Note: Descriptions are shown in the official language in which they were submitted.
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~ Title: Recombinant Viruses Incorpor~ a Protease Cleavable Protein
~ Field of the Invention
The invention relates to recombinant viral particles h~col~old~ g ~rulGase cleavable
p,o~ei,ls and to various applications of the rcco.llbh~ll particles.
Rell UVil al e-,~elo~,es
Retroviral envelope ~1YCO~1VLGinS mPf~i~t~ specific viral att~rhm~ont to cell surface
l~,CG~lUl~ and s~lhseq~ontly trigger fusion b~lweGIl the viral envelope and the target cell
membrane. All leLIovildl envelope spike glyc(~ ltills ex;l~ to date are
homooligomers CO..I;.i.~;,.~ two to four heterodi l~"iC subunits (Doms et al. 1993 Virology
193, 545). Each subunit co.ll~lises a large e~Lldvildl glyco~lulGin moiety (SU)
noncovalently ~tt~rh~ at its C_lG1111i~1US to a smaller L~ .,.hrane polypeptide (TM) that
ancllu,~ the complex in the viral Ill~ ldne,. In the case of murine C-type lGLluvildl
vectors, SU co.l~ es two domains CO~ ''CIr~1 by a proline-rich hinge, the N-
~
domain confc.lil~g l~ce~tor ~L,ecirl~ y and exhibiting a high degree of col~se.vdlionbetween murine le~lk~ viruses (MLVs) with dirr.,.~_..L host ranges (Battini et al. 1992
J. Virol 66, 1468-1475). Moloney MLV envelopes confer an ecoLlopic host range because
they attach selectively to a peptide loop in the murine cationic amino acid ha~pOlL~,.
(CAT-1), found only on cells of mouse and rat origin (Albritton et al. 1989 Cell 57, 659-
666). 4070A MLV envelopes attach to an epitope on the ubiquitous RAM-1 phosphatesyLu~olLt:r that is co~G.ved throughout many ~..~....-.~li~n species, and confer an
amphotropic host range (Miller et al. PNAS 91, 78-82; VaIlZeijl et al. 1994 PNAS 91,
1168-1172). Thus, retroviral vectors with 4070A envelopes infect human cells
promiscuously, whereas vectors with Moloney envelopes fail to infect human cells.
Proteolytic activation
In all retroviruses that have been studied to date, the SU and TM polypeptides are derived
from a single chain precursor glycoproLei-- that undergoes proteolytic ~-ldLuldLion in the
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Gogi compartrnent during its transport to the cell surface. Uncleaved envelope p~ ;u~jo
glyco~-uLeil,s can be incorporated into viruses but are unable to trigger membrane fusion.
The requhc;.ll~--L for proteolytic maturation/activation is a feature comrnon to the fusogenic
membrane gly-;oploteills of many virus families and is most cornmonly m~ t~1 by the
ubiquitous Golgi compamnent serine protease, furin. However, there are well-
~oc-lm~nt~l examples of viral membrane glyco~.oLt:ins tnat resist cleavage by ubiquitous
intral~e~ r proteases and instead are cleaved by secreted proteases available only in a few
host systems (Klenk & Garten 1994 Trends Microbiol. 2, 39). Moreover, there is at least
one example of an inflllen7~ virus strain whose h~rn~ llltinin is activated by a target cell
prûLcase at the stage of virus entry (Boycott et al. 1994 Virology 203, 313). In these
il~iL;.Il~s, the binding reactions of the viral membrane glyuoL,~vL~:ins are ul~fr~chd by
their proteolytic cleavage - only their ability to trigger membrane fusion is affected.
R~ vi~al display of nonviral polypeptides as N-le~ al f ~.. 'nn~ oP SU
A general method has been disclosed which allows the display of a (glyco)polypeptide on
the surface of a l~,Lrù~ .l vector as a gen~tir~lly encoded extension of the SU ~Iyco~l~Lci
(VVO 94/06920~ Medical Research Council). The poly-peptide is fused (by genetic
ilIf~'~illg) to the N-trrmin~l part of the SU glycol,loLeill such that the envelope protein
to which it has been grafted remains sllbst~nti~lly intact and the fused nonviral polypeptide
ligand is displayed on t_e viral surface. To date, the approach has been used to display
many different polyL,e~ide ligands on MLV - based retroviral vectors, inrlllriing single
chain antibodies, cellular growth factors and imm~lnnglobulin binding domains (WO
94/06920, Medical Research Council; WO 96/00294 Medical Research Council; Cossetet al. 1994 Gene Therapy 1, S1; and Nilson et al. 1994 Gene Therapy 1, S17). In
contrast to other rhim~rric retroviral envelope ~IOLt:illS that have been described (CD4
rhim~rra, K~h~ra et al. 1994 Science 266, 1373; Chu & Dol~ul~ 1995 J. Virol. 69,2659; Somia et al. 1995 PNAS 92, 757û) viral incorporation of N-trrrnin~lly extended SU
glyco~oteills does not require the ~ eilce of unmodified envelope glyco~lù~ills.
In principle, a virus displaying such a chimaeric envelope protein might be capable of
multivalent ~tt~rhm~nt both to the natural virus receptor (via the N-terminal domain of
SU) and to the cognate le.,~;~Lor for the displayed polypeptide. We have found that this
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holds true for retroviral vectors displaying epiderrnal growth factor (EGF). However,
depending on its precise nature, its ~io~G~ y to oligomerise and its mode of linkage to
the SU g1yco~loteill? the displayed polypeptide may sterically hinder the interaction
between the N-terminal domain of SU and the natural virus l~cepLur.
Mo~ tiQn of l~Lr-~viluS llo~ by N-tern~inally ~n~le-l SU ~ly~o~~l~leinsWhen dirre.c ~ ,cG~tur-binding domains were displayed on MLV reLlùvildl vectors as N-
termin~l extensions of their intact SU glyco~ioLGins, it was found that host range could be
extended or l~,i.Lli~LGd by the displayed ligand (Cosset et al. 1995 J. Virol. 69, 631~
6322). Thus, as a demonstration of host range extension, murine ecotropic vectors
displaying the RAM-l rcce~Lul-binding domain from 4070A SU were able to bind andinfect RAM-l-positive human cells. In contrast, as a demonstration of host rangei.;Lion, ecc Llu~ic and amphotropic vectors displaying EGF could bind to EGF
.~ce~Lu.~ but were thereafter sequestered into a non-infectious entry ~dLhwdy, giving
greatly reduced titres on EGFl~,ccptor-positive cells, but normal titres on EGF receptor-
l~gaLivG cells. EGF receptor-negative cells, which were fully susceptible to t'ne
P..~j"r~ ,d lGLlu~)ildl vector, showed reduced ~.usce~Lil~ility when they were ~ Li. ~lly
modified to express EGFl~,ceptoli.. The reduction in i.usce~Libility was in p.o~olLion to
the level of EGF l~ce~tul expression. MO1~OVG17 when soluble EGF was added to
colll~eLiLively inhibit virus capture by the EGF rGce~ul~7~ gene Lldl~.re. was restored. In
this latter example, the engineered vector is capable of binding to the natural virus
cel~tùr or to the l~,c~ or for EGF;~tt~r'nmPntto the natural virus lece~Lul leads to
infection of the target cell, whereas the ~tt~rhmrnt to the EGFl~,cep~ul does not lead to
infection of the target cell. Where the target cell e~le~ses both species of lGCG~tùl, the
two binding reactions (4070A envelope protein to RAM-l, and EGF to EGFlGce~or)
proceed in co.l.~c-iLion and the infectivity of the virus for the target cells is reduced in
proportion to the efficiency with which the EGF-EGF receptor binding reaction col~ th,s
virus away from RAM-1.
The degree to which gene Lldl~7ÇGl can be inhibited by this mlQch~ni.cm depends on the
relative ~ffiniri,os of the two binding reactions (envelope protein to natural receptor and
non-viral ligand to its cognate .~ce~or), the relative ri~oncitiPs of the two lGce~Lu~i. on the
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target cell surface, and the relative llen.citi~s of the nonviral ligand and the intact envelope
protein on the viral surface. Inhibition of gene Lldn~r~l is additionally influenced by
intrinsic ~lo~clLies of the receptor for the non-viral ligand, such as the flict~nf~e it projects
from the target cell membrane, its mobility within the target cell membrane and its nalf
life on the cell surface after engagement of ligand.
Steric hindrance of the interaction betwveen the N-terminal domain of SU and the natural
virus ~~,ce~lur provides an 7,ll~ 7rivc ."~ ~h~ni~"~ wlle.LGby polypeptides displayed as N-
r~ ~ " ,;n~l extensions of SU can restrict leLlùvhdl host range. For example, ~h;",7 ic
envelopes displaying the N-l~.".;,~7l domain from 4070A MLV SU as an N-t~rmin~l
e~t~?nci~n of Moloney MLV SU can a~alc,nLly bind to RAM-1 (the re,ce~Lor for 4070A
SU) but not to ecoR (the lccc~Lor for Moloney SU); it may be possible that the displayed
~om~in.c from 4070A SU may form a L-~nelic cap over the Moloney SU trimer,
completely m~cking its ,~ ol binding sites. If this model is correct, then it should also
be possible to gel~latc rhim~ric envelopes in which the rccc~ur bindillg sites of the
intact 4070A SU glycoplo~cul (through which the virus ~ s to human cells) are
m~.c~ l by a displayed polypeptide, such as t'ne N-te~rnin~ m~in of Moloney MLV SU,
that does not bind to human cells.
Phage ~ 15y of cleavable dom~;nc
There are certain similarities 'ocLweell lc.lo\dldl vectors displaying polypeptide ligands as
N-L~lllPillal ext~ncions of their envelope gly~;o~luLcil~. and fil~ .-lous bacteriophage
displaying polypeptide ligands as N-tt~ rnin~l extensions of the gene III proRin. Lib aries
of fil~ us '~snhstr~t~ phage" displaying cleavable binding domains have lccel,tly been
used to identify optimal substrates for known proteases (Matthews & Wells 1993 Science
~, 1113; Matthews et al. 1994 Protein Science 3, 1197; Smith et al. 1995 J. Biol.
Chem. 270, 6440). However, fi~ ous phages do not nanlrally infect ~.;.."."~ n cells
and there has been no demonstration that cleavable domains fused to tne gene III protein
can inflllen~e tne Llu~ l of t'ne phages on which they are displayed.
Suu~ of' the Invention
In a first aspect the invention provides a recombinant viral particle capable of h~ ;Lulg a
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eukaryotic cell, the viral particle colllpli~ g: a subst~nti~lly intact viral glycu~loL~
fused, via an intervening linker region, to a heterologous polypeptide displayed on the
surface of the particle, which heterologous polypeptide mo~ tPs the ability of the viral
particle to infect one or more eukaryotic cell types and is cleavable from the viral
~ glycoprotein by a protease acting selectively on a specific ~loL.,ase cleavage site present
in the linker region, such that cleavage of tne heterologous polypeptide from the viral
glycopLotein allows the glyc~.~loteill to interact normally with its cognate lc:cept~,l on the
surface of a target cell.
Such a particle is of considerable benefit in the targeted delivery of nucleic acid sequences,
which may be present within the particle, to ~ecirlc desired target cells, such as is
required for gene therapy.
In alloLl,er aspect the ill~,.lLioLI provides a nucleic acid col~hu~;L, colllL~lisi~g a sequence
encoding a fusion protein, the fusion co~ i~ a sl-b~ lly intact viral gly~;oploLe~
fused, via an intervening linker region, to a heterologous polypeptide, wherein the fusion
protein is capable of being incorporated into a viral particle capable of i"r~ g an
eukaryotic cell, and further wl~,reill the heterologous polypeptide m~ tPs the ability of
the viral particle to infect one or more eukaryotic cell types, but cleavage of the
heterologous polypeptide from the fusion protein allows the viral glycoprotein to interact
normally with its cognate lcceptor on the surface of the euk~lyoLic cell.
In a further aspect the invention provides a nucleic acid seqllPnre library COlll~ illg a
plurality of the nucleic acid constructs defined above, wherein at least part of the sequence
encoding the hlL~ ulg linker region is r~n~lomi~e~l in each construct, such that each
construct colll~ es one of a plurality of dirr~.~llL linker regions which are lc~ sell~d in
the library.
The invention also provides a library of the viral particles defined above, each particle
comprising a single nucleic acid construct from the nucleic acid library defined above.
The term "subst~nti~lly intact" as used herein is intrrl~ to refer to a viral gl~coL~loL~i
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which retains all of its domains so as to conse;ve post-trancl~tional ~Lucessi.lg,
oligomerisation (if any), viral inco.~o.alion and fusogenic L~-ù~ ~Lies. However, certain
alterations (e.g. point mutations, deletions, additions) can be made to the glyco~r~Jlchl
without ~ignific~ntly ~ffecting these functions, and glycoproteins cont~inin~ such minor
modifications are considered s~hst~nti~lly intact for present ~,lL~oses. In particular, the
gluycolJIùtein may lack a few (e.g. about 1 to 10) amino acid residues, especi~lly at the
N tP~mimls, but will otherwise be generally the same size as the wild-ype protein and
possess subst~nti~lly the same biological ~lu~ ies as tne wild-ype protein.
The ~hlk,. ~ g linker region will preferably be quite short, ypically Colll~ g from 4
to 30 amino acid residues, more ypically 5 to 10 residues. A short linker is preferred,
because this will tend to m~ximi~e the modulation of infection effected by the heterologous
polypeptide. In certain embo~ s, a suitable linker region may be present as a natural
part of the heterologous displayed polypeptide.
The Viral particle may be any virus capable of ilLlt;~;Ling one or more eukaryotic cell types,
but co.lvcl~iel.Lly will be a viral particle suitable for use in gene therapy, such as an
adenovirus or a lC,riUVilUs (especially a C-type rt,LlùviluS).
The viral glyco~loLein will typically col..~ise a viral envelope glyco~lu~chl, or may be
a cl-il--c.ic polypeptide cu...~ h.g se4uel.ces corresponding to different viral glyco~lu
but which, in total, co-~iLuLe a subst~nti~lly intact, functional protein.
The heterologous polypeptide may be a short amino acid sequence (say, a peptide of about
10-20 residues, especially if the sequence undergoes oligomerisation, e.g. a leucine zipper
peptide sequence) but more typically will comprise 30 or more amino acid re~ os
Generally, but not esscllLially, the polypeptide will comprise a functional binding domain.
The heterologous polypeptide, when fused to the viral glycu~.oL~i.. via the linker region,
modulates the ability of the viral particle to infect one or more eukaryotic cell types.
Specifically, the ~.ese.1ce of the heterologous polypeptide serves to inhibit the process of
infection of a eukaryotic target cell m~ t~d by the viral glycuploL~ . The term
"heterologous" is int,onrleri to refer to any polypeptide which is not naturally fused or
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otherwise bound to the viral glyco~.oLehl.
The heterologous polypeptide may or may not possess specific binding affinity for a
surface component of a target cell. In one embodiment, the heterologous polypeptide has
~ affinity for a cell surface colllL,oll~,llt, binding to which will not lead to infection of the cell
by the virus. Within this general embo~lim~ont a variety of different examples (each with
dirrclcll~ ~lo~ ies) can be envisaged. In one example, a eukaryotic cell e~lc~.ses a
l~ce~Lur for the viral glyco~rolcill (binding to which allows the virus to infect the cell) and
a non-pclll~issive l~,ceptor for the heterologous polypeptide, with inhibition of infection
res~llting simply from col,l~eLiLion beL~ the viral glycu~loLcill and the heterologous
polypeptide for binding to their l~ ecLive l~ceptol~. on the target cell. In a different
example, the collÇûlllla~ional arrangement of the l~,~.L,e~,livc rcce~lol~ and their ligands is
such that binding of the heterologous polypeptide to its receptor causes steric hilldla~ce~
such that binding of the viral gl~coplolcill to its lcccL)lor, or fusion of the virus and the
cell, is blocked.
In a second embo-lim~nt the heterologous polypeptide does not bind to a non-p~.lllis~.i~.,
receptc.l on the target cell, but the ~ scllce of the heterologous polypeptide serves to
create steric hi,-.l.,..-~e sufflcient to ~l~,VC.l~ binding of the viral glycoplutein to its
ce~Lur, or may allow billdi~, to occur but inhibits snhseq~llont fusion of the viral particle
with the target cell, such that infection of the cell by the viral particle is inhibited at the
binding and/or fusion stage.
In a particular embodiment the heterologous polypeptide is capable of forming oligomers
when displayed on the surface of the viral particle. Typically the oligomer will be a dimer
or, more preferably, a Llilller. Such oligolllclisation may allow for efficient inhibition of
the interaction between the substantially intact viral glycoprotein and its leceptor, which
inhibition may be removed by proteolytic cleavage of the oligomerised heterologous
polypeptide from the viral glyco~ tehl. The illlc- vel~ g linker may also undergo
oligomerisation.
Where the viral glycoprotein is itself capable of forming oligomers (e.g. retroviral env
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protein), it is plcf~ ,d that the heterologous polypeptide oligomerises with the same
stoichiometry as that of the viral glyco~lùLt~ . Vascular endothelial growth factor (VEGF)
and tumour necrosis factor (TNF) are both proteins which are known to oligol,le,ise and
have high affinity for cell surface ligands. Effective (oligomer-forming, and preferably
ligand-binding) portions of these proteins may be particularly suitable for use as
heterologous polypeptides in accordance with the present invention.
