U.S. patent application number 10/005842 was filed with the patent office on 2002-07-25 for death domain containing receptor 5.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Gentz, Reiner L., Ni, Jian, Rosen, Craig A., Yu, Guo-Liang.
Application Number | 20020098550 10/005842 |
Document ID | / |
Family ID | 26717514 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098550 |
Kind Code |
A1 |
Ni, Jian ; et al. |
July 25, 2002 |
Death domain containing receptor 5
Abstract
The present invention relates to novel Death Domain Containing
Receptor-5 (DR5) proteins which are members of the tumor necrosis
factor (TNF) receptor family, and have now been shown to bind
TRAIL. In particular, isolated nucleic acid molecules are provided
encoding the human DR5 proteins. DR5 polypeptides are also provided
as are vectors, host cells and recombinant methods for producing
the same. The invention further relates to screening methods for
identifying antagonists and antagonists of DR5 activity.
Inventors: |
Ni, Jian; (Rockville,
MD) ; Gentz, Reiner L.; (Silver Spring, MD) ;
Yu, Guo-Liang; (Darnestown, MD) ; Rosen, Craig
A.; (Laytonsville, MD) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
SUITE 600
WASHINGTON
DC
20005-3934
US
|
Assignee: |
Human Genome Sciences, Inc.
|
Family ID: |
26717514 |
Appl. No.: |
10/005842 |
Filed: |
December 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10005842 |
Dec 7, 2001 |
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09042583 |
Mar 17, 1998 |
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60040846 |
Mar 17, 1997 |
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60054021 |
Jul 29, 1997 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 9/10 20180101; A61K
38/00 20130101; A61P 25/28 20180101; C07K 2319/00 20130101; A61P
35/00 20180101; A61P 37/06 20180101; C07K 14/7151 20130101 |
Class at
Publication: |
435/69.1 ;
435/325; 435/320.1; 530/350; 536/23.5 |
International
Class: |
C07K 014/705; C12P
021/02; C12N 005/06; C07H 021/04 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding a polypeptide comprising amino acids from about -51 to
about 360 in SEQ ID NO: 2; (b) a nucleotide sequence encoding a
polypeptide comprising amino acids from about -50 to about 360 in
SEQ ID NO: 2; (c) a nucleotide sequence encoding a polypeptide
comprising amino acids from about I to about 360 in SEQ ID NO: 2;
(d) a nucleotide sequence encoding a polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit
No. 97920; (e) a nucleotide sequence encoding the mature DR5
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97920; (f) a nucleotide
sequence encoding the DR5 extracellular domain; (g) a nucleotide
sequence encoding the DR5 transmembrane domain; (h) a nucleotide
sequence encoding the DR5 intracellular domain; (i) a nucleotide
sequence encoding the DR5 death domain; and (j) a nucleotide
sequence complementary to any of the nucleotide sequences in (a),
(b), (c), (d), (e), (f), (g), (h), or (i) above.
2. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the nucleotide sequence in SEQ ID NO: 1.
3. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the nucleotide sequence encoding the DR5
polypeptide having the amino acid sequence in SEQ ID NO: 2.
4. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the nucleotide sequence in SEQ ID NO: 1 encoding
the mature DR5 polypeptide having the amino acid sequence in SEQ ID
NO: 2.
5. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the complete nucleotide sequence of the cDNA
clone contained in ATCC Deposit No. 97920.
6. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the nucleotide sequence encoding the DR5
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97920.
7. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the nucleotide sequence encoding the mature DR5
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97920.
8. An isolated nucleic acid molecule comprising a polynucleotide
sequence which hybridizes under stringent hybridization conditions
to a polynucleotide sequence having a nucleotide sequence identical
to a nucleotide sequence in (a), (b), (c), (d), (e), (f), (g), (h),
(i), or (j) of claim 1, wherein said polynucleotide which
hybridizes does not hybridize under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence
consisting of only adenosine nucleotides or of only thymidine
nucleotides.
9. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a DR5 polypeptide having an amino acid sequence in (a), (b),
(c), (d), (e), (f), (g), (h), or (i) of claim 1.
10. The isolated nucleic acid molecule of claim 9, which encodes an
epitope-bearing portion of a DR5 polypeptide selected from the
group consisting of: a polypeptide comprising amino acid residues
from about 11 to about 59 in SEQ ID NO: 2; a polypeptide comprising
amino acid residues from about 68 to about 113 in SEQ ID NO: 2; a
polypeptide comprising amino acid residues from about 173 to about
220 in SEQ ID NO: 2; and a polypeptide comprising amino acid
residues from about 224 to about 319 in SEQ ID NO: 2.
11. The isolated nucleic acid molecule of claim 1, which encodes
the DR5 extracellular domain.
12. The isolated nucleic acid molecule of claim 1, which encodes
the DR5 transmembrane domain.
13. The isolated nucleic acid molecule of claim 1, which encodes
the DR5 intracellular domain.
14. A method for making a recombinant vector comprising inserting
an isolated nucleic acid molecule of claim 1 into a vector.
15. A recombinant vector produced by the method of claim 14.
16. A method of making a recombinant host cell comprising
introducing an isolated nucleic acid molecule of claim 1 into a
host cell.
17. A recombinant host cell produced by the method of claim 16.
18. A recombinant method for producing a DR5 polypeptide,
comprising culturing the recombinant host cell of claim 17 under
conditions such that said polypeptide is expressed and recovering
said polypeptide.
19. An isolated DR5 polypeptide comprising an amino acid sequence
at least 95% identical to a sequence selected from the group
consisting of: (a) amino acids from about -51 to about 360 in SEQ
ID NO: 2; (b) amino acids from about -50 to about 360 in SEQ ID NO:
2; (c) amino acids from about 1 to about 360 in SEQ ID NO: 2; (d)
the amino acid sequence of the DR5 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit
No. 97920; (e) the amino acid sequence of the mature DR5
polypeptide having the amino acid encoded by the CDNA clone
contained in ATCC Deposit No. 97920; (f) the amino acid sequence of
the DR5 extracellular domain; (g) the amino acid sequence of the
DR5 transmembrane domain; (h) the amino acid sequence of the DR5
intracellular domain; (i) the amino acid sequence of the DR5 death
domain; (j) the amino acid sequence of an epitope-bearing portion
of any one of the polypeptides of (a), (b), (c), (d), (e), (f),
(g), (h), (i), or (j).
20. An isolated polypeptide comprising an epitope-bearing portion
of the DR5 protein, wherein said portion is selected from the group
consisting of: a polypeptide comprising amino acid residues from
about amino acid residues from about 11 to about 59 in SEQ ID NO:
2; a polypeptide comprising amino acid residues from about 68 to
about 113 in SEQ ID NO: 2; a polypeptide comprising amino acid
residues from about 173 to about 220 in SEQ ID NO: 2; and a
polypeptide comprising amino acid residues from about 224 to about
319 in SEQ ID NO: 2.
21. An isolated antibody that binds specifically to a DR5
polypeptide of claim 19.
22. An isolated nucleic acid molecule comprising a polynucleotide
having a sequence at least 95% identical to a sequence selected
from the group consisting of: (a) the nucleotide sequence of clone
HAPBU13R (SEQ ID NO: 6); (b) the nucleotide sequence of clone
HSBBU76R (SEQ ID NO: 7); (c) the nucleotide sequence of a portion
of the sequence shown in FIG. 1 (SEQ ID NO: 1) wherein said portion
comprises at least 50 contiguous nucleotides from nucleotide 284 to
1,362; and (d) a nucleotide sequence complementary to any of the
nucleotide sequences in (a), (b) or (c) above.
23. An isolated nucleic acid molecule comprising a polynucleotide
encoding a DR5 polypeptide wherein, except for at least one
conservative amino acid substitution, said polypeptide has a
sequence selected from the group consisting of: (a) a nucleotide
sequence encoding a polypeptide comprising amino acids from about
-51 to about 360 in SEQ ID NO: 2; (b) a nucleotide sequence
encoding a polypeptide comprising amino acids from about -50 to
about 360 in SEQ ID NO: 2; (c) a nucleotide sequence encoding a
polypeptide comprising amino acids from about 1 to about 360 in SEQ
ID NO: 2; (d) a nucleotide sequence encoding a polypeptide having
the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. 97920; (e) a nucleotide sequence encoding the mature
DR5 polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97920; (f) a nucleotide
sequence encoding theDR5 extracellular domain; (g) a nucleotide
sequence encoding the DR5 transmembrane domain; (h) a nucleotide
sequence encoding the DR5 intracellular domain; (i) a nucleotide
sequence encoding the DR5 receptor extracellular and intracellular
domains with all or part of the transmembrane domain deleted; (j) a
nucleotide sequence encoding the DR5 death domain; and (k) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or
(j).
24. An isolated DR5 polypeptide wherein, except for at least one
conservative amino acid substitution, said polypeptide has a
sequence selected from the group consisting of: (a) amino acids
from about -51 to about 360 in SEQ ID NO: 2; (b) amino acids from
about -50 to about 360 in SEQ ID NO: 2; (c) amino acids from about
1 to about 360 in SEQ ID NO: 2; (d) the amino acid sequence of the
DR5 polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97920; (e) the amino acid
sequence of the mature DR5 polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.
97920; (f) the amino acid sequence of the DR5 receptor
extracellular domain; (g) the amino acid sequence of the DR5
receptor transmembrane domain; (h) the amino acid sequence of the
DR5 receptor intracellular domain; (i) the amino acid sequence of
the DR5 receptor extracellular and intracellular domains with all
or part of the transmembrane domain deleted; (j) the amino acid
sequence of the DR5 receptor death domain; and (k) the amino acid
sequence of an epitope-bearing portion of any one of the
polypeptides of (a), (b), (c), (d), (e), (f), (g), (h), (i), or
(j).
25. A pharmaceutical composition comprising the polypeptide of
claim 19 and a pharmaceutically acceptable carrier.
26. A pharmaceutical composition comprising the antibody of claim
21 and a pharmaceutically acceptable carrier.
27. A pharmaceutical composition comprising the polypeptide of
claim 24 and a pharmaceutically acceptable carrier.
28. A fusion protein comprising the polypeptide of claim 19 fused
to a heterologeous polypeptide.
29. The isolated nucleic acid of claim 8, wherein said nucleic acid
encodes a protein which is able to be bound by an antibody to a DR5
polypeptide, wherein said polypeptide has the amino acid sequence
in SEQ ID NO: 2;
30. A method for making a recombinant vector comprising inserting
an isolated nucleic acid molecule of claim 8 into a vector.
31. A recombinant vector produced by the method of claim 30.
32. A method of making a recombinant host cell comprising
introducing an isolated nucleic acid molecule of claim 8 into a
host cell.
33. A recombinant host cell produced by the method of claim 32.
34. A recombinant method for producing a polypeptide, comprising
culturing the recombinant host cell of claim 33 under conditions
such that said polypeptide is expressed and recovering said
polypeptide.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a novel member of the tumor
necrosis factor family of receptors. More specifically, isolated
nucleic acid molecules are provided encoding human Death Domain
Containing Receptor 5, or simply "DR5." DR5 polypeptides are also
provided, as are vectors, host cells, and recombinant methods for
producing the same. The invention flier relates to screening
methods for identifying agonists and antagonists of DR5
activity.
[0003] This application claims benefit under 35 U.S.C. .sctn.
119(e) to copending U.S. Provisional Application Ser. Nos.
60/040,846, filed Mar. 17, 1997 and 60/054,021, filed Jul. 29,
1997, both of which are incorporated herein by reference.
[0004] 2. Related Art
[0005] Numerous biological actions, for instance, response to
certain stimuli and natural biological processes, are controlled by
factors, such as cytokines. Many cytokines act through receptors by
engaging the receptor and producing an intra-cellular response.
[0006] For example, tumor necrosis factors (TNF) alpha and beta are
cytokines, which act through TNF receptors to regulate numerous
biological processes, including protection against infection and
induction of shock and inflammatory disease. The TNF molecules
belong to the "TNF-ligand" superfamily, and act together with their
receptors or counter-ligands, the "TNF-receptor" superfamily. So
far, nine members of the TNF ligand superfamily have been
identified and ten members of the TNF-receptor superfamily have
been characterized.
[0007] Among the ligands, there are included TNF-.alpha.,
lymphotoxin-.alpha. (LT-.alpha., also known as TNF-.beta.),
LT-.beta. (found in complex heterotrimer LT-.alpha.2-.beta.), FasL,
CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).
The superfamily of TNF receptors includes the p55TNF receptor,
p75TNF receptor, TNF receptor-related protein, FAS antigen or
APO-1, CD40, CD27, CD30, 4-IBB, OX40, low affinity p75 and
NGF-receptor (Meager, A., Biologicals, 22:291-295 (1994)).
[0008] Many members of the TNF-ligand superfamily are expressed by
activated T-cells, implying that they are necessary for T-cell
interactions with other cell types which underlie cell ontogeny and
functions. (Meager, A., supra).
[0009] Considerable insight into the essential functions of several
members of the TNF receptor family has been gained from the
identification and creation of mutants that abolish the expression
of these proteins. For example, naturally occurring mutations in
the FAS antigen and its ligand cause lymphoproliferative disease
(Watanabe-Fukunaga, R., et al., Nature 356:314 (1992)), perhaps
reflecting a failure of programmed cell death. Mutations of the
CD40 ligand cause an X-linked immunodeficiency state characterized
by high levels of immunoglobulin M and low levels of immunoglobulin
G in plasma, indicating faulty T-cell-dependent B-cell activation
(Allen, R. C. et al., Science 259:990 (1993)). Targeted mutations
of the low affinity nerve growth factor receptor cause a disorder
characterized by faulty sensory innovation of peripheral structures
(Lee, K.F. et al., Cell 69:737 (1992)).
[0010] TNF and LT-.alpha. are capable of binding to two TNF
receptors (the 55- and 75-kd TNF receptors). A large number of
biological effects elicited by TNF and LT-.alpha. acting through
their receptors, include hemorrhagic necrosis of transplanted
tumors, cytotoxicity, a role in endotoxic shock, inflammation,
immunoregulation, proliferation and anti-viral responses, as well
as protection against the deleterious effects of ionizing
radiation. TNF and LT-.alpha. are involved in the pathogenesis of a
wide range of diseases, including endotoxic shock, cerebral
malaria, tumors, autoimmune disease, AIDS and graft-host rejection
(Beutler, B. and Von Huffel, C., Science 264:667-668 (1994)).
Mutations in the p55 Receptor cause increased susceptibility to
microbial infection.
