WO2001000878A9 - Nouveau membre de la famille des facteurs de croissance des cellules endotheliales vasculaires et son utilisation - Google Patents

Nouveau membre de la famille des facteurs de croissance des cellules endotheliales vasculaires et son utilisation Download PDF

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Publication number
WO2001000878A9
WO2001000878A9 PCT/US2000/018085 US0018085W WO0100878A9 WO 2001000878 A9 WO2001000878 A9 WO 2001000878A9 US 0018085 W US0018085 W US 0018085W WO 0100878 A9 WO0100878 A9 WO 0100878A9
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vegf
nucleic acid
protein
polypeptide
seq
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PCT/US2000/018085
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WO2001000878A2 (fr
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David P. Gearing
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Millennium Pharmaceuticals, Inc.
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Priority to AU59036/00A priority Critical patent/AU5903600A/en
Publication of WO2001000878A2 publication Critical patent/WO2001000878A2/fr
Publication of WO2001000878A9 publication Critical patent/WO2001000878A9/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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors

Definitions

  • Angiogenesis is also implicated in physiological and pathophysiological processes such as wound healing and tumor growth and metastasis. Angiogenic factors and growth factors regulate a variety of cellular functions including proliferation, differentiation, migration, and morphogenesis during development.
  • vascular endothelial growth factor is a potent endothelial cell mitogen, survival factor, chemotactic factor and angiogenic factor.
  • the NEGF protein family comprises the structurally and functionally related proteins VEGF-A, VEGF- B/VEGF related factor (VEGF-B/VRF), VEGF-C/VEGF related protein (VEGF- C/NRP), c-fos induced growth factor (FIGF), and placenta growth factor (P1GF) (Erikson, U. et al. (1999) Curr. Topics Microbiol. Immunol., 237:4).
  • VEGF-A is a secreted, dimeric glycoprotein, distantly related in structure to the platelet derived growth factors (PDGF), PDGF-A and PDGF-B.
  • PDGF platelet derived growth factors
  • PDGF-A and PDGF-B are highly conserved cysteine residues important for inter- and intramolecular disulphide bonding.
  • Several VEGF proteins are also heparin binding growth factors, thus, their activity may be regulated by interactions with cell surface and/or extracellular matrix- associated heparan sulfate proteoglycans (Erikson, U. et al. (1999) Curr. Topics Microbiol. Immunol. , 237:41 ; ⁇ eufeld, G. et al., (1999) FASEB J. , 13 :9).
  • VEGF receptors include the high affinity tyrosine kinase receptors, Fit and KDR7flk ( ⁇ eufeld, G. et al, (1999) FASEB J., 13:9; Zachary, I. (1998) Int. J. Biochem. Cell Biol, 30:1169). Additionally, the VEGF- A. 65 protein isoform appears to interact with neuropilin- 1 , a non-protein tyrosine kinase receptor for the semaphorin family ( ⁇ eufeld, G.
  • VEGF proteins vascular endothelial growth factor receptors
  • PKC protein kinase C
  • phosphorylation of proteins such as focal adhesion kinase and paxillin
  • second messengers such as inositol 1,4,5-trisphosphate (IP3) and calcium
  • the present invention is based, at least in part, on the discovery of novel vascular endothelial growth factor family members, referred to herein as "Vascular Endothelial Growth Factor-G” or “VEGF-G” nucleic acid and protein molecules.
  • Vascular Endothelial Growth Factor-G or "VEGF-G” nucleic acid and protein molecules.
  • the NEGF-G molecules of the present invention are useful as modulating agents, or as targets for developing modulating agents to regulate a variety of cellular (e.g., endothelial cell) processes, e.g., cell proliferation, differentiation, migration, angiogenesis, and wound repair.
  • this invention provides isolated nucleic acid molecules encoding NEGF-G proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of NEGF-G-encoding nucleic acids.
  • a NEGF-G nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98%, or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID ⁇ O:l, 3, 10 or 12, or a complement thereof.
  • the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:l or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-212 of SEQ ID NO:l . In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1326-3853 of SEQ ID NO:l. In another embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:l or 3. In another embodiment, the nucleic acid molecule includes a fragment of at least 549 nucleotides (e.g.
  • the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO: 10 or 12, or a complement thereof.
  • the nucleic acid molecule includes SEQ ID NO: 12 and nucleotides 1-164 of SEQ ID NO: 10.
  • the nucleic acid molecule includes SEQ ID NO:12 and nucleotides 1278-3121 of SEQ ID NO:10.
  • the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO: 10 or 12.
  • the nucleic acid molecule includes a fragment of at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 or more nucleotides (e. g. , contiguous nucleotides) of the nucleotide sequence of SEQ ID NO: 10 or 12, or a complement thereof.
  • nucleotides e. g. , contiguous nucleotides
  • a NEGF-G nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID ⁇ O:2 or 11.
  • a VEGF-G nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the entire length of the amino acid sequence of SEQ ID NO:2 or 11.
  • an isolated nucleic acid molecule encodes the amino acid sequence of human or mouse VEGF-G.
  • the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2 or 11.
  • the nucleic acid molecule is at least 50, 100, 200, 300, 400, 500, 549, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2273, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 nucleotides in length.
  • the nucleic acid molecule is at least 50, 100, 200, 300, 400, 500, 549, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2273, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 nucleotides in length and encodes a protein having a VEGF-G activity as described herein.
  • Another embodiment of the invention features nucleic acid molecules, preferably VEGF-G nucleic acid molecules, which specifically detect VEGF-G nucleic acid molecules relative to nucleic-acid molecules encoding non- VEGF-G proteins.
  • such a nucleic acid molecule is at least 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 549, 549-600, 600-650, 650-700, 700-750, 750-800, 800- 850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2200, 2200-2272, 2273, 2273-2400, 2400-2600, 2600-2800, 2800-3000 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 1 or 10.
  • the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-17 and 310-3283 of SEQ ID NO:l.
  • the nucleic acid molecules comprise nucleotides 1-17 and 310-3283 of SEQ ID NO: 1.
  • the nucleic acid molecules consist of nucleotides 1-17 and 310- 3283 of SEQ ID NO: 1.
  • the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to SEQ ID NO: lor 10.
  • the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 11 , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, 3, 10 or 12 under stringent conditions.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a VEGF-G nucleic acid molecule, e.g., the coding strand of a VEGF-G nucleic acid molecule.
  • the invention provides a vector comprising a VEGF-G nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector of the invention.
  • the invention provides a host cell containing a nucleic acid molecule of the invention.
  • the invention also provides a method for producing a protein, preferably a VEGF-G protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell, such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
  • the isolated VEGF-G protein includes at least one VEGF/PDGF superfamily variant motif.
  • the isolated VEGF-G protein includes at least one VEGF-G disulfide knot-like domain.
  • the isolated VEGF-G proteins includes at least one CUB domain.
  • the isolated VEGF-G protein includes a signal sequence.
  • the isolated VEGF-G protein includes at least one VEGF/PDGF superfamily variant motif and at least one CUB domain.
  • the isolated VEGF-G protein includes at least one VEGF-G disulfide knot-like domain and at least one CUB domain. In another embodiment, the isolated VEGF-G protein includes at least one VEGF/PDGF superfamily variant motif and a signal sequence. In another embodiment, the isolated VEGF-G protein includes at least one VEGF-G disulfide knot- like domain and a signal sequence. In a further embodiment, the isolated VEGF-G protein includes at least one CUB domain and a signal sequence. In yet another embodiment, the isolated VEGF-G protein includes at least one VEGF/PDGF superfamily variant motif, at least one CUB domain, and a signal sequence. In yet another embodiment, the isolated VEGF-G protein includes at least one VEGF-G disulfide knot-like domain, at least one CUB domain, and a signal sequence.
  • the VEGF-G protein of the invention has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the amino acid sequence of SEQ ID NO:2 or 11.
  • the VEGF-G protein includes a VEGF/PDGF superfamily variant motif and a CUB domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the amino acid sequence of SEQ ID NO:2 or 11.
  • the VEGF-G protein includes a VEGF-G disulfide knot-like domain and a CUB domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the amino acid sequence of SEQ ID NO:2 or 11.
  • the VEGF-G proteins of the invention play a role in endothelial cell growth, e.g., the regulation of cell proliferation, differentiation, migration, and apoptosis.
  • the VEGF-G proteins of the invention are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 10 or 12.
  • the invention features an isolated VEGF-G protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98% or more identical to a nucleotide sequence of SEQ ID NO:l, 3, 10 or 12, or a complement thereof.
  • This invention further features an isolated VEGF-G protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO : 1 , 3 , 10 or 12, or a complement thereof.
  • the VEGF-G protein has the amino acid sequence of SEQ ID NO:2 or 11.
  • the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:2 or 11, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2 or l l.
  • the proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non- VEGF-G polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
  • a non- VEGF-G polypeptide e.g., heterologous amino acid sequences
  • the VEGF-G proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably VEGF-G proteins.
  • the present invention provides a method for detecting the presence of a VEGF-G nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a VEGF- G nucleic acid molecule, protein or polypeptide such that the presence of a VEGF-G nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of VEGF-G activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of VEGF-G activity such that the presence of VEGF-G activity is detected in the biological sample.
  • the invention provides a method for modulating VEGF-G activity comprising contacting a cell capable of expressing VEGF-G with an agent that modulates VEGF-G activity such that VEGF-G activity in the cell is modulated.
  • the agent inhibits VEGF-G activity.
  • the agent stimulates VEGF-G activity.
  • the agent is an antibody that specifically binds to a VEGF-G protein.
  • the agent modulates expression of VEGF-G by modulating transcription of a VEGF-G gene or translation of a VEGF-G mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a VEGF-G mRNA or a VEGF-G gene.
  • Another aspect of the present invention features methods to treat a subject having a disorder characterized by aberrant VEGF-G protein or nucleic acid expression or activity by administering an agent which is a VEGF-G modulator to the subject.
  • the VEGF-G modulator is a VEGF-G protein.
  • the VEGF-G modulator is a VEGF-G nucleic acid molecule.
  • the VEGF-G modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant VEGF-G protein or nucleic acid expression is a disorder associated with deregulated cell growth such as a prohferative or differentiative disorder, including cancer, e.g., carcinoma, sarcoma, or leukemia, and hypertrophic bone disorders, e.g., opsismodysplasia; and/or a disorder involving aberrant angiogenesis and/or vascularity, e.g., tumor angiogenesis and metastasis, diabetic retinopathy, macular degeneration, psoriasis, endometriosis, Grave's disease, ischemic disease (e.g., atherosclerosis), and chronic inflammatory disease (e.g., rheumatoid arthritis).
  • a prohferative or differentiative disorder including cancer, e.g., carcinoma, sarcoma, or leukemia, and hypertrophic bone disorders, e.g., opsismodysplasia
  • the present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a VEGF-G protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a VEGF-G protein, wherein a wild-type form of the gene encodes a protein with a VEGF-G activity.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a VEGF-G protein, by providing an indicator composition comprising a VEGF-G protein having VEGF-G activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on VEGF-G activity in the indicator composition to identify a compound that modulates the activity of a VEGF-G protein.
