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WO2000009554A1 - Gene humain hspc018 (cbccfe10) - Google Patents

Gene humain hspc018 (cbccfe10) Download PDF

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Publication number
WO2000009554A1
WO2000009554A1 PCT/CN1998/000161 CN9800161W WO0009554A1 WO 2000009554 A1 WO2000009554 A1 WO 2000009554A1 CN 9800161 W CN9800161 W CN 9800161W WO 0009554 A1 WO0009554 A1 WO 0009554A1
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Prior art keywords
polypeptide
identity
seq
compnsmg
subject
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.)
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PCT/CN1998/000161
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English (en)
Inventor
Yaping Yu
Yu Shen
Wei Huang
Baiwei Gu
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Shanghai Second Medical University
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Shanghai Second Medical University
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Priority to PCT/CN1998/000161 priority Critical patent/WO2000009554A1/fr
Priority to CN98810051.7A priority patent/CN1275130A/zh
Publication of WO2000009554A1 publication Critical patent/WO2000009554A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and m 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 present mvention relates to CBCCFEIO, in particular CBCCFEIO polypeptides and CBCCFEIO polynucleotides, recombinant matenals and methods for their production
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of leukemia and aplasuc anemia, hereinafter referred to as "the Diseases", amongst others h
  • the mvention relates to methods for identifying agomsts and ant ⁇ gomsts/rnhibitors usmg the matenals provided by the mvention, and treating conditions associated with CBCCFEIO imbalance with the identified compounds
  • the invention relates to diagnostic assays for detecting diseases associated with inapprop ⁇ ate CBCCFEIO activity or levels
  • the present mvention relates to CBCCFEIO polypeptides
  • CBCCFEIO polypeptides include isolated polypeptides comprising an ammo 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
  • polypeptides include those comp ⁇ sing the ammo acid of SEQ ID NO 2
  • peptides of the present mvention include isolated polypeptides in which the ammo 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 mvention include isolated polypeptides encoded by a polynucleotide comp ⁇ smg the sequence contained in SEQ ID NO 1
  • Polypeptides of the present invention are of interest because HSPC018 is cloned from umbilical cord blood hematopoietic progenitor CD34+ cell and has no homologue Therefore, it is thought to play a role in hematopoiesis
  • CBCCFEIO activity or "CBCCFEIO polypeptide activity” or “biological activity of CBCCFEIO”
  • Also mcluded amongst these activities are antigemc and immunogenic activities of said CBCCFEIO polypeptides, in particular the antigemc and immunogenic activities of the polypeptide of SEQ ID NO 2
  • a polypeptide of the present mvention exhibits at least one biological activity of CBCCFEIO
  • polypeptides of the present mvention 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 characte ⁇ stics Typical such substitutions are among Ala, Val, Leu and lie, 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 CBCCFEIO polynucleotides
  • polvnucleotides include isolated polynucleotides compnsing 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 LD 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 comp ⁇ sing the nucleotide sequence contained in SEQ ID NO 1 encoding the polypeptide of SEQ LD NO 2
  • polynucleotides of the present invention mclude isolated polynucleotides comp ⁇ sing 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 prefe ⁇ ed, 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 comp ⁇ sing 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 prefe ⁇ ed, whilst those with at least 98-99% identity are more highly prefe ⁇ ed, and those with at least 99% identity are most highly prefe ⁇ ed
  • Such polynucleotides include a polynucleotide comp ⁇ sing the polynucleotide of SEQ LD NO 1 as well as the polynucleotide of SEQ LD NO 1
  • the mvention also provides polynucleotides which are complementary to all the above desc ⁇ bed polynucleotides
  • the nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and comp ⁇ ses a polypeptide encoding sequence (nucleotide 149 to 649) encoding a polypeptide of 167 amino acids, the polypeptide of SEQ ID NO 2
  • the nucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encodmg sequence contained in SEQ ID NO 1 or it may be a sequence other than the one contained m 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
  • Prefe ⁇ ed polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, prefe ⁇ ed polypeptides and polynucleotides of the present invention have at least one CBCCFEIO activity
  • Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library de ⁇ ved from mRNA in cells of human umbihcal cord blood, 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 mvention can also be obtained from natural sources such as genomic DNA hbra ⁇ es or can be synthesized usmg well known and commercially available techniques
  • 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 encodmg 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 desc ⁇ bed in Gentz et al , Proc Natl Acad Set USA (1989) 86 821-824, or is an HA tag
  • the polynucleotide may also contain non-coding 5'
  • polypeptide va ⁇ ants which comp ⁇ se the amino acid sequence of SEQ LD NO 2 and rn which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, ammo acid residues are substituted, deleted or added, in any combination
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ID NO 1, may be used as hyb ⁇ drzation 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 mvention and to isolate cDNA and genomic clones of other genes (mclud g genes encoding homologs and orthologs from species other than human) that have a high sequence s mila ⁇ ty 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 comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides Particularly prefe ⁇ ed probes will have between 30 and 50 nu
  • 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 transcnptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template durmg the polyme ⁇ sation reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis
  • Recombinant polypeptides of the present invention may be prepared by processes well known m the art from genetically engineered host cells comp ⁇ sing expression systems Accordingly, in a further aspect, the present mvention relates to expression systems which comp ⁇ se a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs of the present mvention
  • 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 desc ⁇ bed in many standard laboratory manuals, such as Davis et al , Basic Methods m Molecular Biology (1986) and Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989) Prefe ⁇ ed such methods mclude, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection
  • appropnate hosts mclude bactenal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs 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, and plant cells
  • a great vanety of expression systems can be used, for instance, chromosomal, episomal and virus-denved systems, e g , vectors de ⁇ ved from bactenal plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenovrruses,
  • 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 appropnate nucleotide sequence may be inserted mto an expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra)