[0011] Moreover, an about 80 amino acid domain near the C-terminus
of TNFR1 (p55) and Fas was reported as the "death domain," which is
responsible for transducing signals for programmed cell death
(Tartaglia et al., Cell 74:845 (1993)).
[0012] Apoptosis, or programmed cell death, is a physiologic
process essential for the normal development and homeostasis of
multicellular organisms (H. Steller, Science 267:1445-1449 (1995)).
Derangements of apoptosis contribute to the pathogenesis of several
human diseases including cancer, neurodegenerative disorders, and
acquired immune deficiency syndrome (C. B. Thompson, Science
267:1456-1462 (1995)). Recently, much attention has focused on the
signal transduction and biological function of two cell surface
death receptors, Fas/APO-1 and TNFR-1 (J. L. Cleveland et al., Cell
81:479-482 (1995); A. Fraser, et al., Cell 85:781-784 (1996); S.
Nagata et al., Science 267:1449-56 (1995)). Both are members of the
TNF receptor family which also include TNFR-2, low affinity NGFR,
CD40, and CD30, among others (C. A. Smith et al., Science
248:1019-23 (1990); M. Tewari et al., in Modular Texts in Molecular
and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall,
London, 1995). While family members are defined by the presence of
cysteine-rich repeats in their extracellular domains, Fas/APO-1 and
TNFR-1 also share a region of intracellular homology, appropriately
designated the "death domain", which is distantly related to the
Drosophila suicide gene, reaper (P. Golstein, et al., Cell
81:185-186 (1995); K. White et al., Science 264:677-83 (1994)).
This shared death domain suggests that both receptors interact with
a related set of signal transducing molecules that, until recently,
remained unidentified. Activation of Fas/APO-1 recruits the death
domain-containing adapter molecule FADD/MORT1 (A. M. Chinnaiyan et
al., Cell 81: 505-12 (1995); M. P. Boldin et al., J Biol Chem
270:7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588
(1995)), which in turn binds and presumably activates FLICE/MACH1,
a member of the ICE/CED-3 family of pro-apoptotic proteases (M.
Muzio et al., Cell 85:817-827 (1996); M. P. Boldin et al., Cell
85:803-815 (1996)). While the central role of Fas/APO-1 is to
trigger cell death, TNFR-1 can signal an array of diverse
biological activities-many of which stem from its ability to
activate NF-kB (L. A. Tartaglia et al., Immunol Today 13:151-3
(1992)). Accordingly, TNFR-1 recruits the multivalent adapter
molecule TRADD, which like FADD, also contains a death domain (H.
Hsu et al., Cell 81:495-504 (1995); H. Hsu, et al., Cell 84:299-308
(1996)). Through its associations with a number of signaling
molecules including FADD, TRAF2, and RIP, TRADD can signal both
apoptosis and NF-kB activation (H. Hsu et al., Cell 84:299-308
(1996); H. Hsu, et al., Immunity 4:387-396 (1996)).
[0013] Recently, a new apoptosis -inducing TNF ligand has been
discovered. S. R Wiley et al. (Immunity 3:673-682 (1995)) named the
molecule--"TNF-related apoptosis-inducing ligand" or simply
"TRAIL." The molecule was also called "Apo-2 ligand" or "Apo-2L."
R. M. Pitt et al., J. Biol. Chem. 271:12687-12690 (1996). This
molecule was also disclosed in co-pending U.S. provisional
application No. 60/013,405. For convenience, the molecule will be
referred to herein as TRAIL.
[0014] Unlike FAS ligand, whose transcripts appear to be largely
restricted to stimulated T-cells, significant levels of TRAIL are
detected in many human tissues (e.g., spleen, lung, prostate,
thymus, ovary, small intestine, colon, peripheral blood
lymphocytes, placenta, kidney), and is constitutively transcribed
by some cell lines. It has been shown that TRAIL acts independently
from the Fas ligand (Wiley et al., supra). It has also been shown
that TRAIL activates apoptosis rapidly, within a time frame that is
similar to death signaling by Fas/Apo-1L, but much faster than
TNF-induced apoptosis. S. A. Marsters et al., Current Biology
6:750-752 (1996). The inability of TRAIL to bind TNFR-1, Fas, or
the recently identified DR3, suggests that TRAIL may interact with
a unique receptor(s).
[0015] Several unique receptors for TRAIL have already been
identified, In co-pending U.S. provisional patent application No.
60/035,722, DR4, a novel death domain containing receptor for
TRAIL, was disclosed. See, Pan et al., Science 276,111-113 (April
1997). The TR5 receptor, the subject of co-pending U.S. provisional
patent application No. 60/035,496, has now been shown to bind
TRAIL. In co-pending U.S. provisional patent application No.
60/xxxxxx, it was predicted that the TR10 receptor would also bind
TRAIL, owing to sequence homology with DR4.
[0016] The effects of TNF family ligands and TNF family receptors
are varied and influence numerous functions, both normal and
abnormal, in the biological processes of the mammalian system.
There is a clear need, therefore, for identification and
characterization of such receptors and ligands that influence
biological activity, both normally and in disease states. In
particular, there is a need to isolate and characterize additional
novel receptors that bind TRAIL.
SUMMARY OF THE INVENTION
[0017] The present invention provides for isolated nucleic acid
molecules comprising nucleic acid sequences encoding the amino acid
sequence shown in FIG. 1 (SEQ ID NO: 2) or the amino acid sequence
encoded by the cDNA clone deposited as ATCC Deposit No. 97920 on
Mar. 7, 1997.
[0018] The present invention also provides recombinant vectors,
which include the isolated nucleic acid molecules of the invention,
and to host cells containing the recombinant vectors, as well as to
methods of making such vectors and host cells and for using them
for production of DR5 polypeptides or peptides by recombinant
techniques.
[0019] The invention further provides an isolated DR5 polypeptide
having an amino acid sequence encoded by a polynucleotide described
herein.
[0020] The present invention also provides diagnostic assays such
as quantitative and diagnostic assays for detecting levels of DR5
protein. Thus, for instance, a diagnostic assay in accordance with
the invention for detecting over-expression of DR5, or soluble form
thereof, compared to normal control tissue samples may be used to
detect the presence of tumors.
[0021] Tumor Necrosis Factor (TNF) family ligands are known to be
among the most pleiotropic cytokines, inducing a large number of
cellular responses, including cytotoxicity, anti-viral activity,
immunoregulatory activities, and the transcriptional regulation of
several genes. Cellular response to TNF-family ligands include not
only normal physiological responses, but also diseases associated
with increased apoptosis or the inhibition of apoptosis.
Apoptosis--programmed cell death--is a physiological mechanism
involved in the deletion of peripheral T lymphocytes of the immune
system, and its dysregulation can lead to a number of different
pathogenic processes. Diseases associated with increased cell
survival, or the inhibition of apoptosis, include cancers,
autoimmune disorders, viral infections, inflammation, graft versus
host disease, acute graft rejection, and chronic graft rejection.
Diseases associated with increased apoptosis include AIDS,
neurodegenerative disorders, myelodysplastic syndromes, ischemic
injury, toxin-induced liver disease, septic shock, cachexia and
anorexia.
[0022] Thus, the invention further provides a method for enhancing
apoptosis induced by a TNF-family ligand, which involves
administering to a cell which expresses the DR5 polypeptide an
effective amount of an agonist capable of increasing DR5 mediated
signaling. Preferably, DR5 mediated signaling is increased to treat
a disease wherein decreased apoptosis is exhibited.
[0023] In a further aspect, the present invention is directed to a
method for inhibiting apoptosis induced by a TNF-family ligand,
which involves administering to a cell which expresses the DR5
polypeptide an effective amount of an antagonist capable of
decreasing DR5 mediated signaling. Preferably, DR5 mediated
signaling is decreased to treat a disease wherein increased
apoptosis is exhibited.
[0024] Whether any candidate "agonist" or "antagonist" of the
present invention can enhance or inhibit apoptosis can be
determined using art-known TNF-family ligand/receptor cellular
response assays, including those described in more detail below.
Thus, in a further aspect, a screening method is provided for
determining whether a candidate agonist or antagonist is capable of
enhancing or inhibiting a cellular response to a TNF-family ligand.
The method involves contacting cells which express the DR5
polypeptide with a candidate compound and a TNF-family ligand,
assaying a cellular response, and comparing the cellular response
to a standard cellular response, the standard being assayed when
contact is made with the ligand in absence of the candidate
compound, whereby an increased cellular response over the standard
indicates that the candidate compound is an agonist of the
ligand/receptor signaling pathway and a decreased cellular response
compared to the standard indicates that the candidate compound is
an antagonist of the ligand/receptor signaling pathway. By the
invention, a cell expressing the DR5 polypeptide can be contacted
with either an endogenous or exogenously administered TNF-family
ligand.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 shows the nucleotide (SEQ ID NO: 1) and deduced amino
acid sequence (SEQ ID NO: 2) of DR5. It is predicted that amino
acids 1-51 (underlined) constitute the signal peptide (amino acid
residues from about -51 to about -1 in SEQ ID NO: 2); amino acids
52-184 constitute the extracellular domain (amino acid residues
from about 1 to about 133 in SEQ ID NO: 2); amino acids 185-208
(underlined) constitute the transmembrane domain (amino acid
residues from about 134 to about 157 in SEQ ID NO: 2); and amino
acids 209-411 constitute the intracellular domain (amino acid
residues from about 158 to about 360 in SEQ ID NO: 2), of which
amino acids 324-391 (italicized) constitute the death domain (amino
acid residues from about 273 to about 340 in SEQ ID NO: 2).
[0026] FIG. 2 shows the regions of similarity between the amino
acid sequences of DR5 (IILYBX88), human tumor necrosis factor
receptor 1 (h TNFR1) (SEQ ID NO: 3), human Fas protein (SEQ ID NO:
4), and the death domain containing receptor 3 (SEQ ID NO: 5). The
comparison was created with the Megalign program which is contained
in the DNA Star suite of programs, using the Clustal method.
[0027] FIG. 3 shows an analysis of the DR5 amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown. In the "Antigenic
Index-Jameson-Wolf" graph, amino acid residues about 62 to about
110, about 119 to about 164, about 224 to about 271, and about 275
to about 370 as depicted in FIG. 1 correspond to the shown highly
antigenic regions of the DR5 protein. These highly antigenic
fragments in FIG. 1 correspond to the following fragments,
respectively, in SEQ ID NO: 2: amino acid residues from about 11 to
about 59, from about 68 to about 113, from about 173 to about 220,
and from about 224 to about 319.
[0028] FIG. 4 shows the nucleotide sequences (HAPBU13R and
HSBBU76R) of two cDNA molecules which are related to the nucleotide
sequence shown in FIG. 1 (SEQ ID NO: 1).
[0029] FIG. 5A is a bar graph showing that overexpression of DR5
induced apoptosis in MCF7 human breast carcinoma cells. FIG. 5B is
a bar graph showing that overexpression of DR5 induced apoptosis in
human epitheloid carcinoma (Hela ) cells. FIG. 5C is a bar graph
showing that DR5-induced apoptosis was blocked by caspase
inhibitors, CrmA and z-VAD-fmk, but dominant negative FADD was
without effect. FIG. 5D is an immunoblot showing that, like DR4,
DR5 did not interact with FADD and TRADD in vivo. FIG. 5E is a bar
graph showing that a dominant negative version of a newly
identified FLICE-like molecule, FLICE2 (Vincenz, C. et al., J Biol.
Chem. 272:6578 (1997)), efficiently blocked DR5-induced apoptosis,
while dominant negative FLICE had only partial effect under
conditions it blocked. It also shows that TNFR-1 blocked apoptosis
effectively.
[0030] FIG. 6A is an immunoblot showing that DR5-Fc (as well as DR4
and TRID) specifically bound TRAIL, but not the related cytotoxic
ligand TNF.alpha.. The bottom panel of FIG. 6A shows the input
Fc-fusions present in the binding assays. FIG. 6B is a bar graph
showing that DR5-Fc blocked the ability of TRAIL to induce
apoptosis The data (mean.+-.SD) shown in FIG. 6B are the percentage
of apoptotic nuclei among total nuclei counted (n-4). FIG. 6C is a
bar graph showing that DR5-Fc had no effect on apoptosis
TNF.alpha.-induced cell death under conditions where TNFR1-Fc
completely abolished TNF.alpha. killing.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0031] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a DR5 polypeptide
having the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2), or a
fragment of the polypeptide. The DR5 polypeptide of the present
invention shares sequence homology with other known death domain
containing receptors of the TNFR family including human TNFR- I,
DR3 and Fas (FIG. 2). The nucleotide sequence shown in FIG. 1 (SEQ
ID NO: 1) was obtained by sequencing cDNA clones such as HLYBX88,
which was deposited on Mar. 7, 1997 at the American Type Culture
Collection, 12301 Park Lawn Drive, Rockville, Md. 20852, and given
Accession Number 97920. The deposited clone is contained in the
pSport 1 plasmid (Life Technologies, Gaithersburg, Md.).
[0032] Nucleic Acid Molecules
[0033] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0034] Using the information provided herein, such as the nucleic
acid sequence set out in SEQ ID NO: 1, a nucleic acid molecule of
the present invention encoding a DR5 polypeptide may be obtained
using standard cloning and screening procedures, such as those for
cloning cDNAs using mRNA as starting material. Illustrative of the
invention, the nucleic acid molecule of the invention has been
identified in cDNA libraries of the following tissues: primary
dendritic cells, endothelial tissue, spleen, chronic lymphocytic
leukemia, and human thymus stromal cells.
[0035] The determined nucleotide sequence of the DR5 cDNA of SEQ ID
NO: 1 contains an open reading frame encoding a protein of about
411 amino acid residues whose initiation codon is at position
130-132 of the nucleotide sequence shown in FIG. 1 (SEQ ID NO. 1),
with a leader sequence of about 51 amino acid residues. Of known
members of the TNF receptor family, the DR5 polypeptide of the
invention shares the greatest degree of homology with human TNFR1,
FAS and DR3 polypeptides shown in FIG. 2, including significant
sequence homology over multiple cysteine-rich domains. The homology
DR5 shows to other death domain containing receptors strongly
indicates that DR5 is also a death domain containing receptor with
the ability to induce apoptosis. DR5 has also now been shown to
bind TRAIL.
[0036] As indicated, the present invention also provides the mature
form(s) of the DR5 protein of the present invention. According to
the signal hypothesis, proteins secreted by mammalian cells have a
signal or secretory leader sequence which is cleaved from the
mature protein once export of the growing protein chain across the
rough endoplasmic reticulum has been initiated. Most mammalian
cells and even insect cells cleave secreted proteins with the same
specificity. However, in some cases, cleavage of a secreted protein
is not entirely uniform, which results in two or more mature
species on the protein. Further, it has long been known that the
cleavage specificity of a secreted protein is ultimately determined
by the primary structure of the complete protein, that is, it is
inherent in the amino acid sequence of the polypeptide.