  • Figure 1 depicts the cDNA sequence and predicted amino acid sequence of human VEGF-G.
  • the nucleotide sequence corresponds to nucleic acids 1 to 3853 of SEQ ID NOT.
  • the amino acid sequence corresponds to amino acids 1 to 370 of SEQ ID NO: 2.
  • the coding region without the 5' and 3' untranslated regions of the human VEGF-G gene is shown in SEQ ID NO:3.
  • Figure 2 depicts the results of a search which was performed against the HMM database and which resulted in the identification of a "CUB domain" in the human VEGF-G protein.
  • Figure 3 depicts a local alignment of the human VEGF-G protein with the human VEGF-C protein using the GAP program in the GCG software package, using a Blossum 62 matrix and a gap weight of 5 and a length weight of 2. The results showed a 30.357% identity between the two sequences.
  • Figure 4 depicts a local alignment of the human VEGF-G protein with the human VEGF-A protein using the GAP program in the GCG software package, using a Blossum 62 matrix and a gap weight of 5 and a length weight of 2.
  • Figure 5 depicts a local alignment of the human VEGF-G protein with the human TANGO 128 protein using the GAP program in the GCG software package, using a Blossum 62 matrix and a gap weight of 12 and a length weight of 4. The results showed a 51.203% identity between the two sequences.
  • Figure 6 depicts a global alignment of the human VEGF-G protein with the human TANGO 128 protein using the ALIGN program (version 2.0), using a PAM120 scoring matrix, and a gap length penalty of 12 and a gap penalty of 4. The results showed a 42.4% identity between the two sequences.
  • Figure 7 depicts a global alignment of the human VEGF-G nucleic acid sequence with the human TANGO 128 nucleic acid sequence using the ALIGN program (version 2.0), using a PAM120 scoring matrix, and a gap length penalty of 12 and a gap penalty of 4. The results showed a 51.9% identity between the two sequences.
  • Figure 8 depicts the cDNA sequence and predicted amino acid sequence of mouse VEGF-G.
  • the nucleotide sequence corresponds to nucleic acids 1 to 3121 of SEQ ID NO: 10.
  • the amino acid sequence corresponds to amino acids 1 to 370 of SEQ ID NO: 11.
  • the coding region without the 5' and 3' untranslated regions of the mouse VEGF-G gene is shown in SEQ ID NO: 12.
  • panel A depicts the results of a search which was performed against the HMM database and which resulted in the identification of a "CUB domain” in the mouse VEGF-G protein
  • panel B depicts the results of a search which was performed against the SMART database and which resulted in the identification of a "CUB domain” and a "PDGF domain” in the mouse VEFG-G protein.
  • Figure 10 depicts a global alignment of the open reading frame of the human VEGF-G gene with the open reading frame of the mouse VEGF-G gene using the GAP program in the GCG software package, using a nwsgapdna.cmp matrix and a gap weight of 12 and a length weight of 4. The results showed a 85.586% identity between the two sequences.
  • Figure 11 depicts a global alignment of the human VEGF-G protein with the mouse VEGF-G protein using the GAP program in the GCG software package, using a Blosum 62 matrix and a gap weight of 12 and a length weight of 4. The results showed a 84.865% identity between the two sequences.
  • VEGF proteins modulate the proliferation, motility, differentiation and survival of cells, such as endothelial cells or monocytes.
  • VEGF molecules regulate a broad range of cell, e.g., endothelial cell, processes including mitogenesis, permeability, vascular tone, and the synthesis of vasoactive molecules (Zachary, I. (1998) Int. J. Biochem. Cell Biol, 30:1169).
  • VEGF proteins are important in vasculogenesis and angiogenesis, both in normal physiology and the pathophysiology of disease states.
  • the VEGF-G molecules of the present invention are predicted to be growth regulatory proteins that function to modulate cell proliferation, differentiation, motility, ' and apoptosis.
  • the VEGF-G molecules of the present invention may play a role in cellular growth signalling mechanisms.
  • the term "cellular growth signalling mechanism” includes signal transmissions from cell receptors, e.g., growth factor receptors, which regulate one or more of the following: 1) cell transversal through the cell cycle, 2) cell differentiation, 3) cell migration and patterning, and 4) programmed cell death.
  • VEGF-G molecules of the present invention are predicted to be involved in the initiation or modulation of cellular signal transduction pathways that modulate endothelial cell growth, differentiation, migration and/or apoptosis.
  • the VEGF-G molecules by participating in cellular growth signalling mechanisms, may modulate cell behavior and act as therapeutic agents for controlling cellular proliferation, differentiation, migration, and apoptosis.
  • a "cellular prohferative disorder” includes a disorder, disease, or condition characterized by a deregulated, e.g., upregulated or downregulated, growth response.
  • a "cellular differentiative disorder” includes a disorder, disease, or condition characterized by aberrant cellular differentiation.
  • the VEGF-G molecules can act as novel diagnostic targets and therapeutic agents for controlling cellular prohferative and/or differentiative disorders.
  • cellular prohferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; and disorders involving aberrant angiogenesis and/or vascularity, e.g., tumor angiogenesis and metastasis, diabetic retinopathy, macular degeneration, psoriasis, endometriosis, Grave's disease, ischemic disease (e.g., atherosclerosis), and chronic inflammatory diseases (e.g., rheumatoid arthritis).
  • cancer e.g., carcinoma, sarcoma, or leukemia
  • disorders involving aberrant angiogenesis and/or vascularity e.g., tumor angiogenesis and metastasis, diabetic retinopathy, macular degeneration, psoriasis, endometriosis, Grave's
  • VEGF-G-associated or related disorders also include disorders affecting tissues in which VEGF-G protein is expressed.
  • family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally or non- naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins.
  • Members of a family can also have common functional characteristics.
  • members of the VEGF-G family of proteins include at least one "VEGF/PDGF superfamily variant motif in the protein molecule or the nucleic acid molecule encoding the protein molecule.
  • VEGF/PDGF superfamily variant motif includes a protein domain having an amino acid sequence of about 60-130 amino acid residues.
  • a VEGF/PDGF superfamily variant motif includes at least about 70-120, more preferably about 80-110 amino acid residues, and most preferably about 90-100 amino acid residues.
  • the VEGF/PDGF superfamily variant motif is characterized by conserved cysteine residues that form intra- and inter- chain disulfide bonds which may affect the structural integrity of the protein, and in one embodiment has the following signature pattern:
  • VEGF-G has such a signature pattern at about amino acids 268 to 362 of SEQ ID NO:2, and at about amino acids 268-362 of SEQ ID NOT 1.
  • Another VEGF-G family member human TANGO 128 (U.S. Patent Application No.09/223,546, filed Dec.30, 1998), has a VEGF/PDGF superfamily variant motif, suggesting that VEGF-G and TANGO 128 may represent a novel variant subclass of the VEGF/PDGF family of molecules.
  • the VEGF/PDGF superfamily variant motif has the following signature pattern:
  • VEGF-G has such a signature pattern at about amino acids 268 to 362 of SEQ ID NO:2, and at about amino acids 268 to 362 of SEQ ID NO: 11.
  • the VEGF/PDGF superfamily variant motif has the following signature pattern: [RK] -X(2-3) -C-X- [PA] -X(19-20) -P-X-C-X(4) -R-C-G-G-N-C- [GA] - C-X(7) -C-X-C-X(38-41) -C-X-C-X-C (SEQ ID 0.6)
  • VEGF-G has such a signature pattern at about amino acids 268 to 364 of SEQ ID NO:2, and at about amino acids 268 to 364 of SEQ ID NO: 11.
  • a member of the VEGF-G family of molecules includes a VEGF-G disulfide knot-like domain.
  • a VEGF-G disulfide knot-like domain has the following signature pattern :
  • VEGF-G has such a signature pattern at about amino acids 302 to 308 of SEQ ID NO:2, and at about amino acids 302-308 of SEQ ID NO: 11.
  • the VEGF-G disulfide knot-like domain has the following signature pattern :
  • VEGF-G has such a signature pattern at about amino acids 302 to 308 of SEQ ID NO:2, and at about amino acids 302-308 of SEQ ID NOT 1.
  • a member of this novel subfamily of VEGF proteins has a VEGF/PDGF superfamily variant motif which includes at least about 60- 130 amino acid residues and has at least about 50-60% identity with a VEGF/PDGF superfamily variant motif of VEGF-G (e.g., residues 268-362 of SEQ ID NO:2 or 11).
  • a VEGF/PDGF superfamily variant motif includes at least about 70-120 amino acid residues, or about 80-110 amino acid residues, or 90-100 amino acid residues, and has at least 60-70% identity, preferably about 70-80%, or more preferably about 80-90%, identity with a VEGF/PDGF superfamily variant motif of VEGF-G (e.g., residues 268-362 of SEQ ID NO:2 or 11).
  • VEGF-G proteins having at least 50-60% identity, preferably about 60-70%, more preferably about 70-80%, or about 80-90% identity with a VEGF/PDGF superfamily variant motif of human or mouse VEGF-G are within the scope of the invention.
  • VEGF-G family members can be identified based on the presence of a "CUB domain" in the protein or the nucleic acid molecule encoding the protein.
  • the term "CUB domain” includes a protein domain having an amino acid sequence of about 90-140 amino acid residues and having a bit score for the alignment of the sequence to the CUB domain (HMM) of at least about 80.
  • a CUB domain includes at least about 100- 130, more preferably about 110-120 amino acid residues, or 113-117 amino acid residues, and has a bit score for the alignment of the sequence to the CUB domain (HMM) of at least about 85, 90, 100, 120 or greater.
  • the CUB domain (HMM) has been assigned the PFAM Accession PF00431 (http://genome.wustl.edu/Pfam/ .html).
  • the CUB domain is an extracellular domain associated with various developmentally regulated proteins and as such is likely to be involved in developmental processes.
  • a CUB domain contains conserved cysteine residues which are likely to form disulfide bonds that affect protein structure.
  • the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters
  • the hmmsf program which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit.
  • the threshold score for determining a hit can be lowered (e.g. , to 8 bits).
  • a description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et ⁇ /.(1990) Meth. Enzymol.
  • a CUB domain was also identified in the amino acid sequence of mouse VEGF-G at about residues 53-167 of SEQ ID NO.T 1 (see Figure 9).
  • a member of the VEGF-G family of molecules includes a CUB domain with the following signature pattern:
  • VEGF-G has such a signature pattern at about amino acids 62 to 163 of SEQ ID NO:2, and at about amino acids 62 to 163 of SEQ ID NO: 11.
  • a member of this novel subfamily of VEGF proteins has a CUB domain includes at least about 70-130 amino acid residues and has at least about 50-60% identity with a CUB domain of VEGF-G (e.g., residues 62-163 of SEQ ID NO:2 or 11).
