AU2005244575B2 - Human Vascular Endothelial Growth Factor 2 - Google Patents

Description

U

IND

Suman Vascular Endothelial Growrth Factor 2 This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such olynucleotdes and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been. idantified 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 essentia for embryonic development, subsequent growth, and tissue repair. Angiogenesis, however, is an essential part of certain pathological ccnditions such as neoplas-ia, for examle, tumors and glioms, and abnormal angiogenesis is associated with other diseases such as inflamEation, -1/2rheumlatoid arth-_rjti.

5 Psoriasis, and diabeticrenoay c i o l n d l a g b r~ M S c i e n c e 2 3 5 4 4 2 4 4 7 .1 9 8 7 Both acid iC and basic fibroblast growth factor rinol ecules are ?Mitogens for endothelial eJsan te cl ye __Angiotrovin and angiogenin can induce angiogenesis, although their functions are unclear o1c J,13,Cne Vecin n. 153-17o, Lea amd Febiger press). A hicer e ec i e mi o e for vascular endothelial cells is vascular S endothelial grouwth ,factor or v-EGp -raa Nq e ~ocr Rev 1319-32,(92) also known as vascular 'r 'Permleability factor (vpj?) Vascular endothelial rw C) factor is a' secreted ego~~ ioe hs agtcl SDecificity appears to be restricted to vascular endothelial cells.

The murine VErQ? gene has been characterized and its expression pat tern in embryogenesis has been analyzed.

A

Persistent e-Xpression of VEGp was be-din pteia cells adjc todney ng trated endothelilm, in choroid rlexu O nd V kidne _ome3±ujj The data was consistent with a rlel fVG as a multifunctional regulator Of endothelial celgrowth and differentiation (Breier, G. e t al Development. 114:521-.532 (19-92)).

VZGF i s str-ucturally relat~ed to the Uand chain o Platelet -erived growth fatr (DF) tgnS fo rneencYma cells -and placenta grow'th f actor (L?,a eand e l a ce l qioen. These three proteins belong t o) the saefamily and share a. conserved motif. Egtcsen residues contributing to 2i5lfdeht form tion ar str ctl co se~ed n these proteins. Alterntively pi e MIP 4s have been identified for both- -v7Rp, LFadDGan thes d~ffer nt pliing9 products. differ in biologi a activity and in recept'or.-binding 'Specificity. VEsGp and DGc function as homo..dimers or41 hetero-dimers and bind -to receptors Whi~ch elicit intrinsic tyrosine ]cinase jcivt foloig receptor dime-rization.

VSGF has four different forms of 121, 165, 18.9 and 206amino acids due to alternati~ve splicing. VEGF121 and VEGF165 are soluble and are capable of promoting angiogenesis, whereas VEGFl89 and VEGF2'06 are bound to heoarin containing proteoglycans in the cell surface. The temooral and spatial e~ression of VEG? has been correlated with physiolosical S proliferation of the blood vessels (Gajdusek, and Carbon, Cell Physiol., 139:570-579, McNeil, zt Muthukrisbnan, Warder, D'Amore, J. Cell.

N- Biol., 109:81-822, (1989)). Its high a..ffinity bind-ing sites are localized only on endothelial cells in tissue sections (J7akeman, et al., Cin. Invest. 89:244-253, (1989)).

The factor can be isolated from pituitLary cells and several tumor cell lines, and has been imnlicated in somie human gl iomas (Plate, K. a. Nature 359:845-848, (1992)).

Interestingly, e-Nression of VEGF121 or VBGF165 confers on Chinese ham ter ovary cells the ability to form tumors in nude mice (Ferrara, et al., J. Clin. Invest. 91:160-170, (1993)). The inhibition. of VEGP function by anti-VEGF monoclonal antibodies was shown to inhibit tumor growth in imr,=ne-deficient mice (Kim, Nature 362:b43.-844, (1-993)) Further, aL dorainant-negati.ve mutant of the VEC-? receptor has been shown to inhibi t. growth of glioblasto-ms in mice.

Vascular permeability factor, has also been found to be responsible for Poersistent microvascular hyternermeability to plasma proteins even af ter the cessation of injury, which is a chatracteristic feature of normal wound healing. This suggests that VP? is an important factor in wound healing.

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

The expression of VEG? is high in vascularized tissues, lung, heart; placenta and solid tumors) and correlates with angiogenesi1s both temporally and spatially. VEGF has.

also bein shown to induce angiogenesiS in vivo. Since agiogenesis' is essential for the repair of normal iissues, -3es-pecially vascular tissues, VEGF has been proposed f or use in promoting vascular tissue repair in atherosclerosis).

U.S. Patent No. 5,073,492, issued December 17, 199J. to IND Chen et al., discloses a method for synergitclye~acn endothel~ial cell growth in an appropriate environment 'which CULLrises adding to the envrironment, VEGF, effertors and senni-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 pcrotein is a ma~aalian vascular endothelial cell mitogen and, as such, is useful for the promotion of -vascular development and repair, as disclosed in Ituropean Patent Applica-tion No.

92302750.2, Published September 30, 1992.

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

VEG?.

in accordance with one aspect of the present invenrtion, there are Provided novel mature Dolypeptides, as well 'as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The poly-pentides of the present invention are of hunian- 'ori in; In accordance -with -amotfier aspect of the p resent i.nvention, there are provided isolated nucleiLc acid molecules encoding the polypeptides of the present invrention, including mRnks, DRAs, cDN"P.s, 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 pDresent invention, there are provided processes for producing such polypeptidd-s by recombinaent techniques comprising cultuxing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding'a polypeptide of C) the present invention, under conditions promoting exression of said proteins and subseauent recovery of said proteins.

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

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

o In accordance with yet another aspect of the present S 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 teachi gs herein.

The f ollowing drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as enco -assed by the claims.

Fig. I shows the cDN1A sequence and th e corresponding deduced amino acid sequence of the pDolypeptide of the present invention. The standard one letter abbreviations f or amino Sacids are used. Secuencing was performed using 373 Automated DNA Seq-uencer (Applied Biosystems, Inc.) Sequencing dz- ccuracy is predicted to be greater than, 97k.

Fig. 2 is an illuigtration of the a-mino acid- sequence Shomology between the polyoDeptide of the present invei.tion and Sother members of the humnan PDG;F/VEGF family. The boxed areas indicate the conserved sequences and the location of the eight conserved cysteine residues.

Fig. 3 shows a photograph of a gel af ter in vi tro transcription, translation and electr-ophoresis of the polypeptide of the present invention. Lane 1: and rainbow M.W. marker; Lane 2: FGF control; Lane 3: V,7GF2 produced by M13-reverse and forward Primers; Lane 4: V.EGF2 produced by M413 reverse and VEC-F-F4 primers; Lane 5: VEGF2 produced by M13 reverse and VBGF-FS primers.

Fig. 4. VEGF2 polypeptide' is exbressed in a baculovirus system consisting of Sf9 cells. Protein from the mred.i::Lm- and cytoplasm'of cells were analyZed by SDS-PAGE under reducing an o-reducing .conditions.

Rig. 5. The medi~n from SfL9 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 mediumn supernatant and analyzed by SDS-PAGM in the presence or absence of the reducing agent 4-mer-captoethanol and. stained by coomassie brilliant blue.

Fig. 7. Reverse phase EPLC analysis of purified VEC-?2 using a RP 3 00 coltn= 21 x 3 cm, Axpplied Biosystems, 0 -7- IND The column was equilibrated with 0. trifluoropcctic acid. (Solvent A) and. the proteins ehated 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 rnm (Red line-)and 280 nm (Blu- line). The percentage of Solvent B is show.n by Greena line.

Fig. 8 ilstrates t1e effect of partially-purified VEGF2 protein on the -growth of vascular endothelial ctll in comparison to basic fibroblast growt factor.

Fi-. 9 illustrates the effect of purified VEGF2 protein on the growth of vascular endotbelial cells.

The te-rm "-ene" means the segment of DNA involved in producing a polypeptide chain.; it includes regions preceding and following the coding region (leade~r and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

In accordance with one aspect of the present in-vention, there axe pro-vided isolated nucleic acid molecules (poly nuclzotides) which encode for the mature polypeptidcs having the deduced amino acid sequence of Figure I (SEQ MD NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 97149 deposited with the America z Type Culture Collection, 10802 University Boulevard, Manzssas, VA, 20110-2209, United States of America, on 12 May, 1995 or for polypcptidcs which have- fewer amino acid residuaes than those showi-ng in Figure I (SEQ DD NO:2).

A polynucleotide encoding, a polypeptide- of the present invention may be obtained from early stage human embryo (week 8 to 9) osteoclastoinas, adult heart or several breast cancer cell lines. The polynucleotidc of this invention was discovered. in a cDNA .library derived from early stage human embryo week 9. It is sricnarzlly related to the VEGF/PIDGP fami~ly. VtGP contains an open reading frame encoding a protein of 419 amino acid residues of which approxin~ztely the first 23 amino acid residues are -the putative leader Sequzence such that the mature protein comprises 396 amizno acids, mnd which prortein exhibits the highest amino acid sequence Inb oovy t h azcUar "IZendcthe-ia' S-rob.th factor identity)~ followed by PM?or (23t) and PD~pB (2.2k).

