WO2009113965A1 - Isthmin derivatives for use in treating angiogenesis - Google Patents

Isthmin derivatives for use in treating angiogenesis Download PDF

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Publication number
WO2009113965A1
WO2009113965A1 PCT/SG2009/000085 SG2009000085W WO2009113965A1 WO 2009113965 A1 WO2009113965 A1 WO 2009113965A1 SG 2009000085 W SG2009000085 W SG 2009000085W WO 2009113965 A1 WO2009113965 A1 WO 2009113965A1
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seq
polypeptide
sequence
amino acids
amino acid
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PCT/SG2009/000085
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French (fr)
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Ruowen Ge
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National University Of Singapore
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Publication of WO2009113965A1 publication Critical patent/WO2009113965A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to polypeptides and nucleic acids encoding the polypeptides and to the use of these in methods of medical treatment including treatment of conditions involving angiogenesis.
  • Angiogenesis plays a key role in health and diseases. Most angiogenesis occurs in the embryo, where it establishes the primary vascular tree as well as an adequate vasculature for growing and developing organs (Folkman, 1971; 1995; 2007) . Very little turnover of endothelial cells occurs in the adult vasculature except during menstrual cycle and in physiological repair processes such as wound healing. However, a number of disorders are caused by excessive angiogenesis, such as cancer (tumor growth is strikingly angiogenesis dependent) (Carmeliet, 2003) . Therefore, abrogation of angiogenesis is believed to be a promising strategy for treatment of cancer.
  • ECM extracellular matrix
  • TSRs Thrombospondin type-1 repeats
  • TSP-I human Thrombospondin-1
  • the human genome has a large number of proteins that contain TSRs (Adam 2000; Tucker, 2004) . All the TSRs are present either in secreted proteins or in the extracellular portion of transmembrane proteins, implicating possible roles in cell migration, cell-cell and cell-matrix adhesion and communication as well as tissue remodelling " (R. Ge et al., unpublished) .
  • TSRs from TSP-I and other antiangiogenic proteins such as brain-specific angiogenesis inhibitor 1 (BAIl), ADAMTSl and 8, strongly indicate that TSRs play critical roles in the antiangiogenic function of these proteins.
  • Two of the three TSRs in TSP-I are individually antiangiogenic (Iruela- Arispe et al., 1999) .
  • TSRs from properdin and the first TSR of TSP-I are not antiangiogenic (Iruela- Arispe et al. , 1999) .
  • Crystal structure of TSRs from TSP-I revealed a novel, antiparallel, three-stranded fold that consists of alternating stacked layers of tryptophan and arginine residues from respective strands, capped by disulfide bonds on each end (Tan et al., 2002) .
  • Certain motifs within TSR have been shown to be associated with the anti-angiogenic activity of TSRs in TSP-I.
  • One is the conserved wxxwxxw localized at the C-terminal (Iruela-Arispe 1999), another is the conserved region CSVTCG which binds to TSP-I receptor CD36 on endothelial cell membrane (Dawson et al., 1997) .
  • Isthmin is a secreted protein with no known function. It contains two putative domains in the mature protein: a centrally localized TSR and a C-terminal AMOP (adhesion- associated domain in MUC4 and other proteins) (Pera et al., 2002; Rossi et al . , 2004) . It was first identified in Xenopus as a member of the FGF-8 synexpression group and is prominently expressed at the midbrain-hindbrain boundary (Pera et al . , 2002) . Analysis of various genome databases indicated that orthologous Isthmin genes are present in zebrafish (isthminl), mouse and human.
  • Isthmin2 is a homologous gene isthmin2 which is likely the orthologue of the human Isthmin-like gene TAILl (Rossi et al., 2004 and our unpublished data) .
  • Isthmin is a predicted secreted protein of 461 amino acids.
  • Isthmin in Xenopus is expressed in paraxial mesoderm and neural folds at the tailbud stage and in notochord and midbrain-hindbrain boundary (MHB) during neurula stage (Pera et al. 2002) .
  • Isthminl expression could be upregulated by overexpressing of Wnt8 or downregulated by Wnt/ ⁇ -Catenin inhibitor (Weidinger et al. 2005) , indicating its involvement in Wnt signaling regulated process during embryonic development.
  • no function has been identified for this gene in any species.
  • AMOP is a protein domain predicted by bioinformatics that has eight invariant cysteine residues and is only found in secreted proteins or in proteins that also contain extracellular domains involved in cell adhesion. Because of its presence in cell adhesion molecules, AMOP domain is speculated to be involved in adhesion processes (Ciccarelli et al. 2002) . Besides the common cysteine residues that are shared by all AMOPs among different proteins, Isthmin AMOP has a "KGD" motif, which is found in many antagonists of platelet aggregation and integrin-dependent cell adhesion, including tumor cell metastasis.
  • Isthmin and fragments thereof, including the AMOP domain in the absence of the TSR domain potently inhibit angiogenesis .
  • EC endothelial cell
  • Isthmin has a role in zebrafish embryonic angiogenesis. This was investigated by using morpholino antisense oligonucleotide (MO) knockdown technology.
  • MO morpholino antisense oligonucleotide
  • VEGF vascular endothelial growth factor
  • the invention relates to Isthmin and derivatives of Isthmin, to AMOP domains, and also to use of these for the treatment, e.g. inhibition, of angiogenesis.
  • polypeptide comprising:
  • SEQ ID NO: 3 which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3
  • polypeptide comprises an AMOP domain, e.g. a polypeptide sharing at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% identity with amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3.
  • AMOP domain e.g. a polypeptide sharing at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% identity with amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3.
  • the polypeptide inhibits angiogenesis .
  • the polypeptide may have the ability to reduce, impair, disrupt, and/or down-regulate angiogenesis in an individual, e.g. a mammal.
  • the polypeptide may inhibit angiogenic activity.
  • the polypeptide may exhibit anti-angiogenic activity.
  • the polypeptide may have the ability to inhibit growth of new blood vessels from preexisting vessels.
  • the polypeptide may inhibit endothelial cell tube formation, e.g. it may have the ability to reduce, impair, disrupt, and/or down-regulate the assembly of endothelial cells to form tubes.
  • the polypeptide may have the ability to reduce tube length in an endothelial tube formation assay compared to the absence of the polypeptide, e.g. in a given time period. In vitro assays for observing endothelial cell tube formation are described below.
  • the polypeptide may inhibit proliferation of endothelial cells, e.g. it may have the ability to reduce, impair, disrupt, and/or down-regulate production of endothelial cells.
  • the polypeptide may have the ability to reduce the number of endothelial cells produced in an endothelial cell proliferation assay compared to the absence of the polypeptide, e.g. in a given time period.
  • the polypeptide may inhibit proliferation of endothelial cells in the presence of vascular endothelial growth factor (VEGF) .
  • VEGF vascular endothelial growth factor
  • the polypeptide may induce apoptosis of endothelial cells, e.g. it may have the ability to promote, stimulate, and/or generate apoptosis of endothelial cells.
  • the polypeptide may have the ability to increase the number of endothelial cells undergoing apoptosis in an endothelial cell apoptosis assay compared to the absence of the polypeptide, e.g. in a given time period.
  • Apoptosis may be quantitated by measuring DNA fragmentation, such as the number of mono- and oligonucleosomes in the cytoplasmic fraction of polypeptides treated with the polypeptide. In vitro assays for observing apoptosis of endothelial cells are described below.
  • the TSR domain of Isthmin exhibits less anti-angiogenic activity compared to the full length Isthmin polypeptide and other fragments of the Isthmin polypeptide.
  • the full-length polypeptide induced EC apoptosis in the presence of .VEGF.
  • the full-length polypeptide, the N-terminal fragment and the C- terminal fragment disrupted tubular network on Matrigel.
  • the TSR domain alone did not have these activities.
  • the TSR domain alone was not effective at inhibiting endothelial cell proliferation.
  • the polypeptide preferably exhibits one or both of these activities not exhibited by the TSR domain, i.e. the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation.
  • the polypeptide preferably exhibits one or both of these activities when the polypeptide comprises the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence that shares at sequence identity to the TSR domain, e.g. at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent sequence identity.
  • the polypeptide may inhibit proliferation of endothelial cells.
  • polypeptide comprising:
  • polypeptide may not comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 15.
  • the method of medical treatment may be the treatment of angiogenesis in an individual, e.g. by inhibiting angiogenesis .
  • the angiogenesis may be unwanted angiogenesis, e.g. angiogenesis that is associated with disease.
  • the angiogenesis may be tumour angiogenesis, e.g. angiogenesis that is induced by a tumour.
  • Inhibiting tumour angiogenesis may involve inhibiting the blood supply to the tumour, e.g. by inhibiting the formation of the tumour vascular system.
  • the treatment may be treatment of a tumour and/or cancer, e.g. a cancerous condition.
  • Inhibiting angiogenesis may include reducing, impairing, disrupting, and/or down-regulating angiogenesis and/or angiogenic activity in an individual.
  • Inhibiting angiogenesis may involve inhibiting growth of new blood vessels, e.g. from pre-existing vessels. In particular, it may involve one or more of inhibiting endothelial cell tube formation, inhibiting proliferation of endothelial cells and/or inducing apoptosis of endothelial cells.
  • the treatment may be prophylactic. It may be treatment of an individual at risk of unwanted angiogenesis, e.g. tumour angiogenesis, e.g. unwanted proliferation of endothelial cells.
  • polypeptide in the manufacture of a medicament for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis, wherein the polypeptide comprises: (i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:
  • SEQ ID NO: 3 which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
  • a method of inhibiting angiogenesis in an individual or of treating an individual at risk of unwanted angiogenesis comprising administering a polypeptide to the individual, wherein the polypeptide comprises: (i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
  • the method is a method of inhibiting tumour angiogenesis in an individual.
  • the method may be a method of inhibiting proliferation of endothelial cells in an individual .
  • polypeptides of the invention are described in more detail below. These polypeptides may be for use in a method of medical treatment and/or for use in the manufacture of a medicament .
  • nucleic acid comprising a nucleotide sequence, which nucleotide sequence comprises:
  • nucleotide sequence sharing at least 60 percent sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
  • nucleic acid does not comprise the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, or SEQ ID NO: 16.
  • the nucleic acid encodes a polypeptide that inhibits angiogenesis, e.g. tumour angiogenesis .
  • the nucleic acid may encode a polypeptide that inhibits proliferation of endothelial cells.
  • composition e.g. a pharmaceutical composition, comprising a polypeptide or nucleic acid of the invention.
  • the composition may comprise a pharmaceutically acceptable excipient, diluent and/or carrier.
  • kits for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis comprising: (i) a container comprising a polypeptide of the invention, e.g. as a pharmaceutical composition; and optionally
  • a vector comprising a nucleic acid of the invention.
  • the nucleic acid may be operably linked in the vector to a regulatory sequence, e.g. a promoter.
  • a host cell comprising a nucleic acid and/or vector of the invention.
  • a method of producing a polypeptide of the invention comprising: (a) providing a host cell comprising a nucleic acid of the invention, e.g. a nucleic acid comprising a nucleotide sequence that encodes a polypeptide of the invention; and
  • the method may also comprise step (c) isolating the peptide and/or may also comprise, prior to step (a) , introducing the nucleotide sequence that encodes the polypeptide of the invention into the host cell, e.g. on a vector.
  • the step of providing the host cell may also include culturing the host cell. Methods of culturing host cells to express polypeptides and methods of purifying polypeptides are well known in the art.
  • the host cell may express the polypeptide under the control of a constitutive promoter or inducible promoter.
  • SEQ ID NO: 1 refers to the amino acid sequence of mouse Isthmin
  • SEQ ID NO: 3 refers to the amino acid sequence of human Isthmin.
  • the polypeptide may or may not comprise a TSR domain.
  • the polypeptide may or may not comprise an AMOP domain.
  • the polypeptide may comprise a TSR and an AMOP domain.
  • a TSR domain generally refers to a domain of about 40-60 amino acids with six conserved cysteine residues capable of forming 3 disulfide bonds.
  • GGGPCTGEDPETRACNEQPCP has been reported (SEQ ID NO: 17) (Tucker RP., 2004) .
  • Each dot represents any amino acid.
  • WSxW SEQ ID NO: 18
  • CSVTCG SEQ ID NO: 19
  • a polypeptide of the invention may include one or both of these motifs.
  • the CSVTCG motif may occur on the C-terminal side of the WSxW motif.
  • Thrombospondin TSR domains These are W8, S9, WlI, C18, R25, R27, C29, C41, C51 and C56. Some of these residues are shared by the TSR domains of isthmin polypeptides.
  • a polypeptide of the invention may comprise a TSR domain having the following consensus sequence:
  • WSxWxxCxxxCxxxxxxRxRxC (SEQ ID NO: 20) .
  • the consensus sequence is WSXWXXCXXXCXXXXXRXRXCZ 1 CZ 2 CXXXXC (SEQ ID NO: 21), where x is any amino acid, z 1 is 3 to 11 amino acids of any type, and z 2 is 7 to 9 amino acids of any type. More preferably the consensus sequence is
  • the TSR domain may be considered to be amino acids 215 to 259 of SEQ ID NO: 1, in the human Isthmin amino acid sequence (SEQ ID NO: 3) the TSR domain may be considered to be amino acids 218 to 262 of SEQ ID NO: 3.
  • An AMOP domain generally refers to a domain of about 100 amino acids containing eight invariant cysteine residues.
  • An AMOP domain may have a consensus sequence, or may have be a sequence sharing at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity with:
  • the letters a, c, h, p, s, - and + indicate aromatic, charged, hydrophobic, polar, small, negative and positive amino acids respectively. Each dot represents any amino acid. (Ciccarelli et al. 2002) .
  • the AMOP domain may be considered to be amino acids 286 to 449 of SEQ ID NO: 1
  • the AMOP domain may be considered to be amino acids 289 to 452 of SEQ ID NO: 3.
  • Polypeptides comprising or consisting of an amino acid sequence sharing sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3
  • Polypeptides of the invention may comprise or consist of an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the polypeptide may comprise or consist of an amino acid sequence that shares a sequence identity of at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent with SEQ ID NO: 1 or SEQ ID NO: 3, or with a portion of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the sequence identity may be shared over the full length of
  • SEQ ID NO: 1 or SEQ ID NO: 3 refers to the complete amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, respectively.
  • the polypeptide may comprise or consist of an amino acid sequence that shares sequence identity with:
  • a polypeptide comprising such an amino acid sequence may comprise a TSR domain.
  • the polypeptide may comprise or consist of an amino acid sequence that shares sequence identity with:
  • a polypeptide comprising or consisting of such an amino acid sequence may comprise or consist of an AMOP domain.
  • polypeptide may comprise or consist of combinations of the above amino acid sequences.
  • polypeptide may comprise or consist of:
  • the polypeptide may comprise or consist of:
  • polypeptide (b) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
  • the polypeptide may comprise or consist of:
  • the polypeptide may comprise or consist of:
  • a polypeptide comprising or consisting of such an amino acid sequence i.e. an amino acid sequence comprising or consisting of an (a) portion and a (b) portion as described above, may comprise a TSR domain, e.g. in portion (a), and an AMOP domain, e.g. in portion (b) .
  • the polypeptide may comprise a linker that is inserted between the (a) portion and the (b) portion.
  • a linker may be one or more amino acids, e.g. a peptide, polypeptide, polypeptide domain, or a nucleic acid, organic chemical, or any other molecule.
  • the linker may be linked, coupled, bound, and/or attached to the (a) and/or (b) portion via covalent bonding and/or non-covalent bonding, e.g. immunogenic bonding, ionic bonding, dipole-dipole attractions or hydrogen bonding.
  • the (a) portion may be contiguous with the (b) portion, e.g. there may not be any linker molecule.
  • Polypeptides comprising or consisting, of fragments of SEQ ID NO: 1 or SEQ ID NO: 3
  • the polypeptide may comprise or consist of a fragment of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the polypeptide may comprise or consist of a fragment of:
  • Such a fragment may comprise a TSR domain.
  • the polypeptide may comprise or consist of a fragment of:
  • Such a fragment may comprise an AMOP domain.
  • polypeptide may comprise combinations of the above amino acid sequences.
  • the polypeptide may comprise or consist of: (a) a fragment of the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and
  • polypeptide (b) a fragment of the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the amino acid sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
  • the polypeptide may comprise or consist of:
  • the polypeptide may comprise or consist of: (a) a fragment of the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; and
  • the polypeptide may comprise or consist of:
  • a polypeptide comprising, or consisting of, such an amino acid sequence i.e. an amino acid sequence comprising an (a) portion and a (b) portion, may comprise a TSR domain and an AMOP domain.
  • the polypeptide may or may not comprise a linker that is inserted between the (a) portion and the (b) portion.
  • the fragment of SEQ ID NO: 1 or SEQ ID NO: 3 may have a length anywhere between the maximum length and minimum length as indicated below. Preferably the fragment is at least 6 amino acids in length.
  • the fragment may comprise at least one amino acid of SEQ ID NO: 1 that is not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1.
  • the fragment may comprise at least one amino acid of SEQ ID NO: 3 that is not from the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3.
  • the fragment may comprise at least 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.
  • amino acids e.g. contiguous amino acids, of SEQ ID NO: 1 or SEQ ID NO: 3 that are not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or 218 to 262 of SEQ ID NO: 3 respectively.
  • the fragment comprises such amino acids from the N- terminal side of amino acid 215 of SEQ ID NO: 1 or 218 of SEQ ID NO: 3.
  • the polypeptide may comprise the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, from the N-terminal side of the TSR domain in SEQ ID NO: 1 or SEQ ID NO: 3.
  • a polypeptide comprises the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, or otherwise, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation.
  • a polypeptide having one or both of these activities may comprises one or more additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 from outside the TSR domain.
  • polypeptide may comprise at least 46 amino acids, e.g. the polypeptide may comprise more amino acids than the number of amino acids in the TSR domain.
  • Polypeptides comprising or consisting of an amino acid sequence sharing sequence identity with fragments of SEQ ID NO: 1 or SEQ ID NO: 3
  • the polypeptide may comprise or consist of an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the sequence identity shared may be at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity.
  • the polypeptide may comprise or consist of:
  • Such polypeptide may comprise a TSR domain.
  • the polypeptide may comprise or consist of:
  • polypeptide may comprise an AMOP domain.
  • the polypeptide may comprise or consist of combinations of the above amino acid sequences.
  • the polypeptide may comprise :
  • the polypeptide may comprise or consist of:
  • the polypeptide may comprise or consist of:
  • the polypeptide may comprise or consist of:
  • a polypeptide comprising or consisting of such an amino acid sequence i.e. an amino acid sequence comprising an (a) portion and a (b) portion, may comprise a TSR domain, e.g. in the (a) portion, and an AMOP domain, e.g. in the (b) portion.
  • the polypeptide may or may not comprise a linker that is inserted between the (a) portion and the (b) portion.
  • the fragment of SEQ ID NO: 1 or SEQ ID NO: 3 may have a length anywhere between the maximum length and minimum length as indicated below.
  • the fragment is at least 10 amino acids in length when the shared sequence identity is at least 60 percent.
  • any polypeptide of the invention comprises an amino acid sequence sharing sequence identity as specified above with SEQ ID NO: 1 or a portion of SEQ ID NO: 1, it may comprise at least one amino acid that corresponds to an amino acid in the sequence of SEQ ID NO: 1 that is not an amino acid from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1.
  • any polypeptide of the invention comprises an amino acid sequence sharing sequence identity as specified above with SEQ ID NO: 3 or a portion of SEQ ID NO: 3
  • it may comprise at least one amino acid that corresponds to an amino acid in the sequence of SEQ ID NO: 3 that is not an amino acid from the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3.
