WO2001027155A1 - Polypeptides et polynucleotides humains sbgfgf-10a del23 - Google Patents

Polypeptides et polynucleotides humains sbgfgf-10a del23 Download PDF

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WO2001027155A1
WO2001027155A1 PCT/US2000/027956 US0027956W WO0127155A1 WO 2001027155 A1 WO2001027155 A1 WO 2001027155A1 US 0027956 W US0027956 W US 0027956W WO 0127155 A1 WO0127155 A1 WO 0127155A1
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Prior art keywords
polypeptide
sequence
polynucleotide
arg
seq
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Pankaj Agarwal
Frank Barone
Philip D. Hayes
Karen S. Kabnick
Michael S. Mcqueney
Randall F. Smith
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and m identifying compounds that may be agonists, antagonists that 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 as a means to identify genes and gene products as therapeutic targets 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 sbgFGF- 10a_del23 in particular sbgFGF- 10a_del23 polypeptides and sbgFGF- 10a_del23 polynucleotides, recombinant mate ⁇ als and methods for their production.
  • Such polypeptides and polynucleotides are of interest m relation to methods of treatment of certain diseases, including, but not limited to, stroke, traumatic brain injury, cerebral ischemia, cancer, atherosclerosis, rheumatoid arthritis, cirrhosis, pso ⁇ asis, sarcoidosis, ldiopathic pulmonary fibrosis, tumor development, developmental disorders, skeletal disorders, and acrocephaly, hereinafter referred to as " diseases of the invention”.
  • the invention relates to methods for identifying agonists and antagonists (e g., inhibitors) using the mate ⁇ als provided by the invention, and treating conditions associated with sbgFGF- 10a_del23 imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inapprop ⁇ ate sbgFGF- 10a_del 23 activity or levels. Description of the Invention
  • the present invention relates to sbgFGF- 10a_del23 polypeptides.
  • polypeptides include:
  • Polypeptides of the present invention are believed to be members of the Fibroblast Growth Factor (FGF) family of polypeptides. They are therefore of interest because The biological properties of the sbgFGF- 10a_del23 are hereinafter referred to as "biological activity of sbgFGF- 10a_del23" or "sbgFGF-10a_del23 activity.”
  • FGF Fibroblast Growth Factor
  • a polypeptide of the present invention exhibits at least one biological activity of sbgFGF- 10a_del23.
  • Polypeptides of the present invention also include va ⁇ ants of the aforementioned polypeptides, including all allehc forms and splice va ⁇ ants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred va ⁇ ants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comp ⁇ sing an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO: 2, or an isolated polypeptide comp ⁇ sing an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO: 2.
  • Preferred fragments are biologically active fragments that mediate the biological activity of sbgFGF- 10a_del23, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or lmmunogenic m an animal, especially in a human.
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • 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 precursor or a fusion protein. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid m pu ⁇ f ⁇ cation, for instance multiple histidme residues, or an additional sequence for stability du ⁇ ng recombinant production.
  • Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occur ⁇ ng sources, from genetically engmeered host cells comp ⁇ sing expression systems (vide infra) or by chemical synthesis, usmg for instance automated peptide synthesizers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.
  • the present invention relates to sbgFGF- 10a_del23 polynucleotides.
  • Such polynucleotides include:
  • Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comp ⁇ smg an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1, or an isolated polynucleotide comp ⁇ smg an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.
  • Preferred va ⁇ ants of polynucleotides of the present invention include splice va ⁇ ants, allehc va ⁇ ants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
  • Polynucleotides of the present mvention also include polynucleotides encodmg polypeptide va ⁇ ants that comp ⁇ se the ammo acid sequence of SEQ ID NO:2 and in which several, for mstance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination.
  • the present invention provides polynucleotides that are RNA transc ⁇ pts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that:
  • (a) comprises an RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
  • (b) is the RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
  • (d) is the RNA transc ⁇ pt of the DNA sequence of SEQ ID NO: 1 ; and RNA polynucleotides that are complementary thereto.