It ir. particulaxrly importa_-t that a-1-1 eight Cyste-ines a..re c~ncmz--ed with~in all fouz member-3 of rh aiy(e are ofPiure ade-iton, the B:Lcmatlure for the Pnvpfamt±ly, PXCVCmGCtN. (EEO ID O W-6)-iS Cor-e-d 4- V'RG?2 (see iig.~e 2).

The VRG?2 POlypeptide of the present i=renjion is Maeant to include the full lengt~h polyneor.c.e and Polynuclectide sequence whi~ch encodez for any leader sequ1ences and for active fr-agment; of.- thefl length poly-ecd :3iv fragments are =epant 'to include any ;torti-ons of the full lenmtma a-ino acid sequemce which have less than the full 419 aw4,o ac:Lds of the full length amno acid setuence as shownu SZQ ZD NIo. 2 and Piue2, but still contain the elght cys't cine residues show=* cc-servJed in igre2 and such fa-me--t still contalm VR-GF2 activity.

Tlhexe ar-e at least two a~cerzat--ely spliced IrbGF2 m~v sequences present in mocroal tissues. T"he si-ze of the two V3GF2 mP.sequences which correspomd to the full-lexith and truncated version respectively are shown in Figilre 3, lane sho-s two bands imdicatimg the presence of the alternmatively spliced mtxi47 encodjng the VBC?2 polypeptide of the present invention.

The polynucleotide of the presenzt invention .may be in the f orm Of RIC or inm the form of. MQ which fl. includes CDRAI'J. Senomic flMM. and aSythetlc DIINM The MA nay be doublestraded or 5s-glestra-ded, and if single stranded =ay be the coding ztxm-nd or mon-codimmg (an-ti-sense) strand. The codig ateuence whichl encodes the mature polypeptide may be i.de-tical to the coding secn shown in Fig-ure 1 or that of the devosited clone or may be a different coding sequence wluichL coding seaquenCe, as a ramult of the red*und.n'C ordegeneracy of the genetic code, encodes the s-ne =at7=e polype_-tide ac the MA of Pig-are 1 or the deposited =SDk.

a IND The PO]Ynucleotide which encodes,'or the mature Po]Ypeptide of Figur I (SpQ

ID

NOs;2) Or for the Mature Polypeptide encoded by the deposited cDNA May include: only the coding sqeqnenr for the mature POIypeptide; the coding sequence for the MarUIc POlypeptide jj~ coding Seolueflc) an5o'o i eq e c uha intrni or noncoing eque~ 5'and/or 3' Of the coding sequence forth maue ol p j "hus, the term 'POlYncleode encoding 2 Polypeptide3 cncoxnpasse a Plmcetd Which inlcludes onl codig squece fr e a ZII cdizi squeceIo- hePOlypCptide aswell as a polynucleotide which includes additional coding and/or non-codjng sequence.

The Present invention further relates to vari.ants Of the herein above described P-olynucleotides which encode for fragments, analogs and derivatives Of the polypeptide having the deduced amino acid sequence Of rIgUIe 1 (SEQ ID NO: 2) Or the polypepride encoded by the cDNA Of the deposited clone. The variant of the polynuccotide may be a natrallY occurring, allelic variant Of the Polyflucleotide or a non-natuzally occUrring variant of the polynucirotidc.

is Thus, the present invention inllUdes Polynuc-leotides encodingr the same mature polYpeptide as shown in Figure I (S-EQ ID NO: 2) or the same mature Polypeptide encoded by the cDNA of the deposited clone 2s well as variants of such polynucl-oti!s which 'Variants encode for a fragment, derivative or 2nalov, Of th= Polyp erd fFgr SQI O2 or the POIYPePtide encoded by the cD)NA of the deposited clone. Such flfcleotidc variants include deletion variants, substitution1 variants and addit ion or instrtion variants.

As herein above indicated the Polynucleotide may have a coding eunewii a n tur lly occ rr~ af eic var a~ of the coding seq ue-n ce show n in Figure I (SE Q ID N o 1) or of the codingc sequence of the deposited clone. As lMOAM in the art, an allelic variant is an matente form Of a po]Ynuclceotid scqu-ce- which may ave aSbttto eeino addition of one or more flUCleotides, which does not susatal alter the function of the encoded Polypeptide.

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequ.ence ID which allows for purification of the polYpeptide of the present invention. The marker sequience may be a hexahistidine tag sunnied by a pQE-9 vector to provide f or tpurif ication of the mature poly ep Lefsdt h akri the case of a bacterial host, or, f or exaxaole, the marker se~ene m~rbe a bemagglutinin (RA) agwhen a man a host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, et al., Cell, 37:767 (1984)).

The term "~gene" means the segment of DNA inv olved in producing -a polypepDtide chain; it includes regions preceding and following the c oding region (leader and trailer) as well as intervening sequences (introns) between iLndividual coding segments (exons).

Fragments of the full length- gene of the present invention may be used as a hybridization probe for a CDNXA library to isolate the fcull length cDNA, and to isolate other cDNAs which have a high sequence sifrnilarity to the gene or similar, biolocgi ca activity. Probes of this type preferably have at least 30 bases and mLay contain, for examole, 50 or more bases. The probe Tray also be used to identify a cDNA clone correspDonding to a full length transcript and a genomic clone or clones that contain the comolete gene including reg-ulatory and prormotor regions, exons, and intromns. Aun amle of a screen coTrises isolating the coding region of the gene by using the }=own DNA sequence to synthesize an oligonucleotjde probe. Labeled oligonucleotides having a seguence comlementary to that. of the gene of the present :Lavention are used to screen a library ofl h-x-a cDN,, gemoonic DITA or jnIV to deterie which members of the -library the probe hybrictizes to.

SThe present invention further relates to S 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 It 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 S 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either 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 exanmle, 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- Patent Procedure.

The pentinvcntion furthcr relaics to a polypcptide which haVe t:he deduct-d anino acid seauence- of Figure 1 (SEQ D NO: 2) or which has the amino acid sepI--=e encoded by the deposited cDNA, as'well as fragmecnts, analogs and dz-rivatives of such polypep tide.

The terms 'fragrnenf', "derivative" and "analog' when refe-rring to the poly-peptide of Figure I. (SEQ ID NO: 2) or that encoded by the dep'ositcd cDNA, -means a polypeptide which- retains the conserved motif of VBGF proteins as shown in Figure 2 and essentially the same biological ftncdon 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 I (SEQ ID NO:2) or that encoded by the deposited cDNA may be one in which one or more of the anino acid residues arc substitxted with a conserved or non-conserverd a--ino acid residue: (preferably a conserved amnino acid residue) and such substituted amino acid residue may or mnay not be one encoded by the genetic code, or (iH) one in which one or more of the amino acid re-siducs include a substirueni group, or (iii) one in which the mature polypepride is fused with another compound, such as a compound to increase the half-liffe of the polypeptide (for example, polyethylene glycol), or (iv) one-in. which the additional amino acids art fused to the mature polypemtide or one in which comrises f ewer ami~no acid NI residues shown in SEQ ID No. 2 and retains -he conservred motif and yet still retains activity characteristic of th~e VEGF? family of polyp'entides. Such f ragments, derivatives and IND analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and pDolynucleotides of the present invention are pref'erably provided in an isolated f orm, and pref erably are purified to homogeneity.

The term "isolated" means that the material is removed 1fl from its original environient (e the natural environment Sif it is naturally occurring) For exa~le, a naturallyoccurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucfle6tide or poly-Deptide, separated from some or all of the coexisting materials in the natural systeift, is isolated. Such polynucleotides could be Dart of a vector and/or such polynucleotides or polypeptides could be part of a corr_ osition, and still be. isolated in that such vector or CULLosition is. not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQ ID NO: 2 (in Dparticular the mature polyveptide) as well as polvmeptides which have at least similarity (preferably at' Jeast 70t- ide-ntit)) t o the polypeptide of SEQ ID NO:2 and more prefera-bly at least similarity (more Dref-erably at least 951- identity) to the polypePtide of SEQ ID NO: 2 and s till more pDref erably at l~east similarity (still more preferably at least 90% identity) to the poly-peptide of SEQ ID X0:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amnino acids and more preferably:at least 50 amtino acids.

As known in the at "similarity" between two po-lypepDtides is determined by co -aring the amino acid -13sequence and its conserved amino acid substitutes of one po].ypepDtide to the sequence of a second polyveptide.

Fragmnts or nortions of the polypeptides of the present invention may be e~loyed for producin-g the co-resPoondng ID f u 1.-length polypeptide by peptide synthesis; there*fore, the fragments may be employed as internediates for producing the full -length polype-ptjdes. Fragments or Portions of the polynucleotjdes of the present i.nvention may be used to synthesize full-lnt polynucleotides ofth prsn 1nvention.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells Cl which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recomb~inant techniques.

