U.S. patent application number 10/032159 was filed with the patent office on 2002-11-07 for card-domain containing polypeptides, encoding nucleic acids, and methods of use.
Invention is credited to Godzik, Adam, Pawlowski, Krzysztof, Reed, John C..
Application Number | 20020164703 10/032159 |
Document ID | / |
Family ID | 26708055 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164703 |
Kind Code |
A1 |
Pawlowski, Krzysztof ; et
al. |
November 7, 2002 |
Card-domain containing polypeptides, encoding nucleic acids, and
methods of use
Abstract
The invention provides caspase recruitment domain
(CARD)-containing polypeptides and functional fragments thereof,
encoding nucleic acid molecules, and specific antibodies. Also
provided are screening methods for identifying CARD-associated
polypeptides (CAPs), and for identifying agents that alter the
association of a CARD-containing polypeptide with itself or with a
CAP. Further provided are methods of altering a biochemical process
modulated by a CARD-containing polypeptide, and methods of
diagnosing a pathology characterized by an increased or decreased
level of a CARD-containing polypeptide.
Inventors: |
Pawlowski, Krzysztof;
(Malmo, SE) ; Reed, John C.; (Rancho Santa Fe,
CA) ; Godzik, Adam; (San Diego, CA) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Family ID: |
26708055 |
Appl. No.: |
10/032159 |
Filed: |
December 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60257457 |
Dec 21, 2000 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/226; 435/320.1; 435/325; 530/350; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
A01K 2227/105 20130101; A01K 2217/05 20130101; C07K 14/4702
20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/226; 435/325; 530/350; 536/23.2 |
International
Class: |
C12N 009/64; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/435 |
Goverment Interests
[0002] This invention was made with United States Government
support under grant number DBI-0078731 awarded by the National
Science Foundation. The U.S. Government has certain rights in this
invention.
Claims
What is claimed is:
1. An isolated nucleic acid molecule encoding a CARD-containing
polypeptide, wherein said nucleic acid molecule is selected from
the group consisting of: (a) a nucleic acid molecule encoding a
polypeptide comprising the amino acid sequence of CARD-11X (SEQ ID
NO:8) or CARD-12X (SEQ ID NO:16); (b) a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:15;
and (c) a nucleic acid molecule that hybridizes to the nucleic acid
molecule of (a) or (b) under moderately stringent hybridization
conditions.
2. An isolated nucleic acid molecule encoding a functional fragment
of a CARD-containing polypeptide, wherein said nucleic acid
molecule is selected from the group consisting of: (a) a nucleic
acid molecule encoding the CARD domain of CARD-10X (SEQ ID NO:4),
the filament domain of CARD-10X (SEQ ID NO:6), the CARD domain of
CARD11X (SEQ ID NO:10), the ERM (ezrin) domain of CARD-11X (SEQ ID
NO:12), the PDZ domain of CARD-11X (SEQ ID NO:14) and the CARD
domain of CARD-12X (SEQ ID NO:16); (b) a nucleic acid molecule
comprising the nucleotide sequence of the CARD domain of CARD-10X
(SEQ ID NO:3), the filament domain of CARD-10X (SEQ ID NO:5), the
CARD domain of CARD-11X (SEQ ID NO:9), the ERM (ezrin) domain of
CARD-11X (SEQ ID NO:11), the PDZ domain of CARD-11X (SEQ ID NO:13)
and the CARD domain of CARD-12X (SEQ ID NO:15); and (c) a nucleic
acid molecule that hybridizes to the nucleic acid molecule of (a)
or (b) under moderately stringent hybridization conditions, wherein
said nucleic acid molecule does not consist of the nucleotide
sequence set forth as SEQ ID NOS:19 or 21-37.
3. A nucleic acid molecule comprising substantially the same
nucleotide sequence as SEQ ID NO:7 or SEQ ID NO:15.
4. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is cDNA or mRNA.
5. A vector containing the nucleic acid molecule of claim 1.
6. A cell containing the nucleic acid molecule of claim 1.
7. A composition comprising an amount of the nucleic acid molecule
according to claim 1(c) or claim 2(c) effective to inhibit
expression of a CARD-containing polypeptide, and an acceptable
hydrophobic carrier capable of passing through a cell membrane.
8. An oligonucleotide comprising at least 15 contiguous nucleotides
of the nucleotide sequence set forth in any of SEQ ID NOS:1, 7 or
15, or the complement thereof, wherein said nucleic acid molecule
does not consist of the nucleotide sequence set forth as SEQ ID
NOS:19 or 21-37.
9. The oligonucleotide of claim 8, wherein said oligonucleotide is
labeled with a detectable marker.
10. A kit for detecting the presence of a nucleic acid molecule
encoding a CARD-containing polypeptide, comprising at least one
oligonucleotide according to claim 9.
11. A substantially purified CARD-containing polypeptide,
comprising substantially the same amino acid sequence as the amino
acid sequence of CARD-11X (SEQ ID NO:8) or CARD-12X (SEQ ID
NO:16).
12. A substantially purified functional fragment of a
CARD-containing polypeptide, comprising substantially the same
amino acid sequence as the amino acid sequence of the CARD domain
of CARD-10X (SEQ ID NO:4), the filament domain of CARD-10X (SEQ ID
NO:6), the CARD domain of CARD-11X (SEQ ID NO:10), the ERM (ezrin)
domain of CARD-11X (SEQ ID NO:12), the PDZ domain of CARD-11X (SEQ
ID NO:14) and the CARD domain of CARD-12X (SEQ ID NO:16), wherein
said functional fragment is not encoded by a nucleic acid molecule
consisting of the nucleotide sequence set forth as SEQ ID NOS:19 or
21-37.
13. A substantially purified functional fragment of a
CARD-containing polypeptide, comprising at least 10 contiguous
residues of SEQ ID NOS: 2, 8 or 16, wherein said functional
fragment is not encoded by a nucleic acid molecule consisting of
the nucleotide sequence set forth as SEQ ID NOS:19 or 21-37, and
wherein said functional fragment is immunogenic.
14. A method of producing a CARD-containing polypeptide, comprising
expressing the cDNA of claim 3 in vitro or in a cell under
conditions suitable for expression of said polypeptide.
15. An isolated anti-CARD antibody having specific reactivity with
the CARD-containing polypeptide of claim 11 or 12.
16. The antibody of claim 15, wherein said antibody is a monoclonal
antibody.
17. A cell line producing the monoclonal antibody of claim 16.
18. The antibody of claim 15, wherein said antibody is a polyclonal
antibody.
19. A transgenic nonhuman mammal expressing exogenously the nucleic
acid of claim 1.
20. The transgenic nonhuman mammal of claim 19, wherein said mammal
is a mouse.
21. A method for identifying a nucleic acid molecule encoding a
CARD-containing polypeptide, said method comprising: contacting a
sample containing nucleic acids with an oligonucleotide according
to claim 8, wherein said contacting is effected under high
stringency hybridization conditions, and identifying a nucleic acid
molecule that hybridizes to said oligonucleotide.
22. A method for detecting the presence of a CARD-containing
polypeptide in a sample, said method comprising contacting a test
sample with an antibody according to claim 18, detecting the
presence of an antibody:CARD complex, and thereby detecting the
presence of a human CARD-containing polypeptide in said test
sample.
23. A method of identifying a CARD-associated polypeptide (CAP)
comprising the steps of: (a) contacting the CARD-containing
polypeptide of claim 11 or 12 with a candidate CAP; (b) determining
association of said CARD-containing polypeptide with said candidate
CAP, wherein a polypeptide that associates with said
CARD-containing polypeptide is a CAP.
24. A method of identifying an effective agent that alters the
association of a CARD-containing polypeptide with a CARD-associated
polypeptide (CAP), comprising the steps of: (a) contacting the
CARD-containing polypeptide of claim 11 or 12 and said CAP under
conditions that allow the CARD-containing polypeptide and CAP
polypeptides to associate, with an agent suspected of being able to
alter the association of the CARD-containing polypeptide and CAP
polypeptides; and (b) determining association of said
CARD-containing polypeptide with said CAP, wherein an agent that
said alters said association is identified as an effective
agent.
25. A method of altering a biochemical process modulated by a
CARD-containing polypeptide, comprising the steps of: (a)
introducing the nucleic acid of claim 1 or claim 2 into a cell; and
(b) expressing said nucleic acid in said cell, whereby the
expression of said nucleic acid alters a biochemical process
modulated by a CARD-containing polypeptide.
26. A method of diagnosing or predicting clinical prognosis of a
pathology characterized by an increased or decreased level of a
CARD-containing polypeptide in a subject, comprising the steps of:
(a) obtaining a test sample from the subject; (b) contacting said
test sample with a reagent that can bind the CARD-containing
polypeptide of claim 11 or claim 12 under suitable conditions which
allow specific binding of said reagent to said CARD-containing
polypeptide; and (c) comparing the amount of said specific binding
in said test sample with the amount of specific binding in a
reference sample, wherein an increased or decreased amount of said
specific binding in said test sample as compared to said reference
sample is diagnostic or predictive of clinical prognosis of a
pathology.
27. The method of claim 26, wherein said reagent is an anti-CARD
antibody.
28. A method of diagnosing or predicting clinical prognosis of a
pathology characterized by an increased or decreased level of a
CARD-containing polypeptide in a subject, comprising the steps of:
(a) obtaining a test sample from the subject; (b) contacting said
test sample with a reagent that can bind the CARD-containing
nucleic acid molecule of claim 1 or claim 2 under suitable
conditions which allow specific binding of said reagent to said
CARD-containing polypeptide; and (c) comparing the amount of said
specific binding in said test sample with the amount of specific
binding in a reference sample, wherein an increased or decreased
amount of said specific binding in said test sample as compared to
said reference sample is diagnostic or predictive of clinical
prognosis of a pathology.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/257,457, filed Dec. 21, 2000, which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the invention
[0004] This invention relates generally to the fields of molecular
biology and molecular medicine and more specifically to the
identification of proteins involved in programmed cell death,
cytokine processing and receptor signal transduction, and
associations of these proteins.
[0005] 2. Background Information
[0006] Programmed cell death is a physiologic process that ensures
homeostasis is maintained between cell production and cell turnover
in essentially all self-renewing tissues. In many cases,
characteristic morphological changes, termed "apoptosis," occur in
a dying cell. Since similar changes occur in different types of
dying cells, cell death appears to proceed through a common pathway
in different cell types.
[0007] In addition to maintaining tissue homeostasis, apoptosis
also occurs in response to a variety of external stimuli, including
growth factor deprivation, alterations in calcium levels,
free-radicals, cytotoxic lymphokines, infection by some viruses,
radiation and most chemotherapeutic agents. Thus, apoptosis is an
inducible event that likely is subject to similar mechanisms of
regulation as occur, for example, in a metabolic pathway. In this
regard, dysregulation of apoptosis also can occur and is observed,
for example, in some types of cancer cells, which survive for a
longer time than corresponding normal cells, and in
neurodegenerative diseases where neurons die prematurely. In viral
infections, induction of apoptosis can figure prominently in the
pathophysiology of the disease process, because immune-based for
eradication of viral infections depend on elimination of
virus-producing host cells by immune cell attack resulting in
apoptosis.
[0008] Some of the proteins involved in programmed cell death have
been identified and associations among some of these proteins have
been described. However, additional apoptosis regulating proteins
remain to be found and the mechanisms by which these proteins
mediate their activity remains to be elucidated. The identification
of the proteins involved in cell death and an understanding of the
associations between these proteins can provide a means for
manipulating the process of apoptosis in a cell and, therefore,
selectively regulating the relative lifespan of a cell or its
relative resistance to cell death stimuli.
[0009] The principal effectors of apoptosis are a family of
intracellular proteases known as Caspases, representing an
abbreviation for Cysteine Aspartyl Proteases. Caspases are found as
inactive zymogens in essentially all animal cells. During
apoptosis, the caspases are activated by proteolytic processing at
specific aspartic acid residues, resulting in the production of
subunits that assemble into an active protease typically consisting
of a heterotetramer containing two large and two small subunits.
The phenomenon of apoptosis is produced directly or indirectly by
the activation of caspases in cells, resulting in the proteolytic
cleavage of specific substrate proteins. Moreover, in many cases,
caspases can cleave and activate themselves and each other,
creating cascades of protease activation and mechanisms for
"auto"-activation. Thus, knowledge about the proteins that interact
with and regulate caspases is important for devising strategies for
manipulating cell life and death in therapeutically useful ways. In
addition, because capsases can also participate in cytokine
activation and other processes, knowledge about the proteins that
interact with caspases can be important for manipulating immune
responses and other biochemical processes in useful ways.
[0010] One of the mechanisms for regulating caspase activation
involves protein-protein interactions mediated by a family of
protein domains known as caspase recruitment domains (CARDs). The
identification of proteins that contain CARD domains and the
elucidation of the proteins with which they interact, therefore,
can form the basis for strategies designed to alter apoptosis,
cytokine production, cytokine receptor signaling, and other
cellular processes.
[0011] Thus, a need exists to identify proteins that contain CARD
domains. The present invention satisfies this need and provides
additional advantages as well.
SUMMARY OF THE INVENTION
[0012] In accordance with the present invention, there are provided
CARD-containing polypeptides, and functional fragments thereof. The
invention also provides nucleic acid molecules encoding
CARD-containing polypeptides and active fragments thereof, vectors
containing these nucleic acid molecules and host cells containing
the vectors. The invention also provides antibodies that can
specifically bind to CARD-containing polypeptides, and active
fragments thereof.
[0013] The present invention also provides a screening assay useful
for identifying CARD-associated polypeptides (CAPs), and for
identifying agents that can effectively alter the association of a
CARD-containing polypeptide with itself or with other proteins. By
altering the self-association of a CARD-containing polypeptide or
by altering its interaction with other proteins, an effective agent
may increase or decrease the level of caspase proteolytic activity
or apoptosis in a cell.
[0014] The invention also provides methods of altering a
biochemical process modulated by a CARD-containing polypeptide, by
introducing into the cell and expressing a nucleic acid sequence
encoding the polypeptide, or an antisense nucleotide sequence that
is complementary to a portion of a nucleic acid molecule encoding
the CARD-containing polypeptide. Such biochemical processes include
apoptosis, anoikis, cytoskeletal integrity, NF-.kappa.B induction,
cytokine processing, cytokine receptor signaling, and
caspase-mediated proteolysis.
[0015] The invention also provides methods for diagnosing or
prognosing a pathology characterized by an increased or decreased
level of a CARD-containing polypeptide in a cell, by contacting the
test sample with an agent that can specifically bind a
CARD-containing polypeptide or a nucleotide sequence, and
determining the amount of specific binding in the test sample
compared to a reference sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) and
predicted amino acid sequence (SEQ ID NO:2) of CARD-10X. The CARD
domain nucleotide sequence (SEQ ID NO:3) and amino acid sequence
(SEQ ID NO:4), and the Filament domain nucleotide sequence (SEQ ID
NO:5) and amino acid sequence (SEQ ID NO:6), are indicated and
underlined.
[0017] FIG. 2 shows the nucleotide sequence (SEQ ID NO:7) and
predicted amino acid sequence (SEQ ID NO:8) of CARD-11X. The CARD
domain nucleotide sequence (SEQ ID NO:9) and amino acid sequence
(SEQ ID NO:10), the ERM (ezrin) domain nucleotide sequence (SEQ ID
NO:11) and amino acid sequence (SEQ ID NO:12), and the PDZ domain
nucleotide sequence (SEQ ID NO:13) and amino acid sequence (SEQ ID
NO:14), are indicated and underlined.
