EP1009423A1 - Gene de reaction primaire a l'epo, epr3pt - Google Patents

Gene de reaction primaire a l'epo, epr3pt

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Publication number
EP1009423A1
EP1009423A1 EP98939191A EP98939191A EP1009423A1 EP 1009423 A1 EP1009423 A1 EP 1009423A1 EP 98939191 A EP98939191 A EP 98939191A EP 98939191 A EP98939191 A EP 98939191A EP 1009423 A1 EP1009423 A1 EP 1009423A1
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EP
European Patent Office
Prior art keywords
polypeptide
seq
identity
polynucleotide
isolated polynucleotide
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Withdrawn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
EP98939191A
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German (de)
English (en)
Inventor
Kenneth A. Lord
Susan B. Dillon
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SmithKline Beecham Corp
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SmithKline Beecham Corp
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Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Priority claimed from PCT/US1998/016168 external-priority patent/WO1999006063A1/fr
Publication of EP1009423A1 publication Critical patent/EP1009423A1/fr
Withdrawn legal-status Critical Current

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Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agomsts, antagonists and/or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
  • the drug discovery process is currently undergoing a fundamental revolution as it embraces 'functional genomics' , that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on 'positional cloning' .
  • a phenotype that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
  • the present invention relates to EPRG3pt, in particular EPRG3pt polypeptides and EPRG3pt polynucleotides, recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of anemia, polycythemia, cancer, AIDS, and drug- induced anemias, hereinafter referred to as "the Diseases", amongst others.
  • the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with EPRG3pt imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate EPRG3pt activity or levels.
  • the present invention relates to EPRG3pt polypeptides.
  • Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, most preferably at least 97-99% identity, to that of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2.
  • Such polypeptides include those comprising the amino acid of SEQ ID NO: 2.
  • peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2.
  • polypeptides include the polypeptide of SEQ ID NO:2.
  • peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: l .
  • Polypeptides of the present invention are believed to be structurally related to other proteins having homology and/or structural similarity with human p27 (Rasmussen, U.B. , et al. , 1993, Cancer Research 53:4096-4101). They are therefore of interest because the expression of certain proteins is induced by EPO, and the presence of EPO is required for maintenance of expression of such proteins. These EPO-related proteins are involved in the proliferation of EPO-dependent cells and may be important in the growth and development of erythroid and other hematopoietic lineages. These properties are hereinafter referred to as "EPRG3pt activity” or "EPRG3pt polypeptide activity” or "biological activity of EPRG3pt” .
  • a polypeptide of the present invention exhibits at least one biological activity of EPRG3pt.
  • the polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • the present invention also includes include variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
  • Polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • the present invention relates to EPRG3pt polynucleotides.
  • Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
  • polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
  • Such polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ LD NO: 1 encoding the polypeptide of SEQ ID NO:2.
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region.
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99 % identity are more highly preferred, and those with at least 99 % identity are most highly preferred.
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99 % identity are most highly preferred.
  • Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ LD NO: 1 as well as the polynucleotide of SEQ ID NO: 1.
  • the invention also provides polynucleotides which are complementary to all the above described polynucleotides.
  • the nucleotide sequence of SEQ ID NO:l shows homology with human p27 (Rasmussen, U.B., et al., 1993, Cancer Research 53:4096-4101).
  • the nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and comprises a polypeptide encoding sequence
  • nucleotide 595 to 945 encoding a polypeptide of 116 amino acids, the polypeptide of SEQ LD NO:2.
  • the nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO: 1 or it may be a sequence other than the one contained in SEQ ID NO: 1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polypeptide of SEQ LD NO:2 is structurally related to other proteins having homology and/or structural similarity with human p27 (Rasmussen, U.B., et al., 1993, Cancer Research 53:4096-4101).
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one EPRG3pt activity.
  • the present invention also relates to partial or other polynucleotide and polypeptide sequences which were first identified prior to the determination of the corresponding full length sequences of SEQ ID NO: 1 and SEQ LD NO:2.
  • the present invention provides for an isolated polynucleotide comprising:
  • nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, even more preferably at least 97-99% identity to SEQ ID NO: 3 over the entire length of SEQ ID NO:3;
  • nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, even more preferably at least 97-99% identity, to SEQ ID NO: 3 over the entire length of SEQ ID NO: 3; or
  • the present invention provides for an isolated polynucleotide comprising:
  • nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, even more preferably at least 97-99% identity to SEQ ID NO: 4 over the entire length of SEQ ID NO:4;
  • nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99 % identity, to SEQ ID NO: 4 over the entire length of SEQ ID NO:4; or (c) the polynucleotide of SEQ LD NO:4.
  • nucleotide sequences of SEQ ID NOS: 3 and 4 and the peptide sequences encoded thereby are derived from EST (Expressed Sequence Tag) sequences. It is recognised by those skilled in the art that there will inevitably be some nucleotide sequence reading errors in EST sequences (see Adams, M.D. et al, Nature 377 (supp) 3, 1995). Accordingly, the nucleotide sequences of SEQ ID NOS: 3 and 4 and the peptide sequences encoded therefrom are therefore subject to the same inherent limitations in sequence accuracy. Furthermore, the peptide sequences encoded by SEQ ID NOS: 3 and 4 comprise a region of identity or close homology and/or close structural similarity (for example a conservative amino acid difference) with the closest homologous or structurally similar protein.
  • Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of human bone marrow and hematopoietic cells, using the expressed sequence tag (EST) analysis (Adams, M.D. , et al. Science (1991) 252: 1651-1656; Adams, M.D. et al. , Nature, (1992) 355:632-634; Adams, M.D. , et al , Nature (1995) 377 Supp:3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
  • EST expressed sequence tag
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al. , Proc NatlAcad Sci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • polypeptide variants which comprise the amino acid sequence of SEQ LD NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in any combination.
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ LD NO: 1 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to SEQ ID NO: 1.
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95 % identical to that of the referent.
  • the probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ LD NO: 1 or a fragment thereof; and isolating full- length cDNA and genomic clones containing said polynucleotide sequence.
  • Such hybridization techniques are well known to the skilled artisan.
  • Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. lx SSC at about 65°C.
  • the present invention also includes polynucleotides obtainable by screening an appropriate library under stingent hybridization conditions with a labeled probe having the sequence of SEQ LD NO: 1 or a fragment thereof.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA. This is a consequence of reverse transcriptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis.
  • RACE Rapid Amplification of cDNA ends
  • the PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5" in the known gene sequence).
  • primers designed to anneal within the amplified product typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5" in the known gene sequence.
  • the products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5 ' primer.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al. , Basic Methods in Molecular Biology (1986) and Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Sorin-r Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • bacterial cells such as streptococci, staphy locoed, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as streptococci, staphy locoed, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells such as CHO, COS, HeLa, C127, 3T3, BHK, HE
  • expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al. , MOLECULAR CLONING, A LABORATORY MANUAL (supra).
  • Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NO: 1 which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled EPRG3pt nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
  • DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (ee, e.g. , Myers et al. , Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al. , Proc NatlAcad Sci USA (1985) 85: 4397-4401).
  • an array of oligonucleotides probes comprising EPRG3pt nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g. , genetic mutations.
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation in the EPRG3pt gene by the methods described.
  • diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the present invention relates to a diagonostic kit which comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ;
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2.
  • kits may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly anemia, polycythemia, cancer, ALDS, and drug-induced anemias, amongst others.
  • the nucleotide sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • the gene of the present invention maps to human chromosome 14q31-q32. Lesions at this locus have been identified in some lymphoid malignancies.
  • the polypeptides of the invention or their fragments or analogs thereof, or cells expressing them, can also be used as immunogens to produce antibodies immunospecific for polypeptides of the present invention.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C. , Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al. , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985). Techniques for the production of single chain antibodies, such as those described in
  • U.S. Patent No. 4,946,778 can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies.
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
  • Antibodies against polypeptides of the present invention may also be employed to treat the Diseases, amongst others.
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy.
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others.
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, dehvering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • a further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention.
  • the vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • Polypeptides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide.
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned.