Host cells are ge netically engineered (transduced or tran~sformed or transfected) with the vectors of this invention which may be, for examle, a cloning vector or an expression 'vector. The vector may be, for exa=n le, in the form of a plasmid, a' viral particle, a phage, etc. The engineered host cells can be cultured in con-.rentiLonal nutrient media modified as aDprbnriate for activating promoters selecting transfom~ats or anolifying the *VEGB'2 genes of the present invention. The culture conditions, such as tenmeratre, pH and the like, are those previously used with the host cell selected for e.,res sion, and will be apparent to the ordinarily a-killed artisan.

The polyenucleotides of the Presenot invenion may be emuployed for producing polypeptides by recomb-innt techniq-ues. Thus, for exanle, the polynuclectjde may be included in any one of a variety of e:)ression vectors for e~qr=essing a polypeptide. Such vectors include ch-romosml, nonchromosma and syntheti6 DNA sequences, derivatives .of SV4O; bacterial Plasmi-ds; phage DMA; baculoviz-us; yeast Dlasmids, vectors derived from comnbinations~ of Dlasmids and -14 phage DNA, viral DNA such as vaccinia, adenovirus, f.owl Dox CIvir-us, 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 IND vector by a variety of rDrocedures. in general, the DnA secruence is inserted into an approvriate restriction endonuclease site(s) by procedures kown' in' the art. Such procedures and others are deemed to be within the scopoe of those skilled in the art.

The DNA seauence it the exnression vector is operative.1y V) linked to an appropriate exoression control secruence (s) C) (promoter) toc direct mRITA sy-nthesis. As renresentative eramoles of such promoters, there may be mentioned: LTR or promoter, the E._coli. lac or trD, the ph-age Iambda

PL

promoter and other promoters laibwn to control exnression of genes in prokaryotic or eukaryotjic 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 amp~lifying expression.

in addition, the' exo>ression vectors preferably contain one or more selectable marker genes t-o provide a phenotypic trait f~or selection of transformed -host ce lls such. as dihydrofojiate reductase or neomrycin resistance f or- eukaryotic cell culture, or such as tetracycline or axrpicillin resistance in 9.-coli.

The vector containing the appropriate DINA sequence as hereinabove described, as well as an appropriate p-rmoter or control sequence, may be employed to transform an appropriate host to perit the host to ez- ress the protein.

As representative exanmies of appropriate hosts, thaere rmy be mentioned: bacteria!. cells, s'uch as E. coi, StretoLL~ce;, Salmonella t-v-himurii.n; fungal cells, such as yeast; insect cells such as Drosopila Sj2 and Spodontera Sfl animal cells such as HO Cos or Bowes melanoma; aa-OviruLses; Dlant cells, etc. The selection of an appropriate host is deemed to be Within the scope Of those skilled in the art from the teachings herei.

-ore Paticularly, the present invention also includes IND recominat constucts comprising one or more ofth sequences as broadly dsrbdabove. Theth describe constructs cOrrrise a vector, such as a Plasmid- or viral vector, into V) wh~ch a sequence of the invention has bee inred na tn forwa.rd or reverse orientation. In a preferred aspect of thi& embodimnt, the cbnstruct further comnrises sequences, includ~ing, for Iexample, a prootr oeg trly linked to the seqauence. Large numbers of suitable vectors and promoters are known to those of skill in the art. and are cormmercially available. The f ollowing vectors are- pro-,ided by way of examle. Bacterial: pQz7D, pQRG0, PQ-9 Qag pBS, pDiO, Dhagqescrip, z)&-iXl74,. Bluescript SK, pBSKS, P!RSA, PNza, DNH18-2k pNF46A (Stratagene) ptrc99a, PK223- 3, pK233 pDR540, ZPRIT5 (Pharmacia) Bukaryotic: oWqrC) PS"V2CAT, pOG44, pXT1, pSG (Stratagene) .PSVK3, pBV -pMSG, PSVL(Phamaci) _owever, any other plas-mid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloranmhenicol: t+rnsf erase) vectozs or other vectors with selectable markers. Two a-Dprouriate -vectors are pK32-8 and -POY17. Particular named bacterial proniot ers include ladl, lacZ, T3, T7, gct lin p, adt.

Ruka-ryo tic promnoters include QCIV immediate early, H;sv thyrnidjLn kinase, early and late SV4D, LT'-Ps from retro-vjrus and mouse metallothionein 1 Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further emnbodiment, the present i~nventio reas to host cells contaimjng the above-described constructs. The host cell can qbe a higher eukaryotic cell, such Ls a -16mramalian cel~l, or a lower eukaryotic cell, such as a yeast ci cell, or the host cell can be a prokaryoti~c cell, such as a Ubacterial cell. Introduction of the construct into the host cell can be effected by calcium phospDhate transfection,

DRER

ID Dex-zran mediated transfection, or electroporation. (IDavis, Dibner, Battey, Basic Methods in Molecular Biology,(98) The constructs in host cells can be used in a V) conventional manner to produce the gene product encoded by the recombinaAt sequence. Alternatively, the Dolypeptides: of ci the invention can be synthetically produced by conventional pDeptide synthesizers.

c-i Mature proteins can be expressed in ma~nalian cells, yeast, bacteria, or other cells under the cobtrol of appropriate promoters. Cell-free translation .systems can also be errloyed to produce such proteins using RXAz derived f rom the DNA constructs of the pDresent invention.

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

Tramscription of the DIN encoding the poJlypeptideas of the present invention by hiigher eukaryotes is increased by insetting an enhancer sequence into the vector. tnhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its trans cription.

Ranles including the SIV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus: early promoter enhancer, the pojlyom enhancer on the late side of tlle replication origin, and adenovirus enhancers.

G-enerally, recomnbinant eymres sion vectors will incliide origins of replication Anid selectable markers permitting transfomtic n of the host cell, the 'anicill:Ln resistance gepe of 2. coni and S _cere-isiae TR?--l gene.-, and -17a promocter derived from a b.~ghlY-expressed gene to drc transcription ofE a downstream structual sequence. Such U promoters can be derived from operons encoding glycolytic enzymes such as 3 -Dbosphoglycerate kinase (PGK), ce-factcr, IND acid phosmhatase, or heat shock proteins, among others. The heterologous structural sequence is assemhbled in appropriate phase with translation initiation and termination sequences, and preferably, a leader searuence capable of directing V) secretion of translated vrotein into the 'Deriplasmic space or extrace!llar medium. Optionally, the het±ologous sequence can encode afusion protein including an Ntria identification. pe-otide i~arting desired characteristics, stabilization or siirplified purification of expressed recombinant product.

Useful expression vectors f or bacterial use are constructed by inserting a structural D1M sequence encoding a desired protein together with suitable .translation initiation and termination signals In operable reading phase with a functional promoter. The vector will con rise one or more phenotypic selectable markers and an origin of repDlication to ensure maintenance of- the vector and to, if desirable, provide amplif ication with-n the host. Suitable prokar-yrotic hosts for transformation include E. coli, Bacillus subtilis, salm:onefla tv_'iMurium. and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a mrt-ter of choice.

As -a represantativL- but nonlimiting eaeuseful expression vectors for bacterial use can corrise a selectable marker and bacterial origin of replication derived from coimercially available plasmids cormrising.. genetic elements 'of the well known cloning vector.- oDBI322

(ATCC

37017). Such CU&I_"erCal -vectors include, for era,-le, pK-223-3 (Phar-macia Fine Cb.emicals, UDvosala, Sweden) and GEM1 (Promega Biotec, Madison, WI, TUSA). These pB;U.22 "backbone" -18sections are combined with an appropri ate Promoter and the structural sequence to be e~ressed.

Following transformation of a suitable host strain and growth of the host strain to an appDropriate cell density, the IND selected vromo~ter is induced by appropriate means temoerature 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 exctract retained for further oDuzification..

Microbial cells employed in exression- of proteins can be clisrupted by any convenient method, including freeze-thaw Cl cycling, sonication, mechanical disrupDtion, or use of cell1 lysing agents, such methods are well know to those skilled im the art.

Various manunalian cell culture systems can also be employed to express recombinant protein. Exanmles of msrnalian e.xpression 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 co~atiLble vector, for examnle, the C127, 373, CEO, HeLa and BHK cell lines. Manalian e_->=ession vectors will* conrrprise 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 seaences, and 5' flanking nontranscribed seauences. DU sequences derived frcom the splice, and polyadenylation sites may be used to pr-ovide the required nontranscribed genetic elements.

