WO1998034951A1 - Nouvelle famille de cytokines et utilisations correspondantes - Google Patents

Nouvelle famille de cytokines et utilisations correspondantes Download PDF

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Publication number
WO1998034951A1
WO1998034951A1 PCT/AU1998/000078 AU9800078W WO9834951A1 WO 1998034951 A1 WO1998034951 A1 WO 1998034951A1 AU 9800078 W AU9800078 W AU 9800078W WO 9834951 A1 WO9834951 A1 WO 9834951A1
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WIPO (PCT)
Prior art keywords
crp
sequence
seq
polypeptide
cytokine
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PCT/AU1998/000078
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English (en)
Inventor
Douglas J. Hilton
Edouard G. Stanley
Richard P. Harvey
Christine Biben
Louis Fabri
Maria Lah
Andrew D. Nash
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Amrad Operations Pty. Ltd.
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Priority claimed from AUPO5067A external-priority patent/AUPO506797A0/en
Priority claimed from AUPO6420A external-priority patent/AUPO642097A0/en
Priority claimed from AUPO8963A external-priority patent/AUPO896397A0/en
Priority claimed from AUPP0961A external-priority patent/AUPP096197A0/en
Application filed by Amrad Operations Pty. Ltd. filed Critical Amrad Operations Pty. Ltd.
Priority to AU58486/98A priority Critical patent/AU741708B2/en
Publication of WO1998034951A1 publication Critical patent/WO1998034951A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • 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
    • C07K14/52—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

Definitions

  • the present invention relates generally to a new family of cytokine molecules. More particularly, the present invention provides mammalian cytokines which constitute a novel family of cytokines and which are useful in a range of therapeutic and diagnostic applications.
  • Cytokines represent an important class of proteinaceous molecules involved in regulation of a vast array of functions in animals including survival, growth, differentiation and effector function of tissue cells. Cytokines generally fall into particular classes encompassing interleukins, colony-stimulating factors, lymphokines, monokines and interferons amongst many others. The identification of new families of cytokines is an important step in facilitating the use of cytokines in therapy and diagnosis.
  • cytokines comprising mammalian homologues of a protein designated "cerberus" from Xenopus laevis embryos (1).
  • the cerberus molecule is a 270 amino acid protein with a role in inducing head structures in the Xenopus embryo (1).
  • One aspect of the present invention provides an isolated polypeptide of mammalian origin comprising a signal sequence and a domain conforming to a cystine knot and optionally a long N-terminal domain between said signal sequence and cystine knot domain or a derivative of said polypeptide.
  • Another aspect of the present invention is directed to an isolated polypeptide of mammalian origin derivative thereof comprising a signal sequence and a domain conforming to the criteria for a cystine knot and optionally a long N-terminal domain between said signal sequence and said cystine knot domain, said polypeptide comprising the amino acid sequence:
  • ⁇ AA is an amino acid sequence comprising from about 0 to about 50 amino acid residues
  • X 1 is V or I; and is O or 1.
  • cystine knot domain comprising the sequence:
  • x is any amino acid residue and n, is from about 6 to about 10 or comprises a sequence in the cystine knot domain having at least 50% identity to SEQ ID NO: 1 excluding the cystine and x residues.
  • the polypeptide further comprises a long N-terminal domain between said signal sequence and said cystine knot.
  • cystine knot domain comprising the sequence:
  • the polypeptide further comprises a long N-terminal domain between said signal sequence and said cystine knot.
  • X is any amino acid residue; a is from about 2 to about 4; and n2 is from about 6 to about 100.
  • Still yet another aspect of the present invention contemplates an isolated secretable polypeptide or derivative thereof comprising an amino acid sequence having at least 20% homology to the cerberus protein from Xenopus laevis defined in Figure 1.
  • Another aspect of the present invention is directed to an isolated secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO: 4 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined in SEQ ID NO:4 is from a mouse and is referred to herein as "mCRP-1".
  • an isolated secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO: 5 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined in SEQ ID NO:5 is from a rat and is referred to herein as "rCRP-2".
  • Another aspect of the present invention relates to an isolated, secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:6 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined in SEQ LD NO:6 is from a mouse and is referred to herein as "mCRP-2".
  • Yet another aspect of the present invention is directed to a secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:7 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined in SEQ ID NO:7 is from a human and is referred to herein as "hCRP-2".
  • Still yet another aspect of the present invention provides a secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO: 19 and/or 20 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined by SEQ ID NO: 19 and/or 20 is from a human and is referred to herein as "hCRP-1".
  • Even yet another aspect of the present invention relates to a secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:22 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined by SEQ ID NO:22 is hCRP-1 but without the intron and corresponding amino acid translation.
  • nucleic acid molecule comprising a sequence of nucleotides or a complementary sequence of nucleotides which encodes an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ LD NO:7, SEQ ID NO: 19, SEQ ID NO:20 and SEQ ID NO:22 or a sequence having at least 50% similarity thereto.
  • nucleic acid molecule comprising a nucleotide sequence substantially as set forth in SEQ ID NO:3, SEQ ID NO: 18 or SEQ ID NO:27 or a sequence having at least 50% similarity thereto or a sequence capable of hybridizing to one of the above under low stringency conditions at 42°C.
  • Still yet another aspect of the present invention contemplates a method for modulating expression of a CRP cytokine in a mammal such as a human, primate or laboratory test animal, said method comprising contacting a gene encoding said CRP cytokine with an effective amount of a modulator of CRP cytokine expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of the CRP cytokine.
  • Even yet another aspect of the present invention is directed to a method for modulating activity of the CRP cytokine in a mammalian such as a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditio sufficient to increase or decrease CRP cytokine activity.
  • the molecule may be a proteinaceo molecule or a chemical entity and may also be a derivative of a CRP cytokine or its ligand (e a receptor) or a chemical analogue or truncation mutant of a CRP cytokine or its ligand.
  • Still even yet another aspect of the present invention relates to a pharmaceutical compositio comprising one or more CRP cytokines or derivatives thereof or a modulator of CRP cytoki expression or CRP cytokine activity and one or more pharmaceutically acceptable carrie and/or diluents. These components are referred to as the active ingredients.
  • a method for detecting CR in a biological sample from a subject comprising contacting said biological sampl with an antibody specific for a CRP(or group of CRP s) or its derivatives or homologues f a time and under conditions sufficient for an antibody-CRP complex to form, and then detectin said complex.
  • Another aspect of the present invention provides a use of a CRP cytokine or its function derivatives in the manufacture of a medicament for the treatment of defective or deficient CR mediated activities.
  • Figure 1 is a representation of amino acid alignments using Clustal method with PAM25 residue weight table, (a) Alignment of EST AA120122, mCER-1 (mCRP-1) and cerber ( Figure 1); (b) Alignment of mCER-1 (mCRP-1), cerberus and murine DAN (mCRP-2); ( Alignment between (NDP) Norrie disease protein and murine DAN (mCRP-2); (d) Alignme between mCER-1 (mCRP-1) and murine DAN (mCRP-2); (e) Alignment between huma mouse and rat DAN (hCRP-2, mCRP-2 and rCRP-2, respectively); (f) Alignment betwe cerberus and mCER-1 (mCRP-1).
  • Figure 2 is the nucleotide and amino acid sequence of mCRP-1.
  • Figure 3 is the deglycosylation and Western analysis of secreted mouse mCRP-2 protei Supernatant from COS cells transfected with a murine mCRP-2-FLAG expression vector w chromatographed over an anti-FLAG affinity column according to manufacturer's instructio (Eastman Kodak Company, New Haven CT). Monomeric CRP protein was isolated by g filtration and subject to N-linked deglycosylation using N-Glycosidase-F (Boehring Mannheim, Mannheim, Germany). Purified mCRP-2 protein prior to and after deglycosylati was subject to SDS-PAGE gel and visualised by silver staining and Western blot using an ant FLAG antibody.
  • Figure 4 is a graphical representation showing transient DAN expression in cos cells.
  • Figure 5 shows the steps in the purification of mCRP-2 (C-FLAG) using affinit chromatography.
  • Figure 6 is a photographic representation showing SDS-PAGE analysis of mCRP-2 (C-FLAG
  • Figure 7 is a graphical representation showing size exclusion analysis of mCRP-2 (C-FLAG
  • Figure 8 is a photographic representation of Western Blot analysis of mCRP-1 post M2 affini purification.
  • Figure 9 is a representation of SDS PAGE, Western blot and HPLC analysis of purified prote of mCRP-1.
  • A SDS PAGE of mCRP-1 following removal of carbohydrate;
  • B size exclusi analysis of mCRP-1;
  • C regressional analysis of peak fractions containing mCRP-1 compar to standards;
  • D N-terminal amino acid sequence analysis of RP-HPLC purified mCRP-1 [SEQ ID NO:23].