  • the amino acid sequence may comprise at least 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350 amino acids, e.g. contiguous amino acids, that correspond to the same number of amino acids in the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and that are not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or 218 to 262 of SEQ ID NO: 3, respectively.
  • the polypeptide may comprise an amino acid sequence sharing sequence identity as specified above with the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, that correspond to amino acids from the N-terminal side of the TSR domain in SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, that correspond to amino acids from the N-terminal side of the TSR domain in SEQ ID NO: 1 or SEQ
  • polypeptide comprises an amino acid sequence sharing sequence identity as specified above with the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation.
  • a polypeptide having one or both of these activities may comprise one or more additional amino acids that correspond to amino acids in SEQ ID NO: 1 or SEQ ID NO: 3 that are outside the TSR domain.
  • Polypeptides comprising or consisting of portions of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the polypeptide may comprise or consist of a portion of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the polypeptide may comprise or consist of:
  • the polypeptide may comprise or consist of: (4) the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3; or
  • the polypeptide may comprise or consist of:
  • the polypeptide may comprise or consist of:
  • the polypeptide may comprise or consist of: (a) the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and
  • Polypeptides that include the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3 may also include additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 respectively.
  • polypeptides that include the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or amino acids 218 to 262 or SEQ ID NO: 3 may include additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 on the N-terminal and/or C-terminal side of these sequences.
  • polypeptides that include the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3 may also include additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 respectively.
  • polypeptides that include the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3 may include additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 on the N-terminal and/or C-terminal side of amino acids 286 to 449 of these sequences.
  • polypeptide comprises the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation.
  • a polypeptide having one or both of these activities may comprises one or more additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 from outside the TSR domain.
  • a polypeptide that comprises an amino acid sequence sharing sequence identity with a specified sequence from SEQ ID NO: 1, that comprises a fragment of SEQ ID NO: 1, that comprise an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 1, or that comprises a specified sequence from SEQ ID NO: 1 may comprise or consist of: an amino acid sequence sharing sequence identity with, a fragment comprising the amino acid sequence of, an amino acid sequence sharing sequence identity with a fragment comprising the amino acid sequence of, or may comprise, the amino acid sequence: 214-259, 213-259, 212-259, 211-259, 210-259, 209-259, 208-259, 207-259, 206-259, 204-259, 203-259, 202-259, 201-259, 200-259, 190-259, 180-259, 170-259, 160-259, 150-259, 140-259, 140-259, 130-259, 120-259, 110-259, 100-259, 90-259, 80-259, 70-259, 60-259, 50-259, 40
  • a polypeptide that comprises an amino acid sequence sharing sequence identity with a specified sequence from SEQ ID NO: 3, that comprises a fragment of SEQ ID NO: 3, that comprises an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 3, or that comprises a specified sequence from SEQ ID NO: 3, may comprise or consist of: an amino acid sequence sharing sequence identity with, a fragment comprising the amino acid sequence of, an amino acid sequence sharing sequence identity with a fragment comprising the amino acid sequence of, or may comprise, the amino acid sequence: 217-262, 216-262, 216-262, 215-262, 214-262, 213-262, 212-262, 211-262, 210-262, 209-262, 208-262, 207-262, 206-262, 205-262, 204-262, 203-262, 202-262, 201-262, 200-262, 190-262, 180-262, 170-262, 160-262, 150-262, 140-262, 130-262, 120-262, 110-262, 100-262, 90-262, 80
  • the specified sequence includes amino acids X and Y, i.e. the amino acids at the start and end of the specified sequence are included in the sequence.
  • a polypeptide may be a peptide, polypeptide or protein.
  • the polypeptides of the invention may be isolated polypeptides.
  • isolated polypeptide refers to a polypeptide that has undergone some degree of isolation, e.g. by purification.
  • a polypeptide of the invention may be a synthetic peptide, e.g. it may be synthesized by chemical synthesis. Alternatively polypeptides of the invention may be produced recombinantly .
  • the present invention is based on the finding that the Isthmin polypeptide and fragments thereof have anti-angiogenic activity and may be useful in suppressing angiogenesis e.g. in cancer.
  • the Isthmin polypeptide comprises a TSR domain and an AMOP domain. Both of these domains have been shown herein to independently have anti-angiogenic activity.
  • TSR and AMOP domains are generally known and exist in other polypeptides it is expected, based on the results reported herein, that other known TSR and AMOP domains will also exhibit anti-angiogenic activity.
  • the present invention encompasses polypeptides comprising any TSR domain or AMOP domain for use in therapeutic methods of inhibiting angiogenesis .
  • TSR domains are found, for example, in TSP proteins such as TSP-I and TSP- 2.
  • the Isthmin polypeptide and fragments thereof have anti-angiogenic properties that are not shown by the TSR domain alone.
  • the Isthmin polypeptide comprises an AMOP domain and a TSR domain.
  • a polypeptide comprising any AMOP domain and any TSR domain will have useful anti-angiogenic properties and such polypeptides are also encompassed by the present invention.
  • These polypeptides are preferably for use in methods of inhibiting angiogenesis.
  • the polypeptide may be a polypeptide in which the AMOP domain and TSR domain are contiguous or separated by intervening amino acids.
  • the TSR domain and AMOP domain may be separated by a chemical linker which may not be an amino acid linker.
  • Chemical linkers are well-known to the person skilled in the art.
  • at least one of the TSR domain or AMOP domain is an Isthmin TSR or AMOP domain.
  • a polypeptide comprising a TSR domain and the N-terminal portion of Isthmin polypeptide has anti-angiogenic properties that are not shown by the TSR domain alone.
  • the present invention also encompasses polypeptides comprising any TSR domain and the N- terminal portion of an Isthmin polypeptide, e.g. for use in therapeutic methods of inhibiting angiogenesis.
  • the Isthmin N-terminal portion will usually be provided on the N-terminal side of the TSR domain, e.g. contiguously or separated by intervening amino acids.
  • the N-terminal portion and TSR domain may be separated by a non-amino acid linker.
  • the N-terminal portion may, for example, be amino acids 26 to 214 from SEQ ID NO: 1 or amino acids 26 to 217 from SEQ ID NO: 3.
  • a polypeptide comprising a TSR domain and Isthmin N-terminal portion may also comprise an AMOP domain.
  • the present invention also encompasses Isthmin polypeptides in which the TSR domain is replaced with a heterologous TSR domain or in which the AMOP domain is replaced by a heterologous AMOP domain respectively.
  • both the TSR domain and AMOP domain may be replaced by a heterologous TSR and AMOP domain.
  • heterologous means, for example, that the TSR or AMOP domain is not the native Isthmin TSR or AMOP domain, e.g. the AMOP and TSR domains do not occur naturally together in the same polypeptide .
  • the TSR domain, AMOP domain, and/or Istmin N-terminal portion referred to above may share at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity with the mouse or human Isthmin TSR domain, AMOP domain and/or N-terminal portion respectively.
  • the TSR domain over which sequence identity is shared may be amino acids 215 to 259 of SEQ ID NO: 1 or amino acids 218 to 262 of SEQ ID NO: 3
  • the AMOP domain over which sequence identity is shared may be amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3
  • the N-terminal portion over which identity is shared may be amino acids 26 to 214 from SEQ ID NO: 1 or amino acids 26 to 217 from SEQ ID NO: 3.
  • nucleic acid comprising a nucleotide sequence, which nucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or
  • nucleotide sequence sharing at least 60 percent sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
  • the nucleic acids of the invention may be used in methods of medical treatment e.g. inhibition of angiogenesis in an individual, e.g. inhibition of tumour angiogenesis, e.g. inhibition of proliferation of endothelial cells.
  • the method may be prophylactic, e.g. it may be treatment of an individual at risk of unwanted angiogenesis.
  • the nucleic acids may be used in the manufacture of a medicament.
  • nucleic acid in the manufacture of a polypeptide for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis, wherein the nucleic acid comprises a nucleotide sequence comprising:
  • SEQ ID NO: 2 or SEQ ID NO: 4 or (ii) a nucleotide sequence sharing at least 60 percent sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or
  • Nucleic acids of the invention preferably encode polypeptides of the invention.
  • the nucleic acid may or may not encode a TSR domain.
  • the nucleic acid may or may not encode an AMOP domain.
  • the nucleic acid may encode a TSR and an AMOP domain.
  • the nucleic acid may encode a TSR and/or an AMOP domain.
  • Nucleic acids comprising or consisting of a nucleotide sequence sharing sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4
  • Nucleic acids of the invention may comprise a nucleotide sequence sharing at least 60 percent identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the nucleic acid may comprise a nucleotide sequence that shares a sequence identity of at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent with SEQ ID NO: 2 or SEQ ID NO: 4.
  • the sequence identity may be shared over the full length of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the "full length" sequence of SEQ ID NO: 2 or SEQ ID NO: 4 is the complete nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 respectively.
  • the nucleic acid may comprise or consist of a nucleotide sequence that shares sequence identity with:
  • a nucleic acid comprising such a nucleotide sequence may encode a TSR domain.
  • the nucleic acid may comprise or consist of a nucleotide sequence that shares sequence identity with:
  • a nucleic acid comprising such a nucleotide sequence may encode an AMOP domain.
  • the nucleic acid may comprise combinations of the above nucleotide sequences.
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of:
  • a nucleic acid comprising an (a) portion and a (b) portion may encode a TSR domain, e.g. in portion (a), and an AMOP domain, e.g. in portion (b) .
  • the nucleic acid may comprise a nucleotide sequence between portion (a) and (b) , e.g. that encodes a linker.
  • the (a) portion may be contiguous with the (b) portion.
  • the nucleic acid may comprise or consist of a fragment of SEQ
  • the nucleic acid may comprise or consist of a fragment of:
  • Such a fragment may encode a TSR domain.
  • the nucleic acid may comprise or consist of a fragment of:
  • Such a fragment may encode an AMOP domain.
  • the nucleic acid may comprise combinations of the above nucleotide sequences.
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of:
  • a nucleic acid comprising such a combination of nucleotide sequences i.e. a nucleotide sequence comprising an (a) portion and a (b) portion may encode a TSR domain, e.g. in the (a) portion, and an AMOP domain, e.g. in the (b) portion.
  • nucleotide sequence encodes a polypeptide comprising a fragment of SEQ ID NO: 1
  • nucleotide sequence may encode at least one amino acid of SEQ ID NO: 1 that is not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1.
  • nucleotide sequence encodes a polypeptide comprising a fragment of SEQ ID NO: 3
  • nucleotide sequence may encode at least one amino acid of SEQ ID NO: 3 that is not from the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3.
  • the nucleotide sequence may encode at least 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.
  • amino acids e.g. contiguous amino acids, of SEQ ID NO: 1 or SEQ ID NO: 3 that are not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or 218 to 262 of SEQ ID NO: 3 respectively.
  • a nucleotide sequence may encode a polypeptide comprising the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, from the N- terminal side of the TSR domain in SEQ ID NO: 1 or SEQ ID NO: 3.
  • the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation.
  • a nucleotide sequence encoding a polypeptide having one or both of these activities may encode one or more amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 from outside the TSR domain.
  • Nucleic acids comprising or consisting of a nucleotide sequence sharing sequence identity with fragments of SEQ ID WO: 2 or SEQ ID NO: 4
  • the nucleic acid may be a nucleic acid sequence sharing sequence identity with a fragment of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the sequence identity shared may be 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity.
  • the nucleic acid may comprise or consist of:
  • nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 603 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 81 to 1472 of SEQ ID NO: 4; or
  • nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 678 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 1472 of SEQ ID NO: 4; or (3) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; or (4) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4.
  • Such a nucleotide sequence may encode a TSR domain.
  • the nucleic acid may comprise or consist of:
  • nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4; or
  • nucleic acid may encode an AMOP domain.
  • the nucleic acid may comprise combinations of the above nucleic acid sequences.
  • the nucleic acid may comprise or consist of: (a) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and
  • the nucleic acid may comprise or consist of: (a) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and
  • the nucleic acid may comprise or consist of:
  • nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4.
  • the nucleic acid may comprise or consist of:
  • nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
  • a nucleic acid comprising or consisting of such a nucleotide sequence i.e. a nucleotide sequence comprising an (a) portion and a (b) portion, may encode a TSR domain, e.g. in the (a) portion, and an AMOP, e.g. in the (b) portion.
  • nucleotide sequence encodes an amino acid sequence sharing sequence identity as specified above with SEQ ID NO: 1, or a portion of SEQ ID NO: 1, it may encode at least one amino acid that corresponds to an amino acid in the sequence of SEQ ID NO: 1 that is not an amino acid from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1.
  • nucleotide sequence encodes an amino acid sequence sharing sequence identity as specified above with SEQ ID NO: 3, or a portion of SEQ ID NO: 3, it may encode at least one amino acid that corresponds to an amino acid in the sequence of SEQ ID NO: 3 that is not an amino acid from the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3.
  • the nucleotide sequence may encode at least 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, e.g. contiguous amino acids, that correspond to the same number of amino acids in the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and which are not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or 218 to 262 of SEQ ID NO: 3 respectively.
  • a nucleotide sequence may encode a polypeptide comprising an amino acid sequence sharing sequence identity as specified above with the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, that correspond to amino acids from the N-terminal side of the TSR domain in SEQ ID NO: 1 or SEQ ID NO: 3.
  • the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation.
  • a nucleotide sequence encoding a polypeptide having one or both of these activities may encode one or more additional amino acids that correspond to amino acids in SEQ ID NO: 1 or SEQ ID NO: 3 that are outside the TSR domain.
  • nucleic acids comprising or consisting of portions of SEQ ID NO: 2 or SEQ ID NO: 4
  • the nucleic acid may comprise a portion of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the nucleic acid may comprise or consist of:
  • nucleotide sequence consisting of nucleotides 603 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 81 to 1472 of SEQ ID NO: 4.
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of: (a) the nucleotide sequence consisting of nucleotides 1245 to
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of:
  • the nucleic acid may comprise or consist of:
  • Nucleic acids that encode the amino acid sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3 may also encode additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 respectively.
  • nucleic acids that encode the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or amino acids 218 to 262 or SEQ ID NO: 3 may encode additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 on the N-terminal and/or C-terminal side of these sequences.
  • nucleic acids that encode the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3 may also encode additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 respectively.
  • nucleic acids that encode the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3 may encode additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 on the N-terminal and/or C-terminal side of these sequences.
  • nucleotide sequence encodes a polypeptide comprising the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3
  • the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation.
  • a nucleotide sequence encoding a polypeptide having one or both of these activities may encode one or more amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 from outside the TSR domain.
  • a nucleic acid that encodes an amino acid sequence sharing sequence identity with a specified sequence from SEQ ID NO: 1, that encodes a fragment of SEQ ID NO: 1, that encodes an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 1, or that encodes a specified sequence from SEQ ID NO: 1, may encode: an amino acid sequence sharing sequence identity with the amino acid sequence of, a fragment comprising the amino acid sequence of, an amino acid sequence sharing sequence identity with a fragment comprising the amino acid sequence of, or may comprise amino acids:
  • a nucleic acid that encodes an amino acid sequence sharing sequence identity with a specified sequence from SEQ ID NO: 3, that encodes a fragment of SEQ ID NO: 3, that encodes an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 3, or that encodes a specified sequence from SEQ ID NO: 3, may encode: an amino acid sequence sharing sequence identity with the amino acid sequence of, a fragment comprising the amino acid sequence of, an amino acid sequence sharing homology with a fragment comprising the amino acid sequence of, or may comprise the amino acid sequence: 217-262, 216-262, 216-262, 215-262, 214-262, 213-262, 212-262, 211-262, 210-262, 209-262, 208-262, 207-262, 206-262, 205-262, 204-262, 203-262, 202-262, 201-262, 200-262, 190-262, 180-262, 170-262, 160-262, 150-262, 140-262, 130-262, 120-262, 110-262, 100-262
  • nucleotides X and Y a sequence consisting of nucleotides X to Y
  • the specified sequence includes nucleotides X and Y, i.e. the nucleotides at the start and end of the specified sequence are included in the sequence.
  • a nucleic acid may be any nucleic acid, either DNA or RNA.
  • the invention includes nucleic acids that are complementary to the nucleic acids described herein and RNA transcripts of the nucleic acids described herein.
  • the nucleic acids are preferably isolated nucleic acids.
  • isolated nucleic acid refers to a nucleic acid that has undergone some degree of isolation, e.g. by purification.
  • nucleic acid of the invention may be a synthetic nucleic acid, e.g. it may be synthesized by chemical synthesis. Alternatively nucleic acid of the invention may be produced recombinantly .
  • a fragment of SEQ ID NO: 1, 2, 3, or 4 including fragments of sequences within SEQ ID NO: 1, 2, 3, or 4 may be of defined length and/or may have a defined minimum and/or maximum length.
  • a fragment may comprise at least, i.e. have a minimum length of, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the corresponding full length sequence.
  • the fragment may have a maximum length, i.e. be no longer than, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the corresponding full length sequence .
  • a fragment of SEQ ID NO: 1 or SEQ ID NO: 3 may comprise at least, i.e. have a minimum length of, 5, 6, 7, 8, 9, 10, 11,
  • a fragment of SEQ ID NO: 3 may comprise at least 461, 462, or 463 contiguous amino acids of SEQ ID NO: 3.
  • a fragment of SEQ ID NO: 1 or SEQ ID NO: 3 may have a maximum length of, i.e. be no longer than, 5, 6, 7, 8, 9, 10, 11, 12,
  • a fragment of SEQ ID NO: 3 may have a maximum length of 461, 462, or 463 contiguous amino acids of SEQ ID NO: 3.
  • a fragment of SEQ ID NO: 2 or SEQ ID NO: 4 may comprise at least, i.e. have a minimum length of, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
  • a fragment of SEQ ID NO: 4 may comprise at least 2300, 2400, 2500, 2600, or 2666 contiguous nucleotides of SEQ ID NO: 4.
  • a fragment of SEQ ID NO: 2 or SEQ ID NO: 4 may have a maximum length of, i.e. be no longer than, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
  • Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequence (referred to by the SEQ ID No.) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences .
  • sequence identity of the shorter comparison sequence may be determined over the entire length of the longer given sequence or, where the comparison sequence is longer than the given sequence, sequence identity of the comparison sequence may be determined over the entire length of the shorter given sequence.
  • the candidate sequence can only have a maximum identity of 10% to the entire length of the given sequence. This is further illustrated in the following example:
  • the given sequence may, for example, be that encoding SEQ ID NO: 1) .
  • sequence identity may be determined over the entire length of the given sequence. For example:
  • the given sequence may, for example, be that encoding SEQ ID NO: 1
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalW 1.82.
  • the default parameters e.g. for gap penalty and extension penalty, are preferably used.
  • Identity of nucleic acid sequences may be determined in a similar manner involving aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and calculating sequence identity over the entire length of the respective sequences. Where the aligned sequences are of different length, sequence identity may be determined as described above and illustrated in examples (A) and (B) .
  • amino acid in a sequence of interest that is specified as corresponding to a particular amino acid in a given sequence, e.g. SEQ ID NO: 1, means that the amino acid in the sequence of interest is paired with an identical amino acid in the given sequence when the sequences are pairwise aligned.
  • Correspondence is determined as follows: the amino acid sequence of interest and the given sequence are pairwise aligned, for example using the ClustalW 1.82 software, using the default parameters.
  • the ClustalW software may be accessed from the European Bioinformatics institute at http://www.ebi.ac.uk/clustalw/.
  • the pairwise alignment will result in amino acids in the sequence of interest being paired with amino acids in the given sequence.
  • a particular amino acid in the sequence of interest "corresponds" to the amino acid in the given sequence if it is paired with an identical amino acid in the given sequence.
  • operably linked may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide sequence under the influence or control of the regulatory sequence.
  • a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide sequence which forms part or all of the selected nucleotide sequence.
  • the resulting transcript may then be translated into a desired protein or polypeptide.