  • Appropnate secretion signals may be incorporated mto the desired polypeptide to allow secretion of the translated protein mto the lumen of the endoplasmic reticulum, the pe ⁇ plasmic space or the extracellular environment These signals may be endogenous to the polypeptide or they may be heterologous signals If a polypeptide of the present mvention is to be expressed for use in screemng assays, it is generally prefe ⁇ ed that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested p ⁇ or to use in the screenmg assay If the polypeptide is secreted mto the medium, the medium can be recovered in order to recover and punfy the polypeptide If produced intracellularly, the cells must first be lvsed before the polypeptide is recovered Polypeptides of the present mvention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of the gene characterized by the polynucleotide of SEQ LD 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 vanety of techniques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, sahva, tissue biopsy or autopsy mate ⁇ al
  • the genomic DNA may be used directly for detection or may be amplified enzyma ⁇ cally by usmg PCR or other amplification techniques p ⁇ or 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 compa ⁇ son to the normal genotype
  • Pomt mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled CBCCFE 10 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 m electrophoretic mobility of DNA fragments in gels, with or without denatu ⁇ ng agents, or by direct DNA sequencmg (e g , Myers et al
  • the diagnostic assays offer a process for diagnosing or deteimuurung a susceptibility to the Diseases through detection of mutation m the CBCCFEIO gene by the methods desc ⁇ bed
  • diseases may be diagnosed by methods comp ⁇ sing determining from a sample de ⁇ ved 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 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
  • kits may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly leukemia , and aplastic anemia, 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 co ⁇ elating 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 co ⁇ elated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins
  • 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.
  • 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 mvention than their affinity for other related polypeptides m the p ⁇ or art
  • Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a non-human animal, usmg routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell l ne cultures can be used Examples mclude the hyb ⁇ doma technique (Kohler, G and Milstetn, C , Nature (1975) 256 495-497), the t ⁇ oma techmque, the human B-cell hybndoma techmque (Kozbor et al , Immunology Today (1983) 4 72) and the EBV- hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)
  • smgle chain antibodies such as those descnbed in U S Patent No 4,946,778, can also be adapted to produce smgle chain antibodies to polypeptides of this mvention
  • transgenic mice, or other organisms, mcludmg other mammals may be used to express humanized antibodies
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to purify the polypeptides by affinity chromatography
  • Antibodies against polypeptides of the present mvention may also be employed to treat the Diseases, amongst others
  • the present mvention relates to genetically engmeered soluble fusion protems compnsing a polypeptide of the present mvention, or a fragment thereof, and vanous portions of the constant regions of heavy or light chams of l munoglobulms of vanous subclasses (IgG, IgM, IgA, IgE) Preferred as an immunoglobulin is the constant part of the heavy cham 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 clottmg factor Xa
  • this invention relates to processes for the preparation of these fusion protems by genetic engineering, and to the use thereof for drug screemng, diagnosis and therapy
  • a further aspect of the mvention also relates to polynucleotides encodmg such fusion protems Examples of fusion protein technology
  • a further aspect of the mvention relates to an lmrnunological/vaccine formulation (composition) which, when mtroduced mto a mammalian host, mduces an lmmunological response in that mammal to a polypeptide of the present mvention wherem the composition compnses a polypeptide or polynucleotide of the present mvention wherem the composition compnses a polypeptide or polynucleotide of the present mvention
  • the vaccine formulation may further comp ⁇ se a suitable earner Smce a polypeptide may be broken down in the stomach, it is preferably admmistered parenterally (for instance, subcutaneous, intramuscular, mtravenous, or mtradermal mjection)
  • Formulations suitable for parenteral a ⁇ rninistration m include aqueous and non-aqueous stenle mjection solutions which may contain anti-oxidants, buffers, bactenostats
  • the screemng method may simply measure the bmding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound Alternatively, the screemng method may mvolve competition with a labeled competitor Further, these screemng methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, usmg detection systems appropnate to the cells bearing the polypeptide Inhibitors of activation are generally assayed in the presence of a known agomst and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polypeptides may be employed in screemng methods for mverse agomsts or inhibitors, m the absence of an agomst or inhibitor, by testmg whether the candidate compound results m inhibition of activation of the polypeptide Further, the screemng methods may simply
  • the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These mclude, but are not limited to, ligand bmding and crosslinking assays m which the polypeptide is labeled with a radioactive isotope (for mstance, - ⁇ 1), chemically modified (for mstance, biotmylated), or fused to a peptide sequence suitable for detection or punfication, and mcubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy These screemng methods may also be used to identify agomsts and antagomsts of the polypeptide which compete with the binding of the polypeptide to its receptors, if any Standard methods for conductmg such assays are well understood m the art
  • polypeptide antagonists examples 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 polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented
  • the present mvention relates to a screemng kit for identifying agomsts, antagomsts, ligands, receptors, substrates, enzymes, etc for polypeptides of the present mvention, or compounds which decrease or enhance the production of such polypeptides, which compnses
  • polypeptide of the present mvention may also be used in a method for the structure-based design of an agomst, antagomst or inhibitor of the polypeptide, by
  • the present mvention provides methods of treating abnormal conditions such as, for instance, leukemia and aplastic anemia, related to either an excess of, or an under-expression of, CBCCFEIO polypeptide activity
  • an inhibitor compound as hereinabove descnbed, optionally in combination with a pharmaceutically acceptable earner, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc in competition with endogenous polypeptide may be administered Typical examples of such competitors mclude fragments of the CBCCFEIO polypeptide
  • expression of the gene encodmg endogenous CBCCFEIO polypeptide can be inhibited usmg expression blocking techniques
  • Known such techniques mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 ⁇ n Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • ohgonucleotides which form tnple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan ef ⁇ / , Science (1991) 251 1360)