In the present invention the heterologous polypeptide is cleavable from the viral
glycop,vLei" by the selective action of a protease (i.e. a molecule capable of cleaving a
peptide bond) which cleaves the linker region at a protease cleavage site. The cleavage
site ,e~lcsc.,Ls a unique peptide sequence not present, or at least not ~ccPcsible to the
protease, in the viral glycu~oteill, although a similar site may be present in the
heterologous polypeptide (this is generally preferably avoided, as proteolytic attack on the
heterologous polypeptide may affect its functioning). The size, and number, of the
~vL-,ase cleavage sites in the linker region may be varied with advantage. Thus, for
example, the ylc;sellce of two or more cleavage sites, lc;co~ni3ed by the same or by
,~,~e~:~ivc; L~ut~ases could f~cilit~tP cleavage, whilst the use of one long cleavage site will
tend to enh~nre Sp~;irlci~y of cleavage.
Large numbers of specific proteases, and the cleavage sites they recognise, are known to
those skilled in the art (see, for example, Vassalli & Pepper 1994 Nature 370, 14-15, and
ref.,~c;nces cited therein). Proteases are involved in a number of physiological and/or
pathological p,ocesses, such as tissue remodelling, wound healing, infl~mm~tion and
tumour invasion, and such proteases would be of use in the present invention. Specific
classes of protease which would be of use include: serine ~,oL~ases (such as
plasminogen/plasminenzymes);~ Lehleproteases;andmatrixmetallo~,oL~ ases(MMPs)
of various types, (such as Gel~tin~ce A and membrane-type MMP ~or MT-MMP]).
The protease which serves to cleave the heterologous polypeptide from the viral
glycoprotein is preferably selectively secreted by the cell to which it is desired to target
the viral particle, or at least the tissue in which the target cell is located. It is L1l~fellGd
that the protease will be secreted only by cells of the target cell type or, less preferably,
_
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omy by cells (ot'ner than the target cells) remote from the tissue co~ the target cell.
This confers an extra degree of specirlciLy, which is desirable when the particle is used
for targeted gene delivery. Thus the present invention allows for two-step ~ eLillg, in
which a first level of specificity may be imposed by the heterologous polypeptide (e.g.
~ with specific affinity for a ligand on the surface of the target cell), and a second level of
specificity may be imposed by selective cleavage of the heterologous polypeptide by
proteases secreted by, or in the same tissue as, the target cell. ~Ittorn~tively~ the relevant
protease may be added exogenously, such t'nat if the viral particle is used for targeted gene
delivery in a patient, the protease may be ~ d (e.g. by injection) to the tissue in
which the target cell is located.
essibility of the protease cleavage site to the relevaM ~lul~ase (i.e. that which
recognises and cleaves the site) may also be varied. It has been found by the present
hlvcnlul~ that use of a short intervening linker region (e.g. S amino acid residues) tends
to restrict ~rce,~ihility of the cleavage site, and use of a larger linker region (e.g. 15 to
20 residues) tends to incl~asc ~rceccibility of the cleavage site. This phenomenon is
p~c~ ably due to seric hi".l.,....~e of the cleavage site due to the proximity of the viral
gly~;oproleill and/or the heterologous polypeptide. Acco,.li~ly, it should also be possible
to modify arce~cihility of the cleavage site, as desired, by valyillg the size of the
heterologous polypeptide.
In one embodiment, the cleavage site is ~rc~ossihle to the relevant protease before the viral
particle becomes bound to an eukaryotic cell, whilst in an ~lt~rn~tive embodiment the
cleavage site is in~rcescihle to the protease until the viral particle has become bound to
a eukaryotic cell. In t'nis latter embo~1iml-nt the cleavage site may be made ~c cPc~ible by
a conformational change occurring as a result of binding of the heterologous polypeptide
to its cognate receptor. Alternatively, the viral glycup.oLeill binding to its cognate
cc~tor may make the cleavage site arces~ible, cleavage of the heterologous polypeptide
then allowing fusion of the viral particle to the eukaryotic target cell.
As inrlir~te~l above, in another aspect the invention provides for a method of selectively
delivering a nucleic acid to a target eukaryotic cell present among non-target cells,
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Co~ lisil.g: a~lmi"i~l~,ing to the target and non-target cells a recombinant viral particle
capable of infecting eukaryotic cells, the particle cu.n~.isi.lg the nucleic acid to be
delivered, and a fusion protein comprising a substantially intact viral glyco~,oLt:ill fused,
via an intervening linker region, to a heterologous polypeptide displayed on the surface
of the particle, which heterologous polypeptide modulates the ability of the particle to
infect one or more eukaryotic cell types and being cleavable from the glycoprotein by a
protease acting selectively on a specific protease cleavage site present in the linker region,
such that cleavage of the heterologous polypeptide from the glycopl~,~ill occurspreferentially at, or in the vicinity of, the target cell and allows the viral glyco~roLeill to
interact normally with its cognate receptor on the surface of the target cell.
The method may be ~clr~Jlllled in vitro, for example to deliver a lethal nucleic acid to
fibroblasts in tissue culture, which cells often oUL~ W a slower-growing, more
dirr~..,."i~te~l cell type in culture. ~It..~ iv~:ly, the method may be pclro,-lled as a
m.othf -1 of gene therapy, in vivo or may be ~.,.rul-l-ed ~x vivo, on cells which are then re-
introduced into a human or animal subject. Pl~r~lc..Lial cleavage of the protease cleavage
site may occur only when the viral particle is bound to the target cell, or when the viral
particle is ~ ent to the target cell and thus exposed to a protease secreted by the target
cell. It may well be pler~ d to add the relevant protease exogenously, after
~ mini~tration of the viral particle, so as to ensure sufficient co.lc~ dLion of the ~ L~ase
and as another aid to specificity of delivery (by local a~ i"i~L.dLion of the protease). It
is already known that some proteases may be safely given in vivo (e.g. those enzymes,
such as urokinase, streptokinase and tPA, given to patients with myocardial infarcts).
The invention also provides, in a further aspect, a method of screening nucleic acid
sequences for those which encode an amino acid sequence which may or may not be
cleaved by a protease. As already mentioned, many viral envelope glycoproteins are
processed through the cellular export pathway of the eukaryotic cell in which they are
synth.osi~ecl7 generally leading to cleavage, which cleavge is os~ for production of an
infectious viral particle.
The invention the.efolc provides a method of screening nucleic acid sequences for those
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11
which encode an amino acid sequence which may or may not be cleaved by a plutcase
present in the export paLllway of an eukaryotic cell, comprising: c,.l-cing the expression
of a plurality of nucleic acid sequences in eukaryotic cells, each sequence encoding a
s~lbst,.mi~lly in~act viral glycoprotein fused to a heterologous polypeptide via a randomised
intervening linker region, the L"~sence of the heterologous polypeptide serving to inhibit
the (binding or fusion) interaction of the viral glyco~luLcill with its cognate receptor, and
wherein each nucleic acid sequence further colllplises a par'~ging signal allowing for viral
incorporation, such that those hlt~ -~c~ g linkers which are recognised by a protease
present in the export pathway of the eukaryotic cells will allow for cleavage of the
heterologous polypeptide from the viral glycoL~loLcill, reslllting in the production of an
infectious viral particle; and recovering those nucleic acid sequences directing the
expression of such cleavable linker regions from an infected cell.
Nucleic acid seqllPrlre ~lPt~l ".i..~tion may optionally be p~,~ru.ll.ed, to deduce those amino
acid sequences which are recognised by an export prolease.
A modification of the above method will allow for the scl~ ~lg of nucleic acid sequences
for those which encode an amino acid sequence which may or may not be cleaved by a
protease present in the eukaryotic cell import p~Lllw~y. As explained above, the ~lcscl~e
of a heterologous polypeptide may, in some embo-iimPnt~, still allow for binding of t'ne
viral gly~;ol~lutein to its cognate receptor, but will prevent fusion of the viral particle with
the eukaryotic cell to which it is bound. Cleavage of the heterologous polypeptide by a
protease in the cellular import pathway will then allow infection of the cell.
Thus, in a further aspect the invention provides for a method of s~;lc~lPillg nucleic acid
sequences for those which encode an amino acid sequence which may or may not be
cleaved by a protease. comprising: causing the c~lcs~.ion of a plurality of nucleic acid
sequences in eukaryotic cells, each sequence encoding a substantially intact viral
glyco~loLcil~ fused to a heterologous polypeptide via a randomised intervening linker
region, the ~l~sence of the heterologous polypeptide serving to inhibit the fusion of a viral
particle with a eukaryotic cell to which it is bound, and wherein each nucleic acid
sequence further cull-~lises a p~ck~ging signal allowing for viral incorporation; enriching
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the viral particles so produced for those which retain the heterologous polypeptide (and
so are non-infectious); and cont~rting the enriched particles with a susceptible eukaryotic
cell Colllplisil.g, or in the presence of, a protease such that those intervening linkers which
are recognised by the protease will allow for cleavage of the heterologous polypeptide
from the viral glycupl~Jt~ , reS~lt;ng in productive infection of the eukaryotic cell; and
recovering those nucleic acid sequences directing the expression of such cleavable linker
regions from the infectefl cell. As above nucleic acid sequence ~letermin~tion may
optionally be pe.rol,llcd to allow deduction of the corresponding amino acid sequences.
The e... i~ r step is required because of the possibility that the heterologous polypeptide
may be cleaved from the viral glycop,u~eill by an export pathway protease during ~yllLhe.,is
of the particles. A number of possible enrichm~nt techniques will be readily apparerlnt
to those skiled in the art with the benefit of the present ~rh;.,g. For example, prior to
infection of the susceptible cells, the viral particles could be subjected to an affinity
enrirhmPnt technique - the particles could be passed through an antibody affinity column,
whc~ l the antibody has affinity for the heterologous polypeptide. Those particles which
retain the heterologous polypeptide will be bound to the column, whilst those in which the
heterologous polypeptide was cleaved during export from the producing cell will pass
st~ight through the column. After washing, the bound particles may be eluted (e.g. by
competition with free heterologous polypeptide, or the part thereof recognised by the
antibody, or by alteration of pH or other factors) and then used to infect the susceptible
"intlir~tor" cells.
The invention will now be further described by way of illustrative examples, and with
reference to the acco,llpall~dllg figures, in which:
Figure 1 is a s~hrm~tir l~lesf..l~fion of retroviral vector constructs coding for chimeric
envelopes;
Figures 2 and 3 are photographs of Western blots demonstrating viral incorporation of
certain chimeric polypeptides and their sel~iLivi-y to Factor Xa protease;
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13
Figure 4 is a photograph showing the infectivity of various ~-galactosidase tr~n~ cin~
viruses on target cells with or without Factor xa tre~rmPnt as judged by assay on X-gal
cont~ining plates;
Figure 5 is a schPm~tir l~yl~se~ tion of how two-step ~~ iulg of gene delivery might
be achieved using the present invention;
Figure 6A is a photograph of a Western blot demo~ .t;,~y~ viral iucol~o-d~ion of certain
chimeric polypeptides and their se~siLiviLy to Factor Xa ~ ase;
Figure 6B is a bar chart illu~,LldLing the infectivity of certain recombinant viruses in the
~,se~lce or absence of Factor Xa;
Figure 7 is a sch~ lir l~ se~ lion of l~,Ll~vildl vector colL~Ll-lcL~7 coding for cl~ c~ic
envelopes;
Figure 8A is a photograph of two Western blots, the upper one co...~ electrophoretic
mobility of various ch;-ll ~;C polypeptides, the lower one colllpalillg the amount of protein
present;
Figure 8B is a photograph of a Western blot colll~alhlg the sensi~ivily to Factor Xa
protease of various rhimPric polypeptides;
Figure 8C is a photograph of a Western blot colllyalillg procec.~inv of certain chimPric
polypeptides;
Figure 9 is a panel of photographs Culll~illg the growth of of a recombinant virus on
NIH 3T3 and A431 cells, with or without Factor Xa tre~tmPnt
Figure 10 is a schPm~tir represent~tic)n of retroviral vector constructs coding for chimeric
envelopes;
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14
Figure 11 shows three Tables, A, B and C, illustrating the titre (in el~y~llc forrning units,
"e.f.u.") of various recombinant viruses on NIH 3T3 or A431 cells in the absence (-) or
ence (+) of Factor Xa protease;
Figure 12 is a srh~m~tic lcL~-~se~ tion of retroviral vector constructs coding for chimeric
envelopes;
Figure 13A is a photograph of a Western blot demonstrating viral incorporation of various
Ch~ .iC poly~lides;
Figure 13B is a photograph of a Western blot colllpalillg the sensitiviy of various
chimeric polypeptides in the ~l~ sellce (+) or absence (-) of pro-ge!~tin~e A, with (+) or
without (-) pre-activation of the protease by p-~minophenylmercuric acetate (APMA);
Figure 1~ is a bar chart showing how hlLCLiviLy of a l~,colllbilldllt virus is ~lep~nrl-ont upon
con~;ellLlaLion of pro-gel~tin~o A;
Figure 1~ is a bar chart colll~alil g the i~e-;LiviLy of three different recombinant viruses
on HT 1080 or A431 cells;
Figure 15A is a photograph comparing the growth of a recombinant virus on HT 1080 or
A431 cells;
Figure 16 is a panel of four photographs (I, II, III and IV) co~ ~hlg the infectiviy of
various viruses on HT 1080 (H) or A431 (A) cells;
Figure 17 is a photograph of a gel for detection of gelatinolytic activity; and
Figures 18 and 19 are sch~m~tic l.,pl~c..l~tions of retroviral vector constructs coding for
chimeric envelopes.
EXAMPLES
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F,Y~ml-le 1
Su~
Tropism-modifying binding domains were anchored to murine lellkAPmiA vi;us (MLV)envelopes via factor Xa-cleavable linkers to generate leLlovildl vectors whose Llupk.lll
could be regulated by factor Xa protease. The binding domains could not be cleaved from
vector particles by factor Xa when the linker was fused to a~mino acid +7 of Moloney
MLV SU but could be efficiently cleaved when fused to amino acid +1 of Moloney or
4070A MLV SU glycoploL~ s. Vectors displaying a cleavable EGF domain were
selectively seql~Pstpred on EGF l~ce~Lor-t~ ;s~illg cells, but their h~cliviLy was fully
restored when the EGF domain was cleaved from the vector particles with factor Xa.
Partial le~oldtion of il~feuLivi~y was observed when omy a fraction of the envelope
plO~t;illS were cleaved. Collv~.ely, vectors that displayed a cleavable RAM-1 binding
domain fused to Moloney MLV SU had an exrAn~1e~1 host range t'nat was reversible upon
l1~.A~ with factor Xa. It is suggested that retroviral vectors with en~ el~d binding
specificities whose Llupi~.llliS regulated by t~ O..Ul~. to ~.~ecirlc proteases may facilitate
novel strategies for ~ lg retroviral gene delivery.
Intro~ln~tion, results, and ~liS~
MLV-derived l~ uvhdl vectors are versatile gene delivery vehicles whose host range can
be varied by illcolyoldlion of dirr~ envelope spike glyco~luteil~s (Miller, 1992 Curr.
Top. Microbiol. Tm mllnr)l. 158, 1; Vile & Russell, 1995 British Medical Bulletin. 51, 12;
Weiss, in ReLluvilidae7 J. Levy, Ed. (Plenum Press, 1993), pp. 1-108). Retroviral
envelope spike glyco~loLei-ls m~liAtP virus ~ttA(~hmPnt to specific receptors on the target
cell surface and subsequently trigger fusion between the lipid membranes of virus and host
cell. The envelope spike gly~u~uluL~ s of murine lellkAPmi~ viruses (MLVs) are
homotrimers in which each of the three heterodimeric subunits col~lL,lises a large extraviral
glycoprotein moiety (SU) ~ttAl~h~l at its C-l~-. .,.i,..lc to a smaller transmembrane
polypeptide (TM) that anchors the complex in the viral membrane (August et al., 1974
Virology 60, 595; Ikeda et al., 1975 J. Virol. 16, 53; Kamps el al., 1991 Virology 184,
687). SU consists of two domains connPctecl by a proline-rich hinge, the N-terminal
domain co~ .lhlg lece~Lur specificity and exhibiting a high degree of conservation
between MLVs with different host ranges (Battini et al., 1992 J. Virol. 66, 1468; Morgan
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16
et al., 1993 J. Virol. 67, 4712; Battini et al., 1995 J. Virol. 69, 713). Moloney MLV
envelopes confer an ecotropic host range because they attach selectively to a peptide loop
in the murine cationic amino acid transporter (CAT-1), found only on cells of mouse and
rat origin (Albritton et al., 1989 Cell 57, 659; Albritton et al., 1993 J. Virol. 67, 2091).
4070A MLV envelopes attach to an epitope on the ubiquitous RAM-1 phosphate syll~o.Le.
that is conserved throughout many m~mm~ n species and confer an amphotropic hostrange (Miller et al., 1994 Proc. Natl. Acad. Sci. U.S.A. 91, 78; VanZeijl et al., 1994
Proc. Natl. Acad. Sci. U.S.A. 91, 1168; Kavanaugh et al., 1994 Proc. Natl. Acad. Sci.
U.S.A. 91, 7071). Thus, retroviral vectors with 4070A envelopes infect human cells
promiscuously whereas vectors with Moloney envelopes fail completely to infect human
cells.
Cell-selective retroviral gene delivery has recently been achieved by en~ille~,~illg new
binding domains into the envelope glycoproteins of leLl~vildl vectors (Valsesia-Wi~m~nn
et al., 1994 J. Virol. 68, 4609; K~c~h~ra et al., 1994 Science 266, 1373; Chu &
Dornburg, 1995 J Virol. 69, 2659; Nikunj et al., 1995 Proc. Natl. Acad. Sci. USA 92,
7570; Cosset et al., 1995 J. Virol. 69, 6314). When dirrcr~.lL l~,ccpLur-binding domains
were displayed on MLV retroviral vectors as N-terminal extensions of their intact SU
glycoL,l~,Leills (Russell et al., 1993 Nucl. Acids Res. 2I, 1081), it was found that host
range could be extended or restricted by the displayed ligand (Cosset et al., 1995 J. Virol.
