Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W095/31544 21899~ r~l,n~ 4
MODULATOR OF TNF/NGF SUPl~RFAMILY RECEPTORS AND SOLUBLE
OLIGOMleRIC TNF/NGF SUPERFAMILY RECEPTORS
.
Field of the Invention
The pres~nt in~ention is generally in the field of receptors belonging to the TNF~NGF
15 superfatnily of receptors and the control of their biological fimctions. The l~F,~NGF superfamil~
of receprors includes receptors such 2s the p5s and p75 tunnor necrosis factor receptors (l~'F-Rs)
and the FAS li~2nd rec~ptor (alsD called FAS/APO1 or FAS-R and her~inafter will be called FAS-
R) and others. More sp~cifically, the present invention concerns novel proleins ~hich bind to the
intracellular domains (IC) ofthe pS5 and p75 TNF-Rs and the Fas-R~ (these intracellular domains
20 designated p55IC, p751C and Fas-IC, ~,pc~u~ ) and which no~el proteins are capable of
modulating the function of th~ p55 and p75 TNF-Rs and the Fas-R. One of the proteins capab~e
of bindins the p551C of the iMact pS5-TNF-R is the p551C its~f in the form of a p551C molecule
or a portion thereo such as for exar~ple. the so-ealied 'd~ath domain' {DD) of the p55IC. Thus,
the present invention also wneerns ne~ INF-associated effects that c2n be indueed in eells in a
25 lig2nd (lNF) lJ~ i fashion by the h.. ~ domain of the p55 TNF-R (p55IC) or
portions thereof. The preseM invention also concerns the preparation and uses of th~se noYel p55
and p75 lNF-R-bindin proteins, and Fas-R birlding proteins~ referred to herein as p55IC-~
p75IC- and Fas-IC- binting proteins.
In another aspect~ the present inv~ntion a~so eoncerns new soluble oligomeric TNF-Rs~
30 oligomeric FAS-Rs and oligomerie receptors haYin~ a tnixture of TNF-Rs and ~AS-Rs, their uses,
and nnethods for the produetion thereof
r ~ ~ of - T~v~ntion ~ ' Prior.~rt
Tumor Necrosis Factor ~TNF~) and Ly~ Luf.ill (l'NF-O (hereina*~r~ l'NF~ refers to
both TNF- and TNF-O are ~ pro-- '` y c-ytokines formed mainly by
~/~.. ,.. l A- y~ which have many effeets on cells (Wallaeh, D. (1986) in: Interferon 7
(lon Gresser~ ed.), pp. 83-1~2, Acadennic Press~ London; and Beutler and Cerami (1987)). Both
TNF-o ant TNF~ irlitiate their eects by binting to speeifc cell su~face receptors. Some of the
effccts are li;;ely to be beneficial to the organism: they may destroy~ for exarnple tumor cells or
Yirus infected eells and augrnent ' i41 activities of ~ 4....to~y~5. In this way, TN~
40 contributes to the defense of the oruanis~n against tumors and infectious ageMs and contributes to
the recover~U from in,p;rS. Thus. l~lF ean be used as an anti-tumor agent iA ~Yhich application it
binds to its receptors on the surfaee of tumor eells and thereby initiates the events ~eating to the
teath of the tumor eells. TNF can a~so be used as an arlti-infectious agent.
_ _ _ _ _ _ _ _ _ . _ . . _ _ _
W095/~1544 2 1 8q~ 83 ~ 4
However, botr. Il~F-a asld TNF-,G also have deleterious effects. There is evidence that
ov-producion of T~s-a can pla} a major pathogenic role in several diseases. Thus, effeets of
TNF-a~ prima~ on tr- vaseulature, are nov~ kno~vn to be a major eause }or symptoms of septi~
shock (Tracey et al, 1-36~. In some diseases, TI`,F may caus~ excessive loss of weight (cachexia)
'i by suppressing acti~ ec of sdipocytes and boy eausing anorexia, and T'NF-o vYas thus cal~ed
eaehetin. It was also ..escribed as a mediator of the dama~e to tissues in rheumatic diseases
~Beutler and Cerami, l?S7) and as a major mediator cf the damage obser~ed in graft-versus-host
reactions (Piquet et a'... 1987). In addition. TNF is known to be involved in the process of
' '` ' and in mæny other diseeses.
Two distinct, i tA~ A. .Ill,y expressed, receptors, the p55 and p75 T~F-Rs, which bind
both TNF-a and T~ G specificali~, irlitiate andlor mediate the aboie noted biolooical effects of
se rwo recep-ors have structurally dissimilar ;..l.ic~ltulal domains suggesting ~hat they
signal difrerently ~See Hohmann et al., 1989: Engelmann et al., 1990~ I3roelhaus et al., 1990:
Leotscher el al., 199~. Schall et al., 1990; Nophar et al., 199G, Smith OE Gl., 1990; and EIeller el
15 al.. 1990). ~owe~ er, tile cellular, ~ , for example, the various proteins and possibly other
fae~ors, which are in~ei~ed in the irltraccllular signalul~ ofthe pSS M p75 TNTF-Rs have yet to be
elucidated (as set fort}. herein below. there i5 deseribed for the first bme, ne~ proteins capable of
bindin~ to the p?SlC an~ pS5 IC). It is this; ,..o.~,~,llul~ signaiing, which occurs usuaily after the
binding of the ligand, i.e. TNF (a or O, to the receptor, that is responsible for the cv , ,
20 of the caseade of reaetions that ultirnatelS resul~ in the observed response of the cell to TNF.
~ s regards the above mentioned cytocidal effect of TNF, in most eells studied so far, this
effect is triggered mainly by the pS5 TNF-R. Antibodies against the C~LtGC~ UIa~ domain (ligarld
binding domain) of the pS5 ~NF-R ean themselves trig~er the eytocidal effect ~sec s--:P 41~486)
which correlates with the effectivih~ of receptor cross-linking by the antibodies. believed to be the
25 f~rst step in the genera~ion of ~he IllllaCC~t~al signaling process. Further, m~ational sr~!u;ies
~Rr~l~rh~ h et al., 1992 Tartagiia et al., 19g3) have shown that the biological funcbon of the
pSS TNF-R depends on the irltegrih~ of its ;..L.acc:lul~. domain, and ~t ~,O.U; ~ly it has been
suggested that the initia~ion of ;u.. ~lluu~ signaling leaditlg to the eytoeidai effect of Tl~F occurs
~s a .~ of the assoei~tion of h~o or more; " ' domains of the pS5 l~lF-R.
30 l~oreover, TNF ~a and O occurs as a hl ard as such has been suggested to induce
intraeellular si nali~ ~.ia the pS5 TNF-R by wa~, of its abilihy to bind to and to eross-link the
receptor rnolecules, i e. eause reeeptor r.~ OA;U~ Herein below there is described how the
pSSlC and p55DD can self-associate and induce~ in a ligand-; ~ Fashon, TNF-associated
effects in ceils.
35 Another member of the INF/NGF aul~.. L~llv of receptors is the FAS recepto~ (FAS-R)
which has also been called the Fas arltigen, a ceii-surfaee protein expr~ss~d in various tissues and
sharing homology with a number 3f c~il-surfac,e receptors including TNF-R and NGF-R. Th~
FAS-R mediates edl death in the form of apûptosis (Itoh et al., 1,91), and appears to serve as a
neyative seleetor of ~uLU~ ve T ceiis, i.e. durirlg maturation of T eeiis, FAS-R mediates the
40 apoptopic death of T cells J~U~Y~ self-antigens. It has also been found that mutations in the
WO95/31544 21 ~t9 83 r~ r.`0!~o~4
3
FAS-R gene (/pr~ cause a l~ oplvllf~ iu~ disord ,n mice that resembles the i-uman
-- disease sy,stcmic lupus ~ v,~ SLE) (W l -Fukuna~a et al.. 199 ). The
ligand for the FAS-R appears to be a cell-surface associated molecule carried b~, amongst others,
killer T cells (or cytotoxic T Iymphocyt~s - CTLs). and henc~ vJhen such CTLs conract cells
S carrying FAS-R they are capab~e of inducing apoptopic cell death of the FAS-R-carrying ceDs.
Further, a monoclonal antibody has ~een prepar~d that is specific for FAS-R. this monoc~onal
anibodv beir g capable of inducrng apoptopic ce~l death in cells car~}ing FAS-R, includin.~ mouse
cells 1- FL ' by cDNA encoding human FAS-R (Itoh et al., 1991).
It has also been found that various other normal cells, besides T IVI~ V~ S~ express the
FAS-R on their surface and can bc killed b~ the triggerinO ofthis receptor Ul.~ul.~lull~d induction
of such a killing process is suspected ~o conrribute to ussue dama~e in certain diseases, for
examp~e, the destruction of liver cells in acute hepaitis. ~ccordinsly. finding w~ys to restrain the
cytoto~ic acti~,ity of ~AS-R may have therapeutic potential.
Conversely, since it l~as also been found that certain malienant cells and ~I~i-infected cells
1~ carry the FAS-R on their surface, antibodies ag~inst FAS-R or the FAS-R ligzqnd. may be used to
triggcr the F~S-R nnediated cytotoxic effects in these and t~hereby provide a means for combating
such malisnant cells or E~IV-infected cells (see Itoh et al., l,91). Findm~ ~et other wavs for
cnhancing the cytotoxjc activity of FAS-R may therefore also have therapeutic poteMial.
It has been a long felt need to pro~ide a way for modulating the cellular response to TNF (
a or ~) and FAS-R ligand, for e~an~ple, in ~ lo~ ,! Situa~ions as meMioned above. uhere
T~iF or F~S-R ligand is over-expressed it is desirable to inhibit the TNF- or FAS-R livand-
intucet cytocidt~l effects~ while in other situations~ c.g. wound healing ,.~ it iS desirable
to enhance the ~ effect, or in the case of FAS-R in tumor cells or HlV-infected cells it is
tesirable to enhance thc FAS-R mediated effect.
A number of approaches have been made by the present inYentors (see for example,European Application Nos. EP 186833, EP 308378, EP 398327 and EP 412486) to re~,ulate the
deleerious effects of TNF b~ inhibiting the binding of TNF to its receptors usinr~ anti-~NF
~nthodies or by using soluble ~NF receptors (being essentiaDy the soluble e,u~ h. domains
of the receptors~ to compete u~ith the binding of TNF to ~he cdl surface-bound IN~-Rs. Further,
on the basis thtLt INF-binding to its receptors is required for the TNF-induced cellular effetts.
~pproaches by the present ir~ventors (see for example EP0 ;6g9~) h2v.e been made to modulat~
the T~ eff~ct by modulating the acti~ity of the ~-Rs. Briefly, EPO 568g25 relates to a
method of modolatirlg si~nal tl l,J ~ and/or cleavage in ~ Rs wherebv peptides or other
molecules may in~eract either with the receptor itself or uith effector proteins interactinv with the
3~ receptor~ thus modulating the normal functioning of the INF-Rs IrL EPO 568925 there is
described the CV~ - C~.V.l and Cll~ ~Ch~ 01~ o~ ~ atious mutant pSS I~F-~s~ havin~ mutations
in the ~ , and ' acd~ul.u dornains of the pS5 I~-R. In this way
~ions ~vithiD the above domains of the pSS INF-R wer~ ideMified as bcing essential to the
functioning of the receptor, i.e. the bir,ding of the ligand (TNI~) and the subsequent signal
' ~ . and i " l~ sigrlaling which ultimatdy results in the observe~ ect on the
wo95131544 21 899 83 r~ n.. . 4
cells Further, there is also described a number of approaches to isolate an~ id~nri~ proteins~
peptides or other factors which are capable of binùin~ to the various regions ir. the above dornains
of the TNF-R which proteins~ peptides and other factors may be invoi~ed in regulating or
modulating the activ;ty of the TNF-R A number of approaches for isolating ar.d cloDinr~ the D~A
sequences encodirg such proteins and pepùd~s; for cv~uu~,L;~ expression ~ec~ors for the
production of these proteins and peptides: and for the preparation of antibodies or fragtn~Ms
thereof w}lich interact with the TNF-R or with the above proteins and peptides that bind vanous
regions of the TNF-R, are also set forth in EP0 568g~5. However. no description is rnade in EP0
~689, 5 of the actua5 proteins and peptides ~hich bind to the ;...- a~,~ 5U~al domains of the TNF-Rs
1~ (e g pSS TNF-R), nor is anv description made of the yeast two-hybrid approach to isolat~ and
identif~ such proteins or peptides ~hich bind to the intracellular domains of TNF-Rs Similar~,
heretofor~ there has been no disclosure of proteins or peptides capable of binding the intracellular
dornairl of FAS-R
Thus, v.~hen it is desired to inhibit the effect of T!~F~ or the FAS-R iigand~ it would b~
desirable so decrease the zmount or the activity of TNF-Rs or FAS-R at the cell surface. while
incre~se in the arnoum or the activity of INF-Rs or FAS-R would be desired ~vhen an enhanced
TNF or F.~S-R li~and effect is sou~ht To this end the promoters of both th~ r ~ ~ ~NF-R and the
p75 T~F-R have rec~ntl~ been sequenced and analyzed b- the present invenrors and a nu~nber Of
ke~ sequence motifs have been found that are specific to various ~la~ i,u~;oll re~ulating factors,
~Q and as such the expression of these TNF-Rs can be contro~led at th~ir promoter level, i e
inhibition of `, ` from the promoters for a decreasc in the number of receptors, and an
of l. a 7 ~ from the promoters for an increase irl the number of receptors (,seeIL 104355 and 11, 109033 and their cu,l~ r- '` ~, as yet,, ' ' ' - ' FP and PCT
, 1~) Cu.~ ul~d;l~ studies concerning the control of FAS-R at the level of the
2j promotet of the FAS-R gene have yet to be reported.
Further~ it should aiso be menuoned that~ ~hile it is i'~no~vn that the turnor necrosis factor
' ~TNF) receptors, and the structurally-rdated receptor FAS-R, tri~ger in cells, upon stimulation bv
leul~-ocyte-prûduced ligands. destruaive activities that lead to their own dernise, the ~,n. ~
ûf this triggerinY are stiil httie understood. Mutatiûnal studies indicate that in FAS-~ and the pSS
30 'rNF receptor (pSS-R) signaling fûr ~iy~ .id~ty involve distinct regions within their intrar,~ r
domains ~ hll~rh et al., 199~, Tarta~lia et ai., l9g3, Itoh and Nagata. 19g3) These regions
(the 'dcath dornains') have sequence sir~ilarity. The 'death domairls' of both FAS-R and pS5-R
tend to sdf-associate Tneir ~c. r, -rori~ti~n âpparently promotes that receptor a~ eY,ad,ull which
is necessat~ for irlitiation of signaiing (as SQ forth herein below, as well as Song et al., 1991
35 Walla~h. et al, 1994. Boldin Q al., ~995) and at high levels of receptor expression can result in
triggering of ~ie ~ signaling (as set forth hereir~ bdow. and Boldin et al, 199
Thus, prior to the present invention~ there havç not been provided proteins w-hich may
reguiate the effect of ligands belonging to the TNF/NGF superfi~m~ly, such as the TNF or FAS-R
ligarld effect on cells, by mediation of the iuual cllulal signaling process, which signalin~ is
40 probAbly go~erned ~o ~ large extent by the; A- ~ r domains (ICs) of th~ receptors belonging
WO95131544 2 1 89q 83 , J"J~ 4
to the l'N.F~N&F superfarnily of receptors, such as thosc of the lNF-Rs. i e. the p55 and p7c
I~F-R ' " ' domains (pSSIC and p75IC. .c,~ 'y)) as well as the FAS-IC.
Accordingly, it is one aim of the in~eMion to provide proteins ~h:ch are capable of
binding to the i...~ e'lul~ domains of the TNF-Rs and FA~j-iR. which prcteins are presentl~
c believed to be involved in the u~ lluL l signaiing process initizted by the bindiny of TNF to its
receptors, or the bindr~ of F~S ligand tO its receptor.
Another aim of the invention is to pro~ide antagonists (e.g. antibodies) to these
intraceilular domain-binding proteins (IC-binding proteins) which nnay bt used to inhibit the
siynaiing process, when desired, when such IC-binding proteins are positive si~nal effectors ~I.e.
1~ induce signaling), or to enhance the signaling process, ~hen desired. ~hen such IC-bindingz
proteins arc ner~ativ,e signai effectors (i.e. inhibit signaling)
S'et another aim of ehe in~ention is to use such IC-bindiny pro~eins to i~olate and
r~, > - additionai proteins or fac~ors, which rnay, for example, be involved funher
d~ L.e~l in the sigmaiing process, andlor ~o isolate and iden~ify othe: receptors further
I~ upstream in the signaiing process ~o which these IC-binding proteins bind (~ . other l~rF-Rs or
reiated receptors), and hence, in ~hose t^unction the lC-binding proteins rlre ziso in~olved.
Moreover, i1 is zn aim of the present invention to use the abvve-men~ioned IC-bindina
proteins as anti~ens for the prcparation of polyclonzl and/or rnr noclo"~l antibodies there~o. The
r~ntibodies~ in turrl, may be used for the purification of the ne v IC-binding proteins from different
ZG sources, such as celi exkacts or l~all~ i cdl lines.
r.-lh. 1.,.~ , these antibodies may be used for diagnostic purposes. e.g. for identifying
disorders rdated to abnormal functiorling of cellular effects mediated by receptors belongin2 10
the TNF/NGF receptor - ~.p~
A further atrn of the inveMion ;s to provide ~ c~ ,l 05;1iv .~ comprisin2 the
2~ ~bove IC-binding proteins, and ~ ,1 C<J~ D;~ comprising ~he ~C-bindin~r protein
for th~ keatment or prophylaxis of TNF-induced or ~AS lizand-induced conditions,for example, such c - ,~ can be used to enhance the TIYF or FAS li~and effect or to inhibit
the TNF or FA~i ligand elfect depending on the above noted rlature of the IC-binding protein or
antagonist thereof contained in th~ c~
Moreover, in accordance with another aim of the present invention~ there is disclosed
other vays for eliminating or ....l~ forrned or e~c~u~onr~ y a~l.u.fl~ d
TNF or FAS-R ligand, by the use of soluble oligomeric TN.F-Rs, o~igomeric FAS-Rs, or
oli~omers being a mixture of TNF-i~s and FAS-Rs. ~n this respect it shouid be mentioned that one
attempt in titis direction was the isolation and 1 ~ ' ' production of a ~ Binding Protein
35 cailed TBP-~ ~hich was shown to be able to antagoni~e the effeets of TNF. This ,, ' was
determined both by measuring reduction of the cyto~oxic activity of TNF, as wdl as by measurin~
of IN.F binding to its receptors (EP 308 378). TEIP-I w~s shown to protect cells from
TNF toxicity at c. - ~ of a few nanograms per ml and to interfere ~ith the binding of
both TNF~ and TNi~-~ to ccils, when applied ' ' '~ with these cytoicines. Ft~rther
' '' of the meeha~ism b~ which TBP-I functions reveaied that TBP-I does not interact
WO95131544 2 1 89q83 r~ . 4
~ith the targct ceii, but rather blocks the funcrion of TNF by binding TN~ specificaily. thus
competing for l~iF with the ~NF r~ceptor.
r~ y~ ~vith a different purrficaion technique~ the preser.ce of two active
1'/~`' C was ~ound one, TBP-I, and also a second TNF-binding proteu~ ich we calied
TBP-II (first described in EP 398327). Both proteins provide pro~ection agaillst the in vitr~
c~tocidrl effect of TNF arld both bind TNF-~3 less effectively than TNF~ Aithough in Si~S
PAGE anaiysis the two proleirls, TBP-I and TBP-II, appeared to bave a ~ er,v similar molecular
size. they could clearly be ~" ~ ' ' from each other by lack of ;~ cross reactivity,
differin~ N-terrninai amino acid sequences Md differing amino acid ~ C,;L;Oll.
However. the above noted earlier soluble TNF binding proteins are monorneric and being
capabie of bindin~ oniy one monomer of thc INF l1U~ ;CI~ th~ natura! li~and, which still
permits ll~F activit~ (i.e. incomplete n~ ) b~ virtue of the ~NF stil; having two active
monomers unbound by the l~-F binding proteins. Further, heretofore ~her~ has been no disc~osure
of soluble FAS~ s ~solub~e FAS-R ii~and binding proteins) capable of bindina tc F.~S-R ligand
lS which is icnown to be a ~-v ~ ;., .., ceil-surface associated moiecule.
A so-cailed 'death domain' of the pS5-1C (Tartagiia et al., 1993) has been disclosed, but
did not show, in accordance with the present inv~ntion, that the pS5-IC Md the 'death domain'
ti~ereof seif-associat~s. this self-association being primarily responsible for ~he sisnaiing leadins ro
irlduc~iorl of ceii cytotoxis. Moreover. this publication is silent on the possibilit~ of producing the
20 soiuble. oli~omeric TNF-Rs, or the soluble, oiigomeric Fas-Rs. or mixed oli~omeric thereo nor
does it disclose other lNF - ' effects induced b~ tbe p55-IC or portions thereof, e.g. IL-8
8ene e~prsion induction, aii of the present in~ention. Likewise, another p~h~ tinn, publishcd
after the date of the present invention, disclosed the ~ i.e. self-association) ability of
the pS5-IC, but did not reiate, as rloted above, to the usage thereof to prepare soluble, oligomeric
25 TNF-Rs or Fas-Rs nor to the other INF-associated effects induced in a liga.~ d~
m~miter by the p55-lC or poTtions thereof according to the inYention
Summilrv of the T
In accordance with the present inYerltion, we haYe found novel proteins u~hich are capable
of binding to either the i " ` domain of the pS5 TNF-R ~the p55IC-bindin~ proteins), of
30 the p75 T~iF-R (the p751C-binding proteins), arid of the FAS-R (the FAS-IC-binding proteins).
These p551C-, p751C- and FAS-IC- birlding proteins may act as mediators or modulators of thc
lNF or FA~i-R ii~and ei~fect on cells by way of mediating or moduiating the i.,~ il~ signaiin~
process which usu2ily occurs following the binding of TNF to the pS5 andlor p75 TNF-~, or the
binding of the FAS-R ligand at the ceii surfilce. Further, it has been ~,,",. ;~ 2nd ~ ly
35 found that tile pSSlC and FAS-IC ar~ capabl~ of self association and that fra ,ments of the p551C
and ~AS-IC are siniiariy capable of binding to the pS5 IC, particularly the so-ca~led 'death
domsins (DD) withirl the ICs of these receptcrs, i.e. tile p55Di} and F~S-DD. Thus, p55 IC and
FAS-IC and their fragments aiso repreSerlt proteins capabl~ of binding to the p55TC and FAS-IC
2n~i hence may be moduiators of th~ l~TF or FAS-R iigand effect on cel~s.
.. . .. . ..
W0 9~/31544 2 1 8 q 9 8 3 i ~".,~ - ~
7
Fu~ , the nature of the binding of one of the novel proteins of the inveMion. the
herein designated 55.11 proui4 to the ;~ Ctllul~ don~tin of p~5-~NF-R has been more full~
elucidated (see E~ample l).
Moreover. in another aspect, the present invention is based on the finding that the
5 intraceiluiar domain of the pS5 TNF receptor (p55-IC), a region contained ther~in, thc so-called
p~5-lC 'death domain'. the ;, l r~. .-1!~,1,. dor~ain of the FaslAP01 receptor IFas-lC), and a re$ir n
corltaioed therein, the so-caDed Fas-lC 'death domain' are capable of self-associaùon.
Accordinc~ly, it is possible to construct by standard .C.,.,~ l~lL DNA techniques, a soluble,
olii7,omeric TNF recep~or beins a fusion product, containing at Ieast tuo ~t~scc"ul~ domains of
10 a TNF receptor al its one end, and at its other end dt least two of th~ abo~ e noted self-associatin~
iouacellular dornains or pomons thereof, ~vhich self-associate to provide an oligomer having at
[east two such fusion products linked together. Such a soluble, oligomeric T~F-R is thus capable
of binding two monomers of the naturally-occurrinL7 lhF hv~ ... r and as such eff~ctivel~
neutralizes T~ activity. The r.~ul.dli~.";u.l of TNF ac~ivity being, desirable in all of the above
5 mentioned conditions wherein TNF is o ~ " vd~l~,ed ~L; "'~"`' j or is d~ J~7~ d ~.ù c.~vusl~
in high doses resulting in undesirable side effects. Further, the effecti~e binding of l~lF b~ the
soluble~ oligomeric receptors of the invention ma~ aiso save ~o al~o.~ for the binding of
w~ug~ vu~l~ added r~ and its subsequent desired slow-release in conditions where TNF is
. ' ~ c,l for its benefcial effects, e.g. in t~tmor therapy. Like~vise. it is aiso possible to
20 construct by stand2rd .~ DNA techniques an oligomeric FAS-R being a fusion product.
cor~taining at least two ~ . domains of a FAS-R at its one end, and at its other end at
leart two of the above noted self-associating i ~. ' ' domains or portions thercof. which self-
arsociate to provide an oligomer havin_ at least twû such fusion products lini~ed tog7ether. Such
an oli~omeric FAS-R is thus capable of binding tuo monomers of the naturally occurring FAS-R
L~ iigand l~vl.lv~ . and as such effectively neutraiizes FAS-R ligand activit~ The D ,.~a~ sf
FAS-R li~2nd activity beinL7 desirable in ail of the 2bo~e mentioned conditions where excess
amounts thereof are associzted vith undesirable side effects. In a similar fashion, and in view of
recent reports indicating a possible associains~ between rNF aDd FAS-R li~and-induced effects
on cells and hence also a possible associ2tion, g.,v l., ' 'I~ at Ihe cell surface where they attacb
30 to thoir receptors, it is aiso possib3e to construct by standard r~n ~ .l DNA techniques a
mrxed oli$omeric r~ceptor having specificity for both I~F and FAS-R ligand. Such 2 mixed
oligomer would be a mixture of th~ above nûted fusion products containin~ at least one
domain of a TNF-R and at least one e, ll .' ' domain of a FAS-R at its one end,
and at its other end at least two of the above mentioned self-associating intracellular domains or
35 portions theof, which s~if ~Qnci~t~ to provide a rnixed oligomer having at least two .such fusion
products iinl;ed toszetber. Such a mixed oli~omer is ti us capabie of bindinl7, at least one monomer
of INF and one monomer of F.4~S-~ ligand at the same time, thereb~, reducing or effectively
DeutraliZjng the INF and FAS-R ligand activities at the ceil surface in conditiorls, as noted abov.e
where excess amounts of these t~vo cytokines are associated uith, ' ' ' ceDular effects. As
40 noted above, the FAS-~ ii$and is usuaDy cel' . ~ associ~ted, arld recent reports aiso describe
Wo95131544 2 1 ~9 ~ ~3 ~ C~o~
ce31-surface-associat~d forms of TNF. ~ence, these mixed n~F-RtFAS-R oligomers a~e especia31
useful for ~ of TMF and FAS-R 3igand activities at the cell surface.
Accordin~]y, the present invention pro-ides a DNA sequence encodin~- a protein capable
of binding to one or more of thc i,~ .',.l~ domains of one or more receptors be30ngin~ to th~
5 tumor necrosis Lc~v.l.._. ve growth factor (INF~ GF~ superfamily of reCeF~rs
In particular, the present inventiorl provides z DNA sequence se3ected from the ~roup
consistin~ of: .