The polypeptides can be recovered and purified. from recombinant cell cultures by methods including a: onitun sulfkte or ethailol precipitation, acid extraction, aniLon or cation exchange chromatography, P1hosmhocell."lose chromatography, hydrophobic interaction chromatography, *af fini ty chromatography, hydroxylaatite chromatogr-aphy and -19lectin chromat..r 5 Y. Protein ref olding tp a eUe as necessary in gOltn S~fg.rteps ofa te uatede (R'PLC) can be m PrcLu-c liid h.

e loyed for final Pu if ca io OStepsh n thell POlyPePtides. of the present invention myb naturally Purified product, or a pro uc oei a synthetic procedures, Prpoue yrc~nt Of-ceical from a0ro'oic o euiaryotic host (f or axa le b tt bacterial, yeast, higher plant, imscta d marala ell in Deending upon th *n mammla rebinn Proceurethe plz ineto ayb lcSylated Or may be no-qlcsltd m e o nyp e~ 5 o h n e t o a a lso in clu d e an in itia l met~ioineamino acid residue.

As hon n igures 8 and the V3GF2 Polyle,-tide of S8Q ID No., 21 Minus the initial 46 amino acids, is a potent raitogen for vascular endothelial cells and stimnulates. their growth and Proliferation. The results of othCbo analsisPerformed for- the VEGF2' nucleic acid' sequence encoding th-is Polynpeptide wherein 20 Pgq Of RIM from several human tissues were probed with

"'P-.VG

2 illustrates that this -protein is activel~y eXroressed in the heart and lung which is fuirther evid~e =Of*Mtgenlic activity.

Accordingy of? it be enmr ye To DrOmote angiogenesis, for examp~le, to stimulate the grow-th of transplanted tissue where cor'Ona- 1 hypass surger- is Perform~ed. VEG?2 may also be etrmloyed to prcrmotm on heain particularly to re..vaacularize damaged sses or stimujlate collateral blood flow during ischemia and where new capDillary angiogenesis is desired. -v--GP2 may be en~loyed to treat f ull-thickness wounds such as deraiel Ulcers, .Including Pressure sores, venous ulcers, and diabetidc ulcers. In addition# VEGF2 tay be eTMloyed to treat fl~hc~ 5 un a n d n e s h e r a k i n g r a t O r f l a p i s u s e d t o r e p a i r such buxns and injuries. VEC.F2 May also be eInloyed for use in plast-ic surery, for exan~le, for the repair of lacerations from trauma and cuts inscitcn ihSrey Along these same lines, VEGF2 may be loyed to :induce the growth of damaged bone, Periodontim or ligament tiLssue.

VEGF2 may also be employed for regenerating suP_ Orting tissues of the teeth, including cement= and PeriodolltaI ligament, that have been damaged by disease. and trama Since angiogenesjs is i~crtant in keening wounds clean and non-infected, VEGF2 may be emojIoyed i.n associa tion with surgery* and following the* repair of cuts. ,it may also be emp~loyed for the treatment of abdominal wounds where* there is a high risk of infection.

VEGF2 may be e~loyed Z or the promotion of endothelializaticn in vascular graft surgery. in t~e case of vascular grafts using either transplanted or synthetic materiall VZC;2 can be applied to the surface of the graf t or at the junction to promote the growth of vascular endothelial cells. VEGF2 may also be employed to repair da=mage of mYocardja-l tissue as a -result of. myocardial infarction. VEGF2 May also be employe d to repair the cardiac vascular System af ter ischemia. VEG?2 may also be employed to treat damaged vascular tissue as a result of coronary artery disease -and peripheral and MS vascular disease.

VEGF2, may also be loyed to coat artif icia.prosthese or naturaa organs which are to be transplanted in the body to nuinimijze rejection of the transplanted material and to stimulate vascularization of the transplanted mater ials.

V:RGF2 May also be emloyed for vascualar tiss-ue repair, or exantole, that occurring during arteriosclerosis and requLired following balloon angioplasty where vascular tissues are damraged.

-VEGF2 nucleic acid sequences -and VEc-F2 p.Clyeptides may also be emplbyed for -in vitro nuroses related to scientific research, synthesis qf D2.K and manufacture of DNA vectors, and for the production or diagnostics and therapeutic-s to -21 treat humlan disease. For exanjle, VEGF2 may be erLPloyed f or 7n Vtr o cultuin- Of vascular endothelial cells, where it is added to the conditional medium in a concentration fErom- io pg/Ml to i0 ng/tnl.

IND Fragments of the full leng-th VEGF2 gene may be used as a hybridization probe f or a cDNA library to isolate other genes which have a high seamence similarity to the gene or similar biological activity. Probes of this type generally have at least 50 base pairs, alhuhthey may have a grzeater numnber of bases. The probe may also be used to identif a cl)N clone corresponding to a full length transcript and a genomic clone or clones that contain the complete VEGF2 gene including regullatory and promotor rein, axon s an d introns. An exarp le of a screen cou- rises isolating the codingr region of the VEGF2 gqene by using *the known_ DNA sequence to synthesize an oligonucleotjde probe. Labeled oligonuclectides having a sequence corm~plementary to thaat of the gene of the present invention are used to screen a library of human cD2XA, genomic DNA or mPI M to determine which m~embers of the library the probe hybridizes to.

This invention provides methods for identification of VEGF2 receptors. The gene encoding the receptor can be identif ied b-Dy numerous methods known to those of skill in the art, f or ex-amle, ligand nanni-g and FACS sorting (Coligqan, et al., Current -Protocols in Immun., Chapter (199)).Preferably, expression clon-ing is employed wherein pDolyadenylated RNA is prepared from. a cell responsive to VRGF2, and a cDNqA. library created from this R.IMi divided into pools and used to transfect COS cells or other cells that are not responsive to VEG?2. Transfected cells which are g'rown on glass slides are e:)osed to labeled VEC-F2. VEGF2 'can be label-ed "by a*variety of means including, iodination or inclusion of a recognition site for a site- Specific- protein kinase. Following fi-xation and incubation, the slides are subjected to autoradiogr~aphic analysi~s.

-22- Posiive ools are identified and sub-pools are Drepared amd retransfected using an iterative sub-pooling and rescreening Process, eventually yielding a single clone that encodes the Dutative recepotor.

As an-alternative approach for recertor identifi cation, labeled VEGF2 can be PDhotoaffinity linked with cell membrame or extract preparations that express the receptor molecule.

kn Coss-linked material is resolved by PAGE and exposed to xray f ilm. The labeled co; -lex: containing VEC-F2 is then excised, resolved into iPeptide fragments, and subjecteh to Cl protein microsequencing. The amino acid sequence obtained from microseguencing would be used to design a set of degenerate lgnue~±eprobes to screen a cDNA library to identify the gene encoding the putative receptor.

This invention is also related to a method of screening coi~ounds to identify those which are VRGF2 agonist 5 or antagonists. An examle of such a method takes advantage of the ability of VEG?2 to significantly stimulate the proliferation of humnan endothel~ial cells in the Presence of the comitogen 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, polypepti.Aes of the present invention and the conmounad to be screened- are added.

After incubation at 37 0 C, cultures are pulsed with 1 /iCi of thymidine (5 Ci/n ml; 1 Ci 37 BGg; NEq) for a suffiLcient time to incor-oorate the 3 [11 and har-vested onto glass f iber f ilters (Cambridge Technology, Watertown, MA). mean H thyrnidine incorporation (cpm) of triplicate cultures is determ1ined using a 1licuidd scintillation counter (Beckaan Instruments, Irvine, CA). Signif icant 3 [a thyrnidine incorporation.n as -compared to- a control assay where the, or-ound is excluded, indicates stimulation of endothelial cell prolif eration.

-23- To assay f Or antagonists, the assay described above Iis Per'f ored. and the ability of the cormound to ijji 3 H] thymidine incorporation in the pres ence -Of VEGF2 indiLcates that the CO= oumd is an antagonist to VEGF2. A.Alternatively, VEGF2 antagonists may be detected by combining vEGp2 ai Potential antagonist with membrane -bound 'VEGr2 ree no d or recombiat receptors under appropriate condtions for a cO=r-etitive inhibition assay. V3CGF2 can be l~abeled, such as by radioactivity, such that the number Of VEG?2 molecules bound to the receptor can determine the effec± 5 ofth potential antagonist.Lcies Oth Alternatively, the response of a known second messenger system following interaction of VEGF2 and receptor would be measured and conaed in the poresence or absende of the COTPOund. Such second messenger systems include but are not limited to, CAMIP guanylate cyclase, ion channels or pPhpojflositide hydrolysis. In another method, a ma=rrralian cell Or membrane preparation exbressiLng the VEc-F2 receptor is incubated with labeled V'EGP2 in the presence of the COrrpound.

The ability of the cotround to ehneor block this interaction could then be measured.

Potential VRG-F2 antagonist iude an antibody, or in some cases, an Oligonuclect ide, which bind to the DO lvveptide and effectively eliminate V M?2 function. Alter-nativeay, a Potential antagonist may be a closely related pirotein -wbi-ch binds to VEc-F2 receptors, however, they are inactive for=ms of the PDOlypeptide and thereby prevent the action of VEGP2.

E--=les of these antagonists include a negativ~e domnant muta nt o h E I2 ro yo d ,e c n o mutant~ of th IE F "~pt d for example, one c a n o the hetero-dimeric form of VEG?;2 may be dominant and may be mutated such that biological activity is not retained.- An exale of a -negative dominant mut--t includes truxicated versions Of a dimeric VEGF2 which is capable Of iLnteracting with another dimer to form wild type VEGF2, -however, the -24resulting homo-dimer is inactive and fails to e-ii characteristic VEGp activity.

