Note: Descriptions are shown in the official language in which they were submitted.
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MODULATOR OF TNF/NGF SUPERFAMILY RECEPTORS AND SOLUBLE
OLIGOMERIC TNF/NGF SUPERFAMILY RECEPTORS
Field of the Invention
The present invention is generally in the field of receptors belonging to the
TNF/NGF
superfamily of receptors and the control of their biological functions. The
T'I~'FINGF superfamily
of receptors includes receptors such as the p55 .and p75 tumor necrosis factor
receptors ('TI~tF-Rsl
and the FAS ligand receptor (also called FAS/APOI or FAS-R and hereinafter
will be called FAS
R) and others. More specifically, the present invention concerns novel
proteins which bind to the
intracellular domains (IC) of the p55 and p7~ TNF-Rs and the Fas-R, (these
intracetlular domains
designated p55IC, p75IC and Fas~IC, respectively) and which novel proteins are
capable of
modulating the function of the p:~ and p?5 TNF-Rs and the Fas-R. One of the
proteins capable
of binding the p55IC of the imact p~5-TIr'F R is the p55IC itself in the form
of a p55IG molecule
or a portion thereo>~ such as for example. the so-called 'death domain' (DD)
of the p55IC. Thus,
the present inversion also concerns new T's1F-associated effects that can be
induced in cells in a
ligand (TNF)-independent fashion by the intracellular domain of the p55 TNF-R
(p55IC) or
portions thereof. The present invention also concerns the preparation and uses
of these novel p55
and p75 TNF-R-binding proteins, and Fas-R binding proteins, referred to herein
as pSSIC-,
p75IC- and Fas-IC- binding proteins.
In another aspect, the present imrention also concerns new soluble oligomeric
TNF-Rs,
oligomeric FAS-Rs and oligomeric receptors having a mixture ofThl'F-Rs and
I~AS-Rs, their uses,
and methods for the production thereof.
BackøroLad of the Invention and Prior Art ,
Tumor Necrosis Factor (TNF-a) and Lyrnphotoxin (TNF-Vii) (hereinafter, TNF,
refers to
both TNF-a and TNF-p) are multifunctional pro-inflammatory cytolines formed
mainly by
mononuclear phagocytes, which have many effr'ects on ceps (Wallach, D. (1986)
in : Interferon 7
(Ion Dresser, ed.), pp. 83-I22, Academic Press, London; and Beutler and Cerami
(1987)). Both
TNF-cc and TNF-~i initiate their effects by binding to specific cell surface
receptors. Some.of the
effects are likely to be beneficial to the organism : they may destroy, for
example tumor cells or
virus infected cells and augment arsibactaial activities of granulocytes. In
this way, ;TNF
contributes to the defense of the organism against tumors and infectious
agents and contributes to
the recovery from injury. Thus. TNF .can be used as an anti-tumor agent in
which application it
binds to its receptors on the surface of tumor cells and thereby initiates the
events leading to the
death of the tumor cells. TNF can also be used as an anti-infectious agent.
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However, bole. 5?v'F-a and TNF-G also have deleterious er'focts. There is ev
idence that
over-production of T~; -a can play a major pathogenic role in several
diseases. Thus, effects of
TNF-a, primarily on t: vasculature, are now known to be a major cause for
symptoms of septic
shock (Tracey et al., 1~SG). In some diseases, Tiv'Ir' may cause excessive
loss of weight (caehexia)
by suppressinb artivit:a of adipocytes and by causing anorexia, and TNF-a was
thus called
cachetin. h was also ;:eseribed as a mediator of the damage to tissues in
rheumatic diseases
(Beutler and Cerami, 1S?) and as a major mediator of the damage observed in
graft-versus-host
reactions (Piquet et a:., 1987). In addition, TVF is known to be involved in
the process of
inflammation and in mt~tv other diseases.
Two distinct, independently ecpressed, receptors, the p~~ and p7S T~TF-Rs,
which bind
both Th(F-a and T~If-G specifics!!}~, initiate and/or mediate the above noted
biological effects of
TNF. These two receetors have structurally dissimilar intracellular domains
suggesting that they
signal differently (See Hohmann et al., 1989; Engelmann et al., 1990;
Brockhaus et al., 1990;
Leotscher et al., I99C'. Schall et al., 19.90; Nophar et aL, I990; Smith et
al., 1990; and Heller et
al.. 1990). I-3owever, the cellular mechanisms, for example, the various
proteins and possibly other
factors, which are invoiced in the inuacellular signaling of the p55 an p i 5
TIv'F-Rs have yet to be
elucidated {as set forth herein below. there is described for the first time,
new proteins capable of
binding to the p?SIC and p55 IC). It is this intracellular signaling, which
occurs usually after the
binding of the Iigand, i.e. TNF (a or ~), to the receptor, that is responsible
for the commencement
of the cascade of reactions that ultimately result in the observed response of
the cell to TNF.
As regards the above mentioned cytocidal effect of TNF, in most cells studied
so far, this
effect is triggered mainly by the p55 TNF-R Antibodies against the e~.-
tracellular domain {ligand
binding domain) of the p55 TNF-R can themselves trigger the cytocidal effect
(see EP 413486)
which correlates with the effectivitt~ of receptor cross-linking by the
antibodies. believed to be the
first step in the generation of the intracellular signaling process. Further,
mtvational s~.:~dies
(Brakebusch et al., 199; TartagIia et al., 1993) have shown that the
biolo~rical function of the
pS5 TNF-R depends on the intcgrit;~ of its intracellular domain, and
accordingly it has been
suggested that the initiation of intracellular signaling leading to the
cytocidal effect of TNF occurs
as a consequence of the association of two or more intracellular domains of
the p55 TNF-R.
Moreover, TNF (a and Vii) occurs as a homotrimer and as such has been
sugbested to induce
intracellular signaling va the p55 7'Nf-R by way of its ability to bind to and
to cross-link the
receptor molecules, i.e. cause receptor aggregation. Herein below there is
described how the
pSSIC and pSSDD can self associate and induce, in a figand-independent
fashion, T';~F-associated
effects in cells.
Another member of the TNFINGF supecfamily of receptors is the FAS receptor
(FAS-R)
which has also been called the Fns antigen, a cell-surface protein expressed
in various tissues .aid
sharing homology with a numbs of cell-surface receptors including TNF-R and
NGF-R. ?he
FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991 ), and
appears to serve as a
negative selector of autoreactive T cells, i.e. during maturation of T cells,
FAS-R mediates the
apoptopic death of T cells recognizing self antigens. It has also been found
that mutations in the
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PCT/US95/05854
FAS-R gene (Ipr) cause a lymphoprofiieration disorder in mice that resembles
the human
autoimmune disease systemic Iupus erythematosus (SLE) (Wacanabe-Fukunaga et
al.. 1992). The
ligand for the FAS-R appears to be a cell-surface associated molecule carried
by, amongst others,
killer T cells (or cytotoxic T lymphocytes - CTLs), and hence when such CTLs
contact cells
carrying FAS-R, they are capable of inducing apoptopic cell death of the FAS-R-
carrying cells.
Further, a monoclonal antibody has been prepared that is specific for FAS-R,
this monoclonal
antibody being capable of inducing apoptopic cell death in cells carrying FAS-
R, including mouse
cctls transformed by cDNA encoding human FAS-R (Itoh et al., 1991).
It has also been found that various other normal cells, besides T lymphocytes,
e~cpress the
FAS-R on their surface and can be killed by the triggering of this receptor.
Uncontrolled induction
of such a killing process is suspected to contribute to tissue damage in
certain diseases, for
example, the destruction of liver cells in acute hepatitis. Accordingly,
finding ways to restrain the
cytotoxic activity of F AS-R may have therapeutic potential.
Conversely, since it has also been found that certain malignant cells and HIV-
infected cells
carry the FAS-R on their surface, antibodies against FAS-R, or the FAS-R
licrand, may be used to
trigger the );AS-R mediated cytotoxic effects in these and Lhereby provide a
means for combating
such malignant cells or HIV-infected cells (see Itoh et al., 1991 ). Finding s-
et other ways for
enhancins~ the cytotoxic activity of FAS-R may therefore also have therapeutic
potential.
It has been a long felt need to provide a u~ay for modulating the cellular
response to TNF
a or ~) and FAS-R ligand, for a:ampte, in pathological situations as mentioned
above, where
TNF or FAS-R ligand is over-expressed it is desirable to inhibit the TNF- ar
FAS-R lieand-
induced cytocidal effects, while in other situations, e.g. wound healing
applications, it is desirable
to enhance the TNF effect, or in the case of FAS-R in tumor cells or HI'f-
infected cells it is
desirable to enhance the FAS-R mediated eB'ect.
A number of approaches have been made by the present inventors (see for
example,
European Application Nos. EP 186833, EP 308378, EP 398327 and EP 412486) to
regulate the
deleterious effects of TNF by inhibiting the binding of TNF to its receptors
using anti-TNF
antibodies or by using soluble TNF receptors (being essentiaDy the soluble
ea~tracellular domains
of the receptors) to compete with the binding of TNF to the cell surface-bound
TNF-Rs. Further,
on the basis that TNF-binding to its receptors is required for the TNF-induced
cellular effects.
approaches by the present inventors (see for example EPO 568925) have been
made to modulate
the TIvF effect by modulating the actt<~ity of the Tlv'F-Rs. Briefly, EPO
X68925 relates to a
method of modulating signal transduction and/or cleavage in TNF-Rs whereby
peptides or other
molecules may interact either with the receptor itself or with effector
proteins interacting with the
receptor, thus modulating the normal functioning of the TNF-Rs. In EPO 568925
there is
desetibcd the construction and characterization of i~arious mutant p55 TNF-Rs,
having mutation
in the extraceUular, transmembranal, and intracellular domains of the p55 TNF-
R. In this way
regions within the above domains of the p55 TNF-R were identified as being
essential topthe
functioning of the receptor, i.e. the binding of the Iigand (?NF) and the
subsequent signal
ttansduction and intracellular signaling which ultimately results in the
observed TNF-effect on the
4
cells. Further, there is also described a number of approaches to isolate an~
identify proteins,
peptides or other factors which are capable of binding to the various regions
ir. the above domains
of the TNF-R, which proteins, peptides and other facxors may be involu:d in
regulating or
modulating the activity of the TNF-R. A number of approaches for isolating ar
d cloning the DTIA
sequences encoding such proteins and peptides; for constructing expression
vectors for the
production of these proteins and peptides; and for the preparation of
antibodies or fr$emcnts
thereof which interact with the TNF-R or with the above proteins and peptides
that bind various
reaons of the TNF-R, are also stt forth in EPO 568925. However, no description
is made in EPO
X68925 of the actual proteins and peptides which bind to the intracvlluiar
domains of the TNF-Rs
. 1 G (e.g. p55 TNF-R), nor is any description made of the yeast two-hybrid
approach to isolate_ar~d
identify such prottins or peptides which bind to the irnracellular domains of
TNF~Rs. Similarly,
heretofore there has been no disclosure of proteins or peptides capable of
binding the intracellular
domain of FAS R. ._
Thus, when it is desired to inhibit the effect of T'._v'F, or the FAS-R
:igand, it would be
l ~ desirable to decrease the amaum or the activity of ?NF Rs or FAS-R at the
cell surface, while.arl
increase in the amount or the activity of TNF-Rs or FA,S-R would be desired
when an enhanced
TNF or FAS-R tigand effect is sought. Ta this end the promoters of both the
p5~ T~1'F-R and.~the
p75 T13F-R have recently been sequenced and analyzed by the present inventors
and a number of
kcs~ sequence motifs have been found that are specific to various
transcription regulating factors,
2C~ and as such the expression of these TNF Rs can be controlled at their
promoter level, ..i:e.
itdu'bition of transcription from the promoters for a decrease in the number
of receptors, and an
enhancement of transcription from the promoters for an increase in the number
of receptors.
Ca:responding studies eaneernirrg the control of FAS-R at the level -of-.the
25 promoter of the FAS-R gone have yet to be reported.
Further, it should also be mentioned that, while it is known that the tumor
necrosis factor
' (T'N~ receptors, and the structurally-rtlated receptor FAS-R; trigger in
cells, upon stimulation by
leukocyte-produced ligands, destructive activities that Lead to their own
demise, the mechanisms
ef this triggering are still little understood. Mutational studies indicate
that in FAS~R and the:p35
30 TNF receptor (p55-R) signaling for cytotoxiasy imrolve distinct regions
within their intracellular
domains (Brakebusch et al,, 1992; Tartaglia et al., 1993; hoh and Nagata,
1993). These.repons
(the 'death dotnains~ have sequence similarity. The 'death domains' of both
FAS-R and.:p5,5~R
tend to self assoaate. Their self association apparently promotes that
receptor aggregation fyvhich
is necessary for itutiation of signaling (as set forth herein below, as well
as Song at al.,, ~199.i;
3 5 Wallach et aL, 1994; Boldin et al., ~ I 995) and at high Levels of
receptor expression can result .in
trigsering of ligand~ndependern signaling (as set forth herein below, and
Boldin et al., 199,). .
Thus, prior to the present invention, there have not been provided proteins
which may
regulate the effect of Iigands belonging to the TN'1~INGF superfamily, such as
the TNF or.FAS-R
Iigand effect on ~eclls, by mediation of the intracellular signaling process,
which signaling ;is
40 probably governed to a Large extent by the intracellular domains (ICs) of
the receptors below
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to the ThIFINGF superfamify of receptors, such as those of the TNF-Rs, i c.
the p55 and p?~
T:~IF-R intracellular domains (p55IC and p75IC, respectively), as well as the
F AS-IC.
Accordingly, it is one aim of the invention to provide proteins u~h:ch are
capable of
bindinb to the intracellular domains of the TNF-Rs and FAS-R which proteins
are presentlw
believed to be involved in the intracellular signaling process initiated by
the binding of TNF to its
receptors, or the bindinb of FAS ligand to its receptor.
Another aim of the invention is to provide antagonists (e.g. antibodies) to
these
intracellular domain-binding proteins (IC-binding proteins) which may be used
to inhibit the
si~,:naling process, when desired, when such IC-binding proteins are positive
signal effectors (i.e.
lu induce siunaling), or to enhance the signaling process, when desired, when
such IC-bindin_~
proteins are neLative signal effectors (i.e. inhibit signaling),
Yet another aim of the incemion is to use such IC-bindinb proteins to isolate
and
characterize additional proteins or factors, which may, for e:cample, be
involved further
downstream in the signaling process, and/or to isolate and identify othe:
receptors further
1~ upstream in the signalins process to which these IC-binding proteins bind
(e.g.~other T'I~rF-Rs or
related receptors ), and hence, in whose function the IC-binding proteins are
also involved.
Moreover, it is an aim of the present invention to use the above-mentioned IC-
binding
proteins as antigens for the preparation of polyclonal andlor monoclonal
antibodies thereto. The
antibodies, in turn, may be used for the purification of the new IC-binding
proteins from difl'crent
2G sources, such as cell extracts or transformed cell lines.
Furthermore, these antibodies may be used for diagnostic purposes, e.g. for
identifying
disorders related to abnormal functioning of cellular effecu mediated by
receptors belonging to
the TNFINGF receptor superfamily. .
A further aim of the invention is to provide pharmaceutical compositions
comprising the
2~ above IC-binding proteins, and pharmaceutical compositions comprising the
IG-binding protein
amaganists, for the treatment or prophylaxis of TNF-induced or FAS ligand-
induced conditions,
for example, such compositions can be used to enhance the TI\'F or F AS Iigand
effect or to inhibit
the 'fNF or FAS ligand effect depending on the above noted natwe of the IC-
binding protein or
antagonist thereof contained in the composition.
30 Moreover, in accordance with another aim of the present invention, there is
disclosed
other ways for eliminating or antagonizing endogenously formed or exogenously
administered
TNF or FAS-R ligand, by the use of soluble oligomeric TNF-Rs, oligomeric FAS
Rs, or
oligomers being a mixture of TNF-Rs and FAS-Rs. In this respect it should be
mentioned that one
attempt in this direction was the isolation and recombinant production of a
TIFF Binding Protein
35 called TBP-1 which was shown to be able to antagonize the effects of TNF.
This a~rtagonistn was
determined both by measuring reduction of the cytotoxic activity of TNF', as
well as by measuring
interference of ~NF binding to its receptors (EP 308 378). ?BP-I was shown to
protect cells from
TNF toxiaty at concentrations of a few nano~ams per ml and to interfere v~~ith
the binding of
both 'rNF-a and TNF-p to cells, when applied simultaneously with these
cytoltines. Further
40 examination of the mechanism by which THP-I functions revealed that TBP-I
does not interact
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with the target cell, but rather blocks the function of TNF by binding 'I1-F
specifically, thus
competing for TNF with the TNF receptor.
Consequently, with a different purification technique, the presence of two
active
components was found : one, TBP-I, and also a second TIv'F-binding protein
which we called
TBP-II (first described in EP 398327). Both proteins provide protection
zgainst the ire vitro
cyZOCidal effect of TNF and both bind T~1F-p less effectively than TNF-c:
Although in SDS
PAGE analysis the two proteins, TBP-I and TBP-II, appeared to have a wen-
similar molecular
size, they could clearly be distinguished from cach other by Lack of immunolo=
cal cross reactivity,
differing N-terminal amino acid sequences and differing amino acid
composition.
Fiowcver, the above noted earlier soluble TNF binding proteins are manomeric
and being
capable of binding only one monomer of the TNF homotrimer, the natural Iigand,
which still
permits TVF activity (i.e. incomplete neutralization) by virtue of the fNF
stil: having two active
monomers unbound by the TIvTr binding proteins. Further, heretofore there ha:
been no disclosure
of soluble FAS-Rs (soluble FAS-R ligand binding proteins) capable of binding
to 1~AS-R ligand
1~ which is known to be a homotrimeric, cell-surface associated molecule.
A so-called 'death domain' of the pss-IC (Tartaglia et al., 1993) hzs been
disclosed, but
did not show, in accordance with the present invention, that the p55-IC and
the 'death domain'
thereof self associates, this self association being primarily responsible for
the si'naling leading to
induction of cell c5~totoxis. Moreover, this publication is silent on the
possibility of produc'mg the
soluble, oligomeric TNF-Rs, or the soluble, oligomeric Fas-Rs, or mixed
oligomeric thereof nor
does it disclose other TNF-associated effects induced by the p55-IC or
portions thereof, e.g. IL-8
gene expression induction, all of the present invention. Likewise, another
publication, published
after the date of the present invention, disclosed the aggegation (i.e, self
association) ability of
the p55-IC, but did not relate, as noted above, to the usage thereof to
prepare soluble, oligomeric
TNF-Rs or Fas-Rs nor to the other TNF-associated effects induced in a lis;a: d-
indepo~a~t
manner by the p55-IC or portions thereof according to the invention
Summary of the Invention
In accordance with the present invention, we have found novel proteins which
are capable
of binding to either the intracellular domain of the p55 T'!VF-R (the pSSIC-
binding proteins), of
the p75 TNF-R (the p75IC-binding proteins), and of the FAS-R (the FAS-IC-
binding proteins).
Thcsc p55IC-, p75IC- and FAS-IC- binding proteins may act as mediators or
modulators of the
T.NF or FAS-R liband effect on cells by way of mediating or modulating the
intracellular signaling
process which usually occurs following the binding of TNF to the p55 andlor
p75 TNF-R, or the
binding of the FAS-R Iigand at the cell surface. Further, it has been
surprisingly and unexpectedly
found that the p55IC and FAS-IC are capable of self association and that
fragments of the p55IC-
and FAS-IC are similarly capable of binding to the p55 IC, particularly the so-
called 'death
domains (DD) within the ICs of these receptors, i.e. the p55DD and FAS-DD.
Thus, p55 IC and
FAS-IC and their fragnents also represent proteins capable of binding to the
p55IC and FAS-IC
and hence may be modulators of the 'f~TF or FAS-R ligand effect on cell s.
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7
Furthermore, the nature of the binding of one of the novel proteins of the
invention, the
herein designated ~5. i 1 protein, to the intracellular domain of p55-TNF-R
has been more fully
elucidated (see Example 1 ),
Moreover, in another aspect, the present invention is based on the finding
that the
S intracellular domain of the p55 TNF receptor (p55-IC), a region contained
therein, the so-called
p55-IC 'death domain', the intracellular domain of the FasIAPOI receptor fFas-
1C), and a re~~ion
contained therein, the so-called Fas-IC 'death domain' are capable of self
association.
Accordingly, it is possible to construct by standard recombinant DNA
techniques, a soluble,
oligomeric TNF receptor being a fusion product, containing at least two
extracellular domains of
14 a TNF receptor at its one end, and at its other end at least two of the
above noted self associating
invacellular domains or portions thereof, which self associate to provide an
olioomer having at
Ieast two such fusion products linked together. Such a soluble, oligomeric
T1'F-R is thus capable
of binding two monomers of the naturally-occurrinb TTv'F homotrimer, and as
such effectively
neutralizes T'fF activity. The neutralization of TNF activity being desirable
in all of the above
15 mentioned conditions wherein TNF is overproduced endogenously or is
administered exoeenously
in high doses resultin' in undesirable side effects. Further, the effective
binding of TNF by the
soluble, oligomeric receptors of the invention may also serve to allow for the
binding of
exoeenously added TI~rF and its subsequent desired slow-release in conditions
where TNF is
administered for its beneficial effects, e.g. in tumor therapy. Likewise, it
is also poss'bIe to
20 construct by standard rerombittant DNA techniques an oLigomeric FAS-R being
a fusion product,
containing at least two exvaceUular domains of a FAS-R at its one end, and at
its other end at
least two of the above noted self associating intracellular domains or
portions thereof, which self
associate to provide an oligomer having at least two such fusion products
linked together. Such
an oligomcric FAS-R is thus capable of binding two monomers of the naturally
occurring FAS R
25 ligand homotrimer, and as such effectively neutralizes FAS-R ligand
actiZ~iy The n°~itralizatio~. of
FAS-R ligand activity being desirable in all of the above mentioned conditions
where excess
amounts thereof are associated with undesirable side effects. In a similar
fashion.. and in view of
tecent reports indicating a possible associating between 'TNF and FAS-R ligand-
induced effects
on cells and hence also a possible association, geo~aphically at the cell
surface where they attach
30 to their receptors, it is also possible to construct by standard
recombinant DNA techniques a
mixed oligomeric receptor having specificity for both Ti'JF and FAS-R
lic_:and. Such a mixed
oligomer would be a mixture ~of the above noted fusion products containing at
least one
extracellular domain of a TNF-R and at least one extracellutar 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
3 S portions thereof, which self associate to provide a tnixcd oligomer having
at least two such fusion t -
products linked together. Such a mined oligomer is thus capable of binding at
least one monomer
of TI~tF and one monomer of F AS-R ligand at the same time, thereby reducing
or effectively
neutralizing the TNF and FAS-R ligand activities at the cell surface in
conditions, as noted above
where excess amounts of these two cytokines are associated with undesirable
cellular effects. As
40 noted above, the FAS-R ligand is usually cell-surface-associated, and
recent reports also describe
CA 02490080 1995-05-11
WO 95131544 PCTlLjS95105854
s
cell-surface-associated forms of TNF. Hence, these mixed TNF-R~FAS-R oli~omers
are especially
useful for neutralization of TNF and FAS-R ligand activities at the cell
surface.
Accordingly, the present invention pro~~ides a D~1.4 sequence enco3in_ a
protein capable
of binding to one or more of the intracellular domains of one or more
re;.cptors belonging to the
tumor necrosis factorinen~e growth factor (TNFlNGF} superfamily of receptors.