  • Figure 10 is a representation showing an SDS PAGE, Western blot and HPLC analysis of mCRP-2.
  • A SDS-PAGE-Western blot with and without carbohydrate molecules;
  • B SDS PAGE- Silver stain either with or without reduction;
  • D and
  • C size exclusion analysis followed by linear regressional analysis of peak fractions containing mCRP-2;
  • E N-terminal amino acid sequencing of mCRP-2 [SEQ ID NO:24].
  • Figure 11 is a representation showing the complete genomic sequence of hCRP-1 and corresponding amino acid sequences to exons 1 and 2.
  • Figure 12 is a representation showing the coding sequence of hCRP-1 without the intron and corresponding amino acid translation.
  • Figure 13 is a representation showing alignment of the predicted amino acid sequence of hCRP-1, mCRP-1 an ⁇ Xenopus cerberus. The alignment was performed using DNA megalign under default conditions. hCRP-1 shares approximately 68% identity with mCRP-1 and approximately 25% identity with Xenopus cerberus.
  • Figure 14 is a representation showing a comparison of mCRP-1 and cerberus activities in Xenopus animal cap assays.
  • A Formation of cement glands in individualised animal caps (stage 35) after mock injection (panel A), or injection of mRNAs encoding mCRP-1 (panel B), CFLAG-mCRP-1 (panel C) or cerberus (panel D).
  • a single darkly pigmented cement gland is induced by injected cerberus mRNAs in some cases (see Figure 3B), but not by mock injection. Note in panel B the secretion of sticky exudate.
  • B Formation of cement glands in individualised animal caps (stage 35) after mock injection (panel A), or injection of mRNAs encoding mCRP-1 (panel B), CFLAG-mCRP-1 (panel C) or cerberus (panel D).
  • a single darkly pigmented cement gland is induced by injected cerberus mRNAs in some cases (see Figure 3B), but not by mock
  • Nkx2.3 and Nkx2.5 are expressed in cardiac progenitors and anterior pharyngeal endoderm.
  • T4 globulin is specific to blood, a ventral mesodermal derivative .
  • XeHAND marks cardiac and vascular smooth muscle progenitors in lateral mesoderm.
  • NCAM is a pan-neural marker.
  • Otx2 is a marker of anterior tissues, expressed in midbrain, forebrain, placodes, cement gland and anterior mesoderm.
  • Krox20 is expressed in rhombomeres 3 and 5 in the hindbrain.
  • CGI 3 is expressed specific to cement gland. Edd expression is ubiquitous at low levels but enriched in endoderm. Equal mRN A was assessed by expression of EF- 1 alpha (EFla).
  • Figure 15 is a photographic representation showing expression of mCRP-1 during gastrulation.
  • A-C, H and I show lateral views of embryos where anterior is to the left, posterior to the right; the arrowhead represents the embryonic/extra-embryonic junction.
  • A. Three early primitive streak stage embryos showing mCRP-1 expression in a midline stripe on the anterior side (left embryo), then progressively (middle and right embryos) in migrating mesendodermal wings arising in the anterior region of the streak.
  • B Early streak embryo hybridised with probes for both mCRP-1 and brachyury. Nascent mesodermal wings positive for brachyury are seen on the posterior side, while the mCRP-1 strip is seen anteriorly.
  • C Late primitive stage embryos hybridised with an mCRP-1 probe. Expression is confined to anterior mesendoderm.
  • D Distal view of the same embryo as in C, showing lack of mCRP-1 expression in the region of the node.
  • E Anterior view of the same embryo as in C, showing mCRP-1 expression set back from the embryonic/extra-embryonic junction and absent from the cardiac progenitor region (brackets).
  • F Early neurula embryo (anterior view) hybridised with a BMP2A probe. Expression occurs in the domain of the cardiac progenitors (brackets), but also somewhat into the extra-embryonic domain.
  • G Late primitive stage embryos hybridised with an mCRP-1 probe. Expression is confined to anterior mesendoderm.
  • D Distal view of the same embryo as in C, showing lack of mCRP-1 expression in the region of the node.
  • E Anterior view of the same embryo as in C, showing mCRP-1 expression set back
  • Neurula embryo hybridised with Nkx2-5, specific to the cardiac progenitors at that stage (brackets).
  • H Sagittal section through a late streak embryo highlighting expression of mCRP-1 in anterior mesendoderm. The arrowhead indicates the position of the node, where expression is absent.
  • I Enlargement of a section near adjacent to the one shown in H, depicting mCRP-1 expression in both endoderm and associated mesoderm. Note that anterior mesendoderm lacks expression.
  • J Headfold stage embryo (anterior view) showing mCRP-1 expression in a wedge of anterior mesendoderm.
  • K Headfold stage embryo
  • Figure 16 is a photographic representation showing expression in anterior axial mesoderm. Transverse histological sections of an -E7.75 embryo hybridised in wholemount with an mCRP-1 probe at the level of A. prechordal mesoderm; B. notochordal precursos; C. node. Expression is seen in prechordal and notochordal plates, but is absent from the note. Arrows delimit the apparent extent of these structures. The node is clearly recognised by the recessed nature of its innermost cells (the "pit"). Notochordal and prechordal plate cells have a morphology distinct from surrounding endoderm, consistent with their having a smaller surface area ventrally when viewed by scanning electron microscopy. Prechordal plate is wider than notochordal precursors, forming a wedge shape. Note mCRP-1 expression is head mesoderm.
  • Figure 17 is a photographic representation showing expression of mCRP-1 in paraxial mesoderm.
  • A Early headfold stage embryo showing the mCRP-1 anterior expression domain (left; see Figure 15), now faded almost completely, and the first appearance of two strips within paraxial mesoderm.
  • B Ventroanterior view of a late headfold stage embryo showing absence of mCRP-1 expression in anterior mesendoderm, but stronger expression in paraxial mesoderm.
  • C Dorsal view of the tail region of a E9.5 embryo (anterior to the left) showing mCRP-1 expression in four stripes in paraxial mesoderm.
  • D Transverse section through an E8.5 embryo showing that most or all cells with a paraxial strip express mCRP-1.
  • E E.
  • the figures shows three Otx-2 '1' embryos harvested at E6.7 (embryo on the left) and E7.5.
  • E6.7 embryo on the left
  • E7.5 embryos on the left
  • mCER- 1 expression arrows
  • A anterior
  • em embryonic region
  • ex extra-embryonic region
  • P posterior.
  • Figure 19 is a representation showing that mCRP- 1 maps to the central region of mouse chromosome 4 as determined by interspecific backcross analysis.
  • A Segregation patterns of mCRP-1 and linked genes in the 92 backcross animals that were typed for all loci. Each column represents the chromosome identified in backcross progeny that was inherited from the (C57BL/6JxM. spretus) FI parent. The shaded boxes represent the presence of the C57BL/6J allele and the white boxes represent the presence of the M. spretus allele. The number of offspring inheriting each type of chromosome is Usted at the bottom of each column.
  • B Partial chromosome 4 linkage map showing the location of mCRP-1 in relation to linked genes.
  • Recombination distances between loci in cM are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown on the right.
  • References for the human map positions of loci cited in this study can be obtained from GDB (Genome Data Base), a computerised database of human linkage information maintained by the William H. Welch Medical Library of The John Hopkins University (Baltimore, MD).
  • Restriction endonuclease digestion was with EcoRI or Zt ⁇ mHL A schematic representation of the mCRP-1 locus is indicated below the panel. The map was determined by restriction enzyme and sequence analysis of clones isolated from a genomic library of the 129 strain. Restriction enzyme sites relevant to this study are indicated. Coding exons of the mCRP-1 gene are boxed. The star indicates the EcoRI site absent from the mCRP-1 allele of pt.
  • Figure 20 is a representation showing: A. Western blot analysis of CFLAG-mCRP- 1 protein purified from CHO cells (see Materials and Methods) before (-) and after (+) treatment with N-glycosidase F (n-gly). B. Western blot analysis of CFLAG-mCRP-1 protein secreted from 293T cells with (+) and without (-) reduction with lOOmM dithiothreitol (DTT). mCRP-1 protein was detected with M2 anti-FLAG antibody. The mobility of molecular weight standards (size shown in kilodaltons) is indicated on the left of each panel.
  • Figure 21 is a photograph representation of a Western blot analysis of 293T hCRP-1-I-SPY transient expression using I-SPY antibody (Dll).
  • One aspect of the present invention provides an isolated polypeptide of mammalian origin comprising a signal sequence and a domain conforming to the criteria for a cystine knot and optionally a long N-terminal domain between said signal sequence and cystine knot domain or a derivative of said polypeptide.
  • the polypeptide of the present invention is, in its naturally occurring form, secretable and glycosylated.
  • the present invention extends, however, to recombinant, synthetic or other modified forms which have an altered glycosylation pattern and/or which have an altered capacity to be secreted from a cell.
