CA2231400A1 - Vegf-related protein - Google Patents

Abstract

A human VEGF-related protein (VRP) has been identified and isolated that binds to, and stimulates the phosphorylation of, the receptor tyrosine kinase Flt4.
The VRP is postulated to be a third member of the VEGF protein family. Also provided are antibodies that bind to VRP and neutralize a biological activity of VRP, compositions containing the VRP or antibody, methods of use, chimeric polypeptides, and a signal polypeptide for VRP.

Description

VEGF-RFT ~TEn PROTEIN

BACKGROUND OF THE iNVENTlON
Field of th~ Invention The present invention pertains generally to a receptor protein tyrosine kin~e (rPTK) iigand. More 5 I,a licula ly, the invention relates to a novel ligand d~ d VEGF-related protein (VRP) or VH l, which binds to, and ~ ' the ~Lv~ Lu~yldliu~ of. the Flt4 tyrosine kinase receptor (also known as the Sal-SI receptor) and the isolation and .c ' ~ ~ u ~ of the same.
. of iRr~ l Art The ~ of new blood vessels either from dirf..c g ~ cells during e..liJ.y~
10 d.~_lo~ M (~ ) or from p~ cAi~dl~,3 vessels during adult life (P ~GioL. - -: -) is an essentiai feature of organ d.i~l,,~--.- - l. ..,~ .udu-liu--. and wound healing in higher ulL ~ ~ Foikman and Shing, ~jQL
~h~L.i~: 10931-10934(1992);ReynoldsetaL,FA.'~F.RJ ~:886.892(1992);Risaueta1,Dc.clù~ t.
lQZ: 471-478 (1988). AUr ;~ is also - - y for certain I :' - ' O ' processes including i ,, (Foikman,l~l~t-~eM~ r~ 1:27-31[1995])and..,t~u~,aii.y.MilleretaL,Am J.Pathol..145:574-S84(1994).
15 While several growth factors can stimulate ~ ~ " (Kiagsbrun and D'Amore, Ann Rev. Phys~
53: 217-239 [1991]; Foikman and Klagsbrun, ~. ~ E: 442-447 [1987]), vascular ~ 1~ -' " ' growth factor (VEGF)(Ferraraetal.,En~n Rev..13:18-32[1992])isapotent ~ " factorthatactsviathe- ~l--'l--l;-' cell-specific receptor tyrosine kinases ~-like tyrosine kinase (Fltl) (Shibuya et aL, On~n~r"r 5: S19-524 [1990];deVriesetaL,~E~,~:989-991 [1992])andfetal liverkinase(Flkl)(also~ KDR). Quirm 20 etaL, Proc. ~tl A~ Sci. USA. 90: 7533-7537 (1993); MillaueretaL, Cell.12: 835-846 (1993); M - ' .. ;.
etaL,Pr~ N~tl Af-~ Sci.USA.88:9026-9030(1991);TermanetaL,Binrh~m BioDhvc I~PC ('nmm-ln 187:
1579-1586 (1992); Terman etaL, Onr~p~nr- ~: 1677-1683 (1991); Oelrichs et aL, Onco~ene 3~: 11-18 (1993).
ThesetwoVEGFreceptorsandathirdorphanreceptor,Flt4(PajusolaetaL,('~nr~rPec ~:5738-5743[1992];
Gallande~aL,Qnco~ene.~: 1233-1240[1993];Finnertye~aL,Onco~ene.~:2293-2298[1993~)c-~l;U~l-a 2 5 subfamily of class Ill receptor tyrosine kinases that contain seven extracellular ;.. - -- ~gksl~LI;. ~-like domains and a split in~ rlllll ~' tyrosine kinase domain. Mustonen and Alitalo. 1. C~ll Binl ~2: 895-898 ( 1995). See also WO 94/10202 published I I May 1994 and PCT/US93/00586 filed 22 January 1993 (Avraham e~ aL ). These three receptors have 31-36% amino acid identity in their eY~ r ligand-binding domains.
Mice deficient in Fltl (Fong et aL, ~ah~,. ~i: 66-70 [19951) or Flkl (Shalaby et aL, ~, 376: 62-30 66[1995])(generatedbygenetargetingin L~llbl~J1l8, stemcells)haveseveredefectsinv~-. lOo~ anddie in utero at C~lb~ olliC day 8-9. The phenotype of the receptor-deficieM mice differs c- .~ al)ly~ however.
Mice lacking Fltl have a .li~ ~.i~d vascular ~ ull 1;-- . that extends to the major vessels as well as to the u. ' - e. while ~ ~ l cell di~ lLialiull appears to be normal. Fong et aL, supra. Mice lacking Flkl have a major defect in the d~ielu~ of mature . ,d~lhrli~l cells as well as a severe reduction in 35 h -, - cell ~.o~,. -;1---~ Shalaby et aL, s~pra. Thus. VEGF may act on enfinth~ l cells at more than one stage of v~...ks, .... ~:~
Flt4 is also ~ipecifir~lly expressed in ~ 1 cells; it is first observed in day 8.5 mouse embryos in ~< ' ' ' cell ~c~ u~:~. Kaipainen e~ al., Proc. I~l~tl Acad. Sci. USA. ~: 3566-3570 ( 1995); Kaipainen e~

aL, J. Ex~l. M~ ~i 178: 2077-2088 (1993). See also Hatva et aL, Am J P~thnl.. 146: 368-378 (1995). As d~ _lui"l~_~" proceeds. Flt4 eA~ ;un becomes confined to the venous and Iymphatic .. 1.. 1h. 1;- -- and is funally restricted to the Iyll~lJLaLic vessels. Concicrf nt with this finding, adult human tissues show Flt4 eAiJ~ aiùl~ in Iymphatic c ..lf.ll f i - while there is a lack of cA~ aiù-- in arteries, veins. and capillaries. i~ , et aL. Proc.
5 ~:ltl ACZ~fi SC;. USA. supra. Clones encodulg human and mouse Flt4 have been isolated either by PCR with prirners from CO~ d tyrosine kinase regions (Finnerry et al., supra; PCT/US93/00586. supra; Aprelikova et aL, t'~rlr~r Res.. 52: 746-748 tl992]) or by lu.. '5~1UI~ .Y h.~l,...liL..Iio.. with a Flk2 probe. Galland e~ aL, Grnnmir~ ~: 475-478 ( 1992). Altemative splicing of the Flt4 mRNA produces two varianU of the protein differing by 65 amino acids at the C-temminus. Pajusola et aL, Onro~n~ 8: 2931-2937 (1993). These variants 10 migrate as bands of 170-190 kDa that are partially cleaved i~,ut~olyt;~ll.~ in the r " ' domain to produce aformofabout125kDa. PajusolaetoL,Onco~ene~,supra;PajusolaetoL,Oncos~ene~9:354S-3S5S(1994).
E~ i~,.. of the longer spliced form of Flt4 as a chirnera with the extracellular domain of the CSF- I receptor shows that the Flt4 intracellular domain can signal a ligand-~ growth response in rodent ~L...~ ' Pajusola et aL, Onco~ene. 2. supra; Borg et al., Onco~ene. 10: 973-984 (1995). Flt4 has been localized to 15 human . ~ ~ 5q34-q35 (Aprelikova er aL, supra; Galland et aL, G~n~mirc supra); Fltl and Flkl are locatedatl3ql2 (ImbertetaL,Cvto~enet.CcllGenet..67:175-177[1994])and4ql2. SaitetaL,Cvto~enet.
Cell Genet.~ 70: 145-146 (1995); SpritzetaL, Genomics. j~: 431-436 (1994).
VEGF is a I ~ " cysteine-rich protein that can occur in at least four forms due to ' ~_ splicing of its mRNA. Ferrara et aL, supra. While VEGF is a high-affunity ligand for Fltl and Flk 1, it does not 2 o bind or activate Flt4. Pajusola et aL, Oncopene. 9, supro. The only other closely related member of the VEGF
family is placental growth factor (PIGF), which has 47~/O amino acid identity with VEGF. Maglione et aL, Proc.
tl ArS~ri SC;. USA. 88: 9267-9271 (1991). PIGF also occurs in two alt~ ly spliced forms which differ in the presence orabsence of a basic hepatin billding domain of 21 amino acids. Maglione et a/., Onco~n~ 8:
925-931 (1993); Hauser and Weich, Grow~h Factors. 2: 259-268 (1993). PIGF binds to Fltl but not to Flkl 25 (Park et aL, J. Biol. ('h~rn ~: 25646-25654 tl994]); it is believed that its binding to Flt4 has not been ,1~ r ---~ i PIGF fails to duplicate the capillary ~ 1:-l cell ...im~ or vascular p .. - ~ y activities of VEGF, :- -c~ P that these activities are mediated by the Flk I receptor. Park et aL, s~pra.
~ r that modulate the Flkl receptor or r.~ 1; a~.liv~L;u.l of a VEGF receptor are disclosed in the patent literature. For example, WO 95/21613 i,--l-l;~l-~di 17 August 1995 discloses c-, ~l-u~ that 30 modulate KDRJFlkl receptor signal t,~ ;O.. so as to regulate and/or modulate v~ ~.log. ~;~ and _ ,, and disclose using Flkl to evaluate and screen for drugs and analogs of VEGF involved in Flkl ..ori~ I ';--l- by either agonist or ~ a~ , WO 95/21865 ~ h~d 17 August 1995 discloses -' ' ~ ve with anim~al n..u. . - -' ' kinase (NYK)/Flkl. which mol- ~ can be used to provide agents for treatrnent, ~upll~l~i., and diagnosis of an :~ ~r;O~ ppnri~nt phenotype; and WO

3 5 95n 1868 published 17 August 1995 discloses ~- ~ O~ ~I ;l-o~ that cpecifir~lly bind to an extracellular domain of a VEGF receptor and n~l~ii7~ d-Li~ liull of the receptor.

CA 0223l400 l998-03-09 S~MARY OFTHEnYVENTION
cDNA clones have now been identified that encode a novel protein. " _ ' VRP. which binds to and ~ the i~Lu~ llv-yLllir~ n of the receptor tyrosine kinase Flt4. VRP is related in amino acid sequence to VEGF. but does not interact r,tJ,JI~,.;rll.ly with the VEGF receptors. Fltl and Flkl.
In one aspect, the invention provides isolated bin!ogir~lly active human VRP ~c ~~ at least 265 amino acids. In another aspect, the invention supplies isolated ' k O lly active human VEGF-related protein (VRP) ~,u~ .i..g an amino acid sequence cu...~ ,u.g at least residues + I through 29, inelusive. of Figure I .
In furiher aspeet, the invention supplies isolated L ' ~ 'Iy active human VRP CU~ JI _ an amino aeid sequenee shown as residues -20 through 399. inelusive, or residues I through 399. inelusive, of Figure 1.
l û The invention also pcrtains to chirnenis .~ g the VRP fused to another ~ d~ For example, the ;..~ - provides a ehimerie pol~iJ.~,Lidc. co...~ i..g the VRP fused to a tag pol~ tidc ser"r~"e An example of sueh a ehimera is epitope-tagged VRP.
In another aspeet, the inveMion provides a c~ '1'~~ . cv~ h.o l 'oO 'Iy aetive VRP and a lly ? -r ~ ~ earrier. In a more speeifie c ..ho~ the invention provides a l'l -~
15 t - useful for p lu~luliun of vaseularor Iymph e ~ eell growth - . ~ a ll~.................... t; -lly effeetiveamountoftheVRPina~ Iy_ , ' '- carrier. Inanotheraspeet,this~ furthert , i~ another cell growth faetor sueh as VEGF andlor PDGF.
In a further aspect, the invention provides a method of treating vaseular tissue and iJ'~ - e - ~,S;og~ in a mammal cu...~ iu.g to the mammal an effcctivc amount of the ~o~p~
2 0 cu--.,~- e VRP. In another ~ L ~ ' t, the invention provides a method for treating trauma affeeting the vaseular; ' - ~' ~,UllliJI ' ' _ - ' ' ' - ' _ to a mammal suffering from said trauma an effeetive amount of the ~- ~- ~I ~ ' ;~ - e the VRP. The trauma is, for example, diabetie ulcers or a wound of the blood vessels orheart. Inanotherc ~ i~o~ .the inventionprovidesamethod fortreatingady~r-- ~ l statech,~"_,~ ~d by laek of aetivation or lack of ' ' of a receptor for VRP in a mammal CU~IiJI i .:..g P 1 ~ ~; ~ ;~ ~ i..g to the 25 mammal an effective amount ofthe c-----l ~-:~;~,., .-.--l~;--;..g the VRP.
The invention also provides a method which involves c ~ ' - '; E the Flt4 reeeptor with the VRP to cause , ' , ' yhlliun of the kinase domain thereo~ For example, the invention provides a method for ~ ; E the I~I.o:."h~..yla~iullofatyrosinekinasedomainofaFlt4receptorcu...".i:.i..gc .~--l;-~Ean~YtrP~ domain of the Flt4 receptor with the VRP.
30The invention also provides a -' ' antibody which binds to the VRP and ~,.ef~,.,ll;ly also a ~ ' _ ' activity of the protein, one biological activity being .,I.,~,~,t~,.i~d as "-~ - g r . ' ' ' or vascular p . ~ - ~;I;ly or vascular e ,1..I~ cell growth in a mammal. AlI~ _Iy or c ; ~_ly, the invention provides a ~ 1 ~ =' antibody which binds to the ~J I ' portion from residues -20 through I 37~ inclusive, or from residues + I through 137, inclusive, of the arnino acid sequence shown in 35 Figure l The antibody can be used, for example, to detect the presence of the VRP in a l -" _ I sample suspected of having the protein. or to treat patients. The invention c~ t- - pl ~ - a l ~ - l c~
_ such antibody and a 1~ lly ~ -U!~ carrier. as well as a method of treating diseases or disorders cLu~t~ d by u~d~ Lle excessive neovascularization or vascular p~ lity in a mammal cu.. ,.. e ~ i,.. g to said mammal an effective amount of one of the ~I ;l -u ~ - - described above. Further WO 97/09427 PCT~US96/14075 included by the invention is a method for ~reating a d~-~r~ l sute ~,ha~a~ ;~d by exeessive activation or inhibition of a receptor for VRP in a mammal ~,ullltJl ;aulg '- ~ u.g to the mammal an effective amount of one ofthe - ~l;bo~ described above.
In addition. the invention c-- . . ~ a peptide co~ P of an amino acid se4uenee shown as 5 residues -20 through - I . inelusive, of Figure I .
In a further ~ o~ ~l the invention provides an isolated nucleic acid molecule encoding VRP or a VRP ehimera. m one aspect, the nucleic acid molecule is RNA or DNA that encodes a L ~ I o lly active VRP
or is ~ , ' y to nucleic acid sequence encoding such VRP, and remains stably bound to it under stringeM
s- The nucleic acid moleeule optionally ineludes tile regions of the nueleic acid 5~ of Figure û I whieh eneode signal se ~ c In one ~ ~hv~ . the nueleie acid sequence is selected from:
(a) the eoding region of the nueleic acid sequence of Figure I that codes for the p~ r~ from residue -20 to residue 399 or that codes for the ~nature protein from residue I to residue 399 (i.e., ....~ 372 through 1628, inclusive, or . i~ 432 ihrough 1628, inclusive, of the nueleie acid sequenee shown in Figure I as SEQ ID NO: I ); or 15 (b) a sequence cv.. ~ i -e, to the sequenee of (a) within the scope of fi~b. - .- ~ ~ y of the genetic code.
in another aspect, the nucleic acid moleeule ean be provided in a .c, ' ,r' .~ veetor Cv~ ;ah~g the nueleieaeidmoleeuleoperablylinlcedtoeontrol 3~ e~ 'V ;' i byahosteell~ rr~ ~iorh al~fu~lllcd with the veetor. The invention further provides a host eell cullli!l; ,;..g the veetor or the nueleie acid - ' A method of ~-vvu~u~o VRP is also provided whieh cv...,ul ~_s eultuiing a host eell ~.u...~ O the nueleie aeid 2û moleeule and ~~v._g the protein from the host eell culture.
BRlFF DE~;CRIPTION OF TH~ DRAW~NGS
Figures I A- I D depict the . .- ~ ir ~ coding sequence (SEQ ID NO: 1), ~- ~- l u~ , ' Y
sequence (SEQ ID NO: 2), and deduced amino aeid sequenee (SEQ ID NO: 3) of the human VRP deseribed hetein.
Figure 2 depiets binding of Flt4/lgG and of Rse/lgG (an unrelated reeeptor fusion protein~ to the human glioma eell line G61, whieh binding was evaluated by FACS analysis Figures 3A and 3B l~a~J~,.,Li~r~ly depiet a map of eDNA elones eneoding human VRP and an &1'~ "" - '1 of the protein sequenee VRP (SEQ ID NO: 3) with that of VEGF121 (SEQ ID NO: 4) and PIGF131 (SEQ ID
NO: S). Figure 3A shows the extent of four VRP eDNA elones; dashed lines indieate the missing portions of 30 VHI . I and VH I .3. Arrows indieate .c~ liun enzyme sites; the shaded box indicates the putative seeretion signal sequenee; the open box indieates the mature protein; Y-type d~ within the open box indieate the potential N-linked glyeosylation sites; and vertieal lines indieate the eysteine residues. A diagrarn of VEGF121 is shown for ~,u~llyal iavll. The hy il upallly plot (Kyle and Doolittle, J. Mol. Biol.. 157: l û5- 132 [1982]) is for VRP. In Figure 3B. overlining indieates the ~egion eneoded by an ~,A~ICa:>e;l sequence tag (EST) (sequence of 35 aportionofacDNAclone)fromGenBank~ ;r~ dHsclwFllB
Figure 4 depicts a map of the cDNA clone for full-length human VRP herein versus eleven known ESrs. The eleven EST partial amino acid sequence r. a~ n:~ are H07991 and H07899 (5' and 3' ends of the same cloned fragment. .c~,ue~ l,v), H05134 and Hû5177 (3' and 5' ends of the same cloned fragment.
. c~ ), HSC I WF 112 and HSC I WFI I I (3~ and 5~ ends of the same cloned fragment. respectivelv). T81481 W O 97/09427 PCTrUS96/14075 and T81690 (3' and 5' ends of the same cloned fragment. ~y~ Iy), R77495 (a 3' end of a eloned f' _ t) and T84377 and T89295 (5' and 3' ends of the same cloned fragment. respectively).
Figure 5 depicts binding of 1251-Flt4/lgG to purified VRP. The binding was p..L.-..ed in the absence (-) or presence (+) of 100 nM receptor IgG fusion protein (Fig.5A) or with in.,- c~i~g ~ of Flt4/lgG
5 (Fig.5B).
Figure 6 shows a graph of the cell count of human lung .ni-,.u~a ,cular c .~ h~ cells as a funetion of the c~ of VEGF or VRP in the cell culture medium to assess and compare O activity.
DETAlLFn DESCRIPTION OF T~E PRF~ERRFn El~IRODIMF~TS

In d~ ,.;l,;.-g the present inveMion, the following terms will be , ' ~_d, and are intended to be defuled as indicated below.
"Human VRP" is defined herein to be a pol~ ide sequence C~JI~ 5 at least residues -20 to 399, inclusive. or residues +I to 399~ inclusive. of the amino acid sequence shown in Figure I, ineluding residues -5 to 399. inclusive, and residues 1 to 399. inclusive. of the amino acid sequence shown in Figure 1. as well as 15 ~ - ' ,,- 'ly active ~ tinn~ ù~dl. or ~ I variants of the above , having at least 265 amino acids and/or having at least residues + I through 29. inclusive, of Figure 1. In a preferred . ' ' ~ t, the protein sequence has at least residues +I thrûugh 137. inclusive, of Figure 1. more l,.efe.al,ly at least residues -20 through 29, inelusive, of Figure 1, and most p.ef..dl,ly at least residues -20 through 137, inelusive, of Figure 1. In another preferred - ~ ~1 c,- ~; ~ . ~1 the L ~ ' ~" 'Iy active variants have a length of 265 to about 4S0 20 aminoacidresidues,more~ f~,.al,lyabout300-450,evenmore~.. f.. dL,lyabout350-450,andmost~,.c~l ' Iy about 399-419 amino acid residues. Another preferred set of variants are variants that are i..~,.l ' or ~ ~ ' variants, or ci~ tinn:~l variants where the deletion is in the signal sequence and/or is not in the N-terminalregionofthemolecule(ie.,residues 1-29,~ f.,al.1yresidues 1-137). ThedefinitionofVRPexcludes all known EST s~ such as, e.g., H07991, H05134, H05177, HSCIWF112, HSCIWFIII, T81481, 25 R77495. H07899, T84377. T81690. and T89295, as well as all forms of VEGF and PIGF.
"Biologically active" for the purposes herein means having the ability to bind to, and stimulate the phu:~Lu~ldliu~ of. the Flt4 receptor. Generally, the protein will bind to the eYt~nr~ domain of the Flt4 receptor and thereby activate or inhibit the i.,1-~ tyrosine kinase domain thereof. C~ , 'y. binding oftheproteintothereceptormayresultinc ~ ~, orinhibitionofl,.ulif..aLionand/orLrf .c.aialiu..and/or 3 0 a~ ,a~iun of cells having the Flt4 receptor for the VRP in vivo or in vitro. Binding of the protein to the Flt4 receptor can be ~ 1- ~ 1 using C.u~ aiu~dl ~ including cu...u.~ , binding methods, such as RLAs, ELISAs, and other cu...~,~,lilive binding assays. Ligand/receptor c , ' can be i' -~ ~ using such s~lJa. ~ methods as filtration~ c..a.; ~ ;.... flow cytometry (see, e.g., Lyman et aL, ~11, 75: 1157-1167 [1993]; Urdal et aL, 1. Biol. Chem.. ~:2870-2877 tl988]; and Gearing et aL, EMRO J.. ~:3667-3676 rl989]), 35 and the like. Results from binding studies can be analyzed using any cu,,~ ional graphical ~ c~ ~., of the binding data. such as S: ' .1 analysis (Scatchard, Ann NY A~rl Sci..51 :660-672 [1949]; Goodwin et aL, ~L 73:447-456 ~1993]), and the like. Since the VRP induces pl.u:.~.hu,ylation of the Flt4 receptor, cu~ iu~al tyrosine phu~ul-u-ylation assays, such as the assay described in Example 5 herein. can also be used as an i- ~ ;.,.. of the fo. .. ~ ;-... of a Flt4 receptor/VRP complex.

