WO2000044903A1 - Facteur de croissance h de type facteur de croissance d'origine plaquettaire/facteur de croissance de l'endothelium vasculaire et ses utilisations - Google Patents

Facteur de croissance h de type facteur de croissance d'origine plaquettaire/facteur de croissance de l'endothelium vasculaire et ses utilisations Download PDF

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WO2000044903A1
WO2000044903A1 PCT/US2000/001895 US0001895W WO0044903A1 WO 2000044903 A1 WO2000044903 A1 WO 2000044903A1 US 0001895 W US0001895 W US 0001895W WO 0044903 A1 WO0044903 A1 WO 0044903A1
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vegf
pdgf
growth factor
polypeptide
host cell
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PCT/US2000/001895
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WO2000044903A9 (fr
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Ulf Eriksson
Kari Alitalo
Juha Lauren
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Ludwig Institute For Cancer Research
Licentia Ltd.
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Priority to EP00915701A priority Critical patent/EP1147193A4/fr
Priority to AU36930/00A priority patent/AU3693000A/en
Priority to JP2000596145A priority patent/JP2002535006A/ja
Publication of WO2000044903A1 publication Critical patent/WO2000044903A1/fr
Publication of WO2000044903A9 publication Critical patent/WO2000044903A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention relates to growth factors for connective tissue cells, fibroblasts, myofibroblasts and glial cells and/or to growth factors for endothelial cells, and in particular to a novel platelet-derived growth factor/vascular endothelial growth factor-like growth factor, to pharmaceutical and diagnostic compositions and methods utilizing or derived from the factor.
  • vasculogenesis is characterized by the in si tu differentiation of endothelial cell precursors to mature endothelial cells and association of these cells to form vessels, such as occurs in the formation of the primary vascular plexus in the early embryo.
  • angiogenesis the formation of blood vessels by growth and branching of pre-existing vessels, is important in later embryogenesis and is responsible for the blood vessel growth which occurs in the adult.
  • angiogenesis (Pepper et al . , Enzyme & Protein, 49: 138-162, 1996; Breier et al . , Dev. Dyn . , 204: 228-239, 1995; Risau, Na ture, 386: 671-674, 1997).
  • angiogenesis is tightly controlled and limited under normal circumstances to the female reproductive cycle, establishment and maintenance of pregnancy.
  • angiogenesis can be switched on in response to tissue damage and fractures.
  • solid tumors are able to induce angiogenesis in surrounding tissue, thus sustaining tumor growth and facilitating the formation of metastases (Folk an, J., Na ture Med. , 1: 27-31, 1995) .
  • Angiogenesis is also involved in a number of pathologic conditions, where it plays a role or is involved directly in different sequelae of the disease.
  • Some examples include as neovascularization of associated with various liver diseases, neovascular sequelae of diabetes, neovascular sequelae to hypertension, neovascularization in post-trauma, neovascularization due to head trauma, neovascularization in chronic live infection (e.g. chronic hepatitis), neovascularization due to heat or cold trauma, dysfunction related to excess of hormone, creation of hemangiomas and restenosis following angioplasty.
  • Inhibition of angiogenesis would be useful in preventing or alleviating these pathological conditions.
  • promotion of angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis, such as in coronary heart disease and thromboangitis obliterans .
  • Angiogenesis is a physiologically complex process involving proliferation of endothelial cells, degradation of the extracellular matrix, branching of vessels which involves migration and invasion of the surrounding tissue and subsequent cell adhesion events and finally, tube formation.
  • the molecular mechanisms underlying the complex angiogenic processes are far from being understood.
  • FGFs fibroblast growth factors
  • PDGF platelet-derived growth factor
  • TGF transforming growth factor alpha
  • HGF hepatocyte growth factor
  • VEGFs vascular endothelial growth factors
  • RTKs endothelial receptor tyrosine kinases
  • VEGF/VEGF family namely two PDGFs (A and B) , VEGF and six members that are closely related to VEGF.
  • the six members closely related to VEGF are: VEGF-B, described in International Patent Application PCT/US96/02957 (WO 96/26736) and in U.S. Patents 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki; VEGF-C, described in Joukov et al . , EMBO J. , 15: 290-298, 1996 and Lee et al . , Proc . Na tl . Acad. Sci .
  • VEGF-D described in International Patent Application No. PCT/US97/14696 (WO 98/07832), and Achen et al . , Proc . Na tl . Acad . Sci . USA, 95: 548-553, 1998; the placenta growth factor (P1GF), described in Maglione et al . , Proc. Natl. Acad. Sci. USA, 88: 9267-9271, 1991; VEGF2, described in U.S. Patents 5,932,540 and 5,935,540 by Human Genome Sciences, Inc; and VEGF3, described in International Patent Application No.
  • P1GF placenta growth factor
  • VEGF family member has between 30% and 45% amino acid sequence identity with VEGF.
  • the VEGF family members share a VEGF homology domain which contains the six cysteine residues which form the cystine-knot motif.
  • Functional characteristics of the VEGF family include varying degrees of mitogenicity for endothelial cells, induction of vascular permeability and angiogenic and lymphangiogenic properties.
  • VEGF Vascular endothelial growth factor
  • VEGF has strong chemoattractant activity towards monocytes, can induce the plasminogen activator and the plasminogen activator inhibitor in endothelial cells, and can also induce microvascular permeability. Because of the latter activity, it is sometimes referred to as vascular permeability factor (VPF) .
  • VEGF is also che otactic for certain hematopoetic cells. Recent literature indicates that VEGF blocks maturation of dendritic cells and thereby reduces the effectiveness of the immune response to tumors (many tumors secrete VEGF) (Gabrilovich et al . , Blood, 92: 4150-4166, 1998; Gabrilovich et al . , Clinical Cancer Research, 5: 2963-2970, 1999) .
  • VEGF-B has similar angiogenic and other properties to those of VEGF, but is distributed and expressed in tissues differently from VEGF.
  • VEGF-B is very strongly expressed in heart, and only weakly in lung, whereas the reverse is the case for VEGF. This suggests that VEGF and VEGF-B, despite the fact that they are co-expressed in many tissues, may have functional differences .
  • VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique by screening for cellular proteins which might interact with cellular retinoic acid-binding protein type I (CRABP-I) . Its isolation and characteristics are described in detail in PCT/US96/02957 and in Olofsson et al . , Proc . Na tl . Acad. Sci . USA, 93: 2576-2581, 1996.
  • CRABP-I retinoic acid-binding protein type I
  • VEGF-C was isolated from conditioned media of the PC-3 prostate adenocarcinoma cell line (CRL1435) by screening for ability of the medium to produce tyrosine phosphorylation of the endothelial cell-specific receptor tyrosine kinase VEGFR-3 (Flt4), using cells transfected to express VEGFR-3.
  • VEGF-C was purified using affinity chromatography with recombinant VEGFR-3, and was cloned from a PC-3 cDNA library. Its isolation and characteristics are described in detail in Joukov et al . , EMBO J. , 15: 290-298, 1996.