[0018] FIG. 3 shows the nucleotide sequence (SEQ ID NO:15) and
predicted amino acid sequence (SEQ ID NO:16) of CARD-12X. The CARD
domain nucleotide sequence (SEQ ID NO:17) and amino acid sequence
(SEQ ID NO:18) is indicated and underlined.
[0019] FIG. 4 shows the nucleotide sequence (SEQ ID:19) and
predicted amino acid sequence (SEQ ID NO:20) of GI 10436238.
[0020] FIG. 5 shows the nucleotide sequences of CARD-10X ESTs: GI
9094656 (SEQ ID NO:21), GI 7132200 (SEQ ID NO:22) and GI 5884878
(SEQ ID NO:23).
[0021] FIG. 6 shows the nucleotide sequences of CARD-11X ESTs: GI
6926669 (SEQ ID NO:24), GI 6143407 (SEQ ID NO:25), GI 2785620 (SEQ
ID NO:26), GI 1838222 (SEQ ID NO:27), GI 6927709 (SEQ ID NO:28), GI
9720543 (SEQ ID NO:29), GI 9142863 (SEQ ID NO:30), GI 1761194 (SEQ
ID NO:31), GI 8151878 (SEQ ID NO:32), GI 2007639 (SEQ ID NO:33), GI
8042493 (SEQ ID NO:34), GI 2079290 (SEQ ID NO:35), and GI 7044777
(SEQ ID NO:36).
[0022] FIG. 7 shows the nucleotide sequence of a CARD-12X EST: GI
10316320 (SEQ ID NO:37).
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides novel polypeptides involved
in programmed cell death, or apoptosis. The principal effectors of
apoptosis are a family of intracellular cysteine aspartyl
proteases, known as caspases. Caspase activity in the cell is
regulated by protein-protein interactions. Similarly,
protein-protein interactions influence the activity of other
proteins involved in apoptosis. Several protein interaction domains
have been implicated in interactions among some
apoptosis-regulating proteins. Among these is the casapase
recruitment domain, or CARD-containing polypeptide which are so
named for the ability of the CARD-containing polypeptides to bind
caspases. In addition to their ability to bind caspases, numerous
CARD-containing polypeptides bind other proteins, including other
CARD-containing polypeptides. Further, CARD-containing polypeptides
influence a variety of cellular and biochemical processes beyond
apoptosis, including cell adhesion, inflammation and cytokine
receptor signaling.
[0024] In accordance with the present invention, there are provided
isolated CARD-containing polypeptides or functional fragments
thereof.
[0025] The term "CARD-containing polypeptide" as used herein refers
to a protein or polypeptide containing a CARD domain. As used
herein, the term "CARD domain" refers to a Caspase Recruitment
Domain. A CARD domain is a well known protein domain of
approximately 80 amino acids with characteristic sequence
conservation as described, for example, in Hofmann et al., ITrends
Biochem. Sci. 22:155-156 (1997). CARD domains have been found in
some members of the Caspase family of cell death proteases.
Caspases-1, 2, 4, 5, 9, and 11 contain CARD domains near their
NH2-termini. These CARD domains mediate interactions of the zymogen
inactive forms of caspases with other proteins which can either
activate or inhibit the activation of these enzymes.
[0026] For example, the CARD domain of pro-caspase-9 binds to the
CARD domain of a caspase-activating protein called Apaf-1
(Apoptosis Protease Activating Factor-1). Similarly, the CARD
domain of pro-caspase-1 permits interactions with another CARD
protein known as Cardiac (also referred to as RIP2 and RICK), which
results in activation of the caspase-1 protease (Thome et al.,
Curr. Biol. 16:885-888 (1998)). Furthermore, pro-caspase-2 binds to
the CARD protein Raidd (also know as Cradd), which permits
recruitment of pro-caspase-2 to Tumor Necrosis Factor (TNF)
Receptor complexes and which results in activation of the caspase-2
protease (Ahmad et al., Cancer Res. 57:615-619 (1997)). CARD
domains can also participate in homotypic interactions with
themselves, resulting in self-association of polypeptides that
contain these protein-interaction domains and producing dimeric or
possibly even oligomeric complexes.
[0027] CARD domains can be found in association with other types of
functional domains within a single polypeptide, thus providing a
mechanism for bringing a functional domain into close proximity or
contact with a target protein via CARD:CARD associations involving
two CARD-containing polypeptides. For example, the Caenorhabiditis
elegans cell death gene ced-4 encodes a protein that contains a
CARD domain and a ATP-binding oligomerization domain called an
NB-ARC domain (van der Biezen and Jones, Curr. Biol. 8:R226-R227).
The CARD domain of the CED-4 protein interacts with the CARD domain
of a pro-caspase called CED-3. The NB-ARC domain allows CED-4 to
self-associate, thereby forming an oligomeric complex which brings
associated pro-CED-3 molecules into close proximity to each other.
Because most pro-caspases possess at least a small amount of
protease activity even in their unprocessed form, the assembly of a
complex that brings the proforms of caspase into juxtaposition can
result in trans-processing of zymogens, producing the
proteolytically processed and active caspase. Thus, CED-4 employs a
CARD domain for binding a pro-caspase and an NB-ARC domain for
self-oligomerization, resulting in caspase clustering, proteolytic
processing and activation.
[0028] In addition to their role in caspase activation, CARD
domains have been implicated in other cellular processes. Some
CARD-containing polypeptides, for example, induce activation of the
transcription factor NF-.kappa.B. NF-.kappa.B activation is induced
by many cytokines and plays an important role in cytokine receptor
signal transduction mechanisms (DiDonato et al., Nature 388:548-554
(1997)). Moreover, CARD domains are found in some proteins that
inhibit rather than activate caspases, such as the IAP (Inhibitor
of Apoptosis Protein) family members, cIAP1 and cIAP2 (Rothe et
al., Bcl-10 protein (Willis et al., Cell 96:35-45 (1999)). Also,
though caspase activation resulting from CARD domain interactions
is often involved in inducing apoptosis, other caspases are
primarily involved in proteolytic processing and activation of
inflammatory cytokines (such as pro-IL-1.beta. and pro-IL-18).
Thus, CARD-containing polypeptides can also be involved in cytokine
receptor signaling and cytokine production, and, therefore, can be
involved in regulation of immune and inflammatory responses.
[0029] In view of the function of the CARD domain within the
invention CARD-containing polypeptides or functional fragments
thereof, polypeptides of the invention are contemplated herein for
use in methods to alter biochemical processes such as apoptosis,
NF-.kappa.B induction, cytokine processing, cytokine receptor
signaling, and caspase-mediated proteolysis, thus having modulating
effects on cell life and death (i.e., apoptosis), inflammation,
cell adhesion, and other cellular and biochemical processes.
[0030] It is also contemplated herein that invention
CARD-containing polypeptides can associate with other
CARD-containing polypeptides to form invention hetero-oligomers or
homo-oligomers, such as heterodimers or homodimers. In particular,
the association of the CARD domain of invention polypeptides with
another CARD-containing polypeptide, such as Apaf-1, CED-4,
caspases-1, 2, 9, 11, cIAPs-1 and 2, CARDIAK, Raidd, Dark, CARD4,
an invention CARD-containing polypeptide, and the like, including
homo-oligomerization, is sufficiently specific such that the bound
complex can form in vivo in a cell or in vitro under suitable
conditions. Similarly therefore, an invention CARD-containing
polypeptide can associate with another CARD-containing polypeptide
by CARD:CARD form invention hetero-oligomers or homo-oligomers,
such as heterodimers or homodimers.
[0031] In accordance with the present invention, sequences for
CARD-containing polypeptides have been determined. Thus, the
present invention provides CARD-containing polypeptides, including
the newly identified CARD-containing polypeptides designated
CARD-10X (SEQ ID NO:2), CARD-11X (SEQ ID NO:8) and CARD-12X (SEQ ID
NO:16), and functional fragments thereof.
[0032] CARD-10X was identified as an unannotated protein product in
the NR protein sequence database (GI 10436238; SEQ ID NO:20,
encoded by GI 10436237; SEQ ID NO:19). A human EST (GI 9094656; SEQ
ID NO:21) and two ESTs for Gallus homologs (GI 7132200; SEQ ID
NO:22 and GI 5884878; SEQ ID NO:23) from CARD-10X have also been
identified.
[0033] CARD-11X was identified from the nucleotide database of High
Throughput Genomic Sequences (NTGS) (GI 10198542, 9887755) and also
from the NR nucleotide database, GI 9665194). Several human ESTs
from CARD-11X have been identified: GI 6926669 (SEQ ID NO:24), GI
6143407 (SEQ ID NO:25), GI 2785620 (SEQ ID NO:26), GI 1838222 (SEQ
ID NO:27), GI 6927709 (SEQ ID NO:28), GI 9720543 (SEQ ID NO:29), GI
9142863 (SEQ ID NO:30), GI 1761194 (SEQ ID NO:31), GI 8151878 (SEQ
ID NO:32), GI 2007639 (SEQ ID NO:33), GI 8042493 (SEQ ID NO:34), GI
2079290 (SEQ ID NO:35), and GI 7044777 (SEQ ID NO:36).
[0034] CARD-12X was identified from the database of HTGS (GI
8224622). A human EST from CARD-12X has also been identified: GI
10316320 (SEQ ID NO:37).
[0035] The invention CARD-10X, -11X and -12X nucleic acid molecules
do not consist of the exact sequence of the nucleotide sequences
set forth in publically available databases, such as Expressed
Sequence Tags (ESTs), Sequence Tagged Sites (STSs) and genomic
fragments, deposited in public databases such as the nr, dbest,
dbsts, gss and htgs databases, including nucleotide sequences of
the GI accession numbers set forth above. Likewise, the invention
CARD-10X, -11X and -12X polypeptides do not consist of the exact
sequence of the amino acid sequences set forth in publically
available databases, or of the exact amino acid sequence of a
translated product of an EST set forth in the databases. Since one
of skill in the art will realize that the above-recited excluded
sequences may be revised at a later date, the skilled artisan will
recognize that the above-recited sequences are excluded as they
stand on the priority date of this application.
[0036] The CARD domains of these CARD-10X, -11X and -12X are
similar to each other, sharing approximately 40-50% sequence
identity, as well as similarity by fold prediction criteria. The
closest homologs of the CARD domains of the disclosed
CARD-containing polypeptides are found in human B-cell CLL/lymphoma
10 protein, and in the equine herpesvirus 2 hypothethical protein
E10, which have about 25% sequence identity to the disclosed CARD
domains.
[0037] Apart from their CARD domains, both CARD-10X and CARD-11X
contain conserved domains found in proteins that bind the cell
cytoskeleton. Specifically, CARD-10X contains a domain of
approximately 150 amino acids found in many intermediate filament
proteins (Pfam code 00038), designated the "filament" domain (SEQ
ID NO:6). Intermediate filament proteins are distinct but
structurally related proteins that make up intermediate filaments,
which are major components of the cytoskeleton. Many intermediate
filament proteins are developmentally regulated and tissue
specific, and undergo a variety of post-translational
modifications, including phosphorylation.
[0038] CARD-11X contains a domain of approximately 300 amino acids
similar to a domain found in members of the "Ezrin/radixin/moiesin"
or "ERM" family (Pfam code 00769). The ERM domain of CARD-11X is
most similar to the helical linker region of ERM, but there is also
some similarity to the actin-binding region of the ERM domain. ERM
domain proteins are involved in connections between cytoskeletal
structures and the plasma membrane. ERM proteins mediate these
connections by associating both with F-actin and with juxtamembrane
proteins, including extracellular matrix and cell adhesion
receptors, in a regulated fashion.
[0039] CARD-11X also contains a domain designated the "post
synaptic density disc-large zo-1" or "PDZ" domain (Pfam code
00595). PDZ domains are protein interaction modules that mediate
the binding of a class of submembraneous proteins to ion channels
and membrane receptors, including neurotransmitter receptors.
[0040] The structural and functional properties of intermediate
filament domains, ERM domains and PDZ domains, and proteins
containing such domains, can be found in the Pfam database. Pfam is
a publically available large collection of multiple sequence
alignments and hidden Markov models that covers many common protein
domains. Version 5.5 of Pfam (September 2000) contains alignments
and models for 2478 protein families, based on the Swissprot 38 and
SP-TrEMBL 11 protein sequence databases.
[0041] CARD-10X and CARD-11X are the first CARD-containing
polypeptides described to contain cytoskeleton binding domains.
These proteins are likely to provide a link between the cell
cytoskeleton and the apoptosis regulatory machinery. In particular,
CARD-10X and CARD-11X are proposed to play a role in the regulation
and/or execution of anoikis, an apoptotic process that occurs when
cells are deprived of attachments via integrins, causing
disorganization of the cytoskeleton. Specifically, a CARD domain
attached to the cytoskeleton may be involved in bringing a caspase
to filaments in order to facilitate cytoskeleton cleavage.
[0042] This prediction is consistent with results indicating that
at early stages of apoptosis, caspases group near the cytoskeleton,
and microvillar breakdown occurs. Additionally, a caspase-3-like
protease has been show to play a role in the cleavage of the ERM
protein moiesin during platelet activation.
[0043] Accordingly, besides the biochemical processes such as
apoptosis, NF-.kappa.B induction, cytokine processing, cytokine
receptor signaling, and caspase-mediated proteolysis, in which
other known CARD-containing polypeptides are implicated, the
CARD-containing polypeptides of the invention are also predicted to
be involved in cytoskeletal integrity and anoikis.
[0044] As is readily appreciated by one of skill in the art,
different isoforms of a gene can be expressed as differently
spliced gene products. Thus, it is contemplated herein that the
CARD-containing polypeptides of the invention can exist in a
variety of isoforms. As referred to herein, an "isoform" of a
CARD-containing polypeptide is a biologically active
CARD-containing polypeptide that contains at least two contiguous
exon sequences from among the various exon sequences known to code
for the reference polypeptide.
[0045] For example, CARD-11X (SEQ ID NO:8) is encoded by 26 exons,
as evidenced by comparison of SEQ ID NO:7 with the genomic sequence
set forth in the NR nucleotide database as GI accession number
9665194. CARD-12X (SEQ ID NO:16) is encoded by at least 2 exons, as
evidenced by comparison of SEQ ID NO:15 with the genomic sequence
set forth in the HTGS nucleotide database as GI accession number
8224622. It is also contemplated herein that an isoform of a
CARD-containing polypeptide can include additional amino acids not
encoded by the described exon sequences.
[0046] In one embodiment, the invention provides a substantially
purified CARD-containing polypeptide, comprising substantially the
same amino acid sequence as the amino acid sequence of CARD-11X
(SEQ ID NO:8) or CARD-12X (SEQ ID NO:16).
[0047] In another embodiment, the invention provides a
substantially purified functional fragment of a CARD-containing
polypeptide, comprising substantially the same amino acid sequence
as the amino acid sequence of the CARD domain of CARD-10X (SEQ ID
NO:4), the filament domain of CARD-10X (SEQ ID NO:6), the CARD
domain of CARD-11X (SEQ ID NO:10), the ERM (ezrin) domain of
CARD-11X (SEQ ID NO:12), the PDZ domain of CARD-11X (SEQ ID NO:14)
and the CARD domain of CARD-12X (SEQ ID NO:16).
[0048] As employed herein, the term "substantially the same amino
acid sequence" refers to amino acid sequences having at least about
70% or 75% identity with respect to the reference amino acid
sequence, and retaining comparable functional and biological
activity characteristic of the polypeptide defined by the reference
amino acid sequence. Preferably, polypeptides having "substantially
the same amino acid sequence" will have at least about 80%, 82%,
84%, 86% or 88%, more preferably 90%, 91%, 92%, 93% or 94% amino
acid identity with respect to the reference amino acid sequence;
with greater than about 95%, 96%, 97%, 98% or 99% amino acid
sequence identity being especially preferred.