  • the vectors of the invention may include one or more elements that facilitate expression of nucleotide sequence in a host cell.
  • the vector may include an element that allows the vector to replicate in a host cell.
  • the vector may include an element that allows selection of host cells that contain the vector, e.g. a marker gene.
  • Host cells comprising a nucleic acid of the invention.
  • the host cell may be a eukaryotic cell, e.g. a yeast or a CHO cell, or a prokaryotic cell, e.g. E. coli.
  • the host cell may be a cell that is suitable for culturing, i.e. it may be a cell that is not part of a living human or animal body.
  • the nucleic acid of the invention may be present in the host cell as part of the host cell genome.
  • the nucleic acid may be present in the host cell as an autonomously replicating entity, e.g. a plasmid.
  • the nucleic acid is heterologous to the host cell, i.e. the nucleic acid is not naturally present in the host cell.
  • the nucleic acid may be inserted into the genome, e.g. by genetic engineering.
  • Endothelial cell tube formation The polypeptides of the invention may be assessed for their ability to inhibit endothelial cell tube formation.
  • a method of observing this ability may include evaluating, monitoring, measuring, determining, and/or quantitating the ability of the polypeptide to inhibit endothelial cell tube formation.
  • the method may involve culturing endothelial cells on extracellular matrix (ECM) e.g. Matrigel, ECMatrix, which is a solid gel consisting of various basement proteins, such as laminin, collagen type IV and heparin sulphate proteoglycans.
  • ECM extracellular matrix
  • Endothelial cells may be mixed with the polypeptide and added to the ECM. The ECM may then be incubated and tube assembly may be observed after a suitable time period, e.g. 6 hours. This method is described in the Results and Methods below.
  • the extent of tube assembly may be quantitated by measuring the length of tubes and an average length may be calculated.
  • a reduction in tube length relative to the tube length in the absence of the polypeptide is indicative that the polypeptide inhibits endothelial cell tube formation.
  • the polypeptide may reduce endothelial cell tube formation by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or even 90 percent relative to the absence of the polypeptide. Such a reduction may be observed after incubating the endothelial cells with 10OnM polypeptide on ECM for 6 hours.
  • the polypeptides of the invention may be assessed for their ability to inhibit proliferation of endothelial cells.
  • a method of observing this ability may include evaluating, monitoring, measuring, determining, and/or quantitating the ability of the polypeptide to inhibit proliferation of endothelial cells.
  • the method may involve incubating endothelial cells with the polypeptide for a period of time, e.g. 24 hours, and adding Bromodeoxyuridine (BrdU) at the end of the incubation period.
  • BrdU may then be detected immunochemically by partially denaturing double stranded DNA.
  • BrdU is incorporated into newly synthesized strands of DNA of actively proliferating cells and therefore the amount of BrdU detected indicates the number of cells that are synthesizing DNA. This method is described in the Results and Methods below.
  • the extent endothelial cell proliferation may by quantitated by measuring the amount of BrdU in the assay.
  • a reduction in the amount of BrdU relative to the amount in the absence of the polypeptide is indicative that the polypeptide inhibits endothelial cell proliferation.
  • the polypeptide may reduce endothelial cell tube formation by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or even 90 percent relative to the absence of the polypeptide. Such a reduction may be observed after incubating endothelial cells with 10OnM polypeptide .
  • the polypeptides of the invention may be assessed for their ability to induce apoptosis of endothelial cells.
  • a method of observing this ability may include evaluating, monitoring, measuring, determining, and/or quantitating the ability of the polypeptide to stimulate apoptosis of endothelial cells.
  • the method may involve incubating endothelial cells with the polypeptide for a period of time, e.g. 24 hours, together with VEGF. The method may then involve lysing the cells and observing the mononucleosomes and oligonucleosomes in the cytoplasmic fraction of the cell lysate. Mononucleosomes and oligonucleosomes may be observed using ELISA. This method is described in the Results and Methods below.
  • the extent of endothelial cell apoptosis may be quantitated by measuring the amount of mononucleosomes and oligonucleosomes in the cell lysate. An increase in the amount of mononucleosomes and oligonucleosomes relative to the amount in the absence of the polypeptide is indicative that the polypeptide induces apoptosis.
  • the polypeptide may increase apoptosis by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or even 90 percent relative to the absence of the polypeptide. Such an increase may be observed after incubating 10OnM polypeptide with endothelial cells for 24 hours in the presence of lOng/ml VEGF.
  • variants of Isthmin are polypeptides that do not include the exact amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and nucleic acids that do not include the exact nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • variants of Isthmin truncates are polypeptide and nucleic acids that do not include the exact amino acid sequence or nucleotide sequence of the mouse Isthmin truncates described in the Examples, or the corresponding human Isthmin truncates.
  • the mouse Isthmin truncates and corresponding coding sequences used in the Examples are amino acids 26 to 461 of SEQ ID NO: 1; amino acids 26 to 277 of SEQ ID NO: 1; amino acids 215 to 259 of SEQ ID NO: 1; amino acids 271 to 461 of SEQ ID NO: 1;
  • nucleotides 678 to 1985 of SEQ ID NO: 2 nucleotides 678 to 1433 of SEQ ID NO: 2; nucleotides 1245 to 1379 of SEQ ID NO: 2; nucleotides 1413 to 1985 of SEQ ID NO: 2;
  • amino acids 26 to 464 of SEQ ID NO: 3 amino acids 26 to 280 of SEQ ID NO: 3; amino acids 218 to 262 of SEQ ID NO: 3; amino acids 274 to 464 of SEQ ID NO: 3;
  • nucleotides 156 to 1472 of SEQ ID NO: 4 nucleotides 156 to 920 of SEQ ID NO: 4; nucleotides 723 to 866 of SEQ ID NO: 4; nucleotides 902 to 1472 of SEQ ID NO: 4.
  • Variants of the present invention preferably have the ability to inhibit angiogenesis and are preferably for use in methods of medical treatment.
  • the variant molecules of the invention may have the ability to inhibit endothelial cell tube formation, inhibit proliferation of endothelial cells, and/or induce apoptosis in endothelial cells in an in vitro assay.
  • a variant according to the invention has a similar, or at least the same, ability to inhibit angiogenesis e.g. as measured by any of these in vitro assays.
  • a "similar ability" means that the ability of the variant to inhibit angiogenesis is at least 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent of the ability of Isthmin to inhibit angiogenesis.
  • this refers to the activity of the polypeptide encoded by the nucleic acid molecule.
  • Variants of the present invention may be: (i) novel, naturally occurring, molecules, for example obtainable from organisms other than mouse or human, and which may be identified by hybridisation, for example.
  • artificial Isthmin molecule derivatives which can be prepared by the skilled person in the light of the present disclosure. Such derivatives may be prepared, for instance, by site directed or random mutagenesis, or by direct synthesis.
  • a variant nucleic acid for example, is generated either directly or indirectly, e.g. via one or more amplification or replication steps from an original nucleic acid having all or part of the sequence shown herein.
  • Particularly included are truncated variants which include only a distinctive part or fragment, however produced, corresponding to a portion of the sequences described herein, for example functional parts of the polypeptide that are still capable of inhibiting angiogenesis .
  • polypeptides of the invention may also comprise additional amino acids, additional domains, or may be conjugated to additional domains or other molecules. Additional amino acids, domains, or molecules conjugated to the polypeptide may provide an additional function, for example in assisting purification of the polypeptide. Examples of additional domains that may assist in purification of the polypeptide are 6-histidine tag and glutathione S-transferase tag. Polypeptides may be fusion proteins, fused to a peptide or other protein, such as a label, which may be, for instance, bioactive, radioactive, enzymatic or fluorescent.
  • Variant polypeptides may comprise at least one modification substitution, inversion and/or deletion of one or more amino acids .
  • conservative replacements which may be found in such polymorphisms may be between amino acids within the following groups: alanine, serine, threonine; glutamic acid and aspartic acid; arginine and leucine; asparagine and glutamine; isoleucine, leucine and valine; phenylalanine, tyrosine and tryptophan.
  • Variants of Isthmin may be produced by modifying SEQ ID NO: 1, 2, 3, or 4, or any of the Isthmin truncates described above.
  • a polypeptide of the invention may be a fragment of SEQ ID NO: 1, SEQ ID NO: 3 or any one of the truncates described above. Such fragments may be provided in isolated form, i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region with one or two non-naturally contiguous sequences fused to it. Additionally, several fragments may be comprised within a single larger polypeptide.
  • Changes to nucleic acid sequences may be desirable for a number of reasons. For instance, they may introduce or remove restriction endonuclease sites or alter codon usage. Alternatively changes to a sequence may produce a derivative by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.
  • Such changes may modify sites which are required for post translation modification, such as cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for glycosylation, lipolysation etc.
  • Leader or other targeting sequences e.g. membrane or golgi locating sequences
  • Other desirable mutations may be random or site directed mutations in order to alter the activity, e.g. specificity, or stability of the encoded polypeptide. Changes may be by way of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
  • altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining peptide conformation.
  • variants having non-conservative substitutions are also included.
  • substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure.
  • regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.
  • variants of the invention may also be created by chemical modification of Isthmin or any of the Isthmin truncates described above. Methods for chemical modification of polypeptides are well known in the art.
  • a polypeptide of the invention may be obtained by expression of a nucleic acid that encodes the polypeptide using a suitable vector and host organism.
  • suitable vectors and hosts are well known in the art (see e.g. Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York) .
  • Polypeptides, and particularly fragments, of the invention may also be created using chemical synthesis by any suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
  • the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. Many such methods are now commonplace to those skilled in the art.
  • variants of the invention may also be created by modification, using molecular biological techniques, of the nucleic acid that encodes Isthmin or any of the Isthmin truncates described above, or a nucleic acid that encodes a further variant or homologue of the invention.
  • Molecular biological techniques are well known in the art.
  • the invention provides a method of producing and/or identifying a variant of Isthmin, which method comprises the steps of: (i) providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or any of the Isthmin truncates described above, or the nucleic acid sequence encoding these,
  • Step (iii) may involve measuring the ability of the variant to inhibit endothelial cell tube formation, inhibit proliferation of endothelial cells, and/or induce apoptosis in endothelial cells in an in vitro assay.
  • Antibodies to Isthmin, or variants of Isthmin may also be used to screen for Isthmin, which methods are well known to those skilled in the art.
  • the Isthmin amino acid or nucleotide sequence i.e. SEQ ID NO: 1 or 3, or SEQ ID NO: 2 or 4 may be used in a data-base (e.g. of ESTs, or STSs) search to find sequences that share a specified level of sequence identity, such as those which may become available in due course, and expression products of which can be tested for activity as described herein.
  • a data-base e.g. of ESTs, or STSs
  • variants may be provided by standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes.
  • Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter.
  • Labelled probe may be hybridised to the DNA fragments on the filter and binding determined.
  • DNA for probing may be prepared from RNA preparations from cells.
  • Probing may optionally be done by means of so-called “nucleic acid chips”, see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review.
  • nucleic acid molecules may be created which may be screened for nucleic acids that encode a polypeptide of the invention. Such libraries may be created using the nucleic acid molecule that encodes Isthmin. The Isthmin nucleic acid molecule sequence may be mutated, for example using random PCR mutagenesis, or gene shuffling techniques. The resulting mutated nucleic acid molecules may be ligated into a vector. Libraries of polypeptides may be created, by expressing the nucleic acid molecules in a suitable host.
  • the present invention includes nucleic acid that hybridise to SEQ ID NO: 2 or SEQ ID NO: 4, and nucleic acids that hybridise to nucleotides 603 to 1985 of SEQ ID NO: 2; nucleotides 678 to 1985 of SEQ ID NO: 2; nucleotides 678 to 1433 of SEQ ID NO: 2; nucleotides 1245 to 1379 of SEQ ID NO: 2; nucleotides 1413 to 1985 of SEQ ID NO: 2; nucleotides 1458 to 1949 of SEQ ID NO: 2; nucleotides 81 to 1472 of SEQ ID NO: 4; nucleotides 156 to 1472 of SEQ ID NO: 4; nucleotides 156 to 920 of SEQ ID NO: 4; nucleotides 723 to 866 of SEQ ID NO: 4; nucleotides ' 902 to 1472 of SEQ ID NO: 4; and/or nucleotides 945 to 1436 of SEQ ID NO:
  • nucleic acid sequences may be identified by using hybridization and washing conditions of appropriate stringency.
  • Complementary nucleic acid sequences will hybridise to one another through Watson-Crick binding interactions. Sequences which are not 100% complementary may also hybridise but the strength of the hybridisation usually decreases with the decrease in complementarity. The strength of hybridisation can therefore be used to distinguish the degree of complementarity of sequences capable of binding to each other.
  • the stringency of a given reaction may depend upon factors such as probe length, washing temperature, and salt concentration. Higher temperatures are generally required for proper annealing of long probes, while shorter probes may be annealed at lower temperatures. The higher the degree of desired complementarity between the probe and hybridisable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so.
  • hybridizations may be performed, according to the method of Sambrook et al., ("Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 0.5-1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.
  • Hybridization is carried out at 37-42 0 C for at least six hours.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • Tm melting temperature
  • n is the number of bases in the oligonucleotide.
  • Hybridisation under high stringency conditions may involve performing the hybridisation at a temperature of Tm-15 or higher.
  • Moderate stringency may be considered to be Tm-25 to Tm-15.
  • Low stringency may be considered to be Tm-35 to Tm-25.
  • nucleotide sequences can be categorised by an ability to hybridise to a target sequence under different hybridisation and washing stringency conditions which can be selected by using the above equation.
  • the Tm may be used to provide an indicator of the strength of the hybridisation.
  • Sequences exhibiting 95-100% sequence complementarity are considered to hybridise under very high stringency conditions, sequences exhibiting 85-95% complementarity are considered to hybridise under high stringency conditions, sequences exhibiting 70-85% complementarity are considered to hybridise under intermediate stringency conditions, sequences exhibiting 60-70% complementarity are considered to hybridise under low stringency conditions and sequences exhibiting 50-60% complementarity are considered to hybridise under very low stringency conditions.
  • a cancerous condition that be may treated by the present invention may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation) , neoplasm or tumour or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumour.
  • the cancerous condition may be a cancer and may be a benign or malignant cancer and may be primary or secondary (metastatic) .
  • a neoplasm or tumour may be any abnormal growth or proliferation of cells and may be located in any tissue. Examples of tissues include the colon, pancreas, lung, breast, uterus, stomach, kidney, testis, central nervous system
  • peripheral nervous system including the brain
  • skin including the brain
  • blood or lymph including the brain
  • a cancerous condition may be treated by inhibiting angiogenesis .
  • Inhibition of angiogenesis may be inhibition of angiogenesis in a tumour that is, or is not, inducing angiogenesis.
  • Inhibition of angiogenesis in a tumour that is not inducing angiogenesis may prevent the tumour from inducing angiogenesis .
  • Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, sublingual, oral and nasal.
  • the medicaments and compositions may be formulated in fluid or solid form.
  • Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g.
  • the invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
  • Figure 1 shows diagrams illustrating the domain organization of four different forms of truncated Isthmin. These are: Full Length ISM - SEQ ID NO: 1; Recombinant ISM-FL - amino acids 26 to 461 of SEQ ID NO: 1; Recombinant ISM-N - amino acids 26 to 277 of SEQ ID NO: 1; Recombinant ISM-C - amino acids 271 to 461 of SEQ ID NO: 1; Recombinant ISM-TSR - amino acids 215 to 259 of SEQ ID NO: 1.
  • ISM-FL includes TSR and AMOP
  • ISM-N includes TSR and an N- terminal region with no known protein domain
  • ISM-C includes AMOP.
  • Signal peptide was cleaved and His-tag was added at C- terminal of each recombinant protein.
  • Native Isthmin contains a signal peptide when synthesized but no His-tag. The mature, secreted Isthmin should have , no signal peptide as it has been cleaved during the secretion process. A key to the domains is shown at the bottom of the Figure.
  • Figure 2 shows the results of experiments demonstrating that Isthmin inhibits EC tube formation on Matrigel.
  • it shows representative photos showing the inhibitory effect of ISM-C, -N, -FL on EC tube formation assay in the presence of 2%FBS as control, (i) shows control, (ii) shows ISM-FL, (i ⁇ ) shows ISM-N, (iv) shows ISM-C, (v) shows ISM-TSR.
  • ISM- TSR had no effect on EC tube formation.
  • Figure 3 shows results of adding recombinant Isthmin at different time-points during the EC tube formation process (Oh, Ih, 2h, 4h after plating ECs onto Matrigel) .
  • Investigation of ISM-C, -N and -FL showed significant influences to tube assembly when adding Isthmin at the early stage as depicted at 0 hours, (i) shows control, (ii) shows ISM-FL, (iii) shows ISM-N, (iv) shows ISM-C, (v) shows ISM- TSR.
  • ISM-TSR had no observable effect on tube formation.
  • Figure 4 shows the results of experiments investigating the role of Isthmin in EC migration in the presence (A) and absence (B) of VEGF. No significant effect of Isthmin on EC migration was observed with or without VEGF. Normalisation of the data values were performed with respect to the data value for migration with VEGF for (A) and control for (B) .
  • Figure 5 shows the results of experiments demonstrating that Isthmin inhibits VEGF-induced EC proliferation. Normalisation of measured values was performed against the data value for control .
  • Figure 6 shows the results of experiments investigating induction of EC apoptosis by Isthmin. Quantification of apoptosis was performed using cell death ELISA, which measures fragmented DNA in the presence of VEGF. ISM-FL induced EC apoptosis in a dose-dependent manner.
  • Figure 7 shows the results of experiments investigating a role for Isthmin in zebrafish embryonic angiogenesis .
  • Figure 7 shows representative microphotographs of zebrafish Isthmin 1 gene (zlsml) morpholino antisense oligonucleotide (MO) knockdown and Mismatch MO control in Tg (fli-l:GFP) transgenic zebrafish under the same magnification.
  • (A) shows wild-type
  • (B) shows MO control
  • C) shows zlsml MO.
  • the impaired intersegmental vessels in zlsml morpholino injected larvae is indicated by a box in (C) . No abnormal blood vessel can be seen in the wild-type and Morpholino control group.
  • Figure 8 shows representative microphotographs of zebrafish Isthmin 1 gene (zlsml) morpholino antisense oligonucleotide (MO) knockdown and Mismatch MO control in Tg (fl
  • Figure 8 shows amino acid and nucleotide sequences used in the of mouse Isthmin and truncates used during the experiments, as well as human Isthmin.
  • (A) shows the amino acid sequence of mouse Isthmin (SEQ ID NO:
  • (B) shows the nucleotide sequence of mouse Isthmin (SEQ ID NO: 2) .
  • (C) shows the amino acid sequence of human Isthmin (SEQ ID NO: 3) .
  • (D) shows the nucleotide sequence of human Isthmin (SEQ ID NO: 4) .
  • (E) shows the amino acid sequence of mouse ISM-FL (SEQ ID NO: 5) - (F) shows the nucleotide sequence used to generate mouse ISM-
  • Figure 9 shows the amino acid and nucleotide sequences of Zebrafish Isthminl and Xenopus laevis Isthmin.
  • A shows the amino acid sequence of Zebrafish Isthminl (SEQ ID NO: 13) . Accession no. NP_001012376.1 (GI : 59933256) .
  • (B) shows the nucleotide sequence of Zebrafish Isthminl (SEQ ID NO: 14. Accession no. NM_001012376.1 (GI : 59933255) .
  • (C) shows the amino acid sequence of Xenopus laevis Isthmin
  • Figure 10 shows the results of experiments investigating whether Isthmin inhibits tumor growth in vivo.
  • A (i) shows control Bl 6 mouse melanomas (modified with empty vector) and (ii) shows ISM-FL overexpressing mouse melanomas.
