Title of Invention	"A GLYCOSYLATED IMMUNOGLOBULIN"
Abstract	The present invention relates to a glycosylated immunoglobulin or a fragment thereof, in which an immunoglobulin variant, comprising one or more amino acid modifications selected from the group consisting of M160N, A195N, T243N, E265N, Y299T, F331T and Q346N, is additionally glycosylated, and a gene encoding the same. Also, the present invention relates to a glycosylated fusion protein formed as a result of linkage of (a) a glycosylated immunoglobulin or a fragment thereof, in which an immunoglobulin variant having a modified amino acid sequence forming one or more Asn- X-Ser/Thr sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion thereof, a gene encoding the same, a recombination expression vector comprising the gene, a host cell transformed or transfected with the recombinant expression vector, and a method of preparing a glycosylated fusion protein comprising culturing the transformant or transfectant and isolating the glycosylated fusion protein from the culture, and a pharmaceutical composition comprising the glycosylated fusion protein thus prepared.

Title of Invention

"A GLYCOSYLATED IMMUNOGLOBULIN"

Abstract

The present invention relates to a glycosylated immunoglobulin or a fragment thereof, in which an immunoglobulin variant, comprising one or more amino acid modifications selected from the group consisting of M160N, A195N, T243N, E265N, Y299T, F331T and Q346N, is additionally glycosylated, and a gene encoding the same. Also, the present invention relates to a glycosylated fusion protein formed as a result of linkage of (a) a glycosylated immunoglobulin or a fragment thereof, in which an immunoglobulin variant having a modified amino acid sequence forming one or more Asn- X-Ser/Thr sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion thereof, a gene encoding the same, a recombination expression vector comprising the gene, a host cell transformed or transfected with the recombinant expression vector, and a method of preparing a glycosylated fusion protein comprising culturing the transformant or transfectant and isolating the glycosylated fusion protein from the culture, and a pharmaceutical composition comprising the glycosylated fusion protein thus prepared.