[0049] In accordance with the invention, specifically included
within the definition of "substantially the same" amino acid
sequence is the predominant amino acid sequence of a particular
invention CARD-containing polypeptide disclosed herein. The
predominant amino acid sequence refers to the most commonly
expressed naturally occurring amino acid sequence in a species
population. A predominant polypeptide with multiple isoforms will
have the most commonly expressed amino acid sequence for each
isoform. A predominant CARD-containing polypeptide of the invention
refers to an amino acid sequence having sequence identity to an
amino acid sequence disclosed herein that is greater than that of
any other naturally occurring protein of a particular species
(e.g., human).
[0050] Given the teachings herein of the nucleic acid or amino acid
sequences corresponding to the invention CARD-containing
polypeptides, one of skill in the art can readily confirm and, if
necessary, revise the nucleic acid or amino acid sequences
associated with the CARD-containing polypeptides of the invention.
For example, the sequences can be confirmed by probing a cDNA
library with a nucleic acid probe corresponding to a nucleic acid
of the invention using PCR or other known methods. Further, an
appropriate bacterial artificial chromosome containing the region
of the genome encoding an invention CARD-containing polypeptide can
be commercially obtained and probed using PCR, restriction mapping,
sequencing, and other known methods.
[0051] A CARD-containing polypeptide or functional fragment thereof
can have conservative amino acid substitutions as compared with the
reference polypeptide amino acid sequence. Conservative
substitutions of encoded amino acids include, for example, amino
acids that belong within the following groups: (1) non-polar amino
acids (Gly, Ala, Val, Leu, and Ile); (2) polar neutral amino acids
(Cys, Met, Ser, Thr, Asn, and Gln); (3) polar acidic amino acids
(Asp and Glu); (4) polar basic amino acids (Lys, Arg and His); and
(5) aromatic amino acids (Phe, Trp, Tyr, and His).
[0052] A CARD-containing polypeptide or functional fragment can
also be chemically derivatized, provided that the polypeptide
retains a CARD-containing polypeptide biological activity. For
example, chemical derivatization of an invention polypeptide can be
alkylation, acylation, carbamylation and iodination. Derivatized
polypeptides also include, for example, those molecules in which
free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
Free carboxyl groups can be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups can be derivatized to form O-acyl or O-alkyl derivatives.
The imidazole nitrogen of histidine can be derivatized to form
N-im-benzylhistidine.
[0053] A CARD-containing polypeptide or functional fragment can
also be substituted with one or more amino acid analogs of the
twenty standard amino acids, for example, 4-hydroxyproline,
5-hydroxylysine, 3-methylhistidine, homoserine, ornithine or
carboxyglutamate, and can include amino acids that are not linked
by peptide bonds.
[0054] A CARD-containing polypeptide or functional fragment can
also contain mimetic portions that orient functional groups that
provide a function of a CARD-containing polypeptide. Mimetics
encompass chemicals containing chemical moieties that mimic the
function of the polypeptide. For example, if a polypeptide contains
two charged chemical moieties having functional activity, a mimetic
places two charged chemical moieties in a spatial orientation and
constrained structure so that the charged chemical function is
maintained in three-dimensional space. Exemplary mimetics are
peptidomimetics, peptoids, or other peptide-like polymers such as
poly(.beta.-amino acids), and also non-polymeric compounds upon
which functional groups that mimic a peptide are positioned.
[0055] Another embodiment of the invention provides a
CARD-containing polypeptide, or a functional fragment thereof,
fused with a moiety to form a conjugate. As used herein, a "moiety"
can be a physical, chemical or biological entity which contributes
functionality to a CARD-containing polypeptide or a functional
fragment thereof. Functionalities contributed by a moiety include
therapeutic or other biological activity, or the ability to
facilitate identification or recovery of a CARD-containing
polypeptide. Therefore, a moiety will include molecules known in
the art to be useful for detection of the conjugate by, for
example, by fluorescence, magnetic imaging, detection of
radioactive emission. A moiety may also be useful for recovery of
the conjugate, for example a His tag or other known tags used for
protein isolation and/or purification, or a physical substance such
as a bead. A moiety can be a therapeutic compound, for example, a
cytotoxic drug which can be useful to effect a biological change in
cells to which the conjugate localizes.
[0056] The term "functional", when used herein as a modifier of
invention CARD-containing polypeptides, or fragments thereof,
refers to a polypeptide that exhibits biological activities similar
to at least a portion of a CARD-containing polypeptide of the
invention. Biological activities of a CARD-containing polypeptide
of the invention include, for example, the ability to bind to a
CARD-associated polypeptide (e.g. a caspase or pro-caspase), to
another CARD-containing polypeptide, to a cytoskeletal component,
or to another protein, thereby altering apoptosis, NF-.kappa.B
induction, cytokine processing, cytokine receptor signaling,
caspase-mediated proteolysis, cytoskeletal integrity or
anoikis.
[0057] The ability of a CARD-containing polypeptide to bind another
polypeptide such as a CARD-associated polypeptide can be assayed,
for example, using methods well known in the art, such as yeast
two-hybrid assays, co-immunoprecipitation, fluorescence resonance
energy transfer (FRET) assays, GST fusion co-purification, and the
like.
[0058] In accordance with the invention, there are also provided
functional fragments of CARD-containing polypeptides which retain
some, but not all, of the predominant naturally occurring
CARD-containing polypeptide activities. A "functional fragment" is
any truncated form, either carboxy-terminal, amino-terminal, or
both, of the predominant naturally occurring protein.
[0059] For example, a functional fragment of an invention
polypeptide can contain one or more of the following: a CARD
domain, a filament domain, a ERM domain, and a PDZ domain. In a
specific example, a functional fragment of a CARD-containing
polypeptide such as CARD-10X can contain a CARD domain, but lack a
functional filament domain, or vice versa. Such a fragment will
retain certain CARD-10X biological activities (e.g., CARD domain
functionality), but not all such activities (e.g., lack filament
domain functionality).
[0060] In another example, a functional fragment of a CARD-11X
polypeptide can contain one, or any two, of the CARD domain, ERM
domain and PDZ domains, but lack at least one functinal domain.
Such a fragment will retain certain CARD-11X biological activities
(e.g., CARD domain functionality, or association with cytoskeletal
components, or both), but not all such activities.
[0061] In one embodiment, the activity of the functional fragment
will be "dominant-negative." A dominant-negative activity will
allow the fragment to reduce or inactivate the activity of one or
more isoforms of a predominant naturally occurring CARD-containing
polypeptide.
[0062] Another biological activity of a CARD-containing polypeptide
or functional fragment thereof is the ability to act as an
immunogen for the production of polyclonal and monoclonal
antibodies that bind specifically to an invention CARD-containing
polypeptide. Such immunologic activity may be assayed by any method
known to those of skill in the art. For example, a test-polypeptide
encoded by a CARD-encoding cDNA can be used to produce antibodies,
which are then assayed for their ability to bind to an invention
CARD-containing polypeptide. If the antibody binds to the
test-polypeptide and the reference polypeptide with substantially
the same affinity, then the polypeptide possesses the requisite
immunologic biological activity.
[0063] Thus, the invention also provides a substantially purified
functional fragment of a CARD-containing polypeptide, comprising at
least 10 contiguous residues of CARD-10X (SEQ ID NO:2), CARD-11X
(SEQ ID NO:8) or CARD-12X (SEQ ID NO:16), wherein the functional
fragment is immunogenic.
[0064] The length of the functional fragments of the invention can
range from about 10 amino acids up to the full-length sequence of
an invention CARD-containing polypeptide. In certain embodiments,
such as for invention immunogenic fragments, the amino acid lengths
include, for example, at least about 12 amino acids, such as at
least about, or not more than, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60 or 65 amino acids. In other embodiments, such as for longer
invention immunogenic fragments and fragments containing CARD,
filament, ERM or PDZ domains, the functional fragments can contain
at least about, or not more than, 70, 75, 80, 90, 100, 125, 150,
175, 200, 250, 300, 400, 500 or more amino acids in length up to
the full-length CARD-containing polypeptide sequence.
[0065] As used herein, the term "substantially purified" means a
polypeptide that is in a form that is relatively free from
contaminating lipids, polypeptides, nucleic acids or other cellular
material normally associated with a polypeptide in a cell. A
substantially purified CARD-containing polypeptide can be obtained
by a variety of methods well-known in the art, e.g., recombinant
expression systems described herein, precipitation, gel filtration,
ion-exchange, reverse-phase and affinity chromatography, and the
like. Other well-known methods are described in Deutscher et al.,
"Guide to Protein Purification"0 Methods in Enzymology Vol. 182,
(Academic Press, (1990)). The methods and conditions for
biochemical purification of a polypeptide of the invention can be
chosen by those skilled in the art, and purification monitored, for
example, by an immunological assay, binding assay, or a functional
assay.
[0066] An example of a method for preparing the invention
polypeptide(s) is to express nucleic acids encoding a
CARD-containing polypeptide in a suitable host cell, such as a
bacterial cell, a yeast cell, an amphibian cell such as an oocyte,
or a mammalian cell, using methods well known in the art, and
recovering the expressed polypeptide, again using well-known
purification methods. Invention polypeptides can be isolated
directly from cells that have been transformed with expression
vectors as known in the art. Recombinantly expressed polypeptides
of the invention can also be expressed as fusion proteins with
appropriate affinity tags, such as glutathione S transferase (GST)
or poly His, and affinity purified. The invention polypeptide,
biologically functional fragments, and functional equivalents
thereof can also be produced by in vitro transcription/translation
methods known in the art, such as using reticulocyte lysates, as
used for example, in the TNT system (Promega). The invention
polypeptide, biologically functional fragments, and functional
equivalents thereof can also be produced by chemical synthesis. For
example, synthetic polypeptides can be produced using Applied
Biosystems, Inc. Model 430A or 431A automatic peptide synthesizer
(Foster City, Calif.) employing the chemistry provided by the
manufacturer.
[0067] Methods to identify additional invention polypeptides
containing a functional fragment of a CARD-containing polypeptide
are well known in the art. For example, genomic or cDNA libraries
from any species or tissue are commercially available or can be
readily prepared, and can be probed according to methods known in
the art. Full-length polypeptide-encoding nucleic acids, such as
full-length cDNAs can be obtained by a variety of methods
well-known in the art, such as 5' and 3! RACE.
[0068] In another embodiment of the invention, chimeric
polypeptides are provided comprising a CARD-containing polypeptide,
or a functional fragment thereof, fused with another polypeptide or
functional fragment thereof. Polypeptides with which the
CARD-containing polypeptide or functional fragment thereof are
fused can include, for example, glutathione-S-transferase, an
antibody, or other proteins or functional fragments thereof which
facilitate recovery of the chimera. Further polypeptides with which
a CARD-containing polypeptide or functional fragment thereof are
fused will include, for example, luciferase, a green fluorescent
protein, an antibody, or other proteins or functional fragments
thereof which facilitate identification of the chimera. Still
further polypeptides with which a CARD-containing polypeptide or
functional fragment thereof can advantageously be fused include,
for example, the LexA DNA binding domain, ricin, .alpha.-sarcin, an
antibody or fragment thereof, or other polypeptides which have
therapeutic properties or other biological activity.
[0069] Further invention chimeric polypeptides contemplated herein
are chimeric polypeptides wherein a functional fragment of a
CARD-containing polypeptide is fused with a catalytic domain or a
protein interaction domain from a heterologous polypeptide. One of
skill in the art will appreciate that a large number of chimeric
polypeptides are readily available by combining domains of 2 or
more CARD-containing polypeptides of the invention. Further,
chimeric polypeptides can contain a functional fragment of a
CARD-containing polypeptide of the invention fused with a domain of
a protein known in the art, such as CED-4, Apaf-1, caspase-1, and
the like.
[0070] The invention also provides methods for administering
CARD-containing polypeptides to an individual to modulate an
activity associated with a CARD-containing polypeptide, including
induction of apoptosis or anoikis, tumor suppression, modulation of
inflammation or cell adhesion and the like. A CARD-containing
polypeptide can be administered therapeutically to an individual
using expression vectors containing nucleic acids encoding
CARD-containing polypeptides, as described below. In addition,
CARD-containing polypeptides, or a functional portion thereof, can
be directly administered to an individual. Methods of administering
therapeutic polypeptides in the form of a pharmaceutical
composition are well known to those skilled in the art.
[0071] An exemplary method of delivering a CARD-containing
polypeptide to an intracellular target is to fuse a CARD-containing
polypeptide or functional fragment to an intracellular-targeting
peptide that can penetrate the cell membrane or otherwise deliver a
polypeptide to the intracellular environment such as via
internalization, thereby causing the fused CARD-containing
polypeptide to enter the cell. One example of such an
intracellular-targeting peptides is a fusion to the transduction
domain of HIV TAT, which allows transduction of up to 100% of cells
(Schwarze et al., Science 285:1569-1572 (1999); Vocero-Akbani et
al., Nature Med. 5:29-33 (1999)).
[0072] Another example of such an intracellular-targeting peptide
is the Antennapeida homeoprotein internalization domain (Holinger
et al., J. Biol. Chem. 274:13298-13304 (1999)). Still another
intracellular-targeting peptide is a peptide that is specific for a
cell surface receptor, which allows binding and internalization of
a fusion polypeptide via receptor-mediated endocytosis (Ellerby et
al., Nature Med. 5:1032-1038 (1999)). Such intracellular-targeting
peptides that mediate specific receptor interactions can be
advantageously used to target a tumor (see Ellerby et al., supra,
1999). Alternatively, a CARD-containing polypeptide of the
invention can be incorporated, if desired, into liposomes,
microspheres or other polymer matrices (Gregoriadis, Liposome
Technoloqy, Vols. I to III, 2nd ed., CRC Press, Boca Raton Fla.
(1993)).
[0073] In accordance with another embodiment of the invention,
there are provided isolated nucleic acid molecules encoding a
CARD-containing polypeptide or functional fragment thereof. The
invention isolated nucleic acids encoding CARD-containing
polypeptides are selected from:
[0074] (a) a nucleic acid molecule encoding a polypeptide
comprising the amino acid sequence of CARD-11X (SEQ ID NO:8) or
CARD-12X (SEQ ID NO:16);
[0075] (b) a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:7 or SEQ ID NO:15; and
[0076] (c) a nucleic acid molecule that hybridizes to the nucleic
acid molecule of (a) or (b) under moderately stringent
hybridization conditions.
[0077] The invention also provides isolated nucleic acid molecules
encoding functional fragments of a CARD-containing polypeptide
selected from:
[0078] (a) a nucleic acid molecule encoding the CARD domain of
CARD-10X (SEQ ID NO:4), the filament domain of CARD-10X (SEQ ID
NO:6), the CARD domain of CARD-11X (SEQ ID NO:10), the ERM (ezrin)
domain of CARD-11X (SEQ ID NO:12), the PDZ domain of CARD-11X (SEQ
ID NO:14) and the CARD domain of CARD-12X (SEQ ID NO:16);
[0079] (b) a nucleic acid molecule comprising the nucleotide
sequence of the CARD domain of CARD-10X (SEQ ID NO:3), the filament
domain of CARD-10X (SEQ ID NO:5), the CARD domain of CARD-11X (SEQ
ID NO:9), the ERM (ezrin) domain of CARD-11X (SEQ ID NO:11), the
PDZ domain of CARD-11X (SEQ ID NO:13) and the CARD domain of
CARD-12X (SEQ ID NO:15); and (c) a nucleic acid molecule that
hybridizes to the nucleic acid molecule of (a) or (b) under
moderately stringent hybridization conditions.
