U.S. patent application number 11/685096 was filed with the patent office on 2008-07-03 for modulators of the functions of receptors of the tnf/ngf receptor family and other proteins.
This patent application is currently assigned to Yeda Research and Development Co., Ltd.. Invention is credited to Andrei Kovalenko, David Wallach.
Application Number | 20080159986 11/685096 |
Document ID | / |
Family ID | 11069938 |
Filed Date | 2008-07-03 |
United States Patent
Application |
20080159986 |
Kind Code |
A1 |
Wallach; David ; et
al. |
July 3, 2008 |
MODULATORS OF THE FUNCTIONS OF RECEPTORS OF THE TNF/NGF RECEPTOR
FAMILY AND OTHER PROTEINS
Abstract
Proteins which are capable of modulating/mediating the function
of RIP, their preparation and uses are provided.
Inventors: |
Wallach; David; (Rehovot,
IL) ; Kovalenko; Andrei; (Rehovot, IL) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Yeda Research and Development Co.,
Ltd.
Rehovot
IL
|
Family ID: |
11069938 |
Appl. No.: |
11/685096 |
Filed: |
March 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10245593 |
Sep 18, 2002 |
7189535 |
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11685096 |
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09927458 |
Aug 13, 2001 |
7083788 |
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10245593 |
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09381358 |
Sep 20, 1999 |
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PCT/IL98/00125 |
Mar 19, 1998 |
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09927458 |
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Current U.S.
Class: |
424/93.2 ;
435/456 |
Current CPC
Class: |
A61P 37/00 20180101;
A61P 31/10 20180101; A61P 35/00 20180101; A61P 31/00 20180101; A61P
31/14 20180101; A61K 38/00 20130101; A61P 43/00 20180101; C07K
14/4747 20130101 |
Class at
Publication: |
424/93.2 ;
435/456 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/86 20060101 C12N015/86 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 19, 1997 |
IL |
120485 |
Claims
1. A pharmaceutical composition for modulating the RIP effect on
cells, comprising, as active ingredient, a recombinant animal virus
vector encoding a protein capable of binding a cell surface
receptor and encoding the RAP protein encoded by a DNA sequence in
a clone deposited with Collection Nationale de Cultures de
Microorganismes under accession number I-2706.
2. A method of modulating processes that are mediated/modulated by
RIP directly or indirectly and which include the inhibition of
NF-.kappa.B and JNK, comprising treating said cells with one or
more polypeptides capable of binding to RIP, wherein said treating
of cells comprises introducing into cells a DNA sequence encoding
said one or more polypeptides in the form of a suitable vector
carrying said sequence, said vector being capable of effecting the
insertion of said sequence into said cells in a way that said
sequence is expressed in said cells, and said one or more
polypeptides being selected from the group consisting of: (a) a
RIP-associated protein (RAP) encoded by a DNA sequence in a clone
deposited with Collection Nationale de Cultures de Microorganismes
under accession number I-2706; (b) a fragment of (a) which is
capable of binding to RIP or inhibiting the NF-.kappa.B inducing
effect of RIP; and (c) an analog of (a) or (b) which differs from
the sequence of (a) or (b) by no more than 10 substitutions,
deletions and/or insertions of amino acid residues and is capable
of binding to RIP or inhibiting the NF-.kappa.B inducing effect of
RIP.
3. A method for the modulation or mediation of the RIP
modulated/mediated intracellular effects on the inflammation, cell
death or cell survival pathways in which RIP is involved directly,
or indirectly via other modulators/mediators of these pathways,
comprising treating cells with one or more polypeptides, wherein
said treating of cells comprises introducing into said cells a DNA
sequence encoding said polypeptide in the form of a suitable vector
carrying said sequence, said vector being capable of effecting the
insertion of said sequence into said cells in a way that said
sequence is expressed in said cells, and said one or more
polypeptides being selected from the group consisting of: (a) a
RIP-associated protein (RAP) encoded by a DNA sequence in a clone
deposited with Collection Nationale de Cultures de Microorganismes
under accession number I-2706; (b) a fragment of (a) which is
capable of binding to RIP or inhibiting the NF-.kappa.B inducing
effect of RIP; and (c) an analog of (a) or (b) which differs from
the sequence of (a) or (b) by no more than 10 substitutions,
deletions and/or insertions of amino acid residues and is capable
of binding to RIP or inhibiting the NF-.kappa.B inducing effect of
RIP.
4. A method according to claim 3, wherein said treating of said
cells is by transfection of said cells with a recombinant animal
virus vector comprising: (a) constructing a recombinant animal
virus vector carrying a sequence encoding a viral 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 said polypeptide, that when expressed in said
cells is capable of modulating/mediating the activity of RIP; and
(b) infecting said cells with said vector of (a).
5. A method for treating tumor cells or HIV-infected cells or other
diseased cells, comprising: (a) constructing a recombinant animal
virus vector carrying a sequence encoding a viral surface protein
capable of binding to a specific tumor cell surface receptor or
HIV-infected cell surface receptor or receptor carried by other
diseased cells and a sequence encoding a polypeptide, that when
expressed in said tumor, HIV-infected, or other diseased cell is
capable of enhancing the RIP modulated/mediated direct or indirect
killing of said cell, wherein said one or more polypeptides are
selected from the group consisting of: (a) a RIP-associated protein
(RAP) encoded by a DNA sequence in a clone deposited with
Collection Nationale de Cultures de Microorganismes under accession
number I-2706; (b) a fragment of (a) which is capable of binding to
RIP or inhibiting the NF-.kappa.B inducing effect of RIP; and (c)
an analog of (a) or (b) which differs from the sequence of (a) or
(b) by no more than 10 substitutions, deletions and/or insertions
of amino acid residues and is capable of binding to RIP or
inhibiting the NF-.kappa.B inducing effect of RIP.
Description
FIELD OF THE INVENTION
[0001] 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 TNF/NGF superfamily of receptors
includes receptors such as the p55 and p75 tumor necrosis factor
receptors (TNF-Rs, hereinafter called p55-R and p75-R) and the FAS
ligand receptor (also called FAS/APO1 or FAS-R and hereinafter will
be called FAS-R) and others. Specifically, the present invention
concerns novel proteins which bind to other proteins which
themselves bind directly or indirectly to members of the TNF/NGF
receptor family and other intracellular modulatory proteins, and
more specifically, it relates to one such protein, herein
designated RAP (for RIP-associated protein), which binds to RIP
(for `receptor interacting protein`), which, in turn, binds to
itself and to MORT-1, FAS-R, p55-R, TRADD and Traf2.
[0002] Accordingly, the present invention concerns, in general, new
proteins which are capable of modulating or mediating the function
of RIP and thereby also capable of modulating or mediating,
directly or indirectly, the function of other proteins which bind
to RIP directly or indirectly. In particular, the present invention
concerns RAP, its preparation and uses thereof, as well as any
isoforms, analogs, fragments and derivatives of RAP, their
preparation and uses.
DESCRIPTION OF THE RELATED ART
[0003] Tumor Necrosis Factor (TNF-.alpha.) and Lymphotoxin
(TNF-.beta.) (hereinafter, TNF, refers to both TNF-.alpha. and
TNF-.beta.) are multifunctional pro-inflammatory cytokines formed
mainly by mononuclear phagocytes, which have many effects on cells
(Wallach, D. (1986) In: Interferon 7 (Ion Gresser, ed.), pp.
83-122, Academic Press, London; and Beutler and Cerami (1987)).
Both TNF-.alpha. and TNF-.beta. 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 antibacterial
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.
[0004] However, both TNF-.alpha. and TNF-.beta. also have
deleterious effects. There is evidence that overproduction of
TNF-.alpha. can play a major pathogenic role in several diseases.
For example, effects of TNF-.alpha., primarily on the vasculature,
are known to be a major cause for symptoms of septic shock (Tracey
et al., 1986). In some diseases, TNF may cause excessive loss of
weight (cachexia) by suppressing activities of adipocytes and by
causing anorexia, and TNF-.alpha. was thus called cachetin. It was
also described as a mediator of the damage to tissues in rheumatic
diseases (Beutler and Cerami, 1987) and as a major mediator of the
damage observed in graft-versus-host reactions (Piquet et al.,
1987). In addition, TNF is known to be involved in the process of
inflammation and in many other diseases.
[0005] Two distinct, independently expressed, receptors, the p55
and p75 TNF-Rs, which bind both TNF-.alpha. and TNF-.beta.
specifically, initiate and/or mediate the above noted biological
effects of TNF. These two receptors 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., 1990; Schall et al., 1990; Nophar et al.,
1990; Smith et al., 1990; and Heller et al., 1990). However, the
cellular mechanisms, for example, the various proteins and possibly
other factors, which are involved in the intracellular signaling of
the p55 and p75 TNF-Rs have yet to be elucidated. It is this
intracellular signaling, which occurs usually after the binding of
the ligand, i.e., TNF (.alpha. or .beta.), 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.
[0006] 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 extracellular domain (ligand
binding domain) of the p55 TNF-R can themselves trigger the
cytocidal effect (see EP 412486) which correlates with the
effectivity of receptor cross-linking by the antibodies, believed
to be the first step in the generation of the intracellular
signaling process. Further, mutational studies (Brakebusch et al.,
1992; Tartaglia et al., 1993) have shown that the biological
function of the p55 TNF-R depends on the integrity 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 (.alpha.
and .beta.) occurs as a homotrimer, and as such, has been suggested
to induce intracellular signaling via the p55 TNF-R by way of its
ability to bind to and to cross-link the receptor molecules, i.e.,
cause receptor aggregation.
[0007] Another member of the TNF/NGF superfamily of receptors is
the FAS receptor (FAS-R) which has also been called the FAS
antigen, a cell-surface protein expressed in various tissues and
sharing homology with a number of cell-surface receptors including
TNF-R and NGF-R. The 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 FAS-R
gene (lpr) cause a lymphoproliferation disorder in mice that
resembles the human autoimmune disease systemic lupus erythematosus
(SLE) (Watanabe-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, monoclonal antibodies have been prepared that are
specific for FAS-R, these monoclonal antibodies being capable of
inducing apoptopic cell death in cells carrying FAS-R, including
mouse cells transformed by cDNA encoding human FAS-R (Itoh et al.,
1991).
[0008] A number of approaches have been made by the applicants (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 essentially the
soluble extracellular 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 applicants (see for
example EP 568925) have been made to modulate the TNF effect by
modulating the activity of the TNF-Rs.
[0009] Briefly, EP 568925 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 function of the TNF-Rs. In EP 568925, there is described
the construction and characterization of various mutant p55 TNF-Rs,
having mutations in the extracellular, transmembrane, 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 to the functioning of the receptor, i.e., the binding of
the ligand (TNF) and the subsequent signal transduction and
intracellular signaling which ultimately results in the observed
TNF-effect on the cells. Further, there is also described a number
of approaches to isolate and identify proteins, peptides or other
factors which are capable of binding to the various regions in the
above domains of the TNF-R, which proteins, peptides and other
factors may be involved in regulating or modulating the activity of
the TNF-R. A number of approaches for isolating and cloning the DNA
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 fragments
thereof which interact with the TNF-R or with the above proteins
and peptides that bind various regions of the TNF-R, are also set
forth in EP 568925. However, EP 568925 does not specify the actual
proteins and peptides which bind to the intracellular domains of
the TNF-Rs (e.g., p55 TNF-R), nor does it describe the yeast
two-hybrid approach to isolate and identify such proteins or
peptides which bind to the intracellular domains of TNF-Rs.
Similarly, in EP 568925 there is no disclosure of proteins or
peptides capable of binding the intracellular domain of FAS-R.
[0010] While it is known that the tumor necrosis factor (TNF)
receptors, and the structurally-related receptor FAS-R, trigger in
cells, upon stimulation by leukocyte-produced ligands, destructive
activities that lead to their own demise, the mechanisms of this
triggering are still little understood. Mutational studies indicate
that in FAS-R and the p55 TNF receptor (p55-R) signaling for
cytotoxicity involve distinct regions within their intracellular
domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and
Nagata, 1993). These regions (the `death domains`) have sequence
similarity. The `death domains` of both FAS-R and p55-R tend to
self-associate. Their self-association apparently promotes that
receptor aggregation which is necessary for initiation of signaling
(see Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995),
and at high levels of receptor expression can result in triggering
of ligand-independent signaling (Boldin et al., 1995).
[0011] Like other receptor-induced effects, cell death induction by
the TNF receptors and FAS-R occurs via a series of protein-protein
interactions, leading from ligand-receptor binding to the eventual
activation of enzymatic effector functions, which in the case
studies have elucidated non-enzymatic protein-protein interactions
that initiate signaling for cell death: binding of trimeric TNF or
the FAS-R ligand molecules to the receptors, the resulting
interactions of their intracellular domains (Brakebusch et al.,
1992; Tartaglia et al., 1993; Itoh and Nagata, 1993) augmented by a
propensity of the death-domain motifs to self-associate (Boldin et
al., 1995a), and induced binding of two cytoplasmic proteins (which
can also bind to each other) to the receptors' intracellular
domains--MORT-1 (or FADD) to FAS-R (Boldin et al., 1995b;
Chinnaiyan et al., 1995; Kischkel et al., 1995) and TRADD to p55-R
(Hsu et al., 1995; Hsu et al., 1996). Three proteins that bind to
the intracellular domain of FAS-R and p55-R at the `death domain`
region involved in cell-death induction by the receptors through
hetero-association of homologous regions and that independently are
also capable of triggering cell death were identified by the yeast
two-hybrid screening procedure. One of these is the protein, MORT-1
(Boldin et al. 1995b), also known as FADD (Chinnaiyan et al., 1995)
that binds specifically to FAS-R. The second one, TRADD (see also
Hsu et al., 1995, 1996), binds to p55-R, and the third, RIP (see
also Stanger et al., 1995), binds to both FAS-R and p55-R. Besides
their binding to FAS-R and p55-R, these proteins are also capable
of binding to each other, which provides for a functional
"cross-talk" between FAS-R and p55-R. These bindings occur through
a conserved sequence motif, the `death domain module` common to the
receptors and their associated proteins. Furthermore, although in
the yeast two-hybrid test MORT-1 was shown to bind spontaneously to
FAS-R, in mammalian cells, this binding takes place only after
stimulation of the receptor, suggesting that MORT-1 participates in
the initiating events of FAS-R signaling. MORT-1 does not contain
any sequence motif characteristic of enzymatic activity, and
therefore, its ability to trigger cell death seems not to involve
an intrinsic activity of MORT-1 itself, but rather, activation of
some other protein(s) that bind MORT-1 and act further downstream
in the signaling cascade. Cellular expression of MORT-1 mutants
lacking the N-terminal part of the molecule has been shown to block
cytotoxicity induction by FAS/APO1 (FAS-R) or p55-R (Hsu et al.,
1996; Chinnaiyan et al., 1996), indicating that this N-terminal
region transmits the signaling for the cytocidal effect of both
receptors through protein-protein interactions.
