[go: up one dir, main page]

WO2001049845A1 - Variant d'epissage de facteur de croissance - Google Patents

Variant d'epissage de facteur de croissance Download PDF

Info

Publication number
WO2001049845A1
WO2001049845A1 PCT/AU2001/000010 AU0100010W WO0149845A1 WO 2001049845 A1 WO2001049845 A1 WO 2001049845A1 AU 0100010 W AU0100010 W AU 0100010W WO 0149845 A1 WO0149845 A1 WO 0149845A1
Authority
WO
WIPO (PCT)
Prior art keywords
btc
polypeptide
sequence
polynucleotide molecule
variant
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.)
Ceased
Application number
PCT/AU2001/000010
Other languages
English (en)
Inventor
Andrew Jeremy Dunbar
Christopher Goddard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes Biopharma AU Ltd
Original Assignee
Gropep Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gropep Ltd filed Critical Gropep Ltd
Priority to AU24944/01A priority Critical patent/AU2494401A/en
Publication of WO2001049845A1 publication Critical patent/WO2001049845A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a polynucleotide sequence which encodes a naturally occurring splice variant of human betacellulin (BTC) , designated BTC- ⁇ , which lacks polynucleotide sequences normally present in the gene which codes for authentic BTC.
  • BTC- ⁇ a naturally occurring splice variant of human betacellulin
  • the betacellulin variant of the invention may be used as a pharmaceutical for the treatment of certain human diseases.
  • the invention also provides methods for producing BTC- ⁇ by recombinant DNA techniques.
  • peptide growth factors are implicated in a wide variety of physiological and pathological processes, including signal transduction, cell survival, differentiation, cell adhesion, cell migration, immune response, hematopoiesis, inflammation, tissue repair, atherosclerosis and cancer. Consequently there is a great deal of interest in isolating, characterising and defining the functional mechanisms of peptide growth factors, not only in order to understand their role in the basic mechanisms underlying normal mammalian growth and development, but also because of their potential therapeutic use.
  • RTKs transmembrane receptor tyrosine kinases
  • Most RTKs belong to small groups of highly homologous receptors which bind similar ligands and maintain inter-receptor interactions through ligand-induced homo- and hetero-dimer formation.
  • RTKs autophosphorylate on specific tyrosine residues in their cytoplasmic domains.
  • the phosphorylated tyrosine residues serve as high affinity docking sites for proteins which possess SH2 or phosphotyrosine binding (PTB) domains.
  • Such proteins include She, Grb2 and the p85 subunit of phosphoinositide 3 ' -kinase (PI 3-kinase) . This leads to activation of signalling pathways such as the mitogen- activated protein kinase pathway, resulting in a complex cascade of intracellular signals culminating in specific events for target cells.
  • PI 3-kinase phosphoinositide 3 ' -kinase
  • ErbB-1 also called epidermal growth factor receptor (EGFR)
  • ErbB-2 also called HER2 or Neu
  • ErbB-3 ErbB-4
  • EGFR epidermal growth factor receptor
  • peptide growth factors are ligands for the ErbB receptor family. This group of factors shares a high degree of sequence similarity, particularly with respect to a common six-cysteine 36-40 a ino acid residue epidermal growth factor (EGF) motif. This motif has a spacing of
  • CX 7 C 4 CX I QCX I CX 8 C forms three intramolecular disulfide bonds (C1-C3, C2-C 4 , C 5 -C 6 ) and a characteristic three loop structure, including the C1-C3 disulphide loop, C 2 -C 4 disulphide loop and C 5 -C 6 disulphide loop.
  • Mammalian ligands for the ErbB family include EGF (Savage et al . , 1972, J. Biol . Chem. 247: 7612-7621), transforming growth factor- ⁇ (TGF- ⁇ ) (Marquardt et al . , 1984, Science, 223: 1079-1082), heparin-binding EGF-like growth factor (HB-EGF) (Higashiyama et al . , 1991, Science, 251: 936-939), epiregulin (Toyoda et al . , 1995, J. Biol. Chem., 270: 7495-7500), amphiregulin (Shoyab et al .
  • EGF Sevage et al . , 1972, J. Biol . Chem. 247: 7612-7621
  • TGF- ⁇ transforming growth factor- ⁇
  • HB-EGF heparin-binding EGF-like growth factor
  • NRG neural- and thymus-derived activator for ErbB kinases
  • NRG neuregulin subfamily, which includes the products of four genes (NRG1 (Marchionni efc al . , 1993, Nature, 362: 312-318), NRG2 (Chang et al . , 1997, Nature, 387: 509-512; Carraway et al . , 1997, Nature, 387: 512-515), NRG3 (Zhang et al . , 1997, Proc . Natl. Acad. Sci .
  • BTC betacellulin
  • BTC is synthesised as a membrane-anchored precursor protein which can be proteolytically cleaved to release the soluble mature growth factor.
  • Mature BTC binds to and activates the erbBl and erbB-4 homodimers, and in addition, all possible ErbB heterodimers, including the highly oncogenic heterodimeric erbB2-erbB3 receptor complex (Beerli and Hynes, 1996, J. Biol. Chem., 271: 6071-6078; Riese et al .
  • BTC is expressed in a wide range of normal tissues, and is particularly highly expressed in both fetal and adult pancreas (Watanabe et al . , 1994, J. Biol.
  • BTC has been shown to be overexpressed in a human pancreatic cancer (Yokoyama et al . , . 1995, Int. J. Oncology, 7: 825-829) . Recent studies suggest that BTC may play an important role in the formation of pancreatic ⁇ - cells (Mashima et al . , 1996, J. Clin. Invest., 97: 1647- 1654; Watada et al . , 1996, Diabetes, 45: 1828-1831; Huotari et al . , 1998, Endocrinology, 139: 1494-1499; Ishiyama et al . , 1998, Diabetologia, 41: 623-628; Mashima et al . , 1999, Diabetes, 48: 304-309).
  • ErbB3 overexpression in human tumors has not been as frequently observed, ErbB3 has been found to be overexpressed and/or constitutively activated in a subset of mammary tumor cell lines (Alimandi et al . , 1995, Oncogene, 10: 1813-18211995).
  • ErbB receptors may therefore have a central role in both the development and progression of human tumors.
  • these receptors and their ligands are viewed as potential therapeutic targets for the development of drugs which interfere with receptor activation.
  • an ErbB receptor antagonist such as an ErbB ligand-like protein which has receptor binding affinity, but is unable to activate the receptor.
  • the invention provides an isolated polynucleotide molecule selected from the group consisting of