Ante Potential VEGF2 antagonist is an antisense construct prepared using antisene ecoo Atsn tecnolgy an be used to control gene e:,,ression through tr'iPle-helix formation or antisense MNA or. RMA, both Of which methods are based on binding ofE a P)olynucleotide to DN2A. or RNA. For exa=_le, the 5'coding Portion Of the Poyuloid eune which encodes for the mature 'Polypeptides Of the present invention, 'is used to design an antisense RNA oligonuclectide of from about .10 to 40 base Pairs in length. A DMA oligonucleotide is designed to be cu lementary to a region of the _gene involved in tramnscript.lon (triple helix -see Lee et al., 2Thcl. Acids Res., 6:3073 Cooney et al, Science, 241:456 (1988) and Dervan et al. Science, 251: 136.0 thereby Preventing trans-cription and the production of VRGF2. The antisen e: PINA oligonuclectjde hybridizes to the mtnjVL ijn vivo and blocks translation of the mRNAM molecule into the V-RGF2 Polypeptide (Antisense Oka-11o, J. XNeurocherm. 5 6 0 (19913) Oligodeoxynucl eot des as Antis.En~e Inhibitors: of Gene EkiDressiOn, CC Press, Boca Ralton, FL (1988s)) The Olicgonucleotjdes described above can also be delivered to cells suc2h that the antisenS' PHNA or DNMA may be E-=nressed in 'Vjvo to inh-ibit production of VEGF2.

Potential VEGF2 antagonists also include small molecules wh-ich bind to and occ%2py the active site of the polyeptide thereby making the catalytic site inaccessible to substrate such that norml biological activity is prevrented. Rxzamples Of s-Mall molecules include but are not limited to small peptides or Deptide-like molecules.

The antagonj:is 5 may be enmloyed- to 'treat. limit angiOgeneasis necessay for solid txao-r metastasis.

The MPINA encoding for VEGF2 is found to be expressed at modjrate levels In at least two breast tumor cell lineswhc isindi~cative of the role Of VEGF2 POlY-ept ides in the Tfalignant phenotype. Gliormas are also a tY-Pe of neoplasia which may be treated with the a=ntacocnists of the present invention.

IND The antagonists ray also be used to treat chronic inflammation caused by increased vascular permeability.I addition to these disorders,, the antagonit a als nb e~rloyed to treat retinopathy associated with diabetes, rheum'atoid arthritis and psoriasis.

The antagonists may be elbyed in a co-Uosition with a pharmaceutically acceptable ca±rier, as herei:ate described.nfe The "VEC-F2 olyopeptides and agonists and antagonists may be employed in combination with a suitable phat-maceutical carrier. Such comositions co -rise a therapeutically effective amount of the p olype-ptide or agonist or antagronist, and a Pohar-maceutically acceptable carrier or excipient Such a carrier includes but is 'not limited to saline, buffered saline, dextrose, water,, glycerol, ethanol, and combinations thereof. The f ormulation should suit the mode of administration.

The invention also provides a bbarmaceutical pack or kit corrmrising one or more containers f illed with one or more of the ingredients of the zharmaceutical corr-osit-lons OfL 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 Pohaxrmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for humnan adainistration. In addition, the pharmaceutical coI -ositions may loyed in conjunction with other therapeutic co ounds.

The pharmaceutical compositions may be, administered in a convenient manner such as by the topical, intravenous, aLntraiperitoneal, intramuscular.r intratm r, subcutaneous, intranasal or intrademl rop1tes.. The nharmaceutical -26- CO=Ostions are admi.nis-ered in an amounrt Wbhich is effEective for treating and/or pronhylxi of the snecific inaiCation.

In general, the pharmaceutical comositions are adminaLstered in an amount of at least a-bout 10 pg/kg body weight and in Most cases they will be administered in an amont not in exes f~u~8mg~ od eght per dlay. In most cases, the dosage is from about 10 pg/kg to about I mgq/kg body weight daily, taking into account the routes of in administration, sy~toms, etc.

The VEGF2 polyetie and agonistso atgoi~ which are polypeptides may also be enmloyed in accordace with the nresent invention by expression of such polypeptide _n vi vo, which is often referred to as "gene therapy.

Thus, for examnle, cells such as bone marrow cells may be engineered with a polynucleotide (BMA or RNA) encoding for the polypeptide ex -vvo the engineered cells are then provided to a patient to be treated 'with the Polypeptide.

Such methods are well-3,:owm in the art. For ex-.amole, cells maybe egineered by procedures ]aaow in the art by use of a retroviral particle containing RAencodling the Polypeptide of the present invention.

Similarly, cells may be engineered in vi vo for exo:ression of a Dolypeptide in viro, for exazymle, by procedures known in the art.' As known in the art-, a producer cell for producing a retroviral particle containing PMN encoding a rPolyoepotide of the present invention may be adraiistered to a Datient for engineering cells in vivo and ezx_-ression 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 teaching oZ the present invention. For exmle, the exression vehicle for engineering' cells may be other than a retroviral particle, for exam~le, an adenovirus, which may be used to engineer cells in. viro af ter combination with a suitable delivery vehicle.

-27- Retrovi-uses 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 Vir-us, Rarvey Sarcoma Virus, avian leukosis vir-us, gibbon ape leukemia virus, human immunodef iciemcy virus, adenovirs, Myeloproliferative Sarcoma Virus, and marmmary tum or vr U in one embodiment, the retrovira. T)lazsm-id vector is derived.

from Moloney Murine Leukemia Virus.

The vector includes, one or more promoters. Suitable promoters wb.ich may be employed include, but are not limited to., the retroviral LTR; the SV40 promoter; and the human cytomegalovjus (CMV) promoter described in Miller, et al., aio0technicres, Vol. 7, Nio. 9, 980-990 (1.989), any other promoter cellular promoters such as eukaryotic cellular promoters including, but not limited to, the 2iistone, pol 1I11, and -actin promoters) Other viral promoters which may be emp-loyed include, but are not limited to, adenovirus promoters, tbymidjne kinase (TX) promoters, and B19 tParvovirus 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 Polypeptiide 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 adenov-iral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory s yncytial virus (RSV) promoter; inducible promoters, such as the iMMT promoter, the metallothionein promoter; heat sh~ock promoters; the albumin promoter; the AnoAl promoter; human globin promoters; viral thymidine kinase promoters, such as.

the Herpes Simpl~ex thymidine kinase promoter; retroviral

L~

(including the m odi fie d retroviraj. LT~s hereinabove described); the -actin Dromter; and human growth hormone -28- Promoters. The promoter also may be the native -Promoter which controls the gene encoding the polypeptide.

U The retrorviral plasmid vector is e~loyed to transduce packaging cell lines to form producer cell lines. Exannmles IND of packaging cells wh-ich may be transfected include, but are not limited to, the PE5OI, PA317, Ot-AM, PAl12, Tl.9.14X, VT-l9-l7-E2, OCRIP, GP+E-86, GP+envArnl2,, and DA.N cell lines as described in Miller, Human CGene Therany, Vol. 3., pgs. 5-14 (1990), which is incorporated herein by reference in its e-ntirety. !fhe vector +may transduce the packaging cells through any means known in the' art. Such means include, but are not limited to, electro-pora tion, the use of liposomes, and CaPO, precipitation. in one alternati-ve, the retroviral plasmid vector may be encapsulated Into a li-posone, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retrovira.

vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector parti-cles then may be emnoloyed, to transduce euk-aryoti-c cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid seigence encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited -to, embryonic 7sterm cells, embryonic ca-rcinoma cells, as well as hematopoietic stern cells, hepatocytes, fibroblasts, myoblasts, keratino'cytes, 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 VBGF2 nucleic acid sequaences.

Individal carigmtations in the VEGF2 gene may be detected at the 3DRA lev'el by a variety of techniques.

Nuclic cid fo dignosis may be obtained from a patipn-'s cells, such as from.blood, urine, saliva, tissue biop)sy and -29autonsy material. The genomic DNA may be used directly for detection or may be 2i~lified enzymatically by usiLng U (S iki et atu e, 24:163-166 prior to analysis.

RAor cDNA may also be used f or the same pux-mose. As an IND exarmle, PCR rrimers co~lementazy to the nucleic acid encoding \tEGF2 can be used to identify and analyze \TEGF2 mutations. For exa~nle, deletions and insertions can be detected by a change in size of the a lified pro duct in Incomoarison to the normal genotype. Point mutations rcan be identified by hybridizing amplified DRA to radolabeled VRGF2 RAor alternatively, radiolabeled VEC-F2 antisense DNk sequences. Perfectly matched sequences can be distinguished CI from mismatched duplexes by PNase A digestion or by differences in melting temgeratures.