  • the polynucleotide sequence of SEQ ID NO: 1 shows homology with human Kaposi's sarcoma oncogene fibroblast growth factor mRNA (Ml 7446) (An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family. Delli Bovi et al. Cell. 50(5): 729-37, 1987).
  • the polynucleotide sequence of SEQ ID NO: 1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2.
  • polypeptide of SEQ ID NO:2 may be identical to the polypeptide encodmg sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ID NO:l, 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 ID NO:2 is related to other proteins of the Fibroblast Growth Factor (FGF) family, having homology and/or structural sirmla ⁇ ty with rat FGF-10 (g ⁇ l517808) (Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family, Yamasaki et al., J. Biol. Chem. 271(27):15918-15921, 1996) and human FGF10 (g ⁇ 4758360) (Structure and expression of human fibroblast growth factor-10.
  • FGF Fibroblast Growth Factor
  • Preferred polypeptides and polynucleotides of the present mvention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one sbgFGF- 10a_del23 activity.
  • Polynucleotides of the present mvention may be obtained usmg standard cloning and screening techniques from a cDNA library de ⁇ ved from mRNA in cells of human hippocampus, (see for mstance, Sambrook et al, Molecular Clonmg: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N.Y. (1989)). 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.
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide m readmg frame with other codmg sequences, such as those encodmg a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions.
  • a marker sequence that facilitates pu ⁇ fication 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 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, ⁇ bosome binding sites and sequences that stabilize mRNA.
  • Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO: 1 may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers for a nucleic acid amplification reaction (for instance, PCR). Such probes and p ⁇ mers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encodmg paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence simila ⁇ ty to SEQ ID NO: 1 , typically at least 95% identity.
  • Preferred probes and p ⁇ mers will generally comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred p ⁇ mers will have between 20 and 25 nucleotides.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process comp ⁇ sing the steps of screening a library under stringent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • hyb ⁇ dization techniques are well known to the skilled artisan.
  • Preferred stringent hyb ⁇ dization conditions include overnight incubation at 42°C m a solution comp ⁇ sing: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium 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 mvention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stringent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ID NO:l or a fragment thereof, preferably of at least 15 nucleotides.
  • isolated polynucleotides preferably with a nucleotide sequence of at least 100, obtained by screening a library under stringent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ID NO:l or a fragment thereof, preferably of at least 15 nucleotides.
  • PCR Nucleic acid amplification
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existmg 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' p ⁇ mer.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engmeered host cells comp ⁇ sing expression systems. Accordingly, m a further aspect, the present mvention relates to expression systems comp ⁇ sing a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered 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 using 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 mvention.
  • Polynucleotides may be introduced into host cells by methods desc ⁇ bed in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al.(ib ⁇ d).
  • Preferred methods of introducing polynucleotides into host cells include, for mstance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • bacte ⁇ al cells such as Streptococci, Staphylococa, E coli, Streptomyces and Bacillus subhlis 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, HEK 293 and Bowes melanoma cells.
  • a great va ⁇ ety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al 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, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage 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 that is able to maintain, propagate or express a polynucleotide to produce a polypeptide m a host may be used.
  • the approp ⁇ ate polynucleotide sequence may be mserted into an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., (ibid).
  • Approp ⁇ ate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into 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.
  • a polypeptide of the present mvention is to be expressed for use m screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell.
  • the cells may be harvested p ⁇ or to use m the screenmg assay. If the polypeptide is secreted into the medium, the medium can be recovered m order to recover and pu ⁇ fy the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered.
  • Polypeptides of the present invention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, amon 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 pu ⁇ fication. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng lntracellular synthesis, isolation and/or pu ⁇ fication.
  • Polynucleotides of the present mvention may be used as diagnostic reagents, through detecting mutations m the associated gene. Detection of a mutated form of the gene characte ⁇ zed by the polynucleotide of SEQ ID NO:l in the cDNA or genomic sequence and 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 spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a va ⁇ ety of techniques well known m the art.
  • Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy mate ⁇ al.
  • the genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques pnor to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change m size of the amplified product m compa ⁇ son to the normal genotype. Point mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled sbgFGF- 10a_del23 nucleotide sequences.
  • DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments m gels, with or without denatu ⁇ ng agents, or by direct DNA sequencing (see, for instance, 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 Natl Acad Sci USA (1985) 85: 4397-4401).
  • An array of ohgonucleotides probes comp ⁇ smg sbgFGF- 10a_del23 polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Such arrays are preferably high density arrays or g ⁇ ds.
  • Array technology methods are well known and have general applicability and can be used to address a va ⁇ ety of questions m molecular genetics including gene expression, genetic linkage, and genetic va ⁇ abihty, see, for example, M.Chee et al., Science, 274,
  • Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the mvention. Decreased or increased expression can be measured at the RNA level using any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for mstance PCR, RT-PCR, RNase protection, Northern blotting and other hyb ⁇ dization methods.
  • Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present mvention, m a sample de ⁇ ved from a host are well-known to those of skill m the art. Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the present invention relates to a diagnostic kit comp ⁇ sing:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA transc ⁇ pt thereof;
  • kits an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comp ⁇ se a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.
  • the polynucleotide sequences of the present invention are valuable for chromosome localisation studies.
  • the sequence is specifically targeted to, and can hyb ⁇ dize 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, Mendehan Inhe ⁇ tance m Man (available on-line through Johns Hopkins University Welch Medical Library).
  • DNAs Each of these DNAs contains random human genomic fragments maintained in a hamster background (human / hamster hyb ⁇ d cell lines). These PCRs result m 93 scores indicating the presence or absence of the PCR product of the gene of interest. These scores are compared with scores created using PCR products from genomic sequences of known location. This compa ⁇ son is conducted at http://www.genome.wi.mit.edu/.
  • the polynucleotide sequences of the present mvention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present mvention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them.
  • the techniques used are well known m the art and include in situ hyb ⁇ dization techniques to clones arrayed on a g ⁇ d, such as cDNA microarray hyb ⁇ dization (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR.
  • a preferred method uses the TAQMAN (Trade mark) technology available from Perkm Elmer. Results from these studies can provide an indication of the normal function of the polypeptide m the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene can provide valuable insights into the role of the polypeptides of the present mvention, or that of mapprop ⁇ ate expression thereof in disease.
  • mapprop ⁇ ate expression may be of a temporal, spatial or simply quantitative nature.
  • the polypeptides of the present invention are expressed m the hippocampus .
  • a further aspect of the present mvention relates to antibodies.
  • the polypeptides of the mvention or their fragments, or cells expressmg them, can be used as lmrnunogens to produce antibodies that are lmmunospecific for polypeptides of the present mvention.
  • the term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention 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-bea ⁇ ng fragments, or cells to an animal, preferably a non- human animal, using routine protocols.
  • an animal preferably a non- human animal
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hyb ⁇ doma technique (Kohler, G.
  • transgenic mice or other organisms, including other mammals, may be used to express humanized antibodies.
  • the above-desc ⁇ bed 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 diseases of the invention, amongst others.
  • the present invention relates to a method for inducing an immunological response m a mammal that comp ⁇ ses inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokme-producmg T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established withm the individual or not.
  • An immunological response in a mammal may also be induced by a method comp ⁇ ses delive ⁇ ng a polypeptide of the present mvention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo m order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention.
  • One way of admmiste ⁇ ng the vector is by accelerating it into the desired cells as a coating on particles or otherwise.
  • Such nucleic acid vector may comp ⁇ se DNA, RNA, a modified nucleic acid, or a DNA/RNA hyb ⁇ d.
  • a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition).
  • the formulation may further comp ⁇ se a suitable earner. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection).
  • parenterally for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous ste ⁇ le injection solutions that may contain anti-oxidants, buffers, bactenostats and solutes that render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous ste ⁇ le suspensions that 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-d ⁇ ed condition reqm ⁇ ng only the addition of the ste ⁇ le liquid earner immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-m water systems and other systems known m the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expe ⁇ mentation.