  • B shows the growth curve of control and ISM-expressing melanomas. ISM- FL significantly suppressed subcutaneous B16 tumor growth in mouse .
  • Tube formation is a multiple process involving migration, cell adhesion and differentiation.
  • EC rapidly align and form hollow tubular structures when cultured on ECMatrix, which is a solid gel consisting of various basement proteins, such as laminin, collagen type IV and heparan sulfate proteoglycans etc. (Grant et al., 1989; Kubota et al., 1988; Kleinman et al.,1986) .
  • Premixed endothelial cell suspension with different concentrations of Isthmin and its truncated recombinants were added to the top of the ECMatrix. The extent of tube assembly can be monitored after 6 hours incubation in tissue-culture incubator.
  • the EC tube formation time course was investigated to analyze how Isthmin disrupts tube formation at the cell morphological level.
  • the same concentrations of ISM-TSR, -C, -N, -FL were added to the culture medium at Oh, Ih, 2h and 4h respectively after plating cell on Matrigel.
  • Ih the obvious disrupted extension of tube assemblies were seen in the presence of ISM-C, -N and -FL but not ISM-TSR.
  • tubular structures were not much interrupted in the presence of ISM-C, -N and -FL as compared to the control.
  • Endothelial cell apoptosis Fourthly, we asked whether Isthmin restrains angiogenesis by inducing apoptosis. Induction of apoptosis in endothelial cells is one of the mechanisms that angiogenesis inhibitors work. It has been shown that TSPs inhibit angiogenesis through FasL-Fas signalling pathways which activate caspases and apoptosis (Lucas and Paul., 2003) . DNA degradation occurs several hours before plasma membrane breakdown in the cytoplasm of the apoptotic cell, which leads to the enrichment of mono- and oligonucleosomes .
  • apoptosis was analyzed by determination of mono- and oligonucleosomes in the cytoplasmic fraction of Isthmin-treated cell lysates. As shown in Figure 6, EC apoptosis is induced by ISM-FL. ISM-FL induced EC apoptosis dose-dependently .
  • ISM-FL was cloned into a mammalian expression vector where it is expressed as a secreted extracellular protein.
  • the expression vector was transfected into mouse B16 melanoma cells. Cells stably expressing ISM-FL were selected clonally. Clones that expressed a high level of ISM-FL in the culture media were selected. Matching clones containing empty expression vector were selected as controls. Note that B16 cells express a very low level of ISM endogenously . As shown in Figure 10, ISM expression in B16 tumors significantly suppressed tumor growth compared to controls. The ISM producing tumor was also pale with less blood, presumably from suppression of tumor blood vessel formation. Melanin production by this melanoma cells was also suppressed.
  • Isthmin potently inhibits angiogenesis . It interrupted EC tube formation, inhibited EC proliferation, induced EC apoptosis and suppressed tumour formation in vivo. In our transgenic zebrafish model, the impairment of blood vessels was observed in zlsml knockdown zebrafish. All these indicate that Isthmin has potential applications in the treatment of angiogenesis diseases .
  • Proteins expression and purification Recombinant truncated Isthmin and full length Isthmin were cloned to pET-M (derived from pET-32) vector and induced by IPTG. Protein purification was done under denatured conditions (6M urea) according to the QIAGEN manufacturer protocol.
  • Migration assay was performed using Falcon cell culture insert as descried with modifications (24) .
  • HUVECs were started 4 hours.
  • Various concentrations of proteins were pre-incubated with 20000 cells for 30 min at 37°C before seeding onto the gelatin-coated cell culture inserts while lOng/mlVEGF was placed into the lower chamber.
  • the cell culture insert chamber was then incubated at 37 0 C for 6 hours.
  • cells staying on the insert were removed by cotton swab, cells migrating on the lower surface of the inserts were fixed, stained and photographed under a light microscope (40 * ) .
  • Five random fields (upper, down, left, right and centre) were quantified using Image J 1.32 software.
  • HUVECs were starved 3-4 hours, and then pre-treated with different concentrations of proteins for 30 min. Seeded 20000 cells per well onto the surface of the polymerized Matrigel and incubated for 6 hour in tissue-culture incubator. Normal
  • Matrigel purchased from CHEMICON Inc
  • growth factor reduced Matrigel BD. Inc
  • Cell proliferation was determined using the BrdU (bromodeoxyuridine) kit (CHEMICON Inc) . 20000 cells per well was incubated with 2%FBS with or without lOng/ml VEGF and various concentrations of proteins for 24h. After incubation, the following procedure was according to the manufacturer' s protocol .
  • BrdU bromodeoxyuridine
  • Apoptosis Assay Cells were cultured in 6-well-plate overnight and then incubated with proteins for 24 hours in 2%FBS in the presence of 10ng/ml VEGF. After protein treatment, apoptosis was quantified using a cell death detection ELISA kit (Roche Applied Science) .
  • MO morpholino antisense oligonucleotides
  • the concentrated MOs was diluted into working concentrations of ImM, with nuclease-free water and phenol red dye.
  • each experiment included a control MO group with no binding target and an un-injected control group.
  • Each MO was microinjected into wild type zebrafish embryos and GFP labelled blood vessel transgenic zebrafish embryos at the single cell stage. Microinjection volumes were estimated at InI per embryos. There were 30-40 embryos in each trial group. After injection, embryos were incubated at 37C in embryo medium. Post-injection phenotypes were observed at different time points of the development stages.
  • Synthetic antisense MOs were microinjected into fertilized zebrafish eggs at the 1-2 cell stage. Embryos were observed under fluorescent, microscope at 36-48hpf (hours post fetrtilization) . Intersegmental vessels (ISVs) are usually formed between 24-48hpf.
  • Mouse tumor formation assay ISM-FL was cloned into a mammalian expression vector and stably expressed in mouse B16 melanoma cells using standard molecular and cell biology methods.
  • One million Bl 6 tumor cells, control or ISM-expressing, were injected subcutaneously into the dorsal flank of mice.
  • control cells were injected to the left dorsal flank and ISM expressing tumor cells were injected into the right dorsal flank. Tumors appear after about 6 days post injection. Tumor growth was monitored by measuring the tumor size (length and width) with a digital calliper. Experiments were terminated at 14 days post tumor cell injection.
  • TSR thrombospondin type 1 repeat
  • Angiostatin a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79,315-328,1994. (15) Robert Kerbel and Judah Folkman. Clinical translation of angiogenesis inhibitors. Nature Rev 2:727-739,2002.
  • Asch,A.S., et al Thrombospondin sequence motif (CSVTCG) is responsible for CD36 biding. Biochem. Biophys. Res. Commun.182, 1208- 1217.1992.

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Abstract

The present disclosure relates to polypeptides and nucleic acids encoding the polypeptides and to the use of these in methods of medical treatment including the treatment of angiogenesis. The peptide derivatives are obtained from the secreted protein Isthmin and contain the AMOP domain. Compositions comprising the polypeptides and methods of using them are disclosed.

Description

Angiogenesis
Field of the Invention
The present invention relates to polypeptides and nucleic acids encoding the polypeptides and to the use of these in methods of medical treatment including treatment of conditions involving angiogenesis.
Background to the Invention
Angiogenesis plays a key role in health and diseases. Most angiogenesis occurs in the embryo, where it establishes the primary vascular tree as well as an adequate vasculature for growing and developing organs (Folkman, 1971; 1995; 2007) . Very little turnover of endothelial cells occurs in the adult vasculature except during menstrual cycle and in physiological repair processes such as wound healing. However, a number of disorders are caused by excessive angiogenesis, such as cancer (tumor growth is strikingly angiogenesis dependent) (Carmeliet, 2003) . Therefore, abrogation of angiogenesis is believed to be a promising strategy for treatment of cancer. Many endogenous angiogenesis inhibitors have also been identified including the extracellular matrix (ECM) proteins thrombospondin-1 and -2 (TSP-I and -2) (Iruela-Arispe et al . , 1991; Lawler, 2002, Zhang et al . 2007) .
Thrombospondin type-1 repeats (TSRs) were initially identified in human Thrombospondin-1 (TSP-I) (Lawler & Hynes., 1986; Lawler, 2002) . The human genome has a large number of proteins that contain TSRs (Adam 2000; Tucker, 2004) . All the TSRs are present either in secreted proteins or in the extracellular portion of transmembrane proteins, implicating possible roles in cell migration, cell-cell and cell-matrix adhesion and communication as well as tissue remodelling" (R. Ge et al., unpublished) . Studies of TSRs from TSP-I and other antiangiogenic proteins such as brain-specific angiogenesis inhibitor 1 (BAIl), ADAMTSl and 8, strongly indicate that TSRs play critical roles in the antiangiogenic function of these proteins. Two of the three TSRs in TSP-I are individually antiangiogenic (Iruela- Arispe et al., 1999) . On the other hand, TSRs from properdin and the first TSR of TSP-I are not antiangiogenic (Iruela- Arispe et al. , 1999) .
Crystal structure of TSRs from TSP-I revealed a novel, antiparallel, three-stranded fold that consists of alternating stacked layers of tryptophan and arginine residues from respective strands, capped by disulfide bonds on each end (Tan et al., 2002) . Certain motifs within TSR have been shown to be associated with the anti-angiogenic activity of TSRs in TSP-I. One is the conserved wxxwxxw localized at the C-terminal (Iruela-Arispe 1999), another is the conserved region CSVTCG which binds to TSP-I receptor CD36 on endothelial cell membrane (Dawson et al., 1997) .
Isthmin (ISM) is a secreted protein with no known function. It contains two putative domains in the mature protein: a centrally localized TSR and a C-terminal AMOP (adhesion- associated domain in MUC4 and other proteins) (Pera et al., 2002; Rossi et al . , 2004) . It was first identified in Xenopus as a member of the FGF-8 synexpression group and is prominently expressed at the midbrain-hindbrain boundary (Pera et al . , 2002) . Analysis of various genome databases indicated that orthologous Isthmin genes are present in zebrafish (isthminl), mouse and human. Zebrafish has another homologous gene isthmin2 which is likely the orthologue of the human Isthmin-like gene TAILl (Rossi et al., 2004 and our unpublished data) . In mouse, Isthmin is a predicted secreted protein of 461 amino acids. Isthmin in Xenopus is expressed in paraxial mesoderm and neural folds at the tailbud stage and in notochord and midbrain-hindbrain boundary (MHB) during neurula stage (Pera et al. 2002) . In zebrafish, Isthminl expression could be upregulated by overexpressing of Wnt8 or downregulated by Wnt/ β-Catenin inhibitor (Weidinger et al. 2005) , indicating its involvement in Wnt signaling regulated process during embryonic development. However, no function has been identified for this gene in any species.
AMOP is a protein domain predicted by bioinformatics that has eight invariant cysteine residues and is only found in secreted proteins or in proteins that also contain extracellular domains involved in cell adhesion. Because of its presence in cell adhesion molecules, AMOP domain is speculated to be involved in adhesion processes (Ciccarelli et al. 2002) . Besides the common cysteine residues that are shared by all AMOPs among different proteins, Isthmin AMOP has a "KGD" motif, which is found in many antagonists of platelet aggregation and integrin-dependent cell adhesion, including tumor cell metastasis.
Summary of the Invention
It has now been found that Isthmin and fragments thereof, including the AMOP domain in the absence of the TSR domain, potently inhibit angiogenesis . This has been shown using four different cell assays representing in vitro angiogenesis processes. These are endothelial cell (EC) tube formation, migration, proliferation and apoptosis. In addition, it has been shown that Isthmin has a role in zebrafish embryonic angiogenesis. This was investigated by using morpholino antisense oligonucleotide (MO) knockdown technology. Furthermore, overexpression of Isthmin in mouse melanoma cells inhibited tumor growth and melanin production from these tumor cells .
Using these techniques it has been shown that the recombinant mouse Isthmin protein potently inhibited vascular endothelial growth factor (VEGF) -induced EC proliferation in a dose- dependent manner. Its N-terminal and C-terminal fragments (containing the AMOP domain) also mildly inhibited VEGF- induced EC proliferation while the TSR domain alone was not effective.
It has also been shown that both the N-terminal fragment and C-terminal fragment (containing the AMOP domain) as well as the full-length protein disrupted tubular network on Matrigel, with the full-length protein and C-terminal fragment more potent than N-terminal fragment. Also, full-length Isthmin protein induced EC apoptosis in the presence of VEGF. The TSR domain alone did not have these activities. These results demonstrate that isthmins, and particularly AMOP domains, have significant potential as new therapeutic agents for inhibiting angiogenesis and thereby provide promising new treatments for cancer. The discovery that AMOP domains have angiogenic activity is particularly surprising as these domains previously had no proven function and were merely predicted to be involved in cell adhesion processes.
Broadly the invention relates to Isthmin and derivatives of Isthmin, to AMOP domains, and also to use of these for the treatment, e.g. inhibition, of angiogenesis.
In a first aspect of the invention there is provided a polypeptide comprising:
(i) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or
SEQ ID NO: 3; or (ii) a fragment of the amino acid sequence of SEQ ID NO:
1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ
ID NO: 3, or
(iii) an amino acid sequence sharing at least 60 percent sequence identity with the fragment defined in (ii); wherein the polypeptide does not comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 15.
Preferably the polypeptide comprises an AMOP domain, e.g. a polypeptide sharing at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% identity with amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3.
Preferably, the polypeptide inhibits angiogenesis . For example, the polypeptide may have the ability to reduce, impair, disrupt, and/or down-regulate angiogenesis in an individual, e.g. a mammal. The polypeptide may inhibit angiogenic activity. In particular, the polypeptide may exhibit anti-angiogenic activity. The polypeptide may have the ability to inhibit growth of new blood vessels from preexisting vessels.
The polypeptide may inhibit endothelial cell tube formation, e.g. it may have the ability to reduce, impair, disrupt, and/or down-regulate the assembly of endothelial cells to form tubes. The polypeptide may have the ability to reduce tube length in an endothelial tube formation assay compared to the absence of the polypeptide, e.g. in a given time period. In vitro assays for observing endothelial cell tube formation are described below.
The polypeptide may inhibit proliferation of endothelial cells, e.g. it may have the ability to reduce, impair, disrupt, and/or down-regulate production of endothelial cells. The polypeptide may have the ability to reduce the number of endothelial cells produced in an endothelial cell proliferation assay compared to the absence of the polypeptide, e.g. in a given time period. The polypeptide may inhibit proliferation of endothelial cells in the presence of vascular endothelial growth factor (VEGF) . In vitro assays for observing proliferation of endothelial cells are described below.
The polypeptide may induce apoptosis of endothelial cells, e.g. it may have the ability to promote, stimulate, and/or generate apoptosis of endothelial cells. The polypeptide may have the ability to increase the number of endothelial cells undergoing apoptosis in an endothelial cell apoptosis assay compared to the absence of the polypeptide, e.g. in a given time period. Apoptosis may be quantitated by measuring DNA fragmentation, such as the number of mono- and oligonucleosomes in the cytoplasmic fraction of polypeptides treated with the polypeptide. In vitro assays for observing apoptosis of endothelial cells are described below.
We have found that the TSR domain of Isthmin exhibits less anti-angiogenic activity compared to the full length Isthmin polypeptide and other fragments of the Isthmin polypeptide. In particular, we have found that the full-length polypeptide induced EC apoptosis in the presence of .VEGF. In addition, the full-length polypeptide, the N-terminal fragment and the C- terminal fragment disrupted tubular network on Matrigel. The TSR domain alone did not have these activities. Also, in contrast to the full-length, N-terminal and the C-terminal fragments, the TSR domain alone was not effective at inhibiting endothelial cell proliferation.
The polypeptide preferably exhibits one or both of these activities not exhibited by the TSR domain, i.e. the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation. In particular, the polypeptide preferably exhibits one or both of these activities when the polypeptide comprises the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence that shares at sequence identity to the TSR domain, e.g. at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent sequence identity. The polypeptide may inhibit proliferation of endothelial cells.
In a further aspect of the invention there is provided a polypeptide comprising:
(i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(ii) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(iii) a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iv) an amino acid sequence sharing at least 60 percent sequence identity with the fragment defined in (iii); for use in a method of medical, e.g. therapeutic, treatment .
In any aspect of the invention the polypeptide may not comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 15.
The method of medical treatment may be the treatment of angiogenesis in an individual, e.g. by inhibiting angiogenesis . The angiogenesis may be unwanted angiogenesis, e.g. angiogenesis that is associated with disease. In particular, the angiogenesis may be tumour angiogenesis, e.g. angiogenesis that is induced by a tumour. Inhibiting tumour angiogenesis may involve inhibiting the blood supply to the tumour, e.g. by inhibiting the formation of the tumour vascular system.
The treatment may be treatment of a tumour and/or cancer, e.g. a cancerous condition. Inhibiting angiogenesis may include reducing, impairing, disrupting, and/or down-regulating angiogenesis and/or angiogenic activity in an individual. Inhibiting angiogenesis may involve inhibiting growth of new blood vessels, e.g. from pre-existing vessels. In particular, it may involve one or more of inhibiting endothelial cell tube formation, inhibiting proliferation of endothelial cells and/or inducing apoptosis of endothelial cells.
The treatment may be prophylactic. It may be treatment of an individual at risk of unwanted angiogenesis, e.g. tumour angiogenesis, e.g. unwanted proliferation of endothelial cells.
In a further aspect of the invention there is provided use of a polypeptide in the manufacture of a medicament for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis, wherein the polypeptide comprises: (i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID
NO: 3; or
(ii) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or (iii) a fragment of the amino acid sequence of SEQ ID NO:
1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(iv) an amino acid sequence sharing at least 60 percent sequence identity with the fragment defined in (iii) .
In a further aspect of the invention, there is provided a method of inhibiting angiogenesis in an individual or of treating an individual at risk of unwanted angiogenesis, comprising administering a polypeptide to the individual, wherein the polypeptide comprises: (i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(ii) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(iii) a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iv) an amino acid sequence sharing at least 60 percent sequence identity with the fragment defined in (iii) .
Preferably, the method is a method of inhibiting tumour angiogenesis in an individual. The method may be a method of inhibiting proliferation of endothelial cells in an individual .
The polypeptides of the invention are described in more detail below. These polypeptides may be for use in a method of medical treatment and/or for use in the manufacture of a medicament .
In another aspect of the invention, there is provided an isolated nucleic acid comprising a nucleotide sequence, which nucleotide sequence comprises:
(i) a nucleotide sequence sharing at least 60 percent sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or
(ii) a fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, which fragment has at least 18 contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 4, or
(iii) a nucleotide sequence sharing at least 60 percent sequence identity with the fragment defined in (ii) ; or
(iv) a nucleotide sequence encoding a polypeptide of the invention; wherein the nucleic acid does not comprise the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, or SEQ ID NO: 16.
Preferably, the nucleic acid encodes a polypeptide that inhibits angiogenesis, e.g. tumour angiogenesis . The nucleic acid may encode a polypeptide that inhibits proliferation of endothelial cells.
In a further aspect of the invention there is provided a composition, e.g. a pharmaceutical composition, comprising a polypeptide or nucleic acid of the invention. The composition may comprise a pharmaceutically acceptable excipient, diluent and/or carrier.
In a further aspect of the invention, there is provided a kit for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis, wherein the kit comprises: (i) a container comprising a polypeptide of the invention, e.g. as a pharmaceutical composition; and optionally
(ii) instructions for administering the composition to the individual.
In another aspect of the invention, there is provided a vector comprising a nucleic acid of the invention. The nucleic acid may be operably linked in the vector to a regulatory sequence, e.g. a promoter.
In another aspect of the invention, there is provided a host cell comprising a nucleic acid and/or vector of the invention.