[0037] Therefore, the present invention provides a nucleotide
sequence encoding the mature DR5 polypeptide having the amino acid
sequence encoded by the cDNA clone contained in the host identified
as ATCC Deposit No. 97920, and as shown in FIG. 1 (SEQ ID NO: 2).
By the mature DR5 protein having the amino acid sequence encoded by
the cDNA clones contained in the host identified as ATCC Deposit
No. 97920, is meant the mature form(s) of the DR5 protein produced
by expression in a mammalian cell (e.g., COS cells, as described
below) of the complete open reading frame encoded by the human DNA
sequence of the clone contained in the vector in the deposited
host. As indicated below, the mature DR5 having the amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.
97920, may or may not differ from the predicted "mature" DR5
protein shown in SEQ ID NO: 2 (amino acids from about 1 to about
360) depending on the accuracy of the predicted cleavage site based
on computer analysis.
[0038] Methods for predicting whether a protein has a secretory
leader as well as the cleavage point for that leader sequence are
available. For instance, the method of McGeoch (Virus Res.
3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690
(1986)) can be used. The accuracy of predicting the cleavage points
of known mammalian secretory proteins for each of these methods is
in the range of 75-80%. von Heinje, supra. However, the two methods
do not always produce the same predicted cleavage point(s) for a
given protein.
[0039] In the present case, the predicted amino acid sequence of
the complete DR5 polypeptide of the present invention was analyzed
by a computer program ("PSORT"). See, K. Nakai and M. Kanehisa,
Genomics 14:897-911 (1992). PSORT is an expert system for
predicting the cellular location of a protein based on the amino
acid sequence. As part of this computational prediction of
localization, the methods of McGeoch and von Heinje are
incorporated. The analysis by the PSORT program predicted the
cleavage sites between amino acids 51 and 52 in FIG. 1 (-1 and 1 in
SEQ ID NO: 2). Thereafter, the complete amino acid sequences were
farther analyzed by visual inspection, applying a simple form of
the (-1,-3) rule of von Heinje. von Heinje, supra. Thus, the leader
sequence for the DR5 protein is predicted to consist of amino acid
residues from about 1 to about 51, underlined in FIG. 1
(corresponding to about -51 to about 1 in SEQ ID NO: 2), while the
predicted mature DR5 protein consists of residues from about 52 to
about 411 in FIG. 1 (corresponding to about 1 to about 360 in SEQ
ID NO: 2).
[0040] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA may be the coding strand,
also known as the sense strand, or it may be the non-coding strand,
also referred to as the anti-sense strand.
[0041] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0042] Isolated nucleic acid molecules of the present invention
include DR5 DNA molecules comprising an open reading frame (ORF)
shown in SEQ ID NO: 1; DNA molecules comprising the coding sequence
for the mature DR5 protein; and DNA molecules which comprise a
sequence substantially different from those described above, but
which, due to the degeneracy of the genetic code, still encode the
DR5 protein. Of course, the genetic code is well known in the art.
Thus, it would be routine for one skilled in the art to generate
such degenerate variants.
[0043] In another aspect, the invention provides isolated nucleic
acid molecules encoding the DR5 polypeptide having an amino acid
sequence encoded by the cDNA clone contained in the plasmid
deposited as ATCC Deposit No. 97920 on Mar. 7, 1997. In a further
embodiment, nucleic acid molecules are provided that encode the
mature DR5 polypeptide or the full length DR5 polypeptide lacking
the N-terminal methionine. The invention further provides an
isolated nucleic acid molecule having the nucleotide sequence shown
in SEQ ID NO: 1 or the nucleotide sequence of the DR5 cDNA
contained in the above-described deposited clone, or a nucleic acid
molecule having a sequence complementary to one of the above
sequences. Such isolated molecules, particularly DNA molecules, are
useful as probes for gene mapping by in situ hybridization with
chromosomes, and for detecting expression of the DR5 gene in human
tissue, for instance, by Northern blot analysis
[0044] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated DNA molecule having the nucleotide sequence of the
deposited cDNA, the nucleotide sequence shown in SEQ ID NO: 1, or
the complementary strand thereto, is intended DNA fragments at
least about 15 nt, and more preferably at least 20 nt, still more
preferably at least about 30 nt, and even more preferably, at least
about 40, 50, 100, 150, 200, 250, 300, 400, or 500 nt in length.
These fragments have numerous uses which include, but are not
limited to, diagnostic probes and primers as discussed herein. Of
course larger DNA fragments 500-1500 nt in length are also useful
according to the present invention, as are fragments corresponding
to most, if not all, of the nucleotide sequence of the deposited
cDNA or as shown in SEQ ID NO: 1. By a fragment at least 20 nt in
length, for example, is intended fragments which include 20 or more
contiguous bases from the nucleotide sequence of the deposited DNA
or the nucleotide sequence as shown in SEQ ID NO: 1.
[0045] Preferred nucleic acid fragments of the present invention
include, but are. not limited to nucleic acid molecules encoding: a
polypeptide comprising the DR5 extracellular domain (amino acid
residues from about 52 to about 184 in FIG. 1 (from about 1 to
about 133 in SEQ ID NO: 2)); a polypeptide comprising the DR5
transmembrane domain (amino acid residues from about 185 to about
208 in FIG. 1 (from about 134 to about 157 in SEQ ID NO: 2)); a
polypeptide comprising the DR5 intracellular domain (amino acid
residues from about 209 to about 411 in FIG. 1 (from about 158 to
about 360 in SEQ ID NO: 2)); and a polypeptide comprising the DR5
death domain (amino acid residues from about 324 to about 391 in
FIG. 1 (from about 273 to about 340 in SEQ ID NO: 2)). Since the
location of these domains have been predicted by computer graphics,
one of ordinary skill would appreciate that the amino acid residues
constituting these domains may vary slightly (e.g., by about 1 to
15 residues) depending on the criteria used to define each
domain.
[0046] Preferred nucleic acid fragments of the invention encode a
full-length DR5 polypeptide lacking the nucleotides encoding the
amino-terminal methionine (nucleotides 130-132 in SEQ ID NO: 1) as
it is known that the methionine is cleaved naturally and such
sequences maybe useful in genetically engineering DR5 expression
vectors. Polypeptides encoded by such polynucleotides are also
contemplated by the invention.
[0047] Preferred nucleic acid fragments of the present invention
further include nucleic acid molecules encoding epitope-bearing
portions of the DR5 protein. In particular, such nucleic acid
fragments of the present invention include, but are not limited to,
nucleic acid molecules encoding: a polypeptide comprising amino
acid residues from about 62 to about 110 in FIG. 1 (about 11 to
about 59 in SEQ ID NO: 2); a polypeptide comprising amino acid
residues from about 119 to about 164 in FIG. 1 (about 68 to about
113 in SEQ ID NO: 2); a polypeptide comprising amino acid residues
from about 224 to about 271 in FIG. 1 (about 173 to about 220 in
SEQ ID NO: 2); and a polypeptide comprising amino acid residues
from about 275 to about 370 in FIG. 1 (about 224 to about 319 in
SEQ ID NO: 2). The inventors have determined that the above
polypeptide fragments are antigenic regions of the DR5 protein.
Methods for determining other such epitope-bearing portions of the
DR5 protein are described in detail below.
[0048] In addition, the invention provides nucleic acid molecules
having nucleotide sequences related to extensive portions of SEQ ID
NO: 1 which have been determined from the following related cDNA
clones: HAPBU13R (SEQ ID NO: 6) and HSBBU76R (SEQ ID NO: 7). The
nucleotide sequences of HAPBU13R and HSBBU76R are shown in FIG.
4.
[0049] Further, the invention includes a polynucleotide comprising
any portion of at least about 30 nucleotides, preferably at least
about 50 nucleotides, of SEQ ID NO: 1 from residue 284 to 1,362,
preferably from 284 to 681.
[0050] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clones contained in ATCC
Deposit No. 97920. By "stringent hybridization conditions" is
intended overnight incubation at 42.degree. C. in a solution
comprising: 50% formamide, 5.times.SSC (150 mM NaCl, 15mM trisodium
citrate), 50 mM sodium phosphate (pH 7.6), 5.times.Denhardt's
solution, 10% dextran sulfate, and 20 g/ml denatured, sheared
salmon sperm DNA, followed by washing the filters in 0.1.times.SSC
at about 65.degree. C.
[0051] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least, about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 or 80-150 nt, or
the entire length of the reference polynucleotide. These are useful
as diagnostic probes and primers as discussed above and in more
detail below.
[0052] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in SEQ ID
NO: 1).
[0053] Of course, a polynucleotide which hybridizes only to a poly
A sequence (such as the 3' terminal poly(A) tract of the DR5 cDNA
shown in FIG. 1 (SEQ ID NO: 1)), or to a complementary stretch of T
(or U) resides, would not be included in a polynucleotide of the
invention used to hybridize to a portion of a nucleic acid of the
invention, since such a polynucleotide would hybridize to any
nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone).
[0054] As indicated, nucleic acid molecules of the present
invention which encode a DR5 polypeptide may include, but are not
limited to, the coding sequence for the mature polypeptide, by
itself; the coding sequence for the mature polypeptide and
additional sequences, such as those encoding a leader or secretory
sequence, such as a pre-, or pro- or prepro- protein sequence; the
coding sequence of the mature polypeptide, with or without the
aforementioned additional coding sequences, together with
additional, non-coding sequences, including for example, but not
limited to introns and non-coding 5' and 3' sequences, such as the
transcribed, non-translated sequences that play a role in
transcription, mRNA processing--including splicing and
polyadenylation signals, for example--ribosome binding and
stability of mRNA; additional coding sequence which codes for
additional amino acids, such as those which provide additional
functionalities. Thus, for instance, the polypeptide may be fused
to a marker sequence, such as a peptide, which facilitates
purification of the fused polypeptide. In certain preferred
embodiments of this aspect of the invention, the marker sequence is
a hexa-histidine peptide, such as the tag provided in a pQE vector
(Qiagen, Inc.), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci USA
86: 821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. The "HA" tag is
another peptide useful for purification which corresponds to an
epitope derived from the influenza hemagglutinin protein, which has
been described by Wilson et al., Cell 37:767 -778(1984). As
discussed below, other such fusion proteins include the DR5
receptor fused to Fc at the N- or C-terminus.
[0055] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs, or derivatives of the DR5 receptor. Variants may
occur naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a chromosome of an organism. Genes II,
Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0056] Such variants include those produced by nucleotide
substitutions, deletions or additions which may involve one or more
nucleotides. The variants may be altered in coding or non-coding
regions or both. Alterations in the coding regions may produce
conservative or non-conservative amino acid substitutions,
deletions or additions. Especially preferred among these are silent
substitutions, additions, and deletions, which do not alter the
properties and activities of the DR5 receptor or portions thereof.
Also especially preferred in this regard are conservative
substitutions.
[0057] Further embodiments of the invention include isolated
nucleic acid molecules that are at least 90% identical, and more
preferably at least 95%, 96%, 97%, 98% or 99% identical, to (a) a
nucleotide sequence encoding the polypeptide having the amino acid
sequence in SEQ ID NO: 2; (b) a nucleotide sequence encoding the
polypeptide having the amino acid sequence in SEQ ID NO: 2, but
lacking the amino terminal methionine; (c) a nucleotide sequence
encoding the polypeptide having the amino acid sequence at
positions about 1 to about 360 in SEQ ID NO: 2; (d) a nucleotide
sequence encoding the polypeptide having the amino acid sequence
encoded by the cDNA clone contained in ATCC Deposit No. 97920; (e)
a nucleotide sequence encoding the mature DR5 polypeptide having
the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. 97920; (f) a nucleotide sequence that encodes the DR5
extracellular domain having the amino acid sequence at positions
about 1 to about 133 in SEQ ID NO: 2, or the DR5 extracellular
domain encoded by the cDNA contained in ATCC Deposit No. 97920; (g)
a nucleotide sequence that encodes the DR5 transmembrane domain
having the amino acid sequence at positions about 134 to about 157
of SEQ ID NO: 2, or the DR5 transmembrane domain encoded by the
cDNA contained in ATCC Deposit No. 97920; (h) a nucleotide sequence
that encodes the DR5 intracellular domain having the amino acid
sequence at positions about 158 to about 360 of SEQ ID NO: 2, or
the DR5 intracellular domain encoded by the cDNA contained in ATCC
Deposit No. 97920; (i) a nucleotide sequence that encodes the DR5
death domain domain having the amino acid sequence at positions
about 273 to about 340 of SEQ ID NO: 2, or the DR5 death domain
encoded by the cDNA contained in ATCC Deposit No. 97920; and () a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i)
above.
[0058] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a DR5 polypeptide is intended that the nucleotide sequence
of the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence encoding the DR5 polypeptide. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. The reference (query) sequence may be the
entire DR5 nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) or
any polynucleotide fragment as described herein.
[0059] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to,
for instance, the nucleotide sequence shown in SEQ ID NO: 1 or to
the nucleotide sequence of the deposited cDNA clone can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0060] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB alignment of DNA sequences to
calculate percent identiy are: Matrix=Unitary, k-tuple=4, Mismatch
Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff
Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or
the length of the subject nucleotide sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence because of 5' or 3' deletions, not
because of internal deletions, a manual correction is made to the
results to take into consideration the fact that the FASTDB program
does not account for 5' and 3' truncations of the subject sequence
when calculating percent identity. For subject sequences truncated
at the 5' or 3' ends, relative to the the query sequence, the
percent identity is corrected by calculating the number of bases of
the query sequence that are 5' and 3' of the subject sequence,
which are not matched/aligned, as a percent of the total bases of
the query sequence. A determination of whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of this
embodiment. Only bases outside the 5' and 3' bases of the subject
sequence, as displayed by the FASTDB alignment, which are not
matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score. For
example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the
5' end of the subject sequence and therefore, the FASTDB alignment
does not show a matched/alignement of the first 10 bases at 5' end.
The 10 unpaired bases represent 10% of the sequence (number of
bases at the 5' and 3' ends not matched/total number of bases in
the query sequence) so 10% is subtracted from the percent identity
score calculated by the FASTDB program. If the remaining 90 bases
were perfectly matched the final percent identity would be 90%. In
another example, a 90 base subject sequence is compared with a 100
base query sequence. This time the deletions are internal deletions
so that there are no bases on the 5' or 3' of the subject sequence
which are not matched/aligned with the query. In this case the
percent identity calculated by FASTDB is not manually corrected.
Once again, only bases 5' and 3' of the subject sequence which are
not matched/aligned with the query sequnce are manually corrected
for. No other manual corrections are made for the purposes of this
embodiment.
[0061] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NO: 1, the nucleic acid
sequence of the deposited cDNAs, or fragments thereof, irrespective
of whether they encode a polypeptide having DR5 activity. This is
because even where a particular nucleic acid molecule does not
encode a polypeptide having DR5 activity, one of skill in the art
would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present
invention that do not encode a polypeptide having DR5 activity
include, inter alia: (1) isolating the DR5 gene or allelic variants
thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH")
to metaphase chromosomal spreads to provide precise chromosomal
location of the DR5 gene, as described in Verma et al., Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
(1988); and (3) Northern Blot analysis for detecting DR5 mRNA
expression in specific tissues.
[0062] Preferred, however, are nucleic acid molecules having
sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NO: 1, the nucleic acid
sequence of the deposited cDNAs, or fragments thereof, which do, in
fact, encode a polypeptide having DR5 protein activity. By "a
polypeptide having DR5 activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the DR5 protein of the invention (either the
full-length protein or, preferably, the mature protein), as
measured in a particular biological assay. For example, DR5 protein
activity can be measured using the cell death assays performed
essentially as previously described (A. M. Chinnaiyan, et al., Cell
81:505-12 (1995); M. P. Boldin, et al., J Biol Chem 270:7795-8
(1995); F. C. Kischkel, et al., EMBO 14:5579-5588 (1995); A. M.
Chinnaiyan, et al., J Biol Chem 271:4961-4965 (1996)) and as set
forth in Example 5, below. In MCF7 cells, plasmids encoding
full-length DR5 or a candidate death domain containing receptor are
co-transfected with the pLantern reporter construct encoding green
fluorescent protein. Nuclei of cells transfected with DR5 will
exhibit apoptotic morphology as assessed by DAPI staining. Similar
to TNFR-1 and Fas/APO-1 (M. Muzio, et al., Cell 85:817-827 (1996);
M. P. Boldin, et al., Cell 85:803-815 (1996); M. Tewari, et al., J
Biol Chem 270:3255-60 (1995)), DR5-induced apoptosis is preferably
blocked by the inhibitors of ICE-like proteases, CrmA and
z-VAD-fink.
[0063] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence of the deposited cDNA, the nucleic acid sequence shown in
SEQ ID NO: 1, or fragments thereof, will encode a polypeptide
"having DR5 protein activity." In fact, since degenerate variants
of these nucleotide sequences all encode the same polypeptide, in
many instances, this will be clear to the skilled artisan even
without performing the above described comparison assay. It will be
further recognized in the art that, for such nucleic acid molecules
that are not degenerate variants, a reasonable number will also
encode a polypeptide having DR5 protein activity. This is because
the skilled artisan is fully aware of amino acid substitutions that
are either less likely or not likely to significantly effect
protein function (e.g., replacing one aliphatic amino acid with a
second aliphatic amino acid).
[0064] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that proteins are surprisingly tolerant of amino
acid substitutions.
[0065] Polynucleotide Assays
[0066] This invention is also related to the use of the DR5
polynucleotides to detect complementary polynucleotides such as,
for example, as a diagnostic reagent Detection of a mutated form of
DR5 associated with a dysfunction will provide a diagnostic tool
that can add or define a diagnosis of a disease or susceptibility
to a disease which results from under-expression over-expression or
altered expression of DR5 or a soluble form thereof, such as, for
example, tumors or autoimmune disease.
[0067] Individuals carrying mutations in the DR5 gene may be
detected at the DNA level by a variety of techniques. Nucleic acids
for diagnosis may be obtained from a patient's cells, such as from
blood, urine, saliva, tissue biopsy and autopsy material. The
genomic DNA may be used directly for detection or may be amplified
enzymatically by using PCR prior to analysis. (Saiki et al., Nature
324:163-166 (1986)). RNA or cDNA may also be used in the same ways.
As an example, PCR primers complementary to the nucleic acid
encoding DR5 can be used to identify and analyze DR5 expression and
mutations. For example, deletions and insertions can be detected by
a change in size of the amplified product in comparison to the
normal genotype. Point mutations can be identified by hybridizing
amplified DNA to radiolabeled DR5 RNA or alternatively,
radiolabeled DR5 antisense DNA sequences. Perfectly matched
sequences can be distinguished from mismatched duplexes by RNase A
digestion or by differences in melting temperatures.
[0068] Sequence differences between a reference gene and genes
having mutations also may be revealed by direct DNA sequencing. In
addition, cloned DNA segments may be employed as probes to detect
specific DNA segments. The sensitivity of such methods can be
greatly enhanced by appropriate use of PCR or another amplification
method. For example, a sequencing primer is used with
double-stranded PCR product or a single-stranded template molecule
generated by a modified PCR. The sequence determination is
performed by conventional procedures with radiolabeled nucleotide
or by automatic sequencing procedures with fluorescent-tags.
[0069] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments in gels, with or without denaturing agents. Small
sequence deletions and insertions can be visualized by high
resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science 230:1242 (1985)).
[0070] Sequence changes at specific locations also may be revealed
by nuclease protection assays, such as RNase and SI protection or
the chemical cleavage method (e.g., Cotton et al., Proc. Natl Acad.
Sci. USA 85: 4397-4401 (1985)).
[0071] Thus, the detection of a specific DNA sequence may be
achieved by methods which include, but are not limited to,
hybridization, RNase protection, chemical cleavage, direct DNA
sequencing or the use of restriction enzymes, (e.g., restriction
fragment length polymorphisms ("RFLP") and Southern blotting of
genomic DNA).
[0072] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations also can be detected by in situ analysis.
[0073] Vectors and Host Cells
[0074] The present invention also relates to vectors which include
DNA molecules of the present invention, host cells which are
genetically engineered with vectors of the invention and the
production of polypeptidthe invention by recombinant
techniques.
[0075] Host cells can be genetically engineered to incorporate
nucleic acid molecules and express polypeptides of the present
invention. The polynucleotides may be introduced alone or with
other polynucleotides. Such other polynucleotides may be introduced
independently, co-introduced or introduced joined to the
polynucleotides of the invention.
[0076] In accordance with this aspect of the invention the vector
may be, for example, a plasmid vector, a single or double-stranded
phage vector, a single or double-stranded RNA or DNA viral vector.
Such vectors may be introduced into cells as polynucleotides,
preferably DNA, by well known techniques for introducing DNA and
RNA into cells. Viral vectors may be replication competent or
replication defective. In the latter case viral propagation
generally will occur only in complementing host cells.
[0077] Preferred among vectors, in certain respects, are those for
expression of polynucleotides and polypeptides of the present
invention. Generally, such vectors comprise cis-acting control
regions effective for expression in a host operatively linked to
the polynucleotide to be expressed. Appropriate trans-acting
factors either are supplied by the host, supplied by a
complementing vector or supplied by the vector itself upon
introduction into the host.
[0078] A great variety of expression vectors can be used to express
a polypeptide of the invention. Such vectors include chromosomal,
episomal and virus-derived vectors e.g., vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, from viruses such as baculoviruses,
papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl
pox viruses, pseudorabies viruses and retroviruses, and vectors
derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids, all may be used for expression in accordance with this
aspect of the present invention. Generally, any vector suitable to
maintain, propagate or express polynucleotides to express a
polypeptide in a host may be used for expression in this
regard.
[0079] The DNA sequence in the expression vector is operatively
linked to appropriate expression control sequence(s)), including,
for instance, a promoter to direct mRNA transcription.
Representatives of such promoters include the phage lambda PL
promoter, the E. coli lac, trp and tac promoters, the SV40 early
and late promoters and promoters of retroviral LTRs, to name just a
few of the well-known promoters. In general, expression constructs
will contain sites for transcription, initiation and termination,
and, in the transcribed region, a ribosome binding site for
translation The coding portion of the mature transcripts expressed
by the constructs will include a translation initiating AUG at the
beginning and a termination codon (UAA, UGA or UAG) appropriately
positioned at the end of the polypeptide to be translated.
[0080] In addition, the constructs may contain control regions that
regulate as well as engender expression. Generally, such regions
will operate by controlling transcription, such as repressor
binding sites and enhancers, among others.
[0081] Vectors for propagation and expression generally will
include selectable markers. Such markers also may be suitable for
amplification or the vectors may contain additional markers for
this purpose. In this regard, the expression vectors preferably
contain one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells. Such markers
include, but are not limited to, dihydrofolate reductase or
neomycin resistance for eukaryotic cell culture, and tetracycline
or ampicillin resistance genes for culturing E. coli and other
bacteria.
[0082] The vector containing the appropriate DNA sequence as
described elsewhere herein, as well as an appropriate promoter, and
other appropriate control sequences, may be introduced into an
appropriate host using a variety of well known techniques suitable
to expression therein of a desired polypeptide. Representative
examples of appropriate hosts include bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the
art.
[0083] Among vectors preferred for use in bacteria are pQE70, pQE60
and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,
Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from
Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
available from Pharmacia. Among preferred eukaryotic vectors are
pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and
pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors
are listed solely by way of illustration of the many commercially
available and well known vectors available to those of skill in the
art.
[0084] Selection of appropriate vectors and promoters for
expression in a host cell is a well known procedure and the
requisite techniques for expression vector construction,
introduction of the vector into the host and expression in the host
are routine skills in the art.
[0085] The present invention also relates to host cells containing
the above-described constructs discussed above. The host cell can
be a higher eukaryotic cells such as a mammalian cell, or a lower
eukaryotic cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a bacterial cell. The host strain may be
chosen which modulates the expression of the inserted gene
sequences, or modifies and processes the gene product in the
specific fashion desired. Expression from certain promoters can be
elevated in the presence of certain inducers; thus expression of
the genetically engineered polypeptide may be controlled.
Furthermore, different host cells have characteristics and specific
mechanisms for the translational and post-translational processing
and modification (e.g., phosphorylation, cleavage) of proteins.
Appropriate cell lines can be chosen to ensure the desired
modifications and processing of the foeign protein expressed.
[0086] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods in Molecular Biology (1986).
[0087] The polypeptide may be expressed in a modified form, such as
a fusion protein (comprising the polypeptide joined via a peptide
bond to a heterologous protein sequence (of a different protein)),
and may include not only secretion signals but also additional
heterologous functional regions. Such a fusion protein can be made
by ligating polynucleotides of the invention and the desired
nucleic acid sequence encoding the desired amino acid sequence to
each other, by methods known in the art, in the proper reading
frame, and expressing the fusion protein product by methods known
in the art. Alternatively, such a fusion protein can be made by
protein synthetic techniques, e.g., by use of a peptide
synthesizer. Thus,. for instance, a region of additional amino
acids, particularly charged amino acids, may be added to the
N-terminus of the polypeptide to improve stability and persistence
in the host cell, during purification or during subsequent handling
and storage. Additionally, a region also may be added to the
polypeptide to facilitate purification. Such regions may be removed
prior to final preparation of the polypeptide. The addition of
peptide moieties to polypeptides to engender secretion or
excretion, to improve stability and to facilitate purification,
among others, are familiar and routine techniques in the art. A
preferred fusion protein comprises a heterologous region from
immunoglobulin that is useful to solubilize proteins. For example,
EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion
proteins comprising various portions of constant region of
immunoglobin molecules together with another human protein or part
thereof. In many cases, the Fc part in a fusion protein is
thoroughly advantageous for use in therapy and diagnosis and-thus
results, for example, in improved pharmacokinetic properties (EP-A
0232 262). On the other hand, for some uses it would be desirable
to be able to delete the Fc part after the fusion protein has been
expressed, detected and purified in the advantageous manner
described. This is the case when the Fc portion proves to be a
hindrance to use in therapy and diagnosis, for example when the
fusion protein is to be used as an antigen for immunizations. In
drug discovery, for example, human proteins, such as the
hIL5-receptor, have been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5.
See, D. Bennett et al., Journal of Molecular Recognition, 8:52-58
(1995) and K. Johanson et al., The Journal of Biological Chemistry,
270:9459-9471 (1995).
[0088] The DR5 polypeptides can be recovered and purified from
recombinant cell cultures by standard methods which include, but
are not limited to, ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography ("HPLC") is employed for
purification. Well known techniques for refolding protein may be
employed to regenerate active conformation when the polypeptide is
denatured during isolation and/or purification.
[0089] Polypeptides of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host, including, for example, bacterial, yeast, higher
plant, insect and mammalian cells. Depending upon the host employed
in a recombinant production procedure, the polypeptides of the
present invention may be glycosylated or may be non-glycosylated.
In addition, polypeptides of the invention may also include an
initial modified methionine residue, in some cases as a result of
host-mediated processes.
[0090] DR5 polynucleotides and polypeptides may be used in
accordance with the present invention for a variety of
applications, particularly those that make use of the chemical and
biological properties of DR5. Among these are applications in
treatment of tumors, resistance to parasites, bacteria and viruses,
to induce proliferation of T-cells, endothelial cells and certain
hematopoietic cells, to treat restenosis, graft vs. host disease,
to regulate anti-viral responses and to prevent certain autoimmune
diseases after stimulation of DR5 by an agonist. Additional
applications relate to diagnosis and to treatment of disorders of
cells, tissues and organisms. These aspects of the invention are
discussed further below.
[0091] DR5 Polypeptides and Fragments
[0092] The invention further provides an isolated DR5 polypeptide
having the amino acid sequence encoded by the deposited cDNA, or
the amino acid sequence in SEQ ID NO: 2, or a polypeptide or
peptide comprising a portion of the above polypeptides.
[0093] It will be recognized in the art that some amino acid
sequence of DR5 can be varied without significant effect on the
structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity. Such
areas will usually comprise residues which make up the ligand
binding site or the death domain, or which form tertiary structures
which affect these domains.
[0094] Thus, the invention further includes variations of the DR5
protein which show substantial DR5 protein activity or which
include regions of DR5, such as the protein portions discussed
below. Such mutants include deletions, insertions, inversions,
repeats, and type substitutions. As indicated above, guidance
concerning which amino acid changes are likely to be phenotypically
silent can be found in Bowie, J. U. et al., Science 247:1306-1310
(1990).