  • the splice variant of the invention is designated BTC- ⁇ , and polynucleotides encoding BTC- ⁇ are referred to as BTC- ⁇ polynucleotides .
  • the term "authentic" when used with reference to BTC means the normal soluble mature BTC protein.
  • substantially homologous sequence refers to a polynucleotide which has a sequence which encodes a polypeptide which is functionally equivalent to the specific BTC- ⁇ sequences disclosed herein, and encompasses substitutions, deletions and insertions in the specifically-disclosed polynucleotide sequences.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • an allelic variant is an alternative form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the polynucleotide molecule has a sequence of human origin.
  • the molecule may be DNA or RNA.
  • the DNA may be genomic DNA or cDNA.
  • the molecule may be naturally occurring or recombinant, and may be isolated from a cellular source, or may be chemically synthesized. Suitable methods of molecular cloning or of chemical synthesis are well known in the art.
  • the DNA may be double stranded or single stranded, and if single stranded may be the coding (sense) or non-coding (anti-sense) strand.
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence shown in Figure 2, or may be a different coding sequence which, as a result of the redundancy of the genetic code, encodes the same polypeptide.
  • the invention also encompasses a nucleic acid molecule capable of hybridizing to the coding sequence shown in Figure 2, under at least low stringency hybridization conditions, or a nucleic acid molecule with at least 70% sequence identity to SEQ ID NO.6.
  • Methods for assessing ability to hybridize and percentage sequence identity are well known in the art.
  • a suitable program for determining percentage sequence identity is BLAST 2 . 0 Sequence Comparison (NIH) (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html) .
  • the limiting parameters imposed for such a task are the default setting for the program as displayed on this web site.
  • the nucleic acid molecule is capable of hybridizing to the coding sequence shown in Figure 2 under high stringency conditions, or has at least 80%, most preferably at least 90% sequence identity thereto.
  • a nucleic acid molecule having at least 70%, preferably at least 90%, more preferably at least 95% sequence identity to one or more of these sequences is also within the scope of the invention.
  • the invention also includes polynucleotides in which the coding sequence for the polypeptide is fused in frame to a polynucleotide sequence which aids in expression of a polypeptide from a host cell, for example a polynucleotide leader sequence encoding the first 11 or 46 amino acids of pig growth hormone or the first 105 amino acids of thioredoxin.
  • a polynucleotide leader sequence encoding the first 11 or 46 amino acids of pig growth hormone or the first 105 amino acids of thioredoxin.
  • Other suitable leader sequences are known in the art .
  • the polynucleotides of the invention may also have the coding sequence fused in frame to a marker sequence which aids in purification of the polypeptides of the invention.
  • the marker sequence may be a hexa-histidine tag to provide for purification of the polypeptide fused to the marker in the case of a bacterial host or may be a FlagTM (Kodak) tag when a mammalian host, e.g. human embryonal kidney 293 -T cells, is used.
  • polynucleotide sequence which may be DNA or RNA, which encodes (a) a polypeptide having a deduced amino acid sequence as set out in any one of SEQ ID NO 1, NO 2 , NO 3 , NO 4 , NO 5 or NO 6, or (b) a fragment, analogue, derivative or allelic variant thereof which has the ability to bind to an ErbB receptor, and which optionally also comprises one or more additional coding or non-coding sequences.
  • the polynucleotide sequence may comprise the complete coding sequence (SEQ ID No. 1), or may optionally comprise
  • coding sequence for the mature polypeptide (b) the coding sequence for the mature polypeptide, and additional coding sequences, such as a leader or secretory signal sequence (amino acids 1-94, SEQ ID No . 3); the coding sequence for the mature polypeptide and other additional coding sequences (for example amino acids 32-129, SEQ ID No. 4, or amino acids 32-111, SEQ ID No . 5 ) ; or
  • the first 93 nucleotides of the BTC- ⁇ polynucleotide encode amino acids which represent the signal peptide which is cleaved to form the mature polypeptide, for example amino acids 32-129 (SEQ ID No.4).
  • the BTC- ⁇ polynucleotides of the invention have wide utility, including but not limited to their use in the preparation of BTC- ⁇ expression vectors, primers and probes to detect and clone BTC- ⁇ , and diagnostic reagents. Diagnostic methods utilizing BTC- ⁇ polynucleotides include hybridisation and PCR assays utilising BTC- ⁇ polynucleotides as primers or probes, as appropriate.
  • the invention provides an isolated polypeptide molecule which is a naturally occurring splice variant of human BTC, or an analogue, fragment or derivative thereof which has the ability to bind to an ErbB receptor .
  • the polypeptide has the ability to act as an agonist or an antagonist to one or more activities mediated by ErbB receptors.
  • ErbB receptors A person skilled in the art will readily be able to determine the biological activity of a given splice variant of the invention, using methods known in the art .
  • fragment when referring to the polypeptide of the invention mean a molecule which retains essentially the same biological function or activity as this polypeptide.
  • an analogue includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • BTC- ⁇ amino acid sequence is included within the scope of the invention, provided that it is functionally active.
  • the terms “functionally active” and “functional activity” in reference to BTC- ⁇ means that the BTC- ⁇ is able to bind to an ErbB receptor, and/or that the BTC- ⁇ is immunologically cross-reactive with an antibody directed against an epitope of naturally-occurring BTC- ⁇ . Therefore, BTC- ⁇ amino acid sequence variants generally will share at least about 75%, preferably greater than 80%, and more preferably greater than 90% sequence identity with the translated amino acid sequence set out in SEQ ID NO: 2, after aligning the sequences to provide for maximum ho ology, as determined, for example, by the Fitch, et al .