  • VEGF-C was shown to be angiogenic in the mouse cornea model and in the avian chorioallantoic membrane (Cao et al . , Proc . Na tl . Acad . Sci . USA, 95: 14389-14394, 1998) and was able to induce angiogenesis in the setting of tissue ischemia (Witzenbichler et al . , Am . J. Pa thol . , 153: 381-394, 1998). Furthermore, VEGF-C stimulated lymphangiogenesis in the avian chorioallantoic membrane (Oh et al . , Dev. Biol . , 188: 96-109, 1997) and in a transgenic mouse model (Jeltsch et al . , Science, 276: 1423-1425, 1997) .
  • VEGF-D was isolated from a human breast cDNA library, commercially available from Clontech, by screening with an expressed sequence tag obtained from a human cDNA library designated "Soares Breast 3NbHBst" as a hybridization probe (Achen e t al . , Proc . Na tl . Acad. Sci . USA, 95: 548-553, 1998) . Its isolation and characteristics are described in detail in International Patent Application No. PCT/US97/14696 (WO98/07832 ) .
  • VEGF-D ⁇ N ⁇ C a biologically active fragment of VEGF-D
  • This fragment consists of VEGF-D amino acid residues 93 to 201 linked to the affinity tag peptide FLAG ® .
  • the VEGF-D gene is broadly expressed in the adult human, but is certainly not ubiquitously expressed.
  • VEGF-D is strongly expressed in heart, lung and skeletal muscle.
  • Intermediate levels of VEGF-D are expressed in spleen, ovary, small intestine and colon, and a lower expression occurs in kidney, pancreas, thymus, prostate and testis.
  • No VEGF-D mRNA was detected in RNA from brain, placenta, liver or peripheral blood leukocytes.
  • VEGF-D was shown to be angiogenic in the rabbit cornea (Marconcini et al . , Proc . Na tl . Acad . Sci . USA, 96: 9671-9676, 1999) .
  • the lymphangiogenic capacity of VEGF-D has not yet been reported, however, given that VEGF-D, like VEGF-C, binds and activates VEGFR-3, a receptor thought to signal for lymphangiogenesis (Taipale et al . , Cur . Topics Micro . Immunol . 237: 85-96, 1999), it is highly likely that VEGF-D is lymphangiogenic.
  • VEGF-D and VEGF-C may be of particular importance for the malignancy of tumors, as metastases can spread via either blood vessels or lymphatic vessels; therefore molecules which stimulate angiogenesis or lymphangiogenesis could contribute toward malignancy. This has already been shown to be the case for VEGF. It is noteworthy that VEGF-D gene expression is induced by c-Fos, a transcription factor known to be important for malignancy (Orlandini et al . , Proc . Na tl . Acad. Sci . USA 93: 11675-11680, 1996). It has been speculated that the mechanism by which c-Fos contributes to malignancy is the up-regulation of genes encoding angiogenic factors .
  • P1GF was isolated from a term placenta cDNA library. Its isolation and characteristics are described in detail in Maglione et al . , Proc . Na tl . Acad . Sci . USA, 88: 9267-9271, 1991. Presently its biological function is not well understood.
  • VEGF2 was isolated from a cDNA library from a early stage human embryo. While this molecule is stated to have about 22% homology to PDGF and 30% homology to VEGF, the method of isolation of the gene encoding VEGF2 is unclear. VEGF2 has been shown to stimulate human umbilical vein endothelial cells (HUVEC) proliferation.
  • HUVEC human umbilical vein endothelial cells
  • VEGF3 was isolated from a cDNA library derived from colon tissue. VEGF3 is stated to have about 36% identity and 66% similarity to VEGF. The method of isolation of the gene encoding VEGF3 is unclear and no characterization of the biological activity is disclosed.
  • Similarity between two proteins is determined by comparing the amino acid sequence and conserved amino acid substitutions of one of the proteins to the sequence of the second protein, whereas identity is determined without including the conserved amino acid substitutions.
  • the PDGF/VEGF family members act primarily by binding to receptor tyrosine kinases.
  • Five endothelial cell-specific receptor tyrosine kinases have been identified, namely VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt4), Tie and Tek/Tie-2. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction.
  • VEGFR-1 The only receptor tyrosine kinases known to bind VEGFs are VEGFR-1, VEGFR-2 and VEGFR-3.
  • VEGFR-1 and VEGFR-2 bind VEGF with high affinity, and VEGFR-1 also binds VEGF-B and P1GF.
  • VEGF-C has been shown to be the ligand for VEGFR-3, and it also activates VEGFR-2 (Joukov et al . , The EMBO Journal , 15: 290-298, 1996).
  • VEGF-D binds to both VEGFR-2 and VEGFR-3.
  • a ligand for Tek/Tie-2 has been described in International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc. The ligand for Tie has not yet been identified.
  • VEGF vascular endothelial growth factor
  • the VEGF receptor was found to specifically bind the VEGF 165 isoform via the exon 7 encoded sequence, which shows weak affinity for heparin (Soker et al . , Cell , 92: 735-745, 1998).
  • the receptor was shown to be identical to human neuropilin-1 (NP-1), a receptor involved in early stage neuromorphogenesis .
  • NP-1 human neuropilin-1
  • P1GF-2 also appears to interact with NP-1 (Migdal et al., J. Biol. Chem., 273: 22272-22278, 1998).
  • VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelial cells.
  • both VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelia (Oelrichs et al., Oncogene, 8: 11-18, 1992; Kaipainen et al., J. Exp. Med. , 178: 2077-2088, 1993; Dumont et al., Dev. Dyn., 203: 80-92, 1995; Fong et al., Dev. Dyn., 207: 1-10, 1996) and VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., Proc. Natl. Acad. Sci. USA, 9: 3566-3570, 1995) .
  • VEGFR-3 is also expressed in the blood vasculature surrounding tumors.
  • VEGFR-1 is mainly expressed in endothelial cells during development, it can also be found in hematopoetic precursor cells during early stages of embryogenesis (Fong et al., Nature, 376: 66-70, 1995). In adults, monocytes and macrophages also express this receptor (Barleon et al., Blood, 87: 3336-3343, 1995) . In embryos, VEGFR-1 is expressed by most, if not all, vessels (Breier et al., Dev. Dyn., 204: 228-239, 1995; Fong et al., Dev. Dyn., 207: 1-10, 1996).
  • VEGFR-3 The receptor VEGFR-3 is widely expressed on endothelial cells during early embryonic development but as embryogenesis proceeds becomes restricted to venous endothelium and then to the lymphatic endothelium (Kaipainen et al., Cancer Res., 54: 6571- 6577, 1994; Kaipainen et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570, 1995) . VEGFR-3 is expressed on lymphatic endothelial cells in adult tissues. This receptor is essential for vascular development during embryogenesis.
  • VEGFR-1 The essential, specific role in vasculogenesis, angiogenesis and/or lymphangiogenesis of VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos. Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation . Analysis of embryos carrying a completely inactivated VEGFR-1 gene suggests that this receptor is required for functional organization of the endothelium (Fong et al . , Na ture, 376: 66-70, 1995).
  • VEGFR-3 is required for the maturation of primary vascular networks into larger blood vessels.
  • VEGFR-3 plays a role in development of the lymphatic vasculature and lymphangiogenesis given its specific expression in lymphatic endothelial cells during embryogenesis and adult life. This is supported by the finding that ectopic expression of VEGF-C, a ligand for VEGFR-3, in the skin of transgenic mice, resulted in lymphatic endothelial cell proliferation and vessel enlargement in the dermis .
  • VEGF-C may have a primary function in lymphatic endothelium, and a secondary function in angiogenesis and permeability regulation which is shared with VEGF (Joukov et al . , EMBO J. , 15: 290-298, 1996).
  • VEGF-like proteins have been identified which are encoded by four different strains of the orf virus. This is the first virus reported to encode a VEGF-like protein. The first two strains are NZ2 and NZ7, and are described in Lyttle et al . , J. Virol . , 68: 84-92, 1994. A third is D1701 and is described in Meyer et al . , EMBO J.
  • the fourth strain is NZ10 and is described in International Patent Application PCT/US99/25869. It was shown that these viral VEGF-like proteins bind to VEGFR-2 on the endothelium of the host (sheep/goat/human) and this binding is important for development of infection (Meyer et al . , EMBO J. , 18: 363-374, 1999; Ogawa et al . J. Biol . Chem . , 273: 31273-31282, 1988; and International Patent Application PCT/US99/25869.
  • the four VEGF-like proteins have sequence identity to each other as well as to VEGF.
  • the orf virus is a type of species of the parapoxvirus genus which causes a highly contagious pustular dermatitis in sheep and goats and is readily transmittable to humans.
  • the pustular dermatitis induced by orf virus infection is characterized by dilation of blood vessels, swelling of the local area and marked proliferation of endothelial cells lining the blood vessels.
  • VEGF family of growth factors are members of the PDGF family.
  • PDGF plays a important role in the growth and/or motility of connective tissue cells, fibroblasts, myofibroblasts and glial cells (Heldin et al . , "Structure of platelet-derived growth factor: Implications for functional properties", Growth Factor, 8: 245-252, 1993). In adults, PDGF stimulates wound healing (Robson et al . , Lancet, 339: 23-25, 1992).
  • PDGF isoforms are disulfide-bonded dimers of homologous A- and B-polypeptide chains, arranged as homodimers (PDGF-AA and PDGF-BB) or a heterodimer (PDGF-AB) .
  • PDGF isoforms exert their effects on target cells by binding to two structurally related receptor tyrosine kinases (RTKs) .
  • RTKs receptor tyrosine kinases
  • the alpha-receptor binds both the A- and B-chains of PDGF, whereas the beta-receptor binds only the B-chain.
  • RTKs receptor tyrosine kinases
  • the alpha-receptor binds both the A- and B-chains of PDGF, whereas the beta-receptor binds only the B-chain.
  • These two receptors are expressed by many in vi tro grown cell lines, and are mainly expressed by mesenchymal cells in vivo .
  • the PDGFs regulate cell proliferation, cell survival and chemotaxis of many cell types in vi tro (reviewed in Heldin et al . , Biochim Biophys Acta . , 1378: F79-113, 1998).
  • PDGF-A epithelial
  • PDGF-B endothelial cells
  • PDGFs have been observed in several pathological conditions, including maligancies, arteriosclerosis, and fibroproliferative diseases (reviewed in Heldin et al . , The Molecular and Cellular Biology of Wound Repair, New York: Plenum Press, 249-273, 1996) .
  • PDGFs as regulators of cell proliferation and survival are well illustrated by recent gene targeting studies in mice that have shown distinct physiological roles for the PDGFs and their receptors despite the overlapping ligand specificities of the PDGFRs .
  • Homozygous null mutations for either of the two PDGF ligands or the receptors are lethal.
  • Approximately 50% of the homozygous PDGF-A deficient mice have an early lethal phenotype, while the surviving animals have a complex postnatal phenotype with lung emphysema due to improper alveolar septum formation because of a lack of alveolar myofibroblasts (Bostrom et al . , Cell , 85: 863-873, 1996).
  • the PDGF-A deficient mice also have a dermal phenotype characterized by thin dermis, misshapen hair follicles and thin hair (Karlsson et al . , Devel opment , 126: 2611-2, 1999). PDGF-A is also required for normal development of oligodendrocytes and subsequent myelination of the central nervous system (Fruttiger et al . , Development, 126: 457-67, 1999). The phenotype of PDGFR-alpha deficient mice is more severe with early embryonic death at E10, incomplete cephalic closure, impaired neural crest development, cardiovascular defects, skeletal defects, and edemas [Soriano et al .
  • mice develop similar phenotypes that are characterized by renal, hematological and cardiovascular abnormalities (Leveen et al . , Genes Dev. , 8: 1875-1887, 1994; Soriano et al . , Genes Dev. , 8: 1888-96, 1994; Lindahl et al .
  • the invention generally provides an isolated fragment of a PDGF/VEGF-Like Growth Factor H which has the ability to stimulate and/or enhance proliferation or differentiation of endothelial cells and/or growth and/or motility of connective tissue cells, myofibroblasts and glial cells, and compositions useful for diagnostic and/or therapeutic applications.
  • the invention provides an isolated and purified polynucleotide molecule which encodes a portion of a PDGF/VEGF-like growth factor which is structurally homologous to members of the PDGF/VEGF family of growth factors (hereinafter "PDGF/VEGF-Like Growth Factor H" .
  • the polynucleotide molecule is a DNA molecule which comprises the sequence set out in Figure 1 (SEQ ID NO:l) .
  • SEQ ID NO:l the sequence set out in Figure 1
  • This aspect of the invention also encompasses polynucleotide molecules having a sequence such that they hybridize under stringent conditions with the DNA of Figure 1 (SEQ ID NO:l).
  • the encoded polypeptide of the invention has the ability to stimulate and/or enhance proliferation and/or differentiation and/or growth and/or motility of cells expressing a novel growth receptor including, but not limited to, endothelial cells, connective tissue cells, myofibroblasts and glial cells and comprises a sequence of amino acids corresponding to the amino acid sequence set out in Figure 2 (SEQ ID NO:2), or a fragment or analog thereof which has the ability to stimulate and/or enhance proliferation and/or differentiation and/or growth and/or motility of cells expressing the novel PDGF/VEGF-Like Growth Factor H receptor including, but not limited to, endothelial cells, connective tissue cells (such as fibroblasts) , myofibroblasts and glial cells.
  • a novel growth receptor including, but not limited to, endothelial cells, connective tissue cells (such as fibroblasts) , myofibroblasts and glial cells.
  • polypeptides have at least 85% identity, more preferably be at least 90%, and most preferably at least 95% identity to the amino acid sequence of in Figure 2 (SEQ ID NO : 2 ) , or a fragment or analog thereof having the biological activity of the PDGF/VEGF- Like Growth Factor H.
  • percent sequence identity is determined by using the alignment tool of "MEGALIGN” from the Lasergene package (DNASTAR, Ltd. Abacus House, Manor Road, West Ealing, London W130AS United Kingdom) and using its preset conditions. The alignment is then refined manually, and the number of identities are estimated in the regions available for a comparison.