In another aspect of the invention, there is provided a method of producing a polypeptide of the invention comprising: (a) providing a host cell comprising a nucleic acid of the invention, e.g. a nucleic acid comprising a nucleotide sequence that encodes a polypeptide of the invention; and
(b) causing the host cell of (a) to express the polypeptide encoded by the nucleic acid.
The method may also comprise step (c) isolating the peptide and/or may also comprise, prior to step (a) , introducing the nucleotide sequence that encodes the polypeptide of the invention into the host cell, e.g. on a vector. The step of providing the host cell may also include culturing the host cell. Methods of culturing host cells to express polypeptides and methods of purifying polypeptides are well known in the art. The host cell may express the polypeptide under the control of a constitutive promoter or inducible promoter.
Polypeptides
The Results and Methods sections below describe experiments with a number of truncates of Isthmin, schematic diagrams of which are shown in Figure 1. SEQ ID NO: 1 refers to the amino acid sequence of mouse Isthmin, SEQ ID NO: 3 refers to the amino acid sequence of human Isthmin.
The polypeptide may or may not comprise a TSR domain. The polypeptide may or may not comprise an AMOP domain. In some embodiments the polypeptide may comprise a TSR and an AMOP domain.
A TSR domain generally refers to a domain of about 40-60 amino acids with six conserved cysteine residues capable of forming 3 disulfide bonds. A consensus sequence of
WGEWSEWSPCSVTCGGG...VQTRTRS...CCNPPPP.. GGGPCTGEDPETRACNEQPCP has been reported (SEQ ID NO: 17) (Tucker RP., 2004) . Each dot represents any amino acid. It has been reported that the motifs WSxW (SEQ ID NO: 18) (where x is any amino acid) and CSVTCG (SEQ ID NO: 19) are highly conserved although not ubiquitous in TSR domains (Adams et al.) • A polypeptide of the invention may include one or both of these motifs. The CSVTCG motif may occur on the C-terminal side of the WSxW motif.
Adams et al . also reports invariant residues in the
Thrombospondin TSR domains. These are W8, S9, WlI, C18, R25, R27, C29, C41, C51 and C56. Some of these residues are shared by the TSR domains of isthmin polypeptides. A polypeptide of the invention may comprise a TSR domain having the following consensus sequence:
WSxWxxCxxxCxxxxxxRxRxC (SEQ ID NO: 20) . Preferably the consensus sequence is WSXWXXCXXXCXXXXXXRXRXCZ1CZ2CXXXXC (SEQ ID NO: 21), where x is any amino acid, z1 is 3 to 11 amino acids of any type, and z2 is 7 to 9 amino acids of any type. More preferably the consensus sequence is
WSXWXXCXXXCXXXXXXRXRXCZ1CZ2CXXXXC (SEQ ID NO: 22) .
In the mouse Isthmin amino acid sequence (SEQ ID NO: 1) the TSR domain may be considered to be amino acids 215 to 259 of SEQ ID NO: 1, in the human Isthmin amino acid sequence (SEQ ID NO: 3) the TSR domain may be considered to be amino acids 218 to 262 of SEQ ID NO: 3.
An AMOP domain generally refers to a domain of about 100 amino acids containing eight invariant cysteine residues. An AMOP domain may have a consensus sequence, or may have be a sequence sharing at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity with:
.h...h..C.pWhp.ps....pahh....p.ssCPC...p.hh + .Fh.... s s..C.hp h....CC.Ys..h..h R . h . hh .. sh a .. h . s .. hCph .. p ... C cc (SEQ ID NO: 23) .
The letters a, c, h, p, s, - and + indicate aromatic, charged, hydrophobic, polar, small, negative and positive amino acids respectively. Each dot represents any amino acid. (Ciccarelli et al. 2002) . In the mouse Isthmin amino acid sequence (SEQ ID NO: 1) the AMOP domain may be considered to be amino acids 286 to 449 of SEQ ID NO: 1, in the human Isthmin amino acid sequence (SEQ ID NO: 3) the AMOP domain may be considered to be amino acids 289 to 452 of SEQ ID NO: 3.
Polypeptides comprising or consisting of an amino acid sequence sharing sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3
Polypeptides of the invention may comprise or consist of an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The polypeptide may comprise or consist of an amino acid sequence that shares a sequence identity of at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent with SEQ ID NO: 1 or SEQ ID NO: 3, or with a portion of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The sequence identity may be shared over the full length of
SEQ ID NO: 1 or SEQ ID NO: 3. The "full-length" of SEQ ID NO: 1 or SEQ ID NO: 3 refers to the complete amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, respectively.
The polypeptide may comprise or consist of an amino acid sequence that shares sequence identity with:
(1) the sequence consisting of amino acids 215 to 259 of SEQ
ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; or (2) the sequence consisting of amino acids 26 to 277 of SEQ
ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; or
(3) the sequence consisting of amino acids 26 to 461 of SEQ
ID NO: 1 or the sequence consisting of amino acids 26 to 464 of SEQ ID NO: 3. A polypeptide comprising such an amino acid sequence may comprise a TSR domain.
The polypeptide may comprise or consist of an amino acid sequence that shares sequence identity with:
(4) the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3; or
(5) the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
A polypeptide comprising or consisting of such an amino acid sequence may comprise or consist of an AMOP domain.
The polypeptide may comprise or consist of combinations of the above amino acid sequences. For example, the polypeptide may comprise or consist of:
(a) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO : 3 ; and
(b) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
The polypeptide may comprise or consist of:
(a) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and
(b) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3. The polypeptide may comprise or consist of:
(a) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3.
(b) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
The polypeptide may comprise or consist of:
(a) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; and
(b) an amino acid sequence sharing sequence identity with the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
A polypeptide comprising or consisting of such an amino acid sequence, i.e. an amino acid sequence comprising or consisting of an (a) portion and a (b) portion as described above, may comprise a TSR domain, e.g. in portion (a), and an AMOP domain, e.g. in portion (b) . The polypeptide may comprise a linker that is inserted between the (a) portion and the (b) portion. Such a linker may be one or more amino acids, e.g. a peptide, polypeptide, polypeptide domain, or a nucleic acid, organic chemical, or any other molecule. The linker may be linked, coupled, bound, and/or attached to the (a) and/or (b) portion via covalent bonding and/or non-covalent bonding, e.g. immunogenic bonding, ionic bonding, dipole-dipole attractions or hydrogen bonding. Alternatively, the (a) portion may be contiguous with the (b) portion, e.g. there may not be any linker molecule. Polypeptides comprising or consisting, of fragments of SEQ ID NO: 1 or SEQ ID NO: 3
The polypeptide may comprise or consist of a fragment of SEQ ID NO: 1 or SEQ ID NO: 3. The polypeptide may comprise or consist of a fragment of:
(1) the sequence consisting of amino acids 26 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 464 of SEQ ID NO: 3; or (2)' the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; or
(3) the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3.
Such a fragment may comprise a TSR domain.
The polypeptide may comprise or consist of a fragment of:
(4) the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3; or
(5) the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3. Such a fragment may comprise an AMOP domain.
The polypeptide may comprise combinations of the above amino acid sequences. For example, the polypeptide may comprise or consist of: (a) a fragment of the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and
(b) a fragment of the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the amino acid sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3. The polypeptide may comprise or consist of:
(a) a fragment of the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and (b) a fragment of the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
The polypeptide may comprise or consist of: (a) a fragment of the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; and
(b) a fragment of the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the amino acid sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
The polypeptide may comprise or consist of:
(a) a fragment of the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; and
(b) a fragment of the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
A polypeptide comprising, or consisting of, such an amino acid sequence, i.e. an amino acid sequence comprising an (a) portion and a (b) portion, may comprise a TSR domain and an AMOP domain. The polypeptide may or may not comprise a linker that is inserted between the (a) portion and the (b) portion.
The fragment of SEQ ID NO: 1 or SEQ ID NO: 3 may have a length anywhere between the maximum length and minimum length as indicated below. Preferably the fragment is at least 6 amino acids in length. Where any polypeptide of the invention comprises a fragment of SEQ ID NO: 1 the fragment may comprise at least one amino acid of SEQ ID NO: 1 that is not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1. Where any polypeptide of the invention comprises a fragment of SEQ ID NO: 3, the fragment may comprise at least one amino acid of SEQ ID NO: 3 that is not from the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3. The fragment may comprise at least 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, or 350 amino acids, e.g. contiguous amino acids, of SEQ ID NO: 1 or SEQ ID NO: 3 that are not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or 218 to 262 of SEQ ID NO: 3 respectively. Preferably the fragment comprises such amino acids from the N- terminal side of amino acid 215 of SEQ ID NO: 1 or 218 of SEQ ID NO: 3.
The polypeptide may comprise the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, from the N-terminal side of the TSR domain in SEQ ID NO: 1 or SEQ ID NO: 3. Where a polypeptide comprises the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, or otherwise, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation. A polypeptide having one or both of these activities may comprises one or more additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 from outside the TSR domain.
The polypeptide may comprise at least 46 amino acids, e.g. the polypeptide may comprise more amino acids than the number of amino acids in the TSR domain. Polypeptides comprising or consisting of an amino acid sequence sharing sequence identity with fragments of SEQ ID NO: 1 or SEQ ID NO: 3
The polypeptide may comprise or consist of an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 1 or SEQ ID NO: 3. The sequence identity shared may be at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity.
The polypeptide may comprise or consist of:
(1) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting of amino acids 26 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 464 of SEQ ID NO: 3; or
(2) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; or (3) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting' of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3. Such polypeptide may comprise a TSR domain.
The polypeptide may comprise or consist of:
(4) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3; or
(5) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3. Such a polypeptide may comprise an AMOP domain. The polypeptide may comprise or consist of combinations of the above amino acid sequences. For example, the polypeptide may comprise :
(a) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; and
(b) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
The polypeptide may comprise or consist of:
(a) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; and
(b) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
The polypeptide may comprise or consist of:
(a) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and
(b) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
The polypeptide may comprise or consist of:
(a) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and
(b) an amino acid sequence sharing sequence identity with a fragment of the sequence consisting amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
A polypeptide comprising or consisting of such an amino acid sequence, i.e. an amino acid sequence comprising an (a) portion and a (b) portion, may comprise a TSR domain, e.g. in the (a) portion, and an AMOP domain, e.g. in the (b) portion. The polypeptide may or may not comprise a linker that is inserted between the (a) portion and the (b) portion.
The fragment of SEQ ID NO: 1 or SEQ ID NO: 3 may have a length anywhere between the maximum length and minimum length as indicated below. Preferably, the fragment is at least 10 amino acids in length when the shared sequence identity is at least 60 percent.
Where any polypeptide of the invention comprises an amino acid sequence sharing sequence identity as specified above with SEQ ID NO: 1 or a portion of SEQ ID NO: 1, it may comprise at least one amino acid that corresponds to an amino acid in the sequence of SEQ ID NO: 1 that is not an amino acid from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1. Where any polypeptide of the invention comprises an amino acid sequence sharing sequence identity as specified above with SEQ ID NO: 3 or a portion of SEQ ID NO: 3, it may comprise at least one amino acid that corresponds to an amino acid in the sequence of SEQ ID NO: 3 that is not an amino acid from the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3. The amino acid sequence may comprise at least 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350 amino acids, e.g. contiguous amino acids, that correspond to the same number of amino acids in the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and that are not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or 218 to 262 of SEQ ID NO: 3, respectively.
The polypeptide may comprise an amino acid sequence sharing sequence identity as specified above with the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, that correspond to amino acids from the N-terminal side of the TSR domain in SEQ ID NO: 1 or SEQ
ID NO: 3. Where a polypeptide comprises an amino acid sequence sharing sequence identity as specified above with the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation. A polypeptide having one or both of these activities may comprise one or more additional amino acids that correspond to amino acids in SEQ ID NO: 1 or SEQ ID NO: 3 that are outside the TSR domain.
Polypeptides comprising or consisting of portions of SEQ ID NO: 1 or SEQ ID NO: 3.
The polypeptide may comprise or consist of a portion of SEQ ID NO: 1 or SEQ ID NO: 3. The polypeptide may comprise or consist of:
(1) the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; or
(2) the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; or
(3) the sequence consisting of amino acids 26 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 464 of SEQ ID NO: 3.
The polypeptide may comprise or consist of: (4) the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3; or
(5) the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
The polypeptide may comprise or consist of:
(a) the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and
(b) the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
The polypeptide may comprise or consist of:
(a) the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3; and (b) the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
The polypeptide may comprise or consist of: (a) the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3; and
(b) the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
Polypeptides that include the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3 may also include additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 respectively. In particular, polypeptides that include the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or amino acids 218 to 262 or SEQ ID NO: 3 may include additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 on the N-terminal and/or C-terminal side of these sequences.
Likewise, polypeptides that include the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3 may also include additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 respectively. In particular, polypeptides that include the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3 may include additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 on the N-terminal and/or C-terminal side of amino acids 286 to 449 of these sequences.
Where a polypeptide comprises the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation. A polypeptide having one or both of these activities may comprises one or more additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 from outside the TSR domain.
A polypeptide that comprises an amino acid sequence sharing sequence identity with a specified sequence from SEQ ID NO: 1, that comprises a fragment of SEQ ID NO: 1, that comprise an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 1, or that comprises a specified sequence from SEQ ID NO: 1 may comprise or consist of: an amino acid sequence sharing sequence identity with, a fragment comprising the amino acid sequence of, an amino acid sequence sharing sequence identity with a fragment comprising the amino acid sequence of, or may comprise, the amino acid sequence: 214-259, 213-259, 212-259, 211-259, 210-259, 209-259, 208-259, 207-259, 206-259, 204-259, 203-259, 202-259, 201-259, 200-259, 190-259, 180-259, 170-259, 160-259, 150-259, 140-259, 140-259, 130-259, 120-259, 110-259, 100-259, 90-259, 80-259, 70-259, 60-259, 50-259, 40-259, 30-259, 20-259, 10-259, 1-259, or
215-262, 215-263, 215-264, 215-265, 215-266, 215-267, 215-268, 215-269, 215-270, 215-271, 215-272, 215-273, 215-274, 215-275,
215-276, 215-277, 215-278, 215-279, 215-280, 215-290, 215-300,
215-310, 215-320, 215-330, 215-340, 215-350, 215-360, 215-370,
215-380, 215-390, 215-400, 215-410, 215-420, 215-430, or 215- 440, 215-449 or
214-260, 213-261, 212-262, 211-263, 210-264, 209-265, 208-266,
207-267, 206-268, 205-269, 204-270, 203-271, 202-272, 201-273,
200-274, 190-280, 180-290, 170-300, 160-310, 150-320, 140-330,
130-340, 120-350, 110-360, 100-370, 90-380, 80-390, 70-400, 60-410, 50-420, 40-430, 30-440, 20-459, 10-459, or 1-459 of SEQ ID NO: 1.
A polypeptide that comprises an amino acid sequence sharing sequence identity with a specified sequence from SEQ ID NO: 3, that comprises a fragment of SEQ ID NO: 3, that comprises an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 3, or that comprises a specified sequence from SEQ ID NO: 3, may comprise or consist of: an amino acid sequence sharing sequence identity with, a fragment comprising the amino acid sequence of, an amino acid sequence sharing sequence identity with a fragment comprising the amino acid sequence of, or may comprise, the amino acid sequence: 217-262, 216-262, 216-262, 215-262, 214-262, 213-262, 212-262, 211-262, 210-262, 209-262, 208-262, 207-262, 206-262, 205-262, 204-262, 203-262, 202-262, 201-262, 200-262, 190-262, 180-262, 170-262, 160-262, 150-262, 140-262, 130-262, 120-262, 110-262, 100-262, 90-262, 80-262, 70-262, 60-262, 50-262, 40-262, 30- 262, 20-262, 10-262, 1-262, or
218-263, 218-264, 218-265, 218-266, 218-267, 218-268, 218-268, 218-269, 218-270, 218-271, 218-272, 218-273, 218-274, 218-275, 218-276, 218-277 , 218-278 , 218-279 , 218-280 , 218-290 , 218-300 , 218-310 , 218-320 , 218-330 , 218-340 , 218-350 , 218-360 , 218-370 , 218-380 , 218-390 , 218-400 , 218-410 , 218-420 , 218-430 , 218-440 , 218-450 , 218-460 , 218-464 , or
217-263, 216-264 215-265, 214-266, 213-267, 212-268, 211-269, 210-270, 209-271, 208-272, 207-273, 206-274, 205-275, 204-276, 203-277, 202-278, 201-279, 200-280, 190-290, 180-300, 170-310, 160-320, 150-330, 140-340, 130-350, 120-360, 110-370, 100-380, 90-390, 80-400, 70-410, 60-420, 50-430, 40-440, 30-450, 20- 460, 10-464, 1-464 of SEQ ID NO: 3.
Throughout this specification where a particular sequence of amino acids is specified, e.g. "a sequence consisting of amino acids X to Y", the specified sequence includes amino acids X and Y, i.e. the amino acids at the start and end of the specified sequence are included in the sequence.
In this specification, a polypeptide may be a peptide, polypeptide or protein. The polypeptides of the invention may be isolated polypeptides. The term "isolated polypeptide" refers to a polypeptide that has undergone some degree of isolation, e.g. by purification.
A polypeptide of the invention may be a synthetic peptide, e.g. it may be synthesized by chemical synthesis. Alternatively polypeptides of the invention may be produced recombinantly .
The present invention is based on the finding that the Isthmin polypeptide and fragments thereof have anti-angiogenic activity and may be useful in suppressing angiogenesis e.g. in cancer. The Isthmin polypeptide comprises a TSR domain and an AMOP domain. Both of these domains have been shown herein to independently have anti-angiogenic activity. As TSR and AMOP domains are generally known and exist in other polypeptides it is expected, based on the results reported herein, that other known TSR and AMOP domains will also exhibit anti-angiogenic activity. Thus, the present invention encompasses polypeptides comprising any TSR domain or AMOP domain for use in therapeutic methods of inhibiting angiogenesis . TSR domains are found, for example, in TSP proteins such as TSP-I and TSP- 2.
Furthermore, it is shown herein that the Isthmin polypeptide and fragments thereof have anti-angiogenic properties that are not shown by the TSR domain alone. The Isthmin polypeptide comprises an AMOP domain and a TSR domain. Thus, it is expected, based on the results reported herein, that a polypeptide comprising any AMOP domain and any TSR domain will have useful anti-angiogenic properties and such polypeptides are also encompassed by the present invention. These polypeptides are preferably for use in methods of inhibiting angiogenesis. For example, the polypeptide may be a polypeptide in which the AMOP domain and TSR domain are contiguous or separated by intervening amino acids.
Alternatively, the TSR domain and AMOP domain may be separated by a chemical linker which may not be an amino acid linker. Chemical linkers are well-known to the person skilled in the art. Preferably at least one of the TSR domain or AMOP domain is an Isthmin TSR or AMOP domain.
Likewise, it has also been shown herein that a polypeptide comprising a TSR domain and the N-terminal portion of Isthmin polypeptide has anti-angiogenic properties that are not shown by the TSR domain alone. Thus, the present invention also encompasses polypeptides comprising any TSR domain and the N- terminal portion of an Isthmin polypeptide, e.g. for use in therapeutic methods of inhibiting angiogenesis. In such a polypeptide the Isthmin N-terminal portion will usually be provided on the N-terminal side of the TSR domain, e.g. contiguously or separated by intervening amino acids. Alternatively, the N-terminal portion and TSR domain may be separated by a non-amino acid linker. The N-terminal portion may, for example, be amino acids 26 to 214 from SEQ ID NO: 1 or amino acids 26 to 217 from SEQ ID NO: 3. A polypeptide comprising a TSR domain and Isthmin N-terminal portion may also comprise an AMOP domain.