  • the signal sequence may be removed or otherwise inactivated or a signal sequence from another molecule may be fused to the subject polypeptide.
  • signal sequence is used in its broadest sense and includes a hydrophobic leader sequence.
  • the term "cystine knot” is conveniently as described by McDonald and Hendrickson (7) and Isaacs (8).
  • N-terminal domain when present in the polypeptide of the present invention is from about 100 to about 300 amino acids in length and more preferably from about 100 to about 200 amino acids in length.
  • the subject polypeptide comprises the long N-terminal domain flanked by the signal sequence and cystine knot domain.
  • Another aspect of the present invention provides an isolated polypeptide of mammalian origin derivative thereof comprising a signal sequence and a domain conforming to the criteria for a cystine knot and optionally a long N-terminal domain between said signal sequence and said cystine knot domain, said polypeptide comprising the amino acid sequence:
  • ⁇ AA ⁇ is an amino acid sequence comprising from about 0 to about 50 amino acid residues
  • X 1 is V or I; and n is 0 or 1.
  • cystine knot domain comprising the sequence:
  • x is any amino acid residue and n, is from about 6 to about 10 or comprises a sequence in the cystine knot domain having at least 50% identity to SEQ ID NO: 1 excluding the cystine and x residues.
  • the isolated polypeptide comprises an N-terminal domain of from about 100 to about 200 amino acids between the signal sequence and the cystine knot domain.
  • the percentage identity of related molecules to SEQ ID NO:l is at least about 55%, more preferably at least about 60%, still more preferably at least about 65%, even more preferably at least about 70% or greater such as at least about 71-75%, 76-80%, 81-85%, 86- 90% or 91-100%.
  • cystine knot domain comprising the sequence:
  • the isolated polypeptide comprises a N-terminal domain of from about 100 to about 200 amino acids between the signal sequence and the cystine knot.
  • the percentage identity of related molecules to SEQ LD NO: 2 is at least about 55%, more preferably at least about 60%, still more preferably at least about 65%, even more preferably at least about 70% or greater such as at least about 71-75%, 76-80%, 81-85%, 86- 90% or 91-100%.
  • X is any amino acid residue; a is from about 2 to about 4; and n2 is from about 6 to about 100.
  • Particularly preferred embodiments of these aspects of the present invention comprise a long N-terminal domain flanked by a signal sequence and a sequence conforming to the criteria for a cystine knot.
  • sequences of part of the cystine knot domain is:
  • X is any amino acid residue
  • X' is preferably K; and n3 is from about 6 to about 13.
  • Another aspect of the present invention provides an isolated secretable polypeptide or derivative thereof comprising an amino acid sequence having at least 20% similarity to the cerberus protein from Xenopus laevis defined in Figure 1.
  • the secretable polypeptide as hereinbefore defined above constitutes a novel family of cytokines.
  • the present invention extends, therefore, to all members as defined to be part of this family and to derivatives thereof.
  • CRP cerberus related protein
  • Individual members are defined by a lower case letter prefix to CRP to indicate the mammaUan origin. Where more than one CRP cytokine has been identified from a particular species, the abbreviation is followed by a number.
  • rCRP refers to a CRP cytokine from a rat
  • hCRP refers to a human CRP
  • mCRP-1 and "mCRP-2" are murine CRPs.
  • the present invention further provides an isolated secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined in SEQ ID NO:4 is from a mouse and is referred to herein as "mCRP-1".
  • the present invention provides an isolated secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO: 5 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined in SEQ ID NO:5 is from a rat and is referred to herein as "rCRP-2".
  • a further embodiment of the subject invention provides an isolated, secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:6 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined in SEQ ID NO:6 is from a mouse and is referred to herein as "mCRP-2".
  • Still a further embodiment provides a secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:7 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined in SEQ ID NO:7 is from a human and is referred to herein as "hCRP-2".
  • Even yet another embodiment of the present invention is directed to a secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO: 19 and/or 20 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined by SEQ ID NO: 19 and/or 20 is from a human and is referred to herein as "hCRP-1".
  • Another embodiment of the present invention provides a secretable polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:22 or having at least 50% similarity thereto and which polypeptide has the identifying characteristics of a CRP cytokine.
  • the molecule defined by SEQ ID NO: 22 is hCRP-1 but without the intron and corresponding amino acid translation.
  • the percentage similarity is at least about 60%, more preferably at least about 70%, still more preferably at least about 80%, even more preferably at least about 90% and yet even more preferably at least about 95% similar to at least about 5 contiguous amino acids, and more preferably at least about 10 contiguous amino acids of SEQ ID NO:4 or 5 or 6 or 7 or 19 and/or 20 or 22.
  • the present invention further extends to nucleic acid molecules, preferably in isolated form, encoding members of the CRP cytokine family.
  • the nucleic acid molecule comprises a sequence of nucleotides or a complementary sequence of nucleotides which encodes the amino acid sequence set forth in SEQ ID NO:4 or a sequence having at least 50% similarity thereto.
  • the nucleic acid molecule comprises a sequence of nucleotides or a complementary sequence of nucleotides which encodes the amino acid sequence set forth in SEQ ID NO:5 or a sequence having at least 50% similarity thereto.
  • the nucleic acid molecule comprises a sequence of nucleotides or a complementary sequence of nucleotides which encodes the amino acid sequence set forth in SEQ ID NO:6 or a sequence having at least 50% similarity thereto.
  • the nucleic acid molecule comprises a sequence of nucleotides or a complementary sequence of nucleotides which encodes the amino acid sequence set forth in SEQ ID NO:7 or a sequence having at least 50% similarity thereto.
  • nucleic acid molecule comprises a sequence of nucleotides or a complementary sequence of nucleotides which encodes the amino acid sequence set forth in SEQ ID NO: 19 and/or SEQ ID NO:20 or a sequence having at least 50% similarity to either or both thereof.
  • nucleic acid molecule comprises a sequence of nucleotides or a complementary sequence of nucleotides which encodes the amino acid sequence set forth in SEQ ID NO: 22 or a sequence having at least 50% similarity thereto.
  • the nucleotide sequence is as set forth in SEQ ID NO: 3 or a sequence having at least 50% similarity thereto and which is capable of hybridizing under low stringency conditions at 42°C to SEQ ID NO:3.
  • the nucleotide sequence set forth in SEQ ID NO: 3 encodes mCRP-1.
  • the nucleotide sequence is as set forth in SEQ ID NO: 18 or is a sequence having at least 50% similarity thereto and which is capable of hybridizing under low stringency conditions at 42°C to SEQ ID NO: 18.
  • the nucleotide sequence set forth in SEQ ID NO: 18 is a genomic sequence encoding hCRP-1.
  • the nucleotide sequence is as set forth in SEQ ID NO:21 or is a sequence having at least 50% similarity thereto and which is capable of hybridizing under low stringency conditions at 42°C to SEQ ID NO:21.
  • the nucleotide sequence set forth in SEQ ID NO: 18 encodes hCRPl but without the intron.
  • Reference herein to a low stringency at 42 °C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
  • the percentage nucleotide similarity to SEQ ID NO:3 is at least about 60%, more preferably at least about 70%, still more preferably at least about 80%, even more preferably at least about 80% and yet even more preferably at least about 95% or above similarity to at least about 10 and more preferably at least about 20 contiguous nucleotides in SEQ ID NO:3.
  • the percentage nucleotide similarity to SEQ ID NO: 18 or SEQ ID NO:21 is at least about 60%, more preferably at least about 70%, still more preferably at least about 80%, even more preferably at least about 80% and yet even more preferably at least about 95% or above similarity to at least about 10 and more preferably at least about 20 contiguous nucleotides in SEQ ID NO:18 or SEQ ID NO:21.
  • similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels.
  • homoology and “identity” as used herein can be considered equivalent to the term “simUarity”.
  • the CRP cytokines of the present invention are preferably but not exclusively of human, primate, laboratory test animal (eg. rabbit, guinea pig, mouse, rat), companion animal (eg. dog, cat), livestock animal (eg. sheep, cow, horse, donkey, pig) or captive wild animal (eg. deer, fox, kangaroo) origin.
  • laboratory test animal eg. rabbit, guinea pig, mouse, rat
  • companion animal eg. dog, cat
  • livestock animal eg. sheep, cow, horse, donkey, pig
  • captive wild animal eg. deer, fox, kangaroo
  • the present invention encompasses a range of derivatives of the CRP cytokines.
  • Derivatives include fragments, parts, portions, mutants, homologues and analogues of the CRP polypeptide and corresponding genetic sequence.
  • Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to the CRP cytokine or single or multiple nucleotide substitutions, deletions and/or additions to the genetic sequence encoding the CRP cytokine.
  • "Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences.
  • Reference herein to the "CRP" cytokine includes reference to all derivatives thereof including functional and nonfunctional derivatives.
  • Analogues of the CRP cytokines contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde foUowed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5- phosphate followed by reduction with NaBH
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde foUowed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acy
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D- isomers of amino acids.