  • Polypeptides of the present invention have one or more biological functions that are of relevance m one or more disease states, m particular the diseases of the mvention hereinbefore mentioned. It is therefore useful to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, m a further aspect, the present invention provides for a method of screenmg compounds to identify those that stimulate or inhibit the function or level of the polypeptide.
  • Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the mvention as hereinbefore mentioned.
  • Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical hbra ⁇ es, collections of chemical compounds, and natural product mixtures.
  • Such agonists or antagonists so-identified may be natural or modified substrates, gands, receptors, enzymes, etc., as the case may be, of the polypeptide; a structural or functional mimetic thereof (see Coligan et al , Current Protocols in Immunology l(2):Chapter 5 (1991)) or a small molecule.
  • Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes beanng the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve measunng or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g agonist or antagonist).
  • a labeled competitor e.g agonist or antagonist
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropnate to the cells beanng 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. Further, the screening methods may simply comp ⁇ se the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measunng a sbgFGF- 10a_del23 activity m the mixture, and compa ⁇ ng the sbgFGF- 10a_del23 activity of the mixture to a control mixture which contains no candidate compound.
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also m high-throughput screening (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246, 20-29, (1997).
  • Fusion proteins such as those made from Fc portion and sbgFGF- 10a_del23 polypeptide, as hereinbefore descnbed, 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 measunng 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 that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • a polypeptide of the present mvention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known m the art.
  • ligand binding and crosshnkmg assays include, but are not limited to, ligand binding and crosshnkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance, 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 screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any.
  • a radioactive isotope for instance, ⁇ 1
  • chemically modified for instance, biotmylated
  • Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy.
  • antagonists of polypeptides of the present invention mclude antibodies or, m some cases, oligonucleotides or proteins that 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 a small molecule that bind to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • Screenmg methods may also involve the use of transgemc technology and sbgFGF -
  • transgemc animals The art of constructing transgemc animals is well established.
  • the sbgFGF- 10a_del23 gene may be introduced through micromjection mto the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts.
  • Particularly useful transgemc animals are so-called "knock-in" animals in which an animal gene is replaced by the human equivalent withm the genome of that animal. Knock-m transgemc animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target.
  • transgemc animals are so-called "knock-out" animals m which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled.
  • the gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells m the animal Transgemc animal technology also offers a whole animal expression-clonmg system in which introduced genes are expressed to give large amounts of polypeptides of the present invention Screening kits for use in the above described methods form a further aspect of the present invention. Such screening kits comp ⁇ se:
  • polypeptide of the present invention (c) a cell membrane expressing a polypeptide of the present invention; or (d) an antibody to a polypeptide of the present invention.
  • polypeptide is preferably that of SEQ ED NO:2.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an
  • Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its ongmal environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism 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.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said organism, which organism may be living or non-living.
  • Polynucleotide generally refers to any polyribonucleotide (RNA) or polydeox ⁇ bonucleotide (DNA), which may be unmodified or modified RNA or DNA.
  • Polynucleotides include, without limitation, 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 single- and double-stranded regions, hyb ⁇ d molecules comp ⁇ sing DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, t ⁇ tylated bases and unusual bases such as mosme.
  • a variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found m nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Polypeptide refers to any polypeptide 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, oligopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain ammo 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 that are well known m the art.
  • Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini. It will 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 cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation, biotmylation, covalent attachment of fiavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a lipid or hpid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, mynstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as argmylation, and ubiquitination (see
  • “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. “Fragment” of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1.
  • Vanant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retams the essential properties thereof.
  • a typical vanant of a polynucleotide differs in nucleotide sequence from the 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 in ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below.
  • a typical vanant of a polypeptide differs in ammo acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical.
  • a vanant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, insertions, deletions in any combination.
  • a substituted or mserted ammo acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, he, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr.
  • a vanant of a polynucleotide or polypeptide may be naturally occur ⁇ ng such as an allele, or it may be a vanant that is not known to occur naturally.
  • Non-naturally occumng vanants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • polypeptides having one or more post-translational modifications for instance glycosylation, phosphorylation, methylation, ADP nbosylation and the like.