69, 6314). Thus, ecotropic vectors displaying a RAM-l l~ce~or-binding Aom~in from
4070A SU were able to infect RAM-1-positive human cells whereas amphotropic vectors
displaying epidermal growth factor (EGF) could bind to EGF lCC.,ptol~ but were Lhe~ca~L~l
sequestered into a noninfectious entry pathway, giving greatly reduced titres on EGF
~cepLo~-positive cells, but norrnal ti~res on EGF receptor-negative cells. In the current
study, we have explored the possibility of ge"~ldLh1g retroviral vectors whose engin.oered
tropism can be regulated by specific proteases.
Initially, we inserted a short factor Xa protease-sensitive linker (amino acid sequence
IEGR), (LoLLcl~cl~ et al., 1981 Methods Enzymol. 80, 341), into a previously described
EGF-MLV envelope çhim~era (EMo7) in which the EGF domain was fused to amino acid+7 in Moloney MLV SU by a short linker cont~inin~ three ~l~nin,o~ The Xa cleavage
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signal was inserted between the alanine linker and amino acid +7 of Moloney SU to give
the construct EXMo7, described below and illustrated in Figure 1. In Figure 1, the
general format for all of the constructs is shown diagramm~tir~lly and the sequence
surrounding the site of fusion between the displayed ligand (EGF or N-terrninal binding
~lom~in of 4070A-MLV) and the MLV envelope protein (Moloney or 4070A) is shown in
detail for each of the cO~ cl~. Beside each construct is a srh~o~n~tic representation of
the N-terrninal region of the e~ sed envelope glycop.ut~ monomer; Open circles
in-lir~te N-terminal .ecc;~L( l-binding domain of the (ecotropic) Moloney MLV SUglycol,lutei~, filled squares in~lirate the N-telluillal l~,cepLol binding ~1om~in of the
(arnphotropic) 4070A MLV SU glycoplot~ , grey triangles l~ ,senL EGF, and factor Xa
cleavage sites are denoted with arrows. LTR: long terminal repeat, L: envelope signal
peptide, p: polyadenylation sequence. The NotI cloning site is also shown.
The rhim~eric envelopes and a control ecotropic (Moloney) envelope were e~ ,;,sed in
TELCeB6 cells which express MLV gag-pol core particles and an nl~T ~r7 retroviral
vector (Cosset et al., 1995 J. Virol. 69, 7430-7436). Virus-cont~ining ~U~ from
the transfected TELCeB6 cells were hdlv~L~d, filtered (0.45~m), digested with 0 or 4
,ug/ml factor Xa L,luLease for 90 mim-tes and ultracellL.iruged to pellet the viral particles.
R~Llovildl particles incorporating el~ ic envelopes were analyzed by Western
blotting (Figure 2) before (-) or after (+) Ll~ with factor Xa ~loL~ase.
Lanes A, B and C were loaded with pelleted leLlo~ildl vectors i,~olpuldting Mo, EXMol,
and EXMo7 envelopes, respectively. The dirrt 1~,~l- envelope ~lession cul~lu~L5 were
~ldl~Çe~ d (as described in Sambrook et al., Molecular cloning, A laboratory m~ml~l,
(Cold Spring Habour, N.Y., 1989) pp. 16.33-16.36) into TELCeB6 parl~ging cells and
stable phleollly~;in (50 ~g/ml) resistant colonies were exp~nt~ and pooled. Cells were
grown in DMEM supplemented with 10% fetal calf serum and when confluent tral~Çelled
from 37~C to 32~C and inrllb~te~ for 72 hrs. Supernatants con~;.i"i"g retroviral particles
were harvested after overnight (16 hrs) inrub~tion in 10 mls serum-free DMEM at 32~C
and filtered (0.45,um) before being inrllb~te~ with 0 or 4 ~g/ml of factor Xa (Promega)
for 90 mimltrs at 37~C in the L,l~sence of 2.5 rnM CaCl~. The supernatants were
centliruged at 30 000 rpm in a SW40 rotor (Rerkm~n) for 1 hour at 4~C and the pelleted
viral particles were l~i,u~ended in 100}~1 phosphate buffered saline. 20,u1 of each sample
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was se~a,dted on a 10% polyacrylarnide gel under reducing conditions (T ~Pmmli, ~ature
(London, 277, 680 (1970)) followed by transfer of the ~loteins onto nitrocellulose paper.
The SU proteins were ~letPcte~l as previously described (Cosset et al., 1995 J. Virol. 69,
6314) using specific goat antibodies raised against Rausher murine le~lk~tomi~ virus
envelope glycup~uLeills (Qualiy Biotech Inc, USA) followed by Horseradish
peroxidase-conjugated rabbit anti-goat antibodies (DAKO D~nm~rk) and developed using
an enh~nre~ ch~Tnilllmin~srel,ce kit (A ~ l Life Science).
EGF was not cleaved from the EXMo7 envelope by factor Xa (Fig. 2, lane C), sug~e~L.
that the cleavage site was not ~cc~csihle to the protease when inserted in this position.
We therefore made new constructs, EMol and EXMol (described below), coding for
chim~Pric envelopes in which EGF is fused to amino acid +1 (rather than +7) of
Moloney SU by a linker COIllpliSill~ 3 al~ninr~, or 3 ~l~nin~os and the IEGR factor Xa
cleavage site (see Figure 1). EMol and EXMol chi~ P,ic envelopes were incorporated
into virions and analysed on ;~ nblots after t.c~t",~ ,~ with 0 or 4 ,ug/ml factor Xa
~lot~ase for 90 min-ltrS. Figure 2 shows that EXMol envelopes were cleaved by factor
Xa to yield an SU cleavage product whose mobility was in~ictinguishable from unmodified
Moloney SU. Control EMol envelopes which lack the factor Xa cleavage site were not
cleaved. These results in~lir~tto that the precise positioning of the IEGR peptide in the
rhim~rric envelopes is important for its optimal recognition and cleavage by factor Xa.
We have previously demonstrated EGF receptor-mP~ tt~l host range restriction of
retroviral vectors displaying rhim~rric envelopes in which EGF was fused to amino acid
+5 of 4070A SU by a short (AAA) linker (Cosset et al., J. Virol. 69, 1995, cited above).
Retroviruses displaying these chim~rric envelopes could bind to EGF receptors but were
thereafter sequestered into a noninfectious entry pathway, giving greatly reduced titres on
EGF receptor-positive cells, but near-norrnal titres on EGF lcce~Lul-negative cells.
We the.~,fo.e corlstructedpl~mi~lc EA1 and EXA1 (described below) coding for chim~rric
envelopes in which EGF is fused to amino acid + 1 of 4070A SU by AAA or AAAIEGR
linkers lei,ye~;Live:ly (see Figure l).
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19
DNA Co-l~lru-l~
The expression plasmids FBMoSALF and FB4070ASALF (described by Cosset et al.,
1995 J. Virol. 69, cited above) coding for unrnodified Moloney and 4070A MLV
envelopes are referred to in the text as Mo and A ~ ecLively. Construction of EA,
EMo7 (previously called EMO) and AMO expression plasmids was also described by
Cosset et al., (cited above).
To generate EXo7, EMol and EXMol, PCR primers NotXMo7Back, NotMolBack and
NotXMolBack (,e.,~e~;Lively) were used with primer envseq7 to amplify modified envelope
fragments from Mo (FBMoSALF) which were digested with NotI and BamHI and cloned
into the NotI/BamHI-digested backbone of EMo7.
To generate EAl and EXAl, PCR ~ NotAlBack and NotXAlBack (,._~eclively)
were used with primer 4070Afor to amplify mollifi~ envelope fr~gm~ontc from A
(FB4070ASALF) which were digested with NotI and BamHI and cloned into the
NotI/BamHI--ligested backbone of EA.
Finally, the AMol and AXMol co~L.u~ (referred to below) were generated by cloning
the NdeI-NotI fragment from AMO into the NdeI/NotI-digested backbones of EMol and
EXMol, .~,i,L,ecLively. The col~cLIless of all constructs were confirm~ by DNA
sequencing.
Oliogonucleotides used (with restriction sites underline) were:
NotXMo7Back, 5'-GCA AAT CTG CGG CCG CAA TCG AGG GAA GGC CTC ATC
AAG TCT ATA ATA TCA CC (Seq ID No. l);
NotMolBack, 5'-GCA AAT CTG CGG CCG CAG CTT CGC CCG GCT CCA GTC C
(Seq ID No. 2);
NotXMolBack, 5'-GCA AAT CTG CGG CCG CAA TCG AGG GAA GGG CTT CGC
CCG GCT CCA GTC C-3' (Seq ID No. 3);
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NotAlBack S'GCA AAT CTG CGG CCG CAA TGG CAG AGA GCC CCC ATC-3'
(Seq ID No. 4);
NotXAlBack S'-GCA AAT CTG CGG CCG CAA TCG AGG GAA GGA TGG CAG
AGA GCC CCC ATC-3' (Seq ID No. 5);
envseq7, S'-GCC AGA ACG GGG l l'l GGC C-3' (Seq ID No. 6);
4070Afor, S'-CTG CAA GCC CAC ATT GTT CC-3' (Seq ID No. 7).
Figure 3 shows that the IEGR sequence in the interdomain linker of the t~ ,;.sed EXA1
envelopes was COL~ L1Y recognized and cleaved by factor Xa whereas there was no
cleavage of control EA1 envelopes. Refelli~ to Figure 3 (an immlm~lblot of the
recombinant amphotropic l~,Lluvildl particles before (-) or after (+) Ll.~ with factor
Xa protease): lanes A, B and C were loaded with pelleted lc;Lluvil~l vectors illcolL,uldLillg
A, E~A1 and EXA1 envelopes"~es~ec;Livt:ly. The analysis was ~e,rolllled as described
above for Figure 2.
We then titrated vectors illCOl~uldLi~g EA1 and EXA1 rhim~ric envelopes on EGF
rece~to.-lle~Liv~ and EGF receptor-positive human cell lines as follows: EGF
l~,c~,~L-)l-e~re;,~."lg cell lines A431 (ATCC CRL1555), HT1080 (ATCC CCL121), and
EJ (Bubenik, el al., 1973 Int. J. Cancer 11, 765) were grown in DMEM Supplennf nte~l
with 10% fetal calf serum (Gibco-BRL) at 37~C in an atmosphere of 5% C07. Jurkat T
cells (ATCC CRL8805) were grown in RPMI supple ~ ed with 10% fetal calf serum at37~C in an atmosphere of 5% CO2. For infections, target cells were seeded at 2 x 105
cells/well in six-well plates and inrll'c at~-1 at 37~C overnight. Producer cell :jU~
cont~inin~ ,B-g~l~rtosi~l~ce-tr~ncdllring retroviruses were filtered (0.45 f~m) after overnight
inrnb~tion at 32~C in serum free me~linm SU~e1naL~11L dilutions in 2.5 ml serum-free
m-oriillm were inrn~ted with target cells for 2 hours in the presence of 8 ~g/ml polybrene.
The retroviral supernatant was then removed and the cells were inrn~ate(l with regular
m~linm for 48-72 hours. X-Gal st~ininf~ for ~ief~ortinn of ,B-g~l~rtocic1~ce activity was
rolllled as previously described (T~k~llrlli et al., 1994 J. Virol. 68, 8001). Viral titre
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21
(enzyme forming unitslml) was c~lr~ tP~l by counting blue stained colonies
microscopically with the use of a grid place underneath the 6 well plates.
~ Both vectors incorporating EAl or EXAl envelopes could infect EGF receptor-negative
Jurkat cells but were selectively sequestered on EGF receptor-eA~rcssing human cells,
although EXAl was sequestered less completely than EAl (Table 1). When soluble EGF
was added as col"~cLilor to prevent the vectors from binding to EGF rece~Lul~ their
infectivity on EGF .~,ce~Lor positive cells could be fully restored (Table 1), co.,i;.",i.,g
that sequestration was mf~ ttqd specifically through binding of the ~ ;"rel~d envelopes
to EGF .cc~Lo.~.
We then tested whether the restricted host range conferred by EXA 1 envelopes could be
extlorl-lPd (i.e. revert to amphotropic) upon cleavage of the displayed EGF domain.
Vectors inco~ Li~g EAl or EXAl envelopes were treated with increasing doses of
factor Xa and titrated on EGF ~cc~L~,r-e~ ssi~g A431 cells (Table 2). Complete
cleavage of the fused EGF domain with 4 ~g/ml factor Xa for 90 I~ s (Figure 3)
completely restored the h~rc~;LiviLy of vectors with EXAl envelopes but had no effect on
the h~c~iviLy of vectors c~.~ , EAl envelopes. Partial le~L~aLion of vector titre was
seen at lower co"~ ;olIS of factor Xa in-1ir~ting that the vector particles could recover
a low level of illf~;LiviLy when only a fraction of their envelope ~.oLt;i~.s were cleaved.
These data provide further evidence that retroviral vectors displaying EGF are
competitively seqllPstered by EGF receptors, and show that their tropism can be regulated
by a specific protease that cleaves the EGF ~lom~in from the viral surface.
Factor Xa protease is capable of binding directly to procoagulant phospholipid on the
surface of an enveloped virus (Pryzdial & Wright, 1994 Blood 84, 3749-3757) and might
therefore go on to become stably associated with phospholipid in the engin-o~red vector
particles after cleaving their EXAl envelopes. A control experiment was therefore
performed to ~;ol.r.,lll that the restoration of infectivity of vectors incorporating EXAl
envelopes on A43 1 cells was due to cleavage of EGF, and not m~ tPtl by
particle-associated factor Xa protease. We therefore constructed plasmid AXMol and a
control plasmid AMol (described above), coding for chimaeric envelopes in which the
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22
RAM-1 receptor binding domain is fused to amino acid +1 of Moloney SU by a factor
Xa protease-cleavable (AAA~IEGR) or non-cleavable (AAA) linker (Figure 1). As
e~cpect~l, vectors incorporating AMol and AXMol chim~ric envelopes could bind toRAM-1 allowing targeted infectio~ of a variety of human cell lines, and the IEGRseq~l~on~e in the interdomain linker of the AXMol envelope was coll~cLly recognized and
cleaved by factor Xa (data not shown). We thcL~rol~: tested wL~Lh~r the e~t~n~ l host
range conferred by AXMol envelopes could be lci,Lli~Lt:d (i.e. revert to ecotropic) upon
cleavage of the displayed 4070A ~om~in Tr~ with 4 ~g/ml factor Xa for 90
minntes selectively de;,L-uyed the inÇt;~;LiviLy of vectors with AXMol envelopes on human
cells but did not reduce their hlrt;-;LiviLy on mouse cells (Table 2). Vectors callyi~ AMol
envelopes were unaffected by the protease Ll~ These data collrllln that the
extton~ rl Ll~pi~lll of l~lu~dlal vectors displaying AMo envelopes is due to the displayed
4070A dom~in and show that the l~lulalion of illÇ~-;LiviLy of vectors h,col~oldLillg EXA1
envelopes on A43 1 cells was due to cleavage of EGF, and not m~ t~ by
particle-associated factor Xa ~luLcase.
m.~ y, we have g~.-~.,.l~A l~,Lluvhal vectors displaying cleavable binding dom~inc
that are anchored to the viral envelope glycoLJlvt~ by a linker that acts as a substrate for
factor Xa ~lOI~ ase. The displayed binding dom~inc confer novel host range ~ Lies
upon the vectors and these host range alterations are reversible upon treating the vectors
with factor Xa. In principle it should be possible to use linkers that are substrates for
proteases other than factor Xa in colljull~;Lion with binding flom~inc that recognise cell
-
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Table 1.
Infection of human cell lines with retroviral vector particles displaying EGF-4070A cl~..~Lic
envelopes.
Titre (CFU/ml) against cell line:
A431 HT1080 EJ EJ Jurkat
EGF-receptor* + + + +
EGF:, +
Con~
A 10~ lo6 lo6 lo6 10
EA1 5 10 30 2 x 10' 10~
EXA1 '~00 100 310 10~ 104
* EGF-receptor status determined by FACS analysis.
; + in~ t~S incubation of cells with retroviral vectors in the plc:~ence of 1 yM human EGF
(R&D systems, UE~).
SUBSTITUTE SHEET (RULE 26)
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24
Table 2. Regulation of vector tropism by factor Xa protease.
Titre (CFU/ml) against cell line:
NlH3T3 NIH3T3 A431 A431 A431 A431
FXa Gug/ml):* 0 4 0 0.0156 0.75 4
Construct_*
Mo lo6 106 0 0 0 0
A 106 lo6 lo6 lo6 lo6 lo6
EA1 6 x 10~ 3 x 10j 10' 102 10' 10'
EXA1 lo6 2 x 106 1 X 103 4 x 103 6 x 10~ ~; x 105
AMol 2 x 10~ 2 x 10' 103 103 103 103
AXMol 5 x 10~ lo6 5 x 103 , 5 X 103 2 x 103 0
* Filtered supernatants cont~ining ~-g~ tos~ ce-tr~ncd~lcing retroviruses were preinc~lh~t~ with
various concentrations (0, 0.0156, 0.25 and 4 ,ug/ml) of factor Xa (Promega) for 90 minllt~s at
37~C with ~.5 mM added CaCl2 The treated sulJe.llatants were then added to the target cells
and the viral titres were determined as described in (12).
SlJ~;~ JTE SHEET (RULE 26)
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surface ~ce~Lul~ other than those described above. Retroviral vectors with en.~ .ed
binding specificity whose tropism is regulated by exposure to specific ~Ivt~ases may
facilitate novel strategies for targeting retroviral gene delivery.
mrle 2
The inventors sought to establish whether vectors incorporating EXA1 envelopes would
recover their infectivity on EGF ~ce~r-positive cells upon cleavage of their displayed
EGF domain. Vectors h~col~oldli~g EA1 or EXA1 envelopes were the.~r(,.~ treated with
factor Xa and titrated on EGF ~,ce~Lur-e~,e;,si~ A431 cells. Complete cleavage of the
fused EGF domain with 4 ,ug/ml factor Xa for 90 ...;..~ s completely Les~oled the
hlfe-;~iviLy of vectors with EXA1 envelopes but had no effect on the il~;liviLy of vectors
Cdllyil~ EA1 envelopes (Figure 4). Figure 4 illustrates factor Xa~ i;At~-1 infection of
A431 cells with rhim~oric EGF-4070A MLV vector particles. Filtered ~u~e,.
cont~inin~ ~-g~l~rtosiri~e-tran~ cing retroviruses (A, EA1, or EXA1) were preinrllb~trr~
with 0 (-) or 4 (+) ~g/ml CO~ ions of factor Xa (PluluC~d) for 90 Ill;llll~rS at 37~C
with 2.5 rnM added CaCl2. The treated ~u~ t~ntc were then used for target cell
tr~n~ lrtion, as ~esrriberl above. X-gal-stained plates were photographed wilLuuL
fir,.lion.
Partial ~ Lulalion of vector titre was seen a lower col~ce"Ll~Lions of factor Xa (Table 2)
in~lir~ting that the vector particles could l, covt:r a low level of infectivity when only a
fraction of their envelope p,uL~i"s were cleaved. These data provide further evidence that
uvildl vectors displaying EGF are co...l.e~ vely seq~lestered by EGF ,~.,e~tol~, and
show that their Llo~i~lll can be regulated by a specific protease that cleaves the EGF
~lom~in from the viral surface.
In the two step L~getillg strategy outlined above, cleavage of the rhim~ric envelope is
preceded by its ~tt~rhm~ont to the target cell via the eng;--~e.~d binding rlom~in Therefore,
to de~. ll..i.~P wL~,Lller EGF rec~,~Lur-bound vector particles that were cleaved at the cell
surface could go on to infect their target cells, we loaded the vectors ûnto EGF,~Ce~Lol-posiLive A431 cells and EJ cells, washed the cells, and then treated them with
factor Xa protease. Table 3 shows that, when sequestered onto EGF ,ece~u,s and then
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26
cleaved by factor Xa protease, the vectors incorporating EXAl, but not EAl envelopes,
proceeded to infect their target cells.
The availability of a targetable, injectable vector would greatly f~rilit~te the development
of gene Il~ d~y approaches requiring direct in vivo gene delivery to selecte~l target tissues.
In this report we have demo.l.~ r~cl the feasibility of a novel two step~ g ~lld~y
which may allow the ~ on of retroviral vectors ~ ed to infect cells e~ g
specific l.,ce~tor/~loL~ase combinations. There are many membrane-associated ~ cdSeS
,
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Table 3. Factor Xa protease triggering retroviral infection on
the cell-surface of human A431 and E.l cells.
Tltre (CFU/ml) against cell line:
A431 A431 EJ EJ
FXa (~g/ml): o 4 o 4
Constructs*
A ~ 3 x 104 3 x 104 1 x 104 1 x 104
EA1 ~ <5 c5 0 0
EXA1 ~ 4 x 102 1 x 104 <~ 3 x 102
~ A431 and EJ cells were incubated with 2 ml of filtered superna-
tant containing ~-galactosidase-transducing retroviruses for 1 hr
at 4~C. Cells were then washed two times with cold serum-free
medium and incubated with 0 or 4 ~g/ml of factor Xa (Promega)
for 2 hrs at 37~C in serum-free medium. After inc~ ~h~tion for 48
hrs with medium supplemented with 10% fetal calf serum the viral
titres were determined as described in Table 1.
S~J~ 1 l l ~JTE SHEET (RULE 26)
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2 8
that may be of interest in this respect such as the proteases that co-operate in Aegr~Air~g
the extracellular matrix during tumour invasion (Poustis-Delpont et al., 1992 Cancer
Research 52, 3622-3628; Vassalli & Pepper, 1994 Nature 370, 14-15; Sato et al., 1994
Nature 370, 61-65; and Chen et al., 1995 Breast Cancer Res. Treat. 31, 217-226);h~ ro~oietic dirrertlltiation antigens that are also membrane ~luL~ases (Shipp & Look,
1993 Blood 82, 1058-1070) or the membrane protease that has been implicated in the entry
pathway of HIV (Ml~rak~ mi et al., 1991 Biochim. Biophys. Acta 1079, 79-284).
Libraries of fi~ ous "substrate phage" displaying cleavable binding domains haveccc,lLly been used to identify optimal substrates for known ~loleascs (Matthews & Wells,
1993 Science 260, 1113-1117; Matthews et al., 1994 Protein Science 3, 1197-1205; and
Smith el al., 1995 J. Biol. Chem. 270, 6440-6449). In principle, it should be possible to
ge~ 1e similar l'elrOVilUs display libraries e~ s~ulg N-terrnin~ily çxl...A.~A envelopes
with r~nAorni~eA linker sequences. Such libraries might provide the basis for selection
strategies Aesign~oA to identify novel intracellular or membrane-~soc;;~lrd ~luL~.ases or to
isolate c!~ envelopes that target novel cell-specific lcc~,~lur-~luLcasec combinations.
~mrle 3
S~ ~r
As described above, several polypeptides have now been displayed on .elLovilal vector
particles as N-te~nin~l extensions of their envelope spike gly~o~loLeills. Folding,
assembly, transport, viral incorporation, lecc~Lol ~tt~rhment and fusion triggering by the
chim~f ri- envelopes can be variably inflnem~e(i by the N-l~ i polypeptides, depending
on their unique structural and functional characteristics. In this example the illvcllLo~
dclllo~Lldt~ that the RAM-1 binding domain from the homotrimeric 4070A SU
gly~;c~L~luteill can strongly inhibit Rec-1 mlofli~terl infection by the homûtrimeric Moloney
SU gly~;o~ Lein when grafted to its N-~t:....i....c. It is also shown that short trimeric
leucine zipper peptides, but not a monomeric helical peptide, can inhibit RAM-1 m~ t~fl
infection by the 4070A envelope when fused to its N-LI:lllPillus. Cleavage signals were
en~ ee~ed into the chim~eric envelopes such that the displayed polypeptides could be
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cleaved from the vector particles by addition of factor Xa protease. In all of the envelopes
displaying trimeric polypeptides, the steric block to Rec-1 or Ram-l mPf1i~tP~i infection
was reversed when the trimeric N-t~orrnin~l extensions were cleaved from the virally
incorporated envelopes. These data suggest that the m~cking of envelope functions by the
inhibitory N-termin~l extensions is a consequence of their assembly into a L. ~e.ic
complex at the tip of the SU glyco~lutehl trimer to which they were grafted. Theimplications for retroviral vector L~lgeLillg are ~liccllsseA
MLV-derived r~,Lluvilal vectors are versatile gene delivery vehicles whose host range
~,ol~ellies are .i~ . ",;"~rl by membrane glycoproteins which m~ te their ~ 1".~ to
specific rece~Lol~ and subsequently trigger fusion The envelope glyco~luL~ins of the
murine lellk~mi~ virus (MLV) are displayed as a homotrimeric complex on the suRace
of the virus (Fass et al., Nature Structural Biology 3:465-469; Kamps et al., Virology
184:687-694) Each subunit of the trimer consists of two partct SU and TM. The SU(suRace) colll~uLl~,llLis entirely e~LLlavilal and is ~tt~-~h~-l to the retrovirus via the smaller
TM component. which anchors the complex in the viral lllclllbldlle (Pinter et al.,
Virology 91:345-351). The N-t~rrnin~l domain of the SU glyco~LoL~ confers l~,cc~Lor
~ecirlciLy and e~hil,ils a high degree of col~elvation between MLVs with different host
ranges (Battini et al., J. Virol. 69:713-719). Moloney MLV envelopes confer an
ecoL.upic host range because they bind to a murine cationic amino acid Lla~uli~l(Albrittonetal., J. Virol. 67:2091-2096; Albrittonetal., CellS7:659-666). 4070AMLV
envelopes attach to the RAM-l phosphate l.a~ lLt;r which is conserved throughout many
n species, to confer an amphotropic host range (Kavanaugh et al., Proc. Natl.
Acad. Sci. USA 91:7071-7075). After binding to target cell receptors has occurred, the
Llhll~lic SU-TM complex is thought to undergo a large collrolllla~ional rearrangement
which triggers the process of fusion b.,.ween the viral and target cell membranes.
The inventors and their colleagues have been exploring different strategies for Lalgeti
the entry of lc;~lOvilal vectors into selected target cells by ell~hleelillg new dete~nin~m~
into their SU glycoplo~eills (Cosset et al., J. Virol. 69:6314-6322; Nilson et al., Gene
Ther. 3:280-286; Russell et al., Cold Spring Harbour Laboratory, Cold Spring Harbour,
N.Y.; Valsesia-Wi~l...~.... et al., J. Virol. 68:4609-4619; Valsesia-Withn~nn et al., J
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Virol. 70:2059-2064). In the preceding examples is described a novel two-step ~LLdt._~,y
that allows the LdlgeLmg of retroviral vectors through protease-substrate interactions, in
which the retroviral vector ~tt~rhlos to the target cell via an engineered binding domain
(step one), whe~c.l~on the engineered linker that tethers the virus to the binding domain
is cleaved by a specific protease (step two), allowing the virus to go on and infect the
target cell. A disadvantage of this two-step tal~clLLlg ~LLdLc~ y, as set forth above, is that
whilst domin~tin~ the specificity of vector att~r~m~nt the cleavable binding domain does
not completely block the ability of the SU trimer to attach to its natural .cce~Lur on non
target cells. The uncleaved vector the~rù-e retains the ability to infect non target cells
through the Rarn-l L.~e~tor. To overcome this disadvantage, we were il~ L~d to
develop envelope mo~iifir~tinns that would completely inhibit the hlr..,Li~ y of uncleavcd
vectors but would perrnit full l~e~Lu.dLion of i~ccLiviLy upon eA~osule to a selecte~l
~lUL~dse .
In the course of eA~ iL~c~ (described in this specification) to rh~ ;c~ the ecoL~opic
infectivity of Moloney MLV envelope rhi..~ s, it was found that a vector displaying the
Rarn-1 targeted AXMol envelope (Nilson et al., Gene Ther. 3:280-286), could not
efficiently infect cells tbrough the ecoLiupic lcc~,~Lur (Rec-1) unless it was first cleaved by
factor Xa protease. To explain this ~-upeliy of the AXMol envelope we hypothl~-cice(l that
the displayed RAM-1 binding domain might be forming a trimeric complex at the tip of
the Moloney SU glyc~ ù~ein trimer to which it was grafted, thereby blocking its Rec-1
binding site. To further test this hypothesis, we grafted oligo...~ Ieucine zipper
peptides (Harbury et al., Science 262:1401-1407) onto 4070A SU glyco~lut~hls andcharacterised the p,ù~lies of retroviral vectors incolyo-dLhlg the chimaeric envelopes.
MATERIALS AND METHODS
Plasmid Co~L~u~Lion
The unmodified envelopes of 4070A MLV and Moloney MLV were encoded by the
eAp.ei,~ion plasmids FB4070ASALF (A) and FBMoSALF (Mo), l~ s~ecLively (Cosset eta~., 1995 J. Virol. 69, 7430-7436)). The constructs AMol and AXMol, which code for
chim~Pric envelopes in which the RAM-1 l~ce~3Lol binding domain from 4070A SU is
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fused to amino acid +1 of Moloney SU by a factor Xa protease-cleavable (AAAIEGR)or non-cleavable (AAA) linker have been described previously (Nilson et al., Gene Ther.
3:280-286). Constructs EAl and EXAl, coding for cllim~eric envelopes in which EGF
is fused to arnino acid + 1 of 4070A SU by a linker comprising three ~i~nin~5, or three
~l~ninPs and the IEGR factor Xa cleavage site, have also been described (Nilson et a~.,
Gene Ther. 3:280-286).
To construct vectors displaying helical peptides, pl~mi~1~ pEGSlXAl and pEGS3XA1were first produced in which there is a 12 amino acid (AAAGGGGSIEGR, Seq ID No.
8) or 22 amino acid (AAAGGGGSGGGGSGGGGSIEGR, Seq ID No. 9) linker,
u;~ively, between the 4070A MLV envelope and the displayed EGF domain. PCR
~lh~ NotGSlXAlback and NotGS3XAlback (res~e.,~ ely) were used with primer
4070Afor to amplify mo-~ifif ~1 envelope fr~gmPnt~ from EXA1 which were ~igestf~-l with
NotI and BamHI and cloned into the NotI/BamHI-digested backbone of EAl.
Figure 7 is a diagld~ Lic l~ sf ~ ion of plasmid constructs coding for ~1l;",~
envelope glyco~rol~ s in which the helical peptides AA, VL and II were fused to residue
+1 of the 4070A MLV SU. The general format is shown ~i~gr~m~tir~lly and the amino
acid sequence (single letter code) of the helical peptides and the linkers between these
peptides and the SU protein are shown in detail. Ll-~, long ~f ",i,.~l repeat; L, envelope
signal peptide. Amino acid residues at the a and d positions of the heptad repeat are
shown in bold.
To genfld~ pl~cmi-ic pVLXA1, pVLGS1XA1 and pVLGS3XA1, PCR pl~'llC.~ Gal4
VLback and Gal4 VLfor were used to produce PCR fragments by priming off each other
and then outer ~lilllel:, Gal4back and Gal4for were used to amplify the fragment further.
The PCR products were digested with SfiI and NotI and cloned into the SfiI/NotI-digested
backbones of EXA1, pEGSlXAl and pEGS3XA1.
To g~,.~.aL~ plasmids pAAXAl and pAAGS3XA1, PCR primers Gal4 AAback and Gal4
AAfor were used to produce PCR fragments by priming off each other and then outer
~lhll~,lS Gal4back and Gal4for were used to amplify the fragments further. The PCR
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products were digested with sfiI and Notl and cloned into the Sfi ~Not ~igested backbones
of EXAl and pEGS3XAl.
To generate pl~cmi~1c pIIXAl, pIIGS lXAl and pIIGS3XAl, PCR prirners Gal4 IIback and
Gal4 IIfor were used to produce PCR fragments by priming off each other and then outer
prirners Gal4back and Gal4for were used to amplify the fr~gTnPntc further. The PCR
products were digested with SfiI and l~otI and cloned into the S.fiI/NotI-digested backl oncs
of EXAl, pEGSlXAl and pEGS3XAl. The correct sequence of all CO~ uCL~ was
verified by DNA seqllenrin~
The following oligonucleotides (with restriction sites underlined) were used:
NotGSlXAlback, 5'-GCA AAT CTG CGG CCG CAG GTG GAG GCG GTT CAA
TCG AGG GAA GGA TGG CAG AG-3' (Seq ID No. 10);
NotGS3XAlback, 5'-GCA AAT CTG CGG CCG CAG GTG GAG GCG GTT CAG
GCG GAG GTG GCT CTG GCG GTG GCG GAT CGA TCG AGG GAA GAA TGG
CAG AG-3' (Seq ID No. 11);
Gal4 VLback (cont~inin~ SJ~ site), 5'-GGC ATT CAT GCG GCC GCG GCC CAG CCG
GCC ATG AAG CAA CTA GAA GAC AAG GTG GAG GAA CTC CTT AGC AAG
GTA TAC C-3' (Seq ID No. 12);
Gal4 VLfor ~con~inin~ NotI site), 5'-GCA AAT CTG CGG CCG CCT CTC CAA CAA
GCT TCT TCA GTC GAG CGA CTT CGT TCT CAA GAT GGT ATA CCT TGC TAA
~A ~-3' ~5cq 1~ ~o. 13~,
Gal4 AAback (co..~ g SfiI site), 5'-GGC ATT CAT GCG GCC GCG GCC CAG CCGGCC ATG AAG CAA GCA GAA GAC AAG GCA GAG GAA GCT CTT AGC AAG
GCT TAC C-3' (Seq ID No. 14);
Gal4 AAfor (co..l;~ g NotI site), 5'-GCA AAT CTG CGG CCG CCT CTC CAG CAA
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33
GCT TCT TTG CTC GAG CAG CTT CGT TCT CTG CAT GGT AAG CCT TGC TAA
GAG C-3' (Seq ID No. 15);
Gal4 IIback (cont~inin~ SfiI site), 5'-GGC ATT CAT GCG GCC GCG GCC CAG CCG
GCC ATG AAG CAA ATC GAA GAC AAG ATA GAG GAA ATT CTT AGC AAG
ATC TAC C-3' (Seq ID No. 16);
Gal4 IIfor (Cont~inin~ NotI site), 5'-GCA AAT CTG CGG CCG CCT CTC CTA TAA
GCT TCT TGA TTC GAG CAA l-l-l CGT TCT CTA TAT GGT AGA TCT TGC TAA
GAA TTT C-3' (Seg ID No. 17);
Gal4 back, 5'-GGC ATT CAT GCG GCC GCG GC-3' (Seq ID No. 18);
Gal4 for, 5'-GCA AAT CTG CGG CCG CCT CTC-3' (Seq ID No. 19); and 4070Afor
(d~sclibed above).
Target cell lines and ~ o~lu~lion of viruses
GP+Env AM12 cells (Malh~wiL~ et al., Virology 167:400-406) were derived from themurine cell line NIH 3T3 and express the MLV-A envelope which blocks the RAM-l
lcce~L(~I by hlklr~ r~nce. NIH 3T3, GP+Env AM12 and the human cell line A431 (Giard
et al., J. Natl. Cancer Inst. 51, 1417-1421), were grown in DMEM supplemented with
10% fetal calf serum. The different envelope e~,~lc~ion constructs were Lldl~.rccLcd into
TELCeB6 p~cl~ging cells (Cosset et al., J. Virol. 69:7430-7436) by calcium phosphate
iLdLion (T~krllrlli et al., J. Virol. 68:8001-8007) and stable phleomycin (SOmg/ml)
lc~ .L~lL colonies were e~p~n~ cl and pooled. Cells were grown in DMEM supplernelltto~l
with 10% fetal calf serum and when confluent Lld~-~.r.,ll~d from 37'C to 32 C and
inr~lb~tr-l for 72hrs. Supernatants cont~ining retroviral particles were harvested after
overnight (16hrs) incubation at 32'C in 10mls serum-free DMEM for infections, orDMEM suppl~ r~ with 2% fetal calf serum for immnnoblots. All supernatants were
filtered (0.45~Lm) before use.
Tmmnnoblots
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34
Virus producer cells were Iysed in a 20mM Tris-HCI buffer (pH 7.5) cont~ining 1%Triton X-100, 0.05 % SDS, 5mg/ml sodiurn deoxycholate, 150mM NaCI and lmM PMSF.
Lysates were inr~b~nocl for 10 mins at 4'C and were centrifuged for 10 mins at 10,000
x g to pellet the nuclei. Virus sarnples were obtained by ultraceIlLlirugation of filtered
viral supernatants (lOrnl) at 30 000 rpm in a SW40 rotor (Rerkrn~n, USA) for 1 hr at
4 C. The pelleted viral particles were I~aua~ended in 1O0~L1 PBS. Sarnples (3O~LL1 for cell
lysates, or 1O~L1 for pelleted virions) were then separated on a 10% polyacrylamide gel
under redncin~ conditions followed by tlal~rt:r of the proteins onto nitrocellulose paper.
For Factor Xa cleavage, 10~1 of the pelleted viral particles were inrnh~tf tl with O or 4
,ug/ml of Factor Xa (PIulllega, USA) for 90 min at 37 C in the E~lcseIlce of 2.5mM CaCl,
before running on the sc:pa,aLi~g gel. The SU p~oleiIls were ~If~t~ct.o~l as previously
described (Cosset el al., J. Virol. 69:6314-6322) using specific goat antibodies raised
against either Rausher le~-k~f mi~ virus (RLV) gp70 SU or RLV p30 capsid protein (CA)
(Quality Biotech Inc, USA) which were diluted 1/1,000 and 1/10,000 respectively. Blots
were developed with horseradish peroxidase-conjugated rabbit anti-goat antibodies
(DAKO, D~.. ~.l~) and an fnh~n-~ecl ch~mil~ inf~srf.Ire kit (A~ al~ll Life Science,
UK).
Target cell Infection
Target cells were seeded at 2 x 10~ cells/well in six-well plates and inr~lb~t~l at 37 C
overnight Producer cell aupf Il.~t~ntc cont~ining ~-galactosidase-transducing retroviruses
were filtered (0.45~Lm) after overnight inrnk~tion at 32 C in serum-free mf~ m The
haI ~e~Led SUpC. .~ were inr~lb~tf d with O or 4 ~g/ml of factor Xa (PrullIf y,~) for 90
...;..~l~5 at 37 C in the ~I~sence of 2.5mM CaCl~. Su~...,.I~..l dilutions in 2ml serum-free
media were i~ b~rd with target cells for 6 hrs in the presence of 8~g/ml polybrene. The
retroviral au~f . ,~ was then removed and the cells were inr~lb~tf~d with regular Illf~,l;l-~-l
for 48-72 hrs. X-Gal st~ining for detection of ~B-galactosidase activity was performed as
previously described (Tatu el al., EMBO J. 14: 1340-1348). Viral titre (enzyme forrning
units/ml) was r~ tf d by counting blue stained colonies microscopically with the use of
a grid placed nn~lernf~th the 6 well plates.
RESULTS
-
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Rec-l m~ te~ infection by envelopes ~ ''7''7illg a Ram~ e~ g d(~ in
AMol and AXMol are previously described ~him~friC envelopes in which the RAM-l
receptor binding domain from 4070A SU is fused to ~minoarid + 1 of Moloney SU by a
noncleavable (AAA) or factor Xa-cleavable (AAAIEGR) linker (Nilson et al., Gene Ther.
3:280-286). Viruses incoll,oldtillg the AMol and AXMol envelopes were pelleted,
cleaved with 0 or 4~g/ml factor Xa protease and then analysed on i.. l.. r~blots using an
anti-envelope a"Lise~u~ as a probe.
The reversible inhibition of infection by retroviral incolyoldtion and cleavage of chim-~f ric
envelopes e~ si"g a factor Xa-cleavable, N-terrninal RAM-l binding domain is shown
in Figure 6A and 6B. Figure 6A is an ;I~ blot of pelleted recombinant retroviralparticles inco~ulaLil~g Mo, AMol or AXMol envelopes before (-) or after (+) l,~
with factor Xa protease, probed with a~iLisf lull, to the SU gl~co~ioLeil,. Figure 6B shows
the results when the target cell line GP+Env AM12 was infected with harvested producer
cell~.u~ llldL~lL.co~t~ining~B-g~ to~ ce-tr~n~ lcinglc;L~Ovuuses(AMol,AXMol,Mo
and A) with or without Llc~- f -l with factor Xa protease. Detection of ,6'-galactosidase
activity was ~.Ço""ed by X-gal st~ining and titres were expressed as e.f.u.lml.
It is a~d~ from Figure 6A that the ch;.,.,,f,lic envelopes were illco,~o,dted into virions
with equal efficiency (although less efficiently than the unmodified Moloney SU) and that
AXMol, but not AMol envelopes, were cleaved by factor Xa protease to yield an SUcleavage product whose mobility was inr~ hle from unmodified Mo SU.
The infectivity of these Ram-1 targeted veclors was then tested on NIH3T3 cells and on
NIH3T3 Llal~.Çt~;~lL~. (GP+Env AM12) ov~ A~l~ssi,lg the 4070A envelope which blocks
the coll~ ollding Ram-1 receptor by i~llt;lr~ llce. The vectors AMol and AXMol were
fully infectious on the unmodified NIH3T3 cells which express both Rec-1 and Ram-1,
giving titres in excess of 106 efu per ml (not shown), however their infectivity was greatly
reduced on the Ram-1 deficient cells, suggesting that they were unable to utilise the
ecoLlo~ic l~cepLol, Rec-1 (Fig. 6B). This result was unexpected and was in contrast to
results obtained with similar chim~eric Moloney SU glyco~luL~ills displaying monomeric
growth factor domains or single chain antibody fragments in which Rec-1 me~li,.t~d
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36
infection was not seriously co~ olllised by the displayed domains (Ager et al., Human
Gene Ther., in press; Cosset el al., J. Virol. 69:6314-6322). This led to the proposal that
the displayed Ram-l binding domain rnight be forming a trimeric complex at the tip of the
Moloney SU glycoprotein trimer to which it was grafted, thereby blocking the Rec-1
binding site and/or h-L~lrt:lillg with Rec-l m.ofli~tt~f~ fusion trig~ elhlg. Such a block would
be expected to be reversible by cleaving the Ram-1 binding domain from the vector and,
in keeping with this prediction, the infectivity of the AXMol vector was fully l'eoLI)lCC
on Rec-1 positive, Ram-1 deficient cells when the Ram-l targeting ~70m~in was cleaved
from its surface with factor Xa protease (Fig. 6B).
Col~lru~Lion of rhim~Pric 4070A envelopes displaying helical rept~ c
To further test the idea that a trimeric polypeptide could block the functions of a Llh~C~ic
envelope ~ly~;o~luL~ when fused to its N-~ c, and to ~ie~ 0 whether the collce~L
could be applied to an amphotropic MLV SU glycù~loLl:ill, we made a series of CO~.Llu~;L~.
coding for chim~ric envelopes in which mollulll.,Lic or t. i.~ helical peptides were
fused to amino acid +1 of 4070A SU via Factor Xa-cleavable linkers (Fig. 7). Thehelical peptides that were chosen for t_ese studies were v~LL;cL-lL~. of the dimeric GCN4
leucine zipper peptide with ~,y~,le~ ti~ V, L, I or A (single letter ~minnacid code)
Ou~..Li~uLions in the a and d positions of the heptad repeat tnat are known to force t_e
formation of ~ ;c coiled coils (VL and II peptides) or to ~Ic~r~llt oligomerisation (AA
peptide) (Harbury, et al., Science 262:1401-1407). When designing t_ese corlstructs, we
were cullr~ ..rd that t_e oligolnelisation of the displayed VL and II peptides might be
hindered if they were tet_ered too closely to the underlying 4070A SU glyco~,vL~ hl. The
spacing between t_e 4070A SU gly~;O~lvL~ill and the displayed peptide motifs wastherefore varied by insertion of linkers COlll~li'.illg amino acids AAAIEGR, Seq ID No.
20), AAAGGGGSIEGR (Seq ID No. 8) or AAAGGGGSGGGGSGGGGSIEGR (Seq ID
No. 9), where the highlightt-(l sequence is known to be recognised and cleaved by Factor
Xa (Nilson et al., Gene Ther. 3:280-286).
E~ O;on, viral incorporation and cleavage of rhim~ric 4070A envelopes
The AA, VL and II chimaeric envelopes and a control arnphotropic (4070A) envelope
were stably Ll~rec;l~d into TELCeB6 cells which express MLV gag-pol core particles and
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37
an nls LacZ retroviral vector (Cosset et al., J. Virol. 69:7430-7436). Virus-com~ining
SU~CI 11~ were harvested from these stably tral~Çe-;Lced TELCeB6 cells and
ultracellLliruged to pellet the viral particles. Pellets were than analysed on immllnoblots
for the p~ ,cllce of viral core proteins and envelope L~luteills (Fig. 8A).
Figure 8 illustrates the viral incol,uoldtion and cleavage of chi"~ ic envelopes e~lcssi~
factor Xa-cleavable helical peptides as N-tf"";"~l extfn~ions of the 4070A MLV SU.
Figure 8A is an immlmt~blot of pelleted retroviral particles incorporating cllim~f i~
envelopes. The lane COIlh,~lL~ are as follows: l:VLXAl, 2:VLGSlXAl, 3:VLGS3XA1,
4:AAXAl, 5:AAGS3XAl, 6:IIXAl, 7:IIGSlXAl, 8:IIGS3XAl, and 9:A. The top
immnn()blot was probed with an anti-SU a~lLiSc~ulll and the lower one with an anti-p30
allliselulll to detect the p30 CA protein.
Figure 8B shows the Factor Xa-mf ~ tf (l cleavage of cllim~f ric envelopes and takes the
form of an ;Ill~lllloblot of pelleted lccolllbi~t amphotropic IcL-u~ dl ~u~les
iuCOI~OldLiu~ A, VLXAl, AAXAl, IIXAl or EXAl envelopes before (-) or after (+)
Ll~l.llf 11 with factor Xa protease, probed with anti-SU ~Lise~ulll.
Figure 8C is an immlmnblot of cell lysates ple~dl~,d from the virus producing TELCeB6
Lld~rccLdllL~ A, VLXAl, AAXAl, IIXAl and the control, ullLlal~rcclcd TELCeB6,
probed with aMi-Su antiserum.
The number of vector particles present in each sample, de~ellllhled by st~ining with p30
allLisclulll to detect the p30 CA protein, was found to be approximately equivalent (Fig.
8A). However, when the efficiencies of viral hlcol~oldLion of the different chimil~rir
envelopes were colllpdlcd, by st~ining with an anti-SU allLisclulll, it was found that
hlcol~oldLion is greatly inflllen~e~l by the ~ie~ellce of the oli~ ollle-i~ing peptide.
Envelopes displaying the control monomeric peptide (AA) were incorporated almost as
effi- itomly as wild type 4070A envelopes whereas envelopes displaying the VL peptide
were incc,l~uldLcd much less efficiently and there was no visible incorporation of
envelopes displaying the II peptide. To (1et~rmin~o if the helical peptides could be cleaved
from the SU ~ly-;o~luLcills to which they were grafted, viral pellets were digested with 0
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3 8
or 4 ~g/rnl factor Xa protease and then analysed on ;mmllnoblots as before.
Figure 8B shows that there is a mobility shift when expressed envelopes VLXA1, AAXA1
and the control EXA1, have been cleaved with factor Xa protease, inrlir~ting that the
helical peptides are indeed cleaved from the SU. Due to the low levels of incorporation
of the IIXA1 rhim~Pric envelope, cleavage can not be seen for this vector. This
immnnoblot also in~lirstPs that the çhims~ric envelope AAXA1 was incol~o,dl~d 10 times
more efficiently than VLXA1.
To further investigate the poor incorporation of the VL and II rhimsPric envelopes we
~c.roll,led immnnoblots of cell Iysates pl~pd-~,d from the virus producing TELCeB6
Llal~r~LdllL~. Figure 8C shows that the unprocessed plC~ Ol:j of all three chi.l.s .;r
envelopes are ~lPtoct~hle in the cell lysates. However, the VL and II envelope ~ .Ul~
are less al~ul~d~lL than the AA ~l~;LUl~Ol. Also, the ~loce~ g of the VL and II
~r~ to mature SU is severely il~ailed relative to the proce~ing of the AA
~lc~ OL in-lir~ting that these chim~ric envelopes are not erl ;~ y ~ ulhd from the
endoplasmic retirlllnm to the Golgi conlpalL~ L.
I~re~ iLy of v.~Lu.s displaying ~ hi...~. ~ .c ~lv~ es before and after cleavageTo d~ t~ i..P whether the helical peptides were m~C~ing the functions of the 4070A
envelopes to which they were fused we titrated the vectors on Ram-l e~ ssh1g cells,
NIH3T3 and A431 before and after they were cleaved with factor Xa protease (Table 4
and Figure 9). Figure 9 shows the reversible inhibition of infection by cleavage of the
rh;,.,~ic envelope, VLXA1, e~les~hlg a factor Xa-cleavable, N-t~ormin~l oligolll~Li~hlg
peptide and is a m~gnifiPrl view of virally infrctPrl cells after X-gal st~ining. Chim~Pri~
envelope VLXAl shows strong inhibition of h~.;Livi~y on NIH 3T3 and A43 1 cells, which
is reversible on addition of factor Xa.
The control vectors displaying the AA peptide gave titres comparable to that of the wild
type amphotropic vector and the titres did not change after factor Xa cleavage int1ic~t;ng
that the AA peptide does not signifir~ntly hl~,.r.,.l, with the functions of the underlying
4070A envelope. Conversely, the vectors displaying the trimerising VL and II helical
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peptides gave greatly reduced titres on both cell lines which were ~h~-t-ell as much as
2000-fold by factor Xa cleavage. In the case of the VI~A1 c1-;...7~ ~ic envelope, on
cleavage with 4 ,ug/ml factor Xa protease, the titre on NIH3T3 cells hlcleased from 1~1
efu/ml to 3 x 105 efu/ml and on A431 cells from 318 efulml to 105 efulml (Fig. 9).
Vectors displaying the II helical peptide gave generally lower titres than those displaying
the VL peptide, plc~ ably due to the reduced hlcol~o~dlion of ch;...~t.ic envelopes
displaying the II peptide. Interdomain spacing had little a~dlcllL infhlenre on the titre of
the uncleaved vectors, nor on the degree of titre t~.. h~.-~.. l that was observed after
exposure to the factor Xa protease.
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4 0
-- _1. ~ o
~ o o
~ x ~ ~ e ~0~
~ X m m
3 c~ I x ~ e , ~ ~
C X X
+ ~0 0
~0 0
X
~ :1
Z
SUIBSTITUTE SHEET (RULE 26)
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41
DISCUSSION
In the above example we have shown that the Ram-1 binding domain from the
homotrimeric 4070A SU glyCOUluLcL~l can inhibit Rec-l ~ r~ rci infection by the
homoLlilLIclic Moloney SU glycoulùLcL~l when grafted to its N-t~ . We have also
~ shown that short tfiLIleric leucine zipper peptides, but not a monomeric helical peptide, can
inhibit Ram-l m~rii5~t~d infection by the 4070A envelope when fused to its N-lt.In both cases, by using factor Xa ,ul~ûLcase to cleave the trimeric N-l~ ...in~l ex~ ic~
from the virally incorporated envelopes, it was possible to reverse the block to Rec-l or
Ram-l mr~rli~t~od infection. We propose that the ",~cL-;"g of e.l~lo~e functions by these
inlli~iLcjly N-tr-~min~ rtf~n~ions is a con~eq l~onr~e of their assembly into a LlLIll~.ic
complex at the tip of the SU glycolJ~oLcin trimer to which they are ~ftr~rl,
The VL, II and AA peptides that we fused to the 4070A envelope are ...~ of the
GCN4 leucine zipper in which the co~ cd, buried residues that direct dimer fo. ..~ ;o..
have been sl~hstinlteci with valine, l~lçin~, isol~ ciL e or alanine residues (Harbury et al.,
.Sci~n~e 262: 1401-1407). The VL mutant oli~uL,l~.ises to form e~Ll~n~cly stable (Tm 95 ~ C)
two- and three-sn~n~i~ri alpha-helical coiled coil ~Llu;Lul~,S wh~l~,as the II mutant forms
exclusively three-str~n~ cl coiled coils which are even more stable (Tm > 100'C) than the
VL sLlu~;Lulcs. In the AA peptide, all of the hy~o~hol)ic core residues of the GCN4
leucine zipper were sub~ with ~l~nin~ to ~lcvel,L oligulll~.isaLion of the mutant
peptide whilst plese~ g its helical structure.
R~,Lluvilal inccjl~oldLion of c-h;...~ ic envelopes displaying the VL and II peptides was
~i nifi~ntly ill~ah~d relative to ch;...~ ic envelopes displaying the control AA peptide,
which showed only a slight reduction in i lcol~ulalion cuLul,dlcd to unmodified 4070A
envelopes. The VL çhim~ric envelopes were approximately ten-fold less abundant in
viral pellets than the AA ~him~ric envelopes, and the II ch;.n~- ~ic envelopes were so
poorly hlcol~ulalcd that they were not visible on i------~ blots of pelleted virions. By
;,,,,,,.-nnblotting cell lysates from the Llal~rcLLcd TELCeB6 cells with anti-envelope
allli~elulll, it was shown that the intr~ce~ r ablln-l~n~e of the pl~e~ul:,ol polypeptides for
each of the chi~ r.;.- envelopes was closely collelaLcd with their ab...~ e in viral
pellets. The low viral incol~olaLion of the VL and II f~him~.oric envelopes is thc~fc,lc a
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42
conse~uence of their poor expression and/or folding in the virus producing cells.
It is cu~ lLly unclear what is responsible for the impaired expression of the VL and II
chimaeric envelopes. Neither protein appears to be toxic to the virus producing cells since
there was no dir~l~nce in the number or size of stably tr~nC~ recl TELCeB6 clones that
were obtained after tral~recLillg the different (oligomerising or control) chim~toric envelope
expression plasmids (data not shown). ~n alternative possibility might be that the low
intr~c~ell~ r abnn~ nre of the VL and II envelope ~r~,~,ul~ol:~ iS due to their premature
oligomerisation in the endoplasmic reticulum. Premature oligomerisation of the nascent
polypeptide chains via their N-terrninal VL or II peptides might seriously colll~lolllise the
folding of individual subunits leading to their aggregation and accelerated proteoly~ic
destruc~ion. In keeping with this idea, in a related system the ch~elolle-guided folding
of infl~l~n7~ haema~ ;" monomers is known to be completed in the endoplasmic
reti~-hlm before the fully folded subunits can be assembled into homotrimers (Valsesia-
Wittmann etal., J. Virol. 6~:46094619). The fact that the II helical peptide forms very
stable trimers and that the VL peptide forms slightly weaker interactions might then
explain why the ch;lll~l liC envelopes displaying the VL peptides gave better incorporation
than the ~him~ric envelopes displaying the II peptides. To test this idea, we are planning
to ~tn. ".1~ cl-il"~f-lic envelopes displaying trimeric leucine zipper peptides with reduced
stability (i.e. Iower meltin~ t~ dLUleS) co~llpal~d to the VL and II peptides that were
used in t'nis study.
All veclors carrying the VL or II chimaeric envelopes showed inhibition of infection on
NIH3T3 and A431 cells, which was reversible on cleaving the peptides from the vectors
witn factor Xa protease. Titres were not restored completely ~o wild type levels due to
the reduced levels of incorporation of these envelopes. The VL and II peptides therefore
function as oligomerising peptide adaptors which mask the fimctions of the re~roviral
envelope glyeo~ )teill to which they are fused. The innibition of infection may be as a
result of the oligomerizing peptide blocking binding of the vector to its target cells by
m~sking the underlying binding domain. A.lternatively, t-he ~ ;.e.lce of an oligomerizing
peptide may prevent dissociation of the envelope trimer, blocking fusion. UllÇolLulIately7
because of the impaired incorporation of the VL and II chim~-ric envelopes, binding
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43
studies were ulli~o~ aLive so we are unable to ~let~-rmin~o which of these ...~ch,~ ...c is
more du...i..~
A low level of background illrc~liviLy was collsi~lcllLly observed when uncleaved vectors
displaying the VL and II peptides were used to infect NIH3T3 and A431 cells (Table 4).
The back~loulld was slightly higher on the NIH3T3 cells than on the A431 cells and
tended to increase with increasing length of the linker peptide that was inserted between
the 4070A SU and the oligomerizing peptides. We believe that this back~l~,ulld infectivity
occurs because a few of the chim~Pric envelopes are cleaved by endogenous proteases
derived from the target cells. In previous studies using chim~eric envelopes displaying
a cleavable EGF ~om~in we have observed that the IEG~ factor Xa cleavage site can be
cleaved to a small extent by proteases released from NIH3T3 and A431 cells. We also
found that increasing the length of the linker seqnenre bc~weeu the factor Xa cleavage site
and the displayed EGF domain increased the ~rce~ihility of the factor Xa site to these
endogenous pru~ases.
In s~ -n~y, our results ~ Jl~ Le that retroviral vector i~c~LiviLy can be reversibly
inhibited by fusing cleavable trimeric peptide adaptors to the N-te ...;..~ of the 4070A SU
envelope glycupluteill. Infectivity is restored by exposing the vector particles to a
protease that cleaves the adaptor from the SU glycoplvLt ihl. It is anticipated that these
adaptors will be useful to prevent infection of llollL~l~ et cells in the two-step (targeted
~rh",e,~l targeted cleavage) talgeL..lg strategies that we are ~;u-~,nLly developing.
Chirnaeric envelopes displaying the VL and II peptide adaptors that were used in this study
were poorly e"~ ed. We are therefore aLLell,ptill~, to identify similar m~ing adaptors
that do not co~ lllise the expression of chi--~ ~ dc envelopes on which they aredisplayed.
F.Y~mrle 4
Co.~ u~lion of r~ vi~ uses cont~ining cleavable oligomerising adaptors ~,vith an EGF
binding (lom~in
MATERlALS AND METHODS
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44
Plasmid Co~ Lion
Vectors pEGF LVA1 and pEGF LVXA1 display an oligomerising peptide, LV, fused to
residue + 1 of 4070A SU with a non cleavable (SAA) or factor Xa protease-cleavable
(SAAIEGR, Seq ID No. 21) linker and also display the EGF binding domain. To
generate these vectors, PCR primers Gal4 LV, Gal4 LVbak and Gal4 LVfor were usedfor assembly of the PCR fragment coding for the oligoll~isillg peptide, LV (Harbury et
al., 1993 Science 262, 1401-1407). The PCR product was (~igecte-i with NotI and Eagl
and cloned into the NotI-~ligest~i backbones of EAl and EXAl. Figure 10 shows a
diagramatic le~l~,e ~li lion of the two constructs. The correct se~lu~..ce of the con~L~u~;~
was verified by DNA seq~lenrin~.
+The following oligonucleotides (with 1~ 1ion sites nn~PrlinPd) were used:
Gal4 LV, 5'-GAC AAG CTA GAG GAA GTA CTT AGC AAG CTC TAC CAT GTC
GAG AAC GAA CTT GCT CGA GTT AAG AAG-3' (Seq ID No. 22);
Gal4 LVback (cont~inin~ NotI site), 5'-GGC ATT CAT GCG GCC GCA ATG AAG
CAA GTG GAA GAC AAG CTA GAG GAA GTA C-3' (Seq ID No. 23);
Gal4 LVfor (cr ..li.i"i"~ EagI site), 5'-GCA AAT CTG CGG CCG ACT CTC CCA GAA
GCT TCT TAA CTC GAG CAA GTT C-3' (Seq ID No. 24).
Target cell lines and ~ lu~lioll of viruses
The murine cell line NIH 3T3, and the human cell line A431, were grown in DMEM
supplem.ontrd with 10% fetal calf serum. The envelope ~,c~ion constructs were
L~ srt:cLed into TELCeB6 p~r~gjng cells by calcium phosphate p~ tion and stable
phleollly~ l (SOmg/ml) resistant colonies were expanded and pooled. Cells were grown
in DMEM supphPmPntPd with 10% fetal calf serum and when confluent transferred from
37~C to 32~C and inr~1h~tPd for 72hrs. SuL,ell~L~nts cont~inin~ retroviral particles were
hal~,~,;,Led after overnight (16hrs) inrllk~fion at 32~C in lOmls serum-free DMEM for
infections. All sllr~orn~t~ntc were filtered (0.45~m) before use.
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Target cell Infection
Target cells were seeded at 2 x 10' cells/well in six-well plates and inrllh~Af'Ad at 37 C
overnight The harvested su~ue~ tAAntc cont~ining ~-galactosidase-transducing retroviruses
were in~A~lb~tPd with 0 or 4 ,uglml of factor Xa (PlUI11C~ ) for 90 Ill;lllllrs at 37 C in the
presence of 2.5mM CaCl,. SuperniA1t~Ant dilutions in 2ml serum-free media were inr~lb~ted
with target cells for 6 hrs in the ~l~,se,lce of 8~g/ml polybrene. The retroviral ~u~ t;~
was then removed and the cells were inlA-llbiAtpr~ with regular medium for 48-72 hrs. X-Gal
sti ininY for detection of ,B-g~l~rtoci~ P activity was performed and viral titre (el.~yllle
forming unitslml) was calculated by coul~Li~ blue stained colonies microscopically with
the use of a grid placed unde.llealll the 6 well plates.
Host range properties of virus incorporating cl~ ic envelopes
To cletArmin~A wl~Lllcr the oligomerising peptide was m~kin-~ the functions of the 4070A
envelope to which it was fused, we titrated the vectors on Ram-l c~,u-e~s-l~ cells,
NIH3T3 and A431 before and after they were cleaved with factor Xa ~Loltase (Fig. 11).
Both vectors gave greatly reduced titres on NIH3T3 and A43 1 cells colll~a.~d to wildtype
amphotropic vector. On cleavage of EGF LVXAl with 4 ,ug/ml factor Xa ~roL~ase the
titre on NIH3T3 cells and A431 cells ill.~ sed by up to 200 fold. Cleavage of the contol
vector EGF LVAl, which does not carry the factor Xa cleavage signal, however, did not
result in such an increase in titre.
These data de~Au-~Llate that a mollo.llc~ic binding domain can be displayed as part of a
trimerising adaptor which blocks the function of the underlying envelope until it is cleaved
with a ~e-,irlc ~luL~ase~
~mrle 5
Angiogenesis, inflimmiAtion and tumour invasion are linked to the overexpression of
matrix metallo~lotei.~ es (MMPs) which degrade the extr~ce~ kAr matrix. The MMPsare Lh~,lerc,l~ plullli~ g targets for therapy. As an alt~ n;A~tive to using MMP inhibitors,
we are developing MMP-activatable gene delivery systems. Here, we describe the
construction of vectors incorporating inhibiL~ly adaptors that are efficiently cleaved by
activated MMPs. The MMP-sensitive vectors underwent cleavage activation selectively
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46
on target cells eAy~ hlg endogenous membrane-associated MMPs, and gene delivery was
dl,~ f ir~lly L.~hAIl~'e~ MMP-activatable vectors will offer new o~polLu~liLies for ~,.,Li~
of therapeutic genes to sites of disease.
Matrix metallo~okil~ses (MMPs) are important for angiogenesis, tissue remodelling,
infl~mm~tinn and wound healing, and they play a crucial role in various pathological
processes including cancer invasion and mpt~ct~cis and the destruction of articular cartilage
in rhP~lm~foid ~LIlliLis (Liotta et al., 1991 Cell 64, 327; Woessner Jr., 1991 FASEB J.
5, 2145; Ray & Stetler-Stevenson 1994 Eur. Respir. J. 7, 2062; Karelina et al., 1995 J.
Invest. Dermatol. 105, 411). The known MMPs include matrilysin, collagenasesl-3,stromelysinsl-3, ge1~tin~cec A and B and a group of 4 membrane-type MMPs (MT-MMP)
which are anchored to cell membranes (Sato et al., 1994 Nature 370, 61; Takino et al.,
1995 J. Biol. Chem. 270, 23013; Will & F;~ .... 1995 Eur. J. Bioch-m 231, 602;Puente et al., 1996 Can. Res. 56, 944). Most of the MMPs are secreted as zymogenforms and reguire a~;tivaLioll before they can exert their proteolytic activities. The net
activities of the el.~ylllcs are also regulated by the three tissue inhibitors of MMPs (TIMPs
1-3). Once activated, the MMPs co-operate with one another in a cascade pathway to
cause degradation of the çxtrarPllular matrix. Gel~tin~cP A (GLA; MMP-2) and the MT-
MMPs are of special interest with respect to tumour invasion. Pro-GLA is secreted by
stromal fibroblasts and collce~ at~d on turnour cell membranes, especially at the invasive
front of the tumour (Afzal et al., 1996 Lab. Invest. 74, 406; Nomura et al., 1996 Int. J.
Can. (Pred. Oncol.) 69, 9). It binds as a pro-GLA-TIMP-2 complex to MTl-MMP which
then me~ tec its cleavage activation on the surface of the tumour cell (Strongin et al.,
1995 J. Biol. Chem. 270, 5331; Emmert-Buck et al., 1995 FEBS Letters 364, 28; Gilles
et al., 1996 Int. J. Can. 65, 209). Indeed, MTl-MMP-mediated activation of pro-GLA
is considered to be illl~olL~t for the progression of cancer and the cOllc~llL~ ion of active
GLA is often found to be elevated in invasive or mtot~.ct~tic tumours. Hence, there is
considerable interest in the exploitation of MMPs as promising targets for novelth~d~tic agents and there are several general or specific MMP-inhibitors that are
~;ull~,llLly being tested for their usefulness in tre~tmPnt of MMP-linked rlice~ces in a
number of clinical trials (Hodgson 1995 Biotech. 13, 554; Eccles et al., 1996 Can. Res.
56, 2815). Here, as an ~ItPrn~tive to the use of MMP-inhibitors, we propose the use of
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a MMP-activatable gene delivery system.
This example describes the geneldtion of targeted retroviral vectors whose infectivity for
human EGF receptor-e~ cssillg cancer cells is strongly activated by membrane-associa~d
MMPs.
A series of r~ ..sPIiC envelope e~ ssion constructs was gell~ Led in which a cDNA
coding for the 53 amino acid receptor binding domain of EGF was linked to the N-L~ al codon of the 4070A murine lellkPmi~ virus (MLV) SU envelope gly~o~luLcill via
short non-cleavable or protease-cleavable linkers. In brief, the rhim~orjr vectors E.A and
E.X.A, have an EGF cDNA, fl~nk~-l by SfiI and Nod restriction sites, i~c.L~,1 at codon
+ 1 of the N-t~lllillus of wild type 4070A MLV SU (surface protein gp 70) envelope, with
a linker of either 3 ~l~nin~os (E.A.) or 3 ~l~ninrs and the IEGR Factor Xa cleavage
sequence (E.X.A.) beL~ the domains. Figure 12 is a srhpn~tir r~ se.,~ ;o~- of the
cl~ lic envelope ~ e;,~ion constructs, E.A, E.G4S.A, E.X.A and E.MMP.A. The
envelope col~LlueL~ were L~reeLed into TELCeB6 compl~ ;"g cells, virus-producingclones were pooled and e~ n-l~cl in 10% FCS-DMEM selection "~r.~ "~ COl~ 50
~ug/ml phle~,llly~. Arrows in-1ir~te potential site of cleavage by l~,S~e~,~ive proteases.
To obtain col~Llu~:Ls E.MMP.A and E.G4S.A, PCR ~lhllels AlGelA Nb (5' GCA AAT
CTG CGG CCG CAC CTT TGG GAC TTT GGG CAA TGG CAG AGA GCC CCC
ATC, Seq ID No. 27) or NLlAlB (5' GCA AAT CTG CGG CCG CAG GTG GAG GCG
GTT CAA TGG CAG AGA GCC CCC ATC, Seq ID No. 28) r~s~e~;Lively~ were used
with prirner 4070Afor (described above) on E.A to generate NorI-tailed PCR fr~gmf~nt~
of the 4070A SU coding sequence encoding the MMP-cleavable (PLGLWA) or non-
cleavable linker (G4S) as a 5' extension. The PCR fr~rn~ntc were digested with NotI and
BamHI and cloned into the NorI-BamHI digested backbone of E.A to gellclaLe constructs
E.MMP.A and E.G4S.A. The sequences of the Col~LlucL~ were ch~-r~ and verified byDNA se~ e~
The E.A and E.G4S.A chim~ric envelopes contained non-cleavable linkers AAA and
AAAGGGGS (Seq ID No. 25) respectively (single letter amino acid code), the E.X.A
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48
envelope contained a Factor Xa-cleavable linker AAAIEGR and the E.MMP.A envelopecontained the linker AAAPLGLWA (Seq ID No. 26) in which tne hi~hlightt~d sequence
is known to be recognised and cleaved by GLA and by MTl-MMP (Ye et al., 1995
Biochem. 34, 4702; Will et al., J. Biol. Chem. 271, in press) (Figure 12).
The cnimaeric envelope constructs and a wild type 4070A envelope e~le;.~ion col~LL~lcL
were stably L1A~ rC~ into TELCeB6 comple-..~ i..g cells wnich express Moloney MLV
gag-pol ~lolcills and tne nlcT ~r7 lcL uv Udl vector, as described in the L)l~ce~ g examples.
Upon L al~rccLion of these cells with a functional envelope e~ ,ssiou pl~cmi~ lÇccliùus
enveloped vector particles capable of tral~Çcll~ tne lacZ marker gene are rescued into
tne culture sUFt~rn,.t~nt Viral supc~ . were harvested from collLlu~ L plates of pooled
transfected TELCeB6 cells and the viral particles were pelleted by ull . ,.re. .1 . irugation and
i .. , .. rblotted using a l anti-envelope allLisc~ L~lll as probe. r.. ~nblù~ g was ~e-l rul.ued
as described in the preceding examples. The results are shown in Figure 13A, B.
Figure 13A is an i.. r,blot showing COlll~ dtiVc viral i~ olaLion of the EGFrhi~lqt .ic vectors (lane 2=E.A; lane 3=E.X.A; lane 4=E.MMP.A; lane 5=E.G4S.A)
and the wild type 4070A SU (lanes 1 and 6). Figure 13B is an illlll~ blot demo~ till~
cleavage of MMP-cleavable linker in E.MMP.A by ~ùlirle~ p-a~illc,phenylmercuric
acetate (APMA)-acLiv~Lcd gel~tin~e A (GLA). An aliquot of the E.MMP.A viral pellet
was in~llb,qtt~ c~pecLivcly, with PBS only (lane 1), APMA-activated GLA (final
collcellLldLion 32 ,uglml; lane 2) and APMA at a f~ co,-r~ ion of 2 rnM (lane 3).
Lane 4 shows unmodified wild type 4070A-SU.
It is a~a,c.ll from Figure 13A that all four rhim~loric envelopes were c,~ ,;.sed and
incorporated into virions, as i,..i ir~rc~ by the decrease in mobility colllpal~d to wild type
4û70A-SU, and t-h-at the relative efficiencies of envelope incorporation were comparable
in the four different recombinant virus stocks.
To detcllllhle if GLA could recognise the PLGLWA sequence on the E.MMP.A vector
and thus cleave the EGF domain from the chim~l-ric viral envelope without degrading the
underlying 4070A SU gl~;oplotcin, we inrllb~torl aliquots of the E.MMP.A and control
_
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49
viral pellets for 30 min at 37~C with PBS, p-aminophenylmercuric acetate (APMA) or
APMA-activated GLA, after which imm--noblots were performed as before. [GelAtinAcP
A (GLA) was purified as a zymogen form and requires activation by inrllbAtion with
APMA (2 mM) for 1 h at 25~C prior to use. 10~1 of the resuspended E.X.A, E.G4S.Aor E.MMP.A viral pellets were inrllkAtto~l with PBS, APMA (final collce.~LldLion 2 mM)
or APMA-activated GLA (32 ~g/ml) for 30 min at 37~C].
On treatment of E.MMP.A-SU with activated GLA, a band with the same mobility as the
wild type 4070A-SU was recovered, in-ljrAting that the EGF domain could be efficiently
cleaved from this rhi~ iC envelope without further GLA-m~o~iAt~l degradation (Fig.
13B). The E.G4S.A and E.X.A chimAPric envelopes were ..,-hrr~ by treAtrn~r~t with
GLA in-lir~tinsg that cleavage was specific for the MMP-sell~iLive linker (not shown).
Our previous data (Nilson et al., 1996 Gene Therapy 3, 280) i".lir~ rl that the i~ecLiviLy
of the E.X.A vector was minim~l on EGF-l~,c~toi positive A431 cells but could be fully
and selectively I~Loled by cleaving the rhimAPriC envelope with Factor Xa protease. This
result was co~ri~"~ using the E.X.A vector stocks that were g~ll.,.dled in the current
study which bound strongly to EGF l~,C~)Lul~ on A431 cells and gave a titre of 103 efu/rnl
rising to 106 efu/ml after the EGF domain was cleaved from their surface with Factor Xa
protease (data not shown). The i ~ ;Livilies of the E.A, E.G4S.A and E.MMP.A vectors
on A431 cells were low between 102-103 efu/rnl and were not greatly increased bytre~tment with Factor Xa protease (not shown).
To ~leL,~ . ".i.,~ wl..,~ r the ilL[eclivi~y of the MMP-cleavable E.MMP.A vector could be
activated by GLA, we ~ ull~ed infections on A431 cells in the presence of increasing
collce,lLldLions of exogenous pro-GLA. Since A431 cells are known to activate pro-GLA
to GLA, pre-activation of the protease with APMA was not nt~ce~5A,y
For the infection assays, A431 cells in 10% FCS-DMEM were seeded, at a density of 3
x 104 per individual well, in a 24-well tissue culture plate (Corning, New York) overnight
at 37~C. The media were removed the next day and the cells were washed once in serum-
free DMEM. Varying amounts (final concentration 2-40 ~Lg/ml) of pro-GLA were mixed
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5 0
with 200 ~LI of filtered E.MMP.A viral suL~ dL~nt after which the ~ Lul~. was added to
A431 cells and inr~lbi~7ted at 37~C for 6 h. At the end of 6 h, the media was removed and
cells were washed once in serum-free DMEM. The cells were then inr~lh~tPA in 10%FCS-DMEM for 72 h at 37~C before they were washed once in cold PBS, fxed in 0.5%gutaldehyde-PBS for 15 min, washed once again with PBS and i"~ tPd with X-gal
overnight at 37~C. The number of colonies tri~n~ lcecl with the vector (blue colonies)
were counted and the titre e~LL,l~,ssed as efu/ml viral ~.u~ Figure 14 is a graph
showing that increase in titre (efu x 10~4/ml) of the E.MMP.A MMP-sel.~.iLivt: vector on
A431 cells is correlated with the amount of pro-ge1,.tini~e A (pro-GLA) added onto the
cells.
It was found that as the conce.lLldtion of exogenous pro-GLA was increased incremPnti-.lly
from 2 to 40 ~Lg/ml, the il~;LiviLy of the E.MMP.A vector hl,l~dsed in a dose-dependent
nl~l. From 1.3 x 103 efu/ml in the absence of pro-GLA, the titre ill;leased S0-fold
to 6.5 x 104 efu/ml in the ~ c.lce of 40 ,ug/ml pro-GLA. The titre of the noncleavable
E.G4S.A vector was relatively llnrh~n~ed from 1.2 x 102 efu/ml in the ,7hsenre of pro-
GLA to 1.4 x 10~ efu/ml in the ~l~,s~l~ce of 40 ,ug/ml pro-GLA. Activation of il~ecliviLy
was specific to tne vector with the MMP-cleavable linker as hlrec~iviLy of E.X.A increased
only 3-fold in the ~ s~llce of 40 ~g/rnl pro-GLA (not shown).
We next explored the possibility tnat endogenous target cell-derived MMPs could activate
tne E.MMP.A vector in tne absence of exogenous MMP. HT1080 is a human
fibrosarcoma cell line that col~.LiLuLivt:ly produces MT1-MMP and pro-GLA (Okada et al.,
1995 Proc. Natl. Acad. Sci. 92, 2730). We therefore inrllhi~tPd the viral supe~ti~ntc of
E.G4S.A, E.X.A and E.MMP.A on HT1080 and A431 cells for 6 h at 37~C in the absence
of exogenously added pro-GLA: 200~L1 of the filtered E.X.A, E.G4S.A or E.MMP.A viral
~.U~ were added onto A431 or HT1080 cells for 6 h at 37~C with 8 ~g/ml
polybrene, after which t'ne incubation mrAillm was removed and the cells washed once in
serum-free DMEM. The cells were inrllbi~tpA in 10~ FCS-DMEM for 72 h at 37~C
before they were stained with X-gal.
Figure 1~ is a graph showing the titre of EGF rhimi~eric vectors on A431 and HT1080
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cells. Figure 15A shows the high infectivity of E.MMP.A vector on HT 1080 cells
compared to on A431 cells as in~iir~t~-rl by the number of blue ,~-g~i~rtosi~ e positive
colonies. One ml out of 10 ml filtered E.MMP.A viral supern~t~nt was inr~lb~t~d with
5 rnM CaCl~ for 30 min at 37~C before it was inrllk~t~l on the r~ e.;Li~7e cell types for
6 h at 37~C. At the end of 6 h, the cells were washed in serum-free DMEM and
inrl-b~tto~l in 10% FCS-DMEM for 72 h after which they were stained with X-gal. The
respective titres are ~A~Iessed as efu/rnl viral sUpe.~
Con~ictent with previous results, the il~CIiviLy of the vectors on A431 cells was low in
the absence of exogenous pro-GLA. However, on HT1080 cells, the infectivity of the
MMP-cleavable vector E.MMP.A was activated by two orders of m~nih~P co,ll~al~;d to
the MMP-resisLallL control vectors E.G4S.A and E.X.A (Fig. 15, 15A). Thus, in the
~bsPnre of any added exogenous MMP, the higher titre of the MMP-dep~n-~nt E.MMP.A
vector must be due to its cleavage by MMPs produced endogenously by HT1080 cells.
To ~lete....i..~? if the MMP-activatable E.MMP.A vector could selectively target the MMP-
eA~lc;ssillg HT1080 cells in ~lefe~w.ce over A431 cells, we allowed the vector to infect
both cell types on the same petri dish cimlllt~npously~ We grew A431 and HT1080 cells
s~paia~ly on coverslips, placed them in the same petri dish and added sup~
c~ ;--ing the E.MMP.A or control vectors: A431 and HT1080 cells were seeded
sepald~ly, on 25 mm Tk~ ox coverslips (Corning) contained in 6 well plates, in 10%
FCS-DMEM overnight after which the media was removed and the cells washed once in
serum-free DMEM. The coverslips coated with the cells were placed in a 10 cm petri
dish (Falcon) and E.G4S.A (1:1.5 dilution), E.MMP.A (1:1.5) or 4070A (1:20)
~7upellldLanL~. were added onto the petri dishes with 8 ,ug/ml polybrene for 6 h at 37~C. At
the end of the inr~lh~tion period, the media was removed and the cells were incubated in
10% FCS-DMEM for 72 h before X-gal staining. The results are shown in Figure 16:E.MMP.A vector grown on HT1080 (H) cells and A431 (A) cells is shown in I and II,
with the control E.G4S.A vector in III and the wild type 4070A vector in IV.
When L~l~,se.lLed with both cell types, E.MMP.A infected HT1080 cells ~l.,Çel~llLially over
A431 cells. The wild type 4070 A vector and E.G4S.A vector with the non-cleavable
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linker showed no such preference (Fig. 16). In tbese CX~ 7, the MMP-activatable
E.MMP.A vector did not infect A431 cells more efficiently in the ~l~se.lce of HT1080
cells than in their ~bs~onre~ This suggests that soluble GLA released into the n~P~ m from
the HT1080 cells does not play a ~ nifir~nt role in activation of the vector. Tnct~arl, the
results SLLU~1~1Y in-lir~t~ that the cleavage activation of the E.MMP.A vector is localised
to the surface of the HT1080 cells and that it is m~ tf~-~ by l~-~,~bl~le-i~soci~t~d MMPs
acting on vector particles that have bound to the EGF l~.Ce~01~7 on these cells.
The sismifir"nt role that MT-MMP plays in cleavage activation of the MMP-cleavable
vector was ~,u~olL~d by results from e~ t?nt~ using natural MMP inhibitors TIMP-1
and TIMP-2 and a synthetic inhibitor, CT 1339. For the inhibition studies, TIMP-1 at a
final collcel~Ll~Lion of 10 ~g/ml, TIMP-2 (5 ~g/mI) or CT 1339 (1 mM) was used. The
inhibitors were added to 200 ~Ll of diluted (1:10) E.MMP.A or l~n~ f~ofi E.G4S.A viral
~7U~ The llPL~Lulc was then added onto A431 or HT1080 cells, which had bee~
washed once in semm free DMEM, and the cells were i.. h,.~d for 6 h at 37~C. At the
end of the inr-lhatinn period, the cells were washed once in serum free DMEM, inf ~lbi~t~od
for 72 h in 10% FCS-DMEM after which they were stained with X-gal. The E.MMP.A
e. n,.li...l was diluted to obtain a titre that would allow ~ c~ t~ counting of the llu
of tr~n.~ er1 colonies. Inhibition studies on A431 cells were ~,.rulllled with 200 ~l
m~ l-ted E.MMP.A or E.G4S.A in L,lese~ce of 16 ~g/ml pro-GLA.
Table 5: Tnfl~ n~e of MMP inhibitors on the titre of vectors on A431 and HT1080 cells.
Cell type TIMP-1 TIMP-2 CT 1339
HT 1080 16.7 +/- 2.1 72.7 +l- 9.1 73.3 +/- 6.4
A431 79.3 +/- 10.2 83.3 +/- 8.4 83.4 +/- 9.8
MMP-dependent E.MMP.A vector was added to A431 cells in presence of 16 ,L~g/ml pro-
GLA or to HT1080 cells in the absence of exo~enous pro-GLA, with or without tne
addition of natural MMP inhibitors TIMP-l, TIMP-2 or ~7yllLllt;Lic inhibitor, CT 1339.
Values (means + SD, n=3) le~lesell~ p.,~ ~ge decrease in titre (with inhibitors)cOlll~al~d to that of the control (without inhibitors).
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53
All three innibitors have strong activity against GLA and can prevent the activation of
E.MMP.A vectors by exogenous GLA (Table 5). However, unlike TIMP-2 and CT 1339,
TIMP-l could not efficiently block the activation of E.MMP.A by endogenous MMPs on
HT1080 cells (Table 5). An important dirrele.lce between TIMP-l and the other inhibitors
is that it displays only weak activity against the MT1-MMP expressed on HT1080 cells
(Fig. 17. described below). These e~.ul~e~ 7 therefore poiM to a central role for tne
MT-MMP in HT1080-m~ tt~d activation of the E.MMP.A vector.
Figure 17 is a gelatin zymogram showing the effect of TIMP-l or a synthetic MMP-inhibitor, CT 1339 on cellular activation of endogenous pro-GLA on HT 1080 cells. Tne
E.MMP.A viral supernatant was inr~lh,.tt~fl on HT 1080 cells for 6 h at 37~C in the
absence of any inhibitors (lane 1), in the presence of 10 ,ug/ml (lane 2) or 30 ~g/nl
TIMP-1 (lane 3), and 1 ~M (lane 4) or 10 ~M (lane 5) CT 1339. At the end of tne
inr-~h,-tion period, an aliquot of the ~.UL~ t;.llt was loaded onto 7% SDS-PAGE gel
cont~ining 0.5 g/ml denatured type I collagen and electrophoresis was carried out at 4~C
for 1 h, after which the gel was i~l~;u'oated twice for 15 min each in 2.5% Triton-X 100
to remove the SDS, washed in water and then in-~l-b~.tto~l overnight at room ~ll~.,~dLulc
in 100 mM Tris, 30 mM CaCl2 - 0.0015% Brij and 0.001 % NaN3. The gel was then
stained in 0.25% Coom~cci~o Rrilli~nt Blue Green (Sigma). The location of gelatinolytic
activity on the gelatin zymogram is ~'et~ct~'ole as a clear band in the background of blue
st~ining.
There have been many variably sllccescful attempts to target retroviral vectors through
ligand-l~c~:~Lol interactions (Valsesia-Wittm~.nn et al., 1994 J. Virol. 68, 4609; Cosset et
al., 1995 J. Virol. 69, 6314; K,.c~h~ra et al., 1994 Science 266, 1373; Matano et al.,
1995 J. Gen. Virol. 76, 3165; Somia et al., 1995 Proc. Natl. Acad. Sci. 92, 7570). Here
we have adopted a two-step Ldlg.,Lillg strategy that allows us to utilise the specificity of
protease-substrate interactions to activate the infec~iviLy of ~cct~Lor-targeted retroviral
vectors. We previously relied on the addition of exogenous Factor Xa protease for vector
activation, an approach that might have rather limited applications for in vivo gene
tnerapy. Here, we have demol.~.LldLed for the first time a retroviral vector whose
infectiviry can be activated by endogenously produced disease-associated proteases. The
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54
vector is optimally cleaved and activated by membrane-associated MMPs on human
tumour cell lines.
The targeting strategy that we have pursued may have illL~Ic~Lillg parallels with the
mechanism of HIV entry in which primary virus ~tt~rhmPnt to CD4 leads to a
conformational rearrangement or proteolytic cleavage in gp 120, and secondary virus
~tt~rhment to one of the lccellLly characterised HIV co-receptors (Feng el al., 1996
Science 272, 872; Deng et al., 1996 Nature 381, 661; Handley et al., 1996 J. Virol. ~0,
4451). C-type l~Ll~vildl vectors with engineered SU glycoproteins could therefore be
developed as model systems to probe the entry mrrh~n;~mc that are employed by naturally
occurring viruses, such as HIV.
It is hoped that targeted vectors of the type that we have described in this report will open
up new possibilities for gene therapy in MMP-associated ~ es~ for example in cancer,
where elevated MMP production in tumour deposits is required for angiogenesis,
invasiveness and mPr~ct~ric potential and is strongly correlated with poor prognosis
(Murray et a/., 1996 Nature Med. 2, 461).
~mrle 6
Rt:llv~/iral display of L~ c binding ~lonl~inc~ TNF alpha and CD40 ligand.
The following experiments demol~LldLt: that rhim~ric envelopes bearing TNF alpha or
CD40 ligand as an N-t~rmin~l extension can be illcol~olaLt:d into I~Llovildl vector particles
where it appears that the trimeric binding domain forms a cap over the envelope
glycoprotein to which it is fused. The amphotropic infectivity of the vectors incorporating
these chim~ric envelopes is therefore low but is greatly enh~nreri by cleaving the trimeric
ligand from their surface.
Cell Lines
The TELCeB6 cell line has been described in the precrrling examples. The NIH 3T3,
A43 1 (human squamous carcinoma; ATCC CRL1555) and HT1080 (human fibrosarcoma;
ATCC CCL121) cell lines were grown in DMEM (Gibco-BRL, UK) suppl~mpntpcl with
10% fetal calf serum (I~CS; PAA Biologicals, UK), berl7ylpenicillin (60 mg/ml) and
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streptomycin (100 mg/ml) at 37~C in an atmosphere of 5% CO.. The B cell lines, Daudi
(human Burkitt's lymphoma: ATCC CCL 213), Raji (human Burkitt's Iymphoma; ATCC
CCL 86) and K422 (human Non-Hodgkin B cell; Dryer et al., 1990 Blood 75:709-714),
and T cell line, Jurkat (human acute T cell le--kt~ ATCC TIB 152) were grown in
RPMI 1640 (Gibco-BRL) supplement~ with 10% FCS, benzylpenicillin (60 mg/ml) an
tol~-ycin (100 mg/ml) at 37~C in an atmosphere of 5% CO..