_
In particular. the present invention provides a DNA sequence selected from the
group
consisting of
(a) a cDNA sequence derived from the coding region of a native TNF-R
intracellular domain-
bindinb protein;
(b) DNA sequences capable of hybridization to a DNA of (a) under moderately
stringent
conditions and which encode a biologically active TI~TF'-R intracellular
domain-binding
protein; and
(c) DNA sequences which are degenerate as a result of the genetic code to the
DNA
sequences defined in (a) and (b) and which encode a biologically active TNF-R
I 5 intracellular domain-binding protein.
The present invention also provides a DN'A sequence selected from the group
consisting
of
(a) a cDNA sequence derived from the coding region of a native F AS-R
intracellular domain-
binding protein;
(b) DNA, sequences capable of hybridization to a cDNA of (a) under moderately
stringent
conditions and which encode a biolopeally active FAS-R intracellular domain-
binding
protein; and
(c) DNA sequences which are degenerate as a result of the genetic code to the
DNA
sequences def ned in (a) and (b) and which encode a biologically active FAS-R
intracellular domain-binding protein.
In embodiments of the present invention the DNA sequences encode p55 TNF-R,
p75
TNF-R and FAS R irnraceUular domain-binding proteins, such as those encoding
the herein
designated proteins 55.1, 55.3, 55.I I, 75.3, ?5.16, F2, F9 and DD11.
The present invention also provides a protein or analogs or derivatives
thereof encoded by
any of the above sequences of the invemion, said proteins, analogs and
derivatives being capable
of binding to one or more of the intracdlular domains of one or more TNF-Rs or
FA'S ;R.
Embodiments of this aspect of the invention include the herein designated
proteins 55,1, 55.3,
55.11, 75.3, 75.16, F2, F9 and DDI 1, their analogs and their dctivatives. .
Also provided by the present invention are vectors encoding the above proteins
of the
invention, which contain the above DNA sequences of the invention,.these
vectors being capably'
of being expressed in suitable cukaryotic or prokaryotic host cells;
transformed eukaryotic' or
prokaryotic host cells containing such vectors; and a method for produrang the
proteins, analogs
or derivatives of the im~esxtion by growing such transformed host cells under
conditions suitable
for the expression of said protein, analogs or derivatives, efr'ecting post-
translational modifications
of said flrotein . .as necessary for obtention of said protein and extracting
said expressed protein
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9
analogs or derivatives from the culture medium of said transformed cells c:
from cell extracts of
said transformed cells.
In another aspect. the present invention also provides antibodies o: active
derivatives or
fragments thereof specific to the proteins, analogs and derivatives thereof,
of the invention.
By yet another aspect of the invention, there are provided various uses of the
above DNA
sequences or the proteins which they encode. according to the invention, which
uses include
amongst others
(i) a method for the modulation of the TIv'F or FAS-R lisand ~ficct on calls
carrying a
TNF-R or a FAS-R, comprising treating said cells with one or mc~rc proteins,
analogs or
derivatives selected from the group consisting of the proteins, analogs and
derivatives,
according to the invention, and a protein being the p55IC, p~SDD. FAS-IC or
FAS-DD,
analogs or derivative: thereof, all of said proteins being capable of binding
to the
intracellular domain and modulatinL the activity of said T: TF-R or FAS-R
wherein said
treating of the cells comprises introducing into said cells said one or more
proteins.
analogs or derivatives in a farm suitable for intracellular administration or
introducing into
said cells, in the form of a suitable expression vector, the DNA sequence
encoding said
one or more proteins, analogs or dcrivativ es ;
(ii) a method for modulating the TI''F or FAS-R li?and effect on cells
carrying a TNF
R or a FAS-R comprising treating said cells with antibodies or active
derivatives or
20_ fragments thereof according to the invar~tion;
(iii) a method for modulating the TNF or FAS-R ligand effect on cells carrying
a TTIF-
R or FAS-R comprising treating said cells with an oligonueleotide sequence
encoding an
antisense sequence of at least part of the sequence according to the
invention, or
encoding an antisense sequence of the p55IC, pS~DD, FAS-1C, or FAS-DD
sequence.
said oligonucleotide sequence being capable of blocking the e;cpression of at
least one of
the TNF R or FAS-R intraccUular domain binding proteins;
(iv) a method for modulating the TNF or FAS-R iigand efrect on cells carrying
a TNF-
R or FAS-R comprising
(a) constructing a recombinant animal virus vector carrying a sequence
encoding a viral
surface protein that is capable of binding to a specific cell surface receptor
and a sequence
selected from an oligonucleotide sequence encoding an antisrnsc sequence of at
least part
of the sequence according to the invention and an oligonucleotide sequence
encoding an
antisensc sequence of the p~3IC, p55DD, FAS-IC, or FAS-DD sequence, said
oligonucleotide sequence being capable of blocL-ing the expression of at least
one of the
TNF-R or FAS R intracellular domain binding proteins v~~hen introduced into
said cells by
said virus,; and
(b) infecting said cells with said vector of (a).
(v) a method for modulating the TIv'F or FAS-R tigand rffect on cells
carr~ring a TNF
R or a FAS-R, comprising treating said cells with a suitable vector encoding a
ribozymc
X10 having a sequence specific to a sequence selected from an mRI~TA sequence
encoding a
CA 02490080 1995-05-11
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IO
protein, analog: or derivative of the invention and an mR,'A sequer:e
encodin~T the p55IC.
p55DD, F.45-IC or FAS-DD, said :ibozyme sequence capable of interacting with
said
mRNA sequence and capable of cleavin' said mRNA sequence resulting in the
inhibition
of the expression of the protein, anaioe or dert'~ativc of the invention or of
the e~cpression
of the p:SIC, p55DD, FAS-IC or FAS-DD.
(vi) a method for treating tumor cells or HIV-infected cells, or other
diseased cells,
compri sing
(a) constructing a recombinant animal virus vector carrying a sequence
encoding a
viral surface protein that is capable of binding to a tumor cell surface
receptor or HIV
infected cell surface receptor or is capable of binding to another cell
surface receptor of
other diseased cells and a sequence selected from a seauence according to the
invention
encoding a protein, analog or derivative of the invention and a sequence
encoding the
p55IC, p~SDD, FAS-IC, FAS-DD, or a biologically active analot or derivative
thereof.
said protein, analog or derivative ef the invention, p5 SIC, p5 f DD, FAS-JC,
FAS-DD.
IS analob or, derivative, when expressed in said tumor cell or HIS'-infected
cell, or other
diseased cell being capable of killing said cell; and
(b) infecting said tumor cells or HIL'-infected cells or other infected cells
with said vector
of (a).
(vii) a method for isolating and identifying proteins, factors or receptors
capable o
binding to the intracellular domain binding proteins according to the
invention, comprisinc
applying the procedure of a~'miry chromatography in which said protein
according to the
invention is attached to the af~nnity chromatography matrix, said attached
protein is
brought into contact with a cell extract and proteins, factors or receptors
from cell extract
which bound to said attached protein arc then eluted, isolated analyzed;
(W i) a method for isolating and identifying proteins, capable of bindini to
the
intracellular domain binding proteins according to the invention, comprising
applying the
yeast two-hybrid procedure in which a sequence encoding said intracellular
domain
binding protein is carried by one hybrid vector and a sequence from a cDNA or
genomic
DNA library is carried by the second hybrid vector, the vectors then being
used to
transform yeast host cells and the positive transformed cells being isolated,
followed by
exffa,ction of the said second hybrid vector to obtain a sequence encoding a
protein which
binds to said intracellular domain binainst protein; and
('nc) a method for isolating and idettrifying a protein capable of binding to
the
intracellular domains of 'INF-Rs or F AS-R comprising applying the procedure
of non
stringent southern hybridization followed by PCR cloning, in which a sequence
or parts
thereof according to the invention is used as a probe to bind sequences from a
cDNA or
genomic DN.A library, having at least partial homolo~r thereto, said bound
sequences then
amplified and cloned by the PCR procedure to yield clones encoding proteins
having at
least partial homology to said sequences according to the invention.
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The present invention also provides a pharmaceutical composition for the
modulation of
the TNF- or FAS ligand- effect on cells comprising, as active ingredient, any
one of the following
(i) a protein according to the invention, or the protein p55IC, p55DD. FAS-IC
or FAS-DD, its
biologically active fraemcnts, analogs, derivatives or mixtures thereof; (ii)
a recombinant animal
virus vector encodinE a viral surface protein capable of bindinb to~ a 'I i~'F-
R or FAS-R - carrying
cell - or tumor cell-specific receptor and a sequence encoding a protein.
analog or derivative of
the invention or encoding the pS~IC, p55DD, FAS-IC or FAS-DD; (iii) a
recombinant animal
virus vector encoding a viral surface protein as in (ii) above and an
oligonucleotide sequence
encoding an antisense sequence of the p55IC, p55b17, FAS-1C or FAS-DD
sequence: and (iv) a
l G vector encoding a ribozyne of sequence capable of interacting with a
mR.'vA sequence encoding a
protein, analog or derivative of the invention or a mRNA sequence encoding the
p55IC, p55DD.
FAS-IC or FAS-DD.
A specific embodiment of the above aspects of the invention is the use of the
p5~-IC or
DN.A encoding therefor. This embodiment is based on the discovcr5~ that the
p55-IC may in a
1 > ligand {'INF)-independem fashion induce other T1W'-associated effects in
cells. Accordingly, there
is provided a method for inducing TNF-associated effects in cells or tissues
comprisins treating
said cells with one or more proteins, analogs or derivatives thereof said one
or more proteins
being selected from a protein being essentially all of the sclf associatinJ
intracellular domain of the
p55 TNF-R (p55-IC) or portions thereof capable of self associating and
induang, in a ligand
20 ~-independent manner, said TNF effect in the cells, wherein said treating
of the cells
comprises introducing into said cells said one or more proteins, analogs or
deriwtives in a form
suitable for imracellular introduction thereof, or introducing into said cells
a DNA sequence
encoding said one or more proteins, analogs or derivatives in the form of a
suitable vectar
carrying said sequence, said vector being capable of effecting the insertion
of said sequence into
25 said cells in a way that said sequence is expressed in said cells,
Embodiments of the above method of the invention include
{i) a method wherein said treating of cells is by transfection of said cells
with a
recombinant animal virus vector comprising the steps of
(a) constructing a recombinant animal virus vector carrying a sequence
encoding a
30 vial surface protein (ligand) that is capable of binding to a specific cell
surface receptor on the
surface of said cells to be treated, and a second sequence encoding a grotein
being the p55-IC,
portions thereof, analogs and derivatives of all of the foregoing, said
protein when expressed in
said cells being capable of self=association and induction of said one or more
TIv'F-associated
effects; and
35 (b) infecting said cells with the vector of (a).
Vii) a method wherein said x:NF effect to be induced in said cells is the
induction of IL~8
gene e~.~pression, said.vector carrying a sequence encoding essentially all of
said p55-1C, portions
thereo>~ analogs and derivatives of all of the foregoing, which are capable,
when expressed in the
cehs of self association and sigrtaIing for the induction of said IL-8 gene
expression.
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(iii) a method for treating tumor cells or virally-infected cells, or for
augmenting the
antibacterial effect of granulocytes, wherein said viral vector carries a
sequence encoding a viral
Iigand capable of binding a specific cell surface receptor on the surface of
said tumor cells, virallv-
infected cells or pranulocytes and a sequence encoding said p5~-IC. portions
thereof, analogs and
derivatives thereof, which when expressed in said tumor, virally-infected or
granulocytc cells
induces TNF-associated effects leading to the death of these cells.
(iv) a method for treating tumor cells, wherein said p55-IC, portions thereof,
analogs or
derivatives thereof, when expressed in the tumor cells, induce the expression
of IL-8 which leads
to the killing of said tumor cells by its chemotactic activit~~ which attracts
oranulocytes and other
lymphocytes to the tumor cells resulting in the death of the tumor cells.
In this aspect of the invention, there is thus also provided the intracellular
domain of the
p55-R (p55-IC), portions. analogs and derivatives of all of the aforegoing for
use in the treatment
of cells by induction therein of TNF-associated effects; and the following
embodiments thereof
(i) the p55-IC, portions, analogs and derivatives for use in the treatment of
cells by
1, induction therein of IL-8 Gene expression.
(ii) the p55-IC, portions, analogs and derivatives for use in the treatment of
tumor cells by
induction therein of IL-8 gene expression resulting in the killing of the
tumor cells.
Moreover. in this aspeet of the invention there is provided a pharmaceutical
composition
for treating cells by induction therein of TNF-associated effects, comprising,
as active in~,~redient,
p55-IC, portions thereof analogs and derivatives of all of the aforegoing, and
a pharmaceutically
acceptable carrier; and the following embodimerns thereof
(i) a pharmaceutical composition for treating cells by induction therein of
TI~TF-associated
effects, comprising, as active ingredient a recombinant animal virus vector
encoding p55-IC,
portions thereof, analogs and derivatives of all of the aforegoing. and a
protein capable of binding
a cell surface protein on the cells to be treated. _ ., _ .
(ii) a pharmaceutical composition for the treatment of tumor cells.
administration of said
composition leading to the induction of IL-8 expression, and subsequent
killing of the tumor cells.
As yet another aspect, the present invention provides a soluble, vligomeric
tumor necrosis
factor receptor (TIv'F-R) comprising at least two self-associated fusion
proteins, each fusion
protein having (a) at its one end, a TNF binding domain selected from the
extracellular domain of
a TNF-R, analogs or derivatives thereof, said extracellular domain, analogs or
derivatives thereof
being incapable of deleterious self association and being able to bind TIVF;
and (b) at its other
gad, a self associating domain selected from (i) essentially all of the
intracellular domain of the
p55 ThIF-R (p55-IC), extending from about amino acid residue 206 to about
amino acid residue
3~ 426 of the native pS~ TNF-R molecule (p55-R); (ii) the death domain of the
p55-IC extending
from about amino acid residue 328 to about amino acid residue 425 of the
native p55-R{iii)
essentially all of the intracellular domain of the Fas/A,F01 receptor (Fas-
IC); (iv) the death
domain of Fas-1C; and (v) analogs, fractions or derivatives of any one of (i)-
(iv) being capable of
self associaiiott, wherein said at least rivo self associated proteins self
associate only at said ends
~ (b) having said ends (a) capable of binding to at least two TIFF monomers,
each end (a) capable
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13
of binding one TNF monomer; and salts and functional derivatives of. said
soluble, olis~omeric
TNF-R.
Embodiments of this aspect of the invention include all of the above
combinations of ends
(a) with ends(b) ac defined above, for example, a soluble, oligomeric TI~IF-R
comprising as
cxtracellular domain, the p55-R e~.-tracellular domain and as self=associating
intracellular domain,
the p55-IC.
Mareov cr, there is also provided a process for producing the soluble
oligomeric flv'F-R of
the invention comprising
(a) the construction of an expression v ector encoding any one of said fusion
proteins, the
I0 DNA sequence of each of said ends of the fusion protein being obtained from
cloned DNA
sequences encoding essentially all of said e~-tracellular domain of the T-~1F-
R, analogs or
derivatives thereof and from cloned DNA sequences encoding essentially all of
said p~ 5-IC, p55-
1C death domain, Fas-1C, Fas-IC death domain, analogs or derivatives of aII of
the aforegoing,
said ends beinc: Iigated together to form a fusion protein sequence, and said
fusion protein
sequence being inserted into said vector under the comrol .of transcriptional
and translationai
regulatory sequences;
(bj invoduction of the vector of (a) into a suitable host cell in which said
fusion protein-is
expressed; and
(c) purification of the fusion protein expressed in said host cells, said
fusion protein self
associating prior to, during, or following the purification process to Sheld a
soluble, oligomeric
TNF-R
Furthermore, there is also provided a vector encoding the above fusion
proteins, useful in
the above method of the invention; host ctlls containing the vector, as «~cll
as a pharmaceutical
composition comprising the soluble, oligomeric 'T:~F-R, salts or functional
dernatives thereof and
mia-tures of any of the aforegoing according to the invention, as active
in~~redient, together wiuu a
pharmaceutically acceptable carrier. Similarly, the soluble, oligomeric 'fNF-
R, salts, funetiorial
derivatives thereof and mixtures of any of the aforegoing, according to the
invention, are provided
for use in antagonizing the deleterious effect of TNF in mammals, especiall~~
in the treatment of
conditions wherein an excess of Ti'TF is formed endogenously or is e~ogenously
administered; ar
alternatively, for use in maintaining prolonged beneficial effects of T:~1F in
mammals when used
with TNF exogenously administered.
Along the Iines set forth concerning the above aspect of the invention, it has
also been
discovered that it is possible to construct a soluble, oIigomeric FasIAP01
receptor (Fas-R) which
is useful for antagonizing the deleterious effects of the Fas ligand.
Accordingly, in a further
aspect, the present invention provides a soluble, oligomeric Fas/:AI'O1
receptor (Fas-R)
comprising at least two self associated fusion groteins, each fusion protein
having (a) at its -one
end, a Fas ligand binding domain selected from the txtracellular domain of a
Fas-R analogs or
dem~atives thereof being incapable of self associating and bcirie able to bind
Fas ligand; and (b) at
its other end,~a self associating domain selected from (i) esserttially all of
the intracellular domain
of the p55 ?NF-R (p55-IC), extending from about amino acid.rcsidue 206 to
about amino acid
CA 02490080 1995-05-11
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14
residue 426 of the native p~~ TNF-R molecule (p55-R); (ii) the death domain of
the p~5-IG
extending from about amino acid residue 328 to about amino acid residue ,26 of
the native p35-
R; (iii) essentially all of the intracellular domain of the Fas/AP01 r eceptor
(Fas-IC); (iv) the
death domain of Fas-IC; and (v) analogs or derivatives of any one of (i)-(iv)
being capable of self
association, wherein said at least two self associated proteins only self
associate at said ends (b)
having said ends (a) capable of binding to at least two Fas ligand monomers,
each end (a) capable
of binding vne Fas ligand monomer; and salts and functional derivatives of
said soluble,
oligomeric Fas-R.
In accordance with this aspect of the invention, there is also provided a
process for the
production of the soluble, oligomeric Fas-R comprising
(a) the construction of an expression vector encoding any one of said fusion
proteins, the
DNA sequence of each of said ends of the fusion protein being obtained from
cloned DNA
sequences encoding essentially all of said exuacellular domain of the Fas-R.
analogs or derivatives
thereof , and from cloned DNA sequences encoding essentially all of said p5~-
IC, p55-IC death
domain, Fas-IC, Fas-IC death domain, analogs or derivatives thereof of all the
aforegoin~, said
ends being ligated together to for~rn a fusion protein sequence, and said
fusion protein sequence
being inserted into said vector under the control of transcriptional and
translational regulatory
sequences;
(b) introduction of the vector of (a) into a suitable host cell in which said
fusion protein is
expressed; and
(c) purification of the fusion protein expressed in the host cells, said
fusion protein self
associating prior to, during, or following the purification process to meld a
soluble, oligomeric
Fas-R.
Moreover, also provided are an expression vector containing the fusion protein
sequence
encoding the soluble oligomeric Fas-R, useful in the above process; host cells
~.ontaining the
vector; and pharmaceutical compositions comprising the soluble, oligomeric Fas-
R salts or
functional derivatives thereof or mixtures of any of the aforegoins as active
ingredient together
with a pharmaceutically acceptable carrier. Similarly, there is provided a
soluble, oli?omeric Fas-
R, salts or functional derivatives thereof or mixtures of any of the
aforegoing, for use in
antasonizing the deleterious e!~'ect of Fas ligand in mammals, especially in
the treatment of
conditions wherein an excess of the Fas ligand is formed endogenously or is
exogcnously
administered.
In a similar fashion to that noted above concerning the oligomeric TNF-Rs and
oligomeric
FAS-Rs, it is also possible to prepare mixed oligomers having binding
specificity for both TNF
and FAS-R ligand. Thus, the present invention also provides a mixed oligomeric
TNF-RIFAS-R
comprising at least two self associated fusion proteins, one of which fusion
proteins is selected
from any one of the above mentioned fNF-specific fusion proteins; and the
other fusion protein is
selected from any one of the above mentioned FAS-R Iigand-specific fusion
proteins.. to provide a
mixed oligomer having at least one TNF-R extracellular domain and at least one
F.AS-R
extracellular domain associated by vrtuo of the self association between the
intracellular domains
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."
or portions thereof fused to each of these eYUacellular domains. These mixed
olic:omeric
receptors are prepared by preparing, as noted above, the oligomcric TNF-Rs and
the oligomeric
FAS-Rs and then mixing these totether and subsequently selecting, by standard
procedures, those
oligomers having binding specificity for both FAS-R ligand and TIv'F. Another
way for preparing
S the mixed oligomeric receptors is by co-transfecting suitable host cells
«~th vectors, as noted
above, encodinb any of the T1V'F-specific fusion proteins (soluble Tlv'F-Rs)
and encoding any of
the FAS-R ligand-specific fusion proteins (soluble FAS-Rs), purifying the
expressed fusion
proteins which self associate prior to, during. or following the purification
to yield oligomeric
receptors, and then selecting by standard procedures, those oligomeric
receptors which arc
10 capable of binding to both TNF and FAS-R ligand.
Likev~~ise. there is also provided pharmaceutical compositions comprising the
mixed
oligomeric receptors, salts or functional derivatives thereof or mixtures of
aw of the aforegoine
as active in~~rediem together with a pharmaceutically acceptable carrier. In
addition, there is
provided the mixed oligomeric receptors, salts or functional derivatives
thereof or mixtures of any
15 of the aforegoing, for use in antagonizing the deleterious effects of both
T11F and F AS-R Iigand in
mammals, especiahy in the treatment of conditions wherein an excess of TNF and
FAS-R ligand is
formed endogenvusl~~ or is exoLenously administered; or alternatively, for use
in maintaining
prolonged (slow-release) beneficial effects of TNF andlor FAS-R ligand in
mammals when used
with TNF and/or FAS-R Iigand (in soluble form) exogenously administered.
Other aspects and embodiments of the present invention are also provided as
arising from
the following detailed description of the invention.
It should be noted that, where used throughout, the following terms :
"Modulation of the
TIv'F-effect on cells" and "Modulation of the FAS-ligand effect on cells" are
understood to
encompass in virrv as well as tn vivo treatment.
Brief Description of the brawings
Figs la-c depict schematically the partial and preliminary nucleotide sequence
of cDNA clones
encoding the p55IC and p?SIC-binding proteins, wherein Fig. 1(a) is the
sequence of
clone 55.11 encoding the p55IC-binding protein 5.11; Fig. 7 (b) is the partial
and
preliminary sequence of clone ?5.3 encoding the p75IC-binding protein 75.3;
and Fig. 1(c)
is the partial and preliminary sequence of clone ?5.16 encoding the p?5)iC-
binding protein
p?5.16; all as described in Example 1; and Fig. 1(d) depicts the deduced amino
acid
sequence of protein 55.11, deduced from the nucleotide sequence of Fig. 1(a),
as also
described in Example 1.
Fig. 2 is a reproduction of a Northern blot which shows the SS.I 1-specific
mRNAs present in a
number of tested cell lines, as described in Example 1.
Figs. 3A and B are reproductions of autoradiograms depicting the in vitro
binding of the protein
encoded for by the 55.11 cDNA to GST fusion proteins containing portions of
p55-IC,
wherein in Fig. 3A there is depicted the binding of the full-length 55.11
protein (55.11
full) to the various GST fusion proteins; arid in Fig. 38 there is depicted
the bindinb of a
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WO 95/31544 PCTIUS95/05854
16
portion of ~ 5.11 fused to the FLAG octapeptide to the various GST fusion
proteins, all as
described in Example 1.
Fig. 4 shows schematically a comparison of the deduced amino acid sequence of
human 5.11 to
related protein sequences derived from lower organisms, as described in
Example 1.
S Fig. 5 is a reproduction of a Western blot stained with anti-MBP polyelonal
antiserum, showing
the self association of the pSSIC, the Western blot derived from an SDS-PAGE
gel on
which were electrophoresed the interacting bacterially-produced chimeric
proteins p55IC-
MBP and p55IC-GST (lanes 1-4) or the control interaction between the chimeric
protein
pSSIC-MBP and GST alone (lanes ~-8), the interactions between the chimeric
proteins
(and control) being carried out on glutathion-agarose beads prior to SDS-PAGE,
as
described in Example 2.
Fig. 6 is a reproduction of phase contrast micrographs sho~zng the cyrtotoxic
effect of the full-
length pSSIC in HTtal cells transfected with an expression vector encoding
this p~SIC
(right panel); and the inhibition of this cy~totoxic efrect when expression of
the vector is
blocked by treating the cells with tetracycline (left panel), as described in
Example 2.
Fig. 7 depicts the ligand-independent triggering of the cvtocidal effect in
HeLa cells transfected
with the full-lens,~th p55-R, its intracellular domain, or pans of the
intracellular domain
including the 'death domain' where
(i) at the extreme left hand side of Fig. 7 there is depicted schematically
the various
DNA molecules encoding the full-length pS5-R, its intracellular domain and the
portions
of the intracellular domain which were inserted into the vector with cvhich
the HeLa cells
were transfected.
(ii) the left and middle bar graphs show the TVF receptor expression in the
HeLa
cells of each of the types of receptor shown at the extreme left of Fig. ',
the left bar
representing the amounts of receptor in ng/ccll sample and the mid~Ie bar
~~~~~h
representing the amounts of receptor c~cpressed in terms of radioiodinated TNF
bound to
the transfected cells; and
(iii) the right bat graph showing the viability of the HeLa cells expressing
the
various kinds of the receptor;
and wherein in all of the bar graphs the open bars represent cells transfected
in the
presence of tetracycline and the closed bars represent cells transfected in
the absence of
tetracycline; all of the above being described herein in Example 2.
Fig. 8 depicts the ligand-independent induction of IL-8 gene expression in
HeLa cells transfected
with the full-length p55-R or its intracellular domain (p55IC), wherein in
panel A there is
shown a reproduction of a Northern blot representing the Northern analysis of
RNA
extracted from HeLa cells treated or untreated with TNF (tvvo left hand lanes
marked
'control' and "ThIF'), and of RNA extracted from HeLa cells transfeeted with
vectors
encoding the p55-R, p55-IC or the control protein, luciferase (the remaining
lanes marked
'p55-IC', 'p55 R' and Luc, respectively), the cells having been transfected in
the presence
(+) or absence (-) of tetracycline in each case (hence two lanes per
transfection); and
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WO 95/31544 PCT/US95/05854
17
wherein in panel B there is shown the methylene blue stainin' of 18S rRNA in
each of the
HeLa cell sample shown in panel A; all of the abavc being described in Example
2.
Fig. 9 (A and B) depicts graphically the lieand independent triggering of a
c;rtoeidal effect in
HeLa cells transfected with pSSR or parts thereof, or u,~ith FAS-IC, u~hcrein
in Fig. 9A
there is depicted the results with respect to the p55R or parts thereof and in
Fig. 9B there
is depicted the results with respect to the FAS-IC. In the left hand panels of
both Fig. 9A
and B there is depicted schematically the portion of the p~SR or FAS-IC used
in the
transfections ~~hile the right hand panels depict graphically the experimental
results, all as
described in Example 2.
Fig. 10 depicts schematically the partial and preliminary nucleotide sequence
of a cDNA clone,
called T2', which encodes a protein capable of binding to the p55IC and p'AS-
IC, as
described in Example 3.
Fig. 11 depicts schematically the partial and preliminary nucleotide sequence
of a cDNA clone,
called F9, which encodes a protein capable of binding to the p»IC and FAS-IC,
as
described in Example 3.
Fig. 12 depicts sehematicallv the partial and preliminary nucleotide sequence
of a cDl~'A clone,
called DD11, which encodes a protein capable of binding to the p~~IC,
especially the
p55DD, and FAS-IC, as described in Example 3.
Detailed Description of the Invention
The presem invention relates, in one aspect, to novel proteins vwhich are
capable of binding
to the intracellular domain of receptors belonging to the ?NF/NGF superfamily,
such as TNF-Rs
and FAS-R and hence are considered as mediators or modulators of this
superfamily of receptors,
e.g. of the TNF Rs and FAS-R, having a role in, for example, the signaling
process that is initiated
by the binding of TNF to the TNF-R and FAS Iigand to FAS R. Examples of these
proteins are
those which bind to the intracellular domain of the p55 TNF-R (p55IC), such as
the proteins
designated herein as 55.1: 55.3 and SS.11 (Example 1) as well as those encoded
by cDNA clones
F2, F9, and DDII (E.xample 3); those which bind to the intracellular domain of
the p75 TNF-R
(p75IC), such as the proteins designated herein as 75.3 and 75.16 (Example 1
); and those which
bind to the intracellular domain of FAS-R (FAS-IC), such as the proteins
encoded by cDNA
clones F2, F9 and DD11 (Example 3). Proteins 55.1 and ~~.3 have been found to
represent
portions or fragments of the intracellular domain of the p55 TNF-R (p55IC);
other proteins,
55.11, 75.3 and 75.16, represent proteins not described at all prior to the
present invention (75.3,
75.1G) or those that have been described (55.11, see Khan et aL, 1992) but
whose function and
other characteristics, particularly, the ability to bind to a TNF-R, were not
described in any way
(see Example I, below). The new proteins encoded by cDNA clones F2, F9 and
DD11 also
represent proteins pre~.iously not described at all, i.e. their sequence is
not in the'GENEBANK' or
'PROTEIN BANK' data banks of DNA or amino acid sequences.
Thus, the present invention concerns the DNA sequences encoding these proteins
and the
protans encoded by these sequences.
CA 02490080 1995-05-11
WO 95/31544 PCT/US95I05854
18
Moreover, the present invention also concerns the DhA sequences encoding
biolo~eally
active analogs and derivatives of these proteins, and the analogs and
dcrivativcs encoded thereby.
The preparation of such analogs and derivatives is by standard procedure (see
for example,
Sambrook ct al., 1989) in which in the DNA sequences encodins these proteins,
one or more
codons may be deleted, added or substituted by another, to yield analogs
having at least a one
amino acid residue change with respect to the native protein. Acceptable
analogs are those which
retain at least the capability of binding to the intracellular domain of the
T1V'FINGF receptor
superfamily, such as FAS-R or TNF-R, e.g. the p55IC, p75IC or FAS-IC, or which
can mediate
any other binding or enzymatic activit3~, c.g. analogs which bind the p55,
p75IC or FAS-IC but
t 0 which do not signal, i e. do not bind to a further dowstream receptor,
protein or other factor, or
do not catalyze a signal-dependent reaction. In such a way analogs can be
produced v~~hich have a
so-called dominant-negative effect, namely, an analog which is defective
either in binding to the,
for example, p~SIC, p?SIC or FAS-IC, or in subsequent signaling following such
bindinL. Such
analogs can be used, for example, to inhibit the TNF- or FAS-ligand- effect by
competing with the
natural 1C-binding proteins. Likewise, so-called dominant-positive analogs may
be produced
which would sen~e to enhance, for example, the TNF or FAS ligand effect. These
would have the
same or better IC-binding properties and the same or better signaling
properties of the natural IC-
binding proteins. Similarly, deri~~atives may be prepared by standard
modifications of the side
S~raups of one or more amino acid residues of the proteins, or by conjugation
of the proteins to
. another molecule e.g. an anubody, enzyne, receptor, etc., as arc well known
in the art.
The new' ?NF-R and FAS-R intracellular domain - binding proteins, e.g, the
proteins SS.7 ,
55.3, 5.11, 75.3, 75.16 as well as the proteins encoded by cDNA clones F2, F9
and DD11
(hercinafrer, F2, F9 and DD11) have a number of possible uses, for example.
(i) They may be used to mimic or enhance the function of T:~3F or F AS-R
Iigand, in
2~ situations where an enhanced 'T1~TF or FAS-R ligand effe.et is desired such
as :n a~~ti-te.~;or.
anti-inflammatory or anti-FiIV applications where the TNF-or FAS-R ligand-
induced
cytotoxicity is desired. In this case the proteins, e.g. those binding to the
p55IC such as
S.I, 55.3, as well as F?, F9 and DD11, and the free p55IC itself (see below
and Example
2), as v~~ell as the 'death domain' of the p55IC (p55DD), which enhance the
TNF effect; or
proteins F2, F9 and DD 11 as well as FAS-IC and F AS-DD which enhance the FAS-
R
Iigand effect, i.e. cytoto~c effect, may be introduced to the cells by
standard procedures
I:now ger se. For example, as the proteins are intracellular and it is desired
that they be
introduced only into the cells where the TNF or FAS-R ligand effect is wanted,
a rystem
for specific introduction of these proteins into the cells is necessary. Onc
way of doing this
3~ is by creating a recombinant animal virus e.g. one derived froth Vaccinia,
to the D1~A of
which the following two genes will be introduced the gene encoding a Iigand
that binds to
cell surface proteias specifically expressed by the cells e.g. ones such as
the Alas (HIS j
virus gp120 protein which binds specifically to some cells (CD4 lymphocytes
and related
leuketnias) or any other Iigand that binds specifically to cells carrying a
TI'S-R or FAS-R,
such that the recombinant virus vector wit! be capable of binding such T'NF-R-
or FAS-R-
CA 02490080 1995-05-11
WO 95/31544 PCT/US95l05854
19
carrlrin~ cells; and the gene encoding the new intracellula- domain-binding
protein or the
p55IC, p55DD, FAS-IC ar FAS-DD protein. Thus, expression of the cell-surface-
binding
protein on the surface of the virus will target the virus specincally to the
tumor cell or
other TNF-R- or FAS-R- carry~inc; cell, following which the intracellular
domain-binding
S protein encoding sequence or p55IC, pSSDD, FAS-IC or FAS-DD encoding
sequence will
be introduced into the cells via the virus, and once expressed in the cells
will result in
enhancement of the TNF or FAS-R Iigand ctfect leading to the death of the
tumor cells or
other TT~'F-R- or FAS-R- carrying cells it is desired to kill. Construction of
such
recombinant animal ~~ru5 iS by standard procedures (see for example, Sambrook
et al.,
IO 1989). Another possibility is to introduce the sequences of the new
proteins or the pS;IC,
p55DD, FAS-iC or FAS-DD in the form of oligonueleotides which can be absorbed
by the
cells and expressed therein.
(ii) They may be used to inhibit the '1'VF or FAS-R ligand effect, e.g. in
cases such as
tissue damage in septic shock, graft-vs.-host rejection, or acute hepatitis,
in which case it
15 is desired to block the TNF-induced TNF-R or FAS-R ligand induced FAS-R
intracellular
signaling. In this situation it is possible, for example, to introduce into
the cells, by
standard procedures, oligonucleotides having the anti-sense coding sequence
far these
new proteins, or the anti-sense coding sequence for p55IC, p~~DD, FAS-IC ar
FAS-DTI,
which would effectively block the translation of mRN As encoding these
proteins and
24 , thereby block their expression and lead to the inhibition of the T~F- or
FAS-R ligand-
eff'ect.
Such olisonucleotidcs may be introduced into the cells using the above
recombinant virus approach, the second sequence carried by the virus being the
oligonucleotide sequence. Another possibility is to use antibodies specific
for these
25 proteins to inhibit their intracellular signaling activity. It is possible
that these new~
proteins have an c~-tracellular domain as well as an intracellular one, the
latter which binds
to the TNF-R or FAS-R binding domain, and thus antibodies ?enerated to . their
cxtracellular domains can be used to block their TNF- or FAS-R ligand- related
functions.
Yet another way of inhibiting the TNF or FAS-R iigand effect is by the
recently
30 developed ribozvme approach. Ribozymes are catalytic R~'~TA molecules that
specifically
cleave ItNAs. Ribozymes may be engineered to cleave target RNAs of choice,
e.g. the
m~t.NAs encoding the new proteins of the im~ention or the mRNA encoding the
pSSIC,
p55DD, FAS-IC or FAS-DD. Such ribazymcs would have a sequence specific for the
mRUlA of choice and would be capable of interacting therewith (complementary
bindinb)
35 folloR~ed by cleavage of the mRZVA, resulting in a decrease (or complete
loss) in the
expression; of the protein it is desired to inhibit, the level of decreased
ea~pression being
dependent upon the level of ribozyme expression in the target cell. To
introduce
ribozymes into the cells of choice (e.g. those carrying TNF-Rs or FAS-R) gay
suitable
vector may be used, e.g. plasmid, animal virus (retrovirus) vectors, that are
usually used
40 for this purpose (see also (i) above, where the virus has, as second
sequence, a cDl~A
CA 02490080 1995-05-11
WO 95131544 PCTIUS95/05854
2u
' encoding the ribozyme sequence of choice), Moreover, ribozwnes can be
constructed
which have multiple targets (mufti-target riboz;~mes) that can be used, for
example, to
inhibit the expression of one or more of the proteins of the invention and/or
the p~SIC,
psSDD, FAS-IC or FAS-DD as well (For reviews. methods etc. concerning
ribozymes see
Chen et al., 1992; Zhao and Pick, 1993; Shore et al., 199;. Joseph and Burke,
1993;
Shimayama et al., 1993; Cantor of al.., 1993; Barinaga. 199: ; Crisell et al.,
1993 and
Koimmi et al., 1993).
iii) They may be used to isolate. identify and clone other proteins which are
capable of
binding to them, e.g. other proteins involved in the intracellular signaling
process that are
downstream of the TNF-R or FAS-R intracellular domain In this situation, these
options,
namely, the DNA sequences encoding them may be used in the yeast two-hybrid
system
(see Example 1, below) in which the sequence of these protein: will be used as
"baits" to
isolate, clone and identify from cDNA or genomic DNA libraries other sequences
("preys") encoding proteins which can bind to these neH~ Tlv'F-R or FAS-R
intracellular
domaitrbinding proteins. In the same way, it may also be determined whether
the specific
proteins of the present invention, nameE~~, those which bind to the p55IC.
p75IC, or FAS-
IC, can bind to other receptors of the 'I~FIIvIGF superfamiU of receptors, For
example, it
has recently been reported (Schwalb et al., 7993; Baens et al.. 1993; Crowe et
al., 1994)
that there exist other T~1F-Rs besides the p55 and p75 'INTF-Rs. Accordingly,
using the
yeast two-hybrid system it may be specifically tested whether the proteins of
the present
invention are capable of specifically binding to these other TIv'F-Rs or other
receptors of
the ?NF/NGF superfamily. Moreover, this approach may also be taken to
determine
whether the proteins of the present invention are capable of binding to other
known
receptors in whose activity they may have a functional role.
ZS (iv) The new proteins may also be used to isolate, identif~~ and clone
other proteins of
the same class i.e. those binding to TIVF-R or FAS-R intracellular aomains or
to
functionally related receptors, and involved in the intracellular signaling
process. In this
application the above noted yeast two-hybrid system may be used, or there may
be used a
recently developed (fir ilks et al" I 989) system employing non-stringent
southern
hybridization followed by PCR cloning. In the Vililks et al, publication,
there is described
the identification and cloning of two putative protein-tyrosine kinases by
application of
non-stringent southern hybridization followed by cloning by PCR based on. the
lozown
sequence of the ldnase motif, a conceived kinase sequence. This approach may
be used,
in accordance with the preseni im~ention using the sequences of the new
proteins to
identify and clone those of related Ti'iF-R, FAS-R or related receptor
(TNFI'NGF
superfamily receptors) intracellular domain-binding proteins.
(v) Yet another approach to utilising the new proteins of the invention is to
use them
in methods of affinity chromatography to isolate and identify other proteins
or factors to
which they arc capable of binding, e.e. other receptors related to TNF-Rs
(TNF/NGF
receptor superfamily) or other proteins or factors involved in the
intracellular signaling
CA 02490080 1995-05-11
21
process. In this application, the proteins of the present invention, may be
individually
attached to afftnin~ chromatography matrices and thec: brought into contact
with cell
extract: or isolated proteins or factors suspected of being involved in the
imracellutar
signaling process. Following the affinity chromatography procedure, the other
proteins or
factors whaeh bind to the new proteins of the invention, can be eluted,
isolated and
characterized.
(«) As noted above, the new proteins of the invention may also be used as
immuno~ens (antis:ens) to produce specific antibodies thereto. These
antibodies may also
be used for the purposes of purification of the new proteins either from cell
extracts or
from transformed cell lines producing them. Further, these antibodies may be
used -for
diagnostic purposes for identih~ing disorders related to abnormal functioning
of the TNF
or FAS-R Iigand system; e. e. overactive or underactivc TNF- or F ~rS-R ligand-
induced
cellular effects. Thus, should such disorders be related to a malfunctioning
intracellular
signaling system involving the new proteins, such antibodies w outd serve as
an important
1 S diagnostic tool.
It should also be noted that the isolation, identification and
characterization of the nev~
proteins of the invention may be performed using any of the well known
standard screeiiiii~
procedures. For example, one of these screening procedures, the yeast two-
hybrid procedure asf is
set forth in the following examples (Eacamples 1 and 3), was used to identify
the new proteins of
the invention. Likewise as noted above and below, ocher procedures may be
employed such- as
amity chromatography, DNA hybridization procedures, etc. as are well 1,-sown
in the art, to
isolate, identify and characterize the new proteins of the iwention or to
isolate, identif~~ and
characterize additional proteins, factors, receptors, etc, which are capable
of binding to the aew~
proteins of the invention or to the receptors belongdng to the TIv'FlNCF
family of rc:eptors.=a si~~
As regards the antibodies mentioned herein throughout, the term "antibody" is
meant 'to
include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric
antibodies, anti-idiat5~pic
(anti-Id) antibodies to antibodies that can be labeled in soluble or bound
form, as well as
fragnents thereof provided by any known technique, such as, but not limited to
enzymatic
cleavage, peptide synthesis or recombinant techniques.
Polyctvnal antibodies are heterogeneous populations of antt'bady molecules
derived-from
the sera of animals immunized with an antigen. ~. monoclonal antibody contains
a substantially
homogeneous gopulaiion of antibodies specific to antigens, which populations
contains
substantially similar epitope binding sifts. i~4Abs may be obtained by methods
known to those
skilled in the art. See; for exarnplc Kohler and Milstein, Nature, 2~G:495-497
(1975); U.S: Patent
No. 4,376,114; Ausubel et al., gds., I3arIow and Lane ANTI$ODIES ' A
LABORATORY
MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., gds.;
Current Protocols in
Immunology, Greene publishing Assoc, and Wiley Intcrscience N.Y., (1992,
1993).
Such antibodies may be of any
immunollobulin class including fgG. IgM. IgE, IgA, GILD and any subclass
thereof rA
CA 02490080 1995-05-11
hybridoma producing a m.~b of the present invention tnay be cultivated i»
arrn, i» siru or r» viva.
Production of high titers o~ mAbs in viva or in siru makes this the presently
preferred method of
pro.luction.
Chimeric antibodies are tnolecule.s different portions of which are derived
from dif~r'erem
animal species, such as those having the variable region derived from a marine
tnAb and a human
immunoglobulin constant repon. Chimeric antibodies are primarily used to
reduce
immunogenicity in application and to increase yields in production. for
example, where marine
mAbs have hislta yields from hybridomas but higher immunogenicity in humans,
such that
humanlmurine chimeric tttAbs are used. Chimeric antibodies and methods far
their production are
' 10 known in the art (Cabilly et al., Pruc. Natl. Acctd .Sci. t :SA 81:3273-
3277 (1984}; Motrison et al.,
Proc. h'arl. Acid ,Sci. LJ.~A 81:6851-685, (1984); Boulianne et al.,
~'1~'axrrrc 312:643-(i46 (1984);
Cabilly et al., European Patent Application 125023 (published :vovember 14,
1984); Neuberger et
al., ~'Vaturc~ 314:26S-270 ( 1985): Taniguchi et al., European Patent
Application 171496 (published
February 19, 1985); Marrisan et aL, European Patent Application 173494
(published March ~,
1980); ~ieuberger et al., PCT Application WO 8601533, (published March 13,
198G); Kudo et al.,
European Patent Application 184187 (published June 11, 1986); Sahagan 4t al.,
J Immunal.
13 i;lOG6-107: (1986); Rabinson et al., International Patent Application Ire.
W08702671
(published May 7, 198'7); Liu et al., Prac. Natl. Acid Sci tlS.l 84:3439 X443
(1987); Sun et al.,
Proc. Natl. Acac~ Sci L~.4 84:214-218 (1987); Better et al., .Scienrx 240:1041-
1043 (1988); and
Harlow and Lane, ANTIBODIES :A LABORATOR~C' MANUAL, supra.
An anti-idiotypic (anti Id) antibody is an antibody which recoc_ntizes unique
determinants
generally associated with the antigen-binding site of an antibody. An Id
antibody can be prepared
by immunizing an animal of the same species and genetic type (e.g. mouse
strain) as the source of
the mAb with the mAb to which an anti-Id is being prepared. The immunized
animal ' u,~il1
recognize and respond to the idiotypic determinants of the irnrnunizinl;
antibody by producing an
antibody to these idiotypic deteaninants (the anti-Id antibody). See, for
example, Lt.S. Patent No.
4,699, 880.
The anti-Id antibody may also be used as an "immunogen" to induce an immune
respon~e
in yet another animal, producing a so-called anti-anti-Id amibody. The anti-
anti-Id may : be
epitopically identical to the original mAb which induced the anti-Id. Thus, by
using antibodies to
the idiotypic deterntiaartts of a mAb, it is possible to identify other clones
expressing antibodies bf
identical specificity. ,
Accordingly, mAbs generated against the IC-bindinz proteins, analogs or
dcriyativcs
thereof of the present invention or the p55IC, p55DD, FAS-IC, FAS-DD, analogs
or.derivatives
thereof may be used to induce anti-Id antibodies in suitable animals, such as
B ALB/c mice: Spleein
cells from such iatcnunized mice are used to produce anti-Id hybridomas
secreting ami-Id ~iii~bs.
Fuzther, the anti Id m.4bs can be coupled to a carrier such as keJfiole limpet
hemocyanin {IQ.H)
aid used to immunize additional BALBIc mitt. Sera from these mice will contain
anti-anti'=Id
antibodies that have the binding properties of the original mAb specific for
an epitope ,of ;.the
CA 02490080 1995-05-11
WO 95/31544 PCTIUS95/05854
23
about IC-bindin_ proteins, analogs or derivatives yr p»IC, p:SDD, FAS-IC or
F.4S-DD_
analogs or derivatives.
The anti-Id mAbs thus have their ow-n idiot5~pic epitopes, or "idiotopes"
structurall~~
similar to the epitope being evaluated, such as GRB protein-oc.
The term "antibody" is also meant to include both intact molecules a5 well as
fragments
thereof, such as, for example, Fab and Flab'), which are capable of binding
antigen. Fab and
F(ab'r fragments lack the Fc fragment of intact antibody, clear more rapidly
from the circulation,
and may have less non-specific tissue binding than an intact antibody (V~rahl
et al., .I. Nucl. :'I~IcW
24:31b-325 (198;}).
It will be appreciated that Fab and F(ab')' and other fragments of the
antibodies useful in
the present invention may be used for the detection and quantitation of the IC-
binding proteins or
p55IC, p55DD. FAS-IC or FAS-DD according to the methods disclosed herein for
intact
antibody molecules. Such fra_c:ments are typically produced b~~ proteol~nic
cleavage, using
enzymes such as papain (to produce Fab fragments] or pepsin (to produce
F(air')~ fragments}.
l~ An antibody is said to be ''capable of binding" a molecule if it is capable
of specifically
reacting with the molecule to thereby bind the molecule to the antibody, The
term "epitope" is
meant to refer to that portion of any molecule capable of being bound by an
antihody which can
also be rccoenized by that antibody-. Epitopes or "antigenic determinants''
usually consist of
chemically active surface groupings of molecules such as amino acids or sugar
side chains and
have specific three dimensional structural characteristics as well as specific
charge characteristics.
An "antigen" is a molecule or a portion of a molecule capable of being bound
by an
antibody which is additionally capable of inducing an animal to produce
antibody capable of
binding to an epitope of that antigen. An antigen may have one ar more than
one epitope. The
specific reaction referred to above is meant to indicate that the antiben will
react, in a highly
selcctivc manner, with its corresponding antibody and not with the multitude
of other antibcicties
which may be evoked by other antigens.
The antibodies. includin' fragments of antibodies, useful in the present
invention may be
used to quantitatively or qualitatively detect the IC-binding proteins or
p55IC, p55DD, Fr~S-IC,
FAS-DD in a sample or to detect presence of cells which express the IC-binding
proteins of the
preseat invention or the p55IC, p55DD, FrIS-IC, FAS-DD proteins. This can be
accomplished b;~
immunofluorescence techniques employing a fluorescently labeled antibody (see
below) coupled
with light microscopic, flow cytomctric, or fluorometric detection.
The artibodies (or fragments thereof) useful in the present invention may be
employed
histologically, as in inllnunofluorescence or immunoelectron microscopy, for
irt situ detection of
1C-binding proteins of the present invention or the pSSIC, p~3DD, FAS-IC. FAS-
DD. hl sine
detection may be accomplished by remo~~ino a histological specimen fram a
patient, and providing
the labeled antibody of the present invention to such a specimen. The antibody
(or fragment) is
preferably provided by applying or by overlaying the labeled antibody (or
fragment} ~'to a
biological sample. Through the use of such a procedure, it is possible to
determine not only the
presence of the IC-binding proteins or the p55IC, p55DD, FAS-IC, FAS-DD, but
also its
CA 02490080 1995-05-11
WO 95/31544 - PCT/US95105854
24
distribution on the e~:amined tissue. Using the present invention, taose of
ordinay skill will readily
perceive that any of wide variety of histological methods (such 3s staining
procedures) can be
modified in order to achieve such in situ detection.
Such assays for IC-binding proteins of the present im~ention or the p~~IC,
p55DD, FAS
IC, FAS-DD, typically compzises incubating a biological sample. such as a
biological fluid, a
tissue extract. freshly harvested cells such as hmphocrtes or leukocytes, or
ells which have been
incubated in tissue culture, in the presence of a delectably labeled antibody
capably of identifying
the IC-binding proteins or the p55IC, p55DD, FAS-IC. FAS-DD, and detecting the
antibody by
any of a number of techniques well knov~~n in the art.
IO = The biolopcal sample may be treated with a solid phase support or carrier
such as
nitrocellulose, or other solid support or carrier which is capable of
immobilizing cells, cell
particles or soluble proteins. The support or carrier ma~~ then be washed
v~ith suitable buffers
followed by treatment with a detestably labeled antibody in accordance with
the present invention,
as noted above. The solid phase support or carrier may then be washed vvith
the buffer a second
I ~ time to remove unbound antibody. The amount of bound iabel on said solid
support or carrier
may then be detected by conventional means.
By "solid phase support", "solid phase carrier", "solid support", "solid
carrier", "support"
or "carrier" is intended any support or carrier capable of binding antigen or
antibodies. Well-
lmown supports or carriers, include glass, polystyrene, polypropylene,
pol;~ethylene, deatran,
20 nylon amylases. natural and modified celluloses, golyacrvlamides, gabbros
and magnetite. The
nature of the carrier can be either soluble to some ea-tent or insoluble for
the purposes of the
present imrention. The support material may have virtually any possible
structural configuration so
long as the coupled molecule is capable of binding to an antigen or antibody.
Thus, the support or
carrier configuration may be spherical, as in a bead, cylindrical, as in the
inside surface of a test
25 tube, or the external surface of a rod. Alternatively, the surface may be
flat such as a sheet, test
strip, etc. Prcfetted supports or carriers include polystyrene beads. Those
skilled in the art will
know may other suitable carriers for binding antibody or antigen, or will be
able to ascertain the
same by use of routine experimentation.
The bindir~ activity of a gives lot of antibody, of the invention as noted
above, nosy be
30 determined according to well known methods. Those skilled in the art will
be able to determine
operative and optimal assay conditions for each determination by employing
routine
experimentation_
Other such steps as washing, stirring, shaking, filtering and the like may be
added to the
assays as a customary or necessary for the particular situation.
35 One of the ways in which an antibody in accordance with the present
invention can be
deteaably labeled is by Iinldng the same to an enzyme and use in an enzyme
immunoassay (E1A).
This enzyme, in tuna, whoa laxer exposed to an appropriate substrate, will
react with the substrate
in such a manner as to produce a chemical moiety which can be detected, for
example, by
spcctrophotometrie, fluorometric or by ~risual means. Enzymes which can be
used detectably.label
40 the antibody include, but are not limited to, tnalate dehydrogenase,
staphylococcal nuclease, delta-
CA 02490080 1995-05-11
5-steroid isomeras, yeas alcohol dehydrogenase, alpha~~ly,:erophosphate
dehydroeenase. triose
' phosphate. isomerase, horseradish peroudase, alkaline phosphatase,
asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, unease, catalase, glucose-6-
phosphate dehydrogenase,
glucoamylase and acetylcholinesterase. The detection car be accomplished by
colorimctric
methods which employ a chromogenic substrata for the enzyme. Detection may
also be
accomplished by visual comparison of the extent of enzymatic reaction of a
substrate in
comparison with similarly prepared standards.
Detection may be accomplished using any of a ~~arierrr of other immunoassays.
For
example, by radioactivity labeling the antibodies or ancibod~~ fra_~ments, it
is possible to detect R
PTPase through the use of a radioimmunoassay (RIA). A good description of RIA
may be found
in Laboraton Techniques and Biochemistry in Molecular Hiology, by Work, T.S.
et al., Notch
Iiolland Publishing Company. NY (1975) with particular reference to the
chapter entitled "An
Introduction to Radioimmune Assay and Related Techniques" by Ghard, 'T.
The radioacti~ a isotope can be detected by such means ~as the use of a y
counter
or a scintillation counter or by autoradio~~raphy.
It is also possible io label an anribodv in accorda~:ce with the present
invention with a
fluorescent compound. When the fluorescently labeled antioody is exposed to
light of the proper
wavelength, its presence can be then detected due to fluorescence. Among the
most commonly
used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrinc,
pycocyanin, altophycocyanin, o-phthaldehyde and fluorescamine.
The antibody can also be detestably labeled using fluorescence emitting metals
such gas
1528, or others of the lamhanide series. These metals can be attached to the
antibody using such.
metal chelating groups as diethyienetriamine pentaacetic acid (ETPA).
The antibody can also be detestably labeled by coupling it to a
chemiluminescent
compound. The presence of the chetniluminoscont-tagged antibody . is then
deterrriiried ~Fk~y
detecting the presence of luminescence that arises durine the course of a
chemical reaction.
Examples of particularly useful chemih~minescent Labeling compounds are
iuminol, isoluminol,
thcromatic accidinium ester, imidazolt, acridinium salt and o~:alate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the
present
imremion. Bioluminescence is a type of chemiluminescence found in biological
systems in-v~ihich''a
catalytic protein increases the efficiency of the chemiluminescent reaction.
The presence_'of a
bioluminescent protein is determined by detecting the presence of
luminescence. Important
bioluminescent compounds for purposes of labeling are luciferin, luciferase
and aequorin.
An antibody molecule of the present invention may be adapted for utilization
yin 'an
immunometzic assa~,~, also Imow-n~as a "two-site" or "sandwich" assay. in a
t3~pical immunometric
assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to
a solid ~ support ~or
carrier and a Quantity of detestably labeled soluble antibody is added to
permit detection"aridtor
quantitation of the ternary complex formed between, solid-phase antibody,
antigen, and labeled
antibody.
* Trade-mark
CA 02490080 1995-05-11
WO 95/31544 °CT/IIS95/05854
.~.b
Typical, and preferred, immunometric assays include "forward" assays in which
the
antibody bound to the solid phase is first contacted with the sample being
tested to exuact the
antigen from the sample by formation of a binary solid phzse antibody-antigen
complex. After a
suitable incubation period, the solid support or carrier is v~ashed to remove
the residue of the fluid
sample, including unrcacted antigen, if any, and the contacted with the
solution containing an
unknown quantity of labeled antibody (which functions as a "reporter
molecule"). After a second
incubation period to permit the labeled antibody to comply with the antinen
bound to the solid
support or carrier throueh the unlabeled antibody, the solic support or
carrier is washed a second
time to remove the unreacted labeled antibody.
In another type of "sandwich" assay, which may also be useful with the
antigens of the
present invention, the so-called "simultaneous" and "ret~erse" assays are
used. A sirnuItaneous
assay involves a single incubation step as the antibody bound to the solid
support or cattier and
labeled antibody are both added to the sample being tested at the same time.
After the incubation
is completed, the solid support or carrier is washed to remove the residue of
fluid sample and
1 S uncomplexed labeled antibody. The presence of labeled antibody associated
with the solid supper,
or cagier is then determined as it would be in a conventional "fonvard"
sandwich assay.
In the "reverse" assay, stepwise addition nrst of a solution of labeled
antibody to the fluid
sample followed by the addition of unlabeled antibody bound to a solid support
or carrier after a
suitable incubation period is utilized. After a second incubation, the solid
phase is washed in
conventional fashion to free it of the residue of the sample being tested and
the solution of
unrcacted labeled amibody. The determination of labeled antibody associated
with a solid support
or cattier is then determined as in the "simultaneous" and "forward" assays.
The new proteins of the invention once isolated, identified and characterized
by any of the
standard screening procedures, for example, the ~~cast two-hybrid method,
affinity
chromatography, and any other well known method l:nov~m in the art; may then
bP produced :hy
any standard recombinant DNA procedure (see for example, Sambrooh, et al.,
1989) in which
suitable eukaryotic or prokaryotic host cells arc transformed by appropriate
eukaryotic or
prokaryotic vectors containing the sequences encoding for the proteins.
Accordingly, the prcsem
invention also concerns such expression vectors and transformed hosts for the
production of the
proteins of the invention. As mentioned above, these proteins also include
their biolos~cally active
analogs and derivatives, and thus the vectors encoding them also include
vectors encoding
analogs of these proteins, and the transformed hosts include those producing
such analogs. The
derivatives of these proteins arc the derivatives produced by standard
modification of the proteins
or their analogs, produced by the transformed hosts.
In another aspect, the invention relates to the use of the free intracellular
domain of the
p55 TNR-R (pSSIC) or FAS-R (FAS-IC) or their so-called 'death domains' (p~SDD
or FAS DD,
respectively) as an agent for enhancing the TNF or FAS-R ligand effect on
cells, on its ow-n .(see
Example 2). Where it is desired to introduce a ThTF- or FAS-R-ligand- induced
cytotoxic effect in
calls, e.g. cancer cells or HIV-infected cells, the p»IC, p55DD, FAS-IC or FAS-
DD can be
introduced into such cells using the above noted (see (i) above) recombinant
animal vine (e.g.
CA 02490080 1995-05-11
WO 95131544 PCT/US95/05854
27
vaccinia) approach. Mere too, the native p55IC. pS~DD. r AS-IC or FAS-DD,
biololricaiiy active
analogs and derivatives or framnents may be used, all of which can be prepared
as noted above.
Likewise, the present invention also relates to the specific blocking of the
TNF-effect or
FAS-R ligand-effect by blocking the activity of the p55IC, pSSDD, FAS-IC ar
FAS-DD, e.b. anti
s sense oligonucleatides may be introduced intb the cells to block the
expression of the pSSIC,
p55DD, FAS-IC or FAS-DD.
The present invention also relates to pharmaceutical compositions comprising
recombinant
animal virus vectors encoding the TNF-R ar FAS-R intracellular domain binding
proteins
(,including the pSSIC, p55DD, FAS-IC and F.4S-DD), which vector also encodes a
virus surtace
1Q protein capable of bindinb specinc target cell (e.c. cancer cells) surface
proteins to direct the
insertion of the inuacellular domain binding protein sequences into the cells.
In another aspect, the present invention also concerns, specifically, the
effects of the self
associating intracellular domain of the p~5 TNF receptor i g~5-IC, see Example
2~. An example of
such effects, which is an effect normally mediated by T\'F binding to its
receptor and which is
15 mimicked by the signaling activity of the self associating p~5-IC or parts
thereof, is the induction
of expression of the gene encoding IL-8.
h.-8 is a cytokine belonging to the subclass of chemokiaes having primarily
cheinotactic ,
activity, and has been shown to play a major role in the chemotaxis of
~,~ranulocytes and other cell
types associated with a number of patholocdcal states (see for example, Endo
et al., 199; Sckido
20 et aL, 1993; Harada et al., 1993; Fcrrick et al., 1991).
TNF has a beneficial activity, and is used as such, in treatments to destroy
tumor ells and
virus i,~ected cells or to augment antibacterial activities of granulocytes.
However, as noted
above, TNF also has undesirable activities in which case it is desired to
block its activity,
inch~ding those situations where large doses of TNF are used in cancer
therapy, antiviral, therapy
25 or antibacterial therapy.
Accordingly, it is desirable to be able to direct TVF or a substance capable
of mimicking
iu beneficial activity to the cells ar tissues that it is specifically desired
to treat.
In accordance with the present iwention it has been found that the self
associating
intracellular domain of the p55-R (p55-IC) can, in a ligand-independent
manner, mimic a number
30 of effects ofTNF, e.g. the 'death domain' of p55-IC can induce cytotoxic
effects an cells, and that
the p55-IC can induce IL-8 gene expression. Thus, it is possible to utilize
the p55-IC to mimic
TNF function in a site-directed fashion, i.c. to introduce the p55-IC only to
those cells ar tissues it
is desired to treat.
One example of the above approach, as mentioned above, is to specifically
transfect.
3~ (transform) tumor cells ar malignant tissue with a DNA molecule encoding
p~~-IG or a portion
thereof which can induce not only cytotoxic effects on such cells or tissue
but also augment these
effects by the co- -induction of IL-8, which W 11 result in the accumulation
at the site of these cells
or tissue of s~anulacytes and other lymphocytes, which, in turn, will serve to
destroy the tumor
cells or tissue. This approach obviates the need for administration of large
doses of TNF with its
40 as.~cociated deleterious side-effects.
CA 02490080 1995-05-11
WO 95/31544 . , PCT/US95/05$54
as
Using conventional recombinant DNA technoloc,~. it is possible to prepare
various regions
of the p55-IC and to determine which region is responsible for cacti TNF-
induced effect, e.g. we
have determined that the 'death domain' is responsihle far cytotoxicity
(Example 2), and we have
already prepared various other constructs containing f onions of the p55-IC,
which portions
(tobether with part or all of the death domain) may be responsible fox other
TNF-effects, arid
which may be used in a Iigand-independent manner, once self associated for
activity, to induce
these effects, e.g. IL-8 induction.
It should be noted that the sequeace of the p55-IC involved in the induction
of other TNF
associated effects (e.g. IL-8 induction) may be different to that involved in
cytotoxicity, i.e, may
inctude none or only part of the 'death domain' and hav a other sequence
motifs from other regions
of the intracellular domain, or may be the same sequence. different features
of the sequence !same
sequence motif) being involved in the induction of different effects.
Accordingly, as detailed above and below, expression vectors coataining these
p~5-IC
portions, analogs or derivatives thereof may be prepared, expressed in hose
cells, purified and
tested for their activry. In this way, a number of such p~~-IC fragments
having one or more TNF
associated activities may be prepared and used in a ditierential fashion for
the treatment of any
number of pathological conditions, e.g. viral infections, bacterial
infections, tumors, etc. In all of
these situations the speeihe activity can be augmented b~- incorporation (or
co-transfection) with
the p~5-IC fragment responsible for IL-8 gene expression induction, permitting
the desirable IL-8
chcmotactic activity to enhance the destruction of the~cclls or tissues it is
desired to destroy.
Thus, without adcriinistering systemically Th'F, it is possible to induce its
desirable effects
by specifically introducing all or part of the p55-IC into the cells or
tissues it is desired to treat.
The p55-IC may be introduced specifically into the cells or tissues it is
wished to destroy
by any one of the abovementioned procedures. For example, one way of doing
this is by creating
a recombinant animal virus e.g. one derived from Vaccinia, to whose DNA the
followi~ti ~~~
genes will be introduced ' the gene encoding a ligand that binds to cell
surface proteins
specifically expressed by the cells e.g. ones such as the AD7S virus gp120
protein which binds
specifically to some cells (CD4 lymphocytes and related lcukemias) or any
other ligand that binds
specifically to cells carrying a TNF-R such that the recombinant virus vector
will be capable of
binding such TIvTF-R-carrying cells; and the gene encoding the p55-IC or a
portion thereof. Thus,
expression of the cell-surface-binding protein on the surface of the virus W
11 target the virus
specifically to the tumor cell or other TNF-It-carrying cell, following which
the p55-IC, or
portion thereof, encoding sequence will be introduced into the cells via the
virus, and once
expressed in the cells will result in enhancement of the TNF effect leading to
the death of the
3~ tumor cells or other 'ITrF-R-carrying cells it is desired to !:ill or
induction, for example, of IL-8
which will lead to cell death. Construction of such recombinant animal virus
is by standard
procedures (see for example, Sambrook et al., 1989), Another possibility is to
introduce the
sequences of the p55-IC or parts thereof in the form of oligonucleotides which
can be absorbed
by the cells and expressed therein.
CA 02490080 1995-05-11
WO 95/31544 PCT/US95105854
29
The present invention thus also relates speci5cally to pharmaceutical
compositions
comprising the above recombinant animal virus vectors encoding the p55-IC or
portions thereof,
which vector also encodes a virus surface protein capable of binding specific
target cell (e.g.
cancer cells) surface proteins to direct the insertion of the p~5-IC, or
portions thereof, sequence
S into the cells.
The present invention relates, in yet another aspect, to new synthetic T11F
receptors which
are soluble and capable of oligomerization to form dimerie, and possibly also
high order
multimeric, TNF receptor molecules, cacti monomeric part of these receptors
being capable of
binding to a TNF monomer. T~'F occurs naturally as a homotrimer containing
three, active ?NF
monomers, each capable of binding to a single T1'F receptor molecule, while
TNF receptors
occur naturally as monomers each capable of binding only one of the monomers
of the TNF
homotrimeric molecule. Thus, when TNF binds to TyF receptors on the cell
surface, it is capable
of binding to three receptor molecules resulting in the clustering of the TN'F
receptors, which is
believed to be the start of the silrnaling process which ultimately triggers
the observed TNF
effects on the cells.
While T'I'TF has many desirable effects such as its abiliy to destroy, for
example, tumor
cells or virus-infected cells and to augment antibacterial activities of
granulocytes, TNF does
however, have many undesirable effects such as, fur example, in many severe
diseases including
autounmune disorders, rheumatoid arthritis, graft-versus-host reaction (graft
rejection), septic
shock, Ti'1F has been implicated as the major cause for pathological tissue
destruction. TNF may
also cause excessive loss of weight (cachexia) by suppressing the activities
of adipocytes.
T4oreover, even when administered for its desirable activities, e,g. in the
treatment of various
malignant or viral diseases, the dosages of TNF used arc often high enough to
cause within the
patient a number of undesirable cytotouc side-effects; e.g. the destruction of
healthy tissue.
Accordingly, in all of the above instances where TNF action is undesirable, an
effective
inhibitor of TNF has been sought. bZarty TNF-blocking agents have been
proposed, including
soluble proteins capable of binding TNF and inhibiting its binding to its
receptors and hence also
inhibiting the cyZOtoxic effects of TNF (see EF' 3083'78, fiP 398327 and EP
568925). However,
these TNF binding proteins, or soluble TNF receptors are monomeric, each
binding only one of
the TNF monomers of the TNF homotrimer. Hence, the blocking of the 'fl~'F
function may not be
complete, each monomeric receptor-bound T1~IF' molecule still having two TGIF
monomers free to
be able to bind cell-surface TNF receptors and illicit its effects on the
cells.
In order to overcome the above drawbacks in blocldrtg TNF function, there has
been
developed in accordance with the present invention a means for constructing,
as fusion proteins,
soluble oligomeric TNF receptors which ate capable of binding at mast two TTW
monomers of the
naturally occurnng TNF homotrimer molecule. As a consequence, these soluble
oIigomeric 1NF
receptors bind more.avidly to their TNF Iigand than the previously known
monomeric soluble
TNF binding proteins or receptors. For example, v~~hen the soluble T;VF
receptor of the invention
is in the form of a dimer, it is capable of binding two T:VF monomers of a TVF
trimer and hence
do C~,~S°° ~ .»'.e ~.~rnnleya ,~a~trst:~~tt~~ CC rho 'T't~'_
~i~ic ~~utr?'lIZ?ttOr! 11e"1~ mOrP ~llStP!nef~
CA 02490080 1995-05-11
WO 95/31544 . PCTlUS95/05854
because of a lower dissociation rate of the dimeri,: soluble receptors from
the TNF. Moreover,
such soluble, oligomeric receptors are also large: than their monomeric
counterparts and thus,
pharmaceutically, they are also advantageous because of the likelihood of
their having a slower
clearance rate from the body.
5 The basis for the development of the soluble oligomeric TIFF receptors of
the invention,
was the discovery that the intracellular domain of the p55-R TNF receptor was
capable of self
association, and further, that within this intracellular domain (p55-IC) there
exists a region, the
so-called 'death domain'. which is also capable of self association and as
such, in a ligand-
indepcndent fashion, can cause cytotoxic effects en cells (see Example 2)
Utilizing this self
0 association properly of the p55-IC and its 'death domain' it is thus
possible to construct a fusion
protein. using standard recombinant DNA technolo~~y~, containing essentially
alI of the
e~ctracellutar domain of a TNF receptor such as the p75-R or p55-R receptors,
preferably the p5~-
R, and fused thereto, essentially all of the intracellular domain (p55-IC) or
the death domain of
the p5~-IC. Im this way a nea~ fusion product is produced which has ai one end
the TNF binding
S domain i.e., the extracehular domain of the receptor, and at its other end
the intracellular domain
or the death domain thereof which is capable of self association. Accordingly,
such a product can
oligomerize by self association between two (and possible more) p55-1C or
death domains thereof
to yield oligomers (or at least dimers) having at Ieast nvo TNF binding
domains.
Furthermore, it has also been discovered in accordance with the present
invention, that the
?0 FaslA.PO1 receptor has a self associating, intracellular domain inclusive
of a self associating
'death domain' haring certain homolofy to the pSs-IC and death domain thereof
(Example 2).
Accordingly, it is possible to construct the soluble, oligomeric TNF receptors
of the invention by
fusing the exuacellular domain of the TNT' receptor (as noted above) to the
intracellular domain
or the 'death domain' of the FasIAP01 receptor.
~5 In both of the above noted situations, the oligomaric TNF receptors of the
invention are
soluble by virtue of having only the soluble extcacellular domain of the TNF
receptor and the
soluble intracellular domain or death domain .thereof of either the p55 R TN-F
receptor or the
FasIAP01 receptor, i.e. they do not contain the transmembranal {insoluble)
domain of either type
of receptor.
;0 The construction of the about oligomcric TNF receptors of the invention are
detailed
heron below in Example 4. It should however be noted that upon construction of
the oligomeric
TNF receptors of the invention, there may arise a situation, heretofore not
reported, that the
extraceilular domain of the TIv'F receptor is capable of self-association, a
situation that may not be
desirable as it could interfere with the ability of the oligomeric receptor to
bind to t~vo or more
Th'F monomers of the TNF homotrimeric molecules or may lead to less than
optimal binding ,of
such TNF monomers. Accordingly, in such a situation, it is possible, by
standard recombinant
DNA procedures, to modify the eh-tracellular domain of the TNF receptor by,
for example,
. deleting or substituting one or more amino acid residues contained within
the self associating
repon to prevem such self association. Such modifications of the ea-
cracellular domain of the TNF
receptor -are thus also part of the present invention and arc designated
herein as analobs or
CA 02490080 1995-05-11
WO 95131544 . PCTIUS95/05854
31
derivatives of the e~.-cracellular domain of the T_VF receptor. In a similar
fashion, the self
associating intracellular domain (IC) or death domain (DD) Thereof of the p55-
R receptor or the
FasIAP01 receptor used in the oligomeric TNF receptors o~the invention, may
also be analogs or
derivatives thereof i.e. may be any modification of the p55-IC sequence or
portions thereof
including the death domain (p55DD}. or any modification of the Fas/~PO1
intracellular domain
(FAS-iCj sequeace or portions thereof including the death domain (FAS DD},
providing that
these modifications yield a self associating product.
Similarly, once produced and purified, the soluble oligomeric TNF receptors,
analogs or
derivatives thereof. may be further modified b5~ standard chemical means to
provide salts and
functional derivatives thereof for the purposes of preparing pharmaceutical
compositions
containing as active ingredients these TNF receptors of the invention.
For the production of the soluble, oligomeric TiVF receptors of the invention,
the DNA
sequences encoding the e~.-tracellular domain of the TNF receptor are.
obtained from existing
clones of the entire TNF receptor, as is the intracellular domain or death
domain thereof, and as is
also the intracellular domain or death domain of the Fas,%APO1 receptor (see
Example 2 and
Example 5). In this u:ay the DNA sequence of the desired extracellular domain
is ligated to the
DNA sequence of the desired intracellular domain or portion thereof including
the death domain,
and this fused product is inserted (and Iigated) into a suitable expression
vector under the control
of the promoter and other expression control sequences. Once formed, the
expression vector is
introduced (transformation, transfeetion, etc.) into a suitable host cell,
which then expresses the
vector to yield the fusion product of the invention being the soluble self
associating T~1F receptor
molecules. These arc then purified from the host cells by standard procedures
to yield the final
product being the soluble, oligomeric TNF receptors.
The preferred preparation of the fusion product encoding the ea-tracellular
domain and
intracellular domain or portion thereof is by way of PCR technology using
otigonucleorides
specific for the desired sequences to be copied from the clones encoding the
entire TNF receptor
molecule. Other means are also possible, such as isolating the desired
portions encoding the
extracellular domain and the intracellular domain, by restriction
endonucleascs and then splicing
these together in a known fashion, with or W thout modifications at the
terminal ends of the
restriction frar_aments to ensure correct fusion o~ the desired portions of
the receptor (ek-tracellular
and intracellular domains or portions thereofj. The so-obtained fusion
products are then inserted
into the eoprcssion victor of choice. ~-
In a similar fashion, the present invemion also concerns soluble, oligomeric
Fas/AP01
(FAS) receptors containiag the e:ctracellular domain of the FasJAP01 receptor
and the self
associating intracellular domain of the p55-R (p55-IC), the death domain
theceof (p55DD), or the
self associating intracellular domain of the Fas/AP01 receptor (FAS-)iC) or
the death domain
thereof (FAS DD), or any analogs or derivatives thereof (see above}. The
construction of these
soluble, oligomeric FAS receptors is detailed in Example ~ herein below, using
an available cloned
full-length FAS receptor-encoding sequence as starting material and the
appropriate
oligonucleotides for PCR production of the desired extracellular and
intracellular domains,
CA 02490080 1995-05-11
WO 95/31544 PCTIUS95105854
follen:ed by ligation thereof to yield a fusion product. which is then
inserted into a suitable
ea-pression vector. ?~s detailed above and belov.~. prokaryotic or euharyotic
vectors and host cell
may be used to produce the desired soluble, oIieomeric FAS receptors, which
can then be purified
and formulated, as active ingredient. into a phartaaceutical composition.
S The above soluble, oligomeric FAS receptors of the invention are intended
for effective
blocking of the Fas lieand, which may also exist as a trimer (similar to T~fF,
see above), each
oligomeric receptor of the invention capable o: binding two or possible more
Fas ligands and
thereby neutralize their aeti«ty. The Fas ligand is 1.-sown to be
predominantly cell-surface
associated but may also exist in a soluble form. In any event, the oligomcric
F AS receptors of the
invention can bind to at least two monomers of this lictand and thereby
neutralize more effectivel~~
(than monomeric FAS receptors) the activiry of the Fas Iigand. The Fas ligand,
and hence
activation thereby of the FAS receptor, has been implicated in a number of
pathological states,
particularly those relating to liver damage (apoptosis of hepatocwes, for
example), includine liver
damage associated with hepatitis, as well as ir, autoimmune conditions,
including lymphocyte
damage (apoptosis) in HIV-infected humans (see, for example Ogasawara et al.,
1993, Cheng et
al., 1990. Accordingly, the soluble, olieomeric F AS receptors of the
invention are intended for
blocking the activity of Fas ligand and ma~~ be used as active ingredient in
pharmaceutical
compositions for ueating such Fas ligand-associated patholotical states.
Likewise, the present invention also concerns soluble, oligomeric receptors
which have
binding affinity for both TNF and FAS-R Iigand, the so-called "mixed" TNF
RIFAS-R oligomeric
receptors. These mixed oligomeric receptors will contain at least one Tl~'F-R
extracellular domain
and at least one FAS-R extracellular domain which arc associated in the
oligomeric recoptar by
virtue of each of these exuaceIlular domains being fused t~ any one of the
above-mentioned, self
associating, p55IC, p55DD. FAS IC or FAS DD
These mixed oligomeric receptors may be prepared by : (a) providia? a-y of the
a.'~ove
noted fusion products which contain the exuacellular domain of a TNF-R (p75
TNF-R or
preferably, p55 TNF-R) fused to any one of the self associating intracellular
domains p55 IC and
FAS IC or an}~ ono of the self associating 'death domain' p55DD and FAS DD, or
any self
associating portions, analogs or derivatives of any thereof; (b) providing any
of the above noted
fusion products which contain the extracellular domain of FAS-R fused to any
one of the self
associating p55IC, FAS-IC, p55DD, and FAS DD, or any self associating
portions, analogs or
derivatives of any thereof and (c) mixing any of the TNF-specific fusion
products of (a) with any
of the FAS-R ligand-specific fusion products of (b) to provide (following
standard selection and
purification procedures) oligomeric (dimeric or higher order oligomeric)
receptors which have at
least both the extracellular domains ef a T:VF-R and FAS-R that are associated
by.virtue of the
self association capability of their fused IC or DD regions.
Another possibility for the preparation of the above mixed oligomeric
receptors is by co-
transforming suitable host cells with the above-mentioned expression vectors,
one of which
encodes the TNF-specific TNF-R fusion products and one of which encodes the
FAS-R ligand-
specific FAS-R fusion products. Following the e~.~pression of these different
fusion products in the
CA 02490080 1995-05-11
WO 95/31544 PCT/US95/05854
host cells, the mixed oligomeric (TIFF-RT'AS-Ri receptors may be obtained by
standard
purification and selection procedures.
The utility of these mixed affinity oligome~c receptors is primarily for the
neutralization of
both T'!~1F and FAS-R Iigand when these are o~r:r-expressed endogenously or
are at undesirably
high levels folloHZnb exogenous adminisvation. Recent evidence points to a
likelihood that there
exists a synergism in function between the FAS-R ligand (usually cell-surface
associated) and
TNF-a (which may also be cell-surface associate;:). Accordingly, in some
instances it is desired to
neutralize both of these Iibands at the same point on the cell surface, i.e.
such a mixed-amity
receptor can block both the TNF bindine to its r eceptor and the binding of
FAS-R ligand to its
receptor. Accordineiy, these mixed-afl7niy receptors may be used as an active
ingredient in
pharmaceutical compositions for treating such conditions (see above) where
both TIFF and FAS-
R Ii~and effects are undesirable.
Similarly, along the lines mentioned abo~~e concerning the soluble, oligomeric
TNF-R and
FAS-R, and mixed TNF-R/FAS-R olieomers or the invention, it is also possible
to produce
1 ~ soluble, oiigomeric receptors for other receptor., or any mixtures
thereof, in particular those of
any of the other members of the TNFr'NGF super family. In this case, any of
the extracellular
domains of the various receptors can be fused to the above-mentioned self
associating
inuacellular domains or portions thereof or to anv other intracellular domains
of the super family
members also capable of self association.
fixpression of any of the recombinant proteins of the invention as mentioned
herein can be
effected in eukaryotic cells (e.g. yeast, insect or mammalian cells), using
the appropriate
expression vectors. Any method known in the arc may be employed.
For example, the DNA molecules coding for the proteins obtained by any of the
above
methods are inserted into appropriately constructed expression vectors by
techniques well known
in the art (see Sambrook et al., 1989). Double-stranded cDI~'A is linked to
plasr!id vectors by
homopoh~tneric tailing or by rcstzictioo linl.-ing involving the use of
synthetic DNA linkers or
blunt-ended ligation techniques. Dh:~ ligases are used to ligate the DIv'A
molecules and
undesirable joining is avoided by treatment W th alkaline phosphatase.
In order to be capable of expressing the desired protein, an expression vector
should
comprise also specific nucleotide sequences containing transcriptional and
translational regulatory
information linked to tbc DNA. coding for the desired protein in such a way as
to permit gene
expression and production of the protein. First, in order for the gene to be
transcribed, it must be
preceded by a promoter recognizable by RNA polymerise, to which the polymerise
binds and
thus initiates the transcription process. There are a ~~ariety of such
promoters in use, which work
with different efficiencies (strong and weak promoters). They are di~'erent
for prokaryotic and
;:;~;;~.
eukaryotic cells.
The promoters that can be used in the present invention may be either
constitutive; for
example the int promoter of bacteriophage 0, the ~1_a promoter of the ~3-
lactamasc gone ~ of
pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of
pPR325, etc.,
or inducible, such as the prokaryotic promoters including the major right and
left promoters of
CA 02490080 1995-05-11
WO 95/31544 Pt"T/US95/05854
bacteriophage ~ (PL and P~, the trp,, sec 4 la=?. 1~, ~, and ga_l promoters of
E. cell, or the
trpllac, hWrid promoter, etc, lGlick, B.R. ( 198 : . Besides the use of strong
promoters to generate
large quantities of mRNA, in order to achieve f~gh le4els of gene expression
in prokaryotic cells,
it is necessary to use also ribosome-binding sites to ensure that the mRNA is
efficiently translated.
One example is the Shine-Dalgarno sequence (SD sequence) appropriately
positioned from the
initiation codon and complementary to the 3'-terminal sequence of iGS RNA.
For eukaryotic hosts, different transcriptional and transiational regulatoy
sequences may
be employed, depending on the nature of the host. They may be derived from
~~iral sources, such
as adenovrus, bovine papilloma virus, Simian ~:rus, or the like, where the
regulatory signals are
associated with a particular gene which has a high level of expression.
Examples are the ?K
promoter of Herpes virus. the SV44 early promoter, the yeast gall eeae
promoter, etc.
Transcriptional initiation regulacott~ si~ntals may be selected which allow
for repression and
activation. so that expression of the ~:encs can be modulated.
. The DNA molecule comprising the nucleotide sequence coding for the fusion
product
proteins of the invention is inserted into a vec:or having the opeiably linked
transcriptional and
translational regulaton~ signals which is capable ef integrating the desired
gene sequences into the
host cell. The cells which have been stable transformed by the introduced DNA
can be selected by
also introducing one or more markers which allow for selection of host cells
which contain the
expression vector. The marker may pro~~ide for phototrophy to an auxotropic
halt, biocide
resistance, e.g. annbiotics, or hea~~~ metals, such as copper, or the like.
The selectable marker
. gene can either be directly linked to the DNA gene sequences to be
expressed, or introduced into
the same cell by co-transfection. Additional elements may also be needed for
optimal synthesis of
proteins of the invention. These elements ma5~ include transcription
promoters, enhancers, and
termination si~.Tnals. cDNA expression vector s incorporating such elements
include those
described by Okayama, H. (1983).
In a preferred embodiment, the imroduced DNA molecule will be incorporated
into a
plasmid or viral vector capable of autonomous re;pIication in the recipient
host. Factors of
importance in selecting a particular plasmid or viral vector include : the
ease with which recipient
cells that contain the vector may be reco_anized and selected from those
recipient cells which do
not contain the vector; the number of copies of the vector which arc desired
in a particular host;
and whethex it is desirable to be able to "shuttle" the vector between host
cells of different
species.
Preferred prokaryotic vectors include plasmids such as those capable of
replication in $.
toll, for example, pBR322, CaIEI, pSC101, pACYC 184, etc. (see Maniatis et
al., 1982;
Sambrook et al., 1989); Bacillus plasmids such as pC194, pC221, pT127, etc.
(Gryczan, T..
{1982)); Streptomyces plasmids including pIJ101 (Kendall, K.J. et al.,
(1987)); Streptomyces
bacieriophages such as QtC31 (Chalet, K.F. et al., in : Sixtl~Intornational
Syrrl,aosium on
~rc~otrrvcetales Biolosri. (1986)), and Pseudomonas plasmids (lohn, J.F. et
al., (1986), and
Izald, K. (1978)). Preft~rred eukaryotic plasmids include BPV, vaecinia, SV40,
2-micron circles,
etc., or their derivatizes. Such plasmids are well known in the aft (Botstein,
D. et al., (1982);
CA 02490080 1995-05-11
WO 95/31544 PCT/US95105854
JJ
Broach, J.R. in : The Molecular Biology, of the feast Saccharomvc~s : Life
Cvcle and Inheritatics
(1981); Broach, J.R, (1982); Bolton. D.P. et al., (1980); Maniatis, T., in :
Celf Biolo~rv v A
Co~,rehensive Treatis~yol. 3 ' Gene Ex ression, (1980); and Sambrook et al.,
1989).
Once the vector or DhIA sequence containing the constructs) has been prepared
for
expression, the D~1A construet(s) may be introduced into an appropriate host
cell by any of a
variety of suitable means : transformation. transfection, conjugation,
protoplast fusion,
electroporation, calcium phosphate-precipitation, direct microinjection, etc.
Host cells to be used in the invention may be either prokaryotic or
eukaryotic. Preferred
prokaryotic hosts include bacteria such as E. c;nli, Bacillus, Streptomyces,
Pscudomonas,
Salmonella, Serratia, etc. The most preferred prokaryotic host is E. ~uli.
Bacterial hosts of
particular interest include E. evh K1? strain 294 (ATCC 314-46), E. cwli X1776
(A,TCC 31537),
,~ cull ~~'3110 (F-, lambda-, prototropic (_~TGC 27;'?5)), and other
enterobacterium such as
Salmonella typhimurium or Serratia marcescens and various Pseudomonas species.
Under such
conditions, the protein will not be glycosylated. The prokaryotic host must be
compatible with the
replicon and control sequences in the expression plasmid.
Preferred euharyotic hosts are mammalian cells, e.g. human, monkey, mouse and
Chinese
hamster ovan~ (CHO) cells, because they provide post-translational
modincations to protein
molecules including correct folding or glycosylation at correct sites. Also
yeasts cells can carry
out post-translational peptide modifications including I;Iycosylation. ~
number of recomb'uiant
DNA strategies adst which utilize strong promoter sequences and high copy
number of plasmids
which can be utilized for production of the desired proteins in yeast. Xeast
recognizes leader
sequences on cloned mammalian gene products and secretes peptides bearing
leader sequences
(i.e., pre-peptides). '
After the introduction of the vector, the host cells are grown in a selective
medium, which
selects for the growth of vector-containing cells. Expression of the cloned
gene sequences)
results in the production of the desired proteins.
Purification of the recombinant proteins is carried out by any ane of the
methods known
for this purpose, i.e. any convrntional procedure involving extraction,
precipitation,
chromatogaphy, electrophoresis, or the like. A further purification procedure
that may be used in
preference for purifying the protein of the invention is affinity
chromatography using anti-TNI"
receptor monoclonal antibodies, which are produced and immobilized on a gel
matrix contained
within a column. Impure preparations containing the recombinant protein are
passed through the
column. The protein will be bound to the column by the specific antibody while
the impurities will
pass throufih. After washing, the protein is eluted from the gel by a change
in p):I or ,ionic
3 5 strength.
As used herein (see above), the term 'salts' refers to both salts of carboxyl
groups and to
acid addition salts of amino groups of the protein molecule formed by means
known in the art.
Salts of a carboa~yl group include inorganic salts, for example, sodium,
calcium, and salts with
organic bases as those formed, for example, ro~~ith amines, such as
trieihanolamine, arginine or
CA 02490080 1995-05-11
WO 95131544 PGT/US95/05854
lysine. Acid addition salts include, for example, salts with mineral acids and
salts with organic
acids.
"Functional derivatives" as used herein covers derivatives which may be
prepared from the
functional groups which occur as side chains on the residues or the N- or C-
terminal s~~roups, by
S means known in the art, and are included in the invention as long as they
remain pharmaceutically
acceptable, i.c. they do not destroy the activist- of the protein and do not
confer toxic properties
on compositions containing it. These derivatives include aliphatic esters or
amides of the carbox-hl
groups, and N-acyl derivatives of free amino groups of 0-acyl derivatives of
free hydroxyl groups
formed with acyl moieties (e.g. alkanoyl or carboc~~clic aroyl groups).
10 -"Fractions" as used herein refers to am~ part or portion of the receptor.
(intracellular or
extracellular domains thereof), or of the proteins binding to the
intracellular domain of the
receptor, provided it retains its biotol,~ical activity.
As mentioned above, the present invention also relates to various
pharmaceutical
compositions comprising a pharmaeeuticall~~ acceptable carrier and the various
noted active
15 in~edients of the invention or their salts, functional derivatives, or
mixtures of any of the
foregoing. These compositions may be used in am' of the conditions as noted
herein, for example,
in conditions where there is an over production of endogenous TNT', such as in
cases of septic
shock, cachexia, daft-versus host reactions, autoimmune diseases like
rheumatoid arthritis, etc.
The way of administration can be via any of the accepted modes of
administration for similar
20 agents and will depend on the condition to be treated, e.g. when used to
inhibit TNF effects they
may be administered intravenously in case of septic shock or local injection
in case of rhwmatoid
arthritis (for example, into the knee), or continuously by infusion, etc. The
compositions may also
be used, for example, in cases of TNF intoxication caused by exogenous
administration of
excessive amount (overdoses) of TNF, e.g. in the case of cancer therapy or
oral disease therapy.
25 The pharmaceutical compositions of the invention are prepared for
adr~inistratie~ h:~
mixing the protein or its derivatives with physiologically acceptable
carriers, stabilizers and
excipients, and prepared in dosage form, e.g. by lyophilization in dosage
vials. The amount of
active compound to be administered will depend on the route of administration,
the disease to be
veatcd and the condition of the patient. For example, local injection in case
of inflammatory
30 conditions of rhwm$toid arthritis wdl require less'activ~ ingredient on a
body weight basis~than
wt~l iatravcnous infusion in case of septic shock.
Other aspects of the invention will be apparent from the following examples.
The invention will now be described in more detail in the following non-
limiting exacxiples
and the accompanying drawings
.,,,
_.
CloninE and isolation of Qroteins which bind to the intracellular domains of
the p55 and
Q75 T1VF receptors
To isolate proteins interacting with the intracellular domains of the p55 and
p75 TNF
receptors (p~SIC and p75 IC), the yeast two-hybrid system was used (Fields and
Song, 1989).
CA 02490080 1995-05-11
WO 95/31544 pCT/US95/05854
3"
Briefly, this two-hybrid system is a yeast-based genetic assay to detect
specific protein-protein
interactions i~: vivo by restoration of a eukaryotic transcriptional activator
such as GAZA that has
1<vo separate domains, a DNA binding and an activation domain, which domains
when expressed
and bound together to form a restored Gr~t4 protein, is capable of binding to
an upstream
activating sequence which in turn activates a promoter that controls the
expression of a reporter
gene, such as lacZ or HIS3, the expression of which is readily observed in the
cultured cells. In
this rystem the genes for the candidate interacting proteins are cloned into
separate expression
vectors. In one expression vector the sequence of the one candidate protein is
cloned in phase
with the sequence of the G.4L4 DNA-binding domain to generate a hybrid protein
with the GALA
DNA-binding domain, and in the other vector the sequence of the second
candidate protein is
cloned in phase with the sequence of the GAL4 activation domain to ?enerate a
hS~brid protein
with the GrlL4-activation domain. The two hybrid vectors are then co-
transformed into a yeast
host strain having a lacZ or HIS3 reporter gene under the control of upstream
GAL4 binding
sites. Only those transformed host cells (cots ansformants) in which the two
hybrid proteins are
expressed and are capable of interacting with each other. will be capable of
expression of the
reporter gene. In the case of the lacZ reporter gene, host cells e~cpressing
this gene vviIl became
blue in color when 7~-gaI is added to the cultures. Hence, blue colonies are
indicative of the fact
that the two cloned candidate proteins arc capable of interacting with each
other.
Using this two-hybrid system, the intracellular domains p55IC and p75IC were
cloned,
. separately, imo the vector pGBT9 (carrying the GAL4 DNA-binding setaucncc,
provided by
CLONTECH, USA, see below), to create fusion proteins with the G4L4 DIVA-
binding domain
(similarly, the intracellular domain, FAS~IC and a portion of the 55IC,
namely, the 55DD were
also cloned into pGBT9 and used to isolate other IC-binding proteins, see
Example 3 below). For
the cloning of p55111; and p75IC into pGBT9, clones encoding the full-len~zth
cDlrTA sequences of
p55 TNF-R (Schall et al., 1990) and p75 TNF-R (Smith et al., 1990) were used
from which the
intracellular domains (IC) were excised as follows : p55IC was excised using
the enzymes EcoRI
and SaII, the EcoRI-SaII fragment containing the p55IC sequence was then
isolated by standard
procedures and inserted into the pGBT9 vector opened, in its multiple cloning
site region (IvICS),
with EcoRI and SaII. p75 IC was excised using the enzymes BspHI and SalI, the
BspFII-SaIT
franment containing the p75 IC sequence was then isolated by standard
procedures arid filled-in
with the klenow enzwte to generate a fragment which could be inserted imo the
pGBT9 vector
opened with Smal and SaII.
The above hybrid (chimeric) vectors were then cotransfected (separately, ~ one
cotransfection with the p55IC hybrid and one with the p75 IC hybrid vector)
together with a
33 eDNA library from human HeLa cells cloned into the pGAD GH vector, bearing
the GAL4
activating, domain,, into the HF7c yeast host strain (all the above-noted
vectors, pGBT9 and
pGAD GH carrying the HeLa cell eDNA library, and the yeast strain were
purchased 'from
Clontech Laboratories, Inc., USA, as a part of MATCFaviAhER Two-Hybrid System.
#PT1265-
1). The co-transfected yeasts were selected for their ability to grow in
medium lacking Histidine
(Ii'ts- medium), growing colonies being indicative of positive transformants.
The selected yeast
CA 02490080 1995-05-11
WO 95131544 PCT/US95105854
~s
clones were then tested for their abilin~ to ex~rzss the lacZ gent, i.e. for
their LAC Z activity, and
this by adding X-gal to the culture medium. v~~hich is catabolized to form a
blue colored product
bl~ ~-galactosidase, the enzyme encoded by the lacZ gene. Thus, blue colonies
are indicative of an
active lacZ gene. For activity of the IacZ gene, it is necessary that the GAL4
transcription
activator be present in an active form in the transformed clones, namely that
the GAL4 DNA-
binding domain encoded by one of the above h3~brid vectors be combined
propcrlv with the GAL4
activation domain encoded by the other hybrid vector. Such a combination is
only possible if the
two proteins fused to each of the GALS domains are capable of stably
interacting .(binding) to
each other. Thus, the I-bs+ and blue øAC Z~ colonies that were isolated are
colonies which
have been cotransfected with a v actor encoding p~SIC and a vector encoding a
protein product of
human HeLa cell origin that is capable ofbindin~ stably to pas IC; or which
have been transfccted
with a vector encodinb p75IC and a vector encoding a protein product of human
HeLa cell origin
that is capable of binding stably to p75 IC.
The plasmid DNA from the above His", LAC Z" yeast colonies was isolated and
1 S eleccroporatcd into E. coG strainI~ 1 O 1 by standard procedures followed
by selection of Leu+
and ~Ampicillin ~ resistant transformants, these transformants being the ones
cam~ing the hybrid
pGAD GH vector which has both the AmpR and Leu~ coding sequences. Such
transformants
therefore are clones carrying the sequences encoding newly identified proteins
capable of binding
to the p551C or p75IC. Plasmid DNA was then isolated from these transformed E.
coli and
recested by
(a) retransforming them with the original intracellular domain hybrid plasmids
(hybrid
pGTB9 carrying either the p55IC or p7~IC sequences) into yeast strain HF7 as
set forth
hereinabove. As controls, vectors carrying irrelevant protein encoding
sequences., e.g. pACT-
lamin or pGBT9 al5ne were used for corraasformation with the p55IC-binding
protein or p75IC-
binding protein encoding plasmids. The cotransformed yeasts were then tested
for c.~owth on His-
medium alone, or with different levels of 3-aminotriazole; and
(b) recraasforming the plasmi.d DNA and original intracellular domain hybrid
plasmids
and control plasmids described in (a) into yeast host cells of strain SF'Y526
and determining the
LAC Z'~ activity (effectivity of ~-gal formation, i.e. bhxe color formation).
the results of the above tests revealed that the pattern of graw-th of
colonies, in His-
medium was identical to the pattern of LAC Z actiy~, as assessed b; the color
of the colony, i.e.
I3is+ colonies were also LAC Z+. Further, the LAC Z activity in liquid culture
(preferred culture
conditions) was assessed after transfection of the GAL4 DNA-binding and actin
anon-domain
hybrids into the SFX52G yeast hosts which have a better LAC Z inducibilit5r
with the GAL4
transcription activator than that of the HF-7 yeast host cells.
The results of the above co-transfections are set forth in Table 1 below, from
which it is
apparent that a number of proteins were found that were capable of binding to
the p55IC; or the
p75IC, namely, the proteins designated 55.11, which binds to the p55IC; and
75.3 and .75.16
. which bind to the p75IC. All of these p55IC- and p75IC-binding proteins are
authentic human
proteins all encoded by cDNA sequences ori~~inating from the IicLa cell cDNA
library, which .
CA 02490080 1995-05-11
WO 95/31544 PCT/US95/05854
39
were fused to the G.aL,4 activation-domain sequence in the plasmid pG.AD GH in
the above yeast
two-hybrid analysis system
Interestingly, it was also found that fragments of the p55IC, itself, namely,
the proteins
designated 55.1 and 55.3 were capable of binding to g55IC. These are discussed
also in Example
2 below.
~.BLE 1
SUMMARY OF THE CIiARACTERISTICS OF SOME OF THE
cDNA CLONES (SEE ALSO EhAMPLE 3) ISOLATED Bl' THE
TWO-HYBRIDSl'ST~DZ APPROACH
DNA-binding Activation- Colon3~ colorLac Z Rcti<~itc~
; ~ in
domain hybrid domain hybrid li uid culture
assay
GBT9-IC55 --- white ' 0.00
GBT9-iC55 55.1 blue 0.65
GBT9-IC55 _ _ blue 0.04
55.3
--- 5 S. I white 0.00
--- i 5 white 0.00
S.s ~
ACT-Lamzn _ white ' 0.00
_
55.1
ACT-Lamin __ white 0.00
55.3
GBT9 55.1 white 0.00 !
GBT9 ~ 55.3 0.00 w
' white
BT9-IC55 ( 55.11 ; blue ND
55.1 I white ND
ACT-Lamin SS.11 white ND
GBT9 55.11 white 1'TD
GBT9-IC75 75.3 blue Iv'D
GBT9-IC75 -- w-hitc ND
--- 75.3 ; white ND
i
ACT-Lamin 75.3 white ND
GBT9 75.3 white ND
GBT9-IC7> 75.16 blue ND
75.16 white ND~
ACT-Lamin 75.16 white ND
GBT9 75.16 white ND
In the above Table 1, the plasmids and hybrid encoding the G.4L4 DNA-
binding.dornain
and GAL4 activation domain are as follows : ;
pNA-binding domain hybrids
pGBT9-IC55 : full-length intracellular domain of the p55-TNF-R (p55IC)
pACT-Lamin : irrelevant protein - lamin.
pG13T9 : vector alone
pGBT9-IC75 : full-length intracellular domain of the p75-TNF-R (p75IC) . ~-~ ~
>~::~:aax
Activation-domain hybrid : ... ;;~~a;--v~~~
55.1 and 55.3 correspond to fragments of the intracellular domain of the p55-
TIv'F-R.
55.11 : is the novel protein associating with the p55-TIFF-R
75.3 and 75.16 are the novel proteins associating with the p75-TNF-R.
CA 02490080 1995-05-11
' The above noted cloned cDl~:~ encoding the novel p~~IC- and p75IC- binding
proteins,
55.11, ?5.3 and 75.16, were then sequenced using standard DNA sequencing
procedures. The
partial sequence of all of these protein-encoding sequences is set forth in
Figs. 1 a-c, where Fig.
1(a) depicts the sequence of the cDNA encoding protein 55.11; Fig. 1(b)
depicts the partial
S sequence of the eDNA encoding protein 75.3; and Fig. 1(c) depicts the
partial sequence of the
cDNA encoding protein 75.16. In Fig. 1(d) there is shown the deduced amino
acid sequence of
the protein 55.11, as deduced from the nucleotide sequence of Fig. 1(a).
It should be noted. however, that a partial sequence of the eDNA encoding the
55.11
protein has also been reported by Khan et al. (1992), in a study of human
brain cDNA sequences,
which study was directed at the establishment of a new rapid and accurate
method for the
sequencing and physical and genetic mapping ef human brain cDNAs. However,
Khan et al. did
not provide any information as regards the function or any other
characteristics of the protein
encoded by the 55.11 eDN A sequence; such functional or other analysis not
being the intention of
lvhan et aI. in their study.
1 s Analysis and characterization of the 5S 11 protean
fal_ neral Procedures and Materials
ail Ctonin~ of the cD~I4 of 55 11
Upon the analysis {for example, Northern Analysis -see below) of the
cDNA of protein 55.1 I, it was revealed that the above noted 55.11 cDNA cloned
by the two
hybrid scrctn procedure represented only a partial cDNA of 35.11 having
nucleotides 925-2863
(see Fig. 1(a)) which code for amino acids 349-900 (see Fig. 1(d)). The
remaining part of the
55.11 cDNA (nucleotides 1-924 (Fig. 1(a)) which code for amino acids 1-308
(Fig. 1(d))] was
obtained by standard procedures. namely, by cloning by PCR from a human fetal
liver cDNA
library (for more details, see below). The full nucleotide sequence of 55.11
(Fig. 1(a)) was
determined is both directions by the dideoxy chain termination method.
fiil Two-hybrid ~patactosidase expression tests
~i-galactosidase expression tests were performed as described above,
except that in some of the tests, the plfl' I6 vector, which contains the
activation domain of VP16,
. was used instead of pGAD-GH, the Gal4 activation domain vector. Numbering of
residues in the
proteins encoded by the cDNA inserts are as in the Swiss-Prot*data bank.
Deletion mutants were
produced by PCR, and point mutations by oligonucleotide-directed mutagenesis
(Kunkel, 1994).
{iiil Northe n analysis
Total RNA was isolated using TRI REAGENT (Molecular Research
Crnter, Inc., Cincinnati, Oh., U.S.A.), denatured in formaldehyde<'formamide <-
buffer,
electrophoresed through an agaroselformaldehydc gel, and blotted to a
GeneScreen.-..Plus*
membrane (Dupont, Wilminston, De., U.S.A.) in IOxSSPE buffer, using standard
techniques. The
blots were hybridized with the partial cDNA of 55.11 (see above, nucleotides
92~-28G3),
radiolabeled with .the random-prime kit (Boehringer Mannheim Biochcmica,
Mannheim,
Germany), and washed stringently. Autoradiography was performed for I week.
* Trade-mark
CA 02490080 1995-05-11
a~
jivl Ez~re~sion of 55.11 eD'~ 4 in HeLa cells and binding of the 55.11 protein
toil to hione S-transferstse ~us~nroteins of p55'IC
Glutathione S-transferase (GST) fusions with p55-IC (GST-p55IC) and
with p55-IC truncated below amino acid 345 (GST-p55IC:45) were produced and
adsorbed to
glutathione-agarose beads as described in Example ? below (see also Smith and
Corcoran, 1994;
Frangioni and Neel, 1993). The cDNAs of 55.11 (1-2863 nucleotides, i.e. the
full-length SS.i1
cl7NA), of FLAG-55.11, and of fuciferase were expressed in HeLa cells. FLAG-
55.11 is the
region extending; between residues 309 and 900 in the 55.11 protein (the
partial cDNA of 55.11
(nucleotides 925-28b3), originally cloned by the two hybrid screen), N-linked
to the FLAG
octapeptide (Eastman Kodak, New Haven, Ct., U.S.A.). -Expression of the fusion
proteins was
accomplished using a tetracycline-controlled expression vector (HtTA-1 ) in a
HeLa cell clone that
expresses a tetracycline-controhed transa:.tivator (see Example 2 below, and
Gossen and Bujard,
1992). Metabolic labeling of the expressed proteins with j35S] T2et and [3oSj
Cys (Dupont,
Wilmington, De., U.S.A, and Amersham, Buckinghamshire, England), lysis of the
HeLa cells,
immunoprecipitation, and binding of the labeled proteins to the GST fusion
proteins were
performed as described below (Example 2), except that 0.5°~a rather
than 0.1% Nonidet F-40 was
present in the cell lysis buffer. The immunoprecipitations of 55.11 and FLAG-
55.11 were
achieved using a rabbit antiserum (diluted 1:500) raised against a GST fusion
protein containing
the region of 55.11 that ekrtends between amino acids 309 and 900 and a mouse
monoclonal
antibody against the FLAG octapeptide (M2; Eastman Kodak; 5 ~eglml of cell
Iysate).
fbl Binding of the 55.11p tro ein to~55-I~aithin transformed veasts
In this study it w-as sought to ascertain the nature of the binding between
5.11
and p55IC, in particular, the re~dons of both of these proteins involved in
this binding. For this
purpose the above two-hybrid procedure was used in which various full-length
and deletion
2S mutants of p55IC (see also Example 2 below) in "DNA-binding domain"
constructs were used as
"baits" to bind the "preys", being the partial SS,I1 protein encoded in
constructs in which the
partial 55.11 sequence (residues 309-900, as originally isolated) was fused to
the "activation
domain" in the vectors GAL4AD and VP16AD. Further, various deletion mutants of
55.11 were
also constructed and fused to the "activation domain" in the GAL4Al7 vector
(e.g. mutants of
55.11 having only residues 309-6S0 and 457-900). The binding of the various
'binding domain'
constructs to the various 'activation domain' constivcts was examined in
transferred SFY526
yeast cells. The binding was assessed by a two-hybrid ~-galactosidase
expression filter assay. The
non-relevant proteins SNFI and SNF4 screed as positive controls for the
'binding domain' and
'activation domain' constructs, respectively; the empty Gal4 (pGAD-GH) and VP
1 G (pVP 1 G)
vectors sen~ed as nebative controls for the 'activation domain' constructs;
znd the. empty Gal4
(pGBT9) vector served as a nebative control for the bindinb, domain'
constructs. The results of
the assay are set forth in Table 2 below in which the symbols "~" and "J-:-"
indicate the
development of strong color within 20-60 min of initiation of the assay,
respectively (positive
binding results); and "-" indicates no development of color within 24h of
commencement of the
assay (negative results). Blank spaces in the Table indicate binding assays
not tested.
* Trade-mark
CA 02490080 1995-05-11
WO 95131544 4 2 pCf/US95105854
T le 2
Binding of the 55.11 protein to p55-IC
within transformed yeasts
D
o m s~d~1 I
z o'
m o
r
+ + +
a ~ °~' + + + ' t
a >
0
z . ~~,o
q t
Q ~ ~y .
> m ors ~ +
V m °0
a z sos~ t
0
a
r °89,6
t
°°s'sp + + + + + t t t t
+ +
.~
_~
U ~ M c° M ~ ~ Ii ,
9 ~ O O O 'd t~~D N
N N N N N M
2~ ~' r .
(_~ ~_
zm
d~~ .
.~
O~,
;~ a ;~.;
z o . ~,~~
o ~a~
- ,~02 .
...
CA 02490080 1995-05-11
WO 95131544 PCT/US95/05854
43
From the results presented in Table 2 above it may be concluded that ».11
binds to p55-
IC at a site which is distinct from the 'death domain' (residues 328-426) of
p.SS-IC.
The 55. i 1 protein bound to a truncated p5~-IC from which the death domain
had been
deleted (construct 206-328 in 'Table 2). more effectively than to nontruncated
p55-IC. It also
bound to an even further C terminally truncated construct (construct 206-308j
and to a construct
from which both the death domain and a membrane proximal part were deleted
(construct 243-
328), However, the 55.11 protein did not bind to a construct that was N-
terminally truncated
down to amino acid 266 (Table 2). These findings indicate that the binding
site for 55.11 is
located in the regrion that extends between residues 243 and 308 of p55-IC and
that the N
terminus of this binding site is between residues 243 and 266.
Transfer of the cDNA for 5.11 from the originally cloned 'prey' construct,
which
contained the Gal4 activation domain, to a prey construct containing the VP 16
activation domain
did not decrease the binding efbcienc~~ of the 55.11 protein to p55-IC (Table
2). Thus, the
structures) involved in tlus binding appear to reside within the 55.11 molecut
and not to involve
the site of fusion of 55.11 with the acti~~ation domain
However, binding of 55.11 to p55-IC was abolished by even limited truncations
of the
55.11 protein at either its C (55.11 construct 309-680) or N terminus (55.11
construct 457-900).
(residue 309 is the first residue in the 55.1I protein encoded by the partial
cDNA clone oril,~nally
isolated in the two hybrid saecn).
The observed binding between 55.11 and p55-IC appeared to be specific since
SS.I 1 did
not bind to other proteins, including three receptors of the Th~'INGF receptor
family (p75-R,
FaslAPO1 and CD40) and other proteins such as lamin and cyclin D (data not
shown). It should
be noted that of the other TIv'F/NGF receptor proteins tested there was also
tested portions
thereof which include their intracellular domains : human FAS-R (residues 175-
319), CD40
(residues 216-277) and p75-TNF-R (residues 287-461), none of which bound 55.11
(data not
shown).
~cl Northern analysis of the RN4 frarn several call Lines, using the 55.11
cDN4 ac a
probe and cloninE of the full-legit ~ 55.1LcDNA
The cell lines examined were HeLa, CEM, Jurkat, and HepG2 cells derived from
human epithelial carcinoma, as acute lymphoblastic T cell leukemia, an acute T
cell leukemia, and
a hepatocellular carcinoma, respectively. The 55.11 cDNA original isolated
(nucleotides 9? 5
2863) was used as a probe. Samples consisted of 10 ~S of RNA/lane. The results
of the Northern
analysis are shown in Fig. 2, which is a reproduction of a Northern blot.
From Fig. 2 it is thus apparent that the Northern analysis using the 55.11
cDNA as a probe
revealed, in several cell lines, a single hybridizing transcript of about 3
kB, which is larger than the
cDNA (2 kB) of the originally isolated 55.11 cDNA. Using oligonucleotide
primers that
correspond to the 55.11 sequence, we cloned by PCR a 5' extending sequence
whose len~,nh was
about 1 kB. The sum of the length of this 5' e~ctcnding sequence W th that of
the originally cloned
cDNA approximates the length of the 55.11 transcript. The 3 1;B cDNA that
encompassed both
CA 02490080 1995-05-11
WO 95131544 PCT/fJS95105854
44
these portions w~as effectively expressed in transfected HeLa cells (see
belou~) yielding a protein
of about s4 kDa, which suggests that the 3 1~B cDN~, contains a translational
start site.
d In vitro bindino f the 55.11 rotein to GST-fusion roteins containin onions
of 5
S To ascertain that 55.11 can indeed bind to p~5-IC and to exclude involvement
of
yeast proteins in this binding, the in vitro interaction of GST p55-IC fusion
proteins, produced by
bacteria, with the protein encoded by the 3 kB 55.11 cDNA {55.11-full),
produced by transfceted
HeLa cells, was examined. In this study the cDNAs for the full-length 55.11,
FLAG-SS.II
(residues 309-900 of 55:11 encoded by the originally cloned partial cDNA and
fused at the N
terminus with the FLAG octapcptide), and lucifcrase (control) were expressed
in transfected
HeLa cells and metabolically labeled W th (-ASS] Met and [355] Cys. The
following proteins were
fused with GST : full-length pS5-IC (GST-pSS-IC) and pSS-IC C-terminally
uuncated up to
amino acid 345 (GST-p55-IC345) to remo~~e most of the 'death domain' (see
Table ~). GST alone
served as a control. Lysates of the transfected cells were immunoprecipitated
with antibodies
against the 55.1 I protein when the full-lengnh 55.11 protein was used for
binding the GST-fusion
proteins, or with antibodies against the FLAG octapeptide when the FLAG-55.11
fusion product
was used for binding the GST-fusion proteins. The proteins were analyzed by
SDS-
polyacrylart~ide gel electrophoresis (SDS-PAGE; 10°.o acrylamide),
followed b5 autoradiography.
In Figs. 3A and B are shown reproductions of the autoradiograms of the above
SDS-PAGE gels, in which Fig. 3 A depicts the binding of the full-length 55.11
protein (55.11-full)
to the various GST-fusion proteins; and in which Fig 3B depicts the binding of
the Flag-55.11
fusion product to the various GST-fusion proteins. In Fib. 3 A there is shown
in the extreme right
hand lane a control immunoprecipitate of lysates of cells transfected with
only the full-length
55.11 and immunoprecipitated with the anti-55.11 antibodies (x.55.11 Abs). In
Fig. 3B there is
shown in the extreme right band lane a control immunoprccipitatc of lysates of
cells transfected
with only the FLAG-55.11 and immunoprecipitated with the anti-FLAG antibodies
{otFLAG
Abs).
Thus, it is apparent from Figs. 3A and B that the protein encoded by the full-
length
55.11 cDNA can be expressed in HeLa cells and it binds to fusion proteins that
contained the full
p55-IC (GST-p55IC) or a truncated p55-1C that lacked most of the death domain
(GST-
p55IC345) (Fig. 3 A). The full-length 55.11 protein did not bind to GST alone
(control). Similarly,
the HeLa ceU-expressed protein encoded by the initially cloned partial cDNA of
55.1 I in fusion
with the FLAG octapeptide (FLAG-55.11) bound in vitro to GST-p55IC and GST-
p55IC345,
but not to GST (Fig. 3B). The above results also therefore provide additional
evidence (see (b)
above) that the 55.11 binds to a region of the pSSIC upstream of the 'death
domain', i.e. in the
region of the p55-IC that is more proximal to the transmembrane domain.
Moreover, the above study also demonstrates that, in accordance with the
present
invention, antibodies to 55.11 have been successfully produced {Fig. 3A).
CA 02490080 1995-05-11
WO 95/31544 PCT/US95105854
4~
Le) Comparison of the deduced amino acid sequence of human SS.II to that of
related ro~_teins present i tower organisms, and sequence features of the
55.11 rp otein
As mentioned above, in accordance with the present invention, the full-length
;5.11 eDNA has been cloned and sequenced (see nucleotide sequence in Fig. 1
(a)) and the full
amino acid sequence of 55.11 has been deduced from the cDNA sequence (see
amino acid
sequence in Fig. I(d)). Data bank (GenBanhThSBMBL DataBank) searches revealed
that parts of
the sequence of the human 55.11 cD\A (accession numbers T03659, 219559, and
F091~8) and
its mouse homologue (accession numbers X80422 and Z3I147) have alread~~ been
determined
during arbitrary sequencing of cDNA libraries. A cDNA sequence (accession
number U18247)
that encodes for a human protein of ~ 96 amino acids present in cultures of
human hepatoma
HC10 cells is similar to that of 55.11. This hepatoma protein, however, lacks
an N terminal
portion (amino acids 1-297) corresponding to that of 55.11 and also differs
from 55.11 at the
regions that correspond to residues 29%-3?7 and residues 648-G68 in 55.11. The
searches of the
data bank also revealed that proteins with very high sequence homolo~ry~ to
55.11 exist in
Saccharomyces cerevisiae (yeasts), arahi~iup~is rhaliana (plants) and
C'.aenorhahtlitis eleyanv
(worms). Thus, 55.11 appears to fulfill an evolutionat5~ conserved function.
In the yeasts, there
are two known proteins (the open readint frame ~C'HItG27c and SEN3) whose DNA
sequences
resemble that of 55.11. The sizes of both are close to that of 55.11. YHR02?c
is known only by
the sequencing of a genomic clone while SEN3 has been cloned as a cDNA. The
sites within
55.11 that are similar to those in SEN3 correlate to the sites of its
similarity to YHR027c,
although much more similarity is evident between X5.1 ! and YHIZ027c than
between SS.I I and
SEN3. The DNA sequence information available for the Aicthiclopsis thaliana
and
Caenorhahditis elegam proteins, although only partial, clearly shows that
these proteins are as
similar to 55.11 as the 'Y>$027c protein of yeast. The only one of these four
proteins whose
nature has been eluc'sdated so far is the yeast SEN3, whose homology to ~S.II
is limited. SElvT3
has been identified as the yeast ~qui~alent of the p112 subunit of an
activator of the 20S
proteasome (the proteolytic core of the 2GS proteasome [Rechsteiner et al.,
1993; Del\Sartino et
al., 1994j) (M.R. Culbertson and M. Hockstrasser, personal communication).
1n Fig. 4 there is shown schematically a comparison of the deduced amino acid
sequence of human 55.11 to that of the above-mentioned, related proteins
present in lower
organisms. In Fig. 4 the sequences that are compared are the sequences of
amino acids predicted
for : the 55.11 eDNA (sec Fig. 1 (d)); an open reading frame {YT-~t027c)
within a cosmid derived
from the 8th chromosome of Saccharnmvce.~ cerevisiae (nucleotides 21253-24234,
accession
number U10399); SEN3, the cDNA of a Saceharonryces cereui~iac protein
(accession number
L06321); a partial cDNA of a protein of the plant Arcrbidopsis thaliana
(accession number
T21500); and a partial eDNA of a proton of the nematode C.'aenorhahditi.s
elegan.c (accession
number D2?396). The 'KEKE' sequence in 55.11 is marked with a solid line and
the sequence
AI'AGS(x)gLL with broken Iines. The sequences were aligned using the 1?lLIrUP
and
PRET?I'BOX programs of the CrCG package. Gaps introduced to maximize
alignments are
denoted by dashes.
CA 02490080 1995-05-11
WO 95/31544 PCT/US95/05854
46
rls regards the various sequence features or motifs present in the human 55.11
sequence the following has been obsewed : Consewed amino acid sequence motifs
were not
discerned within the protein encoded for by 55.11, except for a repetitive
'KEhE' sequence that
extends between Lys 61.t and Glu 632 (underlined in Fig. 4). Such 'K,)rICE'
sequences, which are
present in many proteins, including proteasonal subunits and chaperonins, may
promote
association of protein complexes (Reaiini ct al., 1994). A sequence
AYAGS(x)gLL appears twice
in the 55.11 protein (at sites 479 590. see Fib. 4); no functional
significance for this sequence has
yet been described.
(f3 ~~guence features of the p5SI~ region invoived in binding to the ~~
~l~rot~in
As described above (see (b) and (d)), the 55.11 protein binds to a region of
the
p55-IC between residues 243 and 308 (the N terminus of this binding site being
between residues
243 and 266), this region being upstream ef the 'death domain' and more
proximal to the
transmembrane domain of the p55-T?v'F'-R. This region within p55-IC to which
55.1 l binds has a
high content of proline, serine, and threonine residues. However, this region
does not contain the
I S RPM/ and RPIvL? proline-rich motifs present in several other cytokine
receptors (OTleal and Y u-
Lee, 1993). In the region that extends ben~~een residues 243 and 266, whose
deletion abolishes
the binding of p55-R to 55.11 (see (b) and (d) above and Table 3), two of the
s~rinas and two of
the threonines are followed by prolinc residues, which makes them potential
sites for
phosphorylation by MAP lcinase, Cl~C2, and other profine-dependent kinases
(Seger and Krebs,
1995). Phosphorylation of this site in the receptors might affect its binding
to the 55.11 protein.
In view of all of the aforementioned with regards to protein 55.11 and its
binding to p55-
IC it can be concluded that in accordance with the present invention, a new
protein has been
found which binds to a distinct region upstream to the 'death domain' of p55-
IC. Such binding
could affect TNF-mediated activities other than induction of cell death. The
region to which 51.11
binds has previously been shown to be involved in induction of nitric oxide
sy~nthase (Tartaglia et
al., 1993), and appears to be involved in the activation of the neutral
sphingomyelinase by ?NF
(Wiegmann et al., 1994). It is thus possible that association (binding) of
55.11 w7th the
intracellular domain of p55-T:~IF-R (p55IC) affects or is involved in : (i)
the signalinb for these
above noted or other TNF effects, (ii) the folding or processing of the
protein (as suggested by
the similarity of 55.I 1 to a subunit of the 26S proteasome), or (iii) the
regulation of the activity or
expression of p55-TNF-R
EXAMPLE 2
if c 'on abili ~ of th i t ace lular domain f th 5'S T'V r . r ~5 n its
capability to reuse cell death and other features and activities thereof. and
a related
~as%APOl receptor's intra~~l~,lar docnain
As set forth in Example 1 above, it was discovered that the intracellular
domain of p55
TNF-R (p55IC) is capable of binding to itself, and further that fragments of
p55IC, namely
proteins 55.1 and 55.3, arc also capable of binding to p55IC.
' - CA 02490080 1995-05-11
~7
It is know chat the binding of T1t to F5S T:~I'1:-R leads to a c~-tocidal
effect on the cells
carn~ing this receptor. Further, antaodies against the ewracellular domain or
this receptor can
themselves nigger this effect, in correlation with the effectivty of receptor
cross-linking by them.
In addition, mutational studies (Tartaglia et al., (1.993); Brakcbusch et al.,
(1992)) showed
that the function of the p5~-R depends on the integrity of its intracellular
domain. It was therefore
suggested that the initiation of signaling for the cytocidal effect of TNF
occurs as a consequence
of association of two or more intraceltuiar domains of the p55-R (p55-IC),
imposed by receptor
aggregation. The results in accordance with the present invention provide
further evidence for this
notion, showing that e~;pression of the intracellular domain of the p55-R
within cells, without the
transmembrane or intracellular domain, triggers their death. Such free
intracellular domains of the
p55-R are shown to self associate, which probably accounts for their ability
to function
independently of TNF. The fact that the si~~naling b; the full length p~5-R
does depend on TNF
stimulation is suggested to reflect acti~~yti(es~ of the transmembrane or
e~ctracellular domain of the
receptor which decrease or prevent this self association.
The ability of the intracellular domain of the p55-R (p55-IC} to self
assaciate was found
serendipitously, in the attempts to clone c~ector proteins which interact with
this receptor (see
Example 1 above). We applied for that purpose the above mentioned "two hybrid"
technique. In
addition to the novel protein, 55.11 found to associate (bind) to the p55IC,
it was also found that
three other cloned HeLa cell cDN.~s contained cDNA sequences ancoding for
parts of the
inuaceIIular domain of the p55-R, implying that the p55-IC is capable of self
association. Two of
these clones were identical, containing an insert which encodes for amino
acids 328-42f
(designated as clone 55.1 encoding protein fragment 55.1 of the p55IC}. The
third contained a
Longer insert, encoding for amino acids 277-426 (designated as clone 55.3
encoding protein
fragment 55.3 of the p55IC).
In addition, we assessed the 'tn_ vitro interaction between two bacterially
produced
chimeras of the p55IC, one, in which it was fused to the maltose binding
protein {MBP) and the
other in which is was fused to the ~,~lutathione-S-transferasc (GST). These
chimeras were
constructed, cloned and expressed by standard methods. Following their
expression, the
assessment of the self interaction of the p55-R intracellular domain (p55IC)
by determining the
interaction of the above bacterially-produced chimeric proteins GST-IC55 (Mr -
SIkD) and
MBP-IC55 (Mr - 6? l;D) with cacti other. Equal amounts of the GST-IC55 chimera
(samples of
lanes 1-4 in Fig. 5) or GST alone (samples of lanes 5-8 in Fig. 3) were bound
to glutathione-
agarose beads (Sigma) and were then incubated with the same amount of MBP-rC55
fusion
protein in one of the following buffer solutions : = -
(i) buffer I (20m1vI Tris-HCI, pH 7.5, IOUmM KCI, 2tnM CaCIZ, 2tnM MgCIZ, SmM
DTT, 0.2°.'o Triton X100*O.SmM PMSF, 5°fo Glycerol). This was
done for the samples of Lanes 1
and 5 of Fig. 5.
(ii) buffer I containing SmM EDTA instead of M,gCIZ. This was done for the
samples of
Lanes 2 and 6 of Fig. 5.
* Trade-mark
CA 02490080 1995-05-11
WO 95131544 PCT/US95/05854
.~8
(iii) buffer I containing ~SOrrLli instead of 100mM KCI. This was done fo: the
samples of
Lanes 3 and 7 of Fig. 5.
(iv) buffer I containing 400m.1I inaead of 100mIVI KC1. This was done for the
samples of
Lanes 4 and 8 of Fig. 5.
After incubation with rotation for ~h at 4°C, the beads were washed
with the same buffers
and then boiled in SDS-PAGE buffer followed by electrophoresis by PAGE. The
proteins on the
gel were then Western blotted to a nitrocellulose membrane which was then
stained with
polyclonal antiserum asainst MBP. A reproduction of this stained Western blot
is shown in Fig. 5,
the samples in lanes 1-8 being those noted above.
From Fig. 5 it is apparent that the p55IC-MBP chimera bind to the pSSIC-GST
chimera
(lanes 1-4) independently of divalent cottons and even at a rather high salt
concentration (0.4M
KCI). Thus, it is concluded that the p~=IC is able to avidly self associate.
To evaluate the functional implications of the propensity of the p55-IG to
self associate,
we attempted to express the p55-IG within the cytoplasm of cells which are
sensitive to the
cytocidal effect of Th'F. Considering the possibility that the p55-IC wzlI
turn to be cvtotaxic, we
chose to express it in an inducible manner, using the recently developed,
tightly regulated
tetracycline-controlled mammalian expression system (Gosscn and Boujard,
1992}. Expression of
the p55-IC resulted in massi~~e cell death (Fig. 6, right panel). The dying
cells displayed cell
surface blabbing as observed in the killing of the cells by TNF. Transfection
of the p53-IC
construct to the cells in the presence of tetracycline, which reportedly
decreases the expression of
pI~lO-3 regulated constructs by as much as IO$ fold, still resulted in some
cell death, although
significantly Iess than that observed in the absence of tetracycline (Fig 6,
left panel). In contrast,
cells transfected with a control construct, containing the lucipherase cDNA,
showed no sums of
death (results not shown).
?5 The ability of the p55-IC to trigger cell death, when expressed without the
tra,nsmembrane
or extracellular domains of the receptor, pro~rides further evidence for the
involvement of this
domain in signaling. Furthermore, it indicates that no other part of the
receptor plays a direct role
in such sirs~naling.. Studies of the effects of mutations, including those
mutations studied in the
present invention, on the function of the p55-IC, indicated that the region
extendinb between
amino acid residues 326 and 40? is most critical for its function. This rc~ton
shows marked
resemblance to sequences within the intracellular domains of two other
receptors, evolutionarily
related to the p55 TIFF-It- namely, the Fas receptor (Itoh et al., 1991; pohm
et al., 1992), which
can also signal for cell death and CD40 -a receptor (Stamenkovic et al., 1989)
which enhances
cell growth; this sequence therefore stems to constitute a conserved motif
which plays some kind
.5 of veneral role in signaling. Since it dots not resemble known motives
characteristic of enzymatic
activities, it seems plausible that it signals in indirect manner, i.e,
possibly by serving as a dockinb
site for signaling enzymes or for proteins which transmit stimulatory signals
to them. The p55-IC,
the Fas receptor and CD 40 can all be stimulated by antibodies against their
cxtracellular domain.
Their stimulation could be shown to correlate with the ability of the
antibodies to cross-fink the
receptors. It therefore stems that the signaling is initiated as a consequence
of interaction of two
CA 02490080 1995-05-11
WO 95131544 PCT/ITS95105854
~9
or more intracellular domains imposed bs- aggregation of the extracellular
domains. Involvement
of such interaction in the initiation osic.~naling of these receptors was also
indicated by studies
(Brahebusch et al., 1992) showing that cspression of receptors made
nonfunctional by mutation
of their intracellular domain, had a ''dominant negative" effect on the
function of co-expressed
normal receptors. Aggregation of the p= ~-R in response to TNF was suggested
to occur in a
passive manner, merely due to the fact that each of the TNF' molecules, which
occur as
homotrimers, can bind two or three receptor molecules. However, the findings
of the present
invention suggest that this process occurs somewhat differently.
The propensity of the p55-IC to self associate indicates that this domain
plays an active
role in its induced abgregation. More~~~er. this activity of the p55-IC seems
to suffice for initiating
its signaling, since when expressed independently of the rest of the receptor
molecule, it can
trigger cell death in the absence of T'.v'F or any other exterior stimuli.
Nevertheless, when
expressed as the full length receptor, the p55-TNF-R does not signal, unless
stimulated by TIvTF.
One must, therefore, assume that when activating the p55-TNF-R, TNp actuall3~
overcomes some
inhibitory mechanisms, which prevent spontaneous association of the
intracellular domains, and
this inhibition is due to the linkage of the p55-IC to the rest of the
receptor molecule. The
inhibition may be due to the orientation imposed on the intracellular domain
by the
transmembrane and extracellular domain, to association of some other proteins
with the receptor
or perhaps just due to restriction of the amounts of receptors that arc
allowed to be placed in the
plasma membrane. Of note, this control mechanism should be rather effective,
since according to
some estimations, the binding of wen just one TN'F molecule to a cell suffices
for the tribgering
of its death.
Spontaneous signaling, independent of iigand can result in extensive
derangement of the
process controlled by this receptor. The best lmow~n example is the
deregulation of growth factor
receptors. h4utations due to which they start signaling spontaneously, for
example those that
cause them to aggregate spontaneously, play an important role in the
deregulated growth of
tumor cells. TNF effects, when induced in ehcess, are well known to contribute
to the patholobry
of many diseases. The ability of free intracellular domains (p55ICs) of the
p$5-TNF-R to signal
independently of TrrF may contribute to such excessive function. It seems
possible, for example,
that some of the cytopathic effects of viruses and other pathogens result, not
from their direct
cytoeidal function, buE from protcolytic detachment of the intracellular
domain of the p55-TIVF-R
and the resulting TNF-like cytotoxic effect.
To further elucidate the re5rion(s) within p55IC which is responsible for its
self association
capability and hence its ligand-independent cell cytotoxicin~, and also to
determine whether other
related members of the TNF/NGF receptor family (e.g. FAS-R) also have
invacellular domains
with self association capabilities and ligand-independent effects, the
following detailed study. was
performed : '
. . ;
CA 02490080 1995-05-11
WO 95/31544 PCT/US95/U5854
SG
~1 General Procedures and A4aterials
(i) Two hybrid screen and two-hybrid d-~alactosidase expression test
cDNA inserts, encoainL the p~5-IC and its deletion mutants, the Fas-IC and
various other proteins (see Table 3), were cloned by PCR, either from the full-
length eDNAs
cloned previously in our laboratory, or from purchased cDNA libraries. ~i-
galactosidase
expression in yeasts (SFY526 reporter strain (Barrel et al., 1993))
transformed with these cDNAs
in the pGBT-9 and pGAD-GH vectors (DNA binding domain (DBD) and activation
domain (AD)
constructs, respectively) was assessed by a liquid test (Guarente, 1983); it
was also assessed by a
fiber assay, yielding qualitatively the same results (not shown). Two-hybrid
screening (Fields and
Song, I9s9) of a purchased Gal4 AD-tagged HeLa cell cDNA library (Clontech,
Palo Alto, Ca.,
U.S.A..) for proteins that bind to the intracellular domain of the pS5-R. (p55-
IC), was performed
using the HF7c yeast reporter strain according to the recommendation of the
p:oducer. Positivity
of the isolated clones was assessed by (a) prototrophy of the transformed
yeasts for histidine
when gown in the presence of 5 rn_'~~ 3-aminotriazole, (b) ~-galactosidase
expression (c)
specificity tests (interaction with SI\'F4 and lamin fused to Gal4 DBD).
(ii) In vitro self association of bacterialfv oroduced~55-IC fusionnrateins
Glutathione S-transierase (GST) and glutathione S-transferase-p55-IC fusion
protein (GST-p55-IC) were produced as described elsewhere (Frangiani and Neel.
19Q3;
Ausubel et al., 1994). Maltose binding protein (lt,~LBP) fusion proteins were
obtained using the
pMalcRI vector (New England Biolabs) and purified on an amylose resin column.
The interaction
of the MBPP and GS? fusion proteins was investigated by incubating glutathione-
agarose beads
sequentially with the GST and N!$PP fusion proteins (5 pg protein I 20 ~tl
beads; first incubation
for 15 min, and the second for 2h, both at 4°C). Incubation with MBP
fusion proteins was carried
out in a buffer solution containing 20 mM Tris-HCI, pH 7.5, 100 mM KCI, 2 mM
CaCl2, 2 mM
MgCl2, 5 mM dithiotreitol, 0.2% Triton X100, 0.5 rnM phenyl-methyl-sulphonyl-
fluoride and 5%
(v/v) glycerol or, when indicated, in that same buffer containing 0.4 M KCI,
or 5 mM F-DTA
instead of MgCl2. .Association of the MBP fusion proteins was assessed by SDS
polyacrylamide
gel electrophoresis (10% acrylamide) of the proteins associated with the
glutathione-agarose
beads, followed by Western blotting. The blots were probed with rabbit
antiserum against MBP
(produced in our laboratory) and with horseradish-peroxidase-linked goat-anti-
rabbit
immunoglobulin.
(iii) induced expression in kleLa cells of the",~35-R and fragments thereof
HeLa cells expressing the. tetracyciinc-controlled transactri~ator developed
by
Gossen and Bujard (the HtTA-1 clone (Gossen and $ujard, 199?)), were grown in
Dulbecco's
modified Eagle's mediurtL containing 10% fetal calf serum, 100 u/mI
penicillin, _ 104 ~tc~/ml
streptomycin and 0.5 mg/ml neomycin. cDN A inserts encoding the p55-R or pans
thereof were
introduced into a tetracycline-controlled expression vector (pL~HD 10-3,
kindly provided by H.
Bujard). The cells wore transfected with the expression construct (5 ug DNAIG
cm plate) by the
calcium phosphate precipitation method (Ausubel et al., 1994). Effects of
transient expression of
the transfected proteins were assessed at the indicated times after
transfcction in the presence or
CA 02490080 1995-05-11
WO 95131544 pCTJUS95105854
sl
absence of tetracycline (1 uglml). Clones of cells stably transfected with the
human p55-IC cDlv'A.
in the pUl-iD 1 Q-3 vector were established by transfecting the cDNA to HtTA-1
cells in the
presence of tetracycline together W th a plasmid conferring resistance to
hygromycin, followed by
selcaed for clones resistant hygromycin (200 p~rJml). Expression of the eDNA
was obtained by
~ removal of tetracycline which was otherwise maintained constantly in the
cell growth medium.
(iv) Assessment of TNF-like effects tr~~~ered b~nduccd expression of the o5~-R
~d fra~,ments thereof
Effects of induced expression of the receptor and of TNF on cell viability
~werc
assessed by the neutral-red uptake method (Wallach, 1984). Induction of IL-8
gene expression
was assessed by Northern analysis. RNA was isolated using TRl REAGENT
(Molecular Research
Center, Inc.), denatured in formaldehyde/formamide buffer, electrophorcsed
through an
agaroselformaldehyde gel and blotted to a GeneScreen Plus membrane (Du Pont)
in IOxSSPE
buffer, using standard techniques. Filters were hybridized with an IL-8 cDNA
probe (Matsushima
et al., 1988), nucleotides 1-392). radiolabeled by the random-prime L-it
(Boehringer Mannheim
Biochemica, Mannheim, Germany) and washed stringently according to the
protocol of
manufacturer. Autoradiography wzs performed for 1-2 days.
(v) Assessment of TNF rece~to; ex~res~ion
TNF receptor expression in samples of 1x106 cells was assessed by measuring
the
binding of TNF, labeled with IZ$I by the chloramine-T method, as previously
described
30 (Holtmann and Vfallach, 1987). It was also assessed by ELISA,, performed as
described for the
quantification of the soluble TNF receptors (Aderka et al., 1991), except for
the use of RIPA
buffer (10 mM Tris-HCI, pH 7.5, 150 mM NaCI. 1% NP-40, 1% deoxycholate,
0.1°lo SDS and 1
mM EDTA) to lyse the cells (70 uUlOb cells) and to dilute the tested samples.
The soluble form
of the p55-It, purified from urine, screed as the standard.
b M tational an 1 sis of t tra el ula d main o a 5 - ~- to d termine t a
regions of the p55-IC involved in itc self accoci~,i~
As noted above, p55-IC can self associate and trigger cytotoxic effects on
cells, and there
are portions of the p55-IC, which themselves were capable of binding to the
full-length p55-IC. In
particular, one of the portions of the p55-IC (designated as protein fragment
55.1 in Example 1
above) was identified that was capable of binding strongly to the full length
p55-IC, this portion
was sequenced and was observed to contain the amino acid residues 328-426 of
the p55-TNF-R
which are within the p55-IC. It has further been discovered (see below) that
the above portion,
protein fragment 5~.1, is itself capable of self association and of triggering
cytoto~ac effects on
cells. Hence this portion of the p55-IC has been called the 'death domain',
and is located in the
rer.~'on beEween amino acid residues 328-426 of the human p55-R, most likely
consisting of amino
acid residues between about residue 328 and 414 thereof.
The fact that the 'death domain' in the p55-IC self associates was found by
happenstance.
On screening a HeLa cell cDNA library by the two-hybrid technique (see Example
1 above) for
proteins that bind to the intracellular domain of this receptor, we detected
among the cDNAs
whose products bound specifically to the intracellular domain-G.~~L,4 DBD
fusion-protein, several
CA 02490080 1995-05-11
WO 95/31544 PCT/US95I05854
52
clones (e.g. 55.1 and 55.3) that themselves encoded for parts of the p55-R
intracellular domain
(p55-IC; marked with asterisks in Table 3).
Applying the two-hybrid test to evaluate the ea-tent of specificity in the
self association of
p55-IC and to define more accurately the region involved Ied to the following
findings (Table 3) : .
S (a) The self association of p55-IC is coned to a region within the 'death
domain'. Its N terminus
is located between residues 328 and 344 and its C terminus, close to residue
404, somewhat
upstream of the reported G terminus of this domain (residue 414). (b) Deletion
of the membrane-
proximal part of p55-IC upstream of the 'death domain' enhanced self
association, suggesting that
this region has an inhibitor effect on the association. (c) Mouse p55-1C self
associates, and also
i0 associates with the 'death domain' of human p55-R. (d) examination of the
self association of the
intracellular domains of three other receptors of the TNF/NGF receptor family
: Fas/APO1 (FAS-
R), CD40 (Fields and Song, 1989) and the p75 T~'F receptor (Smith et al..
1990), showed that
Fas-IC, which signals for cell death by a sequence motif related to the p55-R
'death domain', self
associates, and associates to some extent with the p55-IC. However, CD40-IC,
that provides
IS gowth stimulatory signals (even though also containins a sequence
resembling the 'death
domain'), and p75-IC, that bears no structural resemblance to p55-IC, do not
self associate, nor
do they bind p55-IC or Fas-IG.
CA 02490080 1995-05-11
WO 95/31544 PCTIUS95105854
53
TABLE 3. Self association of the intracellular domains of p55-R and Fas/AP01
within
transformed yeasts : assessment by a two-hybrid ~i-galactosidase expression
test.
U
C I ( I o 0 0 o a o I
C~
I I I I I I I I N I i
cn
..
.U
O =_~ I I I I l ! ! I I i
=~
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'
'
= 1 I I I I I I i ( I I
_ _
O y
I I I I I I ~ o o I o 0
.
c:.
~U
.
I I I i I N ~ a I I I
O
N
N
f'f
I I
L. e-W
< N ~ I 1 1 I I I I I I I I
t,,
h i I I I 1 I I I I I I
.
' ~'
I I I I i I ( ( I 1 I
xt c
...;..~~ r~
L
O G ~ C O O
O
P7 , . , . .
Q
00 V M O O C1 ~.
V
n n; p
N
N
'et .
tV O N O O V ...0 I O .'
O V1 1~7
N
N v
Q
U
U U U
'
'~ - ..
~ U
Z
'~
O In H ~
. G L G
.
V V V IA ': ~ Q G1
V _
< N N N 'C N ::~ V tJ ~ C
"~ "", ' .... G""
r ef Q ~ M M
. t = H H U P.
r_ t 1 1 1 " ..
~ t
V1
r o N ~ o o ~"'~~ ~ z ~ v
o
~ ~ - N - W ~ .
~ N .i
r N f Ih .. .. .-.~. ..
. v S
9Zb ;
=
a ,
s~ o
c~ . . .
,
tc~ D ;
g
a _,
e
,
CA 02490080 1995-05-11
WO 95131544 pCTNS95105854
>4
Table s abo~~e shoms the quantitati~~e assessment of the interaction of Gal4
hybrid
constructs encompassing the follow n~- proteins : the intracellular domain of
human p5>-R and its
various deletion mutants (residues numbered as in (Loetscher et al., 1990));
the intracellular
domains of mouse p55-R (residues 334-454, numbered as in (Goodv~~in et al.,
1991)); mouse
S FasIAP01 f,Fas-IC, 166-306, numbered as in (Watanabe-Fukanaga et al.,
1992)); human CD40
(CD40-IC, 216-277, numbered as in (Stamenkovic et al., 1989)): and human p7S
TI~TF receptor
(p?5-IC, 287-461, numbered as in (Smith et aL, 1990)). SNF1 and S'NF4 were
used as positive
controls for association (Fields and Song, 1989), and lamin as a negative
control (Barrel et al.,
1993). Proteins encoded by the Gal4 DBD constructs (pGPT9) are listed
vertically; those
encoded by thr Gal4 AD constructs (pG.AD-GH), horizontally. The two deletion
mutants denoted
by asterisks were cloned in a two-hybrid screen of a HeLa cell cDNA library
(Clontech, Pato
Alto, Ca., U.S.A.) using p55-IC cloned in pGBT9 as "bait". In that screen,
four of about 4Yt 06
cDNA clones examined were positive. Three of these clones were found to
correspond to parts of
human ps5-R cDNA (two were identical, encoding residues 328-426 and one
encoding residues
277-426). The fourth was found to encode an unknown protein. The (3-
galactosidase expression
data are averages of assays of tv~~o independent transformants and are
presented as amount of (3-
galactosidase product; (a unit of activity being defined as OD42p times 103
divided by OD6pp of
the yeast culture and reaction time, in minutes). The detection limit of the
assay was 0.05 units.
Variation between duplicate samples were in all cases Icss that 25% of the
average (not tested}.
An in vitro test of the interaction of a p55-IC-glutathionc-S-transferase
(GST) bacterial
fusion protein with a p55-IC-maltose binding protein (IvlBP) fusion protein
confirmed that p55-R
self associates and ruled out involvement of yeast proteins in this
association (see above}. The
association was not affected by increased salt concentration, nor by EDTA (see
above).
To evaluate the functional implications of the self association of the death
domain, we
examined the way in which induced expression of p55-R, or of parts of it,
affect cells sensitive to
TNF cytotoxicity. The results of this analysis art set forth in Fig. 7 which
depicts the li~and
independent tribgering of a cy~tocidal effect in HeLa cells transfected with
p55-R, its intracellular
domain (p55-IC) or parts thereof (including the 'death domain').
In Fig. 7 there is shown schematically, the various DNA molecules encoding the
different
types of TNF receptors included in the vectors with which the HeLa cells were
transfected
(extreme left hand side of Fig. 7); and the expression (left and middle bax
graphs) and the viability
(right bar graph) in HeLa cells expressinfi transiently the various full-
length g55-R (p55-R), p55
IC or parts of p55-IC or, as a control, luciferase (LUC) (each being depicted
at the extreme left
side of Fig. 7), using a tetracycline-controlled expression vector. The open
bar graphs (left,
middle and right) represent cells transfected in the presence of tetracycline
(1 pg~ml), which
inhibits expression; and the filled bar graphs (left, middle and right)
represent Celts transfected in
the absence of tetracycline. Tl~t'F receptor expression was assessed 20h after
transfection, both by
EL1SA, using antibodies against the receptor's e~,-tracellular domain {see
schematic illustration on
the left side of Fig. 7), and by determining the binding of radiolabeled TNF
to the cells (middle).
The cytocidal effect of the transfected proteins was assessed 4Sh after
transfection. Daia shown
WO 95!31544 CA 02490080 1995-05-11 pCT~595/05854
$$
are from one of three experiments v~th qualitatively similar results, in which
each construct was
tested in duplicate. ND - not determined.
Thus, from Fig. 7 it is apparent that by using an expression vector that
permits strictly
controlled expression of transfected cDN As bS- a tetracycline regulated
transactivator (Gossen and
Bujard, 1992), a mere increase of p~5-R expression in HeLa cells by expression
of transiently
transfected cDNA for the full-lengnh receptor resulted in quite e.~ctensive
cell death. An even
greater cytotoxicity was observed when expressing just ps5-IC. Significant
cytotoxicity was also
observ,~ed when expressing just a part of p5$-~C comprising essentially the
'death domain' (residues
328-42b) in the HeLa calls. On the other hand, expression of parts of pSS-IC
that lacked the
'death domain' or contained just part of it (or expression of the luciferase
gene, used as an
irrelevant control) had no effect on cell viability. The cytotoxicity of p~3-
IC was further
confirmed using cells stably transformed with its cDNA; these cells continued
to grow when p5$-
1C expression was not induced, but died when p55-IC was e.~cpressed (see
above).
(~,~ Other eff'~, of thg intraceliul r domain of the p55-TNF-R
To examine whether other activities of TNF are triggered by the self
association of the
intracellular domain, including the 'death domain' thereof, we examined tb;e
affect of increased
e~,-pression of the full-length receptor (p55-R) and of the expression of the
intracellular domain of
the receptor (p55-IC), on the transcription of irncrlculdn 8 (IL-8), known to
be activated by TNF
(Matsushima et al., 1988). The results are show in Fig. 8, which depicts the
ligand-independent
induction of iL-8 gene expression in HeLa cells transfected with p55-R or p55-
IC, using a
teuacycline-controlled construct (see also 'General Procedures and Materials'
and Example 1
above). In panel A of Fig. 8 there is shown a reproduction of a Northern blot
representing the
Northern blot analysis (see 'General Procedures and Materials' above) of RNA
{7 ~g/lane)
extracted from HeLa (HTta-1) cells, untreated ('control) or treated ('TNF')
with TNF (500 l.ilml
for 4h), or the HTta-1 cells 24h after transfection (in the presence or
absence of tetracycline) with
p55-IC ('p55-IC'), the p55-R ('p55-R~, or luciferase ('Luc') cDNA. In panel B
of Fig. 8 there is
shown a reproduction of a Northern blot representing the mcthylcne blue
staining of 18S rRNA in
each of the samples shown in panel A of Fig. 8.
Thus, as is apparent from Fig. 8, transfection of HeLa ctlls with a
tetracycline-controlled
construct encoding the p5~-R cDNA induced IL-8 transcription. An even stronger
induction was
observed in cells transfected with the cDNA for p55-IC. In both cases, the
induction occurred
only when tetracycline was excluded from the cell growth medium, indicating
that it occurs as a
consequence of expression of the transfected p5.5-R or p55-IC. Transfection
with luciferase
eDNA, as a control, had no effect on Zh-8 transcription.
3 5 Accordingly, from the above results (Fig. 8), it appears that a mere
increase in p55-R
expression, or even expression of just the inuacellular domain (p55-IC)
thereof is sufficient to
trigger, in a ligand (TNF)-independent fashion, cytotoxicity and other effects
as well, including
that of an increase in the ea-pression of the IL-8 gene w7thin.cells. The
triggering of these effects is
most likely due to the self association of the intracellular domain of the p55-
R (p55-IC). As is set
an f~nh a~~ve. it annear~ that. lln~r celf as~nciation of the n5~-1C. the
'death domain' thereof is
WO 95/31544 CA 02490080 1995-05-11
PCT/US95/05854
5G
primarily responsible for signaling the induction of the intracellular
processes leading to the
triggering of cytotvxicity within the cells, u~hilst the other effects, e.g
the sitnalinw leading to the
induction of 1L-8 gene expression, are likely due to other regions of the p55-
IC as well, following
the self association thereof. It is therefore possible that different regions
of the p55-IC are
responsible for the different T1'F-induced effects (e.g. cytotoxicity, IL-8
induction) within cells,
these effects being a consequence of the intracellular si~~naling upon self
association of the p.SS-
IC.
T'he fact that the pS5-IC, can induce in a ligand (TNF)-independent fashion,
the triggering
of other intracellular effects e.g. IL-8 induction, means that the p55-IC or
specific portions
thereof may be used as a hie;hly specific tool for bringing about such
effrects in cells or tissues that
it is desired to treat, without the need for treating such cells or tissues
with T=VF. In many
pathological conditions (e.g. malignancies), treatment with TNF, especially at
high dosages can
lead to undesirable side-effects due to the number of intracellular effects
induced systemically by
ThtF following its binding to its receptors. By way of the discovery in
a:.cordance with the present
invention that the p55-IC can mimic specific other TNF-induced effects
(besides cytotoxicity),
e.g. II,-8 induction, opens the wa}~ for introducing in a cell- or tissue-
specific manner, p5s-IC or
specific portions thereof, which will be capable of signaling for the
induction of specific desired
intracellular effects, e.g. IL-8 induction, and thereby overcome the systemic
side-effects often
observed during TNF treatment.
~~gand-indsnendent triggering, of cytocidal eff~ct~ in H~La cells bY the
intracellular
domains and. thc'death domains' thereof of p5S TIfF R and FAS-R r[Fas/A~O~,~
As regards the cytotoxic activity of the intracellular domains of the p55 TNF-
R and FAS-
R (p55IC and FAS-IC) it has now also been further elucidated that both the
p551C, its 'death
domain' (p55DA) and the FAS-IC are capable of a Iigand-independent triggering
of a cy~tocidal
effect in HeLa cells. In this study, HeLa cells were transfected with
expression vectors containing
various constructs of either the full-length p55-TIv'F-R, portions thereof
including the p55IC and
p55DD or the FAS-IC. In one set of experiments HeLa cells were co-transfected
with constructs
containing the p55 TNF-R (p55-R) and the FAS-IC (for details of the
constructs, their
preparation, etc. see above). The results of this study arc depicted in Fig. 9
( A and B j, wherein in
both Fig. 9A and B the constructs used for transfecting the HeLa cells are
shown schematically in
the left hand panels; the results of the TNF or FAS receptor expression are.
shown graphically in
the two middle panels (second and third panels from the left); and the results
of transfected cell
viability are shown gaphicallv in the right hand panels. In Fig. 9A there is
shown the results of
transfected HeLa cells transiently expressinb the full-length p55-R, p55-IC or
pans thereof, or as
3~ a control, lucifcrase (LUC), in all cases using a tetracycline-controlled
expression vector. In Fig.
9B there is shown the results of transfected HeLa cells transiently expressing
FAS-1C alone or
together with the p55-R, using a tetracycline-controlled expression vector. In
thegraphic
representation of the results in Fig. 9A and B, the open bars represent cells
transfected in the
presence of tetracycline (1 itg/ml), which inhibits expression, and the closed
bars represent cells
transfected in the absence of tetracycline. TNF receptor expression was
assessed 20h after
CA 02490080 1995-05-11
WO 95/31544 PCTNS95/05854
Si
transfection, both by ELISA usinL antibodies against the ea-tracellular domain
of the receptor {see
left hand ~anelsj, and by determining the binding of radiolabeled TNF to the
cells (middle panels).
The cytocidal effect of the transfected proteins was assessed 48h after
transfection. The data
shown are from one of three experiments with qualitatively similar results in
which each construct
was tested in duplicate The designation ND' in Figs. 9A and B means not
determined. From the
results shown in Figs. 9A and B it is apparent that expression of only the
p»IC results in even
greater cynotoxicity. Sisazificant cwotoxicity also occurs when expressing
just the death domain
(pSSDD), In contrast, expression of parts of p55IC lacking the death domun or
containing only
part thereof, had no effect on cell viability. Expression of the FAS-IC did
not result in significant
cytotoxicity, yet it significantly enhanced the cyotoacit5~ of co-expressed
pS5-R.
~ 'AMPL ~,3
Additional proteins capable of binding to the intracellular domains of p~5
~'1~F-R or FAS-
R
I S Using the same approach and technology set forth in Example 1 above;
'three more
proteins have been isolated and identified which arc capable of binding to the
pSSIC or FAS-IC.
In Fins. 10-12 there is shown schematically the partial and preliminary
'nucleotide
sequence of cDNA clones, called F2, F9 and DD 11, respectively.
Clones F2 and F9 were isolated by screening a marine (mouse) embryonic library
using the
marine FAS-IC as "bait". In Fig. 10 there is shown schematically the partial
nucleotide sequence
from iha F2 cDNA that has been sequenced. 1n Fig. 11 there is shown
schematically the partial
nucleotide sequence of 1724 bases from the F9 cDNA that has been sequenced.
Analysis of the
binding capability of the protein encoded by clones F2 and F9 (F2 and F9,
respectively) has
shown that
2S {a) F2 interacts strongly with human pSSIC and pSSDD and with marine FAS-
IC,
while it interacts weakly with non-rele~-ant {control) proteins SNF1 and Lamin
as well as relevant
protein, human FAS-IC.
{b) F9 interacts strongly with human p5S-IC and marine FAS-IC, while it
interacts
weakly with human FAS-IC (relevant protein) and irrelevant proteins SNF 1 and
Lamin.
(c) Neither F2 nor F9 interacted at all with human p75IC, pGBT9 (empty bait
v ector), or human CD-40.
Further, from 'Gene Bank' and Protein Bank' searches it was revealed that F2
and
F9 represent new proteins.
Thus, F2 and F9 represent new proteins having binding specificity for both FAS-
IC
and pSSIC.
Clone DD11 was isolated by screening a human HeLa library using the human
p55D17 as
"bait". In Fig. 12 there is shown schematically the partial nucleotide
sequence of 42S bases from
the DD11 cDNA that has been sequenced.
The DD11 clone has an approx. length of 800 nucleotides. The full length of
the transcript
.1 f1 ie 41~ ny~ 1 7 t~A rant ant 1,?t~i~~ ~P n nfnl~Pr~ yqine t~A In ..
~,,~ol..esc of the ri:..r
.. , . ~:~.'. t. C. . . . : C. . . . ~ . C . r_ _ .. _ r1!i _. -
CA 02490080 1995-05-11
WO 95!31544 PCT/US95/05854
58
capability of the protein encoded by clone DD 11 has shown that DD11 interacts
strongly with the
pSSDD (a.a. 326-414) (see Fig. 9) and does not interact with deletion mutants
of this domain, e.b.
a.a. 326-40.t. DD 11 also interacts with mouse and human FrIS-IC and to some
extent also with
):.amin. DD 11 does not interact at all with SN'F1 nor with pGBT9 {empty bait
vector). DD 11 is
also not found in the 'Gene Bank' and Protein Bank' databases. Thus DD11
represents a pS5 l;C
(pSSDD) and FAS-IC specific binding protein.
x 1 4
construction of soluble dimeric TNF receptors
~ 0 Based on the findings set forth in Example 2 above, that the intracellular
domain of the
p55-R (p55-IC) and a portion thereof (che'death domain'); and that the
intracellular domain of the
FaslAP01 and a portion thereof (also called the 'death domain') which
resembles the p55-IC
'death domain', are capable of self association, it is possible to construct
new TNF receptors
which are capable of self association (aggregation) and which are soluble.
Such TNF receptors
1 S will be fusion proteins having essentially all of the ex-cracellular
domain of the pSS-R fused to
essentially all of the intracellular domains or 'death domains' thereof of the
p~~-R or Fas/APO1.
Thus, such fusion cc:nstructs n~iil be devoid of the transmembranal domain of
the p55-R (or
FASIAPO1) and hence wilt be soluble. Moreover, by virtue of the self
association capability of
the intracellular domains or 'death domains' thereof these fusion constructs
will be capable of
20 oligomerization to provide at least dimers (and possibly also higher order
multimers) of the p55-
R Consequently, such dimeric TIv'F receptors (p55-R) will be capable of
binding to at least two
TNF monomers of the naturally-occurring Tr'F homotrirner to provide a soluble
TNF receptor
which binds more avidly to its ligand (homotrimeric TNF).
Accordingly, at least four types of p55 TNF receptor fusion proteins will be
constructed
25 each of which will be capable of oligomcrization and will be soluble
(i) A fusion product between the extracellular domain of pSS-R (EC55) and the
intracellular domain of p55-R (p~5-1C);
(ii) A fusion product between the EC55 and the 'death domain' of p55-IC
(DD55);
(iii) A fusion product between the ECSS and the intracellular domain of
Fas/AP01
30 (ICFAS); and
(iv) A fusion product between the EC55 and the 'death domain' of ICFAS
(DDFAS).
In each of the above fusion proteins the T1VF monomer binding capability is
provided by
the EC» portion while the oligomerization (or at least dimerization) of each
kind of fusion
3~ protein is provided by its'taif rer~~on being any of the p55IC, DDSS, ICFAS
or DDFAS portions.
For construction of the above fusion proteins, standard techniques of
recombinant DN.A
technology will be employed that are now well established in the art {see for
example Sambrook
et al., (1989) Molecular Cloning : A Laboratory h4anual, Cold Sprint Harbor
Laboratory Press,
Cold Spring Harbor, N.Y.). Briefly, any suitable bacterial, bacteriophage, or
animal virus
40 expression vector (cloning vehicle or plasmid designed for expression of
the inserted DNA of
VVO 95!31544 CA 02490080 1995-05-11
PCTlUS95105854
$o
choice) may' be employed into which will be inserted in one or more stages the
DNA encoding the
ECSS and one of the 'tails' being the p55-1C, DD55, ICFAS or DDFAS. The so-
inserted DNA
encoding each of the fusion proteins will be placed under the control of the
carious expression
control sequences of the clotting vehicle or plasmid such as promoters,
ribozyme binding sites,
transcriptions) factor binding sites, etc. These expression control sequences
will be chosen
depending on the type of expression vector chosen and hence the type of host
cell {eukaryotic or
prokaryoric) in which it is desired to express the fusion proteins of the
invention. Preferred host
cells (and hence expression vectors) are eukaryotic, in particular, mammalian.
The DNA molecule encodins each of the above noted fusion proteins will be
prepared and
inserted into the expression vector by the followinb procedure
(a) Firstly, a set of oligonucleotides for use in PCR will be constructed by
standard
means, the oligonucIeotides being
1) ACC ATG GGC CTC TCC ACC GTG (ECSS, sense)
2) ACGC GTC G AC TGT GGT GCC TGA GTC CTC (ECS S, antisense)
3) ACGC GTC G:~C CGC TAC C.4A CGG TGG AAG (ICSS, sense)
4) TCA TCT GAG A.4G.ACT GGG (ICSS, antisense
5) ACGC GTC G.~C AAG AGA AAG GAA GTA CAG (IC FAS, sense)
6) CTA GAC C 4~. GCT TTG GAT (IC FAS, antisense)
7) ACGC GTC GAC CCC GCG ACG CTG TAC GCC tDD3S, sense)
8) ACGC GTC GAC GAT GTT GAC TTG AGT AAA (DD FAS, sense)
(b) Plasmids containing the cloned full-length pS5-R and FasJAPOI receptors
which we
have in our laboratory (see also co-pending EPSG8925 and Examples 1-3 above)
will be subjected
to the following manipulations to yield the DIVA fragments encoding each of
the fusion proteins,
which DNA fragments are then ligated into the above noted expression vector of
choice
(i) To produce the ANA fragment coding for EC55 which is a component of all 4
fusion proteins, PCR is performed on a plasmid bearing cDNA of human p55 using
the about
oligonucleotide nos. 1 and 2 (size of fragmtent 640 bp).
(ii) To get a fusion product ECSS-ICSS, PCR is performed on a plasmid bearinb
cDNA for human pSS using oligonucleotide nos. 3 and 4, to obtain a DNA
fragmont coding for
IC55 (size 677 bp} which is then mixed with EC55 digested by Sal i and ligated
by blunt end
ligation into any expression vector for mammalian cells under the control of
an appropriate
promoter. The orientation of the inserted EC55--ICSS in the vector is verified
by restriction
digestion and by sequenang.
{iii) To get a fusion product EC55-IC FAS, IC FAS is produced by PCR an a
3 S plasmid with cDNA for FAS using olieonucleotide nos. S and 6, to obtain a
fracmtent (size 448
bp) which is then cut by Sal I and mixed with EC55 cut by SaII, and
subseducntly is blunt ligated
into a manunalian expression vector under the control of an appropriate
promoter. The
orientation of the inserted ECSS--IC FAS in the vector is verified by
restriction digestion and by
sequencing. ;
CA 02490080 1995-05-11
WO 95/31544 PCT/US95/05854
(iv) To get a fusion product EC55--DDS, a DNA fragment is produced 7th the
DD55 sequence by PGR in cDNA for human p5~ using oligonucleotide nos. 7 and 4.
The product
with a size of 314 by is cut by SaII and mixed with ECSS cut by SaII, and
subsequentl~~ blunt
ligated into the mammalian expression vector. Orientation of the inserted EC55-
-DD55 in the
5 vector is vecificd by restriction digestion and by sequencing.
(v) To get a fusion product ECSS--DD FAS, a DNA fragnent with DD FAS is
produced by PCR on cDNA for FAS usinz oligonucleotide nos. 6 and 8. The
product with a size
of 332 by is cut with SaII, and mixed with EC55 cut by Sal I and subsequently
blunt ligated into
the mammalian expression vector. Orientation of the EC55--DD FAS is then
verified by
10 restriction digestion and sequencing
Once the above expression vectors have been constructed, they will then be
introduced by
standard methods into suitable mammalian cells (e.g. Chinese Hamster Ovar;~
(CHO) or TTonkey
Kidney (COS) cells) for the purposes of expression. The so-expressed fusion
proteins will then be
purified by standard methods (see co-pending EP308378; EP398327; and
EP568925). The
15 purified fusion proteins v~711 then be analyzed for their ability to
oligomerize (and the extent
thereof, i.e. whether they form dimers or higher order multimers) and for
their ability to bind TNF
(aad the affinity or avidiy of binding thereof).
za le 5
20 Constr ction of soluble dimeric FacIAP01 receptors
In a similar fashion to that set forth in Example 4 above, it is possible to
produce the
following four kinds of Fas/AP01 fusion products, each of which will be
capable of
oligomerization and will be soluble
(i) Fusion product betvvecn the extracellular domain of Fas/AP01 (EC FAS) and
25 the intracellular domain of p55-rC;
{ii) Fusion product between the fiC FAS and the 'death domain' of p55-IC
(DD55);
(iii) Fusion product bctv~~een the EC FAS and the intracellular domain of
FasIAP01 (IC FAS); and
30 (iv) Fusion product between the EC FAS and the 'death domain' of IC FAS (DD
FAS).
In each of the above fusion proteins the FAS ligand binding capability is
provided by the
EC FAS portion, while the oligomerization (or at least dimerization) of each
kind of fusion
protein is provided by its 'tail' repon being any of the p5~-IC. DD55, IC FAS
or DD FAS
35 portions.
The construction of the DNA fragments encoding the above fusion proteins and
expression vectors containing them v~~ill be as detailed in Example 4, except
different appropriate
olibonucleotides {not shown) will be used for the preparation of the EC FAS
fragment to be
ligatcd to any of the above noted 'tail' regions. Subsequently, the expression
vectors will be
introduced into the suitable host cells. and the resulting expressed fusion
proteins W II be purified
CA 02490080 1995-05-11
WO 95/31544 pCT/US95/05854
61
and tested for their ability to oligomerize (and the extent thereof, i.e,
whether they form dimers or
higher order multimers) and for their ability to bind the FAS ligand (and the
affinit}~ or avidity of
binding thereofj.
S Example G
Construction of soluble oligomeric'mixed' TNP'fE~S._xeoento~s
To prepare oligomcric receptors having 'mixed' aflanity, i.e. affinity for
both ThIF and the
FAS-R ligand, the above-mentioned (Examples 4 and 5) fusion products may be
utilized in the
followins procedure
i) Providing a fusion product as set forth in Example 4, which contains the
e~,-tracellular domain of a TNF-R (p75 TNF-R or p55 TiVF-R) fused to any one
of : the p55 IC,
FAS-1C, p55 DD or FAS DD;
ii) Providing a fusion product as set forth in Example S, which contains the
extracellular domain of Fas-R fused to any one of : p53 IC, FAS-IC, p55 DD or
FAS-DD; and
iii) mixing any one of the fusion products of i) with anv one of the fusion
products of ii) to provide a new dimeric (or higher order oligotneric)
receptor which has both the
extracellular domains of a Tr'F-R and FAS-R that are joined by their -IC or -
DD regions.
In the above procedure the fusion products of i) and ii) may be provided
separately,
namely, from their purification from transformed cells in which they were
produced, and then
mixed in vitro to obtain the mixed affinity receptors. Alternatively, the host
cells may be co
transfected with vectors carrying sequences encoding both types of fusion
products, in which
case, the mixed affinity receptors may be obtained directly from the co-
transfected cells. The
actual oligomerization of the fusion products into oligomeric receptors may
take place within the
cells or during or following the purification procedure to obtain the fusion
products expressed in
the cells. To specifically select for the mixed affinity receptors any
standard method may be
utilized, for example, affinity chromatography procedures in which antibodies
against the TNF-R
and FAS-R extraceilular domains are used in sequential chromatographic steps
to scf~ct for thQSe
receptors having; both types of extracellular domain.
CA 02490080 1995-05-11
W 0 95/31544 6 2 PCT/US95105854
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