(a) a cDNA sequence derived from the coding region of a native TNF-R ;.-h~ hll~ domain-
binding proteirl;
10 (b) Dl~A sequences capable of ll~v~i.li~l;v,~ to a D~A of (a) under moderately strin~ent
condilions and which erlcode a biolo~ically active TlsTF-R ;ntraccllular domain-binding
protein, and
(c) DNA se~uences which are degenerate as a result of the generic code to the DNA
sequences defined in (a) and ~b) and which encode . a biolo~cal3-~ active TNF-R
;~I~IG~CI ~1~= domain-bindin~ protein.
The preseM inventioD also provides a D~'A sequence selected from the ~roup consistin~
of:
~a) a cDNA se~uence derived from the codin~ region of a native FAS-R ;"~ A~ domain-
binding proteu~;
20 (b) DNA sequences capable of ~.,,b.i li,,GIivn to G cDNA of (a) under moderate3y sv-ingenl
conditions atld which encode a biologica31y active FAS-R intracellular domain-bindiny
protein; and
(c) Dl~A sequences which are degenerate as a result of the genetic code to t31e DNA
sequences def ned in (a~ and (b~ and v~-hich encodc a biolosically active FAS-R
'11 'a domain binding protein.
In . ~ " of the presens irlvention the DNA sequences enc~de p5j TMF-R p75
T."IF-R and FAS-R irltracellular d ' " " proteins, such as those encoding the herein
designMed proteins 55.i, 55.3~ 55.11, 75.3, 75 16, F2, Fg and D~l l.
The present invention also provides a protein or analogs or derivasives ~hereof encoded by
any of the above se~uenc~s of the invension, said proteins. analo~s and derivatives being capable
of binding to one or n~ore of the " ' domains of one or more rNF-Rs or FAS-R
F.. ,~.V~l:.. ~t~ of this tspect of the invention inc~ude the herein designated proteins ~5.1, 55.3,
55.11, 75.3, 75.16, F2, F9 and DDI 1, their analogs and their deri~atives.
Also provided by the present invention are vectors encoding the above proteins of the
35 invention, which contain the above DNA sequenCeS of the inv~nsion, these ~ectors being capable
of being expressed in suitable alkaryotic o~ p.u,k~ot;c host cells; ~.al.~rv~ eukaryotic or
prolcaryotic host cells containing such vectors~ and a method for producing the proteins, analogs
or derivatrves of the invention by grow~ng such ;~ rv~ d host cd}s under condiions suitable
for the expression of said protein, analogs or derivatives, effectin~ post-~ ~ h~ v~ s
40 of said protein as necesstrv for obtention of said protein and extractinr said expressed protein
WO9S/31544 2 1 8 9 9 8 3 ~ S,!:.,.,4
9
2nalogs or derivatives from the culture medium of said ,~,,r~ cells or f~m c~ll extracts of
said trarlsformed ceils.
In another aspect. the presen~ inv~ntion a~so proT~ides antibodies or active denvatives or
fragme~ts thereof specific to the proteins, analogs 2nd derivatives thereo of the inv~ntion.
By yet another aspect of the invention, there are provided various uses of the above DNA
sequeDces or the proteins ~hich they encode. acco~ding to thc invention, whch uses includc
amongst others:
(i) a method for th~ modulation of the T~F or FAS-R ligand e~ect on cells carryin~ a
TNF-R or a FAS-R, comprising treating said cells with one or more proteir~s, analogs or
derivatives selected from the group consisting of the proteins, analo~s and derivatives.
accordins to the invention, and a protein being the pSSIC, p55DD. ~AS-IC or F.4.S-DD,
aualo6s or derivatives thereof, all of said proteins being capable of bindin~ to the
i...,acell.ll~L domain and modulatin~ the activity of said I~ R or FAS-R wherein said
treating of the cells comprises introducing into said cells said one or more proteins
IS analogs or derivati~es in a form suitable for i~lla~e~L~l~ adLl.. ll~llaLLo.l or introducing into
said cdls, in the forln of a suitable expression vector, the l:)NA sequence encodin~ said
one or more proteins, analo~s or derivatives;
(ii) 2 method for modulating the Tl~TF or FAS-R ligand effect on cells carrying a TNF-
R or a FAS-R comprising treating said cells with antibodies or aCIive derivatives or
fragments thereof according to the in~ention;
(iu) a mcthod for modulating the TNF or FAS-R 5gand eff~ct on cdls caT~viny a ~-
R or FAS-R comprising treating said celis with an .~ se~uence encoding an
antisens~ sequence of at least part of th~ sequence according to the invention, or
encoding an antisense sequence of the p55IC, p55DD. FAS-IC, or FAS-D~ sequence.
said ~ 5" ' ' sequence being capable of blocicing the e~pression of at least one of
the INF-R or FAS-R i " ' domsin binding proteins;
v) a mhod for modulating the TNF or FAS-R li~and ef~`ect on cells carryiny 2 TN~-
R or FAS-R comprising:
(a) UU.~.lU~Iillg a ~ animal virus vector carryiny a sequence encoding a viral
surface protein that is capable of binding to a specif c cell surf2ce receptor and a sequence
selected from an ~ 'iL ~c~ ' sequence encoding an antisense s~quence of at least pa~t
of the sequence according to the invention and an c~ ( ' sequence cncoding an
antisense sequence of the p55~C, p55DD, F.iS-lC~ or FAS-DD sequence, said
' _ ' ~ ' sequence beino capable of b~ocicing the expression of at least one of the
~;iF-R or FAS-R il~t~acel~ulal domain binding proteins ~hen introduced into said cdls by
said virus: and
~b) infecting said cells with ssid vector of (a).
(v) a methot for modulâting the ~'F or FAS-R 1igand effect on cells carr~ing a TNF-
R or a FAS-R, comprising treating said cells ~vith a suitable vector encoding a ribozyme
~zving z sequence specif~c to a sequence selccted from zn mRNA se~uence encoding a
W095/31544 2 1 8 9~ 83 ~ U~ -- 4
proteir" analog or deriva~ive of Ihe invemion and an mRNA sequenc~ encoding the p5 SIC~
p55DD, FAS-IC or FAS-DD, said riboz,vme sequence capable of interactin~ with said
mRNA sequence and capable of cleaving said mR~A sequence resullinc in the irhibition
of the expression of the protein, analog or dcri~ative of the invention or of the expression
of the p~51C, p55DD, FAS-IC or FAS-DD.
(V1 a method for treating tumor cells or E~V-infect~d cells, or orher diseased cells,
comprising
(a) co.L.i,u~.Li ~ a .c~,..~;..c,,. animal ~irus vector carryin~ a sequence encodiny a
~iral surface protein that is capable of binding to a rumor cell surface receptor or ~
infected cell surfac~ receptor or is capable of bindin_ to anotha cell surface receptor of
other diseased cells and a sequence select2d from a sequence accordin~ to the invention
encoding a protein, analog or derivatii!e of the invention and a sequenc~ encoding the
p551C, pSSDD~ FAS-IC, FAS-DD, or ~ biologically active analo~ or derh,-ative thereof.
said protein, analog or deriva~ive of the invention, pS5IC, pS~DD, FAS-IC. FAS-DD,
ana~og or derivative, when e~pressed in said rumo~ cell or ~I~'-infected cell, or other
diseased cell being capable of l~lling said cell; and
(b) infecting said tumor cells or E~-infecred cells or other infec~ed cells with said vec~o~
of (a).
(vii) a method for isolating and ideMifyinz proteins, factors or receptors capable of
bindirlg to the ~ ~ul~ domain bindi3g proteins according to the invention, compnsing
applying the procedure of affiniry ~,L,,.,.-~ .,hJ in which said protein according to the
invention is attached to the afrinity ~ J matrix~ said attached protein is
brought into comact with a cell extract and protans, factors or receptors from cell extract
whjch bound to said atlached protein are then eluted, isolared analyzed;
(Yii') a method for isolating and identifyin~ proteins, capable of binding ro th~
" ' domain binting proteins according to the invention, comprisin~ applying thc
yeast two-hybrid procedure in ~vhich a sequence encoding said ~ domain
bLnding proteir~ is carried by one hybrid vector and a sequence from a c~NA or genornic
DNA library is carried by the second hybrid v,ector, the vectors then bein~ used to
transform yeast host cells and the positiv~ i ~ ' cdls beiny isolated, followed by
extraction of the raid second hybrid vector to obtain a sequence encodinr~ a protein wbich
binds to said ...~dlu14, domain binding protein; 4nd
(ix) a method for isolatin~ and iderltifying a protein capable of bindin~ to the
i..LI41,CllU14, domains of TNF-Rs or FAS-R comprising applying the procedure of non-
srringent southem ;lJb~id; ~liol~ follo~ved by ~CR cloning, in ~-hich a sequence or parts
thereof according to thc inYention is used as a probe to bind sequences from a cDNA or
genornic DNA library, having at least partial homology thereto, ssid bound sequences then
~mplined and cloned by the PCR procedure to yield clones encoding proteins havin~ at
le4st partial homology to said sequences according tO the invention.
WO9~/31544 21 8~1983 rc"u~
I~
The present in~entioll also provjcies a p}~ c~ r~ .o~ n for the modulation of
the l'NF- or FAS b~and- effect on cells comprisir~g, as active ingredient~ any one of the fo~iowin~
: (i) a pro1eirl according to the invention~ or the protein pS51C, pS5DD. FAS-IC or FAS-DD, its
biologically active fragrnents, analogs, derivatives or mix~ires thereof; ~ii) a leuvll~b~ animal
S virus vector encodin~ a virai surface protein capable of birldin~ to a ~'F-~ or FAS-R - carrying
cell - or T~or cdl-specific receptor and a sequence encodin~ a protein, an log or derivative of
the invention or encodirlg the pSSIC~ pSSDD, FAS-IC or FAS-DD; (iii) a lc.uluU;ll~lL animal
virus vector encoding a virai surfaGe protein as in tii) above and an r~ - iro~;lr~ sequence
encoding an antisense sequence of the pSSlC, p55D~, ~AS-lC or F~S-DD sequence; and tiV) a
] o vector encoding a riboz~e of sequence capaGle of iMeracting u jth a mF '~TA sequence encoding a
protein, analog or d~rivatite of the invention or a rnRI~lA sequence encodin~ the p5r~1C, pS5DD.
FAS-IC or FAS-DD.
.~ specific l ml~oriim~nt of tile above aspects of tbe itlvention is the use of the p55-1C or
DNA ~ncoding therefor. This ~mho~iim~nt is based on the discover~ that the p55-lC may in a
lS ligand (~ ir: fasnion induce other ~rF-associated effects in cells. Accordingiy, there
is provided a method for inducins ~NF-associared effccts in cells or tiss~es comprising treating
said cells vith one or more proteins, analogs or derivanves thereof, said one or nnore proteins
being selected from a protein beinsg essentially aii of tite self e ~ori ~ I in- iMracel~ular domain of the
pSS TNF-R (pSS-lC) or portions thereof capabie of sei~-associatin~ and inducin~, in a ligand
20 (INF)'~ t~nl manner, said l~F effect in the cdis~ whein said treatinæ of the ceils
comprises introducing into said cells said one or more proteins, analo~s or deri~atives in a forrn
suitab]e for i..i,~c~liul~ .IILl`O~..~iO" thereof, or introducing into szid celis a DNA sequence
encodin~g said one or more proteins, analogs or derivatives in the forn~ of a suitable vector
carr,ving said sequeitce, said vector being capable of effectin~ the insettion of said sequcnce into
25 said ceiis in a way that said setiueûce is expressed in said cells.
F'..,t.o.';- .~ ofthe abo~e method oftile invention include .
~ l) a method whein said treatiDg of cells is by i '` ' of said cells with a
.~U.~ animai virus vector compnsin.~ the rteps of: ~
(a) c~ , a ,c . ' ' arlimal virus ~ector carr~inæ a sequence encodin a
30 vira~ surface protein (lieand) that is capable of bindiAg to a specific ceil surface receptor on the
surface of said cd~s to be treated, and a second sequence encodinr! 2 protein being the pSS-lC.
portions thereof, analogs and derivatives of 311 of the foregoin2, said protein when e~tpressed in
said cells being capable of self &.~,.laLOll and induction of said one or more TNF-associated
ef~ects; and
3~ (b) infecting said cells with the vector of (a).
~ i) a method wherein said INF effec~ ~o b~ induced in said cells is the induction of IL-8
~ene expresrion, said vector carrying a sequence encoding essentially all of said pS5-~C, portions
thereof~ analor~s and deli~,a~ives of all of the foregoing, which are capable, v~hen expressed in the
cells of s~: ' ' and signalin~ for the induction of said IL 8 gene expression.
wo g~/31544 2 1 ~ 9 9 ~ 3 r~l,u~ i t~O~4
(iji) a method for treatin~ turnor ce~s or viraily-infected ceils, or for au~rnentin~ the
arltib2aerjai effect of granulocytes~ whaein said viral veaor carries a se~quence ~ncoding a vjrai
li~and capable of binding a specific cell surface receptor on the surfac~ of said tumor cells, viral~y-
infecled cells or grarlulocytes and a sequence encoding said pS5-lC porlions ther~of. anaio~s 2nd
deriv.atives ~hereof~ which when expressed in said turnor, viraily-infected or p~ iu.,y~c cells
induces lNF: ' effects leading ~o the death of these ceDs
(rv) a metbod for treating tumor eells, wher~jn said pSS-IC, portions thaeof, analogs or
derjvatives thereof, when e~ipressed in the turnor ceDs, induce the expression of IL-8 which leads
to the i;iiling of said tumor cells by its . h ~ L~ activit~ which attracts ~-allU;Ocy~> znd other
10 1) , ' ~,c.~ to the tumor ceDs resulting in the death of the numor cells.
In this aspect of the inven~ion~ there is thus aiso provided the intracellular domain of the
pSS-R v~55-~C)~ portions. anaiogs and derj~-atives of ail of the aforegoing for use in the treatment
of celis by induction therein of TNF-associated effec~s; and the ~ollowiny ~ G/~ thereof:
(i) the pS'i-lC, portior~s, anaiogs and deri-atives for use in the rreatment of cells b}
I ~ induction therein of rL-8 ~ene expression.
~ i) the p~5-1C~ portions, anaiogs and derivatis,es for use in the treatment of tumo~ cells by
induction thereit~ of ~-8 gene e~ipression resuiting in the iiiliing of the tumor cells.
Moreo~er. in this aspect of the invention thae is pro~ided a r~ nl ~,u~ V~l~lùn
for treating ceDs by induction therein of INF-assoaated effects, rnmptic~ as active in~redient,
'O p55-IC, portions thereof. ana~ors rtnd derivarives of all of ti~e aforesoins, and a ~lla~ a.,cu
ac,ceptable carLier; and the foDowing r .h~ thereof:
(i) a ~ rf ~ L~ for treating cells by induction therein of ~'F-.
effects, comprising, as active ingredient a l~v~u;r~u~ aoimal viruS vector encodinL p55-lC,
portions thereof, analogs and derivatives of all of the aforegoing, aod a protein capable of binding
25 a cell surface protein on the cells to be trcatcd.
(ii) a ~ ' ' cv ~ for the treatm~rLt of tumor cells. adnuuu~l. aLiù~l of said
1 ;. ,. . Ieading to the induction of IL-8 expression, and subsequent killing of the tumor ceils.
As yet aoother aspect, the present invetltiOn pro~,ides a solubte, olir~omeric tumor necrosis
factor receptor ~lNF-R) comprisins at least t~o a~2~faw~ ;a~ fusion proteins, ~ach fusion
30 protein having (a) at its one and, a TNF binding domain selected from the ~ r domain of
tl TNF-R, analogs or derivatives thereof, sait . . l ~ ~. ..tli .~ ~ domain. analogs or derivatives thereof
bein~ incapable of deletious self-assoeiation and bein~ able to bind TNI;: and (b) at its other
end, a ~l' -Cco~ tinE domain selected from (i) ess0tially all of the l . a~ ~11"1 domain of the
p55 T~-R (pSS-lC). e~;tending from about amino acid nsidue 206 to about amino acid residue
3~ 426 of the native piS lNF-R molecule (p55-R), (ii) the death domain of the pS5-IC extending
from about arnino acid residue 328 to about amino acid residue 426 of the native p55-R; (iii)
essenially all of the ;~ e~ dornain of the Fa~POI receptor (Fas-IC); (iv) the death
domain of Fas-lC; and (v) anzloss, frac~ions or deriv2tives of any one of (i)-(iv) bein~ capable of
self-assoeiation, whaein said at least two self-associated proteins self-assoeiate only at said ends
40 (b) having said ends ~a) eapable of bindirl~ to at least t~vo TI`lF monomers. each end (a) capable
.. .. . . . .
WO95131544 2 1 8 9 9 8 3 . ,I/VV ~.or 4
13
of binding one TNF monomer; and salts and functional deriv,ati~es of said so~uble~ oligonncric
INF-R.
r~ of this aspect of the inVentiOn include aii of the abo~ e ~ c of ends
(a) with ends(b) as d~fined abo~e, for example, a solubie, oiigomeric TNF-R comprising as
5 . . ~ , domain, the p55-R CALI a~ l;~ domain and as seif-associating ;nrrP,r~ r domain~
the pS5-lC.
Moreo~-er, there is also provided a proeess for producin,g the soluble oligomerie TNF-~ of
the invention eomprising .
(a) the construction of an expression ector encodil~g any one of said fusion proteins~ the
10 ~NA sequence of each of said ends of the fusion protein being obtained from cloned D~A
sequences encodinLt essentialiy all of said ~ domain of the T~F-R, analoDs or
d~rivatives thereof; and ~`rom cloned l:)~A sequences encoding essentially all of said p'5-lC, p55-
IC death domain, Fas-lC, Fas-lC death domain, arlalogs or derivatives of a~ of the aforegoing,
said ends being li~ated together to form a fusion protein sequenc~, and said fusion proteill
15 s~quence beirl~ inserted into said vector under the control of ~ c~ and Lla~ aliùnâl
regulatorS sequences,
(b) ~..1UVL;VI~ of the vector of (a) into a suitable host cdl in which said fusion protein is
expressed; and
(c) purification of the fusion protein expressed in said host cells, said fusion protein sel
20 associating prior to, durin~, or following the puri~ication process to s~ield a so~uble, o}igomeric
TNF-R
Li~ u~c, there is also proYided a veetor encoding the abo~ e fusion proteins, useful in
the above method of the inYention; host cells containing the Yeetor, as ~eli aS a r~
co-~pc~-;vlcomprisinDthesoluble,o~igomericTNF-RsaltsorFunctionalderivativesthereofand
2~ mixtureS of any of the aforegoing aceordinD to the invention, aS active ingredient, tvgether wie~ a
''-' P~ ' acceptabi~ ear~ier. Sin~iiariy, the soluble. oligonneric T~IF-R Salts, functional
derivatives thereof and mixtures of any of the aforegoing, accordin~ to the inYention. are pro~id~d
for use in ,, '1~ the ddetelious effect of INF in man~nais, especiail~ in the trealment of
eonditions wherein an excess of T~F is forrned ..,ric,g. ~ or is ~.AVD_~VU~l~ a~i"u...~t~"c i, or
30 7~ , for use in maintaining prolonged beneficial effects of I~F in mammâls when used
~Yith lNF eA~ -v.,..~ r ~ ' 'i.
Along the iines set Forth concerr' ng the above aspect of rhe in~ention, it has aiso been
discovered that it is possibie to construct a soluble, oligomeric Fas/APOI receptor (Fas-~) which
is usefui for A ~ u ~g the deleterious efrects of the Fas ii~and Accordingiy, in a further
3~ aspect, the present invention proYides a soiuble. oligomPric Fas/.~POl receptor IF c.-R)
comprising at lezst two ~ Cr,ri~Pd fusion proteins, each fusion protein having (a) u its one
end, a Fas E~and birldin domain seiected from the ~ a.eil~i~ domain of a Fas-R anaiogs or
deri~atives thereofbeing incapable of ,_lf ~ori~-ing~ and being able to bind Fas ligand; and (b) at
its othcr end, a ~.f . ~, domain selected from (i) essentially all of the ;.. "~ domain
40 of the p55 INF-R (pSS IC~, eAte~dir~g ~rom about an~no aeid.residu~ 206 ro a~out amino acit
... .
WO 95131544 2 i ~ ~ ~ 8 3 r~l~o~ 4
14
residue 426 of thc nuive p~S TI~ molecule (pSS-R):. (ii) t~ death domain of rhe pSS-lC
extending from about amino acid residue 328 to about amino acid residue ' 6 of the native p55-
R~ (iii) essenfially all of tbe intracellular domain of the Fas/APOl receptor OEas-icj, ~iv) the
de~th domain of Fas-IC; and (v) anaioys or derivati~es of any one of (i)-(iv) beirlg capabie of self-
arsociation~ wherein said at lease two self-associated proteins only self-associa~e at said ends (b)
having said ends (a) capable of bindiDg to at least two Fas ligand monomers. each end (a) capable
of binding one Fas ligand monom~r; and sails and functionai derivatives of said soluble~
oligomenc Fas-R.
In accordance ~vith thig aspect of the invention~ there is also provided a process for the
o productjon of the solubl~, oiigomeric Fas-R comprisulg:
(a~ the v~ of an expression v~ctor encoding any one of said fusion proteins, theDNA sequence of e~ch of said ends of t~le fusion protein beir~ obtained from cloned DNA
sequences encoding essentiall~ all of said ~ '. .1 dornain of the Fas-R anaiogs or derivatives
thereof, and from cloned DNA sequences encoding essentiall~ all of said pSS-IC, p55-lC death
15 domain, Fas-lC. Fas-IC death domain analoss or derivatives eher~of of ail the aforegoing, said
ends being ligated together to for~n a fusion protein sequence~ and said fusion protein sequence
bein~ insetted into said vector under the control of t r 1 and tr~ t1f~n~l regulatory,
sequences;
(b) ud...,~;v., of the ~ector of (a) into a suitable host cdl in which said fusion protein is
20 expressed; and
(c) purrfication of the fusion protein expressed in the hos~ cel~s, said fusion protein self-
associating prior to, during, or follo~ing the purification process to ~ield a soluble, oligomeric
Fas R.
Moreover, l~o pro~,ided are an expression vector containin~ the fusion protein sequence
25 encoding thc soluble oli~omelic Fas-R useful in the above process; host cdls ~,ontainin~ she
vector; ~nd 1~ co~ v~;l;U~ comprisin~ the soluble. oligomeric Fas-R salts or
functional derivatives thereof or rnixtures of any of the afore~oing as active ingredient so~ether
with a; ' ' 'J~ acceptab~e carrier. Similarl~, there is provided a soluble, o~omeric Fas-
R, salts or functional derivatives theof or r~ixtures of any of the aforegoing, for use in
30 ~ ~ _ the deleterious effect of Fas ligand in rnarnrnais, especially in the treatment of
conditions wherein an excess of the Fas li~and is formed ~ ~v_ u ~ly or is ~".v~ ly
In ~ similar fashion to that noted above concerning the oiigomeric l~NF-Rs and oligomeric
FAS-Rs, it is also possible to prepare mixed oligomers having biDding specificity for both ~F
35 and FAS-R ligand. Thus, the present invention also provides a n~ixed oligomeric TI~F-RIFAS-R
comprising at least t~vo self-associated fusion proteins, one of which fusion proteins is selected
from any one of the above merltioned lNF-specific fusion proteins, and the other fusion protein is
selected from arl~ one of the above menioned FAS~R ligand-speci~ic fusion proteins. to provide a
n~LYed oligorner havirlg at least one TNF-R, " ' domain and at least one F.~S-R
40 ~ h ~ d~main associated by ~irtue of the self-association between the i.". 2. ,cl~l1~ domains
_ _ _ = = _ _ _ . . _ _ _ .
W0 95/3~544 2 1 8 9 ~ 8 3 r~"u ~ 4
or portions thereof fused ro cach of these ~ domains These rni~ed olieomeric
receprors are prepared by preparing as noted above~ the oligomeric INF-Rs and the oiigomeric
FAS-i~s and then mKirl6 shese together and ~ .c~ y selecting, b~ standard procedures. those
oiigomers having bindin6 specificity for both FAS-R ligand and T~iF. Another way for preparin~
- 5 the rnixed oligomeric rcceptors is by cO-h~.~r~.,li..~ suitable host cells ~iith vectors, as no~ed
~bove, encodin~ any of th~ specific fusion proteins (soluble Th-F-Rs) and encoding any of
- tile FAS-R iigand-specific fusion proteins ~soluble FAS-Rs), purifyin~ the exprcssed fusion
proteins which self-associate prior to, during. or follo ving the purification to yieid oligomeric
recei~tors, and then seleairlg by standard procedures, those oligomeric receptors which are
10 capab!e of binding ~o both TNF and FA'i-R ligand.
Likewise, th~re is also provided pl~ u~il,al cu~ v~i~iv.ls comprisins th~ mixed
oji~omeric receptors, saits or functionai derivatives thereof or mixtures of any of the aforegoine
as active in redient to~ether wirh a ,ul~ c~ acceptable carrier. In addition, there is
provided ~he mi~ed oligomenc receptors, saits or functionai derivatives thereof or rnixtures of an~
15 of the aforegoins, for use in snts~ni7in~T the deleterious effects ûf both I~IF and FAS-R li~and in
m is, especiaily in the treatnnent of conditions ~vherein an excess of I~IF and FAS-R lis~and i~
forrned ,~o~. "u~ or is e~u~ ou:~ly 6dl.".""~cd, or ' ~e'~ for use in rnsintsining
prolonged (slow-release) beneficial effects of ~NF andlor F.~S-R ligand in mamma~s when used
with lNF and~or FAS-R Ge_nd (in soluble form) ~v~ Ou~ d.
Other aspects and .. .~ " of the present invention are also provided as arising from
the foUo~vine detailed description of the inverltion.
It should be noted that. v~-here used throughout~ the following terrns: "Mn~ stinn of the
ll~F-eff~ct on cdls" and "M~ in~ of the FAS-ligand effect on cells" are urlderstood to
encompass in virro as well 25 in vivo treatment.
Brief l~escr;ption of t~- Drawin~s
Flgs la-c depict ~ , the partizl and prelir~in~y nucleotide sequence of cDNA clones
encoding the p551C and p75IC-binding proteins, ~?herein fig. I(a) is the sequence of
clone 55.11 erlcoding the p55IC-bindirlg proteir~ ~i.ll, Fig. I(b~ is the p6rtial and
preliminss~5r sequence of clone 75.3 encoding the p7~1~-binding protein 75.;, and Flg. I(c)
i5 the parti21 and ~,. c' ~ sequence of clone 75 .16 encoding the p751C-binding protein
p75.16; all as described in ~ample 1; and Fig. I(d) depicts the deduced amino acid
sequence of protein 55.11, deduced from the nucleotide sequence of Fig. I(a), as also
described irl ~xarnple I .
- Fig. 2 is a .e~.o~.~;o" of a Northern blot which shows the 5~.1 I-specific rnRhAs present in a
nurnber of tested cell lines, as described in l~xample 1.
Flss. 3A and B are ~ udu~,~;u.. ~ of ~ol ~diog"~ depicting the in w~ro bindin~ of the protein
encoded for by the 55.11 cDNA to GST fusion proteins containing portions of p55-lC.
wherein in Fig. 3A there is depicted the binding of the full-leneth 55.11 protein (55.~1
fuD) to the various GST fusion proteins; and in Fig. 3B there is depicted the binding of a
WO g5/31544 ~ ,9~ 4
21 8'~q83
porriorl of ~5.11 filsed IO the F~AG ~ r~ e to the vario.us GST fusion proteins. ali as
described in ~xsmple 1.