WO 97/09427 PCTrUS96/14075 The term "epitope tagged" when used herein refers to a chimeric poly~,~,,Jfidc ~ .;..g the entire VRP, or a portion thereof. fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody Ihel Cagail~:" can be made. yet is short enough such that it does not interfere with activity of the VRP. The tag poly~ ,li.;lc ~ ~,al~ly also is fairly unique so that the antibody ~
5 does not a ~ lly cross-react with other epi~opes. Suitable tag pol~,u~ id.!, generally have at least six amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9-30 residues).
"Isolated." when used to describe the various proteins disclosed herein, means protein that has been irbntifipd and separated and/or ~~.~,u._~,d from a c~ of its natural e.,~u~ C~
of its natural e.-vuu....l~.. are materials that would interfere with ~" _ or ~. r uses for 10 the protein. and may include enzyrnes, i~---. ... ~ and other ~., t or r~ p.~ solutes. In prefered --I,~l;~~ theproteinwillbepurified(l)toadegreesufficienttoobtainatleastl5residuesofN-temminal or intemal amino acid sequence by use of a spinning cup ieq ~ ~ , or (2) to ~ g ~ by SDS-PAGE under non-reducing or reducing c~ using Coo~ blue or, I,.efi,.dl,ly, silver stain. Isolated protein ineludes protein in s~tu within .~ cells. sinee at least one ~- r of the VRP natural 15 c..vuu.u..~.,l will not be present. Ordinarily, however, isolated protein will be prepared by at least one step.
~r ~y pure" protein means a c- .. . ~p- .- ;~ ;~,n cu,,"u, iaul~, at least about 90% by weight of the protein, based on total weight of the c ~ ,f;.al,ly at least about 9S% by weight. "r~ ~; -îly l ~ -protein means a c- .--~~ ;,.-. cu...~ u.g at least about 99% by weight of protein, based on total weight of the An "isolated" VRP nueleie aeid molecule is a nueleie aeid moleeule that is identified and separated from at bast one c~ nueleie aeid molecule with which it is ordinarily ~ ~ in the natural source of the VRP nueleie acid. An isolated VRP nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated VRP nueleie aeid -' ' therefore are ~ .t;.~ l from the VRP nueleie acid molecule 25 as it exists in natural cells. However. an isolated VRP nucleic acid moleeule ineludes VRP nueleic acid conuined in cells that ordinarily express VRP where, for example, the nucleic acid molecule is in a ' ~-----~-- - -' location different from that of natural cells.
The isolated VRP polypeptide, VRP nucleic acid, or VRP antibody may be labeled for ~ I: .,~"-- ,~l ;c and probe purposes, using a label as described and defined further below in the . l ;~ on uses of VRP . . ;I -v~l ~
The CAIJI~ ;OII "control SC-l~ - f~'- refers to DNA ~ necessary for the CA~I~a:~;UU of an operably linked coding sequence in a par icular host organism. The eontrol s~ that are suitable for I... k~ ~ . for example, include a promoter, optionally an operator setl~enre a ribosome binding site, and possibly, other as yet poorly ~ od s~ c Eukaryotie eells are known to utilize ~JlU~IlUt~a, pOlyad--",' signals. and L~hà~ a.
Nueleie acid is "operably linked' when it is placed into a fi-nrtion~ with another nucleic acid sequenee. For example. DNA for a p.c~ e or seeretory leader is operably linked to DNA for a ~I~"~.,JIid~,ifitisexpressedasa~ that~Jalli.,;~.,t.~inthesecretionofthepolypeptide;apromoteror enhancer is operably linked to a coding sequence if it affects the llall,~. iyliull of the ae 1" ~ or a ribosome binding site is operably linked to a coding sequence if it is poailiulled so as to facilitate lla~ -'' Generally, W O 97/09427 PCT~US96/14075 "operably linked" means that the DNA Sf ~Iv ~ ~C being linked are r~ v~ and. in the case of a secretory leader, cu.,~;g.~v~ and in reading phase. However. L~hallCc~a do not have to be c ~ o~ Linking is a~ J by ligation at cu,.~.,.. ;.,.. L .~ iu.. sites. If such sites do not exist, the synthetic ûl;gu~ r adaptors or linkers are used in a.,~,u.Ja...,e with con~...iul~al practice.
Thc term "antibody" is used in the broadest sense and crerifir~lly covers single anti-VRP l bc ' (including agonist and ~ g. -- . ;~ ' ' ) and anti-VRP antibody . . with polyt r , ~ ~
S~ ;r.~
The term ~'.~ no~lû~ l antibody" as used herein refers to an antibody obtained from a F, ' of lly hV---5e,. ~v~- ~t;l,o-li c. i.e., the h~v;~;llùal ~ ~.l.o~ coll~Jl;aill~5 the F r ' " are identical except for possible naturally-occurring .~ that may be present in minor amounts. M.~--~ lo -are highly specific, being directed against a single antigenic site. FU~ u~c, in contrast to ~
(pol.~_lu..al) antibody ~ a~ which typically include different a--~' - ~- directed against different (epitopes), each ~ ' ' antibody is directed against 8 single ~ on the antigen.
The .- ..~r" 1-~---1 ~I;l o~{ c herein include hybrid and ~cc ': ~ " ~ produced by splicing a 15 variable (including l.,~ ".,. ~ ' ' ) domain of an anti-VRP antibody with a constant domain (e.g. '1 ~ 1"
~- - ' ), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with h~t.,.,~l~o _ proteins, r~;~dl_a~ of species of origin or ~0'~' " class or subclass A- ~ as well as antibody G."s...l,,~ta (e.g., Fab~ F(ab')2, and Fv), so long as they exhibit the desired ~;-')g ' activity. See, e.g U.S. Pat. No. 4,816,567 and Mage and Lamoyi, in M~ n~ l Antihn~
r~ TPI.;~ _An~ ~VI;-A~ pp.79-97 (Marcel Dekker. Inc.: New York. 1987).
Thus, the modifier ..--. ~ indicates the character of the antibody as being obtained from a 1 -- l; n~/ h.. n~ .. rn.. ~ pnp~ inn of _.. l il .~. l ~ and is not to be cunaLl .. ~d as requiring l.. uJu~,~iu.. of the antibody by any prl li.,u6. method. For example. the .. o. l- .. -1 .l il ~uJ; to be used in accv.J~.,c with the present invention may be made by the l..~ L.. ;.Iu...a method fu st described by Kohler and Milstein, I!l~ ~i495 25 (1975), or may be made by ... ' ~ DNA methods. U.S. Pat. No. 4,816,567. The "....... - ~ ti' ~ ' ' may also be isolated from phage libraries generated using the ~ . described in McCafferty er al.. Nature.
348:S52-554(1990), for example.
Pd~ forms of non-human (e.g. murine) _..l;l.o~l;. e are specific chimeric ;~ glnl~ul: -,lrl,--lin chains, or rl~r.~ thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding 3 o ~ - of - ~ ;I .o~l; -) which contain minimal sequence derived from non-human; ~ .. ..r l~.lJ~llin For the most part, I ~ ' _ ~l ;l ~o~ ~: c are human ;. . - -- ,o~;l.,b l ;. .~ (recipient antibody) in which residues from a _ , ~ y ~ ;..;..g region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody? such as mouse, rat. or rabbit having the desired specificity, affinity, and capacity.
In some jnct-nr. c Fv r.~.... ~ ,.k region (FR) residues of the human ;.. ---.~.~slnb.. lin are replaced by Cull-~l-O '.~. non-human residues. Fu.lL.,....ù.~, the l-- -..- .;,. d antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or Ga~ 'OI ~ rhese m.~I; ri. Al ;1~
aremadetofurtherrefuneandoptimi_eantibodyp~.ru....a..~c. Ingeneral.thel -..--.;- ;lantibodywillcomprise 'ly all of at least one. and typically two. variable domains. in which all or ~ - .l ;-lly all of the CDR
regions <.OII~.a~ulld to those of a non-human ;... -..- ,oglnl~lin and all or 5..1,~l_."i~11y all of the FR regions are CA 02231400 l99X-03-09 W O 97/09427 PCTrUS96/14075 those of a human i.... - .ogl.,l, Iin C- ~ sequence. The 1~ ;,. I antibody optimally also will comprise at least a portion of an i o~ lob~ n constant region (Fc), typically that of a human i.. ~ o~,lob~
As used herein, "vascular - ..~ l cell growth factor," or "VEGF," refers to a ' growth fsctor derived originally from bovine pituitary follicular cells having the amino acid sequence of Fig. 2 of WO
90/13649, and has the human amino acid sequence of Fig. 10 of WO 90/13649. Sec also U.S. Pat. No.
5,194,596, which discloses bovine VEGF of 120 amino acids and human VEGF of 121 amino acids. The biological activity of native VEGF is capable of ~.. u...uLil.g selective growth of vascular ~ cells but not of bovine comeal ~ ~t--~ l cells. Iens epithelial cells. adrenal cortex cells, BE~K-21 ~;L,.. ~' or t.~.
lo The ~AIJ~ a;U~l "trauma affecting the vascular ~ u~ h~ refers to trauma, such as injuries. to the blood vessels or heart, including the vascular network of organs, to which an animal or human. p.cf..d.l~ a mammal, and most preferably a human. is '; ' F . ' of such trauma include wounds, incisions. and ulcers, most ~. ef.. aL Iy diabetic ulcers and wounds or 1-~ r ;. - .~ of the blood vessels or heart. Trauma includes ' caused by intemal events as well as those that are imposed by an extrinsic agent such as a pathogen, which can be improved by l~u~uliun of vascular P--~ l cell growth. It also refers to the treatment of wounds in which nc~ ,ulali~aliùn or re-~ ~riulh li~ qfi~m is required for healing.
of vascular or Iymph ~ h ~ll 1; I cell growth" refers to inducing or i,.~.c~i..g the growth ofvascularorlymphP--I~ l;-lcells,includinghumanlung..~i~..u.~.ular ~ ' ' 'cells."Disorders related to v~ ,~, ~.~ ;~ and A-~ include cancer, diabetes, h -- G;~ ~ -q. and 2 0 Kaposi's sarcoma.
"Diseases or disorders ~hala~ 4d by l..-d~ L 1~ excessive neovascularization or vascular p ~ t ~ Iy" refer to diseases or disorders that include, by way of example, excessive nc~).aa~ulal;~alioll, tumors, and especially solid ~ -I;u-~ ~~ tumors, -' ~ ' atthritis, psoriasis, aLh~uS~ uaia, diabetic and other rc ' , ' let~ ' a, age-related macular dCg~ aliùn, 11~.~ -aa-uh- g~ rnmq ~'-----1"
25 thyroid L~ IJIaaial (including Grave's disease), comeal and other tissue t.,...~ -a~ and chronic ~ '' F . ' of diseasesordisordcrs~l.aa.,h.i~.lby~ i "e eAc..~ vascularp ~ _h~ y include edema ~o- :-t ;I with brain tumors, ascites ~u :-~- d with .. -1;~ --- ;. c Meigs' syndrome. Iung ;- n ~ --- o~ nephrotic syndrome, p~,~h,a~d;al effusion (such as that ~ CO~ d with p~ dili5)~ and pleural effusion.
3 o "D,~r. - l ;. .-- -I states ~ al~ i~d by excessive a~li vdtiull or i--hiL,;liun of a receptor for VRP" (such receptor including Flt4) refer to disorders or diseases that would be b~n~fir~ y treated by ~u~;dh~g to a mammal having such a I ' - ' ~, ' condition an ~ l to VRP, such as a chimera of Flt4 or its e domain (e g. an IgG fusion with Flt4) or an antibody to VRP.
~~Dy~r ~,;-~ ~~1 states ~lla a~ .d by lack of a.,~ivaLiull or lack of inhihi~irn of a receptor for VRP"
(such receptor including Flt4) refer to disorders or diseases that would be b- r;. :_lly treated by providing VRP
or a VRP receptor agonist to a mammal with such a paLllolcgir;~l con~
"Treatment" refers to both 1l ~ ;- treatment and prophylactic or ~ ,.n~ e measures. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be ~ V_.It~l W O 97/09427 PCTrUS96/14075 "Mammal" for purposes of treatment refers to any animal classified as a mammal. including humans, domestic and farm animals. and zoo~ sports. or pet animals, such as dogs, horses. cats. cows, etc. P~ef~,.dllly, the mammal herein is human.
"Effective amount" or ,l. ~~ ly effective amount" of the VRP. VRP c~ --- antibody, or antibody c~ nJ- :~ .. is an arnount that is effective either to prevent, lessen the ~. Jl a~h~g of, alleviate. or eure the treated ennAitinn For example, an effective amount of VRP includes that amount whieh is sufficient to enhance the growth of vascular .-..A..I~ . in vivo or to treat trauma. and an "effeetive amount" of VRP
antibody includes that amount which is s~lffri~nt to reduce excess ncu.~,uLui-aliu~ and v Il. Modes for C~r5dnP Out the Invention The present invention is based on the discovery of a novel VRP which binds to, and ' the phûs}Jl.c..~/ld~ n of, the Flt4 receptor.
Ihree ~ ua l.~,s were ~ ' ' to identify protein that would bind and stimulate the p' . ' ~ Id~iu~
of the Flt4 receptor. First, the full-length receptor was stably c~ a~, i in 293 cells to establish a receptor tyrosine kinase pLoa~Jl.u.ylation assay of Flt4 8~,Lvdliull. This assay was used to screen about 400 eell 15 ~ and tissue extracts, without positive results.
Seeond, the e-l~ r domain of the receptor was ~iA~ ,d as a fusion protein with an ~ , ' ' ' Fc domain. By using this fusion protein (Flt4/lgG) to sereen eell lines for ~ ' - bound ligands by FACS analysis, one positive eell line WdS iA~r ~ifi~A The human glioma line, G6 1, gave about a lO-fold shift in peak lluu.~;..,.,...,e intensity that was specific for Flt4/lgG (Fig. 2). Attempts to ~A~ ' elone this 20 putative ...~...1,. buu..d ligand by the 1l- . r. ~i.,., of pools of cDNA elones into COS eells followed by screening with labeled Flt4/lgG gave no positives from 640 pools of 1000-5000 elones each. Flt4/lgG was also used to generate polyclonal antisera and ....,"~ ..l ;l.oA ~ that had agonistic activity and that were used to develop the Flt4 tyrosine ~JLûa~Lu~ylation assay as described in Example S below.
Third. candidate ligand proteins were tested for their ability to bind to Flt4/lgG or to activate the Flt4 25 phGal,l.u.ylation assay. Labeled VEGF failed to bind to Flt4/lgG, although the expected binding of VEGF to Fltl/lgG or Frkl/lgG was routinely detected. The failure of VEGF to bind or stimulate the pllOa~ l.u-ylation of Flt4 has been reported by Pajusola et aL, 017( nP~n~ 2. s~pra. An aA~ l candidate ligand protein was found by use of cloning ~ - details of which are provided in Exarnple 3 below. The human VRP cDNA
sequence is depicted in Figure l A- I D. The predicted mo' l~ weight of the protein is 44.8 kDa.
3 o A d~.,. i~J~iU~ follows as to how the biologically active human VRP may be prepared.
I . r~ c~ ûf VRP
Most of the .l; ~ below pertains to t~ udu~iu~ of vRp by culturing cells ~ r~ m~ i with a vector c-- ~ L, VRP nucleic acid and recovering the polypeptide from the cell culture. It is further ~ iu..~d that the VRP ofthis invention may be produced by homr~g l~.~.u~bh~a~iO~, as provided for in WO 91/06667, 35 published 16 May 1991.
Briefly,thismethodinvolves~.a...ru....i~.gprimaryhumancellsco~ .;..gahumanVRP~ ,od...ggene with a construct (ie., vector) Cu~ Jflai~g an a~ gene [such as dihydrofolate reductase (DHFR) or others discussed belowl and at least one flanking region of a length of at least about 150 bp that is hnn r ~ lg with a DNA sequence at the locus of the coding region of the VRP gene to provide a~ ,adu~ of the VRP gene.

~~0 97/09427 PCTrUS96/14075 The ..~ 1f gene must be at a site that does not interfere with CAIJIC..a;UII of the VRP gene. The m -- ru~ is ~ t d such that the construct becomes homologously i..t~O. ~ into the genome of the primary cells to define an .---.pl;l~ lP region.
Primary cells cu...~ i-.g the conswct are then selected for by means of the ~ ' ' gene or other 5 marker present in the construct. The presence of the marker gene L ~ 1. c the presence and ;. ~t~ of the construct into the host genome. No fwther selection of the primary cells need be made. since selection will be madeinthesecondhost. If desired theoc.,u..~,..ccoftheh~ O .e~u~ ~ cventcanbed- ~
bye, ' ,i.,gPCRandeither,c-l-- .- :-~etheresultingamplifiedDNAa~ orcl, 5 .--; -~gtheat,l,.u~.
Iength of the PCR fragment when DNA from correct h~ ,c 'c O integrants is present and ~ 1;- e only those 0 cells ~ g such r. ~ Also if desired, the selected cells may be --mplifi~ d at this point by stressing the cells with the a~ ulJI ia~c a ..l,l;ryi..g agent ~such as ~.._LI.ul~ cAate if the ~ . ' - ' ' - gene is DHFR), so that multiple copies of the target gene are obtained. Preferably, however, the ~ r.. ~ step is not L~ d until after the second L a- - r -- l--~ described below.
Afterthe selection step. DNA portions ofthe genome. ~..IT;.: .aly large to include the entire . ' ' ' 15 regiûn~ are isolated from the selected primary cells. Secondary m~ C~}JIC~a;On host cells are then b ~ ' with these genomic DNA portions and cloned. and clones are selected that contain the ~ . ' ' ' region. The . ' ~ '-' region is then amplified by means of an alll~ yil~g agent if not already amplified in the primary cells. Finally, the s__ulldaly cA~JIc;~a;ull host cells now cu...~.; .i..g multiple copies of the . ' ' ' region ~ e VRP are grown so as to express the gene and produce the protein.
A. I~rl~inn of DNA Enf ~AinP VRP
The DNA encoding VRP may be obtained from any cDNA library prepared from tissue believcd to possess the VRP mRNA and to express it at a A - ' '- level. Accu.. ~'y, human VRP DNA can be ~ _..;_...ly obtained from a cDNA libraty prepared from human brain tissue, e.g., a glial cell line. The VRP-encoding gene may also be obtained from a genomic library or by ol;g.. - -~ tid~ synthesis.
Libraries are screened with probes (such as ~ -o~l ; to the VRP or olig~ k~iAf S of about 20-80 bases) designed to identify- the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be c-~A~ I using standard ~ .o~c.lu.~ as described in chapters 10-12 of uol~ et al., Molf~rl~hr Clnnin ~P: A Labor~ rv M~n~ (New York: Cold Spring Harbor Laboratory Press, 1989). An ~ means to isolate the gene encoding VRP is to use PCR ~.._lI.c ' ~Oy as described in 30 section 14OfSambrooketaL,supra.
A preferred method of ~ i.,u.g this invention is to use carefully selected oli~.,..--- I- u(iAf~ 5 to screen cDNA libraries from various human tissues. preferably brain cell lines. The ~~iiC,.. -- 1. ,1;-l- s~.l.. ~c selected as probes should be of sufficient length and ~. .11~ :- . . I Iy ~~la~lb ~,UOu ~ that false positives are ~
The olip..~ lr,~ jA, must be labeled such that it can be detected upon h~,l..;.li~iu.. to DNA in the 3 5 library being screened. The preferred method of labeling is to use 32P-labeled ATP with pol~,....~ Ir~/l;A~ kinase.
as is well known in the art. to radiolabel the oligo- ,- ~ I -,1 ;A. However. other methods may be used to label the ol;2;. --. -- I ~1 jA~ jnrl~Aing, but not limited to, biotinylation or enzyme labeling.
In some preferred . . l~o~ the nucleic acid sequence includes the native VRP signal sequenr~-Nucleic acid having all the protein coding sequence is obtained by screening selected cDNA or genomic libraries W O 97/09427 PCT~US96/1407 using the deduced amino acid sequence disclosed herein for the first time. and. if necessary, using co..~ . ' primer extension plu~edu~ci~ as described in section 7.79 of Sambrook et aL, supra. to detect ~ au~a and p.u.,e;. .;..g i"t~,... - ' of mRNA that may not have been reverse-tla..s~, ;Lcd into cDNA.
Arnino acid sequence variants of VRP are prepared bv u-l u-lu~ g a~ u~- r 1 ~; i changes into 5 the VRP DNA, or by synthesis of the desired VRP poly~,plidc. Such variants represent insertions.
and/or deletions of, residues within or at one or both of the ends of the amino acid sequence shown for the VRP
in Figure l. Preferably, these variants represent insertions and/or s~ c within or at one or both ends of the mature ePql~nrr and/or insertions. s~ and/or deletions within or at one or both of the ends of the signal sequence for VRP shown in Fig. I. Any c~A~n l of insertion, ~ and/or deletion is made 10 to arrive at the funal construct, provided that the funal construct po~ s the desired l '~ ' activity as defined herein. The amino acid changes also may alter post-l~ processes of the VRP, such as changing the nwnber or position of glycosylation sites, altering the ~llc~ a~c a l~.hul i~g clla a~ lil,5, and/or altering the intra-elh~ location of the VRP by inserting, deleting, or otl._, ~. ia~i affecting the leader sequence of the VRP.
Variations in the native sequence as described above can be made using any of the r. I ;-l ~ and g,. A~ for cu..~ _i;;. and non-cu~ a~ set forth in U.S. Pat. No. S,364,934. These include oliU~ mediated (site-directed) l".. lA,~ c alanine s~AAnning and PCR ",.~t.~j. - ' ' See also, for example, Table I therein and the .l;~ -. . surrounding this table for guidance on selecting amino acids to change, add, or delete.
B. !nc~tion of Nucleic Acid intAI Re~licable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding native or variant VRP is inserted into a replicable vector for fwther cloning (A ~ of the DNA) or for t.~ ;O~. Many vectors are available.
The vector c v l .~ generally include. but are not limited to, one or more of the following: a signal sequence, an origin of ,~ . one or more marker genes. an enhancer element, a promoter, and a Ll_lla~ i 25 ~ , (i) Si~nAI Se~uence Cu...~uù...,..~
The VRPs of this invention may be produced ,c~o ~ lly not only directly, but also as a fusion pol~"_pt;dc with a l-~t~. . ' O polypeptide, which is preferably a signal sequence or other poly~,_ptid_ having a specific cleavage site at the N h. .,.u.u, of the mature protein or poly~,_p~i.le. In general. the signal sequence 30 may be a c.. l.. ,.. - l of the vector, or it may be a part of the VRP DNA that is inserted into the vector. The h_t.,,~ ~, signal sequence selected preferably is one that is ~ and ~.,u.~ ._d (i.e., cleaved by a signal peptidase) by the host cell. For p~ukol ~ ~nic host cells that do not l ~,COol~ and process the native VRP signal 5~ 1 the signal sequence is ~~ t~ d by a prokaryotic signal sequence selected, for example, from the group of the alkaline l ~h ~ -. penir~ n~cp Ipp, or heat-stable C.lt~,.u~u~.ul ll leaders. For yeast secretion 35 the native signal sequence may be ' ' by, e.g, the yeast invertase leader, alpha factor leader (including ~ ~ and Kl., " ".,, .v. c~ ~ -factor leaders, the latter described in U.S. Pat. No.5,010.182 issued 23 April l 991), or acid rh~ leader. the C olbicans glucoamylase leader (EP 362,179 p..hlichPd 4 April 1990), or the signal described in WO 90/13646 publ;alled 15 November l990. In r.. ... ~ cell e,.~ ;ol.
the native signal sequence (e.g., the VRP ,u.~ e that normally directs secretion of VRP from human cells in v vo) is ~ r I ..y, although other rn~~mlliAn signal 5~ c may be suitable, such as signal s~
from other animal VRPs. and signal ~ .. r; from secreted polypeptides of the same or related species. as well as viral secretory leaders. for example, the herpes simplex gD signal.
The DNA for such precursor region is ligated in reading frame to DNA encoding the mature VRP.
(ii) Ori~in of Rcvli~ f f~ vu~
Both eAi,.. and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate i ' r ' ''y of the host ~lu~ ,<o-- ~i DNA, and includes origins of ,~pl; ~ , or _ 1~
IC, I g ~ Such ~ are well known for a variety of bacteria, yeast, and viruses. The origin 10 of ~ from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2iu plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma ad~ . .. us, VSV or BPV) are useful for cloning vectors in ~- -------~1; cells. Generally, the origin of . IJI;~ JII C ~ ~p~ l is not needed for eAi..c vectors (the SV40 origin may typically be used only because it contains the early i-,.u..lv~..).
Most eAiJ~ ~ vectors are "shuttle" vectors, i e., they are capable of .~ .~ in at least one class 15 of u.i~a ~ but can be I", r ~ I into another organism for ~AiJ~;U~. For example, a vector is cloned in E.
coliandthenthesamevectorisL.,..af..~cdintoyeastor.. ~.-.~I;-~cellsforeAiJ-. eventhoughitisnot capable of .~ e ~ "~ ly of the host cell ~ c ~
DNA may also be P~nplifirf1 by insertion into the host genome. This is readily ~r~f.- ,1,~ .1 using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is ~ ~ , ' ~ y to a 2 o sequencc found in Bacill~s genomic DNA. T. .~ r~ of Bacillus with this vector results in I - ' ,, .~ ~ ' with the genome and inser~ioa of VRP DNA. However, the recovery of genomic DNA encoding VRP is more complex than that of an ~ - ~c,- ~ iy ~ - . " - ' vector because I c~LI i~,Liùn enzyme digestion is required to excise thc VRP DNA.
(iii) Selection Gene C~ ~uu~
EAiJ~;U~ and cloning vectors should contain a selection gene. also termed a '- ' '- marker. This gene encodes a protein necessary for the survival or growth of 1. A-,~r~ d host cells grown in a selective culture medium. Host cells not LI A ~r. -- . . . ~d with the VeclOr ~f~ .; .g the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer I~Ji ,Lancc to -- ~ or other toxins, Gg., ' -'lin, nco...~,;-., Il._LLuL--,A,,t-, or ~ a~y-,li-.c, (b) c~--Fl ~ y~hic J- r~ or (c) supply 3 o critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are ~ . ~r--~ r .. ~d with a h~ O gene produce a protein cv.. f.. i.. g drug rc~ .~.. ,c and thus survive the selection regimen. Examples of such dominant selection use the drugs n~u~y~,u~ (Southern e~ aL, J Molec.
1. Genet.. 1:327 [1982]), mycophenolic acid (Mulligan et aL, Science~ 209:1422 tl980]) or hy!!;.u...y~;...
35 SugdenetaL,Mol ~ il Bioi.. ~:410-413(1985). Thethreeexamplesgivenaboveemploybacterialgenesunder cukal~ ic control to convey ~Ji:~l.,C to the ~ u~k~t~, drug G418 or n~ ;.. (geneticin), xgpt i....nl:r acid), or hygromycin~ .ly Another example of suitable sc~ markers for mAmmAii~-l cells are those that enable the i~i. ifi. -~;.... of cells co.,l~ .,L to take up the VRP nucleic acid. such as DHFR or Ihyllli-l;..c kinase. The m-~nm~ n cell ll-~ru~ are placed under selection pressure that only the ha~all are uniquely adapted to survive by virtue of having iaken up the marker. Selection pressure is imposed by culturing the halla~ulllla~l a under c~ in which the CO~ ,,lua~iOll of selection agent in the medium is a~ a;~_ly changed, thereby leading to ~ ,,l of both the selection gene and the DNA that encodes VR?.
5 A.. .~ is the process by which genes in greater demand for the p. uJu.,~iun of a protein critical for growth are ncit~ ' in tandem within the eLI~ os ~ r, of successive g_,._,d~iùlls of ~ec bi - cells. Increased of VRP are s~lLh_ai2~d from the amplified DNA. Oiher examples of . ' - ' ' genes include rnl~t~ll..ll.;l),l. ~.-1 and -Il, i-JIefc.diJly primate mrt~ ollr - genes. ~ i - ~- :--- ' oi-nithine dc~.a~ i~UA~ laac~ etc.
1 û For example, cells h ~ ' with the DHFR selection gene are fust identified by culti ing all of the h '' in a culture medium that contains nl_Ll-uilc - (Mtx), a cu...iJ~ c r _ ' ~ of DHFR. An , . u,u. ~ host ccll when wild-type DHFR is e . ' ,~d is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and i,.., _ ' as described by Urlaub and Chasin, Proc. ~ tl A~ ri SC;, USA.
77:4216 (1980). The 1" - r " .- =d cells are then exposed to increased levels of ~ Lh~/i c This leads to the 15 synthesis of multiple copies of the DHFR gene, and, c- ~-.- ~ ~ ~ ~ .l -- .1 iy, multiple copies of other DNA c the cAi,.c;la;un vectors, such as the DNA encoding VRP. This . " - I ~ . can be used with any otherwise suitable host, e.g., ATCC No. CCL61 CHO-Kl, r~v~ g the presence of ~ g. ~ ~ DHFR
if, for example, a mutant DHFR gene that is highly resistant to Mtx is; . ' ,ud (~P I 17,060).
ly, host cells [lJa~ ~iL,~l~ ly wild-type hosts that contain e .~i~g --- ~ - DHFRl l,.-- .~r....~d or 20 co-i ~ ~ with DNA ~r l ~ encoding VRP, wild-type DHFR protein, and another s~ marker such as ~..i..Oc,l.~_uaidc 3'_pl~ ..u.af,.~c (APH) can be selected by cell growth in medium c~ g a selection agent for the s I ' ' marker such as an aminoglycosidic ' e.g., I..u.~..y~,i.., r~_o,,,.~_i.., or G418. See U.S. Patent No. 4,965,199.
A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7.
S,; -l~ e~aL~ ~:39(1979);KingsmanetaL~Gene~1:141(1979);TschemperetaL~Gene~1~:157( 1980). The trp I gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in L~ . for example, ATCC No. 44076 or PEP4-1. Jones, Genetics. ~:12 (1977). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting ha~ ru....aliu.. by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are 3 o , ' ~ ' by known plasmids bearing the Leu2 gene.
In addition, vectors derived from the 1.6 ,um circular plasmid pKD I can be used for 1. -- ~r. -- . .- ~ ;- --- of Kl..r.~ yeasts. Bianchi etaL, C-- r. Genet.. .L~:18S (1987). More recently, an eA}...,Ja;ùl- system for large-scale ~,.uJu~,liu.l of .er ~ ~ calf chymosin was reported for ,Y. Iactis. Van den Berg, Bio/Tl ' - - ' -~.
~:135 (1990). Stable multi-copy ~A~ a;oll vectors for secretion of mature .~ ' ~ human serum albumin byindustrialstrainsofKI~ ,, r~havealsobeen~ r~n~i FleeretaL,Bio/Te.,lll~lc_Y.~:968-975(1991).
(iv) Promoter C....~oll~
EAIJ~ c and cloning vectors usually contain a promoter that is r~ ~ U~ ~1 by the host organism and is operably linked to the VRP nucleic acid. F~ul~lut~,.a are ullhallalaL~d sc-~ c~c located upstream (S') to the start codon of a structural gene (generally within about 100 to lû00 bp) that control the l~allsLI;,ulioll and 1. ' of particular nucleic acid seqllt nrt such as the VRP nucleic acid 5"qUPllt'". tO which they are operably linked. Such yl~ typically fall into two classes, inducible and culladluli~_. Inducible ~u~vt~
are p.~ ~ that initiate increased levels of L~t~..a.,.;pliu.. from DNA under their control in response to some change in culture c~ e.g., the presence or absence of a nutrient or a change in ~ c. At this time 5 a large number of 1,. u...ut~ o~ ;7~ ~ by a variety of potential host cells are well known. These p- ~
are operably linked to VRP~ o.li..g DNA by .c,..u.i..g the promoter from the source DNA by ~.,~hi~,liù..
enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native VRP promoter se4uence and many l.~,t~ may be used to direct ~I.I;ri. _l ;n~ and/or CA~ ' of the VRP
DNA. However, l,~t.,.~ ' ~ p~ are preferred. as they generally permit greater 1. .- -~- ~ ;pl;~ ~ and higher 1 0 yields of VRP as cc ...~.". cd to the native VRP promoter.
I'ru...ot~ suitable for use with prokaryotic hosts include the ~ t-rn--A and lactose promoter systems (Chang er aL, ~as~. ~:615 rl978]; Goeddel ef aL, ~, ~:544 [1979]), alkaline pl~r~p~ a l.~"t~"L_. (trp) promoter system (Goeddel, Nucleic Ar~ Res.. ~:4057 tl980]; EP 36,776), and hybrid p.~ suchasthetacprûmoter. deBoeretal.,Proc.~l~tl A~ I Sci.US~.80:21-25(1983). However,other 15 knownbacterial~lulllvt.,.aaresuitable. Their. I~ havebeenp.. ~ 1. A therebyenablinga skilled worker operably to ligate them to DNA encoding VRP (S ' ' e~ aL, Cell. j~Q:269 [1980]) using linkers or adaptors to supply any required l~alli.,liu.. sites. Pl~ for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding VRP.
Promoter s~ are known for e~y~ Virtually all L..ka. ~llic genes have an AT-rich region 20 located 3~ Y 25 to 30 bases upstream from the site where ha la~ iUII is initiated. Another sequence found 70 to 80 bases upstream from the start of 1. ~la.,l i~Jliull of many genes is a CXCAAT region where X may be any ~ At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these, ~1. . _ c are suitably inserted into .~ttiC CA~JIC ' vectors.
25 F , ' of suitable p~u.. ~v~ g s~, for use with yeast hosts include the plul.. ut.,.a for 3-y~ pl .~l~ycerate kinase (Hitzeman et aL, 1. 13iol. Ch~m 2 S S:2073 t 1980]) or other glycolytic enzymes (Hess et aL, J. Adv. Fn7yme 1~ 149 [1968]; Holland, Bio. ~ y. 17:4900 [1978]), such as enolase, 3 i ' , ' deh~ uS_~aac.l - ~ ~L ;~ pyruvatedecarboxylase,l.l...~ Gu ~ ~L ;~ .glucose-6-1 . ' 3_pl~ ,lyceraternutase,pyruvatekinase,l.: r' . ' - ia 3 o ~ and ~ ,L;
Other yeast p. ~ which are inducible pl ull~U~.:~ having the e~ a.l~a.. ~,c of ll a~a~ io~
controlled by growth c~ are the promoter regions for alcohol d~hyJ.u5~,.la~c 2, iaO~ylû~lllu~C C~ acid ~' .' - de~a~ti~,enzymes r~ - ' withnitrogen~ ol;~ gl~c~.aldehyde-3-phos-phate dch~J.u5_..~c. and enzymes ~c r ' ' for maltose and galactose ~ . Suitable vectors and 35 ~-~ for use in yeast ~AIJI~.. iU~ are further described in EP 73,657. Yeast ~ .h_- ~_, also are al~ v ~Iy used with yeast y-~
VRP lla~a~ iùn from vectors in rn~mn~ host cells is controlled, for example, by ~lu~utc.a obtained from the genomes of viruses such as polyoma virus. fowlpox virus (UK 2.211,504 published 5 July 1989), e ' .~hua (such as Adenovirus 2). bovine p~rillom~ virus. avian sarcoma virus, cytomegalovirus, a ~et-u~i u~, hepatitis-B virus and most pl~,~.aiJly Simian Virus 40 (SV40), from h~t~logo~lc, ~
e.g., the actin promoter or an; .~ ogl~lb~ n promoter, from heat-shock Ulu..lU:~,.a. and from the promoter normally: ~ with the VRP sequence. provided such i,., are ,r , ' ' - witn the host cell systems.
The early and late ~IUIIIUt~,~a of the SV40 virus are cu.. ~.,.. i~,.. lly obtained as an SV40 r~ liull fragment that also contains t e SV4û viral origin of .~ iu. . Fiers et aL. ~, 273 :1 13 ( 1978): Mulligan and Berg, Science. ~2:1422-1427 (198û); Pavlakis et al., Proc. ~l~tl Ar~i Sci. USA. 78:7398-7402 (1981).
The ~ ' earlypromoterofthehumanc~u,,,~,~alv.i uaiscu..~_,,;.,,.llyobtainedasa~dlllE.~i.;~,liu--f~agment. Grc~,....~.~etaL,~as~, 18:355-36û(1982). Asystemfor.,Ai..c gDNAin.~ ;- hostsusing û the bovine l , " virus as a vector is disclosed in U.S. Patent No. 4,419,446. A . ~ l;ri ~ of this system is dvJ~"ib~d in U.S. Patent No. 4,6û1,978. See also Gray et oL, Nattire. _9S:S03-S08 (1982) on CAiJIC ' E, cDNA encoding immune interferon in monkey cells; Reyes et aL, ~a~. ~2 Z:S98-601 (1982) on ~AiJlC~;U~ of human ir~ f~,.u.. cDNA in mouse cells under the control of a 11.,~ " ~ kinase promoter from herpes simplex virus;CanaaniandBerg,Proc.N-tl Ar-~l Sci.USA Z2:5166-5170(1982)oncAy.c ofthehumaninterferon ~ I gene in cultured mouse and rabbit cells; and Gomman et aL, Proc. Natl. ~ r~,l Sci. USA. 79:6777-6781 ( 1982) on~A~.. ~ of bacterialCATs~ inCV-I monkeykidneycells,chickenembryorb..L' Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus long temninal repeat as a promoter.
(v) Fnh~nr~r ElPmPnt Cu..~
2 o T . ~ of a DNA encoding the VRP of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. F ' are cis-acting elements of DNA, usually about from lû to 3ûû bp, that act on a promoter to increase its l~ iull. Fnh~ r~s are relatively u.;~,..~ ion and position ~ ' r ~ t~ having been found 5' (Laimins et al., Proc. Natl. Ar~l Sci. USA. 78:993 [1981]) and 3' (Lusky et aL, Mol. Cell Bio.. ;~:1108 I1983]) to the ll~la~ iOII unit, within an intron (Banerji et aL, ~, ;~:729 [1983]), as well as within the coding sequence itself. Osbome et aL, Mol. Cell Bio.. _:1293 (1984).
Many enhancer ~ are now known from ... ~ 1 . genes (globin, elastase, albumin, cc f~uu.~ and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. F , ' include the SV40 enhancer on the late side of the ~ u origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the Irl~ . origin, and ad~ ..i.u, e ' ~ See also Yaniv, 3 o Nature. i~2~: 17-18 (1982) on . ' ~ elements for activation of eukaryotic ~,.u.. vt~ .. The enhancer may be spliced into the vector at a position 5' or 3' to the VRP-c..cùdi..g SelllJt~nre but is ~. ~,f~. ..bly located at a site 5' from the promoter.
(vi) T~ liù~ T~ ;.... Cu,.~v,l~
~ vectors used in eukaryotic host cells (yeast. fungi, insect, plant. animal, human. or nucleated cells from other ml~ltirell~ r u.~;a.. ;:,.. a) will also contain ~r.l.. _ necessary for the ~ of ion and for ~labili i--g the mRNA Such S-~l ~ts are commonly available from the 5' and, v : ~ - lly 3'. 1 ' ' regions of eukaryotic or viral DNAs or cDNAs. These regions contain ~ k o~
segments L.. u~.. ib~d as polyadenylated Ga~ in the .. .~ 1 -c ;I portion of the mRNA encoding VRP