[0080] Also provided are isolated nucleic acid molecules having
substantially the same nucleotide sequence as the CARD11X (SEQ ID
NO:7) or CARD-12X (SEQ ID NO:15) coding sequence.
[0081] The nucleic acid molecules described herein are useful for
producing invention polypeptides, when such nucleic acids are
incorporated into a variety of protein expression systems known to
those of skill in the art. In addition, such nucleic acid molecules
or fragments thereof can be labeled with a readily detectable
substituent and used as hybridization probes for assaying for the
presence and/or amount of an invention CARD-encoding gene or mRNA
transcript in a given sample. The nucleic acid molecules described
herein, and fragments thereof, are also useful as primers and/or
templates in a PCR reaction for amplifying genes encoding invention
polypeptides described herein.
[0082] The term "nucleic acid molecule" or "polynucleotide"
encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),
probes, oligonucleotides, and primers, and can be single stranded
or double stranded. DNA can be either complementary DNA (cDNA) or
genomic DNA, e.g. a CARD-encoding gene, and can represent the sense
strand, the anti-sense strand, or both. Examples of nucleic acids
are RNA, cDNA, and isolated genomic DNA encoding a CARD-containing
polypeptide.
[0083] One means of isolating a CARD-encoding nucleic acid
polypeptide is to probe a mammalian genomic or cDNA library with a
natural or artificially designed DNA probe using methods well known
in the art. DNA probes derived from the CARD-encoding gene are
particularly useful for this purpose. DNA and cDNA molecules that
encode CARD-containing polypeptides can be used to obtain
complementary genomic DNA, cDNA or RNA from mammalian (e.g., human,
mouse, rat, rabbit, pig, and the like), or other animal sources, or
to isolate related cDNA or genomic clones by screening cDNA or
genomic libraries, using methods described in more detail
below.
[0084] In general, a genomic sequence of the invention includes
regulatory regions such as promoters, enhancers, and introns that
are outside of the exons encoding a CARD-containing polypeptide,
but does not include proximal genes that do not encode a
CARD-containing polypeptide.
[0085] Use of the term "isolated" as a modifier of nucleic acids or
polypeptides means that the molecules so designated have been
produced in such form by the hand of man, and thus are separated
from their native in vivo cellular environment.
[0086] Invention nucleic acids encoding CARD-containing
polypeptides and invention CARD-containing polypeptides can be
obtained from any species of organism, such as prokaryotes,
eukaryotes, plants, fungi, vertebrates, invertebrates, and the
like. A preferred source of invention nucleic acids are mammalian
species, e.g., human, rat, mouse, rabbit, monkey, baboon, bovine,
porcine, ovine, canine, feline, and the like, with human
particularly preferred.
[0087] As employed herein, the term "substantially the same
nucleotide sequence" refers to a nucleic acid molecule having
sufficient identity to the reference polynucleotide, such that it
will hybridize to the reference polynucleotide under moderately or
highly stringent hybridization conditions. In one embodiment, a
nucleic acid molecule having substantially the same nucleotide
sequence as the reference nucleotide sequence encodes substantially
the same amino acid sequence as that set forth in any of SEQ ID
NOS:2, 8 or 16, or its functional fragments. In another embodiment,
DNA having "substantially the same nucleotide sequence" as the
reference nucleotide sequence has at least 60%, such as 65%, 70%,
72%, 74%, 76%, 78%, 80%, 82%, 84%, 86% or 88%, more preferably at
least 90%, 91%, 92%, 93% or 94%, yet more preferably at least 95%,
96%, 97%, 98% or 99% identity to the reference nucleotide
sequence.
[0088] In accordance with the invention, specifically included
within the definition of "substantially the same" nucleotide
sequence is the predominant nucleotide sequence of a particular
invention CARD-containing polypeptide described herein. The
predominant nucleotide sequence refers to the most commonly present
naturally occurring nucleotide sequence in a species population. A
predominant CARD-encoding nucleic acid of the invention refers to a
nucleotide sequence having sequence identity to a nucleotide
sequence disclosed herein that is greater than that of any other
naturally occurring nucleotide sequence of a particular species
(e.g., human).
[0089] A nucleotide sequence that is substantially the same as a
reference nucleotide sequence can include, for example, one or
several nucleotide additions, deletions, or substitutions with
respect to the reference sequence. Exemplary substitutions to a
reference sequence are substitutions that do not change the encoded
amino acid sequence due to the degeneracy of the genetic code, or
that result in a nucleotide sequence that encodes an amino acid
sequence that is "substantially the same" as a reference
polypeptide, as described above. Such additions, deletions and
substitutions can correspond to variations that are made
deliberately, or which occur as mutations during nucleic acid
replication.
[0090] A nucleotide sequence that is substantially the same as a
reference CARD-encoding nucleotide sequence can be a sequence that
corresponds to homologs of other species, including other mammalian
species. The corresponding nucleotide sequences of non-human
species can be determined by methods known in the art, such as by
PCR or by screening genomic, cDNA or expression libraries. A
nucleotide sequence that is substantially the same as a reference
sequence can also correspond to splice variant forms of the
CARD-encoding nucleotide sequence.
[0091] A nucleic acid molecule of the invention can include one or
more non-native nucleotides, having, for example, modifications to
the base, the sugar, or the phosphate portion, or having a modified
phosphodiester linkage. Such modifications can be advantageous in
increasing the stability of the nucleic acid molecule.
[0092] Furthermore, a nucleic acid molecule of the invention can
include, for example, a detectable moiety, such as a radiolabel, a
fluorochrome, a ferromagnetic substance, a luminescent tag or a
detectable binding agent such as biotin. Such moieties can be
advantageous in applications where detection of a CARD-encoding
nucleic acid molecule is desired.
[0093] As used herein, the term "hybridization" refers to the
binding of complementary strands of nucleic acid (i.e.,
sense:antisense strands or probe:target-DNA) to each other through
hydrogen bonds, similar to the bonds that naturally occur in
chromosomal DNA. Stringency levels used to hybridize a given probe
with target-DNA can be readily varied by those of skill in the
art.
[0094] The phrase "stringent hybridization" is used herein to refer
to conditions under which polynucleic acid hybrids are stable. As
known to those of skill in the art, the stability of hybrids is
reflected in the melting temperature (Tm) of the hybrids. In
general, the stability of a hybrid is a function of sodium ion
concentration and temperature. Typically, the hybridization
reaction is performed under conditions of lower stringency,
followed by washes of varying, but higher, stringency. Reference to
hybridization stringency relates to such washing conditions.
[0095] As used herein, the phrase "moderately stringent
hybridization" refers to conditions that permit target-nucleic acid
to bind a complementary nucleic acid. The hybridized nucleic acids
will generally have at least about 60% identity, such as at least
about 75% identity, more preferably at least about 90% identity.
Moderately stringent conditions are conditions equivalent to
hybridization in 50% formamide, 5.times. Denhart's solution,
5.times. SSPE, 0.2% SDS at 42.degree. C., followed by washing in
0.2.times. SSPE, 0.2% SDS, at 42.degree. C.
[0096] The phrase "high stringency hybridization" refers to
conditions that permit hybridization of only those nucleic acid
sequences that form stable hybrids in 0.018M NaCl at 65.degree. C.
Thus, if a hybrid is not stable in 0.018M NaCl at 65.degree. C., it
will not be stable under high stringency conditions, as
contemplated herein. High stringency conditions can be provided,
for example, by hybridization in 50% formamide, 5.times. Denhart's
solution, 5.times. SSPE, 0.2% SDS at 42.degree. C., followed by
washing in 0.1.times. SSPE, and 0.1% SDS at 65.degree. C.
[0097] Other suitable moderate stringency and high stringency
hybridization buffers and conditions are well known to those of
skill in the art and are described, for example, in Sambrook et
al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., (1989), and Current Protocols
in Molecular Biology, F. M. Ausubel et al., eds., (Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (most recent Supplement).
[0098] Identity of any two nucleic acid or amino acid sequences can
be determined by those skilled in the art based, for example, on a
BLAST 2.0 computer alignment, using default parameters. BLAST 2.0
computer searching and sequence alignments are known in the art
(e.g. Tatusova et al., FEMS Microbiol Lett. 174:247-250 (1999) and
Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997)), and are
publicly available.
[0099] One means of isolating a nucleic acid encoding a
CARD-containing polypeptide is to probe a cDNA library or genomic
library with a natural or artificially designed nucleic acid probe
using methods well known in the art. Nucleic acid probes derived
from a CARD-encoding gene are particularly useful for this purpose.
DNA and cDNA molecules that encode CARD-containing polypeptides can
be used to obtain complementary genomic DNA, cDNA or RNA from
mammals, for example, human, mouse, rat, rabbit, pig, and the like,
or other animal sources, or to isolate related cDNA or genomic
clones by the screening of cDNA or genomic libraries, by methods
well known in the art.
[0100] The invention further provides oligonucleotides that
function as single stranded nucleic acid primers for amplification
of a CARD-encoding nucleic acid, or as probes for identification of
a CARD-encoding nucleic acid.
[0101] The invention oligonucleotides comprise at least 15
contiguous nucleotides of SEQ ID NOS:1, 7 or 15, or its complement.
The invention oligonucleotides can include at least, or not more
than, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125,
150, 175, 200, 225, 250, 275, 300, 325, 350 or more contiguous
nucleotides from the reference nucleotide sequence or its
complement.
[0102] The oligonucleotides of the invention are able to hybridize
to the reference nucleic acid molecules of the invention under
moderately stringent, or highly stringent, hybridization conditions
and thus can be advantageously used, for example, as probes to
detect CARD-encoding DNA or RNA in a sample, and to detect splice
variants thereof; as sequencing or PCR primers; as antisense
reagents to block transcription of CARD-encoding RNA in cells; or
in other applications known to those skilled in the art in which
hybridization to a CARD-encoding nucleic acid molecule is
desirable.
[0103] In accordance with another embodiment of the invention, a
method is provided for identifying nucleic acids encoding a
CARD-containing polypeptide. The method includes the steps of
contacting a sample containing nucleic acids with an invention
oligonucleotide, wherein the contacting is effected under high
stringency hybridization conditions, and identifying a nucleic acid
that hybridizes thereto.
[0104] The invention additionally provides a method of detecting a
CARD-encoding nucleic acid molecule in a sample by contacting the
sample with two or more invention oligonucleotides, amplifying a
nucleic acid molecule, and detecting the amplification. The
amplification can be performed, for example, using PCR.
[0105] The isolated nucleic acid molecules an oligonucleotides of
the invention can be used in a variety of diagnostic and
therapeutic applications. For example, the isolated nucleic acid
molecules of the invention can be used as probes, as described
above; as templates for the recombinant expression of
CARD-containing polypeptides; or in screening assays such as
two-hybrid assays to identify cellular molecules that bind
CARD-containing polypeptides.
[0106] The isolated nucleic acid molecules of the invention can be
prepared by methods known in the art. The method chosen will depend
on factors such as the type and size of nucleic acid molecule one
intends to isolate; whether or not it encodes a biologically active
polypeptide (e.g. having a CARD-containing polypeptide biological
or immunogenic activity); and the source of the nucleic acid
molecule.
[0107] An exemplary method for preparing an isolated nucleic acid
molecule involves amplification of the nucleic acid molecule using
invention oligonucleotide primers and the polymerase chain reaction
(PCR) and, optionally, purification of the resulting product by gel
electrophoresis. Using PCR, a CARD-encoding nucleic acid molecule
having any desired boundaries can be amplified exponentially
starting from only a few DNA or RNA molecules, such as from a
single cell. Desired modifications to the nucleic acid sequence can
also be introduced by choosing an appropriate oligonucleotide
primer with one or more additions, deletions or substitutions. PCR
methods, including methods of isolating homologs of a given nucleic
acid molecule in another species using degenerate primers, are well
known in the art.
[0108] Alternatively, an isolated CARD-encoding nucleic acid
molecule can be prepared by screening a library, such as a genomic
library, cDNA library or expression library, with a detectable
CARD-encoding nucleic acid molecule or anti-CARD antibody. Human
libraries, and libraries from a large variety of mammalian species,
are commercially available or can be produced from species or cells
of interest. The library clones identified as containing
CARD-encoding nucleic acid molecules can be isolated, subcloned or
sequenced by routine methods.
[0109] Furthermore, an isolated CARD-encoding nucleic acid molecule
or oligonucleotide can be prepared by direct synthetic methods. For
example, a single stranded nucleic acid molecule can be chemically
synthesized in one piece, or in several pieces, by automated
synthesis methods known in the art. The complementary strand can
likewise be synthesized in one or more pieces, and a
double-stranded molecule made by annealing the complementary
strands. Direct synthesis is particularly advantageous for
producing relatively short molecules, such as probes and primers,
and also for producing nucleic acid molecules containing modified
nucleotides or linkages.
[0110] The invention also provides methods for detecting a
CARD-encoding nucleic acid in a sample. The methods of detecting a
CARD-encoding nucleic acid in a sample can be either qualitative or
quantitative, as desired. For example, the presence, abundance,
integrity or structure of a CARD-encoding nucleic acid can be
determined, as desired, depending on the assay format and the probe
used for hybridization or primer pair chosen for application.
[0111] Useful assays for detecting a CARD-containing nucleic acid
based on specific hybridization with an isolated invention
oligonucleotide are well known in the art and include, for example,
in situ hybridization, which can be used to detect altered
chromosomal location of the nucleic acid molecule, altered gene
copy number, and RNA abundance, depending on the assay format used.
Other hybridization assays include, for example, Northern blots and
RNase protection assays, which can be used to determine the
abundance and integrity of different RNA splice variants, and
Southern blots, which can be used to determine the copy number and
integrity of DNA. A hybridization probe can be labeled with any
suitable detectable moiety, such as a radioisotope, fluorochrome,
chemiluminescent marker, biotin, or other detectable moiety known
in the art that is detectable by analytical methods.
[0112] Useful assays for detecting a CARD-encoding nucleic acid in
a sample based on amplifying a CARD-encoding nucleic acid with two
or more invention oligonucleotides are also well known in the art,
and include, for example, qualitative or quantitative polymerase
chain reaction (PCR); reverse-transcription PCR (RT-PCR); single
strand conformational polymorphism (SSCP) analysis, which can
readily identify a single point mutation in DNA based on
differences in the secondary structure of single-strand DNA that
produce an altered electrophoretic mobility upon non-denaturing gel
electrophoresis; and coupled PCR, transcription and translation
assays, such as a protein truncation test, in which a mutation in
DNA is determined by an altered protein product on an
electrophoresis gel. Additionally, the amplified CARD-encoding
nucleic acid can be sequenced to detect mutations and mutational
hot-spots, and specific assays for large-scale screening of samples
to identify such mutations can be developed.
[0113] Also provided are antisense-nucleic acids having a sequence
capable of binding specifically with full-length or any portion of
an mRNA that encodes CARD-containing polypeptides so as to prevent
translation of the mRNA. The antisense-nucleic acid can have a
sequence capable of binding specifically with any portion of the
sequence of the cDNA encoding CARD-containing polypeptides. As used
herein, the phrase "binding specifically" encompasses the ability
of a nucleic acid sequence to recognize a complementary nucleic
acid sequence and to form double-helical segments therewith via the
formation of hydrogen bonds between the complementary base pairs.
An example of an antisense-nucleic acid is an antisense-nucleic
acid comprising chemical analogs of nucleotides.