  • analog or “functional analog” refers to a modified form of the fragment of the PDGF/VEGF-Like Growth Factor H in which at least one amino acid substitution has been made such that said analog retains substantially the same biological activity as the unmodified fragment the PDGF/VEGF-Like Growth Factor H in vivo and /or in vi tro .
  • the polypeptide or the encoded polypeptide from a polynucleotide has the ability to stimulate one or more of proliferation, differentiation, motility, survival or vascular permeability of cells expressing a novel PDGF/VEGF-Like Growth Factor H receptor including, but not limited to, vascular endothelial cells, lymphatic endothelial cells, connective tissue cells (such as fibroblasts) , myofibroblasts and glial cells.
  • vascular endothelial cells including, but not limited to, vascular endothelial cells, lymphatic endothelial cells, connective tissue cells (such as fibroblasts) , myofibroblasts and glial cells.
  • the polypeptide or the encoded polypeptide from a polynucleotide has the ability to stimulate wound healing.
  • the PDGF/VEGF-Like Growth Factor H can also have antagonistic effects on cells, but are included in the biological activities of the PDGF/VEGF-Like Growth Factor H. These abilities are referred to hereinafter as "biological activities of PDGF/VEGF-Like Growth Factor H" and can be readily tested by methods known in the art.
  • PDGF/VEGF-Like Growth Factor H collectively refers to the polypeptide of Figure 2 (SEQ ID NO:2), and fragments or analogs thereof which have the biological activity of the PDGF/VEGF-Like Growth Factor H as defined above, and to a polynucleotide which can code for the PDGF/VEGF-Like Growth Factor H, or a fragment or analog thereof having the biological activity of the PDGF/VEGF-Like Growth Factor H.
  • the polynucleotide can be naked and/or in a vector or liposome.
  • the invention provides a polypeptide possessing the amino acid sequence:
  • PXCXXXXCGGCCXXE (SEQ ID NO: 11) which is similar to the other members of the PDGF/VEGF family of growth factors (see Figure 3) .
  • Polypeptides comprising conservative substitutions, insertions, or deletions, but which still retain the biological activity of the PDGF/VEGF-Like Growth Factor H are clearly to be understood to be within the scope of the invention.
  • Persons skilled in the art will be well aware of methods which can readily be used to generate such polypeptides, for example the use of site-directed mutagenesis, or specific enzymatic cleavage and ligation.
  • the skilled person will also be aware that peptidomimetic compounds or compounds in which one or more amino acid residues are replaced by a non-naturally occurring amino acid or an amino acid analog may retain the required aspects of the biological activity of the PDGF/VEGF-Like Growth Factor H.
  • Such compounds can readily be made and tested by methods known in the art, and are also within the scope of the invention.
  • allelic variants of the nucleic acid sequence encoding a PDGF/VEGF-Like Growth Factor H are encompassed within the scope of the invention.
  • Allelic variants are well known in the art, and represent alternative forms or a nucleic acid sequence which comprise substitution, deletion or addition of one or more nucleotides, but which do not result in any substantial functional alteration of the encoded polypeptide.
  • Such variant forms of the PDGF/VEGF-Like Growth Factor H can be prepared by targeting non-essential regions of the PDGF/VEGF- Like Growth Factor H for modification. These non-essential regions are expected to fall outside the strongly-conserved regions indicated in Figure 3.
  • the growth factors of the PDGF family including VEGF, are dimeric, and VEGF, VEGF-B, VEGF-C, VEGF-D, P1GF, PDGF-A and PDGF-B show complete conservation of eight cysteine residues in the N-terminal domains, i.e. the PDGF/VEGF-like domains (Olofsson et al . , Proc . Na tl . Acad .
  • cysteines are thought to be involved in intra- and inter-molecular disulfide bonding. In addition there are further strongly, but not completely, conserved cysteine residues in the C-terminal domains. Loops 1, 2 and 3 of each subunit, which are formed by intra-molecular disulfide bonding, are involved in binding to the receptors for the PDGF/VEGF family of growth factors (Andersson et al . , Growth Factors, 12: 159-164, 1995).
  • cysteine residues should be preserved in any proposed variant form, and that the active sites present in loops 1, 2 and 3 also should be preserved.
  • other regions of the molecule can be expected to be of lesser importance for biological function, and therefore offer suitable targets for modification.
  • Modified polypeptides can readily be tested for their ability to show the biological activity of the PDGF/VEGF-Like Growth Factor H by routine activity assay procedures such as the fibroblast proliferation assay.
  • modified PDGF/VEGF-Like Growth Factor H polypeptides will have the ability to bind to novel PDGF/VEGF-Like Growth Factor H receptors on cells including, but not limited to, endothelial cells, connective tissue cells, myofibroblasts and/or glial cells, but will be unable to stimulate cell proliferation, differentiation, migration, motility or survival or to induce vascular proliferation, connective tissue development or wound healing.
  • modified polypeptides are expected to be able to act as competitive or non-competitive inhibitors of the PDGF/VEGF-Like Growth Factor H polypeptides and growth factors of the PDGF/VEGF family, and to be useful in situations where prevention or reduction of the PDGF/VEGF-Like Growth Factor H polypeptide or PDGF/VEGF family growth factor action is desirable.
  • receptor-binding but non-mitogenic, non-differentiation inducing, non-migration inducing, non-motility inducing, non-survival promoting, non- connective tissue development promoting, non-wound healing or non-vascular proliferation inducing variants of the PDGF/VEGF- Like Growth Factor H polypeptide are also within the scope of the invention, and are referred to herein as "receptor-binding but otherwise inactive variant".
  • one monomer comprises the receptor-binding but otherwise inactive variant modified PDGF/VEGF-Like Growth Factor H polypeptide and a second monomer comprises a wild-type PDGF/VEGF-Like Growth Factor H or a wild-type growth factor of the PDGF/VEGF family.
  • These dimers can bind to its corresponding receptor but cannot induce downstream signaling.
  • modified PDGF/VEGF-Like Growth Factor H polypeptides that can prevent binding of a wild-type PDGF/VEGF-Like Growth Factor H or a wild- type growth factor of the PDGF/VEGF family to its corresponding receptor on cells including, but not limited to, endothelial cells, connective tissue cells (such as fibroblasts) , myofibroblasts and/or glial cells.
  • endothelial cells such as fibroblasts
  • connective tissue cells such as fibroblasts
  • myofibroblasts myofibroblasts and/or glial cells.
  • these dimers will be unable to stimulate endothelial cell proliferation, differentiation, migration, survival, or induce vascular permeability, and/or stimulate proliferation and/or differentiation and/or motility of connective tissue cells, myofibroblasts or glial cells.
  • modified polypeptides are expected to be able to act as competitive or non-competitive inhibitors of the PDGF/VEGF-Like Growth Factor H or a growth factor of the PDGF/VEGF family, and to be useful in situations where prevention or reduction of the PDGF/VEGF-Like Growth Factor H or PDGF/VEGF family growth factor action is desirable.
