AU2006252236A1 - Human Vascular Endothelial Growth Factor 2 - Google Patents

Description

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

AUSTRALIA

Patents Act 1990

ORIGINAL

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

Craig A Rosen, Jing-Shan Hu and Liang Cao WRAY ASSOCIATES Level 4, The Quadrant 1 William Street Perth, WA 6000 Attorney code: WR Invention Title: Human Vascular Endothelial Growth Factor 2 This application is a Divisional of Application 2003204052 filed 7 May 2003.

The following statement is a full description of this invention, including the best method of performing it known to me:- H Vasculdx EndOtheliaj. Growth Factor 2 This invention relates to0 newly identif ied Polynucleotides, Poly-_elDtides encoded by such Polynucleotides, the use of such Polynucleotides and.

polypeptides, as well as the production of such polynucleotiLdes and polypeptides. The polypeptide of the present invention has been. Identified as a memhar of the vascular andotheliaj. growth factor family. More particularly, the poly-peptide, of the present invention is vasculax encdothelial, growth factor 2, sometimes hereinafter referred to as RVEGF2.~ The invention also relates to inbibiting the action of such polypeptide The formation of new blood vessels, or angiogenesis, is essentialI for embhryonic develonment, subs equent gro-otLh, and tissue repair. Ansiogenesis, 'however, is an essential part of certain pathological conditions such as neoplasia, for exampnle, t~mors and gliomas, and abnormal angiogenesis is associated with other diseas es such as infla;tin, -1/2c-i rheumatoid arthritis, Psoriasis, and diabetic rt~oah U(FolkInan J_ and Klagsbr-u Science 235:442-447 (1987))*t Both acid.ic and basic f ibroblast grow-,h f actor miolecules are ritogens; for endothelial. cells and other cell types.

Angiot ropjfl and angiogenin can induce angniogenesij 5 although IND their functions are unclear (Folkcan, J. 1-993, Cacer Medicine -oP. 153-170, Lea and Febiger Press).- A high~ly selective mitogen for vascular endotheuial cells is vascular endothe~ial growth ,factor or VRGF (Ferrara, N. etal IND Endocr. Rev. 13:19-32, (19.92)) also known as vascular permeability factor (VPF) 'Vascular endothelial grcowt-h c-i factor is a secreted angiogenic Mitogen whose target cell specificity annears to be restricted to vascular endothelial cells.

The murjne VRQp gene2 has been characterized and its expression pattern in embryogenesis has been analyzed. A persistent exopression Of VE3GF was observed in epit helial cells adjacent to fenestrated endothelium, in choroid ple-xus and kidney glome±-ulj. The data was. consistent with a role Of VEGF as a multifunctional reg-ulator of endothelial cell growth and dif ferentiation (Breier, G. et: al.

Development, 114: S21-S32 VEGF Is structur-ally relat~ed to the ce and 0 chains of Platelet-.derived growth factor (PDGF), a nltogen fo mesenchymal cells and placenta growth factor ,an endothelial cell rr-togan. These three proteins belong to the same family and share a conserved motif. Eight cysteine residues contributing to disulfidebnd formation are strictly conserved in these proteins. Alt~ernatively pie MRNAs: have been identified for both vBG?, PLG? and PDGF and these different spliciLng products. differ in biological activity and in receptor.-binding SpTecificity. VEG? and PDGF function as homo-dimers or- betero-dimers: and bind -to rec eptors which elicit intrinsic tyrosine kinase ac ativity following receptor. ddmerizatin C1 VEGF has four different forms of 121, 165, 189 and 206 am-ino acids due to alternative splicing. VEGF121 and are soluble and are capable of promoting angiogenesis, Cl whereas VEGF189 and V3G?2OG are bound to he-parin containing proteoglycans in the cell surface. The teroral and snatial IND e.ression of VEG? has been correlated with physiological proliferation of the blood vessels (Gajdusek, and Carbon, Cell Physiol., 139:570-S79, (1989)) McNeil, muthukrisbnan, Warder, D'Amore, Cell.

Biol:, 109:811-822, (1989)). Its high affinily binding sites are localized only on endothelial cells in tissue sections CJakeraan, et al., dlin. Invest. 89:244-2S3, (1989)).

The factor can be isolated from pituit ar cells and several ttnor cell lines, and has been i~licated in some -human gliomas (Plate, K. H. Nature 359:845-848, (1992)).

Interestingly, axoression of VEGFl21 or V3GF165 confers on Chinese h=a.nter ovary cells the ability to form. tumtors in nude mice (Ferrara, et al., J_ Clin. Invest. 91:160-170, (1993)). The inhibition of VEG? *function by anti-VEGF monoclonal antibodies was shown to inhibit tumor growth in immune-deficient mice (Kim, Nature 362:841-844, (1993)) Further, a do-ninant -ne gat ive mutant of the VEC-? recentor has been shown to inhibit grow".th of gi ioblastomas in Trice.

Vascular permreability factor, has also been found to be (responsible -for persistent mcrovascular hy-pernermea-bility to plasma proteins even after the cessation of injury, which IS a characteristic f eature of normal wound healing. This suggests that VPF is an important -Eactor in wound healing.

Brown, L.P. et al., J. 3xo. Med., 17G:1373-9 (1992) The exo~ression of V"EG? is high in vascularized tissues, lung, heart, iplacenta and solid tum~ors) and correlates with eangiogenesis both te-uorally and spatially. VEGF has also beitn shown to induce angiogenesis in Vi1vo. Since angiogenesis' is essential for the repair of normal '-issues, especially vascular tissues, VEGF has been proposed for use in Promoting vascular tissue repair i atherosclerosis).

U.S. Patent No. 5,073,492, issued December 17, 1991 to Chen et al., discloses a method for synergistically enhancing endothelial cell growth in an appropriate environment which INO conorises adding to the environment, VEGF, effectors and Cl serum-derjved factor. Also, vascular endothelial cell grow-th f~actor C sub-unit DNA has beer prepared by polymerase chain Cl reaction techniques. The DNM encodes a protein that may exist as either a hetero-dimer or homo-dimer. The Protein is Cl a ma~aalian vascular endothelial cell mitoge-n and, as such, is useful for the promotion of vascular development and repair, as disclosed in European Patenat Application No.

92302750.2, published September 30, 1992.

The polypeptides of the present invention have been putatively identified as a novel vascular endothealial growth factor based on amino acid secruence homology to huanan VEGF.

in accordance with one aspect of the present invention, there are Provided novel mature polypeptides, as well as biologically active and diagnostically or therapeutically useful ffrag-meants, analogs and derivatives thereof. The polyeptides of the present invention are of huTanan *origin.

In accordance -with another aspect of tb~e present invention, there are provided isolated nucleic acid molecules (encodAing the polypeotides of the present :Lventioa, including MP.NULS, DNAS, cDrqA~s, genomic DNA as well as biologically active and diagnostically or therapeuti cally useful fragments, analogs and derivatives thereol'f.

In accordance with still another aspect of the 'oresent invention, there are provided processes for producing such polypeptides by recombinant techniques comrising culturing recombinant zrolkaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding'a polyoeptide of

NO

Ci the present invention, under conditions promoting expression of said proteins and subsequent recovery of said proteins.

SIn accordance with yet a further aspect of the present CI invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, to stimulate angiogenesis,

NO

on wound-healing, and to promote vascular tissue repair.

SIn accordance with yet another aspect of the present VB :invention, there are provided antibodies against such O 'polypeptides.

O In accordance with yet another aspect of the present

C

q invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, to inhibit the growth of tumors, to treat diabetic retinopathy, inflammation, rheumatoid arthritis and psoriasis.

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

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

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

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

CI The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as enco -nassed by the claims.

ClFig. 1 shows the cDNA sequence and th e correspoong deduced amino acid sequence of the poly-eptide of the p resent invention. The standard one letter abbreviations for armuino acids are used. Sequencing was performed using 373 Automated DNA Sequencer (Applied Biosystems, Inc.). Sequencing accuracy is predicted to be greater,than 97k.

IND Fig. 2 is an illu~tration of the amino acid -sequenc~e homology between the polyocotide of the present invenition and other members of the humnan PDGF/VEGF family. The boxed areas indicate the conserved sequences and the location of the eight conser-ved cysteine residues.

Fig. 3 shows a -ohotog-ranDh of a gel af ter in vitro transcripotion, translation and electrooDhoresis of the polypeptide of the present invention. Lane 1: and rainrbow M.W. marker; Lane 2: FGF control; Lane 3: VEGF2 produced by M13-reverse and forward urimers, Lane 4: VEGF2 produced by M13 reverse and VEGF-F4 primers; Lane 5: VEGF2 produced by M13 reverse and VEGF-F5 primers.

Fig. 4. VRGF2 polypeptideis exoressed in a baculovirus system consisting of Sf9 cells. Protein fromP the medi-'" n d cytoplasm of cells were analyzed by SDS-PAC-B under reducing and non-reducing conditions.

F ig. 5. The medi=z from, SfE9 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. G. VEGF2 was purified from the mediumo supernatant and analyzed by SDS-PAGE in the presence or absence of the reducing agent 6-mercaptoethaol and- stained by coomassie brilliant blue.

Fig. 7. Reverse phase HPL C analysis of purified VEGF2 using a RP-300 colun. (0.21 x 3 cm, Appolied Biosystems, IN-7 Inc,). The column was equilibrated with 0. 1% trifluoroacetic. acid (Solvent A) and thc proteins elute-d with a 7.5 mmi gradient from 0 to 605 Solvent B, composed of acetoaitrile INC containing 0. 07%7 TFA. The protein elition was monito-:ed by absorbance at 215 r= (Red line) and 280 rim (Blue ine). The percetage of Solvent B is shown by Green line, NO Fig. 8 illustat:S the effect of partially-purified VEGF2 protein on the -growth of vascular endothtlial cells in comparison to basic fibroblast growth factor.

Fig. 9 illusiza-Las the effect of purified VEGF2 protcin on the gowth of vascular endothelial celIls.

The te-r,7 "gene" means the segmeint of DNA involved in producing a polypeptide chain; it includes regions prece-ding and following thic coding region (leader and trailer), as wcll as intervening sequences (introns) between individual codig segments (exoris).