[0114] The present invention provides means to alter levels of
expression of CARD-containing polypeptides by recombinantly
expressing CARD-containing anti-sense nucleic acids or employing
synthetic anti-sense nucleic acid compositions (hereinafter SANC)
that inhibit translation of mRNA encoding these polypeptides.
Synthetic oligonucleotides, or other antisense-nucleic acid
chemical structures designed to recognize and selectively bind to
mRNA are constructed to be complementary to full-length or portions
of a CARD-encoding strand, including nucleotide sequences
substantially the same as SEQ ID NOS:1, 7 or 15.
[0115] The SANC is designed to be stable in the blood stream for
administration to a subject by injection, or in laboratory cell
culture conditions. The SANC is designed to be capable of passing
through the cell membrane in order to enter the cytoplasm of the
cell by virtue of physical and chemical properties of the SANC,
which render it capable of passing through cell membranes, for
example, by designing small, hydrophobic SANC chemical structures,
or by virtue of specific transport systems in the cell which
recognize and transport the SANC into the cell. In addition, the
SANC can be designed for administration only to certain selected
cell populations by targeting the SANC to be recognized by specific
cellular uptake mechanisms which bind and take up the SANC only
within select cell populations. In a particular embodiment the SANC
is an antisense oligonucleotide.
[0116] For example, the SANC may be designed to bind to a receptor
found only in a certain cell type, as discussed above. The SANC is
also designed to recognize and selectively bind to target mRNA
sequence, which can correspond to a sequence contained within the
sequences set forth as SEQ ID NOS:1, 7 or 15.
[0117] The SANC is designed to inactivate target mRNA sequence by
either binding thereto and inducing degradation of the mRNA by, for
example, RNase I digestion, or inhibiting translation of mRNA
target sequence by interfering with the binding of
translation-regulating factors or ribosomes, or inclusion of other
chemical structures, such as ribozyme sequences or reactive
chemical groups which either degrade or chemically modify the
target mRNA. SANCs have been shown to be capable of such properties
when directed against mRNA targets (see Cohen et al., TIPS, 10:435
(1989) and Weintraub, Sci. American, January (1990), pp.40).
[0118] The invention further provides a method of altering the
level of a biochemical process modulated by a CARD-containing
polypeptide by introducing an antisense nucleotide sequence into
the cell, wherein the antisense nucleotide sequence specifically
hybridizes to a CARD-encoding nucleic acid molecule, wherein the
hybridization reduces or inhibits the expression of the
CARD-containing polypeptide in the cell. The use of anti-sense
nucleic acids, including recombinant anti-sense nucleic acids or
SANCs, can be advantageously used to inhibit cell death.
[0119] Compositions comprising an amount of the antisense-nucleic
acid of the invention, effective to reduce expression of
CARD-containing polypeptides by entering a cell and binding
specifically to CARD-encoding mRNA so as to prevent translation and
an acceptable hydrophobic carrier capable of passing through a cell
membrane are also provided herein. Suitable hydrophobic carriers
are described, for example, in U.S. Pat. Nos. 5,334,761; 4,889,953;
4,897,355, and the like. The acceptable hydrophobic carrier capable
of passing through cell membranes may also comprise a structure
which binds to a receptor specific for a selected cell type and is
thereby taken up by cells of the selected cell type. For example,
the structure can be part of a protein known to bind to a cell-type
specific receptor such as a tumor.
[0120] Antisense-nucleic acid compositions are useful to inhibit
translation of mRNA encoding invention polypeptides. Synthetic
oligonucleotides, or other antisense chemical structures are
designed to bind to CARD-encoding mRNA and inhibit translation of
mRNA and are useful as compositions to inhibit expression of
CARD-encoding genes or CARD-associated polypeptide genes in a
tissue sample or in a subject.
[0121] The above-described nucleotide sequences can be incorporated
into vectors for further manipulation. As used herein, vector
refers to a recombinant DNA or RNA plasmid or virus containing
discrete elements that are used to introduce heterologous DNA into
cells for either expression or replication thereof.
[0122] The invention also provides vectors containing the
CARD-encoding nucleic acids of the invention. Suitable expression
vectors are well-known in the art and include vectors capable of
expressing nucleic acid operatively linked to a regulatory sequence
or element such as a promoter region or enhancer region that is
capable of regulating expression of such nucleic acid. Appropriate
expression vectors include those that are replicable in eukaryotic
cells and/or prokaryotic cells and those that remain episomal or
those which integrate into the host cell genome.
[0123] Promoters or enhancers, depending upon the nature of the
regulation, can be constitutive or regulated. The regulatory
sequences or regulatory elements are operatively linked to a
nucleic acid of the invention such that the physical and functional
relationship between the nucleic acid and the regulatory sequence
allows transcription of the nucleic acid.
[0124] Suitable vectors for expression in prokaryotic or eukaryotic
cells are well known to those skilled in the art (see, for example,
Ausubel et al., supra, 2000). Vectors useful for expression in
eukaryotic cells can include, for example, regulatory elements
including the SV40 early promoter, the cytomegalovirus (CMV)
promoter, the mouse mammary tumor virus (MMTV) steroid-inducible
promoter, Moloney murine leukemia virus (MMLV) promoter, and the
like. The vectors of the invention are useful for subcloning and
amplifying a CARD-encoding nucleic acid molecule and for
recombinantly expressing a CARD-containing polypeptide. A vector of
the invention can include, for example, viral vectors such as a
bacteriophage, a baculovirus or a retrovirus; cosmids or plasmids;
and, particularly for cloning large nucleic acid molecules,
bacterial artificial chromosome vectors (BACs) and yeast artificial
chromosome vectors (YACs). Such vectors are commercially available,
and their uses are well known in the art. One skilled in the art
will know or can readily determine an appropriate promoter for
expression in a particular host cell.
[0125] The invention additionally provides recombinant cells
containing CARD-encoding nucleic acids of the invention. The
recombinant cells are generated by introducing into a host cell a
vector containing a CARD-encoding nucleic acid molecule. The
recombinant cells are transducted, transfected or otherwise
genetically modified. Exemplary host cells that can be used to
express recombinant CARD molecules include mammalian primary cells;
established mammalian cell lines, such as COS, CHO, HeLa, NIH3T3,
HEK 293 and PC12 cells; amphibian cells, such as Xenopus embryos
and oocytes and other vertebrate cells. Exemplary host cells also
include insect cells such as Drosophila, yeast cells such as
Saccharomyces cerevisiae, Saccharomyces pombe, or Pichia pastoris,
and prokaryotic cells such as Escherichia coli. Additional host
cells can be obtained, for example, from ATCC (Manassas, Va.).
[0126] The invention also provides a method for expression of a
CARD-containing polypeptide by culturing cells containing a
CARD-encoding nucleic acid under conditions suitable for expression
of a CARD-containing polypeptide. Suitable culturing conditions for
expression of an encoded nucleic acid molecule are well known in
the art, and described, for example, in Ausubel et al., supra,
2000.
[0127] CARD-encoding nucleic acids can also be delivered into
mammalian cells, either in vivo or in vitro, to modulate an
activity associated with a CARD-containing polypeptide, including
induction of apoptosis or anoikis, tumor suppression, modulation of
inflammation or cell adhesion and the like. Suitable vectors for
delivering a CARD-encoding nucleic acid molecule of the invention
to a mammalian cell include viral vectors such as retroviral
vectors, adenovirus, adeno-associated virus, lentivirus,
herpesvirus, as well as non-viral vectors such as plasmid vectors.
Such vectors are useful for providing therapeutic amounts of a
CARD-containing polypeptide (see, for example, U.S. Pat. No.
5,399,346, issued Mar. 21, 1995). Delivery of CARD nucleic acids
therapeutically can be particularly useful when targeted to a tumor
cell, thereby inducing apoptosis in tumor cells. In addition, where
it is desirable to limit or reduce the in vivo expression of a
CARD-containing polypeptide, the introduction of a vector
expressing the antisense strand of the invention nucleic acid
molecule is contemplated.
[0128] Viral based systems provide the advantage of being able to
introduce relatively high levels of the heterologous nucleic acid
into a variety of cells. Suitable viral vectors for introducing an
invention CARD-encoding nucleic acid into mammalian cells are well
known in the art. These viral vectors include, for example, Herpes
simplex virus vectors (Geller et al., Science, 241:1667-1669
(1988)); vaccinia virus vectors (Piccini et al., Meth. Enzymology,
153:545-563 (1987)); cytomegalovirus vectors (Mocarski et al., in
Viral Vectors, Y. Gluzman and S. H. Hughes, Eds., Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84));
Moloney murine leukemia virus vectors (Danos et al., Proc. Natl.
Acad. Sci. USA, 85:6460-6464 (1988); Blaese et al., Science,
270:475-479 (1995); Onodera et al., J. Virol., 72:1769-1774
(1998)); adenovirus vectors (Berkner, Biotechniques, 6:616-626
(1988); Cotten et al., Proc. Natl. Acad. Sci. USA, 89:6094-6098
(1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991); Li et
al., Human Gene Therapy, 4:403-409 (1993); Zabner et al., Nature
Genetics, 6:75-83 (1994)); adeno-associated virus vectors (Goldman
et al., Human Gene Therapy, 10:2261-2268 (1997); Greelish et al.,
Nature Med., 5:439-443 (1999); Wang et al., Proc. Natl. Acad. Sci.
USA, 96:3906-3910 (1999); Snyder et al., Nature Med., 5:64-70
(1999); Herzog et al., Nature Med., 5:56-63 (1999)); retrovirus
vectors (Donahue et al., Nature Med., 4:181-186 (1998); Shackleford
et al., Proc. Natl. Acad. Sci. USA, 85:9655-9659 (1988); U.S. Pat.
Nos. 4,405,712, 4,650,764 and 5,252,479, and WIPO publications WO
92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829;
and lentivirus vectors (Kafri et al., Nature Genetics, 17:314-317
(1997)).
[0129] For example, in one embodiment of the present invention,
adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes
(Wagner et al., Proc. Natl. Acad. Sci., USA, 89:6099-6103 (1992);
Curiel et al., Hum. Gene Ther., 3:147-154 (1992); Gao et al., Hum.
Gene Ther., 4:14-24 (1993)) are employed to transduce mammalian
cells with heterologous CARD-encoding nucleic acid. Any of the
plasmid expression vectors described herein may be employed in a
TfAdpl-DNA complex.
[0130] Vectors useful for therapeutic administration of a
CARD-encoding nucleic acid can contain a regulatory element that
provides tissue specific or inducible expression of an operatively
linked nucleic acid. One skilled in the art can readily determine
an appropriate tissue-specific promoter or enhancer that allows
expression of a CARD polypeptide or nucleic acid in a desired
tissue. Any of a variety of inducible promoters or enhancers can
also be included in the vector for regulatable expression of a CARD
polypeptide or nucleic acid. Such inducible systems, include, for
example, tetracycline inducible system (Gossen & Bizard, Proc.
Natl. Acad. Sci. USA, 89:5547-5551 (1992); Gossen et al., Science,
268:1766-1769 (1995); Clontech, Palo Alto, Calif.); metalothionein
promoter induced by heavy metals; insect steroid hormone responsive
to ecdysone or related steroids such as muristerone (No et al.,
Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996); Yao et al.,
Nature, 366:476-479 (1993); Invitrogen, Carlsbad, Calif.); mouse
mammary tumor virus (MMTV) induced by steroids such as
glucocortocoid and estrogen (Lee et al., Nature, 294:228-232
(1981); and heat shock promoters inducible by temperature
changes.
[0131] An inducible system particularly useful for therapeutic
administration utilizes an inducible promoter that can be regulated
to deliver a level of therapeutic product in response to a given
level of drug administered to an individual and to have little or
no expression of the therapeutic product in the absence of the
drug. One such system utilizes a Gal4 fusion that is inducible by
an antiprogestin such as mifepristone in a modified adenovirus
vector (Burien et al., Proc. Natl. Acad. Sci. USA, 96:355-360
(1999). Another such inducible system utilizes the drug rapamycin
to induce reconstitution of a transcriptional activator containing
rapamycin binding domains of FKBP12 and FRAP in an adeno-associated
virus vector (Ye et al., Science, 283:88-91 (1999)). It is
understood that any combination of an inducible system can be
combined in any suitable vector, including those disclosed herein.
Such a regulatable inducible system is advantageous because the
level of expression of the therapeutic product can be controlled by
the amount of drug administered to the individual or, if desired,
expression of the therapeutic product can be terminated by stopping
administration of the drug.
[0132] The specificity of viral vectors for particular cell types
can be utilized to target predetermined cell types. Thus, the
selection of a viral vector will depend, in part, on the cell type
to be targeted. For example, if a neurodegenerative disease is to
be treated by increasing the level of a CARD-containing polypeptide
in neuronal cells affected by the disease, then a viral vector that
targets neuronal cells can be used. A vector derived from a herpes
simplex virus is an example of a viral vector that targets neuronal
cells (Battleman et al., J. Neurosci. 13:941-951 (1993), which is
incorporated herein by reference). Similarly, if a disease or
pathological condition of the hematopoietic system is to be
treated, then a viral vector that is specific for a particular
blood cell or its precursor cell can be used. A vector based on a
human immunodeficiency virus is an example of such a viral vector
(Carroll et al., J. Cell. Biochem. 17E:241 (1993), which is
incorporated herein by reference). In addition, a viral vector or
other vector can be constructed to express a CARD-encoding nucleic
acid in a tissue specific manner by incorporating a tissue-specific
promoter or enhancer into the vector (Dai et al., Proc. Natl. Acad.
Sci. USA 89:10892-10895 (1992), which is incorporated herein by
reference).
[0133] For gene therapy, a vector containing a CARD-encoding
nucleic acid or an antisense nucleotide sequence can be
administered to a subject by various methods. For example, if viral
vectors are used, administration can take advantage of the target
specificity of the vectors. In such cases, there in no need to
administer the vector locally at the diseased site. However, local
administration can be a particularly effective method of
administering a CARD-encoding nucleic acid. In addition,
administration can be via intravenous or subcutaneous injection
into the subject. Following injection, the viral vectors will
circulate until they recognize host cells with the appropriate
target specificity for infection. Injection of viral vectors into
the spinal fluid also can be an effective mode of administration,
for example, in treating a neurodegenerative disease.
[0134] Receptor-mediated DNA delivery approaches also can be used
to deliver a CARD-encoding nucleic acid molecule into cells in a
tissue-specific manner using a tissue-specific ligand or an
antibody that is non-covalently complexed with the nucleic acid
molecule via a bridging molecule (Curiel et al., Hum. Gene Ther.
3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987),
each of which is incorporated herein by reference). Direct
injection of a naked or a nucleic acid molecule encapsulated, for
example, in cationic liposomes also can be used for stable gene
transfer into non-dividing or dividing cells in vivo (Ulmer et al.,
Science 259:1745-1748 (1993), which is incorporated herein by
reference). In addition, a CARD-encoding nucleic acid molecule can
be transferred into a variety of tissues using the particle
bombardment method (Williams et al., Proc. Natl. Acad. Sci. USA
88:2726-2730 (1991), which is incorporated herein by reference).
Such nucleic acid molecules can be linked to the appropriate
nucleotide sequences required for transcription and
translation.
[0135] A particularly useful mode of administration of a
CARD-encoding nucleic acid is by direct inoculation locally at the
site of the disease or pathological condition. Local administration
can be advantageous because there is no dilution effect and,
therefore, the likelihood that a majority of the targeted cells
will be contacted with the nucleic acid molecule is increased.
Thus, local inoculation can alleviate the targeting requirement
necessary with other forms of administration and, if desired, a
vector that infects all cell types in the inoculated area can be
used. If expression is desired in only a specific subset of cells
within the inoculated area, then a promoter, an enhancer or other
expression element specific for the desired subset of cells can be
linked to the nucleic acid molecule. Vectors containing such
nucleic acid molecules and regulatory elements can be viral
vectors, viral genomes, plasmids, phagemids and the like.
