U.S. patent application number 11/130223 was filed with the patent office on 2005-12-29 for modulators of intracellular inflammation, cell death and cell survival pathways.
This patent application is currently assigned to Yeda Research and Development Co., Ltd.. Invention is credited to Boldin, Mark, Malinin, Nikolai, Wallach, David.
Application Number | 20050288225 11/130223 |
Document ID | / |
Family ID | 27271818 |
Filed Date | 2005-12-29 |
United States Patent
Application |
20050288225 |
Kind Code |
A1 |
Wallach, David ; et
al. |
December 29, 2005 |
Modulators of intracellular inflammation, cell death and cell
survival pathways
Abstract
A B1 protein, its isoforms, analogs, fragments and derivatives,
are provided. The protein is useful in the modulation of
intracellular inflammation, cell death and/or cell survival
pathways.
Inventors: |
Wallach, David; (Rehovot,
IL) ; Boldin, Mark; (Rehovot, IL) ; Malinin,
Nikolai; (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: |
27271818 |
Appl. No.: |
11/130223 |
Filed: |
May 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11130223 |
May 17, 2005 |
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09445223 |
Dec 6, 1999 |
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09445223 |
Dec 6, 1999 |
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PCT/IL98/00255 |
Jun 1, 1998 |
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Current U.S.
Class: |
530/350 ;
435/226; 514/18.9; 514/19.3; 514/44R |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/4747 20130101; C07K 14/4705 20130101; A61K 38/00
20130101 |
Class at
Publication: |
514/012 ;
514/044; 435/226 |
International
Class: |
A61K 048/00; C12N
009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 1997 |
IL |
121011 |
Jun 30, 1997 |
IL |
121199 |
Sep 11, 1997 |
IL |
121746 |
Claims
What is claimed is:
1. An isolated polypeptide which potentiates cell death, said
polypeptide consisting of: (a) a sequence comprising SEQ ID NO:1;
(b) a sequence comprising an analog of (a) having no more than ten
changes in the amino acid sequence of (a), each said change being a
substitution, deletion or insertion of a single amino acid, which
analog potentiates cell death; or (c) a fragment of the sequence of
SEQ ID NO:1, which fragment potentiates cell death.
2. A polypeptide in accordance with claim 1, consisting of a
sequence comprising SEQ ID NO:1.
3. A polypeptide in accordance with claim 1, consisting of a
sequence comprising an analog of SEQ ID NO:1, having no more than
ten changes in the amino acid sequence of SEQ ID NO:1, each said
change being a substitution, deletion or insertion of a single
amino acid, which analog potentiates cell death.
4. A polypeptide in accordance with claim 1 consisting of a
fragment of the sequence of SEQ ID NO:1, which fragment potentiates
cell death.
5. An isolated polypeptide in accordance with claim 1, wherein said
sequence of (b) comprises an analog of (a) having no more than five
changes in the amino acid sequence of (a), each said change being a
substitution, deletion or insertion of a single amino acid, which
analog potentiates cell death.
6. An isolated polypeptide in accordance with claim 1, wherein said
sequence of (b) comprises an analog of (a) having no more than
three changes in the amino acid sequence of (a), each said change
being a substitution, deletion or insertion of a single amino acid,
which analog potentiates cell death.
7. A composition comprising a pharmaceutically acceptable excipient
and at least one polypeptide according to claim 1.
8. A method of modulating apoptotic processes or programmed cell
death processes (cell death pathways) in which the B1 protein of
SEQ ID NO:1 is involved, in cells in need of such modulation,
comprising causing one or more polypeptides according to claim 1 to
be disposed within said cells.
9. A method in accordance with claim 8, wherein said causing step
comprises introducing into said cells said one or more polypeptide
in a form suitable for intracellular introduction thereof.
10. A method in accordance with claim 8, wherein said causing step
comprises introducing into said cells a DNA sequence encoding said
one or more polypeptide in the form of a suitable vector carrying
said sequence, said vector being capable of effecting the ingestion
of said sequence into said cells in a way that said sequence is
expressed in said cells.
11. A method of modulating cell survival processes in which the B1
protein of SEQ ID NO:1 is involved, in cells in need of such
modulation, comprising causing one or more polypeptides according
to claim 1 to be disposed within said cells.
12. A method in accordance with claim 11, wherein said causing step
comprises introducing into said cells said one or more polypeptide
in a form suitable for intracellular introduction thereof.
13. A method in accordance with claim 11, wherein said causing step
comprises introducing into said cells a DNA sequence encoding said
one or more polypeptide in the form of a suitable vector carrying
said sequence, said vector being capable of effecting the ingestion
of said sequence into said cells in a way that said sequence is
expressed in said cells.
Description
FIELD OF THE INVENTION
[0001] The present invention is generally in the field of
modulators of intracellular cell death and cell survival pathways
mediated by, amongst others, the receptors of the TNF/NGF
superfamily of receptors and their associated intracellular adaptor
proteins, and caspase and kinase enzymes. More specifically, the
present invention concerns a new protein, originally designated
CBK, but now designated B1, its isoforms, analogs, fragments and
derivatives, which appears to be capable of interacting, directly
or indirectly, with various intracellular proteins and enzymes that
belong to the cell death, cell survival and inflammation pathways,
and hence, which is a modulator of these pathways.
BACKGROUND OF THE INVENTION
[0002] The Tumor Necrosis Factor/Nerve Growth Factor (TNF/NGF)
receptor superfamily is defined by structural homology between the
extracellular domains of its members (Bazan, 1993; Beutler and van
Huffel, 1994; Smith et al, 1994). Except for two receptors, the p55
TNF receptor and Fas/APO1, the various members of this receptor
family do not exhibit clear similarity of structure in their
intracellular domains. Nevertheless, there is much similarity of
function between the receptors, indicating that they share common
signaling pathways. One example for this similarity is the ability
of several receptors of the TNF/NGF family to activate the
transcription factor NF-.kappa.B. This common ability was ascribed
to a capability of a cytoplasmic protein that activates
NF-.kappa.B, TNF Receptor Associated Factor 2 (TRAF2) to bind to
the structurally-dissimilar intracellular domains of several of the
receptors of the TNF/NGF family. By what mechanisms TRAF2 acts and
how its responsiveness to the different receptors to which it binds
is coordinated, is not known.
[0003] TRAF2 is a member of a recently described family of proteins
called TRAF that includes several proteins identified as, for
example, TRAF1, TRAF2 (Rothe et al, 1994; WO 95/33051), TRAF3
(Cheng et al, 1995), and TRAF6 (see Cao et al, 1996a).
[0004] All proteins belonging to the TRAF family share high degree
of amino acid identity in their C-terminal domains, while their
N-terminal domains may be unrelated. As shown in a schematic
illustration of TRAF2 (FIG. 1), the molecule contains a ring finger
motif and two TFIIIA-like zinc finger motifs at its C-terminal
area. The C-terminal half of the molecule includes a region known
as the "TRAF domain" containing a potential leucine zipper region
extending between amino acids 264-358 (called N-TRAF), and another
part towards the carboxy end of the domain between amino acids
359-501 (called C-TRAF) which is responsible for TRAF binding to
the receptors and to other TRAF molecules to form homo- or
heterodimers.
[0005] Activation of the transcription factor NF-.kappa.B is one
manifestation of the signaling cascade initiated by some of the
TNF/NGF receptors and mediated by TRAF2. NF-.kappa.B comprises
members of a family of dimer-forming proteins with homology to the
Rel oncogene that, in their dimeric form, act as transcription
factors. These factors are ubiquitous and participate in regulation
of the expression of multiple genes. Although initially identified
as a factor that is constitutively present in B cells at the stage
of Ig.kappa. light chain expression, NF-.kappa.B is known primarily
for its action as an inducible transcriptional activator. In most
known cases NF-.kappa.B behaves as a primary factor, namely the
induction of its activity is by activation of pre-existing
molecules present in the cell in their inactive form, rather than
its de novo synthesis that, in turn, relies on inducible
transcription factors that turn-on the NF-.kappa.B gene. The
effects of NF-.kappa.B are highly pleiotropic. Most of these
numerous effects share the common features of being quickly induced
in response to an extracellular stimulus. The majority of the
NF-.kappa.B-activating agents are inducers of immune defense,
including components of viruses and bacteria, cytokines that
regulate immune response, UV light and others. Accordingly, many of
the genes regulated by NF-.kappa.B contribute to immune defense
(see Blank et al, 1992; Grilli et al, 1993; Baeuerle and Henkel,
1994, for reviews).
[0006] One major feature of NF-.kappa.B-regulation is that this
factor can exist in a cytoplasmic non-DNA binding form which can be
induced to translocate to the nucleus, bind DNA and activate
transcription. This dual form of the NF-.kappa.B proteins is
regulated by I-.kappa.B--a family of proteins that contain repeats
of a domain that has initially been discerned in the erythrocyte
protein ankyrin (Gilmore and Morin, 1993). In the unstimulated
form, the NF-.kappa.B dimer occurs in association with an
I-.kappa.B molecule which imposes on it cytoplasmic location and
prevents its interaction with the NF-.kappa.B-binding DNA sequence
and activation of transcription. The dissociation of I-.kappa.B
from the NF-.kappa.B dimer constitutes the critical step of its
activation by many of its inducing agents (DiDonato et al, 1995).
Knowledge of the mechanisms that are involved in this regulation is
still limited. There is also just little understanding of the way
in which cell specificity in terms of responsiveness to the various
NF-.kappa.B-inducing agents is determined.
[0007] One of the most potent inducing agents of NF-.kappa.B is the
cytokine tumor necrosis factor (TNF). There are two different TNF
receptors, the p55 and p75 receptors (p55-R and p75-R). Their
expression levels vary independently among different cells
(Vandenabeele et al, 1995). The p75 receptor responds
preferentially to the cell-bound form of TNF (TNF is expressed both
as a beta-transmembrane protein and as a soluble protein) while the
p55 receptor responds just as effectively to soluble TNF molecules
(Grell et al, 1995). The intracellular domains of the two receptors
are structurally unrelated and bind different cytoplasmic proteins.
Nevertheless, at least part of the effects of TNF, including the
cytocidal effect of TNF and the induction of NF-.kappa.B, can be
induced by both receptors. This feature is cell specific. The p55
receptor is capable of inducing a cytocidal effect or activation of
NF-.kappa.B in all cells that exhibit such effects in response to
TNF. The p75-R can have such effects only in some cells. Others,
although expressing the p75-R at high levels, show induction of the
effects only in response to stimulation of the p55-R (Vandenabeele
et al, 1995). Apart from the TNF receptors, various other receptors
of the TNF/NGF receptor family: CD30 (McDonald et al, 1995), CD40
(Berberich et al, 1994; Lalmanach-Girard et al, 1993), the
lymphotoxin beta receptor and, in a few types of cells, Fas/APO1
(Rensing-Ehl et al, 1995), are also capable of inducing activation
of NF-.kappa.B. The IL-1 type I receptor, also effectively
triggering NF-.kappa.B activation, shares most of the effects of
the TNF receptors despite the fact that it has no structural
similarity to them.
[0008] The activation of NF-.kappa.B upon triggering of these
various receptors results from induced phosphorylation of its
associated I-.kappa.B molecules. This phosphorylation tags
I-.kappa.B to degradation, which most likely occurs in the
proteasome. The nature of the kinase that phosphorylates
I-.kappa.B, and its mechanism of activation upon receptor
triggering is still unknown. However, in the recent two years some
knowledge has been gained as to the identity of three
receptor-associated proteins that appear to take part in initiation
of the phosphorylation (see diagrammatic illustration in FIGS. 2A
and 6). A protein called TRAF2, initially cloned by D. Goeddel and
his colleagues (Rothe et al, 1994), seems to play a central role in
NF-.kappa.B-activation by the various receptors of the TNF/NGF
family. The protein, which, when expressed at high levels, can by
itself trigger NF-.kappa.B activation, binds to activated p75 TNF-R
(Rothe et al, 1994), lymphotoxin beta receptor (Mosialos et al,
1995), CD40 (Rothe et al, 1995a) and CD-30 (unpublished data) and
mediates the induction of NF-.kappa.B by them. TRAF2 does not bind
to the p55 TNF receptor nor to Fas/APO1; however, it can bind to a
p55 receptor-associated protein called TRADD and TRADD has the
ability to bind to a Fas/APO1-associated protein called MORT1 (or
FADD--see Boldin et al 1995b and 1996). Another
receptor-interacting protein, called RIP (see Stanger et al, 1995)
is also capable of interacting with TRAF2 as well as with FAS/APO1,
TRADD, the p55 TNF receptor and MORT-1. Thus, while RIP has been
associated with cell cytotoxicity induction (cell death), its
ability to interact with TRAF2 also implicates it in NF-.kappa.B
activation and it also may serve in addition to augment the
interaction between FAS/APO1, MORT-1, p55 TNF receptor and TRADD
with TRAF2 in the pathway leading to NF-.kappa.B activation. These
associations apparently allow the p55 TNF receptor and Fas/APO1 to
trigger NF-.kappa.B activation (Hsu et al, 1995; Boldin et al,
1995; Chinnaiyan et al, 1995; Varfolomeev et al, 1996; Hsu et al,
1996). The triggering of NF-.kappa.B activation by the IL-1
receptor occurs independently of TRAF2 and may involve a
recently-cloned IL-1 receptor-associated protein-kinase called IRAK
(Croston et al, 1995).
[0009] By what mechanism TRAF2 acts is not clear. Several
cytoplasmic molecules that bind to TRAF2 have been identified
(Rothe et al, 1994; Rothe et al, 1995b). However, the information
on these molecules does not provide any clue as to the way by which
TRAF2, which by itself does not possess any enzymatic activity,
triggers the phosphorylation of I-.kappa.B. There is also no
information yet of mechanisms that dictate cell-specific pattern of
activation of TRAF2 by different receptors, such as observed for
the induction of NF-.kappa.B by the two TNF receptors.
[0010] In addition to the above mentioned, of the various TRAF
proteins, it should also be noted that TRAF2 binds to the p55
(CD120a) and p75 (CD120b) TNF receptors, as well as to several
other receptors of the TNF/NGF receptor family, either directly or
indirectly via other adaptor proteins as noted above, for example,
with reference to the FAS/APO1 receptor, and the adaptor proteins
MORT-1, TRADD and RIP. As such, TRAF2 is crucial for the activation
of NF-.kappa.B (see also Wallach, 1996). However, TRAF3 actually
inhibits activation of NF-.kappa.B by some receptors of the TNF/NGF
family (see Rothe et al, 1995a), whilst TRAF6 is required for
induction of NF-.kappa.B by IL-1 (see Cao et al, 1996a).