  • a CUB domain includes at least about 80-120 amino acid residues, or about 90-110 amino acid residues, or 98-104 amino acid residues, and has at least 60-70% identity, preferably about 70-80%, more preferably about 80-90% identity with a CUB domain of VEGF-G (e.g., residues 62-163 of SEQ ID NO:2 or 11).
  • VEGF-G proteins having at least 50-60% identity, preferably about 60-70%, more preferably about 70-80%, or about 80-90% identity with a CUB domain of human or mouse VEGF-G are within the scope of the invention.
  • VEGF-G family members can be identified based on the presence of a signal sequence.
  • a signal sequence includes a peptide containing about 19 amino acids which occurs at the N-terminus of secretory and membrane bound proteins and which contains a large number of hydrophobic amino acid residues.
  • a signal sequence contains at least about 10-30 amino acid residues, preferably about 15- 25 amino acid residues, more preferably about 18-20 amino acid residues, and more preferably about 19 amino acid residues, and has at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g., Valine, Leucine, Isoleucine or Phenylalanine).
  • VEGF-G Such a “signal sequence”, also referred to in the art as a “signal peptide”, serves to direct a protein containing such a sequence to a lipid bilayer, and is cleaved in secreted and membrane bound proteins.
  • a signal sequence was identified in the amino acid sequence of human VEGF-G at about amino acids 1-19 of SEQ ID NO:2.
  • a signal sequence was also identified in the amino acid sequence of mouse VEGF-G at about amino acids 1-23 of SEQ ID NOT 1.
  • VEGF-G family members include at least one or more N-glycosylation sites.
  • Predicted N-glycosylation sites are found, for example, from about amino acids 276-279 of SEQ ID NO:2, and from about amino acid residues 14-17 and 276-279 of SEQ ID NO.l l.
  • VEGF-G family members include at least one N- myristoylation site. Predicted N-myristoylation sites are found, for example, from about amino acid residues 100-105, 192-197, and 303-308 of SEQ ID NO:2, and from about amino acid residues 100-105 and 303-308 of SEQ ID NOT 1.
  • VEGF-G family members include at least one cAMP and cGMP dependent protein kinase phosphorylation site.
  • Predicted cAMPand cGMP dependent protein kinase phosphorylation sites are found, for example, from about amino acid residues 268-271 of SEQ ID NO:2, and from about amino acid residues 268- 271 of SEQ ID NOT 1.
  • VEGF-G family members include at least one protein kinase C phosphorylation site.
  • Predicted protein kinase C phosphorylation sites are found, for example, from about amino acid residues 17-19, 29-31, 66-68, 80-82, 150- 152, 243-245, 273-275, 320-322, 323-325, and 365-367 of SEQ ID NO:2, and from about amino acid residues 29-31, 66-68, 141-143, 150-152, 273-275, 320-322, 323-325, and 365-367 of SEQ ID NOT 1.
  • VEGF-G family members include at least one casein kinase II phosphorylation site.
  • Predicted casein kinase II phosphorylation sites are found, for example, from about amino acid residues 17-20, 168-171, 181-184, 199-202, 219-222, 231-234, 250-253, and 256-259 of SEQ ID NO:2, and from about amino acid residues 168-171, 181-184, 199-202, 213-216, 219-222, 231-234, 250-253, and 256-259 of SEQ ID NOT 1.
  • VEGF-G family members include at least one tyrosine kinase phosphorylation site.
  • Predicted tyrosine kinase phosphorylation sites are found, for example, from about amino acid residues 262-270 of SEQ ID NO:2, and from about amino acid residues 262-270 of SEQ ID NO: 11.
  • Isolated proteins of the present invention preferably VEGF-G proteins, have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or 11, or are encoded by a nucleotide sequence sufficiently homologous to SEQ ID NOT, 3, 10 or 12.
  • the term "sufficiently homologous” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently homologous.
  • amino acid or nucleotide sequences which share at least 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently homologous.
  • an "VEGF-G activity”, “biological activity of VEGF-G” or “functional activity of VEGF-G”, refers to an activity exerted by a VEGF- G protein, polypeptide or nucleic acid molecule on a VEGF-G responsive cell or on a VEGF-G protein substrate, as determined in vivo or in vitro, according to standard techniques.
  • a VEGF-G activity is a direct activity, such as an association with a VEGF-G target molecule.
  • a "target molecule” or “binding partner” is a molecule with which a VEGF-G protein binds or interacts in nature, such that VEGF-G-mediated function is achieved.
  • a VEGF-G target molecule can be a non- VEGF-G molecule or a VEGF-G protein or polypeptide of the present invention.
  • a VEGF-G target molecule is a VEGF-G substrate or receptor, e.g., fit or KDR/flk.
  • a VEGF-G activity can also be an indirect activity, such as a cellular signalling activity mediated by interaction of the VEGF-G protein with a VEGF-G substrate or receptor, e.g., fit or KDR/flk.
  • a VEGF- G activity is the ability to act as a growth regulatory factor and to modulate cell proliferation, differentiation, migration, apoptosis, and/or angiogenesis.
  • VEGF-G proteins and polypeptides having a VEGF-G activity.
  • Preferred proteins are VEGF-G proteins including at least one VEGF/PDGF superfamily variant motif, and, preferably, having a VEGF-G activity.
  • Further preferred proteins include at least one VEGF/PDGF superfamily variant motif, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes- under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, 3, 10 or 12.
  • Preferred proteins are VEGF-G proteins including at least one VEGF-G disulfide knot-like domain, and, preferably, having a VEGF-G activity.
  • Further preferred proteins include at least one VEGF-G disulfide knot-like domain, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, 3, 10 or 12.
  • Other preferred proteins are VEGF-G proteins including at least one CUB domain, and, preferably, having a VEGF-G activity.
  • Additional preferred proteins include at least one CUB domain, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, 3, 10 or 12.
  • Preferred proteins are VEGF-G proteins including at least one signal sequence, and, preferably, having a VEGF-G activity.
  • Further preferred proteins include at least one signal sequence, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, 3, 10 or 12.
  • Additional preferred proteins are VEGF-G proteins including at least one VEGF/PDGF superfamily variant motif and at least one CUB domain, and preferably, having a VEGF-G activity.
  • Further preferred proteins include at least one VEGF/PDGF superfamily variant motif and at least one CUB domain and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, 3, 10 or 12.
  • Other preferred proteins are VEGF-G proteins including at least one VEGF/PDGF superfamily variant motif, at least one CUB domain, and at least one signal sequence and preferably, having a VEGF-G activity.
  • Further preferred proteins include at least one VEGF/PDGF superfamily variant motif, at least one CUB domain, and at least one signal sequence and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO , 3, 10 or 12.
  • Additional preferred proteins are VEGF-G proteins including at least one VEGF-G disulfide knot-like domain and at least one CUB domain, and preferably, having a VEGF-G activity.
  • VEGF-G disulfide knot-like domain and at least one CUB domain and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, 3, 10 or 12.
  • Other preferred proteins are VEGF-G proteins including at least one VEGF-G disulfide knot-like domain, at least one CUB domain, and at least one signal sequence and preferably, having a VEGF-G activity.
  • Further preferred proteins include at least one VEGF-G disulfide knot-like domain, at least one CUB domain, and at least one signal sequence and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, 3, 10 or 12.
  • the nucleotide sequence of the isolated human VEGF-G cDNA and the predicted amino acid sequence of the human VEGF-G polypeptide are shown in Figure 1 and in SEQ ID NOs: 1 and 2, respectively.
  • the human VEGF-G gene which is approximately 3853 nucleotides in length, encodes a protein having a molecular weight of approximately 43 kD and which is approximately 370 amino acid residues in length.
  • the nucleotide sequence of the isolated mouse VEGF-G cDNA and the predicted amino acid sequence of the mouse VEGF-G polypeptide are shown in Figure 8 and in SEQ ID NOs. O and 11, respectively.
  • the mouse VEGF-G gene which is approximately 3121 nucleotides in length, encodes a protein having a molecular weight of approximately 43 kD and which is approximately 370 amino acid residues in length.
  • the predicted mature mouse VEGF-G protein lacking the signal sequence has a molecular weight of approximately 40 kD and is approximately 347 amino acid residues in length.
  • nucleic acid molecules that encode VEGF-G proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify VEGF-G -encoding nucleic acid molecules (e.g., VEGF-G mRNA) and fragments for use as PCR primers for the amplification or mutation of VEGF-G nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated VEGF-G nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOT, 3, 10 or 12, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOT, 3, 10 or 12 as hybridization probes, VEGF-G nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NOT, 3, 10 or 12 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NOT, 3, 10 or 12.
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to VEGF-G nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO .
  • the sequence of SEQ ID NOT corresponds to the human VEGF-G cDNA.
  • This cDNA comprises sequences encoding the human VEGF-G protein (i.e., "the coding region", from nucleotides 213-1325), as well as 5' untranslated sequences (nucleotides 1-212) and 3' untranslated sequences
  • nucleic acid molecule can comprise only the coding region of SEQ ID NOT (e.g., nucleotides 213-1325, corresponding to SEQ ID NO:3).
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 10.
  • the sequence of SEQ ID NO: 10 corresponds to the mouse VEGF-G cDNA.
  • This cDNA comprises sequences encoding the mouse VEGF-G protein (i.e., "the coding region", from nucleotides 165-1277), as well as 5' untranslated sequences (nucleotides 1-164) and 3' untranslated sequences (nucleotides 1278-3121).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NOT0 (e.g., nucleotides 165-1277, corresponding to SEQ ID NO: 12).
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NOT, 3, 10 or 12, or aportion of any of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NOT, 3, 10 or 12 is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOT, 3, 10 or 12, such that it can hybridize to the nucleotide sequence shown in SEQ ID NOT, 3, 10 or 12, thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98% or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NOT, 3, 10 or 12, or a portion of any of these nucleotide sequences.
  • the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NOT, 3, 10 or 12, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a VEGF-G protein, e.g., an immunogenic or biologically active portion of a VEGF-G protein.
  • the nucleotide sequence determined from the cloning of the VEGF-G gene allows for the generation of probes and primers designed for use in identifying and/or cloning other VEGF-G family members, as well as VEGF-G homologues from other species.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NOT, 3, 10 or 12, or of a naturally occurring allelic variant or mutant of SEQ ID NO , 3, 10 or 12.
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater than 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 549, 549-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200- 1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900- 2000, 2000-2200, 2200-2272, 2273, 2273-2400, 2400-2600, 2600-2800, 2800-3000 or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NOT, 3, 10 or 12.
  • Probes based on the VEGF-G nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a VEGF-G protein, such as by measuring a level of a VEGF-G- encoding nucleic acid in a sample of cells from a subject e.g., detecting VEGF-G mRNA levels or determining whether a genomic VEGF-G gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of a VEGF-G protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NOT, 3, 10 or 12, which encodes a polypeptide having a VEGF-G biological activity (the biological activities of the VEGF-G proteins are described herein), expressing the encoded portion of the VEGF-G protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the VEGF-G protein.