  • a Ust of unnatural amino acid, contemplated herein is shown in Table 1.
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention further contemplates chemical analogues of the CRP cytokines capable of acting as antagonists or agonists of the CRP cytokine or which can act as functional analogues of CRP cytokines.
  • Chemical analogues may not necessarily be derived from the CRP cytokine but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of the CRP cytokine. Chemical analogues may be chemicaUy synthesised or may be detected following, for example, natural product screening.
  • glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • the identification of a new family of CRP cytokines allows definition of a consensus protein motif which may be used to identify further family members by, for example, PCR analysis of cDNA and/or genomic libraries.
  • the identification of the CRP cytokines of the present invention also permits the generation of a range of therapeutic molecules capable of modulating expression of the CRP cytokine or modulating the activity of the cytokine.
  • Modulators contemplated by the present invention includes agonists and antagonists of CRP cytokine expression.
  • Antagonists of CRP cytokine expression include antisense molecules, ribozymes and co-suppression molecules.
  • Agonists include molecules which increase promoter activity or interfere with negative regulatory mechanisms.
  • Antagonists of the cytokine include antibodies, inhibitor peptide fragments and soluble receptors.
  • Another embodiment of the present invention contemplates a method for modulating expression of a CRP cytokine in a mammal such as a human, primate or laboratory test animal, said method comprising contacting a gene encoding said CRP cytokine with an effective amount of a modulator of CRP cytokine expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of the CRP cytokine.
  • a nucleic acid molecule encoding the CRP cytokine or a derivative thereof may be introduced into a cell to enhance the ability of that cell to produce CRP or, through co-suppression reduce CRP gene expression.
  • CRP cytokine antisense sequences such as oligonucleotides may be introduced to decrease CRP expression in any cell expressing the endogenous CRP cytokine gene.
  • Another aspect of the present invention contemplates a method of modulating activity of the CRP cytokine in a mammalian such as a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease CRP cytokine activity.
  • the molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of a CRP cytokine or its ligand (eg. a receptor) or a chemical analogue or truncation mutant of a CRP cytokine or its ligand.
  • Modulating CRPs may be important in a number of respects, such as but not limited to modulating early embryo development.
  • CRPs may have a role in somite patterning and in muscles of the body and limbs.
  • the function of the CRPs may be in the muscles themselves (autocrine role), or may be to act on neighbouring tissue of the somite, the dermatome (forming skin) and schlerotome (forming bone).
  • CRP may be involved in craniofacial development and useful clinically as an inductive, maintenance, survival, proliferative, anti-proliferative or differentiation factor in pathologies related to muscle, bone and skin.
  • tumour suppressor suggesting a function as an antiproliferative factor in a broad range of cancers, including breast cancer, lymphoma and leukaemia, melanoma, colorectal cancer, pancreatic cancer, lung cancer, stomach cancer and neuroblastoma.
  • the CRPs are also good candidates for a inductive factor that either induce or give anterior character to, early neural tissue. Such as they may be clinically useful as an inductive, maintenance, survival, proliferative, anti-proliferative or differentiation factor in any degenerative neuropathy or in any grafting procedure involving foetal or neural tissue grafted to correct familial or acquired deficiencies, or to repair neural tissue after trauma.
  • Anterior lateral endoderm is also known to be the source of inducers and suppressors of cardiogenesis and the CRPs may comprise these factors.
  • the present invention contemplates a pharmaceutical composition comprising one or more CRP cytokines or derivatives thereof or a modulator of CRP cytokine expression or CRP cytokine activity and one or more pharmaceuticaUy acceptable carriers and/or diluents. These components are referred to as the active ingredients.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as licithin.
  • microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active ingredients When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.01 ⁇ g and about 2000 mg of active compound.
  • Alternative amounts include between about 1.0 ⁇ g and about 1500 ng, between about l ⁇ g and about 1000 mg and between about 10 ⁇ g and about 500 mg.
  • the tablets, troches, pills, capsules and the like may also contain the components as Usted hereafter: A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oU of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavouring agent such as peppermint, oU of
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • the present invention also extends to forms suitable for topical application such as creams, lotions and gels.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.01 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. Alternatively, amounts administered may be represented in terms of amounts/kg body weight.
  • amounts range from about 0.001 ⁇ g to about 1000 mg/kg body weight may be administered 500 mg/kg body weight or about 10.01 ⁇ g to about or above 0.1 ⁇ g to about 250 mg/kg body weight are contemplated by the present invention.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating CRP expression or CRP activity.
  • the vector may, for example, be a viral vector.
  • Still another aspect of the present invention is directed to antibodies to CRP cytokines and their derivatives. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to CRP cytokines or may be specifically raised to CRP cytokines or derivatives thereof. In the case of the latter, a CRP cytokine or its derivative may first need to be associated with a carrier molecule.
  • the antibodies and/or recombinant CRP cytokine or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.
  • a CRP cytokine and its derivatives can be used to screen for naturally occurring antibodies to CRP cytokines. These may occur, for example in some autoimmune diseases.
  • specific antibodies can be used to screen for CRP cytokines.
  • Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of CRP cytokines levels may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols.
  • Antibodies to CRP cytokines of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody” is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis, cancer, tissue regeneration or development, muscle development or health of neural tissue or for monitoring the program of a therapeutic mitin.
  • CRP proteins can be used to screen for CRP proteins.
  • the latter would be important, for example, as a means for screening for levels of a CRP in a cell extract or other biological fluid or purifying a CRP made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of CRP.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of a CRP, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
  • Another aspect of the present invention contemplates a method for detecting CRP in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for a CRP(or group of CRP s) or its derivatives or homologues for a time and under conditions sufficient for an antibody-CRP complex to form, and then detecting said complex.
  • CRP Creactive protein kinase kinase kinase kinase kinase assays.
  • a wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody.
  • the sample is one which might contain CRP including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
  • a first antibody having specificity for the CRP or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • an aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2- 40 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of any subunit present in the antibody.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target- first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme immunoassay an enzyme is conjugated to the second antibody, generaUy by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.
  • suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeUed antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away.
  • a solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody.
  • the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention also contemplates genetic assays such as involving PCR analysis to detect a CRP gene or its derivatives.
  • Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphoms analysis (SSCP) as specific oligonucleotide hybridisation, as methods such as direct protein truncation tests.
  • the nucleic acid molecules of the present invention may be RNA or DNA. When the nucleic acid molecule is in DNA form, it may be genomic DNA or cDNA. RNA forms of the nucleic acid molecules of the present invention are generally mRNA.
  • nucleic acid molecules of the present invention are generally in isolated form, they may be integrated into or ligated to or otherwise fused or associated with other genetic molecules such as vector molecules and in particular expression vector molecules.
  • Vectors and expression vectors are generally capable of replication and, if applicable, expression in one or both of a prokaryotic cell or a eukaryotic cell.
  • prokaryotic cells include E. coli, Bacillus sp and Pseudomonas sp.
  • Preferred eukaryotic cells include yeast, fungal, mammalian and insect cells.
  • another aspect of the present invention contemplates a genetic construct comprising a vector portion and a mammalian such as a human CRP gene portion, which CRP gene portion is capable of encoding a CRP polypeptide or a functional or immunologically interactive derivative thereof.
  • the CRP gene portion of the genetic construct is operably linked to a promoter on the vector such that said promoter is capable of directing expression of said CRP gene portion in an appropriate cell.
  • the CRP gene portion of the genetic construct may comprise all or part of the gene fused to another genetic sequence such as a nucleotide sequence encoding glutathione-S- transferase or part thereof. It is also within the scope of the present invention to include fusions between CRP cytokines. Such fusion molecules may have increased pleiotropy and/or provides a means of, for example, "humanising" a non-human CRP.
  • the present invention extends to such genetic constructs and to prokaryotic or eukaryotic cells comprising same.
  • the present invention also extends to any or all derivatives of CRP cytokines including mutants, part, fragments, portions, homologues and analogues or their encoding genetic sequence including single or multiple nucleotide or amino acid substitutions, additions and/or deletions to the naturally occurring nucleotide or amino acid sequence.
  • the CRP cytokines and their genetic sequences of the present invention will be useful in the generation of a range of therapeutic and diagnostic reagents and will be especially useful in the detection of ligands (eg. receptors) capable of interacting with CRP cytokines.
  • ligands eg. receptors
  • a recombinant CRP may be bound or fused to a reporter molecule capable of producing an identifiable signal, contacted with a cell or group of cells putatively carrying a CRP ligand (eg. a receptor) and screening for binding of the labelled CRP to a ligand.
  • labelled CRP may be used to screen expression libraries of putative ligands.