  • Amino acid sequence variants of BTC- ⁇ are prepared by introducing appropriate nucleotide changes into BTC- ⁇ DNA, and subsequently expressing the resulting modified DNA in a host cell, or alternatively may be prepared by in vi tro synthesis. Such variants include deletions, insert BTC- ⁇ or substitutions of amino acid residues within the BTC- ⁇ amino acid sequence set out in SEQ ID NO: 2.
  • Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of BTC- ⁇ , provided that the variant possesses the desired functional characteristics described herein. Changes made in the amino acid sequence set out in SEQ ID NO: 2 to arrive at an amino acid sequence variant of BTC- ⁇ also may result in further modifications of BTC- ⁇ when it is expressed in host cells, for example, by virtue of such changes introducing or moving sites of glycosylation.
  • this aspect of the invention relates to BTC- ⁇ polypeptides which have the deduced amino acid sequences set out in Figure 4, as well as fragments, analogues, and derivatives of such polypeptides .
  • the polypeptide of the invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, and is preferably a recombinant polypeptide .
  • the fragments, analogues, and derivatives of the polypeptides in Figure 4 include those in which: (i) one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue, preferably a conserved amino acid residue; such a substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one or more of the amino acid residues includes a substituent group, (iii) the mature polypeptide is fused with another compound to increase the half-life of the polypeptide, or (iv) additional amino acids, such as a sequence which is employed for purification of the mature polypeptide, are fused to the mature polypeptide.
  • Such fragments, analogues and derivatives are deemed to be within the scope of the invention.
  • polypeptides and polynucleotides of the invention are preferably provided in an isolated form, and are preferably purified to homogeneity.
  • the invention provides a vector comprising a polynucleotide molecule according to the invention, and host cells transformed by such vectors.
  • the host cell may be prokaryotic or eukaryotic.
  • the vector is an expression vector.
  • Suitable host cells include bacterial, yeast, insect, and mammalian cells.
  • the invention provides a method of producing BTC- ⁇ , comprising the steps of transforming an expression vector according to the invention into a suitable host cell, cultivating the host cell under conditions suitable for expression of BTC- ⁇ , and isolating BTC- ⁇ . It will be appreciated that host cells comprising selected constructs so formed may express the BTC- ⁇ as a fusion protein within inclusion bodies (IB) .
  • IB inclusion bodies
  • BTC- ⁇ fusion protein we mean a polypeptide consisting of two linked protein components, one of which is selected so as to be expressed in the host cell under the control of a suitable promoter, and the other of which comprises the polypeptide bioactive factor incorporating the motif that confers BTC- ⁇ activity.
  • the fusion protein is produced in order to facilitate the expression and/or processing of the amino acid sequence of the BTC- ⁇ activity.
  • the BTC- ⁇ fusion protein is produced by an appropriate host cell in a fermenter by conventional means understood by those skilled in the art.
  • the BTC- ⁇ is isolated from the host cell following disruption of the host cell by homogenisation, and processed to its biologically pure form using conventional methods of protein purification well recognised by those skilled in the art. These include oxidative refolding to achieve correct disulphide bonding, chemical cleavage of the fusion partner (if used) from the BTC- ⁇ , and various chromatographic steps.
  • the BTC- ⁇ may be isolated as a biologically pure form of the fusion protein, and may then be cleaved from its fusion partner, yielding a peptide that is not extended.
  • the invention provides an antibody directed against a polypeptide according to the invention, or a fragment or derivative of such an antibody which retains its ability to bind to BTC- ⁇ .
  • the antibody of the invention may be polyclonal or monoclonal, or may be a (Fab) 2 fragment, a Fab fragment, a Fv fragment, or a ScFV fragment. Methods for preparation of polyclonal and monoclonal antibodies and preparation of the aforesaid fragments are very well known in the art .
  • the invention provides a composition comprising a polynucleotide molecule, polypeptide molecule or antibody according to the invention, together with a pharmaceutically-acceptable carrier.
  • the composition may optionally further comprise one or more additional therapeutic agents, such as cytotoxic agents or inhibitors of ErbB receptor activation.
  • additional therapeutic agents such as cytotoxic agents or inhibitors of ErbB receptor activation.
  • the compounds of the invention are suitable for use in medical treatment of humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as felids, canids, bovids, and ungulates .
  • the compounds and compositions of the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered.
  • the carrier or diluent, and other excipients will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case.
  • the carrier to be used and the dose and route of administration will depend on the nature of the condition to be treated and the age and general health of the subject, and will be at the discretion of the attending physician or veterinarian.
  • the invention provides a method of treatment of a condition mediated or modulated by ErbB receptors, comprising the step of administering an effective amount of a composition according to the invention to a subject in need of such treatment.
  • the condition is associated with overexpression of an ErbB oncogene.
  • the condition relates to cellular proliferation and/or differentiation. More preferably the condition is a hyperproliferative disease such as a cancer. Most preferably the cancer is selected from the group consisting of breast cancer, epidermoid carcinoma, glioblastoma, and pancreatic cancer.