The present invention also encompasses Isthmin polypeptides in which the TSR domain is replaced with a heterologous TSR domain or in which the AMOP domain is replaced by a heterologous AMOP domain respectively. In some cases both the TSR domain and AMOP domain may be replaced by a heterologous TSR and AMOP domain. In this context the term heterologous means, for example, that the TSR or AMOP domain is not the native Isthmin TSR or AMOP domain, e.g. the AMOP and TSR domains do not occur naturally together in the same polypeptide .
The TSR domain, AMOP domain, and/or Istmin N-terminal portion referred to above may share at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity with the mouse or human Isthmin TSR domain, AMOP domain and/or N-terminal portion respectively. For example, the TSR domain over which sequence identity is shared may be amino acids 215 to 259 of SEQ ID NO: 1 or amino acids 218 to 262 of SEQ ID NO: 3, the AMOP domain over which sequence identity is shared may be amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3, the N-terminal portion over which identity is shared may be amino acids 26 to 214 from SEQ ID NO: 1 or amino acids 26 to 217 from SEQ ID NO: 3.
Nucleic acids
In another aspect of the invention, there is provided an isolated nucleic acid comprising a nucleotide sequence, which nucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or
(ii) a nucleotide sequence sharing at least 60 percent sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or
(iii) a fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, which fragment has at least 18 contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 4, or
(iv) a nucleotide sequence sharing at least 60 percent sequence identity with the fragment defined in (iii) ; or
(v) a nucleotide sequence encoding a polypeptide of the invention; for use in a method of medical treatment.
The nucleic acids of the invention may be used in methods of medical treatment e.g. inhibition of angiogenesis in an individual, e.g. inhibition of tumour angiogenesis, e.g. inhibition of proliferation of endothelial cells. The method may be prophylactic, e.g. it may be treatment of an individual at risk of unwanted angiogenesis. The nucleic acids may be used in the manufacture of a medicament.
In another aspect of the invention there is provided use of an isolated nucleic acid in the manufacture of a polypeptide for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis, wherein the nucleic acid comprises a nucleotide sequence comprising:
(i) the nucleotide sequence consisting of nucleotides
SEQ ID NO: 2 or SEQ ID NO: 4; or (ii) a nucleotide sequence sharing at least 60 percent sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or
(iii) a fragment of the nucleotide sequence of SEQ ID NO:
2 or SEQ ID NO: 4, which fragment has at least 18 contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 4, or (iv) a nucleotide sequence sharing at least 60 percent sequence identity with the fragment defined in (iii); or
(v) a nucleotide sequence encoding a polypeptide of the invention.
Nucleic acids of the invention preferably encode polypeptides of the invention. The nucleic acid may or may not encode a TSR domain. The nucleic acid may or may not encode an AMOP domain. In some embodiments, the nucleic acid may encode a TSR and an AMOP domain. The nucleic acid may encode a TSR and/or an AMOP domain.
Nucleic acids comprising or consisting of a nucleotide sequence sharing sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4
Nucleic acids of the invention may comprise a nucleotide sequence sharing at least 60 percent identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The nucleic acid may comprise a nucleotide sequence that shares a sequence identity of at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent with SEQ ID NO: 2 or SEQ ID NO: 4. The sequence identity may be shared over the full length of SEQ ID NO: 2 or SEQ ID NO: 4. The "full length" sequence of SEQ ID NO: 2 or SEQ ID NO: 4 is the complete nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 respectively.
The nucleic acid may comprise or consist of a nucleotide sequence that shares sequence identity with:
(1) the sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4.
(2) the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides
156 to 920 of SEQ ID NO: 4. (3) the sequence consisting of nucleotides 678 to 1985 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 156 to 1472 of SEQ ID NO: 4.
(4) the sequence" consisting of nucleotides 603 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 81 to 1472 of SEQ ID NO: 4.
A nucleic acid comprising such a nucleotide sequence may encode a TSR domain.
The nucleic acid may comprise or consist of a nucleotide sequence that shares sequence identity with:
(5) the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4.
(6) the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
A nucleic acid comprising such a nucleotide sequence may encode an AMOP domain.
The nucleic acid may comprise combinations of the above nucleotide sequences. For example, the nucleic acid may comprise or consist of:
(a) the sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4; and
(b) the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) the sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4; and (b) the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) The sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and
(b) the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) The sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides
156 to 920 of SEQ ID NO: 4; and
(b) the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
A nucleic acid comprising an (a) portion and a (b) portion may encode a TSR domain, e.g. in portion (a), and an AMOP domain, e.g. in portion (b) . The nucleic acid may comprise a nucleotide sequence between portion (a) and (b) , e.g. that encodes a linker. Alternatively the (a) portion may be contiguous with the (b) portion.
Nucleic acids comprising or consisting of fragments of SEQ ID
NO: 2 or SEQ ID NO: 4
The nucleic acid may comprise or consist of a fragment of SEQ
ID NO: 2 or SEQ ID NO: 4. The nucleic acid may comprise or consist of a fragment of:
(1) the sequence consisting of nucleotides 603 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 81 to 1472 of SEQ ID NO: 4; or (2) the sequence consisting of nucleotides 678 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 1472 of SEQ ID NO: 4; or
(3) the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; or
(4) the sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4. Such a fragment may encode a TSR domain.
The nucleic acid may comprise or consist of a fragment of:
(5) the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4; or
(6) the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
Such a fragment may encode an AMOP domain.
The nucleic acid may comprise combinations of the above nucleotide sequences. For example, the nucleic acid may comprise or consist of:
(a) the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and
(b) the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and (b) the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) the sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4; and
(b) the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the sequence consisting of nucleotides 945 to
1436 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) the sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to
866 of SEQ ID NO: 4; and
(b) the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
A nucleic acid comprising such a combination of nucleotide sequences, i.e. a nucleotide sequence comprising an (a) portion and a (b) portion may encode a TSR domain, e.g. in the (a) portion, and an AMOP domain, e.g. in the (b) portion.
Where the nucleotide sequence encodes a polypeptide comprising a fragment of SEQ ID NO: 1, the nucleotide sequence may encode at least one amino acid of SEQ ID NO: 1 that is not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1. Where the nucleotide sequence encodes a polypeptide comprising a fragment of SEQ ID NO: 3, the nucleotide sequence may encode at least one amino acid of SEQ ID NO: 3 that is not from the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3. The nucleotide sequence may encode at least 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, or 350 amino acids, e.g. contiguous amino acids, of SEQ ID NO: 1 or SEQ ID NO: 3 that are not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or 218 to 262 of SEQ ID NO: 3 respectively.
A nucleotide sequence may encode a polypeptide comprising the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, from the N- terminal side of the TSR domain in SEQ ID NO: 1 or SEQ ID NO: 3. Where a nucleotide sequence encodes a polypeptide comprising the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation. A nucleotide sequence encoding a polypeptide having one or both of these activities may encode one or more amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 from outside the TSR domain.
Nucleic acids comprising or consisting of a nucleotide sequence sharing sequence identity with fragments of SEQ ID WO: 2 or SEQ ID NO: 4
The nucleic acid may be a nucleic acid sequence sharing sequence identity with a fragment of SEQ ID NO: 2 or SEQ ID NO: 4. The sequence identity shared may be 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity.
The nucleic acid may comprise or consist of:
(1) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 603 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 81 to 1472 of SEQ ID NO: 4; or
(2) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 678 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 1472 of SEQ ID NO: 4; or (3) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; or (4) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4.
Such a nucleotide sequence may encode a TSR domain.
The nucleic acid may comprise or consist of:
(5) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4; or
(6) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4. Such a nucleic acid may encode an AMOP domain.
The nucleic acid may comprise combinations of the above nucleic acid sequences. For example, the nucleic acid may comprise or consist of: (a) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and
(b) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1458 to
1949 of SEQ ID NO: 2 or the sequence consisting of nucleotides
945 to 1436 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of: (a) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and
(b) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4
The nucleic acid may comprise or consist of:
(a) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1245 to
1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4; and
(b) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1245 to
1379 of SEQ ID NO: 2 or the sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4; and
(b) a nucleotide sequence sharing sequence identity with a fragment of the sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
A nucleic acid comprising or consisting of such a nucleotide sequence, i.e. a nucleotide sequence comprising an (a) portion and a (b) portion, may encode a TSR domain, e.g. in the (a) portion, and an AMOP, e.g. in the (b) portion.
Where the nucleotide sequence encodes an amino acid sequence sharing sequence identity as specified above with SEQ ID NO: 1, or a portion of SEQ ID NO: 1, it may encode at least one amino acid that corresponds to an amino acid in the sequence of SEQ ID NO: 1 that is not an amino acid from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1. Where the nucleotide sequence encodes an amino acid sequence sharing sequence identity as specified above with SEQ ID NO: 3, or a portion of SEQ ID NO: 3, it may encode at least one amino acid that corresponds to an amino acid in the sequence of SEQ ID NO: 3 that is not an amino acid from the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3. The nucleotide sequence may encode at least 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, e.g. contiguous amino acids, that correspond to the same number of amino acids in the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and which are not from the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or 218 to 262 of SEQ ID NO: 3 respectively.
A nucleotide sequence may encode a polypeptide comprising an amino acid sequence sharing sequence identity as specified above with the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3 and one or more additional amino acids, as indicated above, that correspond to amino acids from the N-terminal side of the TSR domain in SEQ ID NO: 1 or SEQ ID NO: 3. Where a nucleotide sequence encodes a polypeptide comprising an amino acid sequence sharing sequence identity as specified above with the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation. A nucleotide sequence encoding a polypeptide having one or both of these activities may encode one or more additional amino acids that correspond to amino acids in SEQ ID NO: 1 or SEQ ID NO: 3 that are outside the TSR domain.
Nucleic acids comprising or consisting of portions of SEQ ID NO: 2 or SEQ ID NO: 4 The nucleic acid may comprise a portion of SEQ ID NO: 2 or SEQ ID NO: 4. The nucleic acid may comprise or consist of:
(1) the nucleotide sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4; or
(2) the nucleotide sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; or
(3) the nucleotide sequence consisting of nucleotides 678 to 1985 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 156 to 1472 of SEQ ID NO: 4; or
(4) the nucleotide sequence consisting of nucleotides 603 to 1985 of SEQ ID NO: 2 or the sequence consisting of nucleotides 81 to 1472 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(5) the nucleotide sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4. (6) the nucleotide sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of: (a) the nucleotide sequence consisting of nucleotides 1245 to
1379 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4; and
(b) the nucleotide sequence consisting of nucleotides 1458 to
1949 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) the nucleotide sequence consisting of nucleotides 1245 to 1379 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 723 to 866 of SEQ ID NO: 4; and (b) the nucleotide sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) the nucleotide sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and
(b) the nucleotide sequence consisting of nucleotides 1458 to 1949 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 945 to 1436 of SEQ ID NO: 4.
The nucleic acid may comprise or consist of:
(a) the nucleotide sequence consisting of nucleotides 678 to 1433 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 156 to 920 of SEQ ID NO: 4; and
(b) the nucleotide sequence consisting of nucleotides 1413 to 1985 of SEQ ID NO: 2 or the nucleotide sequence consisting of nucleotides 902 to 1472 of SEQ ID NO: 4.
Nucleic acids that encode the amino acid sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3 may also encode additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 respectively. In particular, nucleic acids that encode the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or amino acids 218 to 262 or SEQ ID NO: 3 may encode additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 on the N-terminal and/or C-terminal side of these sequences.
Likewise, nucleic acids that encode the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3 may also encode additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 respectively. In particular, nucleic acids that encode the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or amino acids 289 to 452 of SEQ ID NO: 3 may encode additional amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 on the N-terminal and/or C-terminal side of these sequences.
Where a nucleotide sequence encodes a polypeptide comprising the TSR domain of SEQ ID NO: 1 or SEQ ID NO: 3, the polypeptide preferably induces apoptosis of endothelial cells, and/or inhibits endothelial cell tube formation. A nucleotide sequence encoding a polypeptide having one or both of these activities may encode one or more amino acids from SEQ ID NO: 1 or SEQ ID NO: 3 from outside the TSR domain.
A nucleic acid that encodes an amino acid sequence sharing sequence identity with a specified sequence from SEQ ID NO: 1, that encodes a fragment of SEQ ID NO: 1, that encodes an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 1, or that encodes a specified sequence from SEQ ID NO: 1, may encode: an amino acid sequence sharing sequence identity with the amino acid sequence of, a fragment comprising the amino acid sequence of, an amino acid sequence sharing sequence identity with a fragment comprising the amino acid sequence of, or may comprise amino acids:
214-259, 213-259, 212-259, 211-259, 210-259, 209-259, 208-259, 207-259, 206-259, 204-259, 203-259, 202-259, 201-259, 200-259, 190-259, 180-259, 170-259, 160-259, 150-259, 140-259, 140-259, 130-259, 120-259, 110-259, 100-259, 90-259, 80-259, 70-259, 60-259, 50-259, 40-259, 30-259, 20-259, 10-259, 1-259, or
215-262, 215-263, 215-264, 215-265, 215-266, 215-267, 215-268, 215-269, 215-270, 215-271, 215-272, 215-273, 215-274, 215-275,
215-276, 215-277, 215-278, 215-279, 215-280, 215-290, 215-300,
215-310, 215-320, 215-330, 215-340, 215-350, 215-360, 215-370,
215-380, 215-390, 215-400, 215-410, 215-420, 215-430, or 215- 440, 215-449 or 214-260, 213-261, 212-262, 211-263, 210-264, 209-265, 208-266,
207-267, 206-268, 205-269, 204-270, 203-271, 202-272, 201-273,
200-274, 190-280, 180-290, 170-300, 160-310, 150-320, 140-330,
130-340, 120-350, 110-360, 100-370, 90-380, 80-390, 70-400, 60-410, 50-420, 40-430, 30-440, 20-459, 10-459, or 1-459 of SEQ ID NO: 1.
A nucleic acid that encodes an amino acid sequence sharing sequence identity with a specified sequence from SEQ ID NO: 3, that encodes a fragment of SEQ ID NO: 3, that encodes an amino acid sequence sharing sequence identity with a fragment of SEQ ID NO: 3, or that encodes a specified sequence from SEQ ID NO: 3, may encode: an amino acid sequence sharing sequence identity with the amino acid sequence of, a fragment comprising the amino acid sequence of, an amino acid sequence sharing homology with a fragment comprising the amino acid sequence of, or may comprise the amino acid sequence: 217-262, 216-262, 216-262, 215-262, 214-262, 213-262, 212-262, 211-262, 210-262, 209-262, 208-262, 207-262, 206-262, 205-262, 204-262, 203-262, 202-262, 201-262, 200-262, 190-262, 180-262, 170-262, 160-262, 150-262, 140-262, 130-262, 120-262, 110-262, 100-262, 90-262, 80-262, 70-262, 60-262, 50-262, 40-262, 30- 262, 20-262, 10-262, 1-262, or
218-263, 218-264, 218-265, 218-266, 218-267, 218-268, 218-268,
218-269, 218-270, 218-271, 218-272, 218-273, 218-274, 218-275,
218-276, 218-277, 218-278, 218-279, 218-280, 218-290, 218-300,
218-310, 218-320, 218-330, 218-340, 218-350, 218-360, 218-370,
218-380, 218-390, 218-400, 218-410, 218-420, 218-430, 218-440, 218-450, 218-460, 218-464, or -
217-263, 216-264 215-265, 214-266, 213-267, 212-268, 211-269,
210-270, 209-271, 208-272, 207-273, 206-274, 205-275, 204-276,
203-277, 202-278, 201-279, 200-280, 190-290, 180-300, 170-310, 160-320, 150-330, 140-340, 130-350, 120-360, 110-370, 100-380, 90-390 , 80-400 , 70-410 , 60-420 , 50-430 , 40-440 , 30-450 , 20- 460 , 10-464 , 1-464 of SEQ ID NO : 3 .
Throughout this specification where a particular sequence of nucleotides is specified, e.g. "a sequence consisting of nucleotides X to Y", the specified sequence includes nucleotides X and Y, i.e. the nucleotides at the start and end of the specified sequence are included in the sequence.
In this specification, a nucleic acid may be any nucleic acid, either DNA or RNA. The invention includes nucleic acids that are complementary to the nucleic acids described herein and RNA transcripts of the nucleic acids described herein. The nucleic acids are preferably isolated nucleic acids. The term "isolated nucleic acid" refers to a nucleic acid that has undergone some degree of isolation, e.g. by purification.
An nucleic acid of the invention may be a synthetic nucleic acid, e.g. it may be synthesized by chemical synthesis. Alternatively nucleic acid of the invention may be produced recombinantly .
Fragments
A fragment of SEQ ID NO: 1, 2, 3, or 4, including fragments of sequences within SEQ ID NO: 1, 2, 3, or 4, may be of defined length and/or may have a defined minimum and/or maximum length.
Accordingly, a fragment may comprise at least, i.e. have a minimum length of, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the corresponding full length sequence. The fragment may have a maximum length, i.e. be no longer than, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the corresponding full length sequence . A fragment of SEQ ID NO: 1 or SEQ ID NO: 3 may comprise at least, i.e. have a minimum length of, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 459 or 460 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 3. A fragment of SEQ ID NO: 3 may comprise at least 461, 462, or 463 contiguous amino acids of SEQ ID NO: 3.
A fragment of SEQ ID NO: 1 or SEQ ID NO: 3 may have a maximum length of, i.e. be no longer than, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,
250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 459 or 460 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 3. A fragment of SEQ ID NO: 3 may have a maximum length of 461, 462, or 463 contiguous amino acids of SEQ ID NO: 3.
A fragment of SEQ ID NO: 2 or SEQ ID NO: 4 may comprise at least, i.e. have a minimum length of, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,
360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,
960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, or 2299 contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 4. A fragment of SEQ ID NO: 4 may comprise at least 2300, 2400, 2500, 2600, or 2666 contiguous nucleotides of SEQ ID NO: 4.
A fragment of SEQ ID NO: 2 or SEQ ID NO: 4 may have a maximum length of, i.e. be no longer than, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,
250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, or 2299 contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 4. A fragment of SEQ ID NO: 4 may have a maximum length of- 2300, 2400, 2500, 2600, or 2666 contiguous nucleotides of SEQ ID NO: 4. The fragment may have a length anywhere between the said minimum and maximum length.
Sequence identity
Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequence (referred to by the SEQ ID No.) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences .
Where the aligned sequences are of different length, sequence identity of the shorter comparison sequence may be determined over the entire length of the longer given sequence or, where the comparison sequence is longer than the given sequence, sequence identity of the comparison sequence may be determined over the entire length of the shorter given sequence.
For example, where a given sequence comprises 100 amino acids and the candidate sequence comprises 10 amino acids, the candidate sequence can only have a maximum identity of 10% to the entire length of the given sequence. This is further illustrated in the following example:
(A) Given seq: XXXXXXXXXXXXXXX (15 amino acids) Comparison seq: XXXXXYYYYYYY (12 amino acids)
The given sequence may, for example, be that encoding SEQ ID NO: 1) . % sequence identity = the number of identically matching amino acid residues after alignment divided by the total number of amino acid residues in the longer given sequence, i.e. (5 divided by 15) x 100 = 33.3%
Where the comparison sequence is longer than the given sequence, sequence identity may be determined over the entire length of the given sequence. For example:
(B)
Given seq: XXXXXXXXXX (10 amino acids) Comparison seq: XXXXXYYYYYYZZYZZZZZZ (20 amino acids)
Again, the given sequence may, for example, be that encoding SEQ ID NO: 1
% sequence identity = number of identical amino acids after alignment divided by total number of amino acid residues in the given sequence, i.e. (5 divided by 10) x 100 = 50%.
Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalW 1.82. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. The default parameters of ClustalW 1.82 are: Protein Gap Open Penalty = 10.0, Protein Gap Extension Penalty = 0.2, Protein matrix = Gonnet, Protein/DNA ENDGAP = -1, Protein/DNA GAPDIST = 4.