In accordance with one aspect of the present invention, thcr= arc provided isolatzd naucleic acid molecules (polynuclceotidcs) which enicode for the matur: polypcptides having the deduaced amino acid sequence of Figure 1 (SEQ ED) NO:2) or for the mature polypeptide encoded by the cDNA of the clone de-posite.d as ATCC De-posit No. 97149 deposite-d with the 20 American Type Culture Collection, 10801 Univcrsity Boulevard, Manssas, VA, 20110-2209, United States of America, on 12 May, 1995 or for polype-ptdes which have fewer-r axrino acid residues thana those showing in Figure I (SEQ ID NO:2).

A polynucleotide encodinga a polypcptidc of the present invention. may be obtained ftrm early stage human embryo (week~ 8 to 9) osteoclastomas adult heart or several breast cancer cell lines, The polynucicotida of tis invention was discovered in a cDNA library derived from early stage human embryo week 9. it is structurally related to the VEGFIPDGF family. VEGF2 contains an open reading frame encoding a protein. of 419 amino acid residues of which approxinmatelY the firt 23 amino acid residues art: the putative leader sequence such tha the mature -protein comprises 396 amino acids, and which protei exhiits the highest amino acid sequence hcmology to hua= vaac-ular emdmthelial growth !actor (3o~t identizty) Eollowed by P~r.Pt (23k) and PDG?e (22k) Ic ir. partinularly imporrtantr rhac all eight cysceines are cooserved with.in all 'Fou- era of the family (see b-axed areas of Fig,_re 2) .n !addition, the sismat,-e 4or- the /V~zc f ami, PxvxR=Cf CSEQ T:D '!Sc ~)consa-Ved in V2G?2 (Bee F-re2) 7he VRGFZ oyetd of the preaent ~v~nis uMej,_co include the full lentch poly-eptde and polynucleo tide sequence which encodez for any leader requences and for active fragma-nts o, tb-e f~j.1 len-t palypentide. Active fragments are maeant 'to include any Vortions Of the full IND leng-th amino acid se-ence wbhiah have less than zthe full 42.3 p-4-"o acids of the full leo~thi amino acid sequence as chown in SEQ ZD Nio. 2 anad Fig%=.e 2, .1u= still contaiz -he eighc cysteine renidues shotm conserved in re2 -nd such tz-aqmenzs still cmtain V3GF2 activity.

Thexe axe at least two alrernativ ly spliced VrSGF2 rn7Y-\A sequences present imn ormal tissues_.. ha siz;-e of che two VBSG?2 n? LK sequences which corres-pond to the full-lemg7th a-d tr =cate-d version res-pectively are shown In Figurze 3, lane shozst~obads ndcatig the presence of tb alternaiely spl.icned mpjN- encoding the V3C?2 poiypeptide of the present *The polymucleotide of the praeent invenzion may be in *the focrm Of RC or in the form of- DICA. whiclh DRA includes *cDX-. gennmic DNA, and synth!2tic DC. The MT.A =ay be doubleszranded or single-stradd, and if single stranded -ay be ::he coing ztrand or zon-codingq (anzti-sensze) stranid. The codimg aecIuence which-3 encodes the mattzrm poly-peptide may be j" identical. to the coding sequ-nce show= in Figure 1 or thLat O~F zhe deposited cl.one or na y be a different coling se-que-ce wviich coling sequenc e, as a rEs-Ult of tte redundancy or degeneracy of the genetic_ codae, ancodes the same mature polypeptide a& the DIN Of ?ig-ze I or the deposiro-d cnD-:7.

IN

-9- The PO]Ynudl~otide which encodes for the *=ture POlypeptide of Figtar_ I (SEQ

ID

NOs;2) or for the mature polypept.Ide encoded by the deposited cDNA may include: only the NO cod-ig sequence for the MatureM Polypeptide; the codig sequence for the mature polypeptide (and OPTionafly additional coding sequence) and non-c-oding sequencea, such as intro 5 or noncoding 5coucncc 5' and/or 3' of the codinga SIqCefrteauePoypie Thus, tbh- term "polyniiclnotide encoding a polyne)-ptide-" encom-passes a polyzucleotidc which includes only cod.ig sequence for the polypcptidr- as well as a polynucleotide which r~l includes additional coding and/or non-coding scquenace.

The present invention further rtlates to variants of the hertin above described polynuclcotddes which enccode for framents. analogs and derivatives of the polypeptide having the deduced armio acid sequence of Figure 1 (SEQ ID NO: 2) or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucicotide may be a naturally occurring allic variant of the -Dolynucleotide or a non-naturzally occurring variant of the polynuclcotidc.

Thus, the present inve-otion includcs polynucleoddes encoding the same mature polypeptide as shown in Figure I (82Q ID NO.- 2) or the same mature polypeptide encoded by the c-DNA of the deposited clone as w-,ell as variants of such polynucleotid-s which 'variants encode for a fragment, dcrivative or analog of the polypeptide of Fi ure 1 (SEQ ED NO:2) or the polypegtide encoded by' the cDNA of Che deposited clonec. Such aucleotida variants include deletion, variants, substitution variants and addition or instrtion varia-ts. As herein above indicated, th= polyn-tcleo tide may have- a coding sequencc which is a naturalIly oceuTzrog allelic vaz4iant of the coding sequence shown in Figure I (SEQ ID SO: 1) or of the coding sequence of the dtposIted clone. As known in the art, ani alelic variant is 2a altzrAztC form of a polynucleotidc se~que-nce which may have a substitution, deletion or addition of one or more nlucleotides, which does not substantially alter the function of the encoded poly-entide.

The polyrncleotides of the present invention may also have the coding sequence fused in frame to a marker sem~ence which allows for purification of the polypeptide of the present invenrtion. The marker sequence may be a hexa- INO histicline tag supplied by a DQE-9 vector to prov ide for CI pur-ification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for examole, the marker sequence ma r be'a hemagglutinin (HA) -tag when a marrmnalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hamagglutinin protein (Wilson, et al., Cell, 37:767 (1984)) The term ngene" means the segment of DINA inv-olved in producing a polypeentide chain; it includes regions pr eceding and followi-ng the coding region (leader and trailer) as well as inter-veni-ng sequences (introns) b-etween individual coding segments (exons).

Fragments of the full lengthi gene Of the present invention may be used as a hybridization probe for a cDINA library to isolate the full length cDNA and to isolate other cDMAs which have a high seq-uence simnilarity to the gene or simiular-biological activity. Probes of this type preferably have at least 30 bases and may contain, for exanole, 50 or more bases. The probe ray also be used to identify a cDNA (clone corr-esponding to a fLull length transcriot and a genomic clone or clones that contain the coi~lete gene includ-ing regulatory and promotor regions, axon2, and intron~s. An example of a screen commrises isolating the coding region of the gene by using the Jrnown DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotide2s having a sequence co~lementary to that. of the gene of the present imvention are used to screen a library of h'=-zm cDNM,* genomic DNA or ;aPRNL to determai-ne which members of the -library the probe hybriJ~jzes to.

NO

CA The present invention further relates to U polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least CR' and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to

\O

Mr the hereinabove-described polynucleotides. As herein used, C the term "stringent conditions" means hybridization will l r occur only if there is at least 95% and preferably at least \O 97% identity between the sequences. The polynucleotides S 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 example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.

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

The pr-sc-nt invcion further relates to a polypptidt. which have the dcducz.d am io NO ~acid seaucncz of Figure 1 (SEQ D NO: 2) or which has the airn acid semu-nzz encoded by the deposited cDNA, as well as fragments, analogs and dz-rivativcs of such polypcptida.

.3 The te-,ms "fragrment", 'derivative" and "analog" whcn refe-rringr to the polypeptidc IND of Figure 1 (SEQ ID NO: 2) or that encoded by the deposited cDNA, means a polypeptide which retains the onscrvcd motif of VEGF prote-ins as shown in Figure 2 and essentially the same biological Panction or activity, The polypeptides of the present invention may be recombiant polypeptides. natiral polypcptidr-s or synthe-tic polypeptides, preferably rec-ombinant polypeptrides.

Thec fragmznt, derivative or analog of the polypeptide of Figure 1 (SEQ ED NO or that encodtd by the deposited cDNA ay be one in which onc or more of the amino acid rr-sidues arc substituted with a conscrvcd or non-conserve-d azaino acid residue: (prcfcrably a conser-ved amino acid residue) and such substitutzed amino acid residue may or tmay not be one encoded by the genetic code, or (ii) one in. which one- or ore of the amino acid re.sidues include a substitueni group, or (iii) one in whiuch the mature polypepTide is fused with another compound, such as a comound to increase the half-lifet of the polypeptide (for example, polyethylene ;lycol), or (iv) one in which the additional am-,ino acids art fused to the m-ature Cl polypeptide or one in which cotrrises f ewer amino acid residues shown in SEQ ID No. 2 and retains the conserved motifc and yet still retains activity characteristic of the VEGF family of polypertides. Such f ragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

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

IND The term nisolatedn means that the material is removed from its original environment Ce the natural environment if it is naturally occurring) For exan~le, a naturallyoccurring polynucleotide or polype-ptide Dresant in -a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could b-2 part of a cotrosition, and still be. isolated in that such vector or cort-osition is not part ofL its natural environment.

The polynpeptides; of the present invention include the polyfleotide of SEQ ID NO:2 (iLn -uarticular the mature poly-peptide) as well as nolypeotides which have at least similarity (preferably at' *least 70% identit:y) to the polypeptide of SEQ ID NO:2 and more prefera-ly at least (simlarity (more Tpref erably at least 95t identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least si~arity (still more preferably at least 90% identity) to the poJlypeptide of SEQ TD NQ:2 and also include portions or such polypeptides with such portion of the polypentide generally containing at least 30 amino acids and more preferably~at least 50 amino acid.

As known in the art 'similarityn between two poJlypeptides is determined by ccoaring the amino acid -13seqluence and its conserved amino acid substitutes of one U polypeptide to the sequi~ence of a second polypeptide.