  • the BTC- ⁇ polypeptide of the invention may be employed to stimulate the growth, proliferation and/or differentiation of mammalian cells, such as epithelial cells, and more organised structures, such as skin in vitro or in vivo, either alone or in combination with other factors .
  • this aspect of the invention provides a method of promoting mitogenesis in a subject in need of such treatment, comprising the step of administering an effective amount of the composition according to the invention to the subject.
  • this embodiment provides a method of promoting cell replication, for example in stimulating wound repair.
  • the BTC- ⁇ polypeptide of the invention may be employed as an agent for treatment of wound healing and/or to stimulate tissue repair.
  • a wide range of wounds may be treated by the polypeptide, including cutaneous wounds, burns, corneal wounds, and injuries to epithelial cell-lined hollow organs of the body.
  • Other conditions suitable for treatment with the polypeptide of the invention include chronic conditions, such as chronic ulcers, diabetic ulcers and other non-healing (trophic) conditions .
  • the polypeptide of the invention may also be used for the treatment of diabetes.
  • composition of the invention may be administered in conjunction with one or more additional therapeutic agents . It is contemplated that because of the activity of BTC, BTC- ⁇ will find utility as a replacement for serum or for serum components in media for mammalian cell culture .
  • the invention includes within its scope variant forms of other members of the EGF family of peptide growth factors which are capable of acting as ligands for the ErbB family of receptors, herein defined as "analogous growth factor variants", in which amino acid residues normally present in the authentic polypeptide are absent, in a manner analogous to their absence from BTC- ⁇ .
  • analogous growth factor variants in which amino acid residues normally present in the authentic polypeptide are absent, in a manner analogous to their absence from BTC- ⁇ .
  • the C 5 -C 6 disulphide loop normally present in the authentic molecule is absent, in a manner analogous to BTC- ⁇ .
  • Suitable authentic polypeptides selected from the EGF family include epidermal growth factor, transforming growth factor- ⁇ , heparin-binding EGF- like growth factor, epiregulin, amphiregulin, neural and thymus-derived activator for ErbB kinases (NTAK) , and members of the neuregulin subfamily (NRG1, NRG2 , NRG3 and NRG4) . It is expected that such variant forms will function in a manner analogous to that found in the BTC- ⁇ polypeptides of the invention.
  • both BTC- ⁇ and analogous variants of other growth factors may be synthesised by chemical means such as solid phase polypeptide synthesis or by recombinant methods, all of which are well known in the art .
  • the invention also encompasses BTC- ⁇ from non- human mammals; the BTC- ⁇ polynucleotides of the invention may readily be used as probes for isolation of corresponding polynucleotides from cells of other mammals using methods which are routine in the art.
  • the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning.
  • Figure 1A shows the detection of authentic human BTC and splice variant human BTC- ⁇ in MCF-7 cells (Lane 1) and human breast skin fibroblasts (Lane 2) by RT-PCR. Lane 3 is a control without cDNA template .
  • Figure IB shows the corresponding Southern blot using a cDNA probe encompassing polynucleotides encoding amino acids D 32 -Y 11:L of human BTC.
  • Figure 2A compares a partial nucleotide and deduced amino acid sequence (in one letter amino acid code) of authentic human BTC and splice variant human BTC- ⁇ .
  • the RT-PCR products obtained from MCF-7 cDNA in Figure 1A were cloned into pBluescript II SK and the inserts sequenced. Sequences surrounding the point of the 147 bp deletion (indicated by a downward arrow) are shown.
  • Figure 2B shows the complete nucleotide sequence and deduced amino acid sequence of BTC- ⁇ cDNA.
  • Figure 3 compares the complete amino acid sequences of authentic human BTC and splice variant human BTC- ⁇ in a hypothetical alignment.
  • the horizontal dashes within the BTC- ⁇ sequence indicate the sites of the missing amino acids when compared to the authentic hBTC sequence.
  • the schematic below shows the overall structure of authentic human BTC compared to BTC- ⁇ .
  • FIG. 4 shows the amino acid sequences of the BTC- ⁇ polypeptides (BTC ⁇ _ 129 , BTC ⁇ i-94, BTC ⁇ 32 -. 94 , BTC ⁇ 32 - ⁇ 29 , BTC ⁇ 32 . 111 and BTC ⁇ _ 5 -i29) •
  • Figure 5 shows the expression of authentic human BTC (563 bp) and BTC- ⁇ (416 bp) in a variety of normal human tissues by RT-PCR and Southern blot analysis.
  • Figure 6 shows an SDS-PAGE gel showing the bacterial expression p ⁇ T3.2a BTC- ⁇ constructs. Lane 1, molecular weight markers; Lane 2, BTC 32"111 thioredoxin fusion protein, Lane 3, BTC 32"94 thioredoxin fusion protein; Lane 4, BTC 32"129 thioredoxin fusion protein; Lane 5, molecular weight markers .
  • the polynucleotide sequence of the invention was isolated from MCF-7 cells. It contains an open reading frame encoding a polypeptide of 129 amino acids.
  • the polynucleotide sequence is identical to that of hBTC, except for a 147 bp deletion within the open reading frame (encoding 49 amino acids) resulting in the absence of the C 5 -C 6 disulphide loop normally present in the EGF domain (See Figure 2B and 3) : This was generated as a result of alternative mRNA splicing (exon skipping) of one of the exons of the human BTC gene .
  • BTC- ⁇ polynucleotides may be obtained from a variety of cell sources which express BTC- ⁇ encoding mRNA.
  • the inventors have identified a number of suitable human cell sources for BTC- ⁇ polynucleotides, including but not limited to kidney, liver, pancreas, and a variety of breast carcinoma cell lines (e.g., MCF-7).
  • polynucleotides encoding BTC- ⁇ polypeptides may be obtained by cDNA cloning from RNA isolated and purified from cell sources.
  • cDNA libraries of clones may be prepared using techniques well known to those in the art, and may be screened for BTC- ⁇ encoding DNA with nucleotide probes which are substantially complementary to any portion of the BTC gene .
  • Various PCR cloning techniques may also be used to obtain the BTC- ⁇ polynucleotides of the invention.