  • These ohgomers can be administered >er se or the relevant o gomers can be expressed in vivo
  • a polynucleotide of the mvention may be engineered for expression in a replication defective retrovrral vector, as discussed above
  • the retroviral expression construct may then be isolated and mtroduced mto a packaging cell transduced with a retroviral plasmid vector contairung RNA encodmg a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles contairung the gene of interest
  • the present mvention provides for pharmaceutical compositions compnsing a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present mvention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient
  • a polypeptide such as the soluble form of a polypeptide of the present mvention, agonist/antagonist peptide or small molecule compound
  • earners mclude, but are not limited to, sahne, buffered sahne, dextrose, water, glycerol, ethanol.
  • the mvention further relates to pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • composition will be adapted to the route of administration, for instance by a systemic or an oral route
  • Prefe ⁇ ed forms of systemic ac ninistration m clude mjection, typically by intravenous mjection
  • Other mjection routes such as subcutaneous, mtramuscular, or mtrapentoneal
  • Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents
  • oral administration may also be possible
  • Admmistration 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 mvention, 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 va ⁇ ations in the needed dosage, however, are to be expected m view of the vanety of compounds available and the diffe ⁇ ng efficiencies of vanous routes of admmistration For example, oral administration would be expected to require higher dosages than administration by intravenous mjection Vanations m these dosage levels can be adjusted usmg standard empincal routines for optimization, as is well understood m the art
  • Polypeptides used in treatment can also be generated endogenously m the subject, in treatment modahties often refe ⁇ ed to as "gene therapy" as desc ⁇ bed above
  • cells 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 cells are then mtroduced mto the subject
  • polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology This is most easily facilitated by storing the sequence in a computer readable medium and then usmg the stored data to search a sequence database usmg well known searching tools, such as GCC Accordingly, in a further aspect, the present mvention provides for a computer readable medium having stored thereon a polynucleotide compnsmg the sequence of SEQ ID NO 1 and/or a polypeptide sequence encoded thereby
  • Antibodies as used herein mcludes polyclonal and monoclonal antibodies, chrmenc, smgle chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other rmmunoglobulm expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs m nature, it has been changed or removed from its ongmal environment, or both For example, a polynucleotide or a polypeptide naturally present m a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is “isolated", as the term is employed herem
  • Polynucleotide generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of smgle- and double-stranded regions
  • polynucleotide refers to tnple-stranded regions compnsing RNA or DNA or both RNA and DNA
  • polynucleotide also mcludes DNAs or RNAs containing one or more modified
  • Polypeptide refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds, l e , peptide isosteres "Polypeptide” refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chams, generally refened to as protems Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids "Polypeptides” mclude ammo acid sequences modified either by natural processes, such as post-translational processmg, or by chemical modification techniques which are well known in the art Such modifications are well descnbed m basic texts and m more detailed monographs, as well as in a voluminous research literature Modifications may occur anywhere m a polypeptide, including the peptide backbone, the amino acid side-chains and the ammo or carboxyl termini It will be
  • Vanant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties
  • a typical vanant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m am o acid substitutions, additions, deletions, fusions and truncations m the polypeptid
  • polynucleotides and polypeptides are provided m (1) and (2) below
  • Polynucleotide embodiments further mclude an isolated polynucleotide compnsmg a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleotides in the reference
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides m SEQ ID NO 1
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator
  • any non-mteger product of x n and y is rounded down to the nearest mteger pnor to subtractmg it from x n
  • Alterations of a polynucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • a polynucleotide sequence of the present mvention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certain mteger number of ammo 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 consistmg of at least one nucleic acid deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleic acids m the reference sequence or m one or more contiguous groups withm the reference sequence
  • the number of nucleic acid alterations for a given percent identity is determined by multiplymg the total number of ammo acids m SEQ ID NO 2 by the mteger definmg the percent identity divided by 100 and then subtractmg that product from said total number of
  • n n is the number of ammo acid alterations
  • x n is the total number of amino acids m SEQ ID NO 2
  • y is, for mstance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x n and y is rounded down to the nearest mteger p ⁇ or to subtractmg it from x n
  • Polypeptide embodiments further mclude an isolated polypeptide compnsmg 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, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of amino acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non- conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of ammo acid alterations is determined by multiplymg the total number of ammo acids in SEQ ID NO 2 by
  • n a is the number of ammo acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%.
  • • is the symbol for the multiplication operator.
  • any non-mteger product of x a and y is rounded down to the nearest mteger pnor to subtractmg it from x a
  • a polypeptide sequence of the present mvention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certam mteger number of ammo 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 consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the a mo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either individually among the ammo acids m the reference sequence or in one or more contiguous groups within the reference sequence
  • the number of am o acid alterations for a given % identity is determined by multiplymg the total number of ammo acids m SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtractmg that product from said total number of ammo
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ID NO 2
  • y is, for mstance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest mteger pnor to subtractmg it from x a
  • Fusion protem refers to a protem encoded by two, often unrelated, fused genes or fragments thereof
  • EP-A-0 464 discloses fusion protems compnsmg vanous portions of constant region of inrmunoglobulin molecules together with another human protem or part thereof
  • employmg an i munoglobulin Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262]