[0095] Thus, the fragment, derivative, or analog of the polypeptide
of SEQ ID NO: 2, or that encoded by the deposited cDNA, may be (i)
one in which at least one or more of the amino acid residues are
substituted with a conserved or non-conserved amino acid residue
(preferably a conserved amino acid residue(s), and more preferably
at least one but less than ten conserved amino acid residues) and
such substituted amino acid residue may or may not be one encoded
by the genetic code, or (ii) one in which one or more of the amino
acid residues includes a substituent group, or (iii) one in which
the mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as an IgG Fc fusion
region peptide or leader or secretory sequence or a sequence which
is employed for purification of the mature polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0096] Of particular interest are substitutions of charged amino
acids with another charged amino acids and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the DR5
protein. The prevention of aggregation is highly desirable.
Aggregation of proteins not only results in a loss of activity but
can also be problematic when preparing pharmaceutical formulations,
because they can be immunogenic. (Pinckard et al., Clin Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845
(1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993)).
[0097] The replacement of amino acids can also change the
selectivity of binding to cell surface receptors. Ostade et al.,
Nature 361:266-268 (1993) describes certain mutations resulting in
selective binding of TNF-alpha to only one of the two known types
of TNF receptors. Thus, the DR5 receptor of the present invention
may include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation.
[0098] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
1TABLE 1 Conservative Amino Acid Substitutions Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidme
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
[0099] Amino acids in the DR5 protein of the present invention that
are essential for 10 function can be identified by methods known in
the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding or in
vitro, or in vitro proliferative activity. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J Mol. Biol. 224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)).
[0100] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that
have been purified, partially or substantially, from a recombinant
host cell. For example, a recombinantly produced version of the DR5
polypeptide can be substantially purified by the one-step method
described in Smith and Johnson, Gene 67:31-40 (1988).
[0101] The polypeptides of the present invention also include the
polypeptide encoded by the deposited cDNA including the leader; the
mature polypeptide encoded by the deposited the cDNA minus the
leader (i.e., the mature protein); a polypeptide comprising amino
acids about--51 to about 360 in SEQ ID NO: 2; a polypeptide
comprising amino acids about--50 to about 360 in SEQ ID NO: 2; a
polypeptide comprising amino acids about 1 to about 360 in SEQ ID
NO: 2; a polypeptide comprising the extracellular domain; a
polypeptide comprising the transmembrane domain; a polypeptide
comprising the intracellular domain; a polypeptide comprising the
extracellular and intracellular domains with all or part of the
transmembrane domain deleted; and a polypeptide comprising the
death domain; as well as polypeptides which are at least 80%
identical, more preferably at least 90% or 95% identical, still
more preferably at least 96%, 97%, 98%, or 99% identical to the
polypeptides described above, and also include portions of such
polypeptides with at least 30 amino acids and more preferably at
least 50 amino acids.
[0102] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
DR5 polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the DR5
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0103] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2), the amino
acid sequence encoded by deposited cDNA clones, or fragments
thereof, can be determined conventionally using known computer
programs such the Bestfit program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, 575 Science Drive, Madison, Wis. 53711). When using
Bestfit or any other sequence alignment program to determine
whether a particular sequence is, for instance, 95% identical to a
reference sequence according to the present invention, the
parameters are set, of course, such that the percentage of identity
is calculated over the full length of the reference amino acid
sequence and that gaps in homology of up to 5% of the total number
of amino acid residues in the reference sequence are allowed.
[0104] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=1, Window Size=sequence
length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or
the length of the subject amino acid sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence due to N- or C-terminal deletions,
not because of internal deletions, a manual correction is made to
the results to take into consideration the fact that the FASTDB
program does not account for N- and C-terminal truncations of the
subject sequence when calculating global percent identity. For
subject sequences truncated at the N- and C-termini, relative to
the the query sequence, the percent identity is corrected by
calculating the number of residues of the query sequence that are
N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. A determination of
whether a residue is matched/aligned is determined by results of
the FASTDB sequence alignment. This percentage is then subtracted
from the percent identity, calculated by the above FASTDB program
using the specified parameters, to arrive at a final percent
identity score. This final percent identity score is what is used
for the purposes of this embodiment. Only residues to the N- and
C-termini of the subject sequence, which are not matched/aligned
with the query sequence, are considered for the purposes of
manually adjusting the percent identity score. That is, only query
residue positions outside the farthest N- and C-terminal residues
of the subject sequence. For example, a 90 amino acid residue
subject sequence is aligned with a 100 residue query sequence to
determine percent identity. The deletion occurs at the N-terminus
of the subject sequence and therefore, the FASTDB alignment does
not show a matching/alignment of the first 10 residues at the
N-terminus. The 10 unpaired residues represent 10% of the sequence
(number of residues at the N- and C-termini not matched/total
number of residues in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequnce are manually corrected for.
No other manual corrections are made for the purposes of this
embodiment.
[0105] The polypeptide of the present invention could be used as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0106] The present inventors have discovered that the DR5
polypeptide is a 411 residue protein exhibiting three main
structural domains. First, the ligand binding domain was identified
within residues from about 52 to about 184 in FIG. 1 (amino acid
residues from about 1 to about 133 in SEQ ID NO: 2). Second, the
transmembrane domain was identified within residues from about 185
to about 208 in FIG. 1 (amino acid residues from about 134 to about
157 in SEQ ID NO: 2). Third, the intracellular domain was
identified within residues from about 209 to about 411 in FIG. 1
(amino acid residues from about 158 to about 360 in SEQ ID NO: 2).
Importantly, the intracellular domain includes a death domain at
residues from about 324 to about 391 (amino acid residues from
about 273 to about 340 in SEQ ID NO: 2). Further preferred
fragments of the polypeptide shown in FIG. 1 include the mature
protein from residues about 52 to about 411 (amino acid residues
from about 1 to about 360 in SEQ ID NO: 2), and soluble
polypeptides comprising all or part of the extracellular and
intracellular domains but lacking the transmembrane domain, The
invention further provides DR5 polypeptides encoded by the
deposited cDNA clone including the leader and DR5 polypeptide
fragments selected from the mature protein, the extracellular
domain, the transmembrane domain, the intracellular domain, the
death domain, and all combinations thereof.
[0107] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
described herein. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0108] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.,
"Antibodies That React With Predetermined Sites on Proteins,"
Science 219:660-666 (1983). Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals.
[0109] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. See, for instance, Wilson et al., Cell 37:767-778
(1984) at 777. Antigenic epitope-bearing peptides and polypeptides
of the invention preferably contain a sequence of at least seven,
more preferably at least nine, and most preferably between at least
about 15 to about 30 amino acids contained within the amino acid
sequence of a polypeptide of the invention.
[0110] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate DR5-specific antibodies include: a
polypeptide comprising amino acid residues from about 62 to about
110 in FIG. 1 (about 11 to about 59 in SEQ ID NO: 2); a polypeptide
comprising amino acid residues from about 119 to about 164 in FIG.
1 (about 68 to about 113 in SEQ ID NO: 2); a polypeptide comprising
amino acid residues from about 224 to about 271 in FIG. 1 (about
173 to about 220 in SEQ ID NO: 2); and a polypeptide comprising
amino acid residues from about 275 to about 370 in FIG. 1 (about
224 to about 319 in SEQ ID NO: 2). As indicated above, the
inventors have determined that the above polypeptide fragments are
antigenic regions of the DR5 protein.
[0111] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. Hougthen, R.
A., "General Method for the Rapid Solid-Phase Synthesis of Large
Numbers of Peptides: Specificity of Antigen-Antibody Interaction at
the Level of Individual Amino Acids," Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Pat. No. 4,631,211 to
Hougthen et al (1986).
[0112] As one of skill in the art will appreciate, DR5 polypeptides
of the present invention and the epitope-bearing fragments thereof
described above can be combined with parts of the constant domain
of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins facilitate purification and show an increased
half-life in vivo. This has been shown, e.g., for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins (EPA 394,827; Traunecker et
al., Nature 331:84-86 (1988)). Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG part can also be
more efficient in binding and neutralizing other molecules than the
monomeric DR5 protein or protein fragment alone (Fountoulakis et
al., J Biochem 270:3958-3964 (1995)).
[0113] Polypeptide Assays
[0114] The present invention also relates to diagnostic assays such
as quantitative and diagnostic assays for detecting levels of DR5
protein, or the soluble form thereof, in cells and tissues,
including determination of normal and abnormal levels. Thus, for
instance, a diagnostic assay in accordance with the invention for
detecting over-expression of DR5, or soluble form thereof, compared
to normal control tissue samples may be used to detect the presence
of tumors, for example. Assay techniques that can be used to
determine levels of a protein, such as a DR5 protein of the present
invention, or a soluble form thereof, in a sample derived from a
host are well-known to those of skill in the art. Such assay
methods include radioimmunoassays, competitive-binding assays,
Western Blot analysis, and ELISA assays.
[0115] Assaying DR5 protein levels in a biological sample can occur
using any art-known method. By "biological sample" is intended any
biological sample obtained from an individual, cell line, tissue
culture, or other source containing DR5 receptor protein or mRNA.
Preferred for assaying DR5 protein levels in a biological sample
are antibody-based techniques. For example, DR5 protein expression
in tissues can be studied with classical immunohistological
methods. (Jalkanen, M. et al., J Cell. Biol. 101:976-985 (1985);
Jalkanen, M. et al., J Cell. Biol. 105:3087-3096 (1987)). Other
antibody-based methods useful for detecting DR5 protein gene
expression include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
[0116] Suitable labels are known in the art and include enzyme
labels, such as glucose oxidase, radioisotopes, such as iodine
(.sup.125I, .sup.121I), carbon (.sup.14C), sulphur (.sup.35S),
tritium (.sup.3H), indium (.sup.112In), and technetium
(.sup.99mTc), and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0117] Therapeutics
[0118] The Tumor Necrosis Factor (TNF) family ligands are known to
be among the most pleiotropic cytokines, inducing a large number of
cellular responses, including cytotoxicity, anti-viral activity,
immunoregulatory activities, and the transcriptional regulation of
several genes (Goeddel, D. V. et al, "Tumor Necrosis Factors: Gene
Structure and Biological Activities," Symp. Quant. Biol. 51:597-609
(1986), Cold Spring Harbor; Beutler, B., and Cerami, A., Annu. Rev.
Biochem. 57:505-518 (1988); Old, L. J., Sci. Am. 258:59-75 (1988);
Fiers, W., FEBS Lett. 285:199-224 (1991)). The TNF-family ligands
induce such various cellular responses by binding to TNF-family
receptors, including the DR5 of the present invention. Cells which
express the DR5 polypeptide and are believed to have a potent
cellular response to DR5 ligands include primary dendritic cells,
endothelial tissue, spleen, chronic lymphocytic leukemia, and human
thymus stromal cells. By "a cellular response to a TNF-family
ligand" is intended any genotypic, phenotypic, and/or morphologic
change to a cell, cell line, tissue, tissue culture or patient that
is induced by a TNF-family ligand. As indicated, such cellular
responses include not only normal physiological responses to
TNF-family ligands, but also diseases associated with increased
apoptosis or the inhibition of apoptosis. Apoptosis (programmed
cell death) is a physiological mechanism involved in the deletion
of peripheral T lymphocytes of the immune system, and its
dysregulation can lead to a number of different pathogenic
processes (Ameisen, J. C., AIDS 8:1197-1213 (1994); Krammer, P. H.
et al., Curr. Opin. Immunol. 6:279-289 (1994)).
[0119] Diseases associated with increased cell survival, or the
inhibition of apoptosis, include cancers (such as follicular
lymphomas, carcinomas with p53 mutations, and hormone-dependent
tumors, such as breast cancer, prostrate cancer, Kaposi's sarcoma
and ovarian cancer); autoimmune disorders (such as systemic lupus
erythematosus and immune-related glomerulonephritis rheumatoid
arthritis) and viral infections (such as herpes viruses, pox
viruses and adenoviruses), inflammation; graft vs. host disease,
acute graft rejection, and chronic graft rejection. Diseases
associated with increased apoptosis include AIDS; neurodegenerative
disorders (such as Alzheimer's disease, Parkinson's disease,
Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar
degeneration); myelodysplastic syndromes (such as aplastic anemia),
ischemic injury (such as that caused by myocardial infarction,
stroke and reperfusion injury), toxin-induced liver disease (such
as that caused by alcohol), septic shock, cachexia and
anorexia.
[0120] Thus, in one aspect, the present invention is directed to a
method for enhancing apoptosis induced by a TNF-family ligand,
which involves administering to a cell which expresses the DR5
polypeptide an effective amount of DR5 ligand, analog or an agonist
capable of increasing DR5 mediated signaling. Preferably, DR5
mediated signaling is increased to treat a disease wherein
decreased apoptosis or decreased cytokine and adhesion molecule
expression is exhibited. An agonist can include soluble forms of
DR5 and monoclonal antibodies directed against the DR5
polypeptide.
[0121] In a further aspect, the present invention is directed to a
method for inhibiting apoptosis induced by a TNF-family ligand,
which involves administering to a cell which expresses the, DR5
polypeptide an effective amount of an antagonist capable of
decreasing DR5 mediated signaling. Preferably, DR5 mediated
signaling is decreased to treat a disease wherein increased
apoptosis or NFkB expression is exhibited. An antagonist can
include soluble forms of DR5 and monoclonal antibodies directed
against the DR5 polypeptide.
[0122] By "agonist" is intended naturally occurring and synthetic
compounds capable of enhancing or potentiating apoptosis. By
"antagonist" is intended naturally occurring and synthetic
compounds capable of inhibiting apoptosis. Whether any candidate
"agonist" or "antagonist" of the present invention can enhance or
inhibit apoptosis can be determined using art-known TNF-family
ligand/receptor cellular response assays, including those described
in more detail below.
[0123] One such screening procedure involves the use of
melanophores which are transfected to express the receptor of the
present invention. Such a screening technique is described in PCT
WO 92/01810, published Feb. 6, 1992. Such an assay may be employed,
for example, for screening for a compound which inhibits (or
enhances) activation of the receptor polypeptide of the present
invention by contacting the melanophore cells which encode the
receptor with both a TNF-family ligand and the candidate antagonist
(or agonist). Inhibition or enhancement of the signal generated by
the ligand indicates that the compound is an antagonist or agonist
of the ligand/receptor signaling pathway.
[0124] Other screening techniques include the use of cells which
express the receptor (for example, transfected CHO cells) in a
system which measures extracellular pH changes caused by receptor
activation. For example, compounds may be contacted with a cell
which expresses the receptor polypeptide of the present invention
and a second messenger response, e.g., signal transduction or pH
changes, may be measured to determine whether the potential
compound activates or inhibits the receptor.
[0125] Another such screening technique involves introducing RNA
encoding the receptor into Xenopus oocytes to transiently express
the receptor. The receptor oocytes may then be contacted with the
receptor ligand and a compound to be screened, followed by
detection of inhibition or activation of a calcium signal in the
case of screening for compounds which are thought to inhibit
activation of the receptor.
[0126] Another screening technique involves expressing in cells a
construct wherein the receptor is linked to a phospholipase C or D.
Such cells include endothelial cells, smooth muscle cells,
embryonic kidney cells, etc. The screening may be accomplished as
hereinabove described by detecting activation of the receptor or
inhibition of activation of the receptor from the phospholipase
signal.
[0127] Another method involves screening for compounds
(antagonists) which inhibit activation of the receptor polypeptide
of the present invention by determining inhibition of binding of
labeled ligand to cells which have the receptor on the surface
thereof. Such a method involves transfecting a eukaryotic cell with
DNA encoding the receptor such that the cell expresses the receptor
on its surface and contacting the cell with a compound in the
presence of a labeled form of a known ligand. The ligand can be
labeled, e.g., by radioactivity. The amount of labeled ligand bound
to the receptors is measured, e.g., by measuring radioactivity of
the receptors. If the compound binds to the receptor as determined
by a reduction of labeled ligand which binds to the receptors, the
binding of labeled ligand to the receptor is inhibited.
[0128] Further screening assays for agonist and antagonist of the
present invention are described in Tartaglia, L. A., and Goeddel,
D. V., J Biol. Chem. 267:4304-4307(1992).
[0129] Thus, in a further aspect, a screening method is provided
for determining whether a candidate agonist or antagonist is
capable of enhancing or inhibiting a cellular response to a
TNF-family ligand. The method involves contacting cells which
express the DR5 polypeptide with a candidate compound and a
TNF-family ligand, assaying a cellular response, and comparing the
cellular response to a standard cellular response, the standard
being assayed when contact is made with the ligand in absence of
the candidate compound, whereby an increased cellular response over
the standard indicates that the candidate compound is an agonist of
the ligand/receptor signaling pathway and a decreased cellular
response compared to the standard indicates that the candidate
compound is an antagonist of the ligand/receptor signaling pathway.
By "assaying a cellular response" is intended qualitatively or
quantitatively measuring a cellular response to a candidate
compound and/or a TNF-family ligand (erg., determining or
estimating an increase or decrease in T cell proliferation or
tritiated thymidine labeling). By the invention, a cell expressing
the DR5 polypeptide can be contacted with either an endogenous or
exogenously administered TNF-family ligand.
[0130] Agonist according to the present invention include naturally
occurring and synthetic compounds such as, for example, TNF family
ligand peptide fragments, transforming growth factor,
neurotransmitters (such as glutamate, dopamine,
N-methyl-D-aspartate), tumor suppressors (p53), cytolytic T cells
and antimetabolites. Preferred agonist s include-chemotherapeutic
drugs such as, for example, cisplatin, doxorubicin, bleomycin,
cytosine arabinoside, nitrogen mustard, methotrexate and
vincristine. Others include ethanol and -amyloid peptide. (Science
267:1457-1458 (1995)). Further preferred agonist include polyclonal
and monoclonal antibodies raised against the DR5 polypeptide, or a
fragment thereof. Such agonist antibodies raised against a
TNF-family receptor are disclosed in Tartaglia, L. A., et al.,
Proc. Natl. Acad. Sci. USA 88:9292-9296 (1991); and Tartaglia, L.
A., and Goeddel, D. V., J Biol. Chem. 267 (7):4304-4307 (1992) See,
also, PCT Application WO 94/09137.
[0131] Antagonist according to the present invention include
naturally occurring and synthetic compounds such as, for example,
the CD40 ligand, neutral amino acids, zinc, estrogen, androgens,
viral genes (such as Adenovirus ElB, Baculovirus p35 and LAP,
Cowpox virus crmA, Epstein-Barr virus BHRF1, LMP-1, African swine
fever virus LMW5-HL, and Herpesvirus yl 34.5), calpain inhibitors,
cysteine protease inhibitors, and tumor promoters (such as PMA,
Phenobarbital, and alpha-Hexachlorocyclohexane).
[0132] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed, for example, in Okano, J. Neurochem.
56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of
Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix
formation is discussed in, for instance Lee et al., Nucleic Acids
Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and
Dervan et al., Science 251:1360 (1991). The methods are based on
binding of a polynucleotide to a complementary DNA or RNA.
[0133] For example, the 5' coding portion of a polynucleotide that
encodes the mature polypeptide of the present invention may be used
to design an antisense RNA oligonucleotide of from about 10 to 40
base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
thereby preventing transcription and the production of the
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA
in vivo and blocks translation of the mRNA molecule into receptor
polypeptide.
[0134] In one embodiment, the DR5 antisense nucleic acid of the
invention is produced intracellularly by transcription from an
exogenous sequence. For example, a vector or a portion thereof, is
transcribed, producing an antisense nucleic acid (RNA) of the
invention. Such a vector would contain a sequence encoding the DR5
antisense nucleic acid. Such a vector can remain episomal or become
chromosomally integrated, as long as it can be transcribed to
produce the desired antisense RNA. Such vectors can be constructed
by recombinant DNA technology methods standard in the art. Vectors
can be plasmid, viral, or others know in the art, used for
replication and expression in vertebrate cells. Expression of the
sequence encoding DR5, or fragments thereof, can be by any promoter
known in the art to act in vertebrate, preferably human cells. Such
promoters can be inducible or a constitutive. Such promoters
include, but are not limited to, the SV40 early promoter region
(Bernoist and Chambon, Nature 29:304-310 (1981), the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus
(Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidine
promoter (Wagner et al., Proc. Natl. Acad Sci. U.S.A. 78:1441-1445
(1981), the regulatory sequences of the metallothionein gene
(Brinster, et al., Nature 296:39-42 (1982)), etc.
[0135] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a DR5 gene. However, absolute complementarity, although
preferred, is not required. A sequence "complementary to at least a
portion of an RNA," referred to herein, means a sequence having
sufficient complementarity to be able to hybridize with the RNA,
forming a stable duplex; in the case of double stranded DR5
antisense nucleic acids, a single strand of the duplex DNA may thus
be tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the
length of the antisense nucleic acid. Generally, the larger the
hybridizing nucleic acid, the more base mismatches with a DR5 RNA
it may contain and still form a stable duplex (or triplex as the
case may be). One skilled in the art can ascertain a tolerable
degree of mismatch by use of standard procedures to determine the
melting point of the hybridized complex.
[0136] Potential antagonists acccording to the invention also
include catalytic RNA, or a ribozyme (See, e.g., PCT International
Publication WO 90/11364, published Oct. 4, 1990; Sarver et al.,
Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at
site specific recognition sequences can be used to destroy DR5
mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead
ribozymes cleave mRNAs at locations dictated by flanking regions
that form complementary base pairs with the target mRNA. The sole
requirement is that the target mRNA have the following sequence of
two bases: 5'-UG-3'. The construction and production of hammerhead
ribozymes is well known in the art and is described more fully in
Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous
potential hammerhead ribozyme cleavage sites within the nucleotide
sequence of DR5 (FIG. 1). Preferrably, the ribozyme is engineered
so that the cleavage recognition site is located near the 5' end of
the DR-5 mRNA; i.e., to increase efficiency and minimize the
intracellular accumulation of non-functional mRNA transcripts. DNA
constructs encoding the ribozyme may be introduced into the cell in
the same manner as described above for the introduction of
antisense encoding DNA. Since ribozymes, unlike antisense molecules
are catalytic, a lower intracellular concentration is required for
efficiency.
[0137] Further antagonist according to the present invention
include soluble forms of DR5, i.e., DR5 fragments that include the
ligand binding domain from the extracellular region of the full
length receptor. Such soluble forms of the receptor, which may be
naturally occurring or synthetic, antagonize DR5 mediated signaling
by competing with the cell surface DR5 for binding to TNF-family
ligands. Thus, soluble forms of the receptor that include the
ligand binding domain are novel cytokines capable of inhibiting
apoptosis induced by TNF-family ligands. These may be expressed as
monomers, but, are preferably expressed as dimers or trimers, since
these have been shown to be superior to monomeric forms of soluble
receptor as antagonists, e.g., IgGFc-TNF receptor family fusions.
Other such cytokines are known in the art and include Fas B (a
soluble form of the mouse Fas receptor) that acts physiologically
to limit apoptosis induced by Fas ligand (Hughes, D. P. and Crispe,
I. N., J Exp. Med. 182:1395-1401 (1995)).
[0138] The term "antibody" (Ab) or "monoclonal antibody" (mAb) as
used herein is meant to include intact molecules as well as
fragments thereof (such as, for example, Fab, and F(ab').sub.2
fragments) which are capable of binding an antigen. Fab, Fab', and
F (ab').sub.2 fragments lack the Fc fragment of intact antibody,
clear more rapidly from the circulation, and may have less
non-specific tissue binding of an intact antibody (Wahl et al., J.
Nucl. Med. 24:316-325 (1983)).
[0139] Antibodies according to the present invention may be
prepared by any of a variety of standard methods using DR5
immunogens of the present invention. As indicated, such DR5
immunogens include the full length DR5 polypeptide (which may or
may not include the leader sequence) and DR5 polypeptide fragments
such as the ligand binding domain, the transmembrane domain, the
intracellular domain and the death domain.
[0140] Antibodies of the invention can be used in methods known in
the art relating to the localization and activity of the
polypeptide sequences of the invention, e.g., for imaging these
polypeptides, measuring levels thereof in appropriate physiological
samples, etc. The antibodies also have use in immunoassays and in
therapeutics as agonists and antagonists of DR5.
[0141] Proteins and other compounds which bind the DR5 domains are
also candidate agonist and antagonist according to the present
invention. Such binding compounds can be "captured" using the yeast
two-hybrid system (Fields and Song, Nature 340:245-246 (1989)). A
modified version of the yeast two-hybrid system has been described
by Roger Brent and his colleagues (Gyuris, J. et al., Cell
75:791-803 (1993); Zervos, A. S. et al., Cell 72:223-232 (1993)).
Preferably, the yeast two-hybrid system is used according to the
present invention to capture compounds which bind to either the DR5
ligand binding domain or to the DR5 intracellular domain. Such
compounds are good candidate agonist and antagonist of the present
invention.
[0142] By a "TNF-family ligand" is intended naturally occurring,
recombinant, and synthetic ligands that are capable of binding to a
member of the TNF receptor family and inducing the ligand/receptor
signaling pathway. Members of the TNF ligand family include, but
are not limited to, DR5 ligands, TRAIL, TNF-.alpha.,
lymphotoxin-.alpha. (LT-.alpha., also known as TNF-.beta.),
LT-.beta. (found in complex heterotrimer LT-.alpha.2-.beta.), FasL,
CD40, CD27, CD30, 4-1BB, OX40 and nerve growth factor (NGF). An
example of an assay that can be performed to determine the ability
of DR5 and derivatives (including fragments) and analogs thereof to
bind TRAIL is described below in Example 6.
[0143] Representative therapeutic applications of the present
invention are discussed in-more detail below. The state of
immunodeficiency that defines AIDS is secondary to a decrease in
the number and function of CD4.sup.+ T-lymphocytes. Recent reports
estimate the daily loss of CD4.sup.+ T cells to be between
3.5.times.10.sup.7 and 2.times.10.sup.9 cells (Wei X. et al.,
Nature 373:117-122 (1995)). One cause of CD4.sup.+ T cell depletion
in the setting of HIV infection is believed to be HIV-induced
apoptosis. Indeed, HIV-induced apoptotic cell death has been
demonstrated not only in vitro but also, more importantly, in
infected individuals (Ameisen, J. C., AIDS 8:1197-1213 (1994)
Finkel, T. H., and Banda, N. K., Curr. Opin. Immunol.
6:605-615(1995); Muro-Cacho, C. A. et al., J. Immunol 154:5555-5566
(1995)). Furthermore, apoptosis and CD4.sup.+ T-lymphocyte
depletion is tightly correlated in different animal models of AIDS
(Brunner, T., et al., Nature 373:441-444 (1995); Gougeon, M. L., et
al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and, apoptosis
is not observed in those animal models in which viral replication
does not result in AIDS (Gougeon, M. L. et al., AIDS Res. Hum.
Retroviruses 9:553-563 (1993)). Further data indicates that
uninfected but primed or activated T lymphocytes from HIV-infected
individuals undergo apoptosis after encountering the TNF-family
ligand FasL. Using monocytic cell lines that result in death
following HIV infection, it has been demonstrated that infection of
U937 cells with HIV results in the de novo expression of FasL and
that FasL mediates HIV-induced apoptosis (Badley, A. D. et al., J
Virol. 70:199-206 (1996)). Further the TNF-family ligand was
detectable in uninfected macrophages and its expression was
upregulated following HIV infection resulting in selective killing
of uninfected CD4 T-lymphocytes (Badley, A. D et al., J. Virol.
70:199-206 (1996)). Thus, by the invention, a method for treating
HIV.sup.+individuals is provided which involves administering an
antagonist of the present invention to reduce selective killing of
CD4 T-lymphocytes. Modes of administration and dosages are
discussed in detail below.
[0144] In rejection of an allograft, the immune system of the
recipient animal has not previously been primed to respond because
the immune system for the most part is only primed by environmental
antigens. Tissues from other members of the same species have not
been presented in the same way that, for example, viruses and
bacteria have been presented. In the case of allograft rejection,
immunosuppressive regimens are designed to prevent the immune
system from reaching the effector stage. However, the immune
profile of xenograft rejection may resemble disease recurrence more
than allograft rejection. In the case of disease recurrence, the
immune system has already been activated, as evidenced by
destruction of the native islet cells. Therefore, in disease
recurrence the immune system is already at the effector stage.
Agonists of the present invention are able to suppress the immune
response to both allografts and xenografts because lymphocytes
activated and differentiated into effector cells will express the
DR5 polypeptide, and thereby are susceptible to compounds which
enhance apoptosis. Thus, the present invention further provides a
method for creating immune privileged tissues.
[0145] DR5 antagonists may be useful for treating inflammatory
diseases, such as rheumatoid arthritis, osteoarthritis, psoriasis,
septicemia, and inflammatory bowel disease.
[0146] In addition, due to lymphoblast expression of DR5, soluble
DR5 agonist or antagonist mABs may be used to treat this form of
cancer. Further, soluble DR5 or neutralizing mABs may be used to
treat various chronic and acute forms of inflammation such as
rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, and
inflammatory bowel disease.
[0147] Modes of Administration
[0148] The agonist or antagonists described herein can be
administered in vitro, ex vivo, or in vivo to cells which express
the receptor of the present invention. By administration of an
"effective amount" of an agonist or antagonist is intended an
amount of the compound that is sufficient to enhance or inhibit a
cellular response to a TNF-family ligand and include polypeptides.
In particular, by administration of an "effective amount" of an
agonist or antagonists is intended an amount effective to enhance
or inhibit DR5 mediated apoptosis. Of course, where it is desired
for apoptosis is to be enhanced, an agonist according to the
present invention can be co-administered with a TNF-family ligand.
One of ordinary skill will appreciate that effective amounts of an
agonist or antagonist can be determined empirically and may be
employed in pure form or in pharmaceutically acceptable salt, ester
or prodrug form. The agonist or antagonist may be administered in
compositions in combination with one or more pharmaceutically
acceptable excipients (i.e., carriers).
[0149] It will be understood that, when administered to a human
patient, the total daily usage of the compounds and compositions of
the present invention will be decided by the attending physician
within the scope of sound medical judgement. The specific
therapeutically effective dose level for any particular patient
will depend upon factors well known in the medical arts.
[0150] As a general proposition, the total pharmaceutically
effective amount of DR5 polypeptide administered parenterally per
dose will be in the range of about 1 .mu.g/kg/day to 10 mg/kg/day
of patient body weight, although, as noted above, this will be
subject to therapeutic discretion. More preferably, this dose is at
least 0.01 mg/kg/day, and most preferably for humans between about
0.01 and 1 mg/kg/day for the hormone. If given continuously, the
DR5 agonists or antagonists is typically administered at a dose
rate of about 1 .mu.g/kg/hour to about 50 .mu.g/kg/hour, either by
1-4 injections per day or by continuous subcutaneous infusions, for
example, using a mini-pump. An intravenous bag solution may also be
employed.
[0151] Dosaging may also be arranged in a patient specific manner
to provide a predetermined concentration of an agonist or
antagonist in the blood, as determined by the RIA technique. Thus
patient dosaging may be adjusted to achieve regular on-going trough
blood levels, as measured by RIA, on the order of from 50 to 1000
ng/ml, preferably 150 to 500 ng/ml.
[0152] Pharmaceutical compositions are provided comprising an
agonist or antagonist (including DR5 polynucleotides and
polypeptides of the invention) and a pharmaceutically acceptable
carrier or excipient, which may be administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, drops or transdermal patch),
bucally, or as an oral or nasal spray. Importantly, by
co-administering an agonist and a TNF-family ligand, clinical side
effects can be reduced by using lower doses of both the ligand and
the agonist. It will be understood that the agonist can be
"co-administered" either before, after, or simultaneously with the
TNF-family ligand, depending on the exigencies of a particular
therapeutic application. By "pharmaceutically acceptable carrier"
is meant a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. In a
specific embodiment, "pharmaceutically acceptable" means approved
by a regulatory agency of the federal or a state government or
listed in the U.S. Pharmacopeia or other generally recognized
pharmacopeia for use in animals, and more particularly humans.
Nonlimiting examples of suitable pharmaceutical carriers according
to this embodiment are provided in "Remington's Pharmaceutical
Sciences" by E. W. Martin, and include sterile liquids, such as
water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glyceral solutions can be
employed as liquid carriers, particularly for injectable
solutions.
[0153] The term "parenteral" as used herein refers to modes of
administration which include intravenous, intramuscular,
intraperitoneal, intrasternal, subcutaneous and intraarticular
injection and infusion.
[0154] Pharmaceutical compositions of the present invention for
parenteral injection can comprise pharmaceutically acceptable
sterile aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions as well as sterile powders for reconstitution into
sterile injectable solutions or dispersions just prior to use.
[0155] In addition to soluble DR5 polypeptides, DR5 polypeptide
containing the transmembrane region can also be used when
appropriately solubilized by including detergents, such as CHAPS or
NP-40, with buffer.
[0156] Chromosome assays
[0157] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0158] In certain preferred embodiments in this regard, the cDNA
and/or polynucleotides herein disclosed is used to clone genomic
DNA of a DR5 gene. This can be accomplished using a variety of well
known techniques and libraries, which generally are available
commercially. The genomic DNA is then used for in situ chromosome
mapping using well known techniques for this purpose.
[0159] In addition, sequences can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer
analysis of the 3' untranslated region of the gene is used to
rapidly select primers that do not span more than one exon in the
genomic DNA, thus complicating the amplification process. These
primers are then used for PCR screening of somatic cell hybrids
containing individual human chromosomes.
[0160] Fluorescence in situ hybridization ("FISH") of a cDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 50 or 60 bp. For a review of this technique, see
Verma et al., Human Chromosomes: a Manual of Basic Techniques,
Pergamon Press, New York (1988).
[0161] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available on
line through Johns Hopkins University, Welch Medical Library. The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0162] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0163] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLE 1
[0164] Expression and Purification in E. coli
[0165] The DNA sequence encoding the mature DR5 protein in the
deposited cDNA clone (ATCC No. 97920) is amplified using PCR
oligonucleotide primers specific to the amino terminal sequences of
the DR5 protein and to vector sequences 3' to the gene. Additional
nucleotides containing restriction sites to facilitate cloning are
added to the 5' and 3' sequences respectively.
[0166] The following primers are used for expression of DR5
extracellular domain in E. coli: The 5' primer has the sequence
5'-CGCCCATGGAGTCT GCTCTGATCAC-3' (SEQ ID NO: 8) and contains the
underlined NcoI site; and the 3' primer has the sequence
5'-CGCAAGCTTTTAGCCTGATTC TTTGTGGAC-3' (SEQ ID NO: 9) and contains
the underlined HindIII site.
[0167] The restriction sites are convenient to restriction enzyme
sites in the bacterial expression vector pQE60, which are used for
bacterial expression in this example. (Qiagen, Inc. 9259 Eton
Avenue, Chatsworth, Calif., 9131 1). pQE60 encodes ampicillin
antibiotic resistance ("Amp") and contains a bacterial origin of
replication ("ori"), an IPTG inducible promoter, and a ribosome
binding site ("RBS").
[0168] The amplified DR5 DNA and the vector pQE60 both are digested
with NcoI and HindIII and the digested DNAs are then ligated
together. Insertion of the DR5 protein DNA into the restricted
pQE60 vector places the DR5 protein coding region downstream of and
operably linked to the vector's IPTG-inducible promoter and
in-frame with an initiating AUG appropriately positioned for
translation of DR5 protein.
[0169] The ligation mixture is transformed into competent E. coli
cells using standard procedures. Such procedures are described in
Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses lac repressor and confers kanamycin
resistance ("Kan"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing DR5 protein, is available
commercially from Qiagen, supra.
[0170] Transformants are identified by their ability to grow on LB
plates in the presence of ampicillin and kanamycin. Plasmid DNA is
isolated from resistant colonies and the identity of the cloned DNA
confined by restriction analysis, PCR, and DNA sequencing.
[0171] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:100 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-B-D-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from lac
repressor sensitive promoters, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours.
[0172] Cells then are harvested by centrifugation and disrupted, by
standard methods. Inclusion bodies are purified from the disrupted
cells using routine collection techniques, and protein is
solubilized from the inclusion bodies into 8 M urea. The 8 M urea
solution containing the solubilized protein is passed over a PD-10
column in 2.times. phosphate-buffered saline ("PBS"), thereby
removing the urea, exchanging the buffer and refolding the protein.
The protein is purified by a further step of chromatography to
remove endotoxin. Then, it is sterile filtered. The sterile
filtered protein preparation is stored in 2.times. PBS at a
concentration of 95 .mu./ml.
EXAMPLE 2
[0173] Expression in Mammalian Cells
[0174] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular signals
can also be used (e.g. the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pSVL and pMSG (Pharmacia,
Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and
pBC12MI (ATCC67109). Mammalian host cells that could be used
include, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127
cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and
Chinese hamster ovary (CHO) cells.
[0175] Alternatively, the gene of interest can be expressed in
stable cell lines that contain the gene integrated into a
chromosome. Co-transfection with a selectable marker such as dhfr,
gpt, neomycin, hygromycin allows the identification and isolation
of the transfected cells.
[0176] The transfected gene can also be amplified to express large
amounts of the encoded protein. The dihydrofolate reductase (DHFR)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy et al., Biochem. J. 227:277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) cells are often used for the production of
proteins.
[0177] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology 5:438447 (March 1985)), plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g. with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
[0178] Cloning and Expression in CHO Cells
[0179] The vector pC4 is used for the expression of the DR5
polypeptide. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr
(ATCC Accession No. 37146). The plasmid contains the mouse DHFR
gene under control of the SV40 early promoter. Chinese hamster
ovary- or other cells lacking dihydrofolate activity that are
transfected with these plasmids, can be selected by growing the
cells in a selective medium (alpha minus MEM, Life Technologies)
supplemented with the chemotherapeutic agent methotrexate (MTX).
The amplification of the DHFR genes in cells resistant to
methotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,
Kellems, R. M., Bertino, J. R., and Schimke, R. T., J. Biol. Chem.
253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et
Biophys. Acta 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.
1991, Biotechnology 9:64-68(1991)). Cells grown in increasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach may be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained which contain
the amplified gene integrated into one or more chromosome(s) of the
host cell.
[0180] Plasmid pC4 contains, for expressing the gene of interest,
the strong promoter of the long terminal repeat (LTR) of the Rous
Sarcoma Virus (Cullen et al., Molecular and Cellular Biology
5:438-447(March 1985), plus a fragment isolated from the enhancer
of the immediate early gene of human cytomegalovirus (CMV) (Boshart
et al., Cell 41:521-530 (1985)). Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the
integration of the genes: BamHI, Xba I, and Asp718. Behind these
cloning sites the plasmid contains the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high
efficiency promoters can also be used for expression, e.g., the
human .beta.-actin promoter, the SV40 early or late promoters or
the long terminal repeats from other retroviruses, e.g., HIV and
HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and
similar systems can be used to express the DR5 polypeptide in a
regulated way in mammalian cells (Gossen, M., & Bujard, H.,
Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992). For the
polyadenylation of the mRNA, other signals, e.g., from the human
growth hormone or globin genes, can be used as well.
[0181] Stable cell lines carrying a gene of interest integrated
into the chromosomes can also be selected upon co-transfection with
a selectable marker such as gpt, G418, or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
[0182] The plasmid pC4 is digested with the restriction enzyme
BamHI and then dephosphorylated using calf intestinal phosphates by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[0183] The DNA sequence encoding the complete polypeptide is
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the desired portion of the gene. The 5' primer
containing the underlined BamHI site, a Kozak sequence, and an AUG
start codon, has the following sequence:
5'-CGCGGATCCGCCATCATGGAACAACGGGGACAGAAC-3' (SEQ ID NO: 10). The 3'
primer, containing the underlined Asp718 site, has the following
sequence: 5'-CGCGGTACCTTAGGACATGGCAGAGTC-3' (SEQ ID NO: 11).
[0184] The amplified fragment is digested with the endonuclease
BamHI and Asp718 and then purified again on a 1% agarose gel. The
isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment
inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
[0185] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using the
lipofectin method (Felgner et al., supra). The plasmid pSV2-neo
contains a dominant selectable marker, the neo gene from Tn5
encoding an enzyme that confers resistance to a group of
antibiotics including G418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml
of metothrexate plus 1 mg/ml G418. After about 10-14 days, single
clones are trypsinized and then seeded in 6-well petri dishes or 10
ml flasks using different concentrations of methotrexate (50 nM,
100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new 6-well
plates containing even higher concentrations of methotrexate (1
.mu.M, 2 .mu.M, 5 .mu.M, 10 mM, 20 mM). The same procedure is
repeated until clones are obtained which grow at a concentration of
100-200 .mu.M. Expression of the desired gene product is analyzed,
for instance, by SDS-PAGE and Western blot or by reversed phase
HPLC analysis.
[0186] Cloning and Expression in COS Cells
[0187] The expression plasmid, pDR5-HA, is made by cloning a cDNA
encoding the soluble extracellular domain of the DR5 protein into
the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained
from Invitrogen, Inc.). The expression vector pcDNAI/amp contains:
(1) an E. coli origin of replication effective for propagation in
E. coli and other prokaryotic cells; (2) an ampicillin resistance
gene for selection of plasmid-containing prokaryotic cells; (3) an
SV40 origin of replication for propagation in eukaryotic cells; (4)
a CMV promoter, a polylinker, an SV40 and a polyadenylation signal
arranged so that a cDNA can be conveniently placed under expression
control of the CMV promoter and operably linked to the SV40 intron
and the polyadenylation signal by means of restriction sites in the
polylinker. A DNA fragment encoding the extracelluar domain of the
DR5 polypeptide and a HA tag fused in frame to its 3' end is cloned
into the polylinker region of the vector so that recombinant
protein expression is directed by the CMV promoter. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein described by Wilson et al., Cell 37: 767 (1984). The fusion
of the HA tag to the target protein allows easy detection and
recovery of the recombinant protein with an antibody that
recognizes the HA epitope.
[0188] The plasmid construction strategy is as follows. The DR5
cDNA of the deposited clone is amplified using primers that contain
convenient restriction sites, much as described above for
construction of vectors for expression of DR5 in E coli. To
facilitate detection, purification and characterization of the
expressed DR5, one of the primers contains a hemagglutinin tag ("HA
tag") as described above.
[0189] Suitable primers include the following, which are used in
this example. The 5' primer, containing the underlined BamHI site
has the following sequence:
5'-CGCGGATCCGCCATCATGGAACAACGGGGACAGAAC-3' (SEQ ID NO: 10). The 3'
primer, containing the underlined Asp718 restriction sequence has
the following sequence:5'-CGCGGTACCTTAGCCTGATTCTTTTGGAC-3' (SEQ ID
NO: 12).
[0190] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with BamHI and Asp718 and then ligated. The ligation
mixture is transformed into E. coli strain SURE (available from
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La
Jolla, Calif. 92037), and the transformed culture is plated on
ampicillin media plates which then are incubated to allow growth of
ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis or other
means for the presence of the fragment encoding the extracellular
domain of the DR5 polypeptide
[0191] For expression of recombinant DR5, COS cells are transfected
with an expression vector, as described above, using DEAE-DEXTRAN,
as described, for instance, in Sambrook et al., Molecular Cloning:
a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring
Harbor, New York (1989). Cells are incubated under conditions for
expression of DR5 by the vector.