Fig. 4 shows ' ' ".. a comparison ofthe deduced artlino acid sequence of human 55.11 to
r~lated protein sequences derived from lo~er organisms, as described in Example 1.
S Fi~. j is a ~ udu~,liu~l of a Western b~ot stained wjth anti-i~BP poiyclonal antis~rum~ sho~ing
the self associa~ion of the p551C, the Western blot derived from an SDS-PAG~ gel on
which were .,I.~,u,[l~ the interacting bactenally-produced chimic protems p551C-
AIBP arld pS5IC-GST (lanes I 1) or ihe control interactjon between the chimeric protein
p551C-~3P 2nd GST alone (lanes 5-8), the interactions be~ween the chinneric proteins
(and control) being carried out on glutathion-agarose beads prior lo SDS-PAGE~ as
described in E.sa~Tiple 2.
Fi~. 6 is a l~yl~Jdu~,LIull of phase cûntrast micrographs sho ving the C,YtOlOXiC e~fect of the full-
length p551C in HTtal cells transfected with an expression vect~r encoding this p55IC
(ri~ht panel), and the inhibition ~f this cytotoxic effecl when expression of the vector is
blocked b- trealin~ the celi~ with tetracycline (left panel)~ as described in Example ~.
Fig. 7 depicts the li~and i~ rig~ering of the cytocidal eff'ect in HeLa cells transfected
ith the full-len~th pS5-R, its i"..~.ccl'a~;~l domain, or parts of the ;, ll,, -. li;.l ~ domain
including the 'death domain' where:
~i~ at the extreme left hand side of Fig. 7 there is depicted ,. h ~ y the various
D~A molecules encodirl~ the fuD-len~th p55-R~ its ;I,LI.. ,~,I~u;c domain and the portions
ûf the intracellular domain which were inserted jMO the vector with which the HeLa cdls
were Iransfected.
~li) the left and middle bar ~raphs show the TNli receptor expression in the E~eLa
cells of each of the tvpes of receptor shown at the e~treme left of i ig. 7. the left bar
.~yl~ ; the amounts of receptor in ng~cell sa~nple and the rniddle bar ~i.Syh.
Icl..~w~Li..æ the amounts of receptor expressed in te~ns of l,..~ bound tû
the transfected ceDs; and
(iii) the right bar graph showing the viabilit of the Hd' a cells expressing thevarious kinds of the receptor,
and wherein irl aD of the bar graphs the open bars represeM cells transfected in the
presen~ of tetracycline and the closed bars represent cells transfected in the absence of
yuli~e~ aD of the above being described herei~ in Exa;nple 2.
Flg. 8 depicts the hgand ' A~ induction of IL-S gene expression in HeLa cells transfected
~ith the &D-length p55-R or its i,.LIc.~,elluL domain (p55IC), wherein in panel A there is
shoT,vn 2 ley~du.,.;ùl. of a Northern blot IC~ ~GUU~g the ~orthern analysis of RNA
extracted from HeLa celis treated or nMrGsted with ~NF (two lefi ilsnd lsnes Tmarl;ed
'corltror snd TNF'), snd of RNA extracTed from Hei 2 ceDs transfected witb vectors
encoding the p55-R, p55-lC or the comrol pro~ein, iucifer2se (the rernaining lanes marked
'pS5-IC', 'p5S-R' and Luc, r~y~h~,ly), the cells h2vin~ been trarlsfected in the presence
40 (+) or absence (-) of IGh~.' in eacil case (hence t~vo lanes per L~ -); snd
WO 95/31S44 2 1 8 9 9 8 3 r~ 4
17
wherein in panel B there is shown the methylene blue staininr of I ~S r~NA in each of the
EleLa celi sampie shown iD panel A; zll of the abo~e being described in Example 2.
Fig. 9 (A and ~3) depicts graphically ~lle ligand ;~ tri~erins! of a cytocida~ effect in
~IeLa cells eransfected with p55R or parts thereof, or ~ith F.~S-IC. u~herein in Fig. ,A
S there is depicted the results with rcspect to the pSSR or par~s thereof and in Fig. 9B there
is depicted the results with respect to the FAS-IC. in the left. hand panels of both Fi~. 9~
and B there is depicted ' ~ , the portion of the p55R or FAS-IC used in the
~: wili~e the right hand panels depict graphicail~ ~he ~ l results, all as
described irl Example 2.
10 Fig. 10 depicts 5~h~ ly the partial nd pr~liminar} nucleotide sequence of a cDNA clone,
calied F'', which encodes a protein capable of binding tc the p~SJC and FAS-IC, as
described in Example 3.
Fi~. Il depicts ~ lly the partiai and preiiminar~ nucleotide sequence of a cDNA clone,
calied F9~ which encodes a protein capable of bindin~ tc the pS5IC and FAS-IC, as
described irl ~xample 3
Fi~. 12 depicts s~ matir~lly the partial and prelirQinary nucieotide sequence of a cD~A clon~,
cailed DDI ]. which encotes a protein capabl~ o~ bindin~ lO the p551C, especiaily the
p55DD~ and FAS-IC, as described in Exatnple 3
I~etaDed Des~ription of the lnvention
The presem invention relates, in one aspect, to novel proteins ~-hich are capable of binding
to the, ~ domain of receptors belonging to the TNF/NGF superfanuiy, such as T~F-Rs
and FAS-R and hence are considered as mediators or modulators of this ~U~ of receptors,
e,g. of the ~F-Rs and FAS-R having a role in~ for example, the signaling process that is initiated
by the binding of lN~ to the TNF-R and FAS ligand to FAS-R. Exarnples of these proteins are
those which bind to the; -~ dornain of the p55 Tl~'F-R ~p551C), such as the proteins
designated herein as 55.1, 55.3 and 55.11 (Exatnple 1) as well as those encoded by cDNA clones
F2, F9~ and DDI I (~aJnple 3); those which bind to thc ' ~ lul~ domain of the p75 TNF-R
(p75~C), su~h as the proteinr designated herein as 75.3 and 75.16 (El;atnple l); and those which
bind to the .. ~ ll.. IG. domain of ~AS-R (FAS-1C), sueh as the proteins encoded by cDNA
30 clones F2. F9 and DD11 ~Exarnple 3). Proteins 55.1 and jj.3 have been fowld to represent
portions or fra~ments of the ~ dotnain of the pSS lNF-R (pS51C); other proteins,55.11, 75.3 and 75.16, represent proteins not describcd at ail prior to t~le present invention (75.;,
~5.16) or those tbat h2ve been doscribed tSS. I 1, see Khan el al., 1992) but ~hose function and
other t~ , particularly, the abilit~ to bind to a l~-R, were not described in any wa~v
35 (see Example 1, below). The new proteins encoded by cDNA clones F2, F9 and DD~I also
represent proteins pre~iously not described at alL i.e. their sequence is not in the 'G~NEBA~' or
'PROTEIN BANK' data banks of DNA or arnino acid se~uences.
Thus, the present invention concerns the DNA se4uences encoding these proteins and the
proteins encoded by these se~uences.
WO95/31544 21 89q83 r~l~u~ 4
Moreover, the present inveMion aiso concerns the DNA sequences encoding biologicall~
acti~e anaiogs and derivatives ofthese proteins, and the anaiogs and deri~atives encoded thereby.
Ihe preparation of such anaiogs and derivath.es is by standard procedure (see for exampl~,
Ssmbrook et al., l9g9) in which in the ~NA sequences encodin~ these proteins~ one or more
5 codons may be deleted, added or substi1uted by another, ~o yield anaiogs havin~ at least a one
amino acid residue change with respect to the native proleirl. Acceptabl~ analogs are thos~ which
retain at least the capability of binding tO the ;~ .,ellulG, domain of the T~iF/NGF receptor
superfamily, such as FAS-R or lNF-R e.g. Ihe p55IC, p75IC or FAS-IC. or uhich caD mediate
any oth~r bindin~ or ~nymatic activit~, e.~. analogs which bind the p55, p751C or FAS-IC but
10 which do not siynal. i e. do not bind to a further d~..,Di~c_. receptor, protein or other factor, or
do not cataiyze a signal-dependeM reaction. In such a wa~ analogs can be produced which have a
so-calied dominaM-n~gative effect, nameiy~ an anaiog which is def~ctive either in bindin~ to Ihe,
for example~ p55IC~ p751C or FAS-IC, or in subsequent signaiin~T follo~-in~ such bindin~. Such
anaio~s can be used~ for e~cample, to inhibit the l?lF- or F.~S-ligand- effect by competinL~ with the
15 natural IC-binding proteins. I,ikevise, so-cailed dominant-positive anaiogs may be produced
which wouid ser e to enhance, for example, the TNF or FAS ~igand effect. These would have the
s2me or better IC-bindirlg properties and the same or better signaling properties of the natural IC-
bindini~ proteins. Simiiarly, deri~atives may be prepared by srandard ",~ r,~l,n~ of the side
g,roups of one or more amino acid residues of the proteirls, or by coA,il~tion of the proteins to
20 another molecule e. ~. an antlbod~, enz,vme, receptor, c~c., as are well known in the art.
Tbe new TNF-R and FAS-R ;~ ellul_l domain - bindin~ proteins, e.g. the proteins SS, ~,
55.3~ 5~.ll, 7.,.3, 75.16 as well as ~he proteins encoded b~ cD~lA clones F2, Fg and DDI}
(hereinafter, F~, F9 and Ds.)l 1) h~ve a number of possible uses, for example:
(i) They may be used to mimic or enhance the function of INF or F.~S-R li~and, in
situations where an enhanced T~ or FAS-R ligand effec~ is desir~d such ar in anti-~ mcs,
anti-~ n ' r or anti~ -j t; ~ C where the INF-or FASR li~and- induced
~ ylUL~ r is desired. Irl this case the proteins, e.... thos~ binding to the p551C such as
5.1, 55.3, as weD a F2, F9 and DDI l, and the free pS51C rtself ~see below and E~ample
Z). as u~ell as the 'deatb domain' ofthe p55IC (pS~DD), which enhance tbe TNF eftect; or
proteins FZ. F9 and DDII as ~eil as FAS-IC and F~S-DD whicb enhance the FAS-R
ligand effect, i.e. cytotoxic effect, may be introduced to the cells b- s~andard procedur~s
knoun ~ se. For exar,Apie, as the p~oteins are i..L...ce~ and it is desired that thev be
in~roduced only into the cells where the l-NF or FAS-R ligand effect is wanted, a system
for specific ;.~u v~u~,~iu.. of these proteins into the ceiis is necessary. One way of doing this
3~ is by creatinE a .~ anirnai virus e.g. orle deri~ed from Vaccinia, to the DNA of
which the follo~ving two genes wili be introduced: tile gene encoding a iigand that binds to
cell surf2ce proteins specifically expressed by the ceDs e.g. ones such 2s the An~s (E~V)
virUS gp120 protein which bit~ds specific2ily to some cells tCD4 ly~lpl~O~t~ and related
leukernias) or any other ligand that binds specificaily IO cells carrying a TNF-R or FAS-R
such th2t the ~ ,irus vector uill be capable of binding such TNF-R- or FAS-R-
WO 95~31544 2 1 8 9 9 8 3 r~l,u~ - - 4
19
carr~ing cells; and the sene enco i3ng the new intraceilula- domain-binding protein or the
p551C. pS5DD, FAS-IC or FAS-DD protein. Thus~ expression of the ceii-surface-binding
proteirl on th~ surface of the ViIU5 will target the virus specifi~ally to the tumor ce~l or
other TNF-R- or FAS-R- ca~r~ing eell, follo ving which the inrracellular domain-binding
protein encoding sequenee or p55IC. pS5DD, FAS-IC or F~S-DD encoding sequenee will
be introdueed ineo the cdls via the virus, and once expressed in the eells will result in
~..l.~... ~.... : of the TNF or FAS-R ligand effect leading to ~he death of the tumor celis or
other TNF-R- or FAS-R- earryincr eeiis it is desired to i;i~l. ConslrUetion of sueh
.~ ~ `'~ animal ~irus is b~ standard procedures (se~ for exarnple, Sambrook et al.,
1989). Another possibility is to introduce the sequences of the ne~v proteins or th~ p5CIC,
p55DD, FAS-IC or FAS-DD in the form of oli~."., Ir~ r ~ which can be absorbed by the
celis and expressed therein.
(ii) They may be used to inhibit the TNF nr FAS-R li~and effect1 e.g. in cases such as
tissue dama~e in septic shoci;, graft-vs.-host rejection, or acute hepatiris, in which case it
I5 is desired to block the TNF-induced TNF-R or FAS-R li~and induced FAS-R iMracellular
sigrlalin". In this situation it is possible, for ~Aarnple. ro introduce into the cells, b~
standard procedures. rlli7r~rll~rL~otirlrs haing ~he anti-sense coding~ iequence for these
new proteins, or the anti-sense coding sequence for pSSIC, pC5DD. FAS-IC or FAS-D3~
whieh would effectively b]ock the translation of rnRN~s encodinv these proteins and
thereby bloek ti~ei~r expression and lead to tbe inhibition of the T~F- or FAS-R ligand-
effeet.
Sueh v~ 5 may be introdueed into the cells using the ahove
n~,nl",~,l virUs approach, the second sequenee earried by the vitus being the
~!;L ; 7 ~ ' seq~lenee. ~n~ther pos~ibilit~ is to use zntibodies speeific for the~e
proteins to inhibit their ;~ la, signaiirlg aetivit~. It is possible that the~ r~w
proteinS have an ~A~ ~celh.L domain as weil as an; ~ one, the latter which bindsto the TNF-R or FAS-R binding don~ain, and thus anribodies =enerated to their
" ' dornains ean be use(i to bloeic their Ti'lF- or F~S-R iigand- rdated funetions.
Yet anoth way of inhibiting the TNF or FAS-R ligand effeet is by the reeentiy
devdoped ribozyme approaeh. Ribo2~nes are catalytie R~A molecules that specificaily
c7ieave R.N~s. Ribozymes mzy be engineered to eleave tarOret R~As of ehoice, e.g. the
rnRl~As eneodinO tile new proteins of the imention or the rnRNA eneoding the p551C,
p55DD, FAS^IC or FAS-DD. Such ribozyrnes would have a s~qu~nee specifie for the
r~nRNA of ehoice and would be capzble of interaeting therewith (~ bindins~)
foilo~ed by e]ea~a~e of the rr~'~, resuiting in a decrease (or eomplete los~c) in the
expression of the protein it is desired to inilibit, the level of decreased e-~.pression being
dependent upon the level of ribozyme expression in the target eeil. To introdueeribcyrnes into the cells of choice (e.S. those earrying INF-Rs or FAS-R) any suitable
vector rnay be used, e.g. plasrnid, animai virus (retrovirus) veetors, that are usuzily used
for this purpose ~see aiso (i) Jlbove, where the virus has, as seeond sequenee, a cDNA
WO g5/31544 2 1 3 9 9 8 3 J .~ JO.~4
20
encoding the ribozyme sequence of choice). Moreov~r, riboz~mes can be constructed
which have mu tiple targets ~multi-tar~et riboz~;mes~ thar ct~n be used, for example~ to
irLcLibit the expression of one or mor~ of the prot~ins of the invention andlor the pS51C,
p55DD~ FAS-IC or F.4S-DD as ~veil ~For revietvs. methods et;. concerning ribozy.tnes see
S Chen et al., 1992; Zhao and Pici;, 1993, Shor~ et ai., 1~?~. Joseph and Rurke, 1993,
Shimayama et al., ~99~, Cantor et ai., 1993; Barinai~a. 199~.; Crisell et ai., 1993 and
Koizunli et al., 1993),
(ii) They may be used to isolate, iderltify and clon~ other pro~eins ~hich are capable of
binding to them, e.g. other proteins involved in the ;~ a~llu;~ signaling process that are
dvw~ a~ ofthe T?IF-R or FAS-R intraccllula~ domain In this situation~ thcse options,
namely, the DNA sequences encoding them maS be.used in the yeast two-hybrid system
(see ~xample 1, below) in which the sequence of these Froteins wiii be used as "baits" to
isolate, clone and identify from cr,'N.~ or genomic DNA libraries othcr sequences
~`'preys") encodin~ proteins ~hich can bind to these ne~ TlaTF-R or ~AS-R intraceilular
IS domain-bindinE proteins. In the sam~ ~vay~ it m~! also be detertnined ~hether the sFecific
proteins of the present invention, namei~ those vcilich bind to the pSSIC~ p75IC, or FAS-
IC~ can bind to other rec~ptors of the l`NFINGF superfarnily, of rcceptors. For example~ it
has recentl~ been reported (Schwalb et ai., 1993; Baens et al.~ 199~; Crowe et al.~ 199~)
that there exist other I~F-Rs besides the pSS and p75 T~lFRs. Accordingly, using the
yeast two-hybrid systern it may be specifically tested ~hether the proteins of the present
invention are capable of s~ecificaiiy bindin)l to these other rNF-~s or other receptors of
the TNF/N&F superfamily. Moreover~ this approach mzy aiso b~ taken to deterrninevhether the proteins of thc present in~ention are capable of binding to other known
receptors in whose actiYity they may have a functioral roie.
(iY) The ne~ proteins may also be used to isolate, identi~ and clon~ other proteins of
the same ciass i.e. thos~ bindir~g to TNF-R or FAS-R ;~Ll~r~ellLllal aomains or to
functionally reiated receptors, and invol~ed in the i~ a~ u'a~ si~naiing process. ln this
appiication the above noted ~east two-hybrid systeln ma, be used. or t}Lere may be ~Ised a
recently deveioped (~-~lks et al.~ ~ 98g) s,Ystem employing non-stringcnt sou~hern
~0 llybl;lL~aL;oil foilowed by PCR clonin~. In the Wilics d ai. publication, th~re is described
the ;~ ;v,~ and cloning of two putative protein-tyrosine i~inases by application of
rton-stringent southe,~n iIJLJ~ aL;UII foilowed by cloning by PCR based on the iinown
sequence of the i;inase motif, a conceived i~inase sequence. This approach may be used,
in accordance ~vith the present invention using the sequences of the new proteins to
identify and clone those of reated TNF-R, FAS-R or rclated receptor (T~F/~GF
superfamiiy receptors) " ' domain-binding proteins.
Yet another approach to utiiizing the n~ pro~eins of ~he invention is to use them
in methods of afhnity ~ , . ' to isolate and iden~ify other proteins or factors to
which they ar~ capable of bindr~g, e.g. other receptors reiated to lNF-Rs (INFINGF
receptor superfarnily) or oth~r proteins or factors involved in the i~lLla~, lu ~ si~naiin
Wo 95/31544 2 1 (~ ~t 9 8 3 r~ l/V~ 4
21
proceSs. In this applicatiorL the proteins of the preserlt in~ention may be individually
~ttached to affinit~ ch~l.... v ~ matrices and ther brou~ht into contaGt with c~ll
extracts or isolated protcins or factors suspectcd of bcing involved in the i~ cellulG.
sig,naling process Following the af~inity u.~ pl,~ procedure, the otner proteins or
factors which bind to the new proteins of the invention~ can be cluted, isolated and
~L....~rt~ri7~d.
t~ As noted above, the new proteins of the invenrion may also be used as
,.. r,~;.. 1~ (anti(~ens) to produce specific antibodits thereto. These antibodies may also
be used for the purposes of purification of the ne~- protein~ either from cell extracts or
lû from ~,A.. ~r.. ~; ceil lines producing them. Further~ these antibodies may be used for
diagnostic purposes for identifying disorders related to abnormal functioning of the TI~F
or FAS-R li~and s~vstem; e.s. overactive or underactive l NF- or FAS-R ligand- induced
cellular e~fects Thus, should such disorders be reia~ed to a .n~lr".~. l; l~.g ih,.lc..l~ul~l
signaiing system invol~ing t}~e new proteins, such antibodies ~-ould ser~e as an important
diagnos~ic tool.
It should also be noted that the isolation, ;~s. ;rl. ~ ,.. and ...l~a~ vl, of tile ncv:
proteins of the invention may be perfornned using any of the well known standard screer~ing
procedures. IJor example~ one of these screening procedures~ the yeast two-h~brid proGedure as is
20 set forth in the following examples ~Exarnples 1 and 3), was used to identify the new proteins of
the invention Lii;ewise as noted &bove and below, other procedures may be employed such as
af~inity ', ~ , DNA h~ u;~l; s.~ procedurcs, etc. as ~re well known in the art, to
isolate, iderltify and ,l~ t~.;s. the new proteins of the in~-ention or to isolate, identify and
~ ' additional p.roteins~ factors, receptors~ etc. which are capable of bindin~ to the new
15 proteins of the inventior~ or to the receptors belon~,in~ to the l~INGF famiiy of r~^eptors.
As resards the antibodies mentior~ed herein throughout, the term "antibody" is meant to
include polyclonai antibodies, ~ r~ antibodies (mAbs), chimeric antibodies, anti-idiot~pic
(anti-ld) anibodies to anibodies that can be ]abeied in soluble or bound form, as well as
fra~rnents thereof pro~ided by any icnown te~hnique, such as, but not limited to enzymatic
3v deaYage, peptide syntilesis or ,c teci~iques.
Polyclonal antibodies are ~ ,.c.u~ u~ populations of antibody moiecules derived from
the sera of anirnals irnrnunized with an anti~en. A monoclonal antibody contains a ~
l..~.l~..C~ popuiation of antibodies specific to antigens~ which PV~ v . contains
".~ Sirnilslr eprtope bindin~ sites. I~Abs may be obtained b~ methods knowrl to those
skilied irl the art. See, for example Rohler and Milstein, ~ah~. 2rs6:495-497 (19~5); U.S. Patent
No. 4,376,110; Ausube~ et ai., eds.. Harlow and Lane ANTIBODES .~ LABORA~ORY
MANUAL, Coid Sprin~ Harbor Labora~ory (1988~: and Colli~an et al.~ eds., Current Protocols in
T 1~_~, Greene publishul~ Assoc. and Wiie~ T.,t~ N.~., (1992, 1993), tho corleents
of wilicil references are ;.~cc,.l,v. ,.~d entirel~ herein by reference. Such aneibodies may be of any
40 ` ~ " class inciuding IgG. Igh~ A, G~D and any subclass ther~of A
WO95/31544 2 1 899 83 ~ .,. . 4
__ .
hybridoma producing a nL~b of the present invention may be culth, aled in ~irro, in .~J~tl or m ~v~
Production of hi~h titerr of mAbs in Vil~o or in sit~ mal;es this the presently preferred method of
pro.luction.
Chimeric antibodies are molecules di7~erent portiorJs of v.~hich are deri~ed from different
5 animal species, such as those having the variable region d~ri-v~d from a murine rnAb and a human
- ~' ' ' constant region. Chimeric antibodies are prirnarily used to reduce
O v in application and to increase yields in productiorL for exatnple~ where murine
rnAbs ha~e hig~ter yie~ds from hybridomas but htgher i ~ ~ in hutnans, such thatul - chimenc mAbs are used. Chirneric antibodies and methods for their production are
known in the art ~Cabilly. et al., ProL. hatl. Ar. ar~ ci. l~.SA 81:327;-,277 (1984): Motrison et al.
Proc. IhTatl. Acad .~ci. U~ 6Sil-6S5i (19~4)~ Boulianne et al."'~'at/~r~ ;12:64i-646 (1984)~
Cabilly et al., European Patent Application 125023 (published ~ove7nber 14, 1984); Neuberger et
al.l~ar~re314:26S-2~0(1g85~:Taniguchietal. EuropeanPateM.~pplication17]496(published
Februar~ 19~ 198~j; Momson et al., European Patent Application 173494 (published March S~
198G)~ Neuberger et al.~ PCT App~ication WO 860153i~ (published March 1;~ 1986); Kudo et al.
European Patent Applicarion 184187 (published ~une 11~ 1986)~ Sahagan et al.~ J. In2m71nol.
137:10G6-lO74 (1986); Robinson et al.~ T,~ l Patent Application No. W087G2671
(published Ma~ 7, 1987). Liu et al ~ Proc. I~/at~. ~cari Sc~ llS~ 84:;439-344~ ~1987); Sun et al.~
Proc. J~arL Aca~ Sc~ rJ~l 84:2]4-218 (1987)~ Better et al., .'ici~nCL~ 240:1041-104; ~988): and
20 Harlow atld Lane, ANIIBODIES :A LABORA~ORY MA~lJAL. supra. These references are
entirely ;.l~o,~e,.~lcd herein by referenee.
An anti-idiotypic (anti-Id) antibody is an anhbody which recosgnizes unique d~L~ h~
generally associated with the antigen-bindins site of an antibody. An ld antibody can be prepared
b', immuni~in~ an anima~ ofthe same species and &enetic ~ype (e.g. mouse strain) as the sourc~ of
25 the mL~b ~ith the mAb to ~hich an anti-ld is being prepared. The i~nmunized animal v~ill
recognize and respond to the idiotypic dc.~ ;lla.llL~ of the immuniziny; antibody by produciny an
~ntibody to these idiotypic ~' (the an~i-ld antibody). Se~ for exampl~ U. S. Patent No.
4~699,880, which is herein ertirely; \~ ~p~ by reference.
The anti-ld antibody may also be used as an "; ~ to induce an irnmune response
30 in yet another anitnal, producing a so-called anti-anti-Id antibodv. The anti-anti-Id ma~ be
epitopically identical to the original rslAb which induced the ani-Id. Thus. by using antiboùies to
the idiotypic ~ of a rnAb, it is possible to identify other clones expressing antibodies of
identical speci/ictty.
Accordingly, mAbs generated agsinst the IC-binding proteins~ analogs or d~rivatives
35 thereof, of the preseM invention or the pSSlC, pSSDD, FAS-IC~ FAS-DD~ analogs or derivatives
thereof r~ay be used to induce anti-Id antibodies in suitable animals, sueh as B ~LB/e miee. Spieen
cdls from such irn~nunized miee are used to produce anti-Id hybridomas seerelin~ anti-Id mAbs.
Further, the anti-ld m.~bs can be coupled to a carrier such as ke~hole limpet ~.. ac~
and used to irnrnunize additional B~LB/c mice. Sa from these mice will contain anti-anti-ld
40 antibodies that have the binding properties of the orlginal nsAb specific for an ep~ope of the
. .. . . _ . _ . . _ . .
wo 95131544 2 i 8 9 ~ ~ 3 P~~ ~ ~ 4
~bove IC-bindirl~ pro~eins, anaio~s or denvati~es or p55JC. p~Dr,, FA5-IC or FAS-DD.
arlaiogs or derivatives.
The anti-ld rnAbs thus have their o~hn idiot~pic epilopes, or "idiotopes" structurall~
sirrlilar to the epitope bein~ evaiuated, such as GRB prctein~
The term "antibod~" is a~so meaM to irlcluds both intact molecuies as well as fragments
thereof, such as, for exampie, Fab and ~(ab')~, which are capable of bindin~ zntigen. ~ab and
~ab')2 frasmerlts lack the Fc fragment of intact antibody, clear more rapidly from the circulation,
~nd rnay ha~e less non-specific tissue bindins thaQ an intact arltibod~ ahi et al., ,1. A~ l. .4~d.
24:316-32~ (198;)).