  • Such situations include the tissue remodeling that takes place during invasion of tumor cells into a normal cell population by primary or metastatic tumor formation.
  • PDGF/VEGF-Like Growth Factor H or PDGF/VEGF family growth factor-binding but non- mitogenic, non-differentiation inducing, non-migration inducing, non-motility inducing, non-survival promoting, non-connective tissue promoting, non-wound healing or non-vascular proliferation inducing variants of the PDGF/VEGF-Like Growth Factor H are also within the scope of the invention, and are referred to herein as "PDGF/VEGF-Like Growth Factor H-dimer forming but otherwise inactive or interfering variants".
  • the invention provides a purified and isolated nucleic acid encoding a polypeptide or polypeptide fragment of the invention as defined above.
  • the nucleic acid may be DNA, genomic DNA, cDNA or RNA, and may be single-stranded or double stranded.
  • the nucleic acid may be isolated from a cell or tissue source, or of recombinant or synthetic origin.
  • each sequence encodes the amino acid sequence shown in Figure 2 (SEQ ID N0:2), a bioactive fragment or analog thereof, a receptor-binding but otherwise inactive or partially inactive variant thereof or the PDGF/VEGF- Like Growth Factor H-dimer forming but otherwise inactive or interfering variants thereof.
  • a fourth aspect of the invention provides vectors comprising the cDNA of the invention or a nucleic acid molecule according to the third aspect of the invention, and host cells transformed or transfected with nucleic acids molecules or vectors of the invention. These may be eukaryotic or prokaryotic in origin. These cells are particularly suitable for expression of the polypeptide of the invention, and include insect cells such as Sf9 cells, obtainable from the American Type Culture Collection (ATCC SRL-171), transformed with a baculovirus vector, and the human embryo kidney cell line 293-EBNA transfected by a suitable expression plasmid.
  • Preferred vectors of the invention are expression vectors in which a nucleic acid according to the invention is operatively connected to one or more appropriate promoters and/or other control sequences, such that appropriate host cells transformed or transfected with the vectors are capable of expressing the polypeptide of the invention.
  • Other preferred vectors are those suitable for transfection of mammalian cells, or for gene therapy, such as adenoviral-, vaccinia- or retroviral-based vectors or liposomes. A variety of such vectors is known in the art.
  • the invention also provides a method of making a vector capable of expressing a polypeptide encoded by a nucleic acid according to the invention, comprising the steps of operatively connecting the nucleic acid to one or more appropriate promoters and/or other control sequences, as described above.
  • the invention further provides a method of making a polypeptide according to the invention, comprising the steps of expressing a nucleic acid or vector of the invention in a host cell, and isolating the polypeptide from the host cell or from the host cell's growth medium.
  • the invention provides an antibody specifically reactive with a polypeptide of the invention or a fragment of the polypeptide.
  • This aspect of the invention includes antibodies specific for the variant forms, immunoreactive fragments, analogs and recombinants of the PDGF/VEGF-Like Growth Factor H. Such antibodies are useful as inhibitors or agonists of the novel growth and as diagnostic agents for detecting and quantifying the PDGF/VEGF-Like Growth Factor H.
  • Polyclonal or monoclonal antibodies may be used. Monoclonal and polyclonal antibodies can be raised against polypeptide of the invention or fragment thereof using standard methods in the art.
  • polypeptide can be linked to an epitope tag, such as the FLAG ® octapeptide (Sigma, St. Louis, MO), to assist in affinity purification.
  • an epitope tag such as the FLAG ® octapeptide (Sigma, St. Louis, MO)
  • FLAG ® octapeptide Sigma, St. Louis, MO
  • Such antibodies may be further modified by addition of cytotoxic or cytostatic drugs. Methods for producing these, including recombinant DNA methods, are also well known in the art.
  • Polypeptides or antibodies according to the invention may be labeled with a detectable label, and utilized for diagnostic purposes. Similarly, the thus-labeled polypeptide of the invention may be used to identify its corresponding receptor in situ .
  • the polypeptide or antibody may be covalently or non- covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, ecogenic or radioactive agent for imaging.
  • radioactive or non-radioactive labels may be used. Examples of radioactive labels include a radioactive atom or group, such as 125 I or 32 P.
  • non- radioactive labels include enzymatic labels, such as horseradish peroxidase or fluorimetric labels, such as fluorescein-5- isothiocyanate (FITC). Labeling may be direct or indirect, covalent or non-covalent .
  • Clinical applications of the invention include diagnostic applications, acceleration of angiogenesis in tissue or organ transplantation, or stimulation of wound healing, or connective tissue development, or to establish collateral circulation in tissue infarction or arterial stenosis, such as coronary artery disease, and inhibition of angiogenesis in the treatment of cancer or of diabetic retinopathy and inhibition of tissue remodeling that takes place during invasion of tumor cells into a normal cell population by primary or metastatic tumor formation.
  • Quantitation of the PDGF/VEGF-Like Growth Factor H in cancer biopsy specimens may be useful as an indicator of future metastatic risk.
  • the PDGF/VEGF-Like Growth Factor H may also be relevant to a variety of lung conditions.
  • the PDGF/VEGF-Like Growth Factor H assays could be used in the diagnosis of various lung disorders.
  • the PDGF/VEGF-Like Growth Factor H could also be used in the treatment of lung disorders to improve blood circulation in the lung and/or gaseous exchange between the lungs and the blood stream.
  • the PDGF/VEGF-Like Growth Factor H could be used to improve blood circulation to the heart and 0 2 gas permeability in cases of cardiac insufficiency.
  • the PDGF/VEGF-Like Growth Factor H could be used to improve blood flow and gaseous exchange in chronic obstructive airway disease.
  • the invention provides a method of stimulation of angiogenesis, lymphangiogenesis, neovascularization, connective tissue development and/or wound healing in a mammal in need of such treatment, comprising the step of administering an effective dose of the PDGF/VEGF-Like Growth Factor H, or a fragment or an analog thereof which has the biological activity of PDGF/VEGF- Like Growth Factor H to the mammal.
  • PDGF/VEGF-Like Growth Factor H, or fragment or analog thereof may be administered together with, or in conjunction with, one or more of VEGF, VEGF-B, VEGF-C, VEGF-D, P1GF, PDGF-A, PDGF-B, FGF and/or heparin .
  • PDGF/VEGF-Like Growth Factor H antagonists e.g. antibodies and/or inhibitors
  • PDGF/VEGF-Like Growth Factor H antagonists could be used to treat in conditions, such as congestive heart failure, involving accumulations of fluid in, for example, the lung resulting from increases in vascular permeability by exerting an offsetting effect on vascular permeability in order to counteract the fluid accumulation.
  • Administrations of the PDGF/VEGF-Like Growth Factor H could be used to treat malabsorptive syndromes in the intestinal tract, liver or kidneys as a result of its blood circulation increasing and vascular permeability increasing activities .
  • the invention provides a method of inhibiting angiogenesis, lymphangiogenesis, neovascularization, connective tissue development and/or wound healing in a mammal in need of such treatment, comprising the step of administering an effective amount of an antagonist of the PDGF/VEGF-Like Growth Factor H to the mammal.