  • CRP cytokines are important for the regulation, maintenance and survival of a diverse array of cell types such as but not limited to muscle tissue, bone, skin and/or neural tissue. Accordingly, it is proposed that CRP cytokines or their functional derivatives be used to regulate development, maintenance or regeneration in an array of different cells and tissues in vitro and in vivo. For example, CRPs are contemplated to be useful in modulating neuronal proliferation, differentation and survival.
  • Soluble CRP polypeptides are also contemplated to be useful in the treatment of disease, injury or abnormality in the nervous system, e.g. in relation to central or peripheral nervous system to treat Cerebral Palsy, trauma induced paralysis, vascular ischaemia associated with stroke, neuronal tumours, motoneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, Multiple Sclerosis, peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and infectious diseases such as herpes, rubella, measles, chicken pox, HIV or HTLV-1.
  • a membrane bound CRP may be used in vitro on nerve cells or tissues to modulate proUferation, differentiation or survival, for example, in grafting procedures or transplantation.
  • the CRP cytokines of the present invention or their functional derivatives may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers and/or diluents.
  • the present invention contemplates a method of treatment comprising the administration of an effective amount of a CRP of the present invention.
  • the present invention also extends to antagonists and agonists of CRP molecules and their use in therapeutic compositions and methodologies.
  • Figure 1 is an alignment of amino acid sequences comparing Xenopus laevis cerberus and members of the CRP family from mouse, rat and human sources. CRP proteins share approximately 30% identity and 40% similarity over an 88 amino acid region. mCRP-1 and mCer- 1 are used interchangeably in the specification to refer to the same molecule. Xcer refers to cerberus.
  • mouse CRP-2 (mCRP-2) with a FLAG epitope fused to its C-terminus was transiently expressed in COS cells.
  • a single epitope-tagged protein of 29kDa was secreted from mCRP-2-transfected cells ( Figure 4), but not from those transfected with empty FLAG vector.
  • secreted CRP was N-glycosylated ( Figure 4).
  • Secreted mCRP-2 ran at approximately 30 kDa after deglycosylation. N-terminal amino acid sequence determination confirmed the identity of secreted mCRP-2 and the fact that the hydrophobic signal peptide had been processed (see Figure 1).
  • a full length cDNA encoding a mouse CRP (mCRP-1) was isolated using oligonucleotides based on the Genbank EST AA120122, a mouse embryonic day 7.5 cDNA, in combination with oligonucleotides corresponding to vector sequences of the plasmid pSport-1.
  • the nucleotide and corresponding amino acid sequence is shown in Figure 2.
  • a comparison of the mCRP-1 and cerberus sequence is shown in Figure 1.
  • mice embryonic region cDNA library An amount of 50 ng of mouse embryonic region cDNA library was used as template in PCR which contained two primers.
  • the PCR was performed using 1 unit of Promega Taq polymerase in the buffer supplied in the presence of 200 M dNTPs and 2.5 mM MgCl 2 for 30 cycles at [94°C, 60°C, 73 °C] and 10, 30, 60 seconds].
  • the products of these reactions were diluted 1 in 100 in water and a second PCR was then performed on the diluted sample.
  • All PCRs contained 50 ng of each primer.
  • each of these reactions yielded a prominant product of approximately 500 base pairs.
  • the products were gel purified using Bresaclean fragment purification system and cloned into the Hindi site of pBluscript SK+(Stratagene).
  • Recombinant plasmids were purified and their inserts sequenced using the ml 3 forward and reverse primers described above in standard ABI sequencing reactions. The sequence data obtained from these were combined with the EST data to construct a continuous 1000 base pair sequence from which the putative full length protein homologous to Xenopus cerberus could be translated.
  • mouse CRP (mCRP-2) with a FLAG epitope (DYKDDDDK [SEQ ID NO: 18]) fused to its C-terminus was transiently expressed in COS cells.
  • COS cells were transfected with the mCRP-2 cDNA using a polycationic liposome transfection reagent (Lipofetamine, GibcoBRL) or electroporation (Gene Pulser, Biorad).
  • lipofectamine mediated transfection COS cells grown to approximately 70-80% confluence in 100 mm petri-dishes were washed in serum free DMEM media then exposed to a mixture of mCRP cDNA and lipofectamine diluted in DMEM according to the manufacturers instructions.
  • mCRP-2 was monitored by biosensor analysis and western blot analysis.
  • Samples were monitored for expression using a biosensor (BIAcoreTM) employing surface plasmon resonance detection.
  • M2 antibody specific to FLAG sequence, was immobilised to the sensor surface using NHS-EDC coupling chemistry according to the manufacturers instructions.
  • the running buffer for all biosensor analysis was 10 mM HEPES, 150mM NaCl, 3.4mM EDTA, 0.005% v/v Tween 20 (HBS buffer). The buffer was degassed under vacuum for 10 minutes prior to use to prevent bubble formation. The flow rate was 5 ⁇ l/min.
  • M2 antibody For immobilisation of M2 antibody, 50mM N-hydroxysuccinimide and 200mM N-ethyl- 'dimethylaminopropyl carbodiimide were mixed in a 1: 1 ratio and 35 ⁇ l of this mixture injected onto the sensorchip for surface activation. After 7 minutes, 35 ⁇ l of M2 antibody (100 ⁇ g/ml in 20mM Sodium Acetate pH 4.2) was injected. After a further 7 minutes, 75 ⁇ l ethanolamine was injected to quench any remaining free esters generated during the NHS- EDC activation.
  • M2 antibody 100 ⁇ g/ml in 20mM Sodium Acetate pH 4.2
  • Protein expression was confirmed by western blot analysis. Samples were separated on the basis of size using SDS-PAGE analysis. Samples were then blotted onto PVDF using Tris/glycine/Methanol buffer at 30 V for 1 hour using a Novex blot apparatus. Transfers were performed according to manufacturers instructions. PVDF membranes were then blocked overnight in 2% w/v BSA in TBS buffer (20mM Tris pH 7.4, 150 rnM NaCl, 0.02 v/v Tween 20). Membranes were washed extensively between addition of antibodies with 10 washes of TBS. Blots were probed with primary antibody (M2) for lhr at a final concentration of 10 ⁇ g/ml. Bound M2 was detected using an appropriately conjugated secondary antibody (goat anti-mouse HRP, Silenus) at a dilution of 1:5000 for 1 hour. Bound secondary antibody was visualised by autoradiography after addition of ECL reagent.
  • M2 primary antibody
  • Proteins were purified using the strategy outline in Figure 5. Binding was monitored on the Biosensor and elution performed, after extensive washing in TBS (200 column volumes), using 4 x 5 ml fractions of Flag peptide (60 ⁇ g/ml) in TBS. Fractions were analysed by SDS- PAGE analysis under reducing conditions using the PHAST system (Pharmacia) and protein visualised by silver staining and western blot analysis.
  • mCRP-1 with a FLAG epitope fused to is C- terminus was transiently expressed in COS cells.
  • COS cells were transfected with the mCRP- 1 cDNA using a polycationic liposome transfection reagent (Lipofectamine, GibcoBRL). Cells grown to approximately 70-80% confluence in 100 mm petri-dishes were washed in serum free DMEM media then exposed to a mixture of mCRP- 1 cDNA and lipofectamine diluted in DMEM according to the manufacturers instructions.
  • Lipofectamine GibcoBRL
  • mCRP-1 was purified from positive samples by M2 affinity chromatography and then purified fractions analysed by SDS-PAGE and western blot analysis. Preliminary data indicate mCRP-1 under reducing conditions is a 30 kD protein ( Figure 8).
  • mCRP-1 has been studied in mouse embryos. The expression pattern is similar to that of frog cerberus, with some differences, mCRP-1 is first expressed at the onset of gastrulation in midline anterior endoderm extending to the embryonic/extra-embryonic boundary. This expression is likely to be in primitive endoderm, which will eventually be displaced into the extra-embryonic region. By mid-gastrulation, expression is also seen in anterior-lateral endoderm, tissue which is likely to be the migrating wings of definitive endoderm which displaces the primitive endoderm. By the end of gastrulation, endoderm and mesoderm in the anterior half of the embryo expresses mCRP- 1. Endoderm underlying the cardiac progenitors seems not to express mCRP-1. Shortly afterwards, the anterior expression fades, at first laterally, then from the midline, then is lost completely.
  • mCRP-1 is, therefore, a good candidate for a inductive factor that either induces, or gives anterior character to, early neural tissue.
  • it may be clinically useful as an inductive, maintenance, survival, proliferative, anti-proliferative or differentiation factor in any degenerative neuropathy or in any grafting procedure involving foetal or other neural tissue grafted to correct familial or acquired deficiencies, or to repair neural tissue after trauma.
  • Anterior lateral endoderm is also known to be the source of inducers and suppressors of cardiogenesis (4) and mCRP-1 may be one of these factors. It may be useful clinically as an inductive, maintenance, survival, proliferative, anti-proliferative or differentiation factor for cardiomyocytes in any interventionist procedure to correct cardiomyopathy, including any grafting technique aimed at repairing infarcts (cardiomyoplasty) or valvular defects, modification (including inhibition) of familial or secondary hypertrophy, or induction of compensatory cardiomyocyte growth during aging.
