CA2191891A1 - Modified cells and methods for inhibiting hyperacute rejection of xenogeneic transplants - Google Patents

Abstract

Modified cells for transplantation having reduced capacity to stimulate natural antibody-mediated hyperacute rejection of the cell in a transplant recipient are disclosed. In unmodified form, cells for use in xenogeneic transplantation express at least one epitope on a cell surface antigen which is bound by natural antibodies in a recipient. Prior to transplantation, the cells are modified to alter, reduce or substantially eliminate expression of the epitope on the cell surface. Preferably, the cell is a porcine cell and the epitope is a galactosyl (.alpha.1,3) galactose epitope. Methods for reducing the immunogenicity of a cell for transplantation into a recipient are also disclosed. Modified cells of the invention can additionally be treated to alter, reduce or substantially eliminate another surface antigen (e.g., an MHC class I antigen) which stimulates a cellular immune response against the cell in a recipient. Additionally, a recipient can be treated with an agent which inhibits T cell activity, such as an immunosuppressive drug or an anti-T cell antibody.

Description

~ wo 9~i/33828 2 ~ 9 ~ 8 91 r~

MOI)II;IF.D CI~LLS A~D METIIOI)S FOR ll~rlllBlTlNG IIYPERAC'UTI~, REJECTION 01<' XENOGENEIC TR~NSPLANTS

~f " - Invention S The abilit,v to transplsnt cells, tissues and organs from animals into humans as ,~~.1,.. . ",~ .. ~ for diseased human cells, tissues or organs would overcome a key limitation hl clinical ~ ,l,."n~ the shortage of suitsble human donor organs. I-lo-vever, the problem of immune-mediated rejection continues to hamper the clinicnl application of xenogeneic Xenogeneic tissues, similar to tissues from ,,,;~", t 1,. ~I human donors, are 10 subject to rejection by the human cellular immune system. In addi~ion, ~vhen l~ ,I....t. I
into a human, cells from nonprimate animals may be rejected by antibodies that are present in human serum even ~ ithout prior exposure to cells from the animal. The presence of these preexisting, or natural, antibodies results in rapid rejection of xenografts. This mode of destruction of I I .tl 1~ _. i d animal tissues, termed hyperacute rejection, occurs within minutes 15 to hours of L~ and is distinct from reiection i~ia the cellular imnlune pathway which typically occurs over one to several ~ieeks (see Lal't'erty, K.J. et al. in Transplantation:
Approache.s to G? aft Rejection, NewYor~: Alan l.iss, 198~, pp. 87- 117).
Natural antibodies in human serum that react with nonprimate animal cells are of both Ig~v~ and IgG classes. The binding of natural antibodies to epitopes present on nonprimate 20 animal cells is thought to mediate hyperacute rejection of the 1~ t' d cells by attraction and activation of ~ ll ,l..., .l and/or effector cells that cause cell death. Whole organ grafts can fLul even in the absence of antibody-mediated cell death: attack of the endothelial cells lining the vasculature of the ~onogr~ftf d organ can lead to . I 1~ . ~1,1. " .., ,i -activated release of factors that initiate the coagulation cascade. The anti-thrombogenic lining of Ih~ vasculature is disrupted, resulting in thrombus formation and tissue anoxia, as well as passage of Iymphocytes through the endo~helial cell layer into the tissue (see Bach. F.ll. (1993) Trans~
Proc. 2~:25-29; and Platt, J.L. and Bach, F. H. (1~91) 7ranspla?ltation 52:(337-947). lhe barrier imposed by these naturally occurring antibodies must be surrnounted if a n ~ t.
xenogeneic cell, tissue or organ is to engraft su~,~,s~ rully One approach that has been used in an attempt to inhibit hyperacute rejection of a xenc graft is to preclear the recipient serurn of natural antibodies by perfusion ex vivo through one animal organ prior to the I l .~ 1 ,1 ' U~ of a second animal organ inlo the recipient (se~e Ross,J.R.etal.(1993! Trailsplan~atlvn ~i:1144-1150;andTuso,P.J.etal.(1993) Tran.spla?lta~ton~:1375-137g). Inthisprocedure,humanbloodfromarecipientis circulated ex vrvo through a tirst pig li ver and tested t'or any residual reactivity w ith fresh pig tissues prior to 1l ~lU ",I.."I.~Lion of a second pig li- er imo the~ recipient (Tuso, P.J. et al. (1993) Transplantatio?l ~: 1375- 1378). In addition to requiring two donor organs for each tran.splant recipient~ this approach is limited b~ the efficiency ~ ith which natural antibodies can be removed from the circulation of the recipient.

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A second approacll to inhibiting h peracute rejection has been to treat the recipiellt ~ith ;"~ e drugs or inhibitors of ~.. l.l: .,.~ .. l prior to ~ 5ee B~h, F'.E~. ~ 1993) 7rmls~71. Proc. 25:25-29; and Platt, J.L. and Bach. E'.H. ~1991) rr~ 1nriml ~:937-947). 'Ihis approacll has been successfui in prolonging the survival of grafts up to several mmlths but sut't'ers from problems generally associated Witil of high doses of; ~ a~~
Another approach lo inhibiting natural antibody-mediated rejection of organ grafts ha been to administer high doses of low molecular weight haptens that will inhibit natural antibody binding to the i ",~1 l 5 ~I tissue. This approach has been applied to AB0 blood group " ~ allografting~ another clinical situation in which pre-existing natura antibodies can mediate h,vperacute yraft rejection. The survival of am AB0; ~
cardiac allograft was prolonged from minutes to several days by Ihe in~ection into the yraft recipient of synthetic carbohydrate A and B blood yroup antigens ~see Cooper, D.E~.C. et al.
(1993) ~ransplanta~ion ~6:769-777). Studies in humans and nonhuman primates indicate I ~ that natural antibodies reactive ~~ith cells from a number of different species are directed largely against ca:rbohydrate epitopes on the xenogeneic tissue (see E'latt. J.L. (1992) AlSAJ0 .Jotlr~ta~ 8-16; Ciood, A.E~. etal. (~99~1 Irarspl Proc. ~:554-562: and Satake~ IM. et al.
(1993) Clin. Transpl. 1:281 -288). Thus. Cooper et al. suggested that a similar approach could be used to inhibit h~peracute rejection in xenograft ~
Accordingly, a~proaches that have been attempted or suggested to address the probiem of hyperacute rejection of l l ,~ , " l r~ l cells have been based on treatment of the recipient to remove. suppress or neutralize natural ant;bodies in the recipient's senml.

S of the In- ention 'I'his in~entioll pertahls to mhibition of natural antibody-mediated hyperacute rejec~ion of nonprimate xenografts in human or nonhuman primate transplant recipients to thereby improve xenogeneic . ..~,,, n", . ,; The methods of the invention ~ure based upon treatment of the graft rather than treatment of the recipient. According to the inventiol1, a cell to be trRncplRIlt~1, wlli~ll in unmodified form expresses an epitope on its surface which 30 stimulates hyl)eracute rejection of the cell by natural antibodies in a recipient. is treated such Lhat expression of the epitope on the surface of the cell is altered, reduced o r substantially eliminated. This treatment of the graft inhibhs subsequent recognition of the epitc pe by mltural antibodies in a recipient, thereby inhibiting hyperacute rejection. In a preterred embodiment. the epitope is a ~ l bollydldL~ preferably galactosyl(c( l ,3)gaiactose 35 (Gal( c~ I .3)Gal). Preferred cells for treatment include porcine cells. Cell t,vpes for use in the invention hlclude endothelial cells~ hepatocytes, pancreatic islets~ muscle cells (including si~eletal and cardiac myocytes and myoblasts)~ f broblasts, epithelial cells~ neuronal cells~
bone marrow cells~ b.... ~ .;ctic cells and Iymphoid cells. Dispersed cells can be treated or, I~lLelllc,Li~ cells can be treated withil a tissue or organ (e.g., liver. heart. kidney etc.).

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In one ~.,,h.:.~l;." ,1 ofthe inventioll, llatLIral antibody cpitopes are remo~ed trom the surface of a cell, such as by enzymatic or chemical treatment of the cell. For example~
Gal(o I 3 )Gal epitopes can be cleaved from a cell surfilce by treatment of the cell u ith an alpha-g~l~f~tL ~ f In another embodiment, formation of the epitope on the cell surface is 5 inhibited. This can be ~ d by inhibiting the activity of an enzyme ~l/hich forms the epitope. For example, formation of Gal(oc l .3)Gal epitopes on the surface of a cell can be ~ interfered with by inhLibiting the acti~ity of an alpha-l ,3-galacL~, " llld l~r~,. aae w,ithin the cell.
such as by introducing into the cell a nucleic acid which is antisense to a coding or regulatory region of an alpha- I ,3-galactosyliid.~r~,ld~ gene or by treating the cell with a chemical 10 inhibitor of the enzyme. ln yet another rl 1 ~l lol i; ~ 11, epitopes on a cell surface are altered by binding a molecule to the epitope, thereby inhibiLing its subsequent recognition by natural antibodies in a recipient. For example. Iectins, antibodies or antibody fragments can be bound to an epitope to inhibit its subsequent recognition by natural antibodies.Accordingly, one aspect of the in~lention pertains to a cell for Lld~ 61~LdLioll into a 15 recipient, which. in unmodified form expresses at leasL one epitope on a cell surl'ace antigen that is bound by natural antibodies in a recipient~ wher in the cell is modified to alter, reduce or substantiall~ eliminate expression of Lhe epitope on the cell suriace. In a prei'erred embodiment, the cell is a porcine cell and the epitope is a Gali o l ,3~Gal epitope.
In addition to being modified to alter, reduce or substantially eliminate expression of 20 at least one natural antibody epitope, the cell can be furdler modified to alter, reduce or substantially eliminate expression of at least one other antigen on the cell surface which. in unmodified form, stimulates a cellular immune response against the cell in a recipient. A
preferred second antigen to be altered, reduced or substantially eliminated on the cell is an Mh'C class I antigen. For example, the cell can be furthcr modified b~ contacting thf~ cell, 25 prior to l~ f~ ith an anti-MI-lC' class I antibody, or fragment tLtfereof (e~g., F~ab' 12 fragment). In another f mhnf~imf nt the cell is further modified to express a gene product, for example by introducing into the cell a nucleic acid encodillg the gene product (e.g~, for gene therapy).
Another aspect of the in~ention pertains to m thods for reducing the immunogenicity 30 of a cell for ~ ;f~n The methods invol~e contacting tne cell with a first agent which alters reduces or substantially eliminates expression of a naLural antibody epitope on the cell surface. The agent can be, for e xample, an enzyme which cleaves the epitope from the cell surface~ an antisense nucleic acid which inilibits for~nation of the epitope on the cell surface or a molecule which binds to the epitope on the cell surface and inhibits its recognition b~
35 natural anLibodies in a recipient. In addition to the first agent, the cell can be contacted w ith another agent to alter, reduce or substamially eliminate another antigen on the cell surface which. in unmodified form, stimulates a cellular immune response against the cell (e.g. an MHC class I antigen). Following treatment of tne cell to reduce its b. ~ loL,~ i ly, the cell is administered to a recipient. In addition to recei~ ing a cell having re~duced immunogenicity, ~9~89~ ~ ~
Wo gS13382~1 PCTI~IS~SIOS973 a transplant recipient can also treated with ~nother agent which inhibits T cell act;vity in the recipient (e.g., an ;., .. ,.. ,~ ive drug or anti-T cell antibody) to further inl ibit rejection of the tr,~ncrl~ntPA cells.

5 BriefD~ . i, ti of ~
f;'lg2~res IA-ID aregraphicrepresentationsofthereactivilyofhumanandmonkey sera with untreated porcine endothelial cells.
F'igure 2 is a g,raphic representat;on of the effect of alpha ~ u;i~ cc treatment on the ~ iability of porcine endotheliai cells.
F'igures3ai-3F'aregraphicrepresentatiollsofthebindingofnaturalantibodiesto porcine endolhelial cells at'ter alpha ~,,7'1AAf~Cif~'Acf' treatment of the cells.
Figure 4 is a graphic representation of the time coarse for the ~ of aipha galactosyl epitopes on porcine endothelial ceils following treatment of the cells with alpha-AAtn ~;I--IA if Fi~mre 5 is a ~,raphic represcntation of the bindhlg of various human and anhl1al .sera ui porcine endothelial cells, either antreated or treated with alpha g ~ n~ P to remo~ e alpha galactosyl epitopes~ A ~ a ~ e reduced bhlding of hutnan and moniiey IgG and IgM to porcine cells after enzvme treatment.
Figl~re 6 is a graphic ~cl~es~llAAliull of the effect of various cl)nr ~n~ratir~n~ of hanlan A~O serum and ~ u" ,l .1.., ,...: on the viability of porcine endotheiial cells.Figure 7 is a graphic lcy~ _liuU of the variation in levels o f natural antibcldies inhuman serum from seven individuals.
Fig~ure 8 is a graphic l~yl~ L~Ilh~l~ of the viability of porcine endolilelial cells upon incubation wiLh bovine or human sera either in the presence or absence ot' ~ L,li " -1 _~ A~ e naturai antibody-mediated c~luLu~ of hamaiAl serum and ~
Fi~,lurL~ 9 is a graphic representation of the viability of porcine endothelial cells. either antreated or treated with alpha eAlAAt~c;AAAcP, upon incubation with haman serutn and ncr~as~d ~liabilit~ of the porcine cells ~ollo~ing enzyme treatment.
i~etailed 1~ - ' " of the inventfon This invention features methods for inhibiting h~Tjeracute rejection ot ir~ncr~ d cells by pretormed naturai antibodies in a transplant recipient. Moditied cells l;n use h~
n ~ l ,l lu.l ;on are provided which have a reduced capacity tO stimulate hyperacute re jection 3 ~ in a recipient. Cells are modified su h that expression of at least one epitope on the surface of the cell which stimulates hyperacute rejection (i.e., at least one naLurai antibody epitope) is altered, reduced or substantially eliminated. The modified ceils of the invention are particularly useful in xenogeneic ~ n where natural antibody-mediated hyperacute rejection has posed a barrier to successfui L"...',.l.."l l;l ,., l~lodified cells of ~1E~llFIED SHE~ (~Ui E 91) ISA/EP

., 2t9~89~ l WO gS/33X28 1 ~
- s -nonprinlate oligau can thus be ~slncpis3ntf~d inlo humans and nonhuman primates. The modifed cells and methods of the invention are also applicable~ in certain allogeneic transplant situations in whic.h naturai antibody-media~ed hyperacute rejection may occur.
such as with A130 blood group ~ t. '.~ j S The term "hyperacute rejection", as used hereill, refers to an immnnnlogirsll reac~ion hl a recipient against foreign cells ~hich occurs very rapidly (e.fg., within minutes ~o hours) following ~ of the cells. Typically, hyperacute rejection is mediated by naturalantibodies present in the serum of the recipient.
An individuai ~vho has received or is to receive a foreign cell, tissue or organ graft is referred to herein as a "recipient" (or "host"). An individual that supplies Lhe foreign cell tissue or organ graft is refèrred to herein as a "donor".
The term "natural antibody" as used herein refers to preformed (i.e.. preexistillg) aultibodies reactive against foreign donor cells which are present in serum of a recipient without prior exposure to donor cell antigens. A structure on the surface of a donor cell i e.g., 15 a carbohydra~e) ~vhich is specifically reco~nized by (i.e., can be bound b,~-) a natural antibody is referred to herein as a "natural antibody epitope" or simply all "epitope".
To produce modif ed cells ~h ithin the scope of the invention, the expression of at least one epitope on the surface of the cell to be Ll~~ d -~hich stimulates hyperacute rejection of the cell by natural antibodies in a recipient is altered, reduced or substantially eliminated.
20 The temms "altered" or "alteralion" of the expression of an epitope is intended to encompass mn~lif r~nion of the epitope such Lhat iLs recognition by natural antibodies is inhibited or prevented (although the epitope may still be present at normal levels on the cell surface).
Altematively, when expression of the~ epitope is "reduced" or "substantially elimina~ed", ~he level of expression of the epitope on the cell surface (i.e., amount of the epitope on the cell 25 surface) is decreased or substantiaily abolished relative to a normal level of expression of the epitope on the cell surt'ace.
Various aspects of the invention are described in further detail in the following sl~h~:f~tion~

30 1. Epitopes to be Altered. Keduced or Sllh~s n~isllly Fli Epitopes to be altered, reduced or subsLqntiaily eliminated according to the invention are those ~ hich stimuiate hyperacute rejectioll c f a cell in a recipient. Since natural antibodies in humans and nonhuman primates are IJ-~; iul..~ Lly directed againstcarbohydrate epitopes~ preferred epitopes for mnolifirsltinn are carbohydrate moieties. A
35 preferred carbohydra~e epitope to be altered~ reduced or substantially eliminated is a galactosyl(al,3)galactoseepilope(alsoreferredtohereinasGal(cl1,3)Galoranaipha-gaiactosyl epitope ). i Ip to I % of all circulatinfn IgG in human sera has been found to be directed against this epitope (see Galili, lJ. et al. i 1987) Proc. ~vatl. ~icad. Sci. ~ 84: 1369-1373; Cialili, El etal. (1987)J. Biol Cllcn~. 26~:~683 '1688). Anexperimentdescribed in ?,~9~J ~
wo gsl33828 2 1 ~ 1 8 9 1 - 6 -E,xample 2 .i~ that this epitope on porcine cells is a m~jor epitopc recogn;7..ed b7 natural antibodies in hurnan serum. Terminal alpha-galactosyl epitopes are present on the surface of cells from nonprimate marnmals. prosimians and New World monkeys bu~ not on the surface of cells from humans, apes and Old World tnonkeys ~see Galili, ~J. et al. (1988) S Proe. I 'a~l. A-,ud. Sr i. J~ 2~~ 17755-17762). Tile ~enn "t~alactosyl(al,3) galactosr epitope" is intended to encompass ~ bOllydlrlLc structures ~~hich comprise this moiety and which can be bound by natural antibodies~ such as aGal( I -3)~Gal( l -4)pGlcNAc and o ~}al( l -3)pGal(l~)pGlc ~collectiYely referred to as linear B type structures) ~for ti~ cnrjpttinnc of natural antibody binding to various alpha-galactosyl epitopes see Good, A.H . et al. (1992) Tnans/7L Proc._:559-562).
Alternatively, other epitopes recogni~ed by natural antibodies can ke targeted for alteration, reduction om~liminr~tion Other carbohydrate epitopes that have been reported to be buund by llatural antibodies include A or A-like ~r~bullyrlla~a (including A ~
A trisaccharide, A h~p- 4, A type~ 5, A type 6 and linear .~ type 6), i c~rssrnan li;~.1. .1~ ;~lr and Forssman ~ iP o -i,-Rhamnose and lir-acetyl p-D-g~ , as descrihed in Good, A.H. et al. (1992,~ ~rat1~pl. Proc. 24:559-562. and sulphatides such as galactosylcerarnide-3-sulphate and lactoc~lceramide-3-sulpha~c~ as described in l-lolgersson.
J. et al. ~ 1992) Transpl. f 'roc. 2~L:60s-6o8. .4.dditionally, ~17 ~ Ot~ r~ having molecular weights of 115 kD, 125 kD and 135 kD ha~e been reported to express epitopes recogni~d by naturalantibodies~seePlatt,J.L.etal.(1990) Tran.splanfufion~iO:817-8221.

Il. Mrthn-ic for Alt.~rin"~ R~ rin,~ or Flimin~ J a Cell Surf~r-~ rnitnrr .According to the in~ention, a cell for n ,.,~ is tred~ed prior to transpl;mta~ion to alter, reduce or substantially eliminate expression of at leas~ one epitope Oil tlle celi surlace 25 that stimulates hyperacute reJection of the cell in a recipient. In a pretèrred f~mhndimf nt of the in~ ention, expression of a surtace epitope is reduced or substantially eiiminated by remo~ing the epitope from the cell surface. Remo~al of the natural antibody epitope t'rom the cell surface innibits subsequent recognition of the cell by natural antibodies in a transplant recipient. An epitope can be remo~ed from the surface of a cell by treating the c.ell ~ ith an 30 enz,vme or chemical which clea~ es the epitope from the surface oi'the cell. For example.
carbohydrate epitopes can be cleaved from a cell surl'ace by treatment of the cell ~ith one or more endo- or exclfdy~ u. .id~a specific for the ~,cuI,ol,ydl~t~, to he cleaYed. Preferably, alpha-galactosyl epitopes are cieaved .from a cell surface by treatment of the cell Witil an alpha-g~l~rtnc;tirct~ As described in greater detail in Example 1, treatment of cells trt ~ ro prior to 35 ~ ,u~lioll with an alptla-~ rtoe~ cr (e.g., col-'fee beam alpha-g~ tnCi~i lee commercially aYailable f'rom Sigma Chemical Co.. St. Louis. MO) removes surface alpha-galactosyl epitopes. I;ollo~-ing treatment, remoial ofthese epitopes t'rom the celi suri:dce can be assessed, for example, by neacting the cells, with a labelled lectin specific for the alpha-galactosyl epitope (e.g., fJritfonia simplicif{)i t, or GrS- I, lectin; commercially available l'rom ~ W0 9s/33~28 2 1 ~ ~ $ !~ 1 P_ ~ I.JL . , EY Labs) and assessing the arnount of bin.iing . f the labelled lectin to the~ t}eated cells compared to untreated control cells ~see Example 1), As described in the Examples, it has been found that CS-I binding activity on the surface of porcine e~ndothelial cells is ble after aipha-fgpl~rtoei~1nep treatment. Moreo~er, it has been found that alpha-S galactosyl epitopes on the cell surface are not r~ d t'or several hours after treatment and that even 48 huurs after treatment, GS-I binding activity is still diminished by 60%.
Thus, alpha-ga~ treatment of cells is suff cient to remo~ e surface alpha-galactosyl epitopes and this treatment leads to prolonged diminution of expression of these epitopes on the cell surface. FU~LL~ IOI~ aiphu c,.l,~ iA~eP treatment greatly (i.e., >90%) inhibits natural antibody-mediated (human or monkey). . .~ a dependent Iysis of the porcille endothelial cells (see Example 3). In addition to alpha-p.~l~. l. ci.i~e~P treatment, other ~,fllkullydlal~ moieties can be cleaved by a glycosidase havillg specificity for that moiety.
Altematively, a chemical treatment v,hich remo-es one or more specific carbohydrate moieties, while retaining cell viab;lity and fullction. can be Llsed to r. move natural antibody epitopes from the surface of a cell.
To remove cell-surface natural antibody epitopes, a cell is treated v~ith an ammmt of enz~ me (or chemical) and for a period of time sufticient to reduce or substantially eliminate expre~ssion of the epitope on the cell surface such that upon tr~nipl~nt~tion of the cell into a recipient hyperacute rejection of the cell is inhibited. Appropriate dosages and digestion times may vary depending, fbr example, upon the cell type being treated and the type of digestion reagent used. Appropriate digestion conditions can easily be detemmined by one skilled in the art according to the teachings of the invention. A non-limitiny example of digestion conditions for removal of surface alpha-galactosyl epitopes is 500 milliunits of coffee bean alpha-p,~l ~rt~ e~ (Sigma Chemical Co., St. l,ouis, MO) per I x I o6 cells for 2 hours at 37 ~C in a buftèr of 200 ml l sodium acetate in phosphate buffered saline (PBS) (pH
5 fr~).
In another r~ o~ of the in-!ention~ expression of a cell surface natural antibody epitope i9 reduced or substantially eliminated by inhibiting or preventing formation of the epitope on the cell surtàce, e.g., by interfering ~ith the synthesis of the epitope. For example, the activity oi' an enz~me v~ithin the cell which is necessary for formation of the epitope can be inhibited. Carbohydrate moieties are typic:!lly attached to glycoproteins or glycolipids by specific~ u~ lall~f~la~s Thus,expressionofa l~aIbUIIYdI-t~ epitopeonacellsuriàcecan ke reduced or substantially elirminated by inhibiting the activity of a glycosvltranst'erase invol~ed in the s~ nthesis of the e~pitope. For example, the enzyme responsible for attaching g~lactose in alpha linkage to an underlying chain of sugars on both glycoproteins and ~ glycolipids is UDP galactose alpha-l~3-L~aLlctu~lLlall~rerase (also referred to herein as alpha-galactosy lllall~la~) The enz,~me substrale specificity and kinetics of this enzyme have beenstudied(seel~lices,M.J.andGoldstein,l.J.(1989~J.Biol.Chem.~:1375-1380;amd Joziasse, D.H. et al. (1987) J. Biol. Che~il. 262:2025-2033~ and the gene for the enzyme has _ _ , ,, , .. .... ...... _ . . . ....... . .. .

WO ~s/33X28 9 ~.~, 9 ~

been cloned frclm bo-ine (.To7iasse. D.H. et al. (1989).J. Biol. C'hetn. ~ 14290-14297) an~
murine ~I,arsen, RD. ei al. (19893 Proc. ~'a~ Icrrrl. .Sci. lJ~S~ 86:8227-8231~ tissues. A gene is present in humans that displays . o .~ ,lc homology to the murine gene, but, due to a frameshift mutation and several nonsen~se mutations in the human counterpart of the murine gene (l.arsen7 R.D. et al. (1990) J: BioL ~hem. 265:7055-7061), the active enzyme is not s,vlltllesi7.ed in humans suld Old Wurld monkeys. .4s a result1 this carbohydrate epitope whicb is recognizRd by the natural antibodies in human serum is not normally pre.sent on hurr~n cells.
Inaprefenredr,l,l,u.l;",..,l,theactivityofagly~osyl~ r~ ,e.g.analpllrl-l O galactu~y It~ .f..~ is inhibited by introducing into a cell a nucleic acid which is antisense to a regulatory or coding region of tbe glycos~r lLIdl~ . gene. thereby repressing transcription of the gene or translation of the mRNA. An "antisense" nucleic acid molecule comprises a nucleotide sequence which is . .",.~ y to a coding str&nd (i.e., sense strand) of another nucleiC acid~ e~g~ " ,~ y to an mRN~A sequence~ constnucled 15 according to the rules of ~ratson and Crick base pairing~ and thus crm hycirogen bond to the sense strand of the other nucleic acid. An antisense nucleic acid can fonn a duplex witll an mRI~rA strdnd and pre~ent its eff~cient translation. Additionally, antisense mlcleic acids may increase RNase-mediated llPgr.qrl: tion of mRNA and or hlhibit splicing of pre-mRNA. An antisense sequence can be ,~ ., ... ,n.. y to a sequence found in the coding region of an 20 mRNAorcanbeir",.;.l-.,...S~-~ toa5~or3~ n~ dregionofthemxN~4.. Toinhibit translation, the antisense nucleic acid is preferably ~ L ,.- . ,1 y to a region preceding or spauming the translation initiation codon. Altennatively, an antisense nucleic acid crm bhld to [)l~h4 to fonn a triple heliY and prevent gene I I ,.., ~. . il ,1 ;.1, (see e.g., Stein, C.A. and Cheng Y-C. ( 19'93 ~ .S~cience ~1: 1004-1012). Thus, ~m antisense nucleic acid cdn be complernental y in sequence to a regulatory region of a gene encoding a glycos~ l ""~r~ for instance cl~mrlr mr n~nry tc~ a ~ .Liun initiation sequence or regulatory element (e.g., promoter or enhancer sequence). F or a discussion of tùe regulation of gene expression using antisense genes see Weintraub. E~. et al., Antisense E~NA as a molecular tool for genetic anaiysis, Rel iews - ~e~ld.s ir7 Genetics, Vol. 1~1) 19~6.
In one ~.llbu~ t~ a nucleic acid which is antisense to a regulator~ or coding region of a glycosylkallsferase gene (e.g., alpha-~dla~luayl~ld l~f~ e) is an oh~,ul~u~lcuLide.
T ypically, ~ l;rlr~q between about 5 and 50 nucleotides in length are used. More preferably. oligr-~rl~ otirTr c heh~een about S and 35 nucleotides are used. F-en more preterably. oli~on~ lr~-tirl~q about 20 nucleotides in length are used. An antisense oligonucleotide can be constructed using chemical synthesis procedures known in the art. An oligonucleotide can be chemically s,~nthesized using naturally occurring nucleotides or ~ ariously modif ed nucleutides designed to increase the biologic~l stability of the oliTrronlmlr;~otirlr~ortoincreasethephysicalstabilityoftheduplexformedbehseentheantisense and sense~ nucleic acids. For exarnple, pl.o~t h~ L~ t~, methyl ~7hncrhnn~t~ and _ _ _ _ ., . .. .... _ .. .. ...... .. . .. .. . _ . .. _ .. _ _ .. .

~ WO YS/338~X ~ 9' .~ ;
_ 9 _ ethyi pl~ , ;. .t~ . 7mtisense oligonucleotides (revie~Yed in Steill . C.A. ~md Cheng Y-C.
(19g3) Science 261:1004-1012) are within the scope ofthe inventiom Additionally, acridine substituted nucleotides can be used in the~ antisense oliy.,.."~ ofthe invention.
Antisense oli~u~ cl.uLid~ can be used to inhibit the activity of a glycos;ylllal.arcld~. in a cell 5 by incubatiny them with the cell ir vitro and!or ~ ;,,g them to a subject at an amount and for a thne period sufficient to inhibit tr;~nq~ ~iE tion of the gly.~ dll~r~ld ~ gene or translation of the L~ly~ ylL a--~r~la~ mRNA in the cell (see E~ample~ 4).
In another ~ bvli~ L, an antisense nucleic acid is produced biologically wsing an expression vector into which a nucleic acid has been subcloned in an antisense orientation 10 (i.e.7 nucleic acid transcribed from the inserted sequence ~ill be in an antisense orientation relative to a target nucleic acid of interest). The antisense expression v ector is introduced into cells, for example. in the form of a recombinant plasmid, phagemid or attenuated v irus in which antisense~ nucleic acids are produced under the control of a higll ei'ficiency regulatory re~gion of the vector, the activity of which can be determined by the cell type into which the 15 vector is hltroduced. Pret'erably, the lulllb;lldllL e~pression ~ector is a l;~ollll/;llallL viral vector, such as a retroviral, adenovirdl or adeno-associated viral vector. Protocols for producing l~.vlllb;llalll retroviruses and k>r intccting cells ;n titro or ir l~iVO ivith such viruses can be found in Current Protocols in Molecular Biolo~v, Ausubel. F.l~/l. et al. (eds.) GreenePublishingAssocidtes,(1989).Sectiolls9.10-9.14andotherstandardlaboratory manuals. Exarnples of suitable I~Lluvil~ include pLJ, p7.1P, pWI' and pEM which are well known to those skilled in the art. E,xamples of suitable packaging virus lines include lllCrip, ~C're, ~2 and yn~m. Adenoviral vectors are described hl E~erkner et al. ( 1988) B;oTecl7riques6:616:Rosenfeldetal.(1991)5cterce252:431-434:andRosenfeldetal.
(1992) C'ell G8:143-155. Suitable adenoviral vectors d~rived from the adenovirus strahl Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2~ Ad3, Ad7 etc.) are well known k~ those skilled in the art. Adeno-associated vectors (A.AV) are reviewed in Mu7.yc7~a et al. C'urr.
Topicsil7,~Jicro. andlmmunol. (19Y2) 158:97-129). AnexampleofasuitableAAV vectoris described in Tratschin et al. (1985~11ol C'ell. Biol. 5:3251 -326U.
A .. . ~,. . .1.; ., ~, .1 expression v ector containing a nucleic acid in an antisense orientation is introduced into a cell to generate antisense nucleic acids in the cell to thereby inhibit the activity of a y~ly~v ~ r,;laSe in the cell. Th- ~!ect(lr can be introduced into a cell bv a convent;onal method i'or introducing nucleic acid into a cell. When a viral vector is used, the cell can be infected uith the vector by standard techniques. Cells can be infected ;t) ~!itro or ~ in ~ o. ~ hen a non-viral vector, e.g.~ a plasmid~ is used. the vector can be introduced into the cell by. for example. calcium phosphate ~u~;LJiLaLiu~l, DE.AE-dextran l cl~LIv~ laii~Jll or other suitable method for transfection of the cell.
In yet another PmhoriimPnt an antisense nucleic acid used to inhibit a glycosyltranslerase activity in a cell is a ribozyme ~hich is capable of clea- ing a single-stranded nucleic acid encoding the ~Iy~ ylLIdll~ la~c, such as an mRNA transcript. A

~VO gSJ33B2~ 9 ~ 8 ~ .~,7.1 --catalytic RNA ~ribozyme) ha~ ing ribonuclease activity can be designed which has specificity for an mRl~'A encoding a glycosyitr.3ncf rr!s- such as an alpha-~al~ I n ,.. .~rr, ,.~ For exatnple,riderivativeofaTetrahymenaL-191VSRNAcanbeconstructedinv~hichthebase sequence of the active .site is c~ y to the base sequence to be cleaved in an alpha-S ~aL~ o .yl~ aÇc~a~c mRNA. See for example Cech et al. [J.S. Patent IYo. 4.987,071: C~ecll et al. lJ.5. Patent No. 5~116,742 for d. ~ ., .c of designing ribozymes. Alternatively, an alpha-~$ala~u~lLia ~C.a ,e RNA can be used to selecL a catalytic RNA having specific libu~lucl.,dse activity against the alpha-galactua~lllallarclaac RNA from a pool of'RNA
molecules. Seeforexample.Bartel,D.andSzostak,J.W.5'cience~.:1411-1418(1993)for 10 a desc}iplion of selectine ribozymes. A ribozyme can be introduced into a cell by l,~JIIaLI u~ Lh~g a ~ c . . ." .1 .in .1 expressian vector (e.g., a viral v ector ss discussed above) containing nucleic acid which. when transcrihed~ produces the riboz.~me (i.e., [)NA encoding the ribozyme is cloned into a Ic~ LlJ;l~ l expression vector by conventional techniques).
A prefenred antisense nucleic acid of the invention is antisense to a codine or 15 regulatory region of an aipba-gala~.u~ylL~ .c. ai,~, gene. Antisense oligonucleotidcs. or an antisense Ic~ulllb;llallL e~pression vector can be designed based upon nucleotidL sequences of alpha-galactos~,lL allar~;.aac cDNAs known in the art. The nucleotide sequellce of a murine a-I ,3-galactosyltransferase cDNA is disclosed in LarserL K.D. (1989) Proc. PiafL ~ cad. .~cf.
l:l.S'~,6,:8277-82i!. Thenucleotidesequenceofabovine~-1,3-gald,lu:"yll-a.-,rc-~cDNA
isdisclosedinJoz.iasse,D.H.etal.(1989).. /.Biol.C~Iem.~dL:14290-14297. 'I'oinhibitthe acti~ityofanaipha-gaiactos~lLlalaf~laac~inacellfromaspeciesotherthanmollseorcc~w(e.g., pig). antisense c.~ u~levLid~a are designed which are i..".~ y to nucleotide sequences that are consen~ed among alphrd-gala~lua~ Il-a.~ a~e genes in difierent species (e.g.. based upon comparison of the murine and bo- ine sequence.s to identify conserved 25 regions). Additionally, the known murine and bovine cDNA sequences can be used to dcsign hybridization probes or PCR primers which allow isolation oi'cDNA and'or genomic DNA
clones oi' alpha-galactuayl l l u l l~rrl ~ from other species (e.g., pig) by str~uldard techuliques.
An antisense nucleic acid for use in the inventioll can then be designed based upon the nucleoLide sequence of a cDNA or genomic DNA fragment so isolated. Suitable antisense 30 olig- mlcleoti-t- c i'or inhibiting the acti~ ity of an alpha-galactos~ lilallaLlaac in a cell, designed based upon tne murine and bo~ine alpha-galactosyltransferase cDNA sequences, are described hl Exaunple 4 and showll in SEQ ID I\:'OS:1-6.
Alternati~e to antisense nucleic acids, the activity oi'a glycoaylLlallaklaa~: in a cell can be inhibited, for exarnple~ by use of a competitive mono-, di- or ol iL u, . .1. ~ inhibitor 35 or other form of chemiclll inhibitor of the en7yme. A mono-, di~ or oligl .c~ hich mimics the carboh~drate moiety that is transferred by the transÇerase en~yme but which cannot be attached to glycoproteins or,glycolipids can be added in excess to cells lo ~: ul,.~,~ LiLi~ y inhibit the activity of the glycosylLlallaL~ ; in the cells~ For example, an alpha-1,3-galactosyl transferase in cells can be inhibited by incubating the cells in culhlre 21 gl 8gl ~ , I I
with soluble a-methyl-D-galdctosid e ora-Gal(1,3)j~-Gal(1,4)Glc. Alternatively, anon-ua~ dl.,~,chemicalinhibitoroi'at~l~c~\s~lLIall~l;l~ccanbeused. An invit70 assaycan beusedtoscreenforchemic.alinhibitorsofa.specificglyco~lilal.~r~,a~e. Forexarnple.an inhibitor of an alpha-~ala~ slèrase can be identified based upon tlle ability of the 5 compound to inhihit the transfer of galactose to an N-acetyll ~ toer-mine acceptor using a procedure such as that described i n Cummings, R.D. and .Matto~, S.A. (1988) .1. BioL Cl~em.
263:511-519. TOinhibittheacti~ityofanalpha-galactù~ fela~einacellcapahleof expressing alpha-galactosyl epitopes, a chemical inhibitor so identifed can then be contacted with the cell.
An alternative approach to inhibiting the acti ity of an alpha-~ala ~'O ~yllldll~r~ld~ in a cell is to mutate or substantiall,v eliminate the gene encoding the enzyme in the cell, such as by hol~lologvu~ lcuulnbil~ ILion. thereby preventing expression of an alpha-galactù:,ylL a~ f~ a~e in the cell. For exarnple, a homologous 1~ ~I . ,I .i " . . ,t animal can be created in which the alpha-galacto~ all~rela~ gene has been mutated or disrupted. The 15 term l homologous l~uull)l,illal.t animalll as used herein is intended to describe am animal containiny a gene ~ hich has be n moditied by homologou.s l~.ullll,illaLion bet-~een the gene and a DNA molecule introduced into an mbryonic cell of the animal. To create a homologous l uulllbillallL animal. a ector is prepared which contains a portion of the alpha-galacto~yllld Isf.,~ , gene which has been mutated or disrupted, flanked at its 5' and 3 ends 20 by additional regions of the alpha-t ,~1 los~ r~la~ gene of sufficient length for successful homologous IC;C )lub;llaliull to occur between the mutated gene contained within the-ectorandanendogenouswild-t,~pealpha-~dlacto~ylllal.~r~làs;gene. Typically,severâl kilobases of flanking DNA (both at the 5~ and 3 ends) are included in the vector Isee e.g Thomas~lC.R.andCapecchi,h,l.R.(1987,1C'ell51:503foradescriptionol'homologous recombination ~-ectors). I he vector is introduced into an embryonic stem cell line (e.g., by ch,~Llu~Julaliull) and cells in which the introduced DNA has homologously l olllbilled witl the endogenous gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryo7tic Stem Cells. ,4 Practic~al ApproaciT, E .J. Robertson~ ed. (IRL, Oxford, 1987) pp. 113-157). A chimeric embryo can then be implanted into a suitable ,U~UdU~ female foster animal and the embryo brought to term. Progeny harbouring the homologously ~u,uml~ l DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA. Such a homologous l~ulllbhlallL animal, in which an alpha-galactosyltransferase gene has been mutated or disrupted, can be used as a source of donor cells tor ~ ; .n tllat have a reduced capacity to stimulate hyperacute rejection ot'the cells in a recipient.
In yet another ~IIIbUdilllC:)II of th in ention, expression of a cell surfàce natural antibody epitope is altered by binding a molecule to the epitope such that subsequent binding ,, ,, , _, , , _ ,, wogsJ33828 2t9~ 12- r~l,O.,7.~"~ ~--of natural antibodies to the epitope in a recipient is inhibited (i.e.. recognition of the ep;~ope hy natural antibodies hl a recipient is blocked or obscured by the binding of another molecule to the epitope). For example, a lectin which binds to a specific call~vhy~ilaL~ epits~pe can be used to alter that epitope on tne surface of a cell prior to l. .l, .~ ;nn A preferred lectin 5 for altering all,b~ oct~eyl epitopes is the Grtf~o~za sir7~plfclfoia 1 lectin ~referred to herein as GS-I) which specifically binds to this ~albullydlaL~ structure. GS-î lectin is commercially available from Vector l.~hor~t~-rif e ~3mling~m~ CA. Other lectins ~hich specifically recognize ~,~llbully~ Lr~ moieties are kno~hn in the art. A cell to be tr~nTlqntf d which expresses alpha-Lalactosyl epitopes can be incubated pric~r ~o L~ n with GS-1 lectin 10 in an amount and for a period of tirne sufficient to alter cell surface alpha-galactosyl epitopes such that, upon u .., ~1 ! s ~ n, binding of natural antibodies in the recipient to the epitopes is inhibited.
Alternatively, an antibody, or frapment thereof, which binds to the epitope but does not activate cu~ lll or induce Iysis of the cell in a recipient can be used to alter the 15 epitope c)n the cell surface. Such an antibody, or fragment thereoL ~ul~ ..iLi~/rly inllibits the binding of natural antibodies to the epitope Polyclonal antibodies or, more preti,rabh, .
monoclonal antibodies can be used. A mouse Illulluclvnal antibody, termed Gal-l 3, which .specifically recognizes alpha-galactosyl epitopes has been described in the art (see Galili, U.
et al. (1987) .J. Bzol. ~ em. 262:4683~688~. Monoclonal antibodies specific for alpha-20 galactosyl epitopes or other ~ huLydl~ epitopes can be prepared by standard techniqueskno~n in the art. Preferablv, an F(ab')2 fragment specific for the epitope is used to alter the epitope, therehy avoiding activatiou of ...,.,1~1..I....1 in a recipient. F(abl!2 fragments can bc prepared from intact antibodies by conventional techniques. such pepsin treatment. In a standard procedure for generating F(abl)2 fragments, intact ant;bodies are htcubated ~vith 25 immobilized pepsin and the digested antibody mixture is applied to an immobilized protein A
column. The free Fc portion binds to the column while the F(ab')2 fragme1tts passes through the column. 'l'he F~ab')2 fragments can be further purified by HPI.C or FPI.C. F(ab')o fragments can be tteated to reduce disulfide bridges to ptoduce Fab' fragments. A cell to be l ,,l, ,.~ ,I,~. .f~ .I can be incubaLed prior to U ,., .~ n ~ith an antibody~ or fragment thereoE, 30 which binds to a cell surface natutal antibody epitope in an arnount and for a period of t;me suff1cient to alter the surface epitope such that, upon u O . ~ 1 . . 5 . . ;( 1l l binding of natural antibodies to the epitope in Ihe recipient is inhibited.
l'reatment of a cell to be I "" ~ .I r.7 (or treattnent of cells within a tissue or organ~
accurding to one of the above-described approaches results in altered, reduced or 35 substantially eliminated expression of at least one cell surface natura7 antibody epitope. I'he degree of reduction of expression of the epitope on the cell surtace (e.g., by treatment with a glycosidase ot a nucleic acid which is antisense to a ~ly~u~yllla~f~ se genei C~l be determined by assessing the amount of the epitope expressed on the cell surface fclllowing treatment. This can be accomplished, t'or example, by incubating the cell after treatment with _, _ . . , . .. . . . _ _ . .. . _ _ ... .. .. . .. . ... . .. .