(Vii) CV~ U~.Livll an~i Analvsis of Vectors CuliD~lu~,Lioll of suitable vectors con~aining one or morc of the above-listed C~ employs standard ligation t~ Isolated plasmids or DNA rla~....l~ are cleaved. tailored. and re-ligated in the form desired to generate the plasmids required.
For analysis to conftrm correct s~ in plasmids .,o.. ~L. u~,t~,d. tbe ligation mixtures are used to l~a..sru...l ~. coli K12 strain 294 (ATCC 31,446) and s~ r.-l ~ r~ selected by ~ or t~.~a~ lt~ IC~;D~ where a~t~vttl Plasmids from the ~ r.... - ,t~ are prepared. analyzed by Ic;~ iu~
,_ 1~ ---1 A ~ t~ 5ti~n and/or a~ ecl bythemethodofMessing et aL,NucleicAci~Res..23U9(1981) or by the method of Maxam e~ aL, M~thnrlc in Fn7yrnoloev~ 65:499 (1980).
lû (Viii) Tran~i~tlt EY~lr~C~it n Vectors ,ula l~ useful in the practice of this invention are ~A~t,c vectors that provide for the transient eA~,Ic in .- ~ cells of DNA encoding VRP. In general. transient eAt,l. involves the use of an ~A~t-c vector that is able to replicate crrici~ ly in a host cell, such that the host cell I ' many copies of the eAI~Ic vector and. in turn, synthesizes high levels of a desired polypeptide encoded by the CA~tIC vector. S ' uvl~ e~ aL, supra, pp. 16.17 - 16.22. Transient cA~t~.~taivl- systems. CU~ ;Dh~t!t a suitable ~A~t.C vector and a host cell, allow for the co..~_n;~,..l positive ~ of polyp~
encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired L ~ , ' or ~JL~ tlut~ ics. Thus, transient .,AIJI, systems are pa~ ,uhl ly useful in the invention for purposes of i.L,.Ili~t..g analogs and variants of VRP that are L- ' ,,' 'Iy active VRP.
(ix) S--it~l-le EYPt~ VVP-tebratr Cell Vectors Other methods. vectors, and host cells suitable for ~ ,-- to the synthesis of VRP in .c cell culture are described i,n Gething et aL, Nature. ,22~:620-625 (1981); Mantei a aL, ~"~.:40-46 (1979); EP 117,060: and EP 117,058. A pa L~,ulauly useful plasmid for n~ n~ cell culture eA~ .caa;u of VRP is pR~C5 (EP 307.247) or pSVI6B. W0 91/08291 ~,- I,!: .h.~d 13 June 1991. C. Selection anll T~ ru~ uon of ~ostCellc Suitable host cells for cloning or LA~t~ L~ta;..g the DNA in the vectors herein are the prokaryote, yeast, or higher cuLal yulc cells described above. Suitable l~lul~alyut~;t for this purpose include L. ba~,L. i". such as ~~ .I.,~ali~_ or Gram-positive u-ya liD~-D, for example, r uba~,t~ - such as Es~h,, ;"h..J. e.g, E coli, ~ , vku~.." l~rwinia, Klebsiella. Proteus, Salmonella, e.g, .~ ln ty~,.r ' ' , Serrati4 e.g, Serratia 3 o ~ . and Shigella, as well asi Bocilli such as B. subtilis and B. Iich~ v, (e.g, B. Ii.,.~ ..Ju, 4 I P
disclosedinDD266,710publishedl2Aprill989),P2 ' suchasP.~ ;..o,a,andS~ , One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as F. coli B. ~ coli X1776 (ATCC 31,537), and F coli W3110 ~ATCC Z7,325) are suitable. These examples are illustrative rather than lirniting. Strain W3110 is a ~Ja. Iil,ul,~ I.y prefemd host or parent host because it is a common host strain for 35 ~, ' DNA product f~ n~ Preferably, the host cell should secrete minimal amounts of proteolytic enzymes. For exarnple. strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins. with examples of such hosts including E. coli W3110 strain 27C7. The complete genotype of 27C7 istonA~lptr3phoAaE15~1~argF-lac)169cfnpl~1degP41kan~. Strain27C7w_sdepositedon300ctoberl991 in the Arnerican Type Culture Collection as ATCC No.55.244. Alt~.llaLi~ ~Iy, the strain of E. coli having mutant p.,.iylualllic protease disclosed in U.S. Patent No. 4,946.783 issued 7 August 1990 may be e . ' ~,d.
Altematively still. methods of cloning, e.g. PCR or other nucleic acid polymerase reactions. are suitable.
In addition to prokaryotes. eukaryotic microbes such as fil~ fungi or yeast are suitable cloning or cA~ aiu~ hosts for VRP-c~-co.li..g vectors. Sùc~l.,., ,~ Ct:r~ ;ul ~ or common baker's yeast, is thc most s commonly used among lower eukaryotic host Illi-,l UUI ~uUli ~Illa. However, a number of other genera, species, and strains are commonly available and useful herein, such as S.l~ . h ~ ~ pombe (Beach and Nurse, .290:140 [1981]; EP 139,383 published 2 May 1985); Kl,~._,, ~ hosts (U.S. Patent No. 4,943,S29;
Fleer et oL, supra) such as, e.g., K. Iactis [MW98-8C, CBS683, CBS4574; LVU~ ,VU.L et aL, J. Bacteriol.. 737 (1983)], K. fragilis (ATCC 12,424), K buloou~il ~ (ATCC 16,045), K 1~ (ATCC 24,178), K waltii 10 (ATCC 56,50û), K. ,b ~ _r~M ~ (ATCC 36,906; Van den Berg et aL, supra), K . ~ ' u,~, and K
yarrowia [EP 4022:!6]; Pichia pastoris (EP 183,070; Slc.,h ish.u et al., J. B~~;~ Mirrobinl ;~:265-278 [1988]); Candida; Tr;,l~ reesia (EP 244234); N; v~v,u crassa (Case a aL, Proc. N~tl Arl~tl Sci.
~, Z~:S259-5263 [1979]); ~~ ~ such as S~ ~a;~ (EP 394,538 ~ - d 31 October 1990); and ' ' fungi such as, e.g, ~V. v~v~a. Pe - " . Tol.~r ~7 ' ' (WO 91/00357 15 published 10January 1991),andAspergillushostssuchasA. nidulans(Ballanceetal., Binrh~n~ BioDhvs. l~
~'nmmlm ~:284-289 tl983]; Tilburn a aL, ~, ;~:205-221 [1983]; Yelton et al., Proc. Natl. ~ r~t1 Sci.
~,81: 1470-1474 [1984]) and A. niger. Kelly and Hynes, EMRO J.. _:475479 (1985).Suitable host cells for the ~ ion of gly~,uay' ' VRP are derived from rn.llfirell~ll~~ UltSA,,ia...~.
Such host cells are capable of complex ~J.u.,c..au.g and glycosylation activities. In ~JI ,' any higher 20 . ' .~. cell culture is ~ ' . whether from .,_.t~ or i,,~ culture. F ,1 of i"~
cells include plant and insect cells. Nu,l._.uua bU~UIO~d~AI strains and variants and cc,..~ g f"~..",;aa;~_ insect host cells from hosts such as .Cp-~, aft..O,~,Ia (_ ~;IIu.), Aedes aegypti (mosquito), Aedes ~-'', (mosquito), D~ v~v~.~ 17 ~ ' g.~ (fruitfly), and Bomoyx mori have been itit nfifit t~ See, e.g., Luckowaal.,Bio/li--'--'-f~v.~:47-55(1988);MilleretaL,inGeneticEr.~;.. i.. s~ Setlowetal.,eds.,Vol.
25 8 (Plenum PulJliallillg, 1986), pp.277-279; and MaedaetaL, Nature,315:592-594 (1985). A variety of viral strains for 1., ~ r . l ;v. . are publicly available, e.&, the L- I variant of ,41 - O~ u~,l.J . ulilu~ ,.;ca NPV and the Bm-S
strain of Bomb~ mori NPV, and such viruses may be used as the virus herein accu. di..g to the present invention, pu Li-,uhulyforL-- -r~ ~io.~of.~p~ "~f..O.~.,.lacells.
Plant cell cultures of cotton. com, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts.
30 Typically, plant cells are L,A.~r~, d by ;----1.~ , with certain strains of the bu.,;~,.iu..l A~;,ob~~t~, r .,' which has been ~ iuualy , ' ' to contain the VRP-c...,o.l.,.g DNA. During ;~ n of the plant cell culture with A. r ,' . the DNA encoding the VRP is ll uulaf~ ,d to the plant cell host such that it is Ll- -f ~r. ~l and will, under a~ Jlu~J~iat~, cv. ;n~c express the VRP-c..codi"g DNA. In addition.
regulatory and signal ~ / - c- ~IJ -~ ;l 1~ with plant cells are available. such as the nopaline synthase promoter 35 and pol.~ud~.. ylation signal ~ s Depicker et aL, J. Mol. ~ l- G~n 1:561 (1982). In addition, DNA
segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or i~lcuai~g iu~ levels of plant CAI.~ I genes in recombinant DNA-cv -l-; ~;..g plant tissue. EP 321.196 published 21 June 1989.

WO 97/09427 PCT/US96/1407~;

However, interest has been grealest in v.,~ a~ cells. and propaga-ir~n of ~ a~c cells in culture (tissue culture) has become a routine ~u~clu~ See. e g, Tic~ re~ Academic Press. Kruse and Patterson.
editors(l973) F , ~ of useful. .~ .hostcelllinesaremonkeykidneyCVI lineu~ r~ IbySV40 (COS-7, ATCCCRL 1651); human e,..L.yo~ kidney line (293 or 293 cells, ~1 ~~ d for growth in ~
culture, Grâham et al., J. Gen Virol.. 36:59 [1977]); baby hamster kidney cells (BHK. ATCCCCLI0); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Ar~i Sci. USA. 77:4216 11980]); mouse sertoli cells (TM4, Mather, Biol. Reorod.. ~:243-251 rl980]); monkey kidney cells (CVI ATCCCCL 7û);
African grcen monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical ~,au~,UlUI..a cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
lo 1442); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); mouse ~AJ~U~A y tumor (MMT060562,ATCCCCL51);TRIcells(MatheretaL,Ann~l~NY.Ar~A Sci..~:44-68[1982]);MRC5cells; FS4 cells; and a human h, Iine (Hep G2).
Host cells are ~ r ~ ~. A and ~Jlef~.ably~, A. .~r", ~ d with the above-described ~iA~ iu~ or cloning vectors for VRP ~.vdu.,Liull and cultured in cu.,._..~iu..al nutrient media modified as A~l"U~ .t. for inducing 5 ~.~ r . selecting ~ or amplifying the genes encoding the desired se(, T r ' refers to the taking up of an e.~ ,iu-- vector by a host cell whether or not any coding are in fact expressed. I'' uua methods of u ,~ are known to the ordinarily skilled artisan, forexample. CaP04 and elc~,.-~-.--~;.~ - Succpc'fi~ r. 1;.... is generally I~U~ ; d when any ;~
of the operation of this vector occurs within the host cell.
2 ~ T r ~ means i~ DNA into an organism so that the DNA is l L r ~i ~ I e, eithcr as an ~ ' element or by .,L~ integrant. Dcl, .-1; -~ on the host cell used. tl ~ is done using standard ~. h-l;-l- ~.u~.ut,. to such cells. The calcium treatment employing calcium chloride, as described in section 1.82 of Sambrook et aL, s2 pra, or _le~,~lu~ v-~iun is generally used for prokaryotes or other cells that contain ,--h~l ~1;_l cell-wall barriers. Infection with ,~ ù~u~ iS used for k r ' of certain plant cells, as described by Shaw et aL, ~, ~:315 (1983) and WO 89/05859 published 29 June 1989 In addition, plants may be ~ f. ~ ~. A using ultrasound treatment as described in WO
91/00358 pllbliChPd 10 January 1991.
For . -- . - - ' ;-- cells without such cell walls, the calcium ~ -l ;on method of Graham and van der Eb, Virolo~v. 5~:456-457 (1978) is preferred. General aspects of m~~n~ cell host system 3û u~- -r~ have been described in U.S. Pat. No. 4.399.216 issued 16 August 1983. Tlaul:>r~ into yeast are typically carried out - ' g to the method of Van Solingen et al., ~.~a~.. L~Q:946 (1977) and Hsiao et oL, Proc. l~l~tl Ar~rl SCi. (USA). 76:3829 (1979). However, other methods for i~ udu~h~g DNA into cells, such as by nuclear ...i.,.. , clc.,Llv~Julaliull~ bacterial l~lv~v~laa~ fusion with intact cells, or polycations, e.g.,p~ .,c,pol~,."i~hu.c, etC., mayalsobeused. Forvarious~ I,.. ;.lU._ for ~ r.. ;.. ~ m~mm~ n cells, 35 seeKeownetaL~MethodsinEn7vrllolof~v~:527-537(l99o)andMansouretaL~Nature~:348-352(l988)~
D. C~lt~in~ the Host Cells Prokaryotic cells used to produce the VRP polypeptide of this invention are cultured in suitable media as described generally in Sambrook et al., s~pra.

The Tn~mn~ n host cells used to produee the VRP of this invention may be cultured in a variety of media. Cullllll_~-,;ally available media such as Ham's FlO (Sigma), Minimal Essential Medium (~MEM]. Sigma).
RPMI- I 64û (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham and Wallace, Meth. En7 58:44(1979), Barnes and 5 Sato, An~lBioch~rn IOZ:255(1980),U.S. Pat. Nos. 4.767,704;4.657,866;4,927,762;4.560.655; or 5.122.469;
WO90/û3430;WO87/00195; or U.S. Patent Re. 30.985 may be used as culture media for the host cells. Any of these media may be r r ~ as necessary with 1~ and/or other growth factors (such as insulin.
~ or .-~ ' ~ growth factor), salts (such as sodium ehloride, ealeium, ~ r~ , and ~~ , ' ~ ), buffers (sueh as HEPES), .--~ l 7 (sueh as a~ and II.y..-i~ c), -bi (such as G_~ y~ TM
10 drug), trace elements (defned as inorganic ~ , ~ usually present at final Cull~ b ~liùll:~ in the ...i.,. ~ - ' range), and glucose or an equivalent energy souree. Any other necessary ,, ' may also be included at 3~ r ul~l' C~ ~ that would be known to those skilled in the art. The eulture c---~ sueh as - c, pH, and the like, are those U~c-iûu~ly used with the host eell seleeted for eA~c~:~iul~ and will be apparent to the ordinarily skilled artisan.
In general, ~ ........................... ' and practical r I ; l, - for .. -~ the ~udu~,livil~ of cell eultures ean be found in Marnmali~r~ Cell B -~--'- - ' - ~v a Pr~- tir~ roaeh. M. Butler, ed.
(IRL Press, 1991).
The host eells referred to in this di~_lu ,u.c ~ cells in culture as well as eells that are within a host animal.
2û E. Deteetinv Gene A.. U.lj~ rr~ n Gene , ' ~ and/or eA~c;,:~iol~ may be ~d in a sample direetly, for example, by iu.. al Southem blotting, Northem bloning to ~, the l.. u, ,.,, i~Jfiu~ of mRNA (Thomas, Proe. N~l Ar~l Sci. US~. ~:5201-5205[1980]), dot blotting (DNA analysis), or in situ hyl,.i.li~dliù... using an , . ul '~, labeled probe, based on the s~ .c provided herein. Various labels may be . . ' j~l most 2s commonly r~ . c. particularly 32p, However, other ~. ~ I- .i.l - may also be employed. such as using biotin-modified ~- -- I~ for i~uu~lu~,liu~ into a pol~.---- I~ul;~i~ The biotin then serves as the siîe for binding to avidin or .lil~i:- - which may be labeled with a wide variety of labels, such as r~ - nuu..,~cc.:"
enymes, orthe like. Al - ~_ly, ~iL ~ may be; , ' y~d that can ~~-~c~i~ specific duplexes. including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The ~ .I;l.o~l;, in 3 û tum may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the fnr~ in.. of duplex on the surface, the presenee of antibody bound to the duplex ean be deteeted.
Gene eA~ iùll, a:.~,...ali~_ly, may be ~ d by -1~, ' methods, such as ~ ~ ' staining of tissue sections and assay of cell culture or body fluids, to .~ - directly the ~AIJlI,.~.~il)U of gene product. With ;.. nl~i~l.. I.- ---i- -I staining ~ - - a cell sample is prepared.
3 5 typieally by d_h ~ llaliull and fixation, followed by reaction with labeled ~ . I ;l .ofl :- - specific for the gene product coupled. where the labels are usually visually ~ - l-hk such as ~ .rlllaliC labels. Iluu-c;,~c.-~ labels.
I-----; - s<- -l Iabels, and the like. A particularly sensitive staining ~ l suitable for use in the present inveMion is described by Hsu et aL, Am J.CIin Path.. 7S:734-738(1980).

~ ntiho~iiPc useful for ;~ " i .~1 staining and/or assay of sarnple fluids may be either r~ Ai or polyclonal. and may be prepared in any mamrnal. Cu~ ly. the ' - " may be prepared against a native VRP polypeptide or against a synthetic peptide based on the DNA . provided herein as described further in Section 4 below.
F. P~ - of VRP rulYv~vL;d-VRP ~,..,f..al.ly is recovered from the culture mediurn as a secreted polypeptide. although it also may be recovered from host eell Iysates when directly produced without a secretory signal. If the VRP is ~
bound, it can be released from the . . . I.,, ~r using a suitable detergent solution (e.g. Triton-X 100) When VRP is produced in a .~ ' ~ cell other than one of human origin, the VRP is ~ 'y 10 free of proteins or pol~ ;lc.. of hum~n origin. However, it is neeessary to purify VRP from ' eell proteins or polyt,_~l~idcs to obtain ~,.cp -- ,-l ;- ~-.~ that are s .1 ,~ lly h~ ~t~ ' as to VRP. As a frst step, the eulture medium or Iysate is ~ iru6~d to remove ~ eell debris. VRP ll._.wn.. is purified from c~ .; .- -1 soluble proteins and pol~ id_s. with the following ~ beirig ~A~ pla~.y of suitable ~. ~-;r- - ;~ ucclu-~. by r, l;~ .ti..l~011 aniu.. CA~IIall5_eolumn ethanol~ , reversephaseHPLC;
15 ~,L~ r..~ l.l,y on siliea or on a cAI,l.al."_ resin sueh as DEAE; ulu~ ~.,u;,u.E;, SDS-PAGE;
sulfate ~ . ; gel filtration using, for example, SPrh~rleY G-75; and protein A Sc~L~uuae eolumns to remove c sueh as IgG.
Inthepreferred~ -- I theFlt4receptor-IgGfusionisi-------"il;,- ionanaffunity.,L-- r~ y eolumn and the VRP ean be isolated by affinity ~Jul ir.~aliU.. using this eolumn. .A I ~ . _Iy, the VRP is joined 20 at its N t~,.lllulua to a gl~_u~ D sequenee and is passed through an affrnity ~lu~ y column on whieh is i ----I-;I:, ~1 an anti-gD .---. ~ I---- -' antibody sueh as SB6, whieh is speeifie for a gly~,u~,.u:~, . D
sequenee.
VRP variants in whieh residues have been deleted, inserted, or s- ~ t i are .e_u .~ ,.i in the same fashion as native VRP, taking aeeount of any ~ l; ' changes in l,.ut,~.. Ii_., 0~ ' '-'d by the variation. For 25 example, ~ Jaualiu~l of a VRP fusion with another protein or polypeptide, e.g., a bacterial or viral antigen, faeilitates ~- ;ri- ~ an ;. ---- - rri :y column ~ antibody to the antigen ean be used to adsorb the fusioll pol~ ~J-lJlid-~ l. . - - - - - -- -n rri ;l~y columns sueh as a rabbit pOI~Iullal anti-VRP column ean be e ~r I y"d to absorb the VRP variant by binding it to at least one l~llaulh~g immune epitope.
A protease inhibitor sueh as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit 3 o proteolytic 1 v A- ~ J~ ~ during ~uul ifl~-iu.., and ' may be included to prevent the growth of dd~ . -One skilled in the art will alJ~ ;r~ that "U~;r.~..;U.. methods suitable for native VRP may requjre ~-~O~I;IA;- 1;~-- to account for eharges in the charaeter of VRP or its variants upon CA,UI~ ;UI~ in . ~ ' eell eulture.
G. CovAIPnt Mo~l;r~ C of VRP PolvDeDtides 3 5 Covalent ",o~ of VRP polyp_~I.dci are ineluded within the seope of this invention. Both natjve VRP and amino acid sequence variants of the VRP may be covalently mor~ prl One type of covalent .. r~ of the VRP is u-l-udu~.d into the molecule by reacting targeted amino acid residues of the VRP
with an organic ,I..i~ iu.g agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the VRP.

WO 97/09427 PCTrUS96/14075 Cysteinyl residues most commonly are reaeted with a-hA~ (and cu.. 'I"'~ L amines), sueh as -,l.lu.uace~i., acid or ehlc,..,A~ to give earboxymethyl or earboxy- .. i~ hrl d.. i~ . Cysteinyl residues aiso are derivatized by reaetion with b~u~uLlilluù.ua~.,tu..c. a-bromo-i3-(5-imidozoyl)~.~ r aeid, chloroacetyl pl.~ l, N-alkylmsl~ c 3-nitro-2-pyridyl disulfide. methyl 2-pyridyl disulfide, 5 chlc,.u.. _,.,u,;h. -.. -l- 2-chlu.u,,.e,.,,.,i 1-niLlui~l,.,,ol.orchloro-7-nil.ui,_,,~u-2-oxa-1,3-diazole.
Histidyl residues are d~,.iYd~i~d by reaction with dieth~ ,u~L at pH S.S-7.0 beeause this agent is relatively speeific for the histidyl side ehain. Par~ J~ rl bromide also is useful; the reaetion is ,,ef~,,al,ly p_.ru~ d in 0.1M sodium eaeodylate at pH 6Ø
Lysinyl and amino terminal residues are reaeted with sueeinie or other earboxylie aeid: ' ~. ~ ' 10 Dc. i~_ with these agens has the effeet of reversing the eharge of the Iysinyl residues. Other suitable reagens for d~ e ~-amino C ~ e residues inelude; .i~ir~ sueh as methyl i- ' ~ ~ ' pyridoxal i-'- ,' pyridoxal. cLIu~ubu~uL,~.Lidc, h~iLI~b~ ft~nir aeid, O '.~' ~O, 2~4-ir ' e, and Ll. -- .. --eatalyzed reaetion with glyoxylate.
Arginyl residues are modified by reaetion with one or several cu..~. ' reagens, among them 15 pl.~ .Aal, 2,3-L_ " -. 1,2-e~ and ninhydrin. Dc~ d~aLu~l of arginine residues requires that the reaetion be p_.ru~ _d under alkaline ~ c beeause of the high PKa of the r~ ~-r. ,;....~' group. F.ULI.~....U.C, these reagents may reaet with the groups of Iysine as well as with the arginine - group.
The speeifie . o I;r; ~ ;.., of tyrosyl residues may be made. with pa,Li.,ul_. interest in ~' ~ e 20 speetral labels into tyrosyl residues by reaetion with aromatie . l; - - - ; . c- ....l .u ~l~ or t~a..it~ h -- - Most eommonly, N-ace~ - '- and t~,h~u~iLI~ -' ~ are used to form O-aeetyl tyrosyl speeies and 3-nitro d~.i.,_ ~.;., .~._.,li~_ly. Tyrosyl residues are iodinated using 1251 or 1311 to prepare labeled proteins for use in ~ ~ r, the ~ hlr Ir~in~ T method being suitable.
Carboxyl side groups (aspartyl or glutamyl) are selc_Li~_l), modified by reaetion with ca~L ' ' (R-25 N=C=N-R'), where R and R' are different alkyl groups. such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) .,_l,u~ or 1-ethyl-3-(4-azonia-4,4-di..._Ll,~l~,_.,L~I) ca l,o 1:; i~l~ FU~LI~ U~C, asparlyl and glutamyl residues are cu,,~ d to -~1. - a~; yl and ~,Iu~i--rl residues by reaction with ~ ; - ions.
Dc~ d~i~adull with b;r~ . -1 agents is useful for .,...~ ;..g VRP to a water-insoluble support matrix or surface for use in the method for purifying anti-VRP - 1 ;I o~l ~ and vice-versa. C~ Iy used 3o c.~ agentsinclude,e.g.,l,l-bis(dia~ua~,.,Lyl)-2-pl..,..;l.,Ll._.c,~,luLalald~,ll.~dc,N-Ly~LuA~ "~
esters, forexample, esters with 4-oA7i~ 9lirylic acid, ho ol,;r~ - li~- .-1 ;.. i~l~)~ jt~ ~ including .1;~ rl esters such as 3.3'-dithiobis(s ~ and b;r~ ' ' ' such as l,;_ N ~ ' I 8 oetane. D~ aLi~illg agents such as methyl-3-[(p-A~ h yl)dithio]~,.u,u;c. ' yield phutua~,Li~
illt~ ' that are capable of forming ~,IU:~DIilllk~ in the presence of light. Alternatively, reactive water-3s insolublematriees sueh as cyanogen l,.u~ddc sAtivated ~a~buL~/d-~t~ and the reactive :,uI,:,Ll...~,~ described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,33û,44û are e,..~ ,d for protein; ol,:l:, ;"

WO 97/09427 PCTrUS96/14075 Glutaminyl and ~uaua~,ulyl residues are L~u~..lly fiF~ to the cu~ r ~- g glutamyl and aspartyl residues, .~,~u~lh.~ly. These residues are ~irz~nni~ under neutral or basic co~~ The d form of these residues falls within the scope of this invention.
Other...ori;l~ includehydroxylationofprolineandlysine,plu~uhu,ylationofhydroxylgroups 5 of seryl or threonyl residues. methylation of the ~-amino groups of Iysine, arginine, and histidine side chains (T.E. Creighton, Prnt~ c Str~rt~e ~nri Moi~rl~ Prou~ W.H. Freeman & Co., San Francisco, pp. 79-86 [19831), ac.,t~laliuu of the N h.llli~al amine. and P-lir7-~in~ of any C-temminal carboxyl group.
Another type of covalent ..n.i;ri. ~;.... of the VRP polypeptide included within the scope of this invention c- ~ altering the native gl.'F _O:~ylaliull pattem of the polypeptide. By albring is meant deleting l o one or more ~,a~ L.vh F . ' ' moieties found in native VRP, and/or adding one or more r~l 7 _u ~ laliù~ sites that are not present in the native VRP.
Gl.~. ~latiu~ of pol~ id-,~istypicIllyeitherN-linkedorO-linked. N-linkedreferstothe -~ -'~
ofthe-~ul,vh.~. moietytothesidechainûfan~ residue. Thehiu~,1idc~ ,u~ -X-serine and ~u 7~ -X-threonine. where X is any amino acid except proline, are the .c ,, , for 15 c.~ - 1- - - of the ca~ LvLyd moiety to the ~ side chain. Thus, the presence of either of these tripeptidc ~ - in a pol~,.,tJ~i.l~ creates a potential glycuayla~iu~ site. O-linked y,l~ _u~ylaLu~ refers to the ~ l - l - ' of one of tne sugars N . ~ . galactose, or xylose to a 4 ~ uAy l,...d~ù acid most t ~y serine or threonine, although 5-hy~LuA~ uL~c or 5-h-y~Lu~yly .inc may also be used.
Addition of ~ ,uayLliu~ sites to the VRP polyu.,tJ~iJ,, is ~u~ ,...ly . - . ' ~ ' by altering the 20 amino acid sequence such that it contains one or more of the above-described tripeptide s~ (for N-linked latil~.. sites). The alteration may also be made by the addition of, or ~vl.~1 ;o,~ by, one or more serine or threonine residues to the native VRP sequence (for O-linked glycosylation sites). For ease, the VRP amino acid sequence is ~.f~abl~ altered through changes at the DNA level, pa L~ula ly by mutating the DNA
encoding the VRP polypeptide at ~c~le~ted bases such that codons are g ' that will translate into the 25 desired amino acids. The DNA mutation(s) may be made using methods described above and in U.S. Pat. No.
S,364,934, supra.
Another means of i.,..eaa...g the number of carbohydrate moieties on the VRP pol,l.~,t,lidc is by chemical or C.~...d~i~, coupling of ~ u.,iJ~,~ to the polypeptide. These l,.oc~.luul_, dre aJ~ ~, in that they do not require ~ud~_livn of the pol,~ ,lidc in a host cell that has r~ly~ uaylaliu~ ~1 .;lh ;. e for N- or O-linked 3 o ~ _ua~la~iul~. Dep~n~lin~ on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free ,ullL~Lyl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine. or hy~LuA~ uL~c. (e~ aromatic residues such as those of pL_~ylala~Lc~ tyrosine, or L ~ , 'or (f) the amide group of gh c ~; .r These methods are described in WO 87/05330 published I I
S~. ~ 1987, and in Aplin and Wriston, CRC Crit Rev. Biochem.. pp.259-306 (1981).
3 5 Removal of carbohydrate moieties present on the VRP polypeptide may be P~ C~J~ I rd chemically or c~ali~,ally. Chemical deglycosylation requires exposure of the pol~ idc to the c~ pu~
~in.~.,O 1 . .h~ ...;, acid. or an c~ ' - c~mrolmtl This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylgh~ or N-acet~lK~l-r~ while leaving the polypeptide intac~ Chemicaldeglycosylationisdescribedby1' ' '' etal., Arch.Bioch~m~Biot~hvs~ 59:S2(1987) and by Edge et aL, AnAI Biochem 118:131 (1981). Enymatic cleavage of carbohydrate moieties on pol~ lid ~ can be achieved by the use of a variety of endo- and exo-gly~c.aidascs as described by Thotakura e~al., MPth En7vrnol 138:3S0 (1987).
Glycosylation at potential glycosylation sites may be p. ~ ~,.t~,d by the use of the c~ luu.;wu.~ _u~
s as described by Duskin e al., J. Biol. ChP~n ;~:310S (1982). Tunicarnycin blocks the formation of l,-. ',t ~1 glycoside linLages.
Another type of covalent ,...~.I;ri~ A~ of VRP c-~ linking the VRP pol~ tid. to one of a variety of nu~ ~ polymers, e.g., pol.~_~l.,..~, glycol, pol.~,.u~ ,..c glycol, or pol.~JA~alLylenes, in themannersetforthinU.S.PatentNos.4,640,835;4,496,689;4,301,144;4,670,417;4,791,192Or4,179,337.
Since it is oRen difficult to predict in advance the ~ h ~ of a variant VRP, it will be ~ c that somc s.,.c~,..i..g of the ~~.,u._.~,d variant will be needed to select the optimal variant. A change in the ~ ' O ' character of the VRP molecule, such as affinity for a given antibody, is also able to be measured by n ~ -type ~ y. The variant is assayed for changes in the su~ ~ or ~ I of its ~ ~ activity by cu...~,~ iau.. to the activity observed for native VRP in the same assay. For example, 5 one can screen for the ability of the variant VRP to stimulate protein kinase nctivity of the Flt4 receptor as describedinExampleSherein.Otherpotential.---..l:l~. ';----ofproteinorpol.~ ,.ulJ_.Ii_Jsuchasredox orthermalstability,l..~ .b ~: ~y,~ ;h~ ytoproteolyticd ~ orthetendencytoab~,~ with carriers or into ' a are assayed by methods well known in the art.
H. ': ~A~pPrl VRP
2 0 This invention ~ chimeric POI.~ cc,.. ,t" iau.g VRP fused to another poly~ idc. In one preferred e L ' r, the chimeric poly ~ ;dC cu~ _J a fusion of the VRP with a tag pol~_~.lide which provides an epitope to which an anti-tag antibody can s~,l.,_li._ly bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the VRP. Such el r ta~"~_d forms of the VRP are ~' - - --' ' as the presence thereof can be detected using a labeled antibody against the tag pol~ ,lid-. Also, provision of the epitope tag 25 enables the VRP to be readily purified by affnity l, u ifl.,~.liv.. using the anti-tag antibody. Affinity !JU. ifl.~.Lu..
'- ;m - and ~ assays involving ~ .. ;I oA are described later herein.
Tag pul.~ idcs and their r~ __Li~_ .I ;I .ofl; - are well known in the art. F , ' include the flu HA tag pol.~ id. and its antibody 12CAS (Field e~ al., Mol. CPII Biol ~:2159-2165 [1988]); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 .I;h,~; 5 thereto (Evan er aL, Mol~r~ r ~n~l Cell~lh~ Biolo~v.
3 o 5:3610-3616 [1985]); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody. Paborsky er aL, Prl~t~;n El-r;-- ;..P ~(6):547-553 (1990). Other tag poly~ idcs have been rlicrios~P~l F . ' include the Flag-peptide (Hopp e~ aL, BioTF h. olov~/. Ç:1204-1210 [1988]); the KT3 epitope peptide (Martin e~ aL, 4[l992]);ana-tubulinepitopepeptide(skinnereraL~J.Biol.chpm ~:15163-15166 [1991]); and the T7 gene 10 protein peptide tag. Lut~-F~ ,.I..uLh e~ aL, Proc. Natl. Ar~rl Sci. USA. 87:6393-6397 (1990). Once the tag polypeptide has been selected. an antibody thereto can be g_,,_,_t~d using the ~ . disclosed herein.
The general methods suitable for the CullaLIu~liull and ~JIudu~Liull of epitope-tagged VRP are the same as those disclosed h_~cih~bù~_ with regard to (native or variant) VRP. VRP-tag polypeptide fusions are most cu..._.~ ly cullallu~,d by fusing the cDNA sequence encoding the VRP portion in-frame to the tag WO 97/09427 PCTrUS96/1407~

polypeptide DNA sequence and e.~i,.clah~g the resuli-~nt DNA fusion construct in "~.u~ - - host cells.
Ordharily, when preparhg the VRP-tag polypeptide chimeras of the present invention, nucleic acid encoding the VRP will be fused at its 3' end to nucleic acid encoding the N-termhus of the tag pol~,_yliJ., however 5' fusions are also possible.
Epitope-tagged VRP can be cu.. i~,.i_,.lly purified by affinity ~.lu~ -,~,y.i.~ using tbe anti-tag antibody. The matrix to which the affinity antibody is attached is most often agarose, but other matrices are available (e.g. controlled pore glass or pOly(a~ ,.._.l;.. h.yl)benzene). The ., , -; g~ ' VRP can be eluted from the affinity column by varyhg the buffer pH or ionic strength or adding ~,L_ ~h~ r ' agents, for example.
2. Ti~ . ~-1;- Uet~e C~ A~ aI;U~ of VRP
10 VRP is believed to fmd ~ l ~- ~l ;- use for treating .. ~ . ~l~ via ~ or inhibition of growth and/or di~ lLialiùll and/or a~,liV - of cells havhg the Flt4 receptor or one or more other VRP ~-FYn~, VRP may be a ' ~ ~ ~.d to a patient in these cu~ -- - l ~- c The human VRP is clearly useful insofar as it can be ~-l ..;l.:- h ~.,d to a human having d.~ d levels of . i ~, VRP, preferably in the situation where such d.,J.~,;.a_d levels lead to a ~ - ' disorder. or where there is lack of activation or 15 inhibition of the Flt4 receptor or one or more other VRP ~~ , Various potential ~ ; uses of VRP hclude those in which VEGF is riseful. F . ' of these include uses ~ with the vascular - - i. .l i~- l; . . such as the treatment of traumata to the vascular network, in view of the d..-~o.. ,L _- I rapid iJ.V---vLiU-- by VEGF of the p.vlif~ of vascular r -- I--lh- I' I cells that would surround the traumata and in view of the rel ~ il. between VEGF and the VRP ~ l -~.l:_l .~.-i hereh.
20 Exainplesofsuchtiaumatathatcouldbesotr~atedhclude,butarenotlimitedto,surgicalhcisions,i,a L~,ul.. i~
those hvolvhg the heart. wounds, including ~ incisions, and i-,.... h .-~ - - - of blood vesseis, and surface ulcers involving the vascular ~ ' such as diabetic, I ),' ' c, and varicose ulcers. Other yl..~ ~ ",- ' ~ - .- -- 1;l ;~ ~- -~ that could be hmproved based on the selective .. . :l~ .L,. -: character of the VRP are also included hereh.
2 5 For the traumatic ;- ~ referred to above, tie VRP molecule will be fnrtnlll d and dosed in a fashion - - with good medical practice takhg hto account the specific disorder to be treated. the condition of the hdi vidual patient. the site of delivery of the VRP, the method of .- h . . ;- ;~ ;- - . and other factors known to 1", ~
Additional u.~ for the VRP are in the treatment of full-thickness wounds such as dermal ulcers, 3 o includhg the ~,b~ fl_.- of pressure sores, venous ulcers, and diâbetic ulcers, as well as of full-thickness burns and injuries where ~ ~r; ~g. ~ -: - is required to prepare the burn or injured site for a skin graft or flap. In this case the VRP is either applied directly to the site or it is used to soak the skin or flap that is being LIA-~ t ~i prior to grafthg. In a similar fashion, the VRP can be used in plastic surgery when l~,cvn.LIu~,Liùl~ is required following a burn or other trauma, or for cosmetic purposes.
3 5 J~r.~;n~ is also important h keephg wounds clean and r.v.. ;.. f~ .l The VRP can therefore be used h A -~ ,. . with general surgery and following the repair of cuts and lA~ t is lJat li~ulal ly useful in the treatment of ~ .~l. ~ .. ;. ~AI wounds with a high risk of infection. Neov~.,ul.~ iLaliO~ is also key to fracture repair, since blood vessels develop at the site of bone hjury. A~LdldaL aLu~ of the VRP to the site of a fracture is therefore another utility.