  • mCRP-1 is also expressed in developing somites. Expression prefigures a restricted anterior compartment of the two next-to-form somites within the presomitic mesoderm, and occurs in a similar compartment of the two most recently formed somites. This expression is extremely transient and does not occur in more mature somites. mCRP-1 may be involved in establishing compartments within newly forming somites. In developing chick embryos neural crest migrates through only the anterior portion of the somite. Hence, mCRP-1 may initially give anterior character to somites, defining its interaction with neural crest. mCRP-1 may, therefore, be useful as an inductive factor in imparting particular positional qualities to engrafted tissues.
  • RNA encoding mCRP-1 or mCRP-2 was injected into first cleavage stage Xenopus embryos and resulted in induction of ec topic cement gland formation at later embryonic stages.
  • cement gland formation is often an indicator of the presence of impending appearance of neural tissue, it is likely that mCRP-1 has the capability of inducing anterior neural tissue. This conjecture is entirely consistent with the established properties of Xenopus cerberus.
  • the capacity of mCRP-2 to mimic the action of Xenopus cerberus further suggests that this molecule has an underlying biological significance as a secreted molecule and not as transcription factor.
  • a cDNA fragment containing the entire coding sequence of mCRP-1 was cloned into the expression vector pEFBOS-S-FLAG for production of C-terminal FLAG tagged protein.
  • Transient expression following transfection of COS cells resulted in a low yield of predominantly aggregated material which, following purification by affinity and size exclusion chromatography, was found to be heterogenous at the N-terminus, most Ukely as a result of adverse proteolytic activity.
  • the construct and a vector incorporating a gene encoding puromycin resistance were co-transfected into CHO cells (DMEM, 10% v/v FCS, glutamine, penicillin, streptomycin, 37°C, 10% v/v CO 2 ) using Lipofectamine (Gibco BRL, USA) according to the manufacturer's instructions. Following selection in puromycin (25 ⁇ g/ml, Sigma, USA), resistant colonies were picked by micro- manipulation, expanded and assayed for mCRP-1-CFLAG production by binding to immobiUsed M2 antibody (Kodak Eastman, USA; Biosensor 2000, Pharmacia, Sweden). Several potential candidate clones were identified with clone (CL) 47 selected for further analysis. CL47 was recloned by limit dilution.
  • CL47 was expanded into Roller bottles and cultured until 3 days post confluerice. An amount of 2.5 L of conditioned media was concentrated tenfold (Easy flow diafiltration apparatus, 10 kDa cut-off, Sartorius, USA). Concentrated mCRP-1 was applied to 2 ml of M2 affinity resin (Kodak Eastman) and the unbound fraction reloaded onto the column 4 times prior to extensive washing in tris-buffered saline (TBS, 500 ml). Elution (4 x 5ml) was performed using FLAG peptide (60 ⁇ g/ml, Kodak Eastman) in TBS. Fractions were monitored by SDS PAGE and Western Blot analysis for appropriate pooling of samples for further purification.
  • Cerberus has been shown to have an anti-BMP-like activity when expressed in Xenopus animal cap assays, inducing Otx-2, a marker of anterior embryonic structures, as well as markers of neural tissue, cement gland and endoderm.
  • Otx-2 a marker of anterior embryonic structures
  • the activities of mCRP-1 and cerberus were compared in animal cap assays after injection of synthetic mRNAs at the fertilised egg stage.
  • formation of cement glands in individual animal caps was scored at stage 35, since these were easily recognisable as superficial darkly pigmented patches secreting a sticky exudate.
  • Both native mCRP-1 and CFLAG-mCRP-1 were able to induce pigmented cement glands (Figure 14) with equal frequency ( Figure 14), although at only one quarter the frequency of cerberus.
  • RT-PCR analysis was used to assess expression of various markers in pools of 25-30 animal caps derived from injected and uninjected embryos (Figure 14). Both mCRP-1 and cerberus mRNA s showed near identical activities. mCRP-1 induces the pan-neural maker N- CAM (9), although rather weakly, the cement gland marker CGI 3 (10) and the endoderm- enriched marker Edd (11). Induced tissues apparently had anterior character, since Otx-2, a marker of anterior embryonic structures such as forebrain, midbrain and cement gland was strongly induced, while Krox20, a hindbrain marker was not.
  • the related homeobox genes, XNkx-2.5 and -2.3, normally expressed in cardiogenic mesoderm and anterior (pharyngeal) endoderm were strongly induced. There was no induction of the cardiac-specific differentiation marker, XMLC2a, even when caps were cultured beyond the equivalent of stage 40. Thus, the homeobox markers may be expressed in endoderm, in which case they would indicate foregut character (11). However, it is also possible that early stages of cardiogenesis are activated, without realization of the whole program.
  • the inductive activities of mCRP-1 and cerberus in animal caps are characteristic of a partial inhibition of BMP signaUing.
  • BMPs can act as both mesoderm-inducing and ventralising agents, wriilst strongly inhibiting formation of neural tissue.
  • the inventors found that BMP4 induced expression of T4 globin and XeHAND makers of ventral and lateral mesoderm, respectively ( Figure 14C).
  • mCRP-1 and cerberus were co-expressed with BMP4 in animal caps by coinjection of equimolar amounts of their mRNAs into eggs.
  • mCRP-1 transcripts were first evident at or just before the onset of gastrulation in a stripe, several cell diameters in width, along one side of the egg cylinder. This stripe extended proximally from the embryonic/extra-embryonic junction to just short of the distal tip (15A) and was on the opposite side of the egg cylinder to the primitive streak, abutting the future anterior region of epiblast fated to form anterior neurectoderm. This location was confirmed by hybridising early streak embryos with probes for both mCRP-1 and brachyary (bra) which makes prospective mesodermal cells immediately after passage through the streak ( Figure 15B).
  • mCRP-1 expression was analysed in adult tissues by RNase protection, using an mCRP-1 specific probe and a cyclophilin probe to control RNA recovery and integrity. Under conditions employed, no high level expression was detected in adult tissues including brain, skeletal muscle, saUvary gland, tongue, thymus, heart, lung, stomach, spleen, liver, pancreas, intestine, kidney, bladder, uterus, testes and ovary.
  • Anterior primitive endoderm is known to be essential for patterning the anterior epiblast and neural plate as is anterior embryonic mesendoderm at later stages. Since mCRP-1 is expressed in both of these zones, it could participate in the patterning function, either as an inducer, or an inhibitor.
  • Another gene that may be involved is Otx-2, which encodes a homeodomain protein related to Drosophila orthodenticle and empty spiracles, both involved in head development in the fly. Otx-2 is initially expressed throughout the epiblast of the gastrula, before becoming restricted to anterior tissues including chordamesoderm, forebrain and midbrain.
  • Otx-2 '1' mutants show severe abnormalities at gastrulation, including defective formation of chordamesoderm and loss of all brain compartments anterior to rhombomere 3.
  • a possible role for Otx-2 in the patterning function of primitive endoderm was suggested by examination of null embryos carrying a LacZ reporter gene, which showed that while Otx-2 could be expressed in primitive endoderm in the mutant context, it could not be induced and/or maintained in anterior neural plate.
  • mCRP- 1 expression was examined in Otx-2 '1' embryos by wholemount in situ hybridisation ( Figure 17) and found that it was still expressed in an anterior region, although to varying extents. In more severely affected embryos, expression was restricted to the distal tip and was absent at the embryonic/extra-embryonic junction, marker by a prominent constriction. As noted above, mCRP-1 would normally extend up to this point. In the less affected Otx-2 '1' mutant examined ( Figure 17), mCRP-1 expression tended more towards the norla pattern, although, since anterior patterning is still disturbed in these embryos, it is difficult to say to what extent. EXAMPLE 13
  • a cDNA fragment containing the entire coding sequence of mCRP-2 was cloned into the expression vector pEFBOS-S-FLAG for production of C-terminal FLAG tagged protein.
  • Transient expression following transfection of COS cells resulted in good yields of predominantly dimeric mCRP-2.
  • the construct and a vector incorporating a gene encoding puromycin resistance were co-transfected into rat SR-3Y1 cells (DMEM, 10% v/v FCS, glutamine, penicillin, streptomycin, 37°C, 10% v/v CO 2 ) using Lipofectamine (Gibco BRL, USA) according to the manufacturers instructions.