~ WO9~133828 21~189~ ~; t'I

a labelled lectin or antibody which specifically re.coggnizes the epitope on the cell surface and assessing the amoumt of labelled lectin or antibody bound to the cell compared to umtreated control cells. A fluoroisothiocyanate-labelled CiS-I lectin (~,~,.m~ lly available from Vector l.aboratories. Burlingame, CA) can be used to assess the level of ~xpression of alpha-S galactosyl epitopes on the surface of treated cells prior to 1. ~ a ;~ , such as described inExample 1. Preferably, the le~vel of expression of the epitope is reduced by at least 50-70 ~/o , more preferably at least 70-90%, and e~ en more preferably greater than 90% compared to untreated cells. In the most preferred ~ ho~l ~,. ,,I the expression of the epitope on the cell surface is substantially eliminated such that the presence of the epitope on the cell surface is 10 no longer detectable by standard techniques (at least temporarily. although the treatment may not result in permanent removal of the epitope from the cell surface), It may not be necessary to EJ~ alter. reduce or substantially eliminate the e~pression of natural antibody epitopes on the suriàce of ~ ,t- d cells in order to inhibit reiection of the cells in a host due to a 1,1, ~ ,. ,, " ,- " ~ that has been terrned ~- cnmmn is~f i-ln (tor 1~ ~ discussion see Platt, J.l.. et al. (1990) /rt~rntmolog! TodaL~ 11:450-456). It has been mon~t~ d that tempor~ depletion of natural antibodies from the circulation of a transplant recipient can be sufficient to enable prolonged survival of the graft, despite the eventual r~ .J~ of natural antibodies in the circulation (see Ale~andre, G.P.J. et al. in Xenografi 25~ liardy, M.A. (ed.) New Yori~ Elsevier Science Publishers, 1989. pp. 259-266;
and l'latt, J.L. et al. (1991) Trans/71antation '72:214-220). 'I'hus, temporary alterati~m, reduction or elimination of expression of the epitope on the surface of cells lor ion may be suOEcient to escape the init;al hyperacute response against the graftandkillingofthel,.."~l,l....l~lcellsbyanaturalantibody-mediatedm~h~ni~n- Inthe absence of this initial rejection episode7 ~ ~ ...."n~ may occur such that expression of 25 new epitopes on the cell surface, in the presence of natural antibodie~s in the host~ w ill not induce rejection.

1~1 Cellq for T~ ..,n,li....
One aspect of the invention relates to a modified cell suitable for 1. ~
30 Cells which can be modified in accordance ~ ith the methods include those which express at least one epitope on a cell surface which stimulates hyperacute rejection of the cell by natural antibodies when the cell is L~ l~lLed into a recipient. According to the invention, the cell is modified to alter, reduce or substantially eliminate expression of the epitope on the surfàce ofthe cell, thereby inhibiting hyperacuLe rejection ofthe cell when L~ led into a 35 recipient. ~xpression of the epitope on the surface of the cell is alte.red. reduced or substantially eliminated by one or more of the above-described treatments. Accordingly. in various ~mho~iim~nti the epitope is removed from the cell surface, the formation of the epitope on the cell surface is inhibited or the epitope is altered by contacting the celi prior to L. ~.. 1~l ,1~. .l..l i. .l l ~ith at least one molecule ~hich binds to the epitope.

. ........ .. . ... _ . , ~ .. ......... .. ....... .... . .. . _ .. _, . . . . .. .

w0~3sl33x2x ~ ,g~t~ Pt~lUS~10~973 Cells for use in thf's invelltioll encompass cell types ~hhich can be ~ d for ther~tpeutic purposes and which are capable of expressing on their surface at least one epitope ~hich is recognized hy natural antibodies in a recipïent. E~xarnples of such cells include endothelial c,e]ls~ hepatocytes, pancreatic islet cells. muscle cells ('including skeletal and 5 c.ardiac myocytes and myoblasts), fibroblasts. epithelial cells. neuronal cells! bone marro~
cells. h~ll.dlulJu;.tic cells and Iymphoid cells from nonprimate mammals and certaill prhl1ates (e.g.. prosimians and New Vv'orld Monkeys).
In a preferred embodiment~ the epitope on the cell ~ hicll is altered, reduced or substantially eliminated is a galactosyl~al-3)galactose epitope. Cells ~hich are capable of' 10 expressing alpha-galactosyl epitopes include cells from nonprimate mammals (e.g., pigsJ!
prosimiansandNewWorldMoltkeys(seeGalili.U.etal.(198g)J.Bio/.ChL~m.~ 17755-17762). Preferably. the cell is a porcine cell. In one i mhn~fim~ n1 expression of alpha-galactosyl epitopes on a cell surface is reduced or substantially eliminated by introducing into the cell a nucle;c acid which is antisense to a regulator,v or coding region of an alpha-15 galactosyl-transferase gene (e.g.~ a pig alpha-galactosylLIdll~ltla~- gene in a porcine c~''ll)! as descrihed above. Accordingly. the invention ~ c~LlllJa~ a cell which has been modified to contain such an anlisense nucleic acid, e.g., an . .1 i~ uti~lf' or a IC~UII!;;II~IIL expression vector (for example, a retroviral. adenoviral or adeno-associated vector).
Natural antibody epitopes clm be altered. reduced or substantiall, elimhtated on the 20 surfaceofadispersedpopulationofcellswhicharetobe l"~ ""f~cl intoarecipient.
Allernat;vely, a tissue or organ to be ~,al,~l,ldl"~,d can be treated to alter, reduce or substantiall~ eliminate the expression of natural antibody epitopes on the surface of c.ells ~vithin the tissue or organ. I or example. alpha-galaclosyl epitopes can be removed from the surtace of cells ~riLhin a tissue or organ by incubating the tissue in a solution contairling an '1~ alpha-g ~I f~ or by perfusing the organ v~ith a solution containing the alpha-galRrtocirfRcf A,lternatively, a tissue or organ can be contacted with (e.g., incubaled wi~h or perfused ~iith) an ~ nmmlt ntif.4 antisense to a t~4~u~ylfia~ d~ gene, or int'ecteci ~ith a viral vector containhlg nucleic acid antisense to a gly.,o"~ Ç.~d~ gene. to inhibit the activity of an alpha-gal.lcl~1sylL-d,L,ft-d~ in the cells withm the tissu- or organ. Accordingly, 30 Ihe invention is not only applicable to ~ n of dispersed cells. but also to l"~ "lai~llofintacttissuesandvi~holeorgans~suchasheart~liver~kidney.ltmg~pancrea stomach. intestines, skhl and muscle tissue.
E urther mn-lifil~ ~ti(mc ora cell ofthe invention in additioll to alteration. reduction or elimination of aL least one cell surface nahlral antibody epitope. are ~ithin the scope of the 35 invention. For example, ;n additiûn to modifying a cell of the invention to inhibit hyperacute rejection of tbe cell in Q recipient. the cell can also be modified to inhibit a cellular immunc response against the cell in a recipient Accordingl~, at least one antigen on theD cell surface ~ihich s~imulates a cellular immune response against the cell hl a recipient can be altered prior to l~al.~,ldllldlion. A preferred antigen on the cell surface to be altered is an ME~C class .. . .. .... . . . _ . .... . . . .. .. .. . .... . . . ... . _ . _ ~

~ w0 9sl338~x 2191 8 5 ~ I S 7 ' ~'' r~ s~ ~"J

I antigen. In a one ~ UlLllI..llt, the antigcn on the donor cell to be altered is an M~IC' class I
antigen.
At least two different epitopes on the sarne Mi IC' class I rmtigen on the donor cell can be altered prior to L~ ldlio~ IC class I antigens are present on almost all cell types.
In a normal imrnune response, self MFIC molecules function to present antigenic peptides to the T cell receptor (TC'R) on the surface of self T Iymphocytes. In imrnune recognition of allogeneic or xenogeneic cells, foreign M~C antigens (most likely together with a peptide bound thereto) on donor cells are recognized by the I cell receptor on host T cells to elicil an irnrnune response. Epitopes on an Ml IC class I antigen on a donor cell are altered to interfere with recognition of the MHC class I antigen by T cells in an allogeneic or .senogeneic host (e.g., portions of the Ml-IC class I antigen which are norrnally recognized by the r cell receptor are blocked or "masked" such that nomlal recognition of the MI IC class I ant;gen can no longer occur). Additionally, an altered fomn of an MHC class I amtigen which is e~posed to host T cells (i.e., available for ~ dli~n to the host r cell receptor) may deliver an ill~ U,UI;.1LC or insufficient signal to the host T cell such that, rather than stimulating an immune response against the allogeneic or xenogeneic cell. donor cell-specilic I cell non-is induced. For e.~ample, it is knov~n that T cells which receive an hl~ Jlu~Jl;dle or insufficient signal through their T cell receptor (e.g., by binding to an MIHC
antigen in the absence of a co~fimnl:~tnry signal, such as that provided by B7) become anergic rather than activated and can remain refractory to l~ l,u ;~ for long periods of time (see for example Damle et al. (1981) Proc. A'atl. .~cad. Sci. U~5'~ 78:5096-5100; Lesslauer et al.
(198fi) k'ur. ~ Immunol. 16: 1289- 1795; Gimrni, et al. ( 1991 ) Proc. .~latl. Acad. ~Sci. U5'A 88:
6575-6579; Linsley et al. (1991) J. E,YP. Med. 173 :721 -730; l~oulova et cl. (1991) J Exp.
~ed. 173 :759-762 Razi-Wolf, et al. (1992) Proc. A'atl. Acad. SCL USA 89:4210-4214).
Alternativ, e to Ml IC class I antigens, two or more epitopes on other surface antigens on donor cells can be altered. For e~ample. epitopes on Ml IC class Il antigens can be altered. Similar to ME-IC class I antigens, MHC class 11 antigens funclion to present an~igenic peptides to the T cell receptor on T Iyrnphoc~tes. llowever, ME IC class 11 antigens are present on a limited nurnber of cell types (primarily B cells, ll,a~l.",l,ag~" dendritic cells, Langerhans cells and thymic epithelial cells). In addition to or alternative to MHC~ antigens, epitopes on other antigens on a donor cell whicll interact v.~ith mole~cules on host T cells and which are known to be hl- ol- ed in immunological rejection of allogeneic or xenogeneic cells can be altered. Other donor cell antiyens kno~vn to interact with host T cells and to contribute to rejection ol a donor cell include molecules which function to increase the avidit,voftheinteractionbetweenadonorcellandallostTcell. Duetothispropert~,thesemolecules are typicallv referred to as adhesion molecules (although they may ser-e other functions in addition to increasing the adhesion beh~een a donor cell and a host T cell).
Examples of preferred adhesion molecules which can be altered according to the inventioll include l F A-3,1CA:M- I and ICAM-2. These molecules are ligands for the CD2 and LFA- I

wo~s,r33828 .~,~ 9~8~ u J~ J

receplo}s, respectivel5a on T cells. By alterhlg an adhesion molecule on the donor celt, (9u~'h as LFA-3. lCAh'.-l or similarly functionirg molecule), the ability of the host's r cells tc bind to and interact with the donor cell is reduced. Both L.FA-3 and ICAh~l l are found on endothelial cells v~ithin blnod vessels h1 tr~mcrlqnt d organs such as kidney and heart.
5 Altering these antigens may facilitate n " ~ ,ca i. .l~ of any vascularized implant~ by pre~enting recognition of those antigens by CD'~ and LFA-I+ host T-lymphoc~tes.
The presence of MT IC molecules or adhesicJll molecules such as l.FA-3, I'C'AM-~ etc.
un a particular donor cell can be assessed by standard procedures known in the arL. For exa-nple, the donor cell can be reacted with a labeled antibody directed against the molecule 10 to be detected (e.g., ~,~C molecule, ICAM-1, I,FA-I etc.) and the association orthe labeled anlibody ~vith thf cell can be measured by a suitable technique (e.g., ;...,...l...,I,;~l.~f~hPmi~tr~
flc~w c~tometry etc.).
A prei'erred method for altering at least two difterent epitopes on an antigen on a donor cell to inhibit an immune response against the cell is to COlltQCt the cell ~ith at least l i two dii'ierent molecules which bind to the epitopes. It is preferred that the cell be contacted with at least two different molecule~s w hich bind to the different epitopes prior to administering the cell to a recipient (i.e., the cell is contacted with fhe molecule in ~ itro~. For e:Yample, the cell can be incubated ~ith the molecules which bind to the epitopes under conditions whicll allow binding of the molecules to the epitopes and then any unbounù
20 molecule.s can be remo~ed (such as described in the T YPmrlifir~tif)n to foliow). I'olk~wing f~,~ of the donor cell to a recipient7 the molecules remain bound to the epitopes on thc surface antigen for a sufficient time to interfere with immunological recognition by host cells and induce non-responsiveness in the recipient.
Preferably, the molecule for altering an epitope on a donor cell is an antibod y. or 25 fMgment or derivative thereof which retains the ability to bind to the epitope. For use in therapeùtic applications~ it is necessary that an antibody which b~nds the epitopes tu be altered be unable to fix c~ thus preventing donor cell Iysis. Antibody (-omrl~m fixation can be prevented by deletion of an Fc portion of an antibody, bv u.sin~ an antibody isoh~ pe which is not capable of fi.~cing ~ or, less preferably, hy using a ~ . l lrl ll fixing antibody in conjunction with a drug which inhibits ~ .. l .i.. 1 fixati~
Allernatively~ amino acid residues within Lhe Fc region of an antibody vihich are importanl foractivating-..,..l~l .,...,l (seee.g.,Tanetal. (1990)Proc. Natl. Aca~. S<,~ 87:162-166 I)uncan and willter (1988') I'v'ature 33r~: 738-740) can be mutated to reduce or eliminate the compleme~llt-acti~alillg ability of an intact antibody. Likewise. amino acids residues withill 35 the Fc region of an antibody which are necessary for binding of the Fc region to Fc receptors (seee.g.Canfield,S.M.andS.L.Morrisoll(1991!.1.F.xp.Ale~l.173:1483-14~,l:andL~md,J, etal.(l9~1)J.lmmunol. 147:2657-266~jcanalsobemutatedtoreduceorelilllillatel;'c r-c-ptor binding if an intact antibody is to be used.