It wilS be appreciated that Fab and F(ab')~ and other fragments of the antibodies useful in
ti~e present invention ma~ bc used for th~ detection and c~ of the IC-bindin~ prcteins r1r
pSSIC, pS5DD. FAS-IC or F.~S-DD according to tii1e methods disclose~ herein for intact
antibody molecules. Such fra~tncnts are t~pically produced b,~ proteolytlc cleavage, usin~
er~n~s such as papain (to produce Fab fragments) or pt~psir. (to ploduce F(ab'~2 fragments).
An znibody is said to be ' cap~ble ot` binding'' a molecule if it is capable of specificaIi~
reactinc with th~ molec~ie to thereby bind the moJecule to the antibody. The term ''epitope" is
moant to refer to thu portion of an! molecule czpab~e of being bound by an aMibod,v which can
aiso be recor~nized by that antibody. Epitopes or "zntigenic ~S~....,.;,.- :~' usually consist of
chemicaily active surface groupings of moiecules such as amino acids or su~ar side chains and
ZO b2ve specrfic three d - -- .~ structural ~ l; . as well as specific char~e -'An ~antisen" is a molecule or a portion of a molecule capable of bein~t bound by an
arltibody which is .~$ '`~, capable of inducing an animal to produce antibod~ capable of
bindin~ to ~n epitope of that antigen. An at~tigen may h2ve ooe or more than one epitope. Th~
specific reaction r~ferred to abo e is meant to indicate that the anti~en v~ill react, in a hi~hl~
2~ sdective manner, with its CVI~L~ U~d;II~ antibody and not with the multitude of ~ther antihn~lies
which may be evoked by other anti~ens.
Ihe antibodies, including fra~m~nts of antibodEes, useful in tbe present in~ention may ba
us~d to l~ UL~ or i 1 ~ detect th~ IC-bindin~ proteins or p55IC, p55DD, F~S-IC,
FAS-DD in a sample or to detect presence of cel~s ~-hich express the IC-bindins proteins of the
30 present invenion or the p551C, p5'iDD, FAS-IC, FAS-DD proteins. This can be ~ y ~ b~
C~CG techniques emp10ying a nl~VlG~CGlllly labeled antibod~ ~see below) coupled
with E~ht u~.oy;~l~ f~ow cytometric~ or nuu-u.l.~,t.;~. detection.
The antibodies (or fragmerlts thereof~ usefu~ in the present invention may be employed
~ t '~ , as in ;~ C~ G or ~ tIe~I~U-- ~ IUS~U~ for i71 .5it?~ detection of
35 lC-bindins proteirls of the preserlt inve?ltion or the p55IC, p55DD. FAS-IC~ FAS-DD. /~? .5
detection may be ~ v ~1,S:,~ J by remo~in" a hislolo~icaS specimen from a patient~ and providin~
the labeled ?mtibody of the preSent invention to such a specimen. The antibody (or fragment) is
prefably provided by pplyin~ or by overlaying the labeled antibody (or fragment) to ~
biological sasnple. Through the use of such a procedure, it is possible to deterrnine not only the
40 pres~nce of the IC-bintin~ proteins or the p551C, p55DD, FAS-IC, FAS-DD, but also its
wo95/31~44 2~ 39q~3 ~I/U~ ro 4
dislribution on the examined tissue. Usin~ the present inven~iorl, those of ordirlar, si~iU will readil~
perceive that any of wide variet~ of histolDgicai rnethods tsuch as staining procedures) can be
modified in order to achie~e suGh rn ~tu detecsion.
Such assays for IC-binding pro~eins of the preseM in~ eMion or th~ p5~-lC, p55DD. FAS-
5 IC, FAS-D~ typicaliy comprises incubating a biologicai sample~ such as a biologicai fluid~ a
tissue extract~ freshl~ hzn~ested cells Nch as l~"~,l.oc~ej or leukocytes, or cdls which have been
incubated in ~issue culture, in the presence of a detectabi- labeied antibody capably of identifyinL~
the IC-binding proreins or the p~5IC, p55DD, FAS-IC. F.~S-DD. and detect~ng the antibody by
any of a number of techniques well i~nov,~n in the art.
10 The biological sample may be ~reated with a soiid phas~ support or carrier such as
~.,~..l~ul~ or Dther solid support or Garrier which is capable of ;~ ;,. ) cells. cell
particles or soluble proteins. The support or carrier ma~ then be washed wi~h suitable buffers
followed by treatment wi~h a detectably labded antibod~ in accordance with the present invention~
as noted above. The solid phase support or carrier may then be ~ashed ~ith the buffer a second
1~ time to remo~e unbound antibody. The amount of bound label on said sDlid support or carrier
rnay then be detected by c..ll~..;v,lai means.
By "solid phase support", rsolid phase carrier'`~ "soiid support"~ "solid carrier", "support"
or rcarrier" is intended any support or carrier capable of binding antigen or antibodies Well-
knowo supports or carriers, include glass, polystyrene, pO~ v~ , pol,~ethylene, d~ ran,
nylon amylases, naturai and modified celhlloses, pOlr~ y.a.l~i~3, gabbros and rna~netite. The
nature of the carrier can be either soluble to some extent or insoluble for the purposes of the
present irlvention. The suppon material may have vinually any possible structural rv~ 50
lon~ as the coupled molecule is capable of binding to an antigen or antibody. Thus, the suppon or
carlier r~l~jU,2",u" mav be spherical, as in a bead, cylindrical, as in ~he inside surface of a tesr
2~ tube, or the extetnal surface of a rod. AlternatiYely, the surface ma~ be flat such as a sheet~ test
strip~ elc. Preferred supports or carriers include polystyrene beads. Those slblled in the art ~vill
know may other suitable carri~rs for binding antibody or an~igen, or wil~ be able to ascenain the
sa~ne i~y use of routine .
The binding ac~i~ity of a ~iven lot of antibody, of the inventiot~ as noted above, ma~ be
determirled according tO well known methods. Those skilled in ~he art wili be able to determine
operative and optimal assay conditions for each d ;,~ ';ol by employin~ routine
~ r
Other such steps as ~Yashin6 stirring, sh2king, fil~erin~ 2nd the lil~e r~ay be added to the
assays as is custotnary ot necessaty for the particular situa~ion.
One of the vlays in v~hich an an~ibody in accordance v~ith the present invention c2n be
detectably labeled is by lini;ins the same to an enzyme and use in an en~yme ;..I~
This enzyme, in rurn, when lat exposed to at. appropriate substrate~ will teac~ with the substrate
in wch a m2nner as so produce a chemical moiet~ ~hich c2n be detected, for a~ample, by
I ' - . nuul U~ or by isuai tne2ns. l~nzyrnes which can be used detectably label
th~ amibody include, but are not limited to, malate d.~iJl~-.L~ 4cou~t nuclease, delt2-
wo 95/31544 2 1 8 9 9 8 3 r~".~ 4
5-steroid isomeras, yeast aicohol dc~ ,v~ se, alpha-~]J~lu~,i.uD~ c dc~ u~Dc~ triose
phorphale isomease. horseradish peroxidase, aikaiine ~ T~ `Ç. ~iucose
oxidase, beta-Sc'~ , urease, cataiase. yiucose-6-phospilate ic.~ u,~...dac,
"e and ace~vl.,l.ol..~ e,~e. The deteetion caD be A- ~ by ,vloli....,.li-
S methods which emplo~ a .1" v.~.c,c_..,. substrate for th: enzyme. Detection maS i~iso be
, " ' ' by visua~ ersrnrarisr~n of the extent of enzymatic reaction of a substrate in
comparison ~vith similarly prepared standards.
Detecrion may be ~ usinY a~y of a ariety of other ;""'"/'''~ ,1D. For
example. by l,,~iio~.Liv;LJ iabeiin~ the antibodies or antibod~ fra~ments, it is possible to detect R-
10 PTPase throuYh the use of a . .~ n~Ay (E~IA~. A ~ood description of RIA may be foundirl Laborator~ Techniques and Biu~ ,D~ in Molecular Eliolo~y, by Worii, T.S. et ai., ~orth
Holland Pubiishing Connpany. N~ ~197S) with particular r~ference to the chapter entitled "An
L~, t;iu.,~..t,~ to Ri~l;..' `... - Assay and Related Techniques" by Chard, T., ;Il.,ul~uldL~: i by
re~erence herein. The radioactj~ e isotope can be detected b~ uch means ilS the use of a y counter
15 or a scitltiiiation courlter or by au~vl~div~ J.
It is also possible to label an an~ibod~ in accordatlce ~vith the present invention with a
fiuoreâcent compound. When the fluorescentls labeled antir)od~ is exposed to light of the proper
wavelength~ its presence can be then det~cted due to ~UUI~D~ monY the most cornmonly
used f~uoreseent labelins .,....l..~ are fluoreseein :c~ cy~c~ rhodamine, pll~.ylhl;. e.
20 pycocyanin, " . ' ~.,v, , o-T,..,;.ald~.hJIc~, and nuù.c
The antlbody can also be detectably labeled usirlg nuvlT~.~ence emitting metals such as
] 52E, or others of the lanthanide series. These metals ean be attaehed to the antibody using such
metal ehe~ating groups as d~ pentaacetic acid (ETPA).
The antibûd,v ean also be detectably, labeled b~ couplin~T h to a rl...,.~
~5 eompûund. The presence of the ..1~....;1.~...~,.. -~ ' tag~Ted antibody is therl determined h.y
detecting The presence of i.. ;.. ,~ that arises durimz th~ course of a chemieal reaction.
Examples of particularlS useful, ' '~ labdins~ compounds are iuminol, isoluminol.
tberomatic acridinium ester, imid~7ole, aeridinium sa~t and oxalate ester.
~ilcewise, a ~ -' compound may be used to label th~ antlbodS~ of the present
30 irlveniorL ~irl~ r~ j5 a type of ' ' ~,- found in biolo ical systems in vhich a
eatalytic protein inereaSes the effieiency of the ' ' reaetion. The presenee of a
protein is determined by deteeting the presenee of 1..: 5 ~ Important
' compounds for purposes of labelinf are lueiferi~, lueiferase and aequorin.
An arltibody molecule of the present invention may be adap~ed for utilization in an
assay, also ';nown as a "two-site" or "sandwich" assay. In a tS~pical ' .. ....... l
assay, a quantity of unlabeled antibodS (or fragmem of anTibody) is bound to a so}id support or
ca~ r and a quarltity of deteetably labeled soluble antibody is added to permit detection and~or
qt~ hstTi~sn of the ternary eompl~x formed between solid-phase antibody, antigen, and labeled
antibody.
woss/3l544 2 1 899 ~3 ~ 4
Typicai, 2nd pret-erred, ;,., ~ . assays include "for vard" assays in which the
~ntibody bound to the solid phase is fir$ contacted with the sa~nple being tested to ex~ract the
anti~en from the sarnple by forr~ation of a binary soiid phase antibody-anti~zen comple~ After a
suitable incubation period, the soli~i supporr or carri~r is ~ shed to remove the r~sidue of the fluid
S ssmple, inciuding unreacled antigen, if any, and the contacted with the solu~ion containin~ an
uni~nown quarltity of labded antibody ~vhich functions as a "reporrer rnoiccule"~. After a second
incubation period to perrnit the labeled antibody to comp~ uith the antigen bound to the solid
support or carrier throu~h the uniabded antibod- the solid support or carrier is washed a second
time to rernove the u~reacted labeled antibody.
In another type of "sandwich" assay, which ma~ also be useful with the antigcns of the
present invention, the so-caiied ~ . -- v~" and "re~erse'' assays are used. A
assay in~olves a sin~le incubation step as the anribod~ bound ~o the solid support or carrier and
labeled antibody are both added tO the sample bein~ s~ed at the same tinne. After the incubation
is completed, the solid support or mer is uashed to remove th~ residue of fluid sample and
15 1 , ' ' labeled antibody. The presence of labeied anribody associated uith the solid support
or carrier is then determined as it would be in a CU~ iU~ "for vard" sandwich assay.
In the "re~erse" assay, stepwise addition first of a soiution of labded antibody to the ~duid
sample followed by the addition of uniabeied antibody bound to a so3id support or carrier after a
suitab~ incubation period is utilized. .~fter a second incubation, the solid phase is washed in
20 C~ iOIIài fashion to free it of the residue of the sample being tested and the solution of
unrescted labeled anti7~ody. The d~ . ,.. r -~ , of labeled antibody associat~d u~ith a solid support
or carria is then deter~nined as in the " ' " and "forward" assays.
The new proteins of the invention once isoiated, identi~ied and . I .- aC I ~; . ;1 by 2ny of the
standard screenin~ prclcedures, for example, th~ east two-hybrid method, af~mit~25 ~ ~a~u~atJII~, and an~ otiler weii icrJo-vn method 3~no~n in the art, ma~r tilen ~ produced hy
r~ny standard Ic~ b;l~a~ iA procedure (see for exarnple, Sambroo~, et a3.~ i989) in which
suitable euicaryotic or proicalyotic host cells are ~ r.,,. ~ by appropriate euhryotic or
prokaryotic vectc,rs contairling the se~iuenc~s encoding for the proteins. Accordingly, the present
invenion aiso concerns ruch expression vectors arld ' ' hosts for the productior, of t31e
30 proteins of the invention. As mentioned above, these proteins also include their biologicaily active
anaiogs and derivatives, and thus the vectors encoding them aiso include vectors encoding
anaio~s of these proteins, and the L.. r .. Il hosts include those producirlg such analogs. The
derivatives of these proteins are the deri~-atives produced by standard . - ~ of the proteins
or their arlaio~s, produced by the ~ r~ 1 hosts.
In another aspect~ the invention relates to the use of th~ free ;~ ellul~ domain of th~
p~5 TNR-R (p55IC) or FAS-R (FAS-IC) or their so-called 'death donnains' (pS5DD or FAS-DD,
~e~pe~.L;~ ) as an agent for enh7~ncirlg the TNF or FA~R ligand effect on celis, on its o~n (see
Example 2). Where it is desired to introduce a lNF- or FAS-R-ligand- induced cytotoxic ~ffect in
cells, e.g. cancer celis or EII~-infected cells~ the pjiIC, p55DD, FAS-IC or FAS-DD can be
40 introduced into such cells using the above no~ed (see ~i) above) .~ 7: - arimal virus (e.~.
w0 95/3~544 2 1 8 9 9 ~ 3 r "~
~7
Yaceinia) approaeh Here tOO, the native pSSIC, p55DD. FAS-IC or FAS-D'~. ~iolo~icall~, act
ar.a'.o~s and deri~-aives or fra~ments ma~ be used, al'. of ~-.'.ieh can be prepar~d as noted above
Likewise, the present inver~tion a.so re.ztes 10 the specific bloe.sing of the TN~-effeet or
FAS-R .igand-effect by bloc.cin.r the activity of the p55IC, p~ 5DD, ~;.~IC or FAS-DD, e.Lr. anti-
3 sense c'i,, ' ' ma~ be imrodueed into t~e cel~s to biocl; the expression of the p~5IC.p55DD, PAS-IC or FAS-I:)D
The present invemion a'.so re.ales t~ r~ ?~ ,c~ , eomprising cc-",..,..~..
~na'. virus veetors encoding the TNF-R or FAS-R " ' domain binding proteins
{includir..~ the p55IC, p55DD, F.~S-IC and F.~S-A7D), ~hieh ~ector a.so eneodes a ~ir~.s surface
)0 protein capable of binding specif c target cdl (e.g cancer cells) surfaee proteins to direct the
irsertion of 1he inuace.lular domain binding protein sequences into the ce.".s
Ir. another .lspect, t.'.e present invention also concesns. speeifieall~, the effects of the self-
associating ~ G ~ domain of the pS5 TNF receptor ipcs-lc~ see ~xample 2)~ e~xample of
such eftects, ~.~.ich is an effecl norma.l~ mediated b~ Tixr bir.ding to its receptor and ~vhich is
15 mimieked by the sigr.a~in~ activit~ of the self-associatirlE p~5-IC or parts thereof, is the inducuon
of expression of the gene encoding ~L-8
IL 8 is a cytokine belon~ng to the subclass of chemol~nes having primarily ' ,
aetivily, and has been sbown to pia~ a maior role in the chetnotaxis of Lranulocytes and other eel~
types assodated with a nurnber of ~ ol~ s~at~s (see for exampl~, Endo et al., 1994; Sekido
20 a~, 1993; Harada et al, 1993; Ferrick et al., 1991).
TNF h2s a beneficial activily, and is used as sue4 in treatme~ts to destroy lumor cells and
~s infected celis or to augment '~J~rlC ' I a"hvities of ~A~ 1 However, as noeedabove, TNF also ha~s, ' ab~c activities in which case il is desired to bl~ck its activity,
including those sttuations where large doses of TNF are used in cancer therapy, antiviral therap~
25 or ' 'therapy
Accordulgly, it is tesirable ~e be able to direct T~F or a substarce capable of mir~cking
its beneficial activity to the cells or issues that it is specifically desired to treat
In accortance vith t_e presem invention it has been found that the Self~c~ri~ti~irltraoellular domain of the pS5-R ~p55-~C) can, in a li~and - ~ manner, r~imic a number
30 o~e~fects of ~NF, e.g,. the 'death domair.' of p55-lC can intuce cgto~oxic effecrs on cells~ and ~hA1
the p~s-rc can itlduce IL-8 ~ene expression Thus. it is possible to uilize the p55-IC to mimic
~F function in a si~e-directed fashion, i.e to introduce Ihe p55-lC onl, ~o those cells or tissues it
is desired to treat.
One example of the Abo~ approach, as menti~ned above, is to specifcAlly transfect
35 (tratsform) tumor cells or maligmant tissue v~,th a DNA molecule encoding p~-IC or a portion
thereof which can induee t~ot only c~totoxic effects on such edls or tissue but AISO augment thes~
e~ects by the co-induction of IL-8, ~hich will result in the ~ " a~ the site of these cdls
or tissue of _ ' ,.~ and other ~,.",~ , whieh, in turn, will serve to destroy the tumor
cel]s or tissue. This approach obviates the need for ~ of large doses of A~ with its
40 as~cia~ed d~ seriou~ sid~-effe~ts
.. ...
WOgS/31~44 2 1 89983 ~ J51. 4
. 28
Usir,~ conventional IC~ lluA~ DN~ technolo~. i~ is possib~e to prepare various regiorlc
of th~ p55-lC and to deermine whjch re~ion is responsibl~ for each TNF-induced effect, e.~. we
have determined that the 'death domain' is respons~ble fo. C~Ytotoxictty (Example 2), and we have
already prepared various other constructs contairling ~ortions of the p55-IC, which portionS
S (to~ether ~ith part or all of the death domain) may be responsible for other TN~-effects, and
which may be used in a ligand ~ y ~ mann~r~ once self-associaled for activity, to iPduce
these effects, e.g. AIL-8 induction.
It shouid be noted that the se4uePce of the p55-IC involved in the induction of other TNF-
associated eftects (e.A~. iL-8 induction) may be different to that involved iP C Ivlv~h,;ly, i.~. rrlay
10 inc~ude none or only part of the 'death domain' and have other sequenGe motifs from other r~giorls
of the intracellular domain~ or may be the same sequence. diff~rent f~atur~s of Ih~ sequence (same
se~iuence motifl bein~ involved in the induction of different effects.
Accordingiy, as detailed aboYe and belo-Y, expression vectors corAtaining these pS5-IC
portions, analogs or derivatives thereof may b~ pr~par~d, ~xpress~d in hos. celis, purified and
15 t~st~d for their activiry. In this ~ay, a nurnber of such p~ ~-IC fra~menrs having one or more II~F-
arsociated acti~,ities ~nay be prepared and used in a dif~eremial fashion fo~ th~ treatmen~ of any
number of y~ O~, ~l conditions, e.g. virai infections, bacterial infections. tumors, etc. In ail of
these situations the specific activity can be auynePted b~ incorporation (or co~ ).,) with
the p5~-IC fragment responsible for IL-8 sene expression induction, permitting the desirable IL-8
20 1 ,~ activity to enhance the destruction of the cells or tissu~s it is desired to destro~ .
Thus, without ' t ~ TNF, it is possible to induce its desirdble effects
~y specificaily iMroducing aii or part of the p55-~C into the cells or tissues it is desired to treat.
The p55-lC may be introduced specificaiiy into the celis or tissu~s it is wished to destroy
by any one of the dbo;.. ~ ;vn~,~i procedures. For exatnple, one way of doing this is by creating
25 a ~ animal virus e.g. one derived from Vaccinia, to ~,vhose DNA thc folio~vir.z .~.vo
~en~s will be itltroduced tne gene encodin~ d ligand that binds to cell surface proteins
specificaiiy expressed by the ct~lls e.g. ones such as the ArDS virus ~pl20 protein which binds
specifically to some cells (CD4 1,~ vl~J and related leuke~nias) or any other ligand that binds
specifically to cells carrying a INF-R such that the .e,- ' virus vector v~ill be capabl~ vf
30 bindin~ such INF-R-carrying ceiis, anri th~ gene encoding the p~55-lC or a portion thêreof. Thus,
expression of the ceil-surface-binding protein on the surface of the virus wjll target the Yirus
specificaiiy to the tumor celi or other TNF-~-car~ing cdl, follo~ing which the p55-lC, or
portion thereof~ encoding sequence wili be irAtroduced into ~he cells via ti-Ae virus, and once
expressed in the cells will resuit in ~ ' of the INF effect leading to the death of the
3~ tumor celis 0 othu TNF-R-ca-rSing cdls it is desired lo i~ill or induction, for example, of IL-
~~iAich ~ill lead to ceii death. ConstTuction of such ,.. .'' arAimal virus is by standard
procedur~s ~see for example, Sambrook et al.. 1989), Another possibilit~ is to introduce the
se~u~nces of the p55-lC or parts thereof in th~ form of c'iL . .' ~ which can be absorbed
by the ceiis ~And expressed tA~
. .
WO95/~1544 21 8 q q ~ 3 r~.,o~ -- ~4
.
~g
The present invention thus aisc relates specifcally to p~ ,e-lL~ CO~ JV~;L
comprising th~ above l~.,u,.,l,,,~.,~ arlimal virus vec.ors encoding the p55-IC or portions thereof,
which ~eor aiso encodes a irus surface protein capable of binding spccific target ccll (e.g.
cancer celis) surface proteins to direct the insertiorl of the pS~-~C, or porrions thereof, se~uence
s intc the cetis.
- Th~ present invention relates~ in ye~ another aspect, to ne~ synthetic T~F receptors which
are soluble and capable of o'i~,,.,.; I;,~n to form dimeric, and possibly 2iso high order
multimeric, TNF receptcr mol~cules, each monomcric part of these receptors belng cap2ble of
binding to a Tl~ morlomer. INF occurs naturai]y as a homotrimer containing three, active TNF
10 monomers, each capable of i)inding to a sin~le I~F receptor molecule. v~hile ~NF receptors
OCCur naturally as monoms eaci} capable of binding oniy one of the mor~omers of the TNF
h~ , ;.. molecuie. Thus, vhen l'NF binds tO I~F receptors on the cell surface, it is capable
of binding to three receptor moiecules result~ng in the clustcring of ~he ~F receptors, which is
beiieved to be the start of the signaling process v~hich ullimalely triggers ~he observed TNF
1~ effects on the c~lis.
While T~F has many desirable effects such as its abiiit~ ~o destro~, for example, tumor
cells or -~irus-infected cdls and to augmen~ ?nrihR~^t~ri~ acti~,ities of granulocytes, INF does
however~ have rnany undesirable effects such as. for example, in inany scvcre diseas~s including
disorders, rheumatoid arthritis, graft-versus-host reaction (gra~ rejectiorl), septic
20 shocic, TNF has been irnplicated as the major cause for 1 ~ 1~;, t tissue destruction. INF may
also cause excessive loss of weight (cachexia) by suppressinS the activities of adipocytes.
hIoreover, even when ' ed for its desirable activities, e.g. in the treatmem of vatious
malignane or ~,iral diseases, the dosages of TNF used are often high enoush ~o caus~ within the
patient a rlumber of undesirable cytoto ~ic side-effects, e.g. the dest~uction of he?lthy tissue
Accordin~,hr, in all of the above instances where T~F action is .. J~ an effective
inhibitor of TNF has been sought. ~any TNF-biocicing agents have beeri proposed~ including
soluble proteins capable of bindin8 TNF and inhibiting its binding to its receptors and hence also
inhibitin~ the c~totoxic effects of TNF (see EP 3083~8, EP 398327 and EP ~i68szs) ~iowev,
these TNF binding proteins, or soluble lNF receptors are rnonomeric, each bindin~j only one of
30 the INF monomers of the 'iNF l~ci,..vt-;...~. . Hence, the blocicing of the TI`iF function may not be
complete, each monomeric receptor-bound lNF molecule stiil having two T~F monomers free ~o
be able to bind cell-surface lNF receptors and illicit its effects on the ceils. In order to overcome the above drawbaci;s in blociiing T~F furlction, there has been
developed in accordance v~ith the present invention a means for CO.. l-lC~-~g, as fusion proteins,
35 soluble oligomeric TNF receptors which are capable of binding at least two TNF monomers of the
naturally occurring TNF l,u..,vt.i..._. moleculc. As a ~ . , these soluble oligomeric INF
receptoi~ bind more avidly to their TNF ligand than the preYiously knov~n monomenc soluble
INF binding proteins or rec~ptors. For e cample. v~hen the solubie T~F receptor of the invention
is in the fo~m of a dimer, it is capable of binding tlho TN3~ monomers of a ~ trimN and hence
2~se~ ~ ~nor~ ?!et~ ,A ~ of the T~F ~h~ be~ng mo~e ~ict~ned
2 1 8 ~ Qi3 = = =.
WO 95/31S44 r~ 4
.
because of a lower di~r,r;~fir~n rate of the dimeri~ soluble receptors from the TNF. Mcreover,
such soiuble~ oii~omeric receptors are also larger ;han their monomeric ~.uVII~f~l~ua~a and thus~
i ' , . '~, they are aiso ~ because of the likelihûûd ûf their having a slo~Y~r
clei~rance rate from the body.
S The basis for the d~.,lv~.,.... ~,~ of the solub]e oli~omeric TNF receptors of the invention,
was the ~iscover~ that the " ' domain of the p55-R TNF receptor was capab3e of self-
association, and further, that ~hithin this ;Il~)a~ ul~ domain (p55-IC) there exists a region, the
so-called 'death domain'. which is ai'so capable of self sssrlri~fir~n and as such, in a lieând-
.; fashion~ c~n cause cytotoxic effccts on ccl]s (see ~cample ~) Utiiizin~ this self-
iO a sociation propf~ty ofthe p55-lC and its 'deâth domain' it ii thus posslble to construct a fusion
protein, using standard ~r,~u~l~v~lL DNA technolo~, containing essentialiy ail of the
.2 hlhu domain of a ~NF receptor such ilS the p75-R or pS~-R receptors. preferab~y the pS5-
R, and fused thNeto, essentiall-~ . il of the intraceilular domain ~pS5-1C) or the death domairl of
the p55-TC. In this way a ne~ fusion product is produced which has at one end the TNF binding
domain i.e., the eYtrArp~ qr domain ofthe receptcr~ and at its cther end the iMri~celiular domain
or the death dornain thereof wilich is capable of self-association. ~ccording]~, such a product can
oligomerize by self pQcor;qtirn between two (and possible rnor* p5~-lC or death domi~ins thereof
to yi~ld oligomers (or at least dimers) having at ~eas~ two TNF binding domain~i.
