AU2005200574A1 - Human Vascular Endothelial Factor 2 - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Actual Inventors Address for service is: Human Genome Sciences, Inc.

Craig A ROSEN, Liang CAO and Jing-Shan HU WRAY ASSOCIATES Level 4, The Quadrant 1 William Street Perth, WA 6000 Attorney code: WR Invention Title: "Human Vascular Endothelial Growth Factor 2" The following statement is a full description of this invention, including the best method of performing it known to me:- Human Vascular Endothelial Growth Factor 2 This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been. 'idntified as a member of the vascular endothelial growth factor family. More particularly, the polypeptide of the present invention is vascular endothelial growth factor 2, sometimes hereinafter referred to as "VEGF2." The invention also relates to inhibiting the action of such polypeptide.

The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth, and tissue repair. Angiogenesis, however, is an essential part of certain pathological conditions such as neoplasia, for example, tumors and gliomas, and abnormal angiogenesis is associated with other diseases such as inflammation, -1/2rheumatoid arthritis, psoriasis, and diabetic retinopathy S(Folkman, J. and Klagsbrun, Science 235:442-447, (1987)).

Both acidic and basic fibroblast growth factor molecules are mitogens for endothelial cells and other cell types.

Angiotropin and angiogenin can induce angiogenesis, although their functions are unclear (Folkman, 1993, Cancer Medicine pp. 153-170, Lea and Febiger Press). A highly selective mitogen for vascular endothelial cells is vascular endothelial growth factor or VEGF (Ferrara, et al., Endocr. Rev. 13:19-32, (1992)), also known as vascular permeability factor (VPF). Vascular endothelial growth factor is a secreted angiogenic mitogen whose target cell 1 specificity appears to be restricted to vascular endothelial cells.

The murine VEGP gene has been characterized and its expression pattern in embryogenesis has been analyzed.

A

persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, in choroid plexus and kidney glomeruli. The data was consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation (Breier, G. et al.

Development, 114:521-532 (1992)).

VEGF is structurally related to the a and P chains of platelet-derived growth factor (PDGF), a ritogen for mesenchymal cells and placenta growth factor (PLGP) an endothelial cell mitogen. These three proteins belong to the same family and share a conserved motif. Eight cysteine residues contributing to disulfide-bond formation are strictly conserved in these proteins. Alternatively spliced mRNAs have been identified for both VEGF, PLGF and PDGF and these different splicing products differ in biological activity and in receptor-binding specificity. VEGF and PDGF function as homo-dimers or hetero-dimers and bind to receptors which elicit intrinsic tyrosine kinase activity following receptor dimerization.

-2- VEGF has four different forms of 121, 165, 189 and 206 amino acids due to alternative splicing. VEGF121 and VEGP165 S are soluble and are capable of promoting angiogenesis, whereas VEGF189 and VEGF206 are bound to heparin containing proteoglycans in the cell surface. The temporal and spatial expression of VEGF has been correlated with physiological S proliferation of the blood vessels (Gajdusek, and S Carbon, Cell Physiol., 139:570-579, (1989)); McNeil, Muthukrishnan, Warder, D'Amore, J. Cell.

Biol., 109:811-822, (1989)). Its high affinity binding sites are localized only on endothelial cells in tissue sections (Jakeman, et al., Clin. Invest. 89:244-253, (1989)).

The factor can be isolated from pituitary cells and several tumor cell lines, and has been implicated in some human gliomas (Plate, K.H. Nature 359:845-848, (1992)).

Interestingly, expression of VEGF121 or VEGF165 confers on Chinese hamster ovary cells the ability to form tumors in nude mice (Perrara, et al., J. Clin. Invest. 91:160-170, (1993)). The inhibition of VEGF function by anti-VEGF monoclonal antibodies was shown to inhibit tumor growth in immnune-deficient mice (Kim, Nature 362:841-844, (1993)). Further, a dominant-negative mutant of the VEGF receptor has been shown to inhibit growth of glioblastomas in mice. Vascular permeability factor, has also been found to be responsible for persistent microvascular hyperpermeability to plasma proteins even after the cessation of injury, which is a characteristic feature of normal wound healing. This suggests that VPF is an important factor in wound healing.

Brown, L.P. et al., J. Exp. Med., 176:1375-9 (1992).

The expression of VEGF is high in vascularized tissues, lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. VEGF has also been shown to induce angiogenesis In vivo. Since angiogenesis is essential for the repair of normal tissues, -3especially vascular tissues, VEGF has been proposed for use in promoting vascular tissue repair in atherosclerosis).

U.S. Patent No. 5,073,492, issued December 17, 1991 to Chen et al., discloses a method for synergistically enhancing endothelial cell growth in an appropriate environment which comprises adding to the environment, VEGF, effectors and serum-derived factor. Also, vascular endothelial cell growth factor C sub-unit DNA has been prepared by polymerase chain reaction techniques. The DNA encodes a protein that may exist as either a hetero-dimer or homo-dimer. The protein is a mammalian vascular endothelial cell mitogen and, as such, is useful for the promotion of vascular development and repair, as disclosed in European Patent Application No.

92302750.2, published September 30, 1992.

The polypeptides of the present invention have been putatively identified as a novel vascular endothelial growth factor based on amino acid sequence homology to human

VEGF.

In accordance with one aspect of the present invention, there are provided novel mature polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with still another aspect of the present invention, there are provided processes for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention, under conditions promoting expression of said proteins and subsequent recovery of said proteins.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, to stimulate angiogenesis, wound-healing, and to promote vascular tissue repair.

In accordance with yet another aspect of the present invention, there are provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, to inhibit the growth of tumors, to treat diabetic retinopathy, inflammation, rheumatoid arthritis and psoriasis.

In accordance with another aspect of the present .invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to nucleic acid sequences of the present invention.

In accordance with another aspect of the present invention, there are provided methods of diagnosing diseases or a susceptibility to diseases related to mutations in nucleic acid sequences of the present invention and proteins encoded by such nucleic acid sequences.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

SThe following drawings are illustrative of embodiments S of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

e Fig. I shows the cDNA sequence and the corresponding S deduced amino acid sequence of the polypeptide of the present invention. The standard one letter abbreviations for amino acids are used. Sequencing was performed using 373 Automated SDNA Sequencer (Applied Biosystems, Inc.). Sequencing S accuracy is predicted to be greater than 97%.