Full Text	Technical Field The present invention relates to a glyeosykted immunoglobulin and an inmmoadhesn conpiang the same. More partieulariy, the present invention idatK to an imraimog\|Dbulin or a fragment theirot; \viiich is additional!}-gl)iylatedty andaglyojsyiaidfum a ferment thereofwifli atlcaatone biologically active pK or a portion thereofi BattotMindArt Imminwadhesins (or immunoglobulin fusion proteins) ate antibody-like molecules lesuiting fiom the fuskm of a foig region of a receptor or an adhesive mokoile. Tr typicdirmnunoadhednskm\vnintbearthave file stiucture of an antibody in which the variable region, participating in antigen recognition, is replacdwthaUgandmdir For a long time, a large nurriber of patents have described fosioD piotdnsinv in which is nofaphysiok cally active protein is linked to an antibody (US. Pat Nos. 5,521,288,5M095,6,046310, 09014, 6,100383 and 6^25,448), The immunoadbesin has the Mowing advantages over a molecule not containing an 1) toe fusion protein has increased tota!avi%toalipdbecauseithashivabKymadin^ form; 2) the fusion protein is preset virtue of increased molecular stability; 3) effector cdJs ate activated by flic Fc (Fragment oystallizabfe) portion of the immunoglobuJin heavy chain; and 4) the fusion protein is isolated and purified by a convenient method, for example, using protein A For example^ hthecastt of tutOT a cytokine, to suppress TNF-dependent inflammation responses, tumor necrosis factor receptor (hereinafter, referred to simply as "TNFR") may be used as described in POT Publication Nos. W092/16221 and W095&4326, or a TNFR-imimmoglobuIin(Ig) fason protein way be used as described in US. Pat Nos 5,447,851 and PCT PiMcation No. W094A)6476. Accordmg to numerous reports, IM^-IgMonpxrte^ form or Ig«»-iused form of TNFR (Lessiauer W. al E«r. J. tattminoL, 1991, vol21, jx2883; A&ssam A. et aL PNAS USA, 1991, volSS, p.10535; Pefjpd K. et al J. Exp. Med^ 1991, vol.174, p,I483; Matter KM ctal. J, bmurjoL, 1993, vol.151, p.1548). With respect to the inhMm of TNFadcn or the control of in ^on protein, aimMvaleot (vmume TOT receptors, CD2 and CTLA4 in an Ig fusion construct is expected to improve the efficacy of the fusion construct \K a mairaiiBdc fusion extracellular domain and the Ig heavy chain is expressed in a cell line simultaneously with another mooornerk Mon ptotdn Qigjbt Igligtacham, a dirtOTcfusicto praxis pro light chain. Thectmericfiiskttr form, and has remariyirxxcascd efficacy in ccrapar B J. et al CytDkine, 1995, wL7,pJ59). However, such an Ig fusion protein ina dimericfonn is dfficuhto mduslrialize due to Ihe fdBowu problems: two genes which are indiviWyfusd to the Ig heavy and co-introduced into a host cell; when two different fusion proteins are simultaneously expressed in a chain fosionproteiris do iwtpartidpatem the fcmationo protein and a light chain fusion protien are technically difficult to isolate from a mixture with the moromerb heavy cMiftskm In this legaixi, the present inventor of the soluble domain of a biologically active protein is linked to an N-tenninus of the soluble domain crfm identical ccdhlffleniMologic Also, the present inventors prepared a DNA construct encodadimerbpiotehmctwomdeculesofa mwioinericproteffljm the hinge region of an immunogbbulin JFc fragment, ace disuifide-bonded at the hinge region, and pnxkaoMaiamcr-MdojmericfiisionpTotdnu DNA construct As described above, attempts have been made to improve the efficacy and preparation method of immunoglobulin fusion proteins, but almost all efforts have been unable to increase the stability of the iminunoobulinfuskm proteins, h to regard, as disclosed in Korean Pat Application No. 2002-&W5921, the present im glycosyMon mothe to a conjunction region between a functional domain of a protein and an immunoglobulin Fc region. However, vdren an imrflunoadhesin is gccylatd near a fiMonal Disclosure of the Invention In this regari, the present mvato an additional glycosylatian motif into an immurwglobulin, particularly an Fc portion, of an Hnrrttnwgtabulinfusm period of time than the fonn not containing a glycosylation motif, thereby leading to the present invention. Thus, in one aspect, the present invention provides a glycosylated immunogiobulin or a fragment thereof, in which an immunoglobulin variant comprising one or more amino add modifications selected fiom the group consisting of M16QN, A195N, T243N, E265N, Y299T, F331T and Q346N, is additionally grycosylaled In another aspect, the present invention provides a DNA encoding a gtycosyiated inmiurwglobvdm or a iragmerrt thereof, k \vhich an irrmii^ amino acid modifications selected from the group consisting of Ml SON, A195N, T243N, E265N, Y299T, F331T andQ346N, is additionally glycosylated. In a further aspect, the present invention provides a glyrasylated to result of Mage of (a) a gfycosykted irnmunoglobulin or a fiagment thereof, in v^dch an irnmunogjobulm variant having a nooolfied anrino acid seqpu sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion thereof In yet another aspect, flie present invention provides a DNA molecule encoding a glycosylated fosionprotein, which is Sained as aresuft of linkage of (a) agl or a fiagment (hereof, in which an iniraurwgtobulm variant filming one or more Asn-X-Ser/Tbr sequences is additionally glycosylated, with (b) at least one biologically activeprotein OTarjortioa thereof. In still another aspect, the present invention provides a recombinant expression vector comprising a DMA molecule encoding a glycosylated fusion protein, which is Ibrmed as a result of linkage of (a) a glycosylated immunogfobulin or a fragment thereof; in which an immiinogiobulin variant having a modified amino add sequence forming one or more Asn-X-Ser/Thr sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion thereof In still another aspect, the present invention provides a host ceD transfer or tansfbnned with a reccrabinant expression vector comprising a DNA molecule encoding a glycosylated fusion protein, which is formed as a result of linkage of (a) a glycosylated immiinogiobulin or a fragment 1rm)CmwrManiraiminogfo more Aai-X-Ser/Trrr sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion thereof Li still another aspect, the present invention provides a method of preparing a glycosylated fusion protein, comprising cufcuring a host cell transfected or transformed with a recombinant expression vector OMnpri^ as a result of linkage of (a) a glycosylated iramunogjobulin or a fragment thereof in which an imrrnincglobulmvariartkvinga sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion In still another aspect, the present invention provides a pharmaceutical composition omprisiiigaglycosyla irrimunogltobiilkOT add sequence forming one or more AsnrX-likir/Itesecperm least one biologically active protein or a portion thereof Brief Description of the Drawings The above and other objects, features, and other advantages of the preset invention will be mote clearfy understood fiom the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. 1 ^wsglycosyMcm sites of immunoglobulinsacconiing to the present FIG. 2 is a graph showing expression levels of glycosylated CTLA4-IgG fusion proteins according to the present invention; FIG. 3 is a graph showing the results of Western blotting of glycosylated CTLA4-IgG fusion proteins aaxwding to the present invention; and FIG. 4 is a graph showing changes over time in serum levels of glycosylated CTLA4-IgG fusion proteins according to the present invention, in mice intraperitoneally injected with the fusion proteins. Best Mode fbrCanymg Out the Invention Single capital letters representing amino acids, as used herein, represent the following amino acids according to the standard abbreviation defined by the tatemati^ A:Alamne;B:AsparagineorAsparaticacid; C: Cysteine; D: Asparaticacid; E: Gluiarnicacid; F: Phenylalanine; G: Grycine; tt Histidine; I: Isoleucine; K: Lysine; L: Leucine; M MetWonine; N: Aspargine; P: Proline; Q: Glutemine; R: Argmine; S: Serine; T: Threonme; V: Valine; W: Tryptophan; Y: Tyrosine; and Z: Gluramine or Glutamic acid. 7/37 Tlw designation "(out capital fur an amino addXamiiw acid posttionXone capital for another m & the designated amino add position of a given proteui For example, M179N indicates that die mefoiorairei^due at the 179th The aminoaddpositicraisfflBit TJtelimMglycQsyJafi host are nwdified by the attadsnoof sugar chains. Tte attachment of sugar chassis known to ei^ There are two types of glycosyialinn. OJinlned gJycosj'lation Hulas an oligosoccharidc chain to a saiue and/ur tbreoninfi residue. N-linkdgljw^iatiOT links moHgosaccharide chain to an as^^ In pffltkolai; N4infced gjfjw^^ amino add exduding proline). k the present hventk)n,aDNA sequence OTcodingmi^^ © imitated at one or more nudeotidea to form an additional dycosylatiun site at wWdi O-Hnked or Nlinked glycos'kioii occurs, and tile mutated DNA is expressed in a host cdl to dUow spontaneous gjycosjtoioffl. fe(fflcaped,ax)syliiniin pxsetA invention is constructed by mutating a DNA sequence encoding an immunoglohuHn or a ftagpent teecrfto add aod/cr Snas M finked wsylatko occurs, The '%nmunoglobulinsbich are modified to possess a ycosykkxi motif in the present invention, at piotefonralecales to are produced in B cefls and sow as antigen iwitoBSpecaficalfy rexagoizi a vwde variety of antigens. The moleades hsve a Y-sfa^ed strarture consislingoftwo identical light chains (I chains) and two kJeoW heavy chains (Hc hddlosrfrabyanumbofdisulfidebc 8/37 hinge region The L and H(toisc«ipisevariafaieaiKl constant regbiis. According to features of the coBtant regions otH chains, iramunoglobuliris (Ig) are classified into five isotypes, A (IgA), D (IgD), E ( G dgG) and M OgM). The five subtypes possess unique stractural and biological properties. These inrnunogfobulinsn Since an imnumoadhesingaiaaDy contains a figment of R portion a gtyeosryl an immunoglobuJk The torn "Fc portion of an immunogiobulin" as used herein, refers to a fragment having no antigen-binding activity and being easily crystallized, which comprises a binge region and CH2 and CH3 domains, and a portion responsible for binding of an antibody to effector materials and cells. fa the present invention, a glyc amino acid residues at positions 160,195,243,265,299,331 and 346 of an immunogiobulin (all of theseanano acid residues are present attheFcpe8tionofanimmunogkibu3in). Thus, in one aspect, the presertrmm provides a g j y s y uoe or mure amino acid modifications selected from the group consisting of M160N, A195N, 1243N, E265N, Y299T, B31T and Q346N, and a gene encoding the same, In more detail, a glycosylated immunogiobulin or a ftagmenttbereof listed in Table 1 is provided, which contains oombinatiom of one or more of the aforementioned amino acid modifications. (Table Removed) In another aspect, the present invention provides a glycosylated fusion protein formed as a result of linkage of (a) a glycosylated immunoglobulin or a fragment thereof, in which an imimnwgfobulmvariart sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion thereof b a preferred aspect, the fragment of m irmnuiiogbbulin includes aii Fc portion, and the rxjrtionofabiotogicallyacriwprotem In one aspect, the glycosylated fusion protein has a monomer structure in which a angle polypeptide is loaned as a result of linkage of (a) a glycusylalcd uiiuiuuoglobulin or a fiagmeot thereof m \vhkhmimisnjnoglobulm X-Ser/Ik sequences is additbnally ycosylated, with (b) at least one bMogically active protean or a portion fliereof, Therc«ofabiolcgkyactrvpx3temprd(erablyind ctornain of biologically active proteia T\vo rnoleoiles cf such a monomeric glycosjla^ rusiOT protein n be Msedbyao^sulfictebcMKl at the h i r n In ar«^ aspect, the glycosylatd fusion protein has a nx polypeptide is fooned as a resutt of linkage, in a concatameric form, of (a) a $ycosyiated acid sequence forming one or more AsnrX-o/Ik Kquaffies is a^iticually gjycosylated, wifli (b) a first biologically active protean or a portion thereof and (c) a second biologically active protein or a portion thereof. The first and second biologically active pioteteimy be kfentkalcff differed The portion of a biologically active protein preferably includes a soluble extracellular domain of the biologically active protein. Two molecules of such a monomeric glycosyta^ftscra protein may be linked byadisulfidetorKi at the bing MapcdienedasDectoftheglvc immunoglobulin variant comprises one or more arrano acid modifications selected from the group consisting of M160N, A195N, T243N, E265N, Y299T, E331T and Q346N and is giyeosylated. In a more preferred aspect, foe imroiunQgkbulhvaiiMcorr SEQIDNO: 16toSEQE)NO: 23, and m^ most pdkred aspect, ttearninoactt)NO: 19. The term "biologically active protean" as used herein, refers to a protein, peptide or poryr^ptidermvmggeneralfyph activities after forming an imraunoadbean The term "biological activity", as used herein, is not limited in meaning to physiological or pharmaceutical activities. For example, some irarmmoadheans, suchasthose containing an enzyme, Non-limiting examples of the protein, peptide or polypeptide include hemoglobin, serum proteins (e.g., blood factors including factor VE, VSR and factor IX), immunoglobulin, cytokines (e.g interleuldn), a-, p- and y-interferons, cotorry-stirnulating fectars (e.g., G^F and GM-CSFX plateletderived growth fector (PDGF), aand phosphoKpase activating proteins CPLAft). Ofcer typical biological or therapeutic proteins include insulin, plant proteins (eg., lectin and ricinX tumor necrosis fector ffNF) and its mutant alldes, growftj fectors (e.g., tissue growth fictoxs and endothdial growto iactars such as TOFa or TGFp), hormones (e.g, follicle-stimulating hormone, 11/37 JMtmone, atitidiuretic hormone, pignieirt'C«K!eirfrating or disposing hormones and parathyroid hormone, luteinizinghoiinooeHrieleasii^bxMmoneand its derivatives), cddtonin, caldtonia gene related peptide (OGKP), synthetic erfcephaln, somatornedin, eryteopoietin, hypotfialamus releasing fectars, prolactin, chronic gonadotrophin, tissue plasminogoi-acti\'ating agents, growth tormooe-rdeasing peptide (QHRP^ai^%mKhiincial6ctor(THF). Son«raotdmsuchasinterleukin,inteffecdonytiniulating fector may be produced in a ncn-glvcosylated form by using DMA recombmart tecteiques. The nonycosylated proteins may be useful as bbbgically active materials in the In addition, the biologically active materials useful in the present invention include any part of a polypcptidc, which lias bkactivity in vivo. Examples uf lljc bbbgically active materials include peptides or polypeptides, fiagments of an antibody, single chain-binding proteins (see, U.S, Pat No. ), binding molecules including fusion polypeptides of antibodies or their iagments, polydonal antibodies, moiodonal antibodies, and catalytic jmbbodies. Other examples of the biologically active materials include aUagen pioteans, as lagweed, antigen E» honeybee vetwai, or allergen of rates. la addition the biologically active material useful in tExamples of the enzymes include caitetydrate-specifie enzymes, proteinases, oxidoreductases, transfetases, bydtolases, lyases. ksneraaK, and ligases. In detail, NcmJimMng examples of the endotoxinase, catalase, chymotrypsin, lipase, uncase, adenosioe dephospimtase, tyroane, and biKrabm oxidase, Ejamples of the catbot^drate-specific eiKymes include ucose osdase, Tlie term olubte estacdfular domam", as used bm refe to a portba exposed to tbe extraceflular region of m intiegral membrane protein penetrating die cdl membrane comprising 12/37 bydrophilic auiiiw adds, which arc typically positioned at the surt and thus is soluble in an aqueous enviramnent Of nwstcdlsur^recepta1 proteins, extracellular domains serve to bind specific Kgands, vADe intraoeQular domains play an important rok in signal transduction. In one aspect, the gtycosylatod fusion protein according to (he present invention may be pn^anxi by preparing a DN modified to contain a glycosyiation ate and Mdng thereto another DNA sequence encoding a I^k)gicaflyacdwpi»tebcraportim1hereo£ fo anofter asped, tbeycostjtedfiraonproteiQinay be primed by primarily preparing a DNA sequence (fusion