[0192] Expression of the DR5-HA fusion protein is detected by
radiolabeling and immunoprecipitation, using methods described in,
for example Harlow et al., Antibodies: A Laboratory Manual, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York (1988). To this end, two days after transfection, the cells
are labeled by incubation in media containing .sup.35S-cysteine for
8 hours. The cells and the media are collected, and the cells are
washed land the lysed with detergent-containing RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5,
as described by Wilson et al., cited above. Proteins are
precipitated from the cell lysate and from the culture media using
an HA-specific monoclonal antibody. The precipitated proteins then
are analyzed by SDS-PAGE and autoradiography. An expression product
of the expected size is seen in the cell lysate, which is not seen
in negative controls.
[0193] The primer sets used for expression in this example are
compatible with pC4 used for CHO expression in this example,
pcDNAI/Amp for COS expression in this example, and pA2 used for
baculovirus expression in the following example. Thus, for example,
the complete DR5 encoding fragment amplified for CHO expression
could also be ligated into pcDNAI/Amp for COS expression or pA2 for
baculovirus expression.
EXAMPLE 3
[0194] Cloning and expression of the soluble extracellular domain
of DR5 in a baculovirus expression system
[0195] In this illustrative example, the plasmid shuttle vector pA2
is used to insert the cloned DNA encoding the complete protein,
including its naturally associated signal sequence, into a
baculovirus to express the DR5 protein, using standard methods,
such as those described in Summers et al., A Manual of Methods for
Baculovirus Vectors and Insect Cell Culture Procedures, Texas
Agricultural Experimental Station Bulletin No. 1555 (1987). This
expression vector contains the strong polyhedron promoter of the
Autograph californica nuclear polyhedrosis virus (ACMNPV) followed
by convenient restriction sites. For easy selection of recombinant
virus, the plasmid contains the beta-galactosidase gene from E.
coli under control of a weak Drosophila promoter in the same
orientation, followed by the polyadenylation signal of the
polyhedrin gene. The inserted genes are flanked on both sides by
viral sequences for cell-mediated homologous recombination with
wild-type viral DNA to generate viable virus that express the
cloned polynucleotide. Many other baculovirus vectors could be used
in place of pA2, such as pAc373, pVL941 and pAcIM1 provided, as one
skilled in the art would readily appreciate, that construction
provides appropriately located signals for transcription,
translation, secretion, and the like, such as an in-frame AUG and a
signal peptide, as required. Such vectors are described, for
example, in Luckow et al., Virology 170:31-39 (1989).
[0196] The cDNA sequence encoding the soluble extracellular domain
of DR5 protein in the deposited clone (ATCC No. 97920) is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3'
sequences of the gene:
[0197] The 5' primer for DR5 has the sequence
5'-CGCGGATCCGCCATCATGGA ACAACGGGGACAGAAC-3' (SEQ ID NO: 10)
containing the underlined BamHI restriction enzyme site. Inserted
into an expression vector, as described below, the 5' end of the
amplified fragment encoding DR5 provides an efficient cleavage
signal peptide. An efficient signal for initiation of translation
in eukaryotic cells, as described by Kozak, M., J. Mol. Biol.
196:947-950 (1987) is appropriately located in the vector portion
of the construct.
[0198] The 3' primer for DR5 has the sequence 5'-CGCGGTACCTTAGCCT
GATTCTTTGTGGAC-3' (SEQ ID NO: 12) containing the underlined Asp718
restriction followed by nucleotides complementary to the DR5
nucleotide sequence in FIG. 1, followed by the stop codon.
[0199] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.) The fragment then is digested with BamHI and Asp718
and again is purified on a 1% agarose gel. This fragment is
designated "F1."
[0200] The plasmid is digested with the restriction enzymes Bam HI
and Asp718 and optionally can be dephosphorylated using calf
intestinal phosphatase, using routine procedures known in the art.
The DNA is then isolated from a 1% agarose gel using a commercially
available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). The
vector DNA is designated herein "V1."
[0201] Fragment F1 and the dephosphorylated plasmid V1 are ligated
together with T4 DNA ligase. E. coli I B101 cells, or other
suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning
Systems, La Jolla, Calif.) cells, are transformed with the ligation
mixture and spread on culture plates. Bacteria are identified that
contain the plasmid with the human DR5 using the PCR method, in
which one of the primers that is used to amplify the gene and the
second primer is from well within the vector so that only those
bacterial colonies containing the DR5 gene fragment will show
amplification of the DNA. The sequence of the cloned fragment is
confirmed by DNA sequencing. This plasmid is designated herein pBac
DR5.
[0202] 5 .mu.g of the plasmid pBac DR5 is co-transfected with 1.0
.mu.g of a commercially available linearized baculovirus DNA
("BaculoGold.TM. baculovirus DNA", Pharmingen, San Diego, Calif.),
using the lipofectin method described by Felgner et al., Proc.
Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 .mu.g of BaculoGold.TM.
virus DNA and 5 .mu.g of the plasmid pBac DR5 are mixed in a
sterile well of a microliter plate containing 50 .mu.l of serum
free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).
Afterwards 10 .mu.l Lipofectin plus 90 .mu.l Grace's medium are
added, mixed and incubated for 15 minutes at room temperature. Then
the transfection mixture is added drop-wise to Sf9 insect cells
(ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml
Grace's medium without serum. The plate is rocked back and forth to
mix the newly added solution. The plate is then incubated for 5
hours at 27.degree. C. After 5 hours, the transfection solution is
removed from the plate and 1 ml of Grace's insect medium
supplemented with 10% fetal calf serum is added. The plate is put
back into an incubator and cultivation is continued at 27.degree.
C. for four days.
[0203] After four days ,the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, cited above.
An agarose gel with "Blue Gal" (Life Technologies Inc.,
Gaithersburg, Md.) is used to allow easy identification and
isolation of gal-expressing clones, which produce blue-stained
plaques. (A detailed description of a "plaque assay" of this type
can also be found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
Md., pages 9-10). After appropriate incubation, blue stained
plaques are picked with the tip of a micropipettor (e.g,
Eppendorf). The agar containing the recombinant viruses is then
resuspended in a microcentrifuige tube containing 200 .mu.l of
Grace's medium and the suspension containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later, the supernatants of these culture dishes are
harvested-and then they are stored at 4.degree. C. The recombinant
virus is called V-DR5.
[0204] To verify expression of the DR5 gene, Sf9 cells are grown in
Grace's medium supplemented with 10% heat-inactivated FBS. The
cells are infected with the recombinant baculovirus V-DR5 at a
multiplicity of infection ("MOI") of about 2 (about 1 to about 3).
Six hours later, the medium is removed and is replaced with SF900
II medium minus methionine and cysteine (available from Life
Technologies Inc., Gaithersburg, Md.). If radiolabeled proteins are
desired, 42 hours later, 5 .mu.Ci of .sup.35S-methionine and 5
.mu.Ci .sup.35S-cysteine (available from Amersham) are added. The
cells are further incubated for 16 hours and then they are
harvested by centrifugation. The proteins in the supernatant as
well as the intracellular proteins are analyzed by SDS-PAGE
followed by autoradiography (if radiolabeled). Microsequencing of
the amino acid sequence of the amino terminus of purified protein
may be used to determine the amino terminal sequence of the mature
protein and thus the cleavage point and length of the secretory
signal peptide.
EXAMPLE 4
[0205] Tissue distribution of DR5 gene expression
[0206] Northern blot analysis was carried out to examine DR5 gene
expression in human tissues, using methods described by, among
others, Sambrook et al., cited above. A CDNA probe containing the
entire nucleotide sequence of the DR5 protein (SEQ ID NO: 1) was
labeled with .sup.32P using the rediprime.TM. DNA labeling system
(Amersham Life Science), according to manufacturer's instructions.
After labeling, the probe was purified using a CHROMA SPIN-100.TM.
column (Clontech Laboratories, Inc.), according to manufacturer's
protocol number PT1200-1. The purified labeled probe was then used
to examine various human tissues for DR5 mRNA.
[0207] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) were obtained
from Clontech (Palo Alto, Calif.) and examined with labeled probe
using ExpressHyb.TM. hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization
and washing, the blots were mounted and exposed to film at
-70.degree. C. overnight. The films were developed according to
standard procedures. Expression of DR5 was detected in heart,
brain, placenta, lung, liver, skeletal muscle, kidney, pancreas,
spleen, thymus, prostate, testis, uterus, small intestine, colon,
peripheral blood leukocytes (PBLs), lymph node, bone marrow, and
fetal liver.
[0208] Expression of DR5 was also assessed by Northern blot in the
following cancer cell lines, HL60 (promyelocytic leukemia), Hela
cell S3, K562 (chronic myelogeneous leukemia), MOLT4 (lymphoblast
leukemia), Raji (Burkitt's lymphoma), SW480 (colorectal
adenocarcinoma), A549 (lung carcinoma), and G361 (melanoma), and
was detected in all of the cell lines tested.
EXAMPLE 5
[0209] DR5 Induced Apoptosis in Mammalian Cells
[0210] Overexpression of Fas/APO-1 and TNFR-1 in mammalian cells
mimics receptor activation (M. Muzio et al., Cell 85: 817-827
(1996); M. P. Boldin et al., Cell 85:803-815 (1996)). Thus, this
system was utilized to study the functional role of DR5 in inducing
apoptosis. This example demonstrates that overexpression of DR5
induced apoptosis in both MCF7 human breast carcinoma cells and in
human epitheloid carcinoma (Hela ) cells.
[0211] Experimental Design
[0212] Cell death assays were performed essentially as previously
described (A. M. Chinnaiyan, et al., Cell 81:505-12 (1995); M. P.
Boldin, et al., J. Biol Chem 270: 7795-8 (1995); F. C. Kischkel, et
al., EMBO 14:5579-5588 (1995); A. M. Chinnaiyan, et al., J Biol
Chem 271: 4961-4965 (1996)). Briefly, MCF-7 human breast carcinoma
clonal cell lines and Hela cells were co-transfected with vector,
DR5, DR5.DELTA. (52-411), or TNFR-1, together with a
beta-galactosidase reporter construct.
[0213] MCF7 and Hela cells were transfected using the lipofectamine
procedure (GIBCO-BRL), according to the manufacturer's
instructions. 293 cells were transfected using CaPO.sub.4
precipitation. Twenty-four hours following transfection, cells were
fixed and stained with X-Gal as previously described (A. M.
Chinnaiyan, et al., Cell 81:505-12 (1995); M. P. Boldin, et al., J
Biol Chem 270:7795-8 (1995); F. C. Kischkel, et al., EMBO
14:5579-5588 (1995)), and examined microscopically. The data
(mean.+-.SD) presented in FIG. 5 represents the percentage of
round, apoptotic cells as a function of total beta-galactosidase
positive cells (n=3). Overexpression of DR5 induced apoptosis in
both MCF7 (FIG. 5A) and Hela cells (FIG. 5B).
[0214] MCF7 cells were also transfected with a DR5 expression
construct in the presence of z-VAD-fink (20 .mu.l)(Enzyme Systems
Products, Dublin, Calif.) or co-transfected with a three-fold
excess of CrmA (M. Tewari et al., J Biol Chem 270:3255-60 (1995)),
or FADD-DN expression construct, or vector alone. The data
presented in FIG. 5C shows that apoptosis induced by DR5 was
attenuated by caspase inhibitors ,but not by dominant negative
FADD.
[0215] As depicted in FIG. 5D, DR5 did not associate with FADD or
TRADD in vivo. 293 cells were co-transfected with the indicated
expression constructs using calcium phosphate precipitation. After
transfection (at 40 hours), cell lysates were prepared and
immunoprecipitated with Flag M2 antibody affinity gel (IBI, Kodak),
and the presence of FADD or myc-tagged TRADD (myc-TRADD) was
detected by immunoblotting with polyclonal antibody to FADD or
horseradish peroxidase (HRP) conjugated antibody to myc
(BMB)(Baker, S. J. et al., Oncogene 12:1 (1996); Chinnaiyan, A. M.
et al., Science 274:990 (1996)).
[0216] As depicted in FIG. 5E, FLICE 2-DN blocks DR5-induced
apoptosis. 293 cells were co-transfected with DR5 or TNFR-1
expression construct and a fourfold excess of CrmA, FLICE-DN, FLICE
2-DN, or vector alone in the presence of a beta-galactosidase
reported construct as indicated. Cells were stained and examined
25-30 hours later.
[0217] Results
[0218] Overexpression of DR5, induced apoptosis in both MCF7 human
breast carcinoma cells (FIG. 5A) and in human epitheloid carcinoma
(Hela) cells (FIG. 5B). Most of the transfected cells displayed
morphological changes characteristic of cells undergoing apoptosis
(Earnshaw, W. C., Curr. Biol. 7:337 (1995)), becoming rounded,
condensed and detaching from the dish. Deletion of the death domain
abolished killing ability. Like DR4, DR5-induced apoptosis was
blocked by caspase inhibitors, CrmA and z-VAD-fink, but dominant
negative FADD was without effect (FIG. 5C). Consistent with this,
DR5 did not interact with FADD and TRADD in vivo (FIG. 5D). A
dominant negative version of a newly identified FLICE-like
molecule, FLICE2 (Vincenz, C. et al., J Biol. Chem. 272:6578
(1997)), efficiently blocked DR5-induced apoptosis, while dominant
negative FLICE had only partial effect under conditions it blocked.
TNFR-1 induced apoptosis effectively (FIG. 5E). Taken together, the
evidence suggests that DR5 engages an apoptotic program that
involves activation of FLICE2 and downstream caspases, but is
independent of FADD.
EXAMPLE 6
[0219] The Extracellular Domain of DR5 Binds the Cytotoxic
Ligand-TRAIL, and Blocks TRAIL-Induced apoptosis
[0220] As discussed above, TRAIL/Apo2L is a cytotoxic ligand that
belongs to the tumor necrosis factor (TNF) ligand family and
induces rapid cell death of many transformed cell lines, but not
normal tissues, despite its death domain containing receptor, DR4,
being expressed on both cell types. This example shows that the
present receptor, DR5, also binds TRAIL.
[0221] Given the similarity of the extracellular ligand binding
cysteine-rich domains of DR5 and DR4, the present inventors
theorized that DR5 would also bind TRAIL. To confirm this, the
soluble extracellular ligand binding domains of DR5 were expressed
as fusions to the Fc portion of human immunoglobulin (IgG).
[0222] As shown in FIG. 6A, DR5-Fc specifically bound TRAIL, but
not the related cytotoxic ligand TNF.alpha.. In this experiment,
the Fc-extracellular domains of DR5, DR4, TRID, or TNFR1 and the
corresponding ligands were prepared and binding assays performed as
described in Pan et al., Science 276:111 (1997). The respective
Fc-fusions were precipitated with protein G-Sepharose and
co-precipitated soluble
EXHIBIT A
[0223]
Sequence CWU 1
1
* * * * *