  • a nucleic acid fragment encoding a biologically active portion of a VEGF-G protein may comprise a nucleotide sequence which is greater than 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 549, 549-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1800, 1800-2000, 2000-2200, 2273 or more nucleotides in length.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NOT, 3, 10 or 12 due to degeneracy of the genetic code and thus encode the same VEGF-G proteins as those encoded by the nucleotide sequence shown in SEQ ID NOT, 3, 10 or 12.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 or 11.
  • VEGF-G nucleotide sequences shown in SEQ ID NOT, 3, 10 or 12
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the VEGF-G proteins may exist within a population (e.g. , the human population).
  • Such genetic polymorphism in the VEGF-G genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a VEGF-G protein, preferably a mammalian VEGF-G protein, and can further include non-coding regulatory sequences, and introns.
  • VEGF-G e.g., human or mouse VEGF-G
  • Functional allelic variants are naturally occurring amino acid sequence variants of the VEGF-G protein within a population that maintain the ability to bind a VEGF-G receptor or substrate, and/or modulate cell growth and migration mechanisms.
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2 or 11, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.
  • Non-functional allelic variants are naturally occurring amino acid sequence variants of the VEGF-G, e.g., human or mouse VEGF-G, protein within a population that do not have the ability to either bind a VEGF-G receptor or substrate, or modulate cell growth or migration mechanisms.
  • Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO:2 or 11, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.
  • the present invention further provides orthologues of the human and mouse VEGF-G proteins.
  • Orthologues of the human and mouse VEGF-G proteins are proteins that are isolated from non-human and non-mouse organisms and possess the same VEGF-G receptor or substrate binding mechanisms, and/or modulation of cell growth or migration mechanisms of the human and mouse VEGF-G proteins.
  • Orthologues of the human and mouse VEGF-G proteins can readily be identified as comprising an amino acid sequence that is substantially homologous to SEQ ID NO:2 or 11.
  • nucleic acid molecules encoding other VEGF-G family members and, thus, which have a nucleotide sequence which differs from the VEGF-G sequences of SEQ ID NOT, 3, 10 or 12 are intended to be within the scope of the invention.
  • another VEGF-G cDNA can be identified based on the nucleotide sequence of human or mouse VEGF-G.
  • nucleic acid molecules encoding VEGF-G proteins from different species, and which, thus, have a nucleotide sequence which differs from the VEGF-G sequences of SEQ ID NOT, 3, 10 or 12 are intended to be within the scope of the invention.
  • a monkey VEGF-G cDNA can be identified based on the nucleotide sequence of a human or mouse VEGF-G.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the VEGF-G cDNAs of the invention can be isolated based on their homology to the VEGF-G nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the VEGF-G cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the VEGF-G gene.
  • an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, 3, 10 or 12.
  • the nucleic acid is at least 30, 50, 100, 150, 200, 250, 253, 300, 350, 400, 450, 500, 549, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200 or 2273 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non- limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50°C.
  • Another example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55°C.
  • a further example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOT, 3, 10 or 12 corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g. , encodes a natural protein).
  • allelic variants of the VEGF-G sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NOT, 3, 10 or 12, thereby leading to changes in the amino acid sequence of the encoded VEGF-G proteins, without altering the functional ability of the VEGF-G proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOT, 3, 10 or 12.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of VEGF-G (e.g., the sequence of SEQ ID NO:2 or 11) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the VEGF-G proteins of the present invention e.g., those present in the VEGF/PDGF superfamily variant motif, the VEGF-G disulfide knot-like domain, or the CUB domain, are predicted to be particularly unamenable to alteration.
  • additional amino acid residues that are conserved between the VEGF-G proteins of the present invention and other members of the VEGF family are not likely to be amenable to alteration.
  • nucleic acid molecules encoding VEGF-G proteins that contain changes in amino acid residues that are not essential for activity.
  • Such VEGF-G proteins differ in amino acid sequence from SEQ ID NO:2 or 11, yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:2 or 11.
  • An isolated nucleic acid molecule encoding a VEGF-G protein homologous to the protein of SEQ ID NO:2 or 11 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOT, 3, 10 or 12 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NOT, 3, 10 or 12 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • a predicted nonessential amino acid residue in a VEGF-G protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a VEGF-G coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for VEGF-G biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NOT, 3, 10 or 12, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • a mutant VEGF-G protein can be assayed for the ability to (1) interact with a non- VEGF-G protein molecule, e.g., a VEGF-G substrate or receptor; (2) activate a VEGF-G-dependent signal transduction pathway; (3) modulate cell proliferation, differentiation, migration and/or apoptosis mechanisms; or (4) modulate angiogenic processes.
  • a non- VEGF-G protein molecule e.g., a VEGF-G substrate or receptor
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire VEGF-G coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding VEGF-G.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human VEGF-G corresponds to SEQ ID NO:3 and the coding region of mouse VEGF-G corresponds to SEQ ID NO: 12).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding VEGF-G.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of VEGF-G mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of VEGF-G mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of VEGF-G mRNA.
  • An antisense oligonucleotide can be, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5 '-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a VEGF-G protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave VEGF-G mRNA transcripts to thereby inhibit translation of VEGF - G mRNA.
  • a ribozyme having specificity for a VEGF-G-encoding nucleic acid can be designed based upon the nucleotide sequence of a VEGF-G cDNA disclosed herein (i.e., SEQ ID NOT, 3, 10 or 12).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a VEGF-G-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
  • VEGF-G mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • VEGF-G gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the VEGF-G (e.g., the VEGF-G promoter and/or enhancers) to form triple helical structures that prevent transcription of the VEGF-G gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C.
  • the VEGF-G nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
  • PNAs of VEGF-G nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of VEGF-G nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).
  • PNAs of VEGF-G can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of VEGF-G nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn PT. et al (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy- thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Aca
  • oligonucleotides can be modified with hybridization- triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • VEGF-G proteins and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-VEGF-G antibodies.
  • native VEGF-G proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • VEGF- G proteins are produced by recombinant DNA techniques.
  • a VEGF-G protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the VEGF-G protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of VEGF-G protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of VEGF-G protein having less than about 30%) (by dry weight) of non- VEGF-G protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non- VEGF-G protein, still more preferably less than about 10% of non- VEGF-G protein, and most preferably less than about 5% non- VEGF-G protein.
  • a contaminating protein also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of VEGF-G protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of VEGF-G protein having less than about 30% (by dry weight) of chemical precursors or non- VEGF-G chemicals, more preferably less than about 20% chemical precursors or non- VEGF-G chemicals, still more preferably less than about 10% chemical precursors or non- VEGF-G chemicals, and most preferably less than about 5% chemical precursors or non- VEGF-G chemicals.
  • a "biologically active portion" of a VEGF-G protein includes a fragment of a VEGF-G protein which participates in an interaction between a VEGF-G molecule and a non- VEGF-G molecule.
  • Biologically active portions of a VEGF-G protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the VEGF-G protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or 11, which include less amino acids than the full length VEGF-G proteins, and exhibit at least one activity of a VEGF-G protein.
  • biologically active portions comprise a domain or motif with at least one activity of the VEGF-G protein, e.g., modulating cell growth and/or migration mechanisms.
  • a biologically active portion of a VEGF-G protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length.
  • Biologically active portions of a VEGF-G protein can be used as targets for developing agents which modulate a VEGF-G mediated activity, e.g., a cell proliferation, differentiation, migration, apoptosis, or angiogenic signalling mechanism.
  • a biologically active portion of a VEGF-G protein comprises at least one VEGF/PDGF superfamily variant motif, and/or at least one CUB domain. It is to be understood that a preferred biologically active portion of a VEGF-G protein of the present invention may contain at least one VEGF/PDGF superfamily variant motif. In another embodiment, a biologically active portion of a VEGF-G protein comprises at least one VEGF-G disulfide knot-like domain, and/or at least one CUB domain. It is to be understood that a preferred biologically active portion of a VEGF-G protein of the present invention may contain at least one VEGF-G disulfide knot-like domain.
  • Another preferred biologically active portion of a VEGF-G protein may contain at least one CUB domain.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native VEGF- G protein.
  • the VEGF-G protein has an amino acid sequence shown in SEQ ID NO:2 or 11.
  • the VEGF-G protein is substantially homologous to SEQ ID NO:2 or 11, and retains the functional activity of the protein of SEQ ID NO:2 or 11, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the VEGF-G protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:2 or 11.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the VEGF-G amino acid sequence of SEQ ID NO:2 or 11 having 370 amino acid residues, at least 111, preferably at least 148, more preferably at least 185, even more preferably at least 222, and even more preferably at least 259, 296 or 333 amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • a VEGF-G "chimeric protein” or “fusion protein” comprises a VEGF-G polypeptide operatively linked to a non- VEGF-G polypeptide.
  • An "VEGF-G polypeptide” refers to a polypeptide having an amino acid sequence corresponding to VEGF-G
  • a “non- VEGF-G polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the VEGF-G protein, e.g. , a protein which is different from the VEGF-G protein and which is derived from the same or a different organism.
  • a VEGF-G fusion protein can correspond to all or a portion of a VEGF-G protein.
  • a VEGF-G fusion protein comprises at least one biologically active portion of a VEGF-G protein.
  • a VEGF-G fusion protein comprises at least two biologically active portions of a VEGF-G protein.
  • the term "operatively linked" is intended to indicate that the VEGF-G polypeptide and the non- VEGF-G polypeptide are fused in-frame to each other.
  • the non- VEGF-G polypeptide can be fused to the N-terminus or C-terminus of the VEGF-G polypeptide.
  • the fusion protein is a GST- VEGF-G fusion protein in which the VEGF-G sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant VEGF-G.
  • the fusion protein is an alkaline phosphatase-VEGF-G fusion protein in which the VEGF-G sequences are fused to alkaline phosphatase sequences.
  • Such fusion proteins can be used to assess VEGF-G binding to cells and/or tissues.
  • the fusion protein is a VEGF-G protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of VEGF-G can be increased through use of a heterologous signal sequence.
  • the VEGF-G fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the VEGF-G fusion proteins can be used to affect the bioavailability of a VEGF-G substrate.
  • VEGF- G fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a VEGF- G protein; (ii) mis-regulation of the VEGF-G gene; and (iii) aberrant post-translational modification of a VEGF-G protein.
  • VEGF-G-fusion proteins of the invention can be used as immunogens to produce anti-VEGF-G antibodies in a subject, to purify VEGF-G ligands and in screening assays to identify molecules which inhibit the interaction of VEGF-G with a VEGF-G substrate.
  • a VEGF-G chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamphfied to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamphfied to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a VEGF-G- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the VEGF-G protein.
  • the present invention also pertains to variants of the VEGF-G proteins which function as either VEGF-G agonists (mimetics) or as VEGF-G antagonists.