  • polynucleotides encoding BTC- ⁇ polypeptides of the invention may be obtained by PCR, using oligonucleotide primers comprising polynucleotide sequences encoding portions of the BTC gene.
  • the primer comprises the extreme 5' and 3' coding regions.
  • the oligonucleotide primers have the following sequences, or sequences substantially homologous thereto (SEQ ID No. 8) : 5 ' GAGCGGGGTTGATGGACCGG 3 ' 5 ' TTAAGCAATATTTGTCTCTTC 3 '
  • Representative low and high stringency conditions of hybridisation as referred to herein are as follows : High stringency: hybridization at 42°C in 50% formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 ⁇ g/ml salmon sperm DNA overnight and washed with a final wash of 0.1 x SSC, 0.1% SDS at 42°C.
  • Low stringency hybridization at 28°C in 50% formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 ⁇ g/ml salmon sperm DNA overnight and washed with a final wash of 0.1 x SSC, 0.1% SDS at room temperature.
  • Host cells are transformed or transfected with the vectors of this invention, for example a cloning vector or an expression vector.
  • a cloning vector or an expression vector for example a cloning vector or an expression vector.
  • Various expression vector/host systems may be utilised equally well by those skilled in the art for the recombinant expression of BTC- ⁇ polypeptides.
  • Such systems include, but are not limited to micro-organisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors comprising the desired BTC- ⁇ polynucleotide coding sequence; yeast transformed with recombinant yeast expression vectors comprising the desired BTC- ⁇ polynucleotide coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) comprising the desired BTC- ⁇ polynucleotide coding sequence; or animal cell systems transfected with appropriate mammalian expression vectors comprising the desired BTC- ⁇ polynucleotide coding sequence.
  • micro-organisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors comprising the desired BTC- ⁇ polynucleotide coding sequence
  • yeast transformed with recombinant yeast expression vectors comprising the desired BTC-
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures well known to those skilled in the art.
  • the DNA sequence inserted in the expression vector is operatively linked to an expression control sequence (s) (promoter) to direct mRNA synthesis.
  • s expression control sequence
  • any one of suitable transcription/translation elements may be used.
  • promoters isolated from the genome of mammalian cells e.g., mouse metallothionein promoter
  • viruses that grow in these cells e.g., human cytomegalovirus immediate-early (CMV) promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of the inserted sequences.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. Specific initiation signals are also required for efficient translation of inserted coding sequences . These signals include the ATG initiation codon and adjacent sequences. In cases where the entire BTC- ⁇ coding sequence, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vectors, no additional translational control signals may be needed. However, in cases where only a portion of the BTC- ⁇ coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided.
  • initiation codon must be in phase with the reading frame of the BTC- ⁇ coding sequences to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of transcription attenuation sequences, enhancer elements etc .
  • the expression vectors comprise one or more selectable marker genes to provide a phenotypic trait for selection of transformed or transfected host cells, such as neomycin (G418) resistance for eukaryotic cells, or ampicillin resistance for prokaryotic cells such as E. coli .
  • selectable marker genes such as neomycin (G418) resistance for eukaryotic cells, or ampicillin resistance for prokaryotic cells such as E. coli .
  • the vector containing a DNA molecule of the invention may be employed to transform or transfect an appropriate host to permit the host to express the protein.
  • bacterial cells such as E. coli
  • insect cells such as Drosophila and Sf9
  • animal cells such as CHO, COS , or 293 cells.
  • the invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises one or more regulatory sequences, such as a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • the invention relates to host cells containing the above-described constructs .
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be via a variety of methods which are well known to those skilled in the art .
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptide of the invention can be synthetically produced by conventional peptide synthesisers.
  • polypeptide of the invention produced in a variety of different vector/host expression systems as described above, can be recovered and purified from recombinant cell cultures by a wide variety of methods, including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and reverse-phase high performance liquid chromatography (HPLC) . Protein refolding steps can be used, as necessary, in completing the configuration of the desired polypeptide.
  • the polypeptide of the invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques using a prokaryotic or eukaryotic host. Depending upon the host employed in a recombinant production procedure, the polypeptides of the invention may be glycosylated or may be non-glycosylated.
  • the BTC- ⁇ polypeptide of the invention may be employed as research agents and materials for discovery of treatments and diagnostics for human diseases.
  • one suitable method of using the BTC- ⁇ polypeptide as a diagnostic for human disease comprises the step of quantitatively determining the amount of BTC- ⁇ polypeptide present in a sample from a subject, and comparing the amount thus determined with the amount present in a sample from a normal subject, in which the presence of a significantly different amount in the test subject indicates the presence of a disease.
  • the BTC- ⁇ polypeptide may have the ability to mediate or modulate ErbB receptor activity. The person skilled in the art will readily be able to determine the biological activity of the BTC- ⁇ polypeptide mediated by ErbB receptors, using methods known in the art.
  • the methylene blue cell proliferation assay may be used for the characterisation of the cellular proliferation activity of BTC- ⁇ polypeptides.
