinc-binding protein which contains a ring-finger domain close to its carboxy terminus In contrast to other ⁇ ng-finger-containing proteins, CGR19 lacks a ring- finger associated B-box domain (Madden et al., supra).
  • the ring-finger domain of CGR19 has a consensus sequence of C-X 2 -C-X (9 . 27) -C-X ( 1 _ 3) -H-X (2 _ 3) -C-X 2 -C-X (4 _ 4g) -C-X 2 -C for binding two zinc ions.
  • the invention features a substantially purified polypeptide, cell growth regulator (CELR), having the amino acid sequence shown in SEQ ID NO: 1, or fragments thereof.
  • CELR cell growth regulator
  • the invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or fragments thereof and a composition comprising said polynucleotide sequence.
  • the invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO: 1, or fragments of said polynucleotide sequence.
  • the invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, or fragments or variants of said polynucleotide sequence.
  • the invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof.
  • the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
  • the invention also provides a polynucleotide sequence comprising the complement of SEQ LD NO: 2 or fragments or variants thereof.
  • the present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences.
  • the expression vector containing the polynucleotide sequence 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 thereof, the method comprising the steps of a) culturmg the host cell containing an expression vector containing at least a fiagment of the polynucleotide sequence encoding CELR 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 CELR having the amino acid sequence of SEQ ID NO 1 in conjunction with a suitable pharmaceutical carrier
  • the invention also provides a purified antagonist of the polypeptide of SEQ ID NO 1
  • the invention provides a purified antibody which binds to a polypeptide comprising the ammo acid sequence of SEQ ID NO 1 Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO 1
  • the invention also provides a method for detecting a polynucleotide which encodes
  • CELR in a biological sample comprising the steps of a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO 1 to nucleic acid material of a biological sample, thereby forming a hybridization complex, and b) detecting the hybridization complex wherein the presence of the complex correlates with the presence of a polynucleotide encoding CELR in the biological sample
  • the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization
  • FIGURES Figures 1A, IB, 1C, and ID show the amino acid sequence (SEQ ID NO 1) and nucleic acid sequence (SEQ ID NO 2) of CELR The alignment was produced using MacDNASIS PROTM software (Hitachi Software Engineering Co Ltd San Bruno, CA)
  • Figure 2 shows the amino acid sequence alignments between CELR (1630602, SEQ ID NO 1) and a rat cell growth regulator.
  • rCGR19 (GI 1724077, SEQ ID NO 3), produced using the multisequence alignment program of DNASTARTM software (DNASTAR Inc, Madison WI)
  • Figures 3 A and 3B show the hydrophobicity plots for CELR (SEQ ID NO 1) and rCGR19 (SEQ ID NO 3), respectively
  • the positive X axis reflects amino acid position, and the negative Y axis, hydrophobicity (MacDNASIS PRO software)
  • a host cell includes a plurality of such host cells
  • reference to the “antibody” is a reference to one or more antibodies and thereof known to those skilled in the art, and so forth
  • CELR refers to the amino acid sequences of substantially purified CELR obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which, when bound to CELR, increases or prolongs the duration of the effect of CELR.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of CELR.
  • alleles are an alternative form of the gene encoding CELR. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or 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 CELR include those with deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent CELR. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oiigonucleotide probe of the polynucleotide encoding CELR, and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding CELR.
  • the encoded protein may also be "altered” and contain deletions.
  • negatively charged amino acids may include aspartic acid and glutamic acid: positively charged amino acids may include lysine and arginine: and 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 sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Fragments of CELR are preferably about 5 to about 15 amino acids in length and retain the biological activity or the immunological activity of CELR. Where "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.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which, when bound to CELR, decreases the amount or the duration of the effect of the biological or immunological activity of CELR.
  • Antagonists may include proteins, nucleic acids, carbohydrates, antibodies or any other molecules which decrease the effect of CELR.
  • the term "antibody” refers to intact molecules as well as fragments thereof, such as Fa. F(ab ' ) 2 , and Fv, which are capable of binding the epitopic determinant.
  • Antibodies that bind CELR polypeptides can be prepared using intact polypeptides or 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. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled peptide is then used to immunize the animal (e.g.. a mouse, a rat, or a rabbit).
  • 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 nucleotide sequences which are complementary to a specific DNA or RNA sequence.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • Antisense molecules include peptide nucleic acids and 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 block either transcription or translation.
  • negative is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference 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 CELR. or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • complementary or “complementarity”, as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence “A-G-T” binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be “partial”, in which only some of the nucleic acids bind, or it may be complete when 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 hybridization between 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 PNA molecules.
  • composition comprising a given polynucleotide sequence refers broadly to any composition containing the given polynucleotide sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding CELR (SEQ ID NO: 1) or fragments thereof (e.g., SEQ LD NO:2 and fragments thereof) 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., NaCI).
  • Consensus refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, has been extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) in the 5' and/or the 3' direction and resequenced, or has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly (e.g., GELVTEWTM Fragment Assembly system, GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence .
  • correlates with expression of a polynucleotide indicates that the detection of the presence of ribonucleic acid that is similar to SEQ ID NO:2 by northern analysis is indicative of the presence of mRNA encoding CELR in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.
  • a “deletion”, as used herein, refers to a change in the amino acid or nucleotide sequence and results in the absence of one or more amino acid residues or nucleotides.
  • a nucleic acid derivative refers to the chemical modification of a nucleic acid encoding or complementary to CELR or the encoded CELR. Such modifications include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a nucleic acid derivative encodes a polypeptide which retains the biological or immunological function of the natural molecule.
