U.S. patent application number 10/067615 was filed with the patent office on 2002-08-22 for trail receptors, nucleic acids encoding the same, and methods of use thereof.
This patent application is currently assigned to Thomas Jefferson University. Invention is credited to Alnemri, Emad S..
Application Number | 20020115154 10/067615 |
Document ID | / |
Family ID | 22000937 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115154 |
Kind Code |
A1 |
Alnemri, Emad S. |
August 22, 2002 |
Trail receptors, nucleic acids encoding the same, and methods of
use thereof
Abstract
In accordance with the present invention, there are provided
isolated mammalian TRAIL receptor proteins, antibodies thereto,
therapeutic compositions, and nucleic acids encoding such.
Bioassays and therapeutic methods employing invention DR5 and
TRAIL-R3 proteins are also provided.
Inventors: |
Alnemri, Emad S.; (Ambler,
PA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Thomas Jefferson University
Philadelphia
PA
|
Family ID: |
22000937 |
Appl. No.: |
10/067615 |
Filed: |
February 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10067615 |
Feb 4, 2002 |
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09134618 |
Aug 14, 1998 |
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60055906 |
Aug 15, 1997 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 435/6.16; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/7151 20130101;
A01K 2217/05 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5; 435/6 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/705 |
Goverment Interests
[0002] This invention was made with government support under grant
number AG 13487 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. An isolated nucleic acid encoding a mammalian TRAIL receptor
selected from the group consisting of DR5 and TRAIL-R3, splice
variant cDNA sequences thereof, or an active fragment thereof.
2. The nucleic acid according to claim 1, wherein said mammalian
TRAIL receptor is isolated from a mammal selected from the group
consisting of human, rat, mouse, porcine, ovine, canine and
bovine.
3. The nucleic acid according to claim 1 wherein the encoded splice
variant is DR5s, or an active fragment thereof.
4. The nucleic acid of claim 3 comprising SEQ ID NO:5 or an active
fragment thereof.
5. A nucleic acid encoding a mammalian TRAIL receptor, wherein said
nucleic acid is selected from the group consisting of: (a) DNA
encoding the amino acid sequence set forth in SEQ ID NO:2, SEQ ID
NO:4, or SEQ ID NO:6; (b) DNA that hybridizes to the DNA of (a)
under moderately stringent conditions, wherein said DNA encodes
biologically active DR5 or TRAIL-R3; (c) DNA degenerate with
respect to either (a) or (b) above, wherein said DNA encodes
biologically active DR5,or TRAIL-R3; and (d) splice variant cDNA
sequences of any of (a)-(d).
6. The nucleic acid according to claim 5 wherein the splice variant
cDNA comprises SEQ ID NO:5.
7. The nucleic acid according to claim 5, wherein said nucleic acid
hybridizes under high stringency conditions to SEQ ID NO:1, SEQ ID
NO:3, or SEQ ID NO:5.
8. The nucleic acid according to claim 5, wherein the nucleotide
sequence of said nucleic acid is substantially the same as
nucleotides set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID
NO:5.
9. A nucleic acid according to claim 5, wherein the nucleotide
sequence of said nucleic acid is SEQ ID NO:1, SEQ ID NO:3, or SEQ
ID NO:5.
10. A nucleic acid according to claim 5, wherein said nucleic acid
is cDNA.
11. A vector containing the nucleic acid of claim 5.
12. An expression vector comprising the nucleic acid of claim 5,
wherein the nucleic acid encoding the TRAIL receptor is operatively
linked to a promoter.
13. Recombinant cells containing the nucleic acid of claim 5.
14. An antisense oligonucleotide capable of specifically binding to
mRNA encoded by said nucleic acid according to claim 5.
15. An isolated mammalian protein selected from the group
consisting of DR5, a DR5 splice variant, TRAIL-R3, and a TRAIL-R3
splice variant, wherein said protein is characterized by being able
to bind TRAIL ligand.
16. The protein according to claim 15, wherein the amino acid
sequence of said protein comprises substantially the same sequence
as the protein sequence set forth in SEQ ID NO:2, SEQ ID NO:4, or
SEQ ID NO:6.
17. The protein according to claim 16, comprising the sequence set
forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
18. The protein according to claim 15, wherein said protein is
encoded by a nucleotide sequence that is substantially the same as
SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
19. The protein according to claim 18, wherein said protein is
encoded by a nucleotide sequence set forth as SEQ ID NO:1, SEQ ID
NO:3, or SEQ ID NO:5.
20. A method for expression of a protein selected from the group
consisting of DR5, a DR5 splice variant, TRAIL-R3, and a TRAIL-R3
splice variant, said method comprising culturing cells of claim 13
under conditions suitable for expression of said protein.
21. An isolated anti-DR5 or anti-TRAIL-R3 antibody having specific
reactivity with a protein according to claim 15.
22. The antibody according to claim 21, wherein said antibody is a
monoclonal antibody.
23. The antibody according to claim 21, wherein said antibody is a
polyclonal antibody.
24. A composition comprising an amount of the antisense
oligonucleotide according to claim 14 effective to inhibit
expression of a human DR5 or TRAIL-R3 protein and an acceptable
hydrophobic carrier capable of passing through a cell membrane.
25. A transgenic non-human mammal expressing exogenous nucleic acid
encoding a DR5 or TRAIL-R3 protein according to claim 15.
26. A transgenic non-human mammal according to claim 25, wherein
the transgenic non-human mammal is a mouse.
27. A method for detecting the presence of a mammalian DR5 or
TRAIL-R3 protein in a sample, said method comprising contacting a
test sample with an antibody according to claim 21, detecting the
presence of an antibody-DR5 complex or antibody-TRAIL-R3 complex,
and therefrom detecting the presence of a mammalian DR5 or TRAIL-R3
protein in said test sample.
28. A bioassay for evaluating whether test compounds are capable of
acting as agonists or antagonists for DR5 or TRAIL-R3 proteins
according to claim 15, said bioassay comprising: (a) culturing
cells containing: DNA which expresses DR5 or TRAIL-R3 proteins or
functional modified forms thereof, wherein said culturing is
carried out in the presence of at least one compound whose ability
to modulate apoptotic activity of DR5 or TRAIL-R3 protein is sought
to be determined, and thereafter (b) monitoring said cells for
either an increase or decrease in the level of apoptosis.
29. A bioassay for evaluating whether test compounds are capable of
acting as antagonists for DR5 or TRAIL-R3 proteins according to
claim 18, or functional modified forms of said DR5 or TRAIL-R3
proteins, said bioassay comprising: (a) culturing cells containing:
DNA which expresses DR5 or TRAIL-R3 proteins, or functional
modified forms thereof, wherein said culturing is carried out in
the presence of: increasing concentrations of at least one compound
whose ability to inhibit apoptotic activity of DR5 or TRAIL-R3
proteins is sought to be determined, and a fixed concentration of
TRAIL; and thereafter (b) monitoring in said cells the level of
apoptosis as a function of the concentration of said compound,
thereby indicating the ability of said compound to inhibit DR5 or
TRAIL-R3 apoptotic activity.
30. A method for modulating the apoptotic activity mediated by DR5
or TRAIL-R3 protein, said method comprising: contacting said DR5 or
TRAIL-R3 protein with an effective, modulating amount of said
agonist or antagonist identified by claim 28.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from United States
provisional application serial No. 60/055,906, filed Aug. 15, 1997,
now abandoned.
BACKGROUND INFORMATION
[0003] Apoptosis is a normal physiological process of cell death
that plays a critical role in the regulation of tissue homeostasis
by ensuring that the rate of new cell accumulation produced by cell
division is offset by a commensurate rate of cell loss due to
death. It has now become clear that disturbances in apoptosis, also
referred to as physiological cell death or programmed cell death,
that prevent or delay normal cell turnover can be just as important
to the pathogenesis of diseases as are known abnormalities in the
regulation of proliferation and the cell cycle. Like cell division,
which is controlled through complex interactions between cell cycle
regulatory proteins, apoptosis is similarly regulated under normal
circumstances by the interaction of gene products that either
induce or inhibit cell death.
[0004] The stimuli which regulate the function of these apoptotic
gene products include both extracellular and intracellular signals.
Either the presence or the removal of a particular stimuli can be
sufficient to evoke a positive or negative apoptotic signal. For
example, physiological stimuli that prevent or inhibit apoptosis
include, for example, growth factors, extracellular matrix, CD40
ligand, viral gene products, neutral amino acids, zinc, estrogen,
and androgens. In contrast, stimuli which promote apoptosis include
growth factors such as tumor necrosis factor (TNF), Fas, and
transforming growth factor .beta. (TGF.beta.), neurotransmitters,
growth factor withdrawal, loss of extracellular matrix attachment,
intracellular calcium and glucocorticoids, for example.
[0005] Some of the well known regulators of apoptosis are cytokines
of the tumor necrosis factor (TNF) ligand family, such as Fas
ligand (Fas L) and TNF, which induce apoptosis by activation of
their corresponding receptors, Fas and TNFR-1 (Nagata, S. (1997)
Cell 88, 355-36S). These two receptors belong to a rapidly
expanding family (collectively known as the TNF-receptor family)
containing at least eleven known members (Nagata, S. (1997) Cell
88, 355-365; Chinnaiyan, A. M. et al. (1997)
.SIGMA..chi..iota..epsilon..nu..chi..epsilon. 6, 111-113). Members
of this family contain an extracellular ligand-binding domain, of
2-6 cysteine-rich repeats, which is about 25% conserved between
different family members. The cytoplasmic region is less conserved
between various members except for a stretch of about 80 amino
acids present in Fas, TNFR-1, DR3/Wsl-1/Apo-3/TRAMP, CAR-1 and DR4
(Nagata, S. (1997) Cell 88, 355-365; and references therein). This
intracellular region which has been designated the cytoplasmic
"death domain" is responsible for transducing the death signal.
[0006] Activation of Fas results in recruitment of the
Fas-associated death domain-containing molecule FADD/MORT-1, to the
receptor complex (Boldin M. P. et al. (1995) J. Biol. Chem. 270,
7795-7798; Chinnaiyan A. M. et al. (1995) Cell 81, 505-512;
Kischkel F. C. (1995) EMBO J. 14, 5579-5588). The resulting
signaling complex then triggers activation of the caspase apoptotic
pathway through interaction of the N-terminal death effector domain
(DED) of FADD with the corresponding motifs in the prodomain of
caspase-8 (Mch5/MACH/FLICE) and probably caspase-10 (Mch4) (Boldin
M. P. et al. (1996) Cell 85, 803-815; Bretz J. D. et al. (1996)
Cell 85, 817-827; Alnemri E. S. et al. (1996) Proc. Natl. Sci. USA.
93, 7464-7469; Alnemri E. S. etal.(1996)Cell 87, 171.
[0007] In contrast to Fas, activation of TNFR-1 or DR3 results in
recruitment of another death domain-containing adaptor molecule
known as TRAD (Chinnaiyan A. M. et al. (1996) Science 274, 990-991;
Goeddel D. V. et al. (1995) Cell 81: 495-504). TRADD, can associate
with a number of signaling molecules, including FADD, TRAF2, and
RIP and as a result can transduce an apoptotic signal as well as
activate NF-kB (Goeddel D. V. et al. (1996) Cell 84, 299-308;
Baichwal V. et al. (1996) Immunity 4, 387-396). Consequently,
engagement of TNFR-1 or DR3 can signal an array of diverse
biological activities.
[0008] Recently, a new member of the TNF family known as TRAIL or
Apo-2 ligand was identified and shown to induce apoptosis in a
variety of tumor cell lines (Davis T. D. et al. (1995) Immunity 3
673-682: Ashkenazi A. et al. (1996) J. Biol. Chem. 271,
12687-12690; Ashkenazi A. et al. (1996) Curr. Biol. 6, 750-752).
However, it is unclear what physiological control mechanisms
regulate this form of programmed cell death or how the cell death
pathways interact with other physiological processes within the
organism.
[0009] Apoptosis functions in maintaining tissue homeostasis in a
range of physiological processes such as embryonic development,
immune cell regulation and normal cellular turnover. Therefore, the
dysfunction, or loss of regulated apoptosis can lead to a variety
of pathological disease states. For example, the loss of apoptosis
can lead to the pathological accumulation of self-reactive
lymphocytes such as that occurring with many autoimmune diseases.
Inappropriate loss of apoptosis can also lead to the accumulation
of virally infected cells and of hyperproliferative cells such an
neoplastic or tumor cells. Similarly, the inappropriate activation
of apoptosis can also contribute to a variety of pathological
disease states including, for example, acquired immunodeficiency
syndrome (AIDS), neurodegenerative diseases and ischemic injury.
Treatments which are specifically designed to modulate the
apoptotic pathways in these and other pathological conditions can
change the natural progression of many of these diseases.
[0010] Thus, there exists a need to identify new apoptotic genes
and their gene products and for methods of modulating this process
for the therapeutic treatment of human diseases. The present
invention satisfies this need and provides related advantages as
well.
SUMMARY OF THE INVENTION
[0011] In accordance with the present invention, there are provided
novel isolated mammalian members of the TRAIL-receptor family,
designated DR5, TRAIL-R3, and splice variants thereof including
DR5s. These invention proteins, or fragments thereof, are useful as
immunogens for producing anti-DR5 or anti-TRAIL-R3 antibodies, or
in therapeutic compositions containing such proteins and/or
antibodies. The DR5 and TRAIL-R3 proteins are also useful in
bioassays to identify agonists and antagonists thereto.
[0012] In accordance with the present invention, there are also
provided isolated nucleic acids encoding novel DR5 or TRAIL-R3
proteins. Further provided are vectors containing invention nucleic
acids, probes that hybridize thereto, host cells transformed
therewith, antisense oligonucleotides thereto and related
compositions. The nucleic acid molecules described herein can be
incorporated into a variety of recombinant expression systems known
to those of skill in the art to readily produce isolated
recombinant DR5 or TRAIL-R3 proteins. In addition, the nucleic acid
molecules of the present invention are useful as probes for
assaying for the presence and/or amount of a DR5 or TRAIL-R3 gene
or mRNA transcript in a given sample. The nucleic acid molecules
described herein, and oligonucleotide fragments thereof, are also
useful as primers and/or templates in a PCR reaction for amplifying
nucleic acids encoding DR5 or TRAIL-R3 proteins. Also provided are
transgenic non-human mammals that express the invention
proteins.
[0013] Antibodies that are immunoreactive with invention DR5 or
TRAIL-R3 proteins are also provided. These antibodies are useful in
diagnostic assays to determine levels of DR5 or TRAIL-R3 proteins
present in a given sample, e.g., tissue samples, Western blots, and
the like. The antibodies can also be used to purify DP5 or TRAIL-R3
proteins from crude cell extracts and the like. Moreover, these
antibodies are considered therapeutically useful to modulate the
biological effect of DR5 or TRAIL-R3 proteins in vivo.