Transfection vehicles such as liposomes also can be used to
introduce a non-viral vector into recipient cells. Such vehicles
are well known in the art.
[0136] The invention additionally provides an isolated anti-CARD
antibody having specific reactivity with a invention
CARD-containing polypeptide. The anti-CARD antibody can be a
monoclonal antibody or a polyclonal antibody. The invention further
provides cell lines producing monoclonal antibodies having specific
reactivity with an invention CARD-containing protien.
[0137] The invention thus provides antibodies that specifically
bind a CARD-containing polypeptide. CARD-specific antibodies be
used, for example, for the immunoaffinity or affinity
chromatography purification of an invention CARD-containing
polypeptide, as well as for diagnostic and in vivo imaging
procedures.
[0138] As used herein, the term "antibody" is used in its broadest
sense to include polyclonal and monoclonal antibodies, as well as
antigen binding fragments of such antibodies. With regard to an
anti-CARD antibody of the invention, the term "antigen" means a
native or synthesized CARD-containing polypeptide or fragment
thereof. An anti-CARD antibody, or antigen binding fragment of such
an antibody, is characterized by having specific binding activity
for a CARD polypeptide or a peptide portion thereof of at least
about 1.times.10.sup.5 M.sup.-1. Thus, Fab, F(ab').sub.2, Fd and Fv
fragments of an anti-CARD antibody, which retain specific binding
activity for a CARD-containing polypeptide, are included within the
definition of an antibody. Specific binding activity of a
CARD-containing polypeptide can be readily determined by one
skilled in the art, for example, by comparing the binding activity
of an anti-CARD antibody to a CARD-containing polypeptide versus a
reference polypeptide that is not a CARD-containing polypeptide.
Methods of preparing polyclonal or monoclonal antibodies are well
known to those skilled in the art (see, for example, Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1988)).
[0139] In addition, the term "antibody" as used herein includes
naturally occurring antibodies as well as non-naturally occurring
antibodies, including, for example, single chain antibodies,
chimeric, bifunctional and humanized antibodies, as well as
antigen-binding fragments thereof. Such non-naturally occurring
antibodies can be constructed using solid phase peptide synthesis,
can be produced recombinantly or can be obtained, for example, by
screening combinatorial libraries consisting of variable heavy
chains and variable light chains as described by Huse et al.,
Science 246:1275-1281 (1989)). These and other methods of making,
for example, chimeric, humanized, CDR-grafted, single chain, and
bifunctional antibodies are well known to those skilled in the art
(Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al.,
Nature 341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard
et al., Protein Engineering: A practical approach (IRL Press 1992);
Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press
1995)).
[0140] Anti-CARD antibodies can be raised using a CARD immunogen
such as an isolated CARD-containing polypeptide having
substantially the same amino acid sequence as SEQ ID NOS:2, 8 or
15, or an immunogenic fragment thereof, which can be prepared from
natural sources or produced recombinantly, or a peptide portion of
the CARD-containing polypeptide. A non-immunogenic or weakly
immunogenic CARD-containing polypeptide or portion thereof can be
made immunogenic by coupling the hapten to a carrier molecule such
as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
Various other carrier molecules and methods for coupling a hapten
to a carrier molecule are well known in the art (see, for example,
Harlow and Lane, supra, 1988). An immunogenic CARD-containing
polypeptide fragment can also be generated by expressing the
peptide as a fusion protein, for example, to glutathione S
transferase (GST), polyHis or the like. Methods for expressing
peptide fusions are well known to those skilled in the art (Ausubel
et al., supra, (2000)).
[0141] The invention further provides a method for detecting the
presence of a human CARD-containing polypeptide in a sample by
contacting a sample with a CARD-specific antibody, and detecting
the presence of specific binding of the antibody to the sample,
thereby detecting the presence of a human CARD-containing
polypeptide in the sample. CARD-specific antibodies can be used in
diagnostic methods and systems to detect the level of
CARD-containing polypeptide present in a sample. with respect to
the detection of such polypeptides, the antibodies can be used for
in vitro diagnostic or in vivo imaging methods.
[0142] As used herein, the term "sample" is intended to mean any
biological fluid, cell, tissue, organ or portion thereof, that
includes or potentially includes CARD nucleic acids or
polypeptides. The term includes samples present in an individual as
well as samples obtained or derived from the individual. For
example, a sample can be a histologic section of a specimen
obtained by biopsy, or cells that are placed in or adapted to
tissue culture. A sample further can be a subcellular fraction or
extract, or a crude or substantially pure nucleic acid or
polypeptide preparation.
[0143] Immunological procedures useful for in vitro detection of
target CARD-containing polypeptides in a sample include
immunoassays that employ a detectable antibody. Such immunoassays
include, for example, immunohistochemistry, immunofluorescence,
ELISA assays, radioimmunoassay, FACS analysis, immunoprecipitation,
immunoblot analysis, Pandex microfluorimetric assay, agglutination
assays, flow cytometry and serum diagnostic assays, which are well
known in the art (Harlow and Lane, supra, 1988; Harlow and Lane,
Usinq Antibodies: A Laboratory Manual, Cold Spring Harbor Press
(1999)).
[0144] An antibody can be made detectable by various means well
known in the art. For example, a detectable marker can be directly
attached to the antibody or indirectly attached using, for example,
a secondary agent that recognizes the CARD specific antibody.
Useful markers include, for example, radionucleotides, enzymes,
binding proteins such as biotin, fluorogens, chromogens and
chemiluminescent labels.
[0145] An antibody can also be detectable by, for example, a
fluorescent labeling agent that chemically binds to antibodies or
antigens without denaturation to form a fluorochrome (dye) that is
a useful immunofluorescent tracer. A description of
immunofluorescent analytic techniques is found in DeLuca,
"Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis
et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982),
which is incorporated herein by reference.
[0146] In one embodiment, the indicating group is an enzyme, such
as horseradish peroxidase (HRP), glucose oxidase, and the like. In
another embodiment, radioactive elements are employed labeling
agents. The linking of a label to a substrate, i.e., labeling of
nucleic acid probes, antibodies, polypeptides, and proteins, is
well known in the art. For instance, an invention antibody can be
labeled by metabolic incorporation of radiolabeled amino acids
provided in the culture medium. See, for example, Galfre et al.,
Meth. Enzymol., 73:3-46 (1981). Conventional means of protein
conjugation or coupling by activated functional groups are
particularly applicable. See, for example, Aurameas et al., Scand.
J. Immunol., Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al.,
Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.
[0147] In addition to detecting the presence of a CARD-containing
polypeptide, invention anti-CARD antibodies are contemplated for
use herein to alter the activity of the CARD-containing polypeptide
in living animals, in humans, or in biological tissues or fluids
isolated therefrom. Accordingly, compositions comprising a carrier
and an amount of an antibody having specificity for CARD-containing
polypeptides effective to block naturally occurring ligands or
other CARD-associated polypeptides from binding to invention
CARD-containing polypeptides are contemplated herein.
[0148] The present invention further provides transgenic non-human
mammals that are capable of expressing exogenous nucleic acids
encoding CARD-containing polypeptides. As employed herein, the
phrase "exogenous nucleic acid" refers to nucleic acid sequence
which is not native to the host, or which is present in the host in
other than its native environment, for example, as part of a
genetically engineered DNA construct. In addition to naturally
occurring CARD-containing polypeptide levels, a CARD-containing
polypeptide of the invention can either be overexpressed or
underexpressed in transgenic mammals, for example, underexpressed
in a knock-out animal.
[0149] Also provided are transgenic non-human mammals capable of
expressing CARD-encoding nucleic acids so mutated as to be
incapable of normal activity. Therefore, the transgenic non-human
mammals do not express native CARD-containing polypeptide or have
reduced expression of native CARD-containing polypeptide. The
present invention also provides transgenic non-human mammals having
a genome comprising antisense nucleic acids complementary to
CARD-encoding nucleic acids, placed so as to be transcribed into
antisense mRNA complementary to CARD-encoding mRNA, which
hybridizes to the mRNA and, thereby, reduces the translation
thereof. The nucleic acid can additionally comprise an inducible
promoter and/or tissue specific regulatory elements, so that
expression can be induced, or restricted to specific cell
types.
[0150] Animal model systems useful for elucidating the
physiological and behavioral roles of CARD-containing polypeptides
are also provided, and are produced by creating transgenic animals
in which the expression of the CARD-containing polypeptide is
altered using a variety of techniques. Examples of such techniques
include the insertion of normal or mutant versions of nucleic acids
encoding a CARD-containing polypeptide by microinjection,
retroviral infection or other means well known to those skilled in
the art, into appropriate fertilized embryos to produce a
transgenic animal, see, for example, Hogan et al., Manipulating the
Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory,
(1986)). Transgenic animal model systems are useful for in vivo
screening of compounds for identification of specific ligands, such
as agonists or antagonists, which activate or inhibit a biological
activity.
[0151] Also contemplated herein, is the use of homologous
recombination of mutant or normal versions of CARD-encoding genes
with the native gene locus in transgenic animals, to alter the
regulation of expression or the structure of CARD-containing
polypeptides by replacing the endogeneous gene with a recombinant
or mutated CARD-encoding gene. Methods for producing a transgenic
non-human mammal including a gene knock-out non-human mammal, are
well known to those skilled in the art (see, Capecchi et al.,
Science 244:1288 (1989); Zimmer et al., Nature 338:150 (1989);
Shastry, Experentia, 51:1028-1039 (1995); Shastry, Mol. Cell.
Biochem., 181:163-179 (1998); and U.S. Pat. No. 5,616,491, issued
Apr. 1, 1997, U.S. Pat. No. 5,750,826, issued May 12, 1998, and
U.S. Pat. No. 5,981,830, issued Nov. 9, 1999).
[0152] In addition to homologous recombination, additional methods
such as microinjection can be used which add genes to the host
genome without removing host genes. Microinjection can produce a
transgenic animal that is capable of expressing both endogenous and
exogenous CARD-containing polypeptides. Inducible promoters can be
linked to the coding region of nucleic acids to provide a means to
regulate expression of the transgene. Tissue specific regulatory
elements can be linked to the coding region to permit
tissue-specific expression of the transgene. Transgenic animal
model systems are useful for in vivo screening of compounds for
identification of specific ligands, i.e., agonists and antagonists,
which activate or inhibit CARD-containing polypeptide
responses.
[0153] In accordance with another embodiment of the invention, a
method is provided for identifying a CARD-associated polypeptide
(CAP). The method is carried out by contacting an invention
CARD-containing polypeptide with a candidate CAP and detecting
association of the CARD-containing polypeptide with the CAP.
[0154] As used herein, the term "CARD-associated polypeptide" or
"CAP" means a polypeptide that can specifically bind to the
CARD-containing polypeptides of the invention, or to any functional
fragment of a CARD-containing polypeptide of the invention. Because
CARD-containing polypeptides of the invention contain domains which
can self-associate, other CARD-containing polypeptides are
exemplary CAPs. Other exemplary CAPs are proteins and fragments
thereof that can bind a CARD, ERM, PDZ or filament domain of an
invention CARD-containing polypeptide. For example, cytoskeletal
components that bind the filament domain of CARD-10X or the ERM
domain of CARD-11X are exemplary CAPs.
[0155] A CAP can be identified and its binding with a
CARD-containing polypeptide of the invention further characterized,
for example, using in vitro protein binding assays similar to those
described in, for example, Ausubel et al., supra, 2000, including
co-immunoprecipitation assays, sedimentation assays, affinity
chromatography, gel-overlay assays, radiolabeled ligand binding
assays, surface plasmon resonance (SPR) on BIAcore, nuclear
magnetic resonance (NMR) spectroscopy, circular dichroism (CD)
spectroscopy, and mass spectroscopy. A CAP can also be identified
and characterized in vivo using protein-interaction assays and
methods known in the art, including yeast two-hybrid assays and
FRET-based binding assays.
[0156] Normal association of CARD-containing polypeptide and a CAP
polypeptide in a cell can be altered due, for example, to the
expression in the cell of a variant CAP or CARD-containing
polypeptide, respectively, either of which can compete with the
normal binding function of a CARD-containing polypeptide and,
therefore, can decrease the association of CAP and CARD-containing
polypeptides in a cell. The term "variant" is used generally herein
to mean a polypeptide that is different from the CAP or
CARD-containing polypeptide that normally is found in a particular
cell type. Thus, a variant can include a mutated protein or a
naturally occurring protein, such as an isoform, that is not
normally found in a particular cell type.
[0157] As used herein, a "candidate CAP" refers to a polypeptide
containing a sequence known or suspected of binding one or more
CARD-containing polypeptides of the invention. Thus, a CAP can
represent a full-length protein or a CARD-associating fragment
thereof. Likewise, a CAP-encoding nucleic acid need not encode the
full-length protein, but only the CARD-associating fragment of the
CAP.
[0158] Since CARD-containing polypeptides can be involved in
apoptosis and anoikis, the association of a CAP with a
CARD-containing polypeptide can affect the sensitivity or
resistance of a cell to apoptosis or can induce or block apoptosis
induced by external or internal stimuli. The identification of
various CAPs by use of known methods can be used to determine the
function of these CAPs in cell death or signal transduction
pathways controlled by CARD-containing polypeptides, allowing for
the development of assays that are useful for identifying agents
that effectively alter the association of a CAP with a
CARD-containing polypeptide. Such agents can be useful for
providing effective therapy for conditions caused, at least in
part, by insufficient apoptosis, such as a cancer, autoimmune
disease or certain viral infections. Such agents can also be useful
for providing an effective therapy for diseases where excessive
apoptosis is known to occur, such as stroke, heart failure, or
AIDS.
[0159] A further embodiment of the invention provides a method to
identify agents that can effectively alter CARD-containing
polypeptide activity, for example the ability of CARD-containing
polypeptides to associate with one or more CAPs. Thus, the present
invention provides a screening assay useful for identifying an
effective agent, which can alter the association of a
CARD-containing polypeptide with a CARD-associated polypeptide
(CAP), such as a heterologous CARD-containing polypeptide. Since
CARD-containing polypeptides are involved in biochemical processes
such as apoptosis, the identification of such effective agents can
be useful for altering the level of a biochemical process such as
apoptosis in a cell, for example in a cell of a subject having a
pathology characterized by an increased or decreased level of
apoptosis.
[0160] Further, effective agents can be useful for alteration of
other biochemical process modulated by a CARD-containing
polypeptide of the invention, including, for example, NF-.kappa.B
induction, cytokine processing, cytokine receptor signaling,
caspase-mediated proteolysis, cytoskeletal integrity, inflammation
and cell adhesion.
[0161] As used herein, the term "agent" means a chemical or
biological molecule such as a simple or complex organic molecule, a
peptide, a peptido-mimetic, a polypeptide, a protein or an
oligonucleotide that has the potential for altering the association
of a CARD-containing polypeptide with a heterologous protein or
altering the ability of a CARD-containing polypeptide to
self-associate or altering the ligand binding or catalytic activity
of a CARD-containing polypeptide. An exemplary ligand binding
activity is nucleotide binding activity, such as ADP or ATP binding
activity; and exemplary catalytic activities are nucleotide
hydrolytic activity and proteolytic activity. In addition, the term
"effective agent" is used herein to mean an agent that is confirmed
as capable of altering the association of a CARD-containing
polypeptide with a heterologous protein or altering the ability of
a CARD-containing polypeptide to self-associate or altering the
ligand binding or catalytic activity of a CARD-containing
polypeptide. For example, an effective agent may be an anti-CARD
antibody, a CARD-associated polypeptide, a caspase inhibitor, and
the like.
