WO1999020755A2 - Nouveaux recepteurs de cytokine - Google Patents

Nouveaux recepteurs de cytokine Download PDF

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Publication number
WO1999020755A2
WO1999020755A2 PCT/EP1998/006497 EP9806497W WO9920755A2 WO 1999020755 A2 WO1999020755 A2 WO 1999020755A2 EP 9806497 W EP9806497 W EP 9806497W WO 9920755 A2 WO9920755 A2 WO 9920755A2
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WIPO (PCT)
Prior art keywords
polypeptide
amino acid
cytokine
receptor
ilr
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PCT/EP1998/006497
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English (en)
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WO1999020755A3 (fr
Inventor
Greg Elson
Jean-François Gauchat
Marie Kosco-Vilbois
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Glaxo Group Limited
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Priority to AU13347/99A priority Critical patent/AU1334799A/en
Priority to EP98956844A priority patent/EP1027438A2/fr
Priority to JP2000517076A priority patent/JP2002508922A/ja
Publication of WO1999020755A2 publication Critical patent/WO1999020755A2/fr
Publication of WO1999020755A3 publication Critical patent/WO1999020755A3/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates inter alia to novel molecules believed to be cytokine receptors and to uses thereof.
  • Cytokines and " growth factors are secreted molecules controlling important cell functions such as proliferation, differentiation and survival as well as tissue development. These signalling molecules exert their effects via specific receptors located on the target cell surface. These receptors are grouped into families according to both structural and amino acid sequence similarities.
  • the cytokine receptor superfamily is composed of the receptors for many growth factor families including interferon, TNF and haematopoietic growth factors. The largest subclass in this family is that of the type I cytokine receptors, a group characterized by the presence of a conserved extracellular region of approximately 200 amino acids containing two fibronectin type III folds.
  • haematopoietin receptor module This region, known as the haematopoietin receptor module, has been shown to play an essential role in receptor/ligand binding and receptor/receptor dimerization. It is characterized by four conserved cysteine residues in the first domain and a W-S-x-W-S motif in the second domain.
  • cytokine binding chains in the receptor complexes generally have short or even absent intracytoplasmic regions, whereas gpl30 has a longer intracytoplasmic tail which is involved in the activation of the JAK-STAT pathway.
  • soluble forms of these receptor subunits can render cells sensitive to the appropriate cytokine provided the necessary signal transducing chain is expressed on the cell surface.
  • the specific cytokine binding chains of these cytokine receptors can function either as membrane anchored or soluble proteins.
  • the present invention is based upon the identification and characterisation of a previously unknown mouse molecule and of a previously unknown human molecule, both of which are believed to be new type 1 cytokine receptors.
  • polypeptide is used herein in a broad sense to indicate that a particular molecule comprises a plurality of amino acids joined together by peptide bonds. It therefore includes within its scope substances which may sometimes be referred to in the literature as peptides, polypeptides or proteins.
  • a polypeptide of the present invention preferably incorporates a haematopoietin domain since this is believed to be important in ligand / receptor and receptor / receptor complex formation.
  • Polypeptides of the present invention may be produced by techniques known to those skilled in the art. For example, gene-cloning techniques may be used to provide a nucleic acid sequence encoding such a polypeptide. (Gene-cloning techniques are discussed in greater detail later on in relation to nucleic acid molecules of the present invention.) Alternatively, chemical synthesis techniques may be used to produce polypeptides of the present invention. Such techniques generally utilise solid-phase synthesis. Chemical synthesis techniques that allow polypeptides having particular sequences to be produced have now been automated. Apparatuses capable of chemically synthesising polypeptides are available, for example, from Applied Biosy stems. If desired, short polypeptides can be synthesised initially and can then be ligated to produce longer polypeptides.
  • a polypeptide of the present invention may be provided in substantially pure form. Thus it may be provided in a composition in which it is the predominant polypeptide component present. (It may be present e.g. at a level of more than 50%, of more than 75%, of more than 90%, or even of more than 95%; said levels being determined on a weight/weight basis with respect to the total polypeptide content of the composition.)
  • a polypeptide of the present invention either: a) has the amino acid sequence of amino acids 38 to 422 shown in Figure 1 for hGBRI-ILR or has the amino acid sequence shown in Figure 1 for mGBRI- ILR; b) has one or more amino acid deletions, insertions or substitutions relative to a polypeptide as defined in a) above, but has at least 40% amino acid sequence identity therewith; or c) is a fragment of a polypeptide as defined in a) or b) above, which is at least 10 amino acids long.
  • a polypeptide within the scope of a) may consist of the amino acid sequence of amino acids 38 to 422 shown in Figure 1 for hGBRI-ILR or of the amino acid sequence shown in Figure 1 for mGBRI-ILR. Alternatively it may have an additional N-terminal and/or an additional C-terminal amino acid sequence.
  • N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art. These include using gene-cloning techniques to ligate together nucleic acid molecules encoding polypeptides or parts thereof, followed by expressing a polypeptide encoded by the nucleic acid molecule produced by ligation.
  • Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems.
  • an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone somatostatin by fusing it at its N-terminus to part of the ⁇ galactosidase enzyme (Itakwa et al., Science 198: 105-63 (1977)).
  • Additional sequences can also be useful in altering the properties of a polypeptide to aid in directing the polypeptide to a particular location.
  • a signal sequence may be present to direct the transport of the polypeptide to a particular location within a cell or to export the polypeptide from the cell (e.g. the amino acids 1 to 37 shown in Figure 1 for hGBRI-ILR may be used to provide a signal sequence, or another signal sequence may be present).
  • Different signal sequences can be used for different expression systems.
  • Hydrophobic sequences may be provided to anchor a polypeptide in a membrane.
  • the present invention includes within its scope both soluble and membrane-bound polypeptides.
  • Membrane-bound polypeptides may be in hybrid form if desired. They may therefore have a heterologous transmembrane and/or cytoplasmic domain. For example such domains may be derived from a human IL-13 receptor ⁇ chain.
  • Transfected mammalian cells expressing membrane-bound polypeptides can be used for ligand screening and binding assays (e.g. for antibodies or other molecules binding to the receptor).
  • a polypeptide is linked to a moiety aiding in purification / identification, e.g. a moiety capable of being isolated by affinity chromatography.
  • the moiety may be an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments that bind to said epitope (desirably with a high degree of specificity).
  • the polypeptide can then be eluted from the column by addition of an appropriate buffer and may be cleaved from the epitope. His 6 Glu 2 or 179 tags are preferred for use in purification / identification. Polypeptides comprising one or more such tags are therefore within the scope of the present invention.
  • a polypeptide may be linked to an antibody or to a part thereof. For example it may be linked to an F c portion. This results in a molecule with good stability that can be used both in vitro and in vivo. It may be linked to a part of an antibody that binds to a particular epitope of it is desired to target that epitope.
  • additional amino acids may be provided to result in an amino acid sequence closer in length to the length of the 422 amino acid polypeptide shown in Figure 1 for hGBRI- ILR.
  • an additional two amino acids immediately N-terminal to the amino acid sequence shown in Figure 1 for mGBRI-ILR polypeptide may be provided. (These may be A and H, as is the case for hGBRI-ILR).
  • a signal sequence may also/alternatively be provided.
  • N-terminal or C-terminal sequences may be present simply as a result of a particular technique used to obtain a polypeptide of the present invention and need not provide any particular advantageous characteristic.
  • variants of the polypeptide which has a desired property to produce variants (often known as "muteins") that still have said property.
  • variants of the polypeptides described in a) above are within the scope of the present invention and are discussed in greater detail below in sections ( to (Hi). They include allelic and non-allelic variants.
  • substitutions An example of a variant of the present invention is a polypeptide as defined in a) above, apart from the substitution of one or more amino acids with one or more other amino acids.
  • the skilled person is aware that various amino acids have similar characteristics.
  • One or more such amino acids of a polypeptide can often be substituted by one or more other such amino acids without eliminating a desired property of that polypeptide.
  • amino acids gly cine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
  • amino acids having aliphatic side chains amino acids having aliphatic side chains.
  • glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic).
  • amino acids having aromatic side chains include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains).
  • Amino acid deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining a desired property. This can enable the amount of polypeptide required for a particular purpose to be reduced. For example if the polypeptide is to be used in medicine, dosage levels can be reduced.
  • Polypeptides incorporating amino acid changes can be provided using any suitable techniques.
  • a nucleic acid sequence incorporating a desired sequence change can be provided by site-directed mutagenesis. This can then be used to allow the expression of a polypeptide having a corresponding change in its amino acid sequence.
  • preferred polypeptides of the present invention have at least 40% amino acid sequence identity with the amino acid sequence of amino acids 38 to 422 shown in Figure 1 for hGBRI-ILR or with the amino acid sequence shown in Figure 1 for mGBRI-ILR. More preferably the degree of sequence identity is at least 50% or at least 75%. Sequence identities of at least 90% or of at least 95% are most preferred.
  • sequence identity (whether amino acid or nucleic acid) can be determined by using the "BESTFIT" program of the Wisconsin Sequence Analysis Package Genetics Computer Group version 8.0.
  • Feature c) of the present invention therefore covers fragments of the polypeptides a) or b) above which are at least 10 amino acids long. Desirably these fragments are at least 20, at least 50 or at least 100 amino acids long. Fragments may be useful, for example, in raising antibodies against particular antigens. They may also be useful in studying functionally important domains of a fiill length polypeptide. Thus, for example, a fragment comprising all or part of a haematopoietin receptor module may be provided.
  • Polypeptides of the present invention may be used in medicine.
  • Preferred treatments are human treatments, although veterinary treatments are not excluded.
  • the treatment may be prophylactic or may be in respect of an existing condition.
  • polypeptides of the present invention may be useful either as agonists or as antagonists.
  • Agonists will up-regulate a biological function of a naturally occurring receptor, whereas antagonists will down-regulate such a function.
  • Whether or not a given polypeptide acts as an agonist or an antagonist of a particular biological function can be determined by a skilled person using an appropriate assay procedure.
  • Antagonists may be useful in treating disorders associated with an overexpression of a cytokine or with the expression of a moiety having a level of cytokine activity higher than normal.
  • antagonists of one or more of the functions of receptors of the present invention may be useful in treating disorders associated with high levels of cell proliferation (e.g. in treating cancer).
  • Antagonists may also be useful in treating immune disorders, weight disorders and / or developmental disorders. In particular they may be useful in treating obesity (in view of homology of the polypeptides shown in Figure 1 with the leptin receptor), inflammation, septic shock, AIDS and disorders of embryonic development.
  • Agonists may be useful in treating disorders associated with an underexpression of a cytokine or with the expression of a moiety having a level of cytokine activity lower than normal. They may be useful in treating disorders associated with low levels of cell proliferation. Agonists may also be useful in treating immune disorders, weight disorders and / or developmental disorders. In particular they may be useful in treating obesity (in view of homology of the polypeptides shown in Figure 1 with the leptin receptor), inflammation, septic shock, AIDS and disorders of embryonic development.
  • Antagonists or agonists may therefore be used in the manufacture of a medicament for the treatments mentioned above.
  • the medicament will usually be supplied as part of a pharmaceutical composition, which may include a pharmaceutically acceptable carrier.
  • This pharmaceutical composition will generally be provided in a sterile form in a sealed container. It may be provided in unit dosage form, will generally be provided in a sealed container, and can be provided as part of a kit. Such a kit is within the scope of the present invention. It would normally (although not necessarily) include instructions for use.
  • a plurality of unit dosage forms may be provided.
  • compositions within the scope of the present invention may include one or more of the following: preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colorants, odourants, salts, buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to polypeptides of the present invention. They may be provided in controlled release form, e.g. so as to be effective over a period of at least a week or, more preferably, of at least a month.
  • a pharmaceutical composition within the scope of the present invention may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes.
  • Such a composition may be prepared by any method known in the art of pharmacy, for example by admixing one or more active ingredients with a suitable carrier under sterile conditions.
  • Dosages of an active agent can vary between wide limits, depending upon the nature of the treatment, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. A dosage may be repeated as often as appropriate. If side effects develop, the amount and/or frequency of the dosage can be reduced, in accordance with good clinical practice.
  • polypeptides of the present invention can be used in diagnosis. For example they can be used in binding studies to diagnose the presence or absence of a type 1 cytokine or to diagnose abnormalities in the level of such a cytokine.
  • Polypeptides of the present invention can also be used in screening.
  • soluble or membrane bound receptors / variants thereof may be used to screen for agents capable of binding thereto.
  • agents may be the cytokines which normally bind to the receptors in vivo.
  • they may be agonists or antagonists of such cytokines and may be useful in treating one or more of the disorders discussed in A) above.
  • the present invention therefore includes antibodies that bind to a polypeptide of the present invention.
  • Preferred antibodies bind specifically to polypeptides of the present invention and can therefore be used to purify such polypeptides (e.g. they may be immobilised and used to bind to polypeptides of the present invention.
  • the polypeptides may then be eluted by washing with a suitable eluent under appropriate conditions.
  • Antibodies within the scope of the present invention may be monoclonal or polyclonal.
  • Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when a polypeptide of the present invention or a nucleic acid molecule (e.g. cDNA) capable of being used to provide such a polypeptide is injected into the animal. If necessary an adjuvant may be administered together with the polypeptide of the present invention. The antibodies can then be purified by virtue of their binding to the polypeptide.
  • a suitable animal host e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey
  • an adjuvant may be administered together with the polypeptide of the present invention.
  • the antibodies can then be purified by virtue of their binding to the polypeptide.
  • Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and spleen cells which produce the desired antibody in order to form an immortal cell line. Thus the well-known Kohler & Milstein technique (Nature 256 52-55 (1975)) or variations upon this technique can be used.
  • the present invention includes derivatives thereof which are capable of binding to polypeptides of the present invention.
  • the present invention includes antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12 372-379 (September 1994).
  • Antibody fragments include, for example, Fab, F(ab') 2 and Fv fragments (these are discussed in Roitt et al [supra], for example).
  • Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining V h and V] regions, which contributes to the stability of the molecule.
  • Other synthetic constructs which can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings which mimic the structure of a CDR loop and which include antigen-interactive side chains.
  • Synthetic constructs include chimaeric molecules.
  • humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention.
  • An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions.
  • Synthetic constructs also include molecules comprising an additional moiety that provides the molecule with some desirable property in addition to antigen binding.
  • the moiety may be a label (e.g. a fluorescent or radioactive label).
  • it may be a pharmaceutically active agent.
  • antibodies or derivatives thereof of the present invention have a wide variety of uses in addition to their use in purification of polypeptides discussed above.
  • They can be used in therapy. For example they may be used to block undesirable ligand / receptor or receptor / receptor interactions. They can be used in diagnosis. For example they may be used in RIAs or ELISAs in order to identify the presence or absence of the type 1 chemokine receptors that are within the scope of the present invention.
  • the present invention also includes nucleic acid molecules within its scope.
  • nucleic acid molecules a) code for a polypeptide according to the present invention; or b) are complementary to molecules as defined in a) above; or c) hybridise to molecules as defined in a) or b) above.
  • the polypeptides of the present invention can be coded for by a large variety of nucleic acid molecules, taking into account the well-known degeneracy of the genetic code. All of these coding nucleic acid molecules are within the scope of the present invention. They may be administered to an individual and used to express polypeptides of the present invention. Thus they may be used for the same treatments as the polypeptides of the present invention.
  • the nucleic acid molecules may be used directly, e.g. they may be injected into muscle (optionally after being first incorporated into a carrier, e.g. a lipid-based carrier, such as a liposome). Alternatively they may be inserted into vectors. Vectors for use in treatments include replication-deficient adenoviruses, retroviruses or adeno-associated viruses.
  • Vectors may be used in cloning. They may be introduced into host cells to enable the expression of polypeptides of the present invention using techniques known to the person skilled in the art. Alternatively, cell free expression systems may be used. By using an appropriate expression system the polypeptides can be produced in a desired form.
  • the polypeptides can be produced by micro-organisms such as bacteria or yeast, by cultured insect cells (which may be baculovirus-infected), by mammalian cells (such as CHO cells) or by transgenic animals that, for instance, secrete the proteins in milk. Where glycosylation is desired, eukaryotic (desirably mammalian) expression systems are preferred.
  • Polypeptides comprising N-terminal methionine may be produced using certain expression systems, whilst in others the mature polypeptide will lack this residue.
  • Polypeptides may initially be expressed to include signal sequences. Different signal sequences may be provided for different expression systems. Alternatively, signal sequences may be absent .
  • nucleic acid molecules coding for polypeptides of the present invention also includes nucleic acid molecules complementary thereto.
  • both strands of a double stranded nucleic acid molecule are included within the scope of the present invention (whether or not they are associated with one another).
  • mRNA molecules and complementary DNA molecules e.g. cDNA molecules.
  • Nucleic acid molecules which can hybridise to one or more of the nucleic acid molecules discussed above are also within the scope of the present invention. Such nucleic acid molecules are referred to herein as “hybridising" nucleic acid molecules.
  • a hybridising nucleic acid molecule of the present invention may have a high degree of sequence identity along its length with a nucleic acid molecule within the scope of a) or b) above (e.g. at least 50%, at least 75% or at least 90% sequence identity).
  • nucleic acid molecule As will be appreciated by the skilled person, the greater the degree of sequence identity that a given single stranded nucleic acid molecule has with another nucleic acid molecule, the greater the likelihood that it will hybridise to a nucleic acid molecule which is complementary to that other nucleic acid molecule under appropriate conditions.
  • Desirably hybridising molecules of the present invention are at least 10 nucleotides in length and preferably are at least 25 or at least 50 nucleotides in length.
  • Preferred hybridising molecules hybridise under stringent hybridisation conditions.
  • stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35 °C to about 65 °C using a salt solution which is about 0.9 molar.
  • the skilled person will be able to vary such parameters as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc.
  • hybridising nucleic acid molecules of the present invention hybridise to a cDNA molecule having the sequence shown in Figure 3 or Figure 5; to an RNA equivalent thereof; or to a complementary sequence to any of the aforesaid molecules.
  • Hybridising nucleic acid molecules can be useful as probes or primers, for example.
  • Probes can be used to purify and/or to identify nucleic acids. They may be used in diagnosis. For example probes may be used to determine whether or not an individual has a receptor of the present invention by determining whether or not a complete gene coding for a functional receptor is present.
  • Primers are useful in amplifying nucleic acids or parts thereof, e.g. by PCR techniques.
  • hybridising nucleic acid molecules of the present invention can be used as antisense molecules to alter the expression of polypeptides of the present invention by binding to complementary nucleic acid molecules. (Generally this can be achieved by providing nucleic acid molecules that bind to RNA molecules that would normally be translated, thereby preventing translation due to the formation of duplexes.) This technique can be used in antisense therapy.
  • Antisense molecules may be in the form of DNA or RNA molecules.
  • nucleic acid molecules for use in the present invention include not only those with classical DNA or RNA structures, but also variants with modified (non-phosphodiester) backbones.
  • Two successful attempts to replace the entire backbone have been reported - the morpholino derivatives and the peptide nucleic acids (PNAs), which contain an N-(2-aminoethyl)glycine-based pseudopeptide backbone.
  • PNAs peptide nucleic acids
  • Nucleic acid variants with modified backbones can have increased stability relative to unmodified nucleic acids and are particularly useful where long-term hybridisation is desired (e.g. in antisense therapy).
  • Hybridising molecules may also be provided as ribozymes.
  • Ribozymes can be used to regulate expression by binding to and cleaving RNA molecules that include particular target sequences.
  • nucleic acid molecules of the present invention may therefore have one or more of the following characteristics:
  • RNA including forms with non-naturally occurring bases and/or non-naturally occurring backbones e.g. PNAs).
  • They may be provided in recombinant form i.e. covalently linked to a heterologous 5' and/or a 3 1 flanking sequence to provide a chimaeric molecule (e.g. a vector) which does not occur in nature. 4) They may be provided without 5' and/or 3' flanking sequences that normally occur in nature.
  • They may be provided in substantially pure form, e.g. by using probes to isolate cloned molecules having a desired target sequence or by using chemical synthesis techniques. (Thus they may be provided in a form which is substantially free from contaminating proteins and/or from other nucleic acids.);
  • introns e.g. as a full-length gene
  • introns e.g. as cDNA
  • Figure 1 shows an alignment of human and murine receptor amino acid sequences predicted from cDNA sequence information. Identical amino acid residues are boxed in black.
  • the human and murine polypeptides are referred to as hGBRI-ILR and mGBRI-ILR respectively, since they are believed to be interleukin receptors (or at least substantial parts of such receptors).
  • Figure 2 shows an alignment of the hGBRI-ILR and mGBRI-ILR amino acid sequences shown in Figure 1 with members of the IL-6 type cytokine receptor family within the immunglobulin domain and haematopoietin receptor module.
  • GCSF-R is granulocyte colony stimulating factor receptor
  • CNTF-R is ciliary neurotrophic factor.
  • Figure 3 shows the cDNA sequence obtained for hGBRI-ILR
  • Figure 4 shows the predicted amino acid sequence obtained from the cDNA sequence provided in Figure 3.
  • Figure 5 shows the cDNA sequence obtained for mGBRI-ILR
  • Figure 6 shows the predicted amino acid sequence obtained from the cDNA sequence provided in Figure 5.
  • 5'-RACE was used to clone a further 310bp of the murine cDNA upstream of the 5' end of the cDNA clone using the Marathon cDNA amplification kit from Clonetech (Palo Alto, CA) on poly A+ RNA extracted from mouse lung following manufacturer's guidelines.
  • the primer used in the PCR amplification (along with the AP-1 primer provided) was 5'-CGTACCACCTCAGCTTGTACTTG-3'.
  • PCR products were cloned into the vector pCRII (Invitrogen, Leek, The Netherlands) and colonies screened by colony hybridization using the oligonucleotide probe 5 '-AAGGATCTCACGTGCCGCTGGACACCGGGT-3 ' .
  • a portion of the human GBRI-ILR cDNA was amplified by PCR using cDNA derived from poly A 4- RNA from human lung with the primers 5'-ACCGCCGAGGGCCTCTACTG-3' and
  • Genbank database with expressed sequence tags was searched using TBLASTN with a 20 amino acid sequence surrounding the W-S-x-W-S motif of the mouse IL-13 receptor ⁇ l as query. ESTs showing significant homology were then translated, and the open reading frames used to search the Swissprot database using BLASTP for homologous proteins.
  • the amino acid sequence from the murine EST W66776 showed a high level of homology to members of the IL-6-type cytokine receptor family, as well as to the prolactin receptor.
  • sequence of W66776 to search the Genbank database allowed the identification of overlapping homologous sequences (of both murine and human origin) which in turn were run against the Genbank database to identify more overlapping ESTs (Table I). This allowed the assembly of overlapping nucleic acid sequences encoding the human and mouse putative receptor sequences.
  • a 310bp PCR product was amplified from human lung cDNA using primers designed from the human ESTs. The PCR product was in turn used as a probe to screen a human placental cDNA library, allowing the isolation of a fiill length clone of 1740 bp which included a 3' poly A tail.
  • the human cDNA encoded a precursor protein of 422 amino acids with a putative signal peptide of 37 amino acids. In vitro translation revealed that the AUG codon coding the methionine at the start of the putative signal peptide was indeed used to initiate translation (data not shown).
  • TBLASTN with a 20 amino acid sequence surrounding the W-S-x-W-S motif of the mouse IL-13 receptor ⁇ l as query. ESTs showing significant homology were then translated, and the open reading frames used to search the Swissprot database using BLASTP for homologous proteins.
  • the amino acid sequence from the murine EST W66776 showed a high level of homology to members of the IL-6-type cytokine receptor family, as well as to the prolactin receptor.
  • Using the sequence of W66776 to search the Genbank database allowed the identification of overlapping homologous sequences (of both murine and human origin) which in turn were run against the Genbank database to identify more overlapping ESTs (Table I). This allowed the assembly of overlapping nucleic acid sequences encoding the human and mouse putative receptor sequences.
  • the cDNA clone 479043 which gave rise to the mouse EST found furthest 5' in the sequence assembly was obtained from the IMAGE consortium and sequenced and was found to contain an insert of 1 Kb, including a 3' poly A tail.
  • the rapid amplification of 5' cDNA ends (5 '-RACE) on murine lung cDNA allowed the cloning of a further 308 bp upstream.
  • the murine cDNA encoded a protein of 383 amino acids.
  • the mouse cDNA sequence was incomplete at the 5' end as the first amino acid of the translated sequence aligned to amino acid 39 of the putative human receptor sequence, and no starting methionine or putative signal peptide could be identified.
  • soluble forms of receptors in the type I cytokine receptor family being the product of either membrane shedding or alternative splicing. These soluble forms can exhibit either antagonistic effects in terms of ligand signalling such as those shown by soluble gpl30 and the soluble IL-5 receptor ⁇ chain, or agonistic effects, such as those shown by the IL-6 receptor ⁇ chain, the CNTF receptor and the IL-11 receptor ⁇ chain.
  • Human and murine GBRI-ILR show close homology to members of the IL-6-type cytokine receptor family (Table II) as well as to the prolactin receptor when used as query to search the SwissProt database. Alignment of the human and mouse amino acid sequences to members of the IL-6-type cytokine receptor family showed regions of conserved homology within the two functionally important cytokine receptor-like domain, most notably at the highly conserved four cysteine residues and the W-S-x-W- S motif ( Figure 2). The N-terminal domain of both sequences also appears to represent an Ig-like domain, most closely resembling the C2-set sequence.
  • H 14009 One of the human EST's (H 14009) showing homology to GBRI-ILR was a sequence derived from a genomic clone (D2-17). This clone was generated by exon amplification of chromosomal DNA from human chromosome 19pl2-13.1, allowing us to localise the human GBRI-ILR gene to this region.
  • the gene for the erythropoeitin receptor, which shares significant homology with GBRI-ILR, is the only other member of the receptor family which has been shown to be localized to this arm of chromosome 19.
  • the predominantly expressed form of the human mRNA migrated as a 1.7 Kb transcript, a size close to the one predicted from the clone obtained from the library screening.
  • Another transcript of approximately 4.5Kb was seen in several tissues. This form could encode a membrane-anchored form of the receptor, analogous to the two transcripts detected for the IL-5 receptor ⁇ chain. Expression of the 1.7Kb transcript could be detected in several tissues, but was less ubiquitous than those of gpl30 and IL-6R ⁇ . Strongest expression of the human GBRI-ILR mRNA was detected in the spleen, thymus, lymph node, appendix, bone marrow, thyroid, adrenal cortex, stomach, heart, placenta and skeletal muscle.
  • a 1.7Kb transcript In the adult mouse, expression of a 1.7Kb transcript was seen most strongly in the lung, but the transcript could also be detected at lower levels in skeletal muscle as well as heart and brain. Expression of the 1.7Kb transcript was also detected in the lymph node and thymus of immunized and non-immunized mice as well as in mouse bone marrow. In the embryo, the 1.7Kb transcript could first be detected at day 11 post conception, with expression going through to days 15 and 17 post conception. This pattern of expression would appear to coincide with the emergence of the first detectable progenitors of the immune system, at day 10.5 post conception. Taken together, these data indicate a possible role for GBRI-ILR in the immune system and in embryonic development.
  • Recombinant protein expression using the baculovirus expression system was also found to be less than efficient when using partial cDNA for hGBRI-ILR which was lacking the immunoglobulin domain. Higher levels of protein production were observed when cDNA encoding the complete N-terminal region of the protein was used.
  • THP-1 - HUVEC (IL- -/+ l ⁇ /TNF ⁇ 2hr)
  • Soluble human GBRI-ILR cDNA, truncated at amino acid 378, and coding for the 6 histidine and 179 recognition tags at the 3' end was cloned into pFASTBACl.
  • Recombinant virus was produced using the Life Technologies BAC-TO-BAC kit and used to infect SF9 cells expanded in SF900II medium. Protein secreted into the medium was purified using a NI-NTA resin column (which binds the 6 histidines). Purified protein was detected by western blot analysis using a monoclonal antibody recognising the 179 tag (CLEPYTACD).
  • a Balb/c mouse was immunized on day 0, 7 and 28 subcutaneously in the limbs and behind the neck with 100 ⁇ g of protein per injection. Three days after the final injection, the draining lymph nodes were obtained and the tissue digested using a collagenase and DNAse cocktail according to the procedure reported elsewhere (Kosco-Vilbois M.H., Isolation and Enrichment of Follicular Dendritic Cells from Murine Lymphoid Tissue, Immunology Methods Manual, Vol 3, 1997). The resulting cell suspension was resuspended at 10 6 cells per ml and fused with Sp2 myeloma cells using standard "Kohler and Milstein" protocols. The hybridomas were then selected in HAT medium and 7-10 days after fusion, the supernatants harvested for screening.
  • GBRI-ILR horseradish peroxidase labelled goat anti-mouse IgG (Southern Biotechnology Associates, Inc.) Positive supernatants were retested with plastic immobilized GBRI- ILR.

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Abstract

On a identifié un nouveau polypeptide dont on estime qu'il est un nouveau type de récepteur de cytokine 1; cette identification s'est faite à la fois chez des souris et chez des humains et l'on a obtenu les séquences correspondantes d'ADN complémentaire. Le degré de conservation d'acide aminé est élevé dans ces polypeptides humains et murins, ce qui indique que ce récepteur est important du point de vue fonctionnel. Les polypeptides relevant de cette invention peuvent se révéler efficaces s'agissant de traitement du cancer, de l'obésité ainsi que d'affections immunitaires et de troubles du développement. Ils se révèlent également utiles pour des criblages.
PCT/EP1998/006497 1997-10-16 1998-10-14 Nouveaux recepteurs de cytokine WO1999020755A2 (fr)

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WO1999035264A1 (fr) * 1998-01-09 1999-07-15 Immunex Corporation Adn codant h14, et polypeptides h14
WO1999037769A1 (fr) * 1998-01-22 1999-07-29 Regeneron Pharmaceuticals, Inc. Recepteurs orphelins
WO1999053066A1 (fr) * 1998-04-10 1999-10-21 Genetics Institute, Inc. U4, un membre de la superfamille des recepteurs de l'hematopoietine
WO2000053631A1 (fr) * 1999-03-11 2000-09-14 Schering Corporation Cytokines de mammifere : reactifs et procedes associes
WO2000073451A1 (fr) 1999-06-01 2000-12-07 Schering Corporation Proteines de recepteur mammaliennes, reactifs et procedes y relatifs
US6800460B1 (en) 1999-03-11 2004-10-05 Schering Corporation Mammalian cytokine complexes
WO2018109168A1 (fr) * 2016-12-16 2018-06-21 Singapore Health Services Pte Ltd Récepteur de cytokine leurre
US10106603B2 (en) 2015-12-16 2018-10-23 Singapore Health Services Pte Ltd Treatment of fibrosis
US11040053B2 (en) 2015-03-10 2021-06-22 Chinook Therapeutics, Inc. Compositions and methods for activating “stimulator of interferon gene”13 dependent signalling
US11078268B2 (en) 2016-12-16 2021-08-03 Singapore Health Services Pte Ltd IL-11 antibodies
US11078269B2 (en) 2016-12-16 2021-08-03 Singapore Health Services Pte Ltd IL-11Rα antibodies
US11319368B2 (en) 2019-01-21 2022-05-03 Singapore Health Services Pte Ltd. Treatment of hepatotoxicity with IL-11 antibody

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WO1997012037A1 (fr) * 1995-09-26 1997-04-03 Amrad Operations Pty. Ltd. Nouveau recepteur de l'hematopoietine et sequences genetiques codant ledit recepteur
WO1997015663A1 (fr) * 1995-10-23 1997-05-01 Amrad Operations Pty. Ltd. Nouveau recepteur d'hematopoietine et sequences genetiques le codant
WO1998011225A2 (fr) * 1996-09-11 1998-03-19 Amrad Operations Pty. Ltd. Nouveau recepteur d'hematopoietine et sequences genetiques codant ce dernier
WO1998031811A1 (fr) * 1997-01-16 1998-07-23 Genetics Institute, Inc. Membre de la superfamille des recepteurs d'hematopoietine
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WO1997015663A1 (fr) * 1995-10-23 1997-05-01 Amrad Operations Pty. Ltd. Nouveau recepteur d'hematopoietine et sequences genetiques le codant
WO1998011225A2 (fr) * 1996-09-11 1998-03-19 Amrad Operations Pty. Ltd. Nouveau recepteur d'hematopoietine et sequences genetiques codant ce dernier
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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999035264A1 (fr) * 1998-01-09 1999-07-15 Immunex Corporation Adn codant h14, et polypeptides h14
WO1999037769A1 (fr) * 1998-01-22 1999-07-29 Regeneron Pharmaceuticals, Inc. Recepteurs orphelins
WO1999053066A1 (fr) * 1998-04-10 1999-10-21 Genetics Institute, Inc. U4, un membre de la superfamille des recepteurs de l'hematopoietine
WO2000053631A1 (fr) * 1999-03-11 2000-09-14 Schering Corporation Cytokines de mammifere : reactifs et procedes associes
US6800460B1 (en) 1999-03-11 2004-10-05 Schering Corporation Mammalian cytokine complexes
US7390481B2 (en) 1999-03-11 2008-06-24 Schering Corporation Mammalian cytokine complexes
WO2000073451A1 (fr) 1999-06-01 2000-12-07 Schering Corporation Proteines de recepteur mammaliennes, reactifs et procedes y relatifs
EP1950299A1 (fr) * 1999-06-01 2008-07-30 Schering Corporation Protéines de mammifères réceptrices, réactifs associés et procédés
EP2258849A1 (fr) * 1999-06-01 2010-12-08 Schering Corporation Protéines de récepteurs de mammifères, réactifs et procédés associés
US11040053B2 (en) 2015-03-10 2021-06-22 Chinook Therapeutics, Inc. Compositions and methods for activating “stimulator of interferon gene”13 dependent signalling
US10865240B2 (en) 2015-12-16 2020-12-15 Singapore Health Services Pte Ltd. Treatment of fibrosis with interleukin-11 antibody
US10894825B2 (en) 2015-12-16 2021-01-19 Singapore Health Services Pte Ltd Treatment of fibrosis with interleukin-11 antibody
US10865239B2 (en) 2015-12-16 2020-12-15 Singapore Health Services Pte Ltd. Treatment of fibrosis with interleukin-11 antibody
US10865241B2 (en) 2015-12-16 2020-12-15 Singapore Health Services Pte Ltd. Treatment of fibrosis with interleukin-11 antibody
US10106603B2 (en) 2015-12-16 2018-10-23 Singapore Health Services Pte Ltd Treatment of fibrosis
US10870697B2 (en) 2015-12-16 2020-12-22 Singapore Health Services Pte Ltd. Treatment of fibrosis with interleukin-11 antibody
US10870696B2 (en) 2015-12-16 2020-12-22 Singapore Health Services Pte Ltd. Treatment of fibrosis with interleukin-11 antibody
US10889642B2 (en) 2015-12-16 2021-01-12 Singapore Health Services Pte Ltd Treatment of fibrosis with interleukin-11 receptor alpha antibody
US10894827B2 (en) 2015-12-16 2021-01-19 Singapore Health Services Pte Ltd Treatment of fibrosis with interleukin-11 receptor alpha antibody
US10822405B2 (en) 2015-12-16 2020-11-03 Singapore Health Services Pte Ltd. Treatment of fibrosis with IL-11 receptor alpha antibody
US10894826B2 (en) 2015-12-16 2021-01-19 Singapore Health Services Pte Ltd Treatment of fibrosis with interleukin-11 receptor alpha antibody
US10899832B2 (en) 2015-12-16 2021-01-26 Singapore Health Services Pte Ltd Treatment of fibrosis with interleukin-11 receptor alpha antibody
US10927169B2 (en) 2015-12-16 2021-02-23 Singapore Health Services Pte Ltd Treatment of fibrosis with Interleukin-11 receptor alpha antibody
US11939374B2 (en) 2015-12-16 2024-03-26 Singapore Health Services Pte Ltd. Treatment of fibrosis
US11078268B2 (en) 2016-12-16 2021-08-03 Singapore Health Services Pte Ltd IL-11 antibodies
US11078269B2 (en) 2016-12-16 2021-08-03 Singapore Health Services Pte Ltd IL-11Rα antibodies
US11274143B2 (en) 2016-12-16 2022-03-15 National University Of Singapore Decoy cytokine receptor
WO2018109168A1 (fr) * 2016-12-16 2018-06-21 Singapore Health Services Pte Ltd Récepteur de cytokine leurre
US11319368B2 (en) 2019-01-21 2022-05-03 Singapore Health Services Pte Ltd. Treatment of hepatotoxicity with IL-11 antibody

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AU1334799A (en) 1999-05-10
JP2002508922A (ja) 2002-03-26

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