  • Compounds may be identified from a variety of sources, for example, cells, cell- free preparations, chemical libraries, and natural product mixtures. Such a ⁇ onists.
  • antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc. , as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve competition with a labeled competitor.
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • Constitutively active polpypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring EPRG3pt activity in the mixture, and comparing the EPRG3pt activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and EPRG3pt polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al. , J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16): 9459-9471 (1995)).
  • polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells.
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art.
  • ligand binding and crosslinking assays include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids).
  • a source of the putative receptor include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.
  • polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc. , as the case may be, of the polypeptide, e.g. , a fragment of the ligands, substrates, receptors, enzymes, etc. ; or small molecules which bind to the polypetide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises:
  • polypeptide of the present invention (c) a cell membrane expressing a polypeptide of the present invention; or (d) antibody to a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2.
  • kits may comprise a substantial component.
  • a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by:
  • the present invention provides methods of treating abnormal conditions such as, for instance, anemia, polycythemia, cancer, ALDS, and drug-induced anemias, related to either an excess of, or an under-expression of, EPRG3pt polypeptide activity.
  • abnormal conditions such as, for instance, anemia, polycythemia, cancer, ALDS, and drug-induced anemias, related to either an excess of, or an under-expression of, EPRG3pt polypeptide activity.
  • One approach comprises administering to a subject in need thereof an inhibitor compound (antagonist) as hereinabove described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of Mgands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby aUeviating the abnormal condition.
  • an inhibitor compound as hereinabove described
  • a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of Mgands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby aUeviating the abnormal condition.
  • soluble forms of the polypeptides stiU capable of binding the Mgand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide may be administered. Typical examples of such competitors include fragments of the EPRG3pt polypeptide.
  • expression of the gene encoding endogenous EPRG3pt polypeptide can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internaUy generated or separately OMgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • oMgonucleotides which form triple heUces with the gene can be suppMed (see, for example, Lee et al , Nucleic Acids Res (1979) 3:173; Cooney et al , Science (1988) 241:456; Dervan et al , Science (1991) 251:1360).
  • These oMgomers can be administered per se or the relevant oMgomers can be expressed in vivo.
  • One approach comprises administering to a subject a therapeuticaUy effective amount of a compound which activates a polypeptide of the present invention, i.e., an agonist as described above, in combination with a pharmaceuticaUy acceptable carrier, to thereby aUeviate the abnormal condition.
  • gene therapy may be employed to effect the endogenous production of EPRG3pt by the relevant ceUs in the subject.
  • a polynucleotide of the invention may be engineered for expression in a repMcation defective retroviral vector, as discussed above.
  • the retroviral expression construct may then be isolated and introduced into a packaging ceU transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging ceU now produces infectious viral particles containing the gene of interest.
  • These producer ceUs may be administered to a subject for engineering ceUs in vivo and expression of the polypeptide in vivo.
  • Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical carrier.
  • the present invention provides for pharmaceutical compositions comprising a therapeuticaUy effective amount of a polypeptide, such as the soluble form of a polypeptide of the present invention, agonist/antagonist peptide or smaU molecule compound, in combination with a pharmaceuticaUy acceptable carrier or excipient.
  • a polypeptide such as the soluble form of a polypeptide of the present invention, agonist/antagonist peptide or smaU molecule compound
  • a pharmaceuticaUy acceptable carrier or excipient include, but are not Mmited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers fiUed with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • composition wiU be adapted to the route of administration, for instance by a systemic or an oral route.
  • Preferred forms of systemic a ⁇ ministration include injection, typicaUy by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bUe salts or fusidic acids or other detergents.
  • penetrants such as bUe salts or fusidic acids or other detergents.
  • oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.
  • the dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is weU understood in the art.
  • Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modaUties often referred to as "gene therapy" as described above.
  • ceUs from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The ceUs are then introduced into the subject.
  • Polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology. This is most easUy faciUtated by storing the sequence in a computer readable medium and then using the stored data to search a sequence database using weU known searching tools, such as GCC. Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1 and/or a polypeptide sequence encoded thereby.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as weU as Fab fragments, including the products of an Fab or other immunoglobuMn expression Mbrary.
  • Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturaUy present in a Mving animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated” , as the term is employed herein.
  • Polynucleotide generaUy refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without Mmitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be smgle- stranded or, more typicaUy, double- stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stabiUty or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus,
  • polynucleotide embraces chemicaUy, enzymaticaUy or metaboMcaUy modified forms of polynucleotides as typicaUy found in nature, as weU as the chemical forms of DNA and RNA characteristic of viruses and ceUs. "Polynucleotide” also embraces relatively short polynucleotides, often referred to as otigonucleotides. " Polypeptide” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oMgopeptides or oMgomers, and to longer chains, generaUy referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are weU known in the art. Such modifications are weU described in basic texts and in more detaUed monographs, as weU as in a voluminous research Mterature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini. It wiU be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
  • Polypeptides may be branched as a result of ubiquitination, and they may be cycMc, with or without branching. CycMc, branched and branched cycMc polypeptides may result from post-translation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a Mpid or Mpid derivative, covalent attachment of phosphotidyMnositol, cross-Mnking, cyclization, disuU ⁇ de bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, suifation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, PROTEINS - STRUC
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturaUy occurring such as an aUetic variant, or it may be a variant that is not known to occur naturaUy.
  • Non-naturaUy occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity can be readUy calculated by known methods, including but not Mmited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. , ed.
  • Computer program methods to determine identity between two sequences include, but are not Mmited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al. , J. Molec. Biol. 215: 403-410 (1990).
  • the BLAST X program is pubMcly avaUable from NCBI and other sources (BLAST Manual, Altschul, S., et al. , NCBI NLM NLH Bethesda, MD 20894; Altschul, S., et al , J. Mol Biol 215: 403- 410 (1990).
  • the weU known Smith Waterman algorithm may also be used to determine identity.
  • Parameters for polypeptide sequence comparison include the foUowing:
  • a program useful with these parameters is pubMcly avaUable as the "gap" program from Genetics Computer Group, Madison WI.
  • the aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
  • Parameters for polynucleotide comparison include the foUowing:
  • Gap Length Penalty 3 AvaUable as: The "gap” program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons.
  • a preferred meaning for "identity” for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below.
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ LD NO:l, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individuaUy among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ LD
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide foUowing such alterations.
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individuaUy among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: n n ⁇ x n - (x n • y), wherein n n is the number of amino acid alterations, x n is the total number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for 70% , 0.80 for 80% , 0.85 for 85 % etc. , • is the symbol for the multipUcation operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO: 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individuaUy among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ LD NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individuaUy among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO: 2, or: n a ⁇ x a - (x a • y),
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO:2
  • y is, for instance 0.70 for 70% , 0.80 for 80% , 0.85 for 85 % etc.
  • • is the symbol for the multipMcation operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobuMn molecules together with another human protein or part thereof.
  • employing an immunoglobuMn Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262].
  • EPRG3pt is observed as a 3 kb EPO- inducible message.
  • EPRG3pt is induced by EPO in the presence of the protein synthesis inhibitor, cycloheximide, even though there is no induction of EPRG3pt when cycloheximide alone is added.
  • RNA from other tissues were found. The highest levels occur in spleen (4.8, 4.2kb), placenta (4.8, 0.9 kb), and peripheral blood leukocytes (0.9kb).
  • SEQUENCE LISTING FREE TEXT SEQUENCE INFORMATION SEQ ID NO:l
  • SEQ ID NO:4 GATCCACACTGGCTTCCCCTCCGCTTCCTAAGCCGATTGCCCATCTAAAGGGAGGCAGGGGGCTGCCCCAAgTT GGGAGCCACAAGAAGGGATGACTCCTGTCTACCGCTCTTTGCTTCATTTCGGGAAGTGGAAATTCTGGAGGAGG GTGAGGATC

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des polypeptides et des polynucléotides EPRG3pt ainsi que des procédés permettant d'obtenir ces polypeptides par des techniques de recombinaison. L'invention concerne également des procédés d'utilisation des polypeptides et polynucléotides EPRG3pt dans le domaine de la thérapie et des méthodes diagnostiques.
EP98939191A 1997-08-01 1998-08-03 Gene de reaction primaire a l'epo, epr3pt Withdrawn EP1009423A1 (fr)

Applications Claiming Priority (6)

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US127672 1993-09-28
US5446497P 1997-08-01 1997-08-01
US54464P 1997-08-01
US12742498A 1998-07-31 1998-07-31
US12767298A 1998-07-31 1998-07-31
PCT/US1998/016168 WO1999006063A1 (fr) 1997-08-01 1998-08-03 Gene de reaction primaire a l'epo, epr3pt

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