D Fig. 2 is an illustration of the amino acid sequence homology between the polypeptide of the present invention and S other members of the human PDGF/VEGF family. The boxed areas S indicate the conserved sequences and the location of the eight conserved cysteine residues.

Fig. 3 shows a photograph of a gel after in vitro transcription, translation and electrophoresis of the polypeptide of the present invention. Lane 1: "4C and rainbow M.W. marker; Lane 2: FGF control; Lane 3: VBGF2 produced by M13-reverse and forward primers; Lane 4: VEGF2 produced by M13 reverse and VEGF-F4 primers; Lane 5: VEGF2 produced by M13 reverse and VEGF-F5 primers.

Pig. 4. VEGF2 polypeptide is expressed in a baculovirus system consisting of Sf9 cells. Protein from the medium and cytoplasm of cells were analyzed by SDS-PAGE under reducing and non-reducing conditions.

I Fig. 5. The medium from Sf9 cells infected with a nucleic acid sequence of the present invention was precipitated and the resuspended precipitate was analyzed by SDS-PAGE and was stained with coomassie brilliant blue.

Fig. 6. VEGF2 was purified from the medium supernatant and analyzed by SDS-PAGE in the presence or absence of the reducing agent 6-mercaptoethanol and stained by coomassie brilliant blue.

Fig. 7. Reverse phase HPLC analysis of purified VEGF2 using a RP-300 column (0.21 x 3 cm, Applied Biosystems, -6- SInc.). The column was equilibrated with 0.1% trifluoroacetic acid (Solvent A) and the proteins eluted with a 7.5 min gradient from 0 to 60% Solvent B, composed of acetonitrile containing 0.07% TFA. The protein elution was monitored by absorbance at 215 nm (Red t line) and 280 nm (Blue line). The percentage of Solvent B is shown by Green line.

SFig. 8 illustrates the effect of partially-purified VEGF2 protein on the growth of vascular endothelial cells in comparison to basic flbroblast growth factor.

Fig. 9 illustrates the effect of purified VEGF2 protein on the growth of vascular endothelial cells.

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

In accordance with one aspect of the present invention, there are provided isolated nucleic acid molecules (polynucleotides) which encode for the mature polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 97149 deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA, 20110-2209, United States of America, on 12 May, 1995 or for polypeptides which have fewer amino acid residues than those showing in Figure 1 (SEQ ID NO:2).

A polynucleotide encoding a polypeptide of the present invention may be obtained from early stage human embryo (week 8 to 9) osteoclastomas, adult heart or several breast cancer cell lines. The polynucleotide of this invention was discovered in a cDNA library derived from early stage human embryo week 9. It is structurally related to the VEGF/PDGF family. VEGP2 contains an open reading frame encoding a protein of 419 amino acid residues of which approximately the first 23 amino acid residues are the putative leader sequence such that the mature protein comprises 396 amino acids, and which protein exhibits the highest amino acid sequence hOTMOlOgY to human vaScular ezdorthelilal growth factor (3 o, identity) followed by PDGPt (23t) and PDGPR (22t).- It is particularly important that all eight cysteines are conserved within all four mem ers Of the family (Bee bOzxed areas of Figure 2) 1= Oddition. r-he signature for the PDGW/VRGP family, PXCVx=ckCjCOcCN (SEQ ID NO!6)± 5 s COn.serz.ed in VEC72 (see FiuRe 2).

The VEGP2 Polypeptide of the present invention is meant to include the full length polypeptide and polynucleotide sequence which encoder. for any leader sequences and for active fragments of the full length POlypeptide. Active fragments are meant to include any portions of the full.

length a-ino acid sequence which have less than the full 419 amino acids of the full length amino acid sequence as shown in SEQ ID N1o. 2 and Figure 2, but still. contain the eight cysteinae residues shown conserved in Figure 2 and such fragments still contain VRGF2 activity.

There are at least two altermatively spliced VEGF2 inRNA *sequences present in normal tissues. The size of the two VEGF2 mRA sequences which correspond to thefulentad truncated version respectively are shown in Figure lane shows two bands indicating the presence of the alternatively Bpliced m.R2M encoding the VRGF2 polypeptide of the present :nventi on.

hepolynucleotjde of the present invention may be in **the form Of RIC Or in the form of -DNM. which DNA includes cDNA, genomic MMA, and gynthetic DMA. The MlA may be doublestranded or single -stranded, and if single stranded may be the coding stra-d or no-coding (3.ti-senee strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown iLn Figure I or that of p the deposited clo=, or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 or the deposited cDNak.

The polynicleotide which encodes for the mature polypeptide of Figure 1 (SpQ ID NOs;2) or for the mature polypept -ide encoded by the depositcd cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature Polypeptide (and optonally additional coding sequence) and nion-codiug sequence, such as inf-ons or noncoding sequence 5' and/or 3' of the coding sequence for the mature polypepride.

Thus, the term Hpolynucleoide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the herein above described polynucleaides which encode for fragments, analogs and derivatives of the polypeptide having the deduced arnino acid sequence of lPigure 1 (SEQ ID NO: 2) or the polypeptide encoded by the rDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-nanirauly occurring variant of the polynucleotide.

1s Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO: 2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fraginent, derivative or analog of the polypeptide of Figure 1 (SEQ MD NO :2) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variantsinclude deletion variants, substitution variants and addition or insertion variants.

As lierein above indicated. the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID NO: 1) or of the coding sequence of the deposited clone. As known in the arn, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially D alter the function of the encoded polypeptide SThe polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence S which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexahistidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to S an epitope derived from the influenza hemagglutinin protein (Wilson, et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar -biological activity. Probes of this type preferably have at least 30 bases and:may-contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either 1 retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:1) or the deposited cDNA(s).

Alternatively, the polynucleotide may have at least bases, preferably 30 bases, and more preferably at least bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer. Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of -11- -12- Patent Procedure.

The present invention further relates to a polypeptide which have the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.