gaic) llrni aiuxks both an immunooMin or a ftagnent tbsreof and a bbbgically active protein or a potion thereof and mutating the fosion gene to aBowtte The twopreparaonrnethoisdiffofiomeacho^iercxiryinte and are basMy identical to preparatkm roedxxfe, k protein variant Tbi^ bocanafiar, fee present iuveuliun intends to fcos a modifeato an uraiunogtobulincrafi^ment thereof into \vh A DNA setpence enooding tbe glycosylated immunogJobuJia or flie figment thereof acceding to the present invertk» may te These methods include, but ate not limited to, oligonuclwtidenediated mutagcnesis and cassette jBUtagenesis. In pardcul^,tkl^^ sequence oKoding the glycosylated thereof accotding to, the present invtntion is preferably prepared by digonucleotkie-mediated nattagcnesis. ThistechniqwisweUkrttwnhitheanddesa aL Nua Ac. Res, USA, 1982, wLlO, pp.6487^500). fa brieg the ES4A sequenoe enooding fte rthefiagme a plasmid carrying DNA encoding a non-modified or native imiramoglobulin or a fragment thereof) with an oBgonucleotide coding for a desired modification. After hybridization, a second complete strand complementary to the DNA toplateraybesyn&esizedty the second strand may code for ihe desired modification. Typically, oiigonudeotides used in the aftmnmtioned mefexk ZM composed of about 25 oucleotides. Shorter oKgonudeondes can be employed, but oplHnaloligonucleotldes,atboaile£land right regions of rnooedoxk) These oligonucfeoddes can effectively hybridize with a template DMA. These oligonudeotides may be synthesized by the technique USA, 1978,voL75, p.5765 In one aspect, the present invention provides a DNA sequence encoding an inmnmoglobulin or a fiagraent thereof, which carries one amino acid modification (IgG in Table 1). This DMA sequence may be prepared by performing PCR using DNA encoding an immunoglobulin or its fragment as a template and inodifiction-encoding synthetic oligonudeotides as primers, Primers hybridize with tiieir complemertey sangie-stranded DNA produced by denataation of a doublebranded DNA template during heating. DNA polymerase adds nucteotides to the 3'-OH of the mcxfifiotion-encoded primer one by one in a mamer cornplernentary to a template in the S' to 3' direction. ThenevdysynlheazedstiarKiirxwrpo gene encoding a desh^mcxJificadon, TlKtvty synthesized sbaoi is used as a template DNAm the extension step of PCR, resulting in ampEfication of a gene encoding the modification. In another aspect, Ihe present awention provides a DNA sequence encoding an .When two or more amino acids to be modified are spaced dose to each other on a polypeptide, oil desired modificanons are encoded in one oUgonucleotide and thus simultaneously achieved Therefore, a mutated irnmunogtoboliaor a fragment thereof havirigtvvo cr more amino acid modifications may be prepared by the same method used to prepare the mutated inmiunoobulin or fisgmeat thereof amino add modifications as primers. When two or more amino adds to be modified ate spaced far apart (rafhe case that over 10 amino adds are present between t\wamim acids to be n encoded in one oligonudeotide. Thus, diflerent methods should be introduced One method is to prepare indradual oligciriude When the oUgonudeoddes ate annealed simultaneciusly to a single-stranded template DNA, a newly synthesized secondary singlestanded DNA encodes all of the dearedaraino acid modificatwns. Another approach used in the ppssert invention inctodes tw In the primaiy miflagenesis, using natural DNA as a template, oieoligoni the template, and thus bsteroduplex DNA is produced In 1he secondary naaagenesis, the heteroduplex DNA is used as a template. The template atneady carries at least one modification. When one oHgonudecjtideha an additwral amino add n resulting DNA encodes both of the primary and secondary modifications. The cassette mutagenesis is also a preferred method for thepjerMostira gtycosyktedioimimoglcb This method is tjasedonttetedmkiuedesAstartirmaterialisaplasm3d(oranoirjavecfiagtnent hereof to be modified The cassette rautagenesis is preferably used when a specific itsm'dion enzyme ate is pre However, this is not essential. If siHiiarestri(onerj2ymesitedoesaitexist,itcfflbeintr oicodirjganimminx)gtobdm(wafimenttheie After a restriction enzyme site is introduced into the plasmi4 tbs plasniid is lineari restriction enzyme. A double-stianded oligonudeotide having a DNA sequence that contains a desired mufatioa and is tocaid between reslri method. The two strands are individually synthesized and hybridized using a common technique. Such a dcwbJe-stranded oligomfcleotide is tvp The cassette should be pijaj ta the fiMm of possessing 3'-arid plasmid and may be thus diiectly conjugated to the plasnid Tlieplasmid comes to contain a DN A encoding a desired gh/oosylated immunoglobuljn or a fragment thereof through the aforementioned procedure. In addition, the preparation of a DMA sequence encoding a gly(sylatedimmunoglabulinor a fiagment thereof according to tte present invention rnay be achiev In partioilar, such a DNA sequence may te synthesizer. An oligonucleotide is made based on an amino add sequence of an glycosylated iirapGttinaobulmorai producing an glycosyisted immunobbiinorafiagnientmereof. With respect to a DNA sequence encoding a glycosylated immunoglobulin or a fragment thereof according to the present mvratici amino add is specified by more ftancdon, is wdlkno\i Thus, there is apteality of DNA sciences with degeneracy encoding a glycosylated mimunoglobuiin or a fiagment thereof accoidmgtothepnamventb Alternatively, the gfycosylated Mon protein according to the present invention may be prepared as follows. A DNA sequence eoxxlingthefiiskHi protein ^isiemate^rirfaiedtoas^fiiaon gene") is prepared, and is inserted into a vector including one or more expression control sequences regulantheexrffesswnoftheM Hien,ahost is transformed or fransfected with the resulting rccombinant expresskm vector. The resulting trarsformantortramfectartisoj 16/37 oftte&siongene, A substantially pure gjtycosylated fusion protein coded by the Men gene is recovered from the resulting culture. The tarn "vector", as used herein, means a DMA molecule to saves as a vefaidb enable of stably carrying exogeneous genes into host cells. To be useful in implication, a vector should be leplicable, have a system fix- irtrociucing itself into aho In addition, tie term "necxMnbinant expression vector", as used herein, refers to a circular DNA molecule carrying exogeneous genes operably linked thereto to be expressed in a host cell When introduced into a host cell, the recombinant expression vector has the ability to replicate regardless of host chromosomal DMA at a high copy number and to produce heterogeneous DNA. As generally known in the art, in order to increase expresskailevelofatransfededgeneinahost cell, the geM should be operabfy linked to transcripticMi and in the host cell selected as an expression system. Preferably, the expression regulation sequences and the exogeneous genes may be earned in a single expression vector rontararng selectable replication origin. Inthecasethateukarvc^ttDsateusedasan vector should further comprise expiessimmarkfiis useful in tteeukatyot^ In order to express the DNA sequence (Le, fusion gene) encoding the glycosyiated fusion protein according to the present invention, vanVws expression vectors may be employed. Preferably, stnceanirmnunoglobulmorafra eukaryotic host cells should be used Expression vectors use&l for eukaryotic host cells contain expression control sequences derived from, for exampkiSV40, bovine papfflorravii^ cytomegalovirus. In detail, examples of tie vectasindiidepCDNA3J(+y Calif, USA) and pChx» (Stcatagen, La Jolla, Calif, USA). Expression vectors useful tor yeasts include 2\|A plasmid and hs iso&nns, POTl vector (US. Pat No. 4,931,373) and pPICZ A, B, or C (tavitrogen). Expressi(m vectors useful fijr insect orndirfepVL941,pBluebac4.5andpMelbac (Invrtrogen). However, tw present invention is not limited to these examples. 17/37 Be term "expression control sequences", as used herein, refers to nucleotide sequences reeiessaiyOT advantageous for expr Each control sequence may be native or foreign to the fusion gene. Non-limiting examples of the expression control sequences include leader sequences, polyadenylation sequences, propeptide sequences, promoters, enhancers or upstream activating sequences, signal peptide sequences, and transcription terminators. fa order to express the fusion gene of fce present invention, any of the various expression omtrolsequerm may te insetted Examples of expression control sequences suitable for directing protein expression in mammalian cells include SV40 and early and late promoters of adenovirus, MT-1 (metallothionein gene) promoter, human cytomegalovirus immediate-early gene promoter (CMV), Rouse sarcoma virus (RSV) promoter, and human ubiqmtinC(UbQ promoter. In addition, to irnprove expression levels in mammalian cells, a synthetic intron may be inserted into the 5'-untranslated region of the fusion gene. Examples of expression cartrdsequer^siritabte promoter, PIO promoter, bacubvirus 39K delayed-early gene promoter and SV40 polyadeiiylalion sequence. Examples of expression control sequences suitable for directing protein expression in yeasts include the promoter of the yeast a-rnating system, yeast triose-jphospbate isomerase (TPI) promote aodADH24c promoter. Examples of expression control seqimes suitable ifo directing protein expression in fungal cells include ADH3 prontroterandtraninators. lhe team "operabry linked" refeis to a state in which Aefuskm gene of the present invention is arranged with such a control sequence in a functional relationship. That is, a gene and control sequences are linked in sudh a manner (e.& transcnoti-vating-'protein) binds to the control sequences). For example, when a presequence or secretory leader facilitates secretion of a maojre profit is rcfen to the axling sequence of the protein. A pratncto is opa^lyMed nthacodingsequencewhai it regulates transcription of the coding sequence. Ariboscmie4)iadingsiteisoperablylii±edtoacoding seqimwhm it is present at a position alfov Typically, the tssm "operably linked" means tet linked nudeotide sequent are ra contact with each other. In the case of a secretory leader sequence, fhe tern means that it contact a aiding seoeixand is present within a reading fiame of flie coding sequence. However,an enhancer need not necessarily contact a coding sequence. Linkage of the nucleodde sequences may be achieved by ligation at convenient restriction enzyme recognition sites, h the absence of restriction enzyme recognition sites, ou'goniKlectide adaptors OT linkers nm^ On die other hand, host cells having high introduction efficiency of foreign DNA and having high expression levels of an introduced DNA may be used In particular, as a host cell, a eukaryotic cell capable of glycosytating the fusion protein of the present invention should be used Examples of suitable yeast host cells include strains QfSaccharomyces and Hansemda. Examples of suitable fungal host cells include Tricoderma, Fusarium and Aspergittus species. Examples of suitable insect host ceDs include J^^ora cell lines such as Sf9 or Sf21. Examples of suitable mammalian host cells include CHO cell lines, COS cell lines such as COSI or COS7, animal cell lines such as BHK cell line or mouse cells, and tissueiiltui plant lls and human cells. The fusion gene of fhe present invention or a recranbinantejqaressitm vector ccaipisin same may be introduced into a host ceD by the memods described m basic experimental guide books (e.g., Davis et al, Basic Methods in Molecular Biology (1986). The preferred memods for this introduction into a host cell include, for example, calcium phosphate transfection, DEAE-dexfean mediated transfection, microinjection, canonic lipid-mediated transfection, electroporation, viral transduction, scrape loading, ballistic inlroductMD, and rafection. Tn tie preparation method of die present invention, the host cells are cultivated in a nutrient meoium suitable for rmdifction of a pdypepb using me For example, the cells may be cultivated by shake flask oiltrvation,snjan-scafecfflar OTindustrMteietitors perform to be expressed and/or isolated. The cultivation takes place ina suitable nutrient medium cmtaming carbon and nitrogen sources and inorganic salts, wag procedures known in the art. Suitable media ace commercially availabb from commercial suppliers and may be prepared according to published COTpc4tions(ibr example, the catalog of American Tyie (Mure Collection). If the fusion protein is secreted mto the ruitrient medium, it not secreted, itcan be recovered fiom cell lysaies. The glycosylated fusimprctefooftnepresent irrveon'onniaybereaweiedusinganyoneofa number of methods far isolating a polypeptide, which are known in the art For example, the rttly\|)eptidte may be recovenxi from limited to, centrifiigation, filtration, extraction, spray dying, e Further, die polypeptide may be purified by a variety cfptocediires known rathe art incluc chratnatography (e.g, ion exchange, affinity, hydrophobicity, and size exclusion), dedrqphoresis, differential solubility (e.g, ammonium suHate precrpttation), SDS-PAGE, cr extraction. In a further aect, the present invention provides a pharmaceutical composition comprising the glycosylated fosmprotein acosdrngtotepresertiaventioa The term 'treatment1 ', as used herein, refers to a perfect cure, suppression or alleviation of diseases or disorders. Therefore, the torn 'IrterapeiidcaUy effective anwu amount sufficient to achieve the above pharmaceutical effect In the present invention, the merapeuticalh/ effective amount may vary acxxnxlingtDfte fonnulatkmmdbods, administration modes, patienf s age, weight and gender, severity of the illness, diets, administration duration, adrrrinistraticm routes, rates, and res Thoseskilled inthe art may readily detomineand prescribe a dosage capable of achieving a desired treatenent phamiacutieda»nrx) A gjycosylated CTLA4-Ig fijsbn protein as an emrxxiiment of the present invention is applicable to diseases against which it displays therapeutic effects by inhibiting the action of T-celk, for example, autoimmune diseases such as arthritis or psoriasis, various organ transplants including bone marrow transplants, and varicose veins. Also, fosion proteins with receptors for various cancer-associated cell growth factora may be used in the treatment of cancer because they have improved fh^ due to their effects of increasing serum levels of the recerMnrsandbkxddngangbgenicfec^ors. The carrier used in the pharmaceutical composition of the present invention includes the commonly used carriers, adjuvants and vehicles, in the pharmaceutical field, which are as a whole called 'rjharmaceun'calfy acceptable carriers". Non-limiting pharmaceutically acceptable carriers iiscMmtrKpharrriaceuticalcomposh aluminum stearate, lecithin, serum proteins (e.g, human serum albumin), buffering agents (e.g., sodium phosphate, ycine, sc acid, potassium sorbate, partial glyceride mixtures crfsaturated vegetable fefly acids), water, sate or electrolytes (e, protamine sulfate, disodium hydrophosphate, potassium hydrophoshate, sodium chloride, and zinc salts), colloidal silica, magnesium trisQicate, poryvinvltyytR^done, cefiulose4»sed substrates, poryerhylene glycol, sodium carbojQmethylceMose, pofyarylale, wajasi, \|xrfyerrrylenei)oryt»(ypropylefle coporymers, polyethylene glycol, and wool faL The TJbannacxvdc ODQiposition of me present invention may be administered topically, parenteraDy, intraoculariy, transdermally, inftarectally and intrahrminally, and may be formulated into solutions, suspensions, and the like. The term "parenteral"., as used herein, includes subcutaneous, intranasal, intravenous, intraperitoneal, intramuscular, rate-oracular, intra-synovial, intrastemal, intnacardtal, inrrarhecal, mtraleskmal and intracranial injectkxi or raftisiontecriniques. In one aspect, the r±annaceutieal composition ctflhepreKirtmvaaionnMybe aqueous solutions fi)r parenteral admmistration. fteferabfy, a suitable buffer solution, such as Hank's solution, Ringer's solution or physfokcalh/buflered salir