Construction of t~him~ric envelope ~ ion vectors
The human tumour necrosis factor-alpha (TNF-a)-4070A SU c~lim~ric envelope
expression vectors TNF-a.A, TNF-a.GS .A, TNF-a.X.A, TNF-a.XA, and TNF-a.MMP.A,
have an TNF-a cDNA (Wang et al., 1995 Science, 228: 149-154), flanked by Sf~I and
.A~otI restriction sites, inserted at codon + 1 of the N-terminus of wild type 4070A MLV
SU envelope by different linkers (Fig. 18). The TNF-a.A vector is linked via a 3 alanine
(AAA) linker; TNF-a.GS.A via a non-cleavable AAAG4S linker; TNF-a.X.A via FactorXa protease cleavable linker (AAAIEGR) and TNF-a.MMP.A via an MMP-cleavable
linker (AAAPLGLWA) (single letter amino acid code). The Factor Xa protease cleaves
IEGR after the algi~ e residue and the PLGLWA linker is susceptible to gel~tin~ A
(MMP-2) and MT-MMP between the glycine and leucine residues.
The CD40L-4070A SU chimaeric envelope expression vectors have part of the CD40L
cDNA, flanked by Sf~I and NotI restriction sites, inserted at codon + 1 of the N-terminus
of 4070A MLV by the 4 different linkers as mentioned above. The vectors are termed
CD40L.A, CD40L.GS.A, CD40L.X.A and CD40L.MMP.A (Fig. 19).
A PCR derived Sf~l-NotI DNA fragment encoding the 155 amino acids of the trimeric
human TNF-a was generated using a cDNA template and two primers, sTNFback
(S' > CCG GTA CCG GCC CAG CCG GCC TCT TCT TCT CGT ACC CCG, Seq ID
No. 29) with a Sf~I site, and
nTNFfor (5' > AAG TCT TAG CGG CCG CCA GAG CGA TGA TAC CGA AG, Seq
ID No. 30) with a NotI site.
The S~l-NotI PCR fragment encoding the 145 amino acids of the soluble extracellular
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56
domain of the trirneric CD40L (Gly 116-Leu 261; Karpusas et al., 1995 Structure, 3:
1031-1039) was generated using a cDNA template (ATCC 79813) and two ~
sCD40Lb (5' > CCG GTA CCG GCC CAG CCG GCC GGT GAT CAG AAT CCT CAA
ATT GC, Seq ID No. 31) with a SfiI site and
nCD40Lf (5' > AAG TCT TAG CGG CCG CGA GTT TGA GTA AGC CAA AGG, Seq
ID No. 32) with a NotI site. The ~ .e~;Livc PCR fr~mtomc were ~ ost~l with S~faI and
NotI restriction C~yll~s and cloned into the Sf~-NotI digested EA.1 backbone or EXA.1
to obtain TNF-a.A or CD40L.A. and TNF-a.X.A or CD40L.X.A, lc~e~,Lively (Nilson
et al., 1996 Gene Therapy 3: 280-286).
To obtain TNF-a.GS.A or CD40L.GS.A, and TNF-a.MMP.A or CD40L.MMP.A, therespective Sf~-NotI digested TNF-a or CD40L PCR fragmentc were cloned into Sf~-NotI
digested E.GS.A or E.MMP.A backbones, lc~L~ectively (Peng et al., A gene delivery
system activatable by disease-associated matrix metallopn)Lcinases, submitted). The
sequences of the COl~Llu~ were rll~r~(l and verified by DNA sequ~onring.
Pro~ ct~ of v ru es
The various TNF-a and CD40L envelope eA~ ion plasmids were stably Lld~rc~;Lcd bycalcium phosphate pl"ci~iLation (Sambrook et al., 1989, Molecular cloning: A laboratory
manual) into the TELCeB6 pacl~ging cells. Trarlsfected cells, grown in 10% FCS-
DMEM at 37~C, were selected with 50 ~Lg/ml phleomycin (Sigrna, Poole, Dorset, UK).
l~tosi~t~nt colonies were pooled and e~panlle~i~ and before harvest, the cnnflll~nt cells were
~t;llcd to 32~C for 72 h. The viral supe. .~ were then h~ ed and filtered (0.45
,um, Acrodisc, Gelman Sciences MI, USA) after overnight inrllbation of the confluent cells
with serum free DMEM at 32~C. These filtered ~ t~ntc were then used either for
immnnoblotting~ binding or infection assays.
Tmml-noblots
For immnn~blotting7 the viral particles were pelleted by lllrr~rt-ntrifugation of the flitered
viral supernatant (Rerkm~n, USA) at 30,000 rpm for 1 h at 4~C in a SW 40 rotor. The
pellet was then l~.u~yellded in 100 ~l cold PBS and stored at -70~C till further analysis.
An aliquot (10 ~l) of the viral proteins was separated by electrophoresis on a 10% SDS-
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57
PAGE gel, ele~ ul,a~r.~ d onto nitrocellulose me~nbrane (Hybond ECL, Amersham
Life Sciences, UK) and ~tPct~d by immlmostaining with goat antisera raised against
Rausher lellk~mi~ virus gp 70-SU envelope protein (Quality Biotech, USA), followed by
horseradish peroxidase-conjugated rabbit anti-goat immllnnglobulins antibodies (DAKO,
Denm~rk) and developed with an enh~nre~l chlomilllminPscence kit (Amel~.hd,ll).
To detect the ~l~,se~lce of processed (SU) and ull~ucessed (SU + TM) in the cells, the
viral compleTn~nting cells were grown to confl~ ry on petri dishes (10 cm in ~ mpt~r)~
washed once in cold PBS and then ;~ t~l for 10 min at 4~C with cell lysis buffercont~ining 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton-X, 0.05% sodium
dodecyl sulfate, 5 mg/ml sodium doxycholate and 1 mM PMSF. The lysed cells were
scraped from the plates and the suspension cellLliruged at 10,000 xg for 20 min to pellet
the nuclei. Thirty ~Ll of the ~ was used for electrophoresis and il llll ll~ blotting~
~PSllltC
There were no visible bands of envelope ~luteills seen on the immllnoblots for ch;.ll-r.ic
TNF-a-4070A SU in-lir~ting little or no envelope expression. When the cell lysates were
tested, there was unprocessed (SU + TM) chim~ric envelope pruL~ s but little ~etect~ble
pl~ocessed (SU) envelope ~lOL~i~. Results from the immnnoblots for CD40L4070A SUin~ teA that there was chim~eric envelope e~ ,ssion and incorporation, and e~ ,s~iol~
be~weel~ the chim~ric envelopes were colll~aldble except for CD40L.MMP.A-SU which
is most hig'nly e~ ,ssed.
Infection Assays
i. Infection of cells by f~ ic vectors-blo~king infectivity of 4070A by l~ ,.;c
ligand
The TNF-a and CD40L chim~Pric vectors were tested for infectivity on NIH 3T3, A431
~ and HT1080 cells. The cells were seeded overnight at 37~C at a densitv of approximately
1 x 105 cells per individual well in a 6-well tissue culture plate (Corning, New York).
The m~-linm was removed the next day and cells were washed once in serum-free DMEM.
An aliqout (1 ml) of the fltered viral ~.upcll~L~lL was used to infect the cells in the
presence of 8 ,~g/ml polybrene. At the end of the 6 h incubation period, the merlillm was
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58
removed and the cells washed once in serum-free DMEM and 10% FCS-DMEM was
added. The cells were then inr~lb~t~l for 72 h at 37~C before they were stained with X-
gal. The cells were washed once in cold PBS7 fixed in 0.5% gll-t~lr~ yde-pBS for 15
min, washed once in cold PBS and inrllh~t~fl with X-gal overnight at 37~C. Nurnber of
colonies tr~ncrl~-ce-~ with the ,B-galactosidase gene (blue colonies) were counted and the
titre expressed as erl7yme forming units (efu)/rnl viral su~c~
ii. Tr~ of viral sup~ J~ with Factor Xa proteace: r~ l of blockage to
infectivib by L,.o~ease
An aliquot (1 rnl) of the filtered :~Up~11l'l;1lll was incubated with 2.5 mM CaCl7 in the
p,esence or absence of 4 ~Lg/rnl Factor Xa protease (New Fngl~n~i Biolabs, UK) for 1 h
at 37~C. At the end of the inrllh~tion period, the ~u~ t~nt was added onto the NIH
3T3 or HT 1080 cells for 6 h before the ~uL~ ,,l was lcll~vcd, cells washed and
m~int~in~l in 10% FCS-DMEM before they were stained with X-gal as before.
R~cnltc-
TNF-a-4070A ~ dS
The titre of the TNF-a4070A vectors on NIH3T3 and HT1080 cells were low (Table 6).
This low level of i~cc~iviLy could be due to the low level of c~,;",~ ic envelope
,sion. However, it could also be due to the display of the trimeric TNF-a on the4070A-SU. The trirner was able to block the infectivity of the arnphotropic vector, which
would be oLh~,~wise be highly infective on the murine NIH 3T3 cells, which do not bear
the human TNF-a l~,c..~k~r.
Table 6. Titre of TNF-a-4070A vectors on cell lines in ~lcsellce of 8 ~g/ml polybrene
efu/rnl TNF-a.A TNF-a.G4S.A TNF-a.X.A TNF-a.MMP.A 4070A
NIH3T3 0 7 5 41 1 x 107
HT1080 0 0 0 11 1 x 10'
This blockage of infectivity by the trimeric TNF-a could be reversed by the addition of
factor Xa protease to cleave off the TNF-a ligand on the TNF-a.X.A vector and thus,
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allowing the 4070A-SU to bind and infect the cells. As a result, the titre of the TNF-
a.X.A vector increased 4-fold and 60-fold, lc~pe.,ti~ely, on HT1080 cells and NIH 3T3
cells after treatment with Factor Xa protease (Table 7).
Table 7. Titre (efulml) of TNF-a.X.A on cells in absence or ~lcse.~ce of Factor Xa
protease
NIH 3T3 HT1080
-FXa 10 32
+Fxa 595 136
CD40L-4070A chim~-as
The h~c~;LiviLy of the CD40L-4070A cllim~Pras are .~ignifir~ntly lower than that of the
wild type on NIH 3T3, A431 and HT1080 cells (Table 8), in~ that the display of
CD40L on the envelope is bloclcing the h~ccLiviLy of the vector.
Table 8. Titre (efu/ml) of CD40L-4070A vectors on cell lines in p,c;,eilce of 8 ,ug/ml
polybrene
efu/mlCD40L.A CD40L.G4S.A CD40L.X.A CD40L.MMP.A 4070A
NIH3T36.6 x 1031.6 x 104 2.9 x 103 2.2 x 104 1 x 108
A431 122 2.3 x 103 not done 1.4 x 103 1 X 108
HT10804.6 x lOZ8.2 x 103 3.4 x 102 1.9 x 104 1 X 108
Upon Ll~aLlllc~l~ of the CD40L.X.A with Factor xa protease, the h~-;LiviLy of the vector
is il.clcased dr~m~tir~lly (Table 9).
Table 9. Titre (efu/ml) of CD40L.X.A in absence or presence of Factor-Xa protease
NIH 3T3 A431 HT1080
-Fxa 2.9 x 103 116 97
+Fxa 1 x 106 1 x 106 1 x 106
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Medical Research Council
(B) STREET: 20 Park Crescent
(C) CITY: London
(E) COUNTRY: United Kingdom
(F) POSTAL CODE (ZIP): WlN 4AL
(G) TELEPHONE: (0171) 636 5422
(H) TELEFAX: (0171) 323 1331
(ii) TITLE OF INVENTION: Recombinant Viruses Incorporating a Protease
Cleavable Protein
(iii) NUMBER OF SEQUENCES: 32
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1Ø Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GCA M TCTGC GGCCGC M TC GAGGGAAGGC CTCATC M GT CTAT M TATC ACC 53
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GC MM TCTGC GGCCGCAGCT TCGCCCGGCT CCAGTCC 37
(2) INFORMATION FOR SEQ ID NO: 3:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GC MM TCTGC GGCCGC M TC GAGGG M GGG CTTCGCCCGG CTCCAGTCC 49
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GC MM TCTGC GGCCGC M TG GCAGAGAGCC CCCATC 36
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GC MM TCTGC GGCCGC M TC GAGGG M GGA TGGCAGAGAG CCCCCATC 48
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
- GCCAG M CGG GGIIIGGCC 19
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CTGC M GCCC ACATTGTTCC 20
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Ala Ala Ala Gly Gly Gly Gly Ser Ile Glu Gly Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) Y PE: amino acid
(C) STRANDEDNESS:
(D) -OPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
1 5 10 15
Gly Ser Ile Glu Gly Arg
ZO
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GCAAATCTGC GGCCGCAGGT GGAGGCGGTT C M TCGAGGG MGGATGGCA GAG 53
,
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(2) INFORMATION FOR SEQ ID NO~
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 base pairs
( B ) TYPE: nucleic acid
( C ) STRANDEDNESS: s i ngle
( D ) TOPOLOGY: l i near
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GCMMTCTGC GGCCGCAGGT GGAGGCGGTT CAGGCGGAGG TGGCTCTGGC GGTGGCGGAT 60
CGATCGAGGG MGMTGGCA GAG 83
(2) INFORMATION FOR SEQ ID NO: 12:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
( B ) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GGCATTCATG CGGCCGCGGC CCAGCCGGCC ATGMGCMC TAGMGACM GGTGGAGGM 60
CTCCTTAGCA AGGTATACC 79
(2) INFORMATION FOR SEQ ID NO: 13:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS: s i ngle
( D ) TOPOLOGY: l i near
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GCMMTCTGC GGCCGCCTCT CCMCMGCT TCTTCAGTCG AGCGACTTCG rrCTCMGAT 60
GGTATACCTT GCTMGGAG 79
(2) INFORMATION FOR SEQ ID NO: 14:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
( B ) TYPE: nucleic acid
( C ) STRANDEDNESS: single
( D ) TOPOLOGY: l i near
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GGCATTCATG CGGCCGCGGC CCAGCCGGCC ATGMGCMG CAGMGACM GGCAGAGGM 60
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GCTCTTAGCA AGGCTTACC 79
(Z) INFORMATION FOR SEQ ID NO: 15:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS: s i ngle
( D ) TOPOLOGY: l i near
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GCMMTCTGC GGCCGCCTCT CCAGCMGCT TC I I I GCTCG AGCAGCTTCG I I (; I (; I (iCAT 60
GGTMGCCTT GCTMGAGC 79
(2) INFORMATION FOR SEQ ID NO: 16:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
( B ) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l i near
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGCATTCATG CGGCCGCGGC CCAGCCGGCC ATGMGCAM TCGMGACM GATAGAGGM 60
ATTCTTAGCA AGATCTACC 79
(2) INFORMATION FOR SEQ ID NO: 17:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 base pairs
( B ) TYPE: nucleic acid
( C ) STRANDEDNESS: s i ngle
(D) TOPOLOGY: linear
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GCMATCTGC GGCCGCCTCT CCTATMGCT TCTTGATTCG AGCMI I ICG I l(;l(;lATAT 60
GGTAGATCTT GCTMGMTT TC 82
(2) INFORMATION FOR SEQ ID NO: 18:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
( B ) TYPE: nucleic acid
(C) STRANDEDNESS: single
( D ) TOPOLOGY: l i near
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
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GGCATTCATG CGGCCGCGGC 20
(Z) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GCAAATCTGC GGCCGCCTCT C 21
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Ala Ala Ala Ile Glu Gly Arg
1 5
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Ser Ala Ala Ile Glu Gly Arg
1 5
~ (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
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GACMGCTAG AGGMGTACT TAGCMGCTC TACCATGTCG AGMCGMCT TGCTCGAGTT 60
MGMG 66
(2) INFORMATION FOR SEQ ID NO: 23:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS: s i ngl e
(D) TOPOLOGY: linear
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
GGCATTCATG CGGCCGCMT GMGCMGTG GMGACMGC TAGAGGMGT AC 52
(2) INFORMATION FOR SEQ ID NO: 24:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS: s i ngl e
( D ) TOPOLOGY: 1 i nea r
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GCAMTCTGC GGCCGACTCT CCCAGMGCT TCTTMCTCG AGCMGTTC 49
(2) INFORMATION FOR SEQ ID NO: 25:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 ami no aci ds
( B ) TYPE: ami no aci d
( C ) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Ala Ala Ala Gly Gly Gly Gly Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 26:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 ami no aci ds
(B) TYPE: amino acid
( C ) STRANDEDNESS:
( D ) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Ala Ala Ala Pro Leu Gly Leu Trp Ala
1 5
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GCA M TCTGC GGCCGCACCT TTGGGACIII GGGCAATGGC AGAGAGCCCC CATC 54
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
GCA M TCTGC GGCCGCAGGT GGAGGCGGTT C M TGGCAGA GAGCCCCCAT C 51
(2~ INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
CCGGTACCGG CCCAGCCGGC ~I~IICIlCl CGTACCCCG 39
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
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M GTCTTAGC GGCCGCCAGA GCGATGATAC CG MG 35
(2) INFORMATION FOR SEQ ID NO: 31:
(i~ SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
CCGGTACCGG CCCAGCCGGC CGGTGATCAG M TCCTCMM TTGC 44
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sin~le
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
M GTCTTAGC GGCCGCGAGT TTGAGT MGC C MMGG 36