  • a derivative polypeptide is one which is modified by glycosylation, pegylation. or any similar process which retains the biological or immunological function of the polypeptide from which it was derived.
  • low stringency conditions are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity) In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of 10K to 10M in size and contain all of the elements required for stable mitotic chromosome segregation and maintenance (Harrington, J.J. et al. (1997) Nat Genet. 15:345-355).
  • humanized antibody refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the 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 G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions
  • a hybridization complex may be formed in solution (e.g , C 0 t or R () t analysis) or 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)
  • 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 refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule.
  • “Microarray” refers to an array of distinct polynucleotides or ohgonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support
  • modulate refers to a change in the activity of CELR
  • modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional or immunological properties of CELR
  • Nucleic acid sequence refers to an oiigonucleotide, nucleotide. or polynucleotide, and fragments thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • “Fragments” are those nucleic acid sequences which are greater than 60 nucleotides than in length, and most preferably includes fragments that are at least 100 nucleotides or at least 1000 nucleotides, and at least 10,000 nucleotides in length.
  • the term “oiigonucleotide” refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides, which can be used in PCR amplification or a hybridization assay, or a microarray. As used herein, oiigonucleotide is substantially equivalent to the terms “amplimers”, “primers”, “oligomers”, and “probes”, as commonly defined in the art.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oiigonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues which ends in lysine. The terminal lysine confers solubility to the composition.
  • PNAs may be pegylated to extend their lifespan in the cell where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
  • portion refers to fragments of that protein.
  • the fragments may range in size from five amino acid residues to the entire amino acid sequence minus one amino acid.
  • a protein "comprising at least a portion of the amino acid sequence of SEQ ID NO: 1" encompasses the full-length CELR and fragments thereof.
  • sample is used in its broadest sense.
  • a biological sample suspected of containing nucleic acid encoding CELR, or fragments thereof, or CELR itself may comprise a bodily fluid, extract from a cell, chromosome, organelle, or membrane isolated from a cell, a cell, genomic DNA.
  • binding refers to that interaction between a protein or peptide and an agonist, an antibody and an antagonist. The interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) of the protein recognized by the binding molecule. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.
  • stringent conditions refer to the conditions for hybridization as defined by the nucleic acid, salt, and temperature. These conditions are well known in the art and may be altered in order to identify or detect identical or related polynucleotide sequences.
  • Numerous equivalent conditions comprising either low or high stringency depend on factors such as the length and nature of the sequence (DNA, RNA, base composition), nature of the target (DNA, RNA, base composition), milieu (in solution or immobilized on a solid substrate), concentration of salts and other components (e.g., formamide, dextran sulfate and/or polyethylene glycol), and temperature of the reactions (within a range from about 5°C below the melting temperature of the probe to about 20°C to 25°C below the melting temperature).
  • concentration of salts and other components e.g., formamide, dextran sulfate and/or polyethylene glycol
  • temperature of the reactions within a range from about 5°C below the melting temperature of the probe to about 20°C to 25°C below the melting temperature.
  • One or more factors be may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.
  • substantially purified refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 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. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method 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.
  • Such “transformed” cells include 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. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
  • a “variant " of CELR, as used herein, 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 a glycine with a 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.
  • CELR human cell growth regulator
  • Nucleic acids encoding the CELR of the present invention were first identified in Incyte Clone 1630602 from a colon tissue cDNA library (COLNNOT19) 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 1630602 (COLNNOT19), 77444 (SYNORAB01), 1696423 (COLNNOT23), 2903367 (DRGCNOT01). and 2592063 (MUSCNOT07).
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: l, as shown in Figs. 1A, IB, IC, and ID.
  • CELR is 332 amino acids in length.
  • CELR contains a potential ring-finger domain for zinc-binding encompassing residues C274-C308.
  • CELR has three potential N-glycosylation sites encompassing residues N240-S243, N241-S244, and N279-V282; one potential cAMP- and cGMP-dependent protein kinase phosphorylation site encompassing residues K143-S 146; ten potential casein kinase II phosphorylation sites encompassing residues T8-E1 1 , S 146-E149, T158-E161 ,
  • CELR has chemical and structural homology with a mouse cell growth regulator, rCGR19 (GI 1724077; SEQ ID NO:3). In particular, CELR shares 84% identity with rCGR19.
  • Northern analysis shows the expression of CELR in various cDNA libraries, at least 48% of which are cancerous or immortalized cell lines and at least 22% of which are associated with inflammation and immune response.
  • CELR variants A preferred CELR variant is one having at least 80%. and more preferably at least 90%, amino acid sequence identity to the CELR amino acid sequence (SEQ ID NO- 1) and which retains at least one biological, immunological or other functional characteristic or activity of CELR
  • a most preferred CELR variant is one having at least 95% amino acid sequence identity to SEQ ID NO: 1.
  • the invention also encompasses polynucleotides which encode CELR Accordingly, any nucleic acid sequence which encodes the amino acid sequence of CELR can be used to produce recombinant molecules which express CELR.
  • the invention encompasses the polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 as shown in Figure 1A-H
  • nucleotide sequences which encode CELR and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring CELR undei appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CELR 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.
  • Other reasons for substantially altering the nucleotide sequence encoding CELR and its derivatives without altering the encoded amino acid sequences include the production of 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, or fragments thereof, which encode CELR and its derivatives, 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 CELR or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those shown in SEQ ID NO:2, under various conditions of stringency as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507- 511).
  • 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, LL), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/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).
  • the nucleic acid sequences encoding CELR 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.
  • one method which may be employed, "restriction-site" PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322).
  • genomic DNA is first amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • 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 (Triglia. T. et al. ( 1988) Nucleic Acids Res. 16:8186).
  • the primers may be designed using commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-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 hgation and used as a PCR template
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M et al ( 1991) PCR Methods Applic 1 1 1 1-119) In this method
  • Another method which may be used to retrieve unknown sequences is that of Parker, J D et al (1991 , Nucleic Acids Res 19 3055-3060) Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA) This process avoids the need to screen libraries and is useful in finding intron/exon junctions When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs Also, random-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes Use of a randomly primed library may be especially preferable for situations in which an ohgo d(T) library does not yield a full-length cDNA 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 detection of the emitted wavelengths by a charge coupled devise camera Output/light intensity may be converted to electrical signal using appropriate software (e g GenotyperTM and Sequence Navigator 1 M , 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 CELR may be used in recombinant DNA molecules to direct expression of CELR, 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 CELR As will be understood by those of skill in the art, it may be advantageous to produce
  • CELR-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
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter CELR 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 CELR may be ligated to a heterologous sequence to encode a fusion protein
  • a heterologous sequence to encode a fusion protein
  • a fusion protein may also be engineered to contain a cleavage site located between the CELR encoding sequence and the heterologous protein sequence, so that CELR may be cleaved and purified away from the heterologous moiety
  • sequences encoding CELR may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M H et al (1980) Nucl Acids Res Symp Ser 215-223, Horn, T et al (1980) Nucl Acids Res Symp Ser 225-232)
  • the protein itself may be produced using chemical methods to synthesize the amino acid sequence of CELR, or a fragment thereof
  • peptide synthesis can be performed using ⁇ anous solid-phase techniques (Roberge, J Y et al (1995) Science 269 202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer).
  • the newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins. Structures and Molecular Principles. WH Freeman and Co., New York, NY).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • the amino acid sequence of CELR, or any part thereof may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • the nucleotide sequences encoding e.g., the nucleotide sequences encoding
  • CELR or functional equivalents 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 CELR. 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; 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 or "regulatory sequences” are those non-translated regions of the vector— enhancers, promoters, 5' and 3' untranslated regions—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 may be used for example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluesc ⁇ pt® phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluesc ⁇ pt® phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used
  • the baculovirus polyhed ⁇ n 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.
  • promoters trom 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 CELR, 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 CELR
  • vectors which direct high level expression of fusion proteins that are readily purified may be used
  • Such vectors include, but are not limited to, the multifunctional E. coh cloning and expression vectors such as Bluesc ⁇ pt® (Stratagene), in which the sequence encoding CELR may be ligated into the vector in frame with sequences tor the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced, pIN vectors (Van Heeke. G and S.M Schuster (1989) J.
  • pGEX vectois 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 hepa ⁇ n, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will
  • Saccharomyces cerevisiae 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
  • PGH palladium phosphate
  • the expression of sequences encoding CELR may be driven by any of a number of promoters Foi example, 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 (Takamatsu, N. (1987) EMBO J. 3:17)
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G et al. ( 1984) EMBO J. 3: 1671-1680; Brogue, R. et al. ( 1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105).
  • 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. for example, 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 CELR.
  • CELR Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding CELR 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 CELR 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 CELR may be expressed (Engelhard, E.K. et al. (1994) Proc. Nat. Acad. Sci. 91 :3224-3227).
  • a number of viral-based expression systems may be utilized.
  • sequences encoding CELR 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 CELR in infected host cells (Logan. J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81 :3655-3659).
  • 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 6 to 10M 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 CELR. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding CELR, 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 which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162).
  • a host cell strain may be chosen for its ability to modulate the 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.
  • ATCC American Type Culture Collection
  • cell lines which stably express CELR may 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 1-2 days in an enriched media before they are 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.
  • herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 1 1 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes which can be employed in tk “ or aprt " cells, respectively.
  • antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al ( 1981 ) J. Mol. Biol. 150: 1 - 14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad.
  • host cells which contain the nucleic acid sequence encoding CELR and express CELR 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.
  • the presence of polynucleotide sequences encoding CELR can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding CELR.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding CELR to detect transformants containing DNA or RNA encoding CELR.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CELR include oligolabehng, nick translation, end-labeling or PCR amplification using a labeled nucleotide Alternatively, the sequences encoding CELR, 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
  • Suitable reporter molecules or labels 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 CELR 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 CELR may be designed to contain signal sequences which direct secretion of CELR through a prokaryotic or eukaryotic cell membrane
  • Other constructions may be used to join sequences encoding CELR to nucleotide sequence 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 histidme-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 histidme-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) between the purification domain and CELR may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing CELR and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides a means for purifying CELR from the fusion protein.
  • IMAC immobilized metal ion affinity chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281
  • the enterokinase cleavage site provides a means for purifying CELR from the fusion protein.
  • fragments of CELR may be produced by direct peptide synthesis using solid-phase techniques Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of CELR may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • CELR rCGR19
  • GI 1724077 SEQ ID NO:3
  • Northern analysis shows that the expression of CELR is associated with cancer and inflammation.
  • CELR a fragment or a derivative thereof
  • CELR may be administered to a subject to prevent or treat a disorder associated with cell proliferation.
  • disorders include various types of cancer including, but not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma.
  • a pharmaceutical composition comprising purified CELR may be administered to a subject to prevent or treat a disorder associated with cell proliferation including, but not limited to, the types of cancer listed above.
  • an agonist which is specific for CELR, or a fragment or a derivative thereof may be administered to a sugject to prevent or treat a disorder associated with cell proliferation including, but not limited to, the types of cancer listed above.
  • a vector capable of expressing CELR, or a fragment or a derivative thereof may be administered to a subject to prevent or treat a disorder associated with cell proliferation including, but not limited to, the types of cancer listed above.
  • CELR, or a fragment or a derivative thereof may be administered to a subject to prevent or treat inflammation.
  • Such inflammation includes, but is not limited to, that which is associated with disorders such as Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia. irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis. myocardial or pericardial inflammation.osteoarthritis.
  • disorders such as Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomy
  • osteoporosis pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmune thyroiditis; complications of cancer, hemodialysis, extracorporeal circulation; viral, bacterial, fungal, parasitic, protozoal. and helminthic infections; and trauma.
  • a pharmaceutical composition comprising purified CELR may be administered to a subject to prevent or treat inflammation associated with any disorder including, but not limited to, those listed above.
  • an agonist which is specific for CELR, or a fragment or a derivative thereof may be administered to a subject to prevent or treat inflammation associated with any disorder including, but not limited to, those listed above.
  • a vector capable of expressing CELR, or a fragment or a derivative thereof may be administered to a subject to prevent or treat inflammation associated with any disorder including, but not limited to, those listed above. Decreased expression of CELR appears to be associated with increased cell proliferation. Therefore, in one embodiment, an antagonist of CELR, or a fragment or derivative thereof, may be added to cells to stimulate cell proliferation.
  • CELR may be added to a cell or cells in vivo using delivery mechanisms such as hposomes, viral based vectors, or electroinjection for the purpose of promoting regeneration or cell differentiation of the cell or cells
  • CELR may be added to a cell, cell line, tissue or organ culture in vitro or ex vivo to stimulate cell proliferation for use in heterologous or autologous transplantation
  • the cell will have been selected for its ability to fight an infection or a cancer or to correct a genetic defect in a disease such as sickle cell anemia, ⁇ thalassemia, cystic fibrosis, or Huntington's chorea
  • an antibody specific for CELR 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 CELR
  • a vector expressing the complement of the polynucleotide encoding CELR, oi a fragment or a derivative thereof may be administered to a cell to stimulate cell proliferation, as described above
  • an antagonist of CELR or a fragment or derivative thereof may be administered to a subject to prevent or treat a disordei associated with an increase in apoptosis
  • disorders include, but are not limited to, AIDS and other infectious or genetic immunodeficiencies, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retimtis pigmentosa, and cerebellar degeneration, myelodysplastic syndromes such as aplastic anemia, lschemic injuries such as myocardial infarction, stroke, and reperfusion injury, toxin-induced diseases such as alcohol- induced liver damage, cirrhosis, and lathy ⁇ sm, wasting diseases such as cachexia, viral infections such as those caused by hepatitis B and C, and osteoporosis
  • an antibody specific for CELR 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 CELR
  • a vector expressing the complement of the polynucleotide encoding CELR, or a fragment or a derivative thereof may be administered to a subject to prevent or treat a disorder associated with an increase in apoptosis
  • any of the pioteins 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 CELR may be produced using methods which are generally known in the ait
  • purified CELR may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind CELR
  • Antibodies to CELR may be generated using methods that are well known in the art Such antibodies may include, but are not limited to, polyclonal, monoclonal, chime ⁇ c. single chain. Fab fragments, and fragments produced by a Fab expression library Neutralizing antibodies, (I e., those which inhibit dimei formation) are especially preferred for therapeutic use
  • various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with CELR or 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 lysolecithm, pluromc polyols, polyamons, peptides, oil emulsions, keyhole limpet hemocyanm, and dimtrophenol.
  • BCG Bacilli Calmette-Gue ⁇ n
  • Corynebacte ⁇ um parvum are especially preferable.
  • the ohgopeptides. peptides, or fragments used to induce antibodies to CELR have an amino acid sequence consisting of at least five ammo acids and more preferably at least 10 ammo acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule Short stretches of CELR amino acids may be fused with those of another protein such as keyhole limpet hemocyanm and antibody produced against the chime ⁇ c molecule
  • Monoclonal antibodies to CELR 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 hyb ⁇ doma technique, the human B-cell hyb ⁇ doma technique, and the EBV-hyb ⁇ doma technique (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, Cole, S P et al ( 1984) Mol Cell Biol 62 109-120)
  • 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 (Orlandi, R et al (1989) Proc Natl Acad Sci 86 3833-3837, Winter, G et al (1991) Nature 349 293-299)
  • Antibody fiagments which contain specific binding sites for CELR may also be generated
  • fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be 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 (Huse, W D et al ( 1989) Science 254 1275-1281 )
  • the complement of the polynucleotide encoding CELR 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 CELR
  • complementary molecules or fragments may be used to modulate CELR 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 CELR
  • Expression vectors derived from retro viruses, adenovirus. 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 sequence which is complementary to the polynucleotides of the gene encoding CELR These techniques are described both in Sambrook et al (supra) and in Ausubel et al (supra)