Identity of nucleic acid sequences may be determined in a similar manner involving aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and calculating sequence identity over the entire length of the respective sequences. Where the aligned sequences are of different length, sequence identity may be determined as described above and illustrated in examples (A) and (B) .
Corresponding amino acids An amino acid in a sequence of interest that is specified as corresponding to a particular amino acid in a given sequence, e.g. SEQ ID NO: 1, means that the amino acid in the sequence of interest is paired with an identical amino acid in the given sequence when the sequences are pairwise aligned.
Correspondence is determined as follows: the amino acid sequence of interest and the given sequence are pairwise aligned, for example using the ClustalW 1.82 software, using the default parameters. The ClustalW software may be accessed from the European Bioinformatics institute at http://www.ebi.ac.uk/clustalw/. The default parameters of ClustalW 1.82 are: Protein Gap Open Penalty = 10.0, Protein Gap Extension Penalty = 0.2, Protein matrix = Gonnet, Protein/DNA ENDGAP = -1, Protein/DNA GAPDIST = 4. The pairwise alignment will result in amino acids in the sequence of interest being paired with amino acids in the given sequence. A particular amino acid in the sequence of interest "corresponds" to the amino acid in the given sequence if it is paired with an identical amino acid in the given sequence.
Operably linked
In this specification the term "operably linked" may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide sequence under the influence or control of the regulatory sequence. Thus a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide sequence which forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.
Vectors The vectors of the invention may include one or more elements that facilitate expression of nucleotide sequence in a host cell. The vector may include an element that allows the vector to replicate in a host cell. The vector may include an element that allows selection of host cells that contain the vector, e.g. a marker gene. Methods of introducing nucleic acid such as vectors into cells, e.g. by transformation or transfection, are well known in the art.
Host cells The invention relates to host cells comprising a nucleic acid of the invention. The host cell may be a eukaryotic cell, e.g. a yeast or a CHO cell, or a prokaryotic cell, e.g. E. coli.
The host cell may be a cell that is suitable for culturing, i.e. it may be a cell that is not part of a living human or animal body. The nucleic acid of the invention may be present in the host cell as part of the host cell genome.
Alternatively, it may be present in the host cell as an autonomously replicating entity, e.g. a plasmid. Preferably the nucleic acid is heterologous to the host cell, i.e. the nucleic acid is not naturally present in the host cell. For example, the nucleic acid may be inserted into the genome, e.g. by genetic engineering.
Endothelial cell tube formation The polypeptides of the invention may be assessed for their ability to inhibit endothelial cell tube formation. A method of observing this ability may include evaluating, monitoring, measuring, determining, and/or quantitating the ability of the polypeptide to inhibit endothelial cell tube formation. The method may involve culturing endothelial cells on extracellular matrix (ECM) e.g. Matrigel, ECMatrix, which is a solid gel consisting of various basement proteins, such as laminin, collagen type IV and heparin sulphate proteoglycans. ECM is known by the person skilled in the art and it is commercially available. Endothelial cells may be mixed with the polypeptide and added to the ECM. The ECM may then be incubated and tube assembly may be observed after a suitable time period, e.g. 6 hours. This method is described in the Results and Methods below.
The extent of tube assembly may be quantitated by measuring the length of tubes and an average length may be calculated. A reduction in tube length relative to the tube length in the absence of the polypeptide is indicative that the polypeptide inhibits endothelial cell tube formation. The polypeptide may reduce endothelial cell tube formation by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or even 90 percent relative to the absence of the polypeptide. Such a reduction may be observed after incubating the endothelial cells with 10OnM polypeptide on ECM for 6 hours.
Proliferation of endothelial cells
The polypeptides of the invention may be assessed for their ability to inhibit proliferation of endothelial cells. A method of observing this ability may include evaluating, monitoring, measuring, determining, and/or quantitating the ability of the polypeptide to inhibit proliferation of endothelial cells.
The method may involve incubating endothelial cells with the polypeptide for a period of time, e.g. 24 hours, and adding Bromodeoxyuridine (BrdU) at the end of the incubation period. BrdU may then be detected immunochemically by partially denaturing double stranded DNA. BrdU is incorporated into newly synthesized strands of DNA of actively proliferating cells and therefore the amount of BrdU detected indicates the number of cells that are synthesizing DNA. This method is described in the Results and Methods below.
The extent endothelial cell proliferation may by quantitated by measuring the amount of BrdU in the assay. A reduction in the amount of BrdU relative to the amount in the absence of the polypeptide is indicative that the polypeptide inhibits endothelial cell proliferation. The polypeptide may reduce endothelial cell tube formation by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or even 90 percent relative to the absence of the polypeptide. Such a reduction may be observed after incubating endothelial cells with 10OnM polypeptide .
Apoptosis of endothelial cells
The polypeptides of the invention may be assessed for their ability to induce apoptosis of endothelial cells. A method of observing this ability may include evaluating, monitoring, measuring, determining, and/or quantitating the ability of the polypeptide to stimulate apoptosis of endothelial cells.
The method may involve incubating endothelial cells with the polypeptide for a period of time, e.g. 24 hours, together with VEGF. The method may then involve lysing the cells and observing the mononucleosomes and oligonucleosomes in the cytoplasmic fraction of the cell lysate. Mononucleosomes and oligonucleosomes may be observed using ELISA. This method is described in the Results and Methods below.
The extent of endothelial cell apoptosis may be quantitated by measuring the amount of mononucleosomes and oligonucleosomes in the cell lysate. An increase in the amount of mononucleosomes and oligonucleosomes relative to the amount in the absence of the polypeptide is indicative that the polypeptide induces apoptosis. The polypeptide may increase apoptosis by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or even 90 percent relative to the absence of the polypeptide. Such an increase may be observed after incubating 10OnM polypeptide with endothelial cells for 24 hours in the presence of lOng/ml VEGF.
Variants
Variants of Isthmin are polypeptides that do not include the exact amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and nucleic acids that do not include the exact nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Likewise, variants of Isthmin truncates are polypeptide and nucleic acids that do not include the exact amino acid sequence or nucleotide sequence of the mouse Isthmin truncates described in the Examples, or the corresponding human Isthmin truncates.
The mouse Isthmin truncates and corresponding coding sequences used in the Examples are amino acids 26 to 461 of SEQ ID NO: 1; amino acids 26 to 277 of SEQ ID NO: 1; amino acids 215 to 259 of SEQ ID NO: 1; amino acids 271 to 461 of SEQ ID NO: 1;
nucleotides 678 to 1985 of SEQ ID NO: 2; nucleotides 678 to 1433 of SEQ ID NO: 2; nucleotides 1245 to 1379 of SEQ ID NO: 2; nucleotides 1413 to 1985 of SEQ ID NO: 2;
The corresponding human Isthmin sequences are:
amino acids 26 to 464 of SEQ ID NO: 3; amino acids 26 to 280 of SEQ ID NO: 3; amino acids 218 to 262 of SEQ ID NO: 3; amino acids 274 to 464 of SEQ ID NO: 3;
nucleotides 156 to 1472 of SEQ ID NO: 4; nucleotides 156 to 920 of SEQ ID NO: 4; nucleotides 723 to 866 of SEQ ID NO: 4; nucleotides 902 to 1472 of SEQ ID NO: 4.
Variants of the present invention preferably have the ability to inhibit angiogenesis and are preferably for use in methods of medical treatment. For example, the variant molecules of the invention may have the ability to inhibit endothelial cell tube formation, inhibit proliferation of endothelial cells, and/or induce apoptosis in endothelial cells in an in vitro assay. Preferably, a variant according to the invention has a similar, or at least the same, ability to inhibit angiogenesis e.g. as measured by any of these in vitro assays. A "similar ability" means that the ability of the variant to inhibit angiogenesis is at least 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent of the ability of Isthmin to inhibit angiogenesis.
Where the activity of a variant is described, this refers to the activity of the polypeptide encoded by the nucleic acid molecule.
Variants of the present invention may be: (i) novel, naturally occurring, molecules, for example obtainable from organisms other than mouse or human, and which may be identified by hybridisation, for example.
(ii) artificial Isthmin molecule derivatives, which can be prepared by the skilled person in the light of the present disclosure. Such derivatives may be prepared, for instance, by site directed or random mutagenesis, or by direct synthesis. Preferably, a variant nucleic acid, for example, is generated either directly or indirectly, e.g. via one or more amplification or replication steps from an original nucleic acid having all or part of the sequence shown herein. Particularly included are truncated variants which include only a distinctive part or fragment, however produced, corresponding to a portion of the sequences described herein, for example functional parts of the polypeptide that are still capable of inhibiting angiogenesis .
Also included are molecules which have been extended at their termini with non-naturally contiguous sequences, i.e. polypeptides of the invention may also comprise additional amino acids, additional domains, or may be conjugated to additional domains or other molecules. Additional amino acids, domains, or molecules conjugated to the polypeptide may provide an additional function, for example in assisting purification of the polypeptide. Examples of additional domains that may assist in purification of the polypeptide are 6-histidine tag and glutathione S-transferase tag. Polypeptides may be fusion proteins, fused to a peptide or other protein, such as a label, which may be, for instance, bioactive, radioactive, enzymatic or fluorescent.
Variant polypeptides may comprise at least one modification substitution, inversion and/or deletion of one or more amino acids .
Purely as examples, conservative replacements which may be found in such polymorphisms may be between amino acids within the following groups: alanine, serine, threonine; glutamic acid and aspartic acid; arginine and leucine; asparagine and glutamine; isoleucine, leucine and valine; phenylalanine, tyrosine and tryptophan. Production of variants of Isthmin
Variants of Isthmin may be produced by modifying SEQ ID NO: 1, 2, 3, or 4, or any of the Isthmin truncates described above.
A polypeptide of the invention may be a fragment of SEQ ID NO: 1, SEQ ID NO: 3 or any one of the truncates described above. Such fragments may be provided in isolated form, i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region with one or two non-naturally contiguous sequences fused to it. Additionally, several fragments may be comprised within a single larger polypeptide.
Changes to nucleic acid sequences may be desirable for a number of reasons. For instance, they may introduce or remove restriction endonuclease sites or alter codon usage. Alternatively changes to a sequence may produce a derivative by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.
Such changes may modify sites which are required for post translation modification, such as cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for glycosylation, lipolysation etc. Leader or other targeting sequences (e.g. membrane or golgi locating sequences) may be added to the expressed polypeptide to determine its location following expression.
Other desirable mutations may be random or site directed mutations in order to alter the activity, e.g. specificity, or stability of the encoded polypeptide. Changes may be by way of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining peptide conformation.
Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.
The variants of the invention may also be created by chemical modification of Isthmin or any of the Isthmin truncates described above. Methods for chemical modification of polypeptides are well known in the art.
A polypeptide of the invention may be obtained by expression of a nucleic acid that encodes the polypeptide using a suitable vector and host organism. Examples of suitable vectors and hosts are well known in the art (see e.g. Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York) .
Polypeptides, and particularly fragments, of the invention may also be created using chemical synthesis by any suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings. In conventional solution phase peptide synthesis, the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. Many such methods are now commonplace to those skilled in the art.
The variants of the invention may also be created by modification, using molecular biological techniques, of the nucleic acid that encodes Isthmin or any of the Isthmin truncates described above, or a nucleic acid that encodes a further variant or homologue of the invention. Molecular biological techniques are well known in the art.
Thus, the invention provides a method of producing and/or identifying a variant of Isthmin, which method comprises the steps of: (i) providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or any of the Isthmin truncates described above, or the nucleic acid sequence encoding these,
(ii) modifying the amino acid sequence of the polypeptide, or the nucleic acid encoding the amino acid sequence, and
(iii) measuring the ability of the variant to inhibit angiogenesis .
Step (iii) may involve measuring the ability of the variant to inhibit endothelial cell tube formation, inhibit proliferation of endothelial cells, and/or induce apoptosis in endothelial cells in an in vitro assay.
Methods of purifying polypeptides from heterogenous mixtures are well known in the art (e.g. selective precipitation, proteolysis, ultrafiltration with known molecular weight cutoff filters, ion-exchange chromatography, gel filtration, etc.) . Typical protocols are set out in "Protein Purification - principles and practice" Pub. Springer-Verlag, New York Inc (1982), and by Harris & Angal (1989) "Protein purification methods - a practical approach" Pub. O.U.P. UK, or references therein. Further methods which are known to be suitable for protein purification are disclosed in "Methods in Enzymology Vol. 182 - Guide to Protein Purification" Ed. M P Deutscher, Pub. Academic Press Inc..
Antibodies to Isthmin, or variants of Isthmin, may also be used to screen for Isthmin, which methods are well known to those skilled in the art.
The Isthmin amino acid or nucleotide sequence, i.e. SEQ ID NO: 1 or 3, or SEQ ID NO: 2 or 4 may be used in a data-base (e.g. of ESTs, or STSs) search to find sequences that share a specified level of sequence identity, such as those which may become available in due course, and expression products of which can be tested for activity as described herein.
Alternatively, variants may be provided by standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes.
Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.
Probing may optionally be done by means of so-called "nucleic acid chips", see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review.
Libraries of nucleic acid molecules may be created which may be screened for nucleic acids that encode a polypeptide of the invention. Such libraries may be created using the nucleic acid molecule that encodes Isthmin. The Isthmin nucleic acid molecule sequence may be mutated, for example using random PCR mutagenesis, or gene shuffling techniques. The resulting mutated nucleic acid molecules may be ligated into a vector. Libraries of polypeptides may be created, by expressing the nucleic acid molecules in a suitable host.
Hybridisation The present invention includes nucleic acid that hybridise to SEQ ID NO: 2 or SEQ ID NO: 4, and nucleic acids that hybridise to nucleotides 603 to 1985 of SEQ ID NO: 2; nucleotides 678 to 1985 of SEQ ID NO: 2; nucleotides 678 to 1433 of SEQ ID NO: 2; nucleotides 1245 to 1379 of SEQ ID NO: 2; nucleotides 1413 to 1985 of SEQ ID NO: 2; nucleotides 1458 to 1949 of SEQ ID NO: 2; nucleotides 81 to 1472 of SEQ ID NO: 4; nucleotides 156 to 1472 of SEQ ID NO: 4; nucleotides 156 to 920 of SEQ ID NO: 4; nucleotides 723 to 866 of SEQ ID NO: 4; nucleotides' 902 to 1472 of SEQ ID NO: 4; and/or nucleotides 945 to 1436 of SEQ ID NO: 4. Hybridisation may occur under intermediate . stringency conditions, high stringency conditions, and/or very high stringency conditions.
In accordance with the present invention, nucleic acid sequences may be identified by using hybridization and washing conditions of appropriate stringency.
Complementary nucleic acid sequences will hybridise to one another through Watson-Crick binding interactions. Sequences which are not 100% complementary may also hybridise but the strength of the hybridisation usually decreases with the decrease in complementarity. The strength of hybridisation can therefore be used to distinguish the degree of complementarity of sequences capable of binding to each other.
The "stringency" of a hybridization reaction can be readily determined by a person skilled in the art.
The stringency of a given reaction may depend upon factors such as probe length, washing temperature, and salt concentration. Higher temperatures are generally required for proper annealing of long probes, while shorter probes may be annealed at lower temperatures. The higher the degree of desired complementarity between the probe and hybridisable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so.
For example, hybridizations may be performed, according to the method of Sambrook et al., ("Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-420C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules is to calculate the melting temperature Tm (Sambrook et al., 1989) : Tm = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/n
where n is the number of bases in the oligonucleotide.
As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57 °C. The Tm of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in sequence complementarity .
Hybridisation under high stringency conditions may involve performing the hybridisation at a temperature of Tm-15 or higher. Moderate stringency may be considered to be Tm-25 to Tm-15. Low stringency may be considered to be Tm-35 to Tm-25.
Accordingly, nucleotide sequences can be categorised by an ability to hybridise to a target sequence under different hybridisation and washing stringency conditions which can be selected by using the above equation. The Tm may be used to provide an indicator of the strength of the hybridisation.
The concept of distinguishing sequences based on the stringency of the conditions is well understood by the person skilled in the art and may be readily applied.
Sequences exhibiting 95-100% sequence complementarity are considered to hybridise under very high stringency conditions, sequences exhibiting 85-95% complementarity are considered to hybridise under high stringency conditions, sequences exhibiting 70-85% complementarity are considered to hybridise under intermediate stringency conditions, sequences exhibiting 60-70% complementarity are considered to hybridise under low stringency conditions and sequences exhibiting 50-60% complementarity are considered to hybridise under very low stringency conditions.
Cancer A cancerous condition that be may treated by the present invention may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation) , neoplasm or tumour or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumour. The cancerous condition may be a cancer and may be a benign or malignant cancer and may be primary or secondary (metastatic) . A neoplasm or tumour may be any abnormal growth or proliferation of cells and may be located in any tissue. Examples of tissues include the colon, pancreas, lung, breast, uterus, stomach, kidney, testis, central nervous system
(including the brain) , peripheral nervous system, skin, blood or lymph.
A cancerous condition may be treated by inhibiting angiogenesis . Inhibition of angiogenesis may be inhibition of angiogenesis in a tumour that is, or is not, inducing angiogenesis. Inhibition of angiogenesis in a tumour that is not inducing angiogenesis may prevent the tumour from inducing angiogenesis .
Administration
Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, sublingual, oral and nasal. The medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington' s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins .
The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
Brief Description of the Figures
Embodiments and experiments illustrating the principles of the. invention will now be discussed with reference to the accompanying figures in which: Figure 1
Figure 1 shows diagrams illustrating the domain organization of four different forms of truncated Isthmin. These are: Full Length ISM - SEQ ID NO: 1; Recombinant ISM-FL - amino acids 26 to 461 of SEQ ID NO: 1; Recombinant ISM-N - amino acids 26 to 277 of SEQ ID NO: 1; Recombinant ISM-C - amino acids 271 to 461 of SEQ ID NO: 1; Recombinant ISM-TSR - amino acids 215 to 259 of SEQ ID NO: 1.
ISM-FL includes TSR and AMOP, ISM-N includes TSR and an N- terminal region with no known protein domain, ISM-C includes AMOP. Signal peptide was cleaved and His-tag was added at C- terminal of each recombinant protein. Native Isthmin contains a signal peptide when synthesized but no His-tag. The mature, secreted Isthmin should have, no signal peptide as it has been cleaved during the secretion process. A key to the domains is shown at the bottom of the Figure.
Figure 2 Figure 2 shows the results of experiments demonstrating that Isthmin inhibits EC tube formation on Matrigel. In particular it shows representative photos showing the inhibitory effect of ISM-C, -N, -FL on EC tube formation assay in the presence of 2%FBS as control, (i) shows control, (ii) shows ISM-FL, (iϋ) shows ISM-N, (iv) shows ISM-C, (v) shows ISM-TSR. ISM- TSR had no effect on EC tube formation.
Figure 3
Figure 3 shows results of adding recombinant Isthmin at different time-points during the EC tube formation process (Oh, Ih, 2h, 4h after plating ECs onto Matrigel) . Investigation of ISM-C, -N and -FL showed significant influences to tube assembly when adding Isthmin at the early stage as depicted at 0 hours, (i) shows control, (ii) shows ISM-FL, (iii) shows ISM-N, (iv) shows ISM-C, (v) shows ISM- TSR. ISM-TSR had no observable effect on tube formation. Figure 4
Figure 4 shows the results of experiments investigating the role of Isthmin in EC migration in the presence (A) and absence (B) of VEGF. No significant effect of Isthmin on EC migration was observed with or without VEGF. Normalisation of the data values were performed with respect to the data value for migration with VEGF for (A) and control for (B) .
Figure 5
Figure 5 shows the results of experiments demonstrating that Isthmin inhibits VEGF-induced EC proliferation. Normalisation of measured values was performed against the data value for control .