  • CL23 was expanded into Roller bottles and cultured until 3 days post confluence. An amount of 2.5 L of conditioned media was concentrated tenfold (Easy flow diafiltration apparatus, 10 kDa cut-off, Sartorius, USA). The sample was also buffer exchanged after concentration into 20mM Tris, 0.15% w/v NaCl, 0.1% v/v Tween 20 (TBS). Concentrated mCRP-2 was applied to 2 ml of M2 affinity resin (Kodak Eastman) and the unbound fraction reloaded onto the column 4 times prior to extensive washing in Tris-buffered saline (TBS, 500 ml). Elution (4 x 5ml) was performed using FLAG peptide (60 ⁇ g/ml, Kodak Eastman) in TBS. Fractions were monitored by SDS PAGE and Western Blot analysis for appropriate pooling of samples for further purification.
  • Peak fractions were diluted 1:1 with 20mM Tris, 0.02%v/v Tween 20, 0.05% w/v azide pH 7.5 (Buffer A) and applied to a previously equilibrated MonoQ 5/5 column (Pharmacia, 50 x 5 mm I.D.) via a superloop. After sample loading was complete, the column was re-equilibrated into Buffer A and a gradient developed over 50 min from Buffer A to 1.0M NaCl in Buffer A. Fractions containing mCRP-2 were monitored by biosensor analysis and SDS PAGE. Typically 400 ⁇ g of mCRP-2 was recovered in fractions 18/19 which eluted at 0.35-0.4 M NaCl and was essentially pure (>95%).
  • Protein estimations were determined by SDS PAGE analysis (comparison to known standard concentrations), Western blot, and also by comparison of lO ⁇ g Bovine serum albumin under identical ion exchange chromatographic conditions. Reducing conditions on SDS PAGE ( 0.05 % v/v ⁇ mercaptoethanol ) resulted in a single band residing at 27 kD (Panels A and B, Fig 10). Following removal of carbohydrate the protein was reduced to an apparent molecular weight of 24 kDa (Panel A, Fig 10; N-glycosidase F, Boehringer Mannheim, Germany).
  • a human genomic DNA clone encoding a human CRP (hCRP-1) was isolated by screening a human genomic library (lambda DASH II, Stratagene) with cDNA encoding mCRP-1. Library screens were performed using hybridisation conditions described by Sambrook et al (6). Hybridisation and washing conditions were performed at relatively high stringency at 65°C and 65°C, 2 xSSC/0.1% w/v SDS, respectively. The probe was generated by random priming of the cDNA encoding mCRP-1.
  • the genomic clone, hCRP- 1 was sequenced and found to contain a full open reading frame, encompassing two exons and one intron and parts of the 5'UTR and 3'UTR.
  • the sequence data obtained were used to construct a continuous 3,150 base pair sequence from which the putative full length protein could be translated.
  • the complete genomic nucleotide sequence and corresponding amino acid sequence to exons 1 and 2 is shown in Figure 11.
  • the putative coding sequence without intron and corresponding protein translation is shown in Figure 12.
  • the translated sequence shows sequence similarities with mCRP-1 and Xenopus laevis cerberus.
  • a protein comparison of hCRP-1, mCRP-1 and Xenopus laevis cerberus is shown in Figure 13.
  • mice chromosomal location of mCRP-1 was determined by interspecific backcross analysis of a panel of progeny derived from matings of [(C57BL/6J/ x Mus spretus) Fi x C57BL/6J] mice. This interspecific backcross panel has been typed for over 2400 loci that are well distributed among all autosomes as well as the X chromosome (12). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern transfer to Hybond-N+ and hybridisations were performed essentially as described (13). The probe for mCRP-1 encompassed nucleotides 288-640 and was radiolabelled with [ 32 P]-CTP using a random priming kit (Stratagene).
  • mice were analysed with all of these markers, although for some marker pairs up to 175 mice were typed. To calculate recombination frequencies, each locus was analysed in pairwise combinations using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analysed for each pair or loci, and most the likely gene order are: centrometer - Tyrpl - 1/163 -mCer-1 2/116 - Ifna - 0/122 - Jun -5/174 - Pg l.
  • Recombination frequencies (expressed as genetic distances in centiMorgans (cM) ⁇ standard error) are Tyrpl - 0.6 ⁇ 0.6 - mCer-1 - 2.6 ⁇ 1.5 - [Ifna, Jun] - 2.9 ⁇ 1.3. No recombinants were detected between Ifna and Jun in 122 animals types, suggesting that the two loci are within 2. 5 cM of each other (upper 95% confidence limit).
  • the inventors have compared the interspecific map of chromosome 4 with a composite mouse linkage map that reports the location of many uncloned mutations (provided by the Mouse Genome Database, a computerised databased maintained at the Jackson Laboratory, Bar Harbor, ME, USA).
  • a number of mouse mutations map with 5 cM of the chromosomal position determined for mCRP-1: pintail (pt) (16), polysyndactyly (ps) (17, 18), meander tail (mea) (19) and head blebs (heb) (20). All of these mutations result in phenotypes which include defects in head or tail development and could conceivably be associated with mutation of mCRP-1 or perturbation of its expression. To test this possibility, genomic DNA derived from heterozygous or homozygous individuals of each mutant strain was analysed by Southern blotting using an mCRP-1 probe that detected both coding exons (Figure 18).
  • mCRP-1 is a secreted N-terminally processed glycoprotein
  • pEFBOS I FLAG vector carrying the mCRP-1 insert was co-transfected with a puromycin resistance plasmid into CHO cells using Lipofectamine (Gibco BRL) according to manufacturer's instructions. Following puromycin selection (Sigma; 25mg/ml), resistant clones were picked and expanded, and culture supernatant was assayed fro CFLAG-mCRP- 1 - 1 by analysis of binding to immobilised M2 (anti-FLAG) antibody (Kodak Eastman) on a Biosensor 2000 (Pharmacia).
  • CFLAG-mCRP- 1 was purified from conditioned medium of clone CL47 using M2 affinity resin (Kodak Eastman), eluding with FLAG peptide (60 ⁇ g/ml; Kodak Eastman) in Tris-buffered saline. Fractions were monitored by Western blotting probed with M2 antibody.
  • 293T fibroblast cells were transfected (Lipofectamine) with an pEFBOS construct encoding a C terminal flag tagged mCRP-1.
  • mCRP-l-C was affinity purified M2 (anti-falg antibody) resin (1ml).
  • Flag peptide was used to elute mCRP-1 from the column at a concentration of lOO ⁇ g/ml (5ml). Purified fractions were monitored by Biosensor and by SDS-PAGE. Preliminary Western analysis indicated a 38kDa polypeptide under reducing conditions and a 74kDa polypeptide under non-reducing conditions.
  • N-terminal amino acid sequence analysis of mCRP-1 derived from 293T cells was done and the sequence was the same as that derived from mCRP-1 expressed from CHO cells. Results
  • CFLAG-mCRP- 1 could be recovered from culture supematans of both Chinese hamster ovary (CHO) cells, after stable integration of vector, as well as from 293T cells after transient transfection (Fig. 20A, B).
  • CFLAG-mCRP- 1 purified from CHO cells using immobilised M2 (anti-FLAG) antibody and reversed phase-HPLC migrated principally at 38kD on reducing and denaturing gels.
  • CHO cell-derived material may be abnormally processed or secreted.
  • the inventors examined, CFLAG-mCRP- 1 secreted from 293T cells, and in this case found dimers, with no monomer detected (Fig. 20B). These data demonstrate that mCRP-1 is a secreted, N-terminally processed glycoprotein that can form predominantly disulphide bonded dimers when secreted from certain cell types.
  • a gene encoding hCRP-1 was isolated from a human genomic library (see Example 14). PCR was used to generate probes specific for putative hCRP-1 exons 1 and 2 which were used to screen a number of cDNA libraries constructed from various fetal and adult tissues. No cDNA clones encoding hCRP-1 were isolated.
  • hCRP-1 cDNA was constructed using standard techniques such as splice overlap extension PCR (SOE-PCR). Two separate PCR's were performed to generate exon 1 and exon 2 specific hCRP-1 cDNA, the PCR fragments purified and used in a third PCR to generate a full length cDNA from which the intron has been excised. Attempts were made to clone this cDNA into the expression vector pEFBOS I FLAG for production of a recombinant protein carrying a C-terminal FLAG epitope. This construct proved to be unstable so other epitope tags were employed.
  • SOE-PCR splice overlap extension PCR
  • hCRP-1 For transient expression of hCRP-1, 293T human fibroblast cells were transfected (Lipofectamine, Gibco BRL) with the pEFBOS I-SPY-hCRP-1 construct according to the manufacturers instructions. Three days post transfection supernatant was harvested and expression assessed by biosensor analysis where I-SPY antibody (Dl 1) was immobilised to the sensorchip at 100 ng/ml. C'-I-SPY-hCRP-l was affinity purified from the supernatant using an I-SPY antibody coupled resin (AMRAD Biotech). Purified material was analysed by Western blot analysis using the I-SPY antibody conjugated with HRPO and subsequent ECL detection.