  • Genes encoding CELR can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes CELR 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 even longer if appropriate replication elements are part of the vector system As mentioned above, 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 CELR (signal sequence, promoters, enhancers, and introns) Oligonucleotides derived from the transcription initiation site, e g , between positions -10 and +10 from the start site, are preferred Similarly, inhibition can be achieved using "triple helix" base-
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ⁇ bosomes
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA
  • the mechanism of ⁇ bozyme action involves sequence-specific hybridization of the ⁇ bozyme molecule to complementary target RNA, followed by endonucleolytic cleavage Examples which may be used include engineered hammerhead motif ⁇ bozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding CELR
  • RNA sequences of between 15 and 20 ⁇ bonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oiigonucleotide inoperable
  • suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementaiy oligonucleotides using ⁇ bonuclease protection assays
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding CELR 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 oi 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.
  • 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 polycationic amino polymers (Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-66; incorporated herein by reference) may be achieved using methods which are well known in the art.
  • 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.
  • compositions may consist of CELR.
  • agents to CELR mimetics, agonists, antagonists, or inhibitors of CELR.
  • the compositions may be administered alone or in combination with at least one other agent, such as 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 combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores
  • suitable excipients are carbohydrate or piotein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol, starch from corn, wheat, rice, potato or other plants, cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose, gums including arable and tragacanth, and proteins such as gelatin and collagen
  • disintegrating or solubihzing agents may be added, such as the cross-linked polyvinyl pyrrohdone, agar, 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 arable, talc, polyvmylpyrrolidone, caibopol 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
  • Push-fit capsules can contain active ingiedients mixed with a filler 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, oi liquid polyethylene glycol with or without stabilizers
  • suitable liquids such as fatty oils, liquid, oi liquid polyethylene glycol with or without stabilizers
  • Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or t ⁇ glyce ⁇ des, or hposomes Non-hpid polycationic amino polymers may also be used for delivery Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation
  • penetrants are generally known in the art
  • the pharmaceutical compositions of the present invention may be manufactured in a mannei that is known in the art, e g , by means ot conventional mixing, dissolving, granulating, dragee-makmg, levigating, emulsifying, encapsulating, entrapping, or lyophihzmg 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 succinic.
  • the preferred preparation may be a lyophihzed powder which contain any or all of the following 1-50 mM histidine, 0 l%-2% sucrose and 2-7% mannitol, at a pH range of 4 5 to 5 5, that is combined with buffer prior to use
  • pharmaceutical compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for tieatment of an indicated condition For administration of CELR.
  • labeling would include amount, frequency, and method of administration
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e g . of neoplastic cells, or in animal models usually mice, rabbits, dogs, or pigs
  • 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 ingiedient, for example CELR or fragments thereof, antibodies of CELR, agonists, antagonists or inhibitors of CELR, which ameliorates the symptoms or condition
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals e g , ED50 (the dose theiapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population)
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. 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, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100.000 micrograms, up to a total dose of about 1 g, 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 CELR may be used for the diagnosis of conditions or diseases characterized by expression of CELR, or in assays to monitor patients being treated with CELR, agonists, antagonists or inhibitors.
  • the antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics.
  • Diagnostic assays for CELR include methods which utilize the antibody and a label to detect CELR in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules which are known in the art may be used, several of which are described above.
  • CELR ELISA, RIA, and FACS for measuring CELR are known in the art and provide a basis for diagnosing altered or abnormal levels of CELR expression.
  • Normal or standard values for CELR expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to CELR under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, but preferably by photometric, means. Quantities of CELR 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 CELR may be used for diagnostic purposes.
  • the polynucleotides which may be used include oiigonucleotide 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 CELR may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of CELR, and to monitor regulation of CELR levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CELR or closely related molecules, may be used to identify nucleic acid sequences which encode CELR.
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory region, or a less specific region, e.g., especially in the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding CELR, alleles, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the CELR encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of SEQ ID NO:2 or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring CELR.
  • Means for producing specific hybridization probes for DNAs encoding CELR include the cloning of nucleic acid sequences encoding CELR or CELR derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32 P or 35 S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like
  • Polynucleotide sequences encoding CELR may be used for the diagnosis ot conditions or disorders which are associated with expression of CELR Examples of such conditions or disorders include, but are not limited to, disorders associated with cell proliferation such as adenocarcinoma.
  • leukemia lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma
  • bronchitis cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis.
  • CELR osteoporosis
  • the polynucleotide sequences encoding CELR may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies, or in dipstick, pin, ELISA assays or microarr ys utilizing fluids or tissues from patient biopsies to detect altered CELR expression.
  • Such qualitative or quantitative methods are well known in the art
  • nucleotide sequences encoding CELR may be useful in assays that detect activation or induction of various cancers, particularly those mentioned above
  • the nucleotide sequences encoding CELR 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.