The terms "fragment", "derivative" and "analog" when referring to the polypeptide of Figure 1 (SEQ ID NO: 2) or that encoded by the deposited cDNA, means a polypeptide which retains the conserved motif of VEGF proteins as shown in Figure 2 and essentially the same biological function or activity.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.

The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the deposited cDNA may be one in which one or more of the amino acid residues arc substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues include a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide or one in which comprises fewer amino acid residues shown in SEQ ID No. 2 and retains the conserved motif and yet still retains activity characteristic of the SVEGF family of polypeptides. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

SThe term "isolated" means that the material is removed from its original environment the natural environment if it is naturally occurring). For example, a naturallyoccurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least similarity (preferably at "J east 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least similarity (more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least similarity (still more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determined by comparing the amino acid -13sequence and its conserved amino acid Substitutes of one polypeptide to the sequence of a second polypeptide.

jFragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

IThe present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the VEGF2 genes of the present invention. The culture conditions, such as temperature, pH and the like, are those Previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and -14phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox S virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

D The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or promoter, the E. coli. ac or the phage lambda p, promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.

The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.

The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella tvphimurium; fungal cells, such as yeast; insect cells such as Drosohila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of a appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this O embodiment the a preferred aspect of this e the construct further comprises regulatory N sequences, including, for example, a promoter, operably linked to the sequence. Large nmbers of suitable vectors Sand promoters are known to those of skill in the art, and are commercially available. The following vectors are provided pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNHSA, pNHl6a, pNHLSA, pNH46A (Stratagene); ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the pKK232-8 and pC47. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PL and trp. Eukaryotic promoters include C4V immediate early,

HSV

thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a -16mammalian cell, or a lower eukaryotic cell, such as a yeast 1cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host ;cell can be effected by calcium phosphate transfection,

DEAE-

Dextran mediated transfection, or electroporation. (Davis, Dibner, Battey, Basic Methods in Molecular Biology, (1986)).

:The constructs in host cells can be used in a conventional manner to produce the sene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

iMature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.

Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription.

Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, the ampicillin resistance gene of Z li and S. cerevisiae TRPI gene, and -17a promoter derived from a highly-expresse d gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding 9lycolytic enzymes such as 3 -phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide aplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtili, Salmonlja 7vphimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from conmercially available plasmids comprising. genetic elements of the well known cloning vector pBR322

(ATCC

37017). Such conmmercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA). These pBR3.22 "backbone" -18sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mannalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CEO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and -19lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chrofmatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.

Polypeptides of the invention may also include an initial methionine amino acid residue.

As shown in Figures 8 and 9, the VEGF2 polypeptide of SEQ ID No. 2, minus-the initial 46 amino acids, is a potent mitogen for vascular endothelial cells and stimulates their growth and proliferation. The results of a Northern blot analysis performed for the VEGF2 nucleic acid sequence encoding this polypeptide wherein 20 ug of RNA from several human tissues were probed with 3 2 P-VEGF2, illustrates that this protein is actively expressed in the heart and lung which is further evidence of mitogenic activity.

Accordingly, VEGF2 iA Y" be employed -to promote angiogenesis, for example, to stimulate the growth of transplanted tissue where coronary bypass surgery is performed. VEGF2 may also be employed to promote wound healing, particularly to re-vascularize damaged tissues or stimulate collateral blood flow during ischemia and where new capillary angiogenesis is desired. VEGF2 may be employed to treat full-thickness wounds such as dermal ulcers, including pressure sores, venous ulcers, and diabetic ulcers. In addition, VEGF2 may be employed to treat full-thickness burns and injuries where a skin graft or flap is used to repair such burns and injuries. VEGF2 may also be employed for use in plastic surgery, for example, for the repair of lacerations from trauma and cuts in association with surgery.

Along these same lines, VEGF2 may be employed to induce the growth of damaged bone, periodontium or ligament tissue.

VEGF2 may also be employed for regenerating supporting tissues of the teeth, including cementum and periodontal ligament, that have been damaged by disease and trauma.

Since angiogenesis is important in keeping wounds clean and non-infected, VEGF2 may be employed in association with surgery and following the repair of cuts. It may also be employed for the treatment of abdominal wounds where there is a high risk of infection.

VEGF2 may be employed for the promotion of endothelialization in vascular graft surgery. In the case of vascular grafts using either transplanted or synthetic material, VEGF2 can be applied to the surface of the graft or at the junction to promote the growth of vascular endothelial cells. VEGF2 may also be employed to repair damage of myocardial tissue as a result of. myocardial infarction.

VEGF2 may also be employed to repair the cardiac vascular system after ischemia. VEGF2 may also be employed to treat damaged vascular tissue as a result of coronary artery disease and peripheral and CNS vascular disease.

VEGF2 may also be employed to coat artificiaT prostheses or natural organs which are to be transplanted in the body to minimize rejection of the transplanted material and to stimulate vascularization of the transplanted materials.

VEGF2 may also be employed for vascular tissue repair, for example, that occurring during arteriosclerosis and required following balloon angioplasty where vascular tissues are damaged.

VEGF2 nucleic acid sequences and VEGF2 polypeptides may also be employed for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors, and for the production of diagnostics and therapeutics to -21treat human disease. For example, VEGF2 may be employed for in vitro culturing of vascular endothelial cells where it is ,added to the conditional medium in a concentration from I0 pg/iml to 10 ng/nl.

Fragments of the full length VEGF2 gene may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the gene or :similar biological activity. Probes of this type generally have at least 50 base pairs, although they may have a greater number of bases. The probe may also be used to identify a N clone corresponding to a full length transcript and a genomic clone or clones that contain the complete VEGF2 gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the VEGF2 gene by using the known

DNA

sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

This invention provides methods for identification of VEGF2 receptors The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., Chapter (1991)). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to VEGF2, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to VEGF2. Transfected cells which are grown on glass slides are exposed to labeled VEGF2.

VEGF2 can be labeled by a variety of means including iodination or inclusion of a recognition site for a sitespecific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis.

-22- Positive pools are identified and sub-pools are prepared and retransfected using an iterative sub-pooling and rescreening process, eventually yielding a single clone that encodes the putative receptor.