... , ... ...... .. _ . _ _ _ _ ~ 2I~89,~
wo ss~33x2x A preferred antibody Iragment for aliering an epitope is a F(ab')2 fragment.
Antibodies can be fragmented using conventional techniques. For example. the Fc portion of an antibody can be remo-ed by treating an intact antibody ~ith pepsin, thereby generating a F(ab')2 fragment. In a standard procedure for generathlg } (ab')2 fragments, intact antibodies 5 are incubated v~ith i,~lmui,;li~.~f pepsin and the digested antibody mixture is applied to an immobili~d protein A column. The free Fc portion bhlds to the column w hile the F(ab')2 fragments passes through the column. The F(ab')2 f'ragments can be further purified by IIPLC or FPLC. F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab' fragments.
An antibody, or fragment or derivative thereof, to be used to alter multiple epitopes on an antigen can be derived from polyclonal antisera containing antibodies reactive v~ith a number of epitopes on the antigen. IV[ore preferably, however, hvo different epitopes on the same antigen are altered using t~o different ~ JIlocl~1llal antibodies ~hich bind to h~o different epitopes on tne same antigen (e.g.~ an MHC class I antigen). Polyclonal and 15 monnr.lnnrll antibodies which bhld lo diff'erent epitopes on one or more antigens can be prepared by standard techniques kno~n in the art. For example, a mammal. (e.g., a mouse, hamster, or rabbit) can be immunized ~vith an antigen (e~.g., an ~rlHC class I antigen) or with a cell which expresses the antigen (e.g., on the cell surface) to elicit an antibody response against the antigen in the mammal. Alternatively, tissue or a whole organ which expresses 20 the antigen can be used to elicit antibodies. The progress of hlllll...l;~a;..l, can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other h,.. I.ln,~,3 y can be used with the antigen to assess the levels of antibodies. Following immlmi7~rion antisera can be obtained and. if desired, polyclonal amtibodies isolated from the sera. To produce mnnor~ nrll antibodies, antibody producing cells (Iymphocytes) can be harvested 25 from an immunized animal and fused v,ith myeloma cells by standard somatic cell i'usion procedures thus h~ i-lg these cells and yielding hybridoma cells. Such techniques are ~ell kno~n in the art. For example, the hybridoma technique originally developed by Kohler and Milstein ((1975) Nature 256:495-497) as ~vell as other techniques such as the human B-cell hybridoma technique (~:Cozbar et al., (1983) Immunol. Today 4:72), and the EBV-30 hybridoma technique to produce human ~ l/lOCIulldl antibodies (Cole et al. (1985)Mv~locloilal Antibvdie~ in ( ancer ~hrera/7y Allen R 131iss, Inc., pages 77-96) can he used.
Hybridoma cells can be screened irlrlllu~ h~ rlly for production of antibodies specifically reactive v~ith the antigen and mnnrrlrnr~l antibodies isolated.
Another method of generating specific antibodies, or antibody fragments, reactive 35 against epitopes on an antigen is to screen expression libraries encoding immlmoglnhulill gene~s, or portions thereof, expressed in bacteria ~ith the antigen !'~r a portion thereof). ~or example, complete Fab fragments, VH regions, F~v~ regions and single chain antibodies can be expressed in bacteria using phage expression libraries. See for exarnple ~'ard et al., (1989'1Nafllre 341:544-546;i~usr etal.,il9g9)5cir~nce246:1275-1281;and:McCaffertyet . . .

9 ~ ~
~oss/33s2~ /u.,,~.~s, al. (l 990! Nature 348.552-j54. Altematively, the SC~ T-hu mouse can be used to produce antihodies, or fragments thereof' (available from Genpharm). Antibodies of the appropriate bind;ng specificity which are made by these tec}miques can be used to alter an antigen on a donor cell.
An antibody, or fragment thereof. produced in a non-human subject can be recoynized to var" ing degrees as foreign when ~he amtibod~ is ad~ t~ d to a human subject (e.g.~
~hen a donor cell with an antibody bound thereto is ddlilill;a~ d to a human subject) and an immune response against the antibody may be generated in the subject. One approach for minimizing or eliminating this problem is to produce chimeric or hullIallized antibody 10 derivatives, i.eantibody molecules comprising portions which are derived from non-human antibodies and portions which are derived from human antibodies. Chimeric antibody molecules can include, fclr example, tne antigen binding domain from an amtibody of a mouse, rat, or other species, v~ith human constant regions. A variety of approaches for making chimeric ant;bodies havc been described. See, for example. ~qorrison et al.. Proc.
.~atl. .4CCJd. .5il. li.. 5.A. 81, 6851 (1985): Takedaetal.. ~at~lre 314, 45 (1985~, C'abilly et Qh.
U .S. Patent No. 4,816,5fi~; Boss et al., l.bS. Patent No. 4,816.397; Tanaguchi et al., European l'atentPublicationEP171496;EuropeanPatentPublicationO173494,lJnitedl~ingdomPatent GB 2177096B. For use in therapeutic ~ rl~, it is preferred that an antibody used to used to alter dif~erent epitopes on an antigen not contain an Fc portion. Thus, a humallized 20 F(ab')2 f'ragment in which parts of the variable region of the antibody, e~special]y the conser~ed t'raunework regions of the antigen-binding domain, are of human origin and onl-the hyperi ariable regions are of non-human origin is a preferred antibody derivative. Silch altered immunoglobulin molecules can be produced by any of se- eral techniqlle.s known in the art, (e.g., Teng et al., Jlroc. Natl. .4cad. Sc~. U..SIA. 80, 7308-7312 (1983~; Kozbor et al., i~ L~nol0~ 70dav, 4, 7279 (1983~; Olsson et al., A,~eJh. En~mol., 92, 3-16 ~19821), and are preierably produced according to the teachings of PCT Publication ~ 09~.~06193 or El' 0~39400. E-lumanized antibodies can be cornrnercially produced by, ~or example, Scotgen l.imited, ' l lolly Road~ l'wic~;enharn, Middlesexs Great Britam.
The ability of' two different monoclonal antibodies which bind to the same antigen to 3() bind to dift'erelIt epitopes on the antigen can be determined using a .u~ ,.iitiull binding assay as described in the F~nnplifir~tion Briefly, one mrnr,rTrnrl antibody is labeled and used to stain cells wllich express the .mtigen. The ability of'the unlabeled second monoclonal antibod- to inhibit the binding of the first labeled monoclonal antibody to the antigen on the cells is then assessed. If the second monoclonal antibody binds to a dift'erent epitope Im the 35 antigen than does the first antibody, the second antibody will be unable to ~;ulll~Jcii inhibit the binding of the hrst antibody to the antigen.
Each of the cell surface antigens ha~ g h~o or more epitopes to be altered, e.g., the hil-lC class I antigens, Ml-IC~ class Il antigens,1 FA-3 and ICA~ l is well-. h~ t.~ d and antibodies reac~ive witll these antigens are commercially a~ailable. For example, an antibody wl~ gs/33828 ~ ~ g ~
_ 19 _ reactiYe with hurnan MHC class I antigens (i.e., an anti-llLA class I artibody)~W6/32~ is available trom the American Tissue Culture Society (ATCC EIB 95 ). This antibody was raised against human tonsilla} Iymphocyte membranes and binds to IILA-A~ EILA-B and Hl.A-C (Barnstable~ C.J. et al. (1978 ) Cell 14:9-20). AnoLher amti-MElC class I antibody which c.m be used is Pl 85 (see Davis. ~ '.C. et al. (1984) Hi ~r;~oma Technology i7Z
,~grictlltural and I 'etrinar~ Researc~ .J. Stern and I I.R. Gamble~ eds.. Rownmaul and ~ Allenheld Publishers, Totowa. NJ, pl21~ commerciaily available from Veterinary Medicine Research Development~ Pullman WA). This antibody was raised against swine leui~ocS te antigens (SLA) and binds to class I antigens from several dift'erent species (e.g ~ pig~ human, mouse, goat). An anti-lCA~r-l antibody can be obtained from AMAC, Inc., Maine.
Hybridoma cells producing anti-LFA-3 can be obtained from the American Type Culture Collection, Rockville, MarS land.
Two or more suitable antibodies. or fragments or derii atives thereof~ for use in the invention can be identified based upon their ability to inhibit an immllnolflgi~ RI rejection of allogeneic or Yenogeneic celis. Briel]y, the antibodies (omantibod5 fragments) are incubated for a short period of time (e.g.. 30 minutes at room t~ tUlC~ with cells or tissue to be trRnQplRntt rl any unbound antibody is washed away. The cells or tissue are then I ~
into a recipient animal. The ability of the multiple aullibody l~cLI~ l to inhibit or prevenl rejection of the trRnspl~nt- ~ cells or tissue is then determined by monitoring the function of 20 the graft and 'or by monitoring for sigr~s of rejection of the cells or tissue compared to untre_ted controls.
Other molecules uhich bind to an epitope on an antigen c n a donor cell and produce a t'unctionaily similar result as antibodies~ or fragments or derivatives thereof, (e.g., other molecules w hich interfere with the interaction of the antigen with a L~ tu~uic~ic cell and 25 induce illullullùlu~i~dl ~IUIIIC,~UII~ ) can be used to alter the epitope on the donor cell.
One such molecule is a soluble form of a ligand for an antigen (e.g., a receptor) on the donor cell which could be used to alter an epitope on the antigen on the donor cell. For example~ a soluble form of CD2 (i.e.~ comprising the f Ytr ~ r domain of CD2 without the C or ~;yL~LL;~ ;C domain) can be used to alter an epitope on l.FA-3 on the 30 donor cell by- binding to LFA-3 on donor cells in a manner analogous to an antibody.
Alternatively, a soluble forrn of LFA-I can be used to alter an epitope on ICAI~-I on the donor cell. A soluble f'orm of a ligand can be made by standard ~ n",l DNA
procedures, using a ,~,..,,l~il,...,l e.Ypression vector containing DNA encoding the ligand cll~u~ Jas:~illg only the ~ ~ar~ lllllslr domain (i.e., lacking DNA encoding the35 and e~lulJI~ ,lllic domains). The l~ ul~ ll eYpression vector encoding the extracellulal domain of the~ ligand can be introduced into host cells to produce a soluble ligand. which can then be isolated. Soluble ligands of u~se have a binding affinity for the receptor on the donor cell sut'ficient to remain bound to the receptor to interfere ~ith immunological recognition and induce non-l.~ s when the cell is adll,i"i ,t..l,J to a recipient (e.g., preferably~

. _ . .. . . _ .. ... , . .. . .. ... . . ... _ _ .

w0 9sl338~8 g ~
''' 2~g~ - 20 -the affin3ty for binding of the soluble ligand to the receptor is at least about 10-7 ~vr~.
Additionally~ the soluble 3 igand can be ;n the i'orm of'a fusion protein comprising t3ne receptc~r binding portion of the ligand fused to another protein or portion of a protein. For example, an innmnn~gl~huli!l fusion protein which includes an P~tr~PIl~ r domain, o7 S functional portion of CD2 or LFA-I linked to an imrnunoglobulin hea~y chain constant region (e.g., the hinge, CH2 and CT13 regions of a hurnan immnn~7Jlohnlin such as IgG I ) can be used. l~ yl~ .bulill fusion proteins can be prepared, for example, according to the teachings of C'apon, DJ. et al. (1989) ha~ure ~:525-531 and U.S. Patent No. 5,116,9G4 to C~apon and Lasky.
Another type of rnolecule which can be used to alter an MHC antigen (e.g., and 3~I},C
class 1 antigen! is a peptide which binds to the MHC antigen amd interferes with the interaction of the MHC' antigen with a T Iymphocyte. In one C~llbOlliUI~ , the soluble peptide mimics a region of the T cell receptor which contacts the METC antigen . This peptide can be used to intert'ere with the interaction of the intact T cell receptor lon a 'I~
Ivmptlocyte) ~ith t3~e 3\~1HC' antigen. Such a peptide binds to a region vf th:, M3 1(-' molecule w hich is speciiically reco~ni~d by a portion of the T cell receptor (e.g., the alpha- I or a3pha-2 loop of an Ml IC clas.s 1 antigen), thereby7 alterhlg t3~e MHC class I mtigen and inhibiting recognition of the antigen by the T cell receptor. In another ..lllbo.li~ L, the solubl- peptide mimics a region of a T cell surface molecule v.hich contacts the 3.~1EiC antigen, such as a 2U reyion of the CD8 molecule which contacts an MHC class I antigert or a region of'a CD4 molecule which contacts an M3 IC class 11 antigen. For example. a pep~ide uhich binds to a region of the alpha-3 loop of an MHC class I antigen can be used to inhibit binding to CD8 to the ~mtigem thereby inhibithlg recognition of the antigen by 1' cells. T cell receptor-deriv-d peptides have been used to inhibit M:HC class l-restricted irmnune responses (see e.g., Clayberger, C. et al. (1493~ ~ran.splan~ Proc. 25~477-478) and prok~ng allogeneic skin grat't sur~i~al i7~ l~ivo ~hen inJected ~ h~ llr~ y into the recipient (see e.g., Goss, J.A. et al.
(1993) Proc. IVatl. Aead. .S~ci. l,7SA 90:9872-9876).
It is preferred that an antibody, or fragrnent or derivative thereof, which is used to alter an epitope have an aiT;nity for binding to its target epitope of at least 10-7 1~1. The affinity of an antibody or other molecule for binding to an epitope on an antigen can be detennined by conventional tec'miques (see 7,~1asan. D.W. and ~rlllianLs, A.F. ( 1980~
I~oche~n. J. 187:1-10~. Brielly, the antibodyto betestedis laheled~ithll~S and incubated with cells expressing the antigen at increasing con~f ntrati~n~ until e~ln;lihrilmn is reached.
Data are plotted graphically as Lbound antibody]![free antibody] v ersus [bound ar,tibody; and the~ slope of the line is equa] to the kD (Scatchard analysis).
The same or diffcrent types of molecules can be used to alter hvo or more difii,rent epitopes on a donor cell Tn a preferred embodiment, two different antibodies (or fragments thereof) are used to alter two dif~erent epitopes. Alernatively, one epitope can be altered with one type of molecule and a second epitc~pe can be altered with another type of molecule. For ~918~
~o ~sl33828 example. ~o different epitopes on the same MHC' class 1 antigen can be altered using an anti-MHC class I antibodv amd an MHC'-bindillg peptide.
Alternative to binding one or more molecules (e.g., an antibodies) to epitopes on an antigen on a donor cell to inhibit ;,1,l,l"l,.,1~.~,;. ~l rejection ofthe cell, the epitopes can be 5 altered by other means. For example, epitopes can be directly aitered (e.g., mutated) such that they can no longer interact normally with a he~ tu~ùi~Lic cell (e.g., a r Iymphocyte) in an allogeneic or xenogeneic recipient and induces imrnllnnlllgit~ni non-~c~ to tne donor cell in the recipient. For exarnple, an altered form of am I~ C class I antigen or a&esion molecule (e.g., LFA-3 or ICA~-I ), in ~vhich tw-o or more epitopes are mutated, cam 10 be created by, 1 l ~ and selected in in vitro culture based upon the failure of the molecule to contribute to T cell activation. An altered from of an ivlHC class I antigen or adhesion molecule delivers an hl~ u~ or insufficient signal to a T cell upon binding to a receptor on the T cell. A nucleic acid encoding the mutated form of the antigen (i.e.~ the antigen ~ith mutated epitopes) can then be inserted hlto the genome of a non-human animal, 15 either as a transgene or by homologous I ~I~UIIIbhl~lliUII (to replace the clldùg~llOUS gene encoding the ~hild-type antigen). Cells from the non-human animal uhich express the mutated form of the antigen can then be used as a donor cell for ~ n~l ion into an allogeneic or xenogeneic recipient.
A cell ofthe invention which has a natural antibody epilope altered. reduced or 20 substantially elirninated can additionally be modified to express a gene product, such as a gene product to be provided to a recipient for therapeutic purpc ses. The gene product can be.
for example, a secreted protein, a membrane-bound protein or an intr~PIinlnr protein. Other gene products include acti-~e RNA molecules Non-lirniting examples of secreted gene products of therapeutic interest which a cell of the invention can be modified to express 25 include a I -amtitrypsin, apoA I, 'I~F, soluble TNF receptor, human growth hormone. insulin, IlL~/l)uil:Lill, anti-~ ,~ir~c.. ~ factors and i.. l.. ~hi.ls. For example. the secreted protein can replace a missing function in a subject (e.g., insulin in a diabetic subject) or can stimulate a respon~se in a subject (e.g., TNF or Il -2 can be produced in a tumor-bearing subject to stimulate an immune response against the tumor in the subject) Alternatively, the gene 30 product can be a membrane-bound protein In this case, the gene product remains associated with the membrane of the modit;ed donor cell and functions, fc r example, by binding a soluhle substance in a host (e.g., binding of l,DI, cholesterol by an LDL receptor) or b~
binding to another membrane-bound protein (e.g.. a receptor) on cells of the host to trigger a signal within the recipient cells. Non-limiting examples of membrane-bound gene products 35 which a cell can be modified to express include ùhe LDL receptor~ CFTR and CDI 3.
Alternati~ ely~ the gene product can be an ir i rnr~PI Inl~lr protein The intracellular protein within modif ed donor cells can be introduced hlto cells of a recipient by fusion of the donor cells to recipient cells (e.g.~ fusion of modified myc~blasts or myocytes with muscle cells within the recipient, e.g., to deliver dystrophin). An intracellular protein can also function bs .. . . . .

WO 9~/33~i28 ~2 1 9 i ~ PC'TIU8!~5~)5973 - 2~ -acting upon substances within a recipient that are tal;en up by the modified cell (e.g., to detoxil'y substances withiil the recipient~. Non-limiting examples of intr~rr~ r proteins which a cell can be modified to e~cpress include y lu~ b~ ;dc~ 3-t~lu~,ou~ id~;,dystrophin. ,b-globin~ ~he~yl~klll;nt hyd~tJ,~.~las~, tyrosine hydroxylase, ornithine S L..~ l. l.r~ ase, ~ iS synthetase. IJDP-~lut t~ yl transierase and adenosine deaminase. Preferably, a cell is modified to express a gene product by introduciny into the cell a nucle~ic acid encoding the gene product in a form suitable for expression of the gene product in the cell. For exarnple. a ~ .,"1,;1.,..,1 expression vector (e.g., a viral vector) containing a gene of interest can b- prepared and introduced into a cell of the invention by 10 methods described above regarding antisense expression ~ ectors (or by othcr t.~ llLio techmiques).
As used herein, the term "modified to express a gene product" is intellded to include a cel] treated in a manner that results in the production of a gene product by the cell.
Pret'erably, the cell does not express the gene product prior to mnriifir.qtion Altcrnatively, 1:~ mtlt1ifir~tion of the cell may result in ~m increased production of a gene product already expressed by the cell or resul! in production of a gene product (e.g., an antisense Rl'IA
molecule) ~-hich decreases production of another, ~mdesirable gene product no rmally expressed by the cell.
In a prefèrred rml)()~limrl)t a cell is modified to express a gene product by 20 introducing genetic material, such as a nucleic acid molecule (e.g., RNA or, more preferably, DNA) into the cell. The nucleic acid molecule introduced into t'he cell encodes a gene produc~ to be expressed by the cell. The term "gene product" as used herein is intended to include proteins, peptides and functional RNA molecules. Generally, the genc producî
encoded by the nucleic acid molecule is the desired gene product to be supplied to a subject.
~5 Alterllittively, the encclded gene product is one ~~l~ich induces the e?~pression of tlle desired gene product 'oy the cell (L.g., the introduced genetic material cncodes a trtnscription fac~or which il~duces the rranC~ rjptinn of the gene product to be supplied to the subject).
A nucleic acid molecule introduced into a cell is in a form suitable for expression in the cell Or the gene produc~ encoded by the nucleic acid. Accordingly, the nucleic acid 30 molecule includes coding and regulatory sequences required for 1, n~ 11 of a gene (or portion thereof! and~ when the gene product is a protein or peptide, translation of the gene pmduct encoded by the gene. Regulatori sequences ~hich can be included in the nucleic acid molecule include promoters, enhancers and polyadenylation signals, as well as sequences necessary for transport of an encoded protein or peptide, for example N-te.rrninai 3~ signal sequences for transport of proteins or peptides to the surface of ble cell or for secretion.
Nucleotide sequences which regulate expression ot'a gene product ~e.g., promoter ancl enllancer sequences) are selected based upon the type of cell in which the gene product is to be expressed and the desired le~el of expression of the gen- product. For example~ a promoter l;no~n to coni'er cell-type specific e~pression of a gene linked to the promoter can ~ WO gStl}3~28 21 ~ ~ 8 ~ .J~ ,. 7 .i be used. A promoter specirlc for m~oblast gene expression can be linked to a gene of interest to confer muscle-specific expression of that gene product. Muscle-specific regulatory elements wh;ch are known in the art include upstream regions from the dystrophin gene (Klamut et al.~ (1989) .7l ~ol. Cell. Biol 9:2396), the creatine kinase gene (Buskin and Hauschka, (1989) .~ol. C'ell Biol 9:2627) and the troponin gene (Mar and Ordahl~ (1988) Proc. .7~atl. ~f cad. .S'ci. JSA. 85:6404). Regulctory elements specific for other cell types are kno~n in the art ~e.g.. the albumin enhancer for liver-specific expression; insulin regulatory elements for pancreatic islet cell-specific expression: various neural cell-specific regulatory elements~ including neural dystrophin, neural enolase and A4 amyloid promoters).Alternatively, a regulatory element which can direct constitutive expression of a gene in a variety of different cell t~pes, such as a viral regulator,v element, can be used. Examples of viral promoters commonly used to drive gene expression include those derived from polyoma virus~ Adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs.
Alternativel5, a regulatory element wllich provides inducible expressiol1 of a gene linked thereto can be used. The use of'an inducible regulatory element (e.g., an inducible promoter) allows for modulation of the production of' the gene product in the cell. Examples of potentially useful inducible regulatory systems f'or use in eukaryotic cells include hormone-regulated elements (e.g., see ~fader, S. and ~h'hite, J.ll. (1993) Proc. Natl. Acad. ~S'ci. U.S,4 90:5603-5607), synthetic ligand-regulated elements (see, e.g. Spencer, D.M. et al. (1993) ~Science 262:1019-1024) and ionizing radiation-regulated elements (e.g., see Manome. Y. et al. (1993) Biocltemistr~ 32:]0607-10613; Datta, R. et al. (1992) Proc. Natl. ~cad. ~Sci U~f 89:10149-10153). Additional tissue-specific or inducible regulatory systems which may be developed can also be used in accordance with the in~entioa.
There are a number of techniques known in the art for introducing genetic material into a cell that can be applied to modify a cell of the invention. ln one r~ o~ 1, the nucleic acid is in the form of a naked nucleic acid molecule. In this situation, the nucleic acid molecule introduced into a cell to be modified consists only of the nucle~ic acid encoding the gene product and the necessarS regulatory elements. Alternati-!ely, the nucleic acid encoding the gene product (including the necessary regulatory elements) is contained witbin a plasmid vector, Examples of plasm;d expression vectors include CDM8 (Seed, B., .7~ature 329:840 (1987?!andpMT2PC~'Kaufman,etal..f~.~lBOJ: 6:187-lgS(1987))~ Inanother embodiment, the nucleic acid molecule to be introduced into a cell is contained within a viral vector. In this situation, th- nuck,~ic acid encoding the gene product is inserted into the i iral genome (or a partial viral genome~. The regulatory elements directing the expression of the 35 gene product can be included w ith the nucleic acid inserred into the v iral yenome (i.e, linked to the gene inserted into the viral ~enome I or can be provided by the v iral genome itself, Examples of methods which can be used to introduce naked nucleic acid into ce~lls and viral-mediated transfer of nucleic acid into cells are described separately in the subsections below.

.. , . , . . .. , _ _ _ . . . . . .

2~9~
wo 9s~3373273 r~

A Introduction of N~ Nuclr-ic Acid intn C~-llc I . Tnansf ectiort med ta~ed bv ( aP04: Naked DNA can be introdwced i nto cells by tom~ g a precipitate conta;ning the DNA and calciurn phosphate. For example, a HEPES-buffered saline solution can be mixed with a solution containiny calcium choride and DNA to form a S precipitate and the precipitate is then itncubated ~ith cells. A glycerol c1r dimethyl sulfoxide shock step can be added to increase the amount of DNA taken up by certain cells. CaPO4-mediated Llaua~ Livll can be used to stably (or transiently) transfect cells and is only applicable to in l~itro mnrlifirqti- n of cells. Protocols lor CaPO4- mediated transfecti{ n can be found in C~urrent Protocols in Molprnll ~r Biolo~v~ Ausubel, F.M. el al. ieds.) Greene 1 () Publishing As.sociates, (1989)~ Section 9.1 and in Molecular t, lonir~ I S~hnrdtory ~l ~nllq l 2nl1 F iitinn SambrooL et al. Cold Spring Harbor Laboratory Press~ (1989). Sections 16.3'~-16.40 or other standard laboratory manuals.

~. Tran~fectiort medialed b~! DEAE-de.rtrart: Naked DNA can be introduced into cells by 15 t'c1rmil1g a mixtu}e of the DNA and DEAE-dextran and incubating the mixture ~ith the cella.
A dimethylsulfoxide or ~,LIvlu~lu;llc shocL step can be added to increase the amount of DNA
uptalce. Dl:'AE-dextran transfection is only applicable to i77 vifro modification of cells and can be used to introduce DNA transiently into cells but is not preferred for creating stably transfected cells. Thus, this memod can be used for short term product;on of a yene product 2Q hut is not a method of choice for long-term production of a gene product. Protocols for DEAE-dextran-mediated lransfection can be found in Currcnt Prvtocols in Mole~nlq-usub~L F.M. et al. (eds.) Crreene Publishing Associates, (198g!, Section 9.~ auui in MOIeCIllqr C:ll nin~ A T nhoratory ~vtnm~l 2nri FAiti(m Samhrook et al. Cold SprinS~ E-larbor L.ahorfltory E~ress, (1989~, Sections 16.41-16.46 or other standard laboratory manuals 3. ~lectroporatio7t: Nalked DNA can also be introduced ;nto cells by incubating the cells and the DNA together in an appropriate buffer and subjecting the cells to a high-voltage electric pulse. The efficiency ~ith which DNA is introduced into cells by c~ Llv~ aLit~u is influenced by the stren~th of the applied field, the length of the electric pulse, the 30 temperature, the t o~, rt.", ~ s ;~ and ( nn~ Pntratinn of the DNA and the ionic t;ulll~oa; Lion of the media. Electroporation can be used to stably (or transiently) transfect a wide ~ariety of cell types and is only applicable to i~ vilro modification of cells. Protocols for c~ Llu~)olL~Litl~ cells can be found in Current Protocols in Mnle~ r~ r ~iolo~, Ausubel, F.M.
et al. (eds.) Greene Publishing Associates, (1989~, Section 9.3 and in Moh r l~l~r C'loninf~ A
3~ horaL(lry ~Rnn~l ~n i F,dition Sarnbrook et al. Cold Spring Harborl,aboratory Press, ( 1989!. Sections 16.~4-16.5~ or other standard laboratory manuals.

4. Lipo.some-mediated tran~feetion ("lipofection'~: Naked :DNA c~l be introduced into cells by mixing the DNA with a liposome suspension containing cationic lipids. The ,, , .,,, ,, ,, , ,, ,, , , , ,,,,,,, , , , , , ,, , , , ,,,,, _ , , , _ _ _ ~ WO1~sl33828 2~g189~ ~ r.l,L~

DNA/liposome complex is thcn incubated with cells. Liposome mediated irPnsf~rii~m can be used to stably (or transiently) transfect cells in culture in vitro, Protocols can be found in Currrn~ Protocols in ~Io~ nlPr Biolo~. Ausubel, T .~. et al. (eds.) Greene l'ublishing Associates, (1989), Section 9.4 and other standard laboratory manuals. Additionally7 gene deliver~ in vivo has been ,.; ~ 1.. d using liposomes. See for example Nicolau et al.
(1987') ~eth. ~nz 149:157-176; Wang and Huan~ (1987) Prac. .~Tatl. Acad. Sci. USA
84:7851 -7855: Brigham et al. (1989) Am. J. ~Icd. Sci. 298:278; and Gould-E~ogerite et al.
(1989) GL'~Z~' 84:429-438.

10 5. Direcr Injectio~: Naked DNA can be introduced into cells by directly injecting the DNA
into the cells. For an in Tltro culture of cells, DNA can be introduced by ~ ,LiOIl.
Since each cell is l~ .;cctcd hldividually, this approach is ~ery labor intensive when modif}ing large numbers of cells. Howe~er, a situation wherein I~ )h~ ,Livll iS a method of choice is in the production of transgenic animals (discussed in greater detail belo~). In 15 this situation~ the DNA is stably introduced into a t'ert;lized ooc~1e which is then allowed to de~elop into an animal. The resultant animal contains cells carrying the l)NA introduced into the oocyte. Direct injection has also been used to introduce naked DNA into cells in T'iT'o (see e.g., Acsadi et al. (1991) Nahlre 332: 815-818; ~v'olfFet al. (1990) Science 247:1465-1468).
A deli~!ery apparatus (e.g.. a "gen- gun") for injecting DINA into cells in vivo can be used.
Such an apparatus is commercially available (e.g.~ from BioE~ad).

6. Receptor-~l~ediatLd DN. f ~Jptake: NaLed DNA can also be introduced into cells by ~:~".l~.l.,.~;"g the DN A to a cation, suc,h as pol,~lysine, which is coupled to a ligand t'o} a cell-surf'acereceptor (see t'orexarnple Wu, G. and ~VU~ C.E~. (1988)J. Biol. Chem. 263:14621:
Wilson et al. (1992) J. Biol. Cj.Te~rl. 267:963-967; and ll.S. Patent No. 5,166,320). E3inding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endoc~tosis. Receptors to which a ~DNA-Iigand complex have targeted include the transf'errin receptor and the asialoglycoprotein receptor. A DNA-ligand complex linked to adeno~irus capsids which naturaily disrupt endosomes, thereby releasing material into the cytoplasm can be used to a~,oid degradation of the complex by intracellular Iysosomes (see for example Curiel et al. (IY91) Proc. ~Vall Acad. ~Sci. U.SA 88:8850; Cristiano et al. (1993) Proc. .7~iatL
Acad. Sc~i. l SA 90:2122-2126). Receptor-mediated DNA uptake can be used to introduce DNA into cells either i~ . o or in l'il'O and, additionally, has the added feature that DNA can be selectively tar8eted to a particular cell type by use of'a ligand ~hich binds to a receptor selectively expressed on a target cell of interest.
Generally, when naked l)NA is introduced into cells in culture (e.g., by one of the transfe.ction techniques described abovei only a small fraction of'cells (about 1 out of 10~) t~pically integrate the transfected Dlsl'.b into their genomes (i.e., the DNA is maintained in the cell episomally~. Thus, in order to identitv cells which haYe taken up exogenous DNA, it is .. , .. , .. ... . , . . .... . , . . , _ . ,, , ...... , .. , . . , ., _ ., _ . .. .. .. . .

WOgSI33N28 ~9~ 26- ~ '5~1J

.td~ tU:~ to traulsfect nucleic acid encoding a selectable marker into the cell along Witil the nucleic acid(s) of hIterest. Preferred selectable markers include those wh;ch confer resistance to drugs such as G418, hygromycin and metnotrexate Selectable markers may be introduced on Ihe same plasmid as the gene(s) of interest or may be introduced on a separate plasmid.
An alternative method for generating a cell that is modified to express a gene product involving introducing naked DNA into cells is to create a transgenic animal which contains cells modified to express the gene product of interest. A traulsgenic animal is an amimal having cells that contain a transgene. vvherein the transgene ~as introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA
molecule ~vllich is integrated into the genome of a cell from which a transgenic animal develops and ~hhich remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. Thus, a transyenic animal expressing a gene product of interest in oue or more cell types within the animal can be created, t'or example, by introducing a nucleic acid encoding the gene product (typically linked to appropriate regulatory elements. such as a tissue-specit;c enhancer) into the male pronuclei of a fertilized oocvte. e.g., by Ill;~,lViilj.~.~iUll, and allowing tne ooc~te to develop in a pseudv~ female foster an;mal. Methods fi~r generatingtransgenic animals, particularly animals such as mice, have become l~ tiv.l. l in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870.009 and E logan, B.
et al.. (1~86) A Laboratvry ManuaL Cold Spring Elarbor, New York, Cold Spring Harbor Labor.ltor~ . A transgenic t'ounder animal can be used to breed more animals carrying the transgene. Cells of the tnmsgenic animal which express a gene product of interest can the be used to deli~er the gene producl to a sub ject in accordance with the in~ention.
Alternatively, an animal containing a gene which has been modified by ho.mologous ,Vllll ~;llai;OIl can bè constructed to express a gene product of interçst. For çxample, an gene carried in the genome o:t the animal can be altered by T,.). "olog~
~e~"lul .i" ~I ;fnl (for instance~ all or a portion of a gene couTd be replaced by the huma homologue of the gene to "humanize" the gene product encoded by the gene) or an e.ndoyenous gene can be "knocked out" ~i.e., inactivated by mutation). For example~ an gene in a cell can be knoc!ced out to prevent production of that gene product and then nucleic acid encoding a different (preferred) gene product is introduced into the cell. To create am ao mal ivith homologously recomhined nucleic acid. a vector is prepared wh;ch contains the VNA which is to replace or interrupt the endogenous l:)NA tlanked by DNA
homologous to the ~ y~ "~ DNA isee for example Thomas, ~.R. and Capecchi, M. R.
( 1987~ C'ell ~:503). The vector is introduced into an embryonal stem cell line (e.g., by ~,k.L~ul.l..LtLh~n) and cells which have homologously recombined the DNA are selected (see for example Li, E. et al. ~1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see for example .... ,, .. ... _ . . _ . . . . . .. .. .. . .. . .... . ... ... .. . .. ... .. . .. . .. .... ..

WO 9513382x - 27 ~

Bradley~ A. in 7eralocLu ~ and Emhr~ oniL Ster~2 CL~IIS. A Practtcal ,~pproacfl, E.J.
Robertson, ed. (IRL~ Oxford. 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable ~ udu~ allL female foster animal and the embryo brought to term.Progeny harbouring the homologously l~uulbi~ d DNA in their gerrn cells can he used to S breed animals in which all cells of the animal contain the homologously recombined DNA.
Cells ofthe animal containing the h/~m~ g~u~l~rl l.~,ll,l.;"~ d DNA which express a gene ~ product of interest can then be used to deliver the gene product to a subject in accordance v~ith the invention.

10 B. Vir.ql-M,~ t~ ~I G/'~P Trqnsfer A preferred approach for introducing nucleic acid encoding a gene product into a cell is by use of a ~ ;MI vector containing nucleic acid, e.g. a cDNA, encoding the gene product.
Infection of cells v~ith a viral vector has the advantage that a large proportion of cells receive the nucleic acid, which can obviate the need for selection of cells which have recei-ed the 15 nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA
contained in the ~ ;MI vector. are expressed efficiently in cells which have taken up viral vector nucleic acid and viral vector systems can be used either in ~ itro or in vivo.

I . Relroviruses: Defective I~L~ uvi- .1~ are well i l ,.."~, t~ d for use in gene transfer for genetherapypurposes(forareviewseeMiller,A.D.(199û)B/oocl76:271) Al~,~,ullll,ill~tllL
retrovirus can be constructed having a nucleic acid encoding a gene product of interest inserted into the retroviral genome. Additionally, portions ofthe retroviral genome can be removed to render the retrovirus replication defective. IhG replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing rc~ullll);llaLlL 1 ;;LIuvhu~ and for infec.ting cells in vitro or in t~ivo with such viruses can be found in C urrent Protocols in Molec~lqr Biol~Pv. Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Lxamples of suitable retro~iruses include pLJ, pZTP, p~'E and pEM which are well known to those skilled in the art.
E~amples of suitable pacl~agh~,~ virus lines include ~lCrip, ~irCre, ~' and ~IrAm. Retroviruses have been used to introduce a v ariety of genes into many different cell types, including epithelial cells, endothelial cells, lyl.ll,l.(,.yt~, myoblasts, h~aLu~ k:~ bone marrow cells, in virro andlor Irm~h!c~ (see for exarTIple Eglitis, et al. (1985) ~Science 230:1395-1398; I)anos and Mulligan (1988) Proc. Natl. Acad. Sci. /TS.~ 85:6460-6464; Wilson et al. (1988) Proc. i\iarL
~1cad. .Sci USA 85:3014-3018~ Armentano etal. (19901 Proc. I~{a~l. ,tcacl. Sci. l 'S.4 87:6141-6145; Tluber et al. (1991) Proc. .~Vail. A cad Sci. C.TIS.4 88:8039-8043; Ferry et al. (1991) Proc.
Nall.Acad.Sci. ClSA88:8377-8381;Chow&ur,vetal.(l991)Science254:1802-1805;van Beusechemetal.(1992)Proc.~atl.,4cad..Sci. Z1~5~,~89:764û-7(144;Kayetal.(1992)11uman Gene Therap~ 3:641-647; DQ; et al. (1992) ProG. ~"vatl. .4cad. .Sci. C~S~I 89: 10892-10895, Hwu . , . , .. , ... _ .. , . _ _ . . _ . .. . . . . .. . . _ .

~09s/33s2x ?,~ 9~a9~ - 7..1~

etal.(l9931J. lmr,rum)l. 150:4104-4115;U.S.PatentNo.4,868,11~;U,S,PalerltNo.
4.980,286~ PCT Application WO 89107136; PCT Application WO 89102468; PC'I' Application WO 89iO5345; and PCT Application WO g2107573). Retroviral vectors require target cell division in orcier for the retroviral genome (and foreign nucleic acid inserted into S itl to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell .

2. AI~ /St',S: The genome o~' an adenovirus can be ~l~aui; ul ~1 such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a nonnal l,vtic viral life cycle. See for example Berkller et al. (1988) BioTechniques 6:616;
Roseni'eldetal.11991~Science252:431-434;andRosellfeldetal.(1992~Cell68:143-155.
Suitable adenoviral vectors deri~!ed from the adenovirus strain Ad type S dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those sicilled in the art.
Recombinant ~de~ovh ~ a are advantageous in that they do not require dividing cells to be ef.tèctive gene delivery vehicles and can be used to infect a wide variety of cell t,vpes.
including airway epitheli~m~ (Rosenfeld et ai. (1992) cited sZlp7a), endothelial cells (Lemarchandetal.(1992~ Proc. A1atl. .Icad. ,Sci. W3 89:6482-6486),hepatocytes(ilerzand Gerard (1993) Proc. AratL Aca~f. Sci. ~r.S'A 90:2812-2816) and muscle cells l'Quantin et al.
(1992) Proc. ~latl. Acad. 57Ci. US.4, 89:2581-2584). Addi~ionally, introduced adeno-iral D.NA
2n (and foreigm DNA contained therein) is not integrated into the genome of a host cell but remains episomah thereby avoidmg potential problems that can occur as a result of insertional "."~ in situations vvhere introduced DNA becomes integrated into the host genome (e.~., r~troviral [3NA). Moreover, the carr~ing c,apacity of the adeno-,iral genome for foreign Dl~'A is large ~up to 8 kilobases) relat;ve to other gene deliver.~ vectors (Berhl-r et al.
cited 5UI~t'a; Haj-Ahmarld and Crrctham (1986) J. Virol. 57:267). ~lost replication-defective adenovirai vectors currentl~ in use are delet~d for ail or parts of the viral E:.l and E3 genes hut retail1 as much as 80 % of the adenoviral genetic material.

3. .~feno-~lssociated i 'iruses: Adeno-associated virus (.4 AV) is a naturally occurring defectiue virus tha~ requires another ~irus, such ~s an adenovirus or a herpes v irus, as a hélper v irus for eff~cient replication and a producti-e life cycle. (For a re~.iew see Muzyczl;a et al.
Cz/rr. ~opics irz Micro. csrrlf Irr71nu1zvl (1992) 158:97-129). It is also one of the few viruseY
that ma~ integrate its DNA into non-dividing cells, and exhibits a high frequenc y of stable integration Isee for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356, Samulski et al. (1989) .J. ~ ïrol. 63:382. -3828; and Mcl.aughlin et al. (1989) .J. 1'irol.
62:l963-1973~. ~1ectorscontainingaslittleas300basepairsofAA~/canbepaclagedattd can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribedirlTratschinetal.(1985)il~ol. C~ell. Biol.5:3251-3260canbeusedtointroduce Dli'A into cells. .~ variety of nucleic acids have been introduced into difi'erent cell types , _ _ .. . .. ... ... , _ . . , , .. , .. . , . , . , . .. ,, ., . . . _ .. , _,, _ .