  • Results presented in Fig 21 demonstrate that under reducing conditions the major species of protein was a monomer migrating with an apparent molecular weight of approximately 40 kDa. Other minor species present under reducing conditions are non-reduced dimers, as well as a small amount of aggregated material. Under non-reducing conditions the monomeric form was replaced by a dimer of approximately 80 kDa and a large amount of aggregated material. These results indicate that like mCRP-1 and other cystine knot cytokines, hCRP-1 exists primarily as a dimeric molecule.
  • mCRP-2 is expressed from the beginning of organogenesis (embryonic day 8.5) within the segmenting paraxial mesoderm. mCRP-2 expression prefigures the next-to- form somite within unsegmented mesoderm and in the most recently formed somite. Thus, both mCRP-2 and mCRP-1 are expressed during somite formation, and may be involved in the same type of process. In contrast to mCRP-1, however, mCRP-2 expression is strongest in the posterior region of somites. mCRP-2 and mCRP-1 may therefore act in compartmentalisation of the posterior and anterior regions of the somite, respectively. After dropping considerably in newly formed somites, mCRP-2 expression increases as somites mature.
  • mCRP-2 expression in somites is strongest medially and dorsally.
  • mCRP-2 is also expressed in the limb musculature and in craniofacial epithelium and mesenchyme.
  • mCRP-2 may be useful clinically as an inductive, maintenance, survival, proliferative, anti-proliferative or differentiation factor in pathologies related to muscle, bone and skin.
  • tumour suppressor suggests a function as an anti-proliferative factor in a broad range of cancers, including breast cancer, lymphoma and leukaemia, melanoma, colorectal cancer, pancreatic cancer, lung cancer, stomach cancer and nueroblastoma.
  • mCRP-2 is expressed in aU adult tissues examined with the exception of the liver. Strongest expression levels were observed in bladder, uterus and lungs. EXAMPLE 19 mCRP-1 AND mCRP-2 FORM HETERODIMERS
  • mCRP-1 and mCRP-2 are capable of either associating, or existing in a heterodimeric complex, with each other. This possibility is raised by analysis of supernatants recovered from cells which had been transfected with expression vectors for mCRP-1 and mCRP-2. Briefly, DNA expression vectors designed to produce c-terminally flag tagged mCRP-1 and c-terminally myc tagged mCRP-2 were transfected, either individually or together, into 293T cells using the Lipofectamine transfection protocol described previously. 72 hours later, the supernatants from the various transfections were harvested, clarified by low speed centrifugation and then treated in two different ways.
  • heterodimers between mCRP-1 and -2 raises the following possibilities.
  • the activity of the heterodimer may be different from homodimers of either mCRP- 1 or -2.
  • a heterodimer may be able to bind to distinct receptors, if they exist, and elicit novel responses in the cells that possess them.
  • a heterodimer may possess distinct biochemical properties, in terms of its stability and or solubility. It is also possible that the biological potency of a heterodimer may exceed that of molecules being homodimeric examples of its constituents.
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 8 :
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 13:
  • GGC AGG TTC TGG AAG AAG CCT GAG AGA GAA ATG CAT CCA TCC AGG GAC 702 Gly Arg Phe Trp Lys Lys Pro Glu Arg Glu Met His Pro Ser Arg Asp 85 90 95
  • TGTCTACTTC CTCTGGCAAT ATCTACATTC CAAATGTTAA ATTAAAATTG AGAACTTGCA 1782
  • CTGCAGAGAA TGAGCCTCTC CTTTGGGCCT CATCATTTAC AAAAGAAGCT TGGGCCCCTG 60

Abstract

Cette invention se rapporte d'une manière générale à une nouvelle famille de molécules de cytokines. Cette invention se rapporte plus particulièrement à des cytokines de mammifères qui constituent une nouvelle famille de cytokines et qui sont utiles dans une gamme d'applications thérapeutiques et diagnostiques.
PCT/AU1998/000078 1997-02-11 1998-02-11 Nouvelle famille de cytokines et utilisations correspondantes WO1998034951A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000042206A1 (fr) * 1999-01-18 2000-07-20 Yissum Research Development Company Of The Hebrew University Of Jerusalem Systeme de mise au silence de l'expression et ses differentes utilisations
US7316998B2 (en) 2004-05-27 2008-01-08 Acceleron Pharma Inc. Cerberus/Coco derivatives and uses thereof
US7560541B2 (en) 2002-03-22 2009-07-14 Acceleron Pharma, Inc. Heart20049410 full-length cDNA and polypeptides
US7833971B2 (en) 2006-12-08 2010-11-16 Acceleron Pharma Inc. Uses of cerberus, coco and derivatives thereof
US9045553B2 (en) 2004-05-27 2015-06-02 Acceleron Pharma, Inc. Cerberus/Coco derivatives and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997048275A1 (fr) * 1996-06-20 1997-12-24 The Regents Of The University Of California Inducteurs des tissus endodermiques, cardiaques et neuronaux

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997048275A1 (fr) * 1996-06-20 1997-12-24 The Regents Of The University Of California Inducteurs des tissus endodermiques, cardiaques et neuronaux

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 214, No. 2, issued (14 September 1995), TSUJIMURA A. et al., "Developmental and Differential Regulations in Gene Expression of Xenopus Pleiotrophic Factors - alpha and - beta", pages 432-439, MEDLINE, 95408268. *
CHEMICAL ABSTRACTS, 119:21591, OZAKI T. et al., "Molecular Cloning and Characterization of a cDNA Showing Negative Regulation in V-Src-Transformed 3Y1 Rat Fibroblasts"; & PROC. NATL. ACAD. SCI. USA, Volume 90, No. 7, issued (1993), pages 2593-2597. *
CHEMICAL ABSTRACTS, 122:179709, SAKIYAMA S. et al., "Molecular Cloning and Characterization of a cDNA Showing Tumor-Suppressive Activity in V-Src-Transformed 3Y1 Rat Fibroblasts"; & ADV. ENZYME REGUL., Volume 34, issued (1994), pages 247-255. *
CHEMICAL ABSTRACTS, 122:2407, ENOMOTO H. et al., "Identification of Human DAN Gene Mapping to the Putative Neuroblastoma Tumor Suppressor Locus"; & ONCOGENE, Volume 9, No. 10, issued (1994), pages 2785-2791. *
CHEMICAL ABSTRACTS, 124:142381, OZAKI T. et al., "Cloning of Mouse DAN cDNA and Its Down-Regulation in Transformed Cells"; & JAPAN J. CANCER RESEARCH, Volume 87, No. 1, issued (1996), pages 58-61. *
CHEMICAL ABSTRACTS, 125:217338, BOUWMEESTER T. et al., "Cerberus is a Head-Inducing Secreted Factor Expressed in the Anterior Endoderm of Spemann's Organizer"; & NATURE, Volume 382, No. 6592, issued (1996), pages 595-601. *
CURRENT OPINION IN GENETICS AND DEVELOPMENT, Volume 6, No. 4, issued (August 1996), HOGAN B.L., "Bone Morphogenetic Proteins in Development", pages 432-8. *
EMBO JOURNAL, Volume 16, No. 2, issued (15 January 1997), GUENDA A. et al., "Activation of Stress-Activated Protein Kinase-3 (SAPK3) by Cytokines and Cellular Stresses is mediated via SAPKK3 (mKK6); Comparison of the Specificities of SAPK3 and SAPK2", pages 295-305, MEDLINE, 97180932. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 272, No. 5, issued (31 January 1997), NAKAO A. et al., "Identification of Smad 2, a Human Mad-Related Protein in the Transforming Growth Factor Beta Signalling Pathway", pages 2896-2900. *
ROUX'S ARCH. DEV. BIOL., Volume 205, No. 5-6, issued (1996), NAKAMURA H. et al., "Isolation of Xenopus HGF Gene Promoter and its Functional Analysis in Embryos and Animal Caps", pages 300-310. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000042206A1 (fr) * 1999-01-18 2000-07-20 Yissum Research Development Company Of The Hebrew University Of Jerusalem Systeme de mise au silence de l'expression et ses differentes utilisations
US7005559B1 (en) 1999-01-18 2006-02-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Expression silencing system and different uses thereof
US7560541B2 (en) 2002-03-22 2009-07-14 Acceleron Pharma, Inc. Heart20049410 full-length cDNA and polypeptides
US7316998B2 (en) 2004-05-27 2008-01-08 Acceleron Pharma Inc. Cerberus/Coco derivatives and uses thereof
US7981857B2 (en) 2004-05-27 2011-07-19 Acceleron Pharma Inc. Cerberus/coco derivatives and uses thereof
US9045553B2 (en) 2004-05-27 2015-06-02 Acceleron Pharma, Inc. Cerberus/Coco derivatives and uses thereof
US7833971B2 (en) 2006-12-08 2010-11-16 Acceleron Pharma Inc. Uses of cerberus, coco and derivatives thereof
US8796199B2 (en) 2006-12-08 2014-08-05 Acceleron Pharma, Inc. Uses of Cerberus and derivatives thereof

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