[0011] Accordingly, as regards NF-.kappa.B activation and its
importance in maintaining cell viability, the various intracellular
pathways involved in this activation have heretofore not been
clearly elucidated, for example, how the various TRAF proteins, are
involved directly or indirectly.
[0012] Furthermore, as is now known regarding various members of
the TNF/NGF receptor family and their associated intracellular
signaling pathways inclusive of various adaptor, mediator/modulator
proteins (see brief reviews and references in, for example,
co-pending co-owned Israel Patent Application Nos. 114615, 114986,
115319, 116588), TNF and the FAS/APO1 ligand, for example, can have
both beneficial and deleterious effects on cells. TNF, for example,
contributes to the defense of the organism against tumors and
infectious agents and contributes to recovery from injury by
inducing the killing of tumor cells and virus-infected cells,
augmenting antibacterial activities of granulocytes, and thus in
these cases the TNF-induced cell killing is desirable. However,
excess TNF can be deleterious and as such TNF is known to play a
major pathogenic role in a number of diseases such as septic shock,
anorexia, rheumatic diseases, inflammation and graft-vs-host
reactions. In such cases TNF-induced cell killing is not desirable.
The FAS/APO1 ligand, for example, also has desirable and
deleterious effects. This FAS/APO1 ligand induces via its receptor
the killing of autoreactive T cells during maturation of T cells,
i.e., the killing of T cells which recognize self-antigens, during
their development and thereby preventing autoimmune diseases.
Further, various malignant cells and HIV-infected cells carry the
FAS/APO1 receptor on their surface and can thus be destroyed by
activation of this receptor by its ligand or by antibodies specific
thereto, and thereby activation of cell death (apoptosis)
intracellular pathways mediated by this receptor. However, the
FAS/APO1 receptor may mediate deleterious effects, for example,
uncontrolled killing of tissue which is observed in certain
diseases such as acute hepatitis that is accompanied by the
destruction of liver cells.
[0013] In view of the above, namely, that receptors of the TNF/NGF
family can induce cell death pathways on the one hand and can
induce cell survival pathways (via NF-.kappa.B induction) on the
other hand, there apparently exists a fine balance, intracellularly
between these two opposing pathways. For example, when it is
desired to achieve maximal destruction of cancer cells or other
infected or diseased cells, it would be desired to have TNF and/or
the FAS/APO1 ligand inducing only the cell death pathway without
inducing NF-.kappa.B. Conversely, when it is desired to protect
cells such as in, for example, inflammation, graft-vs-host
reactions, acute hepatitis, it would be desirable to block the cell
killing induction of TNF and/or FAS/APO1 ligand and enhance,
instead, their induction of NF-.kappa.B. Likewise, in certain
pathological circumstances it would be desirable to block the
intracellular signaling pathways mediated by the p75 TNF receptor
and the IL-1 receptor, while in others it would be desirable to
enhance these intracellular pathways.
[0014] Recently, the present inventors have isolated a kinase
called NIK (Israel Patent Application Nos. 117800, 119133 and WO
97/37016) that is capable of binding to TRAF2 and is directly
involved in the phosphorylation reactions leading to induction of
NF-.kappa.B activation.
[0015] In addition, a number of caspases have recently been
isolated by a number of researchers (including the present
inventors (see co-pending, co-owned Israel Patent Application No.
IL 120759)), which interact with the above noted adaptor proteins
(e.g. MORT-1/FADD) or with complexes between the adaptor proteins
and the various receptors of the TNF/NGF receptor family and which
effect the proteolytic reactions leading to apoptotic cell death.
Thus, direct modulation of these caspases would be desired in the
situations noted above when it is desired to inhibit or enhance
cell death, for example, when it is desired to inhibit cell death
it would be desirable to inhibit the activity of these caspases. In
this respect it has been reported (see review in Hofmann et al,
1997) that there exists a region called a prodomain in many of
these caspases that is also present in a number of adaptor proteins
such as, for example, RAIDD (which interacts with RIP, TRADD and
thereby with MORT-1/FADD, the p55-TNF-R and FAS/APO1), an adaptor
protein of the cell death pathway; and c-IAP1, c-IAP2, two proteins
which appear to be inhibitors of apoptosis and which themselves
interact with TRAF2, and thereby may be inhibitors of caspases or
may otherwise stimulate TRAF2 involvement in the cell survival
pathway resulting in induction of NF-.kappa.B activation. As such
this prodomain has also been designated as CARD for `caspase
recruitment domain` (see Hofmann et al, 1997). This prodomain
(CARD) therefore represents another target for modulation of the
intracellular signaling pathways associated with cell death
induction.
[0016] Moreover, recently there has been described (see Review by
Yang and Korsmeyer, 1996) another family of proteins, called the
BCL2 protein family, of which the proteins BCL2, its homolog BCL-X
including the two forms thereof being BCL-XL and the alternatively
spliced BCL-XS, MCL1, Al, BAK, BAD, BAG1, BAX, the adenovirus
E1B-19k, and the Caenorhabditis elegans (C. elegans) CED-9 protein
are all members. Of these proteins it has been observed that BCL2,
BCL-X.sub.L, E1B-19k and CED-9 function to inhibit apoptosis, or to
protect against apoptosis induced by various intracellular
signaling pathways (see Yang and Korsmeyer, 1996). BCL2 and
BCL-X.sub.L are also apparently intracellular membrane-bound
proteins localized to mitochondria, as well as smooth endoplasmic
reticulum, and the perinuclear membrane, the C-terminus of these
proteins having a signal anchor sequence responsible for targeting
and insertion thereof into the outer mitochondrial membrane and the
other, above noted, intracellular membranes. Once anchored in the
various intracellular membranes the BCL2 and BCL-XL proteins are
exposed to the cytosol where they can interact with various other
intracellular proteins.
[0017] How BCL2, BCL-X.sub.L, E1B-19k and CED-9 protect cells has
not yet been fully elucidated, but it appears that their effect is
apparently upstream of the cell death effectors being the various
caspases noted above, such as, for example ICE and ICE-like
proteases of the ICE/CED-3 family including CPP32/Yama, ICE-LAP3
(Mch3), ICH-1 and others. In fact, CED-9 was found to be a specific
inhibitor of the C. elegans death effector proteases CED-3 and
CED-4, and BCL2 is apparently an inhibitor of ICH-1 (also called
NEDD2), in particular, the ICH-1.sub.L form which promotes cell
death. Thus, while the precise mechanism of inhibition of apoptosis
by BCL2, BCL-XL, CED-9 and E1B-19k, is not clear, it is apparently
upstream of the ICE-CED-3 proteases that are the death effectors
(see review of Yang and Korsmeyer, 1996, as well as Chinnaiyan et
al, 1996).
[0018] As regards the other BCL2 family members noted above, BAX is
a cell death promoter. BAX binds to itself and in the form of such
BAX homodimers it promotes apoptosis. BAX also binds to BCL2 and
BCL-X.sub.L and such heterodimers are associated with BCL2's
protective effect against apoptosis. Thus the balance between the
amounts of BAX/BAX homodimers and BAX/BLC2 heterodimers determines
whether cells will be susceptible to apoptosis or whether they will
be protected against apoptosis. BAX is also apparently an
intracellular membrane-bound protein also being localized to a
large degree to the outer mitochondrial membrane (for above
mentioned concerning BAX, see also review by Yang and Korsmeyer,
1996). Further, the above noted BAK and BAD proteins also act as
negative regulators of BCL2 and BCL-X.sub.L activity, namely, they
repress the ability of BCL2 and BCL-X.sub.L to protect cells from
apoptosis. It appears that both BAK and BAD bind BCL2 and
BCL-X.sub.L and thereby prevent BAX from binding to BCL2 and
BCL-X.sub.L resulting in increased amounts of BAX/BAX homodimers
and subsequently increased cell death (see review by Yang and
Korsmeyer, 1996). In this regard it also appears that BAK functions
to block the death-repressor activity of BCL2 and BCL-X.sub.L
directly as BAK/BCL2 and BAK/BCL-X.sub.L heterodimers lack the
ability to protect cells from apoptosis. BAD appears to act more
like a competitive inhibitor for BAX binding to BCL2 and
BCL-X.sub.L, as BAD may replace BAX from BAX/BCL2 and
BAX/BCL-X.sub.L heterodimers, thereby providing for increased
amounts of death-promoting BAX/BAX homodimers. While BAK also
appears to be an intracellular membrane bound protein localized to,
amongst others, mitochondrial outer membranes, BAD, however, is
apparently devoid of a membrane anchor sequence and as such is not
a membrane-bound protein (see review by Yang and Korsmeyer,
1996).
[0019] Another of the above members of the BCL2 family is BAG1 (see
Yang and Korsmeyer, 1996), which is a positive modulator of BCL2
activity leading to enhanced BCL2 protective activity against
apoptosis and even providing for BCL2 protective activity against
apoptosis in cells induced to undergo apoptosis by signals not
usually suppressed by BCL2.
[0020] It should also be noted that the above mentioned
alternatively spliced form of BCL-X.sub.L, namely the BCL-X.sub.S
protein is also an antagonist of BCL-X.sub.L and BCL2 activity, and
blocks their protective activity against apoptosis (see also review
of Yang and Korsmeyer, 1996).
[0021] In view of the above mentioned it appears that the BCL2
family of proteins play a role in regulating cell death or cell
survival pathways intracellularly and a shift in the balance from
proteins of this family that actively block apoptosis to those that
promote apoptosis or inhibit anti-apoptotic activity may result in
increased cell death, and likewise, a shift in the balance the
other way may result in increased cell survival.
[0022] Accordingly, when it is desired to increase cell death by
increasing apoptosis in cells under the circumstances noted above,
it would be desirable to block the activity of BCL2, BCL-X.sub.L
and other members of this family which suppress or inhibit
apoptosis, or to increase the activity of BAX, BAK, BAD,
BCL-X.sub.S and other members of this family which promote
apoptosis or inhibit anti-apoptotic activities of BCL2 or
BCL-X.sub.L. Likewise, when it is desired to increase cell survival
in cells by decreasing apoptosis, it would be desirable to increase
the activity of BCL2, BCL-X.sub.L and other members of this family
that suppress or inhibit apoptosis, or to decrease the activity of
apoptosis promoters of this family as noted above.
SUMMARY OF THE INVENTION
[0023] It is an object of the present invention to provide novel
proteins, including isoforms, analogs, fragments or derivatives
thereof which are capable of modulating the intracellular signaling
pathways leading to inflammation, cell death or cell survival, this
modulation being possibly via the prodomain (CARD) of the various
caspases or via kinase domains of the various kinases involved in
NF-.kappa.B activation. Such novel proteins of the invention would
therefore possibly be direct modulators of caspase activity (cell
death pathway) and/or NF-.kappa.B activation via kinase activity
(cell survival pathway). Likewise, the novel proteins of the
invention are possibly indirect modulators of the intracellular
biological activity of a variety of other proteins involved in the
inflammation, cell death or survival pathways (e.g., FAS/APO1, p55
TNF-R, p75 TNF-R, IL-1-R, MORT-1, TRADD, RIP, TRAF2, NIK, and
others). Likewise, this modulation may also possibly be by direct
or indirect interaction with members of the BCL2 family of
proteins, the novel proteins of the present invention may be able
to modulate the activity of BCL2 or other proteins of this family
and in this sense the novel proteins of the invention may be
indirect modulators of the various caspases, which, in turn, are
modulated by members of the BCL2 family of proteins.
[0024] Another object of the invention is to provide antagonists
(e.g., antibodies, peptides, organic compounds, or even some
isoforms) to the above novel proteins, including isoforms, analogs,
fragments and derivatives thereof, which may be used to inhibit the
inflammation, cell death or survival signaling processes, when
desired.
[0025] A further object of the invention is to use the above novel
proteins, isoforms, analogs, fragments and derivatives thereof, to
isolate and characterize additional proteins or factors, which may
be involved in regulation of the inflammation, cell death or
survival pathways and influence their activity, and/or to isolate
and identify other receptors or other cellular proteins 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.
[0026] A still further object of the invention is to provide
inhibitors which can be introduced into cells to bind or interact
with the novel proteins and possible isoforms thereof, which
inhibitors may act to inhibit inflammation, cell death or survival
processes when desired.
[0027] Moreover, it is an object of the present invention to use
the above-mentioned novel 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.
[0028] Furthermore, these antibodies may be used for diagnostic
purposes, e.g., for identifying possible disorders related to
abnormal functioning of cellular effects mediated directly by
caspases, kinases, proteins belonging to the BCL2 family, or TRAF
proteins or mediated by the p55 TNF receptor, FAS/APO1 receptor, or
other related receptors and their associated cellular proteins
(e.g., RAIDD, MORT-1, TRADD, RIP), which act directly or indirectly
to modulate/mediate intracellular processes via interaction with
TRAF proteins, caspases, kinases, or members of the BCL2 family of
proteins.
[0029] A further object of the invention is to provide
pharmaceutical compositions comprising the above novel proteins,
isoforms, or analogs, fragments or derivatives thereof, as well as
pharmaceutical compositions comprising the above noted antibodies
or other antagonists.
[0030] In accordance with the present invention, a new protein
designated B1, (originally designated CBK for "c-IAP-binding
kinase", due to its having some homology with c-IAP, see Example 1
below, but, hereinafter will be called "B1"), has been isolated
which has a prodomain (CARD) region, a kinase domain and an
intermediate region between said CARD and kinase domains, and hence
is possibly involved in the modulation of inflammation, cell death
and cell survival processes as detailed herein below. As is also
explained herein below, the modulation by B1 of cell death or
survival pathways may be positive (augmentory/enhancing) or
negative (inhibitory) depending on the type of intracellular
proteins with which it interacts.
[0031] Accordingly, the present invention provides a DNA sequence
encoding a B1 protein, isoforms, fragments, or analogs thereof,
said B1, isoforms, fragments or analogs thereof being capable of
interacting with intracellular mediators or modulators of
inflammation, cell death or cell survival pathways directly or
indirectly, said B1, isoforms, fragments or analogs being
intracellular modulators of said intracellular inflammation, cell
death and/or cell survival pathways.
[0032] Embodiments of the above DNA sequence of the invention
include:
[0033] (i) A DNA sequence selected from the group consisting
of:
[0034] (a) a cDNA sequence derived from the coding region of a
native B1 protein;
[0035] (b) a fragment of a sequence of (a) which encodes a
biologically active protein capable of modulating the inflammation,
cell death or cell survival pathway, or both;
[0036] (c) a DNA sequence capable of hybridization to a sequence of
(a) or (b) under moderately stringent conditions and which encodes
a biologically active B1 protein, analog or fragment capable of
modulating the intracellular inflammation, death or cell survival
pathway, or both;
[0037] (d) a DNA sequence which is degenerate as a result of the
genetic code to the DNA sequences defined in (a)-(c) and which
encodes a biologically active B1 protein, analog or fragment
capable of modulating the inflammation, cell death or cell survival
pathway or both.
[0038] (ii) A DNA sequence as above, comprising at least part of
the sequence depicted in FIG. 3 and encoding at least one active B1
protein, isoform, analog or fragment.
[0039] (iii) A DNA sequence as above, encoding a B1 protein,
isoform, analog or fragment having at least part of the amino acid
sequence depicted in FIG. 3.
[0040] In another aspect, the invention provides a vector
comprising any of the above DNA sequences of the invention, capable
of being expressed in host cells selected from prokaryotic and
eukaryotic cells; and the transformed prokaryotic and eukaryotic
cells containing said vector.
[0041] By way of another aspect of the invention, there is provided
a B1 protein, isoforms, fragments, functional analogs and
derivatives thereof, encoded by a DNA sequence of the invention, as
above, said protein, isoforms, fragments, analogs and derivatives
thereof, possibly being capable of modulating the intracellular
inflammation, cell death or cell survival pathways, or both,
directly or indirectly, by association with other intracellular
modulators or mediators of these pathways.
[0042] An embodiment of the protein of the invention is, a B1
protein, isoform, fragment, analogs and derivatives thereof,
wherein said protein, isoform, analogs, fragments and derivatives
have at least part of the amino acid sequence depicted in FIG.
3.
[0043] The invention also provides a method for producing a B1
protein, isoform, fragment, analog or derivative thereof, as above,
which comprises growing the aforesaid transformed host cells under
conditions suitable for the expression of said protein, isoform,
fragment, analog or derivative thereof, effecting
post-translational modification, as necessary, for obtaining said
protein, isoform, fragment, analog or derivative thereof, and
isolating said expressed protein, isoform, fragment, analog or
derivative.
[0044] In a further aspect, the invention provides antibodies or
active fragments or derivatives thereof, specific for the B1
protein, isoform, analog, fragment or derivative thereof of the
invention.
[0045] In an additional aspect, the invention provides for various
methods for the modulation of intracellular signaling pathways, for
example, the following:
[0046] (i) A method for the modulation or mediation in cells of the
activity of inflammation, cell death or cell survival pathways or
any other intracellular signaling activity modulated or mediated
directly or indirectly by B1 or by other molecules to which a B1
protein, isoform, analog, fragment or derivative thereof of the
invention binds or otherwise interacts, directly or indirectly,
said method comprising treating said cells by introducing into said
cells one or more of said B1 protein, isoform, analog, fragment or
derivative thereof in a form suitable for intracellular
introduction thereof, or introducing into said cells a DNA sequence
encoding said one or more B1 protein, isoform, analog, fragment or
derivative thereof 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 as above, wherein said treating of cells
comprises introducing into said cells a DNA sequence encoding said
B1 protein, isoform, fragment, analog or derivative 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 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 said cells to be treated and a second sequence encoding
a protein selected from said B1 protein, isoforms, analogs,
fragments and derivatives as above, that when expressed in said
cells is capable of modulating/mediating the activity of the
inflammation, cell death or cell survival pathways, directly or
indirectly, or any other intracellular signaling activity
modulated/mediated by other intracellular molecules with which said
B1 protein, isoforms, analogs, fragments and derivatives interact
directly or indirectly; and
[0050] (b) infecting said cells with said vector of (a).
[0051] (iv) A method for modulating the inflammation, cell death or
cell survival pathways in cells which are modulated directly or
indirectly by B1, comprising treating said cells with antibodies or
active fragments or derivatives thereof, as above, said treating
being by application of a suitable composition containing said
antibodies, active fragments or derivatives thereof to said cells,
wherein when the B1 protein or portions thereof of said cells are
exposed on the extracellular surface, said composition is
formulated for extracellular application, and when said B1 proteins
are intracellular said composition is formulated for intracellular
application.
[0052] (v) A method for modulating the inflammation, cell death,
cell survival or other pathways in cells which are modulated
directly or indirectly by B1, comprising treating said cells with
an oligonucleotide sequence which is an antisense sequence for at
least part of the DNA sequence encoding a B1 protein of the
invention, said oligonucleotide sequence being capable of blocking
the expression of the B1 protein.
[0053] (vi) A method as above wherein said oligonucleotide sequence
is introduced to said cells via a virus noted in (ii) above,
wherein said second sequence of said virus encodes said
oligonucleotide sequence.
[0054] (vii) A method for modulating the inflammation, cell death,
cell survival or other pathways in which cells are modulated
directly or indirectly by B1, comprising applying the ribozyme
procedure in which a vector encoding a ribozyme sequence capable of
interacting with a cellular mRNA sequence encoding a B1 protein of
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 B1 protein in
said cells.
[0055] In a different aspect, the present invention provides for a
method for isolating and identifying proteins, of the invention,
having homology with or being capable of direct or indirect
interactions with any proteins having a prodomain or caspase
recruiting domain (CARD), or other proteins or enzymes involved in
intracellular signaling, via the kinase or intermediate domains
present in the proteins of the invention, comprising applying the
yeast two-hybrid procedure in which a sequence encoding said
protein with said CARD, kinase, and intermediate domains, or at
least one of these domains, is carried by one hybrid vector and a
sequence from a cDNA or genomic DNA library is carried by the
second hybrid vector, the vectors then being used to transform
yeast host cells and the positive transformed cells being isolated,
followed by extraction of the said second hybrid vector to obtain a
sequence encoding a protein which binds to said CARD-, kinase-,
and/or intermediate domain-containing protein.
[0056] In a yet further aspect of the present invention, there is
provided a pharmaceutical composition for the modulation of the
inflammation, cell death, cell survival or other pathways in cells
which are modulated directly or indirectly by B1, comprising, as
active ingredient, at least one B1 protein, of the invention, its
biologically active fragments, analogs, derivatives or mixtures
thereof.
[0057] An embodiment of the above pharmaceutical composition is one
for modulating the inflammation, cell death, cell survival or other
pathways in cells which are modulated directly or indirectly by B1,
comprising, as active ingredient, a recombinant animal virus vector
encoding a protein capable of binding a cell surface receptor and
encoding at least one B1 protein, isoform, active fragments or
analogs.
[0058] Another embodiment of the above pharmaceutical composition
is one for modulating the inflammation, cell death, cell survival
or other pathways in cells which are modulated directly or
indirectly by B1, comprising as active ingredient, an
oligonucleotide sequence which is an anti-sense sequence of the B1
protein mRNA sequence.
[0059] A further embodiment of the above pharmaceutical composition
is one for the prevention or treatment of a pathological condition
associated with the regulation of apoptosis by one or more
molecules to which a B1 protein binds directly or indirectly, said
composition comprising an effective amount of a B1 protein or a DNA
molecule coding therefor, or a molecule capable of disrupting the
direct or indirect interaction of said B1 protein with one or more
molecules to which a B1 protein binds or with which it
interacts.
[0060] A still further embodiment of the above pharmaceutical
composition is one for the prevention or treatment of a
pathological condition associated with the regulation of apoptosis
by one or more molecules to which a B1 protein binds directly or
indirectly, said composition comprising an effective amount of a B1
protein, isoform, fragment, analog or derivative thereof, or a DNA
molecule coding therefor, or a molecule capable of disrupting the
direct or indirect interaction of said B1 protein, isoform,
fragment, analog or derivative thereof with one or more molecules
to which said B1 protein, isoform, fragment, analog or derivative
binds.
[0061] An additional embodiment of the above pharmaceutical
composition is one for the prevention or treatment of a
pathological condition associated with the regulation of apoptosis
by one or more molecules to which the B1 protein binds directly or
indirectly, said composition comprising a molecule capable of
interfering with the protein kinase activity of B1.
[0062] In another different aspect of the invention there are
provided therapeutic methods as follows:
[0063] (i) A method for the prevention or treatment of a
pathological condition associated with the regulation of apoptosis
by one or more molecules to which a B1 protein binds directly or
indirectly, said method comprising administering to a patient in
need an effective amount of a protein or isoform, fragment, analog
and derivative thereof or a mixture of any thereof, or a DNA
molecule coding therefor, or a molecule capable of disrupting the
direct or indirect interaction of said B1 protein or isoform,
fragment, analog and derivative thereof or a mixture of any thereof
with one or more molecules to which said B1 protein or isoform,
fragment, analog and derivative thereof or a mixture of any thereof
binds directly or indirectly.
[0064] (ii) A method of modulating inflammation processes,
apoptotic processes or programmed cell death processes (cell death
pathways) in which the B1 protein is involved directly or
indirectly, comprising treating said cells with one or more B1
proteins, isoforms, analogs, fragments or derivatives, wherein said
treating of said cells comprises introducing into said cells said
one or more B1 proteins, isoforms, analogs, fragments or
derivatives in a form suitable for intracellular introduction
thereof, or introducing into said cells a DNA sequence encoding
said one or more B1 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.
[0065] (iii) A method of modulating cell survival processes in
which the B1 protein is involved directly or indirectly, comprising
treating said cells with one or more B1 proteins, isoforms,
analogs, fragments or derivatives, wherein said treating of cells
comprises introducing into said cells said one or more B1 proteins,
isoforms, analogs, fragments or derivatives in a form suitable for
intracellular introduction thereof, or introducing into said cells
a DNA sequence encoding said one or more B1 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.
[0066] A still further aspect of the present invention, the
following screening methods and methods for the identification and
production of various ligands are provided:
[0067] (i) A method for screening of a ligand capable of binding to
a B1 protein comprising contacting an affinity chromatography
matrix to which said protein is attached with a cell extract
whereby the ligand is bound to said matrix, and eluting, isolating
and analyzing said ligand.
[0068] (ii) A method for screening of a DNA sequence coding for a
ligand capable of binding to a B1 protein, comprising applying the
yeast two-hybrid procedure in which a sequence encoding said B1
protein is carried by one hybrid vector and sequences from a cDNA
or genomic DNA library are carried by the second hybrid vector,
transforming yeast host cells with said vectors, isolating the
positively transformed cells, and extracting said second hybrid
vector to obtain a sequence encoding said ligand.
[0069] (iii) A method for identifying and producing a ligand
capable of modulating the cellular activity modulated/mediated by
B1 comprising:
[0070] (a) screening for a ligand capable of binding to a
polypeptide comprising at least a portion of B1 having at least
some of the amino acid residues of B1 depicted in FIG. 3, which
include essentially all of the prodomain (or CARD) of B1;
[0071] (b) identifying and characterizing a ligand, other than
BCL2, TRAF2, or portions of a receptor of the TNF/NGF receptor
family or other known proteins having a prodomain (CARD), found by
said screening step to be capable of said binding; and
[0072] (c) producing said ligand in substantially isolated and
purified form.
[0073] (iv) A method for identifying and producing a ligand capable
of modulating the cellular activity modulated or mediated by a B1
protein comprising:
[0074] (a) screening for a ligand capable of binding to a
polypeptide comprising at least the carboxy terminal portion of the
B1 sequence depicted in FIG. 3 including the prodomain (CARD);
[0075] (b) identifying and characterizing a ligand, other than
BCL2, TRAF2, or portions of a receptor of the TNF/NGF receptor
family or other known proteins having a prodomain (CARD), found by
said screening step to be capable of said binding; and
[0076] (c) producing said ligand in substantially isolated and
purified form.
[0077] (v) A method for identifying and producing a ligand capable
of modulating the cellular activity modulated/mediated by B1
comprising:
[0078] (a) screening for a ligand capable of binding to at least
the N-terminal portion of the B1 sequence depicted in FIG. 3
including substantially all of the kinase domain of B1;
[0079] (b) identifying and characterizing a ligand, other than
BCL2, TRAF2, or portions of a receptor of the TNF/NGF receptor
family or other known intracellular modulatory proteins, found by
said screening step to be capable of said binding; and
[0080] (c) producing said ligand in substantially isolated and
purified form.
[0081] (vi) A method for identifying and producing a molecule
capable of directly or indirectly modulating the cellular activity
modulated/mediated by B1, comprising:
[0082] (a) screening for a molecule capable of modulating
activities modulated/mediated by B1;
[0083] (b) identifying and characterizing said molecule; and
[0084] (c) producing said molecule in substantially isolated and
purified form.
[0085] (vii) A method for identifying and producing a molecule
capable of directly or indirectly modulating the cellular activity
modulated/mediated by a protein of the invention, comprising:
[0086] (a) screening for a molecule capable of modulating
activities modulated/mediated by a protein of the invention;
[0087] (b) identifying and characterizing said molecule; and
[0088] (c) producing said molecule in substantially isolated and
purified form.
[0089] Other aspects of the invention will be apparent from the
following Detailed Description of the Invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0090] FIG. 1 shows a diagrammatic illustration of the structure of
the TRAF2 molecule.
[0091] FIG. 2 shows a schematic diagram illustrating some of the
proteins involved in inflammation, cell death and cell survival
(NF-.kappa.B activation) pathways.
[0092] FIGS. 3A-3B show schematically the deduced amino acid
sequence (FIG. 3A) (SEQ ID NO:1) of the B1 protein of the present
invention and the determined nucleotide sequence coding therefor
(FIG. 3B) (SEQ ID NO:2), wherein in the amino acid sequence is
shown the kinase domain of B1 (boxed region at N-terminal end) and
the CARD domain of B1 (underlined region at C-terminal end).
[0093] FIG. 4 shows a Northern analysis of B1 expression in
different human tissues, which shows that B1 is expressed in most
human tissue types.
[0094] FIG. 5 shows schematically the different B1 constructs
tested for NF-.kappa.B activity, cell death potentiation and JNK
activation.
[0095] FIG. 6 shows NF-.kappa.B activation results of measurements
carried out with the different constructs of FIG. 6 and Example
3.
[0096] FIG. 7 shows the JNK1 activation results of measurements
carried out with some of the above constructs.
[0097] FIG. 8 shows that B1 self-associates and binds TRAF1 in vi
vo.
DETAILED DESCRIPTION OF THE INVENTION
[0098] The present invention relates, in one aspect to a new B1
protein that has a prodomain or CARD domain (caspase recruiting
domain) and that has a protein kinase domain of similarity to the
RIP-kinase domain. As such the B1 protein of the present invention
is possibly-capable of interacting with a number of intracellular
proteins involved in the inflammation, cell death (apoptosis) and
cell survival (NF-.kappa.B activation) pathways. This interaction
may be by binding various proteins or otherwise interacting with
them via the prodomain (CARD), or it may be by way of the activity
of the kinase domain, or both of these types of interaction may
occur at the same time. For example, B1 may be able to recruit a
number of proteins having prodomains (CARDs) and then to
phosphorylate them via its kinase domain. Likewise, B1 may serve in
some instances as a docking or recruiting protein via its prodomain
(CARD) for various other prodomain-containing proteins, which may
not be substrates for the kinase domain of B1, or B1 may interact
with various proteins only through its kinase domain and not via
its CARD domain.
[0099] In addition, as is detailed herein, binding assay results
indicate that the new B1 protein of the invention is possibly
capable of binding to the BCL2 protein. This finding raises the
possibility that the B1 protein may be a regulator of BCL2
activity, especially as concerns the regulation of apoptosis. In
initial biological activity analyses, the possibility also arises
that the B1 protein may inhibit the protective effect of BCL2
against apoptosis. This in view of the observations that the B1
protein on its own does not cause cell death, but acts to enhance
cell death when added to cells with other inducers of cell death
such as, for example, FAS-R, p55 TNF-R and RIP (said addition to
cells by co-transformation with vectors capable of expressing in
the cells B1, FAS-R, p55 TNF-R or RIP, see Example 2 below). Hence,
the possibility arises that B1 may not act in an analogous way to
BAX or BAK, which on their own, in the form of homodimers, can
cause cell death (see "Background" section above), but rather, B1
may possibly act in an analogous way to BAD which serves to
negatively regulate BCL2 by binding BCL2 and preventing its binding
to BAX or BAK thereby resulting in more free BAX and/or BAK which,
in turn, cause increased cell death (see `Background` section
above).
[0100] Moreover, with respect to the above noted activation of
NF-.kappa.B and cell survival, B1 may possibly also achieve its
observed activity of enhancing cell death by way of possibly
causing a reduction in NF-.kappa.B activation, maybe by way of B1's
kinase activity which may possibly serve to modulate various
proteins (e.g., NIK) necessary for induction of NF-.kappa.B
activation, with the result that reduced NF-.kappa.B activation
will occur and subsequently cell survival will be reduced. In this
respect it is interesting to note that when B1 is added with
inducers of cell death such as FAS-R, p55 TNF-R or RIP it enhances
their cell killing activity. It is known that both p55 TNF-R and
FAS-R, and possibly also RIP besides inducing the cell death
pathways culminating in increased caspase activity, also induce
activation of NF-.kappa.B which, to some extent, negates the
induced cell death. In some cells it has even been observed that
TNF does not kill the cells, this being attributed to the induction
of NF-.kappa.B activation by the TNF receptors and not the failure
of the co-induced cell death pathways by these receptors, so that
in these cells the NF-.kappa.B-mediated cell survival pathways are
apparently more active than the cell death pathways. Thus, by
blocking NF-.kappa.B induction it would be possible to enhance the
cell killing mediated by, for example, FAS-R, p55 TNF, RIP, and the
B1 protein of the invention may possibly serve this function and
give rise to its observed enhancement of cell death when added with
FAS-R, p55-TNF-R or RIP.
[0101] In view of the above, it arises that B2 may possibly
regulate inflammation, cell death or cell survival processes in a
number of ways, and may even do so simultaneously. For example, B2
may possibly inhibit NF-.kappa.B activation, or B1 may possibly
even act on other intracellular proteins involved in the cell death
or cell survival pathways independently of its possible effects on
NF-.kappa.B or in addition to its possible effects on
NF-.kappa.B.
[0102] Hence, B1 appears to possibly have the capability to
modulate a wide range of intracellular proteins, in particular,
those involved in the inflammation, cell death and cell survival
pathways. As is detailed herein below, a number of known
intracellular proteins have prodomains (CARDs), such as, for
example, various caspase enzymes involved in the proteolytic
destruction of cells (cell death pathway) including ICE, ICH-1,
Mch6 and others, as well as various adaptor proteins also involved
in cell death pathways including RAIDD, c-IAP1, c-IAP2 and others.
In this way B1 may possibly interact directly or indirectly with
various caspases via their common CARDs and thereby possibly
modulate their activity. This modulation may be positive, namely,
B1 may possibly serve to concentrate various caspases and thereby
enhance their proteolytic activity leading to increased cell death.
Further, B1 was isolated using the sequence of c-IAP1 and B1 shares
homology with c-IAP1, which itself is an inhibitor of apoptosis.
c-IAP1 has a prodomain (CARD) and may inhibit apoptosis by
recruiting caspases thereby preventing their activity. B1 may
therefore possibly interact directly or indirectly with c-IAP1 and
lead to a suppression of its inhibition of apoptosis, thereby
resulting in increased cell death. Moreover, B1 possibly by
indirectly or direct interaction with various caspases via its
CARD, may also be able to modulate them by phosphorylation via its
kinase domain, and in this way B1 may enhance the caspase
activity.
[0103] In a more indirect fashion, B1 by possibly being able to
interact indirectly or directly with adaptor proteins such as
RAIDD, c-IAP1, c-IAP2 and others having CARDs can thereby possibly
interact with other proteins more upstream in the inflammation,
cell death and cell survival pathways. For example, RAIDD interacts
with other intracellular proteins such as RIP and TRADD via common
death domains, which, in turn interact with proteins such as
MORT-1, the p55-TNF-R and FAS-R. In this way, by possibly
interacting with RAIDD, B1 is thus possibly indirectly linked to
these death-effecting receptors and proteins. Similarly, c-IAP1 and
c-IAP2 interact with the TRAF2 protein, which, in turn, interacts
with the p75-TNF-R, and with MORT-1, p55-TNF-R and FAS-R via the
interaction between TRAF2 and RIP as well as TRADD. Accordingly, B1
may possibly be an indirect modulator of cell death processes by
being indirectly linked to the above noted adaptor proteins,
effector proteins and receptors. This indirect modulation may be
positive, i.e., it may lead to enhanced cell death.
[0104] Moreover, by virtue of B1 possibly being able to interact at
least indirectly (via c-IAP1) with TRAF2 raises the possibility of
the involvement of B1 in the cell survival pathway which is
associated with the induction of NF-.kappa.B activation. It is now
known that TRAF2 binds directly to NIK (Malinin et al, 1997), which
is directly involved in the induction of NF-.kappa.B activation and
thereby cell survival. Accordingly, by possibly being able to
modulate TRAF2 indirectly, B1 may be capable of modulating the cell
survival pathway as well. Further, by virtue of its kinase domain
B1 may possibly be even more directly involved in the MAP kinase
pathway (to which NIK belongs) leading to induction of NF-.kappa.B
activation and cell survival. However, as noted above, in view of
the fact that B1 leads to an enhancement of cell death, it may be
that B1 has a negative role in the modulation of the cell survival
process, namely, B1 may possibly modulate TRAF2 or B1 may possibly
be directly involved in the MAP kinase pathway but in a way that
leads to reduced NF-.kappa.B activation.
[0105] It is also possible that B1 plays a central role in the
modulation of intracellular signaling pathways, in particular, the
inflammation, cell death and cell survival pathways, and as such B1
may serve to modulate these in a way that may shift the balance
from cell survival to cell death induction in agreement with the
observed (see Example 2) enhancing effect B1 has on cell death
induction. B1 may thus be considered as a `modulator of
intracellular signaling` activity directly or indirectly on various
component proteins making up these pathways.
[0106] Thus, when considering the various possible uses of B1
therapeutically, it is important to understand that in all cases B1
may have multiple roles, namely, it may enhance cell death
processes, and at the same time it may possibly actively inhibit
the induction of NF-.kappa.B and hence inhibit the cell survival
pathway, or depending on the actual proteins/enzymes to which B1
binds and their relative amounts in the cell, B1 may possibly, in
some cells, act to inhibit cell survival pathways, and in others,
may possibly act to enhance cell death pathways by suppressing cell
death inhibitors.
[0107] Therefore, in general, as will arise from the following,
when it is desired to increase cell death, e.g., in tumors,
HIV-infected cells and the like, it may be possible to use B1 to
achieve this goal. For example, B1 may be administered to the cells
directly or a DNA molecule encoding B1 may be introduced into the
cells to increase B1 expression.
[0108] Likewise, in situations in which it is desired to save cells
from cell death induced by TNF or FAS-ligand, for example, in
various inflammations, autoimmune diseases, graft-vs-host reactions
and the like, and instead promote cell survival, then B1
antagonists may possibly be used to achieve this goal. For example,
B1 antagonists may be administered, such as, anti-B1 antibodies,
oligonucleotides having anti-sense B1 sequences, ribozymes with B1
sequences, or various peptides or organic molecules designed
specifically to inhibit B1 activity.
[0109] Hence, when the uses of B1 are noted herein below, they will
be set forth in terms of the modulatory effects of B1 on various
intracellular processes or diseases, and it is to be understood in
view of the above mentioned that this modulation may be positive
(augmentory) as is the case when considering cell death pathways,
or negative (inhibitory) as is the case when considering cell
survival pathways.
[0110] The present invention also concerns the DNA sequences
encoding biologically active B1 proteins, as well as DNA sequences
encoding biologically active analogs, fragments and derivatives
thereof, and the B1 proteins, analogs, fragments and derivatives of
the proteins encoded by the DNA sequences. The preparation of such
analogs, fragments and derivatives is by standard procedures (see,
for example, Sambrook et al, 1989) in which in the DNA encoding
sequences, one or more codons may be deleted, added or substituted
by another, to yield encoded analogs having at least a one amino
acid residue change with respect to the native protein. Acceptable
analogs are those which retain at least the prodomain (CARD) or
kinase domain of B1 or at least active portions of either or both
of these domains, with or without mediating any other binding or
enzymatic activity, i.e., do not bind or otherwise interact,
directly or indirectly, to a further downstream protein or other
factor, or do not catalyze a signal-dependent reaction (e.g.,
kinase reaction). 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 or otherwise interacting with other
proteins via the prodomain, or in subsequent signaling (also
possibly kinase activity) following such binding as noted above.
Such analogs can be used, for example, to modulate the
inflammation, cell death or survival pathways as noted above, by
competing with the natural B1 proteins. Likewise, so-called
dominant-positive analogs may be produced which would serve to
enhance the B1 effect. These would have the same or better
B1-related binding properties to the other proteins and the same or
better signaling properties or kinase activities of the natural B1
proteins. In an analogous fashion, biologically active fragments of
the clones of the invention may be prepared as noted above with
respect to the preparation of the analogs. Suitable fragments of
the DNA sequences of the invention are those which encode a protein
or polypeptide retaining the B1 binding capability to other
proteins or which can mediate any other binding or enzymatic
(kinase) activity as noted above. Accordingly, fragments of the
encoded proteins of the invention can be prepared which have a
dominant-negative or a dominant-positive effect as noted above with
respect to the analogs. Similarly, derivatives may be prepared by
standard modifications of the side groups of one or more amino acid
residues of the proteins, their analogs or fragments, or by
conjugation of the proteins, their analogs or fragments, to another
molecule, e.g., an antibody, enzyme, receptor, etc., as are well
known in the art.
[0111] Of the above DNA sequences of the invention which encode a
B1 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 B1 protein, in which such hybridization is
performed under moderately stringent conditions, and which
hybridizable DNA sequences encode a biologically active B1 protein.
These hybridizable DNA sequences therefore include DNA sequences
which have a relatively high homology to the native B1's cDNA
sequence, and as such represent B1-like sequences which may be, for
example, naturally-derived sequences encoding the various B1
protein isoforms, or naturally-occurring sequences encoding
proteins belonging to a group of B1-like sequences encoding a
protein having the activity of B1 proteins. Further, these
sequences may also, for example, include non-naturally occurring,
synthetically produced sequences that are similar to the native B1
protein 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 B1 proteins, all of which have the activity of B1 proteins.
[0112] To obtain the various above noted naturally occurring B1
protein-like sequences, standard procedures of screening and
isolation of naturally-derived DNA or RNA samples from various
tissues may be employed using the natural B1 protein cDNA or
portion thereof as probe (see for example standard procedures set
forth in Sambrook et al, 1989).
[0113] Likewise, to prepare the above noted various synthetic B1
protein-like sequences encoding analogs, fragments or derivatives
of B1 proteins, a number of standard procedures may be used as are
detailed herein below concerning the preparation of such analogs,
fragments and derivatives.
[0114] A polypeptide or protein "substantially corresponding" to B1
protein includes not only B1 protein but also polypeptides or
proteins that are analogs of B1 protein.
[0115] Analogs that substantially correspond to B1 protein are
those polypeptides in which one or more amino acid of the B1
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 B1 protein to which it corresponds.
[0116] In order to substantially correspond to B1 protein, the
changes in the sequence of B1 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 that substantially
correspond to B1 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
various other proteins having, for example, prodomains (CARD),
kinase binding sites, or to B1 itself, and to modulate the activity
of these other proteins or B1 itself in the modulation/mediation of
the intracellular pathways noted above.
[0117] "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.
[0118] Conservative substitutions of B1 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 B1 protein.
1TABLE 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
[0119] Alternatively, another group of substitutions of B1 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 (1979), and FIGS. 3-9 of Creighton
(1983). Based on such an analysis, alternative conservative
substitutions are defined herein as exchanges within one of the
following five groups:
2 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, He, Val (Cys) 5. Large aromatic residues: Phe, Tyr, Trp
[0120] 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 a-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 (1979) 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.
[0121] 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 that 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., .alpha.-helix or .beta.-sheet, as well as changes
in biological activity, e.g., binding to other proteins with
prodomains (CARD), or kinase activity and/or modulation of cell
death or survival pathways as noted above and below.
[0122] Examples of production of amino acid substitutions in
proteins which can be used for obtaining analogs of B1 proteins for
use in the present invention include any known method steps, such
as presented in U.S. Pat. Nos. RE 33,653, 4,959,314, 4,588,585 and
4,737,462, to Mark et al; U.S. Pat. No. 5,116,943 to Koths et al,
U.S. Pat. No. 4,965,195 to Namen et al; U.S. Pat. No. 4,879,111 to
Chong et al; and U.S. Pat. No. 5,017,691 to Lee et al; and lysine
substituted proteins presented in U.S. Pat. No. 4,904,584
(Shaw).
[0123] Besides conservative substitutions discussed above which
would not significantly change the activity of B1 protein, either
conservative substitutions or less conservative and more random
changes, which lead to an increase in biological activity of the
analogs of B1 proteins, are intended to be within the scope of the
invention.
[0124] 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.
[0125] At the genetic level, these analogs are generally prepared
by site-directed mutagenesis of nucleotides in the DNA encoding the
B1 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, (1987-1995); Sambrook et al
(1989).
[0126] Preparation of a B1 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 B1 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 (1983), the disclosure of which is incorporated
herein by reference.
[0127] 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 (1981), the disclosure
of which is incorporated herein by reference. These phages 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, 1987) may be employed to obtain single-stranded DNA.
[0128] 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-contain- ing 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 bear 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.
[0129] After such a clone is selected, the mutated B1 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.
[0130] Accordingly, gene or nucleic acid encoding for a B1 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 (1987-1995) 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.
[0131] 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 the B1 protein or a
fragment thereof to be custom designed for ligation other sequences
and/or cloning sites in vectors.
[0132] 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; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al; U.S.
Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson et
al; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 to
Gyllensten et al; U.S. Pat. No. 4,889,818 to Gelfand et al; U.S.
Pat. No. 4,994,370 to Silver et al; U.S. Pat. No. 4,766,067 to
Biswas; U.S. Pat. No. 4,656,134 to Ringold; and Innis et al (1990),
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, 1993; Sano et al, 1992; Sano et
al, 1991), the entire contents of which patents and reference are
entirely incorporated herein by reference.
[0133] In an analogous fashion, biologically active fragments of B1
or its isoforms may be prepared as noted above with respect to the
analogs of B1 proteins. Suitable fragments of B1 proteins are those
which retain at least the prodomain-related binding ability or the
kinase activity and which can mediate the biological activity of
the various other proteins or intracellular pathways associated
with B1 proteins directly or indirectly. Accordingly, B1 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 B1 proteins derived from the full B1 protein
sequence, each such portion or fragment having any of the
above-noted desired activities. Such fragment may be, e.g., a
peptide.
[0134] Similarly, derivatives may be prepared by standard
modifications of the side groups of one or more amino acid residues
of the B1 protein, its analogs or fragments, or by conjugation of
the B1 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 B1 proteins.
[0135] 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.
[0136] 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.
[0137] A B1 protein 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 B1 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
the B1 protein or can be cleaved to leave a protein or polypeptide
having the biological activity of the B1 protein. Thus, for
example, the present invention is intended to include fusion
proteins of the B1 protein with other amino acids or peptides.
[0138] The new B1 proteins, their analogs, fragments and
derivatives have a number of possible uses as noted above and
below, for example:
[0139] (i) They may be used to modulate cell survival pathways via
direct or indirect modulation of the intracellular proteins to
which they bind. In situations where an enhanced activity of these
pathways is not desired, i.e., it is desired to inhibit them in
favor of cell death pathways, for example, such as in anti-tumor or
immuno-stimulatory applications, then it is desired that this
modulation, by B1, its isoforms, analogs, fragments or derivatives
be inhibitory. In this case the proteins of the invention, their
analogs, fragments or derivatives, when they are inhibitory for
cell survival pathways, may be introduced to the cells by standard
procedures known per se. For example, as the proteins encoded by
the DNA clones of the invention are intracellular and they should
be introduced only into the cells where desired, a system for
specific introduction of these proteins 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 known receptor, such that the recombinant virus vector
will be capable of binding such cells; and the gene encoding the
proteins of the invention. 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
receptor-carrying cell, following which the proteins encoding
sequences will be introduced into the cells via the virus, and once
expressed in the cells will result in inhibition of the cell
survival pathways leading to a desired cell death or
immuno-stimulatory effect in these cells. 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 encoded proteins in the form of
oligonucleotides which can be absorbed by the cells and expressed
therein.
[0140] Similarly, when the B1 proteins, isoforms, analogs,
fragments or derivatives are stimulatory or otherwise enhance cell
death processes, then they may also be administered to cells as
above to provide increased anti-tumor, immuno-stimulatory or other
cell death activity.
[0141] (ii) They may be used to enhance or augment the cell
survival pathways, or, e.g., in cases such as tissue damage as in
AIDS, septic shock or graft-vs.-host rejection, in which it is
desired to block the cell death pathways or stimulate the cell
survival pathways. In this situation it is possible, when the B1
proteins actually inhibit cell survival processes, or are
stimulatory or otherwise augment cell death pathways, to, for
example, introduce into the cells, by standard procedures,
oligonucleotides having the anti-sense coding sequence for the B1
proteins of the invention, which would effectively block the
translation of mRNAs encoding the proteins and thereby block their
expression and lead to the inhibition of the (cell death) undesired
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.
[0142] Another possibility is to use antibodies specific for the
proteins of the invention to inhibit their intracellular signaling
activity.
[0143] Yet another way of inhibiting the undesired 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 B1 proteins of the invention. Such ribozymes would
have a sequence specific for the mRNA of the proteins 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 proteins, 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 the sequence of the B1 proteins) 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).
[0144] (iii) They may be used to isolate, identify and clone other
proteins that are capable of binding to them, e.g., other proteins
involved in the intracellular inflammation, cell death or cell
survival pathways. For example, the DNA sequences encoding the
proteins of the invention may be used in the yeast two-hybrid
system in which the encoded proteins will be used as "bait" to
isolate, clone and identify from cDNA or genomic DNA libraries
other sequences ("preys") encoding proteins which can bind to the
clones proteins. In the same way, it may also be determined whether
the proteins of the present invention can bind to other cellular
proteins, e.g. other receptors of the TNF/NGF superfamily of
receptors, or other members of the BCL2 family.
[0145] (iv) The encoded proteins, their analogs, fragments or
derivatives may also be used to isolate, identify and clone other
proteins of the same class, i.e., those having prodomains (CARDS)
or kinase domains, or to functionally related proteins, and
involved in the intracellular signaling process. In this
application the above noted yeast two-hybrid system may be used, or
there may be used a recently developed system employing
non-stringent Southern hybridization followed by PCR cloning (Wilks
et al, 1989).
[0146] (v) Yet another approach to utilize the encoded proteins of
the invention, their analogs, fragments or derivatives 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., proteins related to B1 proteins or other proteins or factors
involved in the intracellular signaling process. In this
application, the proteins, their analogs, fragments or derivatives
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 proteins, their analogs, fragments or derivatives
of the invention, can be eluted, isolated and characterized.
[0147] (vi) As noted above, the proteins, their analogs, fragments
or derivatives 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 proteins
of the invention either from cell extracts or from transformed cell
lines producing them, their analogs or fragments. Further, these
antibodies may be used for diagnostic purposes for identifying
disorders related to abnormal functioning of the receptor system or
inflammation, cell death or survival pathways in which they
function. Thus, should such disorders be related to a
malfunctioning intracellular signaling system involving the
proteins of the invention, such antibodies would serve as an
important diagnostic tool. 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, such as, for example, Fab and F(ab').sub.2 fragments
lacking the Fc fragment of intact antibody, which are capable of
binding antigen.
[0148] (vii) The antibodies, including fragments of antibodies,
useful in the present invention may be used to quantitatively or
qualitatively detect the clones of the invention in a sample, or to
detect presence of cells which express the clones of the present
invention. This can be accomplished by immunofluorescence
techniques employing a fluorescently labeled antibody coupled with
light microscopic, flow cytometric, or fluorometric detection.
[0149] 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 clones
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 clones, 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.
[0150] Such assays for the clones 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
capably of identifying the encoded proteins, and detecting the
antibody by any of a number of techniques well known in the
art.
[0151] (viii) The encoded proteins of the invention may also be
used as indirect modulators of a number of other proteins by virtue
of their capability of binding to other intracellular proteins,
which other intracellular proteins directly bind yet other
intracellular proteins or an intracellular domain of a
transmembrane protein.
[0152] For the purposes of modulating these other intracellular
proteins or the intracellular domains of transmembranal proteins,
the proteins of the invention may be introduced into cells in a
number of ways as mentioned hereinabove in (i) and (ii).
[0153] It should also be noted that the isolation, identification
and characterization of the proteins 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 which was used to identify the proteins
of the invention. Likewise 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
proteins of the invention or to isolate, identify and characterize
additional proteins, factors, receptors, etc. which are capable of
binding to the proteins of the invention.
[0154] Moreover, the proteins found to bind to the proteins of the
invention may themselves be employed, in an analogous fashion to
the way in which the proteins of the invention were used as noted
above and below, to isolate, identify and characterize other
proteins, factors, etc. which are capable of binding to the
proteins of the invention-binding proteins and which may represent
factors involved further downstream in the associated signaling
process, or which may have signaling activities of their and hence
would represent proteins involved in a distinct signaling
process.
[0155] The DNA sequences and the encoded proteins of the invention
may be produced by any standard recombinant DNA procedure (see for
example, Sambrook et al, 1989) in which suitable eukaryotic or
prokaryotic host cells 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 are the derivatives
produced by standard modification of the proteins or their analogs
or fragments, produced by the transformed hosts.
[0156] The present invention also relates to pharmaceutical
compositions for modulation of the effects mediated by B1. The
pharmaceutical compositions comprising, as an active ingredient,
any one or more of the following: (i) one or more of the DNA
sequences of the invention, or parts of them, subcloned into an
appropriate expression vector; (ii) a protein according to the
invention, its biologically active fragments, analogs, derivatives
or a mixture thereof; (iii) a recombinant animal virus vector
encoding for a protein according to the invention, its biologically
active fragments, analogs or derivatives.
[0157] The pharmaceutical compositions are applied according to the
disease to be treated and in amounts beneficial to the patient,
depending on body weight and other considerations, as determined by
the physician.
[0158] As noted above, B1 may possibly be an indirect modulator of
TRAF2, and as such it may possibly be involved in NF-.kappa.B
activation via the TRAF2-NIK-interaction. B1 thus has a possible
role in cell survival pathways in ways that TRAF2 functions
independently or in conjunction with other proteins (e.g., p55 TNF
and p75 TNF receptors, FAS/APO1 receptor, MORT-1, RIP and TRADD).
In this respect, there has been recognized the importance to design
drugs which may enhance or inhibit the TRAF2-NIK interaction, as
desired. For example, when it is desired to increase the cell
cytotoxicity induced by TNF it would be desired to inhibit
NF-.kappa.B induction, by inhibiting the TRAF2-NIK interaction or
by inhibiting TRAF2 and/or NIK specifically. Likewise, for example,
when it is desired to inhibit the cell cytotoxicity induced by TNF
it would be desired to enhance NF-.kappa.B induction by enhancing
the TRAF2-NIK interaction or by enhancing TRAF2- and/or
NIK-specific NF-.kappa.B induction. There are many diseases in
which such drugs can be of great help. Amongst others, (see above
discussion as well) acute hepatitis in which the acute damage to
the liver seems to reflect FAS/APO1 receptor-mediated death of the
liver cells following induction by the Fas ligand;
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.
[0159] In such cases, it would be desired to inhibit the FAS/APO1
receptor-mediated cell cytotoxicity (apoptosis) pathway and enhance
the FAS/APO1 receptor-mediated induction of NF-.kappa.B via TRAF2
and the TRAF2-NIK interaction. One way of doing this would be to
increase the amount of NIK in the cells or to increase the amount
of TRAF2 and NIK so that the NIK- or TRAF2-NIK-mediated induction
of NF-.kappa.B activation will be increased providing higher levels
of NF-.kappa.B activation and hence cell survival; or so that the
direct or indirect interaction between FAS/APO1 receptor and TRAF2
(or TRAF2-NIK) will be increased resulting in a decrease in
FAS/APO1 receptor interactions with cell cytotoxic mediators (e.g.,
MACH, see scheme in FIG. 2) to provide for an increase in the
induction of NF-.kappa.B activation and cell survival.
[0160] Conversely, in the case of, for example, tumors and infected
cells (see also discussion above) it would be desired to increase
the FAS/APO1 receptor-mediated cell cytotoxicity to bring about
increased cell death. In this case it would be desired to inhibit
FAS/APO1 receptor-TRAF2 (or -TRAF2-NIK) interactions and/or to
inhibit NIK directly, and thereby to decrease the induction of
NF-.kappa.B activity.
[0161] As the B1 protein of the invention may possibly have an
interaction with TRAF2, it may be possible to enhance or to block
this interaction and thereby to enhance or to inhibit the activity
of TRAF2, in particular, the TRAF2-interaction with NIK and the
associated induction of NF-.kappa.B activation. Enhancement or
inhibition of the interaction between B1 and TRAF2 may possibly be
direct or via other proteins (e.g., c-IAP1, c-IAP2) which bind to
TRAF2 and which possibly interact with B1 directly or indirectly.
Thus, by focusing on the B1 protein and modulating its possible
interaction (direct or indirect) with TRAF2 it is possible also to
modulate the activity of TRAF2 and thereby also the effects of
FAS/APO1 (FAS-R) as well as p55-TNF-R as noted above.
[0162] As also noted above, B1 may possibly act directly on the
mediators of cell death, namely, various caspase enzymes whose
proteolytic activity leads to cell death. Accordingly, the above
mentioned FAS/APO1 (FAS-R) or p55 TNF-R effects may be modulated
directly or indirectly by B1 via B1's possible modulation of the
caspases (e.g., MACH and others) which are associated with p55
TNF-R, FAS-R or its binding protein MORT-1 and which apparently
effect the apoptotic reactions mediated thereby. Thus if B1
interacts with these caspases in a way that enhances their
activity, then such an interaction should be augmented when cell
death is desired as noted above, or should be inhibited when cell
death is not desired as noted above.
[0163] Thus, in view of the above various substances such as
peptides, organic compounds, antibodies, etc. may be screened to
obtain specific drugs which are capable of inhibiting the possible
interaction between B1 and the various other proteins, when such an
interaction is not desired. Such drugs are likely to be those which
specifically recognize the prodomain (CARD) of B1, for example,
peptides, organic molecules, antibodies or antibody fragments,
which bind specifically to the B1 CARD and prevent it interacting
with other CARD-containing proteins. Conversely, when such an
interaction between B1 and the other proteins is desired, then this
may be enhanced by increasing the amounts of B1 in the cells by
standard procedures noted in (i) above. Here too, it may also be
possible to screen for various specific drugs that may be capable
of enhancing the activity of B1 intracellularly or of enhancing its
interaction with other proteins.
[0164] Additionally, as noted above, B1 also has a kinase domain
that may be involved in its modulatory effects of inflammation,
cell death or survival pathways. Accordingly, this kinase domain
may serve to bind and phosphorylate various proteins and thereby
increase or decrease their activity and in this way increase or
decrease the activity of the inflammation, cell death or cell
survival pathways, as the case may be. Accordingly, various
peptides, organic compounds, antibodies, etc., may be screened to
obtain specific drugs which are capable of inhibiting the kinase
activity of B1 when this is desired either for inhibiting or
enhancing inflammation, cell death or survival pathways.
[0165] A non-limiting example of how peptide inhibitors of the B1
interaction with other proteins via its prodomain or kinase domain,
as noted above, 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 a 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) (SEQ ID NO:3)
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 apoptotic 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.
[0166] 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, etc. This
method of Geysen's was shown to be capable of testing at least 4000
peptides each working day.
[0167] In a similar way the exact binding region or region of
homology which determines the interaction between B1 and other
proteins 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 B1 for binding
to, or otherwise interacting with other proteins of the cell death
or cell survival pathways, via the CARD or kinase domains, or even
the intermediary domain of B1 between its CARD and kinase
domains.
[0168] Since it may be advantageous to design peptide inhibitors
that selectively inhibit B1 interactions without interfering with
physiological cell death or survival processes in which other
members of the intracellular signaling pathways are involved, the
pool of peptides binding to B1 in an assay such as the one
described above can be further synthesized as a fluorogenic
substrate peptide to test for selective binding of B1 to such other
proteins to select only those specific for B1. Peptides which are
determined to be specific for, for example, the CARD or kinase
domain of B1, can then be modified to enhance cell permeability and
modulate inflammation, cell death or cell survival processes,
reversibly or irreversibly. Thornberry et al (1994) reported that a
tetrapeptide (acyloxy) methyl ketone Ac-Tyr-Val-Ala-Asp-CH.sub.2OC-
(O)-[2,6-(CF.sub.3).sub.2]Ph was a potent inactivator of ICE.
Similarly, Milligan et al (1995) reported that tetrapeptide
inhibitors having a chloromethylketone (irreversibly) or aldehyde
(reversibly) groups inhibited ICE. In addition, a
benzyloxycarboxyl-Asp-CH.sub.2OC(O)-2,6-dic- hlorobenzene (DCB) was
shown to inhibit ICE (Mashima et al, 1995). Accordingly, in an
analogous way, tetrapeptides that selectively bind to, for example,
the CARD or kinase domain of B1, can be modified with, for example,
an aldehyde group, chloromethylketone, (acyloxy) methyl ketone or a
CH.sub.2OC(O)-DCB group to create a peptide modulator of B1
activity. Further, to improve permeability, peptides can be, for
example, chemically modified or derivatized to enhance their
permeability across the cell membrane and facilitate the transport
of such peptides through the membrane and into the cytoplasm.
Muranishi et al (1991) reported derivatizing thyrotropin-releasing
hormone with lauric acid to form a lipophilic lauroyl derivative
with good penetration characteristics across cell membranes.
Zacharia et al (1991) also reported the oxidation of methionine to
sulfoxide and the replacement of the peptide bond with its
ketomethylene isoester (COCH.sub.2) to facilitate transport of
peptides through the cell membrane. These are just some of the
known modifications and derivatives that are well within the skill
of those in the art.
[0169] Furthermore, drug or peptide inhibitors, which are capable
of inhibiting the activity of, for example, the cell death or cell
survival pathways by interfering with the possible interaction
between B1 and any of the proteins it binds to via the CARD, kinase
or intermediate domains, can be conjugated or complexed with
molecules that facilitate entry into the cell.
[0170] 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.
[0171] 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 teach 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.
[0172] ICE is known to have the ability to tolerate liberal
substitutions in the P.sub.2 position and this tolerance to liberal
substitutions was exploited to develop a potent and highly
selective affinity label containing a biotin tag (Thornberry et al,
1994). Consequently, the P.sub.2 position as well as possibly the
N-terminus of the tetrapeptide inhibitor can be modified or
derivatized, such as to with the addition of a biotin molecule, to
enhance the permeability of these peptide inhibitors across the
cell membrane.
[0173] 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.
[0174] As will be appreciated by those of skill in the art of
peptides, the peptide inhibitors of the B1 interaction with other
proteins, as noted above, according to the present invention is
meant to include peptidomimetic drugs or inhibitors, which can also
be rapidly screened for binding to the CARD, kinase or intermediate
domain of B1 to design perhaps more stable inhibitors.
[0175] 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
analogs, fragments or isoforms of B1, as well as other B1-specific
peptides and proteins (including fusion proteins) which exert their
effects intracellularly.
[0176] 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.
[0177] 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 contain substantially similar epitope binding sites.
MAbs may be obtained by methods known to those skilled in the art.
See, for example Kohler and Milstein (1975); U.S. Pat. No.
4,376,110; Ausubel et al, eds., Harlow and Lane (1988); and
Colligan et al (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.
[0178] 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,
1984; Morrison et al, 1984; Boulianne et al, 1984; Cabilly et al,
European Patent Application 125023 (published Nov. 14, 1984);
Neuberger et al, 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, WO
8601533; Kudo et al, European Patent Application 184187 (published
June 11, 1986); Sahagan et al, 1986; Robinson et al, WO8702671
(published May 7, 1987); Liu et al, 1987; Sun et al, 1987; Better
et al, 1988; and Harlow and Lane, 1988). These references are
entirely incorporated herein by reference.
[0179] An anti-idiotypic (anti-Id) antibody is an antibody that
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.
[0180] 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.
[0181] Accordingly, mAbs generated against the B1 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 B1 protein, or analogs, fragments and derivatives
thereof.
[0182] The anti-Id mAbs thus have their own idiotypic epitopes, or
"idiotopes" structurally similar to the epitope being evaluated,
such as GRB protein-a.
[0183] 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, 1983).
[0184] 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 B1 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).
[0185] 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 that 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.
[0186] An "antigen" is a molecule or a portion of a molecule
capable of being bound by an antibody that 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 that may be evoked by other antigens.
[0187] The antibodies, including fragments of antibodies, useful in
the present invention may be used to quantitatively or
qualitatively detect the B1 protein in a sample or to detect
presence of cells that express the B1 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.
[0188] 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 B1
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 B1 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.
[0189] Such assays for the B1 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 B1 protein, and detecting the antibody
by any of a number of techniques well known in the art.
[0190] The biological sample may be treated with a solid phase
support or carrier such as nitrocellulose, or other solid support
or carrier that 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.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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
that 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
acetylcholine-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.
[0195] 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 Work et al (eds) (1978) with particular
reference to the chapter entitled "An Introduction to Radioimmune
Assay and Related Techniques" by Chard, incorporated by reference
herein. The radioactive isotope can be detected by such means as
the use of a y-counter or a scintillation counter or by
autoradiography.
[0196] 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.
[0197] 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).
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] As mentioned above, the present invention also relates to
pharmaceutical compositions comprising recombinant animal virus
vectors encoding the B1 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 B1
protein sequences into the cells. Further pharmaceutical
compositions of the invention comprises as the active ingredient
(a) an oligonucleotide sequence which is an anti-sense sequence of
the B1 protein sequence, or (b) drugs that block the B1 interaction
with other proteins.
[0205] 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 are well known to
those of skill in the art.
[0206] The B1 protein and its isoforms or isotypes are suspected to
possibly 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 (p55-TNF-R). Expression of such
inhibitory isoforms in cells may constitute a mechanism of cellular
self-protection against Fas/APO1- and TNF-mediated cytotoxicity.
The wide heterogeneity of MACH 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. It is also known that BCL2, BCL-X.sub.L and
other members of the BCL2 family are expressed and are active to
varying degrees in different types of cells giving rise to
variations in the susceptibility of different cells to induced
apoptosis, i.e., some cells are more likely to survive than others
(see above noted review by Yang and Korsmeyer, 1996).
[0207] As noted above, the B1 proteins or possible isoforms may
possibly have varying effects in different tissues. For example,
such varying effects may possibly be as regards their interaction
with other proteins of the inflammation, cell death or cell
survival processes and their influence thereby on the activity of
these pathways, in particular the balance between them and whether
or not this balance will be shifted one way or the other.
[0208] It is also possible that some of the possible B1 protein
isoforms serve other functions. For example, B1, its analogs, or
isoforms may also act as docking sites for molecules that are
involved in other intracellular pathways not related to the above
noted cell death or survival pathways.
[0209] Due to the unique ability of Fas/APO1 and TNF receptors to
cause 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 has 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. In view of the suspected important role of the TRAF
proteins, and hence the B1 protein which may possibly interact
directly or indirectly therewith, or the suspected interaction
between B1 and various caspases, it seems particularly important to
design drugs that can influence or modulate the interaction between
B1 and these other proteins with which it interacts, and in this
way to enhance or inhibit cell death or cell survival as is
desired.
[0210] The present invention also concerns proteins or other
ligands which can bind to the B1 proteins of the invention and
thereby modulate/mediate the activity of the B1 proteins. Such
proteins or ligands may be screened, isolated and produced by any
of the above-mentioned methods. For example, there may be isolated
a number of new ligands, including proteins, capable of binding to
the B1 proteins of the invention.
[0211] As detailed above, such new B1-binding proteins/ligands may
serve as, for example, inhibitors or enhancers of B1-mediated
activity, and as such will have important roles in various
pathological and other situations as detailed above. Another
function of such B1-binding proteins/ligands would be to serve as
specific agents for the purification of the B1 proteins by, for
example, affinity chromatography, these new binding
proteins/ligands being attached to the suitable chromatography
matrices to form the solid or affinity support/matrix through which
a solution, extract or the like, containing the B1 proteins, will
be passed and in this way to facilitate the purification thereof.
Such methods of affinity chromatography are now well-known and
generally standard procedures of the art.
[0212] Likewise, all of the above mentioned B1 proteins, analogs,
fragments, isoforms and derivatives of the present invention may be
used to purify by affinity chromatography the various proteins of
the inflammation, cell death or survival pathways to which they
bind. For example, B1 proteins and analogs, fragments and muteins
thereof may be used for the affinity chromatography purification of
B1-binding proteins. Such a method for identifying and producing
these B1-binding proteins, will include a screening step in which
the B1 protein, or at least a specific portion thereof, is used as
a substrate or "bait" to obtain proteins or any other ligand
capable of binding thereto; followed by steps of identifying and
characterizing such proteins/ligands so-obtained; and subsequently
producing such proteins/ligands in substantially isolated and
purified forms. All these steps are well known to those of skill in
the art and are detailed herein above and herein below.
[0213] The invention will now be described in more detail in the
following non-limiting examples and the accompanying drawings:
[0214] 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, as well as other procedures used in the following
Examples have been detailed in previous publications by the present
inventors in respect of other intracellular signaling proteins and
pathways (see, for example, Boldin et al, 1995a, 1995b, and Boldin
et al 1996). These procedures also appear in detail in the co-owned
co-pending Israel Application Nos. 114615, 114986, 115319, 116588,
117932, and 120367 as well as the corresponding PCT application No.
PCT/US96/10521). Accordingly, the full disclosures of all these
publications and patent applications are included herein in their
entirety and at least as far as the detailed experimental
procedures are concerned. 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 p55-R, FAS-R, RIP and other proteins,
these have been detailed by the present inventors in the above
noted co-owned, co-pending IL and PCT applications and in Malinin
et al, 1997.
Example 1
Isolation, Sequencing and Partial Characterization of the New B1
Protein
[0215] Employing various methods as described in the co-owned
patent applications mentioned above, a new cloned DNA sequence has
been isolated, sequenced and partially characterized. This DNA
sequence encodes a new protein, originally designated as a c-IAP
binding kinase (CBK) by virtue of its homology to c-IAP proteins
and of its having a kinase domain, but now is designated as B1.
[0216] Briefly, in order to further elucidate the intracellular
activity of the recently discovered cellular inhibitors of
apoptosis (IAP) homologs c-IAP1 and c-IAP2 (see Rothe et al, 1995;
Uren et al, 1996; Hofmann et al, 1997) and with which intracellular
proteins they interact, the c-IAP sequences were used to screen for
other possibly homologous, or otherwise related sequences in
various databases, including those having uncharacterized (and not
fully sequenced) expressed sequence tags (ests). In this way a
partial sequence of a new clone was found that had high homology to
c-IAP1. Using this partial sequence, which had previously not been
characterized in any way, PCR primers were prepared for the PCR
cloning of the full-length DNA sequence of this new clone using, as
template DNA, cDNA libraries commercially obtained.
[0217] As a result, a new full-length DNA clone was obtained
encoding a heretofore unknown protein, namely, the new protein
designated B1. A sequence was initially determined for B1 (DNA and
amino acid). A further analysis and determination of the initial B1
sequence revealed some differences at the N-terminal part of the
amino acid sequence (the 5' end of the nucleotide sequence), which
involved the first 19 deduced amino acid residues. This further
sequence determination and analysis yielded the deduced B1 amino
acid sequence and the nucleotide sequence coding therefore as shown
in FIGS. 3A and B, respectively.
[0218] Upon analysis of the amino acid sequences of FIG. 3, it
arises that there is a kinase motif at the N-terminal end of the
protein that is encoded by the first approximately 1000 nucleotides
of the open reading frame (ORF) of the nucleotide sequences of FIG.
3. Further, towards the C-terminal end of the amino acid sequence
there is a prodomain (CARD) structure which is common to a number
of intracellular proteins involved in apoptotic signaling pathways,
for example, c-IAP1, RAIDD (see Duan and Dixit, 1997), and other
caspases such as ICE and ICH-1. In the amino acid sequence of B1
depicted in FIG. 3A there is shown the N-terminal kinase domain
(boxed region) and the C-terminal CARD (underlined region). Between
these two domains is the intermediatory domain of the B1
protein.
[0219] The above noted kinase domain of B1 has high homology (or
similarity) to the known RAF-type kinases and the RIP-kinase
domain.
[0220] The above mentioned prodomain of B1 has recently also been
designated as CARD for `caspase recruitment domain` (see Hofmann et
al, 1997) and appears to serve as a region through which various
proteins interact during the apoptotic signaling process
intracellularly. For example, the p55 TNF-R which does not have a
prodomain (or CARD) interacts with another intracellular protein
TRADD (an adaptor protein) via the death domain region present on
both these proteins. In turn, TRADD can interact with RIP and with
RAIDD (additional such adaptor proteins, see also Hofmann et al,
1997; Duan and Dixit, 1997; Wallach, 1997) all of which have death
domains, such that, via the death domain region the p55-TNF-R can
be complexed directly or indirectly to RAIDD. RAIDD has a prodomain
(or CARD) that can interact or bind with one or more caspases,
e.g., ICH-1 (caspase-2), and possibly others, and thereby can link
the p55-TNF-R to such caspases and bring about apoptosis via the
action of the caspases. Likewise, the p75-TNF-R can interact with
the TRAF2 and TRAF1 proteins via common motifs, and the TRAF
proteins can interact with c-IAP1 and c-IAP2. In a similar fashion
(see also Malinin et al, 1997; WO 97/37016), by virtue of the
ability of the FAS-R (Fas/APO1) to be able to interact with MORT1
(FADD), which, in turn, interacts with TRADD (all via their common
death domains), and the ability of TRADD to interact with TRAF2,
MORT1 can thus be so linked to c-IAP1, c-IAP2 (via TRAF2) and
thereby to ICE, Mch6 and other caspases, or be so linked to ICH-1,
FLICE/MACH or other caspases (via the TRADD-RIP-RAIDD interactions,
noted above). It should also be noted that the p55 TNF-R can also
be so linked to ICE, Mch6 and other such caspases via the above
noted TRADD-TRAF2-cIAP1, c-IAP2-ICE, Mch6 interactions, this by
virtue of the ability of p55 TNF-R to interact with TRADD as
well.
[0221] In addition, it is known that TRAF2 is also involved in an
intracellular pathway (or more than one pathway) that promotes cell
survival via the induction of NF-.kappa.B activation. In this
pathway(s) NIK appears to be directly involved in the
phosphorylation of I-.kappa.B that leads to I-.kappa.B dissociation
from NF-.kappa.B and thereby activation of NF-.kappa.B, whereby
NF-.kappa.B can enter the nucleus and initiate transcription of
various genes, the expression of which are linked to cell survival
(see also "Background" section above).
[0222] Thus, TRAF2 is involved in both the cell death and cell
survival pathways and depending on which proteins predominantly
interact with TRAF2 at any given period in response to various
external stimuli (e.g., ligands bind the various receptors), the
cell may undergo cell death or cell survival induction. Clearly,
there is a fine balance between the various intracellular signaling
proteins that can be shifted to either of the opposing cell death
or cell survival pathways, and TRAF2 appears to be one of the key
proteins maintaining this balance and being responsible for any
shift in the balance one way or the other.
[0223] In FIG. 1 there is shown schematically the structure of the
TRAF2 protein with its various domains and in FIG. 2 there is shown
schematically some of the possible interactions between various
cellular receptors and intracellular signaling proteins and their
involvement in cell death or cell survival (NF-.kappa.B activation)
pathways.
[0224] Accordingly, the possibility arises that the new B1 protein
of the present invention may have an important modulatory role in
the inflammation, cell death and cell survival pathways. B1 has a
prodomain (or CARD domain) which may possibly interact even
indirectly with the prodomain of c-IAP1, c-IAP2, RAIDD and various
caspases (ICE, ICH-1, etc.) and thereby it may possibly interact
even indirectly with TRAF2 and the various proteins which interact
directly or indirectly with TRAF2, including RIP, TRADD, p75 TNF-R,
p55 TNF-R, MORT-1 and FAS-R. B1 also has a kinase domain and as
such it may possibly be involved directly or indirectly in the MAP
kinase pathway, of which NIK appears to be a member, and thereby
may also be involved in the NF-.kappa.B activation pathway.
[0225] Moreover, B1 by virtue of its homology to c-IAP1, may
possibly be a modulator of c-IAP1 (and c-IAP2) activity by
modulating c-IAP1's biological activity or by modulating the
binding of c-IAP1 to other proteins. In this regard (see also
Example 2 below), B1 may possibly act to increase apoptosis by
interacting even indirectly with c-IAP proteins (c-IAP1, c-IAP2)
and disrupting or otherwise decreasing their ability to recruit
caspases and restrict their proteolytic activity, with the result
that more caspases will be free to act proteolytically.
[0226] Another possibility is that B1 via its above-mentioned
possible ability to be able to interact with various mediators of
cell death, directly or indirectly, including TRAF-1 and TRAF2,
RAIDD, RIP, TRADD, p55-TNF-R, p75-TNF-R, MORT-1 and FAS-R; and with
various caspases, may possibly serve to link these proteins to the
caspases and thereby possibly serve as an intermediary agent in the
cell death pathway(s) to which these proteins belong. As such, B1
may be an important mediator of apoptosis.
[0227] A further possibility is that by the possible interaction
(even indirect) of B1 with c-IAP proteins noted above, B1 may
possibly prevent c-IAP binding or interaction with TRAF2 and
thereby may possibly block TRAF2 activity with respect to the MAP
kinase pathway, for example, TRAF2-c-IAP interactions may be
important for TRAF2 interactions with NIK, and if this is prevented
by B1 interaction with c-IAP, then TRAF2-mediated NF-.kappa.B
activation may be blocked resulting in less enhancement of cell
survival and possibly an increase in cell death.
[0228] A still further possibility is that B1 may act in a more
direct manner in modulating the activity of the various caspases.
Thus via interactions, direct or indirect, between the prodomains
(CARD domains) of B1 and various caspases, B1 may possibly lead to
an increase in the activity of these enzymes and thereby increase
the cytotoxicity of these enzymes. In this way B1 may be a direct
augmentor of apoptosis by recruiting or otherwise activating
caspases, (see also Example 2 below).
[0229] An additional possibility is that B1 may act to modulate
intracellular signaling pathways mediating cell death or cell
survival by binding to or interacting with other as yet unknown
proteins.
[0230] It is interesting to note (see above) that B1 has a kinase
domain similar to the RIP-kinase. RIP is also a central protein
involved in the balance between the cell death and cell survival
pathways by virtue of its ability to link between the cell death
mediators (e.g., p55 TNF-R, FAS-R, MORT-1, TRADD) and TRAF-2 and
thereby to NF-.kappa.B activation and cell survival (see FIG. 2).
The RIP-kinase activity may also be a factor in this fine balance,
depending on what are the substrates for this kinase, for example,
what proteins are phosphorylated by RIP and whether this influences
their activity towards increase apoptotic activity, decreased
apoptotic activity, increased NF-.kappa.B activation or decreased
NF-.kappa.B activation. By analogy, B1 may possibly also play such
a central role in which the kinase activity thereof may be
important depending on which proteins are substrates for such
kinase activity.
Example 2
Analysis of the Biological Activity of B1 Protein
[0231] (i) Preliminary Binding Assay to Determine which Known
Proteins Can Bind to B1
[0232] Using the methods from WO 97/37016 methods to prepare and
express DNA constructs and the yeast two-hybrid binding assay, a
construct of B1 from which was removed its kinase domain, i.e., a
truncated B1 having only the intermediate region and the C-terminal
CARD region, was employed to test for its ability to bind various
known proteins involved in intracellular signaling pathways (cell
death and survival pathways). The initial, preliminary results (not
shown) seem to indicate that this truncated B1 binds to BCL2.
[0233] (ii) Cell Cytotoxicity Analysis to Determine the Effect of
B1 on Cell Death or Cell Survival
[0234] Using the methods from WO 97/37016 methods for preparing DNA
constructs and transfecting/transforming cells therewith and
determining the effect on cell death or cell survival by the
expressed products of these constructs, a DNA construct encoding
the full-length B1 protein was used to transfect cells in culture.
Further, in another set of experiments the B1-encoding construct
was used to co-transfect cells with other constructs encoding
FAS-R, p55 TNF-R and RIP, amongst others.
[0235] The results obtained from these transfections (not shown)
indicate that the expressed B1 protein on its own does not cause
cell death. However, when B1 is expressed together with FAS-R, p55
TNF-R or RIP, it enhances the level of cell death induced by these
known inducers of cell death.
[0236] These results taken with those of (i) above, that B1 may
bind to BCL2, raise the possibility that B1 may serve as an
inhibitor of BCL2 activity, i.e., that B1 may prevent BCL2's
activity towards protecting cells against apoptosis (see
"Background" section above), and as such B1 apparently is capable
of enhancing the cell death pathways induced by FAS-R, p55 TNF-R
and RIP, and possibly other inducers of cell death (as also noted
above in "Background" section). In this respect, B1 may possibly
act in an analogous way to the BAD protein, a member of the BCL2
family, which binds to BCL2 and BCL-X.sub.L and thereby results in
increased levels of BAX and BAK that are known to be directly
involved in causing cell death. Another possibility may be that B1,
by virtue of its kinase domain, may phosphorylate BCL2 at the
phosphorylation sites present on BCL2 and in this way may effect
BCL2's activity toward protecting cells against apoptosis,
resulting, ultimately, in the observed effect that B1 has on
enhancing induced cell death.
[0237] Moreover, it is also possible that B1 may, in addition to or
independent of its possible interaction with BCL2, effect the
induction of NF-.kappa.B activation and this via B1's kinase
activity acting in the pathway leading to NF-.kappa.B activation,
for example, B1 may possibly interact with NIK or other kinases in
the pathway that NIK is a member, or it may act on other adaptor
proteins related thereto, e.g., TRAF2, in such a way as to lead to
reduced NF-.kappa.B activation, and ultimately reduced cell
survival and increased cell death.
[0238] Therefore, in summary, it appears that B1 plays a role in
the modulation of intracellular signaling pathways whether they are
those leading to inflammation, cell death or cell survival. B1 may
thus be considered as a `modulator of intracellular signaling`, as
it clearly has the ability to influence inflammation, cell death
and cell survival pathways in a number of ways be they direct
(recruitment of various proteins and activation or inhibition
thereof or via kinase activity) or be they indirect (via
interaction with various other intermediates, e.g., BCL2, and
possibly also c-IAP and thereby to TRAF2, etc; or RAIDD and thereby
to RIP, TRADD, etc.).
Example 3
Additional Analysis of B1 Biological Activity
[0239] NF-.kappa.B activity, cell death assay, Northern analysis
and JNK activity assays were carried out with the following B1 and
B1 mutant constructs (see FIG. 6)
[0240] B1 (see Example 1)
[0241] B1 mut, a mutant of B1 in which the lysine at position 47
was replaced with alanine
[0242] .DELTA.CARD, B1 lacking the CARD domain created by PCR and
cloning into expression vectors
[0243] .DELTA.Xba, B1 lacking the CARD domain but shorter at its 3'
end than .DELTA.CARD, created by the use of the restriction site
and cloning into expression vectors
[0244] .DELTA.Bam, similar to .DELTA.Xba and created in the same
manner, using the Bam restriction enzyme,
[0245] .DELTA.Nde, containing part of the kinase domain and the
CARD domain, created by PCR and cloning into an expression vector,
and
[0246] .DELTA.K, created by PCR using the following primers:
3 1. 5'-CAGAATTCCAGAGTGTTTCAAGTGCCATTC; 2.
5'-AACTCGAGACTTACATGCTTTTATTTTGAA.
[0247] The PCR fragment was cloned into expression vectors and
verified by sequencing.
[0248] NF-.kappa.B activation measurements were carried out by
reporter gene assay as described in WO 97/37016. Briefly, cells
were co-transfected with the HIV LTR-luciferase gene plasmid (1
.mu.g) and the B1 and B1 mutant expression vectors (3 .mu.g). The
amount of transfected DNA was kept constant by addition of an
"empty" vector. 24 hours after transfection, the cells were washed
with PBS and lysed. Luciferase assay was performed as described in
Ausubel et al (1987-1995). The results can be seen in FIG. 6.
[0249] Cell death assay was carried out by growing 293-T cells in
Dulbecco's modified Eagle's minimal essential medium supplemented
with 10% fetal calf serum, non-essential amino acids, 100 U/ml
penicillin and 100 pg/ml streptomycin. 293-T cells
(5.times.10.sup.5 cells in 6 cm dishes) were transiently
transfected using the calcium phosphate precipitation method with
the cDNAs of the different constructs together with the
.beta.-galactosidase expression vector. In the experiments, the
results of which are shown in FIG. 5, each dish was transfected
with 1 .mu.g of a p55 TNF-R, RIP or TRADD construct, 1 .mu.g of the
respective B1 or B1 mutant construct (or, as control, an empty
vector), and 1 .mu.g of pSV-.beta.-gal (Promega). The extent of
cell death at the end of the incubation period was assessed by
determination of .beta.-galactosidase expression, as described by
Boldin et al (1996).
[0250] Northern analysis was performed by conventional methods,
see, e.g., Boldin et al (1995), and revealed that B1 is present in
many human tissues (FIG. 4).
[0251] JNK activation was carried out by transiently transfecting
293-T cells (5.times.10.sup.5 cells in 6 cm dishes), using the
calcium phosphate precipitation method with 1.5 .mu.g of
pSR-HA-JNK1 construct (an HA epitope tagged JNK-1 expression
vector) and 4 .mu.g each of B1 and B1 mutant construct expression
vector. After 24 hrs cells were lysed in lysis buffer (20 mM Hepes
pH 7.6, 10 mM EGTA, 40 mM .beta.-glycerophosphate, 2.5 mM
MgCl.sub.2, 1 mM DTT and 1% NP-40) and Ha-JNK1 protein was
immunoprecipitated with anti-HA antibodies (see e.g. Rothe et al,
1995b). (clone 12 CA5). The kinase assay was performed in 30 .mu.l
of kinase buffer (20 mM Hepes pH 7.6, 40 mM
.beta.-glycerophosphate- , 2.0 mM MgCl.sub.2, 2 mM DTT, 3 nmole ATP
and 3 .mu.Ci of .gamma.-P.sup.32-ATP) at 30.degree. C. for 20
minutes. Bacterially produced GST-Jun protein (about 10 .mu.g) was
used as substrate. The reaction was stopped by addition of 2.times.
SDS-loading buffer, boiled for 3 minutes and analyzed by SDS-PAGE
gel. The results are shown in FIG. 7.
[0252] The results in FIG. 6 show that B1 can induce NF-.kappa.B
activation directly. However, seeing that B1 must also induce
NF-.kappa.B, this activation appears to be independent of its
kinase domain, and it is assumed that it may be connected to the
CARD domain, with or without contribution by part or all of the
intermediate domain of B1.
[0253] Further cell cytotoxicity analysis shows that not only B1
(see example 2 (ii)), but also B1 mut, when expressed together with
p55-TNF-R, RIP or TRADD potentiates the level of cell death.
.DELTA.CARD, .DELTA.Nde and .DELTA. K do so to a lesser extent,
while the other constructs do not. This seems to indicate that at
least the CARD domain is involved in the potentiation of cell
death, possibly together with the intermediate domain.
[0254] The results for the JNK activation also seem to indicate
that at least the CARD domain is involved in this activation, again
possibly together with the intermediate domain.
[0255] In addition to the above, tests carried out have shown that
B1 autophosphorylates. This is proof that B1 is indeed a
kinase.
[0256] Furthermore, it has been confirmed that B1 has homology to
RIP. Computer analysis indicates a 37% identity of the two proteins
on the amino acid level and a homology of 47%. RIP is now widely
considered to be mainly an NF-.kappa.B modulator and the above
results indicate that B1 acts similarly.
Example 4
Binding Characteristics
[0257] The binding characteristics of B1, and mutants thereof are
shown in the following Table III:
4 TABLE III DNA-Binding Hybrid Activation Hybrid LacZ B1 B1 +++ B1
.DELTA.K +++ .DELTA.K B1 +++ .DELTA.K .DELTA.K +++ B1 TRAF2 - B1
TRAF3 - B1 TRAF6 - TRAF2 B1 - TRAF6 B1 - TRAF1 B1 + B1 TANK - B1
NIK - NIK B1 - B1 CASH - CASH B1 - B1 RIP - B1 RAIDD - B1 ICE - B1
ICH-1 - B1 MACH.alpha.1 (C360S) - B1 MORT-1 - B1 cIAP-1 - cIAP-1 B1
+ RIP B1 - RAIDD B1 - ICE B1 - ICH-1 B1 - MACH.alpha.1 (C360S B1
-
[0258] From the results shown in the above table it appear that
when B1 functions to induce NF-.kappa.B activation, it may do so
independently of binding to other proteins known to be involved in
NF-.kappa.B activation, such as, e.g., IRAK, TRAF2, NIK, TRAF6 and
RIP. Thus B1 may induce NF-.kappa.B activation directly or
indirectly via interaction with some other proteins forming part of
this activation pathway.
[0259] As far as B1's observed cell death enhancing activities are
concerned, it also appears from the above table, that B1 does so
without direct interaction with various cell death mediators, such
as, e.g., p55 TNF-R, Fas-R, MORT1, TRADD, RIP, ICE, ICH-1, and the
like.
[0260] Hence, B1 may also function to enhance cell death by an
indirect interaction with these various cell death
mediators/modulators or via other proteins.
[0261] In view of B1's involvement in both, NF-.kappa.B activation,
as well as in cell death enhancement, B1 may be a central protein
involved in the fine balance between the intracellular pathways
leading to cell death or cell survival. In this respect B1,
depending on with which protein it interacts, may be capable of
shifting the balance between cell death and cell survival.
[0262] 293 human embryonic kidney cells (5.times.10.sup.6;
2.5.times.10.sup.6/per 10 cm dish) were transiently transfected by
the calcium phosphate procedure with 10 .mu.g of plasmid encoding
HA-tagged B1 protein (HA-B1) and 10 .mu.g of either a plasmid
encoding Flag-tagged B1 protein (FL-B1) or one encoding Flag-tagged
c-IAP-1 protein (FL-IAP1), or one encoding Flag-tagged TRAF1
(FL-TRAF1), or one encoding Flag-tagged TRAF2 (FL-TRAF2) or with 10
.mu.g of the combination (in 1:1 ratio) of Flag-tagged TRAF1 and
non-tagged TRAF2 (FL-TR1+TR2), or with 10 .mu.g of combination
(1:1:1) of Flag-tagged TRAF1, non-tagged TRAF2 and c-IAP-1
(FL-TR1+TR2+IAP1). Seven hours after transfection cells were washed
and 18 hours later cells were lysed in a buffer containing 50 mM
HEPES pH 7.5, 250 mM NaCl, 0.2% NP-40, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 2.0 .mu.g/ml aprotinin and 20
.mu.g/ml leupeptin (lysis buffer). Immunoprecipitation was
performed by incubation (2hours, 4.degree. C.) of 1 ml aliquots of
lysate with anti-FLAG epitope antibody (5 .mu.g/aliquot) and with
protein G-agarose beads (30 .mu.l/aliquot). Immunoprecipitates were
washed three times with lysis buffer and once with PBS,
fractionated by 10% SDS-PAGE and transferred to a nitrocellulose
membrane (Schleicher & Schuell, Dassel, Germany). Western blot
analysis was performed with anti-HA epitope monoclonal antibodies
applied at a dilution of 1:1000, and the ECL kit (Amersham,
Buckinghamshire, England).
[0263] It is apparent from FIG. 8 that B1 is able to self-associate
as well as to interact with TRAF-1. The level of interaction
appears to be about the same as its self-association. No direct
interaction with TRAF-2 or 1AP1 is observed.
Example 5
B1 Binds to the E Subunit of V-ATPase
[0264] Two hybrid screens with CARD domain of B1 as a bait resulted
in cloning the E subunit of V-ATPase (the review by Nelson et al
(1996).
[0265] The E subunit of V-ATPase is labeled fluorescently and
incubated with a sample of the CARD domain of B1 in the presence of
various samples of a library of organic molecules or peptides.
Following incubation, the B1-CARD motif is immunoprecipitated with
specific antibodies and the amount of fluorescence associated with
this precipitate is measured. Molecules found to interfere with
precipitation of the fluorescently labeled E-protein are further
examined as potential lead compound as drugs that affect cell
viability or growth and/or inflammation via the function of the
E-subunit of ATPase.
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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.
[0270] 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
3 1 540 PRT Homo sapiens 1 Met Asn Gly Glu Ala Ile Cys Ser Ala Leu
Pro Thr Ile Pro Tyr His 1 5 10 15 Lys Leu Ala Asp Leu Arg Tyr Leu
Ser Arg Gly Ala Ser Gly Thr Val 20 25 30 Ser Ser Ala Arg His Ala
Asp Trp Arg Val Gln Val Ala Val Lys His 35 40 45 Leu His Ile His
Thr Pro Leu Leu Asp Ser Glu Arg Lys Asp Val Leu 50 55 60 Arg Glu
Ala Glu Ile Leu His Lys Ala Arg Phe Ser Tyr Ile Phe Pro 65 70 75 80
Ile Leu Gly Ile Cys Asn Glu Pro Glu Phe Leu Gly Ile Val Thr Glu 85
90 95 Tyr Met Pro Asn Gly Ser Leu Asn Glu Leu Leu His Arg Lys Thr
Glu 100 105 110 Tyr Pro Asp Val Ala Trp Pro Leu Arg Phe Arg Ile Leu
His Glu Ile 115 120 125 Ala Leu Gly Val Asn Tyr Leu His Asn Met Thr
Pro Pro Leu Leu His 130 135 140 His Asp Leu Lys Thr Gln Asn Ile Leu
Leu Asp Asn Glu Phe His Val 145 150 155 160 Lys Ile Ala Asp Phe Gly
Leu Ser Lys Trp Arg Met Met Ser Leu Ser 165 170 175 Gln Ser Arg Ser
Ser Lys Ser Ala Pro Glu Gly Gly Thr Ile Ile Tyr 180 185 190 Met Pro
Pro Glu Asn Tyr Glu Pro Gly Gln Lys Ser Arg Ala Ser Ile 195 200 205
Lys His Asp Ile Tyr Ser Tyr Ala Val Ile Thr Trp Glu Val Leu Ser 210
215 220 Arg Lys Gln Pro Phe Glu Asp Val Thr Asn Pro Leu Gln Ile Met
Tyr 225 230 235 240 Ser Val Ser Gln Gly His Arg Pro Val Ile Asn Glu
Glu Ser Leu Pro 245 250 255 Tyr Asp Ile Pro His Arg Ala Arg Met Ile
Ser Leu Ile Glu Ser Gly 260 265 270 Trp Ala Gln Asn Pro Asp Glu Arg
Pro Ser Phe Leu Lys Cys Leu Ile 275 280 285 Glu Leu Glu Pro Val Leu
Arg Thr Phe Glu Glu Ile Thr Phe Leu Glu 290 295 300 Ala Val Ile Gln
Leu Lys Lys Thr Lys Leu Gln Ser Val Ser Ser Ala 305 310 315 320 Ile
His Leu Cys Asp Lys Lys Lys Met Glu Leu Ser Leu Asn Ile Pro 325 330
335 Val Asn His Gly Pro Gln Glu Glu Ser Cys Gly Ser Ser Gln Leu His
340 345 350 Glu Asn Ser Gly Ser Pro Glu Thr Ser Arg Ser Leu Pro Ala
Pro Gln 355 360 365 Asp Asn Asp Phe Leu Ser Arg Lys Ala Gln Asp Cys
Tyr Phe Met Lys 370 375 380 Leu His His Cys Pro Gly Asn His Ser Trp
Asp Ser Thr Ile Ser Gly 385 390 395 400 Ser Gln Arg Ala Ala Phe Cys
Asp His Lys Thr Thr Pro Cys Ser Ser 405 410 415 Ala Ile Ile Asn Pro
Leu Ser Thr Ala Gly Asn Ser Glu Arg Leu Gln 420 425 430 Pro Gly Ile
Ala Gln Gln Trp Ile Gln Ser Lys Arg Glu Asp Ile Val 435 440 445 Asn
Gln Met Thr Glu Ala Cys Leu Asn Gln Ser Leu Asp Ala Leu Leu 450 455
460 Ser Arg Asp Leu Ile Met Lys Glu Asp Tyr Glu Leu Val Ser Thr Lys
465 470 475 480 Pro Thr Arg Thr Ser Lys Val Arg Gln Leu Leu Asp Thr
Thr Asp Ile 485 490 495 Gln Gly Glu Glu Phe Ala Lys Val Ile Val Gln
Lys Leu Lys Asp Asn 500 505 510 Lys Gln Met Gly Leu Gln Pro Tyr Pro
Glu Ile Leu Val Val Ser Arg 515 520 525 Ser Pro Ser Leu Asn Leu Leu
Gln Asn Lys Ser Met 530 535 540 2 2098 DNA Homo sapiens 2
ggccattatg gatggatggg cggcgctacg gcgttggcac cagtctctag aaaagaagtc
60 agctctggtt cggagaagca gcggctggcg tgggccatcc ggggaatggg
cgccctcgtg 120 acctagtgtt gcggggcaaa aagggtcttg ccggcctcgc
tcgtgcaggg gcgtatctgg 180 gcgcctgagc gcggcgtggg agccttggga
gccgccgcag cagggggcac acccggaacc 240 ggcctgagcg cccgggacca
tgaacgggga ggccatctgc agcgccctgc ccaccattcc 300 ctaccacaaa
ctcgccgacc tgcgctacct gagccgcggc gcctctggca ctgtgtcgtc 360
cgcccgccac gcagactggc gcgtccaggt ggccgtgaag cacctgcaca tccacactcc
420 gctgctcgac agtgaaagaa aggatgtttt aagagaagct gaaattttac
acaaagctag 480 atttagttac atttttccaa ttttgggaat ttgcaatgag
cctgaatttt tgggaatagt 540 tactgaatac atgccaaatg gatcattaaa
tgaactccta cataggaaaa ctgaatatcc 600 tgatgttgct tggccattga
gatttcgcat cctgcatgaa attgcccttg gtgtaaatta 660 cctgcacaat
atgactcctc ctttacttca tcatgacttg aagactcaga atatcttatt 720
ggacaatgaa tttcatgtta agattgcaga ttttggttta tcaaagtggc gcatgatgtc
780 cctctcacag tcacgaagta gcaaatctgc accagaagga gggacaatta
tttatatgcc 840 acctgaaaac tatgaacctg gacaaaaatc aagggccagt
atcaagcacg atatatatag 900 ctatgcagtt atcacatggg aagtgttatc
cagaaaacag ccttttgaag atgtcaccaa 960 tcctttgcag ataatgtata
gtgtgtcaca aggacatcga cctgttatta atgaagaaag 1020 tttgccatat
gatatacctc accgagcacg tatgatctct ctaatagaaa gtggatgggc 1080
acaaaatcca gatgaaagac catctttctt aaaatgttta atagaacttg aaccagtttt
1140 gagaacattt gaagagataa cttttcttga agctgttatt cagctaaaga
aaacaaagtt 1200 acagagtgtt tcaagtgcca ttcacctatg tgacaagaag
aaaatggaat tatctctgaa 1260 catacctgta aatcatggtc cacaagagga
atcatgtgga tcctctcagc tccatgaaaa 1320 tagtggttct cctgaaactt
caaggtccct gccagctcct caagacaatg attttttatc 1380 tagaaaagct
caagactgtt attttatgaa gctgcatcac tgtcctggaa atcacagttg 1440
ggatagcacc atttctggat ctcaaagggc tgcattctgt gatcacaaga ccactccatg
1500 ctcttcagca ataataaatc cactctcaac tgcaggaaac tcagaacgtc
tgcagcctgg 1560 tatagcccag cagtggatcc agagcaaaag ggaagacatt
gtgaaccaaa tgacagaagc 1620 ctgccttaac cagtcgctag atgcccttct
gtccagggac ttgatcatga aagaggacta 1680 tgaacttgtt agtaccaagc
ctacaaggac ctcaaaagtc agacaattac tagacactac 1740 tgacatccaa
ggagaagaat ttgccaaagt tatagtacaa aaattgaaag ataacaaaca 1800
aatgggtctt cagccttacc cggaaatact tgtggtttct agatcaccat ctttaaattt
1860 acttcaaaat aaaagcatgt aagtgactgt ttttcaagaa gaaatgtgtt
tcataaaagg 1920 atatttatat ctctgttgct ttgacttttt ttatataaaa
tccgtgagta ttaaagcttw 1980 awwraargkt ctttsrktaa atattagtct
ccctccatga cactgcagta ttttttttaa 2040 ttaatacaag taaaaagttg
aatttgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2098 3 4 PRT Artificial
Synthetic 3 Asp Glu Val Asp 1
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