  • Variants of the VEGF-G proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a VEGF-G protein.
  • An agonist of the VEGF-G proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a VEGF-G protein.
  • An antagonist of a VEGF-G protein can inhibit one or more of the activities of the naturally occurring form of the VEGF-G protein by, for example, competitively modulating a VEGF-G-mediated activity of a VEGF-G protein.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the VEGF-G protein.
  • variants of a VEGF-G protein which function as either VEGF-G agonists (mimetics) or as VEGF-G antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a VEGF-G protein for VEGF-G protein agonist or antagonist activity.
  • a variegated library of VEGF-G variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of VEGF-G variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential VEGF-G sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of VEGF-G sequences therein.
  • a degenerate set of potential VEGF-G sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of VEGF-G sequences therein.
  • Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential VEGF-G sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477.
  • libraries of fragments of a VEGF-G protein coding sequence can be used to generate a variegated population of VEGF-G fragments for screening and subsequent selection of variants of a VEGF-G protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a VEGF-G coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the VEGF-G protein.
  • Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify VEGF-G variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 59:7811 -7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
  • cell based assays can be exploited to analyze a variegated VEGF-G library.
  • a library of expression vectors can be transfected into a cell line, e.g., an endothelial cell line, which ordinarily responds to VEGF-G in a particular VEGF-G substrate-dependent manner.
  • the transfected cells are then contacted with VEGF-G and the effect of the expression of the mutant on signalling by the VEGF- G substrate can be detected, e.g., by measuring intracellular calcium and inositol 1,4,5- trisphosphate (IP3) levels, cell growth, and cell migration.
  • IP3 intracellular calcium and inositol 1,4,5- trisphosphate
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signalling by the VEGF-G substrate, and the individual clones further characterized.
  • VEGF-G protein can be used as an immunogen to generate antibodies that bind VEGF-G using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length VEGF-G protein can be used or, alternatively, the invention provides antigenic peptide fragments of VEGF-G for use as immunogens.
  • the antigenic peptide of VEGF-G comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of VEGF-G such that an antibody raised against the peptide forms a specific immune complex with VEGF-G.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of VEGF-G that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • a VEGF-G immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed VEGF-G protein or a chemically synthesized VEGF-G polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic VEGF-G preparation induces a polyclonal anti-VEGF-G antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as VEGF-G.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind VEGF-G.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of VEGF-G.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular VEGF-G protein with which it immunoreacts.
  • Polyclonal anti-VEGF-G antibodies can be prepared as described above by immunizing a suitable subject with a VEGF-G immunogen.
  • the anti-VEGF-G antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized VEGF-G.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against VEGF-G can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody- producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al (1982) Int. J.
  • an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a VEGF-G immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds VEGF-G.
  • lymphocytes typically splenocytes
  • Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-VEGF-G monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
  • the immortal cell line e.g., a myeloma cell line
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind VEGF-G, e.g., using a standard ELISA assay.
  • a monoclonal anti-VEGF-G antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with VEGF-G to thereby isolate immunoglobulin library members that bind VEGF-G.
  • Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27- 9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271 ; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitiing et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No.
  • recombinant anti-VEGF-G antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science
  • An anti-VEGF-G antibody (e.g., monoclonal antibody) can be used to isolate VEGF-G by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-VEGF-G antibody can facilitate the purification of natural VEGF-G from cells and of recombinantly produced VEGF-G expressed in host cells.
  • an anti-VEGF-G antibody can be used to detect VEGF-G protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the VEGF-G protein.
  • Anti-VEGF-G antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 1, 131 I, 35 S or 3 H.
  • vectors preferably expression vectors, containing a nucleic acid encoding a VEGF-G protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g. , non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adeno viruses and adeno- associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adeno viruses and adeno- associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., VEGF-G proteins, mutant forms of VEGF-G proteins, fusion proteins, and the like).
  • the recombinant expression vectors of the invention can be designed for expression of VEGF-G proteins in prokaryotic or eukaryotic cells.
  • VEGF- G proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells.
  • telomeres Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • VEGF-G activity assays e.g., direct assays or competitive assays described in detail below
  • VEGF-G proteins e.g., direct assays or competitive assays described in detail below
  • a VEGF-G fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the VEGF-G expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • VEGF-G proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166.
  • Developmentally- regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a
  • DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to VEGF-G mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • VEGF-G nucleic acid molecule of the invention is introduced, e.g., a VEGF-G nucleic acid molecule within a recombinant expression vector or a VEGF-G nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a VEGF-G protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), Chinese hamster ovary cells (CHO) or COS cells).
  • HMVEC human umbilical vein endothelial cells
  • HMVEC human microvascular endothelial cells
  • COS cells Chinese hamster ovary cells
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a VEGF-G protein or can be introduced on a separate vector.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a VEGF-G protein.
  • the invention further provides methods for producing a VEGF-G protein using the host cells of the invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a VEGF-G protein has been introduced) in a suitable medium such that a VEGF-G protein is produced.
  • the method further comprises isolating a VEGF-G protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which VEGF-G-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous VEGF-G sequences have been introduced into their genome or homologous recombinant animals in which endogenous VEGF-G sequences have been altered.
  • Such animals are useful for studying the function and/or activity of a VEGF-G protein and for identifying and/or evaluating modulators of VEGF-G activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non- human animal, preferably a mammal, more preferably a mouse, in which an endogenous VEGF-G gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing a VEGF-G- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g. , by microinj ection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the VEGF-G cDNA sequence of SEQ ID NO or 10 can be introduced as a transgene into the genome of a non-human animal.
  • a non-human or non-mouse homologue of a human or mouse VEGF-G gene such as a rat or VEGF-G gene, can be used as a transgene.
  • a VEGF-G gene homologue such as another VEGF-G family member, can be isolated based on hybridization to the VEGF-G cDNA sequences of SEQ ID NOT, 3, 10 or 12 (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a VEGF-G transgene to direct expression of a VEGF-G protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of a VEGF-G transgene in its genome and/or expression of VEGF-G mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a VEGF-G protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of a VEGF-G gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g. , functionally disrupt, the VEGF-G gene.
  • the VEGF-G gene can be a human gene (e.g.
  • the cDNA of SEQ ID NO:3) is a non-human homolog of a human VEGF-G gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:l or 10),
  • a mouse VEGF-G gene e.g., the cDNA of SEQ ID NO: 12
  • a homologous recombination nucleic acid molecule e.g. , a vector, suitable for altering an endogenous VEGF-G gene in the mouse genome.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous VEGF-G gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous VEGF-G gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous VEGF-G protein).
  • the altered portion of the VEGF-G gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the VEGF-G gene to allow for homologous recombination to occur between the exogenous VEGF-G gene carried by the homologous recombination nucleic acid molecule and an endogenous VEGF-G gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking VEGF-G nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • homologous recombination nucleic acid molecule typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51 :503 for a description of homologous recombination vectors).
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced VEGF-G gene has homologously recombined with the endogenous VEGF-G gene are selected (see e.g. , Li, E. et ⁇ l.
  • the selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Ter ⁇ toc ⁇ rcinom ⁇ s and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • aggregation chimeras see e.g., Bradley, A. in Ter ⁇ toc ⁇ rcinom ⁇ s and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al; WO 91/01140 by Smithies et al; WO 92/0968 by Zijlsfra et al; and WO 93/04169 by Berns et al.
  • transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage P 1.
  • Cre/loxP recombinase system of bacteriophage P 1.
  • FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.
  • a cre/loxP recombinase system is used to regulate expression of the transgene
  • animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • VEGF-G nucleic acid molecules, fragments of VEGF-G proteins, and anti- VEGF-G antibodies can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • Active compounds may include, but are not limited to, peptides, nucleic acids, antibodies, and small inorganic or inorganic compounds.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, infradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, infradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringabihty exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., a fragment of a VEGF-G protein or an anti-VEGF-G antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a fragment of a VEGF-G protein or an anti-VEGF-G antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules, oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum fragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum fragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penevers appropriate to the barrier to be permeated are used in the formulation.
  • Such penevers are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • An agent may, for example, be a small molecule.
  • such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • organic or inorganic compounds i.e., including heteroorganic and organometallic compounds
  • doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about lmicrogram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguan
  • the conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drag moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, .alpha.-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • an antibody can be conjugated to a second antibody to Form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054- 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g. , diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
  • a VEGF-G protein of the invention has one or more of the following activities: (1) it interacts with a non- VEGF-G protein molecule, e.g., a VEGF-G substrate, such as a VEGF receptor; (2) it activates a VEGF-G-dependent signal transduction pathway; (3) it modulates cell proliferation, differentiation, and/or migration mechanisms; (4) it modulates angiogenesis, and, thus, can be used to, for example, (1) modulate the interaction with a non- VEGF-G protein molecule; (2) to activate a VEGF-G-dependent signal transduction pathway; (3) to modulate cell proliferation, differentiation, and/or migration mechanisms; (4) to modulate angiogenesis.
  • the isolated nucleic acid molecules of the invention can be used, for example, to express VEGF-G protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect VEGF-G mRNA (e.g., in a biological sample) or a genetic alteration in a VEGF-G gene, and to modulate VEGF-G activity, as described further below.
  • VEGF-G proteins can be used to treat disorders characterized by insufficient or excessive production of a VEGF-G substrate or production of VEGF-G inhibitors.
  • the VEGF-G proteins can be used to screen for naturally occurring VEGF-G substrates, to screen for drugs or compounds which modulate VEGF-G activity, as well as to treat disorders characterized by insufficient or excessive production of VEGF-G protein or production of VEGF-G protein forms which have decreased, aberrant or unwanted activity compared to VEGF-G wild type protein (e.g., cell proliferation and/or differentiation disorders, such as disorders characterized by aberrant angiogenesis).
  • the anti-VEGF-G antibodies of the invention can be used to detect and isolate VEGF-G proteins, regulate the bioavailability of VEGF-G proteins, and modulate VEGF-G activity.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to VEGF-G proteins, have a stimulatory or inhibitory effect on, for example, VEGF-G expression or VEGF-G activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a VEGF-G substrate.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to VEGF-G proteins, have a stimulatory or inhibitory effect on, for example, VEGF-G expression or VEGF-G activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a VEGF-G substrate.
  • the invention provides assays for screening candidate or test compounds which are substrates of a VEGF-G protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a VEGF-G protein or polypeptide or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses a NEGF-G protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate VEGF-G activity is determined. Determining the ability of the test compound to modulate VEGF-G activity can be accomplished by monitoring, for example, intracellular calcium and inositol 1,4,5-trisphosphate (IP3) levels, cell growth, and cell chemotaxis.
  • IP3 intracellular calcium and inositol 1,4,5-trisphosphate
  • the cell for example, can be of mammalian origin, e.g., an endothelial cell.