Aqueous injection suspensions may be supplemented with subancescpabk of increasing i s c t y of suspens which arc exemplified by sodium carboxymethylceMose, sorbitol and dexJrm In addition, suspensions of the active components, such as oily injecticm suspension, earners, vAach are exemplified by fety e%loleate,1nyoetidesorlrposoines. Porycationic non-Iipid amino polymers may also be used as vehicles. Optionally, the suspensions may contain suitable stabilizers or drugs to increase the solubility of components and obtain high concentrations of the components. The phannaceutical composition of the present invmtica is preferably in ttefonm of a sterile injectaWeprqjaratic^ such as a stenleinjectable aqueous OT Such suspension may be formulated according to the methods knovm in the art, using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparations may also be a sterieiqedabtesohitionOT suspension ma a solution in U-butanediol. The acceptable vehicles and solvents include rnacnitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or di-glycerides. In addition, ferry acids, such as oleic acid and glyceride derivatives thereof, may be used in the preparation of injectable preparations, like the pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), and particularly, polyoxvethylated derivatives thereof. The atenentioned aqueous cornposhion is sterilized mainly by filtration using a filter to remove bacteria, mixing with disinfectants or in combination with radiation. The sterilized composition can be hardened, for example, by fieezfMhying to obtain a hardened product, and for practical use, the haidendcofflposto to oixkr to increase ability at room temperature, rd temperature, and prolong shelf-life, the pharmaceutical composition of the present invention may be lyophflized A process for fieezeKhying may drying. After freezing, the composition is heated under pressure to evaporate the water. At the second drying step, residual water is rmrovdfrcm the dry product Hie daily effective dosage of the pharmaceutical composition according to the present mventionistjpkyabcxjtlOpgtoato 300 pg per lglxxfywghi,( most pirfera It wfflbeappai^tothoseskiMmtbeartto be administered to a patient imy vary depend desired biotogjcal activity, thepatient's synqjtoms and drug resistance A better understanding of the present invention may be obtained through the following examples. EwfflbsapjjaraitotlMseskiMmthearttete to illuarate the pnoitinvon, and the scope of the present invention Containing the 3' end of the soluble extracellular domain of CILA4 and i IMA encoding a fusion protein in which IgG Fc is linked to the solubk Fuston protein in vidi IgG Fc is linked to foe soU* extracellular domain 01 EXAMPLE 1 A. Preparation of DNA fragment encoding soluble extracellular domain of CTLA4 A DNA fiagment encoding a soluble extracellular dcartam of CII^^ usingapionter(SB5IDNO: 1) having a lecognirion sequence of a restriction enzyme, EcoRI, and a coding sequence fiir a leader sequence of CILA4, and anrjthertairner (SEQ ID NO: 2) having aPstt recognition sequence and an antisensesequoKeco ofCTLA4. AcDNAte^lateinuIIlwasixqbyrever readion(RT-PCR) using mKNA extract mRNA was isolated using a Tri-Reagent mRNA isolation lot (MRC, USA). Hist, 2xl07 human T lymphocytes were washed with phosphate buffered saline (PBS, pH 72) three times and lysed with 1 ml Tri-Reagent by repetitive pipetting. The cdllysate was mixed with 0.2 ml chloroform by vigorous shaking, allowed to stand at room temperate fro 15 mm, and on^ min. The supernatant was transferred to a 1.5-ml tube, mixed with 05 ml isopropanol, and centri&ged at 15,000 ipm at 4°C fix 15 min. After die supernatant was discarded, the pellet was washed with 1 ml of triple-distilled water (TDW) treated with 75% ethanol-25% DEPC The tube was inverted twice or three times and centrimged at 15,000 rpm at 4°C for 15 min. After the supernatant was completely removed, the KNA pellet was afrniried to remove imiaining dissolved in 50 ulDEPC-treated TDW. B. Preparation of DNA fragment encoding Fc region of IgGl A DNA fragment encoding an Fc region of IgGl was prepared by PCR using aprimer(SEQ ID NO: 3) having a Psfl recognition sequence and a sequence coding tor a 5' end of IgGl Fc, and anofoer primer (SEQ D NO: 4) having an Xbal recognition sequence and an antisense sequence coding fcr a 3'end of IgGl Fc. A cDNA template in the PCR was prepared by RT-PCR using mRNA extracted from peripheral Mood cells (B fyn^feocyte) of patients origin, who were in recovery. RT-PCR was earned out using fce same reagents under the same conditions as in Example 1, part A C Preparation of gene encoding non-glvcosvlated CTLA4-IgG The DNA fragment encoding the soluble extracellular domain of CTLA4 and the DNA fragment encoding the Fc region of IgGl were digested with Pstt and ligated using T4 DNA ligase. The ligated DNA contained a leader sapience to facilitate protefe The fusion gene fiagment thus prodixwas digested wirnBWarri sites of pBluescript KS H(+) (Steatagene, USA), "which is a omrniodaDy available doning vector. The whole coding region was identified by DMA squeacing (SEQ ID NO: 7). A iusion protein expressed fiom the fusion gene was designated "CTLA^IgG", whose predicted amino acid sequence is represented by SEQ H) NO: 8. EXAMPLE 2: Preparation of glycosylated CTLA4-IgGMonproteins In older to introduce a glyeosylatkmn^intofte^ nudeotide sequence containing a mutation leading to an amino add substitution were prepared as follows: imhenudeotide sequence ofSEQ ID NO: 5, a 478480 ccxion(ATG, Met) w^ replaced wife AAC (Asa, NX a 583-585 codon (GCC, Ala) wife AAC (Asa, N), a 727-729 codon (ACC, Tk)wife AAC (Asa, N), a 793-795 codon (GAG, Glu) wife AAC (Asn, N), a 895-897 codon (TAG, I» wife ACC (Ik, TU991-993 codon (TTC, Phe) wife ACC (Ik, T), and a 1036-1038 codon (GAG, Gta) wife AAC (Asn, N). Biformationoiifeese primers is given in Table 3, below. Qycosyiated &SIQJI pKrtebi uf the presera feveniion woe prepared by KIR using die cloning vector carrying CTLA4~hIgG"Coding DMA, prepared in Example 1, as a template, and la detail, each gfecosykted fiisionprafcdn was prepared as Mows, (1) CTLAWi^Krl (Gl varianf): ooe ^ycaqtefiainjotuf was cre^ed usingapimer (SEQ E) NO: 9) designed to have a iradeotkte sapie««cantein^ 480midec^des(ATG,Mrt)i)oatJcarfatteI^ie^ (2) CTlA44ifeG (SEQIDNOS: 12 and H) designed to lve nu:l«)tide s^uences conla (Asa,N)and AC (Ihr, 1) for 793-795 nudeolides (GAG, Glu) md 895^97 nudeotidesCTAC, Tj4 rcspecthdy,positioDcdattheFcregicnofIgG(SEQroNa 5). (3) CTIA44iC5-G3 (G3 variant): two gfycosylation motifs were created using primers (SEQ ID NOS: 13 and 14) designed to have nudeotide sequences containing substitutions of ACC (Ik, T) and ACC (Ik, T) for 895-897 nudeotides (TAG, Tyr) and 991-993 niicleoticles (ITC, Phe), re^ecthdy.poalksidatlheFcrcgonof IgG (SEQ ID NO: 5). (4) CTLA4-hIgG-G4 (04 variant): three glycosylation motifs were created using primers (SEQ ID NOS: 9,12 and 13) designed to have nudeotide sequaK^ owMiing substitutions of AAC (Asn,N), AAC (Asn,N) and ACC (Ik, T) for 478480 mdeotides (ATG, Met), 793-795 nudeotides (GAG, Glu) and 895-897 nudeotides (TAG, Tyr), respectively, positioned at the Fc region of IgG (SEQDNO:5). (5) CTtA4-hIgG"G5 (G5 variant): four gjycosylation motife woe created uang primers (SEQ ID NOS: 9,12,13 and 14) designed to have nudeotide sequences containing substitutions of AAC (Asn, NX AAC (Asn, N), ACC (Tk, 1) and ACC (Ik, 1) for 478480 nudeotides (ATG, Met), 793-795 nudeotides (GAG, Ghi), 895-897 nudeotides (TAG, Tyr) and 991-993 nudeotides (ITC, Pte), respectivdy, positioned at the Fc rcgbn of IgG (SEQ ID NO: 5), (6) CTLA4-h^G-G6 (G6 variant): five grycosyJation motifs were created using primers (SEQ ID NOS: 9,10,12,13 and 14) designed to have nudec^ sequence contain AAC (Asn, N), AAC (Asn, N), AAC (Asn, NX ACC (Tfar, T) and ACC (Thr, T) for 478-480 nudeotides (ATG, Met), 583-585 nudeotides (GCC, AlaX 793-795 nudeotides (GAG, GkX 895-897 nudeotides (TAG, Tyr) and 991-993 nudeotides (TTC, Phe), respectivdy, positioned at fee Fc region of IgG (SEQ ID NO: 5). (7) CTLA (G7 variart): six grycom ID NOS: 9,10,11,12,13 and 14) designed to have nudeotide sequences containing substitutions of AAC (Asn, N), AAC (Asn, N), AAC (Asn, N), AAC (Asn, N), ACC (Ik, T) and ACC (Ik, T) for 478480 nudeotides (ATG, Met), 583-585 nudeotides (GCC, Ala), 727-729 793-795 nudeotides (GAG, Giu), 895-897 nudeotides (TAG, Tyr) and 991-993 nucleotides (FTC, Phe), Jfcspectively, positioned atthe Fc region of IgG (SEQ ID NO: 5). (8) CTlA44iIgG-G8 (G8 variant): seven gjycosylation motifs were created using primers (SEQ ID NOS: 9, 10, 11, 12,13, 14 and 15) deagned to have nudeotide sequences containing substitutions of AAC (Asn, N), AAC (Asn, N), AAC (Asn,N), AAC (Asn,N), ACC (Thr, T), ACC (Thr, T) and AAC (Asn, N) for 478480 nucleotides (ATG, Met), 583-585 nucleotides (GCC, Ala), 727-729 nudeotides (ACC, Thr), 793-795 nucleotides (GAG, Glu), 895-897 nudeotides (TAC, Tyr), 991-993 nucleotides (TTC, Phe) and 1036-1038 nudeotides (CAG, Gto), respectively, positioned at the FcregionoflgG(SEQIDNO: 5). The PCR was earned out as follows. To a PCR tube, 1 ul of CTLA4-hJgG DNA C22ng), 125 U Pfii DNA polymerase (Sttatagene, USA), 4U Pfo DNA Hgase (Stratagene, USAX 1 ul of lOx reaction bufier for PfiiDNAligase, 1 u!ofeadiprimer(10pMXand2ulofdNTP(each 10mM)were added, and triple distilfcd water wasadded to a final volume of 20 ul PCR conditions included two cycles of 3 min at 94°C, 1 min at 61°C and 1 rain at 65°C, and then 29 cyctesoflininat940C;iminat 61and7mmat65,fellwbyfelcfigaticnat65 The PCR products thus obtained woe subjected to sequence analysis to determine whether a glycosylation motif was successfully inserted EXAMPLES A Expression and purification of glycosylated CTLA4-IgG fusion proteins To express glycosylated dIA4-IgG fusion proteins in Chinese hamster ovary K-l cells (CHO-K1, ATCC CCL-61, Ovary, Chinese hamster Crteetvbs giseus), pBhiescrt KB E(+) pksmid DNA containing a CTLA4-hIgG fusion gene into which a glycosylation motif was inserted was isolated from transformed £ coli, and digested whh EcoRl and XbaL The thus-obtained CTLA4- hlgG fusion gene was inserted into BcoRI/Xbal sites of an animal expression vector, pCR™ 3 (fovitrogen, USA). TTie resulting expression vectors were designated as rX^4IgX32 to G8 plasroids. Among them, fte pCT4Ig-G2 rccombinant expression vector was deposited at the Korean Culture Gaiter of MtoocMg^^ assigr^aaxssicrarjumberKCCM 10572. B.Transfection and evaluation of expression of fosion genes Chinese hamster ovary K-l cells (CHOK1) were plated onto six-well tissue culture plates (Nunc, USA) at adensity of l-3x!05 cdls per well, and were grown to a 50-80% confluency in 10% FBS^»i^ainingDMEMmediurn. ma serum-free DMEM, lX2\|agDNAofanyoneofpCT4Ig-G2to G8 plasmids was mixed whh 2-25 jjl of lipofectamine (Gibco BRL, USA), and incubated at room temperature tor 15*45 min to form DNA-liposome complexes. Then, the resulting complex was added to the six-well plates. After an incubation period of 5 hrs, the cells were refbdwim 20% FBScontakung DMEM medium and further cultured for 18-24 hrs. Thereafter, the cdls were cultured in 10% FBSx»ntaining DMEM supplemented wife 3 mg/ml genetkan (G418, Gibco BRL, USA) for three weeks. Formedcolonies were selected and isolated, and men propagated. Whether or not a fusion gene was expressed was evaluated by ELJSA using peroxidaselabefcd goat anti-human IgG (KPL, USA). EUSA was carried out as follows. First, 1 mg/ml of goat antHiuman IgG was diluted to 1^2000 wMi 0.1 M sodium bicarbcaale, and 1001 of flw diluent was aliquotted into a 96-wdl flexible plate OFalcon, USA). After being sealed withsaran wrap, the plate was incubated at 4°C for over 16 hrs to allow the bottom of tie plate to be coated with the antibody. Thea, fee plate was washed tec times with a washing buflfer (Ix 0.1% Tween-2 containing phosphate bufife13 ml BBS, 50 ulTween-20) was added to each well 20 )il of a culture supernatant \vasadded to the first well and serially diluted using a ndcropipette. 0.01 ug/ul of human IgG (Sigma, USA) as a positive contol and a culture fluid of non-transfectedCHO-Klc the test sample. Alter diluttoos were completed, the 96-wdl flexible plate (Falcon, USA) was wrapped wife foil, incubated at 37C for 1 hr 30 min and washed with the washing biiflthree times. Peroxidas»4abefcd goat anti-human IgG (KPL, USA) was dih^to 1^000 wife tie diliientbufer, and 100 ul of the diluent was added to each well, wrapped with foil and incubated at 37°C fear one hair. Afler the reaction was completed, the plate was devdoped with a TMBnriooweUperoxidase substrate system (KPL, USA). Ahsorbance was measured at 630 nm using a microplate reader (Bio-RAD, Model 550 Japan) to determine whether a fusion gene was espessed and teejipession levels of flie fusion gene(FEG.2> As shown in FIG, 2, the 01 variant was expressed in the highest levels, followed by G2.G4, GO and G3 variants. The G5,G6,G7 and G8 variants were found to be latety expressed EXAMHJE4 A. Western blot analysis An expressed protein was purified by inmjinwprecipitato Fast, 50 ul of protein A-Sephatose beads were placed intDaU^tube,mkdv^l{X)uiofbufferA (O.WMbc3ricadd94MNaQpH9.0),aiKloantri&gedatl3,000 Aflerthe supernatant was discarded, this step vvas repeated tiaee times. Eadi protein sarajde was mkedwifh fie equUihated protein A-Sepharose beads and incubated at 4°C for 3 hrs wim rotation to induce binding. Then, theieaction mixtme was oentri%ed at 13,000 rpm, and the beads were washed vvhh buffer A three times. The beadswere mixed wifli 20 ul of bufierB (OJ35 M sodium phosphate, 0.05 protein sample was mixed with 5x buffer containing 5% p-mercaptoethanol, boiled for 5 min, and subjected to reduced SDS-PAGE A 3.5% Acrylamide gel (0.5 M TOs-HCl (pH 6.8), 0.4% SDS) was used asastedcing gel, and a 10% Acrylamide gel (15 M Tiis-HCl Oil 8.8), 0.4% SDS) was used as a running gel. After dectrophoresis, proteins were dectro-transferred onto a 0.4-pm Westran (PVDFtrarisfermeinbiane,S^fbr2hrsat350mA The blot was blocked with 5% skimmffle for 1 hr. After being washed with washing buffer (0.1% Tween-20, lx phosphate buffered saline) three times, the Wot was incubated in a 12000 dilution of peroxidase-labeled goat anti-human IgG (KPL, USA) for 1 k The blot was washed with washing buffer three times, and developed at room temperature fcr 10 mm with 15 ml of a coloring agent, which was inadeaccc^g to a recommended iemetlxxJ using a DAB substrate kit (VE The reaction was taminatedwifcti^te-distilledwato The results are grvea in HO. 3. EXAMPLE 5: Measurement of serum talf-lrvesofglyoosylatoiC^^ Serum half-lives of gjycosylated CTLA4-hIgG fusion proteins were measured in mice as follows. Each fusion protein was intraperitoneally injected into mice (ICR, Samtako Inc., Korea) in a doseof02mg/kg, BtocxJ samples were ollec at grvra tax concentrations were detemiinedaccoix 4). As diown in FIG. 4, the G2, G3 and 04 variants had iiweasedsenm levels, whereas the 01 variant displayed reduced blood circulation time compared to the wild type. In particular, the G2 variant exhibited the highest drculationtime. Industrial Applicability As described hereinbefore, the glycosylated fusion pxtoisacconlkto the present invention ate able to reduce dosage and administration frequency in clinical applications because they have high in vivo stability. We claim: 1. A glycosylated immunoglobulin G or a Fc portion thereof, wherein the glycosylated immunoglobulin G is generated by additionally glycosylating a variant of immunoglobulin G in which a methionine residue at a 160th position from an N-terminus of a mature wild-type immunoglobulin G is replaced with asparagines (M160N), a glutamic acid residue at a 265th position therefrom is replaced with asparagines(E265N), a Tyrosine residue at a 299th position therefrom is replaced with Threonine(Y229T), and a Phenylalanine residue at a 331th position therefrom is replaced with Threonine(F331T). 2. The glycosylated immunoglobulin G or the fragment thereof as claimed in claim 1, wherein the immunoglobulin G variant comprises the amino acid sequence of SEQ ID NO: 19.