FIJ, ~ ,re, it has aiso been discovered in accordance with the presen~ invention. that the
'O Fas/A~OI receptor has a seif-associatin~, intracdlular domain inclusive o~ a self~-c~l~istin~
'death domain' having certain homology to the i755-IC and death domain thcreof (~xampie 2).
Accordirlgly, it is possib~e to corlstruct the soluble, ûligomeric TNF receptors of the invention by
fusing the ~ domain of the TNF receptor (as noted above) to the ".L~ ac~l ullLI domain
or the 'death domain' of the i~as/APOI receptor.
'5 In both of the above noted situatior~s, the oiigomeric TN~ receptors of ~he Invention are
soluble by virtue of having vnly the soluble ~ a~,cuui~ domain of the TNF receptor and the
soluble ~h~ u6"viol domain or death dornairt thereof of either the p55-R TNF receptor ûr the
FaslAPOI receptor, i.e the~ do not cor~tain the ~ v~_ ' (insoluble) domain of either type
of receptor.
The ~v-~aLlu-.L;vll of the above oligomeric T~F rec~ptors of the invention are detailed
herein below in Ex~mple ~. It should howe~er be noted that upon cùll~l.u~,~ivll of the oligomeric
ll~F receptors of the irlvention, there may arise a situation, heretofore not reported, that th~
domain of the 1~ receptor is capable of self-Ac~o~ti~n a situation that may not be
desirable as it could interfere with t'ne ability of the o}igomeric receptor to bind to t~Yo or more
T~ monomers of ti}e INF 1 ~ molecules or may lead to less than optimal binding of
wch INI; monomers. Accordingiy, in such a sinlatiOn, it is possible, by standard ~c
DNA proceciures, to modify the e ~ e~ domain of the TNF recep~or by, for example,
ddeti~ or wbstituting one or more ar~ino acid residues contained within ~he self-a~:,v~..~
regiontoprevem such 'r~to~ tn Such, ~J ~ - ;v ~ofthe ~ avellv~L domain ofthe T~7F
40 receptor are thus also rtart of the preserlt invention and are desi~nated herein aâ analo~s or
WO 9S/31S.~ 2 l 8 q 9 8 3 - r~ . 4
;l
derivatives of the ~ AI domain of th~ T~F receptor. In a similar fashion~ the self-
associating i...la~c lul~ domaia (IC) or death domair~ (DD~ thueof of the pS5-R receptor or the
FaslAPO1 receptor used in the oli~omeric TNF receptors ofthe inv~ntion. ma~ aiso be analo~s or
derivati~es thereof i.e. may be aa~ moriifir~tioTl of the pS5-lC sequence or portions theof
5 including the teath domain (p5~DD). or any ~louir~,a~iull of the Fas/APOI illLIGcelh.la, domain
tFAS-IC) sequence or portioas thereof inciudinE the death dom7Lin (FAS DD~, providinf~ that
tilese ,Y ~ r,. ~ . ., yield a self-associatirLg product.
Similarly, once produced and purified, the soluble oli~omeric TNF receptors, anaio~gs or
derivarives thereof may be further modified b~ stantard chemical means to proYide salts and
10 fimctional derivatives thereof for th~ purposes of prepari2~ p~ d~,eusi~,àl .~ .s~
comainin~ as active iagredients these TNF receptors of the invention.
For the produclion of the soluble. oligomeric 11~ receptors of the invention, the DNA
sequences encoding the ~ domain of the TNF r~ceptor are oblairled from existin~
ciones of the erltire TNF receptor~ as is the intra~Pll~ r domain or death domain thereof, and as is
15 also the ll~L~aC~ al domain or death dornain o~ the FaslAPOI receptor (see i xample 2 and
l~xample 5). In this ~ay the 3NA sequence of the desired ~LIO~,. Iula~ domain is ligated to the
DNA sequence of the desired i~lLI a~ lu~a~ domain or portion thereof includin~ the death domain~
2ad this fi~sed product is inserted (and liga~ed) int~ a suitable expressiorl vector under the control
of the promoter ar~d oth expression control se~uerLces. Once formed, the expression vector is
20 ir~troduced (t, ~ ;V~ ' etc.) jMO a suitable host cell, which then expresses the
vecror to yield the fusion protuct of the inv,ention being the soluble self ~corisrin~ TI~TF reccptor
rnolecules. These are Ihen purified frorn Ihe hos~ cells by standard procedures to yield the Snal
product beirJg th~ soluble~ oligomeric TNF receptors.
Ihe preferred preparation of the fusion product encoding t~Le . ,~ domain and
25 intraceLular domain or portion Ihereof is b~ wa~ of PCR technolo~y using o1igc,l~uc~ cs
specific for the desired sequenccs to be copicd from the clones ercoding the entire lNP receptor
molecule. Other means ar~ also possible~ such as isolatirlg trLe desired portions erlcodin~ the
~u~,llu~ dom2in arld the ill~acdhla~ domairl. by restriction ~ and then splicingthese to~ether irL a known fashion, with or v~ithout ~ov'f~ s at the terminal ends of the
30 restriction frar~mants to ensure correct fusion of the desired portions of the receptor ~r . I . ~ ,1~, .1 .
and inttacellular domains or portions thereof) The so-obtained fusion products are then irlserted
into the e~pression vector of choice.
In a similar fashion~ the preseM invemion also concerrls soluble, oli~omeric Fas/APO~
~FAS) receptors containing the ~ ` domain of the Fas/APO1 receptor and the seli-
35 ~ssociatine ~ ulal domain of the pS5-R (pS5-IC), the death dornain thereof (pSSDD), or the
self-associatin~ i".l~cclhlal domain of th~ Fas/~PO~ recei~tor (FAS-IC) or the dealh dornain
theof (~AS ~)D), or any analogs or daivatives thaeof (see above). The cv~ u~.Liv~l of these
soiuble, oligomeric E'AS receptors is detailed in Ex2mple i herein below, using an available cloned
full-lensth FAS reeeptor-encodin~ sequence as startin~ rnateriai and the
40 ~ ' ' for PCR production of the desired C tl~ " ' 2nd ;l~llacL~lular dom2ins~
WO 95131544 2 1 8 9 ~ 3 3 ~ 4
followed b~ 2tion theof to yield a fusion Froduct, which is then inser~ed imo a suitable
e~prer,sion vector. As detalled abo-e and beloY.. prol:arvotic or eui;aryûtic vectors and host eells
ma~ be used tO produce the desire~i soluble, oligGm~ric FAS receptors, ~hich can th~n be purified
and formulateti. as active ingredieM. jMO a rh7~ rir7~ ..,J~ ,;,,"
The above soluble, oiigomenc FAS receptors of the invention aie intended for effective
blociring of the Fas liLand, which ma,v aiso exis; as a trimer (sirniiar to ~NF, s~e above~ each
oligom~ric rec~ptor of the invention capable OL bindini, tuo ar possible more Fas ligands and
ti~ereby neu~raii7~ thei~ acti it~. Ihe Fas ligand is known to be p~J~ ~ n ~l~ ceil-surface
associated but may aiso exist in a soluble form. In any eveM, the oligomic FAS receptors of the
invention can bind to at ieast r~vo n]onomers of ;his ligand and tberei~ neutraiize more effectivel-
~than monomeric FAS reeeptors) the acti~ir of Ihe Fas ligand. ~he F2s ligand~ and hence
activarion thereby of the FAS r~ceptor. has be~n irnplicated in a number of p7thnloriç~ states,
particulari~ those relaring to iiver dama~e (apoptosis of l~ aiu~ s, for example) including li~er
damase associated witb hepatitis, as well as ir. ~ conditions~ including Iymphocyte
darnage (apop~osis) ir~ Xi~ infected humans (see~ for example Ogasawara a al., 1993, Cheng et
ai., 1994). .~ccordingiy, the soiuble, olii~omeric F.~S receptor~ of the inv~ntion are iMended for
blocking tbe acti~ity of Fas iigand and ma ~e used as active ingredieM in i,r,4"..~
r~ for treatinE such Fas liEand-associated r~h~lr~A l stues
Likevise, the present invention aiso concerns soluble, oligomeric receptors which have
20 bindirl~ affinity for both ~iF ard FAS-R ii~and~ the so-caiied "mixed" ~NF-R/FAS-R oligomeric
receptors Tbese mixed oligornelie reeeptors will contain at least one lNF-R e~ r dornain
3nd at l~ast one FAS-R ~ dom_in which are associa~ed in the oiigomeric reeeptor by
vjrtue of each of these . " ' domains bein~7 fused to any one of the 2bGve-.~,..;one~, selt:
~ssociatinb, p55IC, p55DD, FAS IC or FAS DD
These rnixed oligomeric receptors may be prepared by: (a) providin~ a-y of tne ~o~e
Doted fusion produets ~vhieh eontain the ~ - " ' domain ûf a ~iiF-R (p75 TNF-R~ or
preferahly, p55 INF-R) fused to any one of th~ seif-assoeiating ;~ a~di~l ~ dornains pS S ~C 2nd
FAS IC or any one of the seif-associating 'death dornain' pS5DD and FAS DD, or any self-
associatin~, por~ions, anaiogs or derivatives of any thereof; (b) pro ,iding ar~ of the above noted
fusion products wi.ich eontain the ~ IS:.~ dornain of FAS-R filsed to any one of the self-
2ssoeiatjng p55IC, FAS-IC, p55DD, and FAS DD, or any self-associating portions, analo~s or
derivzbves of 2ny thereof; and (c) mixitl~ anS~ of the I~F-speeific fusion produets of (a) with any
of the FAS-R ligand-rpeeific fusion products of (b) to provide ~fol~owin~ s~arldard se~etion and
purification procedures) o~i~omeric (dimeric or higher older oligomerie) reeeptors which have at
least both the ~ . l, A. . Il~ '1~ ' dom2ins of a I~F-R and FAS-R that are assoeiated by virtue of the
sdf-~ssoeiation c2pability of their fused IC or DD re~ior~s.
Anoth possibility for the preparation of the above rnixed oligomeric reeeptors is by eo-
suitable host eells with the dbo.~ LIo"~ expr~ssion veetors, one of which
cncotes the INF-specific TNF-R fusion produets and one of wilieh encodes th~ FAS-R iigand-
specific FAS-R fusion produets. Following the expression of these different fusion products in tile
_ _ .... . . _ . .... ..
WO 95/31544 2 1 8 9 9 ~ 3
host cells, ~he mrxcd oligomeric (T~-R,FA~-R) re~eptors may be obtained b~ s~andard
purification aDd seiection procedures.
The utiiity of these mixed affinity oligome:~c receptors is primarily for the neutraiization of
both TI~F and F.~S-R ligand when these are o--r-expressed ~...1ræ~ a.~ ly or are al undesirably
S high levds fo~lou~ing exogenous allllUli~UG~iVII. Recent e~,idencs points to a likelihood that tilere
exists a synergism Ln function between the FAS-R ii~and (usuaily celi-surface associated) and
l~iF-o (which may aiso be celi-surface associale .~. Accordingly, in some inStaDCeS it is desired to
neutraiize botD of these iig,ands a~ the same pOI.~Lt on the cell surface. i.e such a rnixed-afLinitv
recep~or can block both the TNF bindin~ to its rec~plor and the bindirlg of FAS-R ligand to it~
IQ receptos. .~c ordin_ly, these mixed-af~init receptors may bc used as an active ingredien~ in
pl.~,,..- ~".;. ~ ,,.,..l,Oc;l; ".c for treatin~ such condilions (see above) where both T~ and FAS-
R li~and effects are undesirable.
Similarly, G~ong the lines mentioned abo- e concernirlg the solub~e, oiigomeric TNF-R and
FAS-R, and mixed TNF-R/FAS-R oli~omers or ~he invention, it is aiso possible to produce
15 soluble, o~ omeric rece~otors for other recep~or~, or any mixtures thereof, in particular those of
an.~ of the other members of the TNF/~GF super famii~. In this case, any of the extraceilular
domains of the various receptors can bc fused to the abo~ e-..._l.,iGL~cd sdf-assoclaring
; lna~dlulal donLains or portions thereof or to an ~ other i,.L- G ~llulal domains of the super family
members also capable of..11 a3..~;aLUIl.
Expression of any of the . pro~eins of the in~ntion as mentioned herein can be
effected in eukaryobc cells (e.g. ycast, insect or marnma~ian cd~s), urin~ the appropriate
expression vectors. Any m~thod known in the art may be employed.
For cxat~ple~ rhc DNA molecules coding for the proteins obtained by any of tbe above
methods are inserted into ap!.,up.;Gi~l~ consttuc~ed expression vectors by techniques well known
rn the art (see Sarnbrook et al., 1989). Double-srranded cDNA is linked to plasrnid vectorr ~
h~ulv~ tsilino or by restrictiori linking involvin~ the use of syrLthetic DN~ linkers or
b~unt-ended li~ation techniques. D~A ligases are used to ~igate the D~TA molecules and
undesirable joinin~ is avoided by treatment ~i~h alkaline ~ .
In order to be capable of expressing the desired protein an e~tpression vectot should
comprise also specific nucleotide sequences containing ~ and t._ regulator~
- linked ~o tbe DNA coding for tlte desired protein in such a way as to permit gene
expression and production of the protein. Firs~, in order for the gene to be tr~ncr~ i it must be
preceded by a promoter lPCO~ by RNA po~ymerase, to which the po~ymerase binds and
thus initiates the l. .- ;I,t; l~ proc~ss. There are a ~ariety of such promoters in use, which vY-ork
3~ with differeM eftiaiencies (strong and heai~- pronnoters). Tbey are different for ~uL~y~JLic and
eukaryotic ceiis.
The promoters that can be used in the present invention may be either co~.,li~u~ or
exampie the ~nt promoter of 1.~ , the ~ promoter of the ~-lactamase gene of
pBR32~, and the CAT promoter of the ,~ acetyl t~ansferase gene of pPR325, etc
or indûàble, such as the }" ~L~uyvl;~ promoters including the major right and ieft promo ~ of
WO 9S/31S44 2 ~ ~ 9 9 ~ 3 r~ . 4
;4
t ~ c, !~ ~L and P~ the ~, ~, l~æ 1~1, Qm~E. and c~al promoters of ~;: coll. or tne
tri~-la~ h~brid promoter~ etc. (G~ick, B.R. (Ig~, `,. Besides the use of stron~ promoters to ssenerate
~arge quantities of mRNA. in order to acbieve ~ h levels of gene expression in ~.. uh~ uLiC CellS,
it is necess2ry tO USe aiso ribosome-bindinr! sites to ensure that the mRNA is ef~icientiy translated.
One exarnple is the Shine-Daicc~arno sequence (SD sequence) ~ylu~ positioned from the
initiation codon and ~ .y to the ~'-terminai sequence of 165 RNA.
For eukaryotic hosts, different ~.~ulscl;pLivl,al and ~ .!. rec~ulator) seguences may
be employed, depending on the nature of the hc~st. They may be d~rived from ~irai sources. such
as adeno~irus, bovine papilloma viruS, Simian ~ :rus, or the lilie, svhere the re~ulatory signals are
10 associated with a particular ~ene which has a h~h levei of expression. ~xamples are the TK
promoter of i?;erpes virus. the SV40 early promoter, the yeast gai4 C~erle promoter~ etc.
T, ~ iullol initiation regulator- signais ma~ be seiected whiGh allow for repression and
aciivation. so that expression ofthe ~enes can ~e modulated.
The DNA molecule comprisir~O the nucleotide sequence codin~ for the fusion product
1~ proteins of the invention is inserted into a vector havin~ the operabl~ lin};ed ~lal~cl;~L;vl~ci and
~u.~lc,iioQ~l regulator si6nais which is capable of intei~atin~ the desired ~ene sequences intû the
host cell. The celis which ha~.e been stablv ~ i by the introduced DN.4 can be selected b~
iiso ir,troducircg one or more markers ~vhich allow for selection of hosl cers which con~ain the
expression vector. The rnarker may pro~ide for 1'- ù~ to an 2uxotropic host, biocide
20 resistarlce, e.g. antibiotics, or hea }~ metals, such as copper, or tbe lilce. The selectable marker
~ere can eith be direct'iy linkcd to the DN~4 ~ene scquences to be expressed, or introduced into
the same cell by CO~i~dC.arc~,Liull. Additiûnal elements may aiso be needed for optimai synthesis of
proteins of the invention. These elements ma~ include L~cu~ J~;ul~ promoters, enhancers and
termination sic~nais. cDNA GXpression vectors ;II.,UI~JUld~ C, such elements inc~ude those
25 described by Okayama, ~. (lg83).
In a preferred ~ -u '~ the imroduced DNA molecule will be ;..~u,pu...~'i into a
plasmid or viral vector capab}e of ~ . repliution in the recipient host. Factors of
importance in selecting a particular plasmid or virai vector include: the ease with which recipient
cei'.iS that contairi the vector may be reco~zed and selected iArom those recipient cells which do
;0 ~ot contain the vector; the number of copies of the vector vhich are desired in a particular host,
~nd whether it is desirable to be able to "shuttler the vector berveen host cells of differenr
species.
Preferred p~uikal~uL;-, vectors include plasmids such as those capable of replicztion in ~.
coJi, for example, pBR3~2, CoEI, pSC101, pACYC 184, etc. ~see Maniatis et al.~ 1g82;
Sunbrook et al., 1989); Bzcillus plzsmids such zs pC~94, pCq'11, pT12~, etc. (Gryczan. T
(1982)): SLlcp~ ~ plzsmids ir~clu&n~ plJ]ûl ~endail, K.J. et ai., (19~7)); SLIel~Lul~ .
i'' ' '~r~ l_ ~ such as 0C31 (Chater, KF. et al., i~: ~i~h 1..1,.,.~l,..,.~1 Svm~ncillm on
'(1986)), and ~ plasmids (John. J.F. er al., (1986), and
~z~ki, K (1978)). Preferred eul~otic plasmids include BPV, vaccinia, SV40, 2-r~icron circle,
40 erc.. or their deriv~ti~cs. Such plzsmids are well . nown in the arr (Botstein, D. et ai., (1982);
WO 95/?1544 2 ~ ~3 9 9 8 3 r~ 4
i
~roach,J.R in:Thei~n1~.~o~Biolo~nftha~east~ .""...~ ri~c~r~P an~lJnhoritrrr~
(19~1); Broach, J.R, ~lg82); Boiion. D.P. ai., (1980); Maniatis~ T., in: Cell BioloLrv A
C~ e T~ti~r Vnl; ~ nf~ Fa;,n~rc~ n (~980~; and Sambrook et ai., lg89).
Once the vector or DNA sequence containin~ the construct(s) h~s been prepared for
S expression~ the ~)?`IA construct(s) may be introduced into an appropriate host cell by any of a
variet~ of suitable means: ~" .,ru~ à~iùl~ tr?~f~rtinn cnrlrl?atinn~ protoplast fusion,
cl~"~lv~Ju~dliu~l, caicium phosphate~ c.ij,i~ iu,l, direct l~ u;LLj~lio~. etc.
Host celis to b~ used irl the invention may be eitiler prokaryotic or eukaryotic. Preferr~d
prokaryotic hosts irlclude bacteria such as ~. ca!i~ Baciilus, S~CJJ~UII~?..~S~ P~
10 Salmonella, Scrr_tia. etc. The most preferred prokatyotic host is E ev~/. Baaeriai hosts of
particular interest inciude F coli Kl' straill ''91 (ATCC il~46), ~. coli Xl /76 (~TCC 31s3~),
i~. c~oli ~'3110 (~-, lambda-, prototropic (.~TCC '?3'5)). and other ~ clv;a~,~t~lm such as
Saimonella l~.h;.l.u,;~ or Serratia marcescens and various p~ ln,~ species. Under such
corlditions~ the protein will not be ~ UU ?~la~e i. The prok~Lryotic hûst must be compatibie ~,ith the
15 repiicon and control sequences in the expression plasmid.
Preferred eu~ argotic hosls are mammaliarl cdls, e.Y human, monicey, mouse and Chinese
harnster ov~ (CHO~ cells~ because thç- provide pOSt-~lr ~ Liul~ ,r~ to protàn
molecules including correct folding or gl~cosylaion at correct sites. Also ~easts cells can carry
out post-L., ~ 1 peptide ,..~.I:r;. ~1,....~ including gl~_o~ L;oL~. A number of ~c..u.,.v........
20 DNA strategies exist which uti!ize slrong promoter sequences and high copy number of plasmids
which can be utilized for production of the desired proteins in yeast. Yeast reco~nizes leader
sequences on cloned manunalian gene products and secr~tes peptides bearing leader sequences
(i,e., pre-peptides).
Af~er the introducrion of the vector, the host cells ~re grown in a sel~ctive medium, which
25 selects for the growth of vector-containing cells Expression of the cloned gene sequence(s)
results in the production of the desired proteins.
Purificarion of the .~.GIIl'v;l~ proteins is carried out by any one of the methods Icnown
for this purpose, i.e. any cu~ diullal procedure involving extraction, plc~.;v;~al;OI~,
~,hl~ C~ or the lilce. A furth purification procedure ~hat may be used in
30 preference for purifying the protein of the in~ention is af~init~ cl..v ~ pl y using anti-TNF
rec~ptor monoclonal antibodies, which are produced and ' ' on a gd matrix coMained
within a column. Impure p epLaLivlL; containing the l~i ' ptotein are passed throu~h the
column. Ihe proteirl wiD be bound to th~ colur~n by the specific antibody while the impurities wil~
pass through. A*er washing, the protein is eluted from the gel by a change in pH or ionic
35 strength.
As used herei~ (see above), the ~erm 'salts' refers to both salts of carboxyl groups and IO
~cid addition salts of amino groups of the protein molecule forrncd by means known in t~e art.
Salts of a carbox~.l group include inorganic salts. for example, sodium, calciusn, and s21ts with
organic bases as thos~ for~ned, for e~nple, ~ith amines, such as ` ' , arginine or
wo 95/31544 2 1 8 9 9 8 3
lysirl~. Acid ad&tion salts include, for example, salts with rnineral acids and salts with organic
sàds.
"Functionai d~rivatives" as used herein covers delivatives which may i~e prepared frorn the
functionai groups which occur as side chains on the residues or the N- or C- ~erminai croups~ by
S rneanr known in the arr, and dre inc~uded in the invemiop as long as they remain ~ ly
scceptsble, i.e. the~ do not destrov the acti~ir.~ of the protein and do no~ confer toxic properiies
on rv .j~rJ~ C containing it. These derivati~ es inciude siiphatic esters or amides of the carbox~l
i3roups, and N-acyl derivatives of free amino groups of O-acyl derivatives of free hydroxyl ~;roups
formed with acyl moieties (e.g. aii~anoyl or carboc~clic aroyl groups~.
"Fraction~c" aS used herein refers to an- part or portion of the receptor, (;l~LI-L~; IUI_ or
w.L d.,el~u:ar domairLs thereof), or of the pro~eins bindin~, to the ",l~ .,i domain of the
receptor, provided it retains its bioio~icai 2cti~it~.
As mentioned above, tile present invention aiso re]ates to various ~ f~
c~ ;... comprising a pl.~ C~;.Iu(,dll. acceptable carlier and the various noted aclive
1~ in~redients of the invention or their salts, functionai deri~aives, or mixtures of any of the
foregoing. These . ,.,.~ may be used in an~ ofthe conditions ~s noted herein. for example,
in conditions where there is an over production of endo~enous TNF, such as in csses of septic
shocic, cache~-ia, ~,raft-versus host resctions, ~lu diseases iike rheumatoid arthritis. etc.
Th~ way of a-i ' `I~AI;UII can be via an~ of thc accepted modes of ~ t,r... for simiiar
~O agettts and will depend on the condition to be treated, e.g. woen used to inhibit lNF effects they
may be ~;..,~ L.- ~.,.,u..~'~ in case of septic shoci~ or local injection in case of rheumatoid
arthritis (for example. into the icnee~, or .,u~.. i.. uv.. il~ b~ irmlsion etc. The (.. ~ v,:l ;v ~s may aiso
be used, for e.~iample, in cases of INF :. ~ l;- caused by exogenous ~ ;u,l of
excessive amount (overdoses) of TNF, e.~. in the case of cancer therapy or ~irai disease therapy.
The ,v~ c;.l...................................... of the invention are prepared for adl 111;.7LI~Li.. !! h:
mixing the protein Or its derivati~es ~ith ph~ acceptable carriers, stabiiizers and
excipients, and prepared in dosage form, e.?~. by 1~. ,' " in do.cage viais. The amount of
active compound to be ' c~ wiil depend on the route of .~ , the disease to b~
treated and tite condition of the patient. For example, locai injection in case of ;.,n~ ...y
3û conditior~s of rheumatoid arthriti will require less active in~redieM on a body weight basis than
~vill intravenous infusion in case of septic shock.
Other aspects of the i3vention will be apparent from the follo~ing c carnpies.
The inveoion will now be described in more d~taii in the following non-limi~ing examples
snd rhe ac~,v...y...", ;~.~ drawings
FXAMPLF I
~' n,p ' isolstioo of proteins whi h bind to the ~ o~ ' - p~ and
~75 T~F rec~ptors
To isolate proreins interac~ing wito the ~ " ' domains of the pSS and p75 TNF
40 receptors (p~51C and p75 IC), the yeast two-hybrid system was used (Fi~lds and Song, Ig89).
wo 95/3 1544 2 1 8 9 9 8 3 1 ~ ., .,~ ~T
3,
Briefiy, this two-hybrid system is a yeast-based genelic assay to detect specif~c protein-prOtein
interactions ~n ~vo by restoration of a eukaryotic u 7 ' ;r~ acivator such as GAL4 that hzs
t~vo separate domains, a DNA binding and an acti ~. ztion domain, which domains when expressed
nnd bound together to form a restored GAL4 protein. is capable of binding to an upstream
5 activating sequence which in turn activates a promoter that controls the expression of a reporter
gene. such as lacZ or r~s3, the expression of which is readi~y obsenet in the cultured ceils. In
this system the genes for the candidate interac~inS proteins are cloned jMO separate expression
vectors. In one expression vector the sequence of the one candidate protein is cloned in p.lase
with the sequence of the &.414 ~)NA-binding domain to generate a hybrid protein with the GAL~
10 DN'A-binding domain, and in the other vector the sequence of the second candidate proteiA is
cloned in phase ~vith the sequence of the GAL1 activ,aion domLIin to ,,enerate a hyorid protein
with the GAL4-acti~arion domain. The two hybrid vectors are then co-L.r..~r~ into a yeast
host strain having a lacZ or HIS3 reporter yene under the control of upstream GAL4 binding
sites. Onl5~ those u r ~ host cells (c~4 l~rLJ~lll4llt~) in which the two hybrid proteins are
15 e.~pressed and are capable of intactin~ ~ith each other. will be capable of e~:pression of the
reponer coene. In the case of the lacZ reporter gene, host cells expr~ssing this gene ~vill become
blue in color when X-gai is added to the cultures. Hence, blue colonies are indicative of the fact
that the two cloned candidate proteins are ~apable of interacting with each other.