  • the ability of the test compound to modulate VEGF-G binding to a substrate or to bind to VEGF-G can also be determined. Determining the ability of the test compound to modulate VEGF-G binding to a substrate can be accomplished, for example, by coupling the VEGF-G substrate with a radioisotope or enzymatic label such that binding of the VEGF-G substrate to VEGF-G can be determined by detecting the labeled VEGF-G substrate in a complex.
  • VEGF-G could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate VEGF-G binding to a VEGF-G substrate in a complex.
  • Determining the ability of the test compound to bind VEGF-G can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to VEGF-G can be determined by detecting the labeled VEGF-G compound in a complex.
  • compounds e.g., VEGF-G substrates
  • the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a compound with VEGF-G without the labeling of either the compound or the VEGF-G. McConnell, H. M. et al. (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell expressing a VEGF-G target molecule (e.g., a VEGF-G substrate) with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the VEGF-G target molecule. Determining the ability of the test compound to modulate the activity of a VEGF-G target molecule can be accomplished, for example, by determining the ability of the VEGF-G protein to bind to or interact with the VEGF-G target molecule.
  • a VEGF-G target molecule e.g., a VEGF-G substrate
  • Determining the ability of the test compound to modulate the activity of a VEGF-G target molecule can be accomplished, for example, by determining the ability of the VEGF-G protein to bind to or interact with the VEGF-G target molecule.
  • Determining the ability of the VEGF-G protein or a biologically active fragment thereof, to bind to or interact with a VEGF-G target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the VEGF-G protein to bind to or interact with a VEGF-G target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e., intracellular calcium or IP3), detecting catalytic/enzymatic activity of the target molecule upon an appropriate substrate, detecting the induction of a reporter gene (comprising a target- responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response (i.e., cell growth or migration).
  • a cellular second messenger of the target i.e., intracellular calcium or IP3
  • detecting catalytic/enzymatic activity of the target molecule upon an appropriate substrate detecting the induction of a reporter gene (comprising a target- responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response (i.e., cell growth or
  • an assay of the present invention is a cell-free assay in which a VEGF-G protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the VEGF-G protein or biologically active portion thereof is determined.
  • Preferred biologically active portions of the VEGF-G proteins to be used in assays of the present invention include fragments which participate in interactions with non- VEGF-G molecules, e.g., fragments with high surface probability scores. Binding of the test compound to the VEGF-G protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the VEGF-G protein or biologically active portion thereof with a known compound which binds VEGF-G to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a VEGF-G protein, wherein determining the ability of the test compound to interact with a VEGF-G protein comprises determining the ability of the test compound to preferentially bind to VEGF-G or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which a VEGF-G protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the VEGF-G protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a VEGF-G protein can be accomplished, for example, by determining the ability of the VEGF-G protein to bind to a VEGF-G target molecule by one of the methods described above for determining direct binding.
  • VEGF-G protein Determining the ability of the VEGF-G protein to bind to a VEGF-G target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA).
  • BIOA Biomolecular Interaction Analysis
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • determining the ability of the test compound to modulate the activity of a VEGF-G protein can be accomplished by determining the ability of the VEGF-G protein to further modulate the activity of a downstream effector of a VEGF-G target molecule.
  • the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.
  • the cell-free assay involves contacting a VEGF-G protein or biologically active portion thereof with a known compound which binds the VEGF-G protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the VEGF- G protein, wherein determining the ability of the test compound to interact with the VEGF-G protein comprises determining the ability of the VEGF-G protein to preferentially bind to or modulate the activity of a VEGF-G target molecule.
  • the cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins (e.g., VEGF-G proteins or biologically active portions thereof).
  • isolated proteins e.g., VEGF-G proteins or biologically active portions thereof.
  • a solubilizing agent such that the membrane-bound form of the isolated protein is maintained in solution.
  • non-ionic detergents such as
  • VEGF-G vascular endothelial growth factor-G
  • binding of a test compound to a VEGF-G protein, or interaction of a VEGF-G protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase/VEGF-G fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or VEGF-G protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of VEGF-G binding or activity determined using standard techniques.
  • VEGF-G protein or a VEGF-G target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated VEGF-G protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, IL
  • streptavidin-coated 96 well plates Piereptavidin-coated 96 well plates
  • antibodies reactive with VEGF-G protein or target molecules but which do not interfere with binding of the VEGF-G protein to its target molecule can be derivatized to the wells of the plate, and unbound target or VEGF-G protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the VEGF-G protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the VEGF-G protein or target molecule.
  • modulators of VEGF-G expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of VEGF-G mRNA or protein in the cell is determined. The level of expression of VEGF - G mRNA or protein in the presence of the candidate compound is compared to the level of expression of VEGF-G mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of VEGF-G expression based on this comparison. For example, when expression of VEGF-G mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of VEGF-G mRNA or protein expression. Alternatively, when expression of VEGF-G mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of VEGF-G mRNA or protein expression.
  • the level of VEGF-G mRNA or protein expression in the cells can be determined by methods described herein for detecting VEGF-G mRNA or protein.
  • the VEGF-G proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • VEGF-G-binding proteins bind to or interact with VEGF-G
  • NEGF-G-binding proteins bind to or interact with VEGF-G
  • NEGF-G-binding proteins bind to or interact with VEGF-G
  • NEGF-G-binding proteins are also likely to be involved in the propagation of signals by the NEGF-G proteins or VEGF-G targets as, for example, downstream elements of a VEGF-G-mediated signalling pathway.
  • VEGF-G-binding proteins are likely to be VEGF-G inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a VEGF-G protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey” proteins are able to interact, in vivo, forming a VEGF-G- dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a reporter gene e.g., LacZ
  • Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the VEGF-G protein.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell free assay, and the ability of the agent to modulate the activity of a VEGF-G protein can be confirmed in vivo, e.g., in an animal such as an animal model for angiogenesis, or for cellular transformation and/or tumorigenesis.
  • Cellular models for the study of angiogenesis include models of endothelial cell differentiation on Matrigel (Baatout, S. et al. (1996) Rom. J. Intern. Med. 34:263-269; Benelli, R et al. (1999) Int. J. Biol. Markers 14:243-246), embryonic stem cell models of vascular mo ⁇ hogenesis (Doetschman, T.
  • Models for studying tumorigenesis in vivo include carcinogen- induced tumors, injection and/or transplantation of tumor cells into an animal, as well as animals bearing mutations in growth regulatory genes.
  • Models for studying angiogenesis in vivo include tumor cell-induced angiogenesis and tumor metastasis (Hoffman, RM (1998-99) Cancer Metastasis Rev. 17:271-277; Holash, J et al. (1999) Oncogene 18:5356-5362; Li, CY et al. (2000) J. Natl Cancer Inst.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a V ⁇ GF-G modulating agent, an antisense V ⁇ GF-G nucleic acid molecule, a V ⁇ GF-G-specific antibody, or a V ⁇ GF-G-binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • this sequence can be used to map the location of the gene on a chromosome.
  • This process is called chromosome mapping.
  • portions or fragments of the VEGF-G nucleotide sequences, described herein can be used to map the location of the VEGF-G genes on a chromosome.
  • the mapping of the VEGF-G sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • the VEGF-G gene has been mapped to human chromosome 11.
  • VEGF-G genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the VEGF-G nucleotide sequences. Computer analysis of the VEGF-G sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the VEGF-G sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells).
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the VEGF-G nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a VEGF-G sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci.
  • FISH Fluorescence in situ hybridization
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al, Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping pmposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease.
  • Comparison of affected and unaffected individuals generally involves first looking for stractural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymo ⁇ hisms.
  • the VEGF-G sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the VEGF-G nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the VEGF-G nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses.
  • SEQ ID NOT or 10 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3 or 12 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from VEGF-G nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification of the individual, living or dead can be made from extremely small tissue samples.
  • Use of Partial VEGF-G Sequences in Forensic Biology DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a pe ⁇ etrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene.
  • the amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • the sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • polynucleotide reagents include the VEGF-G nucleotide sequences or portions thereof, e.g. , fragments derived from the noncoding regions of SEQ ID NO or 10 having a length of at least 20 bases, preferably at least 30 bases.
  • VEGF-G nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., a tissue containing endothelial cells. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such VEGF-G probes can be used to identify tissue by species and/or by organ type.
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., a tissue containing endothelial cells. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such VEGF-G probes can be used to identify tissue by species and/or by organ
  • these reagents e.g., VEGF-G primers or probes can be used to screen tissue culture for contamination (i. e. screen for the presence of a mixture of different types of cells in a culture).
  • VEGF-G primers or probes can be used to screen tissue culture for contamination (i. e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) pu ⁇ oses to thereby treat an individual prophylactically.
  • diagnostic assays for determining VEGF-G protein and/or nucleic acid expression as well as VEGF-G activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted VEGF-G expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with VEGF-G protein, nucleic acid expression or activity. For example, mutations in a VEGF-G gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with VEGF-G protein, nucleic acid expression or activity.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of VEGF-G in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of VEGF-G protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting VEGF-G protein or nucleic acid (e.g. , mRNA, genomic DNA) that encodes VEGF-G protein such that the presence of VEGF-G protein or nucleic acid is detected in the biological sample.
  • a preferred agent for detecting VEGF-G mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to VEGF-G mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length VEGF-G nucleic acid, such as the nucleic acid of SEQ ID NOT , 3, 10 or 12, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to VEGF-G mRNA or genomic DNA.
  • a full-length VEGF-G nucleic acid such as the nucleic acid of SEQ ID NOT , 3, 10 or 12, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to VEGF-G mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • a preferred agent for detecting VEGF-G protein is an antibody capable of binding to VEGF-G protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. , Fab or F(ab')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect VEGF-G mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of VEGF-G mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of VEGF-G protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of VEGF-G genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of VEGF-G protein include introducing into a subject a labeled anti-VEGF-G antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting VEGF-G protein, mRNA, or genomic DNA, such that the presence of VEGF-G protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of VEGF-G protein, mRNA or genomic DNA in the control sample with the presence of VEGF-G protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of VEGF-G in a biological sample can comprise a labeled compound or agent capable of detecting VEGF-G protein or mRNA in a biological sample; means for determining the amount of VEGF-G in the sample; and means for comparing the amount of VEGF-G in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect VEGF-G protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted VEGF-G expression or activity.
  • aberrant includes a VEGF-G expression or activity which deviates from the wild type VEGF-G expression or activity.
  • Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression.
  • aberrant VEGF-G expression or activity is intended to include the cases in which a mutation in the VEGF-G gene causes the VEGF-G gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional VEGF-G protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with a VEGF-G substrate, e.g., a VEGF receptor, or one which interacts with a non- VEGF-G substrate.
  • the term "unwanted” includes an unwanted phenomenon involved in a biological response such as pain or deregulated cell proliferation.