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBCCFE10 et des procédés de production de ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et polynucléotides CBCCFE10 dans le traitement de maladies et les doses diagnostiques.
PCT/CN1998/000161 1998-08-11 1998-08-11 Gene humain hspc018 (cbccfe10) Ceased WO2000009554A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN1998/000161 WO2000009554A1 (fr) 1998-08-11 1998-08-11 Gene humain hspc018 (cbccfe10)
CN98810051.7A CN1275130A (zh) 1998-08-11 1998-08-11 人类基因hspc018(cbccfe10)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000161 WO2000009554A1 (fr) 1998-08-11 1998-08-11 Gene humain hspc018 (cbccfe10)

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WO2000009554A1 true WO2000009554A1 (fr) 2000-02-24

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PCT/CN1998/000161 Ceased WO2000009554A1 (fr) 1998-08-11 1998-08-11 Gene humain hspc018 (cbccfe10)

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Country Link
CN (1) CN1275130A (fr)
WO (1) WO2000009554A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2185332C1 (ru) * 2001-06-01 2002-07-20 Ракитин Георгий Валентинович Устройство для очистки водных растворов

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMBL AL008627, 09 January 1998, see the sequence. *
GenBank AC002091, 09 Semtember 1997, see the sequence. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2185332C1 (ru) * 2001-06-01 2002-07-20 Ракитин Георгий Валентинович Устройство для очистки водных растворов

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