W O 97/09427 PCTrUS96/14075 In cases where the VRP is being used for topical wound healing, as drcrrihed above, i~ may be qAminicured by any of the routes described below for the rc-~ A~ qtion of vascular tissue. or more Ç.,.ably by topical means. In these cases~ it will be qAtninictr~ed as either a solution, spray, gel, cream.
ointment. or dry powder directly to the site of injury. Slow-release devices directing the VRP to the injured site 5 will also be used. In topical ~ the VRP will be applied at a cul,.,c,,.i_ ' ranging from about 50 to 1,000 llg/mL, either in a single ~ - or in dosing regimens that are daily or every few days for a period of one week to several weeks. Generally, the amount of topical r.. ~ t cl is that which is sufficient to apply from about 0.1 to 100 ~1g/cm2 of the VRP, based on the surface area of the wound.
The VRP can be used as a post-operative wound healing agent in balloon ' r ~ ~ a ~ ' c in 10 which vascular ~ ~ ' ' cells are removcd or damaged, together with cv...~,.c ~ of ;;.h~.~ua~ . plaques.
The VRP can be applied to inner vascular surfaces by systemic or locahinha~ rr either as i~Sa~ uua bolus injection or jnfi~;rlnc If desired, the VRP can be a-l- -;--;-~ -~,1 over time using a ...;.,.~. --- r- ; e pump. Suitable co...l~n~:~;....~ for i Iha~ uua aJmi~iaha~iu~ comprise the VRP in an amount effective to promote r AuII~rl;- I cell growth and a parenteral carrier material. The VRP can be present in the 15 c- - ~ ;- -.. over a wide range of ~ - -- -- - - .l- - ~ ;- -- .~ for example, from about 50 ~g/mL to about l,OOO ~Lg/mL using injections of 3 to 10 mL per patient, s ' ~ ~ cl once or in dosing regimens that allow for multiple ~ . r ' ~
Any of the known ~>~uc ' carrier vehicles can be used, such as normal saline or 5-10% dextrose.
The VRP can also be used to promote r-.A.J~ :.. in vascular graft surgery. In the case of vascular grafls using either 1.. ~ n. ;I vessels or synthetic material, for example, the VRP can be applied to 2 o the surfaces of the graft and/or at the junctions of the graft and the existing v ' c to promote the growth of vascular; ~ ' " 'cells. Forsuch rl.l.li~ ~;----~,theVRPcanbeapplied il~ha~. Iy asdescribedabove for balloon _' r ~ ~Y or it can be applied directly to the surfaces of the graft and/or the existing vqc~ ~l c either before or during surgery. In such cases it may be desired to apply the VRP in a l 1. :- L ' 1 carrier maurial so that it will adhere to the affected surface. Suitable carrier materials include. for example, 1-5% carbopol. The 2 5 VRP can be present in the carrier over a wide range of c- ~ ~- ~ . for example, from about 50 ,ug/mg to about 1,000 ~lg/mg. ~ ly, the VRP can be delivered to the site by a ...;~,.u...~.i..g pump as a pa c solution.
The VRP can also be, . ' ~1 to repair vascular damage following ~ ,a d;al i~ Liù~ and to ,~... _.lt the need for coronary bypass surgery by ~ the growth of a collaural ~ ld iùn. The VRP
3 o is v ' ~ ~ cl i..ha._"uual~ for this purpose, either in individual ;..j~- l ;O~C or by .";.,.~ . ; -g pump over a period of time as described above or by direct infusion or injection to the site of damaged cardial muscle.
Th- ~.. -l ;. r.. I -~ ;n-~- of VRP are prepared for storage by mixing VRP having the desired degree of puritywithoptional~ ,' 'Iy , ' ~' carriers,e '. ' or~ r...;... ~ sPl -~, 1 6th edition, Osol, A., Ed., [ 1980]), in the form of Iyophilized cake or aqueous 501~tinnc 3 5 carriers, . ~.: or ~t~ 7rrC are nontoxic to .~ '. ' at the dosages and ~,ùnC.c~LI~ ;u~a employed. and include buffers such as I ' , ' citrate. and other organic acids; ~~ .1;.. ;.l .-~ including ascorbic acid; low m~l~ I~~ weight (less than about 10 residues) polypeptides; proteins. such as serum albumin, gelatin, or i.. nrl.. b_linc hydrophilic polymers such as polyvinylp~.. ulidu.. " arnino acids such as glycine. gl~t-~nin~ .
aa~Jala~:illc~ arginine. or Iysine: m.. ~ .1- ;d~. diaa~,llalidcs. and other carbohydrates including glucose.

mannose, or dextrins: chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol: salt-forming counter-ions such as sodium: and/or non-ionic :~ulra~,L~IL~ such as Tween, Pluronics or po~ lc glycol (PEG).
The VRP also may be Pnt~nrped in lld.,.~ c prepared. for example, by co~c~,. v..,iu..
5 or by u.t~,.L_i..l pol.~...~.iL..~iu.. (for example, h~,LuA~ ,i.ylcellulose or gelatin-...i.,.u.,d~ lcs and poly-' ~ ' ] ~ u ~ a~ _ly)~ in colloidal drug delivery systems (for example, 'i, albumin Illi~,lU:~Jh.,.C.7, Illi~lV- '-~ "'' narlo-particles, and r~ f-7), or in Illa~ll ' ~ Such t~ ' ~ . are disclosed in ~r..,j"..~ 's r~ Sri~nr~ sYpra.
VRP to be used for in vivo ~ -- must be sterile. This is readily: . ' ' ' by filtration lo through sterile filtration . ~l ~ prior $o ar following 1~ r ~ ~-- ' ' and 1~ - ~ VRP ordinarily will be stored in l~ hili~d form or in solution.
VRP c~- l -,~ :~ ;- .. ~ gem~rally are placed into 8 container having a sterile access port, for example, an i ltl_~_.lUU, solution bag or vial having a stopper ~: c ' '~ by a l.~,od~,....ic injection needle.
The route of VRP ~ is in accord with known methods. e.g., those routes set forth above 15 for specific ' as well as the general routes of injection or infusion by i~L~ vu~, h~LIo~
h~c~ b~ lA ut~ or ~ ~ ~ ' means, or sustained release systems as noted below. VRP is a ' ~ ~ cd c- ~ y by infusion or by bolus injection. Generally, where the disorder permits, one should ~ ' and dose dle VRP for site-specific delivery. This is cv..~_ in the case of wounds and ulcers.
Suihble examples of sustained-release ~,.c~, r;~ include ~ J ' ' matrices of solid h~.' . ' ' ~ polymers e ~ ~ ~ the protein, which matrices are in the form of shaped articles, e.g., films, or ".;~ F , ' of sustained-releasematricesincludepoly~ hydrogels [eg., poly(2-l~Lu~L~
..._Lha.,.~' ) as described by Langer ef al., J. Binm~A MAt~ c 15:167-277 (1981) and Langer, ~
I~h. 1~:98-105 (1982) or poly(vi..y' I '-')], polylactides (U.S. Patent No. 3,773,919, EP 58,481), 25 cu~ly.. _.~ of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et aL, Biovo~.. _.~. ~:547-556 tl983]), non~ g ' ' ' ~,~h~l~,..e-vinyl acetate (Langer et aL, supra), c'~,, ~ ' ' '- lactic acid-glycolic acid cu~.oly..._.~
such as the Lupron DepotTM (injectable ~..i".u .~L_..~ c-~ ~1 os~ of lactic acid-glycolic acid cu~.oly..._. and l~,..l,.ulid_ acetate), and poly-D-(-~3-h~LvA.~l,ulyric acid (EP 133,988).
While polymers such as eLl.;l_..c vinyl acetate and lactic acid-glycolic acid enable release of -'r 30 for over 100 days, certain hydrogels release proteins for shorter time periods. When e , ' I proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37~ C, resulting in a loss of L:-'~" ' activity and possible changes in i ~ ;- ;(y. Rational strategies c_n be devised for protein 5t-~hili7-~ir n d~ ; L, on the .. ~ involved. For example, if the ~o~.c~Sd iù~
- ' is di~._uve._d to be i~ - S-S bond r--~ through thio-disulfide i..t~..l. ,~_, 3 5 ' " may be achieved by modifying sul~ L~l residues, Iyorhili7ing from acidic so' ~ic~nc controlling moisture content, using ~ ..ul,. additives. and d~,~elu~u~g specific polymer matrix C~
S ~ ' release VRP c ~ also include lipAlcnm-~ly ~ d VRP~ r ;p.~cA,m~c c~ g VRParepreparedbymethodsknownperse: DE3,218,121;EpsteinetaL,Proc.~lAtl Ar-A~l Sci.USA~82:3688-3692 (1985); Hwang et al., Proc. ~A~I ArAr1 Sci. USA. 77:4030-4034 (1980); EP 52.322; EP 36,676: EP

88,046; EP 143,949; EP 142,641; Japanese patent -l-y~ -. 83-118008; U.S. Patent Nos. 4.48S.04S and 4,S44.S4S; and EP 102,324. Ordinarily the l;~.--~- ~ -. - are of the small (about 200-800 Angstroms) . ~- "
typeinwhichthelipidcontentisgreaterthanabout30mol.%~' ' ul,theselected~,.u~,u., beingadjusted for the optimal VRP therapy.
s When applied topically, the VRP is suitably c ' ~ - ' with other il.a.. d;_.lL, such as carriers and/or 'i ~~-b. There are no l on the nature of such other i.~d;~"~b~ except that they must be t;~ ~ lly ~ I and <- r 1 ~ for their intended a~ ;ah _ ~ . and cannot degrade thc activity of the active ~ c ' of the c- .. . ~ F . ' of suitable vehicles include _~ . crcams, gels, or ~ , with or without purified collagen. The ~ r ~ - ~ also may be i...~ ' into 10 patches, plasters, and ~ ' . p.cf~,.dbly in liquid or semi-liquid form.
For obtaining a gel f~ t~ n, the VRP r~ ~ ~ in a liquid _ . may be mixed with an effective amount of a water-soluble Pob~ .-1/ or synthetic polymer such as PEG to form a gel of the proper viscosity to be applied topically. The poly;._.,.,l.~idc that may be used includes, for example, cellulose d..;.d~ such as e.h_.;r.~,d cellulose d..;. ti~_~, including alkyl cell~lo:~s hydroxyalkyl crll~lnc~c~ and 15 ~~' Ylh~l~UA~_" yl ~ .In ~, ~or example, l~ hYI~r~ J~-. II~LUA~ l CeII~InS~ LUA~ ' ~l cellulose, h~ UA~ U~I ' ~ ' and hydroxypropyl cellulose; st_rch and ~ ~ ' starch; agal-, alginic acid and alginates; gum arabic; pullullan; agarose: . _ dextrans; dextrins; fructans; hulin; mannans; xylans;
' ~ . ' ~- . gl.~ _U~ , glucans; and synthetic t- r - I ~ a~ as well as gums such as x_nthan gum; guar gum; locust bean gum; gum arabic; ~ 1. gum; and karaya gum; and d..;~;.~ and mixtures thereof. The 2 o preferred gelling agent herein is one that is inert to ' ', ' systems, nontoxic, simple to prepare. and not too runny or viscous, and will not d- ~ - the VRP held within it.
~ ef~ ly the pOl~a~,~,L_;Il_ is an etherified cellulose d~,.;. ~_, more p.ef,...l.ly one that is well defined, purified, and listed in USP, e.g., ~~._lh~l-,cllulose and the l..~J.uA~al~l cellulose d..;.ati~_~, such as L~UA.~JIU~ I cellulose, hydroxyethyl cellulose, and hydroxypropyl ~.._;1.~ .lose Most preferred herein is The pol.~ _lhyl...c glycol useful for gelling is typically a mixture of low and high ' ' weight PEGs to obtain the proper viscosity. For example, a mixture of a PEG of ",ol~ ~ I weight 400-600 with one of - ' ' weight 1500 would be effective for this purpose when mixed in the proper ratio to obtain a paste.
The term "water soluble" as applied to the poly~-c~.l._;dcs and PEGs is meant to include colloidal 3 0 solutions and d~ In general. the solubility of the cellulosc d~ is d~ ~- .- - ;- -~d by the degree of ~ of ether grOUpS~ alld the ~ J~ aii~ ~ useful hereill should have a sufflciellt qualltity of such ether groups per ~ ' J .L ualu~u:~ unit in the cellulose chain to render the d~ al;~ _,. water soluble. A degree of ether;~ t~ --ofatleastO.35ethergroupspera..1.,~Lualu~.oseunitisgenerallyc~ffirien~ ~AAitinn~lly~the cellulose d~ _J may be in the form of alkali metal salts, for example, the Li, Na, K, or Cs salts.
3 5 If methylcellulose is c . ', ed in the gel".. ~.f~,.al,ly it cu.. y. ;~ ~ about 2-5%, more preferably about 3%, of the gel and the VRP is present in an amount of about 300- 1000 mg per ml of gel.
An effective amount of VRP to be employed IL~ lly will depend, for example, upon thc 5, the route of admi..iahdt;o.., and the condition of the patient. Accc,.di..~;ly, it will be nf ~ for the therapist to titer the dosage and modify the route of ad...i..i~LIdliu.. as required to obtain the W O 97/09427 PCT~US96/1407~

optimal ~ p~ effect. Typically, the clinician will 5~rlnnjnictl~r tne VRP until a dosage is reached that achieves the desired effect. A typical daily dosage for systemic rreatment might range from sbout 1 ug/lcg to up to 10mg/kgormore.~ onthefactors, - ~~ "edabove. Asanalternativegenerall~opoa;liu.~.theVRP
is ~ ' ' and delivered to the tarOet site or tissue at a dosage capable of r-1 .-8i;~ in the tissue a VRP level 5 greater than about 0.1 ng/cc up to a ~- -- ;..--- - dose that is ~rri. ~ but not unduly toxie. This intra-tissue ~- ~- - ~ ~- - ~~t ;- should be ~ d if possible by c- .- l ;- --,- ~~ infusion. sustained release. topieal a, . ' or injeetionat. , ~d .- :- ~dli~ Theprogressofthistherapyiseasily. - Idby.u..~. -assays.
It is within the scope hereof to combine the VRP therapy with other novel or cc,l.~ iùllal therapies 10 (e.g., growth factors such as VEGF. acidie or basie fil,loijlaaL growth faetor (aFGF or bFGF, .~J~platelet-derived growth factor (PDGF), insulin-like growth factor (lGF-l or lGF-ll), nerve g rowth factor (NGF), anabolic steroids, EGF or TGF-a) for r .l- :- g the activity of any of the growth factors. ineluding the VRP, in ~lull.u~ g eell ~ ; F ~ ~ and repair. It is not necessary that such eo-treatment drugs be ineluded per se in the ~ ~ - ~ ~I ~.r~ of this invention. although this will be cOIlV~ ..;. .IL where such drugs are ~ Sueh 15 a~ ~Gi, are suitably ~ ' ~d in the same manner and for the same purposes as the VRP used alone. The useful molar ratio of VRP to such j_.,ulldal y growth factors is typieally 1;0.1-10, with about e ~ ;. . .o' -- amounts being preferlred.
3. Non-Th- ,~ Di~sn--ctic Uses for VRP
The nucleie aeid encoding the VRP may be used as a ~ " _ for tissue-speeifie typing. For example, 20 such ~IUr Gdu.es as in si~u L~l~l;di~al;ull, Nor~ern and Southern blotting, and PCR analysis may be used to ~1. t . .~ whether DNA andlor RNA eneoding VRP is present in the eell type(s) being . ., ' ' VRP nueleic acid or pol~ JL;d- may also be used as ~1;~ ;- markers. For example, the VRP may be labeled. using the e . ~ deseribed herein, and CA~ ;un of nucleic acid ~1 ' encoding a Flt4 receptor or another VRP
reeeptoreanbe~1 ~ ;ri ~ usingtbelabelledVRP.
If the human VRP--.I~ odi.. g nucleic acid is localized to a human ~hl~ r~ the nucleic acid for human VRP can be used as a marker for this human cl.......
VRP nucleic acid is also useful for the plG,/alaLiun of VRP poly~ ,L;d~ by .G. ul~lb .I~.lIL l~- l --i l -. ' ' herein.
Isolated VRP pol~",. ~,L,Ic may be used in r~u~ ;L~ assays as a standard or control against 3 o which samples c~ e unknown quantities of VRP may be prepared.
VRP ~JIG. ~Liuna are also useful in g. . . 8..p _ .l;l-o~ as standards in assays for VRP (e.g., by labelingVRPforuseasastandardina ~' y,~ldiul. , X assay,orc.l~ylllc linked;.. - -. -- y), for deteeting the presence of the Flt4 receptor or one or more other VRP receptors in a biological sample (e.g., using a labeled VRP), in affinity ~ ..Lion u 1~- ;-I ~ and in cu.""~Lhi.,_-type receptor binding assavs when 35 labeled with " ' e~ enzymes, lluù~u~hu~. spin labels. or the like.
The VRP is also useful as a ~ 1: ~L;r ~ tool. For example. the VRP can be produced in prokaryotic cells using the ~ ' , e' ' ' herein and the unglycosylated protein so produced can be used as a ...ol. - ~ 1-weight mar1cer. The deduced molecular weight (mw) of the VRP is about 44.8 kDa. To use the VRP as a- ' ' weight marker. gel filtration ~I..--- .-~i-g. ,~I.I,y or SDS-PAGE. for example. will be used to separate W O 97/09427 PCT~US96/14075 protein(s) for which it is desired to d~ ~- . . . ;. .- their molecular weight(s) in ~ y the normal way. The VRP
and other - ' lAr weight markers will be used as standards to provide a range of l ' ' weights. For example, pllva~hulylase b (mw = 97,4ûû). bovine serum albumin (mw = 68,000), u~,~lbu...i.. (mw = 46,û0û), VRP (mw - 44,800), trypsin inhibitor (mw = 20,100), and Iysozyme (mw = 14,400) can be used a mw markers.
5 The other ' ' weight markers ... ~ d here can be ~u.-,l.~d cv~ y from Amersham CV.IJU.AI;V~. Arlington Heights, IL. for example. Often. the -' ' weight markers will be labeled to enable easy deteetion following s ~ - T. 1 ; ~ for labeling .~ o~ and proteins are diseussed herein and are well known in the art. For example, the I ~' ' weight markers may be b~ and, follv..i..g on SDS-PAGE, for example, the blot can be ~ ' ' with :~LIC~ P ~ L_ '' ' ~,~ ~ ' The lû bands ean then be deteeted by light ~ cti~n It may also be useful to grow eertain cells having the Flt4 receptor or one or more other VRP receptors ex vivo using the VRP as an ~t. ~ factor or growth factor. Thus, for example, the VRP can be used as a growth faetor in the in vitro culturing of . ' ' ' I cells. For such uses, the VRP ean be added to the eell culture medium at a c~ ~- from about lû pg/mL to about 10 ng/mL.
These cells which are ~o be grown ex vivo may r;~lt~"~cly be exposed to other known growth faetors or eytokines. Ex~ ,l u y eytokines inelude the l~ ~i..~ (e.g., lL-3), ~ r ' _ colony-- g factor (GM-CSF), VEGF, ..._.,... ' ~ colony-,l ;.. --1~ p faetor (M-CSF), ,, ' yt~, colony-~ ' ~ ~ faetor (G-CSF), ~ ;. .g factor (GM-CSF), e..~ Uu~r ~ (Epo), 1~ ,' steelfaetor(SLF),~umornecrosisfaetor(TNF),andg ~ f~,u,,.Thisresultsin~.-'~ -20 and/or dirf .c.~i_Lu~ of the cells having the Flt4 reeeptor or one or more other VRP r~ , In yet another aspeet of the i.. ~ . - . the VRP may be used for affunity ~. .. ;1~- ~ ;. .. - of the Flt4 receptor or one or more other VRP receptors. Briefly, this ~ . 1 - ~ - l~ ~ involves co ~ ly attaehing the VRP to an inert and porous matrix (e.g, agarose reaeted with eyanogen bromide). A solution ~ the Flt4 reeeptor or other VRP reeeptor(s) ean then be passed through the clu~ , , ' ~ material and can be: ' . ly released 25 by changing the elution c--- -l:l;- --~ (e.g. by changing pH or ionic strength).
The purified VRP. and the nucleic acid encoding it, may also be sold as reagents for .- ~- - l - - :~- ~ studies of VRP and its cognate . .i . to study the role of the VRP and the Flt4 receptor or other VRP receptors in norrnal growth and d~.~.lo~. l as well as abnormal growth and d.,~ . - - .l. e.g. in ~
The VRP may be used for ~ screening of potential agonists or for binding to the 3 o Flt4 reeeptor or other VRP ll . VRP variants are useful as standards or eontrols in assays for the VRP, provided that they are r.~ o~". ;- ~ i by the analytical system . . ' y ud, e.g. an anti-VRP antibody.
4. VRP Antihodv ~
A d~ ,fi~J~iu~ follows as to the ~udu~,liu~- of ~iA~ ;L ' as defined herein. These CA~ JI Uy ~il-o-l: - inelude poly-,lol~al, m~mnclnnAl l ~ .--.; . ~I b;~,._irlc. or L_tu~uu~ ; ~ - ~l;l~cfl;-3 5 A . r ù l ~ I A n t i ~ 1 i ~ c rol~lul~al A-~ O~ to the VRP generally are raised in animals by multiple ,.~ -- =o~< (sc) or u~u~. ' (ip) i.~ of the VRP and an adjuvant. It may be useful to C~ the VRP to a protein that is;, .---.r.~,. ;. in the species to be ;...- .-~ . e.g., keyhole limpet hemocyanin, serum albumin, bovine ,u~,lol, l;-- or soybean trypsin inhibitor using a b;r.. I;~ or d~ ali~i,,g agent. for example WO 97/09427 PCTrUS96/14075 rn~lri~n;.l~b ,..yl sl.lr..~ ester (cnnj~gPtinn through cysteine residues), N-hydroxy~
(through Iysine residues), glytaraldehyde, succinic ~.I.y~L;de. SOC12. or RIN=C~NR, where R and Rl are different alkyl groups.
Animals are 1- . ~ .- ~~; ~ d against the i.. -- .n", ~ b ' ~ or derivatives by e ' 7 I mg of I ,ug 5 of conjugate (for rabbits or mice. ~ c~ Iy) with 3 volumes of Frcud's complete adjuvant and injecting the solution '1 ' "y at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of conjugate in Frcud's complete adjuvant by ,~ injection at multiple sitcs. Seven to 14 days latcr the animals are bled and the serum is assayed for anti-VRP antibody titer. Anitnals are boosted until the titerplatcaus. rr~,f;,.aL,I~, the animal i5 boosted with a conjugate ofthe samc VRP with a different protein and/or 10 the ~ ; ~ is through a different cros- I 'AI~;rt~ reagent. ~- ~ also can be made in ~. ' cell culture as protein fusions. Also, ~$$ c~ Li~g agents such as alum arc used to enhance the immune response.
B. M ....~ 1 Antihnrl;~c ~1.. Y 1.. 'I - :' " are obtained from a F . ' of ' lly h ~ ~, ~ ' " i.e., the individual ~ ' " CU..I~J~' ' g the P~ are identical except for possible naturally-occurring 5 mutationsthatmaybeprcsentinminoramolmts. Thus,themodifier".--~- -Yl--- ~l"indicatcsthecharacterofthe antibody as not being a mixture of discrcte ~ bc "
For example, the anti-VRP .- ~ ' of the invention may be made using the hybridoma method frst described by Kohler and Milstein, ~a~. ~:49S ( 1975), or may be made by r~ ' ~ DNA
methods. U.S. Pat. No. 4,816,567.
2 o In the l.~b.i IU~A method, a mouse or ather au~ ~V~I ' host animal, such as hamster, is - ' as l._.c~.~u.~ described to elicit l,~ yLu~ t~,~ that produce, or are capable of ~..u.lb.,i,.g, ~ ';ho~ that will :r;, .lly bind to the protein used for ~ t~,.. may be i--- - : d ~n vitro.
L~ . ' ~ then are fused with myeloma cells using a suitable fusing ag~ent, such as PEG, to form a hybridoma cell. Goding, Mr~n~ 1 A. lil.o.l; ~ P~ ntl Practice. pp.S9-103 (Academic Press, 1986).
2 5 The h ~ v~ lu~a cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more ,- ~1 ~- ~ -- ~ - that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme 1.~.--- -U.;"r guanine pLo:~lJhu~;bu:~yllr~-~
(HGPRT or HPRT), the culture medium for the hyb. idu...~ typically will include h~ ~u - ~ .1 h ;. . a . ~ ;.., and LL,~,..idi..~ (HAT medium), which c..h~ prevent thc growth of HGPRT-deficient cells.
3 û Preferred myeloma cells are those that filse; ~ 'y, support stable high-level ~~AI./I C ' of antibody by the selected antibody-p. udu~ g cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-I I
mouse tumors available from the Salk Institute Cell Di~h il~UliU.. Center, San Diego, California USA, and SP-2 cells available from the American Type Culture f'ollrction Rockville. Maryland USA. Human myeloma and 3 s l..u~ ' t~.t~.~ullly~ llla cell lines also have been described for the ,~/IV iul.Livu of human ~
-- ~il~; - Kozbor, J. Imm~nol.. 133:3û01 (1984); BrodeuretaL, Mol-o~ 1 Antihodv Pru iu~Lu~ T~ hni~u~ c ~n~i ~,,UL~ pp.51-63 (Marcel Dekker, Inc., New York, 1987). See, also. Boerner et oL, J. Immllnl~i..
147:86-95(1991)andWO91/17769,published28No~.,.l,." 1991.fortr~ .cforthe~udu~ u~ofhuman - ~ ..il.V.l;. -W O 97/09427 PCTrUS96/14075 Culture medium in which l.. ~l.-idu.. la cells are growing is assayed for 1~ of ~ C ' -o~ directed against VRP. ~ref~,.al,ty, the binding s~ ,ir~;L.y of .. -v ~ ' produced by I.yl,. i-lullla cells is ~- ~- . . . ;- ~-~d by i~ , ~ or by an in vitro binding assay. such as ' ~ .
(RlA) or enzyme-linked; -- ~ ~- -- .-~ s-,~ l,....L assay (ELISA).
s The binding affunity of the . -- -n- lul-~l antibody can, for example, be ' - ~ ~ by the S~ ' J
analysis of Munson and Pollard, Anql Bioch~m 107:220 (1980).
After hybridoma cells are identified that produce ' - ' ~ of the desired a~c_;G~ ;Ly, affinity, andlor activity, the clones may be; ~ ~ by limiting dilution ylucc~ and grown by standard methods. Goding, M-..\n- 1-... 1 Antihncljes: Prinr~les q~-l Pr,.ctice. pp.59-104 (Academic Press, 1986). Suitable culture media for 10 this purpose include, for example, nulh c ~<,'a Modified Eagle's Medium or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
The . ...~ I- .- I -~ ' - " secreted by the ~,1-- 1- ~-. ~ are suitably ~ ' from the culture medium, ascites fluid, or serum by co..~, - ' ;-- -- ~-----nf L~L,ul; ~.u. ;G~aLùn ~.uccdu.~,~ such as, for example, protein A-Sc~ a~ua~ hyJ-u~.yla~atiLc c,l..l O a~Jh.~ gel clc~hu~Jho~ciai~ dialysis, or affinity clu~ ,, .'.~.
~ ly, it is now possible to produce h ~ animals (e.g. mice) that are capable. upon ~ ~ ~ . of ~u~_u~g a full repertoire of human ~ ' ' in the absence of rs ' ~
l ;.... For example, it has been described that the Lû...~,~ deletion of the antibody heavy chain joining region aH) gene in chimeric and germ-line mutant mice results in complete ~ ' ' ~ ~ of r ~ , antibody ~ Transfer of the human germ-line i ~,' L ' gene array in such 1, I...c mutant mice will 2 o result in the 1~ ' ~ of human -~ ' ' upon antigen; ' " ~ See, e g M ' ' . its et aL, Proc. N~l A
US~.2Q:2S51-2SS(1993~;1' L .itseta~ 2ss-2s8(l993).
In a further ~ L ' t~ l ~ l;. - or antibody r~ c_n be isolated from antibody phage libraries generated using the t ~ 1~ 1. - described in McCafferty et al., ~a~. ~:SS2-S54 ( 1990), using the VRP to select for a suitable antibody or antibody fragment. Claclcson et aL, Nature. ~:624-628 ( 1991 ) and Marks et - 25 aL,J.Mol.Binl 22~:S81-597(1991)dcscribetheisolationofmurinessndhumans ~ ,respectively.using phage libraries. S~ ,- bl;- ~ describe the ~.udu~.liu.. of high-affinity (nM range) human ~
by chain shuffiing (Mark et aL, BiolTPrhnnl 10:779-783 [1992]), as well as - ' ~ ial infection and in vivo "; ~ - S~s a strategy for co~ahu~,lh~g very large phage libraries. W ' a aL, Nuc. Ari~l~ P~
~.:2265-2266 (1993). Thus, these ~- h--;-~ are viable all-,..,d i~ . to halilio..al .---~..ocl~ antibody 30 h~lLidu~a ~ -l-- c for isolation of '~monnclnns~ o~ (especially human ' ' ) that are e r ~ by the present invention.
DNA encoding the ..,ono~'c ~ of the invention is readily isolated and s~5 - cd using -iu~ u~u~,e.luu~,~ (e.g, by using nl ;r.~ L probes that are capable of binding specifirS~lly to genes encoding the heavy and light chains of murine .1 .I.o~l: ,). The l.~l,.idc,...a cells of the invention serve as a 3 5 preferred source of such DNA. Once isolated, the DNA may be placed into ~u. ~aaiO~ vectors. which are then h ' ' into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce ;~ oglr~ n protein. to obtain the synthesis of ...--- ocl.")~ ' - ' in the .~ ' - host cells. The DNA also may be rnnrlifi~l for example. by ~ ;-.g the coding sequence for human heavy- and light-chain constant domains in place of the hnmr l-go~c murine sc 1., ~- - ~ (Morrison et aL, CA 02231400 l99X-03-09 W O 97/09427 PCTrUS96/14075 Proc.N~t A.~ Sci.U~ L6851 [1984]),orbycovalentlyjoiningtothe ~ ' codingsequence allorpartofthecodingsequenceforanon-;.. --.. ~i~.l,.. l;.. polypeptide. Inthatmanner,",l,i.,,~,i.,"or"hybrid"
-o~ are prepared that have the binding ~,c~iG~ y of an anti-VRP .. - .lO~ ' antibody herein.
Typically, such non-;--- -~ glol,- lin polypeptides are ~ d for the constant domains of an s antibody of thc invention, or they are ~ t- d for the variable domains of one ~ ti" c ' ~ site of an antibody of the invention to create a chimeric bivslent antibody CG~ ;aillg one ~ i5_n ~ ' ~ ~ e âi~e having S~c~;rc;Ly for an VRP and another antigen 6~ g site having .~J~..,;G-,;1y for a different antigen.
Chimeric or hybrid - ,1;1~; - also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving ...,~ y agents. For example, ~ may be L-- ~h ~~ using 10 a disulfide exchange reaction or by forming a thioether bond. F . ' of suitable reagents for this purpose include - ~ :- ~ I andmethyl 1 ...~ ptub~L~
For d;-~ , the ~ il .o- l;- - of the invention typically will be labeled with a ~' ' ' moiety. The d-~ ' ' moiety can be any one which is capable of ~,.udu.,iu.g, either directly or indirectly, a ~ ' - ' ' signal. For example, the d - ' ' - moiety may be a . ' pe, such as 3H, 14c, 32p. 3Ss, or 12SI;
15 a ~_ or c~ l u- ~h such as fIuul~ce..l i:~ulhiu~i . rl.n~l-. .. ;- -~, or luciferin; or an en~ne, such as aL~caline ~ . - . be~a-eal -- ~ or k-~ l p~"~
Any method known in the art for -r ' ~y ~n- ~j. ~;d' ;~ ~, the antibody to the d~ ~ ' ' moiety may be ~ ,' ~_d, including those methods described by Hunter et aL, Nature. 144:945 (1962); David et aL, R;~. l.. ;~h V. ~:lû14 (1974); Pain etaL, J. Iml~ol. Meth.. 40:219 (1981); and Nygren, J. Ili~lv~h~
20 Cytnt-hrm 30:4û7 (1982).
The ' ~ ' of the present invention may be s r I j~.;l in any known assay method. such as '~_ binding assays, direct and indirec~ sandwich assays, and i~. --- ...1..~, assays. Zola, M~ r~ Al~ rl;- . A M~m~l of T~ pp.l47-158 (CRC Press. Inc.. 1987).
C . ~_ binding assays rely on tho ability of a labeled standard (which may bc a VRP. or an 25 ~ ~'-" 'ly reactive portion thereof) to compete with the test sample analyte (VRP) for binding with a limited amount of antibody. The amount of VRP~ in the test sample is inversely p, ù~,u. Iio..al to the amount of standard that becomes bound to the ~--l;hv-~; c To facilitate ~ . .~ ;..;ng the amount of standard that becomes bound, the ' ' generally are ' ' ' ' before or aRer the c- ~- - ~1 l ;l ;- ~ so that the standard and analyte that are bound to the ' ' may ~ ly be separated from the standard and analyte which remain 3 o unbound.
Sandwich assays involve the use of two ~-- ~ ;l ~o- l; ~ each capable of binding to a different; - ~ - ~c, - - i portion, or epitope, of the protein to be detected (~RP). In a sandwich assay, the test sample analyte is bound by ~ first antibody which is ' ' I on a solid support, and thereaRer a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., Us Pat No. 4,376,110. The second antibody may itself 3 s be labeled with 8 ~' '~ ~' ' ' moiety (direct sandwich assays) or may be measured using an anti ~~,' ' "
antibody that is labeled with a ~ '' moiety (indirect sandwich assay). For example, one type of sandwich assay i5 an ELISA assay, in which case the ~ t~ moiety is an enzyme.