  • Embodiments include methylation of the N-termmal ammo acid, phosphorylations of se ⁇ nes and threonmes and modification of C-termmal glycmes.
  • Allele refers to one of two or more alternative forms of a gene occumng at a given locus in the genome.
  • Polymorphism refers to a va ⁇ ation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population.
  • Single Nucleotide Polymorphism refers to the occurrence of nucleotide va ⁇ abihty at a single nucleotide position in the genome, withm a population. An SNP may occur withm a gene or withm mtergemc regions of the genome. SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 pnmers are required A common p ⁇ mer is used in reverse complement to the polymorphism being assayed.
  • ASA Allele Specific Amplification
  • This common pnmer can be between 50 and 1500 bps from the polymorphic base.
  • the other two (or more) p ⁇ mers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism.
  • Two (or more) PCR reactions are then conducted on sample DNA. each using the common pnmer and one of the Allele Specific Pnmers.
  • RNA molecules produced from RNA molecules initially transcnbed from the same genomic DNA sequence but which have undergone alternative
  • RNA splicing occurs when a pnmary RNA transc ⁇ pt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences.
  • the term splice vanant also refers to the proteins encoded by the above cDNA molecules.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compa ⁇ ng the sequences
  • identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.
  • % Identity For sequences where there is not an exact correspondence, a “% identity” may be determined.
  • the two sequences to be compared are aligned to give a maximum co ⁇ elation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment.
  • a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
  • Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences.
  • similar ⁇ ty means a companson between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other This likelihood has an associated "score” from which the "% similanty" of the two sequences can then be determined.
  • BESTFIT is more suited to companng two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
  • GAP aligns two sequences, finding a "maximum similanty", according to the algonthm of
  • GAP is more suited to compa ⁇ ng sequences that are approximately the same length and an alignment is expected over the entire length
  • the parameters "Gap Weight” and "Length Weight” used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
  • % identities and simila ⁇ ties are determined when the two sequences being compared are optimally aligned.
  • the BLOSUM62 ammo acid substitution matnx (Henikoff S and Hemkoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used m polypeptide sequence compansons including where nucleotide sequences are first translated mto ammo acid sequences before compa ⁇ son.
  • the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore descnbed.
  • Identity Index is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence.
  • a candidate polynucleotide sequence having, for example, an Identity Index of 0.95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
  • a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 ammo acids of the reference sequence. Such differences are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion. These differences may occur at the ammo- or carboxy- termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups withm the reference sequence.
  • an average of up to 5 in every 100 of the ammo acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descnbed.
  • Identity Index may be expressed m the following equation: n a ⁇ x a - (x a • I), m which- n a is the number of nucleotide or ammo acid differences, x a is the total number of nucleotides or ammo acids in SEQ ID NO 1 or SEQ ED NO.2, respectively,
  • I is the Identity Index
  • Homolog is a genenc term used m the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similanty between the two sequences as hereinbefore defined Falling withm this genenc term are the terms "ortholog", and "paralog".
  • “Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species.
  • “Paralog” refers to a polynucleotide or polypeptide that withm the same species which is functionally similar.
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 533-A discloses fusion proteins compnsmg va ⁇ ous portions of constant region of lmmunoglobulm molecules together with another human protein or part thereof.
  • employing an lmmunoglobulin Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting m, for example, improved pharmacokmetic properties [see, e.g , EP-A 0232 262].

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Abstract

On décrit des polypeptides et des polynucléotides sbgFGF-10a del 23 ainsi que des procédés de production de ces polypeptides par des techniques de recombinaison. On décrit également des procédés d'utilisation de polypeptides et de polynucléotides dans des dosages de diagnostic.
PCT/US2000/027956 1999-10-12 2000-10-10 Polypeptides et polynucleotides humains sbgfgf-10a del23 Ceased WO2001027155A1 (fr)

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GALZIE ET AL.: "Fibroblast growth factors and their receptors", BIOCHEM. CELL BIOL., vol. 75, 1997, pages 669 - 685, XP002937609 *

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