Genetic testing based on DXA seoquece differences may b~e achieved by detection of alteration in electrophoretic mobility of IDNA 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 seuences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DXNA fragments are retarded in the gel at different positions according to their snecific Melting or partial melting temperatures (see,' Myers et Science, 230:1242 (1985)).

Sequence changes at specific locations may alIso be revealed by nuclease protection assays, such as R11ase and SI protection or the chemicalJ cleavage method Cotton. et al., PMS UlSA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNM sequence may be achieved by methods such as hybridization, RWase protection, chemical. cleav-agt, -direct DNA sequencing or the use ofrestriction enzymres, Restriction Fragment Length POlYmnphism (RLP)) and Southern blotting of getomic

DNA.

In addition to more conventional gel-electrophoresis and S DNA sequencing, mutations can also be detected by in situ O 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 SVEG?2 prdtein in a sample derived from'a host are well-known m to those of skill in the art and include radioimmunoassays, S competitive-binding assays, Western Blot analysis, ELISA C1 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 sar;ple 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-specific-protein, 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 -31- Protein present in a given volume of patient sarmle when conMpared against a standard curve.

A com~etition assay may be employed wherein antibodies specific to VEGF2 are attached to a solid sur)port.

IND Polypeptides of the Present invention are then labeled, for exaxnle, by radioactivity, and a sale derived f rom the host are passed over the, solid support and thbe amount of label' kn detected, for example by liquid scintillation chromatography, can be correlated to a quantity of VEGF2 in the sample.

*A "sandwich" assay is similar to an ELISA assay. In a CI "sandwich" assay VBC-F2 is Passed over a solid supoport and binds to antibody attached to a solid support. A second antibody is then bound to the VEGF2. A third 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 f or chromosome identification. The sequence is specifically targeted to and can hybridize wit# a particular location on chron EE mosomie Nrev there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based, on actual1 sequence data (repeat -polymoxrpisms s) are nr~s ently available for marking chromosomal location. The 'mapping of' DINAs to cromnosomes according to the. present invention Is an irmortant first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to Chroossres by preparing PCR primers (preferably .15-25 bp) from the cDHA.

Computer analysis of the cDXA is used to rapidly select primers that do not stan more than one axon in the genomic DNA, thus co=licating the amplification 'process. These primers are then used for PCR screening of somatic cell hybrids 5 containing individual human chromosomes Only those -32- O hybrids containing the human gene corresponding to the primer C will yield an amplified fragment.

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

IO Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of In 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 situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct S 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 steD. 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. Pergamon 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 (availafble -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 genes) Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected 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 mappDingr and genetic CImapping techniques, a CDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This IND assumes I megabase rmpping resolution and one gene per kb).

The polypeptides, their fragments or ot her derivatives, r- or analogs thereof, or cells expressing them can be used as V) an immunogen to produce antibodies thereto. These ant-ibodies can be, for example, polyclonal or monoclonal. ant ibodies.

V) The resent 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 f or 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 iLnto an animal. or by administering the polyopeptide to an animal, Pref erably a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this mann er, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide.

Such antib odies can then bi usei to isolate the-polypepti-de from tissue expressing that polyoeptide. For prepa-ration of monoclonal antibod-ies, any technique which nD-rides antibodies produced by continuous cell line cultures can be used. Exanmles include the hybridoma technique (Koh~ler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the huma-n B-cell hybridoma technique (Kozbor et al., 1983, Immnunology Today 4:72), and the ZBV-hybridoma techni-que to produce human monoclonali antibodies (Cole, et 1978s, in Monoclonal Antibodies and Cacer Therapy, Alam R. Liss, Inc., ppr 77-96).

-34- D Techniques described for the production of single chain O antibodies Patent 4,946,778) can be adapted to produce S single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

I The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All' parts or amounts, unless otherwise specified, V are by weight.

In order to facilitate understanding of the following examples, certain frequently occurring methods and/or terms 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 commercially 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 entym! that acts only-at certain sequences in the DNA. The various restriction enzymes used herein are commercially 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 pg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 pl of buffer solution. For the purpose of isolating

DNA

fragments for pl&smid construction, typically 5 to 50 fg 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 manufac-tuzer. Incubation times of about 1 hour at 37*C are ordinarily used, but may vary in accordace with the Su~plier's instructions. Af ter digesto the recio.~ electrophoresed directly on a polyacrylamidn gel to isolate the desired fragment.

Size selaration of the cleaved fragments iS Performed using 8 percent Polyacrylamide gel described by Goeddel, D. et al2., Nucleic Acids Res., 8:4057 (1.980).

'"ligonuclectides" refers to either a single stranded poyeoyncetd or two co -lem_-tary poyOrnaetd strands which may be chemically synthesized- Such synthetic oliagonucleotides have no 5' phosrhate and thus wiLll not ligate to another oligonucleotide 'without adding a Phosphate with an ATID in the iDresence of a kinase. A- synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Liatin" refers to the process of fEorming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, eft Id., p. .146). Unless other-wise p~rovided, ligation may be accoutlished usingq known buffers and conditions with 10 units of 7T4 DNA ligase ("ligaser) per 0.5 pg of approxJima'tely equimolar amounts of the DNA1 fragments to be ligated.

Unles otherwise statea; transfor aio a 9r o m da described by the rethod of Grah~am, F. and Van der Rh,

A.,

Virology, 52:456-437 (1973).

Exoression att-n of EG2 in i ntsusa dbes Northern blot analysis was carried out to exazine the levels of. ex~ression-of the VEGF2 gene in humaxa tiJssues and' hi~an breast cancer cell lines. Total cellular PIM sa~les were isolated with P.NAzol--1 8 system (B±otecx Laboratories, Inc.). About 1o pg of total PI1M isolated frem each breast -36- 0 -37- NOtissue and cell line spcifled was seaae n 1% agarose gel 2nd bloned Onto a nylon filter, (Molecular Cloning, Sambrook Fritsch, and Maniatis, Cold Spring Harbor Press, 1989). The Va eln rato wa d ne c odng to the Sratagene Clonng' Systems, Inc., Prime-It kit with 50 ng DNA fragment. The labeled DNA was purified with a Select-G-50 coum from 5 Prime 3 Prime, Inc., Boulder, CO. USA. Thc filter was then hybridized with radioactively labeled full length VEGF2 gene at 1,000,000 cprnfml in 0.5 M Napo, and 7 SDS overnight at 65'C. After washing twice aI room terperature and twice at 60'C with x SSC, 0.1 SDS, the filt=-s were then exposed at -70"C overnight with an intensitlingscreen. A message of 1.6 Xb was observed in 2 breast cancer cell Lines- Examnple 2 Cloning and expression of VEG-2 using the baculo-vIrus exrpression systeta The DNA sequence encoding the VPGF2 protein without 46 amino acids and the INterminus, see AtCC Accesion No. 97149, was amlificd using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the glene: The 5' primer has the seqtuence TGT AAT ACG ACT CAC TAT AGG GAT CCC GCC ATG GAG GCC ACG GCT TAT GC (SEQ 11 NO: 7) and contains a BamHi restriction enzyme site (in bold) 2zd 17 nucleotide nucleoride sL-quncz complemnentary to the 5' sequence of VEGF2 (mat. 150-166).

Tbe 3' primer has the sequence GATC TCT AGA TThA OCT CAT TTG TGG TCT (SEQ ID NO:8) and contains the cleavage sit: for the restriction enzymite XbaI and 18 nueleotidecs complementary to the 3' seq~uence of VEGF2, including tbe slop codon and ISnt sequence before stop codon.

The amplified sequences were isolated from* a I agarose gel using a commercially available kit ("Geneclean, f BID 101, Inc., La Jolla, CA). The fragment was the-n digested with the endonuclease Bamal and XbaI and then purif ied again on a 1- CIagarose gel. This fragment was ligated to DACGP67A baculoviz-s transfer vector (P~armingen) at the BaMR1 and XbaI sites. Through this ligation, VEC-?2 CZIA 'was cloned in frame with the signal sequence of baculovir.as 9-p67 gene and was located at the 3' end of the signal sequence in the vector. This is designated pAcGP67A-V.GP2' To clone VEGF2 with the signal sequence of gp67 gene to the DRGl vector for ex~ression, VEGF2 with the si L' sequence and some upstream sequence were excised from the DAcGPS7A-VEGF2 plas-mid at the Xho restriction. endonuclease site located upstream of the VEGF2 cDIX. and at the XbaI r-estriction endonuclease site by XhoI and XhaI restriction enzyme. This fragment was separated from the rest of vector on a it agarose gel and was purified using "Geneclean" kit.

It was designated F2.

The PRG1 vector (modification of pVL941 vector) iLs used for the exnression of the VEGF2 vrotein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovimus vectors a-nd insect cell culture Drocedures., Texa=s Agricultural R)xoerimenta. Station Bulletin No. *1555).- This exoression vector contains the strong polyhedrin promoter of the Autographcs californica nuc1IeAtpolyhedrosis vixUas (AcYIT-PV) followed by the recognition sites for the restriction endonucleases BamR1, Sma., U~aI, B9111 and Asp718. A site for restriction endonuclease Xhol is located uostream of Bam.Rl site. The sequence between Xhol and BanRI is the same as that in PAcGp67A (stat-ic on tapme) vector. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinnant, virus the beta -galactosidase gene from E. coli is inserted in the same orientation as the pDolyhedrin promoter followed by the polyadenyla.tion signal of the polyhedrin" gene. The polyhedrin sequences 4re flan-ked at both sides by -38Brviral sequences for the cell-mediated homologous -Irecombination of cotransfected wild-type viral DNA. Many other bacu-lov-ru vectors could he used in place Of pRGI such as nAc373, DVL941 and oAcIMI. (Luckow, V.A. and Suxrmers',

M.D.,

INO virology, 170:31-39).

The plasmid was digested with the restriction enzymes XboI and Xbal and then dephosphorylated using calf intestinal Phosphatase by procedures known in the art. The DRA, was then isolated from a ;t agarose gel using the CO, mercially 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 HBl01 cells were then transformed and bacteria identified that contained the plasmid (pBac gp67-VEGF2) with the VRG?2 gene using the enzymes BamRj. and XbaI. The sequence of the cloned fragment was confirmed by DNA sequencing.

pg of the plas-id pBac gv67-VEGF2 was cotransfected With 1.0 pg of a commrercially available linearized baculovirus (nBaculoGold' baculoviras DNA", ?harrningen, San Diego, CA.) using the lip0ofection method (Felgn er et al.

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

itpg of BaculoGold'" virus DNA and 5 jug of the Dlasrnid pBac gqp67-VEGF2 were rnixed in a sterile well of z microtiter plate containing s0 g1 of serum free Grace's medium (Life Technologies inc., Ga-ithersburg, MD) Afterwards 10 p1 Liofectin plus 90 ji1 Grace's medium. were added, mixed and incubated for 15 minutes at room te~ erature. Then the transfection mixture was added dropwise to the Sf9 insect cells '(ATcc CRL 1711) seeded in a 35 mtissue culture plate with 1 ml Grace's mediumn without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27 0 C. After 5 hours the transfection solution was removed from the plate and I ml of, Grace's insect meditm. supplemented with 1.0% fetal calf s eu -39-.

was added. The pl~ate was put back into an incubator and Cl cultivation continued at 270C for four days.

After four days the supernatant was collected and a plaque assay perfor~ned similar as described by Stumelrs and Smith (supra) As a modi fi cation an agarose gel with "Blue Gal-" (Life Technologies Inc., Gaithersburg) was used wh-ich allows an easy isolation of blue. stained placues.

(A

detailed description of a "plague assay" can also be found in the user's guide for insect cell culture and baculo-rolg distributed by Lifle Technologies Inc., Gaithersburg, page 9- Four days after the serial dilution, the virus was added Cl to the cells., blue stained plaques were picked with the tipD of an Empendorf pipDette. The agar containingq the recombinant viruses was then resuspDended in an ERpendorf tube containing 200 g.l of Grace's medium~. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovarus was used to infect Sf 9 cells seeded in 35 mmn dse.Fourdays later the supernatants--of- theze-cul-tureFdishes were harvrested and then stored at 4 0

C.

Sf 9 cells were grown in Grace's .medi=n supplemented with heat -inactivated ?FBS. The cells were infected with the recombinant baculovirus V-gp97-VEGF2 at a rmltipli city of infection (1401) of 1. Six ours~later the mediu.mr-iwas removed and repDlaced with SP900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersbr)- 42 hours later 5 ACi of 35S-methionine and 5 ur-i -1S cystei-ne (Amersbam) were ~added. The cells were further incubated for 1G hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGR and autoradiography.

Protein from the mediumn and cytoplasm of the Sf9 cells was analyzed by SDS"PAGE under -reducing and non-reducing condi~tions. See Figure 4. The medum was dial.yzed against MM MRS, pE,5.8. Precnitates were obtained af~er dialysis and resuspended in. 3.00 mM RaCitrate, pH 5. 0. The resuspended precipitate was analyzed again by SD)S-PAGE and was stained with Coomassie Brilliant Blue. See Figure The medium surernatant was also diluted 1:10 in 5'J T,* MES, pH 5.8 and applied to an SP-650M colm (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 KaCl. The VEGF2 was obtained using the elution at 500 rn11. The eluate was anayzed by SDS-PAGR in the presence or absence of reducing agent, 0 -mercaptoethanol and stained by Coassit Brilliant Blue. See Figure 6.

Expresgion of Recombinant VRGF2 in COS cells The expression of plasmaid, VEGF2-H-A is derived from a vector pcDHAI/Ap (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene. 3) E. coli replication origin, 4) Q' V promoter followed by a polylin-ker region, an SV40 intron. and polyadenylation site. A DIM fragment encoding the entire VEGF2 precursor and a HA tag fused in frame to its 3' end was cloned into the polyliLnker region of the vector, thereforie', the recombinant protein expression is directed under thie CKV promter. The RA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described Wilson, H.

Ninon, 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 HAk eipitope.

The. plasmi~d construction strategy is described as follo0wNw The DNA sequence enicoding VEGF2, ATCC Accession No. 97149, was constructed by PCR usi:ng two primers: the 5' primer (CGC CGrA TCC ATG ACT GTA CTC TAC CCA) (SEQ MD NO:9) contains a Bamrni site followed by 18 nucleotides of VEGF2 coding sequence starting from the initiation co-don; the 3' sequence (CGC TCT -41- AG~A TCA =ZC GLA G:C: 7rZ GC GTC C"M CI-C QG GC CCA TT T=C TCTr 31 Is D Ol) otj: secl-ences to am xhal sie, tag, xboI-: site, a-d the last nucjlectides of the VEC-F2 coding AeTauemce (not incluclin the stop codon) Therefore, tthe PCR product containz a Ba=R! size, coding sequence folloved by an X2101 restriction endonucleag site amd RA, tag fused in fr=ane, a tranzlation termintionl stop codomnext to tbhe BA tag, a.nd am, Xhal site.

t The PCR aplif-ied ZK Zragment and the vector. pcL/P-= where digested with B=M and XhaI restrictiom =nzyLe an ligced The ligation =axxure uras tranzfrormed into E. coli strai:n SURM (Strrata-ceme Cloming syste-ms, LaX Tolla, CA -92037) the transform d culture was plated on a=icillln media plates an-d resistant co:L--es were selected. Pizasmid DNA was isolated from trrfox~ants and exanined by restriction analysis for the presence of the correct fragment. For e ressicn of the recormbinant VEG72 COS c ells were trazsf ected with the e--:ression vector DA-cT. methmod Sambrook, R. Fritsch. T. Man-latis, Holecu-lar: Cloming:

A

Laboratory M'amual, Cold Sprimg Labocratory Pregs, (1989)) The exoression of the TG2 protein was detected by.

radlolabellimg and i~.moprecivication. metho=d Bar-low,

D.

Lan~e. Antibodi±es; A Laboratory rianual. Cold Spring Saxb2or Laboratory~ Press, (1918))7 reJls were labelled-f-or 8 Bhours with "S-cyrstein.. two days post trnsfectiom. Oltuxe m-edia.

was then collected and cells were lysed with detergent

(?.PA

hnu ffer (150 =M iZ ~P-4it 0.1% SDS, 11- NP-4D, D.5k DOC, Tris, pH 7-5) (Wilson, I1. et al., id. 37:767 (1.984)).

Both Cell lysate and Culture medi a were precipitated wiLth a= RA vpe~ific mon=oclcnal antiLbody. Prot-eiLns prec ipitated were analyzed on ISta SDS-PAG-Z gals- The effect of iatialv-urified 75C-72 ,rotei:, on tbe zt-oith of as.xlar- endotheiial cells -42- '0 f- On day 1, huma~n umbilical vein endothelial cells (EUVEC) were seeded at 2-SX.1 04 cells/35 rim dish density in M199 medium containing 4k fetal bovine serum (FBS) 16 urits/mi heparin, and 50 units/n.1 endothelial cell growth supplements (RCGS, Biotechnique, Inc.). On day 2, the medium was replaced with m199 containing 10% -FBS, 8 units/mi hevarin. VEGF2 pratein 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 Ec 6, the medium was replaced. On day 8, cell number was determined with a C6ulter Counter ($ee Fig-ure Examle The e-ffect of nurified VEGF2 rrotein on the -crowth of vascular endothelial cells on day 1, human umbilical vein endothelial cells (Ov'EC) were seeded at 2-5 X :L04 cells/35 mm dish density in K199 medium containing fetal bovine ser±.m= (FBS), 16 units/mi heparin, 50 units/ml endothelial. cell growth suzpplements (ECGS, Piotechnique, Inc.). On day 2, the mnediumn was replaced with M199 containing 10t FES, 8 units/mi heparin.

Purif ied VEGF2 protein of SEQ ID No. 2 minus initial 45 ami:no acid residues -was added to the medium at this noint. On days 4 6, the medium: was replaced with fresh-medium. and suxoplements. On day 8, cell number was deterainaed with a Coulter Counter (See Figuare 9).