Full Text

Technical Field
The present invention relates to a glyeosykted immunoglobulin and an inmmoadhesn
conpiang the same. More partieulariy, the present invention idatK to an imraimog|Dbulin or a
fragment theirot; \viiich is additional!}-gl)iylatedty
andaglyojsyiaidfum
a ferment thereofwifli atlcaatone biologically active pK or a portion thereofi
BattotMindArt
Imminwadhesins (or immunoglobulin fusion proteins) ate antibody-like molecules
lesuiting fiom the fuskm of a foig
region of a receptor or an adhesive mokoile. Tr typicdirmnunoadhednskm\vnintbearthave
file stiucture of an antibody in which the variable region, participating in antigen recognition, is
replacdwthaUgandmdir For a long time, a
large nurriber of patents have described fosioD piotdnsinv in which is nofaphysiok cally
active protein is linked to an antibody (US. Pat Nos. 5,521,288,5M095,6,046310, 09014,
6,100383 and 6^25,448),
The immunoadbesin has the Mowing advantages over a molecule not containing an
1) toe fusion protein has increased tota!avi%toalipdbecauseithashivabKymadin^
form;
2) the fusion protein is preset
virtue of increased molecular stability;
3) effector cdJs ate activated by flic Fc (Fragment oystallizabfe) portion of the
immunoglobuJin heavy chain; and
4) the fusion protein is isolated and purified by a convenient method, for example, using
protein A
For example^ hthecastt of tutOT
a cytokine, to suppress TNF-dependent inflammation responses, tumor necrosis factor receptor
(hereinafter, referred to simply as "TNFR") may be used as described in POT Publication Nos.
W092/16221 and W095&4326, or a TNFR-imimmoglobuIin(Ig) fason protein way be used as
described in US. Pat Nos 5,447,851 and PCT PiMcation No. W094A)6476. Accordmg to
numerous reports, IM^-IgMonpxrte^
form or Ig«»-iused form of TNFR (Lessiauer W. al E«r. J. tattminoL, 1991, vol21, jx2883;
A&ssam A. et aL PNAS USA, 1991, volSS, p.10535; Pefjpd K. et al J. Exp. Med^ 1991, vol.174,
p,I483; Matter KM ctal. J, bmurjoL, 1993, vol.151, p.1548).
With respect to the inhMm of TNFadcn or the control of in
^on protein, aimMvaleot (vmume
TOT receptors, CD2 and CTLA4 in an Ig fusion construct is expected to improve the efficacy of the
fusion construct \K a mairaiiBdc fusion
extracellular domain and the Ig heavy chain is expressed in a cell line simultaneously with another
mooornerk Mon ptotdn Qigjbt
Igligtacham, a dirtOTcfusicto praxis pro
light chain. Thectmericfiiskttr
form, and has remariyirxxcascd efficacy in ccrapar
B J. et al CytDkine, 1995, wL7,pJ59).
However, such an Ig fusion protein ina dimericfonn is dfficuhto mduslrialize due to Ihe
fdBowu problems: two genes which are indiviWyfusd to the Ig heavy and
co-introduced into a host cell; when two different fusion proteins are simultaneously expressed in a
chain fosionproteiris do iwtpartidpatem the fcmationo
protein and a light chain fusion protien are technically difficult to isolate from a mixture with the
moromerb heavy cMiftskm
In this legaixi, the present inventor
of the soluble domain of a biologically active protein is linked to an N-tenninus of the soluble domain
crfm identical ccdhlffleniMologic Also, the
present inventors prepared a DNA construct encodadimerbpiotehmctwomdeculesofa
mwioinericproteffljm
the hinge region of an immunogbbulin JFc fragment, ace disuifide-bonded at the hinge region, and
pnxkaoMaiamcr-MdojmericfiisionpTotdnu
DNA construct
As described above, attempts have been made to improve the efficacy and preparation
method of immunoglobulin fusion proteins, but almost all efforts have been unable to increase the
stability of the iminunoobulinfuskm proteins, h to regard, as disclosed in Korean Pat Application
No. 2002-&W5921, the present im
glycosyMon mothe to a conjunction region between a functional domain of a protein and an
immunoglobulin Fc region. However, vdren an imrflunoadhesin is gccylatd near a fiMonal
Disclosure of the Invention
In this regari, the present mvato
an additional glycosylatian motif into an immurwglobulin, particularly an Fc portion, of an
Hnrrttnwgtabulinfusm
period of time than the fonn not containing a glycosylation motif, thereby leading to the present
invention.
Thus, in one aspect, the present invention provides a glycosylated immunogiobulin or a
fragment thereof, in which an immunoglobulin variant comprising one or more amino add
modifications selected fiom the group consisting of M16QN, A195N, T243N, E265N, Y299T, F331T
and Q346N, is additionally grycosylaled
In another aspect, the present invention provides a DNA encoding a gtycosyiated
inmiurwglobvdm or a iragmerrt thereof, k \vhich an irrmii^
amino acid modifications selected from the group consisting of Ml SON, A195N, T243N, E265N,
Y299T, F331T andQ346N, is additionally glycosylated.
In a further aspect, the present invention provides a glyrasylated to
result of Mage of (a) a gfycosykted irnmunoglobulin or a fiagment thereof, in v^dch an
irnmunogjobulm variant having a nooolfied anrino acid seqpu
sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion
thereof
In yet another aspect, flie present invention provides a DNA molecule encoding a
glycosylated fosionprotein, which is Sained as aresuft of linkage of (a) agl
or a fiagment (hereof, in which an iniraurwgtobulm variant
filming one or more Asn-X-Ser/Tbr sequences is additionally glycosylated, with (b) at least one
biologically activeprotein OTarjortioa thereof.
In still another aspect, the present invention provides a recombinant expression vector
comprising a DMA molecule encoding a glycosylated fusion protein, which is Ibrmed as a result of
linkage of (a) a glycosylated immunogfobulin or a fragment thereof; in which an immiinogiobulin
variant having a modified amino add sequence forming one or more Asn-X-Ser/Thr sequences is
additionally glycosylated, with (b) at least one biologically active protein or a portion thereof
In still another aspect, the present invention provides a host ceD transfer or tansfbnned
with a reccrabinant expression vector comprising a DNA molecule encoding a glycosylated fusion
protein, which is formed as a result of linkage of (a) a glycosylated immiinogiobulin or a fragment
1rm)CmwrManiraiminogfo
more Aai-X-Ser/Trrr sequences is additionally glycosylated, with (b) at least one biologically active
protein or a portion thereof
Li still another aspect, the present invention provides a method of preparing a glycosylated
fusion protein, comprising cufcuring a host cell transfected or transformed with a recombinant
expression vector OMnpri^
as a result of linkage of (a) a glycosylated iramunogjobulin or a fragment thereof in which an
imrrnincglobulmvariartkvinga
sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion
In still another aspect, the present invention provides a pharmaceutical composition
omprisiiigaglycosyla
irrimunogltobiilkOT
add sequence forming one or more AsnrX-likir/Itesecperm
least one biologically active protein or a portion thereof
Brief Description of the Drawings
The above and other objects, features, and other advantages of the preset invention will be
mote clearfy understood fiom the following detailed description taken in conjunction with the
accompanying drawings, in which:
FIG. 1 ^wsglycosyMcm sites of immunoglobulinsacconiing to the present
FIG. 2 is a graph showing expression levels of glycosylated CTLA4-IgG fusion proteins
according to the present invention;
FIG. 3 is a graph showing the results of Western blotting of glycosylated CTLA4-IgG
fusion proteins aaxwding to the present invention; and
FIG. 4 is a graph showing changes over time in serum levels of glycosylated CTLA4-IgG
fusion proteins according to the present invention, in mice intraperitoneally injected with the fusion
proteins.
Best Mode fbrCanymg Out the Invention
Single capital letters representing amino acids, as used herein, represent the following amino
acids according to the standard abbreviation defined by the tatemati^
A:Alamne;B:AsparagineorAsparaticacid;
C: Cysteine; D: Asparaticacid; E: Gluiarnicacid;
F: Phenylalanine; G: Grycine; tt Histidine;
I: Isoleucine; K: Lysine; L: Leucine; M MetWonine;
N: Aspargine; P: Proline; Q: Glutemine; R: Argmine;
S: Serine; T: Threonme; V: Valine; W: Tryptophan;
Y: Tyrosine; and Z: Gluramine or Glutamic acid.
7/37
Tlw designation "(out capital fur an amino addXamiiw acid posttionXone capital for another
m
& the designated amino add position of a given proteui For example, M179N indicates that die
mefoiorairei^due at the 179th The
aminoaddpositicraisfflBit
TJtelimMglycQsyJafi
host are nwdified by the attadsnoof sugar chains. Tte attachment of sugar chassis known to
ei^ There are two
types of glycosyialinn. OJinlned gJycosj'lation Hulas an oligosoccharidc chain to a saiue and/ur
tbreoninfi residue. N-linkdgljw^iatiOT links moHgosaccharide chain to an as^^ In
pffltkolai; N4infced gjfjw^^
amino add exduding proline).
k the present hventk)n,aDNA sequence OTcodingmi^^
© imitated at one or more nudeotidea to form an additional dycosylatiun site at wWdi O-Hnked or Nlinked
glycos'kioii occurs, and tile mutated DNA is expressed in a host cdl to dUow spontaneous
gjycosjtoioffl. fe(fflcaped,ax)syliiniin
pxsetA invention is constructed by mutating a DNA sequence encoding an immunoglohuHn or a
ftagpent teecrfto add aod/cr Snas M
finked wsylatko occurs,
The '%nmunoglobulinsbich are modified to possess a ycosykkxi motif in the present
invention, at piotefonralecales to are produced in B cefls and sow as antigen iwitoBSpecaficalfy
rexagoizi a vwde variety of antigens. The moleades hsve a Y-sfa^ed strarture consislingoftwo
identical light chains (I chains) and two kJeoW heavy chains (Hc
hddlosrfrabyanumbofdisulfidebc
8/37
hinge region The L and H(toisc«ipisevariafaieaiKl constant regbiis. According to features of
the coBtant regions otH chains, iramunoglobuliris (Ig) are classified into five isotypes, A (IgA), D
(IgD), E ( G dgG) and M OgM). The five subtypes possess unique stractural and biological
properties. These inrnunogfobulinsn
Since an imnumoadhesingaiaaDy contains a figment of
R portion a gtyeosryl
an immunoglobuJk The torn "Fc portion of an immunogiobulin" as used herein, refers to a
fragment having no antigen-binding activity and being easily crystallized, which comprises a binge
region and CH2 and CH3 domains, and a portion responsible for binding of an antibody to effector
materials and cells.
fa the present invention, a glyc
amino acid residues at positions 160,195,243,265,299,331 and 346 of an immunogiobulin (all of
theseanano acid residues are present attheFcpe8tionofanimmunogkibu3in). Thus, in one aspect,
the presertrmm provides a g j y s y
uoe or mure amino acid modifications selected from the group consisting of M160N, A195N, 1243N,
E265N, Y299T, B31T and Q346N, and a gene encoding the same, In more detail, a glycosylated
immunogiobulin or a ftagmenttbereof listed in Table 1 is provided, which contains oombinatiom of
one or more of the aforementioned amino acid modifications.
(Table Removed)
In another aspect, the present invention provides a glycosylated fusion protein formed as a
result of linkage of (a) a glycosylated immunoglobulin or a fragment thereof, in which an
imimnwgfobulmvariart
sequences is additionally glycosylated, with (b) at least one biologically active protein or a portion
thereof b a preferred aspect, the fragment of m irmnuiiogbbulin includes aii Fc portion, and the
rxjrtionofabiotogicallyacriwprotem
In one aspect, the glycosylated fusion protein has a monomer structure in which a angle
polypeptide is loaned as a result of linkage of (a) a glycusylalcd uiiuiuuoglobulin or a fiagmeot thereof
m \vhkhmimisnjnoglobulm
X-Ser/Ik sequences is additbnally ycosylated, with (b) at least one bMogically active protean or a
portion fliereof, Therc«ofabiolcgkyactrvpx3temprd(erablyind
ctornain of biologically active proteia T\vo rnoleoiles cf such a monomeric glycosjla^ rusiOT
protein n be Msedbyao^sulfictebcMKl at the h i r n
In ar«^ aspect, the glycosylatd fusion protein has a nx
polypeptide is fooned as a resutt of linkage, in a concatameric form, of (a) a $ycosyiated
acid sequence forming one or more AsnrX-o/Ik Kquaffies is a^iticually gjycosylated, wifli (b) a
first biologically active protean or a portion thereof and (c) a second biologically active protein or a
portion thereof. The first and second biologically active pioteteimy be kfentkalcff differed The
portion of a biologically active protein preferably includes a soluble extracellular domain of the
biologically active protein. Two molecules of such a monomeric glycosyta^ftscra protein may be
linked byadisulfidetorKi at the bing
MapcdienedasDectoftheglvc
immunoglobulin variant comprises one or more arrano acid modifications selected from the group
consisting of M160N, A195N, T243N, E265N, Y299T, E331T and Q346N and is giyeosylated. In a
more preferred aspect, foe imroiunQgkbulhvaiiMcorr
SEQIDNO: 16toSEQE)NO: 23, and m^ most pdkred aspect, ttearninoactt)NO: 19.
The term "biologically active protean" as used herein, refers to a protein, peptide or
poryr^ptidermvmggeneralfyph
activities after forming an imraunoadbean The term "biological activity", as used herein, is not
limited in meaning to physiological or pharmaceutical activities. For example, some
irarmmoadheans, suchasthose containing an enzyme,
Non-limiting examples of the protein, peptide or polypeptide include hemoglobin, serum
proteins (e.g., blood factors including factor VE, VSR and factor IX), immunoglobulin, cytokines (e.g
interleuldn), a-, p- and y-interferons, cotorry-stirnulating fectars (e.g., G^F and GM-CSFX plateletderived
growth fector (PDGF), aand phosphoKpase activating proteins CPLAft). Ofcer typical
biological or therapeutic proteins include insulin, plant proteins (eg., lectin and ricinX tumor necrosis
fector ffNF) and its mutant alldes, growftj fectors (e.g., tissue growth fictoxs and endothdial growto
iactars such as TOFa or TGFp), hormones (e.g, follicle-stimulating hormone,
11/37
JMtmone, atitidiuretic hormone, pignieirt'C«K!eirfrating or disposing hormones and parathyroid
hormone, luteinizinghoiinooeHrieleasii^bxMmoneand its derivatives), cddtonin, caldtonia gene related
peptide (OGKP), synthetic erfcephaln, somatornedin, eryteopoietin, hypotfialamus releasing fectars,
prolactin, chronic gonadotrophin, tissue plasminogoi-acti\'ating agents, growth tormooe-rdeasing
peptide (QHRP^ai^%mKhiincial6ctor(THF). Son«raotdmsuchasinterleukin,inteffecdonytiniulating fector may be produced in a ncn-glvcosylated form by using DMA recombmart
tecteiques. The nonycosylated proteins may be useful as bbbgically active materials in the
In addition, the biologically active materials useful in the present invention include any part of
a polypcptidc, which lias bkactivity in vivo. Examples uf lljc bbbgically active materials include
peptides or polypeptides, fiagments of an antibody, single chain-binding proteins (see, U.S, Pat No.
), binding molecules including fusion polypeptides of antibodies or their iagments,
polydonal antibodies, moiodonal antibodies, and catalytic jmbbodies. Other examples of the
biologically active materials include aUagen pioteans, as lagweed, antigen E» honeybee vetwai, or
allergen of rates.
la addition the biologically active material useful in tExamples of the enzymes include caitetydrate-specifie enzymes, proteinases, oxidoreductases,
transfetases, bydtolases, lyases. ksneraaK, and ligases. In detail, NcmJimMng examples of the
endotoxinase, catalase, chymotrypsin, lipase, uncase, adenosioe dephospimtase, tyroane, and
biKrabm oxidase, Ejamples of the catbot^drate-specific eiKymes include ucose osdase,
Tlie term olubte estacdfular domam", as used bm refe to a portba exposed to tbe
extraceflular region of m intiegral membrane protein penetrating die cdl membrane comprising
12/37
bydrophilic auiiiw adds, which arc typically positioned at the surt
and thus is soluble in an aqueous enviramnent Of nwstcdlsur^recepta1 proteins, extracellular
domains serve to bind specific Kgands, vADe intraoeQular domains play an important rok in signal
transduction.