Figure 6
Figure 6 shows the results of experiments investigating induction of EC apoptosis by Isthmin. Quantification of apoptosis was performed using cell death ELISA, which measures fragmented DNA in the presence of VEGF. ISM-FL induced EC apoptosis in a dose-dependent manner.
Figure 7
Figure 7 shows the results of experiments investigating a role for Isthmin in zebrafish embryonic angiogenesis . In particular, Figure 7 shows representative microphotographs of zebrafish Isthmin 1 gene (zlsml) morpholino antisense oligonucleotide (MO) knockdown and Mismatch MO control in Tg (fli-l:GFP) transgenic zebrafish under the same magnification. (A) shows wild-type, (B) shows MO control, (C) shows zlsml MO. The impaired intersegmental vessels in zlsml morpholino injected larvae is indicated by a box in (C) . No abnormal blood vessel can be seen in the wild-type and Morpholino control group. Figure 8
Figure 8 shows amino acid and nucleotide sequences used in the of mouse Isthmin and truncates used during the experiments, as well as human Isthmin. (A) shows the amino acid sequence of mouse Isthmin (SEQ ID NO:
1) .
(B) shows the nucleotide sequence of mouse Isthmin (SEQ ID NO: 2) .
(C) shows the amino acid sequence of human Isthmin (SEQ ID NO: 3) .
(D) shows the nucleotide sequence of human Isthmin (SEQ ID NO: 4) .
(E) shows the amino acid sequence of mouse ISM-FL (SEQ ID NO: 5) - (F) shows the nucleotide sequence used to generate mouse ISM-
FL (SEQ ID NO: 6) . The signal peptide was removed in this construct.
(G) shows the amino acid sequence of mouse ISM-TSR (SEQ ID NO:
7) - (H) shows the nucleotide sequence of mouse ISM-TSR (SEQ ID NO:
8) .
(I) shows the amino acid sequence of mouse ISM-N (SEQ ID NO:
9)
(J) shows the nucleotide sequence of mouse ISM-N (SEQ ID NO: 10) . (The signal peptide was cleaved after expression.)
(K) shows the amino acid sequence of mouse ISM-C (SEQ ID NO:
H) •
(L) shows the nucleotide sequence of mouse ISM-C (SEQ ID NO:
12) . (M) shows an alignment of the amino acid sequence of SEQ ID
NO: 1 and SEQ ID NO: 3.
Figure 9
Figure 9 shows the amino acid and nucleotide sequences of Zebrafish Isthminl and Xenopus laevis Isthmin. (A) shows the amino acid sequence of Zebrafish Isthminl (SEQ ID NO: 13) . Accession no. NP_001012376.1 (GI : 59933256) .
(B) shows the nucleotide sequence of Zebrafish Isthminl (SEQ ID NO: 14. Accession no. NM_001012376.1 (GI : 59933255) . (C) shows the amino acid sequence of Xenopus laevis Isthmin
(SEQ ID NO: 15) . Accession no. NP_001082228.1 (GI : 148222188 ) . (D) shows the nucleotide sequence of Xenopus laevis Isthmin (SEQ ID NO: 16) . Accession no. NM_001088759.1 (GI : 148222187) .
Figure 10
Figure 10 shows the results of experiments investigating whether Isthmin inhibits tumor growth in vivo. (A) (i) shows control Bl 6 mouse melanomas (modified with empty vector) and (ii) shows ISM-FL overexpressing mouse melanomas. (B) shows the growth curve of control and ISM-expressing melanomas. ISM- FL significantly suppressed subcutaneous B16 tumor growth in mouse .
Detailed Description of the Invention Specific details of the best mode contemplated by the inventors for carrying out the invention are set forth below, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.
Results
We generated full-length and three truncated forms of mouse Isthmin as a His-tagged recombinant protein by E.coli expression and Ni-NTA affinity chromatography purification to study in vitro angiogenesis assays. The signal peptide was cleaved from the full-length and the N-terminal Isthmin fragments. We tested the acute cytotoxicity level of these purified proteins on ECs and they were not cytotoxic at least up to 10OnM. Endothelial cell tube formation
Tube formation is a multiple process involving migration, cell adhesion and differentiation. EC rapidly align and form hollow tubular structures when cultured on ECMatrix, which is a solid gel consisting of various basement proteins, such as laminin, collagen type IV and heparan sulfate proteoglycans etc. (Grant et al., 1989; Kubota et al., 1988; Kleinman et al.,1986) . Premixed endothelial cell suspension with different concentrations of Isthmin and its truncated recombinants were added to the top of the ECMatrix. The extent of tube assembly can be monitored after 6 hours incubation in tissue-culture incubator. The results indicated that both full-length Isthmin and its truncated products significantly inhibited tube formation in a dose-dependent manner. Figure 2 shows that complex mesh like structures can be seen in the control and that in the presence of ISM-C, -N, -FL endothelial cells failed to migrate and align them to form tubes. However, ISM-TSR is different from other Isthmin truncated proteins in having no ability in inhibiting EC tube formation.
The EC tube formation time course was investigated to analyze how Isthmin disrupts tube formation at the cell morphological level. The same concentrations of ISM-TSR, -C, -N, -FL were added to the culture medium at Oh, Ih, 2h and 4h respectively after plating cell on Matrigel. Hardly any tubular network formed at Oh in the presence of ISM-C, -N, -FL but not ISM-TSR (Figure 3) . At Ih, the obvious disrupted extension of tube assemblies were seen in the presence of ISM-C, -N and -FL but not ISM-TSR. At 2h, tubular structures were not much interrupted in the presence of ISM-C, -N and -FL as compared to the control. These results indicated that ISM-C, -N, -FL inhibited EC tube formation mainly at the beginning stage of EC tube formation.
Endothelial cell migration Next, we studied the potential role of Isthmin in EC migration. VEGF was used as a chemotactic agent to assess the effect of Isthmin on EC migration. EC migration in the presence of ISM-TSR, -C, -N, -FL at varying concentrations was determined. As shown in Figure 4A, however, all Isthmins had no significant effect on EC VEGF-induced migration. We examined the significance of each protein at InM concentration by analyzing the p-value. These were 0.08, 0.13, 0.11 and 0.1 for ISM-TSR, -C, -N, -FL respectively indicating that there is no significant difference between control and ISM-TSR, -C, -N and- FL. In addition, we also investigated the effect of Isthmin on EC migration in the absence of VEGF. The results (Figure 4B) demonstrated that there was no difference for all Isthmin proteins compared to the control.
Endothelial cell proliferation Then, to test whether Isthmin is capable in inhibiting EC proliferation, the BrdU (bromodeoxyuridine) labelling method was used. BrdU is incorporated into newly synthesized DNA strands of actively proliferating cells when added prior to the end of incubation period. Incorporation of BrdU is detected immunochemically by partially denaturing double stranded DNA thereby allowing the assessment of the population of cells that are synthesizing DNA. As shown in Figure 5, ISM-FL potently inhibited VEGF-induced EC proliferation in a dose-dependent manner. Its ISM-N and ISM-C also mildly inhibited VEGF-induced EC proliferation while ISM-TSR alone was not effective. The measured values at 1 μM for all case studies is significantly lower compared to the control.
Endothelial cell apoptosis Fourthly, we asked whether Isthmin restrains angiogenesis by inducing apoptosis. Induction of apoptosis in endothelial cells is one of the mechanisms that angiogenesis inhibitors work. It has been shown that TSPs inhibit angiogenesis through FasL-Fas signalling pathways which activate caspases and apoptosis (Lucas and Paul., 2003) . DNA degradation occurs several hours before plasma membrane breakdown in the cytoplasm of the apoptotic cell, which leads to the enrichment of mono- and oligonucleosomes . Therefore, apoptosis was analyzed by determination of mono- and oligonucleosomes in the cytoplasmic fraction of Isthmin-treated cell lysates. As shown in Figure 6, EC apoptosis is induced by ISM-FL. ISM-FL induced EC apoptosis dose-dependently .
Inhibition of xenograph tumor growth in mouse
ISM-FL was cloned into a mammalian expression vector where it is expressed as a secreted extracellular protein. The expression vector was transfected into mouse B16 melanoma cells. Cells stably expressing ISM-FL were selected clonally. Clones that expressed a high level of ISM-FL in the culture media were selected. Matching clones containing empty expression vector were selected as controls. Note that B16 cells express a very low level of ISM endogenously . As shown in Figure 10, ISM expression in B16 tumors significantly suppressed tumor growth compared to controls. The ISM producing tumor was also pale with less blood, presumably from suppression of tumor blood vessel formation. Melanin production by this melanoma cells was also suppressed.
Zebrafish Isthminl
We asked whether inhibition of in vitro angiogenesis by Isthmin implicated a possible role of Isthmin in embryonic angiogenesis. In zebrafish, formation of intersegmental vessels is considered to represent capillary sprouting. Using Tg (fIi-I : GFP) transgenic zebrafish as a model for zlsml MO knockdown, we observed that both intersegmental and axial vessels are normal in wild type and mismatch MO control zebrafish. However, intersegmental vessels but not axial vessels were impaired in the zIsmlMO knockdown zebrafish group (Figure 7) . This indicated that zlsml was involved in capillary sprouting which indicates a role for zlsml in embryonic angiogenesis. The amino acid and nucleotide sequences of the zebrafish Isthminl are shown in Figure 9A and 9B respectively.
In summary, we have shown in this study that Isthmin potently inhibits angiogenesis . It interrupted EC tube formation, inhibited EC proliferation, induced EC apoptosis and suppressed tumour formation in vivo. In our transgenic zebrafish model, the impairment of blood vessels was observed in zlsml knockdown zebrafish. All these indicate that Isthmin has potential applications in the treatment of angiogenesis diseases .
Methods
Proteins expression and purification Recombinant truncated Isthmin and full length Isthmin were cloned to pET-M (derived from pET-32) vector and induced by IPTG. Protein purification was done under denatured conditions (6M urea) according to the QIAGEN manufacturer protocol.
Cell migration Assay
Migration assay was performed using Falcon cell culture insert as descried with modifications (24) . HUVECs were started 4 hours. Various concentrations of proteins were pre-incubated with 20000 cells for 30 min at 37°C before seeding onto the gelatin-coated cell culture inserts while lOng/mlVEGF was placed into the lower chamber. The cell culture insert chamber was then incubated at 370C for 6 hours. Then cells staying on the insert were removed by cotton swab, cells migrating on the lower surface of the inserts were fixed, stained and photographed under a light microscope (40*) . Five random fields (upper, down, left, right and centre) were quantified using Image J 1.32 software. Cell tube formation Assay
HUVECs were starved 3-4 hours, and then pre-treated with different concentrations of proteins for 30 min. Seeded 20000 cells per well onto the surface of the polymerized Matrigel and incubated for 6 hour in tissue-culture incubator. Normal
Matrigel (purchased from CHEMICON Inc) and growth factor reduced Matrigel (BD. Inc) were used. Formed tubes were fixed with 37% formalin and photos were taken representatively under
5X magnification.
Proliferation Assay
Cell proliferation was determined using the BrdU (bromodeoxyuridine) kit (CHEMICON Inc) . 20000 cells per well was incubated with 2%FBS with or without lOng/ml VEGF and various concentrations of proteins for 24h. After incubation, the following procedure was according to the manufacturer' s protocol .
Apoptosis Assay Cells were cultured in 6-well-plate overnight and then incubated with proteins for 24 hours in 2%FBS in the presence of 10ng/ml VEGF. After protein treatment, apoptosis was quantified using a cell death detection ELISA kit (Roche Applied Science) .
Microinjection of morpholino (MO) antisense oligonucleotides The morpholino antisense (MO) oligomers of zlsml and control, was purchased from Gene Tools and the zlsml MO antisense sequence is 5'-CTCCGCCGCCAGACGCACCATCCTC-S' and the one of mismatch MO control is 5'- CTgCGCCcCCAGAgGCACgATCgTC-3' . Before, injection, the concentrated MOs was diluted into working concentrations of ImM, with nuclease-free water and phenol red dye. In addition to MO treatment group, each experiment included a control MO group with no binding target and an un-injected control group. Each MO was microinjected into wild type zebrafish embryos and GFP labelled blood vessel transgenic zebrafish embryos at the single cell stage. Microinjection volumes were estimated at InI per embryos. There were 30-40 embryos in each trial group. After injection, embryos were incubated at 37C in embryo medium. Post-injection phenotypes were observed at different time points of the development stages.
Synthetic antisense MOs were microinjected into fertilized zebrafish eggs at the 1-2 cell stage. Embryos were observed under fluorescent, microscope at 36-48hpf (hours post fetrtilization) . Intersegmental vessels (ISVs) are usually formed between 24-48hpf.
Mouse tumor formation assay ISM-FL was cloned into a mammalian expression vector and stably expressed in mouse B16 melanoma cells using standard molecular and cell biology methods. One million Bl 6 tumor cells, control or ISM-expressing, were injected subcutaneously into the dorsal flank of mice. For each mouse, control cells were injected to the left dorsal flank and ISM expressing tumor cells were injected into the right dorsal flank. Tumors appear after about 6 days post injection. Tumor growth was monitored by measuring the tumor size (length and width) with a digital calliper. Experiments were terminated at 14 days post tumor cell injection.
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Claims

Claims
1. A polypeptide comprising:
(i) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(ii) a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or
(iii) an amino acid sequence sharing at least 60 percent sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 10 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; wherein the polypeptide does not comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 13, or SEQ ID NO: 15.
2. The polypeptide of claim 1, wherein the polypeptide inhibits angiogenesis .
3. The polypeptide of claim 1 or 2, wherein the polypeptide inhibits proliferation of endothelial cells.
4. A polypeptide comprising:
(i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(ii) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(iii) a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iv) an amino acid sequence sharing at least 60 percent sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 10 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; for use in a method of medical treatment.
5. The polypeptide of claim 4, wherein the polypeptide is for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis.
6. Use of a polypeptide in the manufacture of a medicament for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis, wherein the polypeptide comprises:
(i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(ii) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(iii) a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or (iv) an amino acid sequence sharing at least 60 percent sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 10 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
7. The polypeptide or use of any one of claims 4 to 6, wherein the polypeptide is for inhibiting tumour angiogenesis.
8. A method of inhibiting angiogenesis in an individual or of treating an individual at risk of unwanted angiogenesis comprising administering a polypeptide to the individual, wherein the polypeptide comprises:
(i) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or (ii) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
(iii) a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or
(iv) an amino acid sequence sharing at least 60 percent sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which fragment has at least 10 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
9. The method of claim 8, wherein the method is a method of inhibiting tumour angiogenesis in an individual.
10. The polypeptide, use or method of any one of claims 1 to 9, wherein the polypeptide comprises an amino acid sequence sharing at least 60 percent sequence identity with the' sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3.
11. The polypeptide, use or method of any one of claims 1 to 9, wherein the polypeptide comprises an amino acid sequence sharing at least 60 percent sequence identity with the sequence consisting of amino acids 26 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 464 of SEQ ID NO: 3.
12. The polypeptide, use or method of any one of claims 1 to 9, wherein the polypeptide comprises a fragment of the sequence consisting of amino acids 26 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 464 of SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
13. The polypeptide, use or method of any one of claims 1 to 9, wherein the polypeptide comprises a fragment of the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
14. The polypeptide, use or method of claim 12 or 13, wherein the polypeptide comprises at least 10 contiguous amino acids of SEQ ID NO: 1 from the N-terminal side of amino acid 215 of SEQ ID NO: 1 or at least 10 amino acids from the N-terminal side of amino acid 218 of SEQ ID NO: 3.
15. The polypeptide, use or method of any one of claim 1 to 9, wherein the polypeptide comprises the sequence consisting of amino acids 26 to 277 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 280 of SEQ ID NO: 3.
16. The polypeptide, use or method of any one of claim 1 to 9, wherein the polypeptide comprises the sequence consisting of amino acids 26 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 26 to 464 of SEQ ID NO: 3.
17. The polypeptide, use or method of any one of claims 10 to 16, wherein the polypeptide inhibits endothelial cell tube formation.
18. The polypeptide, use or method of any one of claims 10 to 17, wherein the polypeptide induces apoptosis of endothelial cells.
19. The polypeptide; use or method of any one of claims 1 to 9, wherein the polypeptide comprises an amino acid sequence sharing at least 60 percent sequence identity with the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3.
20. The polypeptide, use or method of any one of claims 1 to 9, wherein the polypeptide comprises a fragment of the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID
NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
21. The polypeptide, use or method of any one of claims 1 to 9, wherein the polypeptide comprises the sequence consisting of amino acids 215 to 259 of SEQ ID NO: 1 or the sequence consisting of amino acids 218 to 262 of SEQ ID NO: 3.
22. The polypeptide, use or method of any one of claim 1 to 9, wherein the polypeptide comprises an amino acid sequence sharing at least 60 percent sequence identity with the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the amino acid sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
23. The polypeptide, use or method of any one of claim 1 to 9, wherein the polypeptide comprises an amino acid sequence sharing at least 60 percent sequence identity with the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
24. The polypeptide, use or method of any one of claim 1 to 9, wherein the polypeptide comprises a fragment of the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
25. The polypeptide, use or method of any one of claim 1 to 9, wherein the polypeptide comprises a fragment of the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
26. The polypeptide, use or method of any one of claim 1 to 9, wherein the polypeptide comprises the sequence consisting of amino acids 271 to 461 of SEQ ID NO: 1 or the sequence consisting of amino acids 274 to 464 of SEQ ID NO: 3.
27. The polypeptide, use or method of any one of claim 1 to 9, wherein the polypeptide comprises the sequence consisting of amino acids 286 to 449 of SEQ ID NO: 1 or the sequence consisting of amino acids 289 to 452 of SEQ ID NO: 3.
28. The polypeptide, use or method of any one of claims 22 to 27, wherein the polypeptide inhibits endothelial cell tube formation.
29. The polypeptide, use or method of any one of claims 1 to 9, wherein the polypeptide comprises a polypeptide as defined in any one of claims 10 to 21, and a polypeptide as defined in any one of claims 22 to 28.
30. The polypeptide, use or method of claim 29, wherein the polypeptide comprises a linker between the polypeptide as defined in any one of claims 10 to 21, and the polypeptide as defined in any one of claims 22 to 28.
31. The polypeptide, use or method of claim 29, wherein the polypeptide as defined in any one of claims 10 to 21 is contiguous with the polypeptide as defined in any one of claims 22 to 28.
32. An isolated nucleic acid comprising a nucleotide sequence, which nucleotide sequence comprises:
(i) a nucleotide sequence sharing at least 60 percent sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or
(ii) a fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, which fragment has at least 18 contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 4, or
(iii) a nucleotide sequence sharing at least 60 percent sequence identity with the fragment defined in (ii); or
(iv) a nucleotide sequence encoding a polypeptide as defined in any one of claims 1 to 31; wherein the nucleic acid does not comprise the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, or SEQ ID NO: 16.
33. A vector comprising a nucleic acid as defined in claim 32.
34. A host cell comprising a nucleic acid as defined claim 32.
35. A method of producing a polypeptide as defined in any one of claims 1 to 31, comprising: (a) providing a host cell comprising a nucleic acid that comprises a nucleotide sequence encoding the polypeptide; and
(b) causing the host cell of (a) to express the polypeptide encoded by the nucleic acid.
36. A composition comprising a polypeptide as defined in any one of claims 1 to 31.
37. A kit for inhibiting angiogenesis in an individual or for treating an individual at risk of unwanted angiogenesis, wherein the kit comprises:
(i) a container comprising a polypeptide as defined in any one of claims 1 to 31; and optionally
(ii) instructions for administering the composition to the individual.
PCT/SG2009/000085 2008-03-14 2009-03-12 Isthmin derivatives for use in treating angiogenesis WO2009113965A1 (en)

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