Ustng this rvo-hybrid syster.l. the ;...._ ' ' domains pS51C and p75IC were cloned,
separately, into tile vector pGBT9 (carrying the GAL4 DN.4.-oinding sequence, provided by
CLONTECTl USA, see below), to create fusion proteins with the G.4L4 D~TiA-bindin$ domain
(sirAilarly, the ;.~il4~ UId,r domain, FAS-IC and a portion of the 55IC, namely~ the 55~D were
also cloned into pGBT9 and used to isolate o~her IC-binding proteins see Fxampie 3 below). For
the cloning of pSSI~ and F~SIC into pGBT9, clones acoding the full-len~th cDNA sequences of
p55 TNF-R (Schail al., 1990) and p75 TNF-R ~Smith et ai., 1990) were used fi~m ~hich the
i.~tl~cellul~ domains (IC) wae excised as follows p55IC was excised using the enzymes ~coRI
and SaiL the Eco~U-SalI fragment containing the p55IC sequance was then isolated by standard
procedures and inserted into the pGBT9 ~ector opened, in its multiple cloning site region (~CS),
wjth EcoRT and SaU. p75 TC was excised usino the a~Aes BspT~ and Sall, the BspElI-Salr
fragment containing the p75 IC sequence was then isolated b~ standard proc~dures and filled-in
with the Benow enzvtne to ~enerate a fragment which could be inserted into the pGBT9 vector
opened with Smal and Sall.
The above hybrid (chimeric) vectors were then ~u~,-..-r . l.1 (separately, one
~,uL r ' with the p55IC h~brid and one with the p75 IC hybrid ~-ectûr) together with a
3~ cDNA library from huln~n HeLa cdls cloned into the pGAI) GTI vector, bearin~ the GAL4
activating domain, iAto the ~7c yeast host strain (all the a~ove-noted vectors, pGBT9 and
pGAD GH catlying the HeT,a cell cDNA library, and the yeast strain were purchased from
Clontech Laboratories. Inc., USA, as a part of MATCHMA~ Two-Hybrid System~ ~PT1265-
1). The co l~f..~.t~ yeasts were selected for their ability to grow in mediurrl lacl~ing HistidiAe
(ETis~ medtum), growing colonies being indicative of positive ' The sdected yeas~
wo s~ 544 2 ~ 8 9 9 ~ 3 ~ -4
clones ~-cre then tested for their ability to ex?reSs the lacZ gene, i.e. for their LAC Z activir~ and
this by addin~ X-gal to thc cu~ture mcriiurr~ hich is cuabolized, to form a blue coiored product
by ~ , the enz~me encoded by the lacZ oene. Ihus, blue colonies are indicati~e of an
~ctjve lacZ ~ene. For activity of the lac~ sene, it is Decessar~ that the GAL4 L~ J.ioll
activator be present in aLt active forrn in the ~ clon~s~ namely that the GAL4 DNA-
binding domain encoded by one of the abov,e hybrid veetors be combinëd properl,v v.~ith the GAL4
aion doroain encoded by the otber hybrid vector. Such a ~ is onl~ possible if the
t~-o proteins fused to each of the GAL4 dornains are capablc of stably interacting (vinding) to
e~ch other. Thus, the His I arld b~ue (LAC ;~) colonies that were isolated are colonies which
10 haYe been ,uL,a,., acd with a tector encoding pSSlC and a vector encodin~ a protein product of
hurnan HeLa ceil orisin that is capable of binding stably to p~ 1C: or ~hich have been uarsfected
with a vector encodin2o p7~1C and a vector encoding a protein product of human HeLa-cell origin
that is capable of binding stably to p75 IC.
Ihe plastnid r)NA from the above His, LAC ~ yeast coloniej was isolated and
v~ , i into E coli strainEiBIûl b~ andard proc~dures folio ved b . selection of Le~+
~nd Ampicillin resistant ll^ \-r~ these r being the ones carr~ing the hybrid
pGAD GH ~eclor which has both the A npR and Leu2 coding sequences. Such ,~"~r.,l,..A..:~
therefore are clones carryins the se iuences encoding nett~ly identified proteins capable of ~indirts
to th pS51C or p751C. P~asrnid Dl~A t as then isolated fror~ these L~^, c.v..l~,.l E. coli and
20 retested by:
(a) ~ them with tbe orisinal - domain hybrid plasmids (hybrid
pGTB9 carr~ins either the p55~C or p75IC sequences) into yeast strain HF7 as set forth
bereinabove. As controls, vec~ors carrying irrelevant protein encodin2 sequences. .g. pACT-
1~min or pGBT9 aione were used for ~vl~ .... tvith the pS51C-bindin~ protein or p751C-
binding proteilt encoding piasmids. The, ~ - yeasts vere then tested for oro~vth on His~
medium aione, or v~ith differerlt levds of 3 . t.ih~V~" and
tb) ~ ~ _ the plasmid DN~ and ori~inai ;.-l~C~ ~ddl domain hyi~rid plasrnids
snoi control plasmids described in ~a) iDto yeast host celis of strain SFY5~6 and ~ _ tbe
~AC Z+ ILCtivity ~effcctiv*y of ~-sai formaliorl, i.e. blue color formation).
Th~ results of tbe abotr tests reveaied that the pdttern of growth of colonies in Elis~
roedium was idenncii to the pattern of LAC Z acti~itS, as assersed by the color of the colony, i.e.
~iis+ colorlies were aiso LAC Z+. Furtoer, the LAC Z activity in iiquid cuiture ~preferred cuiture
conditions) was assessed after traosfection of the GAL4 DNA-bindin~ and actitation-domain
byb~ids into tbe SF~526 yeast hosts ~hich have a better L.9.C Z inducibilitS~ with the GAL4
, actit~dtor than toat oftoe HF-7 yeast host cells.
Tbe results of the above co-L.~ c dre set forth in lable 1 below, from which it is
apparent tost a number of pro~eins were found that were capable of bindirl~ to the pSSlC or the
p75IC, namely, toe proteins desigDated 55.11, which binds to tDe p551C; and 75.3 and 75.16
. whicb bind to tbe p75IC. All of these p55IC- and p751C-binding proteins are authentic hurnan
proteins all encoded by cDNA sequences originating from the HeLa ceil cD~A libralg, which
, . .. . .... .. .. ...... .......... .... ....... .... ....... ..
W095131544 21 8~3 ~ 4
39
~ere filsed 10 the G.~L~ activation-domain sequence in the plasmid pG~D GH in tbe above ~east
wo-hybrid analysis system
L,t~ .i`..ul~, it was also found that fraemems of the p~SlC~ itself, name]~, the proteins
desi~nated SS I and 55 3 ~vcre capable of binding to pSSlC These are discussed also in Example
5 bclow
TABLE I
SUlvll~ARY OF l~IE C~ARACTERISTICS OF SOME OF THE
cDNA CLONES (SEE ALSO EXAMPLE 3) ISOLATE;D B~' 'r~E
~VO-lIYBRIDSYSTEM APP~OAC~
DNA-bindin Actjr.~ation- Colony color Lac Z ACti~it in
doma t v~ d domllin byb~i(l liquid clllture RSSRy
pGB~ w tite
pGB- - b ue
pG13 ' - , b ~ e ~'
wlte
:w~ te
pA '-Lamin w t te
pArT-l - nin : wlte
p : - ~ w-Lte " '
p l : wlte
p~iBT''- ~55 b ~ e
~tte
pAC- ,amin w t te ~
p~-. T9 w ~ te ~ ~'
pGB- -IC75 75 ~ b ~e
pGB'- -IC~S --- wl te
- 3 w Lte
pACT-La~nin ~ 3 u-Lte ~-~
pGBTg ~ 3 w l te ! -`
pGBT9-lC75 - b I e
wlte
pACI-Lamin ~ w l te
pGBTS ~ w t te ~ _
~0 In the above Table l, the plasmids altd hybrid encoding the G.4L4 DNA-binding domain
and GA~4 act~vation domain are as follows
DNA-bindin~ domain hYbri~l~
pGBT9-lCSS full-len~th ;.,.,...~l~a,l~ domain of the pSS-T.~F-R ~p551C)
pACT-Lamin irreleYant protcin - lamin
pGBI`9 vector alone
pGBT9-IC75 fi~ll.lengtlt ;,.tl~cell~la domain of the p75-lNF-R (p75iC)
A~tiv~ti~n~' hy~rid
55.1 and 55.3 correspond to frasrnents of the il~Ll ~,~ cllul~, domain o~the pS5-TNF-~
55 11 is th~ no~el protein associating u~ith the p55-T~l~-R
75 3 and 75 16 are the novd proteins associating with the p75-TNF-R.
.
. . .. .. _ .... . . ... . . . . . ..... .... . . .. . .. ..
W095/31544 2 1 89~ 83
~o
The aboYe no~ed clonet cDNT.~s encoding the novel p-i5IC- and p~5IC. binding proteins,
55.11~ 75.3 and 75.16, were Iben sequenced using standard l)NA soquencing procedures. The
partial sequence of all of th~se protein-encodin~ sequences is set forth in Figs. I a-c, where Fig.
~(a) depicts the sequence of tlle cDNA encoding prolein 55.11, Fi~. I(b) depicts the parlial
S sequence of the cDlss~.~ encodin~ protein 75.3; and Fig. I~c) depicts the partial sequence of the
cDNA encoding protein 75.16. In Fig. I(d) there is shown the deduced amino acid sequence of
the protein 55 ~ I l, as deduced from the nucleotide sequence of Fig. I (a) .
It should be noted. however, that a partiat sequence of the cDNA encoding the 55 11
prolein has also been reported by Khan et al. (1992), in a study of human brain cDNA sequences,
10 which study was ditected at the ~,lc~ of ~ new rapid ~od accurate method for the
sequeDcing and physical and genetic rnapping of human brain cPNAs. However, Khan et al. did
not pro~ide any informalion as regards the function or any other ~ aJa~.Lc~i~Li~,a of the protein
~ncoded b~ the 5~ . I l cDN.~ sequenc~, such functional or other analysis not being the intention of
~han et at in their study5 Ar~v5js ~ha...~l~.;Ldl :rofthe5S.llnrotein
G I P~. r. .1...~, and MP~rria~s
(i) Clonin~ of the cDNA of 5~.11
Upon the analysis (for exanple, Northern Anatysis -see below) of the
cDNA of protei~t 55.11~ it was reveated that the above noted 55.11 cDNA cloned by the tlhO-
70 hybrid screen procecture represeMed onty a partial cDNA of 55.11 having nucleotides 925-2863
(seo Fig. I~a)) which code for amino acids 309-900 (see Flg I(d)). The rcrnainin~ part of the
~5.11 cDNA [n~ titlr~ 1-9~4 (Fig. 1(a)) which code for amino acids 1-308 (:Fig. I(d))] ~vas
obtained by standard procedures, namely, by cloning by PCB. from a human fetal liver cDNA
library (for more details, see below). The fuD nucleotide sequence of 55.11 (~is. I(a)) was
2~ determined irl both directions by the dideoxy chain termination method.
(~) Two-hvhrid ~ ' trct~
los ~ expression tests were performed as described above,
acoept that in ~com~ ofthe tests, the pVP16 vector, which contains the activatioD domain of VP16~
was used itlstead of pGAD-GH, the Gal4 activation domain vector. Numberin,~j of residues in the
30 proSeins encoded by the cDNA inserts are as in the 5wiss-Prot data bank. Deletion mutAnts were
produced by PCR and point mutations by ~ directed ~ c ~Cunkel~ 1994).
~i ~ North~rn ~n~lly5js
Total RNA was isolated using TRI REAGENT (Molecular Research
Center, Inc., Cincinnati. Oh.~ U.S.A.~. denatured in formaldeh~de/formamide buffer~
3~ l.u.cjcd through an a~,GsJ~ J~ gel, and blotted to a GeneScreen Plus
membrane (I:iupont, Wllmin~on, De., U. S.A.) in 1 0xSSPE buffer, using standard techniques. The
blots ~vere hybridized with the panial cDNA of ~.11 (see abovc~ nucleotides 975-2863),
,~Ai~.l l,rl.~.~ with the random-prime kit (Boehringer Mannheim P~io~h~mi~a Mannheim~
Germany). Md waslled strin~ently. Au~u~dio~ waS performed for i ~veek.
WO ~5131544 2 1 8 q 9 8 3 ~""~
~1
(i-) E;~pr~ssion of ~.11 eOX.~ in HeLa cells And ' ' ~iinr~ orthe 'CC II Droteinto o S-LI .-~.if~ of pS~lC
Glutathione S-transferase (GST) fusiorls ~vith p55-lC ~&ST-p551C) and
with p55-lC trunczted belo~v amino acid 345 (GST-p55IC345) were produced and adsorbed to
5 ~ l " D-ag,~ose beads as described in Exarnple 2 below (see also Smith and Corcoran 1994;
Fran~ioni and Neel~ 1993). Thc cDNAs of ~5.11 (1-2863 nucleotides, i.e. the full-length 55.11
cDNA), of FLAG-55.11~ and of luciferase were expressed in Hel,a cells. FLAG-55.11 is the
region extendin~ between residues 309 and 900 in the 55.11 protein (the partial cDNA of 55.11
(n~ ti~lPc 925-2~63), originally cloned by the two hybrid screen), N-linked to the FLAG
10 octapeptide (Eastn~an Kodak, I~iew Haven~ Ct., U.S.A.). Expression of the fusion proteins was
- , " ' ' using a letrac~cline-controlled expression vector (HtTA-I) in a HeLa cel~ clone that
expresses a tetracycline-controlled ~ a~.Liv~LOl (see Example 2 belov~, and Gossen and Bujard,
1992). Metabolic labeling of the e.xpressed proteins with r3js3 I.Ie~ and [35S] Cvs (Dupont,
~rilmington, De.~ U.S.A. and Arnersh2m, ~ CI~ Enyland), I,~sis of the HeLa cells,
15 ;~ Urlo~ ;t~L;.,.., and binding of the labeled proteins to the CrST fusion proleins were
performcd as described below (Example 2), except that 0.5~O rather than 0.1/~ Nonidct P-40 was
present in the cdl Iysis buffer. The r ~ ;l,ali~ of 55.11 and FLAG-55.11 ~vere
achieved using a rabbit antiserur~1 (diluted 1:500) raiscd against a GST fusion protein containing
the region of 55.1i that extends bet~veen ar~LinO ~Lcids 309 and 900 and a rnouse m0 sntibody agatnst the FLAG octapepride (l~q2; Eas~man Kodak; 5 ~Lg,'ml of cell Iysate).
indine o,f th.~ to n55-1C ~ithin tr~n~fnrmed YeAsts
In this study it was sought to ascertain the nature of the bindin betv.~een 5~.11
and pSSIC, in particular, the regions of both of these proteins in~ol~Gd in thts binding. For this
purpose the above tu-o-hybrid procedure ~as used in uhich various full-len~th and deletion
25 mutants of p55IC (see also l~xample 2 below) in UDNA-binding domain" constructs were used as
hbaits" to bind the ''preysU, being the partial 55.~1 protein encoded in constructs in ~hich the
pattial 55.11 sequence (residues 309-900, as originally isolated) was fused to the "activation
domain" in the vectors GAUAr~ and ~P16AD. Further~ various deletion mutants of 55.11 were
also constructed and fused to the hactis.ation domain" in the GAL~ ector (e.~. mutants of
30 55.11 having only residues 309-6S0 ar~d 457-g00). The bindirlg of the various 'binding domatn'
constructs to the various 'activation domain' constructs was exan~ined in transfected SFY5?6
yeast cells The binding was assessed b~ a Iwo-hybrid ~ y~l Iv~ expression filt~r assay. The
non-relevant proteins SNF1 and SNF4 s~ned as positive controls for the 'binding domain' and
'activation domain' constructs, le~ , the empty Gal4 (pGAD-GH) and ~16 (pV~?16~
35 vectors ser~ed as ne~ati~e controls for the 'activation domain' constructs: and the empty Gal4
(pGBT9~ vector served as a ne~atiYe control for the ~indin~ domain' constructs. The results of
the assa~ are set fonh in Table 2 below in ~hich the symbols "~" and " " indicate the
d~ lv~ of strong color within ~0-60 min of initiation of the assay, r~s~ (positiYe
bindin~ results); and "-" indicates no ~velopi~.~."i of color within ~4h of c~, ,." ., 1 of the
40 assay ~ne~stive results). Blanl~ spaccs in the Tablé indicate bindin~, assays not tested.
WO 95131544 2 ~ ~ 9 q 8 3 ~ . 4
- ~kl~
Binding of the 55.11 protein to p55-IC
within transformed yeasts
~n
mC~
~ 6 ~o + + f
o
O `~
F u~
+
m
5 T 6'~
~ ~9
e~ ~ 60~? 1
6`60 + + + + +
~ '' Z
I ~
~ _
Z-- i'
~Wo9~131544 2 1 ~q9 ~3 p ~ 4
43
From the results pres~nted in T~ble 2 a~ove it ma~ bç cor~ciuded that jS.I I ~iDds to p~-
IC at a site which is dislinct from the 'death domain' (residues ;2~26) of p55-IC.
The 55 11 protein bound to a trur,cated pS~-lC trom which the death domain had be~n
deleted (construct ~06-328 in Table 2)~ more effecivd~ than to llo,ll-u"cated pSS-lC. ~t also
S bound to an even further C termina~ly truncated construct (coDstruct 206-308) and to a construct
from which both thc death domain and a membrane proximal pan were delded (construct 243-
3~8). Ho~vcve~, the SS.II protein did not bind to a construct that was N-tcrminally truncatcd
down to anino acid 266 (Table 2). Th~se findings indicate that the bindin~ site for 5511 is
locatod in the region that extends bet~veen residues 243 and 308 of pSj-IC and that thc N
terrninus of this binding site is between residues 243 and 266.
Transfer of the cl:~N~ for 55.11 from the ori~inally cloned 'prey' construct, ~hich
contained ~he Gal4 acti~ation domain~ to a prey construct containing the ~PI6 activation domain
did not decrease the binding efficienc! of the 55.11 protein to p55-IC t~ablc ~. Thus. the
s~ructure(s) involYed in this birlding appear to reside within the 55.11 moleculc and not to involve
the site of fusion of 55 . I l with the acti~ ation domain
However, bindin~ of 55.11 to pSS-IC was abolished by even limited truncations of the
S5.11proteinateitheritsC(SS.II construct309-680)orNterrninus(55.11 construct45?-900J.
(residue 309 is the first residuc in the SS . I l protein encoded by the panial cDNA clone oriL~inally
isolated in the two hybrid rcreen).
The obse~ved birldir.~g botween SS.l ~ and pSS-IC appeared to be spccific sincc 55.11 did
not bind to othcr proteirls, irlcluding three receptors of the T~FINGF rcccptor family (p7s-R
FaslAPO1 and CD40) and other proteins such aS lamin and c;~cEn D (data not shown). It should
be noted that of the othcr T~F~GF receptor proteins tested there was also tested portions
thereof vhich include their il~tl~LdUUL~I domains human FAS-R (residues 175-31g), CD40
(residues 216-2~7) and p75-TNF-R (residues ~87-461), none of T~hich bound 55.11 ~data not
shown).
(c~ ' Pn~ivsis of the RNA from sev~rsl r~n lines. - th~ ~i5.11 cDNA as 8
probe ~ ' ~ of the ~ ' ' 55.11 cn~A
The cell Enes examined were HeLa~ CEM, Jurkat, and HepG2 cells derived from
human epithdial carcinoma, an acute 1~ T cell leukemia~ an acute T cdl leukemia, and
a ~. p~ ' Carcinoma"f~ The SS.II c3NA original isolated (nl~cl~c~t~ .c c~25
2863) was used as a probc. Samples consisted of 10 llg of RNA/lane. The resltlts of the Northern
analysis are shown in Fig. 2, ~vhich is a ~ cl.. o.lu.Lol. of a Northern blot.
From Fl~. 2 it is thus apparent that the Northern analysis using the 55. ~1 cDNA as a probe
3 5 reve21ed, in several cell Enes, a single h~bridizin~ transcript of about 3 kB~ hich is larger than the
c~NA (2 kB) of the ori~inall~ isolated 5~.11 cl~TA. I)sing ~ O~ C primers that
correspcnd to the 55.11 sequence, we cloned by PC~ a 5' eritending sequence whose length was
about I kB. The sum of the len~th of this 5' extending sequence v.ith that of tl~e originally cloned
c~NA ~ u~ les the lensth of th~ SS. l I transcript. The ~ kB cDNA tha~ fl~Ol l ~ ~AC~_~I both
WO95/31544 21 8~9~d3 ~",.~ - 4
44
these portions was ~ffectiYely expressed in ~ransfected HeLa cells ~see belo~ j ~ ie~din~ a protein
of about ~4 kDa~ w-hich su~gests that the ~ I;B cDli~ contains a ~ , I s~art site.
(d~ In vitro bindin~ of the 5C.II protein to GST-fusion proteins containin~ p~rtions
~f r!S~IC
S To ascertain that 55.}1 can indeed bind to pSS-lC and to exclude ;~v~ Kell~ of
,veast proteins in this binding, thc ~n ~ interaction of GST p55-IC fusion proteinS, ptoduced by
bacteria,withth~proteinenGodcdby~he3kB55.11clDNA(SS.I~-full),producedbyt~ansfected
HeLa cells, was exa~nined. In this stud~ the cDNAs for th~ full-length SS.II, FLAG-55.11
(residues 309-900 of 55.11 encoded bv the ori inally cloned partial cDNA and fused at the N
ter~ninus with the FLAG octapeptide). and luciferase (control) ~ver~ expressed in transfected
HeLa ce~ls and mF'tr~ labeled u~ith L3sS] Met and ~35S~ Cys. The fol~owing proteins were
fused wi~h GST: full-length pSS-IC (GST-pSS-IC) and pSS-lC C-ternninally truncated up to
amino acid 345 (GST-p55-lC345) to remo~ e most of the 'death domain' (see Table ~). GST alone
served as a coMrol. L~sates of the transfected cells ~ere i.n,nul.c,y.c.,;yiLd~ed with antibodies
a3ainst the 55.11 protein when the full-length ~5.11 protein ~as used for binding the GST-fusion
proteins~ or with antibodies a~ainst the FLAG octapeptide when the FLAG-55.11 fusion product
was used for bindin~ the GST-fusion proteins. The proteins we~e analyzed by SDS-y~ ..J;~-f.de gel el~ . ulw.c~;~ (SDS-PAGE 10% ~crylamide), followed by, Al 1 I~In Figs. 3A and B are shown reproductions of the Al.n~ A..~ of the above
20 SDS-PAGE gels, in which Fig. ;A depicts the binding of the full-length SS. I I protein (55 .11 -~ull)
to the ~arious GST-fusion proteins; and in ~hich Fig 3B d~picts the binding of the Flag-55. I l
fusion product to the various GST-fusion proteins. In Fic~. 3A there is sho~n in the extreme r~ht
hand lane a control ;~ u.~o~ ;L~-c of Iysates of cells transfected with only the full-length
55.11 and ;-~L~ p-`t~_ipil~L(id ~ith the anti-55.11 antibodies (c~55.11 Abs). In Flg. 3B there is
Z5 shown in thc extreme right hand lane a control Lll~ vl~cu;J~;~a~c of Iysates of cells transfected
with only the ~LAG-S5.11 and , c~,;y;lGLcd with the anti-FLAG antibodies (o~FLAGAbs).
Thus, it is apparent from Fi~s. 3A and B that the protein cncoded by the full-length
55.11 cDNA can be expressed in HeLa cells and it binds to fusion proteins thât contained the full
30 pSS-lC (GST-pS5ICj or a truncated p55-lC that lacked most of the death domain (GST-
pS5IC345) (Fig. 3.~). The full-lcngth S5 ~ I protein did not bind to GST alone (control) Similarly,
the HeLa: " ~A~ d protein encoded by the initially c~oncd partial cDNA of SS . I I in fusion
with the FL.~G octapeptid~ ~FLAG-55.11) bound m ~ to GST-p55IC and GST-pSSIC345,but not to GST (Flg. 3B). The above rcsults also therefore providc additional evidence (see (b)
35 above) that the SS.I I binds to a region of the p55IC upstream of the 'dcath domain', i.e. in the
re~ion of the pSS-IC that is more proximal to the Ll ~ "c domain
Moreover, the above study also d~,Kor.~ Lcs that, in accordance with tlle present
invention, antibodies to SS . I I ha~ e been successfully produced (Fi3. ~A).
WO95/31544 2 1 8 9 ~ 8 3 r~"~ 4
(e) C. 1 . of the deduced ~m;no ~çid sequ~nce of h~ n Is;~ ll t~ th ~1 ~r
rel~fed present in io~r or~ni~mc. ~ ~ Se~l or~ P f~qt.~re~ of th~ sc,l I prot~ioAs mentioned above, in accordance ~ith the present inventior~ the full-lén~th
55.11 cDNA bas been cloned ar~d sequenced (see nucleo~ide sequenGe in ~ (a)) and the full
S amino acid sequence of S5.11 has been deduced from the cDNA sequence (see arnino acid
sequence in Fig. I(d)). 3ata bank ~GenBankT~l/~BL DataBank) searc~ec revea~ed that parts of
the sequence of the human 55.11 cDN.~ (acGession numbers T03659, Z19559, and F09128) and
its mouse homologue (accession numbers X80422 and Z31147) have alread-~ been detcrmined
during arbitrary sequencirlg of cDNA libraries. A cDNA sequence (accession number Ulg241~
10 that encodcs for a huma71 protein of ~96 amino acids present in cultures of human hepatoma
HC10 cells is similar to that of 55.11. This h~patoma protein~ howcver, lacks an ~I terminal
portion (amiDo acids 1-297) co~ ,ul~d;~ to that of SS.ll and also differs from ~5.11 at the
reg~ons that correspond to residues 297-377 and residues 648-668 in 55.11. rne search~s ofthe
data bank also revealed that proteins ~i~h very ~f~gll sequence homolog~ to SS.II exist in
15 .Sû"cl,u,., -- cere-~i.sTae (yeasts), ~ral~ilJcp.~is IhQlia~7a (plants) and ('.~,v,),u~li.) elegans
(worms). Thus, 55.11 appears to fulfill an e~vlu~iu~a~ Gonserved function. In the yeasts, there
are two known proteins (the open readins frame ~G27c and SEN3) uhose DNA sequences
resemble that of SS. I I . Ihe si7es of bo~h are close to that of ~S. I I . YHR027c is known only by
the sequencing of a genomic clone while SEN3 has been cloned as a cDNA. The sites withi
20 55.11 that are sirnilar to those in SEN3 correlate to the sites of its sintilarity to Y~027c,
although tnuch morc sirnilarity is e~ident between SS l l and Y~27c than between 55.11 ~nd
SEN3. The DNA sequence information available for tbe Al abi~lopsis thalia77a andCuc~ u~Suih~ ~lega77s proteins. although only partial. clearly sho~ s that these proteins are as
similar to 55.11 as the YEDl.027c protein of ycast. The only one of these four proteins ~hosc
25 rlahre has been elucidated so far is the yeast SEN3, whose homoloG~,y to ~i. I ] is limited. SEl~T3
has bee7l identified as the ~east equivalent of the pll2 subunit of an acti~ator of the 20S
proteasome (the proteolytic core of the 26S ~., uLc~Ol~-C [R. ' - ~ et al., 1993~ DeMartino et
al., 1994]3 (M.R Culbertron and M. Hockstrasser, personal . ).
In Fig 4 there is shown c. ~ a comparison of the deduced amino acid
30 sequence of human 55.11 to that of the above-mentioned, related proteins present in lower
organjsms. In F;G5 4 The sequenccs that are compared are the sequences of arnino acids prediGted
for: the 55.11 cDNA(see Fig. l(d)); an open readin~s frame (~'HR027G) within a cosmid derived
from the 8th eh.uil~oso..~ of S~.~,.,S..,. ~I,G.~ cBre~ iae (nucleotides 21Z53-24234, accession
number l,T10399), SEN3, the cDNA of a Su~ G~ cerG~ iae protein (Accession numberL06321) a partial cDNA of a prot~in of the plant Arab~L~(7psis t~aliar7a (accession number
T2] 50û), and a partial cONA of a protein of the nematode ~ nv/h~ clega~ (accession
number D2~396) The 'KEKE' sequence in ~.11 is marked with a solid 7ine and the sequence
A~rAGS~x)gLL ~ith broken ~ines. The sequences ~vere ali~ned using the PILEUP andPRLm'BOX programs of the GCG pac.l;age. Gaps introduced to maYirnize alignments are
4û denoted bv dashes.
_
woss/3ls44 21 8~9 83 r~l~.s. -- 4
As regards the various sequence features or motifs prcsent in the human S~.ll
sequence the fo~lowing has been obser~ed: Conser~ed amino acid sequence motifs w~re not
discerned within the protein encoded ~^or b~ 55.11, except for z repetitive KE~E' sequence th2t
extends between Lys 614 and Glu S3~ (underlined in Fig. 4). Such ~CE' sequences, which are
S present in many proteins, includins proteason~l subunits atld .I,ap~lv.f."~, may promote
~ssociation of protein complexes tRealini et al.~ 1994). A sequence AYAGS(x)~L appears twice
iQ the SS. I I protein (at sites 479 590. see Fig. 4); no functional significance for this se~uence has
yet been described
(n c- fe~ltures of th~ V S~:IC I ' invoived in I " p to the ~S.I I protein
As des~ribed above (see (b) alld (d)), the SS.II protein binds to a region of the
pSS-IC between residues 243 and 308 (the N tersQinus of this binding site being between residues
243 aQd 266), this re~ion bein_ u~stream of the 'death domain' and more proximal to the
l, ~. domain of the p55-T~-T~,. This rcgion within pSS-IC to which S j. l l binds has a
high coMent of proline, serine, and threonine residues. Ho~veva, this region does not contain the
15 RPI~I1 and RP~2 proline-rich moti& present in se~eral oth c~tokiQe receptors (Ol~eal and Yu-
Lee, 1993). TQ the region that ~xtends bel~een residues ~43 and 266, whose deletion abolishes
tQe binding of pSS-R to 5~ . I l (see (b) and (d) abovc and Table ~), t~vo of the s~rines and two of
the tbreoniQes are followed b,v proline residues~ which n1akes them potential sites for
I ' , ' .~al;VII by MAP kinase, CDC2, and oth~r ~ .p ."I~,.,t i;inases tSeger and TCrebs,
20 lggs).r' ,' ~lh.;vllofthissiteinthereceptorsmightaffectitsbindingtotheSS.ll protein.
Tn view of all of the ~v.~ .. ~;o.,~d with reguds to protein 55.11 and its binding to p55-
TC it carl be concluded t'Qat in accordance ~ith the prcsert Qnvention, a new prvtein has been
found ~vhich binds to a distinct rcgion upstream to the 'death do~nain' of pSS-lC. Such binding
cou~d affect l~F-mediated activities other Iharl induction of cell death. The region to which SS. I I
25 biDds llas previously been shown to be involved in induction of nitric oxide s~nthase (Tartaglia et
al., 1993), and appears to be involved in the acti~alion of the neutral ~ .. t~",.~.,li..~c by TNF
(Wiegmann et al., 1994). It is thus possible that association (binding) of SS.II ~ith th~
intracellular domain of pSS-lN~-R (pS5IC) affects or is involved in ~ he signalir~g for these
above noled or other TNF effects, ~li) th~ foldin~ or processing of the protein (as suggested b~
30 the sirDilarity of S j . l l to a subunit of th~ ~6S proteasome), or ~lii) the re~ulation of the activity or
e~tpression of pSS-TNF-R
Fy~MpLF 2
oft~A intr~ - Df ' D~ TNP receptor ~r~S~lC~ and jtc
35 caplbilih to ~ause c~ll death alld other f~tl~res: ' activities thereo Dnd a related
FDCI,~POI .... ... dn._,
As set fonh in Example I above, it was disco~,ered that the ;,,~.~.ccll~.l~ domain of pSS
ll~F-R (pSSIC) is capable of binding to itsel and filrther that fragments of pSSIC, namely
proteirls SS.I and 55.3, are also capable of binding to pSSIC.
WO 95/~1544 2 1 8 q 9 8 3 ~ _,'0! 4
.
~7
It is kno~s~ that the bindin~ of T~ to FS5 T.~F-R ieads to a c~ tocidal effec~ on ~he ceils
c~rrying this receptor. Further~ amibodies against the ~ ac~:lulG~ donnain of lilis recepto~ can
themselves trigger this effect, in corre~ati~n vith the effecti~ity of receptor cross-linicing b~ them.
In addition. mutational studies (Tartaglia et al., (1993), ~;. ' ' ' et al., (1992)) showed
5 ~hat the function of the p5i-R dep~nds on tile int~grit~ of its ;",~ ac~iLllai dornain. It wts therefore
suggested that the initiation of si~naiin~ for the cytocidai effect of TNF occurs as ~
of association of two or more llltlac~l'.d~ domains of the pS5-~ ~pSS~lC)~ imposed by receptor
aggregation. The results in accordance ~ith the present inYention provide further evidence for this
notion~ showin~ that expression of the irltraceiluiar domain of the p55-R within cells, without the
0 Ll~ l~le or;..~,a.~'luLdornain~tri~eerstheirdeath.Suchfreeil~ celluial domainsofthe
p55-R are sho-vn to seif associate, ~hich probably accounts for their ability to function
ly of TNF. The fact that the signaling by the full length p55-R does depend on TNF
stimulation is sugg~sted to reflect ac~ i(es) of the ~ or ~ p~ domain of th~
receptor which decrease or pre ~nt ~ilis seif association.
The ability of the ;.-t~ Uh:l domairJ of the pS5-R (p55-IC) to seif associate was found
se.c.,J.~;luu~ly~ in the attempts to clone effector proteins which interact ~hith this receptor (see
Example I above). We applied for that purpose the above men~ioned "t-vo hvbrid" technique. In
~ddition to the novel protein, 55.11 found ~o asSociate (bind) to the pS5IC, it was aiso found that
tbree other cloned HeLa celi cDN.~s contâined cDNA sequences ~ncoding for parts of ~e
~ " ` dornain of the p55-R, implying that ~he p55-lC is capable of self-association. Two of
tbese ciones were identical~ con~aining an insert which encodes for amino acids 328426
(desi~nated as clone 55.1 encoding protein frâgment 55.1 of the p551C). The third contained a
longer insert~ encoding for amino acids 277-426 (desi~nated as clone 55.3 encoding protein
fraement SS.3 of the pS5IC).
In addrtion, ue assesæd th~ in itrQ interaction between two bacte!ially produced
chimeras of the p5SIC~ one, in which i~ was fused to the maltose bindin~ protein (MBP) and the
other in which is was fused to the 3' ' ~rS-transferGse (GST~. These chimeras were
constructed~ cloned and expressed by stantard methods. Following their expression~ the
assess~nent of the S '' ' ' ' of the p55-R ;.ltlG~ l domain (pS51C) by d .~ , the
interaction of the above bacterially-produced chirneric proteins GST-IC55 (l~r - 51kD) and
Mi3P-ICSS (Mr - 671;~) with each other. Equal amounts of the GST-IC~S chimera (samples of
lanes 1-4 in Fig. S) or G5T alone (samples of lanes S-8 in ~ig. 5) were bound to g~utathione-
a~arose beads (Sigma) and ~Yere then incubated ~ith the same arnount of M3P-IC55 fusion
protein in one of the foUowing buff~r solutions:
(i) buffer 1 (20mM Tris-HCI~ pH 7.5 lOOrnM KCI, 2rnM CaC12. 2rrM MgC12, SmM
DTT, 0 2~,'o Triton X100. O.SrnM PMS~, 5/~ Gl~cerol~. This wa~ done for the sampies of Lanes 1
and 5 of Flg. S.
~li) buffer I containin~ Smi~ i~DTA instead of MgCI~. This u~s done for the sa~npies of
Lanes 2 and 6 of Fi~. 5.
.. . . . . _ _ _ .
WO95/31~4~ 2~ 8~83 I~ JO~4
18
(iii) buffer I contamin~ ,SOm~I instead of loom~ KCI. This was done for the s~mples of
Lanes 3 and ~ of Fig. 5.
(iv) buffer I contairlin~ 400m~ instead of 1 00m.~I KCI. This was dont for the satnp~es of
Lanes 4 and 8 of Fig. 5.
S After incubation with rotation ~^or ~h at 4C, the beads were washed ~ ~ith the same buffers
and then boi~ed in SDS-PA&E buffer foll~-Yed by .I~ ' e~i~ by PAGE. The proteirls on the
~el were then Westem blotted to a ."..~cell,lose membrsne which was then stained ~-vith
polyclonal fJntiserum against MBP. A reproduction of this stained Western blot is shown in Fig. 5,
the samples in lanes ~-8 bang those noted above.
Io From Fig. S it is apparent thAt the pSSIC-M~3P chirnera bind to the pSSlC-GST chimera
~lanes J 1) i~ 7 of divalent cati~nS and even at a ruher high salt CUl~ ld~iull (0.4M
KCI~. Thus, it is concluded that the p~5IC is able to avidly self-associate.
To evaluate the functional iTnpli~RTioTl~ of the propensity of the p5~-IC to self associate.
we attempted lO express the pSS-IC ~it~in the c~toplasm of ceDs which are sensitive to the
cytocidal effect of lNF. Considering the possibility that the p55-IC uill turn to be CytotOXiC, we
chose to express it in an inducibl~ manner? using, the recently deYeloped~ tightly re~ulated
leL.acr"l;..T~controlled mammalian expression s~stem (Gossen and Boujard, 19g~). Expression of
the p55-I~; resulted in masSi~e cell death (Fig. 6, right panel). The dying cells displayed cell
surface blabbing as observed in the kil1ing of the cells by TNF. Transfection of the pSS-IC
.0 construct to the CT~IIS in the preserJce of tetracycline, which reportedly decreases the expression of
pHDI0-3 regulated constructs by as much as 105 fold, stiD resulted in some cell death. althou~h
..f~ less than that observed in the absence of tetracycline ~ig 6, left panel). In contrast,
cells transfected with a control construct, containing the lucipherase cDN~, showed no Si,,nS of
death (results not shown).
,'5 The ability of the p55-lC to tri~ger cell deat4 ~vhen expressed without the ~ 7~ t
or: ~. " ' domains of the receptor, pro~jdes further e~tidence for the invol-ement of t'nis
domain in signaling. r~ ..v~, it indicatGs that no other part of the receptor plays a direct role
in such signaling.. Studies of the effects of mutatiorls, includin~ those mutations studied in the
preSent invention, on the function of the pS5-lC, indicated that the region extendinO between
30 amino acid residues 326 aDd 407 is most critical for its fi,mction. This reOion sho~s marked
I,I"....e to sequences within the ;~.~. o.c~ l~ domains of two other receptors, evvl..liv,.~
related to the pSS T~F-}I, - narnely~ the Fas receptor (Itoh et al., 1991; Oehm et al., 19g2)~ which
can also signal for cell death and CD40 -a receptor ~Starnenl:ovic d al.~ 1989) ~vhich enhances
cell gro~vth; this seouence therefore secms to constitute a conserved motif which plzys some kind
5 of general role in sisnaling. Since it does not resemble kno~vn motives cl.~ f .i~ , of enzymatic
activilies, it seems plausible that it signals in indirect manner, i.e. possibly by servin~ as a dockin~
site for signaling ereymes or for proteins ~vhich transmit stimulatory siOnals to them. The pSS-lC,
the ~as receptor and CD 40 can all be stunulated by antibodies against their ~ domain.
Their stimulation could be sho~ to correlate ~vith the ability of the antibodies to cross-link the
40 recep-ors. It lherefore seems that the siOnaling is irlitiated as a ~O~f I ' ~ f of interaction of two
.
~ WO 95/31544 2 t 8 q q ~ 3 r ~ 4
or more ;~ a~ J~i domains imposei b! aggregation of the ~AL~.... Ilula~ domains. Involvement
of such interaction in the initiation ol sier.alin~ of these receptors was also indicated by studies
(I3rakebusch et _i., 1992) showing that expression of receptors made ~v"r,.,~ IAl by mutation
of their ;lltll~l;ulDJ dom2in, had a ' dorninant neL~Ative" effect or~ the function of co-expressed
S normal receptors. Aggregation of the p~-R in response to TNF was suggested to occur in a
passive msnner~ merely due to the facl that each of the TNF molecules, which occur as
hU...~ , can bind two or three rec~ptor molecuies. However, the fîndings of the present
invention suggest 1hat this process occurs somewhat differentl~.
The propensity of the p55-1C to self associate indicates that this domain plays an active
10 role in its induced aggregation. More~ er. this activity of the p55-IC seems to suftice for initiating
its signaling, since when expressed ;".~ A~ ~ly of ~he rest of the receptor molecule, it can
trigger cell death in the absence o' ~ or any other exterior stimuli. Nevertheless, Yhen
expressed as the fuli length receptor, the pSS-TNF-R does not signal, unless stimulated by Tl~'F.
One must, ther~fore, assume that v hen 2cth,ating the pS~-TNF-R, T~F acruall~ overcomes some
1~ inhibitory ~ which pre-en~ association of the i~ rccllulal domains, and
this inhibitior~ is due to the ~inii2g~ of the pSS-IC to the rest of the receptor molecule. The
inhibition may be due to the orientation imposed on the ;III~D-~CIl'ul~ donnain by the
- and ~Ytr~r~ Dr domain, to association of some other proteinc with the receptoror perhaps iust due to restriction of the arnounts of receptors that are allowed to be pl2ced in the
20 plasma membrane. Of note, this control mech2nism should be rather effective, since according to
some estimations, th~ binding of e~en just one T~l~ molecule ~o a cell suffices for the triggering
of its death.
~ r signaling~ in.~ t of ligand can result in extensive d~, 14 ' of the
process eontrolled by this receptor. The best kno~n exarnple is the d~e~ul~;2l~ of growth factor
ZS receptors. I~utations due to which they start sisn2iing, 1~" for example those that
e2use them to aggregate ~ J~l~', play 2n important role in the dere~ulated growth of
tunnor ceiis. TNF effeets, when induced in excess, are well icnown to contribute to the patholo~,~
of many diseases. The 2bility of free " ' domains (p55ICs) of the pSS-TNF-R to signal
lr - ~,y of T~ may contribute to such excessi~e func~ion. It seems possibie, for exarnple,
30 that some of the eytopathic effects of viruses and other pathogens result, not from their direct
cytocidai function, but from proteoiytic detachment of the intracellular domain of the pS5-TNF-R
and the resultu~ iike cytotoxic effect.
Io furtb elu~idate the region(s) within p55IC which is responsible for its self-association
capabiiity and hence its li~and-; ~ cell Uy~u~u~uc;iJ~ and aiso to determine whether other
35 re~ated members of the l~iF/NGF receptor famiiy (e.~. FAS-R) aiso have ;"~.~c~ domains
with self-association capabilities and ligand-; ~irl 1 ,l effects, the following detailed stud~ was
perfor!ned:
wo 95/31~44 3v r~
(a) Gener31 Procedllres . nd M~terhlls
~) Two hvbrid screen . nd ruo-hybrid B-~PI~ PreSSiOn test
cDNA inserts, enco-inE the pj5-IC and its deletion mutants~ the ~as-lC and
various other proteins (see Table 3), uere cloned b~ PCP., either from the fuil-length cDNAs
S cloned previously in our lavorator}~ or from purchased cDNA libraries ~ t~ C~
expression in veasts ~SFYS't6 reporter strair~ (Bart~l ~t al, 1993)) ~ r,.. "\PA ~ith these cDNA~-
iD the pGBT-9 and pGAD-G~ vectors ~DNA bindin~ domain (DBD) and activ2tion domain (AD)
constructs. ,es~ecl;~(.ly) was assess~d b~ a liquid test (Guarente~ 1~83); it was also assess~d b~ a
fiit~r assa~ yielding, " cir the sa~De results (not shown). Two-hybrid screenin~ (Fields and
10 Song, i989) of a purchased &al4 .~D-tag~ed HeLa cell cDN~ library ~CIontech~ Palo Alto. Ca,
U.S~A~ ) for proteins that bind to the intracellular donnain of the p55-R (p55-IC), was performed
using the HF7c yeast reporter strain according to the ~e~ of the p.oducer Positivity
of the isolated c}ones was assessed b~ ~a) ~IV~U~iV~ of the, ~f'~Y~ easts for histidine
when gro~n in t~e presence of 5 rnl~ 3-~ vL,iA,ulc, ~b) ~-sPl~-t~-~s~ e e~;pression ~c)
13 specificity tests (tnteraction with Sls~4 and larnin fused to Gal4 DBD)
vifro se~f- - - - - of bacteriall- Droduced pS~lC fusion protein~
&lutathione S-transferase (GST) and ~slutathione S-traDsferase-p55-IC fusion
protein (GST-pS5-~C) were produced as described e!sewhere (Frangiorli and Neel~ ] 993~
Ausubel et al.~ IY,4). Maltose biDding protein (l~ P) fusion proteins were obtained using the
20 pMalcRI ~e.,tor (New En~dand Bioiabs) and purified on an amylose resin colurnn. The interaction
of the M~PP and GST fusion proteins was ;..~,sl;~ d by incubating ~,lu~Lùl.c ~ ose beads
with the GST arld MBPP fusion proteins (S ~g protein / 20 111 beads; first incubation
for l S min, and the second for 2h. both at 4C) Incubation with l~iBP fusion proteins was carlied
out in a buffer solution coMainin~ 20 rnl~ Tris-HCI, pH 7 5, lvO n~M KCI, 2 rslM CaClA,l 2 rnl~I
M~C12, 5 mM dithiotrehol, 0.2% Triton XlO0, O S r~I phonyl-methyl-sulp;.u.,~: fluoride and 5%
(vlv) ~Iycerol or, when indicated~ in that same buffer containing 0 4 M KCl, or S mM EDl`A
instead of MgC12. Association of the MBP fusion proteins was assessed by SDS pvl~_~l yl~l~l;de
gel ciccl.e~l.v.csi~ (10~0 aerylamide) of the proteins associated with the glutathione-asarose
beads, follo Yed bv Western blotting. 'rhe blots were probed with rabbit antiserum against MBP
3v (produced in our laborators~) and ~ith horseradish-~ù,did~s~ ~' ' ' sost-anti-rabbit
. .
(iii) ~ ' ' ~vression in ~el.a celis of the p55-~ and fra~ments tllereof
HeLa cells expressin~ the tetracycline-controlled transacti~ator de~eloped by
Gossen and Bujard (the HtTA-I clone (Gossen and Bujard, 1,9~)), were grown in Dulbecco's
modified Easle's medium, containing 10% f~tal caif serum, 100 u/rni penici~lin, 100 ug/ml
~h~,vtu.l.~.,;.. and 0 ~ ms~ml neomycin cDN.~ insert~ encoding the pS5-R or parts thereof were
introduced into a tetracycline-cûnrrolled expression vector (plJHD 10-3, kindly provided by ~E
Bujard). The cells were transfected wilh the e:~pressiot~ construct (5 ~g DN~6 cm p~ate) by the
caicium phosphate pl~ Liv,. method (Ausubel et al, 1994) Effects of transient expression of
40 the transfected proteins w~re asses~ed at the indicated times after IIA~r~ in the presence or
... .. . ..
WO 95/31544 2 1 8 9 q 8 3 ~ 4
51
2Ibsence of tetrac~clin~ g/ml) Clones of cells st;lbl~ transfec~ed ~vjth Ihe human p'iS-IC cDr~A
i~ the plJ~ID 10-3 ~ector were estab~ished bS~ translèCIing the cDN~ to HtTA-I celis in th~
presence of telraCycline to~ether ~ith a Flasmid conferring resistance to hygromycin, followed b~
sdected for clones resistant hygrom~cin ~200 ~ ml). El;pression of the cDNA was obtained b~
S removai of tetracycline wilich was other-~ ise maintained constant3~ in the cell ~rowth medium.
(iv) _~ç~" Or T~TF~ ffPrt~ tri~z.~ered bv induced eA-preasinn of the p55-R
P ' '-~ ' therP~of
Effects of induced expression of the receptor and of TNF oll cdl viability ~vereassessed by the neutrai-red uptaice method (Wailach, Ig84). Induction of IL-~ gene expression
10 was 2ssessed by Northern an2iysis RNA was isoiated using TRi REAG~ENT (~Iolecular Research
Center, Inc.), den2tured in ro.-"~id~ dc~ru, ' '- buffer, ~ uy~l~u~ed through an&~;_ua~ ~ rde gel and blotted to a GeneScreen Plus m~mbrane (Du Pont) in 10xSSPEbuffer, usin~ standard techniques. Filters were hybridi2ed with an IL-8 cDNA probe (MZ~tQ~;mzl
et al., 198~), nucleotides l-392). Ia~ ,d by the random-prime kit (~oehrin~,~er Mannheim
15 ~I;rlrh~miça M&nnileim, Gman~ ) and ~Yashed stringently, according to the protocol of
.~a~,~u-cl. Aulul~l;u4-a~ ,y wzs performed for 1-2 days.
(v) A - ~'TNF ~ - r
TNF reeeptor expression in samples of lX106 cells was 2ssessed by measuring the
birlding of TNF, labeled with iZ5I by the "I.lola~ eT method, as previously described
ZO ~oltmarm and ~Tailach, 1987). It was aiso assessed by ELISA, perforrned as described for the
Of the solllb]e TNF reeeptors (Aderka el ai., Ig91), exc2pt for the usc of RIPA
buffer~IûmMTris-HCI. pH7.j, ISOrnMNaCL 1%NP 10, 1% d~ ycllGl~e, 0.1% SDS and I
rnM~DTA) to Iyse the celis (70 ul/lo6 cells~ a~-zd to dilute the tested samples. The soluble forrn
of the Ps5-R purified from urine, served as the standard.
25 (bl ~ . ' Or ' - ' ~ ' - ' ' ' - p~S-~ (pSS-~C3 to ' - - thP
of th~ pS5-lC involved in its ~.lr z. - ..~
As noted sbove, p55-lC can self z r ~ and trigger cytotoxic effects on eells, and there
are portions of the p55-IC, whz'ch themselves were c2pable of binding to the full-length p55-ïC. In
pattieular. one of the portions of the pS5-IC ~designated as protein fragrn~nt 55.1 in xamp~e ~
30 above) was identif ed that v,as capable of binding strongly to the full length p~S-IC, this portion
W7A'S sequeneed and was oi~ser~ed to eont2in the âmino 2cid residues 328 126 of the pSS-TNF-R
which are ~vithin the p55-JC. It has further been disco~ered (see below) that the above portion,
protein fraYment 5~.1, is itself c2p2ble of self-association and of triggering cytotoxic effects on
cehs Hence this portion of the p55-lC has been called the 'death domain', and is located in the
3 ~ region bet~een amino acid residues 32S-426 of the hum~n P55-R most lilcely consisting of amino
acid residues between about r~sidue 32~ snd 414 thereo
The fsct that the 'death domain~ in the p55-IC self-associates was found by ~
On screenin~ 2 HeLa cell cDNA librar~ by Ihe two-hybrid lechnique (see Example I above) for
proteins that bind to the i.".acelhll~ domain of this receptor, we detected r~morlg the c~)NAs
40 whûse products bound specifically to the il~t ~cell~ , dûtnain-G.4L4 DBD fusion-protein, sevetal
WO 95/31544 2 ~ ~ q ~ ~ 3 P ~
52
clon~s (e.g..~5.1 and 5~.3) tllat Ihem~c~ s encoded for Farts of the p5~-R intracellular domain
(pS5-lC; mark~d wit~ asterisks in Table 3).
Applyin~ the t~vo-h~brid test to evaluate the e~tent of specificity in the self-association of
pSS-lC and to define more accurately the region invo~ved lcd to th~ follo~ findings (Table 3):
(a) Th~ An of pS5-IC is confined to a region within the 'death domain'. Its N terminus
is located between residues 328 and ~44 arld its C terminus~ close to residue 404. somewhat
upstrea~n ofthe reported C tcrrninus of this domain (residue 414). (b) Deletion of the membrane-
proximal part of pS5-IC upstream of the 'death domain' enhanced ~ ri t~ l, suggesting that
this region has atl inhibitory effect on ~he association. (c) ~ouse pS5-TC self-associates, and also
1~ associates ~ith the 'death dom2in' of human p55-R. (d) F~ ;"" of the self-association of the
intracellular domains of three other reccptors of the TNF~'NGF recep~or family: Fas/APO1 (FAS-
R)~ CD40 (Fields and Song, Ig8g) and the p75 ~7F receptor (Snuth et al.. 1990). showed that
Fac-IC~ which signals for cell death by a sequcnce motif related to the pS5-~ 'death domain'~ self-
t~ssociates and associates to some extent with the p5~-lC. However, CD40-IC, that proYides
15 growth stimulatory si~nals (even thou~h also coMaining a sequence resernblin~ the 'death
domain'), arld p75-IC, that bears no strlctural ,~lnl,l~l.,c to pS5-IC, do not self associatc~ nor
do tlle,Y bind pS5-lC or Fas-IC.
WO95/3l544 21 89983 ~ "~ 4
~ABT-~: 3. s.lr of the " '~ domains of p55-R and Fas/APOI within
f~. ' yeasts:: by a two-hybrid ~ ' e2~pression test.
-
~_ O o I I I O O O O O O
I I I o I I ~ I I
m
=~
n ~ ¦ O ¦ O l l I
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~) I I I I I ¦ ¦ O
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o ~ I I I I I I ~ O o I o o
C~U
~ C I I I I I r~. o o I I I O
~or~ o I I I o
o I I I I I I I I I I I
C) Z ~ o
~ ~ rq ~
Z ~ O I I I I I I I I I I I
~ O ~ I o o ~: 0 I I O O
~ O o I I O O
~--r o
"' ~ ~ o o o I O O
C.1
~
. 3
~vt~ E ~
u~O,,~, 2 ~ _
,~ ~/,,
wo95/31544 2 1 8q9 ~3 ~ ' 4
54
Table 3 abo~e shows the quanritalive assessment of the interaction of Gal4 h~brid
constructs r~ A~ a the foliouing proteins the intracellular domain of human pS5-R and its
various deletion mulants ~residues numbered as irl (Loetscher et al, 19,0)); the intracellular
domains of mouse p55-R (residues 334454, numbered as in (Good~in et al., 19~1)); mouse
5 Fa2JAPO~ ~Fas-IC, 166-306, numbered as in (~' bc F,J~r.,.rK et aL., 1992)), human CD40
(CD40-IC~ 216-~77, numbered as iD (.r'' ' ~i.. et ai., 1989)); and human p75 T~F receptor
(p75-lC, 287-461, numbercd as in (Smith et al., 1990)). SNFI and SNF4 ~vere used as positive
controls for association (Fields and Sons, 1989), snd lamiD as a negative corltrol tBartel et al.,
lg93). Proteins encoded by the Gai4 DBD constructs (pGPT9) are listed vertically; those
10 encoded b)~ th~ Gal4 AD constructs (pGAD-GE:i), horizontall)~. The two deletion mutants denoted
b)~ asterisks were cloned in a t~o-hybrid screen of a HeLa cdl cDNA library (Clontech, Palo
Aito, Ca., U.S.A.) usin~ p55-IC cloned in pGBTs as "bait". In that screen, four of about 4xl o6
cDNA clones examined were positive. Three of these clones were found to correspond to parts of
lluman p~r-R cDNA (two were identical, encoding residues 32~26 and one encodin residues
'~77-426). The fourth was found to encode an unknown protein. Ihe 13-r~lort~si~C~ expression
dua are averages of assays of t~o ir,~ t ~ rO~ 2 and are pres~nted as amount of ,b-
~;r~ n;J- ~ product; (a unit of acti~it) bein~ defined as OD420 times i 03 divided by OD600 of
the yeast culture and roaction time~ in minutes). The detection limit of the assay was 0.05 units.