In one aspect, the gtycosylatod fusion protein according to (he present invention may be
pn^anxi by preparing a DN
modified to contain a glycosyiation ate and Mdng thereto another DNA sequence encoding a
I^k)gicaflyacdwpi»tebcraportim1hereo£ fo anofter asped, tbeycostjtedfiraonproteiQinay
be primed by primarily preparing a DNA sequence (fusion gaic) llrni aiuxks both an
immunooMin or a ftagnent tbsreof and a bbbgically active protein or a potion thereof and
mutating the fosion gene to aBowtte The
twopreparaonrnethoisdiffofiomeacho^iercxiryinte
and are basMy identical to preparatkm roedxxfe, k
protein variant Tbi^ bocanafiar, fee present iuveuliun intends to fcos a modifeato
an uraiunogtobulincrafi^ment thereof into \vh
A DNA setpence enooding tbe glycosylated immunogJobuJia or flie figment thereof
acceding to the present invertk» may te
These methods include, but ate not limited to, oligonuclwtidenediated mutagcnesis and cassette
jBUtagenesis.
In pardcul^,tkl^^ sequence oKoding the glycosylated
thereof accotding to, the present invtntion is preferably prepared by digonucleotkie-mediated
nattagcnesis. ThistechniqwisweUkrttwnhitheanddesa
aL Nua Ac. Res, USA, 1982, wLlO, pp.6487^500). fa brieg the ES4A sequenoe enooding fte
rthefiagme
a plasmid carrying DNA encoding a non-modified or native imiramoglobulin or a fragment
thereof) with an oBgonucleotide coding for a desired modification. After hybridization, a second
complete strand complementary to the DNA toplateraybesyn&esizedty
the second strand may code for ihe desired modification.
Typically, oiigonudeotides used in the aftmnmtioned mefexk ZM composed of about 25
oucleotides. Shorter oKgonudeondes can be employed, but oplHnaloligonucleotldes,atboaile£land
right regions of rnooedoxk) These
oligonucfeoddes can effectively hybridize with a template DMA. These oligonudeotides may be
synthesized by the technique USA, 1978,voL75,
p.5765
In one aspect, the present invention provides a DNA sequence encoding an inmnmoglobulin
or a fiagraent thereof, which carries one amino acid modification (IgG in Table 1). This DMA
sequence may be prepared by performing PCR using DNA encoding an immunoglobulin or its
fragment as a template and inodifiction-encoding synthetic oligonudeotides as primers, Primers
hybridize with tiieir complemertey sangie-stranded DNA produced by denataation of a doublebranded
DNA template during heating. DNA polymerase adds nucteotides to the 3'-OH of the
mcxfifiotion-encoded primer one by one in a mamer cornplernentary to a template in the S' to 3'
direction. ThenevdysynlheazedstiarKiirxwrpo
gene encoding a desh^mcxJificadon, TlKtvty synthesized sbaoi is used as a template DNAm the
extension step of PCR, resulting in ampEfication of a gene encoding the modification.
In another aspect, Ihe present awention provides a DNA sequence encoding an
.When two
or more amino acids to be modified are spaced dose to each other on a polypeptide, oil desired
modificanons are encoded in one oUgonucleotide and thus simultaneously achieved Therefore, a
mutated irnmunogtoboliaor a fragment thereof havirigtvvo cr more amino acid modifications may be
prepared by the same method used to prepare the mutated inmiunoobulin or fisgmeat thereof
amino add modifications as primers.
When two or more amino adds to be modified ate spaced far apart (rafhe case that over 10
amino adds are present between t\wamim acids to be n
encoded in one oligonudeotide. Thus, diflerent methods should be introduced One method is to
prepare indradual oligciriude When the oUgonudeoddes ate
annealed simultaneciusly to a single-stranded template DNA, a newly synthesized secondary singlestanded
DNA encodes all of the dearedaraino acid modificatwns. Another approach used in the
ppssert invention inctodes tw In the primaiy miflagenesis, using natural
DNA as a template, oieoligoni
the template, and thus bsteroduplex DNA is produced In 1he secondary naaagenesis, the
heteroduplex DNA is used as a template. The template atneady carries at least one modification.
When one oHgonudecjtideha an additwral amino add n
resulting DNA encodes both of the primary and secondary modifications.
The cassette mutagenesis is also a preferred method for thepjerMostira
gtycosyktedioimimoglcb This method is
tjasedonttetedmkiuedesAstartirmaterialisaplasm3d(oranoirjavecfiagtnent hereof to be modified The cassette rautagenesis is preferably used when a specific
itsm'dion enzyme ate is pre However, this is not essential. If
siHiiarestri(onerj2ymesitedoesaitexist,itcfflbeintr
oicodirjganimminx)gtobdm(wafimenttheie After a
restriction enzyme site is introduced into the plasmi4 tbs plasniid is lineari
restriction enzyme. A double-stianded oligonudeotide having a DNA sequence that contains a
desired mufatioa and is tocaid between reslri
method. The two strands are individually synthesized and hybridized using a common technique.
Such a dcwbJe-stranded oligomfcleotide is tvp The cassette should be
pijaj ta the fiMm of possessing 3'-arid
plasmid and may be thus diiectly conjugated to the plasnid Tlieplasmid comes to contain a DN A
encoding a desired gh/oosylated immunoglobuljn or a fragment thereof through the aforementioned
procedure.
In addition, the preparation of a DMA sequence encoding a gly(sylatedimmunoglabulinor
a fiagment thereof according to tte present invention rnay be achiev In
partioilar, such a DNA sequence may te
synthesizer. An oligonucleotide is made based on an amino add sequence of an glycosylated
iirapGttinaobulmorai
producing an glycosyisted immunobbiinorafiagnientmereof.
With respect to a DNA sequence encoding a glycosylated immunoglobulin or a fragment
thereof according to the present mvratici
amino add is specified by more ftancdon, is wdlkno\i Thus, there is apteality of
DNA sciences with degeneracy encoding a glycosylated mimunoglobuiin or a fiagment thereof
accoidmgtothepnamventb
Alternatively, the gfycosylated Mon protein according to the present invention may be
prepared as follows. A DNA sequence eoxxlingthefiiskHi protein ^isiemate^rirfaiedtoas^fiiaon
gene") is prepared, and is inserted into a vector including one or more expression control sequences
regulantheexrffesswnoftheM Hien,ahost
is transformed or fransfected with the resulting rccombinant expresskm vector. The resulting
trarsformantortramfectartisoj
16/37
oftte&siongene, A substantially pure gjtycosylated fusion protein coded by the Men gene is
recovered from the resulting culture.
The tarn "vector", as used herein, means a DMA molecule to saves as a vefaidb enable of
stably carrying exogeneous genes into host cells. To be useful in implication, a vector should be
leplicable, have a system fix- irtrociucing itself into aho
In addition, tie term "necxMnbinant expression vector", as used herein, refers to a circular
DNA molecule carrying exogeneous genes operably linked thereto to be expressed in a host cell
When introduced into a host cell, the recombinant expression vector has the ability to replicate
regardless of host chromosomal DMA at a high copy number and to produce heterogeneous DNA.
As generally known in the art, in order to increase expresskailevelofatransfededgeneinahost
cell, the geM should be operabfy linked to transcripticMi and
in the host cell selected as an expression system. Preferably, the expression regulation sequences and
the exogeneous genes may be earned in a single expression vector rontararng selectable
replication origin. Inthecasethateukarvc^ttDsateusedasan
vector should further comprise expiessimmarkfiis useful in tteeukatyot^
In order to express the DNA sequence (Le, fusion gene) encoding the glycosyiated fusion
protein according to the present invention, vanVws expression vectors may be employed. Preferably,
stnceanirmnunoglobulmorafra
eukaryotic host cells should be used Expression vectors use&l for eukaryotic host cells contain
expression control sequences derived from, for exampkiSV40, bovine papfflorravii^
cytomegalovirus. In detail, examples of tie vectasindiidepCDNA3J(+y
Calif, USA) and pChx» (Stcatagen, La Jolla, Calif, USA). Expression vectors useful tor yeasts
include 2|A plasmid and hs iso&nns, POTl vector (US. Pat No. 4,931,373) and pPICZ A, B, or C
(tavitrogen). Expressi(m vectors useful fijr insect orndirfepVL941,pBluebac4.5andpMelbac
(Invrtrogen). However, tw present invention is not limited to these examples.
17/37
Be term "expression control sequences", as used herein, refers to nucleotide sequences
reeiessaiyOT advantageous for expr Each
control sequence may be native or foreign to the fusion gene. Non-limiting examples of the
expression control sequences include leader sequences, polyadenylation sequences, propeptide
sequences, promoters, enhancers or upstream activating sequences, signal peptide sequences, and
transcription terminators.
fa order to express the fusion gene of fce present invention, any of the various expression
omtrolsequerm may te insetted Examples
of expression control sequences suitable for directing protein expression in mammalian cells include
SV40 and early and late promoters of adenovirus, MT-1 (metallothionein gene) promoter, human
cytomegalovirus immediate-early gene promoter (CMV), Rouse sarcoma virus (RSV) promoter, and
human ubiqmtinC(UbQ promoter. In addition, to irnprove expression levels in mammalian cells, a
synthetic intron may be inserted into the 5'-untranslated region of the fusion gene. Examples of
expression cartrdsequer^siritabte
promoter, PIO promoter, bacubvirus 39K delayed-early gene promoter and SV40 polyadeiiylalion
sequence. Examples of expression control sequences suitable for directing protein expression in
yeasts include the promoter of the yeast a-rnating system, yeast triose-jphospbate isomerase (TPI)
promote aodADH24c promoter. Examples of expression control seqimes suitable ifo directing
protein expression in fungal cells include ADH3 prontroterandtraninators.
lhe team "operabry linked" refeis to a state in which Aefuskm gene of the present invention
is arranged with such a control sequence in a functional relationship. That is, a gene and control
sequences are linked in sudh a manner
(e.& transcnoti-vating-'protein) binds to the control sequences). For example, when a presequence
or secretory leader facilitates secretion of a maojre profit is rcfen
to the axling sequence of the protein. A pratncto is opa^lyMed nthacodingsequencewhai it
regulates transcription of the coding sequence. Ariboscmie4)iadingsiteisoperablylii±edtoacoding
seqimwhm it is present at a position alfov Typically, the
tssm "operably linked" means tet linked nudeotide sequent are ra contact with each other. In the
case of a secretory leader sequence, fhe tern means that it contact a aiding seoeixand is present
within a reading fiame of flie coding sequence. However,an enhancer need not necessarily contact a
coding sequence. Linkage of the nucleodde sequences may be achieved by ligation at convenient
restriction enzyme recognition sites, h the absence of restriction enzyme recognition sites,
ou'goniKlectide adaptors OT linkers nm^
On die other hand, host cells having high introduction efficiency of foreign DNA and
having high expression levels of an introduced DNA may be used In particular, as a host cell, a
eukaryotic cell capable of glycosytating the fusion protein of the present invention should be used
Examples of suitable yeast host cells include strains QfSaccharomyces and Hansemda. Examples of
suitable fungal host cells include Tricoderma, Fusarium and Aspergittus species. Examples of
suitable insect host ceDs include J^^ora cell lines such as Sf9 or Sf21. Examples of suitable
mammalian host cells include CHO cell lines, COS cell lines such as COSI or COS7, animal cell
lines such as BHK cell line or mouse cells, and tissueiiltui plant lls and human cells.
The fusion gene of fhe present invention or a recranbinantejqaressitm vector ccaipisin
same may be introduced into a host ceD by the memods described m basic experimental guide books
(e.g., Davis et al, Basic Methods in Molecular Biology (1986). The preferred memods for this
introduction into a host cell include, for example, calcium phosphate transfection, DEAE-dexfean
mediated transfection, microinjection, canonic lipid-mediated transfection, electroporation, viral
transduction, scrape loading, ballistic inlroductMD, and rafection.
Tn tie preparation method of die present invention, the host cells are cultivated in a nutrient
meoium suitable for rmdifction of a pdypepb using me For example, the
cells may be cultivated by shake flask oiltrvation,snjan-scafecfflar
OTindustrMteietitors perform
to be expressed and/or isolated. The cultivation takes place ina suitable nutrient medium cmtaming
carbon and nitrogen sources and inorganic salts, wag procedures known in the art. Suitable media
ace commercially availabb from commercial suppliers and may be prepared according to published
COTpc4tions(ibr example, the catalog of American Tyie (Mure Collection). If the fusion protein is
secreted mto the ruitrient medium, it
not secreted, itcan be recovered fiom cell lysaies.
The glycosylated fusimprctefooftnepresent irrveon'onniaybereaweiedusinganyoneofa
number of methods far isolating a polypeptide, which are known in the art For example, the
rttly|)eptidte may be recovenxi from
limited to, centrifiigation, filtration, extraction, spray dying, e Further, die
polypeptide may be purified by a variety cfptocediires known rathe art incluc
chratnatography (e.g, ion exchange, affinity, hydrophobicity, and size exclusion), dedrqphoresis,
differential solubility (e.g, ammonium suHate precrpttation), SDS-PAGE, cr extraction.
In a further aect, the present invention provides a pharmaceutical composition comprising
the glycosylated fosmprotein acosdrngtotepresertiaventioa
The term 'treatment1 ', as used herein, refers to a perfect cure, suppression or alleviation of
diseases or disorders. Therefore, the torn 'IrterapeiidcaUy effective anwu
amount sufficient to achieve the above pharmaceutical effect In the present invention, the
merapeuticalh/ effective amount may vary acxxnxlingtDfte fonnulatkmmdbods, administration modes,
patienf s age, weight and gender, severity of the illness, diets, administration duration, adrrrinistraticm
routes, rates, and res Thoseskilled inthe art may readily detomineand
prescribe a dosage capable of achieving a desired treatenent
phamiac*utieda»nrx) A
gjycosylated CTLA4-Ig fijsbn protein as an emrxxiiment of the present invention is applicable to
diseases against which it displays therapeutic effects by inhibiting the action of T-celk, for example,
autoimmune diseases such as arthritis or psoriasis, various organ transplants including bone marrow
transplants, and varicose veins. Also, fosion proteins with receptors for various cancer-associated cell
growth factora may be used in the treatment of cancer because they have improved fh^
due to their effects of increasing serum levels of the recerMnrsandbkxddngangbgenicfec^ors.
The carrier used in the pharmaceutical composition of the present invention includes the
commonly used carriers, adjuvants and vehicles, in the pharmaceutical field, which are as a whole
called 'rjharmaceun'calfy acceptable carriers". Non-limiting pharmaceutically acceptable carriers
iiscMmtrKpharrriaceuticalcomposh
aluminum stearate, lecithin, serum proteins (e.g, human serum albumin), buffering agents (e.g., sodium
phosphate, ycine, sc acid, potassium sorbate, partial glyceride mixtures crfsaturated vegetable fefly
acids), water, sate or electrolytes (e, protamine sulfate, disodium hydrophosphate, potassium
hydrophoshate, sodium chloride, and zinc salts), colloidal silica, magnesium trisQicate,
poryvinvltyytR^done, cefiulose4»sed substrates, poryerhylene glycol, sodium carbojQmethylceMose,
pofyarylale, wajasi, |xrfyerrrylenei)oryt»(ypropylefle coporymers, polyethylene glycol, and wool
faL
The TJbannacxvdc ODQiposition of me
present invention may be administered topically, parenteraDy, intraoculariy, transdermally, inftarectally
and intrahrminally, and may be formulated into solutions, suspensions, and the like. The term