W O 97/09427 PCTrUS96/14075 C. H~ dAntihf~fliPc Methods for 1--- . ---;,;- ~ nf~n ~ lm_n ~ ;ho~ , are well known in the art. Generally, a 1 --.- ---; i antibody has one or more armino acid residues u~hudu~,CIi into it from a source which is - ' Th~ese non-human amino acid residues are oflen referred to as "import" residues, which are typically taken from an "import"
5 variable domain. I I~ can he essentially p~,, fo~ ed following the method of Winter and co-workers (Jones et al., ~, ~:522-S25 [1986]; Ripf hrnAnn et al., ~a~. ~:323-327 tl988]; Verhoeyen et aL, Science. ~2:1534-1536 [1988]), by t b~it- ;~e rodent CDRs or CDR 1 for the cu--~ r --of a hum, n antibody. A~.~,uldL~r,ly, such ~ ,--- - - ;, . ~, A -l ;ho~ I ; are chimeric - .fil Of l: - (U.S. Pat.
No.4,816,567, supra), wherein ~ ly less than an intact human variable domain has been ' - ~ - ' by 10 the c ull~ r ' ~ sequence frorn a r.u.. ' species. In practice, l ~ ' - bc '' are typically human ~ ~fil-o-l: - in which some CDR residues, and possibly some FR residues, are ' ' by residues from ' ,, sites in rodent ~ l-o-l: c It is U~ U~ i that ' ' '' be 1~- --- ~ -- ~ :-- i with retention of high affinity for the antigen and other favorable ~ ~ k, ' ~u~ d.,i. To achieve this goal, accu..lu.g to a preferred method, l are prepared by a process of analysis of the parental 'f ~ 1. - ~- - - and various c .. "t. ~ I I ' ' products using th~.'' ' 'modelsoftheparentalandI, ----~ s~ Three~" ' '' ,,' ' " models are familiar to those skilled in the art. Cc . programs are available which illustrate ~md display probable three--" ' 'c ~ ~' ~'aLu.,lu,~Jofselected ~1;' - ' ~,' L " s~ T ~ f of these displays permits analysis of the likely role of the residues in the r..--- ,;.,, ~ .~ of thc ' ' 2û r~' ~ " Sf-rin~nr~ i.e.,theanalysisofresiduesthatinfluencetheabilityofthecandidate; n~
to bind its antigen. In this way, FR residues can be selected and _ ' ' ' from the c----~ and import sequence so that the desired antibody ~.L~ ialh,~ such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most c.,h,l -~ ly involved in i ~ e antigen binding. For further details see WO 92/22653, iuvl l ~ d 23 December 1992.
D. Bie~rrific Antih.-fiit c Riererifir ~ - " are .. .- ~ . ~,. L ~- - -i ~,.cf~.al,ly human or 1 ~- ~- .: ,. ~-1 .l il .,"1;. s that have binding - for at least two different antigens. In the present case, one of the binding ~1 ~ : r;- ~ ;. c is fior the VRP, the other one is for any other antigen, and i~J.~,f~.di,ly for a receptor or receptor subunit. For example. b;~ irc cr--cifir~lly binding the Flt4 receptor and the VRP are within the scope of the present invention.
Methods for making bispecific ~ ~;1-"-1; - are known in the art. T~ ly, the ~. ' ' yl. ' " of bispecific -- ~ isbasedontheco CAy-~aiu~oftwo ;~ o~lob 1: heavy-chainAight-chain pairs, where the two heavy chain; have different el .~ ;fi - Millstein and Cuello, ~![ag~, ~Q~:537-539 ( 1983).
Because of the random ~u~u..~L of ;- - ~- .. .~ ,bul;~ ~ heavy and light chains, these h~iJ. idullldc (u,ua~
produce a potential mixture of ten different antibody ~ -' - i~c of which only one has the correct "i~"e ir~
35 structure. The ~-,--- ;ri~ - - of the correct mo' I~ which is usually done by affnity cl..~ JI.y steps, is raih~~ andtheproductyieldsarelow. Similarp.u.,eluu~a_redisclosedinWO93/û8829.published 13 May 1993, and in Traunecker et aL, EMRO J.. 10:3655-3659 (1991).
According to a different and more preferred approach. antibody variable domains with the desired bindingel~:f;-:~;--(antibody -'~, c---'' ' ~sites)arefusedto;~ o~ nconstantdomainc~ c The fusion preferably is with an i. ~gloL. ~li heavy-chain constant domain, ~u...~J.;ai..g at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy- chain constant region (CH 1) - ~ _ the site ncc.,~aa y for light-chain binding present in at least one of the fusions. DNAs encoding the ~ ,,' L ~- heavy-chain fusions and, if desired, the; . ~ lot i:. ~ light chain. are inserted into separate 5 ~.AIJ~C..~;U~ vectors, and are cO-I.a..af~,_t~d into ~'A suitable host u.~, Tbis provides for great flexibiliy in adjusting the mutual ~,,o~u. Liu, a of the three poly~ , r. -~, t~ jn ~ when unequal ratios of the thre pol~ , chains used in the ~,U~Iallu~liUn provide the optimum yields. It is, however, possible to insert the eoding .~ l, - - -- ~ for two or all three POIJ~ - chains in one e~,.~aiu.. vector when the eA~.c of at least two pol~,u~ idc ehains in equal ratios results in high yields or when the ratios are of no pa~ ' 10 _ - In a preferred . L ~ " of this approaeh, the l ~ ri. ~ " are C i~ - A of a hybrid ~ "' ' " heavy ehain with a first binding a~ ir..,ity in one arm, and a hybrid ... -- ~ t, l;-- heavy-ehain/light-ehain pair(l,~ u. " e a seeond binding a~ ir..i y) in the other arm. It was found thatthis ~ - i., strueture facilitates the ~ r ' ' of the desired biQperifir <~ ~l-u A from I .. i; - , ~,,1~1, 1; ehain ': s~ as the presence of an i , oJf~L 1: light ehain in only one half of the b;~ ;ri- moleeule provides for a faeile way of ~ ~j This approaeh is diselosed in WO 94/04690 p,~ 3 Mareh 1994.
For further details of g. - ~; g bispeeifie ~ bc ' see, for exa nple, Suresh et aL, Mrthnrlc in En7vmoloev 210 (1986).
E. Il t 'UCulli''~ AntihoAi~Q
lI_t~ P .I;l.g.l; ~ are also within the seope of the present invention. IIeh,.- ;
' ' arec~. ~p-~-doftwo.u. ' ~joih~ed " Sueh~ ,Ji -have~forexArnple~beenproposed to target irnmune system eells to I ~. . ' eells (US Patent No.4,676,980), and for treatment of HIV infeetion.
WO 91/00360; WO 92noo373; EP 03089. 11"~.. ; _ lil~ùAi may be made using any cu~ .;_ut cross 1 ~ _ methods. Suitable eross-linking age~nts are well known in the art, and are rli~rlosrA for example, in U.S. Pat. No.4,676,980. along with a number of eross-linking t 2 5 5. Usre of VRP Antiborli~Q
i. Th~ Uses VRP ~ oA:- ~ may be useful in eertain ~ ~ t; ' to bloek aetivity of the VRP (forexarnple, to block exeess aetivation or inhibition of the Flt4 reeeptor or another reeeptor that binds to VRP, and tobloekrt . .-I~ A1;~ ,C ~ ' ' " " ~ and _ _ ). Sperifirqlly, theVRP~ ' ~ areuseful~0 in the treatment of various ~- r~ ~' and - ne~ -u;~ diseases and disorders. N~ and related ' ~ that are ' '~ to l.c inelude breast cal~,i..ull.as, lung ~al.,;..u...as, gastrie ~ ,ulu~
,a~ colorectal ca.~ liver ca~ u~aa~ ovarian .,a.~,i..u...aa, thre~l~AQ
cervical ~a~ r~. e,.~u,,,_~ ial ~.a~ u~a, L.. ~<J.. _Il idl h~_. r l ~ ~ iOaia, rlblu:lr LUI~aa~ .,I.u.io.,au~;..u...a, head and neck cancer. nasopharyngeal cau.,;..u...a, Iaryngeal .,a.-,;,,u..-a~,~5 ', L' a, Kaposi's sareoma, -' skin ~,aU~,u~u~aa~ ~ _ ca~ u~A
5 ~
L pancreas ~au-,u,u~ aallucylu~7A~ Scl~.. a, o!;gc ' ' u~ a. me~ r~b! l~ 1 ub' . rhabdom~uaau~u~a. 0~ ~e,-- ;~ sareoma.I ~ ~.Ja u. urinary tract .a,.,i..u...~, thyroid ~.,;.lu...aa. Wilm's tumor, renal cell ca,.,;..u,,,~ prostate .,a -,i..ul--a, abnormal vascular ~.uI;f .-~i... r - I with ~ m-~-aspc edema (such as that ~ccO; - I with brain tumors), and Meigs' syndrome.
N: nc ~Ic ~ that are ~ ' ' to Irtam._.,L include .' 1 arthritis. psoriasis, aLI._.uscl~.uaia. diabetic and other ~ :; op-~l.ipc ~c~ ' ' fiv.u~,laaid. ncoi rJ agc-,c' ~ ' 5 macular J~ 6 ~ S thyroid hy~- r I ' (including Grave's disease), corneal and other tissue 1. ,~ y,' ~
chronic; n -... ~;..-~, lung i, n- . ~ , nephrotic s~..d.ù...c,, ~,.c~<l ~ ascites, "_. _ ' ' effusion (such as that ~--- J with p_~;~Jilia)~ ~ p~UAral Pffi~ri~A -Age-related macular d gf,r - (AMD) is a leading cause of severe visual loss in the elderly ~ . ~ Thec.~AJd~ formofAMDis~ aa~,h.i~dbychoroidal .~ .~ ului~oliu,,andretinalpigment 10 epithelial cell d ~,- I - ~J Because choroidal n_v~àaculaui,aLioll is c ~ with 8 dramatic ~ a~.l;n,5 in IJlo~ the VRP ~ " of the present invention are expected to be especially useful in reducing the severity of AMD.
For i' . rS~ p~ the VRP lil.o~ of the invention are r~ - ~,.;lto a mammal, ~,.efc.dvly a human, in a ~ lly: I - ' I dosage form, including those that may be ~ ,d to 15 a human ;~IhO~ AJI~ as a bolus or by c - infusion over a period of tirne, by ~ h . ', - 1, intra-c.,vv.~ ' intra liAUla~. i It~ .V~ ;--t - ~ r al, oral, topical, or ' ' routes. ThcVRP~ - ' - alsoaresuitably~ ,dby; ~ I, p_. - I. ' 1, or ~. ' ' routes or to the Iymph, to exert local as well as systemic Ih_.a"~ . effects. The i--ha~J.. -route is expected to be paA li._ula. Iy useful, for example, in the treatment of ovarian tumors.
zo Such dosage forms; . pl. . --~ lly a ~ r ~ '- carriers that are i--l._.c.-lly nua ~u, i., and F . ~ of such carriers include ion - ' ,,~ alumina, _' stearate, lecithin, serumproteins, such as human serum alburnin, buffer ' - such as, ' . ~ glycine, sorbic acid, F - -sorbate, partial glyccride mixtures of saturated vegetable fatty acids, water, salts, or clc hol~t~,~ such as p..; sulfate, disodium hydrogen j ~ . ' p~ ---.. hydrogen ~',r, ' - sodium chloride"~inc salts, 25 colloidalsilica, ~ h ' polyvinyl~y~uliJu~_,cellulose-based~ -- f~ andPEG. Carriersfor topical or gel-based forms of VRP o~Ati' - " include pol~ Acl.a. id_~ such as sodium carboxymethylcclluloac or.,.~.~hf!( ' ' . polyvi..yll,~.-ûliJu.._. polyacrylates, polyu,.~_ll.yl.,.,c-polyoxy~,.u~,fl.,..c block polymers, PEG, and wood wax alcohols. For all ~ ;-. s, c~ _miv..al depot forms are suitably used. Such forms include, for example, .. i.,.u~ nano-capsules, 1;~ 5.~ ~ c plasters. '-' forms, nose sprays, 3 o ' ~ ~ tablets, and ~ A3~ The VRP antibody will typically be r~ ~ in such vehicles at a c . .-~ ~ of about 0.1 mg/ml to l OO mg/ml.
Suitable examples of -' ._lc~_ ~J.c~.a. include - - I - ' '- matrices of solid polymers ~ - g the VRP antibody, which matrices are in the form of shaped articles, e.g. films, or ~ r ~ F . "! of ~ rele~e matrices include polyesters, hydrogels (for example, poly(2-~ 35 I.~.Lu.~ L~l ' - jL~tA) as described by Langer et al., s~pra and Langer, slpra, or poly(vinylalcohol), p~Jlyla.,lidf s (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-gl ~ (Sidman et aL, supra), non~' ~ ' ' '- ethylene-vinyl acetak (Langer et aL. supra), dcE,, I~ Iactic acid-glycolic acid c~ _.a such as the Lupron DepotTM (i..jc.lablc ~--i,-u~,h_.e. CO~ J.fd of lactic acid-glycolic acid l,u~ ..,.. a,.d l,_. u.ulidc acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as cll-yle.-e vinyl _ acetate and lactic acid-glycolic acid enable release of ~ 5 for over 100 days, certain l.j.l.u~ release proteins for shorter time periods. When ~ V'RP r ~ s~ remain in the body for a long time. they may denature or aggregate as a result of exposure to moisrure at 37~C, resulting in a loss of L ~ - log ~ ' activity and possible changes in ;... .r,~, .ir :l y. Rational strategies can be devised for ~ ', ' g on the 5 . ~ - involved. Forexample,ifthea~ ,6dtiu~ ..isdiscoveredtobe - ~' ' S-Sbond formation through thio-disulfide i "I.r~.5~ ". may be achieved by l..Odiry;..g sulfhydryl residues, Iyorl~ g from acidic solutions, controlling moisture content, using a~J~IU~J~ additives. and d.,. ~ . n, specific polymer matrix c S ~ -' release VRP antibody r....~ ..- also include l;pos~ y . ,. -d û r 1 the VRP r~ ~ " a;e prepared by methods Icnown in the art, such as described in Epstein etal., Proc. ~:~tl Arqrl SCi.U~ 3688(1985); Hwang etal., Proc. ~ tl Ar~ SCi. USA.~:4030(1980);
and U.S. Pat. Nos. 4,485.045 and 4,544,545. Ordinarily, the 1 l are the small (about 200-800 Angstroms) type in which the lipid content is greater than about 30 mol.% :' ~ ul, the selected p~U,JU~ Iiùl.
being adjusted for the optimal VRP antibody therapy. T r with enh--~r~d ~,;..,..ldiolr time are disclosed 15 in U.S. Patent No.5,013,556.
Anotheruseofthepresentinvention~. .1..;- .ill-u.,JvlaLi..gVRP~ -' ' intofornedarticles. Such articles can be used in ~..o.l~.l ~t; ~g r- dU~h- I; I cell growth and ~ _ " In addition, tumor invasion and , t- ~t: ~ may be - ' ' ~ with these a;ticles.
Forthep.~,~_.a;u..ortreatmentofdisease,thea~,,,.u~,. dosageofVRPantibodywilldependonthe 2 o type of disease to be treated, as defuned above, the severity and cou;se of the disease, whether the be ' are ~ ' ~ - ' for p.~ _ or 1l ~1. ti- purposes, p.~cvious therapy, the patient's clinical histo;y and response to the VRP antibody, and the discretion of the attending pllj The VRP antibody is suitably P ' .d to the patient at one time or over a series of Ll cdtm~.ta.
'~ ' _ on the type and severity of the disease, about I llgAcg to 15 mgAcg of V*P antibody is an 25 initial candidate dosage for r ' ' ' to the patient, whether, for examplc, by one or more separate a 1~ ;- - c, or by r~ infusion. A tyl~ical daily dosagc might range from about I llglkg to l OO mg/kg or more, d r " _ on the factors .. . ti- ..~. J above. For repeatcd a~iu~;al~aliu~a ovcr scvcral days or longer, d, ' _ on the conrlitir n the trcatmcM is sustained until a desired ,u~/.c~aiu.. of disease Sy~,u vl--a occurs.
However. other dosage rcgimens may be useful. The progress of this therapy is easily ed by ~,u~ tiu..dl 30 i ' ~. andassays,inrl~unL forexample,.,I; L,.,-1l;~tumorimaging.
According to anothcr e L " - ofthc invcntion, the el~ ofthe VRP antibody in ~,.c~ i--g or treating disease may be improved by ' - ,, the VRP antibody scrially or in ~ ' - with another ..- agent that is effective for those purposes, such as those c..u....,._ I below, or one or more ~ _ ' i' , - agents such as. for example, alkylating agents, folic acid - _ ~ - ' ' -35 of nucleicacid ' ' ~ ' pyrimidireanalogs,5-nuu~uu~a~,il,cisplatin,purineIr~.- 1. ~, arnines, amino acids. triazol ~ or cu. - . ' Such other agents may be present in the ~ ~. .I .. .- ~ ;ml being - ' ~ ~ - ' or may be a ' - ~.1 separatcly. Also, the VRP antibody is suitably a- 1 .; : .t ~ cd serially or in c ' - with 'i 1~, 1 llcdtl..C..~, whether involving i..adid~io.. or a.l...;.,;~".l;.~ of .adiùa~
~,,, ,~, . = .., In one ~ o~ va~. uLui,aLiùof turnors is attacked in c~ h~ -- therapy. One or more VRP
-- l;ho~ are a-~ d to tumor-bearing patients at ~ .u.-lly effective doses as ~ for example, by ul,~ i g necrosis of the turnor or its .- ~ foci, if any. This therapy is c~--~1; ~ ~ d until such time as no further b-onefi- i--l effect is observed or clinical ~ ;.... shows no trace of the tuml)r or any 5 ~- - ~ ;- foci. Then the ,u-u~ vu~ auxiliary agent is .--~lminic~red, alone or in ~ ~: - with another auxiliary agent. Such agents inciude, e g . TNF, an antibody capable of inhibiting or r.~ , ;"g the ~ ~L; - ~, . - ;-activity of aFGF or bFGF or hepatocyte growth factor (HGF), an rhVEGF ~ - - as described in WO
94/10202, supra, alpha-, beta-, or~, ~ v~, anti-HER2 antibody, heregulin, anti h_..,6. 1i.. antibody, D-factor, i - ' ' I (IL-10 1), ~ 1~,~i.. -2(1L-2),GM-CSF,oragentsthatpromote~fi~u~ uLu CQ'~ 1";.~: intumors,suchasanti-protein C antibody, anti-protein S antibody, or C4b binding protein (WO 91/017S3 ~ _' ' ' ~ ' 21 February 1991), or heat or ' ~ -Since the auxiliary agents will vary in their c[F. ~ ,.. ~ it is desirable to compare their impact on the tumor by matrix screening in cu., ~/~miu~al fashion. The ~ ;.,-~ of VRP antibody and TNF and/or other 15 auxiliary agent is repeated until the desired clinical effect is achieved. Al- ~ ~_ly, the VRP antibody or - -5' " are r ' .i.l togel her with TNF and~ o~liù..-lly, other auxiliary agent(s). In instances where solid tumors are found in the limbs or in other locations r-~ ~~ to isolation from the general c i.~ the i' . - agents dc~,- ib ~d herein are - ' ~d to the isolated tumor or organ. In other e t ' a FGF or platelet-derived growth factor (PDGF) - - ~" t~ such as an anti-FGF or an anti-PDGF ~ - ' g 2 0antibody, is ' - .i.i to the patient in ~ with the VRP antibody. Treatment with VRP
optimally may be -r ~ ~ during periods of wound healing or desirable nov.~_ul~i~aliu ii Oth~ Uses The VRP ~ - of the inveMion also are useful as affinity ~u~ ifi. ~fiùll agents. In this process, the against VRP are; ~---ol,:l: - d on a suitable support. such a S~r~ Y resin or filter paper, using 25 methods well known in the art. The ;--- - ~ 1 antibody then is contacted with a sample ~ ;" ''L the VRP
to be purified, and lI._,~art., the support is washed with a suitable solvent that will remove ~ lly all the material in the sample except tl1e VRP, which is bound to the imn~obili7l~ antibody. Finally, the support is washed with another suitable sol~ent, such as glycine buffer, pH 5.û, that will release the VRP from the antibody.
VRP ~;hu~l; c may also be useful in ,1 ~, - - assays for VRP, e.g., detecting its ~A,~ .ion in 3 o specific cells. tissues. or serum. The ~..tj~ - I' are labeled in the same fashion as VRP described above and/or are ' " ' on an insoluble matrix. VRP - - ~ ;l ~l;- ~ also are useful for the affinity ~" firlw-iu.. of VRP from .. ' cell culture or natural sources. VRP ~ ;l .o~;- c that do not d.t.. ldl,ly cross-react with other proteins can be used to purify VRP free from these other known proteins. Suitable ~l;-~;,.u.li~ assays for VRP and its _ ~L ' are dc;.. . ;l.cd above.
35 111. Eu~ ;--. - ~-l Below are examples of specific . ~I-o~ for carrying out the present invention. The examples are offered for ill~llali~ _ purposes only, and are not intended to limit the scope of the present invention in any way.
All ~ palents and patent a~"~licalh~ns cited herein. whether supra or infra. are hereby i..~u-r - ' by reference in their entirety.