[0014] Methods and diagnostic systems for determining the levels of
DR5 or TRAIL-R3 proteins in various tissue samples are also
provided. These diagnostic methods can be used for monitoring the
level of therapeutically administered DR5 or TRAIL-R3 proteins or
fragments thereof to facilitate the maintenance of therapeutically
effective amounts. These diagnostic methods can also be used to
diagnose physiological disorders that result from abnormal levels
or abnormal structures of the DR5 or TRAIL-R3 proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1D illustrate sequence analysis and tissue
distribution of DR5 and TRAIL-R3. Predicted amino acid sequence of
human DR5 (A) and TRAIL-R3 (B). The mature DRS and TRAIL-R3 are
predicted to start at Glu+1 and Tyr+1 (indicated by black
diamonds), respectively. The putative signal peptide and
transmembrane domains are single- and double-underlined,
respectively. The five identical repeats in the extracellular
domain of TRAIL-R3 (B) are marked by black triangles. The
intracellular cytoplasmic death domain of DR5 (A) is boxed. C,
Colinear alignment of the death domains of members of the TNF
receptor family. Identical residues in at least three out of six
sequences are shaded. The death domain of DR5 is 64, 30, 30, 20,
31% identical to the corresponding domains in DR4, DR3, TNFR-1, Fas
and CAR1 respectively.
[0016] FIGS. 2A and 2B illustrate that the extracellular domains of
DR5 and TRAIL-R3 bind TRAIL and can block TRAIL-induced apoptosis
as set forth in Example III herein.
[0017] FIGS. 3A-3F illustrate that expression of DR5 but neither
TRAIL-R3 nor DR5s induces apoptosis in human cells as set forth in
Example IV herein.
[0018] FIGS. 4A and 4B illustrate the in vivo interactions of DR5
as set forth in Example V herein.
[0019] FIG. 5 illustrates the nucleotide and predicted amino acid
sequence of the splice variant DR5s.
[0020] FIG. 6 illustrates a colinear alignment of DR5s and DR5. The
dotted lines indicate sequences that are spliced in DR5 or
DR5s.
DETAILED DESCRIPTION OF INVENTION
[0021] In accordance with the present invention, there are provided
novel mammalian members of the TRAIL receptor protein family
referred to herein as "DR5" and "TRAIL-R3", and active fragments
thereof. DR5 is also referred to herein as "TRAIL-R2". As used
herein, the phrases "DR5" and "TRAIL-R3" refer to isolated and/or
substantially pure mammalian proteins, preferably human, that are
able to bind to the cytotoxic ligand "TRAIL" (Wiley et al., 1995,
Immunity, 3:673; and Marsters et al., 1996, Curr. Biol., 6:750),
also known as Apo-2L. Invention DR5 and TRAIL-R3 proteins are
further characterized by having the ability to mediate apoptosis.
In their native environment, invention DR5 and TRAIL-R3 proteins
are cell-surface receptor proteins.
[0022] Invention DR5 and TRAIL-R3 proteins include naturally
occurring variants thereof encoded by mRNA generated by alternative
splicing of a primary transcript (e.g., splice variant DR5s), and
further including active fragments thereof which retain at least
one native biological activity, such as, for example, the
immunogenic ability to generate anti-DR5 or anti-TRAIL-R3
antibodies, the ability to bind TRAIL ligand, the ability to
modulate apoptosis, and the like. In isolated form, invention
isolated DR5 and TRAIL-R3 proteins are free of cellular components
and/or contaminants normally associated with a native in vivo
environment. As used herein, the term "polypeptide" refers to full
length DR5 and TRAIL-R3 proteins or fragments thereof.
[0023] It has been found that DR5 but not TRAIL-R3, contains a
cytoplasmic "death domain" necessary for induction of apoptosis,
and engages the apoptotic pathway independent of the adaptor
molecule FADD/Mort1. TRAIL-R3 on the other hand, can bind TRAIL but
does not induce apoptosis, and thus is contemplated to function as
an antagonistic receptor. Similarly, DR5s contains a truncated
death domain of DR5 and thus also functions as an antagonistic
decoy receptor.
[0024] The invention DR5 proteins are further characterized by
being expressed in at least the following cells: heart, brain,
placenta, lung, liver, skeletal muscle, kidney and pancreas (see
FIG. 1D and Example II). Other normal tissues such as testes,
ovary, colon, small intestine and lymphoid tissues show detectable
but low expression of DR5 transcript. In addition, DR5 mRNA
transcript was detected in the following tumor cell lines: HL-60,
promyelocytic leukemia; HeLa cell S3, K-562, chronic myelogenous
leukemia; MOLT-4, lymphoblastic leukemia; Raji, Burkitt's lymphoma;
SW480, colorectal adenocarcinoma; A549, lung carcinoma; G361,
melanoma (FIG. 1D). Surprisingly, it was found that the amount of
DR5 transcript is at least 100-fold higher in most tumor cell lines
than in normal tissues. Thus, a correlation is contemplated between
the high sensitivity of tumor cells to TRAIL and the elevated
levels of DR5 in these cells.
[0025] In addition, transient expression of DR5 triggers
cytoplasmic death domain-dependent apoptosis (see FIGS. 3A and B).
Similar to other TNF-receptor family members, DR5-induced apoptosis
is efficiently blocked by the caspase inhibitors z-VAD-fmk and CrmA
(FIG. 3D) and by the dominant negative inhibitors FLAME-1,
caspase-8-DN and caspase-10-DN (FIG. 3E). Thus, it is contemplated
herein that the upstream caspases-8 and -10 are involved in both
the invention DR5 death signaling pathways. Moreover, unlike Fas,
DR5 does not interact with the death-domain containing adaptor
proteins FADD, CRADD, RIP or TRADD. Surprisingly, however, full
length DR5, but not death domain-deleted mutants, are capable of
forming complexes with caspase-8, caspase-10 and FLAME-1. Because
these proteins do not interact directly, it is likely that the
formation of these DR5-related complexes requires an adaptor
molecule distinct from FADD.
[0026] In a particular embodiment of the present invention, an
invention DR5 is a protein of 411 amino acids (SEQ ID NO:2) with an
overall .about.59% identity to DR4 (FIG. 1A). DR5's domain
structure is highly related to DR4 and the other members of the
TNF-receptor family. An invention DR5 contains a putative
N-terminal signal peptide (amino acids -51 to -1 of FIG. 1A)
followed by an extracellular domain containing two cysteine-rich
pseudorepeats. Following the extracellular domain is a
transmembrane domain (amino acids 132-152 of FIG. 1A) and a
cytoplasmic domain. Within the cytoplasmic domain there is a
stretch of 67-amino acids (amino acids 273-339 of FIG. 1A)
comprising a death domain homology region (FIG. 1C). The death
domain of DR5 is 64, 30, 30, 20, 31% identical to the corresponding
domains in DR4, DR3, TNFR-1, Fas and CAR1, respectively. Based on
these criteria and its apoptotic activity (see Example IV below)
the novel TRAIL-R2 protein is also designated death receptor-5
(DR5).
[0027] In an additional embodiment of the present invention splice
variants are provided. Upon inspection of genomic clones of a TRAIL
receptor, those skilled in the art are able to identify a variety
of potential splice junctions. One such alternative splicing event
yields DR5s which is an alternatively spliced isoform of DR5. DR5s
is a protein of 350 amino acids (SEQ ID NO:6) which is encoded by a
cDNA of 1053 nucleotides (SEQ ID NO:5). The mature DR5s protein
contains a cytoplasmic region with a truncated death domain (FIGS.
5 and 6) that provides a protein having functionality opposite to
that of DR5. To this end, DR5s substantially inhibits apoptosis by
TRAIL, but not by TNF-.alpha. (FIG. 3F). Without wishing to be
bound by theory, DR5s apparently acts as an inhibitory TRAIL-decoy
receptor and thus may be able to protect cells against
TRAIL-induced apoptosis.
[0028] The invention TRAIL-R3 proteins may be characterized as
being expressed in at least the following cells: heart, brain,
placenta, liver, skeletal muscle, kidney and pancreas (see FIG. 1D
and Example II). In addition, TRAIL-R3 mRNA transcript was detected
in the following tumor cell lines: HeLa cell S3, K-562, chronic
myelogenous leukemia; MOLT-4, lymphoblastic leukemia; Raji,
Burkitt's lymphoma; SW480, colorectal adenocarcinoma; A549, lung
carcinoma; G361, melanoma (FIG. 1D). Surprisingly, relative to DR5
mRNA, a significantly elevated expression of TRAIL-R3 mRNA was
observed in normal cells compared to tumor cells.
[0029] In addition, it has been found that transient expression of
TRAIL-R3, which does not naturally contain a death domain, was
incapable of inducing apoptosis (FIGS. 3A and B). Surprisingly,
transient expression of TRAIL-R3 in MCF7 cells significantly
blocked TRAIL-induced apoptosis (FIG. 3C), suggesting that TRAIL-R3
functions as an antagonistic decoy receptor.
[0030] Thus, since TRAIL-R3 does not contain a cytoplasmic death
domain, and is capable of attenuating the cytotoxicity of TRAIL,
TRAIL-R3 is contemplated as functioning physiologically as an
antagonist to DR4 and DR5.
[0031] In a particular embodiment of the present invention, an
invention TRAIL-R3 (SEQ ID NO:4) is a protein of 299 amino acids
with an overall .about.40 and 36% identity to DR4 and DR5,
respectively (FIG. 1B). This protein contains a putative N-terminal
signal peptide (amino acids -63 to -1 of FIG. 1B) followed by an
extracellular domain containing two cysteine-rich pseudo repeats
and five nearly identical PAAEETMN (T) TSPGTPA repeats (amino acids
139-153, 154-168, 169-183, 184-198, and 199-213 of FIG. 1B, which
corresponds to amino acids 202-216, 217-231, 232-246, 247-261, and
262-276 of SEQ ID NO:4). Following the extracellular domain is a
C-terminal transmembrane domain (amino acids 217-236 of FIG. 1B).
Unlike DR4 and DR5, this TRAIL-receptor does not contain a
cytoplasmic domain. Based on these criteria and its ability to bind
TRAIL (see Example III below), this protein is designated
TRAIL-R3.
[0032] As used herein, the term "apoptosis" refers to the
well-known process of programmed cell death. There is a variety of
well-known, generally accepted in-vitro indicia of apoptosis,
including nuclear morphological changes, internucleosomal
fragmentation of DNA, the selective proteolysis of substrates, and
the activation of CPP32-like caspases. For example, the substrates
of the caspase family of cysteine proteases have received
considerable attention because cleavage of these substrates offers
molecular mechanisms for many of the hallmark morphological and
functional changes exhibited by apoptotic cells (Casiano et al., J.
Exp. Med. 184:765-770, (1996).
[0033] Use of the terms "isolated" and/or "purified" in the present
specification and claims as a modifier of DNA, RNA, polypeptides or
proteins means that the DNA, RNA, polypeptides or proteins so
designated have been produced in such form by the hand of man, and
thus are separated from their native in vivo cellular environment.
As a result of this human intervention, the recombinant DNAs, RNAs,
polypeptides and proteins of the invention are useful in ways
described herein that the DNAs, RNAs, polypeptides or proteins as
they naturally occur are not.
[0034] As used herein, "mammalian" refers to the variety of species
from which invention DR5 or TRAIL-R3 proteins are derived, e.g.,
human, rat, mouse, rabbit, monkey, baboon, bovine, porcine, ovine,
canine, feline, and the like.
[0035] Presently preferred DR5 and TRAIL-R3 proteins of the
invention include amino acid sequences that are substantially the
same as the protein sequence set forth in SEQ ID NO:2 and SEQ ID
NO:4, respectively, as well as biologically active, modified forms
thereof. Those of skill in the art will recognize that numerous
residues of the above-described sequences can be substituted with
other, chemically, sterically and/or electronically similar
residues without substantially altering the biological activity of
the resulting protein species. In addition, larger or smaller
polypeptide sequences containing substantially the same sequence as
SEQ ID NO:2 or SEQ ID NO:4 therein (e.g., splice variants including
but not limited to SEQ ID NO:6, active fragments of AT, and the
like) are contemplated.
[0036] As employed herein, the term "substantially the same amino
acid sequence" refers to amino acid sequences having at least about
70% identity with respect to the reference amino acid sequence, and
retaining comparable functional and biological activity
characteristic of the protein defined by the reference amino acid
sequence. Preferably, proteins having "substantially the same amino
acid sequence" will have at least about 80%, more preferably 90%
amino acid identity with respect to the reference amino acid
sequence; with greater than about 95%, about 97%, about 99%, up to
100% amino acid sequence identity being especially preferred. Such
amino acid sequence identity measurements may be determined by
standard methodologies, including use of the National Center for
Biotechnology Information BLAST search methodology available at
www.ncbi.nlm.nih.gov. The identity methodologies most preferred are
those described in U.S. Pat. No. 5,691,179 and Altschul et al.,
Nucleic Acids Res. 25:3389-3402, 1997, both of which are
incorporated herein by reference. It is recognized, however, that
DR5 and TRAIL-R3 proteins arising as splice variants (or DR5 and
TRAIL-R3 nucleic acids referred to herein) containing less than the
described levels of sequence identity or that are modified by
conservative amino acid substitutions, or by substitution of
degenerate codons, are also encompassed within the scope of the
present invention. A preferred DR5 protein disclosed herein, is
human DR5 set forth as SEQ ID NO:2. A preferred TRAIL-R3 protein
disclosed herein, is human TRAIL-R3 set forth as SEQ ID NO:4. A
preferred DR5 splice variant protein disclosed herein, is human
DR5s, set forth as SEQ ID NO:6.
[0037] The term "biologically active" or "functional", when used
herein as a modifier of invention DR5 and TRAIL-R3 proteins, or
polypeptide fragment thereof, refers to a polypeptide that exhibits
at least one of the functional characteristics attributed to TRAIL
receptors. For example, one biological activity of invention DR5
and TRAIL-R3 proteins is the ability to bind to the TRAIL ligand.
Other biological activities of invention DR5 and TRAIL-R3 proteins
include the ability to modulate apoptosis (i.e., increasing or
decreasing the level of apoptosis), the ability to bind
intracellular adaptor proteins, and the like.
[0038] Yet another biological activity of invention DR5 or TRAIL-R3
proteins is the ability to act as an immunogen for the production
of polyclonal and monoclonal antibodies that bind specifically to
DR5 or TRAIL-R3. Thus, an invention nucleic acid encoding DR5 or
TRAIL-R3 will encode a polypeptide specifically recognized by an
antibody that also specifically recognizes the DR5 or TRAIL-R3
proteins (preferably human) including the sequences set forth in
SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Such activity may be
assayed by any method known to those of skill in the art. For
example, a test polypeptide encoded by a DR5 or TRAIL-R3 cDNA can
be used to produce antibodies, which may then be assayed for their
ability to bind to the protein including the sequence set forth in
SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. If the antibody binds to
the test-polypeptide and the protein including the sequence set
forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 with
substantially the same affinity, then the polypeptide possesses the
required biological activity.
[0039] The invention DR5 or TRAIL-R3 proteins can be isolated by a
variety of methods well-known in the art, e.g., the recombinant
expression systems described herein, precipitation, gel filtration,
ion-exchange, reverse-phase and affinity chromatography, and the
like. Other well-known methods are described in Deutscher et al.,
Guide to Protein Purification: Methods in Enzymology Vol. 182,
(Academic Press, (1990)), which is incorporated herein by
reference. Alternatively, the isolated polypeptides of the present
invention can be obtained using well-known recombinant methods as
described, for example, in Sambrook et al., (supra., 1989).
[0040] An example of the means for preparing the invention
polypeptide(s) is to express nucleic acids encoding the DR5 or
TRAIL-R3 in a suitable host cell, such as a bacterial cell, a yeast
cell, an amphibian cell (i.e., oocyte), or a mammalian cell, using
methods well known in the art, and recovering the expressed
polypeptide, again using well-known methods. Invention DR5 or
TRAIL-R3 proteins may be isolated directly from cells that have
been transformed with expression vectors as described herein. The
invention polypeptide, biologically active fragments, and
functional equivalents thereof can also be produced by chemical
synthesis. For example, synthetic polypeptides can be produced
using Applied Biosystems, Inc. Model 430A or 431A automatic peptide
synthesizer (Foster City, Calif.) employing the chemistry provided
by the manufacturer.
[0041] Also encompassed by the term DR5 or TRAIL-R3 are active
fragments or polypeptide analogs thereof. The term "active
fragment" refers to a peptide fragment that is a portion of a full
length DR5 or TRAIL-R3 protein, provided that the portion has a
biological activity, as defined above, that is characteristic of at
least one function of the corresponding full length protein. For
example, an active fragment of an DR5 or TRAIL-R3 protein, such as
an extracellular domain can have an activity such as the ability,
for example, to bind TRAIL ligand or to modulate the level of
apoptosis after binding to TRAIL. The characteristic of an active
fragment of invention DR5 or TRAIL-R3 proteins to elicit an immune
response is useful for obtaining an anti-TRAIL receptor antibody.
Thus, the invention also provides active fragments of invention DR5
and TRAIL-R3 proteins, which can be identified using the binding
and bioassays described herein.
[0042] The term "polypeptide analog" includes any polypeptide
having an amino acid residue sequence substantially identical to a
sequence specifically shown herein in which one or more residues
have been conservatively substituted with a functionally similar
residue and which displays the ability to mimic DR5 or TRAIL-R3 as
described herein. Examples of conservative substitutions include
the substitution of one non-polar (hydrophobic) residue such as
isoleucine, valine, leucine or methionine for another, the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine,
between glycine and serine, the substitution of one basic residue
such as lysine, arginine or histidine for another, or the
substitution of one acidic residue, such as aspartic acid or
glutamic acid for another.
[0043] The amino acid length of active fragments or polypeptide
analogs of the present can range from about 5 amino acids up to the
full-length protein sequence of DR5 or TRAIL-R3. In certain
embodiments, the amino acid lengths include, for example, at least
about 10 amino acids, at least about 20, at least about 30, at
least about 40; at least about 50, at least about 75, at least
about 100, at least about 150, at least about 200, at least about
250 or more amino acids in length up to the full-length DR5 or
TRAIL-R3 protein sequence.
[0044] As used herein the phrase "conservative substitution" also
includes the use of a chemically derivatized residue in place of a
non-derivatized residue, provided that such polypeptide displays
the required binding activity. The phrase "chemical derivative"
refers to a subject polypeptide having one or more residues
chemically derivatized by reaction of a functional side group. Such
derivatized molecules include, for example, those molecules in
which free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
Free carboxyl groups may be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups may be derivatized to form O-acyl or O-alkyl derivatives.
The imidazole nitrogen of histidine may be derivatized to form
N-im-benzylhistidine. Also included as chemical derivatives are
those peptides which contain one or more naturally occurring amino
acid derivatives of the twenty standard amino acids. For examples:
4-hydroxyproline may be substituted for proline; S-hydroxylysine
may be substituted for lysine; 3-methylhistidine may be substituted
for histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine. Polypeptides of the
present invention also include any polypeptide having one or more
additions and/or deletions of residues, relative to the sequence of
a polypeptide whose sequence is shown herein, so long as the
required activity is maintained.
[0045] The present invention also provides compositions containing
an acceptable carrier and any of an isolated, purified DR5 or
TRAIL-R3 polypeptide, an active fragment or polypeptide analog
thereof, or a purified, mature protein and active fragments
thereof, alone or in combination with each other. These
polypeptides or proteins can be recombinantly derived, chemically
synthesized or purified from native sources. As used herein, the
term "acceptable carrier" encompasses any of the standard
pharmaceutical carriers, such as phosphate buffered saline
solution, water and emulsions such as an oil/water or water/oil
emulsion, and various types of wetting agents.
[0046] In accordance with another embodiment of the present
invention, there are provided isolated nucleic acids, which encode
invention DR5 or TRAIL-R3 proteins, and fragments thereof. The
nucleic acid molecules described herein are useful for producing
invention proteins, when such nucleic acids are incorporated into a
variety of protein expression systems known to those of skill in
the art. In addition, such nucleic acid molecules or fragments
thereof can be labeled with a readily detectable substituent for
use as hybridization probes to assay for the presence and/or amount
of an DR5 or TRAIL-R3 gene or mRNA transcript in a given sample.
The nucleic acid molecules described herein, and fragments thereof,
are also useful as primers and/or templates in a PCR reaction for
amplifying genes encoding the invention protein described
herein.
[0047] The term "nucleic acid" (also referred to as
polynucleotides) encompasses ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA), probes, oligonucleotides, and primers.
DNA can be either complementary DNA (cDNA) or genomic DNA, e.g. a
gene encoding an DRS or TRAIL-R3 protein. One means of isolating a
nucleic acid encoding a DR5 or TRAIL-R3 polypeptide is to probe a
mammalian genomic or cDNA library with a natural or artificially
designed DNA probe using methods well known in the art. DNA probes
derived from nucleic acid encoding DRS or TRAIL-R3 proteins are
particularly useful for this purpose. DNA and cDNA molecules that
encode DR5 or TRAIL-R3 proteins can be used to obtain complementary
genomic DNA, cDNA or RNA from mammalian (e.g., human, mouse, rat,
rabbit, pig, and the like), or other animal sources, or to isolate
related cDNA or genomic clones by the screening of cDNA or genomic
libraries, by methods described in more detail below. Such nucleic
acids may include, but are not limited to, nucleic acids having
substantially the same nucleotide sequence as set forth in SEQ ID
NO:1 or SEQ ID NO:3, or splice variant cDNA sequences thereof.
[0048] Also encompassed by the terms DR5 or TRAIL-R3 may be "splice
variant" or "alternatively spliced" proteins thereof. These terms
are used herein to describe a particular nucleotide sequence
encoding an invention receptor and refers to a cDNA sequence or
protein encoded thereby that results from the well known eukaryotic
RNA splicing process. The RNA splicing process may involve the
removal of introns and the joining of exons from eukaryotic primary
RNA transcripts to create mature RNA molecules of the cytoplasm.
Methods of isolating splice variant nucleotide sequences are well
known in the art. For example, one skilled in the art can employ
nucleotide probes derived from the DR5 or TRAIL-R3 encoding cDNA of
SEQ ID NO:1 or SEQ ID NO:3 to screen a cDNA or genomic library as
described herein. A preferred splice variant is the alternatively
spliced isoform of DR5, or DR5short (DR5s). The cDNA encoding this
splice variant is set forth in SEQ ID NO:5.
[0049] Alternative splicing may play an important role in
regulation of apoptosis. For example, alternative splicing of the
Bcl-x, Ced-4, and Ich-1 pre-mRNA produces products that play
opposite roles in apoptosis. In this regard, as described herein,
DR5s may be similar in that this isoform may be capable of
inhibiting apoptosis while the larger form (DR5) may promote
apoptosis (see FIG. 3F).
[0050] In one embodiment of the present invention, cDNAs encoding
the invention DR5 and TRAIL-R3 proteins disclosed herein include
substantially the same nucleotide sequence as set forth in SEQ ID
NO:1, SEQ ID NO:3, and SEQ ID NO:5.
[0051] As employed herein, the term "substantially the same
nucleotide sequence" refers to DNA having sufficient identity to
the reference polynucleotide, such that it hybridizes to the
reference nucleotide under moderately stringent hybridization
conditions. In one embodiment, DNA having substantially the same
nucleotide sequence as the reference nucleotide sequence encodes
substantially the same amino acid sequence as that set forth in SEQ
ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 or a larger amino acid
sequence including SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In
another embodiment, DNA having "substantially the same nucleotide
sequence" as the reference nucleotide sequence has at least 60%
identity with respect to the reference nucleotide sequence. DNA
having at least 70%, at least 80%, more preferably at least 90%,
yet more preferably at least 95%, with up to at least 97%, and at
least 99% identity to the reference nucleotide sequence is
preferred.
[0052] The present invention also encompasses nucleic acids that
differ from the nucleic acids shown in SEQ ID NO:1, SEQ ID NO:3, or
SEQ ID NO:5, but which have the same phenotype. Phenotypically
similar nucleic acids are also referred to as "functionally
equivalent nucleic acids". As used herein, the phrase "functionally
equivalent nucleic acids" encompasses nucleic acids characterized
by slight and non-consequential sequence variations that function
in substantially the same manner to produce the same protein
product(s) as the nucleic acids disclosed herein. In particular,
functionally equivalent nucleic acids may encode polypeptides that
are the same as those disclosed herein or that have conservative
amino acid variations, or that encode larger polypeptides that
include SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. For example,
conservative variations include substitution of a non-polar residue
with another non-polar residue, or substitution of a charged
residue with a similarly charged residue. These variations include
those recognized by skilled artisans as those that do not
substantially alter the tertiary structure of the protein.
[0053] Further provided are nucleic acids encoding DR5 and TRAIL-R3
proteins that, by virtue of the degeneracy of the genetic code, do
not necessarily hybridize to the invention nucleic acids under
specified hybridization conditions. Preferred nucleic acids
encoding the invention DR5 or TRAIL-R3 polypeptide are comprised of
nucleotides that encode substantially the same amino acid sequence
set forth in SEQ ID NO:2, SEQ ID NO:4, or splice variants thereof,
respectively.
[0054] Thus, an exemplary nucleic acid encoding an invention DR5 or
TRAIL-R3 protein may be selected from:
[0055] (a) DNA encoding the amino acid sequence set forth in SEQ ID
NO:2 or SEQ ID NO:4;
[0056] (b) DNA that hybridizes to the DNA of (a) under moderately
stringent conditions, wherein said DNA encodes biologically active
DR5 or TRAIL-R3; or
[0057] (c) DNA degenerate with respect to either (a) or (b) above,
wherein said DNA encodes biologically active DR5 or TRAIL-R3.
[0058] Hybridization refers to the binding of complementary strands
of nucleic acid (i.e., sense:antisense strands or probe:target-DNA)
to each other through hydrogen bonds, similar to the bonds that
naturally occur in chromosomal DNA. Stringency levels used to
hybridize a given probe with target-DNA can be readily varied by
those of skill in the art.
[0059] The phrase "stringent hybridization" is used herein to refer
to conditions under which polynucleic acid hybrids are stable. As
known to those skilled in the art, the stability of hybrids is
reflected in the melting temperature (T.sub.m) of the hybrids. In
general, the stability of a hybrid is a function of sodium ion
concentration and temperature. Typically, the hybridization
reaction is performed under conditions of lower stringency,
followed by washes of varying, but higher, stringency. Reference to
hybridization stringency relates to such washing conditions.
[0060] As used herein, the phrase "moderately stringent
hybridization" refers to conditions that permit target-DNA to bind
a complementary nucleic acid that has about 60% identity,
preferably about 75% identity, more preferably about 85% identity
to the target DNA; with greater than about 90% identity to
target-DNA being especially preferred. Preferably, moderately
stringent conditions are conditions equivalent to hybridization in
50% formamide, 5.times.Denhart's solution, 5.times.SSPE, 0.2% SDS
at 42.degree. C., followed by washing in 0.5.times.SSPE, 0.2% SDS,
at 42.degree. C.
[0061] The phrase "high stringency hybridization" refers to
conditions that permit hybridization of only those nucleic acid
sequences that form stable hybrids in 0.018M NaCl at 65.degree. C.
(i.e., if a hybrid is not stable in 0.018M NaCl at 65.degree. C.,
it will not be stable under high stringency conditions, as
contemplated herein). High stringency conditions can be provided,
for example, by hybridization in 50% formamide, 5.times.Denhart's
solution, 5.times.SSPE, 0.2% SDS at 42.degree. C., followed by
washing in 0.1.times.SSPE, and 0.1% SDS at 65.degree. C.
[0062] The phrase "low stringency hybridization" refers to
conditions equivalent to hybridization in 10% formamide,
5.times.Denhart's solution, 6.times.SSPE, 0.2% SDS at 37.degree.
C., followed by washing in 1.times.SSPE, 0.2% SDS, at 50.degree. C.
Denhart's solution and SSPE (see, e.g., Sambrook et al., Molecular
Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press,
1989) are well known to those of skill in the art as are other
suitable hybridization buffers.
[0063] As used herein, the term "degenerate" refers to codons that
differ in at least one nucleotide from a reference nucleic acid,
e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, but encode the same
amino acids as the reference nucleic acid. For example, codons
specified by the triplets "UCU", "UCC", "UCA", and "UCG" are
degenerate with respect to each other since all four of these
codons encode the amino acid serine.
[0064] Preferred nucleic acids encoding the invention
polypeptide(s) hybridize under moderately stringent, preferably
high stringency, conditions to substantially the entire sequence of
the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, or
SEQ ID NO:5.
[0065] Site-directed mutagenesis of any region of DR5 or TRAIL-R3
cDNA is contemplated herein for the production of mutant DR5 or
TRAIL-R3 cDNAs. For example, the Transformer Mutagenesis Kit
(available from Clontech, Palo Alto, Calif.) can be used to
construct a variety of missense and/or nonsense mutations to DR5 or
TRAIL-R3 cDNA, and the like.
[0066] The invention nucleic acids may be produced by a variety of
methods well-known in the art, e.g., the methods described herein,
employing PCR amplification using oligonucleotide primers from
various regions of SEQ ID NO:1 or SEQ ID NO:3, and the like.
[0067] In accordance with a further embodiment of the present
invention, optionally labeled DR5-encoding and TRAIL-R3-encoding
cDNAs, or fragments thereof, can be employed to probe library(ies)
(e.g., cDNA, genomic, and the like) for additional nucleic acid
sequences encoding related novel mammalian DR5 and TRAIL-R3
proteins. Construction of mammalian cDNA and genomic libraries,
preferably a human library, is well-known in the art.
[0068] Screening of such a cDNA or genomic library is initially
carried out under low-stringency conditions, which comprise a
temperature of less than about 42.degree. C., a formamide
concentration of less than about 50%, and a moderate to low salt
concentration.
[0069] In one embodiment, probe-based screening conditions comprise
a temperature of about 37.degree. C., a formamide concentration of
about 20%, and a salt concentration of about 5.times.standard
saline citrate (SSC; 20.times.SSC contains 3M sodium chloride, 0.3M
sodium citrate, pH 7.01). Such conditions will allow the
identification of sequences which have a substantial degree of
similarity with the probe sequence, without requiring perfect
homology. The phrase "substantial similarity" refers to sequences
which share at least 50% homology. Preferably, hybridization
conditions will be selected which allow the identification of
sequences having at least 70% homology with the probe, while
discriminating against sequences which have a lower degree of
homology with the probe. As a result, nucleic acids having
substantially the same (i.e., similar) nucleotide sequence as SEQ
ID NO:1, SEQ ID NO:3, or splice variants thereof including SEQ ID
NO:5, are obtained.
[0070] As used herein, a nucleic acid "oligonucleotide", also
referred to herein as a probe or primer, is single-stranded DNA or
RNA, or analogs thereof, that has a sequence of nucleotides that
includes, for example, at least 14, at least 20, at least 50, at
least 100, at least 200, at least 300, at least 400, or at least
500 contiguous nucleotide bases that are the same as (or the
complement of) any contiguous bases set forth in any of SEQ ID NO:1
or SEQ ID NO:3. Preferred regions from which to construct probes
include 5' and/or 3' coding regions of SEQ ID NO:1, SEQ ID NO:3, or
splice variants thereof. In addition, the entire cDNA encoding
region of an invention protein, such as the entire sequence
corresponding to SEQ ID NO:1 (DR5) or SEQ ID NO:3 (TRAIL-R3), may
be used as a probe. Further, when probing a splice variant, the
splice junctions or intervening sequences may be used to construct
a probe (see FIG. 6). Probes may be labeled by methods well-known
in the art, as described hereinafter, and used in various
diagnostic kits.
[0071] As used herein, the terms "label" and "indicating means" in
their various grammatical forms refer to single atoms and molecules
that are either directly or indirectly involved in the production
of a detectable signal. Any label or indicating means can be linked
to invention nucleic acid probes, expressed proteins, polypeptide
fragments, or antibody molecules. These atoms or molecules can be
used alone or in conjunction with additional reagents. Such labels
are themselves well-known in clinical diagnostic chemistry.
[0072] Also provided are antisense oligonucleotides having a
sequence capable of binding specifically with any portion of an
mRNA that encodes DR5 or TRAIL-R3 proteins so as to prevent
translation of the mRNA. The antisense oligonucleotide may have a
sequence capable of binding specifically with any portion of the
sequence of the cDNA encoding DR5 or TRAIL-R3 proteins. As used
herein, the phrase "binding specifically" encompasses the ability
of a nucleic acid sequence to recognize a complementary nucleic
acid sequence and to form double-helical segments therewith via the
formation of hydrogen bonds between the complementary base pairs.
An example of an antisense oligonucleotide is an antisense
oligonucleotide comprising chemical analogs of nucleotides.
[0073] Compositions comprising an amount of the antisense
oligonucleotide, described above, effective to reduce expression of
DR5 or TRAIL-R3 proteins by passing through a cell membrane and
binding specifically with mRNA encoding DR5 or TRAIL-R3 proteins so
as to prevent translation and an acceptable hydrophobic carrier
capable of passing through a cell membrane are also provided
herein. Suitable hydrophobic carriers are described, for example,
in U.S. Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and the like.
The acceptable hydrophobic carrier capable of passing through cell
membranes may also comprise a structure which binds to a receptor
specific for a selected cell type and is thereby taken up by cells
of the selected cell type. The structure may be part of a protein
known to bind to a cell-type specific receptor, e.g., FGF and other
growth factors.
[0074] Antisense oligonucleotide compositions are useful to inhibit
translation of mRNA encoding invention polypeptides. Synthetic
oligonucleotides, or other antisense chemical structures are
designed to bind to mRNA encoding DR5 or TRAIL-R3 proteins and
inhibit translation of mRNA and are useful as compositions to
inhibit expression of DR5 or TRAIL-R3 associated genes in a tissue
sample or in a subject.
[0075] In accordance with another embodiment of the invention, kits
are provided for detecting the presence of a DR5 or TRAIL-R3
nucleic sequence comprising at least one oligonucleotide, e.g., a
probe or antisense oligonucleotide, according to the present
invention. Such kits can be used for detecting mutations,
duplications, deletions, splice variant transcripts, rearrangements
or aneuploidies in a DR5 or TRAIL-R3 gene.
[0076] The present invention provides means to modulate levels of
expression of DR5 or TRAIL-R3 proteins by employing synthetic
antisense oligonucleotide compositions (hereinafter SAOC) which
inhibit translation of mRNA encoding these polypeptides. Synthetic
oligonucleotides, or other antisense chemical structures designed
to recognize and selectively bind to mRNA, are constructed to be
complementary to portions of the DR5 or TRAIL-R3 coding strand or
nucleotide sequences shown in SEQ ID NO:l or SEQ ID NO:3. The SAOC
is designed to be stable in the blood stream for administration to
a subject by injection or by direct tumor site integration, or
stable in laboratory cell culture conditions. The SAOC is designed
to be capable of passing through the cell membrane in order to
enter the cytoplasm of the cell by virtue of physical and chemical
properties of the SAOC which render it capable of passing through
cell membranes, for example, by designing small, hydrophobic SAOC
chemical structures, or by virtue of specific transport systems in
the cell which recognize and transport the SAOC into the cell. In
addition, the SAOC can be designed for administration only to
certain selected cell populations by targeting the SAOC to be
recognized by specific cellular uptake mechanisms which bind and
take up the SAOC only within select cell populations.
[0077] For example, the SAOC may be designed to bind to a receptor
found only in a certain cell type, as discussed supra. The SAOC is
also designed to recognize and selectively bind to target mRNA
sequence, which may correspond to a sequence contained within the
sequence shown in SEQ ID NO:1, SEQ ID NO:3, or splice variants
thereof. The SAOC is designed to inactivate target mRNA sequence by
either binding thereto and inducing degradation of the mRNA by, for
example, RNase I digestion, or inhibiting translation of mRNA
target sequence by interfering with the binding of
translation-regulating factors or ribosomes, or inclusion of other
chemical structures, such as ribozyme sequences or reactive
chemical groups which either degrade or chemically modify the
target mRNA. SAOCs have been shown to be capable of such properties
when directed against mRNA targets (see Cohen et al., TIPS, 10:435
(1989) and Weintraub, Sci. American, January (1990), p.40; both
incorporated herein by reference).
[0078] In accordance with yet another embodiment of the present
invention, there is provided a method for the recombinant
production of invention DR5 or TRAIL-R3 proteins by expressing the
above-described nucleic acid sequences in suitable host cells.
Recombinant DNA expression systems that are suitable to produce DR5
and TRAIL-R3 proteins described herein are well-known in the art.
For example, the above-described nucleotide sequences can be
incorporated into vectors for further manipulation. As used herein,
vector (or plasmid) refers to discrete elements that are used to
introduce heterologous DNA into cells for either expression or
replication thereof.
[0079] Suitable expression vectors are well-known in the art, and
include vectors capable of expressing DNA operatively linked to a
regulatory sequence, such as a promoter region that is capable of
regulating expression of such DNA. Thus, an expression vector
refers to a recombinant DNA or RNA construct, such as a plasmid, a
phage, recombinant virus or other vector that, upon introduction
into an appropriate host cell, results in expression of the
inserted DNA. Appropriate expression vectors are well known to
those of skill in the art and include those that are replicable in
eukaryotic cells and/or prokaryotic cells and those that remain
episomal or those which integrate into the host cell genome. In
addition, vectors may contain appropriate packaging signals that
enable the vector to be packaged by a number of viral virions,
e.g., retroviruses, herpes viruses, adenoviruses, resulting in the
formation of a "viral vector."
[0080] As used herein, the term "operatively linked" refers to the
functional relationship of DNA with regulatory and effector
nucleotide sequences, such as promoters, enhancers, transcriptional
and translational stop sites, and other signal sequences. For
example, operative linkage of DNA to a promoter refers to the
physical and functional relationship between the DNA and the
promoter such that the transcription of such DNA is initiated from
the promoter by an RNA polymerase that specifically recognizes,
binds to and transcribes the DNA.
[0081] Suitable transformation vectors are well-known in the art
and include Blueskript and phage Lambda ZAP vectors (Stratagene, La
Jolla, Calif.), and the like. Other suitable vectors and promoters
are disclosed in detail in U.S. Pat. No. 4,798,885, issued Jan. 17,
1989, the disclosure of which is incorporated herein by reference
in its entirety.
[0082] In accordance with another embodiment of the present
invention, there are provided "recombinant cells" containing the
nucleic acid molecules (i.e., DNA or mRNA) of the present
invention. Methods of transforming suitable host cells, preferably
bacterial cells, and more preferably E. coli cells, as well as
methods applicable for culturing said cells containing a gene
encoding a heterologous protein, are generally known in the art.
See, for example, Sambrook et al., Molecular Clonina: A Laboratory
Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., USA (1989).
[0083] Exemplary methods of introducing (transducing) expression
vectors containing invention nucleic acids into host cells to
produce transduced recombinant cells (i.e., cells containing
recombinant heterologous nucleic acid) are well-known in the art
(see, for review, Friedmann, Science, 244:1275-1281 (1989);
Mulligan, Science, 260:926-932 (1993), each of which are
incorporated herein by reference in their entirety). Exemplary
methods of transduction include, e.g., infection employing viral
vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764),
calcium phosphate transfection (U.S. Pat. Nos. 4,399,216 and
4,634,665), dextran sulfate transfection, electroporation,
lipofection (see, e.g., U.S. Pat. Nos. 4,394,448 and 4,619,794),
cytofection, particle bead bombardment, and the like. The
heterologous nucleic acid can optionally include sequences which
allow for its extrachromosomal (i.e., episomal) maintenance, or the
heterologous DNA can be caused to integrate into the genome of the
host (as an alternative means to ensure stable maintenance in the
host).
[0084] Host organisms contemplated for use in the practice of the
present invention include those organisms in which recombinant
production of heterologous proteins has been carried out. Examples
of such host organisms include bacteria (e.g., E. coli), yeast
(e.g., Saccharomyces cerevisiae, Candida tropicalis, Hansenula
polymorpha and P. pastoris; see, e.g., U.S. Pat. Nos. 4,882,279,
4,837,148, 4,929,555 and 4,855,231), mammalian cells (e.g., HEK293,
CHO and Ltk-cells), insect cells, and the like.
[0085] In one embodiment, nucleic acids encoding the invention DR5
or TRAIL-R3 proteins can be delivered into mammalian cells, either
in vivo or in vitro using suitable viral vectors well-known in the
art (e.g., retroviral vectors, adenovirus vectors, and the like).
In addition, where it is desirable to limit or reduce the in vivo
expression of the invention DR5 or TRAIL-R3 proteins, the
introduction of the antisense strand of the invention nucleic acid
is contemplated.
[0086] Viral based systems provide the advantage of being able to
introduce relatively high levels of the heterologous nucleic acid
into a variety of cells. Suitable viral vectors for introducing
invention nucleic acid encoding an DR5 or TRAIL-R3 protein into
mammalian cells (e.g., vascular tissue segments) are well known in
the art. These viral vectors include, for example, Herpes simplex
virus vectors (e.g., Geller et al., Science, 241:1667-1669 (1988)),
Vaccinia virus vectors (e.g., Piccini et al., Meth. in Enzmology,
153:545-563 (1987); Cytomegalovirus vectors (Mocarski et al., in
Viral Vectors, Y. Gluzman and S. H. Hughes, Eds., Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84),
Moloney murine leukemia virus vectors (Danos et al., PNAS USA,
85:6469 (1980)), adenovirus vectors, (e.g., Logan et al., PNAS,
USA, 81:3655-3659 (1984); Jones et al., Cell, 17:683-689 (1979);
Berkner, Biotechniques, 6:616-626 (1988); Cotten et al., PNAS, USA,
89:6094-6098 (1992); Graham et al., Meth. Mol. Biol, 7:109-127
(1991)), adeno-associated virus vectors, retrovirus vectors (see,
e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764), and the like.
Especially preferred viral vectors are the adenovirus and
retroviral vectors.
[0087] For example, in one embodiment of the present invention,
adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes
(Wagner et al., PNAS, USA, 89:6099-6103 (1992); Curiel et al., Hum.
Gene Therapy, 3:147-154 (1992); Gao et al., Hum. Gene Ther.,
4:14-24 (1993)) are employed to transduce mammalian cells with
heterologous DR5 or TRAIL-R3 nucleic acid. Any of the plasmid
expression vectors described herein may be employed in a TfAdpl-DNA
complex.
[0088] As used herein, "retroviral vector" refers to the well-known
gene transfer plasmids that have an expression cassette encoding an
heterologous gene residing between two retroviral LTRs. Retroviral
vectors typically contain appropriate packaging signals that enable
the retroviral vector, or RNA transcribed using the retroviral
vector as a template, to be packaged into a viral virion in an
appropriate packaging cell line (see, e.g., U.S. Pat. No.
4,650,764).
[0089] Suitable retroviral vectors for use herein are described,
for example, in U.S. Pat. No. 5,252,479, and in WIPO publications
WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829,
incorporated herein by reference, which provide a description of
methods for efficiently introducing nucleic acids into human cells
using such retroviral vectors. Other retroviral vectors include,
for example, the mouse mammary tumor virus vectors (e.g.,
Shackleford et al., PNAS, USA, 85:9655-9659 (1988)), and the
like.
[0090] In accordance with yet another embodiment of the present
invention, there are provided anti-DR5 and anti -TRAIL-R3
antibodies having specific reactivity with DR5 and TRAIL-R3
proteins, respectively, of the present invention. Active fragments
of antibodies are encompassed within the definition of "antibody".
Invention antibodies can be produced by methods known in the art
using invention DR5 or TRAIL-R3 proteins, or portions thereof as
antigens. For example, polyclonal and monoclonal antibodies can be
produced by methods well known in the art, as described, for
example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold
Spring Harbor Laboratory (1988)), which is incorporated herein by
reference.
[0091] Invention DR5 and TRAIL-R3 proteins can be used as
immunogens in generating such antibodies. Alternatively, synthetic
peptides can be prepared (using commercially available
synthesizers) and used as immunogens. Amino acid sequences can be
analyzed by methods well known in the art to determine whether they
encode hydrophobic or hydrophilic domains of the corresponding
polypeptide. Altered antibodies such as chimeric, humanized,
CDR-grafted or bifunctional antibodies can also be produced by
methods well known in the art. Such antibodies can also be produced
by hybridoma, chemical synthesis or recombinant methods described,
for example, in Sambrook et al., supra, and Harlow and Lane, supra.
Both anti-peptide and anti-fusion protein antibodies can be used.
(see, for example, Bahouth et al., Trends Pharmacol. Sci. 12:338
(1993); Ausubel et al., Current Protocols in Molecular Biology
(John Wiley and Sons, N.Y. (1989) which are incorporated herein by
reference).
[0092] Antibody so produced can be used, inter alia, in diagnostic
methods and systems to detect the level of DR5 or TRAIL-R3 protein
present in a mammalian, preferably human, body sample, such as
tissue or vascular fluid. Such antibodies can also be used for the
immunoaffinity or affinity chromatography purification of the
invention DR5 and TRAIL-R3 proteins. In addition, methods are
contemplated herein for detecting the presence of DR5 or TRAIL-R3
proteins either on the surface of a cell or within a cell, which
methods comprise contacting the cell with an antibody that
specifically binds to DR5 or TRAIL-R3 proteins, under conditions
permitting binding of the antibody to DR5 or TRAIL-R3 proteins,
detecting the presence of the antibody bound to DR5 or TRAIL-R3,
and thereby detecting the presence of invention polypeptides on the
surface of, or within, the cell. With respect to the detection of
such polypeptides, the antibodies can be used for in vitro
diagnostic or in vivo imaging methods.
[0093] Immunological procedures useful for in vitro detection of
target DR5 or TRAIL-R3 proteins in a sample include immunoassays
that employ a detectable antibody. Such immunoassays include, for
example, ELISA, Pandex microfluorimetric assay, agglutination
assays, flow cytometry, serum diagnostic assays and
immunohistochemical staining procedures which are well known in the
art. An antibody can be made detectable by various means well known
in the art. For example, a detectable marker can be directly or
indirectly attached to the antibody. Useful markers include, for
example, radionucleotides, enzymes, fluorogens, chromogens and
chemiluminescent labels.
[0094] Invention anti-DR5 or TRAIL-R3 antibodies are contemplated
for use herein to modulate activity of the DR5 or TRAIL-R3
polypeptide in living animals, in humans, or in biological tissues
or fluids isolated therefrom. The term "modulate" refers to a
compound's ability to increase (e.g., via an agonist) or inhibit
(e.g., via an antagonist) the biological activity of DR5 or
TRAIL-R3 protein, such as the apoptosis mediating activity of DR5
or TRAIL-R3. Accordingly, compositions comprising a carrier and an
amount of an antibody having specificity for DR5 or TRAIL-R3
proteins effective to block naturally occurring ligands, such as
TRAIL, or other DR5-binding or TRAIL-binding proteins from binding
to invention DR5 or TRAIL-R3 proteins are contemplated herein. For
example, a monoclonal antibody directed to an epitope of DR5 or
TRAIL-R3 present on the surface of a cell that has an amino acid
sequence substantially the same as an amino acid sequence shown in
SEQ ID NO:2, SEQ ID NO:4, or splice variant thereof (e.g., SEQ ID
NO:6), can be useful for this purpose.
[0095] The present invention further provides transgenic non-human
mammals that are capable of expressing exagenous nucleic acids
encoding DR5 or TRAIL-R3 proteins. As employed herein, the phrase
"exagenous nucleic acid" refer to nucleic acid sequence which is
not native to the host, or which is present in the host in other
than its native environment (e.g., as part of a genetically
engineered DNA construct). In addition to naturally occurring
levels of invention TRAIL receptors, invention DR5 or TRAIL-R3
proteins can either be overexpressed, underexpressed, or expressed
in an inactive mutated form (such as in the well-known knockout
transgenics) in transgenic mammals.
[0096] Also provided are transgenic non-human mammals capable of
expressing nucleic acids encoding DR5 or TRAIL-R3 proteins so
mutated as to be incapable of normal activity, i.e., do not express
native DR5 or TRAIL-R3. The present invention also provides
transgenic non-human mammals having a genome comprising antisense
nucleic acids complementary to nucleic acids encoding DR5 or
TRAIL-R3 proteins, placed so as to be transcribed into antisense
mRNA complementary to mRNA encoding DR5 or TRAIL-R3 proteins, which
hybridizes to the mRNA and, thereby, reduces the translation
thereof. The nucleic acid may additionally comprise an inducible
promoter and/or tissue specific regulatory elements, so that
expression can be induced, or restricted to specific cell types.
Examples of nucleic acids are DNA or cDNA having a coding sequence
substantially the same as the coding sequence shown in SEQ ID NO:1,
SEQ ID NO:3, or SEQ ID NO:5. An example of a non-human transgenic
mammal is a transgenic mouse.
[0097] Animal model systems which elucidate the physiological and
behavioral roles of DR5 or TRAIL-R3 proteins are also provided, and
are produced by creating transgenic animals in which the expression
of the DR5 or TRAIL-R3 polypeptide is altered using a variety of
techniques. Examples of such techniques include the insertion of
normal or mutant versions of nucleic acids encoding a DR5 or
TRAIL-R3 polypeptide by microinjection, retroviral infection or
other means well known to those skilled in the art, into
appropriate fertilized embryos to produce a transgenic animal.
(See, for example, Hogan et al., Manipulating the Mouse Embryo: A
Laboratory Manual (Cold Spring Harbor Laboratory, (1986)).
[0098] Also contemplated herein, is the use of homologous
recombination of mutant or normal versions of genes encoding DR5 or
TRAIL-R3 proteins with the native gene locus in transgenic animals,
to alter the regulation of expression or the structure of (see,
Capecchi et al., Science, 244:1288 (1989); Zimmer et al., Nature,
338:150 (1989); which are incorporated herein by reference).
Homologous recombination techniques are well known in the art.
Homologous recombination replaces the native (endogenous) gene with
a recombinant or mutated gene to produce an animal that cannot
express native (endogenous) protein but can express, for example, a
mutated protein which results in altered expression of DR5 or
TRAIL-R3 proteins.
[0099] In contrast to homologous recombination, microinjection adds
genes to the host genome, without removing host genes.
Microinjection can produce a transgenic animal that is capable of
expressing both endogenous and exagenous DR5 or TRAIL-R3 protein.
Inducible promoters can be linked to the coding region of nucleic
acids to provide a means to regulate expression of the transgene.
Tissue specific regulatory elements can be linked to the coding
region to permit tissue-specific expression of the transgene.
Transgenic animal model systems are useful for in vivo screening of
compounds for identification of specific agents, i.e., agonists and
antagonists, which activate or inhibit protein responses.
[0100] Invention nucleic acids, oligonucleotides (including
antisense), vectors containing same, transformed host cells,
polypeptides and combinations thereof, as well as antibodies of the
present invention, can be used to screen compounds in vitro to
determine whether a compound functions as a potential agonist or
antagonist to invention DR5 or TRAIL-R3 proteins. These in vitro
screening assays provide information regarding the function and
activity of invention DR5 or TRAIL-R3 proteins, which can lead to
the identification and design of compounds that are capable of
specific interaction with one or more types of polypeptides,
peptides or proteins.
[0101] Apoptosis plays a significant role in numerous pathological
conditions in that programmed cell death is either inhibited,
resulting in increased cell survival, or enhanced which results in
the loss of cell viability. Examples of pathological conditions
resulting from increased cell survival include cancers such as
lymphomas, carcinomas and hormone dependent tumors. Such hormone
dependent tumors include, for example, breast, prostate and ovarian
cancer. Autoimmune diseases such as systemic lupus erythematosus
and immune-mediated glomerulonephritis as well as viral infections
such as herpes virus, poxvirus and adenovirus also result from
increased cell survival or the inhibition of apoptosis.
[0102] In contrast, apoptotic diseases where enhanced programmed
cell death is a prevalent cause generally includes, for example,
degenerative disorders such as Alzheimer's disease, Parkinson's
disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, and
Cerebellar degeneration. Other diseases associated with increased
apoptosis include, for example, myelodysplastic syndromes such as
aplastic anemia and ischemic injury including myocardial
infarction, stroke and reperfusion injury.
[0103] The DR5 or TRAIL-R3 encoding nucleic acids and polypeptides
of the invention can be used to diagnose, treat or reduce the
severity of cell death mediated diseases such as those described
above as well as other diseases mediated by either increased or
decreased programmed cell death. Additionally, the DR5 or TRAIL-R3
encoding nucleic acids and polypeptides of the invention can be
used to screen for pharmaceutical compounds and macromolecules
which inhibit or promote DR5 or TRAIL-R3 mediated apoptosis.
[0104] For example, the DR5 or TRAIL-R3 encoding nucleic acids,
polypeptides and functional fragments thereof can be used to
diagnose, or to generate reagents to diagnose diseases mediated or
characterized by programmed cell death. Diagnosis can be by nucleic
acid probe hybridization with DR5 or TRAIL-R3 containing nucleotide
sequences, antibody or ligand mediated detection with DR5 or
TRAIL-R3 binding agents or by enzyme catalysis of detectable DR5 or
TRAIL-R3 substrates. Such methods are routine to those skilled in
the art. Detection can be performed ex vivo, for example, by
removing a cell or tissue sample from an individual exhibiting or
suspected of exhibiting a cell death mediated disease. Correlation
of increased DR5 or TRAIL-R3 expression or activity may be
indicative of diseases characterized by enhanced programmed cell
death whereas correlation of decreased DR5 or TRAIL-R3 expression
or activity may be indicative of diseases characterized by the
inhibition of programmed cell death.
[0105] Thus, in accordance with still another embodiment of the
present invention, there is provided a method for identifying
compounds that may bind to DR5 or TRAIL-R3 proteins such as, for
example, antibodies, binding agents, and the like. For example, the
invention DR5 or TRAIL-R3 proteins may be employed in a competitive
binding assay. Such an assay can accommodate the rapid screening of
a large number of compounds to determine which compounds, if any,
are capable of binding to DR5 or TRAIL-R3 proteins. Subsequently,
more detailed assays can be carried out with those compounds found
to bind, to further determine whether such compounds act as
modulators, e.g., agonists or antagonists, of invention
proteins.
[0106] Thus, in another embodiment of the invention, there is
provided a bioassay for identifying compounds that modulate the
activity of invention DR5 or TRAIL-R3 proteins. According to this
method, invention DR5 or TRAIL-R3 proteins, preferably membrane
bound, may be contacted with TRAIL ligand in the presence and in
the absence of a test-compound; the activity of the DR5 or TRAIL-R3
proteins is monitored subsequent to the contact with the test
compound, and those test-compounds that may cause either the
increase or decrease of apoptosis in cellular systems having
membrane bound DR5 or TRAIL-R3 proteins therein may be identified
as functional agents for modulating DRS or TRAIL-R3 proteins.
[0107] In accordance with another embodiment of the present
invention, transformed host cells (either completely-intact or
semi-intact cells) that recombinantly express invention DR5 or
TRAIL-R3 proteins can be contacted with a test compound, and the
modulating effect(s) thereof can then be evaluated by comparing the
relative levels of apoptosis modulation in the presence and absence
of test compound, or by comparing the response of test cells or
control cells (i.e., cells that do not express DR5 or TRAIL-R3
proteins), to the presence of the compound.
[0108] As used herein, a compound or a signal that "modulates the
activity" of invention DR5 or TRAIL-R3 proteins refers to a
compound or a signal that may alter the activity of DRS or TRAIL-R3
proteins so that the activity of the invention DR5 or TRAIL-R3
proteins is different in the presence of the compound or signal
than in the absence of the compound or signal. In particular, such
compounds or signals include agonists and antagonists. An agonist
encompasses a compound or a signal that may activate or increase
the function of invention DR5 or TRAIL-R3 proteins. Alternatively,
an antagonist includes a compound or signal that may interfere
with, inhibit or otherwise decrease DR5 or TRAIL-R3 protein
function. Typically, the effect of an antagonist may be observed as
a blocking of agonist-induced protein activation. Antagonists
include competitive and non-competitive antagonists. A competitive
antagonist (or competitive blocker) interacts with or near the site
specific for TRAIL binding. A non-competitive antagonist or blocker
inactivates the function of the polypeptide by interacting with a
site other than the apoptosis modulating region of invention DR5 or
TRAIL-R3 proteins.
[0109] As understood by those of skill in the art, assay methods
for identifying compounds that modulate DR5 or TRAIL-R3 activity
generally require comparison to a control. One type of a "control"
is a cell or culture that is treated substantially the same as the
test cell or test culture exposed to the compound, with the
distinction that the "control" cell or culture is not exposed to
the compound. For example, a type of "control" cell or culture may
be a cell or culture that is identical to the transfected cells,
with the exception that the "control" cell or culture do not
express native proteins. Accordingly, the response of the
transfected cell to compound may be compared to the response (or
lack thereof) of the "control" cell or culture to the same compound
under the same reaction conditions.
[0110] In another embodiment of the present invention, there is
provided a bioassay for evaluating whether test compounds are
capable of acting as agonists or antagonists for DR5 or TRAIL-R3
proteins, wherein said bioassay comprises:
[0111] (a) culturing cells containing: DNA which expresses a TRAIL
receptor selected from DR5 or TRAILR3, or functional modified forms
thereof, wherein said culturing is carried out in the presence of
at least one compound whose ability to modulate apoptotic activity
of TRAIL receptors is sought to be determined, and thereafter
[0112] (b) monitoring said cells for either an increase or decrease
in the level of apoptosis.
[0113] Such an assay can be carried out in the presence or absence
of TRAIL ligand. Methods are well-known in the art for measuring
apoptosis can be employed in bioassays described herein to identify
agonists and antagonists of DR5 or TRAIL-R3 proteins. For example,
the methods described in Example IV can be used to evaluate the
apoptotic activity of recombinant DR5 or TRAIL-R3 proteins or
mutants and/or analogs thereof, expressed in mammalian host
cells.
[0114] In addition, the occurrence of apoptosis in cell-free
systems can be assessed by detecting the relative levels of:
caspase processing (i.e., the cleavage of the pro-caspase to active
forms; see, e.g., Casciola-Rosen et al., 1996, J. Exp.
Med.,183:1957-1964; Tewari et al, 1995, J. Biol. Chem.,
32:18738-18741; Tewari et al, 1995, Cell, 81:801-809), caspase
activation, cytosolic substrate cleavage, the release of
cytochrome-c from mitochondria, and the like. Exemplary cytosolic
substrates that are cleaved as a result of apoptosis are set forth
in Table 1, and include: fodrin, CPP32, PKC.delta., and the
like.
[0115] When nuclei are present in the bioassays described herein,
the occurrence of apoptosis can be assessed by, in addition to the
methods described above, detecting: chromatin condensation,
shrinkage and fragmentation of the nuclei, and the like (see, for
example, Zanzami et al., J. Exp. Med., 183:1533-1544 (1995);
Newmeyer et al., Cell. 79:353-364 (1994)). In addition, nuclear
substrates that are cleaved as a result of apoptosis are also set
forth in Table 1, and include: DNA topoisomerase (Liu, Ann. Rev.
Biochem. 58, 351-375 1989), lamin B (Lazebnik et al., Proc. Natl.
Acad. Sci. USA 92, 9042-9046. 1995), NuMA (Compton, 1994), PARP
(Lazebnik et al., Supra, 1994), and Ul-70 kDa (Casciola-Rosen et
al., J. Biol. Chem. 49, 30757-30760. 1994), and the like.
[0116] As used herein, "ability to modulate apoptotic activity of
TRAIL receptors" refers to a compound that has the ability to
either induce (agonist) or inhibit (antagonist) apoptosis mediated
by DR5 or TRAIL-R3 proteins.
[0117] In another embodiment of the present invention, the bioassay
for evaluating whether test compounds are capable of acting as
antagonists for DR5 and TRAIL-R3 proteins of the invention, or
functional modified forms of said DR5 and TRAIL-R3 proteins,
comprises:
[0118] (a) culturing cells containing: DNA which expresses DR5 and
TRAIL-R3 proteins, or functional modified forms thereof, wherein
said culturing is carried out in the presence of: increasing
concentrations of at least one compound whose ability to inhibit
apoptotic activity of DR5 and TRAIL-R3 proteins is sought to be
determined, and a fixed concentration of TRAIL; and thereafter
[0119] (b) monitoring in said cells the level of apoptosis as a
function of the concentration of said compound, thereby indicating
the ability of said compound to inhibit DR5 or TRAIL-R3 mediated
apoptotic activity.
[0120] In step (a) of the above-described antagonist bioassay,
culturing may also be carried out in the presence of:
[0121] fixed concentrations of at least one compound whose ability
to inhibit apoptotic activity of DR5 and TRAIL-R3 proteins is
sought to be determined, and
[0122] an increasing concentration of TRAIL.
[0123] Host cells contemplated for use in the bioassay(s) of the
present invention include MCH6 cells, 293 cells, CV-1 cells, COS
cells, HeLa cells, and the like. Presently, preferred host cells
for carrying invention bioassays are HeLa cells as described in
Example VI.
[0124] Also contemplated in yet another embodiment of the present
invention, is a method for modulating the apoptotic activity
mediated by DR5 or TRAIL-R3 proteins, said method comprising:
[0125] contacting an DR5 or TRAIL-R3 protein with an effective,
modulating amount of an agonist or antagonist identified by the
above-described bioassays.
[0126] The present invention also contemplates therapeutic
compositions useful for practicing the therapeutic methods
described herein. For example, the above described DR5 or TRAIL-R3
polypeptides can also be formulated into pharmaceutical
compositions known within the art for the treatment of programmed
cell death mediated diseases characterized by increased or
decreased cell survival and proliferation. Functional fragments and
peptides such as the extracellular TRAIL-binding domains and the
cytoplasmic death domain of DR5 or TRAIL-R3 can similarly be
formulated for the treatment of such diseases associated with
increased or decreased cell survival and proliferation.
Additionally, molecules that interact with DR5 or TRAIL-R3 can
additionally be used to modulate DR5 or TRAIL-R3 mediated
apoptosis. Administration of DR5 polypeptides and functional
fragments thereof may induce or inhibit apoptosis in treated cells,
and may eliminate those cells characterized by increased cell
survival or proliferation.
[0127] Treatment or reduction of the severity of cell death
mediated diseases can also be accomplished by introducing
expressible nucleic acids encoding DR5 or TRAIL-R3 polypeptides or
functional fragments thereof into cells characterized by such
diseases. Elevated synthetic rates of DR5 or TRAIL-R3 may be
achieved, for example, by using recombinant expression vectors and
gene transfer technology. Similarly, treatment or reduction of the
severity of cell death mediated diseases may also be accomplished
by introducing and expressing antisense DR5 or TRAIL-R3 nucleic
acids so as to inhibit the synthesis rates of DR5 or TRAIL-R3. Such
methods are well known within the art and are described herein with
reference to recombinant viral vectors. Other vectors compatible
with the appropriate targeted cell can accomplish the same goal and
therefore can be substituted in the methods described herein in
place of recombinant viral vectors.
[0128] Therapeutic compositions of the present invention contain a
physiologically compatible carrier together with DR5 or TRAIL-R3
polypeptides or functional fragments thereof, a DR5 or TRAIL-R3
modulating agent, or an anti-DR5 or anti-TRAIL-R3 antibody, as
described herein, dissolved or dispersed therein as an active
ingredient.
[0129] As used herein, the terms "pharmaceutically acceptable",
"physiologically acceptable" and grammatical variations thereof, as
they refer to compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials are capable
of administration to a mammal without the production of undesirable
physiological effects such as nausea, dizziness, gastric upset, and
the like.
[0130] The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
known in the art. Typically such compositions are prepared as
injectables either as liquid solutions or suspensions; however,
solid forms suitable for solution, or suspension, in liquid prior
to use can also be prepared. The preparation can also be
emulsified.
[0131] The active ingredient can be mixed with excipients that are
pharmaceutically acceptable and compatible with the active
ingredient in amounts suitable for use in the therapeutic methods
described herein. Suitable excipients are, for example, water,
saline, dextrose, glycerol, ethanol, or the like, as well as
combinations of any two or more thereof. In addition, if desired,
the composition can contain minor amounts of auxiliary substances
such as wetting or emulsifying agents, pH buffering agents, and the
like, which enhance the effectiveness of the active ingredient.
[0132] The therapeutic composition of the present invention can
include pharmaceutically acceptable salts of the components
therein. Pharmaceutically acceptable nontoxic salts include the
acid addition salts (formed with the free amino groups of the
polypeptide) that are formed with inorganic acids such as, for
example, hydrochloric acid, hydrobromic acid, perchloric acid,
nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid,
acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic
acid, oxalic acid, malonic acid, succinic acid, maleic acid,
fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic
acid, sulfanilic acid, and the like.
[0133] Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium
hydroxide, ammonium hydroxide, potassium hydroxide, and the like;
and organic bases such as mono-, di-, and tri-alkyl and -aryl
amines (e.g., triethylamine, diisopropyl amine, methyl amine,
dimethyl amine, and the like) and optionally substituted
ethanolamines (e.g., ethanolamine, diethanolamine, and the
like).
[0134] Physiologically tolerable carriers are well known in the
art. Exemplary liquid carriers are sterile aqueous solutions that
contain no materials in addition to the active ingredients and
water, or contain a buffer such as sodium phosphate at
physiological pH, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous carriers can
contain more than one buffer salt, as well as salts such as sodium
and potassium chlorides, dextrose, polyethylene glycol and other
solutes.
[0135] Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary additional
liquid phases include glycerin, vegetable oils such as cottonseed
oil, and water-oil emulsions.
[0136] As described herein, an "effective amount" is a
predetermined amount calculated to achieve the desired therapeutic
effect, e.g., to modulate the TRAIL mediated apoptotic activity of
an invention DR5 or TRAIL-R3 protein. The required dosage will vary
with the particular treatment and with the duration of desired
treatment. Such dosages are known or can be easily determined by
those skilled in the art. Administration may be accomplished, for
example, by intravenous, interperitonal or subcutaneous injection.
Administration may be performed in a variety of different regimes,
which include single high dose administration or repeated small
dose administration, or a combination of both. The dosing may
depend on the cell type, progression of the disease and overall
health of the individual, and will be known or can be determined by
those skilled in the art.
[0137] It is contemplated that dosages between about 10 micrograms
and about 1 milligram per kilogram of body weight per day may be
used for therapeutic treatment. It may be particularly advantageous
to administer such compounds in depot or long-lasting form as
discussed herein. A therapeutically effective amount is typically
an amount of an DR5- or TRAIL-R3-modulating agent or compound
identified herein that, when administered in a physiologically
acceptable composition, is sufficient to achieve a plasma
concentration of from about 0.1 .mu.g/ml to about 100 .mu.g/ml,
preferably from about 1.0 .mu.g/ml to about 50 .mu.g/ml, more
preferably at least about 2 .mu.g/ml and usually 5 to 10
.mu.g/ml.
[0138] All U.S. patents and all publications mentioned herein are
incorporated in their entirety by reference thereto. The invention
will now be described in greater detail by reference to the
following non-limiting examples.
EXAMPLES
[0139] Unless otherwise stated, the present invention was performed
using standard procedures, as described, for example in Maniatis et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et
al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);
Davis et al., Basic Methods in Molecular Biology, Elsevier Science
Publishing, Inc., New York, USA (1986); or Methods in Enzymology:
Guide to Molecular Cloning Techniques Vol 152, S. L. Berger and A.
R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987).
Example I
Cloning and Characterization of DR5 and TRAIL-R3
[0140] This example demonstrates the identification and cloning of
DR5 and TRAIL-R3 cDNA. Briefly, several EST clones were identified
and their 3' and 5' sequences were compiled. Based on the compiled
sequences, PCR primers were generated and used to clone two cDNAs
which encode two novel members of the TRAIL-receptor family (FIGS.
1A and 1B).
[0141] To identify novel members of the TNF-receptor family, an
approach combining information from the GenBank database of human
expressed sequence tags (ESTs) and PCR was employed (see Alnemri E.
S. et al. (1996) Proc. Natl. Sci. USA. 93, 7464-7469;
Fernandez-Alnemri et al., Cancer Res. 5S:2737-2742 (1995); and
Fernandez-Alnemri et al., Cancer Res. 55:6045-6052 (1995)).
Initially, a search of the GenBank EST data base for sequences
related to TRAIL receptor-1, also referred to as "DR4", identified
several EST clones, which were used to derive 5' and 3' PCR primers
to clone related cDNAs.
[0142] The EST sequences used to clone DR5 were identified as human
GenBank EST clone 650744 (Accession Nos. AA223122 and AA223238) and
clone 664665 (Accession Nos. AA232424 and AA232440). These EST
clones were used to design primers at the extreme 5' and 3' ends of
the DR5 coding region. The 5' primer employed to amplify DR5 cDNA
corresponded to the oligonucleotide set forth as nucleotides 1-18
of SEQ ID NO:1. The 3' primer used corresponds to the
oligonucleotide complementary to nucleotides 1219-1236 of SEQ ID
NO:1.
[0143] Similarly, the EST sequences used to clone TRAIL-R3 were
identified as human GenBank EST clone 470799 (Accession No.
AA031883), clone 129137 (Accession Nos. R10995 and R10996) and
clone 504745 (Accession Nos. AA150849 and AA150541). These EST
clones were used to design primers at the extreme 5' and 3' ends of
the TRAIL-R3 coding region. The 5' primer employed to amplify
TRAIL-R3 cDNA co-responded to the oligonucleotide set forth as
nucleotides 1-18 of SEQ ID NO:3. The 3' primer used corresponds to
the oligonucleotide complementary to nucleotides 883-900 SEQ ID
NO:3.NO:1.
[0144] A 10 .mu.l aliquot of human Jurkat .lambda. Uni-Zap.TM. XR
cDNA library (Fernandez-Alnemri et al., J. Biol. Chem.
269:30761-30764 (1994)) containing approximately 10.sup.8 pfu was
denatured at 99.degree. C. for 5 min. and used as a template for
PCR amplification with the above-described primer pairs for cloning
DR5 and TRAIL-R3 cDNA. The full-length amplification products were
cloned into a small-cut pBluescript II KS.sup.+ vector and
sequenced.
[0145] To confirm the sequence of the PCR-amplified DR5 and
TRAIL-R3 cDNA, the cloned cDNA was then excised from the vector,
radiolabeled and used to screen the original Jurkat .lambda.
Uni-Zap.TM. XR cDNA library for full length cDNA clones. Positive A
plones were purified, rescued into the pBluescript II SK.sup.-
plasmid vector and sequenced for confirmation.
[0146] FIGS. 1A-1D show the sequence analysis and tissue
distribution of invention DR5 and TRAIL-R3 proteins. The predicted
amino acid sequence of human DR5 and TRAIL-R3 is set forth in FIGS.
1A and 1B, respectively. The mature DR5 and TRAIL-R3 proteins start
at Glu+1 and Tyr+1 (indicated by black diamonds), respectively. The
signal peptide and transmembrane domains are single- and
double-underlined, respectively. The five identical repeats in the
extracellular domain of TRAIL-R3 (FIG. 1B) are marked by black
triangles. The intracellular cytoplasmic death domain of DR5 (FIG.
1A) is boxed.
[0147] The DR5 cDNA encodes a protein of 411 amino acids (SEQ ID
NO:2) with an overall--59% amino acid identity to DR4 (FIG. 1A).
Its domain structure is highly related to DR4 and the other members
of the TNF-receptor family. DR5 contains a putative N-terminal
signal peptide (amino acids -51 to -1 of FIG. 1A) followed by an
extracellular domain containing two cysteine-rich pseudorepeats.
Following the extracellular domain is a transmembrane domain (amino
acids 132-152 of FIG. 1A) and a cytoplasmic domain. Within the
cytoplasmic domain there is a stretch of 67-amino acids (amino
acids 273-339 of FIG. 1A) comprising a death domain homology region
(FIG. 1C). Based on these criteria and its apoptotic activity (see
Example IV below) this TRAIL-receptor protein was designated death
receptor-5 (DR5) (also referred to herein as TRAIL-R2).
[0148] The TRAIL-R3 cDNA encodes a protein of 299 amino acids with
an overall .about.40% and 36% amino acid identity to DR4 and DR5,
respectively (FIG. 1B). TRAIL-R3 contains an N-terminal signal
peptide (amino acids -63 to -1 of FIG. 1B) followed by an
extracellular domain containing two cysteine-rich pseudo repeats
and five nearly identical PAAEETMN(T)TSPGTPA repeats (amino acids
139-153, 154-168, 169-183, 184-198, and 199-213 of FIG. 1B).
Following the extracellular domain is a C-terminal transmembrane
domain (amino acids 217-236 of FIG. 1B). Unlike DR4 and DR5,
TRAIL-R3 does not contain a cytoplasmic domain. Based on these
criteria and its ability to bind TRAIL (see Example III below),
this protein was designated TRAIL-R3.
[0149] FIG. 1C shows a colinear alignment of the death domains of
members of the TNF receptor family. Identical residues in at least
three out of six sequences are shaded. The death domain of DR5 is
64, 30, 30, 20, 31% identical to the corresponding domains in DR4,
DR3, TNFR-1, Fas and CAR1, respectively.
Example II
Expression Analyst. of DR5 and TRAIL-R3 mRNA
[0150] This example demonstrates the mRNA expression patterns of
DR5 and TRAIL-R3 in normal and tumor cells.
[0151] FIG. 1D shows a Northern blot analysis of the expression of
DR5 (upper panels) and TRAIL-R3 (lower panels) mRNAs in normal
tissues and tumor cell lines. X-ray film exposure time in the two
lower panels and the upper left panel was for 48 hours, whereas in
the upper right panel was for 2 hours. The cell lines assayed were:
HL-60, promyelocytic leukemia; HeLa cell S3, K-562, chronic
myelogenous leukemia; MOLT-4, lymphoblastic leukemia; Raji,
Burkitt's lymphoma; SW480, colorectal adenocarcinoma; A549, lung
carcinoma; G361, melanoma. Numbers on the right in FIG. 1D indicate
kilobases.
[0152] Northern blot analysis of equivalent amounts of mRNA samples
from normal human tissues and tumor cell lines, with a DR5
riboprobe, detected a .about.4 kb transcript in all the samples
(FIG. 1D, upper panels). Surprisingly, the amount of DR5 transcript
was at least a 100-fold more in most tumor cell lines than in
normal tissues. Autoradiography for less than 2 h was sufficient to
detect the DR5 message in tumor cell lines, compared to 48 h in the
case of the normal tissues. Other abnormal tissues such testes,
ovary, colon, small intestine and lymphoid tissues had detectable
but low expression of DR5 transcript, similar to that observed in
the normal tissues shown in FIG. 1D.
[0153] The TRAIL-R3 riboprobe detected .about.5 kb message in both
normal human tissues and tumor cell lines (FIG. 1D, lower panels).
A significantly elevated expression of TRAIL-R3 mRNA in normal
compared to tumor cells was observed. Given the activities of these
two receptors (see below), this could explain the high sensitivity
of tumor cell lines to TRAIL compared to normal cells (Davis T. D.
et al. (1995) Immunity 3, 673-682; Ashkenazi A. et al. (1996) J.
Bio. Chem. 271, 12687-12690; Ashkenazi A. et al. (1996) Curr. Biol.
6, 750-752).
Example III
TRAIL Binding Assay
[0154] This example demonstrates that DR5 and TRAIL-R3 are
receptors for the cytotoxic ligand TRAIL and can block
TRAIL-induced apoptosis.
[0155] To generate C-terminal Fc-tagged receptors, PCR generated
cDNAs encoding Fas (residues -16-158), DR4 (residues 86-217, with
N-terminal Fas signal peptide-Flag tag), DR5 (residues -51-133) and
TRAIL-R3 (residues -63-217) extracellular domains were inserted
into a modified pcDNA3 vector that allowed for inframe fusion with
the Fc portion of the mouse IgG.
[0156] Recombinant soluble TRAIL with N-terminal T7 and His6 tags
was obtained by Ni-affinity purification from bacteria transformed
with a pET28c-TRAIL (residues 95-281) vector. Receptor-Fc chimeras
were obtained by harvesting conditioned media of 293 cells
transfected with constructs encoding Fas-, DR4-, DR5- or
TRAIL-R3-Fc fusion proteins as described (Chinnaiyan, A. M. et al.
(1997) Science 276, 111-113). Binding of TRAIL to the receptor-Fe
chimeras was performed as described (Chinnaiyan, A. M. et al.
(1997) Science 276, 111-113).
[0157] FIG. 2 shows that the extracellular domains of DR5 and
TRAIL-R3 bind TRAIL and can block TRAIL-induced apoptosis. FIG. 2A
shows the results of an assay in which conditioned media from
cultures of 293 cells transfected for 72 h with empty vector (lane
1), or DR5 (lane 2), DR4 (lane 3), Fas (lane 4) or TRAIL-R3
(TR3-Fc) (lane 5) extracellular domain-Fe fusion proteins, were
incubated with purified soluble T7-His6-TRAIL and then
immunoprecipitated with anti-mouse IgG-agarose. After extensive
washing the samples were analyzed by SDS-PAGE and immunoblotted
with a horseradish peroxidase (HRP)-conjugated T7-antibody (upper
panel). The corresponding receptor-Fc fusions in the conditioned
media were also immunoblotted with anti-mouse Fc antibody (lower
panel).
[0158] FIG. 2B shows the results of an assay in which aliquots of
conditioned media containing receptor-Fc fusion proteins or no
fusion protein (vector) were incubated with equivalent amount of
soluble TRAIL (250 ng/ml) and then added to MCF7 cells. Cells were
stained 8 hours later with propidium iodide and the nuclei were
examined by fluorescence microscopy. The graph shows the percentage
of apoptotic nuclei (mean.+-.SD) as a function of total nuclei
counted under each condition (n=3).
[0159] As set forth above, the extracellular-ligand binding domains
of Fas, DR4, DR5 and TRAIL-R3 were expressed as fusion proteins
with the Fc region of mouse IgG (FIG. 2A, lower panel). As shown in
FIG. 2A, upper panel, DR4-Fc, DR5-Fc and TRAIL-R3-Fc were all
capable of binding TRAIL to the same extent (lanes 2, 3 and 5). As
expected Fas-Fc was unable to bind TRAIL (lane 4). Furthermore,
DR4-Fc, DR5-Fc and TRAIL-R3-FC, but not Fas-Fc, were capable of
blocking TRAIL-induced apoptosis in MCF7 cells (FIG. 2B). These
data indicate that, like DR4, DR5 and TRAIL-R3 are receptors for
TRAIL.
Example IV
Apoptotic Activity of DR5 and TRAIL-R3
[0160] This example demonstrates that DR5 but not TRAIL-R3 induces
apoptosis in human cells.
[0161] For apoptosis assays the mammalian double expression vector
pRSC (Akporiaye E. T. et al. (1997) BioTech. 22, 68) was used,
which allows for expression of lacZ under the RSV promoter and the
test cDNA (DR4, DR4.DELTA., DR5, DR5A, TRAIL-R3) under the CMV
promoter. CrmA, FLAME-1, caspase-8-DN (C345A) or caspase-10-DN
(C358A) (Alnemri E. S. et al. (1997) J. Biol. Chem. 272,
18542-18545) were expressed using pcDNA3 (Invitrogen).
[0162] FIG. 3 illustrates that expression of DR5 but not TRAIL-R3
induces apoptosis in human cells. MCF7 (FIG. 3A) and 293 (FIG. 3B)
cells were transfected with the indicated pRSC-lacZ constructs.
Thirty hours after transfection cells were stained with .beta.gal
and examined for morphological signs of apoptosis. The graphs show
the percentage of round blue apoptotic cells (mean.+-.SD) as a
function of total blue cells under each condition (.gtoreq.23).
[0163] FIG. 3C shows that ectopic expression of TRAIL-R3 attenuates
TRAIL-induced apoptosis in MCF7 cells. MCF7 cells were transfected
with TRAIL-R3 or vector alone for 36 h, then treated with soluble
TRAIL (250 ng/ml) for 8 h. The data are represented as in FIGS. 3A
and 3B, after subtracting the background killing (12-15%) as a
result of transfection.
[0164] FIGS. 3D and 3E show that DR4-induced and DR5-induced
apoptosis is inhibited by the caspase inhibitors, z-VAD-fmk and
CrmA (FIG. 3D), and by the dominant negative inhibitors,
caspase-8-DN, caspase-10-DN and FLAME-1 (FIG. 3E). MCF7 cells were
transfected with DR4 or DR5 expression constructs in the presence
of z-VAD-fmk (20 .mu.M) or co-transfected with a four-fold excess
of a CrmA, caspase-8-DN, caspase-10-DN or FLAME-1 construct or an
empty vector. The data are represented as in FIGS. 3A and 3B.
[0165] It is known that ectopic expression of death
domain-containing members of the TNF-receptor family induces
apoptosis in a ligand-independent manner. Consistent with this
observation, it has been found that transient expression of DR5 in
MCF7 cells or 293 cells triggers apoptosis (FIGS. 3A and 3B). The
level of apoptosis induction was similar to that observed with DR4
(FIG. 3A). DR5 induction of apoptosis was dependent on the presence
of the cytoplasmic death domain, as deletion of this domain
abolished the ability of DR4 and DR5 to induce apoptosis (FIGS. 3A
and 3B). In contrast, TRAIL-R3 which does not naturally contain a
death domain was incapable of inducing apoptosis (FIGS. 3A and 3B).
Interestingly, transient expression of TRAIL-R3 in MCF7 cells
significantly blocked TRAIL-induced apoptosis (FIG. 3C), suggesting
that TRAIL-R3 functions as an antagonistic decoy receptor.
[0166] Similar to DR4 and other TNF-receptor family members,
DR5-induced apoptosis was efficiently blocked by the caspase
inhibitors z-VAD-fkm and CrmA (FIG. 3D). DR4-induced and
DR5-induced apoptosis was also significantly inhibited by the
dominant negative inhibitors FLAME-1 (also known as Casper, FLIP,
I-FLICE, CASH) (Alnemri E. S. et al. (1997) J. Biol. Chem. 272,
18542-18545; Wallach, D. (1997) Nature 388, 123-125; and references
therein), caspase-8-DN and caspase-10-DN (FIG. 3E). Among these,
caspase-10-DN was the most effective in blocking DR4-included and
DR5-induced apoptosis. Inhibition of DR4-induced and DR5-induced
apoptosis by FLAME-1 is consistent with recent observations that
TRAIL-induced apoptosis is blocked by expression of FLIP (FLAME-1)
(Bodmer J. L. et al. (1997) Nature 388, 190-195). These data also
suggest that the upstream caspases-8 and -10 are involved in both
the DR4 and DR5 death signaling pathways.
Example V
In Vivo Interactions of DR5
[0167] This example demonstrates that DR4 and DR5 recruit
caspase-8, caspase-10 and FLAME-1 to the death signaling
pathway.
[0168] T7-epitope tagging was done as described recently (Alnemri
E. S. et al. (1997) J. Biol. Chem. 272, 18542-18545. To generate
N-terminal Flag-tagged receptor and receptor mutants, PCR generated
cDNAs encoding Fas, DR4, DR4.DELTA. (residues 86-351), DR5, DR56
(residues 1-268) were inserted in a modified pcDNA-3 vector that
allowed for inframe fusion with a Flag-epitope tag that is preceded
by Fas-signal peptide.
[0169] Death domain containing adaptor molecules such as
FADD/MORT1, CRADD/RAIDD, TRADD and RIP are recruited by some
members of the TNF-receptor family to engage the upstream caspases
(Nagata, S. (1997) Cell 88, 355-365); Alnemri E. S. et al. (1997)
J. Biol. Chem. 272, 18542-18545). Using co-immunoprecipitation
experiments, DR5 was assayed to determine whether it could interact
with these molecules to transmit the apoptotic signal.
[0170] FIG. 4 illustrates the in vivo interactions of DR5. FIG. 4A
shows that DR5 does not recruit FADD or CRADD. 293 cells were
transfected with expression plasmids encoding T7-epitope tagged
CRADD or FADD and Flag-epitope tagged Fas or DR5. After 36 h,
extracts were prepared and immunoprecipitated with a monoclonal
antibody to the Flag-epitope. The immunoprecipitates (upper panel)
and the corresponding cellular extracts (lower panel) were analyzed
by SDS-PAGE and immunoblotted with a HRP-conjugated
T7-antibody.
[0171] FIG. 4B shows that caspase-8, caspase-10 and FLAME-1 are
recruited to the Fas, DR4 and DR5 signaling complexes. Briefly, 293
cells were co-transfected with the indicated Flag constructs and
plasmids encoding T7-caspase-10 (upper panel), T7-caspase-8 (middle
panel) or T7-FLAME-1 (lower panel) and then immunoprecipitated and
detected as in FIG. 4A.
[0172] Unlike Fas, DRS did not interact with FADD or CRADD (FIG.
4A), nor with RIP or TRADD. A similar observation was reported with
DR4 (Pan et al., 1997, Science 276, 111-113). Interestingly, full
length Fas, DR4 and DR5, but not death domain-deleted mutants, were
all capable of forming complexes with caspase-8, caspase-10 and
FLAME-1 (FIG. 4B). Since these proteins do not interact directly,
it is believed that formation of these complexes would require an
adaptor molecule distinct from FADD.
[0173] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
Sequence CWU 1
1
6 1 1236 DNA Homo sapiens CDS (1)..(1233) 1 atg gaa caa cgg gga cag
aac gcc ccg gcc gct tcg ggg gcc cgg aaa 48 Met Glu Gln Arg Gly Gln
Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys 1 5 10 15 agg cac ggc cca
gga ccc agg gag gcg cgg gga gcc agg cct ggg ctc 96 Arg His Gly Pro
Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu 20 25 30 cgg gtc
ccc aag acc ctt gtg ctc gtt gtc gcc gcg gtc ctg ctg ttg 144 Arg Val
Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45
gtc tca gct gag tct gct ctg atc acc caa caa gac cta gct ccc cag 192
Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50
55 60 cag aga gtg gcc cca caa caa aag agg tcc agc ccc tca gag gga
ttg 240 Gln Arg Val Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly
Leu 65 70 75 80 tgt cca cct gga cac cat atc tca gaa gac ggt aga gat
tgc atc tcc 288 Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp
Cys Ile Ser 85 90 95 tgc aaa tat gga cag gac tat agc act cac tgg
aat gac ctc ctt ttc 336 Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp
Asn Asp Leu Leu Phe 100 105 110 tgc ttg cgc tgc acc agg tgt gat tca
ggt gaa gtg gag cta agt ccc 384 Cys Leu Arg Cys Thr Arg Cys Asp Ser
Gly Glu Val Glu Leu Ser Pro 115 120 125 tgc acc acg acc aga aac aca
gtg tgt cag tgc gaa gaa ggc acc ttc 432 Cys Thr Thr Thr Arg Asn Thr
Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 cgg gaa gaa gat tct
cct gag atg tgc cgg aag tgc cgc aca ggg tgt 480 Arg Glu Glu Asp Ser
Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 160 ccc aga
ggg atg gtc aag gtc ggt gat tgt aca ccc tgg agt gac atc 528 Pro Arg
Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175
gaa tgt gtc cac aaa gaa tca ggc atc atc ata gga gtc aca gtt gca 576
Glu Cys Val His Lys Glu Ser Gly Ile Ile Ile Gly Val Thr Val Ala 180
185 190 gcc gta gtc ttg att gtg gct gtg ttt gtt tgc aag tct tta ctg
tgg 624 Ala Val Val Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu
Trp 195 200 205 aag aaa gtc ctt cct tac ctg aaa ggc atc tgc tca ggt
ggt ggt ggg 672 Lys Lys Val Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly
Gly Gly Gly 210 215 220 gac cct gag cgt gtg gac aga agc tca caa cga
cct ggg gct gag gac 720 Asp Pro Glu Arg Val Asp Arg Ser Ser Gln Arg
Pro Gly Ala Glu Asp 225 230 235 240 aat gtc ctc aat gag atc gtg agt
atc ttg cag ccc acc cag gtc cct 768 Asn Val Leu Asn Glu Ile Val Ser
Ile Leu Gln Pro Thr Gln Val Pro 245 250 255 gag cag gaa atg gaa gtc
cag gag cca gca gag cca aca ggt gtc aac 816 Glu Gln Glu Met Glu Val
Gln Glu Pro Ala Glu Pro Thr Gly Val Asn 260 265 270 atg ttg tcc ccc
ggg gag tca gag cat ctg ctg gaa ccg gca gaa gct 864 Met Leu Ser Pro
Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala 275 280 285 gaa agg
tct cag agg agg agg ctg ctg gtt cca gca aat gaa ggt gat 912 Glu Arg
Ser Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp 290 295 300
ccc act gag act ctg aga cag tgc ttc gat gac ttt gca gac ttg gtg 960
Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val 305
310 315 320 ccc ttt gac tcc tgg gag ccg ctc atg agg aag ttg ggc ctc
atg gac 1008 Pro Phe Asp Ser Trp Glu Pro Leu Met Arg Lys Leu Gly
Leu Met Asp 325 330 335 aat gag ata aag gtg gct aaa gct gag gca gcg
ggc cac agg gac acc 1056 Asn Glu Ile Lys Val Ala Lys Ala Glu Ala
Ala Gly His Arg Asp Thr 340 345 350 ttg tac acg atg ctg ata aag tgg
gtc aac aaa acc ggg cga gat gcc 1104 Leu Tyr Thr Met Leu Ile Lys
Trp Val Asn Lys Thr Gly Arg Asp Ala 355 360 365 tct gtc cac acc ctg
ctg gat gcc ttg gag acg ctg gga gag aga ctt 1152 Ser Val His Thr
Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu 370 375 380 gcc aag
cag aag att gag gac cac ttg ttg agc tct gga aag ttc atg 1200 Ala
Lys Gln Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met 385 390
395 400 tat cta gaa ggt aat gca gac tct gcc atg tcc taa 1236 Tyr
Leu Glu Gly Asn Ala Asp Ser Ala Met Ser 405 410 2 411 PRT Homo
sapiens 2 Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala
Arg Lys 1 5 10 15 Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala
Arg Pro Gly Leu 20 25 30 Arg Val Pro Lys Thr Leu Val Leu Val Val
Ala Ala Val Leu Leu Leu 35 40 45 Val Ser Ala Glu Ser Ala Leu Ile
Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60 Gln Arg Val Ala Pro Gln
Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu 65 70 75 80 Cys Pro Pro Gly
His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95 Cys Lys
Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105 110
Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115
120 125 Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr
Phe 130 135 140 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg
Thr Gly Cys 145 150 155 160 Pro Arg Gly Met Val Lys Val Gly Asp Cys
Thr Pro Trp Ser Asp Ile 165 170 175 Glu Cys Val His Lys Glu Ser Gly
Ile Ile Ile Gly Val Thr Val Ala 180 185 190 Ala Val Val Leu Ile Val
Ala Val Phe Val Cys Lys Ser Leu Leu Trp 195 200 205 Lys Lys Val Leu
Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly 210 215 220 Asp Pro
Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp 225 230 235
240 Asn Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro
245 250 255 Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly
Val Asn 260 265 270 Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu
Pro Ala Glu Ala 275 280 285 Glu Arg Ser Gln Arg Arg Arg Leu Leu Val
Pro Ala Asn Glu Gly Asp 290 295 300 Pro Thr Glu Thr Leu Arg Gln Cys
Phe Asp Asp Phe Ala Asp Leu Val 305 310 315 320 Pro Phe Asp Ser Trp
Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp 325 330 335 Asn Glu Ile
Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr 340 345 350 Leu
Tyr Thr Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala 355 360
365 Ser Val His Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu
370 375 380 Ala Lys Gln Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys
Phe Met 385 390 395 400 Tyr Leu Glu Gly Asn Ala Asp Ser Ala Met Ser
405 410 3 900 DNA Homo sapiens CDS (1)..(897) 3 atg caa ggg gtg aag
gag cgc ttc cta ccg tta ggg aac tct ggg gac 48 Met Gln Gly Val Lys
Glu Arg Phe Leu Pro Leu Gly Asn Ser Gly Asp 1 5 10 15 aga gcg ccc
cgg ccg cct gat ggc cga ggc agg gtg cga ccc agg acc 96 Arg Ala Pro
Arg Pro Pro Asp Gly Arg Gly Arg Val Arg Pro Arg Thr 20 25 30 caa
gac ggc gtc ggg aac cat acc atg gcc cgg atc ccc aag acc cta 144 Gln
Asp Gly Val Gly Asn His Thr Met Ala Arg Ile Pro Lys Thr Leu 35 40
45 aag ttc gtc gtc gtc atc gtc gcg gtc ctg ctg cca gtc cta gct tac
192 Lys Phe Val Val Val Ile Val Ala Val Leu Leu Pro Val Leu Ala Tyr
50 55 60 tct gcc acc act gcc cgg cag gag gaa gtt ccc cag cag aca
gtg gcc 240 Ser Ala Thr Thr Ala Arg Gln Glu Glu Val Pro Gln Gln Thr
Val Ala 65 70 75 80 cca cag caa cag agg cac agc ttc aag ggg gag gag
tgt cca gca gga 288 Pro Gln Gln Gln Arg His Ser Phe Lys Gly Glu Glu
Cys Pro Ala Gly 85 90 95 tct cat aga tca gaa cat act gga gcc tgt
aac ccg tgc aca gag ggt 336 Ser His Arg Ser Glu His Thr Gly Ala Cys
Asn Pro Cys Thr Glu Gly 100 105 110 gtg gat tac acc aac gct tcc aac
aat gaa cct tct tgc ttc cca tgt 384 Val Asp Tyr Thr Asn Ala Ser Asn
Asn Glu Pro Ser Cys Phe Pro Cys 115 120 125 aca gtt tgt aaa tca gat
caa aaa cat aaa agt tcc tgc acc atg acc 432 Thr Val Cys Lys Ser Asp
Gln Lys His Lys Ser Ser Cys Thr Met Thr 130 135 140 aga gac aca gtg
tgt cag tgt aaa gaa ggc acc ttc cgg aat gaa aac 480 Arg Asp Thr Val
Cys Gln Cys Lys Glu Gly Thr Phe Arg Asn Glu Asn 145 150 155 160 tcc
cca gag atg tgc cgg aag tgt agc agg tgc cct agt ggg gaa gtc 528 Ser
Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly Glu Val 165 170
175 caa gtc agt aat tgt acg tcc tgg gat gat atc cag tgt gtt gaa gaa
576 Gln Val Ser Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys Val Glu Glu
180 185 190 ttt ggt gcc aat gcc act gtg gaa acc cca gct gct gaa gag
aca atg 624 Phe Gly Ala Asn Ala Thr Val Glu Thr Pro Ala Ala Glu Glu
Thr Met 195 200 205 aac acc agc ccg ggg act cct gcc cca gct gct gaa
gag aca atg aac 672 Asn Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu
Glu Thr Met Asn 210 215 220 acc agc cca ggg act cct gcc cca gct gct
gaa gag aca atg acc acc 720 Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala
Glu Glu Thr Met Thr Thr 225 230 235 240 agc ccg ggg act cct gcc cca
gct gct gaa gag aca atg acc acc agc 768 Ser Pro Gly Thr Pro Ala Pro
Ala Ala Glu Glu Thr Met Thr Thr Ser 245 250 255 ccg ggg act cct gcc
cca gct gct gaa gag aca atg acc acc agc ccg 816 Pro Gly Thr Pro Ala
Pro Ala Ala Glu Glu Thr Met Thr Thr Ser Pro 260 265 270 ggg act cct
gcc tct tct cat tac ctc tca tgc acc atc gta ggg atc 864 Gly Thr Pro
Ala Ser Ser His Tyr Leu Ser Cys Thr Ile Val Gly Ile 275 280 285 ata
gtt cta att gtg ctt ctg att gtg ttt gtt tga 900 Ile Val Leu Ile Val
Leu Leu Ile Val Phe Val 290 295 4 299 PRT Homo sapiens 4 Met Gln
Gly Val Lys Glu Arg Phe Leu Pro Leu Gly Asn Ser Gly Asp 1 5 10 15
Arg Ala Pro Arg Pro Pro Asp Gly Arg Gly Arg Val Arg Pro Arg Thr 20
25 30 Gln Asp Gly Val Gly Asn His Thr Met Ala Arg Ile Pro Lys Thr
Leu 35 40 45 Lys Phe Val Val Val Ile Val Ala Val Leu Leu Pro Val
Leu Ala Tyr 50 55 60 Ser Ala Thr Thr Ala Arg Gln Glu Glu Val Pro
Gln Gln Thr Val Ala 65 70 75 80 Pro Gln Gln Gln Arg His Ser Phe Lys
Gly Glu Glu Cys Pro Ala Gly 85 90 95 Ser His Arg Ser Glu His Thr
Gly Ala Cys Asn Pro Cys Thr Glu Gly 100 105 110 Val Asp Tyr Thr Asn
Ala Ser Asn Asn Glu Pro Ser Cys Phe Pro Cys 115 120 125 Thr Val Cys
Lys Ser Asp Gln Lys His Lys Ser Ser Cys Thr Met Thr 130 135 140 Arg
Asp Thr Val Cys Gln Cys Lys Glu Gly Thr Phe Arg Asn Glu Asn 145 150
155 160 Ser Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly Glu
Val 165 170 175 Gln Val Ser Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys
Val Glu Glu 180 185 190 Phe Gly Ala Asn Ala Thr Val Glu Thr Pro Ala
Ala Glu Glu Thr Met 195 200 205 Asn Thr Ser Pro Gly Thr Pro Ala Pro
Ala Ala Glu Glu Thr Met Asn 210 215 220 Thr Ser Pro Gly Thr Pro Ala
Pro Ala Ala Glu Glu Thr Met Thr Thr 225 230 235 240 Ser Pro Gly Thr
Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser 245 250 255 Pro Gly
Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser Pro 260 265 270
Gly Thr Pro Ala Ser Ser His Tyr Leu Ser Cys Thr Ile Val Gly Ile 275
280 285 Ile Val Leu Ile Val Leu Leu Ile Val Phe Val 290 295 5 1053
DNA Homo sapiens CDS (1)..(1050) 5 atg gaa caa cgg gga cag aac gcc
ccg gcc gct tcg ggg gcc cgg aaa 48 Met Glu Gln Arg Gly Gln Asn Ala
Pro Ala Ala Ser Gly Ala Arg Lys 1 5 10 15 agg cac ggc cca gga ccc
agg gag gcg cgg gga gcc agg cct ggg ctc 96 Arg His Gly Pro Gly Pro
Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu 20 25 30 cgg gtc ccc aag
acc ctt gtg ctc gtt gtc gcc gcg gtc ctg ctg ttg 144 Arg Val Pro Lys
Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45 gtc tca
gct gag tct gct ctg atc acc caa caa gac cta gct ccc cag 192 Val Ser
Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60
cag aga gtg gcc cca caa caa aag agg tcc agc ccc tca gag gga ttg 240
Gln Arg Val Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu 65
70 75 80 tgt cca cct gga cac cat atc tca gaa gac ggt aga gat tgc
atc tcc 288 Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys
Ile Ser 85 90 95 tgc aaa tat gga cag gac tat agc act cac tgg aat
gac ctc ctt ttc 336 Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn
Asp Leu Leu Phe 100 105 110 tgc ttg cgc tgc acc agg tgt gat tca ggt
gaa gtg gag cta agt ccc 384 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly
Glu Val Glu Leu Ser Pro 115 120 125 tgc acc acg acc aga aac aca gtg
tgt cag tgc gaa gaa ggc acc ttc 432 Cys Thr Thr Thr Arg Asn Thr Val
Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 cgg gaa gaa gat tct cct
gag atg tgc cgg aag tgc cgc aca ggg tgt 480 Arg Glu Glu Asp Ser Pro
Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 160 ccc aga ggg
atg gtc aag gtc ggt gat tgt aca ccc tgg agt gac atc 528 Pro Arg Gly
Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175 gaa
tgt gtc cac aaa gaa tca ggt aca aag cac agt ggg gaa gcc cca 576 Glu
Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro 180 185
190 gct gtg gag gag acg gtg acc tcc agc cca ggg act cct gcc tct ccc
624 Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro
195 200 205 tgt tct ctc tca ggc atc atc ata gga gtc aca gtt gca gcc
gta gtc 672 Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala
Val Val 210 215 220 ttg att gtg gct gtg ttt gtt tgc aag tct tta ctg
tgg aag aaa gtc 720 Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu
Trp Lys Lys Val 225 230 235 240 ctt cct tac ctg aaa ggc atc tgc tca
ggt ggt ggt ggg gac cct gag 768 Leu Pro Tyr Leu Lys Gly Ile Cys Ser
Gly Gly Gly Gly Asp Pro Glu 245 250 255 cgt gtg gac aga agc tca caa
cga cct ggg gct gag gac aat gtc ctc 816 Arg Val Asp Arg Ser Ser Gln
Arg Pro Gly Ala Glu Asp Asn Val Leu 260 265 270 aat gag atc gtg agt
atc ttg cag ccc acc cag gtc cct gag cag gaa 864 Asn Glu Ile Val Ser
Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280 285 atg gaa gtc
cag gag cca gca gag cca aca ggt gtc aac aaa acc ggg 912 Met Glu Val
Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Lys Thr Gly 290 295 300 cga
gat gcc tct gtc cac acc ctg ctg gat gcc ttg gag acg ctg gga 960 Arg
Asp Ala Ser Val His Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly 305 310
315 320 gag aga ctt gcc aag cag aag att gag gac cac ttg ttg agc tct
gga 1008 Glu Arg Leu Ala Lys Gln Lys Ile Glu Asp His Leu Leu Ser
Ser Gly 325 330 335 aag ttc atg tat cta gaa ggt aat gca gac tct gcc
atg tcc taa 1053 Lys Phe Met Tyr Leu Glu Gly Asn Ala Asp Ser Ala
Met Ser 340 345 350 6 350 PRT Homo sapiens 6 Met Glu Gln
Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys 1 5 10 15 Arg
His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu 20 25
30 Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu
35 40 45 Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala
Pro Gln 50 55 60 Gln Arg Val Ala Pro Gln Gln Lys Arg Ser Ser Pro
Ser Glu Gly Leu 65 70 75 80 Cys Pro Pro Gly His His Ile Ser Glu Asp
Gly Arg Asp Cys Ile Ser 85 90 95 Cys Lys Tyr Gly Gln Asp Tyr Ser
Thr His Trp Asn Asp Leu Leu Phe 100 105 110 Cys Leu Arg Cys Thr Arg
Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125 Cys Thr Thr Thr
Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 Arg Glu
Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155
160 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile
165 170 175 Glu Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu
Ala Pro 180 185 190 Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr
Pro Ala Ser Pro 195 200 205 Cys Ser Leu Ser Gly Ile Ile Ile Gly Val
Thr Val Ala Ala Val Val 210 215 220 Leu Ile Val Ala Val Phe Val Cys
Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 Leu Pro Tyr Leu Lys
Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255 Arg Val Asp
Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu 260 265 270 Asn
Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280
285 Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Lys Thr Gly
290 295 300 Arg Asp Ala Ser Val His Thr Leu Leu Asp Ala Leu Glu Thr
Leu Gly 305 310 315 320 Glu Arg Leu Ala Lys Gln Lys Ile Glu Asp His
Leu Leu Ser Ser Gly 325 330 335 Lys Phe Met Tyr Leu Glu Gly Asn Ala
Asp Ser Ala Met Ser 340 345 350
* * * * *
References