~133828 2 1 ~ 1 8 ~ 1 ~ r~

using AA~1 vectors (see for example ~lermonat et al. ( i g84) Proc. NCI/I. ~cad. SL j. U~SIA
81:6~66-64~0: Tratschin e~ al. (1985) .l~ol. C'ell. Biol. 4:2072-2081; ~ondisford et al. (1988) .llol. r~ndocrinol. 2:32-39;Tratschinetal.~1984).1. ~ïrol. 51:611-619;andFlotteetal.
(1993)J. Biol. Chem. 268:3781-3790).
S The efficacy of a particular expression vector syste~m and method of introducing nucleic acid into a cell can be assessed by standard approaches routinely used in the art. For example. l~NA introduced into a cell can be detected by a filter hybridi7ation technique (e.g., Southern blotting) and RNA produced by ~ , of introduced DNA can be detected, for example. by Northenn blo~ting, R~rase protection or reverse ~ d 1 ~ -polymerase chain re~tion (RT-PCR). rhe gene product can be detected by an appropriate assay, for example by ;mmunological deteclion of a produced prote;n, such as with a specific antibody.
or by a functional assay to detect a functional activity of the g-ne product. such as an enzymatic assay. If the gene product of interest to be expressed by a cell is not read;ly assaYable, an expression system can first be optimized using a reporter gene linLied to the regulatory elements and vector to be used. 1 he reporler gene encodes a gene product which is easily detectable and, thus, can be used to evaluate the efficacy of the system. Standard reporter genes used in the art include genes encoding 13-gl:~rtocifl~cf- chll~,~,.,l.l,...,: ~)1 aceh~ l transferase, luciferase and human gro~ith hormone.
~ hen the method used tc~ int.roduce nucleic acid into a population of cells results in mn~1ifi-~iirm of a large proportion of ~he cells and efi;cient expression of the gene product by the cells (e.g.~ as is otten the case w hen using a viral expression vector), the modified population of cells may be used v~ithout f'urther isolation or subcloning of individual cells ~ithin the population. l'hat is, there ma- he sui'ficient production of the gene product by the population of cells such that no further cell isolation is needed. Alternatively, it may be desirable to grow a 1~ population of identically modified cells from a single modified cell to isolate cells ~hich efficiently express the gene product. Such a population of uniform cells can be prepared by isolating a single modified cell by limiting dilution cloning follo~hed by expanding the single cell in culture into a clonal population of cells by standard techniques.
Alternative to introducing a nucleic acid molecule inlo a cell to modify the cell to express a gene product, a cell can be modified by inducing or increasing the level of expression of the gene product by a cell. For example, a cell may be capable of expressing a particular gene product but fails to do so without additional treatment of the cell. Similarhn the cell may express insufficienl am~ounts of the gene product f'or th- desired purpose. Thus, an agent which stimulates expression of a gen e product can be used to induce or increase expression of a gene product by the cell. For example, cells can be contacted with an agcnt i l~irro in a culture medium. rhe agent which st;mulates expression of a gene product may function. for instance, by increasiny transcription of the gene encoding the product, by increasing the rate of translation or stability (e.g., a post transcriplional modification such as a ~ 9~.89~
~o ~s/33x2x poly A tail) of an mRNA encoding the product or by increasing stability, transport or ~7r~1i7 ninn of the gene product. Exarnples of agents ~ilhich can be used to induce expression of a gene product include cytokines and growth fàctors.
Another t Lpe of agent which can be used to induce or increase expression of a gene :; product by a cell is a transcription factor which upregulates transcription of the gene encoding the product. A transcription factor which upregulates the expression of a yene encoding a gene product of interest can be provided to a cell, for example~ bJu introducing into the cell a nucleic acid molecule encoding the lla~ iull factor. 1hus~ this appro~h represents an alternati~e type of nucleic acid molecule uhich can be in~roduced into the cell 10 (for example by one of the preYiously discussed methods). In this caie, the introduced nucleic acid does not directly encode the gene product of interest but rather causes production of the gene product by the c-ll indirectly by inducing expression of the gene product.
In yet another method. a cell is modified to express a gene product by coupling the geIle product to the celL pret'erably to the surface of the cell. For exarnple~ a protein can be 15 oblained by purii~,ing the cell from a biological source or expressing the protehl r~ cnmhin:~ntly using standard l~ billaLll DNA technology. The i.solated protein caul then be coupled to the cell. The terms ''coupled" or "coupiing" refer to a chemical~ enzymatic or other means (e.g.~ by binding to an antibody on the surface of the cell or genetic engineering of linkages) by which a gene product can be linked to a cell such that the gene product is in a 20 form suitable t'or delivering tne gene product to a subject. For example~ a protein can be chemically crosslinked to a cell surface using ~ ,ially a~failable crosslhlking reagents (Pierce~ Rocktord 1I.~ hher approaches to coupling a gene product to a cell hlclude the use of a bispecific untibody ~ hich binds both the gene product and a cell-surface molccule on the cell or mo~ n of tne gene product to include â lipophilic tail (e.g.~ b~ inositol phosphate 2~ linl;age) whicll can insert into a cell membrane.

1~'. Methn-l~ o f th( Invention Another aspect of the ins~ention pertains to methods for reducing the; ~
of a celi ior ~ ,l,.. s 11;. ,n A cell for use in this method is one which has at least one 30 epitope on i~s surface ~hich stimulates hyperacute rejection of the cell by natural rmtibodies hl a recipient subject. The imnmlnogPnit~i1y of the cell is reduced by contacting the cell with an agent which alters. reduces or substantially eliminates expression ot the epitope on the cell surtace, thereby reducing the capacity of the cell to stimulate naturai antibody-mediated hyperacute rejection of the cell in a recipient. Preferably~ the epitope is a calbohyLil atc:, such 3~ as a galactosyl(~al-3)galactose epitope.
An epitope on the surface of the cell is altered, reduced or substantially eliminated bs one of the treatments pre~iously described in Section 11. Accordingly~ in one f-rnhotlim~.nt the agent is one wh;ch clealies the epitope from the cell surface~ such as an enzyme (e.g., an alpha-gal~nt~ ) or a chemical. In another r~h~ ,.l the agent is one which inhibits .. .. ..... .. . . . . ..... .. .. . ...... ..

~ WO9~/33112~ 8~1 ~ I/~1",!i, v~3Jt~

the forrnation of the epitope on the cell surfiace, for example b, inhibiting the acti~!ity of a gly~,ur~ylLIall~L,la ,e in the cell ~e.g., an al-3-galact~ lL a ~artilaa~). [hus, the agent can be an aultisense nucleic acid or a chemical inhibitor of the en~yme. In yet another embodiment, the agent is one wh;ch binds to the epitope and inhibits binding of natural antibodies to the S epitope in a recipient, such as a leclin or an antibody (or fragment thereof) which does not acti~ate romrl~rnpnt or cause Iysis of the cell.
An epitope on a cell sun'ace can be altered, reduced or substantially eliminated in ~ itro or rn v ivo. Accordingly, the term "contacting" is intended to encompass either incubating a cell with the agent in vitro or ~ h~ ,L.Iillg the agent to a subject (e.g., a 10 transplant recipient). Alternativeh;, a cell can be treated i n vif70, adll~ cd to a subject and then further treated in vivv in the subject (e.g., a cell to be L~ lall~r~d is incubated in 1~itt O with antisense oli~ lLid~ the cells are a.hl~ . cd to a subject and thenadditional antisense nlig.. ~ otid~ are a imilli~L.,.ed to the subject; see i~xample 4~. The agent is contacted with the cell in an arnount and for a period of time sufficient to alter, I S reduce or substantially eliminate expression of Lhe epitope such that hyperacute rejection of the cel] is inhibited h~ a recipient.
After a cell is treated in vitr(3 to alle.r, reduce or substcmtially eliminate the expression of at least one naturai antibody epitope on the cell surlace, the cell is administerr d to a recipient. Accordingly, another aspect of the in~ention pertains to methods for n ~~ l,.. .l i. .g 20 à cell into a recipient subject such that hyperacute rejection of the cell by the subject is inhibited. The term "subject" is intended to include humans and nonhuman primates (e.g., Old World Monkeys~. The method in- oi- es contacting the cell, prior to ~ , with an agent which alters, reduces or substantially eliminates expression of at least one epitope on the cell surface which stimulates hyperacute rejection of the cell in the subject and then 25 rl~1rninictPring the cell to the subject. Agents which are used to alter, reduce or substantially eliminate expression of the epitope are as described abo~e. The cLII is adlllillist~.r-d to the subject in an amount and by a route which is suitable for the desired therapeutic result. The cell used in the method can be within a tissue or organ. Accordingly, in these l;ilnl,c,dilllr IIL~
the tissue or organ is 1 l .~ 1 into the recipient by conventiollal techniques for 30 Ll~ ;.... Acceptance of Llall ,~lallLr i cells, tissues or organs can be determined morphologicaily (e.g.. with sicin grafts by examinillg the Ir..~ 5 d tissue or by biopsy) or by assessment of the functionai activity of the graft. For example, acceptance of pancreatic islet cells can be deLennined by measuring insulin production. acceptance of li~er cells can be detemlined by assessing albumin production and acceptance of neural cells can be35 deterrnined by assessing neural cell function.
In addition to treatment of a cell to be ~ pl.~s .1 to reduce h-peracute rejecLion of the cell in a recipient, the method for Llal~.>t~lallL~Lion of the invention can include additional in vitro treatment of the cells prior to ~ andior additional in vivo treatment of the recipient follo- ing n ~~ to further inhibit immunological rejection of the ... . , .. , . , . . _ . , .. , , _, ,,, .. , ... ,, ..... _, .. _ .. . _ _ .. . . . . .

Qv~09~133828 ~9~9 -32- r~ c~,a~ --Lla ~ JlautLLi cel]s. For example, prior to ~ ua ;~ tlle cell can be cont3cted vvith a second agent which aiters expression of at least one antigen on the cell surface which is capable of stimulating a cellular imrnune response against the cell in the subject. Antigens to be altered, and methods for akeration of the alttigen, are as described in Section 111 above.

5 Preferably, an Ml IC class I antigen is altered on the cell surface by contacting the cell prior to 1 . ;" ,~l.l .. ,1..1 ;"" vvith an anti-MEiC class I antibody~ or fragment lhereof (e.g., a n anti~ li lC
class I F (ab')2 fragment).
Additionally or alternati~el,v, a recipient subject can be treated prior to. during smd;or folloi~ing ~ 1 inn v.. ith another agent which inhibits 'r cell activity in the subject.
Thus, in one embodiment, a cell to be transplanted is treated with a first agent whictl alters, reduces or substantially eliminates expression of a cell surface natural antibody epitope and the transplant recipient is treated with a second agent which inhibits T cell activity. In another embodimenL a cell to be llaLla~lLtLlLcd is treated both vvith a first agent which alters, reduces or substantially e}imina~es expression of a cell surface natural antibody epitope and with a second agent whicll alters an antigen on Ihe cell surtace which stimulates a cellular immune response against the cell. and the transplant recipient is treated with a third agent which inhibits T celi ~ti~ity. As used herein, an agent which inilibits ~1 cell activit~ is defined as an agent which results in removal (e.g., ieqnrc~rsltinn) or destructiosl of r cells within a subject or inhibits T cell functions ~,rjthjn the subject (i.e., T cells may still b-present in the subject but are hI a non-funct;onal state, such that tdley are unable to proliferate or elicit or perform effector t uIctions, e.g. cytol;ine production, cytotoxicity etc.). The term "T cell" r~ mature peripheral blood T cells Iymphoc~tes. The agent which inhibits r cc 11 activity ms3y also inhibit the activity or maturation of immature ~r cells (e.g., thymocytes).
The agent wllich inhibits T cell stc~ ity in a subject can be an immnnocltrpressi v~e dn~c. I'he term "in-mlmr~nrpressive drug" is intended to include ~ agents wlIich inhibit or interfe~re ~hith normal immune function. A preferred h~ t..c~:f,~e drugiscyclosporinA. Other;.l..,.ul..~ .,caaivedrugswhichcanbeusedincludeE.i5U6 auld RS-61443. In one ~ullb~dil~ lL, the; .~ll.n~ c drug is administered in30 rnnjlmrtiL~n with at leastone other therapeutic agent. Additional therapeutic agents ~vhich can be administered include steroids (e.g., glu~oco~licoids such as prednisone, methyl prednisolone and ~h~Y 3mrths3cl~n~ ) and rhrm~nht rs3reutic ayents (e.g., a~Llll;L~ hle and c~-clorhncrll 3mil1t~). In another t mho~iimrnt~ an hLIIIIUIIO:~U~IJL~h/C~ drug is administe3ed in conjunctionYsit.lbothasteroidandachemotherapeuticagent, Suitable;,...,,....-.-nl.l,,.a.,ive 35 drugs are commercially available (e.g., cyclosporin A is available from Sandoz, Corp., E~ast IHanover~ NJ).
An i~ ull~l~u~ a;~e drug is u~llllilli:,i.. ,:d hI a firrmnT 3~ir,n vvhich is compatible with the route of .~.1" . h l, ,.. ;~"~ Suitable routes of administration include hItravenous injection (either as a single hlfusion. multiple in~usions or as an intravenous drip over time), , ... , . ... _ .. , , . _ .. .. . ... .. ... , . , .. ..... , . , .. . , .. .. .. , . .. , ... . , .. . ., ..
, . ....... . , .. _, .

~ ~/V0 9~il33828 1 91 8~ 7/~
~ 33 ~
..iu~ injection.;~ iniectionandoral,.~ u,.ii,." Forinlravenous injection~ the drug can be d;ssolved in a phvsiolvgicall$r acceptable carrier or diluent (e.g., a buffèred saline solution) which is sterile and allc ws i'or syringability. Dispersions of'drugs can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
S Con~ enient routes of ~.1., .h~; ~l, ,.1 ;on and carriers for imm n l~ L ,;,;v~ drugs are kno~n in the art. For example. cvclosporin A can be adl~ lhl~l intravenously in a saline solution~ or orally, il,~ f ~lly or hlu~l~ ly in olive oil or other suitable carrier or dilu-nt An i~ c drug is ~ld~ .cd to a recipient subject at a dosage sufficient to achieve the desired therapeutic el'i'ect (e g . inhibition of rejection oF I ~ t. d 10 cells). Dosage ranges for i,,"" ~ ivc drugs~ and other agents v~hich can be , 1, .1",;";~,. .r d there~ith (e.g., steroids and ~h~noLllcla~eutic agents), are kno~in in the art (see e.g., I reed et al. (1992) h~r~lv Eng~l. J. ~red 327:1549: Spencer et al. ( 1992) A~eu EngL J.
A~ed. 3~7:1541;~,'v'idneretal.(1992)NcwEngL.I: ,~fierl.~1:1556;1illdvalletal.(1992)~fnn.
Ne7rroC 31:155; and Lindvall et al. (1992) Arch. J'~eurol. 46:615). A preferred dosage range 15 foriu"~"",n~"l,l,l~ edrugs,suitablefortreatmentofhumans.isaboutl-30mg,~kgofbody vveight per day. A preferred dosage ran~- for cyclosporin A is abouL l - l O mg/kg of body weight per day, mor preferably about l-S mg/kg of body weight per day. Dosages can be adjusted to maintain an optimal level of tbe i, l " ., ..l .. ~ll ,lll .~i~;VL' drug in the serum of the recipient subject. For example, dosages can be adjusted to maintain a preferred serum level for cyclosporin A in a human subject of about 100-200 ng/ml. It is to be noted that dosage values may vary according to factors such as Ihe disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted over time to provide the optimwn therapeutic response according to the individual need and the professional judgment of the person g or supervising the ~ lll 0f the l nl ll~ ll lc and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practicc of the claimed in vention~
In one ~nnhl~liim~-nt of tne invention, an ;. . ~ nc~ ll c~ r drug is alnlill;st.,ied to a subject transiently for a sufficient time to induce T cell tolerance to the transplanted cells in the subject. Transient ~ of an; ~ , c~ c drug has been found lo induce long-term graft-specific tolerance in a grafl recipient (see Bnmson et al. (1991?
Tran~ lartaJiot7~:s4s;~lllt~hin~nnetah(l98l)1ral!splan~aticin~2:2lo:(-TreenetaL(l979) Tartce~ 123;Halletal (1985)J Erp.,l~ed.1~:1683). Adlllilli~LIa~ llofthedrugtOtlle subject can begin prior to I l ~ a ,l i~", of the cells into the subject. For example, initiation of drug "".";" i~ll..l;011 can be a few days (e.g., one to three days) before l~..,l~lll..~ll ~linn.
35 Alternatively, drug ~rirninictrntion can begin the day of ~ 1 ..lu1l;nrl or a fe~ days (generallynotmorethanthreedays)afterll.."~l,1,1,,l"l;-." A-l",;";~ l;nllofthedrugis continued for suflicient time to induce donor cell-specitic tolerance in the recipient such ~hat donor cells will continue to be accepted by the recipient ~hen drug a~l~llh~ aliull ceases.
For e~sample, the drug can be ddlll;.l;~ .i for as short ~s tbree days or as long as three ... ... , .. . . .. , . _ _ _ _ . . . . .

wo ss~3382x 2 ~ 34 - P~

months follov~ing L~ li.n~ pica3h-7 the drug is a~lll.ill.~t~,lc.l tor .at least one week but not more than one month following L~ ."~ .n induction of tolerance to the t~ cells in a subject i5 indicated by the continued acceptance ofthe trRncpl~ntqd cellsafter,.,l.";";~l...l;~,rloftheimtnllr-l~sllrrressivedrughasceased. Accept.qnceo3' S I~L~ 3~1alll~d tissue can be determined morphologically or f'unctionally, as described above.
.4nother t~pe of agent which can be used to inhibit r cell activity in a subject is an antibody~ or stagment or deri77ative thereof7 which depletes or sequeseers r cells in a recipient. Antibodies which are capable of depleting or SP~7~tlP~ttqring T cells Jn s i~o ~hen ~IIll;lli ti.lcd to a subject are known in the art. I'ypicall~, these antibodies bind to an antig-n 10 onthesurf'aceofa'rcell. Polyclonalantiseracanbeused,iorexampleallti-ly,ll~l,o~,~L~
serum. Alternalively~ one or more monoclonal antibodies can be used. Preferred T cell-depleting antibodies include mor-~lc lt7 ql antibodies which bind to CD2. CD3, CD4 or Cr)8 on the surface of T cells. Antibodies which bind to these antigens are knov~n in the art and are available (e~.g.~ from Arnerical7 Tissue Type Collection). A preferred mntnn( 1nni71 antibody for binding to C1)3 on human T cells is OKT3 (ATCCl Cs~L 8001~. The binding of an antibody to surface antigens on a T cell can facilitate ~r~l... ,~U..I;..n of T CLI15 in a subject andior destn3ction of T cells in a subject by endogenous, . .~ ~1,". .; ~. . .~ Altemati vely. a 'I' cell-depleting antibody which binds to an antigen on a T cell surface can be conjugated to a loxin (e.g., ricin) or other cytotoxic molecu3e (e.g., a radioacti~e isotope) to facilitate destruction of T cel Is upon binding of the antibody to the T cells.
Another type of antibody which can be used to inhibit T cell acti- ity in a recipient subject is an antibody which inhibits T cell proliferation. For example, an antibody directed against a ~s~' ceil gro~th factor, such as IL-2, or a T cell gro~th factor receptor, such as the IL-~ receptor, can inhibit prolileration of T cells (see e.g., DeSilia. D.R. et al. (1991) J.
2~ Im~n~nol. 1~7:3261-3267). Accordingly. aul anti-IL-2 or an anti-IL-2 receptor antibocly Cal be adrninistered to a recipient to inhibiL rejection of a L~ /L;L.J cell (see e.g. ~'ood et al.
( 1992) ~ / o~ "~ 49:4l 0). Additionally. both an anti-IL-2 and an anti-lL-2 receptor antibody can be . i . - l, . .;. ,: u, . cd to inhibit T cell activity or can be aJI~illis~ rith anothcr antibody (e.g., which binds to a surface antigen on T cells).
An antibody which depleles. sequesters or inhibits T cells witslin a recipient can be adl.lil.; ,i~lcd al a dose and for an appropriale time to inhibit rejectio n of cells upon tr:mqpl~ntr~ti~n Antibodies are preferably iddlllill;st~.~d h~ nou~ly hl a pls~ ll.~..llically ~scceptable carlier or diluent (e.g., a sterile saiine solution)~ Antibody ;~ u .a ;~ can begin prior to ~ e.g.. one to five days prior to l~ ion) and can continue 35 on ~l daily basis after ~ ion to achie~-e the desired effect (e~sg~ up lo fourteen days after l,...,~l,l~."l..li~nn) A preferred dosage range for A-l- ,;.,i~ n of an antibody to a human subje~ct is about 0.1-0.3 mg,q~g of body weight per day. AlLem~stively, a single high dose of antibody (e.g.. a bolus at a dosage of about 10 mg~g of body weight) can be a.IIll;ll; ,ltl.,.l to ahumansubjectonthedayofi,i~ ,l-'iQn Theeffectivenessofantibodytreatmentin . . , _, ... .. .. ... .. _ . . .. .. ... .. ..... .. ... .... .. . .. .. . . .. ... . .. .

~I W09~C13382R 1~189,~ r~l~u~, u.~

depleting T cells from the peripheral blood can be determincd by cormparing 'r cell counts h blood samples taken from the subject before and after antibody treatment. Dosage regimens may be adjusted over time to provide the optimum therapeutic response according to the individual need and the professional judgment of the person f~Llilli7~.1;11g or supervising the 5 ~ u ~ii.."ofthe ~ mp()~itinns Dcsagerangessetforthhereinareexemplar~ onlyand are not intended to limit the scope or practice of the claimed invention.
This invention is further illustrated by- the follov~ing P,xaTnples which should not be construed as limiting. The contents of all ref'erences and published patents and patent ~prlir~rion~ cited throughout the application are hereby ilTGo~l~uldt~ d by reference.
.X~I~fPl,F I: Remo~al of Galacto.syl(al,3)Galactose Epitopes from Ti'~- ' " ' ' Cells b~ Alpha-C' ' ' ' ' - Treatment In this example~ porcine endothelial cells were treated ~~ith the enzyme alpha-f to remo~e terminal galactosyl(~l,3)galactose epitopes on the cell surtace ~vhich 15 are recognized hy natural antibodies present in sera from humans and other primates.
I-lo~-e- er. prior to treating the cells ~vith alpha-~a~ . the tollowing experiment v.~as performed to determine whether human and other primate ~e.7g., monkey) serum contained antibodies that reacted ~ith porcine endothelial cells.
Porcine endothelial cells were isolated from a swine aorta by collagena.se digestion 20 and cloned by limiting dilution in DMEM (high glucose; cu~ c;.lbr obtahIed from Gibco, Grand Island, NY) ~u~ ,.l.cllted with 10~~o fetal calf'serurn (Intergen. Purchase, N~r') and 1~/o penicillh,/'~lly"u.l.y. hl (Bio~Iitaker, ~ivalkers~ille, MD). Afier isolation. the cells were incubated at 37~C with 5% C'O~. The cells were harves~ed from culture by Lly~hli~.ltiu (trypsin obtained trom Bio~T~'itaker, Wall;ersville, MD), neutralized v~ith culture medium~
25 washed twice with PBS and Ic~ lde :1 in PBSJ0.5~/a bovine serurn albumhI (BSA). Fifty ,u l(lxlO5cells)~veremixedwith50~10fl:10dilutedhumanorcynomolgusmonkey serum (New England Regional Primate Research Center, Southborough, MA) and incubated on iCf for I hour. After the primary incubation, cells viere ~ashed 3 t;mes ~vith PBS/0.5~~a BSA
and 50 7ul of a 1:50 dilution ofthe follc)~ing fluorescein conjugated antibodies were added to 3() the appropriale tubes: goat anti-human IgG (Jackson InmIunoResearch, ~h~est Grove, PA~I, goat anti-human IgM (.Tackson IrnmunoResearch), rabbit anti-monkey IgG (AccurateChemical, Westbury, NY), goat anti-monkey IgM (Nordic, Netherlands) and CJS I B4 (E'T' Laboratories, San ~fateo, CA). I or detection of swine Iymphocyte antigen (SLA) the monoclonal antibody PT-85 (VMRTD, Pullman, U7A) was employed ~-ith a goat anti-mouse 35 IgG secondary antibody (Jackson ImmulIoE~esearchl. The cells were incubated ~hith secondary antibody for 30 minutes on ice After incubation, the tubes were washed 3 times ~ith PBS/0.5~~o BSA and resuspended in 500 111 PBS,/O.Sa/a BSA. All satnples were analyzed by flow cytometry at 488 nrn. The results are illustrated in 7~igures IA-ID. The data indicate that RECrIFIED SllEET ~RULE 91 ISAIEP

.. . . . . ..... ... . .... .. ..... ... . ... .. . .. ... ..... .. . . .. . .. . . .

~g 3,.~9 wo ~sl338~s 2 ~ r ,~

antibodies in moniiey and human serum recognize detenninants present on porcine endothelial cells. Both IgG and IgM antibodies displayed strong reactii ity witti porcine endothelial cells ~hen detected Witi1 anti-human or anti-monkey IgG or IgM secondary antibodies.
S To identify the epitope recognized by anttbodies in human and monkey serum~ alpila-linked galacto.se was remo~ed t'rorn the cells surface by treatment with aiphn g~ to~ ric~
Specifically, primary porcine endot'nelial cells uere 'harvested Irom culture bv tr~ ps;nization (trypsin obtained from Bio~,'itaker, Walkersville~ MD)~ neutralized with culture med;a~
washed once with phosphate buffered saline (PBS) and once ~it'il 20 mM sodium acetate in i'BS (pH 5.8). Coffee bean alpha-g, ~ -''f (Sigma. St. I,ouis, MO) ~as added to cells (500 milliunits of enzyme.~l x I o6 cells') and incubation was carried uut for 2 hours at 37~C' in 200 n~l sodium acetate in PBS (pH 5.83. After enz,vme incubatnon, the cells were washed tv~ ice uith PBS and I ~u~,.".dcd at 2 x I o6 cells/ml in culture mediwm The cells werc analyzed tor i~iability hy assay ~ith MTT as described in Example 3. '['he results of'this 1:~ assay are illustrated in Figure 7. The data rlr~rn~lnctr:~t~? thltt the enzvme treatrment did IlC)t ha -e an effect on the viability of t'ne porcine endothelial cells.
Incubation of enzyme-treated cells with fluorescein labeled G'r~.~oniu ,simpTicifoia I
(GS-I Fl'l'C) a lectin ~~11ich binds specifically to terminal alpha-galactosyl epitopcs~ sho~ed tha~ the enzyme was effective in removing the alpha-galactosyl groups on the cell surfàce using 0.5 unitslml with 1X106 cells at 37''C for 2 hours. Binding ot'Fl'l'C labeled GS-19 was assessed directly (CiS-I) by flOw cvtometrv7 whereas binding of anti-SLA was assessed with a I ITC' labeled anti-mouse IgG (ANTI-SLA), and interaction of natur.tl antibodies was deterrnined by employing species-specific FlTC-labelell anti-lgCI or anti-lgM secondary aultibodies (~II,'~MAN IgG, HUM.4N Ig!~,1, MONKEY IgCi and MONK.E.~'~'' IgM). A control incubation ~ith secondary antibody alone was performed to determine backgrouna.
Onl- the untrealed cells bound GS-I FITC as revealed by flou cytometry (.See F iyure 3A~
CiS-I). lJnaltered binding of an aulti-SLA " ,....~ " ".1 antibody to Ml IC class I molecules (Se~e Figure 3B, AINTI-SI.A) after enz!,me treatment indicated that protease cont~nlin~tioil clf the en~vme did not account for the decreased GS-I binding. The removal of the alpha-galactosyl epitope by enzymatic digestion diminished the bhlding of tne natural aultibodies present in human and monkey sera. Both IgG and IgM binding was decreased in human senim (liee Fiy~ures 3C' and 3D~ Hl.JrMAN IgG and HIUMAN Igl~,1) and ;n monkey serum (5~e Figures 3F and 3F. MONKEY IgG and MON~EY Ig~f) when detected with species specific anti-IgG FITC' or anti-lgh,l FITC labeled secondary antibodies.
The e~tent of removal by alpha-t~ tlac~ idas~ of epitopes recogni~d b~, natural antibodies is shown in 1'able 1. Removal of alpha-linlced galactose from endothelial cells by alpba-galactosidase decreased their reactivih~ ~ ith human and monkey n~ttural antibodies by ~9~O to 90~ o. The reacti- ity of' both IgG and IgM in the human and monkey sera were markedly affected by tbe remo~al of this shlgle epitope.
RECTIFIED S't IEET (RULE 91 WO ~s~3382s 2 1 9 t ~ Pt,~T~usssAl~s73 I'ABLE I
Uedian fluorescence intensity of treated and untreat-d porcine endothelial cells incubated ~ith human or monl;ey serum and detected with anti-lgG-FITC' or anti-lgM-FlTC secondary antibody.

Fluu~.~ccc Treatment Serum Secondary AntibodY Intensity None human anti-lgG 126.55 Alpha-galase human anti-lgG 51.90 (590~o) None human anti-lgM 69.gl Alpha-galase human anti-lgh,~ 20.~4 (71%) None monkey anti-lgG 846.92 Alpha-galase monkey anti-igG 83.40 (90~,h) None monkey ant;-lgG 57.01 Alpha-galase monkey anti-lgM 11.47 (80%) J Nurnbers in parentheses represent the decrease in median lluul~ t intensity observed after treatment with alpha g I t~

Followingenzymetreatment~theefficacyofremovaloi'thealpha-galactosylepitopes was also assessed. The cells were stained with ftuorescein-labelled Griffonia simplfcifoia I
(GS-I). For staining. cells were incubated v~,ith labelled GS-I (~,u~ lally obtained l;om E''r' Labs) for 30 minutes on ice. The stained cells were subjected to FACS analysis to determine the density of the reactive epitope on the cell surface at increasing times after 1 5 digestion.
The results of the experiment are sho-~n in Figure 4. The data indicates that alpha-~,qJ~/~focj-J.qqe treatment of porcine endothelial cells can remove greater than 95~.'o of the cell surface alpha-galacto.syl epitopes. This greatly reduced level of expression of the epitope ûn the cell surface persists for several hours follov~ ing enzyme treatment. Even by 48 hours 20 after treatment. the level of surface expression of the epitope is still diminished by about 60~o compared to untreated controls. This example ,i~m~nqtrqt~ s that a'tpha-g~lq~f~-Yi~JqqP
- treatment of porc.ine endothelial cells is efi'ective at removing cell surface alpha-galactosyl epitopes, and that the epitopes are not reexpressed for several hours to days t'ollowing enzS!me treatment.
~5 . _ _ ~os~c~33s~x ~, - 38 -F..XA! 1PI ,~ 2: Removal of Galactosyl(al,3)Galactose Epitopes Inhibits Binding of Natural .~ntibodies to ~ ' ' Cells In this exarnple, tbe effect of removing alpha-galactosyl epitopes from the surl'ace o f 5 porcine endothelial cells on subse~uent bindimg of human and animal sera to the cells W~5 examined. Porcine~ endothelial cells were isolated and treated with alpha~ o~ toremove cell surface alpha-galactosyl residues, as described in E~zample I . I~ ..liat ly follo-ving treatment. the treated cells and untreated control cells were blcubated ~ith serum from cynomolgus monkey. human. mouse and pig. Sera were typically used at a 1:1010 dilution. Bhlding of anùbodies to th- cells was assessed using spec;es-specific fluoroscein-labelled anti-lgC} or anti-lgM secondary antibodies (to assess the binding of IgG or IgM
antibodies, respectivel~, within the sera). A control incubation with secondary amtibody alone ~as performed to deterrnine background labelling. Staining of the cells w ith the labelled secondary nntibody was assessed by FACS analysis.
The results of the eYperiment are illustrated in Figure 5. Neither mouse nor porcine sera exhibited readily detectable binding to either untreated or enzyme treated porcine endothelial cells. tn contrast, both human and monkey sera exhibited strong binding to untreated porcine endothelial cells. The l~lcdu~ Jlll isotype detected was IgG, although low levels of IgM binding were also observed. These results confirm the presence of natural antibodies specific for porcine cells hl human and monkey sera. Significantly~ treatment of the porcine cells with alpha-g~ to~ P prior to incubation with the sera greatly reduced the subsequent binding of natural antibodies to the porcine cells. This effect was seen for both IgG ano I~M isotypes. These results ~ L~. that antiboo;es dire ,ted agah~st alpha-galactosyl epitopes represent a major component of anti-porcine natu.ral antibodies in human and nonhuman primate sera and, moreover, indicate that treatment of porcine cells with alpha-e~ rto~i-lrc~ c:~n inhibit their recogrtition by these natural antibodies.
E,~CAMPLE, 3: Remo- al of Galactosyl(al ,3)Gnlactosc Epitopes lohihits l~'~atural Antibody-~lediated Cytoto~icity Sera t'rom humans was found to be cytotoYic to porcine endothelial cells in the presence of exogenously added rabbit ronlrlPrnpllt as detected with a colorimet.ric assay for cell viabilit~ employing ~ITT as described in this Example. Porcine endothelial cells were incubated with alpha-g~l 3rtnci~lr~p for hours at 37~C betore treatment with heat-inactivat-d serum with or with(3ut 10" u rabbi~ .... ll Cell -~iability was measured by the M~
35 assay as described belo~ . The results of the e:~periment are illustrated in Figure 6. 'Ihe absorbance obtained for cells treated ~ ith bo~ine serum and complement (CONTROL I is taken as 100%. The absorbance obtained for cells killed with alcohol (DEAD) is also given.
C'- rnplPll-Pnt alone~ or in the presence of boi~ine serum had no effect on cell viability. 'I'he ,, . , .. , _ . ,, _ . , . , . , . , . ... , . , .. . , , , ,,,, ... . , .. .. . ,,, . . , . . ,, . _ . , .,, , . _ .

~I9~5 1 wo ~sl33x28 c~totoxic effect of serum is dependent upon concentration I'hus, natural antibodies bind to porcine cells and are capable of killing the cells in the presence of ~ , ,pl 1,Variat;ons among se- en individuals ~humans) uere apparent in the amount of natural anb'bodies in the sera Porcine endothelial cells vwere incubated uith 10% human serum i'rom S severl individuals followed by detection by ilou cytometric analysis with human specific IgG
or IgM FITC labeled secondary antibody By flow c~tometric analysis, seven different individuals displayed various degrees of IgG and IgM reactivity (mean lluol~sc~ .l.e intensit~-, Figure 7) Variations m the ability of various human sera to kill porcine endothelial cells were also obser ed~ but did not clearly correlate to the levels of'natural antibodies in these I 0 individuals In this example, the eff'ect of removing alpha-galactosyl epitopes from the suri'ace of porcine endothelial cells on the abilit of human sera, together with ;(~, pl., ~ to mediate c~totoxicity was determined For the~ cytotoxicih~ assay, porcine endothelial cells, either untreated or treated with alpha-gal~rtos~ r as described in F,xample I ~ were aliquoted into 1 5 ml lld~lU-cl"~iru~c tubes (I x I o6 cells in 0 5 ml) Serum (either human or, as a control, bovh~e~ uas added to the tubes at an appropriate crrr~ntr~tinn (e g ~ 10-20U/u) folloued by additionofrabb;t.u..,~ . .1(obtainedfromPel-Free~,Rogers,NY)atadil~ltionofl:5.
DMEM (high glucose) uith heat-inactivated fetal calf serum was added to bring the final volume to I ml The tubes were incubated for 4 hours at 3 7~C in 5U/o C02 At the end of the 20 incubation period the tubes were centrifuged at 800 g for 5 minutes The medium uas aspirated off gently to avoid disruption ofthe pellet Cells uere then washed uith I ml of Hanks buffered salt solution (Gibco, Grand Island, NY) The pelle~t was then le~u~ lded in 0 5 ml of a 2 5 mg'ml stock solution ol' MTT (3-(4,5-dimethylthiszol-2-yl)-2,5-di~ll.ll~ Iic~l "olium bromide; Sigma7 St Louis~ M0) and an additional 0 5 ml of ~anks 25 buffered salt solution was then added to each tube MTT was prepared fresh on the day of the assay in DMr.M (high glucose) and 10~ o iètal calf serum Cells w re incubated u ith M'rl' for 4 hours at 37~C in 5% C02 After incubation, cells were centrifuged and washed tuice ~vith PBS The MTT crystals uere solubili~d in 0 5 ml of acidified isopropanol (pH 3 0) and 100 ~I rrom each tube was transferred to a 96--vell microtiter plate Each well uas measured 30 using an automatic plate reader with a 562 nm ~est wavelength In a first experiment using untreated porcille endothelial cells, the cells wereincubated uith ei~her no serum, heat inactivated bo-ine selum or heat inactivated human serum in the presence or absence of rabbit ~ JI~ L as described above Cell viability was measured using the tria701ium salt MTT, as described above As shown in Figure 8, 35 neither complement alone nor bovine serum in the presence or absence of . ", ,~ werL
cytotoxic In con~rast, human serum in the presence (but not the absence) of ~ 1 "i was capable of greatly reducing cell viability, indicating that natural antibodies within the serum are c jtotoxic to the untreated porcine cells in a ~UIII~ llll,lli dependent manner . , . .. . . . .. , _ .. , _ _ . . .. . . . .

wossn382x 9~ r~l,u.,,~

ln the next experiment, porcine endothelial cells were treated uith alpha-t".l ~ as described in E~ample 1~ to remove cell surface alpha-galactosyl residues.
Following treatment, the treated cells or unkeated control cells ~rere incubated w;th human serum (I 0~o or 20%) in the presence of rabbit f ~ ,. - "I for 4 hours as described ahove S Control cells were incubated ~ith bo-ine serum and ~ul~ul~ . Cell viabili~y was measured by the l~/frr assay, as described above. The absorbancy readhlg obtained tvr untreated cells incubated with bovine serum was ta.;en as 1 00~,~'u, and viability of cells incubated witll human serum was measured relati-,e to this. As illustrated in Figure 9, incubation vf untreated porcine cells with human sera and e~ ll reduced their viabilit-10 by d~ o~ lut;ly 70% relative to control cells. However, treatment of ~he cells with Illpha-r RlRrtcliir~ f prior to incubaLion with human sera and ~. " "I.l.., ... ,l restored full cell viability, indicating that removal of alpha-galactosyl residues from the surface of the porcine cells substantially eliminates the cytoto?~ic;ty of human natural antibodies, in combinatiosl ~vith ~;ull~ llclll, against porcine cells.
~X~l~lPl,1~ Inhibition of Alpha-1,3-Gal.~. .lt~ ,e Acthlity in a Cell for Tl , ' ' " lTsing Antisense Nucleic Acid Expression of galactosyl(~l,3)galactose epitopes on the suriace of Q cell can be20 reduced or substantially eliminated by inhibiting the acti~ity of an alpha- 1,3-galac~osyl~l~ul~r~ld ,e en~me in the cell. One method for inhibiting ~he activity of the enzyme utilizes a nucleic acid which is antisense lo a region of the mRNA encoding the enz me ~i.e.. antisense to a coding region of the gene tor the enyme~. An olit ~lu~,leutidc hdving a sequence whicll is antisense to mT~TA encoding a UDP galactose-alpha-1,3-25 galactosyltrRncfiPr~ iP, is designed based upon the rules of Watson-Crick base pairing and synthesized by standard tcchniques. e.g usirtg an automated DNrA~ synthesi~r. For example, three ''0-mer nlit,.,.. T~ - hai~ing sr.quenceS which ere antisense to tbe followint~
m~cleotide positions of an dl~ mRNA can be synthesiz:ed (nucleotide positions are relative tû the start site oftranslation at position 0): nucleotide positions -15 to +5 l~ulluu~ g the translaLion start site), +6 to +2~ and +101 to +1~0. Suitable antisense ~lignm~rlPn~i~lP sequences. designed based upon the sequence of either the murille alpha-~;.lld~lu~ dul~reld~c cDNA (disclosed in l.arsen, R.D. et al. (1989) Proc l~ia~l. A.cad. Si i.
Zl~S'.~ ~i.8''27-8'~3 1 1 or the bovine alpha-galacto~ u~f~l d~ cDNA (disclosed in Joziasse.
D.ll. et al (1989) .1. Biol ('he~m 2~L:l4~4t~-14~971. are as follows (in 5' to 3' orientQtio.n) ~n~
( I (-15 to +5 ): A l CATGAAAATCTTAGGTCC (SF.Q ID NO: I) ~ i+6 to +"5~ GGAGAlC'rTCiAACiC.A.TAGTG (SEQ ID NO: ~

219I8~
~ ~'0 9Sf338~8 . PCT~US"~fO5973 3 (+ 101 to +1~0): TCCCTTGACATTCATTA'I'T'r (SEQ ID NO: 3 ) Co-~
I (-15 to t-5 ): TTCATTATTTl'CTC'CTCA'r~' (SEQ ID NO: 4) 2 (+6 to +25): GAA'rCAC'1-r'l''rCCTTTGACA (SF,Q ID NO: S) 3 (+101 to +120): GTTTCTTGATCIGGTTTA'rCC (SEQ ID NO: 6) To inhibit the activity of the enzyme in porcine hepatocytes, the three nli ,~ lr~ each at a ~ ".~ ;. ", of S 11~1. are added to porcine hepatocyte cultures.
10 After 2 days. the expression of cell surtace aipha-galactosyl epitopes is evaluated by FACS
analysis using FlTC-labelled GS-I lectin as described in E.xample 3. The cells (I X 108) are r~ 1 into a monkey by infusion into an indwelling catheter in the portal vein (in a suspension of 100 ml over 30 minutes!. At days I through j after surgery~ a solution of the antisense nucleotides ( I ml of a 5 ~IM solu~ion of each nucleotide) is infused through the 15 catheter. The success of the transplant can he assessed by ~IPtPnnining the level of porcine albumin in the monkey serum at inte~rvals after LL~ kLliull hy conventional techniques.
At termination of the experiment. the porcine h~ It~J~ ~ L~ caul be Iocalized byimmunohistochemistry using pig specific primary antibodies and biotinylated secondary antibody follo-ved by detection ~ ith streptavidin peroxidase.
Other F~
While the invention has been described in particular with regard to xenogeneic ion~ the invention can be applied to other clinical situations involving naturalantibody-mediated hyperacule rejection. I;or example, natural ant;bodies play a role in the 25 rejeclion of certain allografts, such as ailografts L~ ". d across an ABO blood group mismatch, Similar to the epitopcs on nonprimate cells recognized by nalural antibodies in humans and other nonhuman primates, the A and B blood ~roup antigens are composed of carbohydrate epitopes. Accordingly. the methods of the invention can similarly be applied to altering, reducing or substantially- eliminating the expression of A and'or B blood group 30 antigens on an allogeneic cell to be l~ Lu~ into an ABO incompatible recipien~.
Additionally, in humans the~ exposure of crs~ptic alpha-galactosyl epitopes on the surface of certain ce]ls is thought to be involved in rlnt~immlmP responses. While terminal alpha-gaiactosyl epitopes are not normally expressed on hum~an cells~ internal alpha-gaL~ctosyl epitopes can become exposed on certain cell types, such as erSthroid cells or 35 thyroid cells. either as a result of aging or disease (e.g.~ exposure on erythrocyes as a result of a hematological disorder). I~ lu"l;.Jt. exposure ot'these cryptic epitopes leads to destruction of the the cells (e.g., L l ~ tl,. ucytcs)~ presumably mediated by natural antibodies in the individual directed against the epitope Moreover, this mechau~ism has been directly implicated in the premature destruciion of erythroc ytes in sickle cc ll anemia. (Galili, tJ. et al.

W0 95~33X28 P~

(1987) J Biol. C~1en~ 4683-4688; Galili, i 1. et al. (1986) J. C~in /n~est. 11:27-33).
Accordinglv, the methods of the invention for altering, reducing or eliminating the expressio of alpha-galactosyl epitopes on the surfàce o I cells can also be applied Ih. .~ .lly in sickle cell amemia and other disordens associated ~ith h~ Jt~lulJ- ;dtC expressiûn of alpha-5 galactosyl residues on cells to inhibit the binding of natural antibodie~s to this cryptic epitope For example~ for tre~atment of a 1.. f -,1. ,gi. ~I disorder, Cl,~ LLu~,y~ can be removed from a subject, treated in vi~no with an alpha-~ u~ and returned to the subject.
Alternatively. a nucleic acid (e.g.~ cul..l,hl~uli expression vector) which is antisense to an alpha-talac~ f..~e gene can be introduced into a hematopoietic stem cell of the 10 subject to inhibit the exyression ûf the epitope on h.~mAf~\logi~ cells.

EQI IIVAI FNTS
Those skilled ;n the art will recogni~. or be able to ascertain using no more than routine ~ a ;~n many equivalents tû the .specific ~ b.Jdi~ llt~ of the inventiorl 15 described herein. Such equivalents are intended to he c ". n~ d by the following claims ~ W095~33828 21~1 8 ~1 r~

SEQUENCE LISTING

(1) GENERAL INFORMATION:
(il APPLICAhT: Albert Edge (ii) TITLE OF INVENTION: Modified Cells and Methods for Inhibiting dyperacute Rejection of V~n~g n~;~
Transplants (iii1 NUMBER OF SE2UEh-CE8: 6 ti~) UU~ ~U/~ ADDRESS:
(A) ADDRESSEE: LAHIVE & COC~FIELD
(B) STREET: 60 State Street suite 510 (C) CITY: Boston (D1 STATE: Massachusetts (E1 COUNTRY: USA
(F) ~IP: 02109-1875 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (c) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII text (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILTNG DATE: 17-MAY-1995 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A~ APPLICATION NUMBER: US 08/253 782 (B) FILING DATE: 03-~UN-1994 (C) CLASSIFICATION:
(viii~ ATTO.RNEY/AGENT INFORMATION:
(A) NAME: ~ean M. Silveri (B) REGISTRATION NUMBER: P-39 030 (C) REFERENCE/DOC~ET NUMBER: DNI-007PC
(ix) TELECOMMLl~ICATION INFORMATIOh-:
(A) TELEP~ONE: (617)227-7400 (B~ TELRFA.Y.: (617)227-5941 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) sTRA~mFnNF~s: single (D) lOPOLOGY: linear (ii) MOLECULE TYPF.: oligonucleotide W095133828 - 2 1 9 1 8 9 1 P~IUS95~05973 (xi) 3EQUENCE DESCRIPTION: SEQ ID NO:l:

(2) INFORMATION FOR SEU ID NO:2:
~il 5EQUF.NCE CHARACTERISTICS:
~A) I,FNGTH: 20 base palrs (B) TYPE: nucleic acld (C) STR~NDFn~lEc~ single (3) TOPOLOGY: linear ~ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGhGATCTTG AAGCATAGTG 20 2~
(2~ INFO~MATION FOR SEQ ID NO:3:
(i~ SEQUENCE rp~R~rTFR~3TIcs:
(A) LENGTH: 20 base pairs ~B~ TYPE: nucleic acid (C~ STF~N3E3NESS single (O~ TOPOLOGY: linear (ii~ MOLECULE TYPE oligonucle~tide (xil SEQUENCE DESCP.IPTION: SEQ ID NO:3:

2) INF0RM~TION FOR SEQ ID N0:4:
(i) SEQUENCE CEARAC'TERISTICS:
(A) LENGTB: 20 base pairs (B) TYPE: nucleic acid (C') STRAh~EDNESS: Yingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi¦ SEQUENCE DESCP~IPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
li) SEQUENCE CHARACTERISTICS:
(A) LENGTB: 20 base pairy (S) TYPE: nucleic acid (C) STRANDEDNESS: single (3) ToPoLoaY: linear ~ WO9~133R28 21~18g~ r~~ 73 MOLECULE TYPE: O1ig~

(X1) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) 1N~ LU~ FOR SEQ ID NO:6:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid lC) STRANDEDNESS: single (D) TOPOLOGY: linear (i1) MOLECULE TYPE: ~1;g~n~ ~tide (Xi) SEQUENCE DESCRIPT}ON: SEQ ID NO:6:
GTTTCTTGAT ~G~~ CU~ 20

Claims (62)

1. A cell which, in unmodified form, expresses at lease one epitope on a cell surface antigen which is bound by natural antibodies when the cell is transplanted into a recipient, wherein the cell is modified by removal of the epitope from the cell surface.

2. The cell of claim 1, which is a porcine cell.

3. The cell of claim 1, which is a nonprimate mammalian cell.

4. The cell of claim 1, which is a prosimian or New World Monkey cell.

5. The cell of claim 1, wherein the epitope is a carbohydrate.

6. The cell of claim 5, wherein the carbohydrate is galactosyl(~ 1-3)galactose.

7. The cell of claim 1, which is selected from the group consisting of an endothelial cell, an hepatocyte, a pancreatic islet cell, a skeletal myocyte, a skeletal myoblast, a cardiac myocyte, a cardiac myoblast, a fibroblast, an epithelial cell, a neuronal cell, a bone marrow cell, a hematopoietic cell and a lymphoid cell.

8. The cell of claim 1, which is within a tissue or an organ.

9. The cell of claim 1, which is further modified to express a gene product.

10. The cell of claim 1, which, in unmodified form, further expresses at least one second cell surface antigen which stimulates a cellular immune response against the cell in a recipient, wherein the cell is further modified to alter, reduce or substantially eliminate expression of the second cell surface antigen.

11. The cell of claim 10, wherein the second cell surface antigen is an MIIC class I antigen.

12. The cell of claim 11, which is further modified by contacting the cell prior to transplantation with an antibody, or fragment thereof, which binds to the MIIC class I
antigen.

13. The cell of claim 1, which is modified by treatment with an agent which cleaves the epitope from the cell surface.

14. The cell of claim 13, wherein the epitope is galactosyl(~1-3)galactose and the agent is an alpha-galactosidase.

15. A porcine cell which is modified by removal of at least one galactosyl (~ 1-3) galactose epitope on a cell surface antigen.

16. A cell, isolated from a nontransgenic animal, which, in unmodified form, expresses at least one epitope on a cell surface antigen which is bound by natural antibodies when the cell is transplanted into a recipient, wherein the cell is modified by introduction of a nucleic acid which is antisense to a regulatory or coding region of a gene encoding an enzyme which is necessary for the formation of the epitope on the cell surface.

17. The cell of claim 16, wherein the epitope is galactosyl(~ 1-3)galactose and the enzyme necessary for the formation of the epitope on the cell surface is ~1-3-galactosyltransferase.

18. A cell which, in unmodified form, expresses at least one epitope on a cell surface antigen which is bound by natural antibodies when the cell is transplanted into a recipient, wherein the cell is modified by introduction of an oligonucleotide which is antisense to a regulatory or coding region of a gene encoding an enzyme which is necessary for the formation of the epitope on the cell surface.

19. The cell of claim 18, wherein the epitope is galactosyl(~ 1 -3)galactose and the enzyme necessary for the formation of the epitope on the cell surface is ~1-3-galactosyltransferase.

20. The cell of claim 18, wherein the oligonucleotide is at least about 5-35 nucleotides in length.

21. The cell of claim 20, wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5 and SEQ ID NO:6.

22. A cell which, in unmodified form, expresses at least one epitope on a cell surface antigen which is bound by natural antibodies when the cell is transplanted into a recipient, wherein the cell is modified by contact with at least one molecule which binds to the epitope.

23. The cell of claim 22, wherein the at least one molecule binds to the galactosyl(~
1-3)galactose epitope and inhibits binding of natural antibodies to the epitope in a recipient.

24. The cell of claim 23, wherein the at least one molecule is a lectin.

25. The cell of claim 23, wherein the at least one molecule is an antibody, or fragment thereof, which binds to the epitope but does not activate complement or cause lysis of the cell.

26. A cell which, in unmodified form, expresses at least one epitope on a cell surface antigen which is bound by natural antibodies when the cell is transplanted into a recipient, wherein the cell is modified by contact with a chemical inhibitor of an enzyme necessary for formation of the epitope.

27. A method for reducing the immunogenicity of a cell for transplantation into a recipient, wherein the call in unmodified form expresses at least one epitope on a cell surface antigen which is bound by natural antibodies in the recipient, comprising contacting the cell with a first agent which removes the epitope from the cell surface such that when the cell is transplanted into a recipient, hyperacute rejection of the cell is inhibited.

28. The method of claim 27, wherein the epitope is a carbohydrate.

29. The method of claim 28, wherein the carbohydrate is galactosyl(~ 1-3)galactose.

30. The method of claim 29, where the first agent cleaves the galactosyl(~ 1-3)galactose epitope from the cell surface.

31. The method of claim 30, wherein the first agent is an alpha-galactosidase.

32. The method of claim 27, further comprising, after contacting the cell with the first agent, administering the cell to a recipient.

33. The method of claim 32, further comprising administering to the recipient a second agent which inhibits T cell activity in the recipient.

34. The method of claim 27, wherein the cell is unmodified form expresses at least one second epitope on a second cell surface antigen which stimulates a cellular immune response against the cell in the recipient, the method further comprising contacting the cell with a second agent which alters, reduces or substantially eliminates expression of the second cell surface antigen.

35. The method of claim 34, wherein the second cell surface antigen is an MIIC class I antigen.

36. The method of claim 35, wherein the second agent is at least one antibody, or fragment thereof, which binds to the MIIC class I antigen.

37. The method of claim 34, further comprising, after contacting the cell with the first and second agent, administering the cell to a recipient.

38. The method of claim 37, further comprising administering to the recipient a third agent which inhibits T cell activity in the recipient.

39. A method for reducing the immunogenicity of a cell, isolated from a nontransgenic animal, for transplantation into a recipient, wherein the cell in unmodified form expresses at least one epitope on a cell surface antigen which is bound by natural antibodies in the recipient, comprising introducing into the cell a nucleic acid which is antisense to a regulatory or coding region of a gene encoding an enzyme which is necessary for the formation of the epitope on the cell surface such that when the cell is transplanted into a recipient, hyperacute rejection of the cell is inhibited.

40. The method of claim 39, wherein the epitope is galactosyl(~ 1-3)galactose and the enzyme necessary for the formation of the epitope on the cell surface is ~
1-3-galactosyltransferase.

41. The method of claim 39, wherein the nucleic acid is contained within a recombinant expression vector.

42. A method for reducing the immunogenicity of a cell for transplantation into a recipient, wherein the cell in unmodified form expresses at least one epitope on a cell surface antigen which is bound by natural antibodies in the recipient, comprising introducing into the cell an oligonucleotide which is antisense to a regulatory or coding region of a gene encoding an enzyme which is necessary for the formation of the epitope on the cell surface such that when the cell is transplanted into a recipient, hyperacute rejection of the cell is inhibited.

43. The method of claim 42, wherein the epitope is galactosyl(~ 1-3)galactose and the enzyme necessary for the formation of the epitope on the cell surface is ~
1-3-galactosyltransferase.

44. The method of claim 42, wherein the oligonucleotide is at least about 5-35 nucleotides in length.

45. The method of claim 44, wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5 and SEQ ID NO:6.

46. A method for reducing the immunogenicity of a cell for transplantation into a recipient, wherein the cell in unmodified form expresses at least one epitope on a cell surface antigen which is bound by natural antibodies in the recipient, comprising contacting the cell with a first agent which binds to the epitope such that when the cell is transplanted into a recipient, hyperacute rejection of the cell is inhibited.

47. The method of claim 46, wherein the first agent binds to the galactosyl(~ 1-3)galactose epitope and inhibits binding of natural antibodies to the epitope in a recipient.

48. The method of claim 47, wherein the first agent is a lectin.

49. The method of claim 46, wherein the first agent is an antibody, or fragment thereof, which binds to the epitope but does not activate complement or cause lysis of the cell.

50. The method of claim 46, further comprising, after contacting the cell with the first agent, administering the cell to a recipient.

51. The method of claim 46, further comprising administering to the recipient a second agent which inhibits T cell activity in the recipient.

52. The method of claim 46, wherein the cell in unmodified form expresses at least one second epitope on a second cell surface antigen which stimulates a cellular immune response against the cell in the recipient, the method further comprising contacting the cell with a second agent which alters, reduces or substantially eliminates expression of the second cell surface antigen.

53. The method of claim 52, wherein the second cell surface antigen is an MIIC class I antigen.

54. The method of claim 53, wherein the second agent is at least one antibody, or fragment thereof, which binds to the MIIC class I antigen.

55. The method of claim 52, further comprising, after contacting the cell with the first and second agent, administering the cell to a recipient.

56. The method of claim 55, further comprising administering to the recipient a third agent which inhibits T cell activity in the recipient.

57. An isolated nucleic acid comprising a nucleotide sequence which is antisense to a coding or regulatory region of a porcine alpha-1,3-galactosyltransferase gene which, when introduced into a porcine cell, inhibits the activity of a porcine alpha-1,3-galactosyltransferase in the cell.

58. The nucleic acid of claim 57; which is an oligonucleotide is at least about 5-35 nucleotides in length.

59. The nucleic acid of claim 58, wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5 and SEQ ID NO:6.

60. The nucleic acid of claim 57, which is contained in a recombinant expression vector.

61. A method for transplanting a cell into a recipient subject wherein the cell in unmodified form expresses at least one epitope on a cell surface antigen which is bound by natural antibodies in the recipient, comprising:
a) contacting the cell with a first agent which alters, reduces or substantiallyeliminates expression of the epitope on the cell surface;
b) administering the cell to the recipient subject; and c) administering to the recipient subject a nucleic acid which is antisense to aregulatory or coding region of a gene encoding an enzyme which is necessary for the formation of the epitope on the cell surface such that when the cell is transplanted into the recipient subject, hyperacute rejection of the cell by the recipient subject is inhibited.

62. A method for transplanting a cell into a recipient subject, wherein the cell expresses at least one epitope on a cell surface antigen which is bound by natural antibodies in the recipient, comprising:
a) administering the cell to the recipient subject; and b) administering to the subject a nucleic acid which is antisense to a regulatory or coding region of a gene encoding an enzyme which is necessary for the formation of the epitope on the cell surface such that when the cell is transplanted into the recipient subject, hyperacute rejection of the cell by the recipient subject is inhibited.