"parenteral"., as used herein, includes subcutaneous, intranasal, intravenous, intraperitoneal,
intramuscular, rate-oracular, intra-synovial, intrastemal, intnacardtal, inrrarhecal, mtraleskmal and
intracranial injectkxi or raftisiontecriniques.
In one aspect, the r±annaceutieal composition ctflhepreKirtmvaaionnMybe
aqueous solutions fi)r parenteral admmistration. fteferabfy, a suitable buffer solution, such as Hank's
solution, Ringer's solution or physfokcalh/buflered salir Aqueous injection
suspensions may be supplemented with subancescpabk of increasing i s c t y of suspens
which arc exemplified by sodium carboxymethylceMose, sorbitol and dexJrm In addition,
suspensions of the active components, such as oily injecticm suspension,
earners, vAach are exemplified by fety
e%loleate,1nyoetidesorlrposoines. Porycationic non-Iipid amino polymers may also be used as
vehicles. Optionally, the suspensions may contain suitable stabilizers or drugs to increase the
solubility of components and obtain high concentrations of the components.
The phannaceutical composition of the present invmtica is preferably in ttefonm of a sterile
injectaWeprqjaratic^ such as a stenleinjectable aqueous OT Such suspension
may be formulated according to the methods knovm in the art, using suitable dispersing or wetting
agents (e.g., Tween 80) and suspending agents. The sterile injectable preparations may also be a
sterieiqedabtesohitionOT suspension ma
a solution in U-butanediol. The acceptable vehicles and solvents include rnacnitol, water, Ringer's
solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be
employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be
employed, including synthetic mono- or di-glycerides. In addition, ferry acids, such as oleic acid and
glyceride derivatives thereof, may be used in the preparation of injectable preparations, like the
pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), and particularly, polyoxvethylated
derivatives thereof.
The atenentioned aqueous cornposhion is sterilized mainly by filtration using a filter to
remove bacteria, mixing with disinfectants or in combination with radiation. The sterilized
composition can be hardened, for example, by fieezfMhying to obtain a hardened product, and for
practical use, the haidendcofflposto
to oixkr to increase ability at room temperature, rd
temperature, and prolong shelf-life, the pharmaceutical composition of the present invention may be
lyophflized A process for fieezeKhying may
drying. After freezing, the composition is heated under pressure to evaporate the water. At the
second drying step, residual water is rmrovdfrcm the dry product
Hie daily effective dosage of the pharmaceutical composition according to the present
mventionistjpkyabcxjtlOpgtoato
300 pg per lglxxfywghi,( most pirfera It
wfflbeappai^tothoseskiMmtbeartto
be administered to a patient imy vary depend
desired biotogjcal activity, thepatient's synqjtoms and drug resistance
A better understanding of the present invention may be obtained through the following
examples. EwfflbsapjjaraitotlMseskiMmthearttete
to illuarate the pnoitinvon, and the scope of the present invention
Containing the 3' end of the soluble extracellular domain of CILA4 and i
IMA encoding a fusion protein in which IgG Fc is linked to the solubk
Fuston protein in v*idi IgG Fc is linked to foe soU* extracellular domain 01
EXAMPLE 1
A. Preparation of DNA fragment encoding soluble extracellular domain of CTLA4
A DNA fiagment encoding a soluble extracellular dcartam of CII^^
usingapionter(SB5IDNO: 1) having a lecognirion sequence of a restriction enzyme, EcoRI, and a
coding sequence fiir a leader sequence of CILA4, and anrjthertairner (SEQ ID NO: 2) having aPstt
recognition sequence and an antisensesequoKeco
ofCTLA4. AcDNAte^lateinuIIlwasixqbyrever
readion(RT-PCR) using mKNA extract
mRNA was isolated using a Tri-Reagent mRNA isolation lot (MRC, USA). Hist, 2xl07 human T
lymphocytes were washed with phosphate buffered saline (PBS, pH 72) three times and lysed with 1
ml Tri-Reagent by repetitive pipetting. The cdllysate was mixed with 0.2 ml chloroform by vigorous
shaking, allowed to stand at room temperate fro 15 mm, and on^
min. The supernatant was transferred to a 1.5-ml tube, mixed with 05 ml isopropanol, and
centri&ged at 15,000 ipm at 4°C fix 15 min. After die supernatant was discarded, the pellet was
washed with 1 ml of triple-distilled water (TDW) treated with 75% ethanol-25% DEPC The tube
was inverted twice or three times and centrimged at 15,000 rpm at 4°C for 15 min. After the
supernatant was completely removed, the KNA pellet was afrniried to remove imiaining
dissolved in 50 ulDEPC-treated TDW.
B. Preparation of DNA fragment encoding Fc region of IgGl
A DNA fragment encoding an Fc region of IgGl was prepared by PCR using aprimer(SEQ
ID NO: 3) having a Psfl recognition sequence and a sequence coding tor a 5' end of IgGl Fc, and
anofoer primer (SEQ D NO: 4) having an Xbal recognition sequence and an antisense sequence
coding fcr a 3'end of IgGl Fc. A cDNA template in the PCR was prepared by RT-PCR using
mRNA extracted from peripheral Mood cells (B fyn^feocyte) of patients
origin, who were in recovery. RT-PCR was earned out using fce same reagents under the same
conditions as in Example 1, part A
C Preparation of gene encoding non-glvcosvlated CTLA4-IgG
The DNA fragment encoding the soluble extracellular domain of CTLA4 and the DNA
fragment encoding the Fc region of IgGl were digested with Pstt and ligated using T4 DNA ligase.
The ligated DNA contained a leader sapience to facilitate protefe The
fusion gene fiagment thus prodixwas digested wirnBWarri
sites of pBluescript KS H(+) (Steatagene, USA), "which is a omrniodaDy available doning vector.
The whole coding region was identified by DMA squeacing (SEQ ID NO: 7). A iusion protein
expressed fiom the fusion gene was designated "CTLA^IgG", whose predicted amino acid sequence
is represented by SEQ H) NO: 8.
EXAMPLE 2: Preparation of glycosylated CTLA4-IgGMonproteins
In older to introduce a glyeosylatkmn^intofte^
nudeotide sequence containing a mutation leading to an amino add substitution were prepared as
follows: imhenudeotide sequence ofSEQ ID NO: 5, a 478480 ccxion(ATG, Met) w^ replaced wife
AAC (Asa, NX a 583-585 codon (GCC, Ala) wife AAC (Asa, N), a 727-729 codon (ACC, Tk)wife
AAC (Asa, N), a 793-795 codon (GAG, Glu) wife AAC (Asn, N), a 895-897 codon (TAG, I» wife
ACC (Ik, TU991-993 codon (TTC, Phe) wife ACC (Ik, T), and a 1036-1038 codon (GAG, Gta)
wife AAC (Asn, N). Biformationoiifeese primers is given in Table 3, below.
Qycosyiated &SIQJI pKrtebi uf the presera feveniion woe prepared by KIR using die
cloning vector carrying CTLA4~hIgG"Coding DMA, prepared in Example 1, as a template, and
la detail, each gfecosykted fiisionprafcdn was prepared as Mows,
(1) CTLAWi^Krl (Gl varianf): ooe ^ycaqtefiainjotuf was cre^ed usingapimer (SEQ
E) NO: 9) designed to have a iradeotkte sapie««cantein^
480midec^des(ATG,Mrt)i)oatJcarfatteI^ie^
(2) CTlA44ifeG (SEQIDNOS: 12 and H) designed to lve nu:l«)tide s^uences conla
(Asa,N)and AC (Ihr, 1) for 793-795 nudeolides (GAG, Glu) md 895^97 nudeotidesCTAC, Tj4
rcspecthdy,positioDcdattheFcregicnofIgG(SEQroNa 5).
(3) CTIA44iC5-G3 (G3 variant): two gfycosylation motifs were created using primers
(SEQ ID NOS: 13 and 14) designed to have nudeotide sequences containing substitutions of ACC
(Ik, T) and ACC (Ik, T) for 895-897 nudeotides (TAG, Tyr) and 991-993 niicleoticles (ITC, Phe),
re^ecthdy.poalksidatlheFcrcgonof IgG (SEQ ID NO: 5).
(4) CTLA4-hIgG-G4 (04 variant): three glycosylation motifs were created using primers
(SEQ ID NOS: 9,12 and 13) designed to have nudeotide sequaK^ owMiing substitutions of AAC
(Asn,N), AAC (Asn,N) and ACC (Ik, T) for 478480 mdeotides (ATG, Met), 793-795 nudeotides
(GAG, Glu) and 895-897 nudeotides (TAG, Tyr), respectively, positioned at the Fc region of IgG
(SEQDNO:5).
(5) CTtA4-hIgG"G5 (G5 variant): four gjycosylation motife woe created uang primers
(SEQ ID NOS: 9,12,13 and 14) designed to have nudeotide sequences containing substitutions of
AAC (Asn, NX AAC (Asn, N), ACC (Tk, 1) and ACC (Ik, 1) for 478480 nudeotides (ATG, Met),
793-795 nudeotides (GAG, Ghi), 895-897 nudeotides (TAG, Tyr) and 991-993 nudeotides (ITC,
Pte), respectivdy, positioned at the Fc rcgbn of IgG (SEQ ID NO: 5),
(6) CTLA4-h^G-G6 (G6 variant): five grycosyJation motifs were created using primers
(SEQ ID NOS: 9,10,12,13 and 14) designed to have nudec^ sequence contain
AAC (Asn, N), AAC (Asn, N), AAC (Asn, NX ACC (Tfar, T) and ACC (Thr, T) for 478-480
nudeotides (ATG, Met), 583-585 nudeotides (GCC, AlaX 793-795 nudeotides (GAG, GkX 895-897
nudeotides (TAG, Tyr) and 991-993 nudeotides (TTC, Phe), respectivdy, positioned at fee Fc region
of IgG (SEQ ID NO: 5).
(7) CTLA (G7 variart): six grycom
ID NOS: 9,10,11,12,13 and 14) designed to have nudeotide sequences containing substitutions of
AAC (Asn, N), AAC (Asn, N), AAC (Asn, N), AAC (Asn, N), ACC (Ik, T) and ACC (Ik, T) for
478480 nudeotides (ATG, Met), 583-585 nudeotides (GCC, Ala), 727-729
793-795 nudeotides (GAG, Giu), 895-897 nudeotides (TAG, Tyr) and 991-993 nucleotides (FTC,
Phe), Jfcspectively, positioned atthe Fc region of IgG (SEQ ID NO: 5).
(8) CTlA44iIgG-G8 (G8 variant): seven gjycosylation motifs were created using primers
(SEQ ID NOS: 9, 10, 11, 12,13, 14 and 15) deagned to have nudeotide sequences containing
substitutions of AAC (Asn, N), AAC (Asn, N), AAC (Asn,N), AAC (Asn,N), ACC (Thr, T), ACC
(Thr, T) and AAC (Asn, N) for 478480 nucleotides (ATG, Met), 583-585 nucleotides (GCC, Ala),
727-729 nudeotides (ACC, Thr), 793-795 nucleotides (GAG, Glu), 895-897 nudeotides (TAC, Tyr),
991-993 nucleotides (TTC, Phe) and 1036-1038 nudeotides (CAG, Gto), respectively, positioned at the
FcregionoflgG(SEQIDNO: 5).
The PCR was earned out as follows. To a PCR tube, 1 ul of CTLA4-hJgG DNA C22ng),
125 U Pfii DNA polymerase (Sttatagene, USA), 4U Pfo DNA Hgase (Stratagene, USAX 1 ul of lOx
reaction bufier for PfiiDNAligase, 1 u!ofeadiprimer(10pMXand2ulofdNTP(each 10mM)were
added, and triple distilfcd water wasadded to a final volume of 20 ul PCR conditions included two
cycles of 3 min at 94°C, 1 min at 61°C and 1 rain at 65°C, and then 29 cyctesoflininat940C;iminat
61and7mmat65,fellwbyfelcfigaticnat65 The PCR products thus
obtained woe subjected to sequence analysis to determine whether a glycosylation motif was
successfully inserted
EXAMPLES
A Expression and purification of glycosylated CTLA4-IgG fusion proteins
To express glycosylated dIA4-IgG fusion proteins in Chinese hamster ovary K-l cells
(CHO-K1, ATCC CCL-61, Ovary, Chinese hamster Crteetvbs giseus), pBhiescrt KB E(+) pksmid
DNA containing a CTLA4-hIgG fusion gene into which a glycosylation motif was inserted was
isolated from transformed £ coli, and digested whh EcoRl and XbaL The thus-obtained CTLA4-
hlgG fusion gene was inserted into BcoRI/Xbal sites of an animal expression vector, pCR™ 3
(fovitrogen, USA). TTie resulting expression vectors were designated as rX^4IgX32 to G8 plasroids.
Among them, fte pCT4Ig-G2 rccombinant expression vector was deposited at the Korean Culture
Gaiter of MtoocMg^^
assigr^aaxssicrarjumberKCCM 10572.
B.Transfection and evaluation of expression of fosion genes
Chinese hamster ovary K-l cells (CHOK1) were plated onto six-well tissue culture plates
(Nunc, USA) at adensity of l-3x!05 cdls per well, and were grown to a 50-80% confluency in 10%
FBS^»i^ainingDMEMmediurn. ma serum-free DMEM, lX2|agDNAofanyoneofpCT4Ig-G2to
G8 plasmids was mixed whh 2-25 jjl of lipofectamine (Gibco BRL, USA), and incubated at room
temperature tor 15*45 min to form DNA-liposome complexes. Then, the resulting complex was
added to the six-well plates. After an incubation period of 5 hrs, the cells were refbdwim 20% FBScontakung
DMEM medium and further cultured for 18-24 hrs. Thereafter, the cdls were cultured in
10% FBSx»ntaining DMEM supplemented wife 3 mg/ml genetkan (G418, Gibco BRL, USA) for
three weeks. Formedcolonies were selected and isolated, and men propagated.
Whether or not a fusion gene was expressed was evaluated by ELJSA using peroxidaselabefcd
goat anti-human IgG (KPL, USA). EUSA was carried out as follows. First, 1 mg/ml of
goat antHiuman IgG was diluted to 1^2000 wMi 0.1 M sodium bicarbcaale, and 1001 of flw diluent
was aliquotted into a 96-wdl flexible plate OFalcon, USA). After being sealed withsaran wrap, the
plate was incubated at 4°C for over 16 hrs to allow the bottom of tie plate to be coated with the
antibody. Thea, fee plate was washed tec times with a washing buflfer (Ix 0.1% Tween-2
containing phosphate bufife13 ml
BBS, 50 ulTween-20) was added to each well 20 )il of a culture supernatant \vasadded to the first
well and serially diluted using a ndcropipette. 0.01 ug/ul of human IgG (Sigma, USA) as a positive
contol and a culture fluid of non-transfectedCHO-Klc
the test sample. Alter diluttoos were completed, the 96-wdl flexible plate (Falcon, USA) was
wrapped wife foil, incubated at 37C for 1 hr 30 min and washed with the washing biiflthree times.
Peroxidas»4abefcd goat anti-human IgG (KPL, USA) was dih^to 1^000 wife tie diliientbufer, and
100 ul of the diluent was added to each well, wrapped with foil and incubated at 37°C fear one hair.
Afler the reaction was completed, the plate was devdoped with a TMBnriooweUperoxidase substrate
system (KPL, USA). Ahsorbance was measured at 630 nm using a microplate reader (Bio-RAD,
Model 550 Japan) to determine whether a fusion gene was espessed and teejipession levels of flie
fusion gene(FEG.2>
As shown in FIG, 2, the 01 variant was expressed in the highest levels, followed by G2.G4,
GO and G3 variants. The G5,G6,G7 and G8 variants were found to be latety expressed
EXAMHJE4
A. Western blot analysis
An expressed protein was purified by inmjinwprecipitato
Fast, 50 ul of protein A-Sephatose beads were placed intDaU^tube,mkdv^l{X)uiofbufferA
(O.WMbc3ricadd94MNaQpH9.0),aiKloantri&gedatl3,000 Aflerthe
supernatant was discarded, this step vvas repeated tiaee times. Eadi protein sarajde was mkedwifh
fie equUihated protein A-Sepharose beads and incubated at 4°C for 3 hrs wim rotation to induce
binding. Then, theieaction mixtme was oentri%ed at 13,000 rpm, and the beads were washed vvhh
buffer A three times. The beadswere mixed wifli 20 ul of bufierB (OJ35 M sodium phosphate, 0.05
protein sample was mixed with 5x buffer containing 5% p-mercaptoethanol, boiled for 5 min, and
subjected to reduced SDS-PAGE A 3.5% Acrylamide gel (0.5 M TOs-HCl (pH 6.8), 0.4% SDS)
was used asastedcing gel, and a 10% Acrylamide gel (15 M Tiis-HCl Oil 8.8), 0.4% SDS) was used
as a running gel. After dectrophoresis, proteins were dectro-transferred onto a 0.4-pm Westran
(PVDFtrarisfermeinbiane,S^fbr2hrsat350mA The blot was blocked with 5% skimmffle for 1
hr. After being washed with washing buffer (0.1% Tween-20, lx phosphate buffered saline) three
times, the Wot was incubated in a 12000 dilution of peroxidase-labeled goat anti-human IgG (KPL,
USA) for 1 k The blot was washed with washing buffer three times, and developed at room
temperature fcr 10 mm with 15 ml of a coloring agent, which was inadeaccc^g to a recommended
iemetlxxJ using a DAB substrate kit (VE The reaction was
taminatedwifcti^te-distilledwato The results are grvea in HO. 3.
EXAMPLE 5: Measurement of serum talf-lrvesofglyoosylatoiC^^
Serum half-lives of gjycosylated CTLA4-hIgG fusion proteins were measured in mice as
follows. Each fusion protein was intraperitoneally injected into mice (ICR, Samtako Inc., Korea) in a
doseof02mg/kg, BtocxJ samples were ollec at grvra
tax concentrations were detemiinedaccoix
4).
As diown in FIG. 4, the G2, G3 and 04 variants had iiweasedsenm levels, whereas the 01
variant displayed reduced blood circulation time compared to the wild type. In particular, the G2
variant exhibited the highest drculationtime.
Industrial Applicability
As described hereinbefore, the glycosylated fusion pxtoisacconlkto the present invention
ate able to reduce dosage and administration frequency in clinical applications because they have high
in vivo stability.