Fragments or portions of the polypeptides of the Poresent invention may be emoloyed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as interediates for producing the fkull-length polypeptides. Fragments or portions of the polynucleotides of the -oresert invention may be used to synthesize full-leng-th nolnmucleotides of the present invention.

The present invention also relates to vectors which Cl include polynucleotides of the present invention, host cells w'riich are genetically engineered with vectors of the invention and the production of polypeptides of the Invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the v,,ectors of this invention which mray be, for examople, a cloning vector or an e-=ression vector. The vector may be, for exanmle, in the form of a plas-mid, a viral narticle, a phage, etc. The engineered host cells can be cultured in convlentional nutrient media modified as aunpr-oriate for activating Dromoters, selecting transfEormanmts or amplifying the VEGF2 genes of the present invention. The culture conditions, such as tenmeratre, pH and the like, are those Prevuiously used with the host call selected for a~ression, and will1 be apparent to the ordinarily skilled artisan..

The polynuc eot:ides of the present Invention may -be enployed for producing polypeptides by reccmbnpnt techniq-ues. Thus, for examnle, the polynucleotide may be included in any one of a variety of expression vectors: for expessing a polyPeptide. Such vectors include chr-omosomal, noncromosomal and synthetic DNA sequences, derivat-ives of SV4O; bacterial Dlas-zuds; phage DM4_; baculovirus; yeast plasmids; vectors derive d fCr-L combinations of plas-mids and -14- CI phage DNA, viral DNA such as vaccinia, adenovirus, .fowl. pox virus, and pseudorabies. However, any other vector may be used as long as it is reolicable and viable in the host.

Cl The approuriate DNA sequence may be inserted into the vector by a variety of procedures. in general, the DN~A sequence is inserted into an apnrooriate restriction endonuclease site(s) by procedures known' in the art. Such urocedures and others are deemed to be within the scope of those skilled in the art.

N 4 INO The DIN secruence it the exnression vector is operatively linked to an av rouriate e~ression control sequence(s) Cl(promoter) to direct mR? A synthesis. As representative exarrles of such promoters, there may be mentioned: LTR or promoter, the S. coli. lac or tr, the phage lambcla p, promoter and other uromoters known to control exnress ion of genes in prokaryotic or eukaryotic cells or their viruses.

The axoression vector also contains a ribosome binding site for translation initiation and a transcriotion terminator.

The vector may also include approoriate sequences for amplifying expression.

In addition, the exmrassion vectors preferably contain one or more selectable marker genes to provide a orlenotypic trait for selection of transformed host cells such as diJhydrof olate reductase or aeomycin resistance for- eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coi.

The vector containing the appropriate DIN seotience as here inabove described, as well as an annroDriate p-rmoter or control seq-uence, may be e~loyed to transform an appropriate host to Dermit the host to e-ress the protein.

As reuresentative exa~les of appropriate hosts, there may be mentioned: bacterial1 cells, s uch as E. coli, St'reoto-nces, Salmonella tvoh-muriunz; fungal cells, such as yeast; insect cells such as Drosonhila S2 and Soodootera Sf9 a-nimal cells such as C-4, COS or Bowes melanoma; -isadenoviz-uses; plant cells, etc. The sel~ection of an appropriate host is deemed to be with-in the scope of those skilled in the art from the teach-ings herein.

More particulazlJy, the present invention also includes recombinant constructs comrising one or more of- the seq-uences as broadly described above. The constru~cts IND comrise a vector, such as a nlasmid, or viral vector, into which a sequence of the invention has been inserted, in a C~tforward or reverse orientation. in a preferred aspect of tbhir embodiment, the cbnstruct, fuirther co~rises regtrldtor IND sequences, including, f or exa~le, a promoter, operably linked to the seaence. Large numibers of suitable vectors and promoters are know-n to those of skill in the art, and are coimmercially available. The following vectors are- rovided by way of exanmle. Bacterial: pQE7O, p)QEGO, PQE-9 (Qiagen), pBS, DDO, phagescrint, zSIXl74 .P~luescript SK, rpBSKS, pNH8A, pNHl~a, pDNE1BA, DNR46A (Stratagene) ptrc99a, pKK223- 3, p)KK233-3, pDR54O, pPJIT5 (Pharmacia) Eukaryotic:

DWLNEO,

pSV2CAT, P0044, 'oXTI, p)SG (Stratagene) on-K3, p2PV,

PDMSG,

pSVL (Pharmacia) 3oeeany other plasmd 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 Cchloraumhenicol: trahs ferase) vectors. or other vectors with selectable markers. Two appropriate vectors are DK232-8 and Pa'.17. Particular -named bacterial nrorftotars include ladl, lacZ, T3, T7, gnt, lambda PX1 and tro.

Eukaryotic promoters include CXV imrnecdiate early,EV thymidi-ne kinase, early and late SV4O, LT-Rs from retro-;irus, and mouse metal lothionainr 1 Selection of the appropriate %rector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host Cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast U ~cell, or the host cell can be a prokar-yotic cell, such as a bacterial cell. Introduction of the construct into t he host cell can be effected by calcium phosphate transfection,

DEAE-

Ct Dextran mediated transfection, or electro-ooration. (Davis, Dibner, Battey, Basic Methods in Molecular INO Biology, (1986)) The constru~cts in host cells can be used in a conventional manner to produce the gene -oroduct encoded by I the recombinant seo-uence. Alternatively, the polypeptides of 0 ~the iLnvention can be synthetically produced by conventional peptide synthesizers.

Mature Droteins can be axnressed in ma~nlian cells, yeast, bacteria, or other calls under the cottrol of appropriate promoters. Cell-ffree translation .systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the mrasent invention.

Appmropriate cloning and expression vectors for use with prokaryotic and eukar-yotic hosts are described by Sambhrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, (198.9), the disclosure of which is hereby incorporated by reference.

Transcription of the DINA enco-ding the polypertides of the present ijavention by higher eukaryotes is increased by insetting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promter to increase its trans cri t ion.

Exanlesinclud-ing the EV40 enhancer on the lL.sdeo h reolication origin bo 100 to 270, a cytomegalovirus: early promoter en'hancer, the Dolyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant e~mression vectors will inclUde origins of replication and selectable m-arkers parmittiLng transfoation of the host cell, the -amicillin resistance gepe of E. coli and S. cerevisiaa TRPl gen-e, and -17a promoter derived from a highly-expressed gene to direct U transcrintion of a downstream structural. sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosnhoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The haterologous structural sequence is assembled in appropriate IND phase with translation initiation and termination sequences, and preferably, a leader seq-uence capable of directing V) secretion of translated protein into the Deriiolasmic space or extracellular medium. Optionally, the hetetologous seqluence can encode a fusion protein including an N-terminal identif ication. iectide imparting desired characteristics, stabilization or simplified purif;ication of expressed recombi-nant. nroduct.

Useful exnression vectors for bacterial use are constructed by inserting a structural DU sequence encoding a desired DroteiLn together with suitable translation initiation and termination signals in operable reading phase with a functional TPromoter. The vector will corrorise one or more phenotypic selectable markers and an origin of renlication to ensure maintenance of. the vector and to, if desirable, provide armlificaticn with-in the host. Suitable orokarvotic hosts for transformation include E. coli, Bacillus 5ubtilis, Salmonellai tv-ohimuriun and vari.ous species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter or' choice.

As a representativ-2 but nonlictiting exaumle, usefrul exoression vectors for bacterial use can cotrirse a selectable marker and bacterial origin of replication derived from cournercially available plasmids cutr-rising. genetic elements *of the well ]mown cloning vector. p3k322 (ATCC 37017),. Such commercial vectors include, for example, DIM223-3 (Pharmacia Fine Chemicals, Utcosala, Sweden) and GEMl (Prornega Biotec, Madison, WI, USA) .These oB?322 "backbone" c-i sections are combined with an appropriate promoter and the U structural. sequ.ence to be e.:.ressed.

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

Cells are typically harvested by centrifugation, cisrupted by physical or chemical means,, and the resulting IND crude extract retained for further nur3ification..

Microbial cells emnloyed in exoression of proteins can c-i be disrupted by any convenient method, includin2g freeze-thaw cycling, sonication, mechanical disruntion, or use of cell lysing agents, such methods are well }mow to those skilled i= the art.

Various marmaLlian cell culture systems can also be erloyed to express recombinant protein. Exairtles of mannalia-n expression systems include the COS-7 lines of monkey idney fibroblasts, described.'by Gluzman, Call, 23:175 (1981) and other cell lines canable of e~ressing a comatible vector, for exaumle, the C127, 3T3, CEO, HeLa and BHK cell lines. Mammalian e:ressicn vectors will' co~rise an. origin of7 reulication, a suitable promoter and enan11cer, and also any necessary ribosome bind±ng sites, polyadenylation site, .solice donor and acceptor sites, (trans cri-otional termination sequences, and 51 flanking nontranascribed secaences. DIUL sequences derived from the spli ce, and Tolyadenylation sites -ay be used to oride the required nontra nscribed genetic elents.

The oolypeptides can be recovered and purified Erom recombinant cell cultures by methods including a,=oninn sulfaLte or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic iztera.ction chromatography, *affinity chromatography, hydroxyla~atite chromatograp~hy and -19lectiJn chrOmatogranhy. Protein refolding steps cam be used, U as necessar, in Cor-eting cofigurati~on of the mature protein. Finally, Ighperformance liudChromatogr~aphy (aPLC) can be employed for final purification steps.

ClThe nolypeptidea of the present invention may be a naturally purified product, or a product of .chemical IND synthetic procedures, or produced by recombinant tecbniaqes from a prokaryotic or euk-aryotic host (for exarle, by bacterial, yeast, higher plant, insect and manmmalian cells in C.culture). Depending upon the host em~loyad in a recobiat Droduction procedure, the polyeptides of the =resent Cl imvention may be glycosylated or may be non-glycosy-iated Polypeptidas of the invention may also include an initial meth-ionine amino acid residue.

As shown in Figures 8 and 9, the V3GF2 polyoeptide of SEQ ID No. 2, mi-nus the initial 4G amino acids, is a potent mitogen for vascular endothelial cells and stiMfllteS their grow.'th and proliferation. The results of a Nor-thern blot analysis performed for the VEGF2 nucleic acid sequence encoding this polyoeptide wherein 20 jig of RNIA from several hu.man tissues were probed with 22 -T)-VEGF2, illustrates that this pDrotein is actively expressed in the heart and lung Which is further ev.Yid--)ce of mitogenic activity.

Accordingly, VEGF2 may,- be emloyea tfo Promote angiogenesij 5 for examp~le, to stimulate the grow-th of (transpDlanted tissue where corona~- 1 bypass su-rgery is performed. VEGF2 may also be errmloyed to prormote wound.,healing, particularly to re-v-ascularize damaged tissues or stimulate collateral blood flow during ischemjia and where new capillary angiogenesis is desired. VEGF2 may be errmloyed to treat full-thic 0 ness wounds such as dermal ulce rs, including Pressure sores, venous ulcera, and diabeticc ulcers. In addition, V-EGF2 may be eTmloyed to treat full-thickess burns and injuries where a skin. graft or flap is used to re-pair such burns and injuries. VEC-F2 may also be emtlyd o s in plastic surgery, for exca= 1e f or the repair of U ~lacerations from trauma and cuts in association with Surgery.

Along these same lines, VEGF2 may be emn2.oyed to Induce the grow-th of damaged bone, neriodontim. or ligament tissue.

V2072 may also be e=-loyed for regenerating .suPuorting tissues of the teeth, including cementum and periodor~tal IND ligament, that have been damaged by disgease and trauma.

Sinc -nige i isiprtant in keening wounds clean V) and non-infected, VRGF2 may bemprloyed inassociation with IND srger andfollowing the' repair of cuts. It may also be emnloyed for the treatment of abdominal wounds where. there is CI a high risk of infection.

VEGF2 may be e=-loyed f-or the promotion, of endothelialization, in vascular graft surgery. in thie case of vascular grafts using either transplanted or synthetic material, VBGF2 can be applied to the surf ace of the graft or at the junction to promote the growth of vascular endothelial cells. VEGF2 may also be employed to repair d~amage of myocardial tissue as a result of. myocardial infarction.

VEGF2 may also be employed to repair the cardiac vascular systemt after isohemia. VEG?2 may also be empnloyed to treat damaged vascular tissue as a result of coronary arte2ry disease'and Peripheral. and 0NS vascular disease.

VEGF2 may also be errnloyed tro coat artificiatz prostheses or natural Organs which are to be transplanted in the body to "u-fiflize rejection Of the transplanted material and to Stimulate ,rascularization of the transplanted material2.

V3C-F2 may als=o be e~loyed for Vagc-Llar tissue repair, for: axamrple, that occurring during arteriosclerosis a-nd required following balloon anzgionlasty where vascular tissues are dan-aged.

-VEGF2 nucleic acid sequences and VEGF2 polypentides may also be eumloyed for in vitro Purooses related to sinii research, synthesis qf DIN and manufactu-re of DNA vectors, and for the production o; diagnostics and theraneutica to -21treat human disease. For example, VEGF2 may be eTMloyed f or in 'vitro culturing of vascular endothelial calls, where it is added to the conditional meclium. in a concentrtion from io Pg/MJ. to 10 ng/Ml.

Fragments of the full length VEGF2 game may be used as a hybridization probe for a cDNA~ library to isolate other INDgenes which have a high seocL..ence sim'ilarity to the gene or Clsimilar biological activity. Probes of this type generally Cl have at least 50 base uairs, although they may have a greater mm-nuber of bases. The probe may also be used to identify a c-DNA clone corresvonig to a full leng-th tramscrirn and a genomic clone or clones that contain the comlete VEG?2 gene including regulatory and promotor regions, exons, an-d introns. An exar~le of a screen co~rises isolating the coding region of the VEGF2 gene by using the known

DNA.

sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence co~lementary to that of the genle of the nDresent invention are used to scree-n a library of hum-ain cDN\A, genomaic DIN or mPIT- to determine which members of the library the probe hybrid-izes to.

This invention prov,,ides methods fA-or identification of VEGF2 r-eceptors. The gene encod-ing the receptor can be identified bDy numerous methods knmown to those of skill in the art, for exa=)le, liganad panning and FACS sorting (Coliganm, et Current 'Protocols in Irmuun., rhaipter ((1991)) .Preferably, exoression cloning is e~loyed wherein polyadsnylated IMA is prepared from a cell responsive to VEGF2, and a cDTMA library created from this RIJk is divided into pDools and used to transfect COS cells or other cells that are not resoonsive to VEGF2. Transfected cells which are grown on glass slides are exiose d to labeled VEGF2.

VEG?2 canm be labeled by a variety of means including* iod-ination or- inclusion of a recognition site -for a sitespecific protein kinase. Following fixation and incubation, the slides are subjected to autoradiog-raphic analysis.

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

C~tAs an alternative armroach for rece-Dtor identification, labeled VEGF2 can be photoaffinity linked with call mem~brane or extract preparations that excoress the receptor molecule.

c-iCross-linked material is resolved by PAGE and e.xoosed to X- (Ni ray f ilm. The labeled co~lex containing VEGF2 is then (Ni excised, resolvAed into peptide fragments, and subjecr-eh to protein ricroseouencing. The amino acid sequence obtained from microseguencig would be used to design a set of degenerate 'oligonucleotide probes to screen a cDNA librar to identify the gene encoding the putative recentor.

This invention is also related to a method of screening cotrounds to identify those which are VRGF2 agonists or antagonists. An example of such a method takes advantage of the ability of VEG?2 to significantly stimulate the proliferation of hxuan endothelial cells in the presence of the comitogen Con A. Endothelial cells are obtained and cultured in G-.well flat-bottomed culture plates (Costar, Cambridge, MA) in a reaction mixture supplemented with Con-A (Calbiochem, La Jolla, CA) Con-A, polypeptides of the present invention and the co~ound to be screened- are added.

After incubation at 37 0 C, cultuxres are pulsed with I jiCi of 3 [H)thym-idine (5 Ci/mnol; I Ci 37 BGq; for a su-ffIcient time to incoroorate the and h~arv~ested onto glass fiber filters (Cambridge Technology, Watertown, MA) .Mean [HI thymidline incorporation (cpm) of triplicate cultures is determined using a liquid scintillation counter (Becu-L=ona Instruments, Irvine, CA). Significant thyrnidine incorporation, .as -conmared to- a control assay where tha co=ound is excluded, indicates stimulation of endothelial call proliferation.

-23- To assay f or antagonists, the assay described above is performed and the ability of the co ound to inhibit thymidine incorporation in the presence of VEGF2 indicates that the comoound is an antagonist to VEGF2. Alternatively, ciVEGF2 antagonists may be detected by co-mbining VEGF2 a-nd a potential antagonist with membrane-bound VRGF2 receptors or IND recombinant receptors under appropriate conditions for a comroetitive inhibition as say. VEGF2 can be labeled, such as by radioactivity, such that the number of VEC;?2 molecules c-i boun~d to the receptor can determine the effectiveness of the IND potential antagonist.

Al1ternatively, the response of a known second messenger system following interaction of VEGF2 and receptor would be measured and conmared in the ioresence or abs-nde of the comDound. Such second messenger systems include but are not lim~i ted to, cAMPi guanylate cyclase, ion channels or phosphoinositide hydrolysis. In another method, a iaitnalian cell or membrane nreparation expressiLng the VEG?2 receptor is incubated with labeled VEGF2 in the presence of the corrmound.

The ability of the co~mound to enh.ance or block this interaction could then be measured.

Potential VEGF2 antagonists include an antibody, or in some cases, an oligonurcltide which bind to the polvoDe-tide and effectively eliminate V G?2_ function. Alternatively, a potential antagonist may be a closely related ptotein which binds to VEc-F2 receptors, however, they are inactive forms of the polypeptide and theraby prevent the action of V-EGF2.

Exam.l e s of these antagonists include a negative do=Lnant mutant of the VEGF2 polyvaptide, for eaale, one chain of the hetero-dimeric for"M of VEGB'2 may be dominant and may be mutated such that biological activity is not retained. An example of a .negative dominmant mutant includes Lr-uicated versions of a dimeric VRGF2 which is capable of interacting with another dimer to form wild type VEGF2, -however, the -24c-i resulting homo-dimer is inactive and fails to exhibit characteristic VEGF activity.

Another potential VEGF2 antagonist is an antisens cIconstruct prepared using antisense technology. Aniserse technology can be used to control gene ex~ression through triple-helix formation or antisense DNA or RNA both Of which IND methods are based on binding of a polynucleotide to DNA. or M i RNA. For exa~le, the 5' coding portion of the V) TDolynucleotide sequence, which encodes for the mnature IC Dolypeptides of the present in-;ention, *is used to design an antisense RUNA oligonaucleotide of from about 10 to 40 base p- airs in length. A DNA- oligonucleotide is designed to be complementary to a region of the gene involved in turpn~cripticn (trinle helix -see Lee et al., T~l cd Res., 6:3073 (1979) Cooney et al, Science, 241:456 (1988) and Der-van et al-, Science, 251- 1360 (1991)), thereby preventing trans crip tion and the production of VEGF2. The antisens a PM oligonucleotide hybridizes to the rnRNA in vivo and blocks translation of the mIUM. mnolecul~e into the VEGF2 polypeptide (Antisense Okano, J. Neurocheam., 56:560 (1991); O1ligodeoxynuc 1 eo tides as Antisenrse inhibitors of Gene Eicoression, CRC Press, 3oca Raton, FL (1988) )The oligonucleotides described above can also be delivered to cells such that the antisemse P-1A or DIRA rray be Fxnressed in vivo to inh-ibit production of 'V3GF2.

Potential VEGF2 antagonists also include smrall molecules which bind to and occupy the active site of the pol1 peptide thereby Traking the catalytic site inaccessible to substrate such that noml biological activity is prevented. REcamp1es of =,all molecules include but are not limited to small peptides or nentide-like molecules.

The antagonIsts may be ermloyed- to treat .limit angiogenesis necessar-! fmor solid t-mnnor metastasis.

The mRNA encoding for VEGF2 is fou-nd to be expressed at rnod,~rate lev,.els in at least two breast tumor cell lines which is ndicative of the role of V3GF2 poolypeptides in the U mlignant phenotype. Gliomas are also a type of neoplasia which may be treated with the antagonists of the Present invention.

The antagonists may also be used to treat chronic inflarmmation caused by increased vascular Permea-bility. In add-ition to these disorders, the antagonists may also be emp~loyed to treat retinopathy associated with diabetes, rheu.matoid arthritis and psoriasi.s.

The antagonists may be e_=lIyed i n a coruositi on with a Pharmaceutically acceptable carr-ier, as herei-nafter described.

The VEC-F2 'Doly-oeptides and agonists and antagonists may be emp~loyed in combination with a suitable uhaf-maceutical carrier. Such conositions co~rise a therapeutically effective amount of the polypeptide or agon-ist or antagonist, and a pharmceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water,. glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also p~rovides a Phalrmaceutical p~ack or k-it comprising one or more containers filled with one Or maore Of the ingredients of the ph.rrnaceutical coositons of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of noharmaceuticals or biological products2, which notice reflects aooroval by the agen-Lcy Or Lmanufacture, use or al fo anadministration.

I

addition, the pharmaceutical coT=Dsiti ons may be er~loyed in conjunction with other therapDeutic co -oun ds.

The phamceutical compositions may be administemred in a .conven ient manner such as by the topical, intravenous, intraperitomeal, intramuscular,- intratinn r, subcuta-neous, *intranasal or intrader-mal rop.tes.- The nhar-macautical -26- CoflOsjtjQns are adm~inistered in an amou-nt -which is effective U for treating and/or prophylaxis of the specific indication.

In general, the pharmaceutical co=-Osjtions are admintstered inl anl amount of at least about 10 pg/kg body weight. and in most. cases they will be administered in an amount not in excess of about 8 mg/Kg body weight Per day. In most cases, INO the dosage is from about 10 j.g/kg to about 1 mg/kg body Ctweight daily, taking into account t he routes of administration, symptoms, etc.

The VEGF2 polypnticles, and agonists or antagonists which are polypeptides may also be employed in accordance with the uresent. invention by express ion of such oolyDeptide an vavo, which is often ref erred to as "gene therapy." Thus, for examole, cells such as bone marrow- cells may be engineered with a polynucleotide (DU or RNA) encodi-ng for the Dolyoeitide ex vivo, the engineered cells are then provided to a patient to be treated with the poly-peptide.

Such methods are well-mown in the art. For axa~le, cells may be. engineered by procedures kniown in the art by use of a retroviral particle containing IRH encoding the polypeptide of the nresent invention.

Simiularly, cells may be engineered in vivo f or exoression of A ~oyet n vi vo, f,:or earb procedures known in the art:' s knaown in the art7 a inroducer cell for producing a retroviral particle containing

RNA

(encoding a oolyot-i de of the present inveantion may be ad:-instered to a matient for engineering cells in vivo and expression of the Doly-e~tida in vivo. These and other methods for administering a polypeptide of the present invention by such methods should be apparent to those skilled in the art from the teachings of the present invention. For example, the exoression vehicle for enginearing' cells may be other than a retroviraj. particle, for axample, an adenovir-us, which may be used to engineer cells in viv ,o after combination With a suitable delivery -Zeicle.

-27- Retroviruses from which the retroviral plasm-id vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Marine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey c-iSarcoma Virus, avian leukosis virus, gibbon aDpe leuke-mia virus, human i~mmodeficiency virus, adenovirus, Myeloroliferative Sarcoma Virus, and mrrUrLary tumor -VirUS.

in one embodiment, the retroviral Dlasmdid vector is derived c-i from Moloney Murine Leukema Virus.

CIThe vector includes, one or more promoters. Suitabl~e promoters which may be e~loyed include, but are not limited the retroviral LTR; the SV40 promoter; and the human cy-tomegalovirus (CMV) promoter described in Miller, et al., Biotechniques., Vol. 7, No. 9, 980-990 (1989), or- any other prormoter cellular promoters such as eukaryotic cellular mromoters including, but not limited tLo, the histone, po 1 111, and -actin promoters) Other viral promoters which may be enmloyed include, but are not limited to, adenovirus nromoters, thymidine kinase (TXr) promoters, and 219 narvovirus oromoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequence encodling the polypeptide of the Doresent invention is u~nder the control of-a suitable promoter. Suitable promoters which may be emoloyed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (PSV) promoter; inducible -oromoters, such as the MM promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the AmoAI pDromoter; human globin promoters; viral tbymidine kinase promoters, such as the Heroes Simp lex thymidine kiLnase promoter; retroviral L71P- (including the m odi-Ei e d retroviral LTP-s hereiLnabove described) the -actin -promoter; and human g-rowth hormnone -28nromoers.The promoter also may be the native p~romoter U which controls the gene encodling the polypeptide.

The retrorviral -olasmid vector is e -loyed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but- are not limited to, the PES0l, PA.317, 4'-AM, PA-12, T19-14X, VT-19*-17-H2, P 1 E, IP, G+-86, GP+enavAx-,ad A l lines as described in Miller, Ruman Gene Therapy, Vol. 1, kn ugs. 5-14 (1990), wh~ich is incorporated herein by reference IND in its entirety. TIhe vector may transduce the packaging cells through any means known in the* art. Such means include, but are not limited to, electr-onoration, the use of liposomes, and CaPQ 4 precipitation. in one alternati-ve, the retroviral plasmid vector may be orncansulated into a liposorme, or coupled to a lipid, and then administered to a host:.

The producer cell line generates infectious retroviral vector particles which include the nucleic acid seqruence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic: cells, either in vitro or in vivo. The transduced eukaryotic cells will excpr e ss the nucleic acid sequence Cs) encoding the poJlypeptide. Eukaryotic cells which may be transduced include, but are not lim ited to, embryonic -s-tem cells, embryonic carcinoma cells, as well as hematooietiLc stem cells, hepatocytes, fibroblasts, myoblaats, keratinocytes, endothelial cells, and bronchial eoithelial cells.

This inveantion is also related to the use of the VE-G?2 gene* as part of a d-iagnostic assay for detecting diseases or susceptibility to diseases related to the presence or mutations in VEGF2 nucleic acid sequ-ences.

Individuals carrying mutations- in the VEGF2 gene ray be detected at the DNA level by a variety of techaniqueS.

Nucleic acids for diagnosis may be obtained from a patient's cells, such as from.blood, urine, saliva, tissue bio-osy and.

-29autopsy material. The genomic DNA may be used directly for detection or may be alifi ed enzymnatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.

PM or cDNA may also be used f or the same purpose. As azn exarrole, PCR urimers complementary to the nucleic acid encoding VEGF2 can be used to identify and analyze V-3GF2 IND mutations. For axa~1e, deletions a-nd insertions can be Cl detected by a change in size of the alif ied product in comoarison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled VEGF2 RNA. or alternatively, radiolabeled V3GF2 antisense

DNA

secuences. Perfectly matched sequences can be distinguished from mismatched duplexes by PNase A digestion or by differences in meliting tcemeratures.

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

DNA

fragments of different seq-uences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DIUA fragments are retarded in the gel at different posiion;according to their Sneci-Fic melting or Dartial melting temneratures (see,* Myers et Science, 230:1242 (1925)).

(Sequence changes al; specific locations may also be revealed by nuclease protection assays, such as ?-Nase and S1 protection Or the chemical cleavage method Cotton et: al., PNA S, UJSA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNU sequence may be achieved by methods such as hybridization, RNase nrotection, chemnical. cleavage, -direct DNA sequencing or the use o"E restriction enzymes, Restriction Fragment Length POlymorohisms (RFLP)) and Southern blotting of gelnomic DNA.

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

The present invention also relates to a diagnostic: assay for detecting altered levels of VEGF2 protein *in various tissues since an over-expression of the proteins CULL -ared to INO normal control tissue sampnles may detect the presence of a C~t disease or susceptibility to a disease, for exnle, abnormal cellu-lar dif ferentiation. Assays used to detect levels of VEG?2 'or~tein in a sa le derived 4f'roma host are'well-kmown to those of skill in the art and include radioi~riunoas says, Cl cot~etitive-binding assays, Western Blot analysis, ELISA assays and nsandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Immunology, Chapter 6, (1991) initially comtorises prepa-ring an antibody speci-fic to the VEGF2 antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the maonoclonal antibody. To the reporter antibody is attached a detectable reagent sucha as radioactivity, fluorescence or, in this examole, a horseradish peroxidase enzyme. A: sairrole is removed from a host and incubated on. a solid sunocrt, e.g. a polystyrene d-ish, that binds the proteins in the sa-rple. Any free mrotein binding sites on *the dish are then covered by incubating with a non- specific -orotein, such as, bovine serum albumnen. Next, the mnonoclonal antibody is incubated in the dish during which time the monoclonal antibod-ies attach to any VEGF2 proteins attached to the. polystyrene dish. Al'i unbound monoclonal antibody is washed out with buffer Tr'e reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any muonoclonal antibody bound to VEGF2. Unattached reporter antibody is -then washed-out. Peroxidase substrates are -then added to the dish and the amount of color develo-Ded in a given time period is a measureme.nt of the amount of \TEG?2 -31-

IO

O protein present in a given volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to VEGF2 are attached to a solid support.

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

S* A "sandwich" assay is similar to an ELISA assay. In a S"sandwich" assay VEGF2 is passed over a solid support and binds to antibody attached to a solid support. A second antibody is then bound to the VEG?2. 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 for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an ndi-vidual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphism's) are presently available for marking chromosomal location. The 'mapping of DNAs to-chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

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

Computer analysis of the cDNA is used to rapidly select primers that do not span more than one axon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those -32-

NO

c- hybrids containing the human gene corresponding to the primer 0 will yield an aimlified fragment.

d) SPCR mapping of somatic cell hybrids is a rapid procedure C for assigning a particular DNA to a particular chromosome.

Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of N fragments from specific chromosomes or pools of large genomic C clones in an analogous manner. Other mapping strategies that I) can similarly be used to map to its chromosome include in \O situ hybridization, pretcreening with labeled flowsorted O chromosomes and preselection by hybridization to construct l chromosome specific-cDNA libraries.

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

For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques. 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 examole, in V. McKusick, Mendelian Inheritance in Man (available -on line through Johns Hopkins University Welch Medical Library). The relationship between Sgenes 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 manning and genetic U mapping techniques, a cDNA precisely localized to a chromsomal region associated with the disease could be one o'f between 50 and 500 potential causative genes. (Thi s assumnes 1 megabase mapping resolution and one gene Per kb).

IND The rolypentides, their fragments or other derivatives, or analogs thereof, or cells expmressing them ca-n be used as an inmunogen to produce antibodies thereto. These, ant-ibodies Clcan be;' for exar=-le, polyclonal or monoclonal antibodiaes.

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

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

Such anti~bodies can then be': us~ei to isolate the-p-lypeptide from, tissue exoressing that Dolyoeptide. For preparation of (ofoonl atibodies, any technniorue which Drovides antibodies pDroduced by continuous cell line cultures can be used. Examoles include the hybridona technique (Kohler and Milstein, 1975, Nature, 256:495-497), the triom technique, the humran 3-cell hybridoma technique CKozbor et al., 1983, Iu~nunology Today 4:72), and the =V-hybridoma technique to produce human moonoclonal antibodies (Cole, et al.* 1985, in Monoclonal Antibodies an-d Can~cer Thh'-apy, A-lan R. Liss, Inc., PP?. 77-96).

-34c-i Techniques described for the production of single chain U antibodies Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products c-iof this invention. Also, transgenic mice may be used to exuress humranized anti-bodies to itrnnuogenic polypeptide Vroducts of this invention.

INO The mresent invention will be further described with M i reference to the following examples; however, it is to be kn understood that the tDresent invention is not limited t-o such eaales. All: parts or amounts, unless otherwise s-oecified, are by weight.

CiIn order to facilitate understandi-ng of the ollowing exarmles, certain freantly occurring methods and/or- tern will be described.

"?lasrnids" are designated by a lower case p) preceded and/or followed by capital letters and/or numibers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmnids- in accord with published procedures. In addition, eo~uivalentL Dlasmids to those described a-xe Imown in the art and will be an-Darent to the ordinarily skilled artisan.

"Digestion" of DM.A refer s to catalytic cleava-e of the DNMA with a restrict-on entym~a that acts only-at certain seq-uences in the DNA. The various restriction enzymes used herein are carmercially available and their reaction conditions, cof actors and other remuiraments were used- as would be knaown to the ordinarily skilled artisan. For analytical purposes, typically 1 gzg of olasmid or DNA fragment is used with about 2 units of enzyme in a-bout 20 91L of buffer solution. For the Tuxoose of isolating DNA fragments for olasmaid construction, typically 5 to 50 y9i orL DNA are d-igested with 20 to 250 units of enzyme in a larger volumne. .Appropriate buffers and substrate amounts for particular restriction enzymes are specified the c-imanuf acturer. Incubation times of about J. hour at 37 0 C are U ordinarily used, but may vary in accordance Wt h sUixJlier's instructions. A-fter digestion the reaction is c-i electro~horesed directly on a pnolyacrylanide gel to isolate the desired fragment.

Size se~aration of the cleaved fragments is performed using 8 pDercent polyacryamde gel descri-bed by Goeddel,

D.

et al., Nucleic Acids: R~es., 8:4057 (1980).

V)."lionclo-ids refers to either a single Gtranded INO Dpolydeoxynuclectide or two co -lementary polydeoxyn-ucleotjde strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligomucleotide 'without adding a phosphate with an ATIO in the presence of a kinase. A synthetic oligonuclectide will ligate to a fragment that has not been depho sphorylated.

"Ligation" refers to the process of f crarng phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, et Id., p. 146) Unless otherwise provided, ligation may be acco~lis hed using ]=iown buffers and conditions with 10 units of T4 DNA ligase ("ligasen) per 0.5 pg of approximately equ-imolar amounts of the DNA fragmet tbelgted.

Unless otherwise stated,' tans for mat ion was 'aerformed as described by the -method of Gra-ham, F. and Van der Rb, A., Virology, 52:456-457 (1973).

Exairmle 1 ~resionDaternof VEGF2 in h=L;enT tissues and breast cancer cell lines Northern blot analysis -was carried. out to exazine the levels of: e;,mwession.of the V3GF2 gene in 22=Lan tissues and human breast cancer cell lines. Total cellular P.NAu samnles were isolated with P1ZAzol"" B system (Biotecx Laboratories, *Inc.) About 10 pg of total PM isolated frmm each breast -3G-

UN

-37tissue and cell lie specified was separated- on I1 azarose gel and bloted onto a nylon filter, (Molecular Cloning, Sam-brook Fritsch, and Maniatis, Cold Spvr"g Harbor Prcs3, 1989). The INO Labeli-ngC Traction was dont according to the Sti-atag"ene Cloning Systems, Inc., Prime-It kit with 50 ng DNA fragment. The labeled DNA was purified wit a Select-G-5O colujn from 5 Prime 3 Primne, Inc., Boulder, CO, USA. The filtcr was then hybridized with INO radioactively labe-led Mul lngth VEGF2 gene at 1,OO0,CO0Ocpin/rl in 0.5 M Na.P0 4 and 7% SDS overnight at 65'C. Aftcr washing twice at room teroperanire and twice at 60'C wirh x SSC, 0.15% SDS, the fdltm- were- then exposed at -70'C overnight with. ani intensifyingscreen. A message of 1.6 Kb was obser-ved in 2 breast cancer cell lines.

Example 2 Cloning and expression of VECGF2 using the baculo-,irus; expression systenm Th3e DNA sequence =2coding the VEGF2 protein without 4-6 am-ino acids arnd the Nterminus, see ATCC Acce-ssion No. 97149, was amplified using PCR oligonuclcotidt primers corresponding to the 5' and 3' sequences of the tyene: Tae 5' primer has The sequence TGT AAT ACG ACT CAC TAT AGG GAT CCC GCC ATG GAG 0CC ACG OCT TAT GC (SEQ ED NO:7) and contains a BamH I re.striction enzyme site (in bold) and 17 nucleotidz nucleotide sequzncc complerneatary to zthe 5' sequcnce of YEGF2 (nt. 150-166).

Thbe 3' Drimer has the- sequence GATC TCT AGA TIA GCT CAT T TG TGG TCT (SEQ ID NO:8) and contains the cleavage sit= for the restrictiom. enzyme- XbaI and 18 nucleotides comple-mentary to the 3' sequence of VEGF2, including te sionD codon and 15 nt sequence before stop codon.

The amplified sequences were isolated ,ro*M a 1 agarose gel using a commercially available kit ("Geneclean," BIO 101, Inc., La Jolla, CA). The fragment was thrn di.-ested with thc CI endonucjlease BarnHj and )2aI and then purified again on a 1% U agarose gel. This fragment was ligated to pAcGPG7A baculovirus transfer vector (P~armingen1) at the BamEl and XbaI sites. Through this ligation, VEC-?2 c DIN was cloned in frame with the signal sequence of baculovirtis gDO-7 gene and INO was located at the 3' end of the signal sequence in the vector. This is designated npkcGP67A-V"EGF2.

To clone VEGF2 with the signal sequence of gp67 gene to the rRGl vector for exnress ion, VEGF2 with the signal INO sequence and some upstream sequ~nce were excised from the p)AcGP67A-VEGF2 olasmid at the Xho restriction endonuclease site located unstream of the VEGF2 cDNA and at the XhaI restric"ion endonuclease site by XhoI and XbaI restriction enzyme. Th11is fragment was separated from the rest of vector on a agarose gel and was purified using "Geneclean" kit.

It was designated F2.

The PRGl vector (modification of pVL941 vector) is used for the e--,ression of the VEGF2 protein using the baculovirus exoression system (for review see: St~~ers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture oroc edures, Texas Agricultural Exoerimental Station Bulletin -No.*1555) Tlis exoression vector contains the stronu polyhedr-4n Promoter of the Autogranha californica nucleat polyhedzosis vir-us (AcYNPV) followed by the recognition sites for the restriction endonucleases Bam~l, Sinal, 72baI, BglII and Asn7lB. A site for restriction endonuclease Xhol is located unstream of BamH1 site. The sequence between Xhol and 3anRI is the same as that in PAcGp67A (static on ta-pe) vector. Thne polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant virUS the beta -galactos idase6 gene from E.coli is inserted in the same orientation as the polyhedrin p~romoter followed by the polyadanylation signal of the p~olyhedr2.fLn gene. The polyhedrin sequences qre flanked at bot-h sides by -3B- -Viral sequences for the cell-mediated homologous recombination of cotransfected wild-ty-pe viral DNA. many other baculovirus vectors could be used in place of. pRG1 such as DAc373, _VL -1 and nAcIM1 (Luckow, V.A. and Sunuers, M. D., Virology, 170:31-39).

INDThe 'olasraid was digested with the restriction enzymes Xb~oI and XbaI and then denhosphoryla'ced using calf intestinal phosphatase by procedures known in the art. The DRA was then isolated from a agarose gel using the comrmercial~ly IND available kit ("Genecleen"l BIO 101 Inc.,I La Jolla, Ca.).

This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli H3101 cells were then transformed and bacteria identified that contained the plasmid (pBac gpG7-VEGF2) with the VEGF2 gene using the enzymes Bamal and XbaI. The sequence ofE the cloned fragment was confirmed by DNA sequencing.

p~g of the plas-mid p~ac gD67-VEGF2 %,as cotransfected with 1. 0 pg ofL a commercially available linearized baculovirus ("BaculoGold" baculovirus DNA" ?harmningen, San Diego, CA.) using the lioofection method (Felgn er et al.

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

of BaculoGold"' vir-us DNA and 5 juzg of the plasraid p-lac goG7-VEGF2 were mixed in a sterile well of a-microtiter plate containing 50 4l of serum free Grace's medium (Life (Technologies Inc., Gaithersburg, M'D) Afterwards 10 p1l Lipofectin plus 90 4i Grace's mediu were added, mirxed anid.

Incubated for 15 minutes at room temoerature. Then the transfection mixture was added dropDwise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with I ml Grc s medium -without serumn. Tae plate was rocked back and fcorth 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 mli Of, Grace's insect medItm, suv),lemented with 10%- fetal calf seruzr -39- CIwas added. The plate was put back into an incubator and cultivation continued at 27WC for four days.

After four days the supernatant was collected and a plaque assay performed similar as described by Suimers and Smith (suiora) As a modification an agarose gel with "Blue IND Gal" (Life Technologies Inc., Gaithersburg) was used which M allows an easy isolation of blue stained pla-ues. (A detailed description of a "-olaque assayn can also be found in the user's guide for insect cell culture and baculovirolo 9 -y IND distributed by LiJf6 Technologies Inc., Gaithersburg, page 9- Four days after the serial dilution, the virus was added to the cells, blue stained plaques were picked with the ti-D of an Epne-ondorf pipette. The agar containing thercobnt viruses was then resuspended in an -Eopendorf tube containing 200 Al of Grace's mediumi. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect Sf9 cells asded in 35 mmn dishes. Four days later the supernatants of these culture dishes were harvested and then stored at 4'C.

Sf 9 cells were grown in Grace' s medium supplemented with heat -inactivated -FBS. The cells were infected with the recomrbinant baculovirus V-9-,G7-VREGF2 at a mutpiiyof infection (MOI) of 1. Six hours later the mediumi-was removed and replaced with SF900 II medium minus rnethionine and cysteine (Life Technologies Inc., Gaithersburg-) 42 hours later 5 uCi of S-mrethionine and 5 Ar-i "S cysteine (Amersham) were ~added. The cells were further incubated for IS hours before they were harvested by centrifugation and the la-belled mroteins visualized by SDS-PAGE and autoradiography.

Protein from the mediumn and cytoplasm of the Sf9 cells was analyzed by SIDS-PAGE under -reducing and non-reducing conditions. See Figure 4. The medium was dialyzed against mM MES, pH,5.8. Precuitates were obtained after dialysis and resuspended in. 100 mm NaCitrate, pH 5. 0. The resuspanded

UN

precipitate was analyzed again by SDS-PAG3 and was stained with Coomassie Brilliant Blue. see Figudre The medium supernatant was also diluted 1:10 in 50 rM pH 5.8 and applied to an SP-650M coltm= (1.0 x 6.6 cm, Toyopearl) at a flow rate of 1 Tal/flif. Protein was eluted C1with step gradients at 200, 300 and 500 mM K{aCl. The VEGF2 was obtained using the elutioi at 500 rmm. The eJluate was C~t anayzed by SDS-?AGR in the presence or absence of reducing agent, -mercaptoetbanol and stained by Coomassi Brilliant Blue. See Figure 6.

E>Mression of Rec~bnft VG2 i O el The emression of p1as-mid, VBEGF2-HrA is derived from a vector orcDNAI/Amp (Invitrogen) conltai~ning: 1) SV40 origin of replication, 2) a~icillin resistance gene, 3) E coJli replication origin, 4) Q'MV promoter followed by a poly.ilker region, an SV40 intron and polyadenylazion site. A DNA fragment encoding th e entire VEGF2 precursor and 2L HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore*, the recombinlant protein expression is directed under th~e C14V prowter. The. HA tag corresponds to an epitope derived f ro th e influenza hemagglutiflin protein as previously described Wilsonl,

H.

(Niman, R. Heighten, A Cerelson, M. Connolly, and R.Lrnr 1984, Cell 37:767, (1984) The illfusion of HA tag to the target protein allows easy detection of the recombinant protein with an a-ntibody that recO'nazes the HA. epit~ope.

The. plas~id ccns t--uction strategy is described as The DNA s3eie ncoding VEGF2, ATCC Acceuion No. 97149, was consmiuctt-d by PCR using two primers: the 5' rorimcr (CGC GOA TCC ATG ACT GTA CTC TAC CCA) (SEQ ID NO:9) contains a BamrH! site followed by 18 Pucletidr-s of VEGF2 coding sequencc starting from the initiation codon; the 3' sequencc (CGC TCT Ar-A TCA. AC:C GTA GTC TS GAC 9rC CTAA 7?G GIL c~A GA r C:% C1Mi T=C T= (320 1D NO: 10) Conta.ins iena=a=y sequ-cesto an X :62I site, ZA tag, X-b=3 site, a--d the last: is nucleotides of the VEG-F2 coding mem-ce (not including the stop codon) -heref ore, tbe PCR Product containzi a Bar-nzi site, coding seuence folloved by n X-h0I r~str-ic- Jon endo nucleage site &od IRA tag fused im frane, a tralzaion IND teriati o oon nex to the H;A tag, and a= XThaI site.

The ?CR =plified Ma frImagent anmd the vector'. pcrz~/zp Warm digested wit~h B and X~al restriction ny ligated. The ligation =Ixzure ums zraz~forned into 3. coji strain SURS (St aqee Clomn~ig Syurte-s, Jolla, CA -92037) the zran-sforad cltu~e was plated on a~icill-in media places and rtgir-tant colon4-es wreselected. Plasmid EVA vas isolated fro= renfox-ma-cs and examin:ed by restriction analysis for nuie presence of =he correct fragment. ?or a~ressirn of! che .recombinant v3G?2, CO~S cells war-e transfecced vit the expression vector- DF.A-D7AN mechIod Sambrook, R. Fr-itsch, T. Mamatis, K'nlec'.2.lar- Clominmg A Laboratory m"amual, Cold Sp-ring La-hcrarory Presa, (1989)) 1he e.-cDression of the V2ZG?2-EA proteim was demected bry radinlabelling mmd j neiia nmethmd 2ar-low, D.

Lane, Antibodies; A Laboratory manua, Cold Spring -:az!bor Laborator-y Lresa, 'Cells were labelled-for S bn'ars wIth '"S-cy-steime two days pastr- .etn Citu.z-e media was =hncollected and cells wert- lysed vith dete rgemc (RIPA buffer (1S0 =4 Xa~l, it SP-40, O.l't SDS, It 0.5t- DOC, 50=L1 TriS, pH 7-5) (Wilson, 1. et al., 1d. 37:76*7 (1994)) Both cell lysate and cultur-e media were precipitated wiLthi an HaA -pe :fic =ooclcmal ancb .Pr-tEm4n= pr~eciPita-Zed were analyzed on 15t SDS-PAG3 gals.

The sf fect of Dartialiv--mrified 'EG?2 .nrotein on te~o~ of vpsc-ulax endonthelial cells -42- Cl on dlay 1, human umbilical vein endothelial cells (HUVEC) were seeded at 2-5x.104 ceJlls/35 T-mn dlish density in M199 medium containing 4 fetal bovine serm (173S) 16 units/m1 heparin, CI and S0 units/rn1 endotbelial cell growth supplemnents

(ECGS,

Biotechnigtle, Inc.) on day 2, the medium was replaced with M199 containing 10% F3S, 8 units/mfl he-par.n. VBGF2 protein of SEQ ID NO. 2 minus the initial 45 amino acid residues, Cl (VEGF) and basic FGF (bFGF) were added, at the concentration shown. On days 4 6, the medium was repDlaced. on clay 8, N INDcell number was determnfed with a C04ulter Counter (See Figure Exatrole The effect of pourified VEG?2 nroteafl on the ocrrow th of vascular endothelial cells on day 1, human umbilical vein endothelial cells (R1JVEC) were seeded at 2-5 x 104 cells/35 =dish density in M199 medium containing 4% fetal bovine serum (F 16 units/mid heparin, S0 units/ml endothelial, cell growth supplements (ECGS, Biotechniq~ue, On day 2, the medium was replaced with M199 containing 10% FES, B units/ml hevarin.

Purif ied VEGF2 urotein of SEQ ID No. 2 minus initial 45 amino acid residues was added to the medium at this point. On days 4 6, the medium- was rtplaced with fresh-mtedium~f and supplements. On day 8, cell number was determ-ined with a Coulter Counter (See Figure 9) Expression via Gene hrapy Fibroblasts are obtained from a subject by skin bio-psy.

The resulting tissue is placed in ti-ssua-culture med-ium and separated into sml ieces. Small chunks of tile tissue are placed on a wet sur face of a tissue culture flask, app'roximately ten pieces are.vplaced in each flask. The flask is turned upside down, closecl tight and left at room -43-

NO

c- temperature over night. After 24 hours at room temperature, U the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media Ham's ?12 C( media, with 10% F3S, penicillin and streptomycin, is added.

This is then incubated at 37 0 C for approximately one week.

At this time, fresh media is added and subsequently changed

\O

pq every several days. After an additional two weeks in q culture, a monolayer of fibroblasts emerge. The monolayer is in trypsinized and scaled into larger flasks.

\NO pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine C-i sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and 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 EindIII 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 ligation-mixture is used to transform bacteria B3101, 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 (DMM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is than 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- "?resh M.edia is added to the rtransductd producer cells, and subsequentlY, the media is 1.a=rvested fr= a 10 cm plate Uof conifluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remve detached produce~r cells and this media is then used to infect fibro-blast cells. Media is removed from a sub-con-FlJuent Dlate Of fibroblasts and qu2ickly replaced wit-h the med-ia fr;. tbi- producer cells. ThiS media is remv-ed and rEmlaced %i-th fresh medl!a. if the titer of *i=-us ciis high, thenP- virtujAllY all fibroblas5ts will be- infected and no selection is required. If the tite-r is v--ry low, then it is necessary to use a'retrovi.a~vco hthsaslcal maker, such as neo or The engineered fibr-oblasts are- th'e" inj ected into the host, either a-lone or after having 91en grn to co=-flu ence on cyrzodex 3 microcar-rier b--ad-s The fiLbrblasts now pro~duce the protein product.

Surnprous modifications a-nd variationls of the present invention are oossible in light of the above teacbhings and, therefore, with-in the scape Of the appended- clains, the invention May be practiced otherwise than &2 P&Zrti--u-a~rlY described.

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

Claims (41)

1. 2. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes the full length polypeptide as set forth in SEQ ID NO: 2.
2. 3. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes the mature protein portion of SEQ ID NO:2.
3. 4. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes the proprotein portion of SEQ ID NO: 2. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes the human VEGF-2 polypeptide encoded by the cDNA in ATCC Deposit No. 97149.
4. 6. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes Sa polypeptide comprising amino acids -46 to 373 of SEQ ID NO: 2.
5. 7. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a polypeptide comprising amino acids -23 to 373 of SEQ ID NO: 2.
6. 8. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a polypeptide comprising amino acids 1 to 373 of SEQ ID NO: 2. 9: An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a portion of the mature VEGF-2 polypeptide comprising amino acids 24 to 373 of SEQ ID NO: 2. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes N O c a polypeptide comprising amino acids -46 to 24 of SEQ ID NO: 2. S11. An isolated polynucleotide according to claim 1 wherein the polynucleotide encodes a polypeptide comprising amino acids -23 to 24 of SEQ ID NO: 2.
7. 12. An isolated polynucleotide according to claim 1 wherein the polynucleotide comprises Qa polypeptide comprising amino acids 1 to 24 of SEQ ID NO: 2.
8. 13. An isolated polynucleotide according to claim I wherein the polynucleotide comprises a fragment of the polynucleotide according to any one of a) to d) with the proviso that said polynucleotide fragment comprises at least 30 contiguous nucleotides of the polynucleotides of j) or k).
9. 14. An isolated polynucleotide according to claim 1 wherein the polynucleotide comprises a fragment which hybridises to at least 30 contiguous nucleotides of the polynucleotide encoding amino acids -46 to 24 of SEQ ID NO: 2 under the following conditions: hybridisation in 0.5 M sodium peroxide NaPO, 7% sodium dodecyl sulfate (SDS) at 0 C and washing with 0.5 x SSC, 0.1% SDS at 60°C or equivalent hybridisation Sstringency. An isolated polynucleotide according to claim 1 wherein the polynucleotide comprises the complementary form of the polynucleotide according to any one of claims 2 to claim 14.
10. 16. An isolated polypeptide comprising at least 30 amino acid residues and having VEGF2 biological activity further comprising a member selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of the full length polypeptide of SEQ ID NO:2; b) a polypeptide comprising the amino acid sequence of the mature protein of SEQ ID NO: 2; c) a polypeptide comprising the amino acid sequence of the proprotein portion of (O o -58- C SEQ ID NO: 2; d) a polypeptide comprising the amino acid sequence of the mature polypeptide ,O encoded by the cDNA contained in ATCC Deposit No. 97149; a polypeptide comprising amino acids -46 to 373 of SEQ ID NO: 2; f) a polypeptide comprising amino acids -23 to 373 of SEQ ID NO: 2; g) a polypeptide comprising aniino acids 1 to 373 of SEQ ID NO: 2; h) a portion of the mature VEGF-2 polypeptide comprising amino acids 24 to 373 of SEQ ID NO:2; i) a polypeptide comprising amino acids -46 to 24 of SEQ ID NO: 2; j) a polypeptide comprising amino acids -23 to 24 of SEQ ID NO: 2; k) a polypeptide comprising amino acids 1 to 24 of SEQ ID NO: 2; 1) a polypeptide comprising an active fragment of the VEGF2 polypeptides according to any one of a) to d) with the proviso that part of said polypeptide fragment is encoded by at least 30 contiguous nucleotides of the polynucleotide encoding the polypeptide of any one of j) or k); m) a polypeptide fragment comprising an amino acid sequence encoded by a polynucleotide sequence which hybridises to at least 30contiguous nucleotides of the polynucleotide encoding any one of the polypeptides of j) or k) under the following conditions: hybridisation in 0.5 M sodium peroxide NaPO 7% sodium dodecyl sulfate (SDS) at 65 C and washing with 0.5 x SSC, 0.1% SDS at 60°C or equivalent hybridisation stringency.
11. 17. An isolated polypeptide according to Claim 15 comprising the amino acid sequence of the full length polypeptide of SEQ ID NO:2.
12. 18. An isolated polypeptide according to Claim 16 comprising the amino acid sequence of the mature portion of SEQ ID NO: 2.
13. 19. An isolated polypeptide according to Claim 16 comprising a polypeptide comprising the amino acid sequence of the proprotein portion of SEQ ID NO: 2. IO S.-59- c 20. An isolated polypeptide according to Claim 16 comprising the amino acid sequence of the mature VEGF-2 polypeptide encoded by the cDNA contained in ATCC Deposit SNo. 97149. (N l
14. 21. An isolated polypeptide according to Claim 16 comprising amino acids -46 to 373 of §SEQ ID NO: 2.
15. 22. An isolated polypeptide according Claim 16 comprising amino acids -23 to 373 of SEQ ID NO: 2.
16. 23. An isolated polypeptide according to Claim 16 comprising amino acids 1 to 373 of SEQ ID NO: 2.
17. 24. An isolated portion of the mature VEGF-2 polypeptide according to Claim 16 comprising amino acids 24 to 373 of SEQ ID NO: 2. An isolated polypeptide according to Claim 16 comprising amino acids -46 to 24 of SEQ ID NO: 2.
18. 26. An isolated polypeptide according to Claim 16 comprising amino acids -23 to 24 of SEQ ID NO: 2.
19. 27. An isolated polypeptide according to Claim 16 comprising amino acids 1 to 24 of SEQ ID NO: 2.
20. 28. An isolated polypeptide according to Claim 16 comprising a fragment of any one of the polypeptides of Claims' 16 to 20 with the proviso that part of said polypeptide fragment is encoded by at least 30 contiguous nucleotides of the polynucleotide encoding the polypeptide of any one of Claims 25 to 27.
21. 29. An isolated polypeptide according to Claim 16 comprising an amino acid sequence \O 0 CK CU N encoded by a polynucleotide sequence which hybridises to at least 30 contiguous nucleotides of the polynucleotide encoding any one of the polypeptides of Claims \0 to 27 under the following conditions: hybridisation in 0.5 M sodium peroxide NaPO, Y7% sodium dodecyl sulfate (SDS) at 65 C and washing with 0.5 x SSC, 0.1% SDS tn at 60 0 C or equivalent hybridisation stringency. O An isolated polypeptide according to any one of Claims 16 to 29 further comprising S a heterologous polypeptide.
22. 31. An isolated polypeptide according to any one of Claims 16 to 30 further comprising a homodimer.
23. 32. An isolated polypeptide according to any one of Claims 16 to 31 wherein the polypeptide is glycosylated.
24. 33. A composition comprising the polypeptide according to any one of Claims 16 to 32 or 40 and one or more pharmaceutically acceptable carriers and /or diluents.
25. 34. A vector comprising the polynucleotide according to any one of Claims 1 to A recombinant vector comprising the polynucleotide according to any one of Claims 1-15 operatively associated with a regulatory sequence that controls gene expression.
26. 36. A host cell comprising the polynucleotide according to any one of Claims 1-15 operably associated with a heterologous regulatory sequence or a vector comprising same.
27. 37. The polynucleotide sequence according to any one of Claims 1-15 further comprising a heterologous polynucleotide.
28. 38. The polynucleotide sequence of Claim 37 further comprising a polynucleotide which IO O O -61- encodes a heterologous polypeptide. \0 39. A method for producing a VEGF-2 polypeptide at least comprising the step of Ce¢ C culturing the genetically engineered host cell of Claim 36 for a time and under V conditions suitable for the expression of the polypeptide encoded by said i polynucleotide to occur. O A polypeptide produced by the method of Claim 39.
29. 41. A composition comprising the polynucleotide according to any one of Claims 1-15, 37, or 38 and one or more pharmaceutically acceptable carriers and/or diluents.
30. 42. Use of the polynucleotide according to any one of Claims 1 to 15, 37 or 38 or the polypeptide according to any one of Claims 16 to 32 or 40 in the preparation of a medicament for the treatment of a patient having need of human VEGF-2 polypeptide.
31. 43. An antibody which is capable of binding to the polypeptide according to any one of Claims 16 to 29 with the proviso that the antibody is not capable of binding to a polypeptide consisting of amino acid residues 24 to 373 of SEQ ID NO: 2 or a fragment thereof.
32. 44. An antisense construct capable of binding to the polynucleotide according to any one of Claims 1-15 or a complementary form thereof with the proviso that the antisense construct is not capable of binding to a polynucleotide sequence encoding amino acids 24 to 373 of SEQ ID NO: 2 or a fragment thereof. A method of stimulating proliferation of endothelial cells in a patient comprising administering to the patient the polypeptide according to any one of Claims 16 to 32 or 40 for a time and under conditions sufficient for the proliferation of endothelial cells to occur. IO O U 62 C 46. The method of Claim 45 wherein the patient has vasculature tissue damage. O 47. The method of Claim 45 wherein the patient has a wound, tissue damage or bone Sdamage.
33. 48. The method of Claim 45 wherein the patient has ischemia. O
34. 49. The method of Claim 45 wherein the patient has myocardial infarction. The method of Claim 45 wherein the patient has coronary artery disease, peripheral vascular disease or CNS vascular disease.
35. 51. The method according to any one of Claims 46 to 50 wherein the stimulation of endothelial cell proliferation is capable of further stimulating angiogenesis.
36. 52. The isolated polynucleotide according to any one of Claims 1 to 15, 37, 38 or 44 substantially as hereinbefore described with reference to the Figures and /or Examples.
37. 53. The isolated polypeptide according to anyone of Claims 16 to 32 or 40 substantially as hereinbefore described with reference to the Figures and/or Examples.
38. 54. The vector of Claim 34 substantially as hereinbefore described with reference to the Figures and/or Examples. The host cell of Claim 36 substantially as hereinbefore described iith reference to the Figures and/or Examples.
39. 56. The method according to any one of Claims 39, 45 to 51 substantially as hereinbefore described with reference to the Figures and/or Examples.
40. 57. The use according to Claim 42 substantially as hereinbefore described with reference 0 -63 to the Figures and/or Examples.
41. 58. The conposition of Claima 33 or 41 substantially as hereiDbefore describced wi th ci refrence: to the Figures and/or Examples.