[0162] As used herein, the term "alter the association" means that
the association between two specifically interacting polypeptides
either is increased or decreased due to the presence of an
effective agent. As a result of an altered association of
CARD-containing polypeptide with another polypeptide in a cell, the
activity of the CARD-containing polypeptide or the CAP can be
increased or decreased, thereby altering a biochemical process, for
example, the level of apoptosis in the cell. As used herein, the
term "alter the activity" means that the agent can increase or
decrease the activity of a CARD-containing polypeptide in a cell,
thereby modulating a biochemical process in a cell, for example,
the level of apoptosis in the cell. Similarly, the term "alter the
level" of a biological process modulated by a CARD-containing
polypeptide refers to an increase or decrease a biochemical process
which occurs upon altering the activity of a CARD-containing
polypeptide. For example, an effective agent can increase or
decrease the CARD:CARD-associating activity of a CARD-containing
polypeptide, which can result in decreased apoptosis. An effective
agent can also increase or decrease the association between the ERM
domain, filament domain or PDZ domain of a polypeptide of the
invention and a cellular component, thereby altering cytoskeletal
organization.
[0163] An effective agent can act by interfering with the ability
of a CARD-containing polypeptide to associate with another
polypeptide, or can act by causing the dissociation of a
CARD-containing polypeptide from a complex with a CARD-associated
polypeptide, wherein the ratio of bound CARD-containing polypeptide
to free CARD-containing polypeptide is related to the level of a
biochemical process, such as, apoptosis, in a cell. For example,
binding of a ligand to a CAP can allow the CAP, in turn, to bind a
specific CARD-containing polypeptide such that all of the specific
CARD-containing polypeptide is bound to a CAP, and can result in
decreased apoptosis. The association, for example, of a
CARD-containing polypeptide and a CARD-containing polypeptide can
result in activation or inhibition of the NB-ARC:NB-ARC-associating
activity of a CARD-containing polypeptide. In the presence of an
effective agent, the association of a CARD-containing polypeptide
and a CAP can be altered, which can, for example, alter the
activation of caspases in the cell. As a result of the altered
caspase activation, the level of apoptosis in a cell can be
increased or decreased. Thus, the identification of an effective
agent that alters the association of a CARD-containing polypeptide
with another polypeptide can allow for the use of the effective
agent to increase or decrease the level of a biological process
such as apoptosis.
[0164] An effective agent can be useful, for example, to increase
the level of apoptosis in a cell such as a cancer cell, which is
characterized by having a decreased level of apoptosis as compared
to its normal cell counterpart. An effective agent also can be
useful, for example, to decrease the level of apoptosis in a cell
such as a T lymphocyte in a subject having a viral disease such as
acquired immunodeficiency syndrome, which is characterized by an
increased level of apoptosis in an infected T cell as compared to a
normal T cell. Thus, an effective agent can be useful as a
medicament for altering the level of apoptosis in a subject having
a pathology characterized by increased or decreased apoptosis. In
addition, an effective agent can be used, for example, to decrease
the level of apoptosis and, therefore, increase the survival time
of a cell such as a hybridoma cell in culture. The use of an
effective agent to prolong the survival of a cell in vitro can
significantly improve bioproduction yields in industrial tissue
culture applications.
[0165] It will be appreciated that a functional fragment or peptide
of a CARD-containing polypeptide or a CAP can be an effective
agent, so long as it alters the association between a
CARD-containing polypeptide and a CAP. Such peptides, which can be
as small as about five amino acids, can be identified, for example,
by screening a peptide library (see, for example, Ladner et al.,
U.S. Pat. No.: 5,223,409, which is incorporated herein by
reference) to identify peptides that can bind a CARD-containing
polypeptide or a CARD-associated polypeptide.
[0166] Such peptide effective agents can act by decreasing the
association of a CARD-containing polypeptide with a CAP in a cell
by competing for binding to the CARD-containing polypeptide. A
non-naturally occurring peptido-mimetic also can be useful as an
effective agent. Such a peptido-mimetic can include, for example, a
peptoid, which is peptide-like sequence containing N-substituted
glycines, or an oligocarbamate. A peptido-mimetic can be
particularly useful as an effective agent due, for example, to
having an increased stability to enzymatic degradation in vivo.
[0167] In accordance with another embodiment of the present
invention, there is provided a method of identifying an effective
agent that alters the association of an invention CARD-containing
polypeptide with a CARD-associated polypeptide (CAP), by the steps
of:
[0168] (a) contacting the CARD-containing polypeptide and CAP
polypeptide under conditions that allow the polypeptides to
associate, with an agent suspected of being able to alter the
association of the CARD-containing polypeptide and CAP
polypeptides; and
[0169] (b) determining association of the CARD-containing
polypeptide and the CAP polypeptide, where an agent that alters the
association is identified as an effective agent.
[0170] Methods are well-known in the art for detecting the altered
association of the CARD-containing polypeptide and CAP
polypeptides, for example, measuring protein:protein binding,
protein degradation or apoptotic activity can be employed in
bioassays described herein to identify agents as agonists or
antagonists of CARD-containing polypeptides. As described herein,
CARD-containing polypeptides have the ability to self-associate.
Thus, methods for identifying effective agents that alter the
association of a CARD-containing polypeptide with a CAP are useful
for identifying effective agents that alter the ability of a
CARD-containing polypeptide to self-associate.
[0171] As used herein, "conditions that allow a CARD-containing
polypeptide and a CAP polypeptide to associate" refers to
environmental conditions in which a CARD-containing polypeptide and
CAP specifically associate. Such conditions will typically be
aqueous conditions, with a pH between 3.0 and 11.0, and temperature
below 100.degree. C. Preferably, the conditions will be aqueous
conditions with salt concentrations below the equivalent of 1 M
NaCl, and pH between 5.0 and 9.0, and temperatures between
0.degree. C. and 50.degree. C. Most preferably, the conditions will
range from physiological conditions of normal yeast or mammalian
cells, or conditions favorable for carrying out in vitro assays
such as immunoprecipitation and GST protein:protein association
assays, and the like.
[0172] The present invention also provides in vitro screening
assays. Such screening assays are particularly useful in that they
can be automated, which allows for high through-put screening, for
example, of randomly or rationally designed agents such as drugs,
peptidomimetics or peptides in order to identify those agents that
effectively alter the association of a CARD-containing polypeptide
and a CAP or the catalytic or ligand binding activity of a
CARD-containing polypeptide and, thereby, alter a biochemical
process modulated by a CARD-containing polypeptide such as
apoptosis. An in vitro screening assay can utilize, for example, a
CARD-containing polypeptide including a CARD-containing fusion
protein such as a CARD-glutathione-S-transferase fusion protein.
For use in the in vitro screening assay, the CARD-containing
polypeptide should have an affinity for a solid substrate as well
as the ability to associate with a CARD-associated polypeptide. For
example, when a CARD-containing polypeptide is used in the assay,
the solid substrate can contain a covalently attached anti-CARD
antibody. Alternatively, a GST/CARD fusion protein can be used in
the assay and the solid substrate can contain covalently attached
glutathione, which is bound by the GST component of the GST/CARD
fusion protein. Similarly, a CARD-associated polypeptide can be
used in such screening asays.
[0173] An in vitro screening assay can be performed, for example,
by allowing a CARD-containing polypeptide to bind to the solid
support, then adding a CARD-associated polypeptide and an agent to
be tested. Reference reactions, which do not contain an agent, can
be performed in parallel. Following incubation under suitable
conditions, which include, for example, an appropriate buffer
concentration and pH and time and temperature that permit binding
of the particular CARD-containing polypeptide and CARD-associated
polypeptide, the amount of protein that has associated in the
absence of an agent and in the presence of an agent can be
determined. The association of a CARD-associated polypeptide with a
CARD-containing polypeptide can be detected, for example, by
attaching a detectable moiety such as a radionuclide or a
fluorescent label to a CARD-associated polypeptide and measuring
the amount of label that is associated with the solid support,
wherein the amount of label detected indicates the amount of
association of the CARD-associated polypeptide with a
CARD-containing polypeptide. An effective agent is determined by
comparing the amount of specific binding in the presence of an
agent as compared to a reference level of binding, wherein an
effective agent alters the association of CARD-containing
polypeptide with the CARD-associated polypeptide. Such an assay is
particularly useful for screening a panel of agents such as a
peptide library in order to detect an effective agent.
[0174] Various binding assays to identify cellular proteins that
interact with protein binding domains are known in the art and
include, for example, yeast two-hybrid screening assays (see, for
example, U.S. Pat. Nos. 5,283,173, 5,468,614 and 5,667,973; Ausubel
et al., supra, 2000; Luban et al., Curr. Opin. Biotechnol.
6:59-64(1995)) and affinity column chromatography methods using
cellular extracts. By synthesizing or expressing polypeptide
fragments containing various CARD-associating sequences or
deletions, the CARD binding interface can be readily
identified.
[0175] Another assay for screening of agents that alter the
activity of a CARD-containing polypeptide is based on altering the
phenotype of yeast by expressing a CARD-containing polypeptide. In
one embodiment, expression of a CARD-containing polypeptide can be
inducible (Tao et al., J. Biol. Chem. 273:23704-23708 (1998), and
the compounds can be screened when CARD-containing polypeptide
expression is induced. CARD-containing polypeptides of the
invention can also be co-expressed in yeast with CAP polypeptides
used to screen for compounds that antagonize the activity of the
CARD-containing polypeptide.
[0176] Also provided by the present invention are assays to
identify agents that alter CARD-containing polypeptide expression.
Methods to determine CARD-containing polypeptide expression can
involve detecting a change in CARD-containing polypeptide abundance
in response to contacting the cell with an agent that modulates
CARD-containing polypeptide expression. Assays for detecting
changes in polypeptide expression include, for example,
immunoassays with CARD-specific antibodies, such as immunoblotting,
immunofluorescence, immunohistochemistry and immunoprecipitation
assays, as described herein.
[0177] As understood by those of skill in the art, assay methods
for identifying agents that alter CARD-containing polypeptide
activity generally require comparison to a reference. One type of a
"reference" is a cell or culture that is treated substantially the
same as the test cell or test culture exposed to the agent, with
the distinction that the "reference" cell or culture is not exposed
to the agent. Another type of "reference" cell or culture can be a
cell or culture that is identical to the test cells, with the
exception that the "reference" cells or culture do not express a
CARD-containing polypeptide. Accordingly, the response of the
transfected cell to an agent is compared to the response, or lack
thereof, of the "reference" cell or culture to the same agent under
the same reaction conditions.
[0178] Methods for producing pluralities of agents to use in
screening for compounds that alter the activity of a
CARD-containing polypeptide, including chemical or biological
molecules such as simple or complex organic molecules,
metal-containing compounds, carbohydrates, peptides, proteins,
peptidomimetics, glycoproteins, lipoproteins, nucleic acids,
antibodies, and the like, are well known in the art and are
described, for example, in Huse, U.S. Pat. No. 5,264,563; Francis
et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al.,
Curr. Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998);
Eichler et al., Med. Res. Rev. 15:481-496 (1995); and the like.
Libraries containing large numbers of natural and synthetic agents
also can be obtained from commercial sources. Combinatorial
libraries of molecules can be prepared using well known
combinatorial chemistry methods (Gordon et al., J. Med. Chem. 37:
1233-1251 (1994); Gordon et al., J. Med. Chem. 37: 1385-1401
(1994); Gordon et al., Acc. Chem. Res. 29:144-154 (1996); Wilson
and Czarnik, eds., Combinatorial Chemistry: Synthesis and
Application, John Wiley & Sons, New York (1997)).
[0179] The invention further provides a method of diagnosing or
predicting clinical prognosis of a pathology characterized by an
increased or decreased level of a CARD-containing polypeptide in a
subject. The method includes the steps of (a) obtaining a test
sample from the subject; (b) contacting the sample with an agent
that can bind a CARD-containing polypeptide or nucleic acid
molecule of the invention under suitable conditions, wherein the
conditions allow specific binding of the agent to the
CARD-containing polypeptide; and (c) comparing the amount of the
specific binding in the test sample with the amount of specific
binding in a reference sample, wherein an increased or decreased
amount of the specific binding in the test sample as compared to
the reference sample is diagnostic of, or predictive of the
clinical prognosis of, a pathology. The agent can be, for example,
an anti-CARD antibody, a CARD-associated-polypeptide (CAP), or a
CARD-encoding nucleic acid.
[0180] Exemplary pathologies for diagnosis or the prediction of
clinical prognosis include any of the pathologies described herein,
such as neoplastic pathologies (e.g. cancer), autoimmune diseases,
and other pathologies related to abnormal cell proliferation or
abnormal cell death (e.g. apoptosis), as disclosed herein.
[0181] The invention also provides a method of diagnosing cancer or
monitoring cancer therapy by contacting a test sample from a
patient with a CARD-specific antibody. The invention additionally
provides a method of assessing prognosis (e.g., predicting the
clinical prognosis) of patients with cancer comprising contacting a
test sample from a patient with a CARD-specific antibody.
[0182] The invention additionally provides a method of diagnosing
cancer or monitoring cancer therapy by contacting a test sample
from a patient with a oligonucleotide that selectively hybridizes
to a CARD-encoding nucleic acid molecule. The invention further
provides a method of assessing prognosis (e.g., predicting the
clinical prognosis) of patients with cancer by contacting a test
sample from a patient with a oligonucleotide that selectively
hybridizes to a CARD-encoding nucleic acid molecule.
[0183] The methods of the invention for diagnosing cancer or
monitoring cancer therapy using a CARD-specific antibody or
oligonucleotide or nucleic acid that selectively hybridizes to a
CARD-encoding nucleic acid molecule can be used, for example, to
segregate patients into a high risk group or a low risk group for
diagnosing cancer or predicting risk of metastasis or risk of
failure to respond to therapy. Therefore, the methods of the
invention can be advantageously used to determine, for example, the
risk of metastasis in a cancer patient, or the risk of an
autoimmune disease of a patient, or as a prognostic indicator of
survival or disease progression in a cancer patient or patient with
an autoimmune disease. One of ordinary skill in the art would
appreciate that the prognostic indicators of survival for cancer
patients suffering from stage I cancer can be different from those
for cancer patients suffering from stage IV cancer. For example,
prognosis for stage I cancer patients can be oriented toward the
likelihood of continued growth and/or metastasis of the cancer,
whereas prognosis for stage IV cancer patients can be oriented
toward the likely effectiveness of therapeutic methods for treating
the cancer. Accordingly, the methods of the invention directed to
measuring the level of or determining the presence of a
CARD-containing polypeptide or CARD-encoding nucleic acid can be
used advantageously as a prognostic indicator for the presence or
progression of a cancer or response to therapy.
[0184] The present invention also provides therapeutic compositions
useful for practicing the therapeutic methods described herein.
Therapeutic compositions of the present invention, such as
pharmaceutical compositions, contain a physiologically compatible
carrier together with an invention CARD-containing polypeptide (or
functional fragment thereof), an invention CARD-encoding nucleic
acid, an agent that alters CARD activity or expression identified
by the methods described herein, or an anti-CARD antibody, as
described herein, dissolved or dispersed therein as an active
ingredient. In a preferred embodiment, the therapeutic composition
is not immunogenic when administered to a mammal or human patient
for therapeutic purposes.
[0185] As used herein, the terms "pharmaceutically acceptable",
"physiologically compatible" and grammatical variations thereof, as
they refer to compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials are capable
of administration to a mammal without the production of undesirable
physiological effects such as nausea, dizziness, gastric upset, and
the like.
