Note: Descriptions are shown in the official language in which they were submitted.
~ 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.
Wo95133828 2191891 r "u. ~
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.).
21gl8~
W0 95133818 ~ j r~ l ~ L ~ v l~
- 3 -
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
- 4 -
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 --
- 10-
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 ~ ,
WO 95/33828
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
- 13 -
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
- 17-
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,
- 18 -
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
- 21 -
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
-23 -
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~
-24 -
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~
-25 -
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~
-28 -
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~
-29 -
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
-30 -
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~
-31 -
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 ,~
-36-
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
- 39 -
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.,,~
40 -
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
- 41 -
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~
42 -
(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~
-43 -
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
-44-
(xi) 3EQUENCE DESCRIPTION: SEQ ID NO:l:
ATCATGAAAA TCTTAGGTCC 20
(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:
35 TCCCTTGACA TTCATTATTT 20
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:
TTCATTATTT TCTCCTCATC 20
(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
-45 -
MOLECULE TYPE: O1ig~
(X1) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GAATCACTTT TCCTTTGACA 20
(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