  • the sample is washed and the signal is quantitated and compared with a standaid value If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding CELR in the sample indicates the presence of the associated disease.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring 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, oi a fragment thereof, which encodes CELR, under conditions suitable for hybridization or amplification
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease Deviation between standard and subject values is used to establish the presence of disease.
  • hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient 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
  • CELR may involve the use of PCR.
  • Such ohgomers may be chemically synthesized, generated enzymatically, or produced in vitro
  • Ohgomers will preferably consist of two nucleotide sequences, one with sense orientation (5'->3') and another with antisense (3' ⁇ -5' ), employed under optimized conditions for identification of a specific gene or condition The same two ohgomers.
  • nested sets of ohgomers, or even a degenerate pool of ohgomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences
  • Methods which may also be used to quantitate the expiession of CELR include radiolabehng or biotinylating nucleotides. coamphfication of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby, P C et al (1993) J Immunol Methods, 159 235-244. Duplaa, C et al ( 1993) Anal Biochem 229-236)
  • the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the ohgomer of interest isscreenednted in various dilutions and a spectrophotomet ⁇ c or colo ⁇ met ⁇ c response gives rapid quantitation
  • an oiigonucleotide derived from any of the polynucleotide sequences described herein may be used as a target in a microarray
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image), and to identify genetic variants mutations and polymorphisms This information will be useful in determining gene function, understanding the genetic basis of disease, diagnosing disease, and in developing and monitoring the activity of therapeutic agents (Heller, R et al ( 1997) Proc Natl Acad Sci 94 2150-55)
  • the microarray is prepared and used according to the methods described in PCT application WO95/1 1995 (Chee et al ), Lockhart, D J et al (1996. Nat Biotech 14 1675-1680) and Schena, M et al ( 1996, Proc Natl Acad Sci 93 10614- 10619), all of which are incorporated herein in their entirety by reference
  • the microarray is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs. fixed to a solid support
  • the oligonucleotides are pieferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length Foi a certain type of microarray, it may be preferable to use oligonucleotides which are only 7-10 nucleotides in length
  • the microarray may contain oligonucleotides which cover the known 5 . or 3'.
  • Polynucleotides used in the microarray may be oligonucleotides that are specific to a gene or genes of interest in which at least a fragment of the sequence is known or that are specific to one or more unidentified cDNAs which are common to a particular cell type, developmental or disease state.
  • oligonucleotides In order to produce oligonucleotides to a known sequence for a microarray, the gene of interest is examined using a computer algorithm which starts at the 5' or more preferably at the 3' end of the nucleotide sequence.
  • the algorithm identifies ohgomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray.
  • the "pairs" will be identical, except for one nucleotide which preferably is located in the center of the sequence.
  • the second oiigonucleotide in the pair serves as a control.
  • the number of oiigonucleotide pairs may range from two to one million.
  • the ohgomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oiigonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251 1 16 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference.
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides. or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.
  • RNA or DNA from a biological sample is made into hybridization probes.
  • the mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA).
  • aRNA antisense RNA
  • the aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray so that the probe sequences hybridize to complementary oligonucleotides of the microarray.
  • the biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc ).
  • a detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously This data may be used foi large scale correlation studies on the sequences, mutations, variants, or polymorphisms among samples.
  • CELR may also be used to generate hybridization probes which are useful for 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, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs) bacterial artificial chromosomes (BACs), bacterial PI constructions or single chromosome cDNA libraries as reviewed in Price, C M (1993) Blood Rev 7 127-134, and Trask. B J (1991) Trends Genet 7 149-154
  • Fluorescent in situ hybridization FISH as described in Verma et al (1988) Human Chromosomes A Manual of Basic Techniques. Pergamon Press, New York, NY
  • FISH Fluorescent in situ hybridization
  • OMIM Online Mendehan Inheritance in Man
  • 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, or paits thereof, by physical mapping This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 1 lq22-23 (Gatti, R.A. et al.
  • 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.
  • CELR its catalytic or immunogenic fragments or oligopeptides thereof
  • 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 CELR 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 as described in published PCT application WO84/03564.
  • large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with CELR, or fragments thereof, and washed.
  • Bound CELR is then detected by methods well known in the art.
  • Purified CELR can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • nucleotide sequences which encode CELR 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.
  • 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 COLNNOT19 cDNA library was constructed using 1 microgram of polyA RNA isolated from the unaffected cecal tissue of an 18-year-old Caucasian female with irritable bowel syndrome (IBS)
  • IBS irritable bowel syndrome
  • the cecum and appendix were unremaikable, and the margins were uninvolved
  • the frozen tissue was homogenized and lysed using a Brmkmann Homogenizer Polytron PT-3000 (Brmkmann Instruments, Westbury, NJ) in guanidinium isothiocyanate solution
  • the lysate was cent ⁇ fuged over a 5 7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70M
  • the mRNA was handled according to the recommended protocols in the Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat #18248-013, Gibco/BRL) cDNAs w ere fractionated on a Sepharose CL4B column (Cat #275105-01 , Pharmacia), and those cDNAs exceeding 400 bp were ligated into pINCY 1 The plasmid pINCY 1 was subsequently transformed into DH5 ⁇ TM competent cells (Cat #18258-012. Gibco/BRL)
  • Plasmid Kit (Catalog #26173, QIAGEN) This kit enabled the simultaneous purification of 96 samples in a 96-well block using multi-channel reagent dispensers
  • the recommended protocol was employed except for the following changes 1 ) the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog #2271 1 GIBCO/BRL) with carbenicilhn at 25 mg/L 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 the reading frame was determined.
  • nucleotide sequences of the Sequence Listing or amino acid sequences deduced from them were used as query sequences against databases such as GenBank, SwissProt, BLOCKS, and Pima II. These databases which contain previously identified and annotated sequences were searched for regions of homology (similarity) using BLAST, which stands for Basic Local Alignment Search Tool (Altschul SF ( 1993) J. Mol. Evol. 36:290-300; Altschul, SF et al. (1990) J. Mol. Biol. 215:403-10).
  • BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is 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 such as the one described in Smith RF and TF Smith (1992 Protein Engineering 5:35-51), incorporated herein by reference, can be used when dealing with primary sequence patterns and secondary structure gap penalties. As disclosed in this application, the sequences have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C. G, or T).
  • threshold was set at 10 "25 for nucleotides and 10 "14 for peptides.
  • 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 (Sambrook et al., supra).
  • Analogous computer techniques using BLAST Altschul, S.F., supra; Altschul, S.F. et al., supra
  • BLAST Altschul, S.F. et al., supra
  • Altschul, S.F. et al., supra are used to search for identical or related molecules in nucleotide databases such as GenBank or the LEFESEQTM 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: % sequence identity x % maximum BLAST score
  • 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 1-2% error; and at 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 CELR 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 the Incyte Clone 1630602 was used to design oiigonucleotide primers for extending a partial nucleotide sequence to full length.
  • One primer was synthesized to initiate extension in the antisense direction, and the other was synthesized to extend sequence in the sense direction.
  • 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).
  • High fidelity amplification was obtained by following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. Beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, PCR was performed using the Peltier Thermal Cycler (PTC200; M.J. Research, Watertown. MA) and 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 7 Repeat step 4-6 for 15 additional cycles
  • Step 8 94° C for 15 sec
  • Step 9 65 ° C for 1 min
  • Step 10 68° C for 7: 15 min
  • Step 1 Repeat step 8-10 for 12 cycles
  • reaction mixture A 5-10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQuickTM (QIAGEN Inc., Chatsworth, CA), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
  • QIAQuickTM QIAGEN Inc., Chatsworth, CA
  • Step 2 94° C for 20 sec
  • Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6 72° C for 180 sec
  • nucleotide sequence of SEQ ID NO 2 is used to obtain 5' regulatoiy sequences using the procedure above, oligonucleotides designed for 5' extension and an appropriate genomic library
  • Hybridization probes derived from SEQ ID NO 2 are employed to screen cDNAs, genomic DNAs, or mRNAs
  • 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), labeled by combining 50 pmol of each ohgomer and 250 ⁇ Ci of [ ⁇ - ⁇ 2 P] adenosine t ⁇ phosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN ® , Boston, MA)
  • the labeled oligonucleotides are substantially purified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn) A aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digest
  • oligonucleotides for a microarray the nucleotide sequence described herein is examined using a computer algorithm which starts at the 3' end of the nucleotide sequence.
  • the algorithm identifies ohgomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that would interfere with hybridization.
  • the algorithm identifies 20 sequence- specific oligonucleotides of 20 nucleotides in length (20-mers). A matched set of oligonucleotides is created in which one nucleotide in the center of each sequence is altered.
  • a chemical coupling procedure and an ink jet device are used to synthesize ohgomers on the surface of a substrate (Baldeschweiler, J.D. et al., PCT/WO95/251 16. incorporated herein by reference).
  • a "gridded" array analogous to a dot (or slot) blot is used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array may be produced by hand or using available materials and machines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.
  • the microarray is washed to remove nonhybridized probes, and a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oiigonucleotide sequence on the micro-array.
  • Sequence complementary to the CELR-encoding sequence, or any part thereof, is used to decrease or inhibit expression of naturally occurring CELR.
  • oligonucleotides comprising from about 15 to about 30 base-pairs is described, essentially the same procedure is used with smaller or larger sequence fragments.
  • Appropriate oligonucleotides are designed using Ohgo 4 06 software and the coding sequence of CELR, SEQ ID NO.1
  • a complementary oiigonucleotide is designed from the most unique 5 ' sequence and used to prevent promoter binding to the coding sequence
  • a complementary oiigonucleotide is designed to prevent ⁇ bosomal binding to the CELR-encoding transcript
  • CELR is accomplished by subclomng the cDNAs into appropriate vectors and transforming the vectors into host cells
  • the cloning vector is also used to express CELR in E coh Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing the ammo-terminal Met, and the subsequent seven residues of ⁇ -galactosidase. Immediately following these eight residues is a bacte ⁇ ophage promoter useful for transcription and a linker containing a number of unique restriction sites.
  • Induction of an isolated, transformed bacterial strain with IPTG using standard methods produces a fusion protein which consists of the first eight residues of ⁇ -galactosidase, about 5 to 15 residues of linker, and the full length protein.
  • the signal residues direct the secretion of CELR into the bacterial growth media which can be used directly in the following assay for activity
  • CELR can be expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with an eukaryotic expression vector encoding CELR 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 are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expiession of CELR Then, phase microscopy is used to compare the mitotic index of transformed versus control cells. An decrease in the mitotic index indicates CELR activity.
  • CELR that is substantially punfied using PAGE electrophoresis (Sambrook, supra), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols
  • the amino acid sequence deduced from SEQ ID NO 2 is analyzed using DNASTAR software (DNASTAR Inc) to determine regions of high lmmunogenicity and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions, is described by Ausubel et al (supra), and others
  • the ohgopeptides are 15 residues in length, synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry, and coupled to keyhole limpet hemocyanm (KLH, Sigma, St.
  • CELR Naturally occurring or recombinant CELR is substantially purified by immunoaffinity chromatography using antibodies specific for CELR
  • An immunoaffinity column is constructed by covalently coupling CELR 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
  • CELR Media containing CELR is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of CELR (e g., high ionic strength buffers in the presence of detergent)
  • the column is eluted under conditions that disrupt antibody/CELR binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion), and CELR is collected
  • CELR or biologically active fragments thereof are labeled with 12 T Bolton-Hunter reagent (Bolton et al (1973) Biochem J 133: 529)
  • 12 T Bolton-Hunter reagent 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 CELR, washed and any wells with labeled CELR complex are assayed
  • Data obtained using different concentrations of CELR are used to calculate values for the number, affinity, and association of CELR with the candidate molecules