[0012] Thus, the `death domain` motifs of the receptors p55-R and
FAS-R as well as their three associated proteins MORT-1, RIP and
TRADD appear to be the sites of protein-protein interactions. The
three proteins MORT-1, RIP and TRADD interact with the p55-R and
FAS-R intracellular domains by the binding of their death domains
to those of the receptors, and for both RIP and TRADD their death
domains also self-associate, although MORT-1 differs in this
respect in that its death domain does not self-associate. Further,
MORT-1 and TRADD bind differentially to FAS-R and p55-R and also
bind to each other. Moreover, both MORT-1 and TRADD bind
effectively to RIP. Accordingly, it would seem that the interaction
between the three proteins MORT-1, RIP and TRADD is an important
part of the overall modulation of the intracellular signaling
mediated by these proteins. Interference of the interaction between
these three intracellular proteins will result in modulation of the
effects caused by this interaction. For example, inhibition of
TRADD binding to MORT-1 may modulate the FAS-R-p55 TNF-R
interaction. Likewise, inhibition of RIP in addition to the above
inhibition of TRADD binding to MORT-1 may further modulate
FAS-R-p55 TNF-R interaction.
[0013] Monoclonal antibodies raised against the `death domain` of
p55-R, specifically against the binding site of sites of TRADD and
RIP can also be used to inhibit or prevent binding of these
proteins and thus cause modulation of the interaction between FAS-R
and p55-R.
[0014] Moreover, it has also recently been found that besides the
above noted cell cytotoxicity activities and modulation thereof
mediated by the various receptors and their binding proteins
including FAS-R, p55-R, MORT-1, TRADD, RIP, MACH, Mch4, and G1, a
number of these receptors and their binding proteins are also
involved in the modulation of the activity of the nuclear
transcription factor NF-.kappa.B, which is a key mediator of cell
survival or viability, being responsible for the control of
expression of many immune- and inflammatory-response genes. For
example, it has been found that TNF-.alpha. can actually stimulate
activation of NF-.kappa.B and thus TNF-.alpha. is capable of
inducing two kinds of signal in cells, one eliciting cell death and
another that protects cells against death induction by inducing
gene expression via NF-.kappa.B (see Beg and Baltimore, 1996; Wang
et al., 1996; Van Antwerp et al., 1996). A similar dual effect for
FAS-R has also been reported (see reference to this effect as
stated in above Van Antwerp et al., 1996). It would therefore
appear that there exists a delicate balance between cell death and
cell survival upon stimulation of various types of cells with
TNF-.alpha. and/or the FAS-R ligand, the ultimate outcome of the
stimulation depending on which intracellular pathway is stimulated
to a greater extent, the one leading to cell death (usually by
apoptosis), or the one leading to cell survival via activation of
NF-.kappa.B.
[0015] In addition, the present inventors have also recently
further elucidated the possibly pathway by which members of the
TNF/NGF receptor family activate NF-.kappa.B (see Malinin et al.,
1997 and the various relevant references set forth therein; and
co-owned, co-pending Israel Patent Application Nos. IL 117800 and
IL 119133). Briefly, it arises that several members of the TNF/NGF
receptor family are capable of activating NF-.kappa.B through a
common adaptor protein, Traf2. A newly elucidated protein kinase
called NIK (see above Malinin et al., 1997 and IL 117800 and IL
119133) is capable of binding to Traf2 and of stimulating
NF-.kappa.B activity. In fact, it was shown (see aforesaid Malinin
et al. and IL applications) that expression in cells of
kinase-deficient NIK mutants results in the cells being incapable
of having stimulation of NF.kappa.B in a normal endogenous manner
and also in the cell having a block in induction of NF-.kappa.B
activity by TNF, via either FAS-R, and a block in NF-.kappa.B
induction by TRADD, RIP and MORT-1 (which are adaptor proteins that
bind these p55-R and/or FAS-R receptors). All of the receptors
p55-R, p75-R, FAS-R and their adaptor proteins MORT-1, TRADD and
RIP bind directly or indirectly to Traf2, which by its binding
ability to NIK apparently modulates the induction of
NF-.kappa.B.
[0016] Of the above modulator proteins involved in the fine balance
between cell death and survival following stimulation of FAS-R
and/or p55-R, the protein RIP appears to have an important role.
RIP (see Stanger et al., 1995 and also Malinin et al., 1997) has a
`death domain` in its C-terminal region which enables it to induce
cell cytotoxicity in an independent way and also by association
with the death domains of MORT-1, p55-R, FAS-R and TRADD. RIP also
has a protein kinase domain at its N-terminal region and an
intermediate domain which is believed to enable its intersection
(binding) with Traf2 and thereby its involvement in NF-.kappa.B
induction. Accordingly, details concerning the characteristics and
sequences (DNA and amino acid) of RIP are set forth in the above
noted publications (in particular, Stanger et al., 1995) which are
incorporated herein in their entirety by reference.
[0017] The clone of about 2.2 kB, from which the DNA sequence
encoding the RAP protein according to the present invention, as
presented in parent application Ser. No. 09/381,358, filed Sep. 20,
1999, which is a 371 national stage of PCT/IL98/00125, filed Mar.
19, 1998, was obtained, was resequenced. The laboratory of the
present inventors discovered upon resequencing this clone that the
originally obtained cDNA encoding the RAP protein was not correctly
sequenced. Accordingly, both the DNA and the amino acid sequences
of RAP protein, which are resequenced and deduced, respectively,
and which are inherent from the same deposited clone of about 2.2
kB, are disclosed herein and are entitled to the benefit of
priority based on the deposit of the clone of about 2.2 kB which
was originally sequenced.
SUMMARY OF THE INVENTION
[0018] It is an object of the invention to provide a novel protein
RAP, including all isoforms, analogs, fragments or derivatives
thereof, capable of binding to the RIP protein (herein after
`RIP`). As RIP is capable of interacting directly or indirectly
with the intracellular mediators of inflammation, cell
cytotoxicity/cell death, such as p55-R and FAS-R and their
associated adaptor or modulator proteins such as, for example,
MORT-1, TRADD, MACH, Mch4, G1 and others, the novel proteins of the
present invention by binding to RIP are therefore capable of
affecting the intracellular signaling process initiated by the
binding of the FAS ligand to its receptor, and TNF to its receptor
(p55-R), and as such the new proteins of the present invention are
modulators of the p55-R and FAS-R-mediated effect on cells. As RIP
is also capable of interacting with Traf2 and thereby is capable of
interacting directly or indirectly with NIK and as such RIP acts as
a modulator of inflammation and of cell survival pathways involving
NF-.kappa.B induction, and hence the new proteins of the present
invention are modulators of RIP-related inflammation and cell
survival activity. Likewise, by way of the FAS-R, p55-R and their
modulator proteins MORT-1 and TRADD being capable of inducing
NF-.kappa.B and cell survival either directly or indirectly by
binding to RIP or by binding to Traf2, to which RIP binds the
proteins of the present invention may also be mediators or cell
survival processes by way of operating via common or related
intracellular signaling pathways in which the various above
proteins operate to induce cell survival. Similarly, as p75-R binds
to Traf2 to which RIP binds, the novel proteins of the invention
may also be modulators of RIP-related mediation of p75-R mediated
activity.
[0019] Another object of the invention is to provide antagonists
(e.g., antibodies, peptides, organic compounds, or even some
isoforms) to the above novel RAP proteins, isoforms, analogs,
fragments and derivatives thereof, which may be used to inhibit the
signaling process, or, more specifically, the inflammation
cell-cytotoxicity, or cell-survival processes, when desired.
[0020] A further object of the invention is to use the above novel
RAP proteins, isoforms, analogs, fragments and derivatives thereof,
to isolate and characterize additional proteins or factors, which
may be involved in regulation of receptor activity, e.g., other
proteins which may bind to RAP proteins and influence their
activity, and/or to isolate and identify other receptors further
upstream or downstream in the signaling process(es) to which these
novel proteins, analogs, fragments and derivatives bind, and hence,
in whose function they are also involved.
[0021] A still further object of the invention is to provide
inhibitors which can be introduced into cells to bind or interact
with RAP and possible RAP isoforms which inhibitors may act to
inhibit RIP-associated activity in cell cytotoxic processes and
hence, when desired, to enhance cell survival, or which may act to
inhibit RIP-associated activity in cell-survival processes and
hence, when desired, to enhance cell cytotoxicity.
[0022] Moreover, it is an object of the present invention to use
the above-mentioned novel RAP proteins, isoforms and analogs,
fragments and derivatives thereof as antigens for the preparation
of polyclonal and/or monoclonal antibodies thereto. The antibodies,
in turn, may be used, for example, for the purification of the new
proteins from different sources, such as cell extracts or
transformed cell lines.
[0023] Furthermore, these antibodies may be used for diagnostic
purposes, e.g., for identifying disorders related to abnormal
functioning of cellular effects mediated by the p55-R, FAS-R or
other related receptors.
[0024] A further object of the invention is to provide
pharmaceutical compositions comprising the above novel RAP
proteins, isoforms, or analogs, fragments or derivatives thereof,
as well as pharmaceutical compositions comprising the above noted
antibodies or other antagonists.
[0025] In accordance with the present invention, a novel protein
RAP has been isolated. RAP is capable of binding to, or interacting
with, RIP, and hence is a modulator or mediator of RIP
intracellular activity. RIP is involved in the modulation or
mediation of intracellular signaling pathways, e.g. the cell
cytotoxicity or cell death associated pathway in which RIP has
cytotoxic activity by itself and in association, directly or
indirectly, with a number of other cell-death associated proteins,
such as, for example, MORT-1, TRADD, MACH, Mch4, G1, p55-R and
FAS-R, with which RIP can associate or bind to in a direct or
indirect fashion via the `death domain` motif/module present in RIP
and in all the aforesaid proteins; another pathway being the
inflammation, cell survival or viability pathway in which RIP may
have an activation role, directly or indirectly by virtue of the
presence of a kinase motif or domain present in RIP and RIP's
ability to be capable of binding to Traf2 which can bink NIK which,
in turn, is directly involved in activation of NF-.kappa.B which
plays a central role in inflammation and cell survival. Further,
p55-R and TRADD are also capable of interaction with Traf2 and are
also implicated in NF-.kappa.B activation and thereby in the cell
survival pathway, and hence RIP by being capable of binding to or
interacting with p55-R, FAS-R and TRADD (as well as Traf2) may also
be implicated in the modulation of inflammation, cell survival
activation by these proteins. Accordingly, RIP is a modulator or
mediatior of these pathways, and likewise, the new RAP of the
present invention by binding to RIP is a modulator or mediator of
these intracellular pathways.
[0026] RAP has been isolated and cloned using the yeast two-hybrid
system, sequenced and characterized, and as is detailed herein
below, RAP appears to be a highly specific RIP-binding protein and
hence a specific RIP modulator/mediator. RAP does not bind to
TRADD, MORT-1, p55-R, p75-R and MACH. Further, it appears that RAP
does not have a characteristic death domain module or motif, this
being consistent with the finding that RAP does not induce cell
cytotoxicity on its own.
[0027] As will be used herein throughout, RIP activity is meant to
include both its activity in modulation/mediation in the
inflammation and cell death/survival pathway and its activity in
the modulation/mediation of the cell survival pathway. These
activities are indicated hereinabove and hereinbelow as well as in
all the above-mentioned publications and patent applications, the
full contents of which are incorporated herein by reference.
Likewise, as used herein throughout RAP activity is meant to
include its modulation/mediation of RIP activity by virtue of its
specific binding to RIP, this modulation/mediation of RIP by RAP
including modulation/mediation of the inflammation, cell death and
cell survival pathways in which RIP is involved directly or
indirectly, and as such RAP may be considered as an indirect
modulator/mediator of all the above mentioned proteins and possibly
a number of others which are involved in inflammation, cell death
or cell survival and to which RIP binds, or with which RIP
interacts in a direct or indirect fashion.
[0028] Thus, in accordance with the present invention, a new
protein designated RAP is provided. As noted above, RAP was
isolated and cloned by the two-hybrid screening assay and
characterized as a molecule which binds RIP. Sequencing of RAP has
revealed that it is a novel protein as sequence comparisons
performed between the RAP sequence and those in the various
databases, for example, Genebank `dbest` and Human Genome Database
level 1 databases, did not show any apparent homology between RAP
sequences and any known sequences. Actually two DNA sequences,
differing only in their 5'-non coding regions were found, probably
arising from the same gene by alternative splicing.
[0029] It still remains to be elucidated how RAP binds to RIP, in
particular, there needs to be determined the homology or binding
regions between RAP and RIP which permit their binding to each
other. RAP apparently does not have a death domain module nor any
other known region of enzymatic or other activity, for example, a
kinase or protease domain.
[0030] In view of the above-mentioned, it therefore arises, as
noted above and as set forth hereinbelow, that RAP is apparently a
specific RIP-binding protein and hence a modulator/mediator of RIP
intracellular activity.
[0031] Thus, as RAP apparently has a role in modulating/mediating
inflammation, cell survival and/or cell death activities in which
RIP is involved directly or indirectly especially those related to
cytotoxicity and inflammation caused or induced by various stimuli
including those transmitted via receptors of the TNF/NGF receptor
family and possibly others as well. (For a scheme of RIP's
involvement in these intracellular events and hence RAP's
involvement, see FIG. 1 in Malinin et al., 1997).
[0032] RAP may also serve as an inhibitor of cell cytotoxicity and
inflammation by virtue of its being present as part of a complex of
other proteins, e.g. RIP and proteins bound to RIP, and as such may
affect the cytotoxicity or inflammatory effects of these other
proteins (e.g. p55-R, FAS-R, MACH, Mch4, G1 and MORT-1), ultimately
resulting in an inhibition of their cytotoxic activity or their
activity in inflammation.
[0033] RAP may yet also serve as an enhancer or augmentor of cell
cytotoxicity and inflammation and this by augmenting the activity
of other proteins, e.g. RIP and other proteins bound to RIP as
noted above aiding in the recruitment of these proteins by RIP, the
recruitment serving to augment the cytotoxic activity of the
various proteins or to augment their inflammatory effects.
[0034] Likewise, in an analogous fashion RAP may also serve as an
inhibitor or an augmentor of the cell-survival pathway as noted
above by virtue of RIP's involvement in this pathway.
[0035] Accordingly, the present invention provides a DNA sequence
encoding a RIP-associated protein (RAP) isoforms, analogs or
fragments thereof, capable of binding to RIP and modulating or
mediating the intracellular activity of RIP, said intracellular
activity being a modulation/mediation of inflammation and/or cell
death and/or cell survival.
[0036] In particular, the present invention provides a DNA sequence
selected from the group consisting of:
[0037] (a) a cDNA sequence derived from the coding region of a
native RAP protein;
[0038] (b) DNA sequences capable of hybridization to a sequence of
(a) under moderately stringent conditions and which encode a
biologically active RAP protein; and
[0039] (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 RAP protein.
[0040] Another specific embodiment of the above DNA sequence of the
invention is a DNA sequence comprising at least part of the
sequence encoding at least one isoform of the RAP protein. Another
embodiment of the above DNA sequence is the sequence encoding the
RAP protein as depicted in FIG. 1 (SEQ ID NO:1).
[0041] The present invention provides RAP proteins, and analogs,
fragments or derivatives thereof encoded by any of the above
sequences of the invention, said proteins, analogs, fragments and
derivatives being capable of binding to RIP and
modulating/mediating its biological activity in cell death and/or
cell survival pathways intracellularly.
[0042] A specific embodiment of the invention is the RAP protein,
analogs, fragments and derivatives thereof. The RAP protein
sequence as deduced from the DNA sequence of FIG. 1 is shown in
FIG. 2 (SEQ ID NO:2). Another embodiment is any isoform of the RAP
protein, analogs, fragments and derivatives thereof.
[0043] Also provided by the present invention are vectors encoding
the above RAP protein, and analogs, fragments or derivatives of the
invention, which contain the above DNA sequence of the invention,
these vectors being capable of being expressed in suitable
eukaryotic or prokaryotic host cells; transformed eukaryotic or
prokaryotic host cells containing such vectors; and a method for
producing the RAP protein, or analogs, fragments or derivatives of
the invention by growing such transformed host cells under
conditions suitable for the expression of said protein, analogs,
fragments or derivatives, effecting post-translational
modifications of said protein as necessary for obtaining said
protein and extracting said expressed protein, analogs, fragments
or derivatives from the culture medium of said transformed cells or
from cell extracts of said transformed cells. The above definitions
are intended to include all isoforms of the RAP protein.
[0044] In another aspect, the present invention also provides
antibodies or active derivatives or fragments thereof specific for
the RAP protein, and analogs, fragments and derivatives thereof, of
the invention.
[0045] 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:
[0046] (i) A method for the modulation of the intracellular
inflammation, cell death and/or cell survival pathways modulated or
mediated by the protein RIP, comprising treating said cells with
one or more RAP proteins, isoforms, analogs, fragments or
derivatives thereof, capable of binding to RIP wherein said
treating of said cells comprises introducing into said cells said
one or more proteins, isoforms, analogs, fragments or derivatives
thereof in a form suitable for intracellular introduction thereof,
or introducing into said cells a DNA sequence encoding said one or
more proteins, isoforms, analogs, fragments or derivatives in the
form of a suitable vector carrying said sequence, said vector being
capable of effecting the insertion of said sequence into said cells
in a way that said sequence is expressed in said cells.
[0047] (ii) A method for the modulation of the inflammation, cell
death and/or cell survival pathways mediated by ligands of the TNF
family by effect on cells via the action of the RIP protein,
according to (i) above, wherein said treating of cells comprises
introducing into said cells said RAP protein, or isoforms, analogs,
fragments or derivatives thereof, in a form suitable for
intracellular introduction, or introducing into said cells a DNA
sequence encoding said G1 protein, or isoforms, analogs, fragments
or derivatives in the form of a suitable vector carrying said
sequence, said vector being capable of effecting the insertion of
said sequence into said cells in a way that said sequence is
expressed in said cells.
[0048] (iii) A method as in (ii) above wherein said treating of
said cells is by transfection of said cells with a recombinant
animal virus vector comprising the steps of:
[0049] (a) constructing a recombinant animal virus vector carrying
a sequence encoding a viral surface protein (ligand) that is
capable of binding to a specific cell surface receptor on the
surface of a FAS-R- or p55-R-carrying cell and a second sequence
encoding a protein selected from RAP protein, and isoforma,
analogs, fragments and derivatives thereof, that when expressed in
said cells is capable of modulating/mediating the intracellular
inflammation, cell death and/or cell survival pathways; and
[0050] (b) infecting said cells with said vector of (a).
[0051] (iv) A method for modulating the inflammation, cell death
and/or cell survival pathways mediated by the ligands of the TNF
family effect on cells via the action of the RIP protein comprising
treating said cells with antibodies or active fragments or
derivatives thereof, according to the invention, said treating
being by application of a suitable composition containing said
antibodies, active fragments or derivatives thereof to said cells,
wherein when at least part of the RAP protein is exposed on the
extracellular surface, said composition is formulated for
extracellular application, and when said RAP proteins are entirely
intracellular, said composition is formulated for intracellular
application.
[0052] (v) A method for modulating the inflammation, cell death
and/or cell survival pathways mediated by the ligands of the TNF
family effect on cells via the action of the RIP protein comprising
treating said cells with an oligonucleotide sequence encoding an
antisense sequence of at least part of the RAP protein sequence of
the invention, said oligonucleotide sequence being capable of
blocking the expression of the RAP protein.
[0053] (vi) A method as in (ii) above for treating tumor cells or
HIV-infected cells or other diseased cells, comprising:
[0054] (a) constructing a recombinant animal virus vector carrying
a sequence encoding a viral surface protein capable of binding to a
specific tumor cell surface receptor or HIV-infected cell surface
receptor or receptor carried by other diseased cells and a sequence
encoding a protein selected from RAP protein, analogs, fragments
and derivatives of the invention, that when expressed in said
tumor, HIV-infected, or other diseased cell is capable of killing
said cell via the action of the RIP protein; and
[0055] (b) infecting said tumor or HIV-infected cells or other
diseased cells with said vector of (a).
[0056] (vii) A method for modulating the cell death and/or cell
survival pathways mediated by ligands of the TNF family effect on
cells via the action of the RIP protein comprising applying the
ribozyme procedure in which a vector encoding a ribozyme sequence
capable of interacting with a cellular mRNA sequence encoding a RAP
protein according to the invention, is introduced into said cells
in a form that permits expression of said ribozyme sequence in said
cells, and wherein when said ribozyme sequence is expressed in said
cells it interacts with said cellular mRNA sequence and cleaves
said mRNA sequence resulting in the inhibition of expression of
said RAP protein in said cells.
[0057] (viii) A method selected from the above methods according to
the invention, wherein said RAP protein encoding sequence comprises
at least one of the RAP isoforms, analogs, fragments and
derivatives of any thereof according to the invention which are
capable of binding to RIP.
[0058] (ix) A method for isolating and identifying proteins,
according to the invention capable of binding to the RIP protein,
comprising applying the yeast two-hybrid procedure in which a
sequence encoding said RIP protein or is carried by one hybrid
vector and 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 extraction of the said second hybrid vector
to obtain a sequence encoding a protein which binds to said RIP
protein.
[0059] (x) A method according to any of the (i)-(ix) above wherein
said RAP protein is any one of the isoforms of RAP, analogs,
fragments and derivatives of any thereof.
[0060] (xi) A method according to any of the above (i)-(x) wherein
the RAP protein or any of its isoforms, analogs, fragments or
derivatives is involved in the modulation of the cellular effect
mediated or modulated by any other mediator or inducer to which
said RAP protein, isoform, analog, fragment or derivative is
capable of binding directly or indirectly.
[0061] The present invention also provides a pharmaceutical
composition for the modulation of inflammation, the cell death
and/or cell survival pathways mediated by the TNF family effect on
cells via the action of the RIP protein or the effect of any other
mediator or inducer on cells as noted above, comprising, as active
ingredient any one of the following:
[0062] (i) a RAP protein according to the invention, and
biologically active fragments, analogs, derivatives of mixtures
thereof;
[0063] (ii) a recombinant animal virus vector encoding a protein
capable of binding a cell surface receptor and encoding a RAP
protein or biologically active fragments or analogs, according to
the invention;
[0064] (iii) an oligonucleotide sequence encoding an anti-sense
sequence of the RAP protein sequence according to the invention,
wherein said oligonucleotide may be the second sequence of the
recombinant animal virus vector of (ii) above.
[0065] The present invention also provides:
[0066] I. a method for the modulation of the inflammation,
intracellular cell death and/or cell survival pathways
modulated/mediated by the RIP protein, or the effect of any other
mediator or inducer, or any other NF-.kappa.B inducer or inhibitor,
on cells comprising treating said cells in accordance with a method
of any one of (i)-(x) above, with RAP proteins, isoforms, analogs,
fragments or derivatives thereof or with sequences encoding RAP
proteins, isoforms, analogs or fragments thereof, said treatment
resulting in the enhancement or inhibition of said RIP-mediated
effect, and thereby also of the FAS-R or p55-R-mediated effect, or
of said other mediator or inducer, or other NF-.kappa.B inducer or
inhibitor.
[0067] II. a method as above wherein said RAP protein, analog,
fragment or derivative thereof is that part of the RAP protein
which is specifically involved in binding to RIP, or said other
mediator or inducer, or other NF-.kappa.B inducer or inhibitor, or
said RAP protein sequence encodes that part of RAP protein which is
specifically involved in binding to RIP, or said other mediator or
inducer, or other NF-.kappa.B inducer or inhibitor.
[0068] III. a method as above wherein said RAP protein is any one
of the RAP isoforms, said isoforms capable of enhancing the
RIP-associated effect.
[0069] IV. a method as above wherein said RAP protein is any one of
the RAP isoforms, said isoforms capable of inhibiting the
RIP-associated effect, or other mediator or inducer associated
effect on cells and thereby also of inhibiting the FAS-R- or
p55-R-associated effect on cells, or the other cytotoxic mediator
or inducer effect on cells.
[0070] V. a method as above wherein said RAP protein, isoform,
analog, fragment or derivative capable of enhancing or inhibiting
the RIP-associated effect on the inflammation and cell survival
pathway by way of direct or indirect inhibition of NF-.kappa.B, or
direct or indirect activation of JNK or p38 kinase.
[0071] Isolation of the RAP proteins, their identification and
characterization may be carried out by any of the standard
screening techniques used for isolating and identifying proteins,
for example, the yeast two-hybrid method, affinity chromatography
methods, and any of the other well-known standard procedures used
for this purpose.
[0072] Other aspects and embodiments of the present invention are
also provided as arising from the following detailed description of
the invention.
[0073] It should be noted that, where used throughout, the
following terms: "Modulation/Mediation of the RIP, or FAS-ligand,
or TNF effect on cells"; and any other such "Modulation/Mediation"
mentioned in the specification are understood to encompass in vitro
as well as in vivo treatment and, in addition, also to encompass
inhibition or enhancement/augmentation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] FIG. 1 shows the nucleotide sequence of RAP (SEQ ID NO:1)
with the ATG initiation codon and the TGA step codon indicated in
bold.
[0075] FIG. 2 shows the deduced amino acid sequence of RAP (SEQ ID
NO:2).
DETAILED DESCRIPTION OF THE INVENTION
[0076] The present invention relates, in one aspect, to novel RAP
proteins which are capable of binding to the RIP protein and
thereby of mediating or modulating the intracellular activity of
RIP especially where RIP is involved in modulation or mediation of
inflammation, the cell death and/or cell survival pathways as
detailed herein above. Thus RAP may inhibit RIP activity in the
cell death/inflammation survival pathway, RAP may enhance RIP
activity in the inflammation or cell death survival pathway, or it
may enhance RIP activity in one of these pathways while inhibiting
it in the other.
[0077] More particularly, in accordance with the present invention,
a new protein RAP is provided. RAP has been sequenced and
characterized and it was found that RAP is a RIP-binding protein
having high specificity for RIP, but does not show binding towards
a number of proteins known to be involved in the intracellular
signaling pathways which lead to inflammation, cell death or to
cell survival. RAP also apparently has none of the domains common
to proteins which are active in either of these pathways, i.e. RAP
does not have a `death domain` motif or module, it does not have a
kinase motif or domain and it does not have a protease domain or
motif. The RAP sequence determined is also a unique sequence as
arises from a comparison with sequences in a number of databases
including the Genebank, Human Genome level 1 and `dbest` databases.
As detailed above (also with reference to all publications and
patent applications as noted) RIP is involved in the inflammation,
cell death and cell survival pathways intracellularly. Hence,
regulation or control of the activity of RIP can regulate either or
all of these pathways when such pathways are initiated, by for
example, the binding of TNF or Fas-ligand to their receptors (for
TNF, the p55-R in particular). RIP may play a key role in
determining which pathway is activated to a greater extent and this
by virtue of its being able to bind a number of cytotoxic proteins
having death domains and also a number of proteins having kinase
activity. Accordingly, proteins, such as the RAP protein of the
present invention, which can bind specifically to RIP may play an
important role in modulating RIP activity and thereby modulating
the extent of induction of the one pathway in comparison to the
others. Thus, the RAP protein of the present invention represents
an important intracellular signal modulator or mediator.
[0078] Due to the unique ability of FAS-R and the TNF receptors to
cause cell death, as well as the ability of the TNF receptors to
trigger various other tissue-damaging activities, aberration of the
function of these receptors can be particularly deleterious to the
organism. Indeed, both excessive and deficient function of these
receptors have been shown to contribute to the pathological
manifestations of various diseases. Identifying molecules that take
part in the signaling activity of these receptors, and finding ways
to modulate the function of these molecules, constitutes a
potential clue for new therapeutical approaches to these diseases.
In view of the suspected important role of RIP in FAS-R and p55-R
toxicity, and hence the suspected important regulatory role of RAP
in FAS-R and TNF via modulation of RIP, it seems particularly
important to design drugs that can block the cytotoxic function of
RIP, possibly by way of blocking the binding of RAP to RIP or
otherwise inhibiting the interaction between RAP and RIP under
those conditions in which RAP serves to enhance RIP-mediated
cytotoxicity (as noted above RIP is cytotoxic on its own and in
conjunction with other proteins have death domain regions).
[0079] Likewise, it is also known (see above) that FAS-R and p55-R
are involved in the activation of NF-.kappa.B and thereby of cell
survival. Accordingly, when it is desired to kill cells, for
example cancer cells, HIV-infected cells and the like, it would be
desirable to enhance the cytotoxic effects of FAS-R and p55-R (and
their associated proteins such as, for example, MORT-1, MACH, Mch4,
G1, TRADD), while at the same time to inhibit their ability to
induce NF-.kappa.B. Hence, when the RAP interaction or binding to
RIP results in an augmentation of RIP's possible role in enhancing
NF-.kappa.B induction (possibly via Traf2 and possibly via the
kinase domain and/or intermediate domain of RIP), then it would be
desirable to block this interaction between RAP and RIP to inhibit,
or at least to prevent augmentation, of NF-.kappa.B activation and
thereby shift the balance of TNF- or FAS-ligand-induced effects to
the side of cell cytotoxicity to ultimately provide for increased
cell death.
[0080] Similarly, in the opposite situation (to that noted above)
where RAP's binding to RIP actually causes inhibition of FAS-R and
p55-R inflammatory or cytotoxic effects and it is desired to block
these cytotoxic effects, e.g. in inflammation, various autoimmune
diseases and the like where increased cell survival is sought, then
it is important to design drugs which would enhance the interaction
between RAP and RIP to enhance the overall inhibition of cell death
and shift the balance towards cell survival. It also follows in
light of the above that in the event that RAP's interaction with
RIP causes an inhibition in RIP's function in augmenting
NF-.kappa.B activation, then when cell survival is desired, it is
necessary to block this interaction between RAP and RIP thereby
enhancing RIP's activity in augmenting NF-.kappa.B activation.
[0081] In view of all of the aforementioned, it arises that RIP has
a key role in the balance between induction or mediation of
inflammation, cell death or cell survival pathways and hence RAP
has an equally important role by being a modulator of RIP.
Influencing the RAP-RIP interaction/binding using various drugs or
treatments as noted above and below will possibly allow for a shift
in the intracellular signaling pathways from cell death to cell
survival or vice versa as is desired.
[0082] The present invention also concerns the DNA sequence
encoding a RAP protein and the RAP proteins encoded by the DNA
sequences.
[0083] Moreover, the present invention further concerns the DNA
sequences encoding biologically active analogs, fragments and
derivatives of the RAP protein, and the analogs, fragments and
derivatives encoded thereby. The preparation of such analogs,
fragments and derivatives is by standard procedure (see for
example, Sambrook et al., 1989) in which in the DNA sequences
encoding the RAP protein, one or more codons may be deleted, added
or substituted by another, to yield analogs having at least one
amino acid residue change with respect to the native protein.
[0084] Of the above DNA sequences of the invention which encode a
RAP protein, isoform, analog, fragment or derivative, there is also
included, as an embodiment of the invention, DNA sequences capable
of hybridizing with a cDNA sequence derived from the coding region
of a native RAP protein, in which such hybridization is performed
under moderately stringent conditions, and which hybridizable DNA
sequences encode a biologically active RAP protein. These
hybridizable DNA sequences therefore include DNA sequences which
have a relatively high homology to the native RAP cDNA sequence and
as such represent RAP-like sequences which may be, for example,
naturally-derived sequences encoding the various RAP isoforms, or
naturally-occurring sequences encoding proteins belonging to a
group of RAP-like sequences encoding a protein having the activity
of RAP. Further, these sequences may also, for example, include
non-naturally occurring, synthetically produced sequences, that are
similar to the native RAP cDNA sequence but incorporate a number of
desired modifications. Such synthetic sequences therefore include
all of the possible sequences encoding analogs, fragments and
derivatives of RAP, all of which have the activity of RAP.
[0085] To obtain the various above noted naturally occurring
RAP-like sequences, standard procedures of screening and isolation
of naturally-derived DNA or RNA samples from various tissues may be
employed using the natural RAP cDNA or portion thereof as probe
(see for example standard procedures set forth in Sambrook et al.,
1989).
[0086] Likewise, to prepare the above noted various synthetic
RAP-like sequences encoding analogs, fragments or derivatives of
RAP, a number of standard procedures may be used as are detailed
herein below concerning the preparation of such analogs, fragments
and derivatives.
[0087] A polypeptide or protein "substantially corresponding" to
RAP protein includes not only RAP protein but also polypeptides or
proteins that are analogs of RAP.
[0088] Analogs that substantially correspond to RAP protein are
those polypeptides in which one or more amino acid of the RAP
protein's amino acid sequence has been replaced with another amino
acid, deleted and/or inserted, provided that the resulting protein
exhibits substantially the same or higher biological activity as
the RAP protein to which it corresponds.
[0089] In order to substantially correspond to RAP protein, the
changes in the sequence of RAP proteins, such as isoforms are
generally relatively minor. Although the number of changes may be
more than ten, preferably there are no more than ten changes, more
preferably no more than five, and most preferably no more than
three such changes. While any technique can be used to find
potentially biologically active proteins which substantially
correspond to RAP proteins, one such technique is the use of
conventional mutagenesis techniques on the DNA encoding the
protein, resulting in a few modifications. The proteins expressed
by such clones can then be screened for their ability to bind to
RIP and to modulate RIP activity in modulation/mediation of the
intracellular pathways noted above.
[0090] "Conservative" changes are those changes which would not be
expected to change the activity of the protein and are usually the
first to be screened as these would not be expected to
substantially change the size, charge or configuration of the
protein and thus would not be expected to change the biological
properties thereof.
[0091] Conservative substitutions of RAP proteins include an analog
wherein at least one amino acid residue in the polypeptide has been
conservatively replaced by a different amino acid. Such
substitutions preferably are made in accordance with the following
list as presented in Table IA, which substitutions may be
determined by routine experimentation to provide modified
structural and functional properties of a synthesized polypeptide
molecule while maintaining the biological activity characteristic
of RAP protein.
TABLE-US-00001 TABLE IA Original Exemplary Residue Substitution Ala
Gly; Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly
Ala; Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu
Met Leu; Tyr; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr
Trp; Phe Val Ile; Leu
[0092] Alternatively, another group of substitutions of RAP protein
are those in which at least one amino acid residue in the
polypeptide has been removed and a different residue inserted in
its place according to the following Table IB. The types of
substitutions which may be made in the polypeptide may be based on
analysis of the frequencies of amino acid changes between a
homologous protein of different species, such as those presented in
Table 1-2 of Schulz et al., G. E., Principles of Protein Structure
Springer-Verlag, New York, N.Y., 1798, and FIGS. 3-9 of Creighton,
T. E., Proteins: Structure and Molecular Properties, W.H. Freeman
& Co., San Francisco, Calif. 1983. Based on such an analysis,
alternative conservative substitutions are defined herein as
exchanges within one of the following five groups:
TABLE-US-00002 TABLE IB 1. Small aliphatic, nonpolar or slightly
polar residues: Ala, Ser, Thr (Pro, Gly); 2. Polar negatively
charged residues and their amides: Asp, Asn, Glu, Gln; 3. Polar,
positively charged residues: His, Arg, Lys; 4. Large aliphatic
nonpolar residues: Met, Leu, Ile, Val (Cys); and 5. Large aromatic
residues: Phe, Tyr, Trp.
[0093] The three amino acid residues in parentheses above have
special roles in protein architecture. Gly is the only residue
lacking any side chain and thus imparts flexibility to the chain.
This however tends to promote the formation of secondary structure
other than .alpha.-helical. Pro, because of its unusual geometry,
tightly constrains the chain and generally tends to promote
.beta.-turn-like structures, although in some cases Cys can be
capable of participating in disulfide bond formation which is
important in protein folding. Note that Schulz et al., supra, would
merge Groups 1 and 2, above. Note also that Tyr, because of its
hydrogen bonding potential, has significant kinship with Ser, and
Thr, etc.
[0094] Conservative amino acid substitutions according to the
present invention, e.g., as presented above, are known in the art
and would be expected to maintain biological and structural
properties of the polypeptide after amino acid substitution. Most
deletions and substitutions according to the present invention are
those which do not produce radical changes in the characteristics
of the protein or polypeptide molecule. "Characteristics" is
defined in a non-inclusive manner to define both changes in
secondary structure, e.g. a-helix or .beta.-sheet, as well as
changes in biological activity, e.g., binding to RIP and/or
mediation of RIP's effect on cell death.
[0095] Examples of production of amino acid substitutions in
proteins which can be used for obtaining analogs of RAP proteins
for use in the present invention include any known method steps,
such as presented in U.S. Pat. RE 33,653, 4,959,314, 4,588,585 and
4,737,462, to Mark et al.; 5,116,943 to Koths et al., 4,965,195 to
Namen et al.; 4,879,111 to Chong et al.; and 5,017,691 to Lee et
al.; and lysine substituted proteins presented in U.S. Pat. No.
4,904,584 (Shaw et al.).
[0096] Besides conservative substitutions discussed above which
would not significantly change the activity of RAP protein, either
conservative substitutions or less conservative and more random
changes, which lead to an increase in biological activity of the
analogs of RAP proteins, are intended to be within the scope of the
invention.
[0097] When the exact effect of the substitution or deletion is to
be confirmed, one skilled in the art will appreciate that the
effect of the substitution(s), deletion(s), etc., will be evaluated
by routine binding and cell death assays. Screening using such a
standard test does not involve undue experimentation.
[0098] Acceptable RAP analogs are those which retain at least the
capability of binding to RIP, and thereby, as noted above mediate
the activity of RIP in the intracellular pathways as noted above.
In such a way, analogs can be produced which have a so-called
dominant-negative effect, namely, an analog which is defective
either in binding to RIP, or in subsequent signaling or other
activity following such binding. Such analogs can be used, for
example, to inhibit the effect of RIP, or to inhibit the
NF-.kappa.B inducing (direct or indirect) effect of RIP, depending
on which of these activities is the major one modulated by the
interaction of RAP and RIP (see above), and this by such analogs
competing with the natural RAP for binding to RIP.
[0099] At the genetic level, these analogs are generally prepared
by site-directed mutagenesis of nucleotides in the DNA encoding the
RAP protein, thereby producing DNA encoding the analog, and
thereafter synthesizing the DNA and expressing the polypeptide in
recombinant cell culture. The analogs typically exhibit the same or
increased qualitative biological activity as the naturally
occurring protein, Ausubel et al., Current Protocols in Molecular
Biology, Greene Publications and Wiley Interscience, New York,
N.Y., 1987-1995; Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989.
[0100] Preparation of a RAP protein in accordance herewith, or an
alternative nucleotide sequence encoding the same polypeptide but
differing from the natural sequence due to changes permitted by the
known degeneracy of the genetic code, can be achieved by
site-specific mutagenesis of DNA that encodes an earlier prepared
analog or a native version of a RAP protein. Site-specific
mutagenesis allows the production of analogs through the use of
specific oligonucleotide sequences that encode the DNA sequence of
the desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 20
to 25 nucleotides in length is preferred, with about 5 to 10
complementing nucleotides on each side of the sequence being
altered. In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by publications such as
Adelman et al., DNA 2:183 (1983), the disclosure of which is
incorporated herein by reference.
[0101] As will be appreciated, the site-specific mutagenesis
technique typically employs a phage vector that exists in both a
single-stranded and double-stranded form. Typical vectors useful in
site-directed mutagenesis include vectors such as the M13 phage,
for example, as disclosed by Messing et al., Third Cleveland
Symposium on Macromolecules and Recombinant DNA, Editor A. Walton,
Elsevier, Amsterdam (1981), the disclosure of which is incorporated
herein by reference. These phage are readily available commercially
and their use is generally well known to those skilled in the art.
Alternatively, plasmid vectors that contain a single-stranded phage
origin of replication (Veira et al., Meth. Enzymol. 153:3, 1987)
may be employed to obtain single-stranded DNA.
[0102] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector that
includes within its sequence a DNA sequence that encodes the
relevant polypeptide. An oligonucleotide primer bearing the desired
mutated sequence is prepared synthetically by automated
DNA/oligonucleotide synthesis. This primer is then annealed with
the single-stranded protein-sequence-containing vector, and
subjected to DNA-polymerizing enzymes such as E. coli polymerase I
Klenow fragment, to complete the synthesis of the mutation-bearing
strand. Thus, a mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli JM101 cells, and
clones are selected that include recombinant vectors bearing the
mutated sequence arrangement.
[0103] After such a clone is selected, the mutated RAP protein
sequence may be removed and placed in an appropriate vector,
generally a transfer or expression vector of the type that may be
employed for transfection of an appropriate host.
[0104] Accordingly, gene or nucleic acid encoding for a RAP protein
can also be detected, obtained and/or modified, in vitro, in situ
and/or in vivo, by the use of known DNA or RNA amplification
techniques, such as PCR and chemical oligonucleotide synthesis. PCR
allows for the amplification (increase in number) of specific DNA
sequences by repeated DNA polymerase reactions. This reaction can
be used as a replacement for cloning; all that is required is a
knowledge of the nucleic acid sequence. In order to carry out PCR,
primers are designed which are complementary to the sequence of
interest. The primers are then generated by automated DNA
synthesis. Because primers can be designed to hybridize to any part
of the gene, conditions can be created such that mismatches in
complementary base pairing can be tolerated. Amplification of these
mismatched regions can lead to the synthesis of a mutagenized
product resulting in the generation of a peptide with new
properties (i.e., site directed mutagenesis). See also, e.g.,
Ausubel, supra, Ch. 16. Also, by coupling complementary DNA (cDNA)
synthesis, using reverse transcriptase, with PCR, RNA can be used
as the starting material for the synthesis of the extracellular
domain of a prolactin receptor without cloning.
[0105] Furthermore, PCR primers can be designed to incorporate new
restriction sites or other features such as termination codons at
the ends of the gene segment to be amplified. This placement of
restriction sites at the 5' and 3' ends of the amplified gene
sequence allows for gene segments encoding RAP protein or a
fragment thereof to be custom designed for ligation other sequences
and/or cloning sites in vectors.
[0106] PCR and other methods of amplification of RNA and/or DNA are
well known in the art and can be used according to the present
invention without undue experimentation, based on the teaching and
guidance presented herein. Known methods of DNA or RNA
amplification include, but are not limited to polymerase chain
reaction (PCR) and related amplification processes (see, e.g., U.S.
Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis et
al.; 4,795,699 and 4,921,794 to Tabor et al.; 5,142,033 to Innis;
5,122,464 to Wilson et al.; 5,091,310 to Innis; 5,066,584 to
Gyllensten et al.; 4,889,818 to Gelfand et al.; 4,994,370 to Silver
et al.; 4,766,067 to Biswas; 4,656,134 to Ringold; and Innis et
al., eds., PCR Protocols: A Guide to Method and Applications) and
RNA mediated amplification which uses anti-sense RNA to the target
sequence as a template for double stranded DNA synthesis (U.S. Pat.
No. 5,130,238 to Malek et al., with the tradename NASBA); and
immuno-PCR which combines the use of DNA amplification with
antibody labeling (Ruzicka et al., Science 260:487 (1993); Sano et
al., Science 258:120 (1992); Sano et al., Biotechniques 9:1378
(1991)), the entire contents of which patents and reference are
entirely incorporated herein by reference.
[0107] In an analogous fashion, biologically active fragments of
RAP proteins (e.g. those of any of the RAP or its isoforms) may be
prepared as noted above with respect to the analogs of RAP
proteins. Suitable fragments of RAP proteins are those which retain
the RAP capability and which can modulate or mediate the biological
activity of RIP or other proteins associated with RIP directly or
indirectly. Accordingly, RAP protein fragments can be prepared
which have a dominant-negative or a dominant-positive effect as
noted above with respect to the analogs. It should be noted that
these fragments represent a special class of the analogs of the
invention, namely, they are defined portions of RAP proteins
derived from the full RAP protein sequence (e.g., from that of any
one of the RAP or its isoforms), each such portion or fragment
having any of the above-noted desired activities. Such fragment may
be, e.g., a peptide.
[0108] Similarly, derivatives may be prepared by standard
modifications of the side groups of one or more amino acid residues
of the RAP protein, its analogs or fragments, or by conjugation of
the RAP protein, its analogs or fragments, to another molecule e.g.
an antibody, enzyme, receptor, etc., as are well known in the art.
Accordingly, "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 groups, by means
known in the art, and are included in the invention. Derivatives
may have chemical moieties such as carbohydrate or phosphate
residues, provided such a fraction has the same or higher
biological activity as RAP proteins.
[0109] For example, derivatives may include aliphatic esters of the
carboxyl groups, amides of the carboxyl groups by reaction with
ammonia or with primary or secondary amines, N-acyl derivatives or
free amino groups of the amino acid residues formed with acyl
moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl
derivatives of free hydroxyl group (for example that of seryl or
threonyl residues) formed with acyl moieties.
[0110] The term "derivatives" is intended to include only those
derivatives that do not change one amino acid to another of the
twenty commonly occurring natural amino acids.
[0111] RAP is a protein or polypeptide, i.e. a sequence of amino
acid residues. A polypeptide consisting of a larger sequence which
includes the entire sequence of a RAP protein, in accordance with
the definitions herein, is intended to be included within the scope
of such a polypeptide as long as the additions do not affect the
basic and novel characteristics of the invention, i.e., if they
either retain or increase the biological activity of RAP protein or
can be cleaved to leave a protein or polypeptide having the
biological activity of RAP protein. Thus, for example, the present
invention is intended to include fusion proteins of RAP protein
with other amino acids or peptides.
[0112] The new RAP protein, their analogs, fragments and
derivatives thereof, have a number of possible uses, for
example:
[0113] (i) RAP protein, its analogs, fragments and derivatives
thereof, may be used to modulate the function of RIP in either of
the inflammation, cell death or the cell survival pathways as noted
above. For example, if RAP can modulate RIP's effect on activation
of NF-.kappa.B, JNK (June kinase) or p38 kinase, both such RAP
effects leading to enhance such a RAP-RIP effect when it would be
desirable in anti-tumor, anti- or pro-inflammatory, anti-HIV
applications, etc. In this case the RAP protein, its analogs,
fragments or derivatives thereof, which modulate inflammation,
enhance the cytotoxic effect, or block the cell survival effect,
may be introduced to the cells by standard procedures known per se.
For example, when the RAP protein is entirely intracellular (as
suspected) and should be introduced only into the cells where the
FAS-R ligand or TNF or other cytotoxic protein effect, mediated by
RIP, is desired, a system for specific introduction of this protein
into the cells is necessary. One way of doing this is by creating a
recombinant animal virus, e.g., one derived from Vaccinia, to the
DNA of which the following two 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 AIDs (HIV) virus
gp120 protein which binds specifically to some cells (CD4
lymphocytes and related leukemias), or any other ligand that binds
specifically to cells carrying a FAS-R or p55-R, such that the
recombinant virus vector will be capable of binding such FAS-R- or
p55-R-carrying cells; and the gene encoding the RAP protein. Thus,
expression of the cell-surface-binding protein on the surface of
the virus will target the virus specifically to the tumor cell or
other FAS-R- or p55-R-carrying cell, following which the RAP
protein encoding sequence will be introduced into the cells via the
virus, and once expressed in the cells, will result in enhancement
of the RIP mediation of the FAS-R ligand or TNF effect or
independent RIP. 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 RAP
protein (e.g., any one of the RAP or its isoforms) in the form of
oligonucleotides which can be absorbed by the cells and expressed
therein.
[0114] (ii) They may be used to inhibit the FAS-R ligand or TNF or
related protein effect, mediated by RIP or independent RIP effect,
e.g., in cases such as tissue damage in septic shock,
graft-vs.-host rejection, or acute hepatitis, in which it is
desired to block the FAS-R ligand or TNF induced FAS-R or p55-R
intracellular signaling or independent RIP effect, or other
protein-mediated signaling and at the same time to increase the
cell survival pathway. In this situation, it is possible, for
example, to introduce into the cells, by standard procedures,
oligonucleotides having the anti-sense coding sequence for the RAP
protein, which would effectively block the translation of mRNAs
encoding the RAP protein and thereby block its expression and lead
to the inhibition of the FAS-R ligand- or TNF- or RIP or other
protein-effect. Such oligonucleotides may be introduced into the
cells using the above recombinant virus approach, the second
sequence carried by the virus being the oligonucleotide
sequence.
[0115] Likewise, as noted above, depending on the nature of the
RAP-RIP interaction, it may be possible by the ways of (i) and (ii)
above to enhance or inhibit cell inflammation and survival pathways
where desired.
[0116] Another possibility is to use antibodies specific for the
RAP protein to inhibit its intracellular signaling activity.
[0117] Yet another way of inhibiting the RIP-mediated effects or
RIP independent effect is by the recently developed ribozyme
approach. Ribozymes are catalytic RNA molecules that specifically
cleave RNAs. Ribozymes may be engineered to cleave target RNAs of
choice, e.g., the mRNAs encoding the RAP protein of the invention.
Such ribozymes would have a sequence specific for the RAP protein
mRNA and would be capable of interacting therewith (complementary
binding) followed by cleavage of the mRNA, resulting in a decrease
(or complete loss) in the expression of the RAP protein, the level
of decreased expression being dependent upon the level of ribozyme
expression in the target cell. To introduce ribozymes into the
cells of choice (e.g., those carrying FAS-R or p55-R), any suitable
vector may be used, e.g., plasmid, animal virus (retrovirus)
vectors, that are usually used for this purpose (see also (i)
above, where the virus has, as second sequence, a cDNA encoding the
ribozyme sequence of choice). (For reviews, methods etc. concerning
ribozymes see Chen et al., 1992; Zhao and Pick, 1993; Shore et al.,
1993; Joseph and Burke, 1993; Shimayama et al., 1993; Cantor et
al., 1993; Barinaga, 1993; Crisell et al., 1993 and Koizumi et al.,
1993). This approach is suitable when the RAP-RIP interaction
enhances cell cytotoxicity in situations when it is desired to
block this cytotoxicity, or when the RAP-RIP interaction inhibits
NF-.kappa.B activation in the same situation when it is desired to
block this inhibition to increase such NF-.kappa.B activation, i.e.
in both cases it is desired to increase cell survival as in (ii)
above.
[0118] (iii) The RAP protein, its analogs, fragments or derivatives
may also be used to isolate, identify and clone other proteins of
the same class, i.e., those binding to RIP or to functionally
related receptors or proteins, 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 system employing non-stringent Southern hybridization
followed by PCR cloning (Wilks et al., 1989). In the Wilks 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 known sequence of the kinase motif, a conceived kinase
sequence. This approach may be used, in accordance with the present
invention using the sequence of the RAP protein to identify and
clone those of related RIP-binding proteins.
[0119] (iv) Yet another approach to utilizing the RAP protein, or
its analogs, fragments or derivatives thereof, of the invention is
to use them in methods of affinity chromatography to isolate and
identify other proteins or factors to which they are capable of
binding, e.g., other proteins or factors involved in the
intracellular signaling process. In this application, the RAP
protein, its analogs, fragments or derivatives thereof, of the
present invention, may be individually attached to affinity
chromatography matrices and then brought into contact with cell
extracts or isolated proteins or factors suspected of being
involved in the intracellular signaling process. Following the
affinity chromatography procedure, the other proteins or factors
which bind to the RAP protein, or its analogs, fragments or
derivatives thereof of the invention, can be eluted, isolated and
characterized.
[0120] (v) As noted above, the RAP protein, or its analogs,
fragments or derivatives thereof, of the invention may also be used
as immunogens (antigens) to produce specific antibodies thereto.
These antibodies may also be used for the purposes of purification
of the RAP protein (e.g., RAP or any of its isoforms) either from
cell extracts or from transformed cell lines producing RAP protein,
or its analogs or fragments. Further, these antibodies may be used
for diagnostic purposes for identifying disorders related to
abnormal functioning of the RIP-mediated FAS-R ligand or TNF
system, or independent RIP activities, e.g., overactive or
underactive FAS-R ligand- or TNF-induced cellular effects mediated
by RIP or RIP's own specific cellular effects. Thus, should such
disorders be related to a malfunctioning intracellular signaling
system involving the RIP protein, or various other, above noted
RIP-binding proteins or RAP protein itself, such antibodies would
serve as an important diagnostic tool.
[0121] It should also be noted that the isolation, identification
and characterization of the RAP protein of the invention may be
performed using any of the well known standard screening
procedures. For example, one of these screening procedures, the
yeast two-hybrid procedure as is set forth herein below, was used
to identify the RIP protein (see Stanger et al., 1995) and
subsequently the various RAP proteins of the invention (besides
various other new proteins of the above and below noted co-owned
co-pending patent applications). Likewise as noted above and below,
other procedures may be employed such as affinity chromatography,
DNA hybridization procedures, etc. as are well known in the art, to
isolate, identify and characterize the RAP protein of the invention
or to isolate, identify and characterize additional proteins,
factors, receptors, etc. which are capable of binding to the RAP
proteins of the invention.
[0122] As set forth hereinabove, the RAP protein may be used to
generate antibodies specific to RAP proteins, e.g., RAP and its
isoforms. These antibodies or fragments thereof may be used as set
forth hereinbelow in detail, it being understood that in these
applications the antibodies or fragments thereof are those specific
for RAP proteins.
[0123] Based on the findings in accordance with the present
invention that RAP binds specifically to RIP and as such is a
mediator/modulator of RIP and can thus mediate/modulate RIP's
activity in inflammation, cell death or cell survival pathways in
ways that RIP functions independently or in conjunction with other
proteins (e.g. FAS-R, p55-R, MORT-1, MACH, Mch4, G1 and TRADD in
cell death pathways, or with Traf2 in cell survival pathways) it is
of importance to design drugs which may enhance or inhibit the
RAP-RIP interaction, as desired and depending on which of these
pathways are enhanced/inhibited by the RAP-RIP interaction. There
are many diseases in which such drugs can be of great help. Amongst
others, acute hepatitis in which the acute damage to the liver
seems to reflect FAS-R ligand-mediated death of the liver cells;
autoimmune-induced cell death such as the death of the .beta.
Langerhans cells of the pancreas, that results in diabetes; the
death of cells in graft rejection (e.g., kidney, heart and liver);
the death of oligodendrocytes in the brain in multiple sclerosis;
and AIDS-inhibited T cell suicide which causes proliferation of the
AIDS virus and hence the AIDS disease.
[0124] It is possible that RAP or one or more of its possible
isoforms may serve as "natural" inhibitors of RIP in one or more of
the above pathways and these may thus be employed as the above
noted specific inhibitors of RIP. Likewise, other substances such
as peptides, organic compounds, antibodies, etc. may also be
screened to obtain specific drugs which are capable of inhibiting
the RAP-RIP interaction.
[0125] A non-limiting example of how peptide inhibitors of the
RAP-RIP interaction would be designed and screened is based on
previous studies on peptide inhibitors of ICE or ICE-like
proteases, the substrate specificity of ICE and strategies for
epitope analysis using peptide synthesis. The minimum requirement
for efficient cleavage of peptide by ICE was found to involve four
amino acids to the left of the cleavage site with a strong
preference for aspartic acid in the P.sub.1 position and with
methylamine being sufficient to the right of the P.sub.1 position
(Sleath et al., 1990; Howard et al., 1991; Thornberry et al.,
1992). Furthermore, the fluorogenic substrate peptide (a
tetrapeptide), acetyl-Asp-Glu-Val-Asp-a-(4-methyl-coumaryl-7-amide)
abbreviated Ac-DEVD-AMC, corresponds to a sequence in poly
(ADP-ribose) polymerase (PARP) found to be cleaved in cells shortly
after FAS-R stimulation, as well as other apoptopic processes
(Kaufmann, 1989; Kaufmann et al., 1993; Lazebnik et al., 1994), and
is cleaved effectively by CPP32 (a member of the CED3/ICE protease
family) and MACH proteases (and likewise also possibly by G1
proteases--see for example co-owned co-pending IL 120367).
[0126] As Asp in the P.sub.1 position of the substrate appears to
be important, tetrapeptides having Asp as the fourth amino acid
residue and various combinations of amino acids in the first three
residue positions can be rapidly screened for binding to the active
site of the proteases using, for example, the method developed by
Geysen (Geysen, 1985; Geysen et al., 1987) where a large number of
peptides on solid supports were screened for specific interactions
with antibodies. The binding of MACH proteases to specific peptides
can be detected by a variety of well known detection methods within
the skill of those in the art, such as radiolabeling of the G1
proteases, etc. This method of Geysen's was shown to be capable of
testing at least 4000 peptides each working day.
[0127] In a similar way the exact binding region or region of
homology which determines the interaction between RAP and RIP can
be elucidated and then peptides may be screened which can serve to
block this interaction, e.g. peptides synthesized having a sequence
similar to that of the binding region or complementary thereto
which can compete with natural RAP for binding to RIP.
[0128] Drug or peptide inhibitors, which are capable of inhibiting
inflammation or the cell death activity of RAP by inhibiting the
RAP-RIP interaction can be conjugated or complexed with molecules
that facilitate entry into the cell.
[0129] U.S. Pat. No. 5,149,782 discloses conjugating a molecule to
be transported across the cell membrane with a membrane blending
agent such as fusogenic polypeptides, ion-channel forming
polypeptides, other membrane polypeptides, and long chain fatty
acids, e.g. myristic acid, palmitic acid. These membrane blending
agents insert the molecular conjugates into the lipid bilayer of
cellular membranes and facilitate their entry into the
cytoplasm.
[0130] Low et al., U.S. Pat. No. 5,108,921, reviews available
methods for transmembrane delivery of molecules such as, but not
limited to, proteins and nucleic acids by the mechanism of receptor
mediated endocytotic activity. These receptor systems include those
recognizing galactose, mannose, mannose 6-phosphate, transferrin,
asialoglycoprotein, transcobalamin (vitamin B.sub.12), .alpha.-2
macroglobulins, insulin and other peptide growth factors such as
epidermal growth factor (EGF). Low et al. teaches that nutrient
receptors, such as receptors for biotin and folate, can be
advantageously used to enhance transport across the cell membrane
due to the location and multiplicity of biotin and folate receptors
on the membrane surfaces of most cells and the associated receptor
mediated transmembrane transport processes. Thus, a complex formed
between a compound to be delivered into the cytoplasm and a ligand,
such as biotin or folate, is contacted with a cell membrane bearing
biotin or folate receptors to initiate the receptor mediated
trans-membrane transport mechanism and thereby permit entry of the
desired compound into the cell.
[0131] In addition, it is known in the art that fusing a desired
peptide sequence with a leader/signal peptide sequence to create a
"chimeric peptide" will enable such a "chimeric peptide" to be
transported across the cell membrane into the cytoplasm.
[0132] As will be appreciated by those of skill in the art of
peptides, the peptide inhibitors of the RAP-RIP interaction
according to the present invention is meant to include
peptidomimetic drugs or inhibitors, which can also be rapidly
screened for binding to RAP/RIP protease to design perhaps more
stable inhibitors.
[0133] It will also be appreciated that the same means for
facilitating or enhancing the transport of peptide inhibitors
across cell membranes as discussed above are also applicable to the
RAP or its isoforms themselves as well as other peptides and
proteins which exert their effects intracellularly.
[0134] As regards the antibodies mentioned herein throughout, the
term "antibody" is meant to include polyclonal antibodies,
monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic
(anti-Id) antibodies to antibodies that can be labeled in soluble
or bound form, as well as fragments thereof provided by any known
technique, such as, but not limited to enzymatic cleavage, peptide
synthesis or recombinant techniques.
[0135] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen. A monoclonal antibody contains a substantially
homogeneous population of antibodies specific to antigens, which
populations contains substantially similar epitope binding sites.
MAbs may be obtained by methods known to those skilled in the art.
See, for example Kohler and Milstein, Nature, 256:495-497 (1975);
U.S. Pat. No. 4,376,110; Ausubel et al., eds., Harlow and Lane
ANTIBODIES A LABORATORY MANUAL, Cold Spring Harbor Laboratory
(1988); and Colligan et al., eds., Current Protocols in Immunology,
Greene Publishing Assoc. and Wiley Interscience N.Y., (1992-1996),
the contents of which references are incorporated entirely herein
by reference. Such antibodies may be of any immunoglobulin class
including IgG, IgM, IgE, IgA, GILD and any subclass thereof. A
hybridoma producing a mAb of the present invention may be
cultivated in vitro, in situ or in vivo. Production of high titers
of mAbs in vivo or in situ makes this the presently preferred
method of production.
[0136] Chimeric antibodies are molecules of which different
portions are derived from different animal species, such as those
having the variable region derived from a murine mAb and a human
immunoglobulin constant region. Chimeric antibodies are primarily
used to reduce immunogenicity in application and to increase yields
in production, for example, where murine mAbs have higher yields
from hybridomas but higher immunogenicity in humans, such that
human/murine chimeric mAbs are used. Chimeric antibodies and
methods for their production are known in the art (Cabilly et al.,
Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al.,
Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al.,
Nature 312:643-646 (1984); Cabilly et al., European Patent
Application 125023 (published Nov. 14, 1984); Neuberger et al.,
Nature 314:268-270 (1985); Taniguchi et al., European Patent
Application 171496 (published Feb. 19, 1985); Morrison et al.,
European Patent Application 173494 (published Mar. 5, 1986);
Neuberger et al., PCT Application WO 8601533, (published Mar. 13,
1986); Kudo et al., European Patent Application 184187 (published
Jun. 11, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986);
Robinson et al., International Patent Application No. WO8702671
(published May 7, 1987); Liu et al., Proc. Natl. Acad. Sci USA
84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci USA
84:214-218 (1987); Better et al., Science 240:1041-1043 (1988); and
Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra. These
references are entirely incorporated herein by reference.
[0137] An anti-idiotypic (anti-Id) antibody is an antibody which
recognizes 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 to which an anti-Id is being
prepared. The immunized animal will recognize and respond to the
idiotypic determinants of the immunizing antibody by producing an
antibody to these idiotypic determinants (the anti-Id antibody).
See, for example, U.S. Pat. No. 4,699,880, which is herein entirely
incorporated by reference.
[0138] The anti-Id antibody may also be used as an "immunogen" to
induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody. The anti-anti-Id may be
epitopically identical to the original mAb which induced the
anti-Id. Thus, by using antibodies to the idiotypic determinants of
a mAb, it is possible to identify other clones expressing
antibodies of identical specificity.
[0139] Accordingly, mAbs generated against the RAP proteins,
analogs, fragments or derivatives thereof, of the present invention
may be used to induce anti-Id antibodies in suitable animals, such
as BALB/c mice. Spleen cells from such immunized mice are used to
produce anti-Id hybridomas secreting anti-Id mAbs. Further, the
anti-Id mAbs can be coupled to a carrier such as keyhole limpet
hemocyanin (KLH) and used to immunize additional BALB/c mice. 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 above RAP protein, or analogs, fragments and derivatives
thereof.
[0140] The anti-Id mAbs thus have their own idiotypic epitopes, or
"idiotopes" structurally similar to the epitope being evaluated,
such as GRB protein-a.
[0141] The term "antibody" is also meant to include both intact
molecules as well as fragments thereof, such as, for example, Fab
and F(ab')2, which are capable of binding antigen. Fab and F(ab')2
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 (Wahl et al., J. Nucl. Med.
24:316-325 (1983)).
[0142] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies useful in the present invention may be
used for the detection and quantitation of the RAP protein
according to the methods disclosed herein for intact antibody
molecules. Such fragments are typically produced by proteolytic
cleavage, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab')2 fragments).
[0143] 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
antibody which can also be recognized 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.
[0144] 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 or more than one
epitope. The specific reaction referred to above is meant to
indicate that the antigen will react, in a highly selective manner,
with its corresponding antibody and not with the multitude of other
antibodies which may be evoked by other antigens.
[0145] The antibodies, including fragments of antibodies, useful in
the present invention may be used to quantitatively or
qualitatively detect the RAP protein in a sample or to detect
presence of cells which express the RAP protein of the present
invention. This can be accomplished by immunofluorescence
techniques employing a fluorescently labeled antibody (see below)
coupled with light microscopic, flow cytometric, or fluorometric
detection.
[0146] The antibodies (or fragments thereof) useful in the present
invention may be employed histologically, as in immunofluorescence
or immunoelectron microscopy, for in situ detection of the RAP
protein of the present invention. In situ detection may be
accomplished by removing a histological specimen from 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 RAP protein, but
also its distribution on the examined tissue. Using the present
invention, those of ordinary skill will readily perceive that any
of wide variety of histological methods (such as staining
procedures) can be modified in order to achieve such in situ
detection.
[0147] Such assays for the RAP protein of the present invention
typically comprises incubating a biological sample, such as a
biological fluid, a tissue extract, freshly harvested cells such as
lymphocytes or leukocytes, or cells which have been incubated in
tissue culture, in the presence of a detectably labeled antibody
capable of identifying the RAP protein, and detecting the antibody
by any of a number of techniques well known in the art.
[0148] The biological 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 may then be washed with
suitable buffers followed by treatment with a detectably labeled
antibody in accordance with the present invention, as noted above.
The solid phase support or carrier may then be washed with the
buffer a second time to remove unbound antibody. The amount of
bound label on said solid support or carrier may then be detected
by conventional means.
[0149] 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-known supports or carriers, include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon amylases, natural and
modified celluloses, polyacrylamides, gabbros and magnetite. The
nature of the carrier can be either soluble to some extent or
insoluble for the purposes of the present invention. 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 tube, or the external surface of a rod. Alternatively, the
surface may be flat such as a sheet, test strip, etc. Preferred
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.
[0150] The binding activity of a given lot of antibody, of the
invention as noted above, may be 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.
[0151] Other such steps as washing, stirring, shaking, filtering
and the like may be added to the assays as is customary or
necessary for the particular situation.
[0152] One of the ways in which an antibody in accordance with the
present invention can be detectably labeled is by linking the same
to an enzyme and used in an enzyme immunoassay (EIA). This enzyme,
in turn, when later 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 spectrophotometric,
fluorometric or by visual means. Enzymes which can be used to
detectably label the antibody include, but are not limited to,
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomeras, yeast alcohol dehydrogenase, alpha-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholin-esterase. The detection can be accomplished by
calorimetric methods which employ a chromogenic substrate 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.
[0153] Detection may be accomplished using any of a variety of
other immunoassays. For example, by radioactive labeling the
antibodies or antibody fragments, it is possible to detect R-PTPase
through the use of a radioimmunoassay (RIA). A good description of
RIA may be found in Laboratory Techniques and Biochemistry in
Molecular Biology, by Work, T. S. et al., North Holland Publishing
Company, NY (1978) with particular reference to the chapter
entitled "An Introduction to Radioimmune Assay and Related
Techniques" by Chard, T., incorporated by reference herein. The
radioactive isotope can be detected by such means as the use of a g
counter or a scintillation counter or by autoradiography.
[0154] It is also possible to label an antibody in accordance with
the present invention with a fluorescent compound. When the
fluorescently labeled antibody 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, phycoerythrine, pycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine.
[0155] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152E, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriamine pentaacetic
acid (ETPA).
[0156] The antibody can also be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0157] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which 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.
[0158] An antibody molecule of the present invention may be adapted
for utilization in an immunometric assay, also known as a
"two-site" or "sandwich" assay. In a typical immunometric assay, a
quantity of unlabeled antibody (or fragment of antibody) is bound
to a solid support or carrier and a quantity of detectably labeled
soluble antibody is added to permit detection and/or quantitation
of the ternary complex formed between solid-phase antibody,
antigen, and labeled antibody.
[0159] 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 extract the antigen
from the sample by formation of a binary solid phase
antibody-antigen complex. After a suitable incubation period, the
solid support or carrier is washed to remove the residue of the
fluid sample, including unreacted antigen, if any, and then
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
complex with the antigen bound to the solid support or carrier
through the unlabeled antibody, the solid support or carrier is
washed a second time to remove the unreacted labeled antibody.
[0160] In another type of "sandwich" assay, which may also be
useful with the antigens of the present invention, the so-called
"simultaneous" and "reverse" assays are used. A simultaneous assay
involves a single incubation step as the antibody bound to the
solid support or carrier 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 uncomplexed labeled antibody. The
presence of labeled antibody associated with the solid support or
carrier is then determined as it would be in a conventional
"forward" sandwich assay.
[0161] In the "reverse" assay, stepwise addition first 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 unreacted labeled antibody. The determination of labeled
antibody associated with a solid support or carrier is then
determined as in the "simultaneous" and "forward" assays.
[0162] The RAP proteins of the invention may be produced by any
standard recombinant DNA procedure (see for example, Sambrook, et
al., 1989 and Ansabel et al., 1987-1995, supra) in which suitable
eukaryotic or prokaryotic host cells well known in the art are
transformed by appropriate eukaryotic or prokaryotic vectors
containing the sequences encoding for the proteins. Accordingly,
the present 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
biologically active analogs, fragments and derivatives, and thus
the vectors encoding them also include vectors encoding analogs and
fragments of these proteins, and the transformed hosts include
those producing such analogs and fragments. The derivatives of
these proteins, produced by the transformed hosts, are the
derivatives produced by standard modification of the proteins or
their analogs or fragments.
[0163] The present invention also relates to pharmaceutical
compositions comprising recombinant animal virus vectors encoding
the RAP proteins, 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 RAP protein
sequences into the cells. Further pharmaceutical compositions of
the invention comprises as the active ingredient (a) an
oligonucleotide sequence encoding an anti-sense sequence of the RAP
protein sequence, or (b) drugs that block the RAP-RIP
interaction.
[0164] Pharmaceutical compositions according to the present
invention include a sufficient amount of the active ingredient to
achieve its intended purpose. In addition, the pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically and which can stabilize such preparations for
administration to the subject in need thereof as well known to
those of skill in the art.
[0165] The RAP protein and its isoforms or isotypes are suspected
to be expressed in different tissues at markedly different levels
and apparently also with different patterns of isotypes in an
analogous fashion to the expression of various other proteins
involved in the intracellular signaling pathways as indicated in
the above listed co-owned co-pending patent applications. These
differences may possibly contribute to the tissue-specific features
of response to the Fas/APO1-ligand and TNF. As in the case of other
CED3/ICE homologs (Wang et al., 1994; Alnemri et al., 1995), the
present inventors have previously shown (in the above mentioned
patent applications) that MACH isoforms that contain incomplete
CED3/ICE regions (e.g., MACH.alpha.3) are found to have an
inhibitory effect on the activity of co-expressed MACH.alpha.1 or
MACH.alpha.2 molecules; they are also found to block death
induction by Fas/APO1 and p55-R. Expression of such inhibitory
isoforms in cells may constitute a mechanism of cellular
self-protection against Fas/APO1- and TNF-mediated cytotoxicity. A
similar inhibitory effect of at least some G1 isoforms is also
suspected (G1 being a recently isolated new Mch4- and possibly
MACH-binding protein, and also MORT-1-binding protein that has MORT
MODULES and a protease domain--see co-owned co-pending IL 120367).
The wide heterogeneity of MACH isoforms, and likewise the
suspected, analogous heterogeneity of G1 isoforms, which greatly
exceeds that observed for any of the other proteases of the
CED3/ICE family, should allow a particularly fine tuning of the
function of the active MACH isoforms, and by analogy also the
active G1 isoforms. Hence, as noted above, the RAP proteins or
possible isoforms may have varying effects in different tissues as
regards their interaction with RIP and their influence thereby on
the balance between activation of cell death or cell survival
pathways, as described above.
[0166] It is also possible that some of the possible RAP isoforms
serve other functions. For example, RAP or some RAP isoforms may
also act as docking sites for molecules that are involved in other,
non-cytotoxic effects of Fas/APO1 and TNF receptors via interaction
with RIP or even independently of RIP.
[0167] Due to the unique ability of Fas/APO1 and TNF receptors to
cause inflammation, cell death, as well as the ability of the TNF
receptors to trigger other tissue-damaging activities, aberrations
in the function of these receptors could be particularly
deleterious to the organism. Indeed, both excessive and deficient
functioning of these receptors have been shown to contribute to
pathological manifestations of various diseases (Vassalli, 1992;
Nagata and Golstein, 1995). Identifying the molecules that
participate in the signaling activity of the receptors, and finding
ways to modulate the activity of these molecules, could direct new
therapeutic approaches. Other aspects of the invention will be
apparent from the following examples.
[0168] The invention will now be described in more detail in the
following non-limiting examples and the accompanying drawings.
[0169] It should also be noted that the procedures of: i)
two-hybrid screen and two-hybrid .beta.-galactosidase expression
test; (ii) induced expression, metabolic labeling and
immunoprecipitation of proteins; (iii) in vitro binding; (iv)
assessment of the cytotoxicity; and (v) Northern and sequence
analyses, (see also Boldin et al., 1995b) 2, 3 (see also Boldin et
al., 1996) and 4, below, with respect to MORT-1 and a MORT-1
binding protein, (e.g. MACH), as well as the newly isolated protein
G1 (see IL 120367) are equally applicable (with some modifications)
for the corresponding isolation, cloning and characterization of
RAP and its possible isoforms of the present invention. These
procedures are thus to be construed as the full disclosure of the
same procedures used for the isolation, cloning and
characterization of RAP in accordance with the present invention,
as detailed e.g. in the same or equivalent form in the co-owned
co-pending Israel Application Nos. 114,615, 114,986, 115,319,
116588, 117,932, and 120367 as well as the corresponding PCT
application No. PCT/US96/10521. Further, as regards the NIK protein
and its role in activating NF-.kappa.B and hence cell survival and
the role played by Traf2 in this cell survival pathway, for example
the interaction between Traf2 and RIP and other proteins, these
have been detailed by the present inventors in co-pending co-owned
IL 117800, IL 119133 and Malinin et al., 1997.
EXAMPLE
Cloning and Isolation of the RAP Protein which Binds to the RIP
Protein
(i) Two-Hybrid Screen Sequencing and Preliminary Analysis
[0170] Using the two-hybrid screen (see e.g. Fields and Song, 1989,
WO/96/18641) with RIP devoid of its death domain as the bait in a
B-cell library, a clone of about 1.9 Kb size was isolated and
sequenced.
[0171] Primers from the 5' end of this sequence were designed and
prepared. Employing PCR several cDNA libraries were screened. From
both, a colon cDNA library and a heart cDNA library a clone of
about 0.3 kB was obtained and ligated to the about 1.9 kB clone
obtained from the B-cell library.
[0172] The new clone of about 2.2 kB, designated RAP-1 and
deposited with Collection Nationale De Cultures De Microorganismes
(CNCM), Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cedex
15, France, on Jul. 26, 2001, under accession number I-2706, was
sequenced (SEQ ID NO:1) and its deduced amino acid sequence
determined (SEQ ID NO:2). Although the laboratory of the present
inventors believes that the obtained sequences are correct, the
possibility that there is an anomaly that leads to an error in
sequencing cannot be ruled out completely. Nevertheless, the
nucleotide and amino acid sequences are inherent in the deposit
clone.
[0173] Analysis of the sequence shows that the RAP protein
apparently does not have a `death domain`, it does not have a MORT
MODULE, it does not have a protease domain like those of the ICE
family, it does not have a kinase domain, nor does it have TRAF
domains (see above noted co-pending, co-owned patent applications
and the various references, especially Malinin et al., 1997 with
respect to all the various domains present in the intracellular
signaling pathways). Binding studies revealed that RAP essentially
only binds to RIP, RAP being unable to bind to TRADD, MORT-1,
p55-R, p75-R and MACH.
[0174] Therefore, it appears that RAP is a specific RIP-binding
protein that interacts/binds to RIP in a very specific way,
possibly via a binding domain region which is not present in other
intracellular signaling proteins isolated to date. As RAP appears
to be a specific modulator/mediator of RIP intracellular activity
having an important role in RIP's modulation/mediation of the
inflammation and the cell death/cell survival pathways.
[0175] Briefly, a clone of the RAP protein was obtained following
two-hybrid screening of a human peripheral blood lymphocyte
two-hybrid cDNA library using a fragment of RIP protein devoid of
RIP's "death domain" region as `bait`. The RIP sequence was
available from previous publications (e.g. Stanger et al., 1995)
and as present in the GenBank database under accession No. U 25994
which is the human RIP sequence (also present was the mouse RIP
sequence under accession No. U 25995). Using this sequence
information appropriate PCR-primers were designed by OLIGO4.TM.
software and the DNA fragment corresponding to the coding part of
RIP, but without its C-terminal `death domain` region was obtained
by PCR using as template cDNA from the total RNA Human Fibroblast
Cell library (using standard procedures). This coding part of RIP,
devoid of RIP's `death domain` region was then cloned into the
pGBT-9 vector (Clontech) and used as a bait, as noted above, in the
two-hybrid screening procedure. In this two-hybrid screen a number
of clones were obtained all coding for proteins which are
RIP-binding protein that interact with RIP at a region in RIP
outside of the `death domain` (C-terminus region of RIP), this not
being present in the RIP `bait`. After further proceedings as
described above, the cDNA clone, the sequence of which is shown in
FIG. 1 (SEQ ID NO:1), was obtained.
[0176] Analysis of the above sequences of the RAP clone and
sequences in the `dbest` database, Human Genome Database level 1
and GenBank database revealed that the RAP sequence is a unique
(novel) sequence as no known sequence showed any significant
homology to this RAP sequence.
[0177] Binding assay tests were performed to determine whether RAP
can bind to any of the other known intracellular signaling
proteins. In these tests the proteins TRADD, MORT-1, p55-R, p75-R,
MACH were tested for their ability to bind to RAP. However, it was
found that RAP was incapable of binding to any of these proteins.
RAP also did not bind to any of the irrelevant control proteins,
e.g. lamin, cyclin D.
[0178] All of the above results therefore indicate that the new RAP
protein possibly interacts with RIP in a very specific manner and
as such it represents a specific modulator/mediator of RIP. The
exact site of interaction between RAP and RIP is yet to be
determined but it seems that this site is one specific to RIP and
RAP and not shared by other proteins known to interact with RIP,
e.g. MORT-1, TRADD, FAS-R and possibly also Traf2 (see Malinin et
al., 1997). It also arises that (from sequence analysis and
comparison with sequences in various databases as noted above) that
RAP does not have a `death domain`, a MORT MODULE, a protease
domain (e.g. ICE/CED3 motif), a kinase domain/motif nor TRAF
domains. In line with this, preliminary biological activity
analysis also revealed that RAP apparently has the following
characteristics:
[0179] (i) RAP is not toxic to cells on its own when
overexpressed;
[0180] (ii) RAP does not protect cells from TNF killing and thus is
apparently not an inhibitor of TNF-induced cell cytotoxicity;
[0181] (iii) RAP does not induce NF-.kappa.B on its own:
[0182] (iv) RAP does block NF-.kappa.B activation by TRADD, RIP and
p55 TNF-R;
[0183] (v) RAP was also found to block JNK (Jun kinase) induction
caused by RIP.
[0184] In view of the aforementioned RAP therefore appears to be a
highly specific RIP-binding protein and hence RIP
modulator/mediator, that is likely to be involved in the
RIP-mediated intracellular signaling pathways.
[0185] In light of the above it appears that RAP is involved in
modulation/mediation of RIP's activities intracellularly, these
being its involvement in the inflammation and cell death pathway
(independently via its `death domain` or via interaction with other
proteins such as MORT-1, TRADD, p55-R, FAS-R and associated
proteases such as MACH, Mch4, G1 and the like) and RIP's
involvement in the cell survival pathway (NF-.kappa.B activation,
possibly via interaction with Traf2). The possible ways in which
RAP may modulate/mediate RIP's activity are detailed hereinabove,
for example the RAP-RIP interaction may lead to enhancement of
either the cell death or cell survival pathways, or it may lead to
the inhibition of either the cell death or cell survival pathways,
this enhancement or inhibition possibly being dependent on the
relative activities of other members of these two opposing
intracellular pathways. RAP may also act as a docking protein to
provide for an aggregation of a number of RIP molecules and other
RIP- or RAP-binding proteins, which aggregate may then function
either in the direction of cell death or cell survival (or even
both) depending on the relative activities/amounts of the other
members of these pathways in the cell.
[0186] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0187] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the inventions
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended
claims.
[0188] All references cited herein, including journal articles or
abstracts, published or corresponding U.S. or foreign patent
applications, issued U.S. or foreign patents, or any other
references, are entirely incorporated by reference herein,
including all data, tables, figures, and text presented in the
cited references. Additionally, the entire contents of the
references cited within the references cited herein are also
entirely incorporated by reference.
[0189] Reference to known method steps, conventional methods steps,
known methods or conventional methods is not in any way an
admission that any aspect, description or embodiment of the present
invention is disclosed, taught or suggested in the relevant
art.
[0190] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art (including
the contents of the references cited herein), readily modify and/or
adapt for various applications such specific embodiments, without
undue experimentation, without departing from the general concept
of the present invention. Therefore, such adaptations and
modifications are intended to be within the meaning and range of
equivalents of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance presented herein, in
combination with the knowledge of one of ordinary skill in the
art.
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Sequence CWU 1
1
212119DNAHomo sapiensCDS(217)..(1782) 1tagggagacc caagcttctc
gacggccatt accaatcgcg aaaccggcag ggcggccact 60gtggcggggc tctttccccg
tttcgcctca gctacccctc agctccggta gtcgccagtc 120cggggtcgtc
gccgtttggg gcgggagctg ctcggccccg ccgccgtccc cgtcgccgct
180tccgggtcca ggcccctcgg gccgcctgcc gccgtc atg agg ctg cgg gtg cgg
234 Met Arg Leu Arg Val Arg 1 5ctt ctg aag cgg acc tgg ccg ctg gag
gtg ccc gag acg gag ccg acg 282Leu Leu Lys Arg Thr Trp Pro Leu Glu
Val Pro Glu Thr Glu Pro Thr 10 15 20ctg ggg cat ttg cgc tcg cac ctg
agg ctg tcc ctg ctg tgc acc tgg 330Leu Gly His Leu Arg Ser His Leu
Arg Leu Ser Leu Leu Cys Thr Trp 25 30 35ggg tac agt tct aat acc cga
ttt aca att aca ttg aac tac aag gat 378Gly Tyr Ser Ser Asn Thr Arg
Phe Thr Ile Thr Leu Asn Tyr Lys Asp 40 45 50ccc ctc act gga gat gaa
gag acc ttg gct tca tat ggg att gtt tct 426Pro Leu Thr Gly Asp Glu
Glu Thr Leu Ala Ser Tyr Gly Ile Val Ser55 60 65 70ggg gac ttg ata
tgt ttg att ctt caa gat gac att cca gcg cct aat 474Gly Asp Leu Ile
Cys Leu Ile Leu Gln Asp Asp Ile Pro Ala Pro Asn 75 80 85ata cct tca
tcc aca gat tca gag cat tct tca ctc cag aat aat gag 522Ile Pro Ser
Ser Thr Asp Ser Glu His Ser Ser Leu Gln Asn Asn Glu 90 95 100caa
ccc tct ttg gcc acc agc tcc aat cag act agc atg cag gat gaa 570Gln
Pro Ser Leu Ala Thr Ser Ser Asn Gln Thr Ser Met Gln Asp Glu 105 110
115caa cca agt gat tca ttc caa gga cag gca gcc cag tct ggt gtt tgg
618Gln Pro Ser Asp Ser Phe Gln Gly Gln Ala Ala Gln Ser Gly Val Trp
120 125 130aat gac gac agt atg tta ggg cct agt caa aat ttt gaa gct
gag tca 666Asn Asp Asp Ser Met Leu Gly Pro Ser Gln Asn Phe Glu Ala
Glu Ser135 140 145 150att caa gat aat gcg cat atg gca gag ggc aca
ggt ttc tat ccc tca 714Ile Gln Asp Asn Ala His Met Ala Glu Gly Thr
Gly Phe Tyr Pro Ser 155 160 165gaa ccc atg ctc tgt agt gaa tcg gtg
gaa ggg caa gtg cca cat tca 762Glu Pro Met Leu Cys Ser Glu Ser Val
Glu Gly Gln Val Pro His Ser 170 175 180tta gag acc ttg tat caa tca
gct gac tgt tct gat gcc aat gat gcc 810Leu Glu Thr Leu Tyr Gln Ser
Ala Asp Cys Ser Asp Ala Asn Asp Ala 185 190 195ttg ata gtg ttg ata
cat ctt ctc atg ttg gag tca ggt tac ata cct 858Leu Ile Val Leu Ile
His Leu Leu Met Leu Glu Ser Gly Tyr Ile Pro 200 205 210cag ggc acc
gaa gcc aaa gca ctg tcc atg ccg gag aag tgg aag ttg 906Gln Gly Thr
Glu Ala Lys Ala Leu Ser Met Pro Glu Lys Trp Lys Leu215 220 225
230agc ggg gtg tat aag ctg cag tac atg cat cct ctc tgc gag ggc agc
954Ser Gly Val Tyr Lys Leu Gln Tyr Met His Pro Leu Cys Glu Gly Ser
235 240 245tcc gct act ctc acc tgt gtg cct ttg gga aac ctg att gtt
gta aat 1002Ser Ala Thr Leu Thr Cys Val Pro Leu Gly Asn Leu Ile Val
Val Asn 250 255 260gct aca cta aaa atc aac aat gag att aga agt gtg
aaa aga ttg cag 1050Ala Thr Leu Lys Ile Asn Asn Glu Ile Arg Ser Val
Lys Arg Leu Gln 265 270 275ctg cta cca gaa tct ttt att tgc aaa gag
aaa cta ggg gaa aat gta 1098Leu Leu Pro Glu Ser Phe Ile Cys Lys Glu
Lys Leu Gly Glu Asn Val 280 285 290gcc aac ata tac aaa gat ctt cag
aaa ctc tct cgc ctc ttt aaa gac 1146Ala Asn Ile Tyr Lys Asp Leu Gln
Lys Leu Ser Arg Leu Phe Lys Asp295 300 305 310cag ctg gtg tat cct
ctt ctg gct ttt acc cga caa gca ctg aac cta 1194Gln Leu Val Tyr Pro
Leu Leu Ala Phe Thr Arg Gln Ala Leu Asn Leu 315 320 325cca gat gta
ttt ggg ttg gtc gtc ctc cca ttg gaa ctg aaa cta cgg 1242Pro Asp Val
Phe Gly Leu Val Val Leu Pro Leu Glu Leu Lys Leu Arg 330 335 340atc
ttc cga ctt ctg gat gtt cgt tcc gtc ttg tct ttg tct gcg gtt 1290Ile
Phe Arg Leu Leu Asp Val Arg Ser Val Leu Ser Leu Ser Ala Val 345 350
355tgt cgt gac ctc ttt act gct tca aat gac cca ctc ctg tgg agg ttt
1338Cys Arg Asp Leu Phe Thr Ala Ser Asn Asp Pro Leu Leu Trp Arg Phe
360 365 370tta tat ctg cgt gat ttt cga gac aat act gtc aga gtt caa
gac aca 1386Leu Tyr Leu Arg Asp Phe Arg Asp Asn Thr Val Arg Val Gln
Asp Thr375 380 385 390gat tgg aaa gaa ctg tac agg aag agg cac ata
caa aga aaa gaa tcc 1434Asp Trp Lys Glu Leu Tyr Arg Lys Arg His Ile
Gln Arg Lys Glu Ser 395 400 405ccg aaa ggg cgg ttt gtg atg ctc ctg
cca tcg tca act cac acc att 1482Pro Lys Gly Arg Phe Val Met Leu Leu
Pro Ser Ser Thr His Thr Ile 410 415 420cca ttc tat ccc aac ccc ttg
cac cct agg cca ttt cct agc tcc cgc 1530Pro Phe Tyr Pro Asn Pro Leu
His Pro Arg Pro Phe Pro Ser Ser Arg 425 430 435ctt cct cca gga att
atc ggg ggt gaa tat gac caa aga cca aca ctt 1578Leu Pro Pro Gly Ile
Ile Gly Gly Glu Tyr Asp Gln Arg Pro Thr Leu 440 445 450ccc tat gtt
gga gac cca atc agt tca ctc att cct ggt cct ggg gag 1626Pro Tyr Val
Gly Asp Pro Ile Ser Ser Leu Ile Pro Gly Pro Gly Glu455 460 465
470acg ccc agc cag ttt cct cca ctg aga cca cgc ttt gat cca gtt ggc
1674Thr Pro Ser Gln Phe Pro Pro Leu Arg Pro Arg Phe Asp Pro Val Gly
475 480 485cca ctt cca gga cct aac ccc atc ttg cca ggg cga ggc ggc
ccc aat 1722Pro Leu Pro Gly Pro Asn Pro Ile Leu Pro Gly Arg Gly Gly
Pro Asn 490 495 500gac aga ttt ccc ttt aga ccc agc agg ggt cgg cca
act gat ggc cgg 1770Asp Arg Phe Pro Phe Arg Pro Ser Arg Gly Arg Pro
Thr Asp Gly Arg 505 510 515ctg tca ttc atg tgattgattt gtaatttcat
ttctggagct ccatttgttt 1822Leu Ser Phe Met 520ttgtttctaa actacagatg
tcaactcctt ggggtgctga tctcgagtgt tattttctga 1882ttgtggtgtt
gagagttgca ctcccagaaa ccttttaaga gatacattta tagccctagg
1942ggtggtatga cccaaaggtt cctctgtgac aaggttggcc ttgggaatag
ttggctgcca 2002atctccctgc tcttggttct cctctagatt gaagtttgtt
ttctgatgct gttcttacca 2062gattaaaaaa aagtgtaaat taaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaa 21192522PRTHomo sapiens 2Met Arg Leu
Arg Val Arg Leu Leu Lys Arg Thr Trp Pro Leu Glu Val1 5 10 15Pro Glu
Thr Glu Pro Thr Leu Gly His Leu Arg Ser His Leu Arg Leu 20 25 30Ser
Leu Leu Cys Thr Trp Gly Tyr Ser Ser Asn Thr Arg Phe Thr Ile 35 40
45Thr Leu Asn Tyr Lys Asp Pro Leu Thr Gly Asp Glu Glu Thr Leu Ala
50 55 60Ser Tyr Gly Ile Val Ser Gly Asp Leu Ile Cys Leu Ile Leu Gln
Asp65 70 75 80Asp Ile Pro Ala Pro Asn Ile Pro Ser Ser Thr Asp Ser
Glu His Ser 85 90 95Ser Leu Gln Asn Asn Glu Gln Pro Ser Leu Ala Thr
Ser Ser Asn Gln 100 105 110Thr Ser Met Gln Asp Glu Gln Pro Ser Asp
Ser Phe Gln Gly Gln Ala 115 120 125Ala Gln Ser Gly Val Trp Asn Asp
Asp Ser Met Leu Gly Pro Ser Gln 130 135 140Asn Phe Glu Ala Glu Ser
Ile Gln Asp Asn Ala His Met Ala Glu Gly145 150 155 160Thr Gly Phe
Tyr Pro Ser Glu Pro Met Leu Cys Ser Glu Ser Val Glu 165 170 175Gly
Gln Val Pro His Ser Leu Glu Thr Leu Tyr Gln Ser Ala Asp Cys 180 185
190Ser Asp Ala Asn Asp Ala Leu Ile Val Leu Ile His Leu Leu Met Leu
195 200 205Glu Ser Gly Tyr Ile Pro Gln Gly Thr Glu Ala Lys Ala Leu
Ser Met 210 215 220Pro Glu Lys Trp Lys Leu Ser Gly Val Tyr Lys Leu
Gln Tyr Met His225 230 235 240Pro Leu Cys Glu Gly Ser Ser Ala Thr
Leu Thr Cys Val Pro Leu Gly 245 250 255Asn Leu Ile Val Val Asn Ala
Thr Leu Lys Ile Asn Asn Glu Ile Arg 260 265 270Ser Val Lys Arg Leu
Gln Leu Leu Pro Glu Ser Phe Ile Cys Lys Glu 275 280 285Lys Leu Gly
Glu Asn Val Ala Asn Ile Tyr Lys Asp Leu Gln Lys Leu 290 295 300Ser
Arg Leu Phe Lys Asp Gln Leu Val Tyr Pro Leu Leu Ala Phe Thr305 310
315 320Arg Gln Ala Leu Asn Leu Pro Asp Val Phe Gly Leu Val Val Leu
Pro 325 330 335Leu Glu Leu Lys Leu Arg Ile Phe Arg Leu Leu Asp Val
Arg Ser Val 340 345 350Leu Ser Leu Ser Ala Val Cys Arg Asp Leu Phe
Thr Ala Ser Asn Asp 355 360 365Pro Leu Leu Trp Arg Phe Leu Tyr Leu
Arg Asp Phe Arg Asp Asn Thr 370 375 380Val Arg Val Gln Asp Thr Asp
Trp Lys Glu Leu Tyr Arg Lys Arg His385 390 395 400Ile Gln Arg Lys
Glu Ser Pro Lys Gly Arg Phe Val Met Leu Leu Pro 405 410 415Ser Ser
Thr His Thr Ile Pro Phe Tyr Pro Asn Pro Leu His Pro Arg 420 425
430Pro Phe Pro Ser Ser Arg Leu Pro Pro Gly Ile Ile Gly Gly Glu Tyr
435 440 445Asp Gln Arg Pro Thr Leu Pro Tyr Val Gly Asp Pro Ile Ser
Ser Leu 450 455 460Ile Pro Gly Pro Gly Glu Thr Pro Ser Gln Phe Pro
Pro Leu Arg Pro465 470 475 480Arg Phe Asp Pro Val Gly Pro Leu Pro
Gly Pro Asn Pro Ile Leu Pro 485 490 495Gly Arg Gly Gly Pro Asn Asp
Arg Phe Pro Phe Arg Pro Ser Arg Gly 500 505 510Arg Pro Thr Asp Gly
Arg Leu Ser Phe Met 515 520
* * * * *