[0186] The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
known in the art. Typically such compositions are prepared as
injectibles either as liquid solutions or suspensions; however,
solid forms suitable for solution, or suspension, in liquid prior
to use can also be prepared. The preparation can also be
emulsified.
[0187] The active ingredient can be mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient in amounts suitable for use in the therapeutic methods
described herein. Suitable excipients are, for example, water,
saline, dextrose, glycerol, ethanol, or the like, as well as
combinations of any two or more thereof. In addition, if desired,
the composition can contain minor amounts of auxiliary substances
such as wetting or emulsifying agents, pH buffering agents, and the
like, which enhance the effectiveness of the active ingredient.
[0188] The therapeutic composition of the present invention can
include pharmaceutically acceptable salts of the components
therein. Pharmaceutically acceptable nontoxic salts include the
acid addition salts (formed with the free amino groups of the
polypeptide) that are formed with inorganic acids such as, for
example, hydrochloric acid, hydrobromic acid, perchloric acid,
nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid,
acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic
acid, oxalic acid, malonic acid, succinic acid, maleic acid,
fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic
acid, sulfanilic acid, and the like.
[0189] Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium
hydroxide, ammonium hydroxide, potassium hydroxide, and the like;
and organic bases such as mono-, di-, and tri-alkyl and -aryl
amines (e.g., triethylamine, diisopropyl amine, methyl amine,
dimethyl amine, and the like) and optionally substituted
ethanolamines (e.g., ethanolamine, diethanolamine, and the
like).
[0190] Physiologically tolerable carriers are well known in the
art. Exemplary liquid carriers are sterile aqueous solutions that
contain no materials in addition to the active ingredients and
water, or contain a buffer such as sodium phosphate at
physiological pH, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous carriers can
contain more than one buffer salt, as well as salts such as sodium
and potassium chlorides, dextrose, polyethylene glycol and other
solutes.
[0191] Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary additional
liquid phases include glycerin, vegetable oils such as cottonseed
oil, and water-oil emulsions.
[0192] As described herein, an "effective amount" is a
predetermined amount calculated to achieve the desired therapeutic
effect, i.e., to alter the protein binding activity of a
CARD-containing polypeptide or other biological activity, resulting
in altered biochemical process modulated by a CARD-containing
polypeptide. The required dosage will vary with the particular
treatment and with the duration of desired treatment; however, it
is anticipated that dosages between about 10 micrograms and about 1
milligram per kilogram of body weight per day will be used for
therapeutic treatment. It may be particularly advantageous to
administer such agents in depot or long-lasting form as discussed
herein. A therapeutically effective amount is typically an amount
of an agent identified herein that, when administered in a
physiologically acceptable composition, is sufficient to achieve a
plasma concentration of from about 0.1 .mu.g/ml to about 100
.mu.g/ml, preferably from about 1.0 .mu.g/ml to about 50 .mu.g/ml,
more preferably at least about 2 .mu.g/ml and usually 5 to 10
.mu.g/ml. Therapeutic invention anti-CARD antibodies can be
administered in proportionately appropriate amounts in accordance
with known practices in this art.
[0193] Also provided herein are methods of treating pathologies
characterized by abnormal cell proliferation, abnormal cell death,
or inflammation said method comprising administering an effective
amount of an invention therapeutic composition. Such compositions
are typically administered in a physiologically compatible
composition.
[0194] Exemplary abnormal cell proliferation diseases associated
with CARD-containing polypeptides contemplated herein for treatment
according to the present invention include cancer pathologies,
keratinocyte hyperplasia, neoplasia, keloid, benign prostatic
hypertrophy, inflammatory hyperplasia, fibrosis, smooth muscle cell
proliferation in arteries following balloon angioplasty
(restenosis), and the like. Exemplary cancer pathologies
contemplated herein for treatment include, gliomas, carcinomas,
adenocarcinomas, sarcomas, melanomas, hamartomas, leukemias,
lymphomas, and the like. Further diseases associated with
CARD-containing polypeptides contemplated herein for treatment
according to the present invention include inflammatory diseases
and diseases of cell loss. Such diseases include allergies,
inflammatory diseases including arthritis, lupus, Schrogen's
syndrome, Crohn's disease, ulcerative colitis, as well as allograft
rejection, such as graft-versus-host disease, and the like.
CARD-containing polypeptides can also be useful in design of
strategies for preventing diseases related to abnormal cell death
in conditions such as stroke, myocardial infarction, heart failure,
neurodegenerative diseases such as Parkinson's and Alzheimer's
diseases, and for immunodeficiency associated diseases such as HIV
infection, HIV-related disease, and the like.
[0195] Methods of treating pathologies can include methods of
modulating the activity of one or more oncogenic proteins, wherein
the oncogenic proteins specifically interact with a CARD-containing
polypeptide of the invention. Methods of modulating the activity of
such oncogenic proteins will include contacting the oncogenic
protein with a substantially pure CARD-containing polypeptide or an
active fragment (i.e., oncogenic protein-binding fragment) thereof.
This contacting will alter the activity of the oncogenic protein,
thereby providing a method of treating a pathology caused by the
oncogenic protein. Further methods of modulating the activity of
oncogenic proteins will include contacting the oncogenic protein
with an agent, wherein the agent alters interaction between a
CARD-containing polypeptide and an oncogenic protein.
[0196] Also contemplated herein, are therapeutic methods using
invention pharmaceutical compositions for the treatment of
pathological disorders in which there is too little cell division,
such as, for example, bone marrow aplasias, immunodeficiencies due
to a decreased number of lymphocytes, and the like. Methods of
treating a variety of inflammatory diseases with invention
therapeutic compositions are also contemplated herein, such as
treatment of sepsis, fibrosis (e.g., scarring), arthritis, graft
versus host disease, and the like.
[0197] The present invention also provides methods for diagnosing a
pathology that is characterized by an increased or decreased level
of a biochemical process to determine whether the increased or
decreased level of the biochemical process is due, for example, to
increased or decreased expression of a CARD-containing polypeptide
or to expression of a variant CARD-containing polypeptide. As
disclosed herein, such biochemical processes include apoptosis,
NF-.kappa.B induction, cytokine processing, caspase-mediated
proteolysis, transcription, inflammation, cell adhesion,
cytoskeletal integrity and the like.
[0198] The identification of such a pathology can allow for
intervention therapy using an effective agent, nucleic acid
molecule, antisense oligonucleotide or polypeptide as described
herein. In general, a test sample can be obtained from a subject
having a pathology characterized by having or suspected of having
increased or decreased apoptosis and can be compared to a reference
sample from a normal subject to determine whether a cell in the
test sample has, for example, increased or decreased expression of
a CARD-encoding gene. The level of a CARD-containing polypeptide in
a cell can be determined by contacting a sample with a reagent such
as an anti-CARD antibody or a CARD-associated polypeptide, either
of which can specifically bind a CARD-containing polypeptide. For
example, the level of a CARD-containing polypeptide in a cell can
determined by well known immunoassay or immunohistochemical methods
using an anti-CARD antibody (see, for example, Reed et al., Anal.
Biochem. 205:70-76 (1992); see, also, Harlow and Lane, supra,
(1988)).
[0199] As used herein, the term "reagent" means a chemical or
biological molecule that can specifically bind to a CARD-containing
polypeptide or to a bound CARD/CARD-associated polypeptide complex.
For example, either an anti-CARD antibody or a CARD-associated
polypeptide can be a reagent for a CARD-containing polypeptide,
whereas either an anti-CARD antibody or an anti-CARD-associated
polypeptide antibody can be a reagent for a CARD/CARD-associated
polypeptide complex.
[0200] Increased or decreased expression of a CARD-encoding gene in
a cell in a test sample can be determined, for example, by
comparison to an expected normal level of CARD-containing
polypeptide or CARD-encoding mRNA in a particular cell type. A
normal range of CARD-containing polypeptide or CARD-encoding mRNA
levels in various cell types can be determined by sampling a
statistically significant number of normal subjects. In addition, a
reference sample can be evaluated in parallel with a test sample in
order to determine whether a pathology characterized by increased
or decreased apoptosis is due to increased or decreased expression
of a CARD-encoding gene. The test sample can be examined using, for
example, immunohistochemical methods as described above or the
sample can be further processed and examined. For example, an
extract of a test sample can be prepared and examined to determine
whether a CARD-containing polypeptide in the sample can associate
with a CARD-associated polypeptide in the same manner as a
CARD-containing polypeptide from a reference cell or whether,
instead, a variant CARD-containing polypeptide is expressed in the
cell.
[0201] In accordance with another embodiment of the present
invention, there are provided diagnostic systems, preferably in kit
form, comprising at least one invention CARD-encoding nucleic acid,
CARD-containing polypeptide, and/or anti-CARD antibody described
herein, in a suitable packaging material. In one embodiment, for
example, the diagnostic nucleic acids are derived from any of SEQ
ID NOS:1, 7 or 15. Invention diagnostic systems are useful for
assaying for the presence or absence of CARD-encoding nucleic acid
in either genomic DNA or in transcribed CARD-encoding nucleic acid,
such as mRNA or cDNA.
[0202] A suitable diagnostic system includes at least one invention
CARD-encoding nucleic acid, CARD-containing polypeptide, and/or
anti-CARD antibody, preferably two or more invention nucleic acids,
proteins and/or antibodies, as a separately packaged chemical
reagent(s) in an amount sufficient for at least one assay.
Instructions for use of the packaged reagent are also typically
included. Those of skill in the art can readily incorporate
invention nucleic acid probes and/or primers into kit form in
combination with appropriate buffers and solutions for the practice
of the invention methods as described herein.
[0203] As employed herein, the phrase "packaging material" refers
to one or more physical structures used to house the contents of
the kit, such as invention nucleic acid probes or primers, and the
like. The packaging material is constructed by well known methods,
preferably to provide a sterile, contaminant-free environment. The
packaging material has a label which indicates that the invention
nucleic acids can be used for detecting a particular CARD-encoding
sequence including the nucleotide sequences set forth in SEQ ID
NOS:1, 7 or 15 or mutations or deletions therein, thereby
diagnosing the presence of, or a predisposition for a pathology
such as cancer or an autoimmune disease. In addition, the packaging
material contains instructions indicating how the materials within
the kit are employed both to detect a particular sequence and
diagnose the presence of, or a predisposition for a pathology such
as cancer or an autoimmune disease.
[0204] The packaging materials employed herein in relation to
diagnostic systems are those customarily utilized in nucleic
acid-based diagnostic systems. As used herein, the term "package"
refers to a solid matrix or material such as glass, plastic, paper,
foil, and the like, capable of holding within fixed limits an
isolated nucleic acid, oligonucleotide, or primer of the present
invention. Thus, for example, a package can be a glass vial used to
contain milligram quantities of a contemplated nucleic acid,
oligonucleotide or primer, or it can be a microtiter plate well to
which microgram quantities of a contemplated nucleic acid probe
have been operatively affixed.
[0205] "Instructions for use" typically include a tangible
expression describing the reagent concentration or at least one
assay method parameter, such as the relative amounts of reagent and
sample to be admixed, maintenance time periods for reagent/sample
admixtures, temperature, buffer conditions, and the like.
[0206] A diagnostic assay should include a simple method for
detecting the amount of a CARD-containing polypeptide or
CARD-encoding nucleic acid in a sample that is bound to the
reagent. Detection can be performed by labeling the reagent and
detecting the presence of the label using well known methods (see,
for example, Harlow and Lane, supra, 1988; chap. 9, for labeling an
antibody). A reagent can be labeled with various detectable
moieties including a radiolabel, an enzyme, biotin or a
fluorochrome. Materials for labeling the reagent can be included in
the diagnostic kit or can be purchased separately from a commercial
source. Following contact of a labeled reagent with a test sample
and, if desired, a control sample, specifically bound reagent can
be identified by detecting the particular moiety.
[0207] A labeled antibody that can specifically bind the reagent
also can be used to identify specific binding of an unlabeled
reagent. For example, if the reagent is an anti-CARD antibody, a
second antibody can be used to detect specific binding of the
anti-CARD antibody. A second antibody generally will be specific
for the particular class of the first antibody. For example, if an
anti-CARD antibody is of the IgG class, a second antibody will be
an anti-IgG antibody. Such second antibodies are readily available
from commercial sources. The second antibody can be labeled using a
detectable moiety as described above. When a sample is labeled
using a second antibody, the sample is first contacted with a first
antibody, then the sample is contacted with the labeled second
antibody, which specifically binds to the first antibody and
results in a labeled sample.
[0208] In accordance with another embodiment of the invention,
there are provided methods for determining a prognosis of disease
free or overall survival in a patient suffering from cancer. For
example, it is contemplated herein that abnormal levels of
CARD-containing polypeptides (either higher or lower) in primary
tumor tissue show a high correlation with either increased or
decreased tumor recurrence or spread, and therefore indicates the
likelihood of disease free or overall survival. Thus, the present
invention advantageously provides a significant advancement in
cancer management because early identification of patients at risk
for tumor recurrence or spread will permit aggressive early
treatment with significantly enhanced potential for survival. Also
provided are methods for predicting the risk of tumor recurrence or
spread in an individual having a cancer tumor; methods for
screening a cancer patient to determine the risk of tumor
metastasis; and methods for determining the proper course of
treatment for a patient suffering from cancer. These methods are
carried out by collecting a sample from a patient and comparing the
level of CARD-encoding gene expression in the patient to the level
of expression in a control or to a reference level of CARD-encoding
gene expression as defined by patient population sampling, tissue
culture analysis, or any other method known for determining
reference levels for determination of disease prognosis. The level
of CARD-encoding gene expression in the patient is then classified
as higher than the reference level or lower than the reference
level, wherein the prognosis of survival or tumor recurrence is
different for patients with higher levels than the prognosis for
patients with lower levels.
[0209] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Identification of CARD-containing Polypeptides
[0210] CARD-10X, -11X and -12X proteins were identified using
Saturated Blast searches (Li et al., "Saturated BLAST: An automated
multiple intermediate sequence search used to detect distant
homology," Bioinformatics (2000), in press). A representative set
of CARD domains was used as queries and a cascade of TBLASTN and
PSI-BLAST searches was performed on nucleotide databases at NCBI
(htgs, gss, dbest) and the NR protein database.
[0211] The new candidate CARD-domains were confirmed by:
[0212] 1) Determining whether the identified nucleotide sequence
falls within an exon as predicted by the GENSCAN program.
[0213] 2) Identifying EST sequences corresponding to the novel CARD
domain.
[0214] 3) Performing a FFAS fold prediction calculation against a
database of proteins of known structures (PDB) enriched in
apoptotic domains (Rychlewski et al., Protein Science 9:232-241
(2000)).
[0215] 4) Performing a PSI-BLAST search against the NR protein
database.
[0216] The novelty of the discovered CARD domain was assessed
by:
[0217] 1) Performing a PSI-BLAST search against the NR protein
database at NCBI.
[0218] 2) Performing a PSI-BLAST search against a database of known
CARD domains.
[0219] 3) Performing a BLASTN search (using the nucleotide
sequences of CARD-10X, -11X, -12X) against the NR nucleotide
database at NCBI.
[0220] Additional domains in CARD-10X, 11X, 12X (e.g. PDZ, domain,
filament domain) were identified by performing an exon prediction
(GENSCAN) analysis for full genomic contigs in which the CARD
domains were found.
[0221] The protein sequences obtained in this way were used as
queries for FFAS searches against the PDB, PFAM and COG databases,
as well as for HMM searches in the PFAM database (Bateman et al.,
Nucleic Acids Res. 27:260-262 (1999)).
[0222] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0223] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
* * * * *