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un régulateur de la croissance cellulaire humaine (CELR) et des polynucléotides qui identifient et codent CELR. Elle porte aussi sur des vecteurs d'expression, des cellules hôtes, des agonistes et des antagonistes, ainsi que sur des méthodes de traitement de troubles associés à l'expression de CELR.
EP98953187A 1997-09-22 1998-09-22 Regulateur de la croissance cellulaire Withdrawn EP1015588A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US93484597A 1997-09-22 1997-09-22
US934845 1997-09-22
PCT/US1998/020001 WO1999015661A1 (fr) 1997-09-22 1998-09-22 Regulateur de la croissance cellulaire

Publications (1)

Publication Number Publication Date
EP1015588A1 true EP1015588A1 (fr) 2000-07-05

Family

ID=25466171

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98953187A Withdrawn EP1015588A1 (fr) 1997-09-22 1998-09-22 Regulateur de la croissance cellulaire

Country Status (5)

Country Link
EP (1) EP1015588A1 (fr)
JP (1) JP2001517442A (fr)
AU (1) AU1062199A (fr)
CA (1) CA2304126A1 (fr)
WO (1) WO1999015661A1 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3228197A (en) * 1996-05-29 1998-01-05 Genzyme Corporation Cell growth regulatory genes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9915661A1 *

Also Published As

Publication number Publication date
CA2304126A1 (fr) 1999-04-01
JP2001517442A (ja) 2001-10-09
WO1999015661A1 (fr) 1999-04-01
AU1062199A (en) 1999-04-12

Similar Documents

Publication Publication Date Title
US5849528A (en) Polynucleotides encoding a human S100 protein
US5888792A (en) ATP-dependent RNA helicase protein
US5866119A (en) Human ribonuclease
US5871973A (en) Cell division regulators
US5874248A (en) Glutathione S-transferase homolog
US6265550B1 (en) Insulin receptor tyrosine kinase substrate
WO1999023220A1 (fr) Suppresseur de la signalisation cytokinaire
US5869259A (en) Carboxypeptidase inhibitor
US5858717A (en) Human formin binding protein
US5871971A (en) Human developmentally regulated GTP-binding protein
US5858714A (en) Human metaxin protein
US5863766A (en) Human sigma receptor
US7947804B1 (en) Vesicle trafficking proteins
US6527689B1 (en) Maternally transcribed protein
US5919661A (en) Polynucleotides encoding cytokine inducible regulatory protein
US5914249A (en) Cell-cycle phosphoproteins
US6326158B1 (en) SH3-containing proteins
US6077688A (en) Transducin beta-1 subunit
US5932443A (en) Human antigens
WO1999015554A2 (fr) Proteine de type ras
US6677126B2 (en) Antibody for human translational regulator
EP1021540A1 (fr) Proteine de type p23 de complexe de recepteur de progesterone humain
EP1015588A1 (fr) Regulateur de la croissance cellulaire
WO1998055616A2 (fr) Proteine associee au site brca2
EP1086234A1 (fr) Nouvel inhibiteur de carboxypeptidase d'origine humaine

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000327

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): BE DE ES FR GB IT NL

17Q First examination report despatched

Effective date: 20040218

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040331