  • the antagonist may be any agent that prevents the action of the PDGF/VEGF-Like Growth Factor H, either by preventing the binding of the PDGF/VEGF-Like Growth Factor H to its corresponding receptor on the target cell, or by preventing activation of the receptor, such as using receptor-binding PDGF/VEGF-Like Growth Factor H variants.
  • Suitable antagonists include, but are not limited to, antibodies directed against the PDGF/VEGF-Like Growth Factor H; competitive or non-competitive inhibitors of binding of the PDGF/VEGF-Like Growth Factor H to the PDGF/VEGF-Like Growth Factor H receptor (s), such as the receptor-binding or PDGF/VEGF-Like Growth Factor H dimer-forming but non-mitogenic PDGF/VEGF-Like Growth Factor H variants referred to above; compounds that bind to the PDGF/VEGF-Like Growth Factor H and/or modify or antagonize its function, and anti-sense nucleotide sequences as described below.
  • s such as the receptor-binding or PDGF/VEGF-Like Growth Factor H dimer-forming but non-mitogenic PDGF/VEGF-Like Growth Factor H variants referred to above
  • the method comprises contacting a polypeptide having the amino acid sequence of SEQ ID NO: 2 with a test agent and monitoring binding by any suitable means .
  • Agents can include both compounds and other proteins .
  • the invention provides a screening system for discovering agents that bind a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the screening system comprises preparing a polypeptide having the amino acid sequence of SEQ ID NO:2, exposing the polypeptide to a test agent, and quantifying the binding of said agent to the polypeptide.
  • This screen system provides a means to determine compounds that may alter the biological function of the PDGF/VEGF-Like Growth Factor H.
  • This screening method may be adapted to large-scale, automated procedures such as a PANDEX ® (Baxter-Dade Diagnostics) system, allowing for efficient high- volume screening of potential therapeutic agents.
  • a polypeptide having the amino acid sequence of SEQ ID NO: 2 is prepared as described herein, preferably using recombinant DNA technology.
  • a test agent e.g. a compound or protein
  • a test agent is introduced into a reaction vessel containing the polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • Binding of the test agent to the polypeptide having the amino acid sequence of SEQ ID NO: 2 is determined by any suitable means which include, but is not limited to, radioactively- or chemically-labeling the test agent. Binding of the polypeptide having the amino acid sequence of SEQ ID NO: 2 may also be carried out by a method disclosed in U.S. Patent 5,585,277, which is incorporated by reference.
  • binding of the test agent to the polypeptide having the amino acid sequence of SEQ ID NO: 2 is assessed by monitoring the ratio of folded protein to unfolded protein.
  • this monitoring can include, but are not limited to, monitoring the sensitivity of the polypeptide to a protease, or amenability to binding of the polypeptide by a specific antibody against the folded state of the polypeptide.
  • IC 50 values are dependent on the selectivity of the agent tested. For example, an agent with an IC 50 which is less than 10 nM is generally considered an excellent candidate for drug therapy. However, an agent which has a lower affinity, but is selective for a particular target, may be an even better candidate. Those skilled in the art will recognize that any information regarding the binding potential, inhibitory activity or selectivity of a particular agent is useful toward the development of pharmaceutical products.
  • a PDGF/VEGF-Like Growth Factor H or a PDGF/VEGF-Like Growth Factor H antagonist is to be used for therapeutic purposes, the dose(s) and route of administration will depend upon the nature of the patient and condition to be treated, and will be at the discretion of the attending physician or veterinarian. Suitable routes include oral, subcutaneous, intramuscular, intraperitoneal or intravenous injection, parenteral, topical application, implants etc . Topical application of the PDGF/VEGF-Like Growth Factor H may be used in a manner analogous to VEGF.
  • an effective amount of the PDGF/VEGF-Like Growth Factor H is administered to an organism in need thereof in a dose between about 0.1 and 1000 ⁇ g/kg body weight.
  • the PDGF/VEGF-Like Growth Factor H or a PDGF/VEGF-Like Growth Factor H antagonist may be employed in combination with a suitable pharmaceutical carrier.
  • the resulting compositions comprise a therapeutically effective amount of the PDGF/VEGF-Like Growth Factor H or a PDGF/VEGF-Like Growth Factor H antagonist, and a pharmaceutically acceptable non-toxic salt thereof, and a pharmaceutically acceptable solid or liquid carrier or adjuvant.
  • Such a carrier or adjuvant examples include, but are not limited to, saline, buffered saline, Ringer's solution, mineral oil, talc, corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, dextrose, water, glycerol, ethanol, thickeners, stabilizers, suspending agents and combinations thereof.
  • Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, salves, elixirs, syrups, wafers, ointments or other conventional forms. The formulation to suit the mode of administration.
  • Compositions which comprise the PDGF/VEGF-Like Growth Factor H may optionally further comprise one or more of PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C, VEGF-D, P1GF and/or heparin.
  • Compositions comprising PDGF/VEGF-Like Growth Factor H will contain from about 0.1% to 90% by weight of the active compound (s), and most generally from about 10% to 30%.
  • a sterile formulation preferably a suitable soluble salt form of the PDGF/VEGF-Like Growth Factor H, such as hydrochloride salt
  • a pharmaceutical diluent such as pyrogen-free water (distilled), physiological saline or 5% glucose solution.
  • a suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
  • the invention provides diagnostic/prognostic devices typically in the form of test kits.
  • a diagnostic/prognostic test kit comprising an antibody to the PDGF/VEGF-Like Growth Factor H and a means for detecting, and more preferably evaluating, binding between the antibody and the PDGF/VEGF-Like Growth Factor H.
  • a second antibody directed against antibodies of the same isotype and animal source of the antibody directed against the PDGF/VEGF-Like Growth Factor H (the primary antibody) is provided.
  • the secondary antibody is coupled to a detectable label, and then either an unlabeled primary antibody or the PDGF/VEGF-Like Growth Factor H is substrate-bound so that the PDGF/VEGF-Like Growth Factor H/primary antibody interaction can be established by determining the amount of label bound to the substrate following binding between the primary antibody and the PDGF/VEGF-Like Growth Factor H and the subsequent binding of the labeled secondary antibody to the primary antibody.
  • the diagnostic/prognostic device may be provided as a conventional enzyme-linked immunosorbent assay (ELISA) kit.
  • a diagnostic/prognostic device may comprise polymerase chain reaction means for establishing sequence differences of the PDGF/VEGF-Like Growth Factor H of a test individual and comparing this sequence structure with that disclosed in this application in order to detect any abnormalities, with a view to establishing whether any aberrations in the PDGF/VEGF-Like Growth Factor H expression are related to a given disease condition.
  • a diagnostic/prognostic device may comprise a restriction length polymorphism (RFLP) generating means utilizing restriction enzymes and genomic DNA from a test individual to generate a pattern of DNA bands on a gel and comparing this pattern with that disclosed in this application in order to detect any abnormalities, with a view to establishing whether any aberrations in the PDGF/VEGF-Like Growth Factor H expression are related to a given disease condition.
  • RFLP restriction length polymorphism
  • the invention relates to a method of detecting aberrations in the PDGF/VEGF-Like Growth Factor H gene in a test subject which may be associated with a disease condition in the test subject.
  • This method comprises providing a DNA or RNA sample from said test subject; contacting the DNA sample or RNA with a set of primers specific to the PDGF/VEGF-Like Growth Factor H DNA operatively coupled to a polymerase and selectively amplifying the PDGF/VEGF-Like Growth Factor H DNA from the sample by polymerase chain reaction, and comparing the nucleotide sequence of the amplified the PDGF/VEGF- Like Growth Factor H DNA from the sample with the polynucleotide sequences shown in Figure 1 (SEQ ID NO:l) .
  • the invention also includes the provision of a test kit comprising a pair of primers specific to the PDGF/VEGF-Like Growth Factor H DNA operatively coupled to a polymerase, whereby said polymerase is enabled to selectively amplify the PDGF/VEGF-Like Growth Factor H DNA from a DNA sample.
  • the invention also provides a method of detecting the PDGF/VEGF-Like Growth Factor H in a biological sample, comprising the step of contacting the sample with a reagent capable of binding the PDGF/VEGF-Like Growth Factor H, and detecting the binding.
  • a reagent capable of binding the PDGF/VEGF-Like Growth Factor H is an antibody directed against the PDGF/VEGF-Like Growth Factor H, particularly preferably a monoclonal antibody.
  • the binding and/or extent of binding is detected by means of a detectable label; suitable labels are discussed above.
  • the invention in another aspect, relates to a protein dimer comprising the PDGF/VEGF-Like Growth Factor H polypeptide, particularly a disulfide-linked dimer.
  • the protein dimers of the invention include both homodimers of the PDGF/VEGF-Like Growth Factor H polypeptide and heterodimers of the PDGF/VEGF-Like Growth Factor H and VEGF, VEGF-B, VEGF-C, VEGF-D, P1GF, PDGF-A or PDGF-B.
  • a method for isolation of the PDGF/VEGF-Like Growth Factor H comprising the step of exposing a cell which expresses the PDGF/VEGF-Like Growth Factor H to heparin to facilitate release of the PDGF/VEGF-Like Growth Factor H from the cell, and purifying the thus-released PDGF/VEGF-Like Growth Factor H.
  • Another aspect of the invention involves providing a vector comprising an anti-sense nucleotide sequence which is complementary to at least a part of a DNA sequence which encodes the PDGF/VEGF-Like Growth Factor H or a fragment or analog thereof that has the biological activity of the PDGF/VEGF-Like Growth Factor H.
  • anti-sense nucleotide sequence can be to the promoter region of the PDGF/VEGF-Like Growth Factor H gene or other non-coding region of the gene which may be used to inhibit, or at least mitigate, the PDGF/VEGF-Like Growth Factor H expression.
  • such a vector comprising an anti-sense sequence may be used to inhibit, or at least mitigate, the PDGF/VEGF-Like Growth Factor H expression.
  • the use of a vector of this type to inhibit the PDGF/VEGF-Like Growth Factor H expression is favored in instances where the PDGF/VEGF-Like Growth Factor H expression is associated with a disease, for example where tumors produce the PDGF/VEGF- Like Growth Factor H in order to provide for angiogenesis, or tissue remodeling that takes place during invasion of tumor cells into a normal cell population by primary or metastatic tumor formation. Transformation of such tumor cells with a vector containing an anti-sense nucleotide sequence would inhibit or retard growth of the tumor or tissue remodeling.
  • Polynucleotides of the invention such as those described above, fragments of those polynucleotides, and variants of those polynucleotides with sufficient similarity to the non-coding strand of those polynucleotides to hybridize thereto under stringent conditions all are useful for identifying, purifying, and isolating polynucleotides encoding other, non-human, mammalian forms of the PDGF/VEGF-Like Growth Factor H.
  • polynucleotide fragments and variants are intended as aspects of the invention.
  • Exemplary stringent hybridization conditions are as follows: hybridization at 42°C in 5X SSC, 20 mM NaP0 4 , pH 6.8, 50% formamide; and washing at 42°C in 0.2X SSC.
  • Those skilled in the art understand that it is desirable to vary these conditions empirically based on the length and the GC nucleotide base content of the sequences to be hybridized, and that formulas for determining such variation exist. See for example Sambrook et al , "Molecular Cloning: A Laboratory Manual", Second Edition, pages 9.47-9.51, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1989).
  • purified and isolated polynucleotides encoding other, non-human, mammalian PDGF/VEGF-Like Growth Factor H forms also are aspects of the invention, as are the polypeptides encoded thereby, and antibodies that are specifically immunoreactive with the non-human PDGF/VEGF-Like Growth Factor H variants.
  • the invention includes a purified and isolated mammalian PDGF/VEGF-Like Growth Factor H polypeptide, and also a purified and isolated polynucleotide encoding such a polypeptide .
  • nucleic acids and polypeptides of the invention may be prepared by synthetic means or by recombinant means, or may be purified from natural sources.
  • Figure 1 shows a partial nucleotide sequence of human cDNA (135 nucleotides) (SEQ ID N0:1) encoding a part of PDGF/VEGF-Like Growth Factor H;
  • Figure 2 shows the partial deduced amino acid sequence (45 amino acid residues) of PDGF/VEGF-Like Growth Factor H (SEQ ID NO: 2) derived from the nucleotide sequence of Figure 1; and
  • Figures 3A-3C show an amino acid sequence alignment of the partial deduced amino acid sequence of PDGF/VEGF-Like Growth Factor H with several growth factors belonging to the PDGF/VEGF family (SEQ ID NOs:3-10).
  • Figure 1 shows a nucleotide sequence of partial human cDNA which encodes a portion of a novel growth factor, referred to herein as PDGF/VEGF-Like Growth Factor H.
  • PDGF/VEGF-Like Growth Factor H is a new member of the PDGF/VEGF family.
  • the nucleotide sequence of Figure 1 (SEQ ID NO:l) was identified from the human genomic nucleotide sequence database at the NCBI in Washington, DC.
  • the DNA of Figure 1 encodes a polypeptide of 45 amino acids (SEQ ID NO: 2) which shows significant similarities to the known members of the PDGF/VEGF family.
  • the partially deduced amino acid sequence of the PDGF/VEGF- Like Growth Factor H (SEQ ID NO:2) was aligned to other members of the PDGF/VEGF family of growth factors (SEQ ID NOs:3-10) (see Figures 3A-3C) .
  • the alignment shows that PDGF/VEGF-Like Growth Factor H is related to the PDGF/VEGF family of growth factors and that the protein containing this amino acid sequence is a novel putative member of that family.
  • This partially deduced amino acid sequence of PDGF/VEGF-Like Growth Factor H has some of the cysteines which constitute the signature of the PDGF/VEGF family.
  • Assays are conducted to evaluate whether the PDGF/VEGF-Like Growth Factor H has similar activities to PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C and/or VEGF-D in relation to endothelial cell function, mitogenesis angiogenesis and wound healing. Further assays may also be performed, depending on the results of receptor binding distribution studies.
  • the PDGF/VEGF-Like Growth Factor H polypeptide is introduced into cell culture medium containing 5% serum and applied to bovine aortic endothelial cells (BAEs) propagated in medium containing 10% serum.
  • BAEs bovine aortic endothelial cells
  • the BAEs are previously seeded in 24-well dishes at a density of 10,000 cells per well the day before addition of the PDGF/VEGF-Like Growth Factor H.
  • Three days after addition of the PDGF/VEGF-Like Growth Factor H polypeptide the cells are dissociated with trypsin and counted.
  • Endothelial cell growth assays are performed by methods well known in the art, e.g. those of Ferrara & Henzel, Na ture, 380: 439-443, 1989; Gospodarowicz et al . , Proc . Na tl . Acad. Sci . USA, 86: 7311-7315, 1989; and/or Claffey et al . , Biochem . Biophys . Acta , 1246: 1-9, 1995.
  • the standard Boyden chamber chemotaxis assay is used to test the effect of the PDGF/VEGF-Like Growth Factor H on chemotaxis.
  • Endothelial cells are tested for the effect of the PDGF/VEGF-Like Growth Factor H on plasminogen activator and plasminogen activator inhibitor production, using the method of Pepper et al . , Biochem . Biophys . Res . Commun . , 181: 902-906, 1991.
  • PDGF/VEGF-Like Growth Factor H The ability of the PDGF/VEGF-Like Growth Factor H to induce an angiogenic response in chick chorioallantoic membrane is tested as described in Leung et al . , Science, 246: 1306-1309, 1989.
  • the rat cornea assay of Rastinejad et al . , Cell , 56: 345-355, 1989 may be used; this is an accepted method for assay of in vivo angiogenesis, and the results are readily transferrable to other in vivo systems.
  • mice capable of repopulating the bone marrow of lethally irradiated mice, and have the Lin " , Rh hl , Ly-6A/E + , c- kit + phenotype.
  • the PDGF/VEGF-Like Growth Factor H is tested on these cells either alone, or by co-incubation with other factors, followed by measurement of cellular proliferation by H-thymidine incorporation .
  • the PDGF/VEGF- Like Growth Factor H is incubated with these cells for a period of time, injected into lethally irradiated recipients, and the number of D13 spleen colonies enumerated.
  • Smooth muscle cells play a crucial role in the development or initiation of atherosclerosis, requiring a change of their phenotype from a contractile to a synthetic state. Macrophages, endothelial cells, T lymphocytes and platelets all play a role in the development of atherosclerotic plaques by influencing the growth and phenotypic modulations of smooth muscle cell.
  • An in vi tro assay using a modified Rose chamber in which different cell types are seeded on to opposite cover slips measures the proliferative rate and phenotypic modulations of smooth muscle cells in a multicellular environment, and is used to assess the effect of the PDGF/VEGF-Like Growth Factor H on smooth muscle cells .
  • the ability of the PDGF/VEGF-Like Growth Factor H to inhibit metastasis is assayed using the Lewis lung carcinoma model, for example using the method of Cao et al . , J. Exp. Med. , 182: 2069- 2077, 1995.
  • the effects of the PDGF/VEGF-Like Growth Factor H on proliferation, differentiation and function of other cell types, such as liver cells, cardiac muscle and other cells, endocrine cells and osteoblasts can readily be assayed by methods known in the art, such as H-thymidine uptake by in vi tro cultures.
  • Expression of the PDGF/VEGF-Like Growth Factor H in these and other tissues can be measured by techniques such as Northern blotting and hybridization or by in si tu hybridization.
  • the PDGF/VEGF-Like Growth Factor H is a member of the PDGF family of growth factors which exhibits a high degree of homology to the other members of the PDGF family.
  • VEGF family members contain eight conserved cysteine residues which are characteristic of this family of growth factors. These conserved cysteine residues form intra-chain disulfide bonds which produce the cysteine knot structure, and inter-chain disulfide bonds that form the protein dimers which are characteristic of members of the PDGF family of growth factors.
  • the PDGF/VEGF-Like Growth Factor H will interact with protein tyrosine kinase growth factor receptors .
  • a person skilled in the biotechnology arts can design PDGF/VEGF-Like Growth Factor H mutants with a very high probability of retaining the PDGF/VEGF- Like Growth Factor H activity by conserving the eight cysteine residues responsible for the knotted folding arrangement and for dimerization, and also by conserving, or making only conservative amino acid substitutions in the likely receptor sequences in the loop 1, loop 2 and loop 3 region of the protein structure.
  • the endothelial cell proliferating activity of the PDGF/VEGF-Like Growth Factor H mutants can be readily confirmed by well established screening procedures. For example, a procedure analogous to the endothelial cell mitotic assay described by Claffey et al . , ⁇ Biochem . Biophys . Acta . , 1246: 1-9, 1995) can be used. Similarly the effects of the PDGF/VEGF-Like Growth Factor H on proliferation of other cell types, on cellular differentiation and on human metastasis can be tested using methods which are well known in the art.

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Abstract

L'invention concerne une partie du facteur de croissance H de type PDGF/VEGF, un nouveau membre de la famille des facteurs de croissance; la séquence nucléotidique codant pour ladite partie; des techniques permettant de la produire; des anticorps et d'autres antagonistes de ladite partie; des cellules hôtes transfectées et transformées exprimant ladite partie; des compositions pharmaceutiques la contenant; ainsi que ses utilisations dans des applications médicales et diagnostiques.
PCT/US2000/001895 1999-01-29 2000-01-28 Facteur de croissance h de type facteur de croissance d'origine plaquettaire/facteur de croissance de l'endothelium vasculaire et ses utilisations WO2000044903A1 (fr)

Priority Applications (3)

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EP00915701A EP1147193A4 (fr) 1999-01-29 2000-01-28 Facteur de croissance h de type facteur de croissance d'origine plaquettaire/facteur de croissance de l'endothelium vasculaire et ses utilisations
AU36930/00A AU3693000A (en) 1999-01-29 2000-01-28 Platelet-derived growth factor/vascular endothelial growth factor-like growth factor h, and uses thereof
JP2000596145A JP2002535006A (ja) 1999-01-29 2000-01-28 血小板由来増殖因子／血管内皮増殖因子様増殖因子ｈとその利用法

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Publication number Priority date Publication date Assignee Title
GB2408507A (en) * 2003-10-06 2005-06-01 Proimmune Ltd Chimeric MHC molecule and complex
GB2408507B (en) * 2003-10-06 2005-12-14 Proimmune Ltd Chimeric MHC protein and oligomer thereof for specific targeting
GB2409456A (en) * 2003-10-30 2005-06-29 Proimmune Ltd Oligomeric receptor-ligand pair member complex
GB2409456B (en) * 2003-10-30 2006-01-04 Proimmune Ltd Oligomeric receptor ligand pair member complexes

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AU3693000A (en) 2000-08-18
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EP1147193A1 (fr) 2001-10-24

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