WO 97/09427 PCT/US96/1407~;

FXAMPLE I
Iso~ion of cDNA Ciones Encodin~ Hl-m~n Flt4 Receptor cDNA synthesized from mRNA purified from the human megakaryoc,vte leukemia cell line CMK 11 -5 was amplified with l~,lullJall~ PCR primers based on the cu..~ el regions of tyrosine kinase receptors. Wilks.
Pr~c N~tl Ar~rl Sci USA 86: 1603-1607(1989). Onearnplified~agrnentofabout 180 bpwithauniqueDNA
sequence (~' 'i e ' SAL-S I or tkl; PCTtUS93/00586. supra) was used to screen (lanssen e~ al. . supra) cDNA
libraries from CMKI I-S and DAMI cells to obtain o-~,.lal)"..lg clones that encoded the full-length short form of Flt4 receptor (1298 amino wids). The sequence of the r~~~ ' ' ~ ~ Fltq ~ clones matched that reported from an a~._.y~ ' ' cell line (Pajusola et aL, r~nrPr R~ supra); it encodes 8 amino acid dirr~.~,...,e;.
10 from another reported Flt4 sequence. Gallarld e~ al., Onco~-n~ supra. Clones encoding the long form of Flt4 (1363 amino wids) were cullav u~d by sy. ~ the differing 3' DNA sequence of about 200 bp based on the ~vI,l; hr;i se~ PnrP Pajusola et aL, Onco~ene.3~, supra.
FxAMpLF 2 12~rPntor IPG Fl~cinn Protein~ Flt4nl2G AnticPrvm.
;mfl ~61 FACS An~ lvsi~
Fltl/lgG (Park et aL, supra), Flkl/lgG (Park et aL. supra), Rse/lgG (Godowski et aL, Cell. ~: 355-358 [1995]), and HtkngG (BennettetaL, J. Biol. ~'hPm ~2: 14211-14218 tl994]) were produced as described in these-ef~ ,es. ForFlt4/lgG, DNA encoding the ~ rll~U-- domainofthe Flt4 receptor(amino acids I-775) was spliced to the Fc region of a human IgG heavy chain at the unique ~s~EII site in the plasmid pBSSK-Fc (pBSSK-CH2CH3). BennettetaL,J.Biol.Chem.. 266:23060-23067(1991). Theopenreadingframeencoding Flt4/lgG was cloned in the . ~ ~ : -- . CiA~ ' vector pRK5 (Suva et aL, Science. 2~ Z: 893-896 ~ I 9871) to yieldtheplasmidpRK5.tkligl.1. Thisplasnlidwashall~r~ byelc~ upold~ioll(JanssenetaL~supra)into 293 cells (ATCC CRL 1651), and after 3-4 days, FW/lgG was purified from the serum-free ~ " ~ medium with protetn A agarose (('~' ' ). Flt4 antiserum was . - ~ ~.i by injection of purified Flt4/lgG into 2 5 rabbits.
By using this fusion protein to screen cell lines for membrane-bound VRP by FACS analysis. one positive cell line was iri~ntifiPr~ G61. described below.
The human glioma cell line. G61 (Hamel et aL, J. NP--rosci R-~ 34: 147-157 tl993]), was cultured in F12:DMEM (50:50) (high glucose) ~u ~ g 10% fetal bovine serum, 2 mM L-~ -- .i- - and ~
3 o For FACS analysis of Flt4/lgG binding to G61 cells. I million cells were ~ with 70 nM receptor-lgG
fusion protein in I ' , ' .-buffered saline (PBS)I, 5% goat serum. 2% rabbit serum for 60 min. at 4~ C and then stained with 10 llg/mL biotin-SP- ~ ~ - ' goat anti-human Fc antibody and 10 ~lg/mL R-phycoerythrin-streptavidin (Jackson Immuno Research). G61 caused about a 10-fold shift in peak nuu.~s.,~,.ce intensity that was specific for Flt4/lgG as compared to Rse/lgG, an unrelated tyrosine kinase receptor complex 3 5 (Fig.2). Attempts to CA~ ;UII clone this putative ~ -- ~h, - ~-bound VRP by the l.. -f ~ of pools of cDNA
clones into COS cells followed by screening with labeled Flt4/lgG yielded no positives from 640 pOO1S of 1000-5000 clones each.

T:lxAMpLE 3 tion of cDNA Clones EncodinP, Human VRP
A cDNA library was prepared from polyA+ RNA isolated as described in Cathala et aL, ~e~ 329-33S (1983) and Aviv and Leder, Proc. ~ 1 A~i Sci. USA. 69: 1408- 1412 (1972) from the human glioma cell 5 line. G61. Hamel et al., s~pra. cDNA was prepared from this RNA with reagents from GIBCO/BRL
(S~."t~.Sc. i~,L) and cloned in the plasmid pRKSB (Holmes et aL, Sçi~encet 253: 1278- 1280 [1991]) digested with ~hQI and ~1. Clones encoding VRP were isolated by 5~ ._..illg the cDNA library with synthetic ol;p~ ;- 4 probes based on an ESTsequence (GenBank locus HSCIWFI I I), which showed a ~c ' ' match to VEGF. The EST sequence of HSCIWFI I I is Z99 bp and is 36% identical to VEGF over 50 residues, including an 11 of 13 residue match 15-~ r ;~ . at VEGF arnino acid S6. The sequence is as follows:
5'-CCGTCTACAGATGTGGGGGTTGCTGCAATAGTGAGGGGCTGCAGTGCATGAACACCAGCACG
AGCTACCTCAGNAAGACGTTATTTGAAATTACAGTGC~ ~ l CAAGGCCCCAAACCAGTAA
CAATCAGTTTTGCCAATCACACTTCCTGCCGATGCATGTCTAAACTGGATGTTTACAGACAAGT
TCATTCCATTATTAGACGTTCCCTGCCAGCAACACTACCACAGTGTCAGGCAGCGAACAAGACC
TGCCCCACCAATTACATGTGGAATAATCACATCTGCAGATGCCTG (SEQ ID NO: 6).
The , ofthe ol;g.~ L probes ovhl.4 and ovhl.5; . ' .,_d are indicated below.
ovhl.4: 5'-CTGGTGTTCATGCACTGCAGCCCCTCACTATTGCAGCAACCCCCACATCT (SEQ ID NO:
7) ovh 1.5: 5'-GCATCTGCAGATGTGATTATTCCACATGTAATTGGTGGGGCAG~ I ~ I I ~i I (SEQ ID NO:
8) These two probes were 32p labeled and 1-~. i.li~d in 20% ~ L at 42~ C with a final wash in 30 mM NaCI/3 mM trisodium citrate at 55~ C. Janssen, ('~rent Protocols in Mol~ hr BioloPv. lohn Wiley ~ Sons (199S). Seven positives were identified and .,I.~t~,.i~d from 6S0,000 clones screened. The positives fell into three groups by ~c~u;~,liu~ mapping and DNA 5~~ ~ e 2 s Clones VH 1.4 (pRK.vh I .4.1) and VH 1.6 included the full coding region (Fig. 3A) and were . 1 cd . ' 'y. rhey differ only in length and the lack of two rS ~ eel...g the 3' poly A sequence in VHI.6.
Clone VHI.2 is collinear with VHI.4. Clones VHI.3, VHI.5, and VHI.7 are identical and have a 557 bp deletionwhencu...~,~ucdwithVHl.4(adeletionofbp519-1075),andcloneVHl.l hasa 152bpdeletionwhen compared with VH 1.4 (a deletion of bp 924- 1075). The . ~ and deduced amino acid s~ of VH 1.4 30 are shown in Figure 1.
The sequence cv..l-; ~rd an open reading frame of 419 amino acids bf G;"";"~, with an ATG codon preceded by a purine residue at position -3 as expected for a ~ initiation site. Kozak, Nucl. ~ riri~ Res..
L~: 857-872 (1984). About 250 bp 5' of this ATG are two in-frame ATG codons followed shortly (4 or 10 amino acids) by a stop codon. Both of these ATG's have a pyrimidine at position -3 and would not be expected to 3 s function as a strong U.ul .latiu.. initiation site. Kozak, sl~pra. The encoded amino acid sequence i...edi~t~,ly following the start of the 419 amino acid reading frame is hy.l. ~ r ' IF indicative of an amino-terminal secretion signal sequence. Perlman and Halvorson, J. Mol. Biol.. 167: 391-409 (1983). See Figure 3A. The most likely cleavage site for this sequence would be after amino acid 20, although cleavage following residues 15 or 16 cannot be excluded. von Heijne, Nucl. Acirk Res.. 14: 4683-4690 (1986). The open reading frarne is preceded by a GC-rich 5' ....~ d region of about 380 bp and followed by a 3' ~ region of about 400 bp.
The predicted mature amino acid sequence of human VRP contains 399 amino acid residues (translated Mr ~ 44.8 kDa), of which 37 (9.3%) are cysteine residues: there are three potential N-linked glJ_ua~ldtiu~l sites s (Fig. 3A). An ~ .. a of the amino acid sequence of VRP with the six forms of VEGF and PIGF shows that itismostsimilartoVEGFI2l(32%identical).andPlGFl3l(27%identical)(Fig.3B);thelocationsof80fthe 9 cysteine residues are .,u.~_. ied. While VRP does not contain the regions of basic amino acids found in some forms of VEGF and PIGF, it is cu~aid~,~dbly larger than VEGF and contains a cysteine-rich C-terminal half of the molecule that is not found in VEGF. This cysteine-rich domain has four copies of the pattern Cys followed 10 by ten non-Cys residues followed by Cys-X followed by Cys-X and then by Cys (Fig. 3B), a repeat found more than 50 times in a diptran Balbiani ring 3 prote;n. Paulsson et aL, J. Mol. Biol 21 1: 331-349 (1990). Without being limited to any one theory, VRP may interact with other .. ..i... - -bu....d proteins on these cells via the cysteine residues; such an ~ ' ' ~ ~ ~ has been proposed for the Balbiani protein. Paulsson et al..
supra.
15Two of the cDNA clones (VH I . I and VH I .3 ) contained a 152 or 557 bp deletion when compared with VH 1.4 (Fig. 3A). Both these deletions end ~t the same . ~ ;- i and are presumed to be the result of ~
splicing. Both deletions would be expected to encode the same frame-shifted protein 3' of the deletion which ~ at a stop codon within 15 amino acids. The protein encoded by VH 1.3 would include none of the core cysteine region similar with VEGF. VH I . I contains much of the region that is similar to VEGF; its deletion.
2 0 however, is not ~ ' ~ to the various known forms of VEGF or PIGF. Ferrara et alsupra; Maglione et aL.
supra; Hauser and Weich, supra.
FigLLre 4 discloses an ~ o~VHI.4 (top) with 11 EST cDNA s~ from GenBank. It is noted that the 3' ESrs arc at the polyA end and that the ESrs cover only a little more than half of the full-length sequence of VH 1.4.
25ExAMpLF 4 ReceDtor I~G P~ ;aliun of 35S-Lslh~l-rl VRP
To ~1. r . -; .~ whether VRP is a ligand fior Flt4, t,AIJ~C plasmids c ~ g the VH 1.4 cDNA clone, as wcll as control plasmids (the ~A~ aaiU~ vector alone or with VEGF or PIGF DNA), were L a.. .L_t.d into COS7 cells and the proteins labeled with 35S amino acids. C- n~' -1 media from these cells was ~,.. , ' with Flt4/lgG and Flkl/lgG.. Specifir~lly~ the VRP ~AIJ~.. iU~ plasmid, pRK.vhl.4.2, was cu.. ~uu.,t.,d by deleting about 360 bp of 5' l ' ' sequence (5' of the Aeel site (Fig. 3A) from VH 1.4). This DNA and control plasmids encoding VEGF 165 (Houck ~.t al., Mol. En-l~rrinol.. ~: 1806-1814 tl99 1]), PIGF 152 (Park et al., supra), or the vector alone (pRK5; Suva et al., supra) were Lla~ar~,~t~d into COS7 cells with DEAE-dextran. Ianssen et aL, supra. Two days after u .~ ft ~ - the cells were pulse-labeled in 1 0-cm dishes for S
hourswith5mLof.. - l1;--.;.. ~andcysteine-freeDMEM:,ut",' 'with100,uCi/mLof35Saminoacids (Pro-MixTM brand: ~m~reh- n #SJQ0079) at 37~C. and then chased with DMEM for 7 hours. The laboled ' medium was . ). ' 10-fold by spin ~n. ~ .n.~ .. (Centricon-lOTM brand: Amicon #4203).
Fifty ~lL of the collc.i..LI~-t~d medium was in: ' ~ with 3 ~Lg of receptor IgG and 80 ~lL of a 50% slurry of protejnAagarose(CUhjorh-nl)overnightat4~C. The!~, - werewashedwithPBS/0.1%TritonX-100, W O 97/09427 PCTrUS96/1407~

boiled in SDS sample buffer. and cl._hv~Jl.v.c~d on 12% SDS polyacrylamide gels (Novex #EC60052). The gels were treated with auLu~ io~ .y enhancer (duPont #NEF974) and exposed u~u...i~l.t at -70~C.
Two specific bands of 53 kDa and 33 kDa were ~ r ~ from the VRP h ~- ~ f~ by the Flt4/lgG;
these bands were absent in the vector h - ~r~ "- Little or no specific ~ ;O~ of these two bandc was 5 found with Fltl/lgG or Flkl/lgG. At times. some VRP ~lc 'r - ' was detected with Flkl/lgG, __ ~ g that VRP may have a low-affunity i with Flkl . T..- f ~ with a VEGF-cA~.~ e plasmid showed the expected ~ ;on of a strong band of about 22 kDa with Fltl/lgG and Flkl/lgG (DeVries et al.,supra;
Quinn et aL,supra; Millauer et aL,supra; Terman et aL, Bin~hPm B;O,DhYS.I~C(~nm~ n supra), but no ~IC ', '' -' with Flt4/lgG. Sirnilar cA~ h~ 2a with labeled PIGF showed no IJIC '. ' '' by Flt4/lgG, but 10 did give the expected l,.c . by Fltl/lgG, but not by Flkl/lgG. Park ef al.,supra. These data indicate that the VRP binds to the rY~ domain of the Flt4 receptor, but does not interact (or does so much more weakly) with the VEGF receptors Fltl or Flkl . They also confum the lack of an i~ ;n-, of VEGF with Flt4 (Pajusola et aL, Oncoeene. 2. sJlpra) and indicate that PIGF is also not a ligand for this receptor.
F.XAMPLE S
15 Tvrnc~ Pl.. ~1.. yl~l;n.. of Flt4 ReceDtor To assay Flt4 tyrosine phua~Jllu~ylation (also described in PCT/US93/00586, supra), Flt4 was expressed in 293 cells and Flt4 ~.I.o~l.u.yl~Liull ~d by pLua~,lluLyrosine i _ b! t. .SID ~ ;~ IIY, DNA encoding the long form of human Flt4 was cloned into the ' .,A~I C~ iOII vector pRKS (Suva et aL,supra) to give the plasmid pRK.tkl-3.1. This plasmid was co-~ f '-;1 with a plasmid ~ - g a ~lhu_lyuoaiJ~
20 1 ' . ' t~allar~.~e (neo) haula~ liu~l unit into 293 cells by calcium pl~ pl. ~- ~lC ',: ' (Janssen et aL, supra), and stably 1, .~ i lines were selected by growth on G418 (Gibco). One clonal cell line CA~I c Flt4 (clone 31), as ~ -~~~; I by FACS analysis with Flt4AgG all~ c,l and !"'1"- - f ~ ~ 293 cells were used in the Flt4 tyrosine pllOSIJIlul~ laLiùll assays. One million cells in 100 ~L of PBS/0. 1% bovine serum albumin (BSA) were mixed with 100 ,uL of sample and incubated at 37~C for 15 minutes. The cells were then collected 2s bycc.-tiru~ja.ivllandlysedin250LlLof0.15MNaCl, 10%glycerol. 1%TritonX-100,SOmMHEPESpH7.3, 4 llg/mL PMSF, 0.02 u/mL aprotinin (Sigma A6279), and 20 mM sodium UIILU~ ' ' Flt4 was r ~ by the addition of 8 !lL of rabbit Flt4/lgG allLi l~,~ulll and 30 IlL of protein A agarose.
Washed ~ , - were boiled in SDS sample buffer, clc~hu~JIIûlescd on pol~a~l~lal..ide gels (Novex), .,d to nitroc~ lose (Janssen et aL,supra), and probed with an anti-~JI.ua~,l.u~yrosine ~ o~
3 o aMibody (Upstate F ' ' ~ gy) and an alkaline ~ detection system (Promega).
Samples c-~ - ;-.e VRP or VEGF were prepared by the cle_hul~ of eA~,.c~ion plasmids encoding VHI.4 (pRK.vhl.4.2) or VEGF (Houck e~ aL, supra) into 293 cells and 20-fold cun~,c~-hd i (Centricon-10, Amicon) of the 3-day serum-free c..- 1;l;~ d medium. In the receptor IgG c ~
~A,U_~ i.. _.lLa. the c.ull~,c~lh _ ~ C~ d media were pre-;.. h ,t~ .i I hour at 4~ C with receptor IgG.
Without ctim~ inn 293 cells CA~JlC~aillg or not eAy~c~:~illg Flt4 showed little or no Flt4 tyrosine phua~,llul~laLiull. Stim~ nn of the Flt~l eA,U~C..~ g cells by Flt4/lgG al.L;a~ ..l showed the tyrosine phosphorylation of two bands of 180 and 120 kDa. No increase above basal pLv~Jllul ylation was observed with ~JIC' - serum. and no bands were found with Flt4/lgG antiserum ~ ;OI~ of nv.. eA~ a;llg cells. Two Flt4 bands of about this size have been reported as being ~A~ ed by DAMI and HEL cells. Pajusola et aL, WO 97/09427 PCTrUS96/14075 Oncoeene. 8, supra. In addition. SDS gel analysis of purified Flt4/lgG shows that it is ~ v ~l v,~l of peptides of 150, 80, and 70 kDa. 1'1 t~ --u.al amino acid sequence of the Flt4/lG peptides shows that the 150 and 70 kDa bands have the amino acid sequence YSMTPPTL (SEQ ID NO: 9) (matching the Flt4 sequence starting at residue 25) and that the 80 kDa band has the sequence SLRRRQQQD (SEQ ID NO: 10) (matching the Flt4 5 sequence b~ e at residue 473). Thus. both the Flt4/lgG and full-length Flt4 appear to be partially cleaved in the ~ t~ lAr domain, and the tyrosule plluaylluly' - ~ bands of 180 and 120 kDa observed in the Flt4 phva~Jllul.~laliu~ assays wouldcu.l~undto the 150 and 80 kDapeptides of Flt4/lgG. Addition of apolyc' antiserum to the Flt4 ~,~tJIc~ahl& cells showed the tyrosine pllGa~,llulylation of two Flt4 bands of 180 and 120 kDa; no bands were observed in no-- CAIJ~C ~ ~, cells. These data show that polyclonal ' ' generated to 1 0 the e ~ - domain of the Flt4 receptor are capable of activating Flt4 tyrosine ~I.c.alJhvl ~ ll,Liu...
To: - ~ whether VRP could activate the tyrosine ~ILGatJLvl ,~ hLion of Flt4, c ' ~ ~ ' media from cells 1"-- F r- ;I with the VRP CAIJI. ~ plasmid was assayed. This ~ ' ~ ' medium ~ ~ ' ~
tne tyrosine IJhCIalJIIul ylation of the same 180 and 120 kDa bands found with the agonist polyclonal ~
~1 -- --- ~.. -~~; ~g that VRP is able to stimulate the phc,a~llu.ylation of. as well as bind to, Flt4. C~ rd mediutn 15 from VEGF-cA~ g cells failed to activate Flt4 tyrosine pllua~Lvl.~la~iull.
To eonftrm the a~ y of VRP binding to the receptors of the VEGF family, Flt4/lgG, Fltl/lgG, Flkl/lgG, and Htk/lgG were tested for their ability to compete for VRP-Qtim~l ' Flt4 phua~ l.v-ylation. As expected if VRP is a ligand for Flt4, Flt4/lgG ~I C ~ cd the VRP-Ctim~ I ' phval.llul~la~iu.., while Fltl/lgG, Flkl/lgG, and Htk/lgG, a fusion protein from an unrelated tyrosine kinase receptor, had little or no effect. These~0 data show that VRP is able to induce the tyrosine yl.ua~hulylaliull of Flt4.
FXAMPL~ 6 1~ - ifi~aliull of VRI' Anrl Bin-linP to LAAh~ fl Flt4/lA G
The reading frame encoding the ~ t~,...lh.al secretion signal sequence and about 30 amino acids of the herpes ~ ,u~ ~ D (Lasky and Dowbenko, DNA~ 3: 23-29 [1984]: Pennica et aL, Proc. ~AAtl ~ SC;~
2 5 5E~ : 1142-1146 rl995]) were fused witll a short linker sequence to the putative mature sequence of VRP.
Following secretion from l~ cells, this construct is expected to give the ~ t~....inal gly~u~,.ut~;,. D
sequence:
KYALADASLKMADPNRFRGKDLPVLDQLLEGGAAHYALLP (SEQ ID NO: I I ) followed by the mature VRP sequence GPREAPAAAAAFE (SEQ ID NO: 12). DNA encoding this fusion protein was cloned into the 3 0 vector pRK5 to give the plasmid pRK.vh l .4.5. This plasmid was 1. .- .~r. ~ r- d into 293 cells by elc_llu~vulaliu (Janssen et al., supra), and VRP purified from the 3-4 day serum-free C~ d medium by . ~..c ' antibody (SB6) affinity CIU~ and ~ S d by co' h~ assay (Bio-Rad). This antibody is specific for the gly~,ùplut-;.l D sequence fused to the l'i t~,llliulua of VRP.
Flt4/lgG was iodinated to a specific activity of I000-1500 Ci/mmol with ~ TM brand iodinated beads (Pierce). Binding was p~.ru.. cd with -20,000 cpm 1251-Flt4/lgG and 12 ng VHI.4 gD fusion protein in PBS, 0.5% BSA, 0.02% Tween-20TM auLL~Lull, I llg/mL heparin (binding buffer) co ~ g 20 ~L of a 50% slurry of glass beads cullj_~ ' to ~30 ~g anti-gD mu .. r iu. ~AI antibody (5B6) in a final volume of 100 IlL
for 4-6 hours at 22~C. Beads were collected by filtration (Millipore Multiscreen-HV), washed five times with 200 uL binding buffer. and counted. For binding at ul~lca ~UIg Cv~ lllatiulla of Flt4/lgG (Fig. 5B) the binding W O 97/09427 PCTrUS96/14075 buffer was DMEM (low glucose):F12 (50:50), 20 mM sodium HEPES, pH 7.2. 10% fetal bovine serum. 0.2%
gelatin. and I ug/mL heparin.
The purified VRP cpecifir~lly bound to 1251-Flt4/lgG, and the binding was not competed by unlabeled Fltl/IgG or Flkl/lgG (Fig. 5A). Binding c~,.. l. Iil;.,,~ with i...,.~ ~ cv'.~ ~ .m,.l;...~c of I ' ' ' ' Flt4/lgG (Fig.
5B) gave an EC50 for this of -0.7 nM. s- ~ ; g that the binding of VRP to Flt4 is of high affinity as would be expected if VRP is a ti~!~girz lly relevant ligand for Flt4.
RNA Blots Blots cc ~-l~-;--;..g poly(A)+ human RNA were from Clontech. For the G61 glioma cell line, 5 ug of poly(A)+ and poly(A~ RNA were cle_LIuyllulc~_d on a 1% agarose/2.2 M r..., ~ . h.~d~, gel and l~a l ,f.,..~,d to lû niuv~ ,c~ (Janssen et aL, supra). Blots were l..~l,.;.li~d with 32P-labeled ptobes ovhl.4 and ovhl.5 and washed in 3û mM NaCI/3 mM trisodium citrate at SS~ C.
The G61 glioma cell line used in the cloning of VRP expresses a major VRP RNA band of about 2.4 kb. A minor band of about 2.2 kb may also be present. A 2.4 kb band was cA~..,~_d in adult human tissues from heart, placenta, ovary, and small intestine; a weaker band was found in lung, skeletal muscle. spleen. prostrate, 15 testis, and colon. EAIJIC ~ of a 2.4 kb mRNA was also found in fetal lung and kidney.
ExAMpLF 7 MitoPenic Activitv of VRP
To test whether VRP has ~ o - activity like that found for VEGF, tho growth of human lung h l; ~ cells was ~ 1 at i..~,.c g ~ of VRP or VEGF (Figure 6). !Sp~ifir~lly, human 2û lung mi.,..,~ la, - .~1--1l - 1;-l cells (HMVEC-L, Clnn~tit~s, San Diego, CA) were ~ in the .~ ' ' growth medium (EGM-MV with S% fetal calf serum). For the assay of O low passage (<o) cells were seeded at 6Sûû cells/well in 48-well plates (Costar) and ~r~c,l.;Ol,li.. the .~ ~ .. . - - - . .. Ir rl growth medium. The medium was removed. and the cells were - ' in the growth medium (2% fetal calf serum) without bovine brain extract and . rl - ' with VEGF or VRP. After four days, the cells were 25 removed with trypsin and counted with a Coulter counter (Hialeah, FL).
VRP promoted the growth of these c ~L ~Ih~ 1 cells (see Fig. 6), and thus shares this I ~ activity with VEGF. This is in contrast to PIGF, which has been reported to lack such mi-ngeni~- activity (at < 35 nM).
Park et aL, sl pra. While an effective ~ , . - if agent, VRP was about I û0 fold less potent than VEGF in this assay.
3û In co,\~ ----. a novel secreted protein, VRP. has now been i ' ~-' that is a Flt4 ligand and that the tyrosine pl.o~,~,l.o. ylation of the receptor tyrosine kinase Flt4. VRP is a third member of the VEGF
protein family and has about 30% amino acid identity with VEGF and PIGF. In addition to the VEGF-like domain, VRP contains a ~180 amino acid C-terminal, cysteine-rich domain not found in other members of the ~ - VEGF family. VRP fails to interact a~ . ;ably with the VEGF receptons Fltl and Flkl.

DeDosit of Material The following plasmid has been deposited with the American Type Culture Coll~ctinn 12301 Parklawn Drive, Rockville, MD. USA (ATCC):
Plasmid ATCC DeD. No. DeDosit Date 5 pRK.vhl.4.1 97249 e, ' 6, 1995 This deposit was made under the provisions of the Budapest Treaty on the l. .t~ R~coE,n;~
oftheDepositofMi~,~uu~;a~ sfortheFurp~oseofPatentPrucclLucandthe~ Ih~C~ lC~ (Budapest Treaty). This assures of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subjcct to an a~,~...~.-0 between Grn~ntrrh Inc. and ATCC, which assures p. and L u~ d availability of the progeny of the culture of the deposit to the public upon issuance of the per~inent U.S. patent or upon laying open to the public of any U.S. or foreign pateM Sl; .l,l;- ~ ;. . , ~,Li.,h_~ _. comes first~ and assures availability of the progeny to one ~i. r ~ d by the U.S. C~ -- ~ of Patents and T.rtL -- ~ to be entitled thereto acculdulg to 35 USC
122andtheC--- ~---;~- -"- 'srulespursuantthereto(including37cFRl.l4withpalli~ulu referenceto886 15 OG 638).
The assignee of the present a, .l .i i. ~ ., has agreed that if a culture of the plasmid on deposit should die or be lost or destroyed when ..ulli~_ ~ under suitable c~ the plasmid will be promptly replaced on r- ~1;~ . with another of the same plasmid. Availability of the deposited plasmid is not to be construed as a license to practice the invention in CC ~ua~ ion of the rights granted under the authority of any gu ~/~,. 1l~ll.,.~ in 20 a~,cu~daul~ with its patent laws.
The rul~ written ~ -- is cull .id~...,d to be sufficient ~o enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deros~ i since the deposited ~ is intended as a single ;ll. ~ ;. .- of certain aspects of the invention and any CUII:~II u~,L
that are fimrtjnn~lly c~ .ll are within the scope of this invention. The deposit of material herein does not 25 constitute an ~ that the written d~ iull herein cnntain~d is ~ , to enable the practice of any aspect of the invention, including the best mode thereof. nor is it to be cull .l. u., i as limiting the scope of the clairns to the specific ill~.~l. r~ that it lc~,l.,~.,.lL~. Indeed, various ..-o~ of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the ~UIC~,~ g ~i~ - ~ ;l .l ;., ~ and fall within the scope of the arprn~ d claims.

CA 0223l400 l99X-03-09 WO 97t094Z7 PCTrUS96/l4075 ~UukNuk LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: G~nte~h, Inc.
(ii) TITLE OF lNv~NLlON: VEGF-Related Protein (iii) NUMBER OF S~uu~Nu~S: 12 (iv) CuKK~uN~ENCE ADDRESS:
(A) ~nn~S~: G~n~ntech, Inc.
(B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Fr~nc~co (D) STATE: California (E) UUUN L~Y: USA
(F) ZIP: 94080 (v) COMPUTER ~n~RD~ FORM:
(A) MEDIUM TYPE: 3.5 inch, 1.44 Mb ~loppy disk (B) UU.. ~UL~K: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WinPatin (G~n~nterh) (vi) UUKK~ APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) A~N~:Y/AGENT lN~u.~L!TION:
(A) NAME: Lee, Wendy M.
(B) REGISTRATION NUMBER: P-40,378 (C) Rb~KkNub/DOCKET NUMBER: P0963PCT
(ix) TEL~uu. r ~NlCATION lN kU.~ ~TION:
(A) TELEPHONE: 41S/225-1994 (B) TELEFAX: 415/952-9881 (C) TELEX: 910/371-7168 (2) INFORMATION FOR SEQ ID NO:1:
(i) SkUUkNUk CH~RACTERISTICS:
(A) LENGTH: 2031 base pairs (B) TYPE: Nucleic Acid (C) STR~N~ N~: Single (D) TOPOLOGY: Linear (xi) ~kUu~NL'~ DESCRIPTION: SEQ ID NO:l:

CGC~GG~l~L 'LU~ L~lCC CCCGCCCCGC ~lulC~AAAA AGCTACACCG 50 ArGCG~CCG CGGCGGCGTC ulCCulCGCC ~~U~Ull~AC ~lCGCGGGul 100 CCGAATGCGG GGAGCTCGGA L~LCU~L 11 Cul~L~AGGC TTTTACCTGA 150 CACCCGCCGC ullLCCCCGG CAulGG~l~G GAGGGCGCCC TGCAAAGTTG 200 CA 02231400 l99X-03-09 GGAACGCGGA GCCCCGGACC CG~LCCCGCC GC~l~GCT CGCCCAGGGG 250 GGGTCGCCGG GAGGAGCCCG GGGGAGAGGG ACCAGGAGGG GCCCGCG~CC 300 TCGCAGGGGC GCCCGCGCCC CCACCC~ ac CCCC'GC~AGC GGAC~G~lCC 350 CCCACCCCCG ~lC~ L C~AC CATGCACTTG ~lGGG~ll~l l~I.l~lGGC 400 ~L~l'l~l~l~ ~lCGCC~lG CG~L~1CCC G~..~l~GC GAGGCGCCCG 450 CCGCCGCCGC CGC~-ll~AG TCCGGACTCG A~L~1~'~A CGCGGAGCCC 500 r~c~Gr~ AGGCCACGGC TTATGCAAGC AAAGATCTGG AGGAGCAGTT 550 A~ ~ TCCAGTGTAG ATGAACTCAT GACTGTACTC TACCCAGAAT 600 TGr~rr~r~T T~T~AT~r~r AGATCTTGAA AAGTATTGAT AATGAGTGGA 750 GAAAGACTCA ATGCATGCCA rGGr~-GTGT GTATAGATGT GGGGAAGGAG 800 TTTGGAGTCG CGACAAACAC ~~ .AAA CCTCCATGTG ~ l Cc~ l ~ lA 850 CAGATGTGGG G~11~-1~ A ATAGTGAGGG GCTGCAGTGC ATGAACACCA 900 Gr~rr-~r~CTA CCTCAGCAAG ACGTTATTTG AAATTACAGT GC~L~1 950 r~P~~C'CC~ AACCAGTAAC AATCAGTTTT GCCAATCACA ~l..~la~CG 1000 ATGCATGTCT AAACTGGATG TTTArAr~r~ AGTTCATTCC ATTATTAGAC 1050 .GCC AGCAACACTA CCACAGTGTC AGGCAGCGAA CAAGACCTGC 1100 CCr~rr~TT ACATGTGGAA TAATCACA~C TGCAGATGCC TGGCTCAGGA 1150 AGATTTTATG llLl.~l~GG Al~l~AGA TGACTCAACA GATGGATTCC 1200 GAG CGGGG~llCG GC~.~AGC TGTGGACCCC ACAAAGAACT 1300 ~r~AC TCATGCCAGT ~l~l~l~A AAACAAACTC Ll~CC-AGCC 1350 AA~laGGGc CAACCGAGAA TTTGATGAAA ACACATGCCA ~.~l~lATGT 1400 AAAAGAACCT GCCCCAGAAA TCAACCCCTA AAlC-l~AA AAl~l~C~lG 1450 G~ll~l'~AGC CAGGATTTTC ATATAGTGAA GAA~l~l~l-' ~ll~l~lC~C 1600 TTCATATTGG AAAAGACCAC AAATGAGCTA AGATTGTACT ~llll~AGT 1650 -~6-W O 97/09427 PCTrUS96/t4075 TCATCGATTT TCTATTATGG AAAACTGTGT TGCCACAGTA GAA~-~-~l~ 1700 TGAACAGAGA GAC~ ~ GGTCCATGCT AACAAAGACA AAA~-- ~L~ 1750 ~--u~AAC CATGTGGATA ACTTTACAGA AATGGACTGG AGCTCATCTG 1800 CAAAAGGCCT CTTGTAAAGA ~ GCCAATGACC AAACAGCCAA 1850 GA~ C~C TTGTGATTTC TTTAAAAGAA TGACTATATA ATTTATTTCC 1900 ACTAAAAATA ~ ~C ATTCATTTTT ATAGCAACAA CAATTGGTAA 1950 A~TGAAAATT GTATTAAAAA AAA~LAAAA A 2031 (2) INFORMATION FOR SEQ ID NO:2:
(i) ~Uu~N~ CHARACTERISTICS:
(A) LENGTH: 2031 ba~e pairs (B) TYPE Nucleic Acid (C) STR~N~ )N~ S Single (D) TOPOLOGY: Linear (xi) ~Uu~N~ DESCRIPTION: SEQ ID NO:2:

...... ~.~. .. ~.. AATA CAATTTTCAT TTTATTTTAA ACATATTTTG 50 CATGATATAA AAATATTGAT CACAGTGAGT TTTACCAATT ~.~ G~ ~A 10 0 A~ AA AGAAATCACA AGAGGAAAAT ~-L~G~-~ TGGTCATTGG 200 CAGAAAACCA ~-~---ACAA GAGGC~ GCAGATGAGC TCCAGTCCAT 250 ~-~-~-AAAG TTATCCACAT GGTTCAGGAA AGACAGACTT ll~L~L~ 300 TAGCATGGAC CCACAAGGGT ~...~..~ ACAGACAGTT CTA~GGC 350 TTAGCTCATT ~L~.~LL~L TCCAATATGA AGGGACACAA CGACACACTT 450 CTTCACTATA TGAAAATCCT GGCTCACAAG C~-L~-GGCG ~ ACAT 500 GGCO~.~.~ AACAGCTGCA ~--L~L~G TGGAACTTCT L ~ AA 550 TTAGG~~~~ A~L.~GGGG CAG~ LL TACATACACA CTGGCATGTG 650 TTTTCATCAA A'~ 'CG~'~'L GG~CC~ACAT ~GG~A AGA~-~L~ 700 TTTACAGACA CACTGGCATG A~'L~'~'~'L~'~C TA~LL~LLLG ~G~C~'AC 750 AG~L~GcAGG CCGAAGCCCC GCTCTGCAGA CAC~ACTGACA G~'L~'L~L-'A 800 TCCAGCTCCT 1~7111~1~C ACAGATGTCA TGGAATCCAT U L~'l L~jAGTC 8 s o ATCTCCAGCA TCCGAGGAAA ACATAAAATC 1 LC~1~7AGCC AGGCATCTGC 900 AGATGTGATT ATTCCACATG TAA~1G~7~G GGCAGGTCTT GTTCGCTGCC 950 TGACACTGTG GTA~1~ L'L~C TGGCAGGGAA CGTCTAATAA TGGAATGAAC 1000 ~ ~7~i'L~7~AA ACATCCAGTT TAGACATGCA TCGGCAGGAA GTGTGATTGG 1050 CAAAACTGAT TGTTACTGGT ~1GGGGCU~ GAGAGAGAGG CACTGTAATT 1100 TCAAATAACG ~u~LGAG GTAGCTCGTG ~lW'L~A TGCACTGCAG 1150 ~C'C~1-ACTA TTGCAGCAAC CCCr~r~TCT GTAGACGGAC ACACATGGAG 1200 GTTTAAAGAA G-,-~,---~-C GCGACTCCAA A~-.~1...C CACATCTATA 1250 TTTCAAGATC TCTGTATTAT AA- ~ ~ .,u . ~,C AGCAAATTTT ATA~U L'L 1350 U~UU~7A GTTGAGGTTG GCUL~7~LC TGTTATGTTG CCAGC-.~C.. 1400 LUU~AGCT GACACTTGTA CA~1C~AA TAL~G~ AGAGTACAGT 1450 CATGAGTTCA TCTACACTGG Ar~r~r~CCG TAA~L~-CC TCCAGATCTT 1500 ~7~ L~'ATA AGC.. 71.,GCC .C.,CC.GC~,. CGGG.-UCGC GTCCGAGAGG 1550 TCGAGTCCGG ACTCGAAGGC GGUGG.-G~CG GU WGCGC~ CGCGAGGACC 16 0 0 Crr,r-~r~r~GC ACAG~GC~,A GCAGAGAACA CGCCACAGAG AAGAAGCCCA 1650 GCAAGTGCAT GGTGGAAGGA CC~GG~7.~G gGr~rCGGTC U~U1~GU~GG 1700 GC7rr~r~,TG GGGC,CGCGGG CGCCCU1GC'G AGGCCGC~GG CCC..C~-GG 1750 20 . Cu~ . ULUCc CCGGG~1 C~'L CCC'GGU~ACC C~C'Cu l W GC GAGCCGGAGG 180 o ~GGC~7G~7AGC GG~.CU~GGG CTCCGCGTTC CCAACTTTGC AGGGCGCCCT 1850 CCr~r.rr~-T A~C~,~;~AA GG~GUGG~L GTCAGGTAAA AGCCTCACAG 1900 GAAACCGGAC ATCCGAGCTC CCCGCATTCG GAGC.~GC.,A GGTGAAGCGA 1950 GGGCGAGGGA GGAU~CUGCU G~;1UCGCG lCG-7l.7~AGC .l..~,iAGA 2000 25 ~G~7~Ck7~GG GGGACACCAG AACACCCCGC G 2031 (2) 1N~U.~ ~TION FOR SEQ ID NO 3:
~U~NU~ CHARACTERISTICS
(A) LENGT~ 419 aminO acids (B) TYPE AminO Acid tD) TOPOLOGY L1near (Xi) 7~UU~NU~ DESCRIPTION SEQ ID NO 3 CA 0223l400 l998-03-09 W 0 97/09427 PCT~US96/14075 Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys Cy8 Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala Hi3 Thr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys Cy8 Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cy8 Val Cy5 Arg Ala Gly Leu Arg CA 0223l400 1998-03-09 Pro Ala Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cy8 Lys Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys 10 Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe His Hi~ Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser G1U Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro Gln Met Ser (2) INFORMATION FOR SEQ ID NO:4:
( i ) ; h'yU~ CHARACTERISTICS:
(A) LENGTH: 147 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) s~yu~;~; DESCRIPTION: SEQ ID NO:4:
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu 251 5 lo 15 Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp 30Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Ly~ Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cy23 Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln loO 105 Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Cys Asp Lys Pro Arg Arg (2) INFORMATION FOR SEQ ID NO:5:
(i) Y~Uu~-N~ CHARACTERISTICS:
(A) LENGTH: 149 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) ~UU~N~ DESCRIPTION: SEQ ID NO:5:
Met Pro Val Met Arg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly Leu Ala Leu Pro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu Tyr Pro Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu Leu Arg Cys Thr Gly Cys Cy5 Gly Asp Glu Asn Leu His Cys Val Pro Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Ly~ Ile Arg Ser Gly Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys Glu Cys Arg Pro Leu Arg Glu Lys Met Lys Pro Glu Arg Cys Gly Asp Ala Val Pro Arg Arg (2) lN~.~L'TION FOR SEQ ID NO:6:
(i) ~UU~:N~: CHARACTERISTICS:
(A) LENGTH: 299 baRe pairs (B) TYPE: Nucleic Acid (C) STR~N~ N~:~S: Single (D) TOPOLOGY: Linear (xi) ~Uu~ DESCRIPTION: SEQ ID NO:6:

CC~l~lACAG Al~lGGGG~l TGCTGCAATA GTGAGGGGCT GCAGTGCATG 50 -W O 97/09427 PCT~US96/1407 AACACCAGCA CGAGCTACCT CAGNAAGACG TTATTTGAAA TTACAGTGCC lOO
AA GGC~C~AAAC CAGTAACAAT CA~ ~CC AATCACACTT 150 ~-GCC~ATG CATGTCTAAA CTGGATGTTT ACAGACAAGT TCATTCCATT 200 ATTAGACGTT CC~L~C~AGC AACACTACCA CAGTGTCAGG CAGCGAACAA 25O
GAC~L~CCCC ACCAATTACA TGTGGAATAA TCACATCTGC AGATGCCTG 299 (2) lN~O~ ~.TION FOR SEQ ID NO:7:
(i) S~UU~NC~ C~ARACTERISTICS:
(A) LENGTH: 50 base pair3 (B) TYPE: Nucleic Acid 1 0 (C) ST~NI ll.:l )N ~c;:S: Single (D) TOPOLOGY: Linear (xi) ~byu~N~ DESCRIPTION: SE52 ID NO:7:

~G~l~rll~A TGCACTGCAG CCC~-~ACl'A TTGr~ ~r~ CCCCACATCT 50 (2) lNrO~ ~TION FOR SEQ ID NO:8:
(1) ~~YU~ N' ~ CHARACTERISTICS:
(A) LENGTH: 50 base pair~
(B) TYPE: Nucleic Acid (C) S~N~ Il.:l ~N l~:~S: SingLe (D) TOPOLOGY: Linear (xi) ~yu N~ DESCRIPTION: SEQ ID NO:8:

GCATCTGCAG ATGTGATTAT TCCACATGTA A -G~l~GG CAG~ l~- 50 (2) lN~O~ ~TION FOR SEQ ID NO:9:
(i) ~yu~:N~ CHARACTERISTICS:
(A) LENGTH: 8 a~ino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) S~:YU~:N-~: DESCRIPTION: SEQ ID NO:9:
Tyr Ser Me~ Thr Pro Pro Thr Leu l 5 8 (2) INFORMATION FOR SEQ ID NO:lO:
(i) S~yu N~ C~ARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) 8~yu~Nu~: DESCRIPTION: SEQ ID NO:lO:
Ser Leu Arg Arg Arg Gln Gln Gln Asp (2) INFORMATION FOR SEQ ID NO:11:
(i) ~yU~N~'~ CHARACTERISTICS:
(A) LENGTH: 40 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) ~Uu~:Nu~: DESCRIPTION: SEQ ID NO:ll:
Lys Tyr Ala Leu Ala A~p Ala Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro Val Leu Asp Gln Leu Leu Glu Gly Gly Ala Ala ~is Tyr Ala Leu Leu Pro (2) INFORMATION FOR SEQ ID NO:12:
15 (i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (Xi) 8~yU~N~ DESCRIPTION: SEQ ID NO:12:
Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe Glu

Claims (39)

WHAT IS CLAIMED IS:

1. Isolated biologically active human VEGF-retated protein (VRP) containing at least 265 amino acids.

2. The protein of claim 1 containing 265 to about 450 amino acids.

3. The protein of claim 1 containing about 300-450 amino acids.

4. The protein of claim 1 containing about 350-450 amino acids.

5. The protein of claim 1 containing about 399-419 amino acids.

6. The protein of claim 1 comprising an amino acid sequence having at least residues + 1 through 29, inclusive, of Figure 1.

7. The protein of claim 6 comprising an amino acid sequence having at least residues + 1 through 137, inclusive, of Figure 1.

8. The protein of claim 6 comprising an amino acid sequence having at least residues -20 through 29, inclusive, of Figure 1.

9. The protein of claim 6 comprising an amino acid sequence having at least residues -20 through 137, inclusive, of Figure 1.

10. Isolated biologically active human VEGF-related protein (VRP) comprising an amino acid sequence comprising at least residues +1 through 29, inclusive, of Figure 1.

11. The protein of claim 10 comprising an amino acid sequence having at least residues + 1 through 137, inclusive, of Figure 1.

12. The protein of claim 10 comprising an amino acid sequence having at least residues -20 through 29, inclusive, of Figure 1.

13. The protein of claim 10 comprising an amino acid sequence having at least residues -20 through 137, inclusive, of Figure 1.

14. Isolated biologically active human VEGF-related protein (VRP) comprising an amino acid sequence shown as residues -20 through 399, inclusive, or residues 1 through 399, inclusive, of Figure 1.

15. The protein of claim 14 wherein the sequence is shown as -20 through 399, inclusive, of Figure 1.

16. The protein of claim 14 wherein the sequence is shown as 1 through 399, inclusive, of Figure 1.

17. A composition comprising the protein of claim 1 and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition useful for promotion of vascular endothelial cell growth comprising a therapeutically effective amount of the protein of claim 1 in a pharmaceutically acceptable carrier.

19. The composition of claim 18 further comprising a cell growth factor other than said protein.

20. A method for treating trauma affecting the vascular endothelium comprising administering to a mammal suffering from said trauma an effective amount of the composition of claim 18.

21. The method of claim 20 further comprising administering to said mammal an effective amount of a cell growth factor other than said protein.

22. A method for treating a dysfunctional state characterized by lack of activation or lack of inhibition of a receptor for VRP in a mammal comprising administering to the mammal an effective amount of the composition of claim 17.

23. A method for stimulating the phosphorylation of a tyrosine kinase domain of a F1t4 receptor comprising contacting an extracellular domain of the F1t4 receptor with the protein of claim 1.

24. A chimeric polypeptide comprising the protein of claim 1 fused to a tag polypeptide sequence.

25. A monoclonal antibody which binds to the protein of claim 1 and neutralizes a biological activity of the protein.

26. The antibody of claim 25 wherein the biological activity of the protein is promoting neovascularization or vascular permeability or vascular endothelial cell growth in a mammal.

27. A composition comprising the antibody of claim 25 and a pharmaceutically acceptable carrier.

28. A method of treating diseases or disorders characterized by undesirable excessive neovascularization or vascular permeability in a mammal comprising administering to said mammal an effective amount of the composition of claim 27.

29. A method for treating a dysfunctional state characterized by excessive activation or inhibition of a receptor for VRP in a mammal comprising administering to the mammal an effective amount of the composition of claim 27.

30. A monoclonal antibody which binds to the N-terminal portion from residues -20 through 137, inclusive, or from residues 1 through 137, inclusive, of the amino acid sequence shown in Figure 1.

31. A peptide consisting of an amino acid sequence shown as residues -20 through -1, inclusive, of Figure 1.

32. A method for treating a dysfunctional state characterized by excessive activation or inhibition of a receptor for VRP in a mammal comprising administering to the mammal an effective amount of a VRP
antagonist.

33. A method for treating Kaposi's sarcoma in a mammal comprising administering to the mammal an effective amount of a VRP antagonist.

34. An isolated nucleic acid molecule encoding the protein of any one of claims 1-16

35. The nucleic acid molecule of claim 34 further comprising a promoter operably linked to the nucleic acid molecule.

36. A vector comprising the nucleic acid molecule of claim 34.

37. An expression vector comprising the nucleic acid molecule of claim 34 operably linked to control sequences recognized by a host cell transformed with the vector.

38. A host cell comprising the nucleic acid molecule of claim 34

39. A method of producing VRP comprising culturing the host cell of claim 38 and recovering VRP from the host cell culture.