Variation between duplicate samples were jG all cases less that 25% ofthe averase (not tested).
An in Yrtro test of the interaction of a pS5-IC ~ L.t~Lh J.. ~-S-transferase (GST) bacterial
fusion protein with a pS5-lC-maltose binding protein tMBP) fusior~ protein confirmed that pS5-R
seif-associates and ruied out i...~,l._,....~ of yeast proteins in this association (see above). The
association W85 not affected by increased saLt ~ " nor by I~DIA (see above).
To evaLuate the functionai , " of the self-association of the death domain, we
25 examined the way in which induced cxpr~ssion of pS5-R, 3r of parts of it, a~ect cells sensitive to
~NF cytotoxicity. The results of this analysis are set forth in Fip. 7 which depicts the ligand-
i J~ trig~erin~ of a c)~tocidal effect in ~eLa cells transfected with p5j-R, its ;.lL, ~,cllul~
domain (p55-lC~ or parts thcreof (including the 'death domain~.
In Fig. 7 there is sho~n s~ h .. -1;. Ally, the various DNA molecules encodin~ the diffcrent
30 types of TNF receptors included in the vectors with which the E~ieLa cells were transfected
(extreme left hand side of Fip. 7); and the expression (left and middle bar graphs) and the viability
(risht bar graph) in Hel,a cells expressing transiently the various full-len~th p55-R (p55-R). pS5-
IC or parts of p55-IC or, as a control, luciferase (LUC) (each beinp depicted at the extreme left
~ide of Flg. 7), using a lcLl~y~,];~,., controiied expression vector. The open bar ~raphs (left.
35 middie and right) represent cells transfected in the presence of letracycline tl lag/ml), which
inhibits expression; and the filled bar sraphs tleft, middle and risht) represent cells transfected in
the absence of tetracycline. T~F receptor expression v~as assessed 20h after ~ I`e~,L~/Il, bot~l by
EL~SA, using antibodies against the receptor's ~ - domain (se~ sGhematic illustration on
the left side of ~ig. 7), and by d~....,~,g Ihe binding of l ~ l / le~ ~NF ro the cells (middle)
40 The cytocidal effect of the transfected proteins ~vas assessed 4S~l after l.~ e~,lio". Data sllown
Wo 95/31544 2 1 8 9 9 3 3 ~ ~4
ere from onc o~three ~Yr~rirn~nlC v.i~h qualita~ively similar results~ in uhich each construct ~as
~es~ed in duplicate. ND - not determined.
Thus~ from Fijs~. 7 it is apparent that by using arl expression vector that permits strictl-
~consro~led ~xpression of tr2nsfected cDNAs b~- G tetracycline regulated transactivator (Gossen and
S Eiujard, 1992)~ a mere increase of pSS-R expression in TlcLa cells by expr~ssion of transientl~
transfected cDNA for the full-length receptor resulted in quite xtensive cell death. An e~en
greater cytotoxicity was observed when expressing just pS5-lC. SiL~nificant ~,y~ulu~d~ y W8S also
obser~ed when expressing just a part of p55-lC comprising essentially the 'death domain' (residues
32B426) in the HeLa celis. On the other hand~ expression of p2rts of p5j-IC that iacked the
10 'death domain' or contained just part of it (or expression of the luciferase gene. used as an
irrelevant c~ntrol) had no effect on cell viabiiity. The cytotoxicity of pS5-IC was furtha
confirmed using cells stably llG~Drv~ vith its cDNA; these ceiis continued to grow when p55-
~C expression was no~ induced, but died when pSS-IC ~as expressed (s~e abov~).
(c) O~h-r eff~ of the intr~P~ lqr dQmain Qf th~ p~ R
IS To e~ta~nine whether other activities of TNF are triggered by the seif-association of the
i"~ cei'. l,. dom2in, including the 'death domain' thereof, we examined the effect of increased
expression of rhe ful~-~ength receptor (pSS-R) and of the expression of the i--L~ e~ Gl domain of
the receptor (pSS-IC), on the ~. GliS.l ;yL;vii of in~leuiiin 8 (IL~g), kno vrl to be activated by TNF
( ~ ' et ai.~ 198B). The results are shou~r-i in Fig. 8, ~vhich depicts the li~2nd ~
induction of IL-8 gene expression in HeLa celis trarisfected v~ith p55-~ or p55-IC, using a
lti.,t~ -controlled construct (see atso 'G~neral Proc~dures and Materinis' and Exnmple I
above). In pand A of Fig. 8 there is shov n a l c~.v~ v" of a Northun bl~t l~yiCD~"~i"j~ the
Northern blo~ anniysis ~see 'Generat Procedures and ~iateriGts' above) of RNA ~7 ~ llnne)
extrncted from HeLa (Hi~ta-l) cells, untreated ~'control') or treattd ~TNF') vith TNF (500 ~/rnt
for 4h), or the HTta-l cells 24h after ~, ~ Crn the presence or nbseric~ of t~tracycline) with
p55-IC ('p55-IC'), the pS~-R ('p55-R?, or lucifaase (~uc~ cDNA ~in pnnet B of Fig. 8 there is
shown a . ~ ' of ~i Northern biot I C~ D~ ; the meti-iylene blue stair~ing of I ~S rRNA in
each ofthe sarnples shown in panel A of ~ig. 8.
Thus, as is apparent from Fi~. B, trarlsfection of HeLa cells with a Lch~ Ll~e-controlled
construct encodine the pS~-R cDNA induced IL-8 ilGl~ JtiL~ n even stronger induction was
observed in cells trarisfected wi~h the cDNA for p55-lC. In both cases, the induction occurred
oniy when tetracycline was excluded from the cell grov~th medium, indicatinj~ that it occurs as fl
Of expression of the transfected pS5-R or pS5-IC Transfection with luciferase
cD~'A, as a controi, had no effect on II,-8 ~ t~o,
J~ccorriingly~ from the above results (Fi,,. 8), it appears that a mere increase in p55-R
expression, or even expresSion of just the ;~L~ ul~ domain (pS5-1C) thereof is sufricient to
trigOer, in a li4oand (INF)-, ~ 1 fashion. u,yLvLu~i~iL,y and otner e~Fects as well, includin~
that of an increase in the expression of the IL-8 ~ene ~ ithn ce~ls The triggerjng of these effec~s is
most iikely due to the self-association of the ;I~ C~I~aIIGI dom~in of tbe pSS-R (p55-JC). As is set
A~') f^!th ah.A~e. i~ anrear~ tha-. l}r~or ce~f-a~ct-ciatinn of the nSj-rC. the 'death domain' thereof is
, =, , .. . . .. , = .. _ ...... . . ~ .
wo 95/31~4 2 1 ~ 9 q ~ 3
. 56
primarily responsible fnr signalinr ti)e induclion of tl~e intrace~iui2r processes leading to the
trjg~/erinL~ of cytotoxicit~ ~,ithin the cells, ~llilst the other effects~ e.g the signaiing leadinL~ lO the
induction of L-~ gene exprcssion~ ar~ ~ikei~ du~ to oth~r rei~ions of the pS5-IC as ~ ~]1. fo~io~ins
the self-association thereof It is ~herefore possible that different regions of tlle pS5-lC are
5 responsible for the different I~lF-induced effects ~e.~. cytotoxicity, L-8 induction~ within cells,
these effects bein8 a c~A~eq'' " '' of the intracellular signalin6 upon self-association of the pS5-
IC.
The fact that the pS5-lC~ can induce in a ligand (TNF~ ', ' fashion~ the trig~ering
of other ...i~s~;lulA, effects e.g. IL-~ tnduction~ means that the pS5-IC or specific portions
10 tbereof may be used as a lliuhly specific tool for bringing about such effects in cells or lissues that
it is desir~d to ~rear, without th~ need for treating suci~ celis or lissUes ~ith TNF. In many
p- 1~ jr~l conditions (e~ ~,Al;L~ r~)~ treatmene witb T~F, especially at high dosa~es can
lead to undesirable side-eftects due to the number of iMracelluiar effects induced systemicall~ ~y
TNF following its birding to its rec~ptors. By way of the discovery in accordance ~ilh the present
15 inveMton that the pS5-IC can mimic specific other INF-Wuced effects (besides cytotoxicity),
e.~. ~-8 induction~ opens the wa~ for imroducing in a cell- or tissue-specific manner, pS5-IC or
specific portions thereof~ ~hich will be capab~e of si$naiing for the induction of specific desired
il~t,~llul~, effects~ e.g. IL-8 induction~ and thereby overcorne the systemic side-effects often
observed durrng TNF treatment.
20 ~d) ~ ' ' of c~toc~dsll effects in HeLa ~rll~ by the t
domains and the 'denth domnins' thereof o~ D'iS l~-R and FAS-R (F~/APOl)
As reeards the cytotoxjc activity of the intrsceilular domains of the p'i 5 TNF-R and FAS-
R (pSSIC and FAS-IC) it hns now niso been further elucidated that both the pS5~C. its 'death
domain' (pSSDD) and the FA~i-lC nre capable of a ligand 'A~ A~l' 'l tri~ering of a cytocidal
25 effect in HeLa cells. In tbis study, HeLa cells were transfected with eApression vectors contnining
various constructs of either the fuii-length p55-TNF-R, portions thereof including the pSSIC and
p55~1:) or the FAS-lC. In one set of w.~.w i..l~,.,.~ HeLa cells ~,ere co-transfected wi~h conslructs
containing the p'iS T~iF-R (pS5-R3 and the FAS-IC (for details of the constructs, their
preparation, etc. see nbove). Tbe results of this sludy are depicted in Fig. 9 (.4 and Bj, wherein in
30 both Fig. 9A nnd B the constructs used for transfecting the HeLa cells are shown sAh~Am~ir~lly in
the lefl hnnd parleis; the results of the ll~F or FAS receptor e~;pression are shown ~raphically in
the two middie pnnels (second and third panels from the lefl), and the results of transfected cell
viability are shown ~raphicaiiv in the ri~ht hand pnnels. In Fig. 9A there is shown the resuits of
transfected ~{eLa cells transiently expressing the full-length pS5-R pS5-IC or parts thereof. or as
35 a control, luciferase (Ll,'C), in all cases using a l~n~ controlled expression vector In Fi~.
9B there is sho~vn the resuks of transfected HeLr, celis transiently expressing ~AS-lC alone or
together with the pS5-R, using a tetracycline-controll~d expression veclor In the graphic
..S,.Il~a~iOl~ of the resu~ts in Fig. 9A and B, the open bars represent cells transfected in the
presence of tetracycline (I ~tg/n~ hich inllibits expression, and the closed bMs repres~nt cells
40 transfected in the absence of tetrac~cline. TN~ receptor expression ~as assessed 20h afle~
WO 95~31s~4 2 1 8 9 9 8 3 ~u~ ~ 4
tr~n~fP~ n, bolh by 3~LISA usir,~ anhbodies against the .~ u.ll.d~ dom~ir~ of the receptor (see
left hand panels)~ and b~ T~ the bindin~ of I ~..liol~ l.d TNF to the cells (midd~e panels),
The cytocidal eft^ect of the transf~cted proteins was assessed 4~h after transfection. The data
shown are from one of thr~e ~ is with qllLi;.~ similar results in which each construct
5 . was tested in duplieate The designation `ND' in Figs. 9A and B me2ns not d~termined. From the
results shown in Figs. 9A and B i~ is apparent that expression of only the pjjlC results in even
greater ~ UtUAiC;ly. Signifieant c~totoxicity a~so occurr when e~;pressing just the death domain
(p55DD). In eorltrast~ expression of parts of pS51C lackin~ the death domail~ or eontainulg onl~
part thereof. had no effect OD cell viability, Expr~ssion of the FAS-IC did not result in si~nifieant
10 cytûtoxicity, yet it significAnti~ enhanced the cytoto~icity of co-expressed pS5-R.
F'XAIUPI.F~ 3:
Additiona~ er~tP;n~ eApal~le of b " to th- intrarellul~r domains vf p~5 Tl~F-R or FA~-
IS Using the same Approach and teehnology set forth in Example 1 above, three more
proteins have been isolated and identified whieh 2re capable of binding to the pS51C or FAS-IC.
In Figs. Iû-12 there is shown ~ the partial and preliminary nucleotide
sequenee of cDNA elones, called F2, F9 arld DDI 1, Ic~,u~ 'y.
Clones F2 and F9 were isolated by Screenin~ a murine (mouse) embryonie library using the
20 murine FAS-IC as ~bait~. In Fig. 10 there is sho~vn ~ the partial nueleotide sequenee
from the F2 eDNA that has been sequeneed. In Fig. 1~ there is shown, '.~ lly th6 partial
nucleotide sequence of 1724 bases from the F9 eDNA that h2s been sequenced. Anaiysis of the
biodirlg c2pability of the protein encoded by clones F2 and F9 (F2 and F~"~ .,ly) has
showrl that
(a) F2 interacts strongly with human p~SlC and pSSDD and ~vith murine FAS-IC~
whii~ it interacts weakly with non-rele~ant (control) proteins SNFI and Larnin as well 2s relevant
protein, hurnan FAS-IC.
(b) F9 interaets strongly with human p5 5-IC and murine FAS-IC, while it interacts
wea3dy with ho FAS-IC (rdevant protein) and irre3e~ant proteins SN3 1 ard Lamin.(e) Neither F2 nor Fg interacted 2t all with human p751C, pGBT9 (empty bait
veetor), or human CD ~10,
3~urth~r, from '&ene Bank' and ~rotein Bank' searches it was revealed that F7 and
F9 represent ne~v prot~ins,
Thus, F2 and F9 represent new proteins h2ving binding speeificity for both FAS-IC
and p551C.
Clone DDI 1 w2s isolated by screening a human 31eLa librar~ using th~ humarl p55D3~ as
"bait". In Fig. 12 thae is shown ~ Rlly the partial nucleotid~ sequence of 4~5 bases frorn
the DD I I cDNA that has c~e~n s~qu~nc~d.
Th~ DDI I clone has an approx. Ien~th of ~00 nucleotides. The full len~th of the transcript
2~,.t l ~ k~. ~he tr2n~cr~n~ nn ~eer -rr.~-A ~ h- c!nr,- ~.n~ r!~ hin~
... _ ... . . . . . .. . ... . _ .
wo 95131544 ~ 1 8 ~ ~ 8 3 ~ ,S.'~OJ~
capabi~it5~ of the protein encoded b~ clone DD 11 has shown tl~at DD 11 iMeracls stronsl~ with the
pSSDD (a.s. 326-414) (see Fi~. 9~ snd does not interact with deletion mutants of this domain, e B
a.G. 32610~. DD11 also interacts with mouse and human FAS-IC and to some eAtent also with
l,amin. DDI I does not interact at all with SNFI nor with pG13T~ (~mpry bait vect~r). DDI I is
5 GlSo not found in the 'Gene Bani;' and 'Protein BanlA' databases. Thus DDI 1 represents a pSS IC
(pSSDD) and FAS-IC specific bindin6 protein.
E~nmple 4
of soluble ~ P - -
0 Based on the findings set forth in ~xample ~ above, that the ;.. L~ allGI domain of the
pSS-~ (pSS-IC) Gnd a portion th~reof (the 'death domain'), and that the ;~ GcellulGI domain of the
Fas~POI and a portion thereof ~also called the 'death domain') which resembles the pSS-lC
'death domain', are capable of self-association~ it is possible to construct new lNF rec~ptors
which are capable of self-association (a~regation) and which are solubl~. Such TNF receptors
~S will be fusion proteins ha~in~ essentially all of the ~AL~dccllul4, domain of the pSS-R fused to
~ssentially Gll of she illtl~c~llulG~ dom2ins or 'death domains' thereof of the pj~-R or Fasl~POI.
Thus. such fusion G~.~structs ~ill be devoid of the t,,."~.. '.. ~' domain of the p~S-R (or
FAS/APO1) and hence will be soluble. Moreover. by ~irtue of the self-associasion capability of
the i.~ cc',lul~r domailts or Ideath domains' sherEof. thesE ftsion constr~ucts wil~ be capable o~'
70 e~i,, ' ' to pro ide at least dimers (arld possibly also higher order multimers~ of the pS5-
R C~ 1y~ such dimeric TNF receptors tpSS-R) will be capable of bindin~ to at least two
TNF monomers of the naturally-occurrint~ TF homotrimer to pro~ide a soluble lNP r~ceptor
which binds more a~idly to its lir~altd ti.. v~ ic TNF).
~ccordingly, at least four rypes of pSS TNF receptOr fusion proteins will be consrructed
Z5 each of whieh will be capable of :'i., and will be soluble:
(i) A fusion product bet~veen the e~ L dornain of pSS-R t~C55) and the
~t avel~ul~ domain of pSS-R (p55-TC);
(ii) ~ fusion product between the EC55 and the 'death domain' of pS5-IC (DD55):
(iii) A fusion product bet~een the EC~ and the illU~,,CII.,I~ domain of Fas/APO130 (IC~AS); and
(v) A fusion product betv.~een the EC55 and the Ideath domain' of ICF~S
(DDFAS)
In each of the above fusion proteins the T~F monomer binding capa~ility is pro~ided by
the EC55 portion while the Ol;~v....,~l~,dL;vll (or at least ,I;~ ;vll) of each kind of fusion
35 protein is provided by its 'tail' re~ion bein~ an~ ofthe p551C, DD~5, ICFAS or DDPAS portions
For cv~ . c~i.,-~ of the above fusion proteinS, standard techniques of ~tCo~ DNAtechnolog~ will be employed that are now well established in the a~t (see for exampie Sambrook
et al, tl9~,) ~olecular Clonirg: A Laborator~ l-lanual~ Cold Sprin~ Harbor Laboratory Press,
Cold Spring Harbor, N.Y,), Briefly, arly suitable bacterial bdclc~;v~ g~, or anirnal ~irus
40 expression vector (cloning ~ehicle or p!asmid designed for expression of the inserted DNA of
WO 95/31544 2 1 8 9 9 8 3 F. ~ - 4
59
choice) maS be emplQyed intc ~hich ~ill be ins~ned in on~ or more stages the D~ encodin~ the
EC55 aDd one of the 'tails' bei~l~ the p5~-iC, DD55. ICFAS or DDFAS. The so-inserted DNA
encoding each of the filsion p[oteins will be placed under the control of the arious expression
control sequences of the clonin~ ~ehicle or plasmid such as pron~oters, ribozS~me binding sites,
s ~ "","-l factor bindiny sites~ etc. These expression control sequences will be chosen
depending on the type of expression vector chosen and hence the type of host cell teu};aryotic or
prokaryotic) in whicb it is desired to expresS the fusion proteins of the invention. Preferred host
cel~s (and hence e~cpression vectors) are eukaryotic~ in particular, m~~~m~;7~
The DNA molecule encodin~g each of the above noted fusion proteins will be prepared and
inserted into the expression ecror by the following procedure:
(a) Firstly, a set of ~I;cr,,,~lcv~ for use in PCR uill ~e conscructed by standard
means, the ~ v~ r c being:
l) ACC ATG GGC CTC TCC ACC GTG (EC55, sense)
2) ACGC GTC G.~C ~GT GGT GCC TGA &TC CTC (EIC55~ antisense)
1 'i 33 ACGC GTC G.~C CGC T.~C C.~A CGG T~G AAG (IC55, sense)
4) TCA TCT GAG A.~G ACT GGG (IC55~ antisense
5) ACGC GTC G.~C AAG AGA AAG G~A &TA CAG ~IC FAS~ sense)
~i) CTA GAC C.~ GCT TTG GAT (IC FAS, antisense)
7) ACGC GTC GAC CCC GCG ACG CTG TAC GCC tDD35, sense)
8) ACGC GTC GAC GAT GTT GAC TTG AGT AAA (DD FAS, sense)
(b) P1asmids containing the cloned full-len~h p55-R and Fas/APOI receptors which we
have in our laboratory ~see also co-pending ~P~6~925 and Examples 1-3 above) will be subjected
to the following, ;~ to yield the Dr~;A fragments encoding each of the fusion proteins,
which DNA fragments are then lisated into the above noted expression vector of choice:
~3 To produce the DNA fragment coding for i~C55 which is a component of all 4
fusion proteins, PCR is performed on a plasmid beariDg cDNA of human p55 usin~ the above
'i" ' - ' nos. I and 2 (size oFfra~nent 640 bp).
(il~ To get a fusion product EC55-1C55, PCR is perforrned on a plasrnid bearin~
cDNA for human p55 using ~ ;J~ nos. 3 and 4, to obtain a DNA fragment coding forIC55 (size 677 bp) which is th~n n~ixed with EC55 dG~ested by Sal I and ligated by blunt ond
li~ation into any expr~ssion vector for manun~Gan cells under the control of an ~ v~ a~
prornoter. The orientation of the inserted EC55--IC55 in the vector is ~erified by restriction
dig~stion and by sequencing.
~lii) To g~t a fusion product EC55-IC FAS, IC FAS is produced by PCR on a
35 plasn~id with cDNA for FAS usin~ ~lig "~rl. .~ lr nos. 5 and 6, to obtain a fra~neM (size 4~8
bp) which is then cut by Szl I and mi~ed vith ECj5 cut by SalI, and ~ c~ ly is blunt ~i~atcd
into a mammalian expression vector under the control of an zppropriate promoter. The
orientation of the inserted EC55--lC FAS in the vector is verif ed by restriction digestion and by
sequencing.
... .. , , . , . , . ... . . . . .. .. . _ _ . _ _ . _ _ _
WO95/31544 2 1 ~q9 83 P~ 4
(is) To ~el a fusion product ~C5j--DD~, a DNA fragment is produced ~ith the
DD55 sequencc by PCR in cDNA for human pS5 using rl;_. ,1 .~1~". ;.1~ nos. 7 and 4. The product
~ith a size of 314 bp is cut b~ Sall and mixed with EC55 cut by SalI, and ~,.1,,~.1,.. .,11!, blunt
Ggated imo the mammalian expression vector. Orientation of the inser~.ed EC55--D355 in the
5 vector is ~erified by restriction digestion and by scquencjng.
(v) To ~et a fusion product EC55--DD FAS, a DI~A fra~ment with DD FAS is
produced by PCR on cDNA for FAS usinr! c' ~ ' nos. 6 and 8. Th~ product with a si~e
of 332 bp is cut with Sall. and mixcd with EC55 cut by Sal I and ~ ..c~.~ly blunt li~ated into
thc mammaiian expression vector. OrieMation of the EC55--DD FAS is then verified by
10 restriction digestion and sequencin~,
Once the above expression vectors have been constructed~ they will then be introduced by
standard methots into suitable mamma~ian cells (e.g. Chinese Hamster OvarS (CHO) or l\Ionkcy
Kidney (COS) cells) for the purposes of expression. The so-expressed fusion proteins will then be
purified by standard methods (see co-pending EP30~37~; EP39~327~ and EP56~25). T~e
15 purified fusion proteins uill then be anal~zed for their ability to oli~Tom~n~f~ (and the exterlt
thereof~ i.e. whether they form dimers or hiBh order multimers) arld for their ability to birld TNF
(and the affinitj or avidity of binding thereof).
WP~
ZO ~ troctio~l of ~ - Fs~/APOI l -
In a similar fashion to that set forth in E~cample 4 above, it is possible to produce thefollowing four kinds of Fas/A~OI fusion products, cach of ~vhich will bc capable of
~i" and will be soluble:
(i) Fusion product betu~een the ~ - n~ domain of FaslAPOl (EC FAS) and
the " domain of p55-lC;
(ii) Fusion product between the EC FAS and the 'death domain' of p55-lC
(DD55);
(iii) Fusion product bet~cen the EC FAS and the ;,.t.~ lu:~ domain of
Fas/APOI (IC FAS); and
(rv) Fusion product between the EC FAS and the 'death domain' of IC FAS Q:)D
FAS).
In each of the above fusion proteins the FAS Ggand binding capabilit~ is provided by the
EC FAS portion, while the .~ ' _ ;~L;oll (or at least " ;~I;o,l) of each kind of fusion
protein is provided by its 'tail' region bein~ any of the p5~-IC. DD55, IC FAS or DD FAS
35 portions.
The C~....~u~,liO~ of the D~T~ fragments encoding the abo~e filsion proteins andexprcssion vectors containing thcm will be as detailed in Example 4. except dif~rent appropriate
~'J~ ;df ~ (not shown) will be used for the pr~paration of the EC F~S fra~ment to be
Ggated to anST~ of the above noted 'tair re~ions. Su~s~lu~ y~ the expreision vectors will be
~" introdl~ced into the suitable host cells. and the resulting expressed fusion proteins uill be purified
21 8~983
W0 95/31544 ~ 4
.
61
and tested for their ability to o~i~omaiz~ (and the extent thereof, i.e. whether they form dimers or
higher order multimers) and for Iheir abilil~ to bind the FAS ligand (and the affinif~ or a~idity of
binding thereof ~.
S EA~lmple G
C~ - of Ssb~ ed' TNF~A.~ e rs
To prcpare oligomeric receptors having 'mixedl affirlity, i.e. affini~v for both l`NF ~nd ehe
FAS-R li6and, the abv~ rd (~xamples 4 and 5) fusion products may be ulilized in the
following procedure:
i) Providing a fusion producl as set forth in ~xample 4, v.hich contains th~
e~l, A~clhPal domain of a IANF-R ~p75 TNF-R or pS5 ~IF-R) fused to any one of the pS5 IC,
FAS-IC, pSS DD or FAS DD,
ii~ ProYiding a fusion product as set forth in E~xample 5, which contains the
e~. a~whlal domain of Fas-R fused to any one of: pS5 IC, FAS-IC, pS S DD or F~S-DD; and
iii) mixing an~ one of the fusion products of i) with ans, one of the fusion
products of ii) to pro~ide a ne~v dimeric (or higher order oligomeric) receptor which has both Ihe
domAins of a T~F-R and FAS-R that are joined b~ their -IC or -DD regions.
In the above procedure Ihe fusion products of i) ~nd ii) may be proYided separalely,
namely, from th~ir purification from lIA.~ cells in which they were produced, and then
20 raixed in vi~o to obtairl th~ mixed affinity receptors. AlternatiYely, the host cells may be c~
transfected Y~ith ~ectors curying seo,uerlces encoding both ~pes of fusion products, in which
case, the mixed affinit,Y receptors mav be obtairled directly from the co-transfected cells. The
actuAI ~ of th~ fusion producls into oligomeric receplors may lal;e place ~vithin the
cells or during or followin~ the purification procedur~ to obtain the fusion products exprused in
25 the cells. To spccifically select for the mixed aff~nitY receptors an~ standard method ~nay be
utilized, for examp~e, affinity ~h.OlllAa~ P1~ procedures in which antibodies againsl Ihe TNF-R
and FAS-R ~ domains ue used in sequemial, lu~ grar~;r sleps to select for those
rewp~orsbhv~otht~esof~ h' ~
21 8q~3
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