Excression via Ge-ne Therav 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, closecl tight and left at room -43- 0 temperature 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 Eam's ?12 o media, with 10% F3S, penicillin and streptomycin, is added.

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

At this time, fresh media is added and subsequently changed Severy several days. After an additional two. weeks in Sculture, 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 (198B) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindII 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 murine 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 fragments. The ligatior mixture is used to transform- bacteria Hi101, 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- 00 'Fresh media is added to the transBduced producer cells, and subsequently, the media is hzarested fr= a 10 cm plate of con-fluen~t producer cells. The spoent media, containing the infectious viral particles, is filtered through a millipore filter to reove detached producez- cells amd this media is then used to infect fibrobhlast cells. Media is removed from a sub-con-Fluent plate Of fibrohlasts and clu.ickly replaced with the media the prod,-ucer cells. This media is remved and rerplaced wit-h fresh media.- If the titer of vri=,s is high, then virtually a-11 fibroblasts will infected and no selection is required. If the tite= is ver-y low, then it Clis necessary to use a'retro-vira-l vector that b~as a selectable mnarker, such as 212 or hS The engineered fibrablasts are then~ injected inmto the host, either alone or after- having b'ean grownr to con1fluence on cy-todex 3 microcarrier beads. The fibroablasts no- pr*oduce the protein product.

N-Prous modif icationsg and variations of the present invention are possible in light of the above teach~ings and, therefore, within the sco-pe of the avoenderd claim, the invention may be practiced other-wise than a" particu].arly 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, 00 Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as Cc part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known Sin Australia or any other country.

Claims (26)

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 acid residues 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 acid residues 24 to 373 of SEQ ID NO:2; i) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acid residues -46 to 24 of SEQ ID NO:2; j) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acid residues -23 to 24 of SEQ ID NO:2; k) a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising amino acid residues 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 hybridizes to at least 30 contiguous nucleotides of the polynucleotide encoding amino acid residues -46 to 24 of SEQ ID NO:2 under the following conditions: hybridization in 0.5 M sodium peroxide NaPO 4 7% sodium dodecyl sulfate (SDS) at 65 0 C and washing with x SSC, 0.1% SDS at 60 0 C or equivalent hybridization stringency; and n) a polynucleotide comprising the complementary form of the polynucleotide according to any one a) to m). 00

2. The isolated polynucleotide according to claim 1, wherein the polynucleotide c 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 polynucleotide of j) or k). t 3. The isolated polynucleotide according to claim 1 wherein the polynucleotide comprises a fragment which hybridizes to at least 30 contiguous nucleotides of the polynucleotide I encoding amino acids -46 to 24 of SEQ ID NO:2 under the following conditions: hybridization 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 hybridization stringency.

4. An isolated polypeptide comprising at least 30 amino acid residues and having VEGF2 biological activity, wherein the polypeptide comprises 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 portion of SEQ ID NO:2; c) a polypeptide comprising the amino acid sequence of the proprotein portion of SEQ ID NO:2; d) a polypeptide comprising the amino acid sequence of the mature VEGF-2 polypeptide encoded by the cDNA in ATCC Deposit No. 97149; e) a polypeptide comprising amino acid residues -46 to 373 of SEQ ID NO:2; f) a polypeptide comprising amino acid residues -23 to 373 of SEQ ID NO:2; g) a polypeptide comprising amino acid residues 1 to 373 of SEQ ID NO:2; h) a portion of the mature VEGF-2 polypeptide comprising amino acid residues 24 to 373 of SEQ ID NO:2; i) a polypeptide comprising amino acid residues -46 to 24 of SEQ ID NO:2; j) a polypeptide comprising amino acid residues -23 to 24 of SEQ ID NO:2; k) a polypeptide comprising amino acid residues 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 00 fragment is encoded by at least 30 contiguous nucleotides of the polynucleotide encoding the polypeptides of any one of j) or and i, m) a polypeptide fragment comprising an amino acid sequence encoded by a polynucleotide sequence which hybridizes to at least 30 contiguous nucleotides of the polynucleotide encoding any one of or k) under the following conditions: hybridization in 0.5 M sodium peroxide NaPO 4 7% sodium t' dodecyl sulfate (SDS) at 65 0 C and washing with 0.5 x SSC, 0.1% SDS at 60 0 C or equivalent hybridization stringency. In The isolated polypeptide according to claim 1 or the isolated polypeptide according to Sclaim 4, wherein the polynucleotide encodes or the polypeptide comprises the amino acid sequence of the full length polypeptide of SEQ ID NO:2.

6. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises the amino acid sequence of the mature protein portion of SEQ ID NO:2.

7. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises a polypeptide comprising the amino acid sequence of the proprotein portion of SEQ ID NO:2.

8. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises the amino acid sequence of the mature VEGF-2 polypeptide encoded by the cDNA in ATCC Deposit No. 97149.

9. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises amino acid residues -46 to 373 of SEQ ID NO:2. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises amino acid residues -23 to 373 of SEQ ID NO:2. 00 U

11. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises amino acid c residues 1 to 373 of SEQ ID NO:2.

12. The isolated polypeptide according to claim 1 or the isolated polypeptide according to t claim 4, wherein the polynucleotide encodes or the polypeptide comprises an isolated t portion of the mature VEGF-2 polypeptide comprising amino acid residues 24 to 373 of SEQ ID NO:2. In

13. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises amino acid residues -46 to 24 of SEQ ID NO:2.

14. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises amino acid residues -23 to 24 of SEQ ID NO:2. The isolated polypeptide according to claim 1 or the isolated polypeptide according to claim 4, wherein the polynucleotide encodes or the polypeptide comprises amino acid residues 1 to 24 of SEQ ID NO:2.

16. The isolated polypeptide according to claim 4 comprising an active fragment of the polypeptide according to any one of claims 5 to 8, with the proviso that part of said polypeptide fragment is encoded by at least 30 contiguous nucleotides of the polynucleotide encoding the polypeptide according to any one of Claims 13 to

17. The isolated polypeptide according to claim 4 comprising an amino acid sequence encoded by a polynucleotide sequence which hybridizes to at least 30 contiguous nucleotides of the polynucleotide encoding the polypeptide according to any one of Claims 13 to 15 under the following conditions: hybridization in 0.5 M sodium peroxide NaPO 4 7% sodium dodecyl sulfate (SDS) at 65 0 C and washing with 0.5 x SSC, 0.1% SDS at 60°C or equivalent hybridization stringency.

18. The isolated polypeptide according to any one of claims 4 to 17, further comprising a heterologous polypeptide. 00

19. The isolated polypeptide according to any one of claims 4 to 18, further comprising a c homodimer. The isolated polypeptide according to any one of claims 4 to 19, wherein the polypeptide is glycosylated.

21. The isolated polynucleotide according to claim 1, wherein the polynucleotide I comprises the complementary form of the polynucleotide according to any one claims O 2, 3 and 5 to

22. The isolated polynucleotide according to any one of claims 1 to 3 and 5 to 15, further comprising a heterologous polynucleotide.

23. The isolated polynucleotide according to claim 22, further comprising a polynucleotide which encodes a heterologous polypeptide.

24. A vector comprising the polynucleotide according to any one of claims 1 to 3 and 5 to A host cell comprising the polynucleotide according to any one of claims 1 to 3 and to 15 operably associated with a heterologous regulatory sequence or a vector comprising same.

26. A method for producing a VEGF-2 polypeptide at least comprising the step of culturing the genetically engineered host cell according to claim 25 for a time and under conditions suitable for the expression of the polypeptide encoded by said polynucleotide to occur.

27. A composition comprising the polynucleotide according to any one of claims 1 to 3, to 15, 22 and 23, and one or more pharmaceutically acceptable carriers and/or diluents.

28. Use of the polynucleotide according to any one of claims 1 to 3, 5 to 15, 22 and 23, or the polypeptide according to any one of claims 4 to 20, in the preparation of a medicament for the treatment of a patient having need of human VEGF-2 polypeptide. 00 vv

29. An antibody which is capable of binding to the polypeptide according to any one of claims 4 to 17, 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. t 30. An antisense construction capable of binding to the polynucleotide according to any one of claims 1 to 3 and 5 to 15, or a complementary form thereof, with the proviso Itn that the antisense construct is not capable of binding to a polynucleotide sequence encoding amino acids 24 to 373 of SEQ ID NO:2 of a fragment thereof.

31. A method of stimulating proliferation of endothelia cells in a patient comprising administering to the patient the polypeptide according to any one of claims 4 to 20 for a time and under conditions sufficient of the proliferation of endothelial cell to occur.

33. The method of claim 31, wherein the patient has a condition or disorder selected from the group consisting of vasculature tissue damage, a wound, tissue damage, bone damage, ischemia, myocardial infarction, coronary artery disease, peripheral vascular disease and CNS vascular disease.

34. An isolated polynucleotide according to claim 1 as herein before described with reference to the examples.