As an alternative approach for receptor identi'fication labeled VEGF2 can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule Cross-linked material is resolved by PAGE and exposed to xray film. The labeled complex containing VEGF2 is then excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor. This invention is also related to a method of screening compounds to identify those which are VEGF2 agonists or antagonists. An example of such a method takes advantage of the ability of VEGF2 to significantly stimulate the proliferation of human endothelial cells in the presence of the comnitogen Con A. Endothelial cells are obtained and cultured in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) in a reaction mixture supplemented with Con-A (Calbiochem, La Jolla, CA). Con-A, polypeptides of the present invention and the compound to be screened-are added.

After incubation at 37 0 C, cultures are pulsed with 1 iCi of 3 thymidine (5 Ci/mmol; 1 Ci 37 BGq; NEN) for a sufficient time to incorporate the 3 and harvested onto glass fiber filters (Cambridge Technology, Watertown, MA). Mean thymidine incorporation (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA) Significant 3 [H thymidine incorporation, as compared to a control assay where the compound is excluded, indicates stimulation of endothelial cell proliferation.

-23- To assay for antagonists, the assay described above is performed and the ability of the compound to inhibit [H thymidjne incorporation in the presence of VEGF2 indicates that the compound is an antagonist to VEGF2. Alternatively, VEGF2 antagonists may be detected by combining VEGF2 and a potential antagonist with membrane-bound VEGF2 receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. VEGF2 can be labeled, such as by radioactivity, such that the number of VEGF2 molecules bound to the receptor can determine the effectiveness of the potential antagonist.

Alternatively, the response of a known second messenger system following interaction of VEGF2 and receptor would be measured and compared in the presence or absende of the compound. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis. In another method, a mammalian cell or membrane preparation expressing the VEGF2 receptor is incubated with labeled VEGF2 in the presence of the compound.

The ability of the compound to enhance or block this interaction could then be measured.

Potential VEGF2 antagonists include an antibody, or in some cases, an oligonucleotide, which bind to the polypeptide and effectively eliminate VEGF2 function. Alternatively, a potential antagonist may be a closely related protein which binds to VEGF2 receptors, however, they are inactive forms of the polypeptide and thereby prevent the action of VEGF2.

Examples of these antagonists include a negative dominant mutant of the VEGF2 polypeptide, for example, one chain of the hetero-dimeric form of VEGF2 may be dominant and may be mutated such that biological activity is not retained. An example of a negative dominant mutant includes truncated versions of a dimeric VEGF2 which is capable of interacting with another dimer to form wild type VEGF2, however, the -24resulting homo-dimer is inactive and fails to exhibit characteristic VEGF activity.

Another potential VEGF2 antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 136.0 (1991)), thereby preventing transcription and the production of VEGF2. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the VEGF2 polypeptide (Antisense Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be Expressed in vivo to inhibit production of VEGF2.

Potential VEGF2 antagonists also include small molecules which bind to and occupy the active site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.

The antagonists may be employed to treat limit angiogenesis necessary for solid tumor metastasis.

The mRNA encoding for VEGF2 is found to be expressed at moderate levels in at least two breast tumor cell lines which is indicative of the role of VEGF2 polypeptides in the malignant phenotype. Gliomas are also a type of neoplasia which may be treated with the antagonists of the present invention.

The antagonists may also be used to treat chronic inflanation caused by increased vascular permeability. In addition to these disorders, the antagonists may also be employed to treat retinopathy associated with diabetes, rheumatoid arthritis and psoriasis.

The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, as hereinafter described.

The VEGF2 polypeptides and agonists and antagonists may be employed in combination with a suitable phatmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide or agonist or antagonist, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water,. glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a Pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composit-ins of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, intratum r, subcutaneous, intranasal or intradermal routes. The pharmaceutical -26compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication.

In general, the pharmaceutical compositions are administered in an amount of at least about 10 yg/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 yg/kg to about I mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

The VEGF2 polypeptides, and agonists or antagonists which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptide in vivo, which is often referred to as "gene therapy." Thus, for example, cells such as bone marrow cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the engineered cells are then provided to a patient to be treated with the polypeptide.

Such methods are well-known in the art. For example, cells may be. engineered by procedures known in the art by use of a retroviral particle containing RNA encoding the polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo, for example, by procedures known in the art.' As known in the art7 a producer cell for producing a retroviral particle containing

RNA

encoding a polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such methods should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retroviral particle, for example, an adenovirus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

-27- Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus Myeloproliferative Sarcoma Virus, and mammary tumor virus.

In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to., the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (OMV) promoter described in Miller, et al., Biotechniaues, Vol. 7, No. 9, 980-990 (1989), or any other promoter cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and 5-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of-a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAl promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the R-actin promoter; and human growth hormone -28promoters. The promoter also may be the native promoter which controls the gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, PAl2, T19-14X, VT-19-17-H2, #CRE, #CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO, precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the VEGF2 gene as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in VEGF2 nucleic acid sequences.

Individuals carrying mutations in the VEGF2 gene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and -29autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using

PCR

(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.

RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding VEGF2 can be used to identify and analyze VEGF2 mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled VEGF2 RNA or alternatively, radiolabeled VEGF2 antisense

DNA

sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis.

DNA

fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, 'e Myers et al-, Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic

DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detecting altered levels of VEGF2 protein in various tissues since an over-expression of the proteins compared to normal control tissue samples may detect the presence of a disease or susceptibility to a disease, for example, abnormal cellular differentiation. Assays used to detect levels of VEGF2 protein in a sample derived from a host are well-known to those of skill in the art and include radioimnunoassays, competitive-binding assays, Western Blot analysis,

ELISA

assays and "sandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Immunology, Chapter 6, (1991)) initially comprises preparing an antibody specific to the VEGF2 antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme. A saurple is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on *the dish are then covered by incubating with a non-specificm'rotein, such as, bovine serum albumen. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any VEGF2 proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to VEGF2. Unattached reporter antibody is then washed out. Peroxidase substrates are -then added to the dish and the amount of color developed in a given time period is a measurement of the amount of VEGF2 -31protein present in a given volume of patient sairp1e when conpared against a standard curve.

A competition assay may be empl~oyed wherein antibodies specific to VEGF2 are attached to a soli~d support.

Polypeptides of the present invention are then labeled, for example, by radioactivity, and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatogr.aphy, can be correlated to a quantity of VEGF2 in the sample.

A "sandwich" assay is Similar to an ELIS.A assay. In a "sandwich" assay VBGF2 is passed over a solid support: and binds to antibody attached to a solid support. A second antibody is then bound to the VEGF2. A th-ird antibody which is labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with.-a particular location on an individual human chromosome. Morec~er, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat, polymorphism's) are presently available for marking chromosomal location. The* ma~n fD~ t-hoooe according to the- present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosmes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those -32hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.

Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in Si tu hybr idization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can b& used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases.

For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques. Pergainon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian.

Inheritance in Man (available -on line through Johns Hopkins University Welch Medical Library) -The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent gen es).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between af fected and unaf fected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

-33- With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosoal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes I megabase mapping resolution and one gene Per kb).

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an iumuunogen to produce antibodies thereto. These antibodies can be, for exanple, polyclonJl or monoclonal antibodies.

The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptide corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptide into an animal or by adinistering the polypeptide to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide.

Such antibodies can then b: useid to isolate the-polypeptide from tissue expressing that polypeptide For preparation of monoclonal antibodies any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497) the trioma technique, the hunan B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., PP. 77-96).

-34- Techniques described for the production of single chain antibodies Patent 4,946,778) can be adapted to Produce N single chain antibodies to immnunogenic polypeptjide products -0 of this invention. Also, transgenic mice may be used to express htumani.zed antibodies to irmmunogenic polypeptide products of this invention, The present invention will be further described with reference to the following examples; however, it is to be 71- understood that the present invention is not limited to such examples. All1 parts or amounts, unless otherwise specified, are by weight.

fl in order to facilitate understanding of the following examples, certain frequently occurring methods and/or terms N ~will be described.

"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either corznercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids- in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction entymiE that acts only-At certain sequences in the DNA. The various restriction enzymes used herein are commnercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1. jig of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 p1 of buffer solution. For the purpose of isolating

DXA

fragments for plasmjid construction, typically 5 to 50 yg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for Particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 0 C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamde gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.

et al., Nucleic Acids Res., 8:4057 (1980).

*"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two coplementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 pg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described by the method of Graham, F. and Van der Eb,

A.,

Virology, 52:456-457 (1973).

ExaMnple i ression attern of EG2 in human tissues and breast cancer cell lines Northern blot analysis was carried out to examine the levels of expression of the VEGF2 gene in human tissues and human breast cancer cell lines. Total cellular RNA samples were isolated with RNAzolT" system (Biotecx Laboratories, Inc.). About of total RNA isolated from c x L aboratories, t10 p of total RNA isolated from each breast -36- -37tissue and cell line specified was separated on 1% agaxrose gel and blotted onto a nylon filter, (Molecular Cloning, Sambrook Fritsch, and Maniatis, Cold Spring Harbor Press, 1989). The labeling reaction was done according to the Siratagene Cloning Systems, Inc., Prime-it kit with 50 ng DNA fragment. The labeled DNA was purified with a Select-G-50 column from 5 Prime 3 Prime, Inc., Boulder, CO, USA. The filter was then hybridized with radioactively labeled fa~f length VEGF2 gene at 1,000,000 cpm/rnl in 0.5 M NaPO, and 7% SDS overnight at 65*C. After washing twice at room teriperarue and twice at 60*C with x SSC, 0. 1% SDS, the filters were then exposed at -70'C overnight with. an intensifying screen. A message of 1.6 Xb was observed in 2 breast cancer cell lines.

Example 2 Cloning and expression of 'VEGIP2 using the baculovirus expression systern The DNA sequence encoding the VEGF2 protein without 46 amino acids and the Nterminus, see ATCC Accession No. 97149, was amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene: The 5' primer has the sequence TGT AAT ACG ACT CAC TAT AC3G GAT CCC GCC ATG GAG GCC ACO GCT TAT GC (SEQ ID NO:7) and contans a Bainiji restriction enzyme site (in bold) anid 17 nucleotide nucleodide sequence comnplementary to the 5' sequence of VEGF2 (nit. 150-166).

The 3' primer has thc sequence GATC TCT AGA TIA OCT CAT TTG TGC3 TCT (SEQ ID NO:8) and contain the cleavage site for the restriction enzyme XbaI and 18 nucleotixles complementary to the 3' sequence of VEGF2, including the stop codon and IS nt sequence before stop codon.

The amplified sequences were isolated from a I agarose gel using a commercially available kit ("Geneclean," BID 101, Inc., La Jolla, CA). The fragment was then digested with the endonuclease BamHl and Xbal and then purified again on a 1% agarose gel. This fragment was ligated to pAcGP67A baculovirus transfer vector (PHarmingen) at the BamHI and XbaI sites. Through this ligation, VEGF2 cDNA was cloned in frame with the signal sequence of baculovirus gp67 gene and was located at the 3' end of the signal sequence in the vector. This is designated pAcGP67A-VEGF2.

To clone VEGF2 with the signal sequence of gp67 gene to the pRG1 vector for expression, VEGF2 with the signal sequence and some upstream sequence were excised from the pAcGP67A-VEGP2 plasmid at the Xho restriction endonuclease site located upstream of the VEGF2 cDNA and at the XbaI restriction endonuclease site by XhoI and Xbal restriction enzyme. This fragment was separated from the rest of vector on a 1% agarose gel and was purified using "Geneclean" kit.

It was designated F2.

The PRG1 vector (modification of pVL941 vector) is used for the expression of the VEGF2 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa californica nucleaf "polyhedrosis vir-us (AcMNPV) followed by the recognition sites for the restriction endonucleases BamH1, Smal, XbaI, BglII and Asp718. A site for restriction endonuclease Xhol is located upstream of BamEl site. The sequence between Xhol and BamHI is the same as that in PAcGp67A (static on tape) vector. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant virus the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by -38viral sequences for the cell-mediated homologous recombination of cotransfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRG1 such as pAc373, pVL941 and pAcIM 1 (Luckow, V.A. and Summers,

M.D.,

Virology, 170:31-39).

The plasmid was digested with the restriction enzymes XboI and XbaI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1% agarose gel using the commercially available kit ("Geneclean, BIO 101 Inc., La Jolla, Ca.) This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli HB101 cells were then transformed and bacteria identified that contained the plasmid (pBac gp67-VEGF2) with the VEGF2 gene using the enzymes BamH1 and XbaI. The sequence of the cloned fragment was confirmed by DNA sequencing.

tg of the plasmid pBac gp67-VEGF2 was cotransfected with 1.0 pg of a commercially available linearized baculovirus ("BaculoGold' baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al.

Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

lpg of BaculoGold' virus DNA and 5 pg of the plasmid pBac gp67-VEGF2 were mixed in a sterile well of a microtiter plate containing 50 4l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 p1 Lipofectin plus 90 Al Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with i ml Grace's medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27oC. After 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum -39was added. The plate was put back into an incubator and cultivation continued at 27 0 C for four days.

After four days the supernatant was collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation of blue stained plaques. (A detailed description of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc. Gaithersburg, page 9- Four days after the serial dilution, the virus was added to the cells, blue stained plaques were picked with the tip of an Eppendorf pipette. The agar containing the irecombinant viruses was then resuspended in an Eppendorf tube containing 200 pl of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes were harvested and then stored at 4 0

C.

Sf9 cells were grown in Grace's medium supplemented with heat-inactivated FBS. The cells were infected with the recombinant baculovirus V--p67-VEGF2 at a multiplicity of infection (MOI) of 1. Six hours"later the medium-was removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 pCi of 35 S-methionine and 5 gCi "S cysteine (Amersham) were -added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Protein from the medium and cytoplasm of the Sf9 cells was analyzed by SDS-PAGE under reducing and non-reducing conditions. See Figure 4. The medium was dialyzed against mM MES, pH 5.8. Precpitates were obtained after dialysis and resuspended in 100 mM NaCitrate, pH 5.0. The resuspended C- precipitate was analyzed again by SDS-PAGB and was stained Swith Coomassie Brilliant Blue. See Figure [i The medium supernatant was also diluted 1:10 in 50 mM 0 MES, pH 5.8 and applied to an SP-650M column (1.0 x 6.6 cm, Toyopearl) at a flow rate of 1 ml/min. Protein was eluted with step gradients at 200, 300 and 500 mM NaC1. The VEGF2 was obtained using the elution at 500 mM. The eluate was Ianalyzed by SDS-PAGE in the presence or absence of reducing o agent, f-mercaptoethanol and stained by Coommassie Brilliant C- Blue. See Figure 6.

0 0 Example 3 Expression of Recombinant VBGF2 in COS cells The expression of plasmid, VEGF2-HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CWV promoter followed by a polylinker region, an SV40 intron and polyadenylation site. A DNA fragment encoding the entire VEGF2 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described Wilson, H.

Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as followw: The DNA sequence encoding VEGF2, ATCC Accession No. 97149, was constructed by PCR using two primers: the 5' primer (CGC GGA TCC ATG ACT GTA CTC TAC CCA) (SEQ ID NO:9) contains a BamHl site followed by 18 nucleotides of VEGF2 coding sequence starting from the initiation codon; the 3' sequence (CGC TCT -41- AQA TCA ACC GrA GTC TGG GAC GTC GTA 7=G GTA CTC GAG GCr OkT rrc TCC TC? 21) (sro iD Nc2:1o) COntAixi ~mlmezx sequenceS to an Xbal site, RA tag, YbnI site, and the last mucleotideE of the VEGF2 coding sequence (not including the stop codon) Therefore, the PCR product contai=. a BanHI site, coding Bequence followed by an Xho restriction endo nuclease site and HA tag fused in frame, atrnlio trmination stop codon next to the HA tag, and an Abalt sire.

The PCR amplified MM fragment and the vector. pcD/~p ~were di~gested with Ban: and XbaI restic tion enzyme and ligated. The ligation mixture was t-ranfomed into E. coli straim SflRE (Stratagene Cloning Systems, L~a Jolla, CA .92037) the transform d culture was plated =n a~picillim media plates and resistant colonies were selected. Plas-mid DNA was isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the -recombinant VEGP'2, MOS c ells were transfecred with the expression vector by DEAR-DEXRA method Sambrook, E. Fritsch, T. Maiatis, Molecular Cloni:ng:

A

Laboratory Manual, Cold Spring Laboratory Pregs, (1-989) The expression of the VEGP2-BA protein was detected by.

radiolabellimg and i~mnaprecLipction method Barlow,

D.

Lane. Antibodies A Laboratory anu&1, cold Spring Earbor Laboratory, Press, rCells were labelled-for 8 hours with 3'S-cyste-ine two days post transfection. Cultu~re media was then collected and cells were lysed with detergent (RIP2A bu~ffer (150 M XaC:L, it NP-4a, 0.2V SDS, It NP-40, 0.5t DOC, Tris, pH 7.5) (Wilson. 1. et a2., Id. 37:767 (1984)).

Both cell lysate and culture media were precipitated wiLth an HA spe~fic monoclonal antibody. Proteins precipitated were analyzed on 151 SDS-PAGE gels- The ffect of artially-urified GF2 rotein on the- c'rovth of s laedo elacells -42- On day 1, human umbilical vein endothelial cells (HUVEC) were seeded at 2-5x10 4 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (PBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements

(BCGS,

Biotechnique, Inc.). On day 2, the medium was replaced with M199 containing 10% FBS, 8 units/ml heparin. VEGF2 protein of SEQ ID NO. 2 minus the initial 45 amino acid residues, (VEGF) and basic FGF (bFGF) were added, at the concentration shown. On days 4 6, the medium was replaced. On day 8, cell number was determined with a Coulter Counter (See Figure 8).

Example The effect of purified VEGF2 protein on the 'crrowth of vascular endothelial cells On day 1, human umbilical vein endothelial cells (EUVEC) were seeded at 2-5 x 104 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium was replaced with M199 containing 10% FBS, 8 units/ml heparin.

Purified VEGF2 protein of SEQ ID No. 2 minus initial 45 amino acid residues was added to the medium at this point. On days 4 6, the medium was replaced with fresh-medium and supplements. On day 8, cell number was determined with a Coulter Counter (See Figure 9).

Fxample 6 Expression via Gene Therapy Fibroblasts are obtained from a subject by skin biopsy.

The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room -43temperature over night. After 24 hours at room temperature the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media Ha's F12 media, with 10% FBS, penicillin and streptomycin, is added.

This is then incubated at 37C for approximately one week.

At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIIl and subsequently treated with calf intestinal phosphatase The linear vector is fractionated on agarose gel ana purified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer containing an EcoRI site and the 3' primer further includes a HindIII site.

Equal quantities of the Moloney 'rine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two frigmients. The ligation- mixture is used to transform bacteria EBl01, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.

The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum

(CS),

penicillin and streptomycin. The Msv vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

-44- Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipcre filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retrovirai vector that has a selectable marker, such as pe2 or The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrler beads. The fLbroblasts now produce the protein product.

Nu-erous modifications and va-riations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as Particularly described.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims (48)

1. An isolated polynucleotide comprising a member selected from the group consisting of: a) a polynucleotide encoding the full length polypeptide as set forth in SEQ ID NO: 2; b) a polynucleotide encoding the mature protein portion of SEQ ID NO:2; c) a polynucleotide encoding the proprotein portion of SEQ ID NO: 2; d) a polynucleotide encoding the human VEGF-2 polypeptide encoded by the cDNA in ATCC Deposit No. 97149; e) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acid residues -46 to 373 of SEQ ID NO: 2; f) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acid residues -23 to 373 of SEQ ID NO: 2; g) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acids 1 to 373 of SEQ ID NO: 2; h) a polynucleotide comprising the nucleotide sequence encoding a portion of the mature VEGF-2 polypeptide comprising amino acids 24 to 373 of SEQ ID NO: 2; i) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acids -46 to 24 of SEQ ID NO: 2; j) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acids -23 to 24 of SEQ ID NO: 2; k) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acids 1 to 24 of SEQ ID NO: 2; 1) a polynucleotide fragment of the polynucleotide according to any one of a) to d) with the proviso that said polynucleotide fragment comprises at least contiguous nucleotides of the polynucleotide of j) or k); m) a polynucleotide fragment which hybridises to at least 30 contiguous nucleotides of the polynucleotide encoding amino acids -46 to 24 of SEQ ID NO: 2 under the following conditions: hybridisation in 0.5 M sodium peroxide -56- SNaPO, 7% sodium dodecyl sulfate (SDS) at 65 0 C and washing with 0.5 x SSC, 0.1% SDS at 60 0 C or equivalent hybridisation stringency; n) a polynucleotide comprising the complementary form of the polynucleotide according to any one of a) to m).

2. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes Sthe full length polypeptide as set forth in SEQ ID NO: 2. 0

3. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes the mature protein portion of SEQ ID NO:2.

4. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes the proprotein portion of SEQ ID NO: 2. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes the human VEGF-2 polypeptide encoded.by the cDNA in ATCC Deposit No. 97149.

6. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a polypeptide comprising amino acids -46 to 373 of SEQ ID NO: 2.

7. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a polypeptide comprising amino acids -23 to 373 of SEQ ID NO: 2.

8. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a polypeptide comprising amino acids 1 to 373 of SEQ ID NO: 2.

9. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a portion of the mature VEGF-2 polypeptide comprising amino acids 24 to 373 of SEQ ID NO: 2. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes -57- a polypeptide comprising amino acids -46 to 24 of SEQ ID NO: 2.

11. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a polypeptide comprising amino acids -23 to 24 of SEQ ID NO: 2.

12. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a polypeptide comprising amino acids 1 to 24 of SEQ ID NO: 2.

13. An isolated polynucleotide according to claim 1 wherein the polynucleotide comprises a fragment of the polynucleotide according to any one of a) to d) with the proviso that said polynucleotide fragment comprises at least 30 contiguous nucleotides of the polynucleotides of j) or k).

14. An isolated polynucleotide according to claim 1 wherein the polynucleotide comprises a fragment which hybridises to at least 30 contiguous nucleotides of the polynucleotide encoding amino acids -46 to 24 of SEQ ID NO: 2 under the following conditions: hybridisation in 0.5 M sodium peroxide NaPO 4 7% sodium dodecyl sulfate (SDS) at 0 C and washing with 0.5 x SSC, 0.1% SDS at 60 0 C or equivalent hybridisation stringency. An isolated polynucleotide according to claim 1 wherein the polynucleotide comprises the complementary form of the polynucleotide according to any one of claims 2 to claim 14.

16. An isolated polypeptide comprising at least 30 amino acid residues and having VEGF2 biological activity further comprising a member selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of the full length polypeptide of SEQ ID NO:2; b) a polypeptide comprising the amino acid sequence of the mature protein of SEQ ID NO: 2; c) a polypeptide comprising the amino acid sequence of the proprotein portion of -58- SSEQ ID NO: 2; d) a polypeptide comprising the amino acid sequence of the mature polypeptide encoded by the cDNA contained in ATCC Deposit No. 97149; e) a polypeptide comprising amino acids -46 to 373 of SEQ ID NO: 2; f) a polypeptide comprising amino acids -23 to 373 of SEQ ID NO: 2; g) a polypeptide comprising amino acids 1 to 373 of SEQ ID NO: 2; h) a portion of the mature VEGF-2 polypeptide comprising amino acids 24 to 373 Sof SEQ ID NO:2; i) a polypeptide comprising amino acids -46 to 24 of SEQ ID NO: 2; j) a polypeptide comprising amino acids -23 to 24 of SEQ ID NO: 2; k) a polypeptide comprising amino acids 1 to 24 of SEQ ID NO: 2; 1) a polypeptide comprising an active fragment of the VEGF2 polypeptides according to any one of a) to d) with the proviso that part of said polypeptide fragment is encoded by at least 30 contiguous nucleotides of the polynucleotide encoding the polypeptide of any one of j) or k); m) a polypeptide fragment comprising an amino acid sequence encoded by a polynucleotide sequence which hybridises to at least 30 contiguous nucleotides of the polynucleotide encoding any one of the polypeptides of j) or k) under the following conditions: hybridisation in 0.5 M sodium peroxide NaPO, 7% sodium dodecyl sulfate (SDS) at 65 C and washing with 0.5 x SSC, 0.1% SDS at 60*C or equivalent hybridisation stringency.

17. An isolated polypeptide according to Claim 15 comprising the amino acid sequence of the full length polypeptide of SEQ ID NO:2.

18. An isolated polypeptide according to Claim 16 comprising the amino acid sequence of the mature portion of SEQ ID NO: 2.

19. An isolated polypeptide according to Claim 16 comprising a polypeptide comprising the amino acid sequence of the proprotein portion of SEQ ID NO: 2. -59- An isolated polypeptide according to Claim 16 comprising the amino acid sequence of the mature VEGF-2 polypeptide encoded by the cDNA contained in ATCC Deposit No. 97149.

21. An isolated polypeptide according to Claim 16 comprising amino acids -46 to 373 of SEQ ID NO: 2.

22. An isolated polypeptide according Claim 16 comprising amino acids -23 to 373 of SEQ ID NO: 2.

23. An isolated polypeptide according to Claim 16 comprising amino acids 1 to 373 of SEQ ID NO: 2.

24. An isolated portion of the mature VEGF-2 polypeptide according to Claim 16 comprising amino acids 24 to 373 of SEQ ID NO: 2. An isolated polypeptide according to Claim 16 comprising amino acids -46 to 24 of SEQ ID NO: 2.

26. An isolated polypeptide according to Claim 16 comprising amino acids -23 to 24 of SEQ ID NO: 2.

27. An isolated polypeptide according to Claim 16 comprising amino acids 1 to 24 of SEQ ID NO: 2.

28. An isolated polypeptide according to Claim 16 comprising a fragment of any one of the polypeptides of Claims 16 to 20 with the proviso that part of said polypeptide fragment is encoded by at least 30 contiguous nucleotides of the polynucleotide encoding the polypeptide of any one of Claims 25 to 27.

29. An isolated polypeptide according to Claim 16 comprising an amino acid sequence encoded by a polynucleotide sequence which hybridises to at least 30 contiguous nucleotides of the polynucleotide encoding any one of the polypeptides of Claims to 27 under the following conditions: hybridisation in 0.5 M sodium peroxide NaPO 7 sodium dodecyl sulfate (SDS) at 65 C and washing with 0.5 x SSC, 0.1 SDS at 60 0 C or equivalent hybridisation stringency. An isolated polypeptide according to any one of Claims 16 to 29 further comprising S a heterologous polypeptide.

31. An isolated polypeptide according to any one of Claims 16 to 30 further comprising a homodimer.

32. An isolated polypeptide according to any one of Claims 16 to 31 wherein the polypeptide is glycosylated.

33. A composition comprising the polypeptide according to any one of Claims 16 to 32 or 40 and one or more pharmaceutically acceptable carriers and /or diluents.

34. A vector comprising the polynucleotide according to any one of Claims 1 to A recombinant vector comprising the polynucleotide according to any one of Claims 1-15 operatively associated with a regulatory sequence that controls gene expression.

36. A host cell comprising the polynucleotide according to any one of Claims 1-15 operably associated with a heterologous regulatory sequence or a vector comprising same.

37. The polynucleotide sequence according to any one of Claims 1-15 further comprising a heterologous polynucleotide.

38. The polynucleotide sequence of Claim 37 further comprising a polynucleotide which -61 Sencodes a heterologous polypeptide.

39. A method for producing a VEGF-2 polypeptide at least comprising the step of culturing the genetically engineered host cell of Claim 36 for a time and under conditions suitable for the expression of the polypeptide encoded by said 0 polynucleotide to occur. A polypeptide produced by the method of Claim 39.

41. A composition comprising the polynucleotide according to any one of Claims 1-15, 37, or 38 and one or more pharmaceutically acceptable carriers and/or diluents.

42. Use of the polynucleotide according to any one of Claims 1 to 15, 37 or 38 or the polypeptide according to any one of Claims 16 to 32 or 40 in the preparation of a medicament for the treatment of a patient having need of human VEGF-2 polypeptide.

43. An antibody which is capable of binding to the polypeptide according to any one of Claims 16 to 29 with the proviso that the antibody is not capable of binding to a polypeptide consisting of amino acid residues 24 to 373 of SEQ ID NO: 2 or a fragment thereof.

44. An antisense construct capable of binding to the polynucleotide according to any one of Claims 1-15 or a complementary form thereof with the proviso that the antisense construct is not capable of binding to a polynucleotide sequence encoding amino acids 24 to 373 of SEQ ID NO: 2 or a fragment thereof. A method of stimulating proliferation of endothelial cells in a patient comprising administering to the patient the polypeptide according to any one of Claims 16 to 32 or 40 for a time and under conditions sufficient for the proliferation of endothelial cells to occur. S- 62

46. The method of Claim 45 wherein the patient has vasculature tissue damage.

47. The method of Claim 45 wherein the patient has a wound, tissue damage or bone damage.

48. The method of Claim 45 wherein the patient has ischemia.

49. The method of Claim 45 wherein the patient has myocardial infarction. The method of Claim 45 wherein the patient has coronary artery disease, peripheral vascular disease or CNS vascular disease.

51. The method according to any one of Claims 46 to 50 wherein the stimulation of endothelial cell proliferation is capable of further stimulating angiogenesis.

52. The isolated polynucleotide according to any one of Claims 1 to 15, 37, 38 or 44 substantially as hereinbefore described with reference to the Figures and /or Examples.

53. The isolated polypeptide according to anyone of Claims 16 to 32 or 40 substantially as hereinbefore described with reference to the Figures and/or Examples.

54. The vector of Claim 34 substantially as hereinbefore described with reference to the Figures and/or Examples. The host cell of Claim 36 substantially as hereinbefore described with reference to the Figures and/or Examples.

56. The method according to any one of Claims 39, 45 to 51 substantially as hereinbefore described with reference to the Figures and/or Examples.

57. The use according to Claim 42 substantially as hereinbefore described with reference -63 to the Figures and/or Examples.

58. The composition of Claim 33 or 41 substantially as hereinbefore described with reference to the Figures and/or Examples. Dated this TENTH day of FEBRUARY

2005. Human Genome Sciences. Inc. Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant