CA2224002A1 - Human vascular endothelial growth factor 3 - Google Patents

Abstract

Disclosed is a human VEGF3 polypeptide and DNA (RNA) encoding such VEGF3 polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques and antibodies and antagonist against such polypeptide.
Also disclosed is a method of using such polypeptide for stimulating wound healing and for vascular tissue repair. Also provided are methods of using the antagonists to inhibit tumor growth, inflammation and to treat diabetic retinopathy, rheumatoid arthritis and psoriasis. Diagnostic methods for detecting mutations in the VEGF3 coding sequence and alterations in the concentration of VEGF3 protein in a sample derived from a host are also disclosed.

Description

W O 96/39421 PCTrUS95/07283 ~um~n Vascular E~dothelial Growth Factor 3 This invention relates to newly ;~nt;fied polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present inv~nt~on has been i~nt;fied as a m~mh~ of the vascular endoth~l~l growth factor ~amily. More particularly, the polypeptide o~ the present inv~nt~on is vascular endo~h~ l growth factor 3, sometimes hereina~ter re~erred to as ~ ." The invention also relates to ;nh, ht ting the action o~ such polypeptide.
The ~orm~t;nn o~ new blood vessels, or angiogenesis, is ess~nt~l for c~.~ yO~iC development, subsequent growth, and tissue repair. Angiogen~ci~ however, is an ess~nt~l part of certain pathological conditions such as neoplasia, ~or example, L~-wl~ and gliomas, and abnormal angiog~n~s is associated with other diseases such as in~lammation, rheumatoid arthritis, psoriasis, and diabetic ret~nnp~thy (Folkman, J. and Klagsbrun, M., Science 235:442-447,(1987)).
Both acidic and basic ~ibroblast growth ~actor molecules are mitogens ~or endoth~l;~l cells and other cell types.
Angiotropin and angiogenin can induce angiogenesis, although their ~unctions are unclear (Folkman, J., 1993, ~nc~

W O 96/39421 PCTrUS95/07283 Medicine pp. 153-170, Lea and Febiger Press). A highly selective mitogen for vascular endoth~l;Al cells is vascular endothPl;Al growth factor or V~GF (Ferrara, N., et al ., Endocr. Rev. 13:19-32, (1992)), also known as vascular permPAh;l;ty factor (VPF). Vascular endothel;Al growth factor is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to ~ascular endothPl;~l cells.
The murine VEGF gene has been characterized and its expression pattern in e,.~ yOy.-..Pc;~ has been analyzed. A
persistent expression of VEGF was observed in epithP~
cells adjacent to fenestrated endothelium, e.g., in choroid plexus and kidney glomeruli. The data was consistent with a role of VEGF as a multifunctional regulator of endoth~l;Al cell growth and differPnt;At;on (Breier, G. et al .
Develo~",e~L, 114:521-532 (1992)).
V~GF is structurally related to the ~ and ~ rhAtn~ of platelet-derived growth factor (PDGF), a mitogen for mesenchymal cells and plACPntA growth factor (PLGF), an endothPl;~l cell mitogen. These t-hxee proteins h~long to the same ~amily and share a conserved moti~. Eight cysteine residues contr; hllt; ng to disulfide-~ond ~ormation are strictly conserved in these proteins. AltP~nAt;vely spliced mRNAs have been ;~nt;fied for both VEGF, PLGF and PDGF and these different splicing products differ in biological activity and in receptor-ht n~; ng speci_icity. VEGF and PDGF
function as homo-dimers or hetero-dimers and bind to receptors which elicit intrinsic tyrosine kinase activity following receptor dimerization.
VEGF has four different forms of 121, 165, 189 and 206 amino acids due to alternative splicing. VEGF121 and VEGF165 are soluble and are c~r~hle of ~l~,.,oLing angiogenesis, whereas VEGF189 and VEGF306 are bound to hepar~ contAtn;ng proteoglycans in the cell surface. The temporal and spatial expression of VEGF has been correlated with physiological W O 96~9421 PCTrUS95/07Z83 proliferation of the blood vessels (Gajdusek, C.M., and ~Arhon, S.J., Cell Physiol., 139:570-579, (1989)); McNeil, P.L., Mnthnkr;Shn~n, L., Warder, E., D'Amore, P.A., J. Cell.
Biol., 109:811-822, (1989)). Its high affinity htn~tng sites are localized only on endoth~l;Al cells in tissue sections (Jakeman, L.B., et al., Clin. Invest. 89:244-253, (1989)).
The factor can be isolated from pituitary cells and several tumor cell lines, and has been implicated in some human gliomas (Plate, K.H. Nature 359:845-848, (1992)).
Interestingly, expression of VEGF121 or VEGF165 confers on ~h;ne~e hamster ovary cells the ability to form L~..~ in nude mice (Ferrara, N., et al., J. Clin. Invest. 91:160-170, (1993)). The ;nh;h;tion of VEGF function by anti-VEGF
monoclonal antibodies was shown to ;nh;h;t tumor growth in ;~l-ne-defiCient mice (Kim, K.J., Nature 362:841-844, (1993)). Further, a ~nm;n~nt-negative mntAnt of the VEGF
receptor has been shown to tnh;htt growth of glioblastomas in mice.
Vascular per~-h;l;ty factor, has also been found to be responsible for persistent microvascular hyperperm~Ah;l;ty to plasma proteins even after the cessation of injury, which is a characteristic feature of normal wound heAl;ng This suggests that VPF is an important ~actor in wound h~l ;ng Brown, L.F. et al., J. Exp. Med., 176:1375-9 (1992).
The expression o~ VEGF is high in vascularized tissues, ~e.g., lung, heart, placenta and solid L~IIVLS) and correlates with angiogenesis both t~-.~ dlly and spatially. VEGF has also been shown to induce angiogenesis in ~ivo. Since angiogenesis is ess~nt;~l for the repair of no~l tissues, especially vascular tissues, VEGF has been proposed for use in ~v---~Ling vascular tissue repair (e.g., in atherosclerosis).
U.S. Patent No. 5,073,492, issued December 17, 1991 to Chen et al., discloses a method for synergistically ~nh~nctng endoth~l;~l cell growth in an a~l~Liate envilv~ e~t which W O 96139421 PCT~US95/07283 comprises AAA~ng to the envi.~ .t, V~GF, effectors and serum-derived factor. Also, vascular endoth~l;Al cell growth ~actor C sub-unit DNA has been prepared by polymerase chain reaction techniques. The DNA Gnro~s a protein that may exist as either a hetero-dimer or homo-dimer. The protein is a ~ n vascular endoth~l;Al cell mitogen and, as such, is use~ul ~or the ~l~--.oLion o~ vascular dev~lorm~nt and repair, as disclosed in European Patent Application No.
92302750.2, pllhli~h~ September 30, 1992.
The polypeptides of the present invention have been putatively i~nt;fied as a novel vascular endoth~l; Al growth factor based on amino acid sequence homology to human VEGF.
In accordance with one aspect of the present invention, there are provided novel mature polypeptides, as well as biologically active and diagnostically or therapeutically useful frA~r~nt~, AnAlogs and derivatives thereof. The polypeptides o~ the present inv~nt;on are o~ human origin.
In accordance with another aspect of the present inv~nt;on, there are provided isolated nucleic acid molecules ~nroA;ng the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and A~;~gn~stically or therapeutically use~ul ~ra~ -nts, analogs and derivatives thereo~.
In accordance with still another aspect o~ the present invention, there are provided processes ~or pro~nc;ng such polypeptides by r~ro~h;n~nt techn~ques comprising culturing recomh;nAnt prokaryotic and/or eukaryotic host cells, contA;n;ng a nucleic acid sequence ~nro~;ng a polypeptide o~
the present invention, under conditions promoting expression o~ said proteins and subse~uent recovery o~ said proteins.
In accordance with yet a further aspect of the present invention, there is provided a process _or utilizing such polypeptide, or polynucleotide ~n~g~;ng such polypeptide ~or therapeutic purposes, ~or example, to st~m~ te angiogenesis, wound-h~Al;ng, and to ~ ~",uLe vascular tissue repair.

W O 96/39421 PCT~US95/07283 In accordance with yet another aspect of the present inv~ntion, there are provided ~nt;hoAtes against such polypeptides.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to ;nh;h;t the action of such polypeptides, for ~-mrle~ to ;nh; h; t the growth of L~..~
to treat diabetic ret;nor~thy, inflammation, rheumatoid arthritis and psoriasis.
In accordance with another aspect o~ the present invention, there are provided nucleic acid probes c~...~Lising nucleic acid molecules of ~uf f icient length to speci~ically hybridize to nucleic acid sequences o~ the present inv~nt~n.
In accordance with another aspect of the present invention, there are provided methods o~ A;~gnosing diseases or a suscept; h;l; ty to diseases related to mutations in nucleic acid seqllPnc~ of the present inv~nt;on and proteins ~nroA~A by such nucleic acid seqll~nc~.
In accordance with yet a ~urther aspect of the present inv~nt;on, there is provided a process ~or utilizing such polypeptides, or polynucleotides ~ncoA;ng such polypeptides, for in vitro purposes related to scienti~ic research, synthesis o~ DNA and manu~acture o~ DNA vectors.
The~e and other aspects o~ the present inv~nt;on ch~lllA
be apparent to those skilled in the art ~rom the teachings herein.
The ~ollowing drawings are illustrative o~ emboA;m~nts o~ the invention and are not meant to limit the scope o~ the invention as ~ncomr~ssed by the rl ~;mc, Fig. 1 shows the cDNA sequence and the corresronA;ng deduced amino acid sequence o~ the polypeptide o~ the present invention. The st~nA~d one letter abbreviation~ ~or amino acids are used. Seqll~nc;ng was per~ormed using 373 ~ntom~ted DNA Sequencer (Applied Biosystems, Inc . ) . Seqll~nc; ng accuracy is predicted to be greater than 97%.

WO 96~9421 PCT~US95/07283 _ Fig. 2 is an illustration of the amino acid sequence homology between the polypeptide of the present invention and human VEGF.
In accordance with one aspect of the present invention, there are provided isolated nucleic acid molecules (polynucleotides) which Pnro~P for the mature polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID
NO:2) or for the mature polypeptide ~nro~P~ by the cDNA of the clone deposited as ATCC Deposit No. on May 26, 1995. A polynucleotide Pnro~;ng a polypeptide of the present inv~nt~nn may be obt~;nGA from early stage human ' yu (week 8 to 9) osteoclastomas, adult heart or several breast r~ncP~ cell lines. The polynucleotide of this invention was discovered in a cDNA library derived from human colon tissue. It is structurally related to the VEGF/PDGF
family. VBGF3 ront~n~ an open r~;ng frame ~nco~ing a protein of 221 amino acid residues. The protein P~h;h;ts the highest amino acid sequence homology to human vascular endothPl;~l growth factor with 36.199 % ~ntity and 66.063 s~m;l~ity.
It is particularly important that all eight cysteines are conserved within all the polypeptide of the present inv~nt;nn and the signature for the PDGF/VEGF family, PX~vxxx~XGCCN, is conserved in VEGF3 (see Figure 2).
The VEGF3 polypeptide of the present inv~nt~on is meant to include the full length polypeptide and polynucleotide sequence which encodes for any leader seql~ncPs and for active fr~Ant~ of the full length polypeptide. Active ~ra~r~nt~ are meant to include any portions of the full length amino acid sequence which have less th~n the full 221 amino acids of the full length amino acid sequence as shown in SEQ ID No. 2 and Figure 1, but still nnnt~n the eight cysteine residues shown conserved in Figure 1 and such fra~mPnts still cnnt~n VEGF3 activity.

W O 96/39421 PCTrUS95/07283 The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be do7~ble-stranded or single-stranded, and if single stranded may be the coding strand or non-coA;ng (anti-sense) strand. The rr~A;ng sequence which r~ncoA~F: the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID
NO:1) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the r~Al7nr7-7ncy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 (SEQ ID NO:l) or the deposited cDNA.
The polynucleotide which ~ncor7~s for the mature polypeptide of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the roA; n~
sequence for the mature polypeptide (and optionally additional coA;ng sequence) and non-coding sequence, such as introns or non-roA;nrJ sequence 5' and/or 3' of the coAing sequence for the mature polypeptide.
Thus, the term 'Ipolynucleotide r~ncoAing a polypeptide"
r~ncomr~ses a polynucleotide which includes only co~.7; ng sequence for the polypeptide as well as a polynucleotide which includes additional r oA; ng and/or non-coA; ng sequence.
The present invention further relates to variants of the her~;n~hove described polynucleotides which ~nco~le for fra~m~nt~, ~n~l ogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptide ~ncoA~ by the cDNA of the deposited clone.
The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides ~-nco~ling the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) or the same mature polypeptide ~nco~ l by the WO 96/39421 PCTrUS95/07283 cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID
NO:2) or the polypeptide ~nro~A by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As her~in~hove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID
NO:1) or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alt~n~te form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not subst~nt;~lly alter the function of the encoded polypeptide.
The polynucleotides of the present inv~nt;on may also have the ro~;ng sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present inv~nt;~n. The m~k~ sequence may be a hexa-hist;~;n~ tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the m~k~ in the case of a bacterial host, or, for ~m~l e, the m~k~
sequence may be a hemagglnt;n;n (HA) tag when a m~mm~ n host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived $rom the influenza hemaggl-lt;n~n protein (Wilson, I., et al., Cell, 37:767 (1984)).
The term "gene" means the s~gm~nt of DNA involved in pro~llc~ng a polypeptide chain; it includes regions pr~r~;ng and following the ro~;ng region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Fragm~nt~ of the full length gene of the present invention may be used as a hybridization prob~ for a cDNA
library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence sim;l~ity to the gene or W O 96/39421 PCT~US95/07283 5imtl~r biological activity. Probes of this type preferably have at least 30 bases and may cont~tn~ for example, 50 or more bases. The probe may also be used to identify a cDNA
clone correspon~ng to a full length transcript and a genomic clone or clones that ront~t n the complete gene including regulatory and ~v...vLor regions, exons, and introns. An example o~ a screen ~ ises isolating the ro~ ng region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complement~ry to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to detP~m;n~ which ~mhPrs of the library the probe hybridizes to.
The present invention further relates to polynucleotides which hybridize to the her~tn~hove-described seqll~nrPs if there is at least 70%, preferably at least 90%, and more preferably at least 95% t~nttty between the seqllPnc~s The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the her~tnAhove-described polynucleotides. As herein used, the term "stringent conditionsll means hybridization will occur only i~ there is at least 95~ and preferably at least 97% ;~nt~ty between the sequences. The polynucleotides which hybridize to the her~tn~hove described polynucleotides in a pre~erred ~mho~m~nt encode polypeptides which either retain subst~ntt~lly the same biological ~unction or activity as the mature polypeptide encoded by the cDNAs o_ Figure 1 (SEQ ID NO:l) or the deposited cDNA(s).
Alternatively, the polynucleotide may have at least 20 bases, pre_erably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide o_ the present invention and which has an identity thereto, as her~in~hove 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:l, _or example, for recovery W O 96~9421 PCTAU59J~'~7>8~
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~ ~APnt;ty, preferably at least 90~ and more preferably at least a 95~ ;A~nt~ty to a polynucleotide which ~ncoAe~ the polypeptide of SEQ ID NO:2 as well as fr~~nt~ thereof, which fr~gm~nts have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.
The deposit(s) re~erred to herein will be maint~;n~A
under the terms of the BllA~p~-ct Treaty on the Int~n~t;on~l Recognition of the Deposit of Micro-org~ntcmc for purposes of Patent Procedure. These deposits are provided merely as convenience to those o~ skill in the art and are not an admission that a deposit is required under 35 U.S.C. 112.
The seguence of the polynucleotides rQnt~n~A in the deposited materials, as well as the amino acid seguence o~
the polypeptides ~n~oA~A thereby, are in~l~-~ted herein by re~erence and are controlling in the event of any conflict with any description of se~lPnc~C herein. A license may be reguired to make, use or sell the deposited materials, and no such license is hereby granted.
The present invention ~urther relates to a polypeptides which have the deduced amino acid sequence o~ Figure 1 (SEQ
ID NO:2) or which has the amino acid sequence ~n~oAGA by the deposited cDNA, as well as fra-J, ~ c, analogs and derivatives of such polypeptide.
The terms "~ragment,~ derivative~ and ~analog~ when referring to the polypeptide of Figure 1 (SEQ ID NO:2) or that ~ncoA~A by the deposited cDNA, means a polypeptide which retains the conserved motif of VEGF proteins as shown in Figure 1 (SEQ ID NO:2) and ess~nt~l1y the same biological function or activity.

The polypeptides of the present invention may be rec~hin~nt polypeptides, natural polypeptides or synthetic polypeptides, preferably rec~mhin~nt polypeptides.
The fragment, derivative or analog of the polypeptide of Figure 1 (SBQ ID NO:2) or that -nco~-~ by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one _nro~ by the genetic code, or (ii) one in which one or more o~ the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is ~used with another ~lll~o~d, such as a compound to increase the half-li~e o~ the polypeptide (~or example, polyethylene glycol), or (iv) one in which the addit~onAl amino acids are ~used to the mature polypeptide or (v) one in which co...~lises ~ewer amino acid residues shown in SBQ ID No. 2 and retains the conserved motif and yet still retains activity characteristic o~ the VBGF ~amily o~ polypeptide~. Such frA~rAntS, derivatives and analogs are ~m~ to be within the scope o$ those skilled in the art ~rom the t-Ach~ngA
herein.
The polypeptides and polynucleotides o~ the present invention are pre~erably provided in an isolated form, and pre~erably are puri~ied to h~ Gye~leity.
The term "isolated~' means that the material is ~e...J~ed _rom its original envi~ .t (e.g., the natural envi~ ..t i~ it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living ~n~m~l iS not isolated, but the same polynucleotide or polypeptide, separated ~rom some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a .

W O 96~9421 PCTrUS95/07283 composition, and still be isolated in that such vector or composition is not part of its natural envilc - t.
The polypeptides of the present inv~nt;Qn include the polypeptide of SEQ ID N0:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70~
s;m;l~ity (preferably at least 70% ;~nt;ty) to the polypeptide of SEQ ID N0:2 and more preferably at least 90~
s~m;l~ity (more preferably at least gO~ ;~nt;ty) to the polypeptide of SEQ ID N0:2 and still more preferably at least 95% s;m;l~ity (still more preferably at least 95~ nt;ty) to the polypeptide of SEQ ID N0:2 and also include portions of such polypeptides with such portion of the polypeptide generally ront~n;ng at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art '~s~m;l~ity~ between two polypeptides is determined by romr~ing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
Fr~ ts or portions of the polypeptides of the present invention may be employed for pro~r;ng the corresp~n~;ng full-length polypeptide by peptide synthesis; therefore, the fra~nt-~ may be employed as int~rm~ tes for pro~llr~ng the full-length polypeptides. Fra~_ -nt~ or portions of the polynucleotides of the present inv~nt;~n may be used to synthesize full-length polynucleotides of the present invention.
The present inv~nt;~n also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the inv~nt~n by rer~m~;n~nt techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the W O 96/39421 PCTrUS95/07283 form o~ a pl~$mi~1, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modi~ied as d~~ iate ~or activating ~u~uLers, selecting trans~ormants or amplifying the VEGF3 genes o~ the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected ~or expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed ~or pro~nr; ng polypeptides by r~romh;n~nt techniques. Thus, ~or example, the polynucleotide may be included in any one o~ a variety o~ expression vectors ~or expressing a polypeptide. Such vectors include chrom~cn~-l, nonrhromosomal and synthetic DNA seqn~nre~ e.g., derivatives o~ SV40; bacterial pl ~cm; ~c; phage DNA; baculovirus; yeast plasmids; vectors derived ~rom co~h; n~t; on~ 0~ r~ ~cmt ~c and phage DNA, viral DNA such as vaccinia, adenovirus, ~owl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.
The a~lv~iate DNA sequence may be inserted into the vector by a variety o~ procedures. In general, the DNA
sequence is inserted into an d~l~' iate restriction ~n~nnl-~lease site(s) by procedures known in the art. Such procedures and others are ~mDA to be within the scope o~
those skilled in the art.
The DNA sequence in the expression vector is operatively ;nke~ to an d~LV~' iate expression control sequence(s) (~lv",~Ler) to direct mRNA synthesis. As repres~nt~t;ve ~x~mrles o~ such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or tr~, the phage l~mhrl~ PL
~ ,..~Ler and other promoters known to control expression o~
genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also cnnt~;n~ a ribosome h;n~;ng site ~or translation initiation and a transcription terminator.

W O 96/39421 PCTrUS95/07283 The vector may also include a~u~Liate sequences for amplifying expression.
In addition, the expression vectors preferably cont~ ~ n one or more selectable m~k~ genes to provide a phenotypic trait for selection of transfonmed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampic;ll ~n resistance in E. coli.
The vector cont~;n~ng the a~u~iate DNA sequence as her~n~hove described, as well as an ~u~ iate promoter or control sequence, may be employed to transform an a~Lu~liate host to permit the host to express the protein.
As repres~nt~t~ve examples of d~lu~iate hosts, there may be m~ntton~: bacterial cells, such as E. coli, stre~tomYces, ~~A 1 m~n~ tY~h,m~-nium; fungal cells, such as yeast; insect cells such as Drosn~h;l~ S2 and S~odu~Lera Sf9;
~nim~l cells such as CHO, COS or Bowes mol~nn~~;
adenoviruses; plant cells, etc. The selection of an iate host is ~r~ 1 to be within the scope of those skilled in the art from the t~rh~ngs herein.
More particularly, the present inv~nt~on also includes recomh~n~nt constructs comprising one or more of the seqll~nc~fi as broadly described above. The constructs ~..~-ise a vector, such as a plasmid or viral vector, into which a sequence of the inv~nt;nn has ~een inserted, in a forward or reverse orientation. In a preferred aspect of this ~ m~nt, the construct further ~..-~lises reg~ to~y sequences, including, for example, a promoter, operably l;nke~ to the sequence. Large nnmh~s of suitable vectors and ~u---~Lers are known to those of skill in the art, and are commercially av~ hl e. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQ~-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript~ S~, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pRK223-3, pKR233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, .

W O 96/39421 PCTrUS95/07283 pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as lony as they are replicable and viable in the host.
~ u...~Ler regions can be selected ~rom any desired gene using CAT (chlor~mphPnicol transferase) vectors or other vectors with selectable mArkP~s~ Two a~o~iate vectors are pKR232-8 and pCM7. Particular named bacterial ~u---uLers include lacI, lacZ, T3, T7, gpt, 1 Amhtl;~ PR~ PL and trp.
Eukaryotic promoters include CMV immP~iAte early, HSV
thym~nP kinase, early and late SV40, LTRs ~rom retrovirus, and mouse metallothionein-I. Selection of the d~Iu~Liate vector and ~ u---oLer is well within the level of ordinary skill in the art.
In a further PmhO~mPnt, the present invpnt~ on relates to host cells rontA~n~ng the above-described constructs. The host cell can be a higher eukaryotic cell, such as a cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction o~ the construct into the host cell can be effected by calcium phosphate trans~ection, DEAE-Dextran mediated trans~ection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986~).
The constructs in host cells can be used in a conventional ~=nnP~ to produce the gene product encoded by the re~omh~nAnt se~uence. AltP~n~tvely, the polypeptides o~
the invention can be synthetically produced by co.lve~Lional peptide synthesizers.
Mature proteins can be expressed in mA~l; An cells, yeast, bacteria, or other cells under the control o~
d~lu~Liate promoters. Cell-~ree translation systems can also be employed to produce such proteins using RNAs derived ~rom the DNA constructs o~ the present invention.
A~u~iate cloning and expression vectors ~or use with prokaryotic and eukaryotic hosts are described by Sam~rook, et al., MO1~C1~1A~ rl~ning A Laboratory ~An~ , Second Edition, Cold Spring ~A~hon, N.Y., (1989), the disclosure o$
which is hereby incorporated by reference.
Transcription of the DNA Pnco~; ng the polypeptides of the present invention by higher eukaryotes is increased by inserting an Pnh~nrer sequence into the vector. Rnh~nC~S
are cis-acting elPm~nts of DNA, usually about from 10 to 300 bp that act on a promoter to increa~e its transcription.
RxAmrles including the SV40 Pnh~nc~ on the late side o$ the replication origin bp 100 to 270, a cyt~ ~g~lovirus early promoter PnhAncer~ the polyoma PnhAn~Pr on the late side of the replication origin, and adenovirus ~nhAnrr~s.
Generally, recomh;nAnt expression vectors will include origins of replication and selectable m~rk~s permitting trans$ormation o$ the host cell, e.g., the ampic;lli n resistance gene of E. coli and S. cerevisiae TRP1 gene, and a ~,.,~Ler derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), cY-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in d~' ~liate phase with translAt;~n initi~t;~n and term;nAt;Qn se~lPnrPs, and preferably, a leader sequence cApAhle o$ directing secretion of translated protein into the per;~lAF~;c space or extracellular medium. Optionally, the heterologous sequence can Pnro~P a fusion protein including an N-terminal ;~Pnt;fication peptide imparting desired characteristics, e.g., stAh;l;zation or simplified purifirAt;on of expressed recomh;n~nt product.
Useful expression vectors $or bacterial use are constructed by inserting a structural DNA sequence PnCo~; n~
a desired protein together with suitable translation initiation and termination signals in operable r~i ng phase W O 96/39421 PCTrUS95/07283 with a functional promoter. The vector will comprise one or more phenotypic selectable m~k~s and an origin of replication to ensure maint~n~nce of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, S~lm~n~l la tY~h;mllnium and various species within the genera Psell~r.~n~, Streptomyces, and Staphylororrllc, although others may also be employed as a matter o~ choice.
As a representative but nonl ;m;ting example, useful expression vectors for bacterial use can comprise a selectable m~kPr and bacterial origin of replication derived from commP~cially av~ hl e plasmids comprising genetic el~ nts of the well known cl nn; ng vector pBR322 (ATCC
37017). Such c -~cial vectors include, ~or example, pKR223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (P' ~..~d Biotec, Madison, WI, USA). These pBR322 ~'h~ckhonPn sections are combined with an a~liate ~~ oLer and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth o~ the host strain to an a~Liate cell density, the selected ~- -Ler is ;n~llrP~ by dy~ iate means (e.g., temperature shift or chemical induction) and cells are cultured ~or an addit; on~ 1 period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract ret~;nP~ ~or ~urther purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mPrh~n;cal disruption, or use o~ cell iyQ~ng agent~ ~h m~th~s are w~ll kr.~w to ~cse skilled in the art.
Various l;~n cell culture systems can also be employed to express reromh;n~nt protein. ~xamples of m~mm~ n expression systems include the COS-7 lines o~

-- ~ =

WO 96/39421 PCT~US95/07283 monkey kidney fibroblast~, described by Gluzman, Cell, 23:175 (1981), and other cell lines cApAhle of expressing a compatible vector, for example, the C127, 3T3, CH0, HeLa and B~K cell lines. ~ n expression vectors will comprise an origin of replication, a suitable promoter and ~nhAncer, and also any necessary ribosome h; n~; n~ sites, polyadenylation site, splice donor and acceptor sites, transcrip~;QnAl t~rmin~tion se~l~nr~s, and 5~ flAnk;ng nontranscribed se~nc~c DNA se~-~nr~s derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic el~ tc.
The polypeptides can be recovered and purified from rerom~;n~nt cell cultures by methods including A~n;um sulfate or ethanol precipitation, acid extraction, anion or cation ~rhAnge chromatography, phosphocellulose chromatography, l~d~ ,h~h;c interaction cl~ tngraphy, af~inity chromatography, hy~ylapatite chromatography and lectin cLll -tog~aphy. Protein refol~;ng steps can be used, as necessary, in completing configuration o~ the mature protein. Finally, high perform~n~ uid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic prs~e~es, or pro~--c~ by rers~;n~nt t~hn;~ues ~rom a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and m~ n cells in culture). Dep~n~;ng upon the host employed in a r~cnmhinAnt production procedure, the polypeptides o$ the present inv~nt;Qn may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial methionine amino acid residue.
VEGF3 polypeptide ~ay be employed to ~ ~",~Le angiogenesis, for example, to st~m~lAte the growth of transplanted tissue where coronary bypass surgery is performed. VBGF3 may also be employed to st;m~ te wound W O 96/39421 PCTrUS95/07283 h~l;ng, particularly to re-vascularize damaged tissues or where new capillary angiogenesis is desired. VEGF3 may be employed to treat full-thickness wounds such as dermal ulcers, including pressure sores, venous ulcers, and diabetic ulcers. In addition, VEGF3 may be employed to treat ~ull-thickness burns and injuries where a skin graft or ~lap is used to repair such burns and injuries. VEGF3 may also be employed ~or use in plastic surgery, ~or example, for the repair o_ lacerations _rom trauma and cuts in association with surgery.
Along these same lines, VEGF3 may be employed to ;n~
the growth of damaged bone, perio~ntium or li~Am~nt tissue.
VEGF3 may also be employed for regenerating SU~Ol Ling tissues of the teeth, including ce..._~L~I. and perio~nt~
lig~m~nt, that have been damaged by disease and trauma.
Since angiogenesis is important in k~ptng wounds clean and non-infected, VEGF3 may be employed in association with surgery and following the repair o~ cuts. It may also be employed for the tr~t~-nt of :~h~rlm;n~l wounds where there is a high risk o~ infection.
VEGF3 may be employed ~or the ~l~...~Lion o_ endoth~ tion in vascular graft surgery. In the case o$
vascular grafts using either transplanted or synthetic material, VEGF3 can be applied to the sur~ace of the graft or at the junction to promote the growth o~ vascular endoth~
cells. VEGF3 may also be employed to repair damage o_ myocardial tissue as a result of myocardial in~arction.
VEGF3 may also be employed to repair the cardiac vascular system a~ter is~h~m;~. VFGF3 may also be employed to treat damaged vascular tissue as a result of coronary artery disease and peripheral and CNS vascular disease.
VEGF3 may also be employed to coat artificial prostheses or natural organs which are to be transplanted in the body to ,m;n;mi ze rejection o~ the transplanted material and to st;m~ te vascularization o~ the transplanted materials.

' CA 02224002 1997-12-08 W O 96~9421 PCT~US95/07283 VEGF3 may also be employed for vascular tissue repair, for example, that required during arteriosclerosis and following balloon angioplasty where vascular tissues are damaged.
VEGF3 nucleic acid seqllenc~s and VEGF3 polypeptides may also be employed for in ~itro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors, and ~or the production of ~ nsctics and therapeutics to treat human disease. For example, VEGF3 may be employed for in vi tro culturing of vascular endothPl~l cells, where it is added to the conditional medium in a concentration from 10 pg/ml to 10 ng/ml.
This inVPntt nn provides methods ~or i~Pnt~fication o~
VEGF3 receptors. The gene Pnco~ing the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand p~nn~ng and FACS sorting (Coligan, et al., Cur le~lL Protocols in Immun., 1(2), Chapter 5, (1991)). Pre~erably, expression rlon~ng is employed wherein polyadenylated RNA is prepared ~rom a cell responsive to VEGF3, and a cDNA library created ~rom this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to VEGF3. Transfected cells which are grown on glass slides are exposed to labeled VEGF3.
VEGF3 can be labeled by a variety of means including iodination or inclusion of a rPco~n~tion site for a site-specific protein kinase. Following f;xAt~on and ~nr~ tion, the slides are subjected to autoradiographic analysis.
Positive pools are i~Pnt~fied and sub-pools are prepared and retransfected using an iterative sub-pooling and rescrPPn;ng process, evellLually yiPl~i ng a single clone that Pn~O~Pc the putative receptor.
As an altPrn~t~ve approach for receptor i~Pnt~fication, labeled VEGF3 can be photoaffinity l~nk~ with ç~ll ...~ e or extract preparations that express the receptor molecule.
Cross-l~nke~ material is resolved by PAGE and exposed to X-W O 96~9421 PCTrUS95/07283 ray ~ilm. The labeled complex ContA t n;ng VEGF3 is then excised, resolved into peptide ~rAgm~nts, and subjected to protein microseqll~nc~ng. The amino acid sequence obtA;
~rom microseqll~n~ng would be used to design a set o~
degenerate oligonucleotide probes to screen a cDNA library to ~nt;fy the gene ~nro~;ng the putative receptor.
This invention is also related to a method o~ screening c~".~ounds to i~Pnt;fy those which are VEGF3 agonists or antagonists. An example of such a method takes advantage o~
the ability of VEGF3 to significantly st;mlllAte the proliferation of human endoth~l;Al cells in the presence of the comitogen Con A. Endoth~l~Al cells are obtA~n~ and cultured in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) in a reaction mixture supplemented with Con-A
(Calbiochem, La Jolla, CA). Con-A, polypeptides of the present inV~nt;on and the c~..~G~d to be screened are added.
A~ter ~n~llhAt;nn at 37~C, cultures are pulsed with 1 ~Ci o~
3tH]thym;~;ne (5 Ci/mmol; 1 Ci = 37 BGq; NEN) for a sufficient time to incorporate the 3 [H] and harvested onto glass i~iber ~ilters (Cambridge Technology, Watertown, MA). Mean 3 [H]-thym;~;ne inco ~dtion (cpm) o~ triplicate cultures is determined using a liquid sr~nt~llAtion counter (Beckman In~tL t~, Irvine, CA). Signi~icant 3 [H]thymidine incol~o~dtion, as cQ~r~ed to a control assay where the compound is excluded, indicates s~ tion o~ endoth~l;
cell proli~eration.
To assay for antagonists, the assay described above is per~ormed and the ahility of~ the c.,...~ou~.d to ;nh; h; t 3 tH]thym;~l;ne incorporation in the presence o~ VEGF3 indicates that the c~..~d is an antagonist to VEGF3. Alternatively, VEGF3 antagonists may be detected by cn~h;n;ng VBGF3 and a pot~nt;~l antagonist with ' d le-bound VEGF3 receptors or reco~h~nAnt receptors under d~l~Liate conditions ~or a competitive ;nh; h~ tion assay. VBGF3 can be labeled, such as by radioactivity, such that the number o~ VBGF3 molecules O 96/39421 PCTrUS9~ 8 bound to the receptor can determine the effectiveness o~ the pot~nt;~l antagonist.
Alt~rn~t~vely, the response of a known second messenger system following interaction of VEGF3 and receptor would be measured and compared in the presence or absence of the compound. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion ~h~nn~l Q or phosphoinositide hydrolysis. In another method, a m~mm~ n cell or ~ e preparation expressing the V_GF3 receptor is incubated with labeled V_GF3 in the presence of the cu...~o~ld.
The ability of the compound to ~nh~n~ or block this interaction could then be measured.
Pot~nt;~l V_GF3 antagonists include an ~nt;ho~y~ or in some cases, an oligonucleotide, which bind to the polypeptide and effectively ~l;m~n~te VEGF3 function. Alt~rn~t;vely, a pot~nt~l antagonist may be a closely related protein which binds to V_GF3 receptors, ho.reve~, they are inactive forms of the polypeptide and thereby ~ev~llL the action of V_GF3.
RY~ 1 es of these antagonists include a negative ~ n~nt m~lt~nt of the VEGF3 polypeptide, for ~m~l e, one chain of the hetero-dimeric form of VEGF3 may be ~nm;n~nt and may be mutated such that biological activity is not ret~; n~ . An example of a negative ~n~; n~nt mlltAnt includes tr~n~t~
versions of a ~; - ic VEGF3 which is c~r~hle o~ interacting with another dimer to form wild type V_GF3, however, the resulting homo-dimer is inactive and fails to ~h; h; t characteristic V_GF activity.
Another pot~nt;~l VEGF3 antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression t}-uuyh triple-helix formation or antisense DNA or RNA, both of which methods are based on h; n~; ng of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which ~nco~Q for the mature polypeptides of the present inv~nt;on, is used to design an W O 96/39421 PCTrUS~J/~ 3 antisense gNA oligonucleotide of ~rom about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be compl~m~nt~ry to a region o~ the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 ~1979); Cooney et al, Science, 241:456 (1988);
and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production o~ VEGF3. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation o~ the mRNA molecule into the VEGF3 polypeptide (Ant;cense - Okano, J. Neurochem., 56:560 ~1991);
Oligodeoxynucleotides as Antisense Tnh; h; tors oi~ Gene ~xpression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to ;nh;h;t production o~ VEGF3.
Pot~nt;~l VEGF3 antagonists also include small molecules which bind to and occupy the active site o~ the polypeptide thereby m~k;ng the catalytic site inaccessible to substrate such that normal biological activity is prevented. Fxamples of small molecules include but are not limited to small peptides or peptide-like molecules.
The antagonists may be employed to treat L~..~l~ since angiog~n~s;~ and neovascularization are ess~nt;~l steps in tumor growth. The mRNA encoding ~or VEGF3 is ~ound to ~e expressed at moderate levels in at least two breast tumor cell lines which is indicative o~ the role of VEGF3 polypeptides in the malignant phenotype. Gliomas are also a type o~ neoplasia which may be treated with the antagonists o~ the present inv~nt,nn.
The antagonists may also be used to treat in~lammation caused by increased vascular perm~h;l;ty. In addition to these disorders, the antagonists may also be employed to treat diabetic retinopathy, rheumatoid arthritis and psoriasis.

WO 96/39421 PCTrUS95/07283 The ~nt~gonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
The V~GF3 polypeptides and agonists and antayonists may be employed in cn~; n~t~ on with a suitable pharmaceutical carrier. Such r~mrocitions comprise a therapeutically effective amount of the polypeptide or agonist or antagonist, and a rh~r~~reutically acceptable carrier or excipient. Such a carrier includes but is not limited to c~l ;ne, buffered c~l;n~, dextrose, water, glycerol, eth~nol, and ~o~h; n~tions thereof. The formnl~t;on shnlll~ suit the mode of ;n;stration.
The invention also provides a pharmaceutical pack or kit comprising one or more ~nnt~; n~s filled with one or more o~
the ingredients of the phAr~-r~--t;cal CQ~rOQ,; tions of the inv~ntion. ~QQoc;~ted with such ~nn~ine~(S) can be a notice in the form prescribed by a yOv~7 ~ agency reg~ t~ng the ~-nl~f~cture, use or sale of rh~rm~c~uticals or biological products, which notice reflects a~luv~l by the agency of ~-nnf~cture, use or sale for human ~;n;ctration~ In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic _ _uunds.
The pharm-ce~t;cal c~rocitions may be ~m;n;ctered in a convenient ~-nn~ ~uch as by the topical, intravenous, intraperiton~l, intramuscular, intratumor, s~hc~t~n~ous, intr~n~Q~l or intradermal routes. The ph~rr-ceutical compositions are ~m;n;.ctered in an amount which is effective ~or tre~ t; n~ and/or prophylaxis o~ the specific indication.
In general, the pharmaceutical rnmroQitions are ~;n~ctered in an amount of at least about 10 ~g/kg body weight and in most cases they will be ~m;n;ctered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from a~out 10 ~g/kg to about 1 mg/kg body weight daily, t~k;ng into account the routes of ;niQtration, symptoms, etc.

The VEGF3 polypeptides, and agonists or antagonists which are polypeptides may also be employed in accordance with the present invention by expression o_ such polypeptide in vivo, which is o_ten referred to as "gene therapy."
Thus, _or example, cells such as bone marrow cells may be engineered with a polynucleotide (DNA or RNA) ~nco~ing ~or the polypeptide ex vivo, the engineered cells are then provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use o~ a retroviral particle ront~ining RNA Pnro~; ng the polypeptide of the present invention.
Simil~ly, cells may be engineered in vivo _or expression of a polypeptide in vivo, ~or example, by procedures known in the art. As known in the art, a producer cell for pro~l~ring a retroviral particle rnnt~ining RNA
Pnco~ing a polypeptide o~ the present invPntion may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods ~or ~ini~tering a polypeptide of the present invention by such methods should be apparent to those skilled in the art from the t~hings o~ the present invention. For example, the expression vehicle ~or engineering cells m.ay be other than a retroviral particle, for example, an adenovirus, which may be used to engineer cells in vivo a~ter comhin~tion with a suitable delivery vehicle.
Retroviruses ~rom which the retroviral plasmid vectors herPin~hove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape lel~k~
virus, human imm~lno~ficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and m~m~y tumor virus.
In one ~mhn~im~nt, the retroviral plasmid vector is derived _rom Moloney Murine Tellkemi~ Virus.

WO 96/39421 PCTrUS95/07283 The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) ~"oLer described in Miller, et al., Biotechni~ues, Vol. 7, No. 9, 980-990 (1989~, or any other promoter (e.g., cellular ~~ oters such as eukaryotic cellular ~...uLers including, but not limited to, the histone, pol III, and ~-actin ~l~--,oLers). Other viral promoters which may be employed include, but are not limited to, adenovirus ~ ~..-oLers, thym;~;nP kinase (TK) ~ o..-oters, and B19 parvovirus promoter~. The selection o$ a suitable promoter will be apparent to those skilled in the art $rom the tGAch;ngs contA;ne~ herein.
The nucleic acid se~uence Pnco~; ng the polypeptide o$
the present invention is under the control o$ a suitable ..,oLer. Suitable ~l~--.oLers which may be employed include, but are not limited to, adenoviral ~. Lers, such as the adenoviral major late promoter; or hetorologous ~lo...oLers, such as the cytQ-egAlovirus (CMV) ~l~-.-uLer; the respiratory syncytial virus (RSV) ~l~...~Ler; ;n~~~c;hle promoters, such as the MMT ~~ uLer, the metallothionein ~-~I--~Ler; heat shock ~r~---uLers; the Alhl~;n ~ ~...~Ler; the ApoAI ~.--oLer; human globin ~ Lers; viral thym;~; ne kinase promoters, such as the Herpes Simplex thy~;~in~ kinase ~---uLer; retroviral LTRs (including the modi$ied retroviral LTRs herPinAhove described); the ~-actin ~l~--.uLer; and human growth hormone Lers. The ~- Ler also may be the native promoter which controls the gene ~n~orl; ng the polypeptide.
The retroviral plA~mi~ vector is employed to transduce packaging cell lines to ~orm producer cell lines. Examples ~~ p~rkAging cells which may be trans~ected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-86, GP+envAml2, ~and DAN cell lines as described in Miller, Human Gene Thera~Y, Vol. 1, pgs. 5-14 (1990), which is incol~uldted herein by re~erence W O 96/39421 PCTrUS95/07283 in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use o~
liposomes, and CaPO4 precipitation. In one alternative, the retroviral pl~Fm;~ vector may be encapsulated into a l;rosom~, or coupled to a lipid, and then ~m; n; ~tered to a host.
The pro~llc~ cell line generates in~ectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence~s) ~nro~;ng the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, ~ yO~iC stem cells, C.~JlyO~iC carc;nn~- cells, as well as hematopoietic stem cells, h~r~tocytes, fibroblasts, myoblasts, ker~t;nosytes, endoth~ l cells, and bronch;~l epith~ l cells.
This invention is also related to the use o~ the VEGF3 gene as part of a diagnostic assay ~or detecting diseases or suscept; h;l; ty to diseases related to the presence of mutations in VEGF3 nucleic acid seqll~nc~c.
Indiv; ~11~1 s carrying mutations in the VEGF3 gene may be detected at the DN~ level by a variety of t~rhn;ques.
Nucleic acids ~or diagnosis may be obt~;n~ ~rom a patient's cells, such as ~rom blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly ~or detection or may be ampli~ied enzymatically by using PCR
(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.
RNA or cDNA may also be used ~or the same purpose. As an example, PCR primers compl~m~nt~ry to the nucleic acid ~n~o~i ng VEGF3 can be used to identi~y and analyze VEGF3 mutations. For example, deletions and insertions can be detected by a change in size of the ~mrl; fied product in comp?rison to the normal genotype. Point mutations can be W O 96~9421 PCTAJS95/07283 i~nt;fied by hybridizing amplified DNA to radiolabeled VEGF3 RNA or alternatively, radiola~eled VEGF3 antisense DNA
seqll~nc~s. Per~ectly matched sequences can be disting~ ch~
~rom mismatched duplexes by RNase A digestion or by di~erences in melting tem.~peratures.
~ enetic testing based on DNA sequence dif~erences may be achieved by detection of alteration in electrophoretic m~h;l ity of DNA fr~_ ts in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA
fr~m~ntc o~ dif~erent seqllPnc~C may be distingll;ch~ on denaturing for~m;~P gradient gels in which the mnhilities o~
dif~erent DNA ~r~3 c are retarded in the gel at di~erent positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)).
Sequence changes at speci~ic locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection o~ a speci~ic DNA sequence may be achieved by methods such as hybri~;7~t;on, RNase protection, chemical cleavage, direct DNA se~l~nc; ng or the use o~
restriction enzymes, (e.g., Restriction Fragment Length Polymor~hi~ (RFLP)) and Sollth~n blotting o~ genomic DNA.
In addition to more ~v----t~nn~l gel-electrophoresis and DNA seqll~ncing, mutations can also be detected by in situ analysis.
The present invPntion also relates to a ~ nostic assay for detecting altered levels o~ VFGF3 protein in various tissues since an over-expression o~ the proteins romr~ed to normal control tis~ue samples may detect the presence o~ a disease or suscept; h;l; ty to a disease, ~or example, ~no~ 1 cellular differentiation. Assays used to detect levels of VEGF3 protein in a sample derived ~rom a host are well-known =-- --W O 96139421 PCTrUS95/07283 to those of skill in the art and include radio;mmllno~csays, competitive-h;n~;ng assays, Western Blot analysis, ELISA
assays and ~Isandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Tmm~lnology, 1~2), Chapter 6, (1991)) initially comprises preparing an ~nt;hoAy specific to the VEGF3 antigen, preferahly a monoclonal Ant;hoAy. In addition a l~U' Ler ~nt;hsAy is prepared Ag~;n~t the monoclonal antibody. To the le~u~Ler ~nt;hoAy is attached a detectable reagent such as radioactivity, ~luorescence or, in this example, a horseradi~h ~e~ ;AARe enzyme. A sample is L~ vved _rom a host and ;ncllhAted on a solid ~o~L, e.g. a poly~Ly~eL~e dish, that hinds the proteins in the sample. Any free protein h;nA;ng sites on the dish are then covered by ;n~llhAting with a non-speci~ic protein, such as, bovine serum ~lhllm~n Next, the monoclonal Ant;hoAy is ;nrllh~ted in the dish during which time the monoclonal Antihodies attach to any VEGF3 proteins att~ch~A to the polystyrene dish. All unhound monorlQn~l ~nt;hoAy is washed out with buffer. The Ler ~nt;hoAy l;nkeA to horseradish pero~;AARe is placed in the dish resulting in h;nA;ng of the l~V Ler ~nt;hoAy to any monocl~n~l Ant~hoAy bound to VFGF3. UnattAch~A e~vlLer Ant;hsAy is then washed out. Peroxidase substrates are then added to the dish and the amount o~ color developed in a given time period is a mea~u-~ - t o~ the amount o~ VEGF3 protein pre~ent in a given volume of patient sample when compared ~g~;n~t a st~nA~d curve.
A competition assay may be employed wherein Ant;hoA;es speci~ic to VEGF3 are att~rh~A to a solid ~u~ulL.
Polypeptides o~ the present invention are then labeled, for example, by radioactivity, and a sample derived ~rom the host are passed over the solid su~u~L and the amount o~ label detected, ~or example by liquid ~r;nt;ll~tion chromatography, can be correlated to a quantity o~ VEGF3 in the sample.
A ~Isandwich~ assay is s;m; 1~ to an ELISA assay. In a "sandwich" assay VEGF3 is passed over a solid support and W O 96/394ZI PCT~US95/07283 binds to Ant;ho~y att~rhP~ to a solid support. A second Antiho~y is then bound to the VEGF3. A third AntihoAy which is labeled and specific to the second AntihoAy is then passed over the solid support and binds to the second ~nt; hnAy and an amount can then be ql~Ant;fied.
The seqll~nrPs of the present invention are also valuable for chromosome i~Pntification. The seguence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a ~ e,~t need for iAPntifying particular sites on the chromosome. Few cl~ ome m~t-king reagents based on actual sequence data (repeat polymorphism~s) are presently avA;l~hle for m~nking c~ omal location. The mapping of DNAs to chromosomes according to the present invPntion is an important first step in corr~lAting those seguences with genes associated with disease.
Briefly, seqnPnr~ 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 exon in the genomic DNA, thus complicAting the amplification process. These primers are then used ~or PCR scr~n;n~ o~ c tic cell hybrids rontA;n;ng individual human chromosomes. Only those hybrids rontAining the human gene corresp~nAing to the primer will yield an ampli~ied fragment.
PCR mapping o~ somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular c~ ~some.
Using the present invPnt;nn with the same oligonucleotide primers, sublocalization can be achieved with pAn~lc of ~rA~mPnt~ ~rom specific chromosomes or pools of large genomic clones in an analogous m~nnP~ Other mapping strategies that can s;mil~Tly be used to m~lp to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome speci~ic-cDNA libraries.

W O 96~9421 PCT~US95/07283 Fluorescence in situ hybridization (FISH) o~ a cDNA
clone to a metAph~ce chromosomal spread can be used to provide a preci~e chromo~omal location in one ~tep. Thi~
technique can be used with cDNA as ~hort a~ 50 or 60 ba~e~.
For a review of this technique, ~ee Verma et al., Human Ch~u,,losome~: a ~n~ of Ba~ic Techniques. Pe1yd,-,o-- Press, New York (1988).
Once a ~equence ha~ been mapped to a preci~e chromo~omal location, the phy~ical po~ition of the sequence on the cl~u---osome can be correlated with genetic map data. (Such data are found, ~or example, in V. Mc~usick, M~n~ n Inheritance in Man (av~ hl e on line through ~ohn~ ~ork;nc Univer~ity Welch Medical Library). The relationch~r between genes and diseases that have been mapped to the ~ame chromo~omal region are then ;~nt;fied tl--o~yll l;nk~ge analy~is (coinheritance of physically adjacent gene~).
Next, it i~ nece~sary to determine the differences in the cDNA or genomic sequence between affected and unaf~ected indiv;~ll~lc. I$ a mutation is ob~erved in some or all o~ the af~ected individuals but not in any normal indiv; ~ , then the mutation i~ likely to be the cau~ative agent of the disease.
With current re~olution o~ physical mapping and genetic mapping t~rhn;que~, a cDNA preci~ely 1 orA l; zed to a chromo~omal region a~~ociated with the disease could be one o~ between 50 and 500 pot~nt;~l causative genes. (This a~sume~ 1 ~?g~h~e mapping resolution and one gene per 20 kb).
The polypeptides, their fra~m~nt~ or other derivatives, or analogs thereof, or cells expressing them can be used a~
an ~ nogen to produce ~nt;hoAies thereto. These ~nt; ho~; es can be, for example, polyclonal or monoclonal ~nt;hodies.
The present invention also includes ~h;m~ic, single chain, and hnm~n;zed antibodies, as well as Fab fragments, or the product o~ an Fab expression library. Various procedures W O 96~9421 PCT~US95/07283 known in the art may be used for the production of such Ant;hodies and frA~m~nt~.
~ nt;ho~;es generated against the polypeptide corresron~; ng to a sequence of the present invention can be obtAin~A by direct injection of the polypeptide into an An;~~l or by A~m;n;stering the polypeptide to an An~m~
preferably a nnnh~ n The ~nt;ho~y so obt~;n~ will then bind the polypeptide itself. In this ~nnen, even a sequence ~ncoA;ng only a fragment of the polypeptide can be used to generate Ant;hn~;es h;n~;ng the whole native polypeptide.
Such Ant; ho~; es can then be used to isolate the polypeptide from tissue expressing that polypeptide. For preparation of monoclonal Ant;hodies, any t~rhn;~ue which provides Ant;ho~;es pro~llc~ by ront;nllnus cell line cultures can be used. RX~rl es include the hybridoma t~rhn;que (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma t~chn;~ue, the human B-cell hybridoma terhn;~ue (Rozbor et al ., 1983, T~llnology Today 4:72), and the _BV-hybridoma technique to produce human I norlnn~l Ant;ho~;es (Cole, et al., 1985, in Monoclon~l Ant;hoA;es and Cancer Therapy, Alan R. Liss, Inc., pp 77-96).
Techniques described for the production of single chain Antiho~;es (U.S. Patent 4,946,778) can be adapted to produce single chain Ant;hoA;es to immunogenic polypeptide products of this inv~nt;on. Also, transgenic mice may be used to express ~ - ;zed Ant;ho~;es to ;r-~n~genic polypeptide products of this invention The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or A~nts, unless otherwise speci~ied, are by weight.
In order to fAc;l;tate underst~nA;ng of ~e ~ollowing examples, certain frequently occurring methods and/or terms will be described.

W O 96~9421 PCT~US9~ 7 ~
"PlAsmi~ll are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially avA~l~hle, publicly avA~lAhle on an unrestricted basis, or can be constructed from av~ilAhle plA~m;~c in accord with pllhl~Sh~
procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially avA;lAhle and their reaction conditions, cofactors and other requir~m~nt~ were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ~g of plAcm~ or DNA
~ragment is used with about 2 units of enzyme in about 20 ~l of buffer solution. For the purpose of isolating DNA
fragments for pl AF~ ~ construction, typically 5 to 50 ~g of DNA are digested with 20 to 250 units of enzyme in a larger volume. A~~ iate buffers and substrate amounts for particular restriction enzymes are specified by the manu~acturer. Incubation times of about 1 hour at 37~C are ordinarily used, but may vary in accordance with the supplier's instructions. A~ter digestion the reaction is electrophore~ed directly on a polyacrylAm~ gel to isolate the desired ~ragment.
Size separation o~ the cleaved fra_ nts is performed using 8 percent polyacry~A~;~ gel described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" re~ers to either a single stranded polydeoxynucleotide or two compl~m~nt~y polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5~ phosphate and thus will not ligate to another oligonucleotide without A~ ng a phosphate with an ATP in the presence of a kinase. A synthetic W O 96~9421 PCTAUS95/07283 oligonucleotide will ligate to a ~ragment that has not been horylated .
"LigAt;on~ re~ers to the process o~ ~orming phosphodiester bonds between two double stranded nucleic acid fra~~nts (Maniatis, T., et al., Id., p. 146). Unle~s otherwise provided, ligation may be accompl;~h~ using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 ~g of a~Lo~;mAtely equimolar amounts of the DNA ~rA~m~nt~ to be ligated.
Unless otherwise stated, transformation was performed as described by the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Exam~le 1 Cloninq and ex~ression of VEGF3 usinq the baculovirus ex~ression sYstem The DNA cnntA;ne~ in the ~roc;ted clone which ~nro~
the VEGF3 protein, was A~rl; fied using PCR oligonucleotide primers corresrnn~ing to the 5' and 3' seqll~nr~fi of the gene:
The 5' primer has the sequence 5' GCATGGATCCCAGCCTGA
TGC~-l~CC (SEQ ID NO:4) and cnntA;n~ a BamH1 restriction enzyme site and nucleotide sequence compl~m~ntA~y to the 5' sequence of VEGF3 (nt. 150-166).
The 3' primer has the sequence 5' GcATT~T~ r~TGcTGAG
TCTGAAAAGC 3' (SEQ ID NO:5) and ront~;n~ the cleavage site ~or the restriction enzyme XbaI and nucleotides compl~m~ntA~y to the 3' sequence of VEGF3.
The ~mpl;fied sequences were isolated from a 1~ agarose gel using a r- -~cially av~;lAhle kit (nGeneclean," BIO 101, Inc., La Jolla, C~). The fragment was then digested with the ~n~onllClease BamH1 and XbaI and then purified again on a 1 agarose gel. This ~ragment was ligated to A2GP baculovirus trans~er vector with leader peptide sequence (PHarmingen) at the BamH1 and XbaI sites. Tl~ouyh this ligation, VEGF3 cDN~
was cloned in frame with the signal sequence o~ baculovirus W O 96/39421 PCT~US95/07283 gp67 gene and was located at the 3' end of the signal sequence in the vector. This is designated pA2GP-VEGF3.
To clone VEGF3 with the signal sequence of gp67 gene to the pRG1 vector for expression, VEGF3 with the signal sequence and some upstream se~uence were excised from the pA2GP-VEGF3 plasmid at the Xho restriction ~n~nnllclease site located upstream of the VEGF3 cDNA and at the XbaI
restriction ~n~nnl~clease site by XhoI and XbaI restriction enzyme. This fragment was separated from the rest of vector on a 1% agarose gel and was puri~ied using "Geneclean" kit.
It was designated F2.
The A2PG vector (modification of pVL941 vector) is used for the expression of the VEGF3 protein using the baculovirus expression system (for review see: Sl -~s, M.D. and Smith, G.E. 1987, A m-nll~l o~ methods ~or baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector ront~n~ the strong polyhedrin promoter o~ the Autographa cali~ornica nuclear polyhedrosis virus (AcMNPV) ~ollowed by the recognition sites ~or the restriction cleases Bam.~l, Smal, XbaI, BglII and Asp718. A site for restriction ~n~nnllclease Xhol is located upstream o~
BamH1 site. The sequence between Xhol and Bam.~I is the same as that in pAcGp67A vector. The polyadenylation site o~ the simian ~irus (SV)40 is used ~or e~icient polyadenylation.
For an easy selection o~ reco~h~n~nt virus the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin se~l~nr~s are flanked at both sides by viral sequences for the cell-mediated homologous reromh~n~tion of cotrans_ected wild-type viral DNA. Many other baculovirus vectors could be used in place of A2GP such as pRG1, pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).

W O 96~9421 PCTAUS9J/~
The pl;~sm;rl was digested with the restriction enzymes XboI and XbaI and then ~ph~ phorylated using cal~ intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1~ agarose gel using the co~m~cially avA;l~hle kit ("~nerlean" BIO 101 Inc., La Jolla, Ca ).
This vector DNA is designated V2.
Fragment F2 and the ~rhocphorylated pl ~F~ ~ V2 were ligated with T4 DNA ligase. E.coli HB101 cells were then transformed and bacteria ;~nt;fied that ront~;n~ the plasmid (pBac gp67-VEGF3) with the VEGF3 gene using the enzymes BamH1 and XbaI. The sequence of the clon~ fr~m~nt was confirmed by DNA se~nc;n~.
5 ~g of the pl ~F~i ~ pBac A2GP-VEGF3 was cotrans~ected with 1.0 ~g of a commercially av~ hl e line~ized baculovirus ("BaculoGold~ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipo~ection method (Felgner et al.
Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
l~g o~ BaculoGold~ virus DNA and 5 ~g of the pl ~F~ ~
pBac A2GP-VEGF3 were mixed in a sterile well o~ a microtiter plate r~nt~in~n~ 50 ~l o~ serum ~ree Grace~s medium (Li~e Technologies Inc., Gaithersburg, MD). Afterwards 10 ~1 Lipofectin plus 90 ~l Grace's medium were added, mixed and incubated ~or 15 minutes at room temperature. Then the trans~ection mixture was added d~ ise to the S~9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate was rocked back and ~orth to mix the newly added solution. The plate was then inrllh~ted ~or 5 hours at 27~C. A~ter 5 hours the trans~ection solution was ~ lov~d from the plate and 1 ml of Grace's insect medium supplemented with 10~ fetal cal~ serum was added. The plate was put back into an incubator and cultivation rontin~ at 27~C ~or ~our days.
A~ter ~our days the sup~rn~t~nt was collected and a plaque a~say per~ormed simil~ as described by Summers and Smith (supra). As a modi~ication an agarose gel with "Blue W O 96/39421 PCT~US95/07283 Gal" (Life Technologies Inc., Gaithersburg) was used which allows an easy isolAt;on o~ blue st~;nP~ pla~ues. ~A
detailed description of a "plaque assay" can also be ~ound in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10) .
Four days after the serial dilution, the virus was added to the cells, blue stA;nP~ pla~ues were picked with the tip o~ an Eppendorf pipette. The agar c~nt~;n;ng the recomh;nAnt viruses was then resuspended in an ~ppendorf tube rontA~n;ng 200 ~l of Grace's medium. The agar was removed by a brie~
centrifugation and the SupPrn~tAnt contA;n;ng the rPromh;n~nt hACll~ovirus was used to infect S~9 cells seeded in 35 mm h~ , Four days later the supernatants of these culture ~;Shpc were harvested and then stored at 4~C.
Sf9 cells were grown in Grace's medium supplemented with 10~ heat-inactivated FBS. The cells were infected with the rer~m~;n~nt baculovirus V-A2GP-VEGF3 at a multiplicity of infection (MOI) of 1. Six hours later the medium was removed and replaced with SF900 II medium minus meth; on; nP and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 35S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centri~ t;on and the l~h~lled proteins visualized by SDS-PAGE and autoradiography.
Protein ~rom the medium and cytorl A~m 0~ the S~9 cells was analyzed by SDS-PAGE under re~llc; ng and non-r~llr; ng conditions. The medium was dialyzed against 50 mM MES, pH
5.8. Precpitates were obtA; n~ after dialysis and resuspended in 100 mM NaCitrate, pH 5Ø The resuspended precipitate was analyzed again by SDS-PAGE and was stA; n~
with ~o~m~sie Br;~ nt Blue.
The medium supernatant was also diluted 1:10 in 50 mM
MES, pH 5.8 and applied to an SP-650M column (1.0 x 6.6 cm, Toyopearl) at a flow rate o~ 1 ml/min. Protein was eluted CA 02224002 l997-l2-08 W O 96~9421 PCTrUS9S/07283 with step gradients at 200, 300 and 500 mM NaCl. The VEGF3 was obt~;neA using the elution at 500 mM. The eluate was analyzed by SDS-PAGE in the presence or absence o~ r~ c; ng agent, ~ .elca~Loethanol and st~;nPA by Coommassie Br;~ nt Blue.

Exam~le 2 ExPression via Gene TheraPv Fibroblasts are oht~;n~A ~rom a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small rhllnkc of the tissue are placed on a wet sur~ace o~ a tissue culture ~lask, d~ u~imately ten pieces are pl~r~A in each ~lask. The ~lask is turned upside down, closed tight and left at room temperature over night. A~ter 24 hours at room temperature, the ~lask is inverted and the rhllnkc o~ tissue remain ~ixed to the bottom o~ the ~lask and ~resh media (e.g., Ham~s Fl2 media, with 10% ~3S, penic;ll ~n and sL~Lomycin, is added.
This is then ;nc~lh~ted at 37~C ~or d~ '~ tely one week.
At this time, ~resh media is added and subsequently changed every several days. A~ter an additional two weeks in culture, a monolayer o~ ~ibroblasts y~. The monolayer is trypsinized and scaled into larger ~lasks.
pMV-7 (Rirschmeier, P.T. et al, DNA, 7:219-25 (1988) ~lanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with cal~ intestinal phosrh~t~ce. The 1; ne~n vector is ~raction~t~A on agarose gel and puri~ied, using glass beads.
The cDNA ~ncsA;ng a polypeptide o~ the present invention is amplified using PCR primers which correspond to the 5~ and 3~ end se~uences respectively. The 5~ primer cnnt~;n~ an ~coRI site and the 3' primer includes a HindIII_site. Equal quantities o~ the Moloney murine sarcoma virus l; ne~
h~rkhgn~ and the ampli~ied EcoRI and HindIII ~r~sm~nt are W O 96/39421 PCTrUS95/07283 added together, in the presence of T4 DNA ligase. The resulting mixture is m~nt~n~ under conditions appropriate for ligation of the two fra~rAntc. The ligation mixture is used to trans~orm bacteria HB101, which are then plated onto agar-ront~in;ng kanamycin ~or the purpose of confirm;ng that the vector had the gene o~ interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tisAue culture to con~luent density in Dulbecco's Modified Eagles Medium (DMEM) with 10~ calf serum tCS), penir;~lin and streptomycin. The MSV vector cont~ning the gene is then added to the media and the packaging cells are tr~nA-~llced with the vector. The packaging cells now produce in~ectious viral particles cnntA;n;ng the gene (the p~ck~ging cells are now referred to as pro~llr-~ cells).
Fresh media is added to the transduced pro~llr-~ cells, and subsequently, the media is harvested ~rom a 10 cm plate of confluent pro~llc-~ cells. The spent media, cont~n;ng the infectious viral particles, is filtered tl-v~yh a m; 1 1; iAn~e filter to ....~v~ detAch~ pro~llc-~ cells and this media is then used to infect fibroblast cells. Media is removed ~rom a sub-confluent plate of fibroblasts and quickly replaced with the media ~rom the producer cells. This media is ...~ed and replaced with ~resh media. I~ the titer o~ virus is high, then virtually all fibroblasts will be in$ected and no selection is required. I~ the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered ~ibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The ~ibroblasts now produce the protein product.

Exam~le 3 Bacterial ExPression and Puri~ication o~ VEGF3 -CA 02224002 l997-l2-08 W O 96~9421 PCTAUS95/07283 The DNA of deposited clone which ~ncoAe~ VEGF3, is initially amplified using PCR oligonucleotide primers correspon~;ng to the 5' sequences of the processed VEGF3 protein (minus the signal peptide sequence) and the vector se~l~nces 3' to the VEGF3 gene. Additional nucleotides correspon~ ng to VEGF3 were added to the 5~ and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' GAcTGcATGr~rr~r~
r~A~l~-l-~l~ (SEQ ID NO:6) c~nt~; n~ a SphI restriction enzyme site followed by VEGF3 co~; n~ sequence starting from the presumed t~-m;n~l amino acid of the processed protein codon. The 3' sequence 5' GACTAGAl~-l~-l-l~CAG~-l-l~w CAC 3~
(SEQ ID NO:7) c~nt~nC com.plem~nt~y se~l~nr~C to BglII site located 3' to the VEGF3 DNA insert. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-70 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE-70 ~nro~fi ~nt;h~otic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable ~- -Ler operator (P/O), a ribos~ - h;n~;ng site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 was then digested with SphI and BglII. The amplified sequences were ligated into pQE-70 and were inserted in frame with the sequence ~n~o~ng for the hist;~;ne tag and the RBS.
The ligation mixture was then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in S~..~-ook, J. et al., Molecular ~'lon;n~: A Laboratory Cold Spring Laboratory Press, (1989). M15/rep4 rnnta;n~
multiple copies of the r~ d pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kanr).
Transformants are ;~nt;fied by their ability to grow on LB
plates and ampic;ll;n/kanamycin resistant colon;es were selected. P~ DNA was isolated and con~irmed by restriction analysis. Clones cont~; n; ng the desired constructs were grown overnight (0/N) in liquid culture in LB
media supplemented with both Amp (100 ug/ml) and Kan ~25 -W O 96~9421 PCTrUS95/07283 ug/ml). The O/N culture i~ used to inoculate a large culture at a ratio o~ 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG
(''Isu~lu~yl-~-D-thiogalacto pyranoside") was then added to a ~inal conc~nt~ation of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O le~ng to increased gene expression. Cells were grown an extra 3 to 4 hours.
Cells were then harvested by centri~ugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCl. A~ter clari~ication, solubilized VEGF3 was puri~ied ~rom this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight h~ n~i ng by proteins cont~;n~ng the 6-His tag (Hochuli, E. et al., J.
~h~omatography 411:177-184 (1984)). VEGF3 was eluted ~rom the column in 6 molar gll~n; ~; n~ HCl pH 5.0 and ~or the purpose o~ renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glllt~th~one ~r~llr~) and 2 mmolar glllt~th~one (oxidized). A~ter ; nrllh~t; on in this solution f or 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.
Numerous modi~ications and variations o~ the present invention are possible in light o~ the above teachings and, there~ore, within the scope o~ the appended cl~;mc~ the inv~nt~on may be practiced otherwise th~n as particularly described.

SEQu~NL~ LISTING

(1) r.RNRRAT. IN~O~ ~TION:
(i) APPLICANT: Olsen, et al.
(ii) TITLE OF lNV~N-llON: Vascular Endothel~ Al Growth Factor 3 (iii) NUMBBR OF SE~u~N~:
(iv) ro~R~poNDEN OE Ann~R.~s (A) AnnRR-~SEE: r~Rr.T.A, BYRNE, BAIN, GILFILLAN, OE CCHI, STEWART & OLSTEIN
(B) STREET: 6 BEC ~ R FARM ROAD
(C) CITY: RO.~RT.ANn (D) STATE: NEW JERSEY
(E) ~UUNlKY: USA
(F) ZIP: 07068 (v) COM~ul~ READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DI~K~l-(B) COM~ul~K: IBM PS/2 (C) OPERATING :jY~ll~ : MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1 (Vi ) ~ 'IJ~ 'l' APPLICATION DATA:
(A) APPLICATION NUMB~R:
(B) FILING DATE: con~~ tly (C) CLASSIFICATION:

(vii) Al-lOKN~Y/AG~Nl lN~O~ ~TION:
(A) NAME: FERR~RO, GREGORY D.
(B) REGISTR~TION NUMBER: 36,134 (C) ~K~NL~/DOC ~ T NUMBER: 325800-(viii) TELECOMMUNICATION lN~O~ ~TION:
(A) TEh~U..~: 201-994-1700 (B) TELEFAX: 201-994-1744 W O 96/39421 PCTrUS95/07283 (2) lN~O~ ~TION FOR SEQ ID NO:l:

(i) SEQU~;Nl :~; CH~RACTERISTICS
(A) LENGTH: 666 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRAN~ S: SINGLE
(D) TOPOLOGY: T.TNRAT~

(ii) MOLECULE TYPE: cDNA

(xi) SE~Uh~ DESCRIPTION: SEQ ID NO:l:

ATGAGA~GGT GTAGAATAAG TGGGAGGCCC CCGGC&CCCC CC~l~lCCC CGCCCAGGCC 60 C~-l~l~-lCCC AGCCTGATGC CCCTGGCCAC C~GAGGAAAG L~l~ATG GATAGATGTG 120 TATACTCGCG CTAC~aC~A GCCC~r:~G ~L~l~l~C CCTTGACTGT GGAGCTCATG 180 TGCCCTGACG A~GC~-l~A ~ GCCC A~-l~G~AGC ACCAAGTCCG GATGCAGATC 30O
CTCATGATCC GG~CCCr~G CAGTCAGCTG GGGGAGATGT CC~-l~AAGA ACACAGCCAG 360 TGTGAATGCA GACCTAAAAA A~Gr~r~T GCTGTGAAGC CAGACAGGGC TGCTACTCCC 420 CACCACCGTC CCCAGCCCCG 'l~ l~ll'~L~ ~G~-L~ACT ~-l~CCCC~ AGCACCCTCC 480 CCAGCTGACA Tr~CCr~TC CCACTCr~r-C CCCAGGCCCC ~ ~CC~ACG CTGr~rCr~5 540 CACr~Cr~r-T GCC~L~ACCC CCGGACCTGC CG~-lGCCG~-L GTCGACGCCG CAG~-l-l~-lC 600 ~l-l~l~AAG GGC~G~ -L AGAGCTCAAC CCAGACACCT GCAGGTGCCG GAAG~-lGC~A 660 (2) lN~O.~TION FOR SEQ ID NO:2:
(i) SEQu~l.~ CHARACTERISTICS
(A) LENGTH: 221 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) ST~2ANI ~:1 )N~;SS:
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

WO 96/394Zl PCTAUS95/07Z83 (xi) SE~u~N~ DESCRIPTION: SBQ ID NO:2:
~et Arg Arg Cys Arg Ile Ser Gly Arg Pro Pro Ala Pro Pro Gly 15~al Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His 30~ln Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr ~5 4û 45~ys Gln Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met 60~ly Thr Val Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln 75~rg Cys Gly Gly Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro 90~hr Gly Gln His Gln Val Arg Met Gln Ile Leu Met Ile Arg Tyr 10û 105~ro Ser Ser Gln Leu Gly Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys Lys Asp Ser Ala Val Lys Pro Asp 125 . 130 135 Arg Ala Ala Thr Pro His His Arg Pro Gln Pro Arg Ser Val Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro Ser Pro Ala Asp Ile Thr Gln Ser His Ser Ser Pro Arg Pro Leu Cys Pro Arg Cys Thr Gln His His Gln Cys Pro Asp Pro Arg Thr Cys Arg CYE; Arg Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly Arg Gly Leu Glu Leu Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg

Claims (20)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide as set forth in SEQ ID NO:2;
(b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or (b).

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 2 which encodes the polypeptide as set forth in SEQ ID NO:2.

4. The polynucleotide of Claim 2 which encodes the polypeptide as set forth in SEQ ID NO:2.

5. The polynucleotide of Claim 2 which encodes the polypeptide as set forth in SEQ ID NO:2.

6. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide which encodes a mature polypeptide encoded by the DNA contained in the deposited clone;
(b) a polynucleotide which encodes a polypeptide expressed by the DNA contained in the deposited clone;
(c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).

7. A vector containing the DNA of Claim 2.

8. A host cell genetically engineered with the vector of Claim 7.

9. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 8 the polypeptide encoded by said DNA.

10. A process for producing cells capable of expressing a polypeptide comprising transforming or transfecting the cells with the vector of Claim 7.

11. A polypeptide selected from the group consisting of (i) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof; (ii) a polypeptide comprising amino acid 1 to amino acid 221 of SEQ ID NO:2; and (iii) a polypeptide encoded by the cDNA of the deposited clone and fragment, analogs and derivatives of said polypeptide.

12. A compound effective as an agonist for the polypeptide of claim 11.

13. A compound effective as an antagonist against the polypeptide of claim 11.

14. A method for the treatment of a patient having need of PGSG-1 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 11.

15. The method of Claim 14 wherein said therapeutically effective amount of the polypeptide is administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo.

16. A method for the treatment of a patient having need of VEGF3 comprising: administering to the patient a therapeutically effective amount of the compound of claim 12.

17. A method for the treatment of a patient having need to inhibit VEGF3 comprising: administering to the patient a therapeutically effective amount of the antagonist of Claim 13.

18. A process for diagnosing a disease or a susceptibility to a disease related to expression of the polypeptide of claim 11 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.

19. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 11 in a sample derived from a host.

20. A method for identifying compounds which bind to and activate or inhibit a receptor for the polypeptide of claim 11 comprising:
contacting a cell expressing on the surface thereof a receptor for the polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a compound to be screened under conditions to permit binding to the receptor; and determining whether the compound binds to and activates or inhibits the receptor by detecting the presence or absence of a signal generated from the interaction of the compound with the receptor.