  • unwanted includes a VEGF-G expression or activity which is undesirable in a subject.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in VEGF-G protein activity or nucleic acid expression, such as a cell proliferation and/or differentiation disorder.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in VEGF-G protein activity or nucleic acid expression, such as a cell proliferation and/or differentiation disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted VEGF-G expression or activity in which a test sample is obtained from a subject and VEGF-G protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of VEGF-G protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted VEGF-G expression or activity.
  • a test sample refers to a biological sample obtained from ai subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drag candidate) to treat a disease or disorder associated with aberrant or unwanted VEGF-G expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drag candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a cell proliferation and/or differentiation disorder.
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted VEGF-G expression or activity in which a test sample is obtained and VEGF- G protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of VEGF-G protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted VEGF-G expression or activity).
  • the methods of the invention can also be used to detect genetic alterations in a VEGF-G gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in VEGF-G protein activity or nucleic acid expression, such as a cell proliferation and/or differentiation disorder.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a VEGF-G-protein, or the mis-expression of the VEGF-G gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a VEGF-G gene; 2) an addition of one or more nucleotides to a VEGF-G gene; 3) a substitution of one or more nucleotides of a VEGF-G gene, 4) a chromosomal rearrangement of a VEGF-G gene; 5) an alteration in the level of a messenger RNA transcript of a VEGF-G gene, 6) aberrant modification of a VEGF-G gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a VEGF-G gene, 8) a non- wild type level of a VEGF-G-protein, 9) allelic loss of a VEGF-G gene, and 10) inappropriate post- translational modification of a VEGF-G-protein.
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 :360-364), the latter of which can be particularly useful for detecting point mutations in the VEGF-G-gene (see Abravaya et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a VEGF-G gene under conditions such that hybridization and amplification of the VEGF-G-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • primers which specifically hybridize to a VEGF-G gene under conditions such that hybridization and amplification of the VEGF-G-gene (if present) occurs
  • detecting the presence or absence of an amplification product or detecting the size of the amplification product and comparing the length to a control sample.
  • PCR and/or LCR may be desirable to use as a preliminary amplification
  • mutations in a VEGF-G gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control D ⁇ A is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control D ⁇ A indicates mutations in the sample D ⁇ A.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in VEGF-G can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753- 759).
  • genetic mutations in VEGF-G can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the VEGF-G gene and detect mutations by comparing the sequence of the sample VEGF-G with the corresponding wild-type (control) sequence.
  • sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the VEGF-G gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type VEGF-G sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in VEGF-G cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a VEGF-G sequence e.g.
  • a wild-type VEGF-G sequence is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in elecfrophoretic mobility will be used to identify mutations in VEGF-G genes.
  • single strand conformation polymo ⁇ hism SSCP
  • SSCP single strand conformation polymo ⁇ hism
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in elecfrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in elecfrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing pre- packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a VEGF- G gene.
  • any cell type or tissue in which VEGF-G is expressed may be utilized in the prognostic assays described herein.
  • Monitoring the influence of agents (e.g., drags) on the expression or activity of a VEGF-G protein can be applied not only in basic drug screening, but also in clinical trials.
  • agents e.g., drags
  • the effectiveness of an agent determined by a screening assay as described herein to increase VEGF-G gene expression, protein levels, or upregulate VEGF-G activity can be monitored in clinical trials of subjects exhibiting decreased VEGF-G gene expression, protein levels, or downregulated VEGF-G activity.
  • the effectiveness of an agent determined by a screening assay to decrease VEGF-G gene expression, protein levels, or downregulate VEGF-G activity can be monitored in clinical trials of subjects exhibiting increased VEGF-G gene expression, protein levels, or upregulated VEGF-G activity.
  • the expression or activity of a VEGF-G gene, and preferably, other genes that have been implicated in, for example, a VEGF-G-associated disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes including VEGF-G, that are modulated in cells by treatment with an agent (e.g., compound, drag or small molecule) which modulates VEGF-G activity (e.g., identified in a screening assay as described herein) can be identified.
  • an agent e.g., compound, drag or small molecule
  • VEGF-G activity e.g., identified in a screening assay as described herein
  • agents on VEGF-G-associated disorders e.g. , disorders characterized by deregulated cell growth, differentiation and/or migration mechanisms
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of VEGF-G and other genes implicated in the VEGF-G-associated disorder, respectively.
  • the levels of gene expression can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of VEGF-G or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a VEGF-G protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the VEGF- G protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the VEGF-G protein, mRNA, or genomic DNA in the pre-administration sample with the VEGF-G protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent
  • an agent
  • VEGF-G expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted VEGF-G expression or activity.
  • treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market.
  • the term refers the study of how a patient's genes determine his or her response to a drag (e.g., a patient's "drug response phenotype", or "drug response genotype”.)
  • a drag e.g., a patient's "drug response phenotype", or "drug response genotype”.
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the VEGF-G molecules of the present invention or VEGF-G modulators according to that individual's drag response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drag-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted VEGF-G expression or activity, by administering to the subject a VEGF-G or an agent which modulates VEGF- G expression or at least one VEGF-G activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted VEGF-G expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the VEGF-G aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a VEGF-G, VEGF-G agonist or VEGF-G antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein.
  • the modulatory method of the invention involves contacting a cell with a VEGF-G or agent that modulates one or more of the activities of VEGF-G protein activity associated with the cell.
  • An agent that modulates VEGF-G protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a VEGF-G protein (e.g.
  • the agent stimulates one or more VEGF-G activities.
  • stimulatory agents include active VEGF-G protein and a nucleic acid molecule encoding VEGF-G that has been introduced into the cell.
  • the agent inhibits one or more VEGF-G activities.
  • inhibitory agents include antisense VEGF-G nucleic acid molecules, anti-VEGF-G antibodies, and VEGF-G inhibitors.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a VEGF-G protein or nucleic acid molecule.
  • the method involves administering an agent (e.g. , an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) VEGF-G expression or activity.
  • the method involves administering a VEGF-G protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted VEGF-G expression or activity.
  • Stimulation of VEGF-G activity is desirable in situations in which VEGF-G is abnormally downregulated and/or in which increased VEGF-G activity is likely to have a beneficial effect.
  • stimulation of VEGF-G activity is desirable in situations in which a VEGF-G is downregulated and/or in which increased VEGF-G activity is likely to have a beneficial effect.
  • inhibition of VEGF-G activity is desirable in situations in which VEGF-G is abnormally upregulated and/or in which decreased VEGF-G activity is likely to have a beneficial effect.
  • VEGF-G molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on VEGF-G activity (e.g., VEGF-G gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) VEGF-G- associated disorders (e.g., cell proliferation and/or differentiation disorders, or disorders characterized by aberrant angiogenesis) associated with aberrant or unwanted VEGF-G activity.
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a VEGF-G molecule or VEGF-G modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a VEGF-G molecule or VEGF-G modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drag disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11) :983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymo ⁇ hisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • a genome-wide association relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi-allelic” gene marker map which consists of 60,000-100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants.)
  • gene-related markers e.g., a "bi-allelic” gene marker map which consists of 60,000-100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drag trial to identify markers associated with a particular observed drug response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymo ⁇ hisms (SNPs) in the human genome.
  • SNP single nucleotide polymo ⁇ hisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drug response.
  • a gene that encodes a drag's target e.g., a VEGF-G protein of the present invention
  • all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drag response.
  • the activity of drag metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite mo ⁇ hine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drug e.g., a VEGF-G molecule or VEGF-G modulator of the present invention
  • a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a VEGF-G molecule or VEGF-G modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • the invention is based, at least in part, on the discovery of genes encoding novel members of the VEGF family.
  • the human VEGF-G cDNA was isolated from a human osteoblast library.
  • the nucleotide sequence encoding the human VEGF-G protein (clone jthbb079f06) is shown in Figure 1 and is set forth as SEQ ID NOT.
  • the full length protein encoded by this nucleic acid comprises about 370 amino acids and has the amino acid sequence shown in Figure 1 and set forth as SEQ ID NO:2.
  • the coding region (open reading frame) of SEQ ID NOT is set forth as SEQ ID NO:3.
  • the nucleotide sequence encoding the mouse VEGF-G protein is shown in
  • FIG. 8 is set forth as SEQ ID NO: 10.
  • the full length protein encoded by this nucleic acid comprises about 370 amino acids and has the amino acid sequence shown in Figure 8 and set forth as SEQ ID NO 1.
  • the coding region (open reading frame) of SEQ ID NO: 10 is set forth as SEQ ID NO: 12.
  • a search was performed against the HMM database resulting in the identification of a CUB domain in the amino acid sequence of human VEGF-G (SEQ ID NO:2) at about residues 53-167 of SEQ ID NO:2.
  • the results of the search are set forth in Figure 2.
  • a CUB domain was also identified in the amino acid sequence of mouse VEGF-G (SEQ ID NOT 1) at about residues 53-167 of SEQ ID NO L
  • the results of the search are set forth in Figure 9.
  • the human VEGF-G protein is predicted to have a signal peptide from amino acid residues 1-19 of SEQ ID NO:2. Accordingly, a mature human VEGF-G protein is predicted to include amino acid residues 20-370 of SEQ ID NO:2.
  • the mouse VEGF-G protein is predicted to have a signal peptide from amino acid residues 1-23 of SEQ ID NO 1. Accordingly, a mature mouse VEGF-G protein is predicted to include amino acid residues 24-370 of SEQ ID NO: 11.
  • the VEGF-G protein is also predicted to have at least one N-glycosylation site, at about amino acid residues 276-279 of SEQ ID NO:2, and at about amino acid residues 14-17 and 276-279 of SEQ ID NO: 11.
  • the VEGF-G protein is predicted to have at least one N-myristoylation site, at about amino acid residues 100-105, 192-197, and 303-308 of SEQ ID NO:2, and at about amino acid residues 100-105 and 303-308 of SEQ ID NO L
  • the VEGF-G protein is predicted to have at least one cAMP and cGMP dependent protein kinase phosphorylation site, at about amino acid residues 268-271 of SEQ ID NO:2, and at about amino acid residues 268-271 of SEQ ID NO: 11.
  • the VEGF-G protein is predicted to have at least one protein kinase C phosphorylation site, at about amino acid residues 17-19, 29-31, 66-68, 80-82, 150-152, 243-245, 273-275, 320-322, 323-325, and 365-367 of SEQ ID NO:2, and at about amino acid residues 29-31, 66-68, 141-143, 150-152, 273-275, 320-322, 323-325, and 365-367 of SEQ ID NO L
  • the VEGF-G protein is predicted to have at least one casein kinase II phosphorylation site, at about amino acid residues 17-20, 168-171, 181-184, 199-202, 219-222, 231-234, 250-253, and 256-259 of SEQ ID NO:2, and at about amino acid residues 168-171, 181-184, 199-202, 213-216, 219-222, 231-234, 250-253, and 256-259 of SEQ ID NO L
  • the VEGF-G protein is predicted to have at least one tyrosine kinase phosphorylation site, at about amino acid residues 262-270 of SEQ ID NO:2, and at about amino acid residues 262-270 of SEQ ID NO: 11.
  • the open reading frame of the human VEGF-G gene was globally aligned with the open reading frame of the mouse VEGF-G gene using the GAP program in the GCG software package, using a nwsgapdna.cmp matrix and a gap weight of 12 and a length weight of 4. The results showed a 85.586% identity between the two sequences (see Figure 10).
  • the nucleotide sequence of the human VEGF-G gene was locally aligned with the nucleotide sequence of the mouse VEGF-G gene using the GAP program in the GCG software package, using a nwsgapdna.cmp matrix and a gap weight of 12 and a length weight of 4.
  • the results showed a 74.592% identity between the two sequences.
  • the human VEGF-G protein was globally aligned with the mouse VEGF-G protein using the GAP program in the GCG software package, using a Blosum 62 matrix and a gap weight of 12 and a length weight of 4. The results showed a 84.865% identity between the two sequences (see Figure 11).
  • VEGF-G mRNA was strongly expressed in the ovary, heart, pancreas, and fetal kidney, moderately expressed in the prostate, testis, small intestine, spinal cord, trachea, placenta and kidney, and weakly expressed in the colon, brain, skeletal muscle, and liver.
  • VEGF-G mRNA was strongly expressed in the adrenal medulla and the adrenal cortex.
  • VEGF-G mRNA was weakly expressed in various immune system tissues such as spleen, lymph node, thymus, peripheral blood leukocytes, bone marrow, and fetal liver.
  • VEGF-G mRNA was also expressed in the thyroid and stomach, but was not expressed in adult or fetal lung, or fetal brain.
  • Northern blot analysis of mouse tissues revealed strong expression of VEGF-G mRNA in the heart and kidney, moderate expression in the brain, and no expression in the spleen, lung, liver, skeletal muscle and testis.
  • VEGF-G expression was also determined by PCR analysis of cDNA libraries from various tissues and cell lines. Detecting expression by a library array procedure entailed preparing a PCR mixture including Taq Polymerase, dNTPs, and PCR buffer, and adding a vector primer, a primer internal to the gene of interest, and an aliquot of a library in which expression was to be tested. This procedure was performed with many libraries at a time in a 96 well PCR tray, with 80 or more wells containing libraries and a control well in which the above primers were combined with the clone of interest itself. The control well served as an indicator of the fragment size to be expected in the library wells, in the event the clone of interest was expressed within.
  • VEGF-G was expressed in choroid plexus (MCPdL) and lung Bleomycin model day 7, and weakly expressed in long term bone marrow cells, MLTC-1 (mouse Ley dig tumor cells), lung, brain, and lung Gonzolo day 15, 3 hours.
  • VEGF-G The expression of VEGF-G in human endothelial cells was analyzed by TaqMan® Quantitative Polymerase Chain Reaction.
  • Probes were designed by PrimerExpress software (PE Biosystems) based on the sequence of the VEGF-G gene. Each VEGF-G gene probe was labeled using FAM (6- carboxyfluorescein), and the ⁇ 2-microglobulin reference probe was labeled with a different fluorescent dye, VIC. The differential labeling of the target gene and internal reference gene thus enabled measurement in same well. Forward and reverse primers and the probes for both ⁇ 2-microglobulin and target gene were added to the TaqMan ® Universal PCR Master Mix (PE Applied Biosystems). Although the final concentration of primer and probe could vary, each was internally consistent within a given experiment.
  • a typical experiment contained 200nM of forward and reverse primers plus lOOnM probe for ⁇ -2 microglobulin and 600 nM forward and reverse primers plus 200 nM probe for the target gene.
  • TaqMan matrix experiments were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). The thermal cycler conditions were as follows: hold for 2 minute at 50°C and 10 minute at 95°C, followed by two-step PCR for 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute.
  • a comparative Ct method is used for the relative quantitation of gene expression.
  • the following method was used to quantitatively calculate VEGF-G gene expression in the various samples relative to ⁇ -2 microglobulin expression in the same sample.
  • the threshold cycle (Ct) value is defined as the cycle at which a statistically significant increase in fluorescence is detected. A lower Ct value is indicative of a higher mRNA concentration.
  • the Ct value of the VEGF-G gene is normalized by subtracting the Ct value of the ⁇ -2 microglobulin gene to obtain a ⁇ Ct value using the following formula:
  • ⁇ Ct Ct VEGF .
  • Expression is then calibrated against a cDNA sample showing a comparatively low level of expression of the VEGF-G gene.
  • the ⁇ Ct value for the calibrator sample is then subtracted from ⁇ Ct for each tissue sample according to the following formula:
  • HVS Human umbilical vein endothelial cells
  • HUVEC monolayers were exposed to laminar sheer stress by culturing the cells in a specialized apparatus containing liquid culture medium. Static cultures grown in the same medium served as controls. The in vitro LSS treatment at 10 dyns/cm 2 was designed to simulate the shear stress generated by blood flow in a straight, healthy artery such as the internal mammary artery. Alternatively, HUVEC cultures were treated with human IL-l ⁇ , a factor known to be involved in the inflammatory response, in order to mimic the physiologic conditions involved in the atherosclerotic state. Stimulation of endothelial cells with IL-1 induces the expression of several inflammatory markers. Experimental and control cells were harvested and analyzed for gene expression at 1, 6 and 24 hours.
  • Exposure of HUVECs to LSS resulted in a reduction in VEGF-G gene expression at 1 and 6 hours, and an induction in VEGF-G levels following 24 hours of LSS treatment, as compared to static controls.
  • IL-1 stimulation of HUVECs resulted in a time dependent decrease in VEGF-G expression.
  • HMVEC human microvascular endothelial cells
  • VEGF-G may be involved in the regulation of endothelial cell processes such as growth, proliferation, differentiation, migration and tube formation.
  • This example describes a secretion assay that was perfonned to determine if VEGF-G is secreted from cells. Briefly, the secretion assay was performed as follows: 8x10 5 293T cells were plated per well in a 6-well plate and the cells were incubated in growth medium (DMEM, 10% fetal bovine serum, penicillin/strepomycin) at 37°C, 5% CO 2 overnight. 293T cells were transfected with 2 ⁇ g per well of full-length VEGF-G in the pMET7 vector and 10 ⁇ g per well of LipofectAMINE (GIBCO/BRL Cat. # 18324- 012) according to the manufacturer's instructions.
  • DMEM 10% fetal bovine serum, penicillin/strepomycin
  • the transfection media was removed and fresh growth medium was added to allow the cells to recover overnight.
  • the medium was removed and each well was gently washed twice with DMEM without methionine and cysteine (ICN Cat. # 16-424-54).
  • 1 ml DMEM without methionine and cysteine supplemented with 50 ⁇ Ci Trans- 35 S-Label 35 S-L-methionine/ 35 S-L-cysteine; ICN Cat. # 51006 was added to each well and the cells were incubated at 37°C, 5% CO 2 .
  • a 150 ⁇ l aliquot of conditioned medium was obtained and 150 ⁇ l of 2X SDS sample buffer was added to the aliquot.
  • VEGF-G is a secreted protein.
  • EXAMPLE 4 VEGF-G BINDING TO ENDOTHELIAL CELLS
  • VEGF-G alkaline phosphatase- VEGF-G fusion protein
  • bACE cells bovine adrenal cortical capillary endothelial cells
  • BAE bovine aortic endothelial cells
  • Phospha-Light chemilluminescent assay system Tropix, Inc. Bedford, MA
  • bACE cells were plated into gelatinized 96-well plates (3000 cells/well) and allowed to grow to confluence. The cells were then fixed with acetone.
  • AP-VEGF- G was incubated with the cells for 1 hour, and specific binding was detected with a microplate luminometer according to the manufacturer's instructions.
  • VEGF-G binds to endothelial cells with high affinity, and may thus selectively exert its effects on endothelial cells.
  • EXAMPLE 5 EXPRESSION OF RECOMBINANT VEGF-G PROTEIN IN BACTERIAL CELLS
  • VEGF-G is expressed as a recombinant glutathione-S- transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized.
  • GST glutathione-S- transferase
  • VEGF-G is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
  • Expression of the GST- VEGF-G fusion protein in PEB 199 is induced with IPTG.
  • the recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB 199 strain by affinity chromatography on glutathione beads.
  • the pcDNA/Amp vector by Invitrogen Co ⁇ oration (San Diego, CA) is used.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire VEGF-G protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
  • the VEGF-G DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the VEGF-G coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the VEGF-G coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the VEGF-G gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HB101, DH5 ⁇ , SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
  • COS cells are subsequently transfected with the VEGF-G-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • VEGF-G polypeptide The expression of the VEGF-G polypeptide is detected by radiolabelling ( 35 S-methionine or 35 S- cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells are labelled for 8 hours with 35 S-methionine (or 35 S- cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCI, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5).
  • detergents RIPA buffer, 150 mM NaCI, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5).
  • Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-P AGE.
  • DNA containing the VEGF-G coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the VEGF-G polypeptide is detected by radiolabelling and immunoprecipitation using a VEGF-G specific monoclonal antibody.

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Abstract

La présente invention concerne des molécules d'acide nucléique isolées, désignées molécules d'acide nucléique VEGF-G, codant de nouveaux membres de la famille des facteurs de croissance des cellules endothéliales vasculaires (VEGF). Cette invention concerne également des molécules d'acide nucléique anti-sens, des vecteurs d'expression de recombinaison contenant des molécules d'acide nucléique VEGF-G, des cellules hôtes dans lesquelles les vecteurs d'expression ont été introduits, ainsi que des animaux transgéniques non humains dans lesquels un gène VEGF-G a été introduit ou introduit par disruption. De plus, cette invention concerne des protéines VEGF-G isolées, des protéines de fusion, des peptides antigéniques et des anticorps anti-VEGF-G. Cette invention concerne également des méthodes diagnostiques utilisant les compositions de cette invention.
PCT/US2000/018085 1999-06-30 2000-06-29 Nouveau membre de la famille des facteurs de croissance des cellules endotheliales vasculaires et son utilisation WO2001000878A2 (fr)

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AU59036/00A AU5903600A (en) 1999-06-30 2000-06-29 A novel vascular endothelial growth factor family member and uses thereof

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US09/343,671 1999-06-30

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US6630142B2 (en) 1999-05-03 2003-10-07 Zymogenetics, Inc. Method of treating fibroproliferative disorders
US6468543B1 (en) 1999-05-03 2002-10-22 Zymogenetics, Inc. Methods for promoting growth of bone using ZVEGF4
CA2386383A1 (fr) * 1999-10-07 2001-04-12 Curagen Corporation Polypeptides agissant comme facteur de croissance et acides nucleiques codant pour ces derniers
WO2001089450A2 (fr) * 2000-05-19 2001-11-29 Eli Lilly And Company Traitement de pathologies musculosquelettiques par le polypeptide lp85 et des analogues de celui-ci

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