  • Cell lines suitable for this assay include, but are not limited to, Balb/c 3T3, HaCaT, IEC-6, SF3169, MvlLu and CalOst cells.
  • the cell lines are subcultured at a density of 10-20 x 10 4 cells/ml -1 and incubated overnight at 37°C in the presence of 5% C0 2 .
  • the cells are then washed extensively with DMEM to remove any residual medium, after which BTC- ⁇ can be added at various concentrations.
  • Authentic BTC can optionally be assayed in parallel for comparison.
  • BTC- ⁇ polypeptides can be analysed by the competitive displacement of 125 ⁇ - betacellulin from receptors present on various cell lines including, but not limited to, AG2804 fibroblasts and
  • Chinese hamster ovary cells transfected with ErbB-2, ErbB-3 and ErbB-4 receptor homodimers Preferably cells are grown to 70-80% confluence in DMEM supplemented with 10% (v/v) foetal bovine serum in 24-well plates. The cells are washed twice with binding buffer; [100 mM Hepes (pH 7.6), 120 mM NaCl, 5 mM KCI, 1.2 mM MgS04.7H 2 0, 8 mM glucose, 0.1% bovine serum albumin] and then incubated with appropriate concentrations of each peptide and 125 I-betacellulin in binding buffer for 18 h at 4°C.
  • binding buffer [100 mM Hepes (pH 7.6), 120 mM NaCl, 5 mM KCI, 1.2 mM MgS04.7H 2 0, 8 mM glucose, 0.1% bovine serum albumin]
  • BTC- ⁇ polypeptide may be incorporated in physiologically or pharmaceutically-acceptable carriers to provide compositions for application to the affected area.
  • the nature of the carriers may vary widely, and will depend on the condition to be treated and the intended site of application. For application to the skin, a cream or ointment base is preferred.
  • the BTC- ⁇ polypeptides of the invention may be impregnated into bandages and other wound dressings to provide for continuous exposure of the wound to the polypeptide.
  • the concentration of polypeptide in the treatment composition is not critical but should be an effective amount to induce epithelial proliferation.
  • effective amount as used herein in all methods of use for BTC- ⁇ polypeptides, means an amount sufficient to elicit a statistically significant response at a 95% confidence level (p ⁇ 0.05 that the effect is due to chance alone) .
  • the amount of polypeptide employed can be determined empirically, based on the response of cells in vitro and response of experimental animals to the subject polypeptide or formulations containing the subject polypeptide. Additionally, the polypeptide of the invention may be used to prevent or inhibit proliferation of adenocarcinoma cell proliferation.
  • BTC- ⁇ polypeptides may be used for systemic and targeted therapy of certain cancers overexpressing ErbB receptors, such as metastatic breast cancer, with minimal toxicity to normal tissues and organs.
  • the BTC- ⁇ polypeptides of the invention can be conjugated with various toxins that are cytotoxic to cancerous cells. More particularly, ErbB- expressing tumor cells may be specifically targeted and killed by contacting such tumor cells with a fusion protein comprising a cytotoxic polypeptide covalently linked to any one of the BTC- ⁇ polypeptides of the invention. Examples include, but are not limited to, ricin, daunorubicin, methotrexate and various bacterial toxins, such as
  • Toxin-conjugated-BTC- ⁇ polypeptides for targeted cancer therapy may be administered by any route which will result in polypeptide interaction with the target cancer cells, including systemic administration and injection directly to the site of the tumor.
  • Another therapeutic strategy is administration by sustained-release systems, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • a further therapeutic strategy is the administration of BTC- ⁇ polypeptides with an agent which inhibits ErbB receptor activation. Such agents would include antibodies directed to an epitope of the ErbB receptor.
  • the invention also encompasses polyclonal and monoclonal antibodies which recognise epitopes which are specific to BTC- ⁇ polypeptides.
  • Anti-BTC- ⁇ antibodies may be used for the detection and quantification of BTC- ⁇ polypeptide expression in cultured cells, tissue samples, and in vivo .
  • monoclonal antibodies recognising epitopes from different parts of the BTC- ⁇ structure may be used to detect and/or distinguish binding from non-binding regions of the ligand.
  • the antibody is a polyclonal or monoclonal antibody which rt ⁇ - rt ⁇ tf ⁇ ⁇ g cr ⁇ - > ⁇ Cfi ii M g r ⁇ ⁇ 1 Hi J • ⁇ 1 ⁇ Q ⁇ Hi P rt W rt SB Hi tf ⁇ tf p ) 3 rt PJ 0 ⁇ ⁇ X rt ⁇ ii PJ ii H 0 ⁇ ⁇ ii H ⁇ o 0 ⁇ tf j tf ⁇ ⁇
  • anti-BTC- ⁇ antibodies as affinity reagents for the purification of BTC- ⁇ polypeptides, and as immunological probes for elucidating the biosynthesis, metabolism and biological functions of BTC- ⁇ polypeptides .
  • Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of BTC- ⁇ polypeptides.
  • polyclonal antibodies a number of host animals are acceptable for the generation of anti-BTC- ⁇ antibodies by immunisation with one or more injections of a BTC- ⁇ polypeptide preparation, including but not limited to rabbits, mice, rats, goats, etc.
  • Various adjuvants may be used to increase the immunological response in the host animal, depending upon the host species, including but not limited to alum, isco s, or complete or incomplete Freund's adjuvant.
  • Monoclonal antibodies to epitopes of BTC- ⁇ polypeptides may be prepared by using any technique -which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not restricted to, the original hybridoma technique (Kohler and Milstein, Nature 1975, 256: 495-497) and the human B- cell hybridoma technique (Kosbor et al . 1983, Immunology Today, 4:72) .
  • Humanised antibodies may be prepared according to procedures described in the literature (e.g., Verhoeyen et al . , 1988, Science 239: 1534-1536) .
  • Example 1 Detection of BTC- ⁇ by RT-PCR and cloning into pBluescript II SK
  • Normal human breast skin fibroblasts were prepared from a piece of skin obtained during surgery for breast reduction. The skin was cultured as an explant for 5 days in DMEM supplemented with 10% fetal calf serum and penicillin-streptomycin sulphate until the fibroblasts had grown into a monolayer.