We claim:
1. A glycosylated immunoglobulin G or a Fc portion thereof, wherein the glycosylated
immunoglobulin G is generated by additionally glycosylating a variant of immunoglobulin G
in which a methionine residue at a 160th position from an N-terminus of a mature wild-type
immunoglobulin G is replaced with asparagines (M160N), a glutamic acid residue at a 265th
position therefrom is replaced with asparagines(E265N), a Tyrosine residue at a 299th
position therefrom is replaced with Threonine(Y229T), and a Phenylalanine residue at a
331th position therefrom is replaced with Threonine(F331T).
2. The glycosylated immunoglobulin G or the fragment thereof as claimed in claim 1, wherein
the immunoglobulin G variant comprises the amino acid sequence of SEQ ID NO: 19.

Documents:

82-DELNP-2007-Abstract (30-11-2009).pdf

82-DELNP-2007-Abstract-(11-11-2010).pdf

82-delnp-2007-abstract.pdf

82-DELNP-2007-Claims (30-11-2009).pdf

82-DELNP-2007-Claims-(11-11-2010).pdf

82-delnp-2007-Claims-(29-10-2010).pdf

82-delnp-2007-claims.pdf

82-delnp-2007-correspodence-others.pdf

82-DELNP-2007-Correspondence-Others (30-11-2009).pdf

82-delnp-2007-Correspondence-Others-(11-11-2010).pdf

82-delnp-2007-Correspondence-Others-(29-10-2010).pdf

82-delnp-2007-correspondence-others-1.pdf

82-delnp-2007-description (complete).pdf

82-delnp-2007-drawings.pdf

82-delnp-2007-Form-1-(11-11-2010).pdf

82-DELNP-2007-Form-1.pdf

82-delnp-2007-form-18.pdf

82-delnp-2007-Form-2-(11-11-2010).pdf

82-delnp-2007-form-2.pdf

82-DELNP-2007-Form-3.pdf

82-DELNP-2007-Form-5.pdf

82-delnp-2007-pct-210.pdf

82-DELNP-2007-PCT-237.pdf

82-delnp-2007-pct-304.pdf

82-delnp-2007-pct-373.pdf

82-DELNP-2007-Petition-137 (30-11-2009).pdf

82-DELNP-2007-Petition-138 (30-11-2009).pdf

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Patent Number

246647

Indian Patent Application Number

82/DELNP/2007

PG Journal Number

10/2011

Publication Date

11-Mar-2011

Grant Date

08-Mar-2011

Date of Filing

02-Jan-2007

Name of Patentee

MEDEXGEN INC.

Applicant Address

2ND FLOOR, MEDICAL BLDG 1, HANYANG UNIVERSITY, COLLEGE OF MEDICINE, 17 HAENGDANG-DONG, SEONGDONG-GU, SEOUL 133-791 KOREA

Inventors:

#	Inventor's Name	Inventor's Address
1	CHUNG, YONG-HOON	#212-1201, SAMIK APT., SONGPA-DONG, SONGPA-GU, SEOUL 138-170, KOREA
2	YI,KI-WAN	# 560501, GARAK APT., GARAK 1-DONG, SONGPA-GU, SEOUL 138-161, KOREA
3	CHO,HOON-SIK	# 410-604, JOOGONG 4-DANGI, CHANG 1-DONG, DOBONG-GU, SEOUL, 132-041, KOREA
4	PARK, HONG-GYU	## 522-7, MOK 2-DONG, YANGCHEON-GU, SEOUL, 158-052, KOREA
5	KIM, KWANG-SEONG	## 306-2103, ECO-TOWN, SINJANG 2-DONG, HANAM-SU, GYEONGGI-DO, 465-715

PCT International Classification Number

C07K 16/42

PCT International Application Number

PCT/KR2005/001627

PCT International Filing date

2005-05-31

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10-2004-0038833	2004-03-31	Republic of Korea