  • Total RNA (2 ⁇ g) was subsequently reverse transcribed to cDNA with Superscript II enzyme according to the manufacturer's instructions using oligo dT primers (Gibco BRL, Gaithersburg, MD) .
  • cDNA corresponding to human BTC- ⁇ was amplified by PCR with sense primer
  • PCR was carried out in 50 ⁇ l of 60 mM Tris-S0 4 , 18 mM (NH 4 ) 2 S0 4 , 1.5 mM MgS0 4 (pH 9.1), 0.2 mM dNTPs, 200 ng each primer, 1 U eLONGase (Gibco BRL, Gaithersburg, MD) and 1 ⁇ l cDNA.
  • the blot was incubated in prehybridization buffer (5x SSC, 5x Denhardt's, 0.1% sodium dodecyl sulphate (SDS), and 50 ⁇ g/ml salmon sperm DNA) at 55°C for 2 h and hybridized for 16 h in the same buffer containing 32 P-dCTP-labelled human BTC cDNA probe (D 32 -Y 11:L ) generated by the random priming method using a Gigaprime kit (Geneworks, Sydney, Australia) . Two washes were performed in 2x SSC, 0.1% SDS for 5 min at room temperature, followed by two more 15 min washes in 0.5x SSC, 0.1% SDS at 55°C.
  • prehybridization buffer 5x SSC, 5x Denhardt's, 0.1% sodium dodecyl sulphate (SDS), and 50 ⁇ g/ml salmon sperm DNA
  • the recombinant plasmids were propagated and sequenced by the dideoxynucleotide chain terminator method, using a Thermosequenase cycle sequencing kit (Amersham-Pharmacia Biotech, Sydney, Australia) with M13 T7 and T3 promoter primers. PCR products were sequenced in both directions to confirm the sequence; this was done twice from two independent clones ( Figure 2A and B) .
  • Glyceraldehyde-3 -phosphate dehydrogenase (G3PDH) specific 5' and 3' primers (sense: 5' were used as a control to verify the presence of cDNA in each reaction. The results are illustrated in Figure 5. Particularly strong expression was observed in kidney, liver and pancreas, and heart, skeletal muscle and lung were also positive.
  • G3PDH Glyceraldehyde-3 -phosphate dehydrogenase
  • BTC- ⁇ polypeptides constituting amino acids 32-94 (BTC- ⁇ 32-94 ) (see Figur -3 and Figure 4, polypeptide 3) or
  • BTC- ⁇ 32""129 32-129 (BTC- ⁇ 32""129 ) (see Figure 3 and Figure 4, polypeptide 4) or BTC constituting amino acids 32-111 (BTC 32-111 ) (see Figure 3) were expressed as thiororedoxin fusion proteins in the expression vector pET3.2a.
  • the open reading frame sequence of BTC- ⁇ 32"94 was amplified by PCR using the primer set
  • BTC- ⁇ 32"129 the above sense primer was used in conjunction with the antisense primer 5 ' -CGTCTCGAGTCATTAAGCAATATTTGTCTCTTC-3 ' (Xhol site is underlined) and pBlue-BTC- ⁇ as template.
  • BTC 32"111 was amplified with sense primer 5 ' -CGTCCATGGCTGATGGGAATTCCACCAGAAGT-3 '
  • Example 4 Construction of BTC- ⁇ expression plasmid for mammalian expression
  • Full length BTC- ⁇ was cloned into the vector pcDNA3.1 (Invitrogen) to generate expression vectors for the mammalian production of BTC- ⁇ as follows. Briefly, pBlue-BTC- ⁇ (see Example 1) was digested with ApaJ and Bam ⁇ .1 and the released insert purified following agarose gel electrophoresis . The digested and purified insert was then cloned into Apal/BamHI-digested pcDNA3.1, and recombinant clones identified following transformation in E. coli TOP10 cells (Invitrogen) .
  • the pBlue-BTC- ⁇ was used as a template in PCR using the primers 5 ' -CTCGGGAATTCCGACTACAAGGACGACGATGACAAGACCAGAAGTCCTGAA-3 ' (sense; underlined nucleotides correspond to an ⁇ OoRI restriction site; double underlined nucleotides encode the FLAG epitope tag) and
  • Example 5 Large scale bacterial expression and purification of BTC- ⁇ polypeptides
  • the recombinant plasmids pET-BTC- ⁇ 32"94 , pET-BTC- ⁇ 32"129 and pET- BTC 32"111 were transformed into the E. coli strain BL21trxB (DE3 ) (Novagen) .
  • This strain is deficient in the protein thioredoxin reductase, thus favouring disulphide bond formation in the cytoplasm of E. coli .
  • Ni-NTA agarose resin Qiagen
  • Requilibration buffer 50 mM NaH 2 P0 4 , 0.3 M NaCl, 10 mM imidazole, pH 8.0
  • the resin was then pelleted by centrifugation (1500 rpm, 5 min) and washed 4 times in 50 mM NaH 2 P0 4 , 0.3 M
  • BTC fusion proteins were then eluted from the resin by four successive washes in 8 ml 50 mM NaH 2 P0 4 , 0.3 M NaCl, 250 mM imidazole, pH 8.0) .
  • BTC 32 " 111 or B C _ ⁇ 3 2 -94 , 3 2 - ⁇ 2 9 p ept j_ des were released from the thioredoxin fusion partner by proteolytic cleavage with recombinant enterokinase. Briefly, the eluted fractions containing BTC 32"111 or BTC- ⁇ 32"94 ' 32 129 fusion proteins (approximately 32 ml) were dialysed against 2 x 8L of 50 mM Tris-Cl, 1 mM CaCl2, 0.1% Tween-20 (pH 7.4) (Spectrapore membrane, 3500 MW cut-off) at 4°C.
  • the dialysed sample was then centrifuged to remove any precipitated material and enterokinase (Invitrogen) added to a final concentration of 0.1 units/20 ⁇ g protein.
  • the cleavage reaction was then allowed to proceed for 16 hours at 37°C before termination by adjustment of the pH to 3.0 with cone, trifluoroacetic acid (TFA) .
  • the cleavage reaction mixture was applied to a C4 reverse-phase HPLC column (Aquapore BU-300, 2.1mm x 100 mm,Brownlee Lab) at a flow rate of 0.5 ml/min.
  • the column was washed with 0.1% TFA until OD ⁇ 4nm returned to baseline, and protein was eluted with a gradient of 0.1% TFA-0.08% TFA/80% CH 3 CN over 40 min at 0.5 ml/min.
  • Fractions containing authentic BTC 32"111 or BTC- ⁇ 32"94 ' 32 ⁇ 129 peptides were pooled and lyophilised. The purity of the final preparation (s) was confirmed by SDS- PAGE and electrospray ionisation mass spectroscopy .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une séquence polynucléotidique codant pour un variant d'épissage naturel de la bétacelluline (BTC) humaine, appelée BTC-β. La séquence polynucléotidique de la BTC-β est privée de la séquence codant pour la dernière boucle bisulfure C5-C6 du motif CX7CX4C10CX1CX8C du facteur de croissance épidermique, normalement présente dans le gène codant pour la BTC type. La BTC-sg(b) peut être utilisée pour traiter des affections induites ou contrôlées par des récepteurs ErbB. L'invention concerne également des procédés permettant de produire, d'une part, la BTC-ß par des techniques de recombinaison de l'ADN et, d'autre part, des anticorps contre la BTC-β.
PCT/AU2001/000010 2000-01-06 2001-01-05 Variant d'epissage de facteur de croissance Ceased WO2001049845A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU24944/01A AU2494401A (en) 2000-01-06 2001-01-05 Growth factor splice variant

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPQ4969 2000-01-06
AUPQ4969A AUPQ496900A0 (en) 2000-01-06 2000-01-06 Growth factor splice variant

Publications (1)

Publication Number Publication Date
WO2001049845A1 true WO2001049845A1 (fr) 2001-07-12

Family

ID=3819103

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2001/000010 Ceased WO2001049845A1 (fr) 2000-01-06 2001-01-05 Variant d'epissage de facteur de croissance

Country Status (2)

Country Link
AU (1) AUPQ496900A0 (fr)
WO (1) WO2001049845A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006012707A1 (fr) * 2004-08-06 2006-02-09 Gropep Limited Procédé de traitement du diabète
WO2006128125A3 (fr) * 2005-05-27 2007-08-02 Five Prime Therapeutics Inc Methodes et compositions pour stimuler le captage du glucose dans des cellules musculaires et pour traiter des maladies
WO2019212279A1 (fr) * 2018-05-03 2019-11-07 연세대학교 산학협력단 Composition pour l'excrétion de sucre
US11524998B2 (en) 2020-07-16 2022-12-13 Novartis Ag Anti-betacellulin antibodies, fragments thereof, and multi-specific binding molecules

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999062955A1 (fr) * 1998-05-29 1999-12-09 Biomolecular Research Institute Limited Conception d'agonistes et d'antagonistes de la famille des recepteurs egf

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999062955A1 (fr) * 1998-05-29 1999-12-09 Biomolecular Research Institute Limited Conception d'agonistes et d'antagonistes de la famille des recepteurs egf

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ABE Y. ET AL.: "Disulfide bond structure of human epidermal growth factor receptor", J. BIOL. CHEM., vol. 273, 1998, pages 11150 - 11157 *
DATABASE GENBANK [online] 14 January 1995 (1995-01-14), Database accession no. AAA61158 *
DATABASE GENBANK [online] 14 January 1995 (1995-01-14), Database accession no. K03220 *
DATABASE GENBANK [online] 14 January 1995 (1995-01-14), Database accession no. K03221 *
DUNBAR A. ET AL.: "factor in milk: Purification, characterization and molecular cloning of bovine betacellulin", BIOCHEM. J., vol. 344, 1999, pages 713 - 721 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006012707A1 (fr) * 2004-08-06 2006-02-09 Gropep Limited Procédé de traitement du diabète
WO2006128125A3 (fr) * 2005-05-27 2007-08-02 Five Prime Therapeutics Inc Methodes et compositions pour stimuler le captage du glucose dans des cellules musculaires et pour traiter des maladies
WO2019212279A1 (fr) * 2018-05-03 2019-11-07 연세대학교 산학협력단 Composition pour l'excrétion de sucre
US11524998B2 (en) 2020-07-16 2022-12-13 Novartis Ag Anti-betacellulin antibodies, fragments thereof, and multi-specific binding molecules

Also Published As

Publication number Publication date
AUPQ496900A0 (en) 2000-02-03

Similar Documents

Publication Publication Date Title
TOYODA et al. Distribution of mRNA for human epiregulin, a differentially expressed member of the epidermal growth factor family
AU676502B2 (en) HER4 human receptor tyrosine kinase
Lee et al. Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor
US5811098A (en) Antibodies to HER4, human receptor tyrosine kinase
JP3039802B2 (ja) 繊維芽細胞成長因子のための受容体
CZ208197A3 (en) Side protein of human receptor for interleukin 1
CZ302689B6 (cs) Izolovaná molekula nukleové kyseliny, fúzní polypeptid jí kódovaný, vektor s jejím obsahem, systém vektor-hostitel pro produkci fúzního polypeptidu a zpusob jeho prípravy, antagonista dimerního VEGF a použití
TW201446262A (zh) 活化素-ＡｃｔＲＩＩａ拮抗劑及其治療或預防乳癌之用途
WO2007115571A2 (fr) Fragments peptidiques dérivés du récepteur erbb
JPH07506488A (ja) ｎｅｕレセプターの組換え刺激因子
US7705117B2 (en) EGFH2 genes and gene products
US6544759B1 (en) Polynucleotides encoding a novel growth factor which acts through ErbB-4 kinase receptor tyrosine
WO2001049845A1 (fr) Variant d'epissage de facteur de croissance
CN102134275B (zh) 表皮生长因子受体变异体
JP2001517445A (ja) 甲状腺刺激ホルモンの突然変異体およびそれに基づく方法
CA2351522A1 (fr) Acides nucleiques et polypeptides et utilisations correspondantes
AU2004265512B2 (en) Splice variants of ErbB ligands, compositions and uses thereof
CN1477117A (zh) 新型人磷脂酰乙醇胺结合蛋白，其编码序列及用途
JP2003510029A (ja) Ｅｇｆｈ２遺伝子および遺伝子産物
AU730816B2 (en) Vitamin D response element binding protein
US7544485B2 (en) Baldness related gene and the polypeptide encoded thereby, and uses
WO1996012019A2 (fr) Recepteur humain her4 de type tyrosine-kinase appartenant a la famille des facteurs de croissance de l'epiderme
WO1996012019A9 (fr) Recepteur humain her4 de type tyrosine-kinase appartenant a la famille des facteurs de croissance de l'epiderme
CN1288057A (zh) 一种编码新的人血管紧张素ⅱ－1受体相关蛋白的基因及其应用和制备方法
Stortelers The role of EGF-like growth factors in selective ErbB receptor dimerisation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP