U.S. patent application number 11/457765 was filed with the patent office on 2007-05-24 for novel apoptosis-modulating proteins, dna encoding the proteins and methods of use thereof.
This patent application is currently assigned to TANOX, INC.. Invention is credited to Philip J. Barr, Michael C. Kiefer.
Application Number | 20070117748 11/457765 |
Document ID | / |
Family ID | 26856568 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070117748 |
Kind Code |
A1 |
Kiefer; Michael C. ; et
al. |
May 24, 2007 |
NOVEL APOPTOSIS-MODULATING PROTEINS, DNA ENCODING THE PROTEINS AND
METHODS OF USE THEREOF
Abstract
The present invention provides a novel family of
apoptosis-modulating proteins. Nucleotide and amino acid residue
sequences and methods of use thereof are also provided.
Inventors: |
Kiefer; Michael C.;
(Clayton, CA) ; Barr; Philip J.; (Berkeley,
CA) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY
SUITE 1200
DENVER
CO
80202
US
|
Assignee: |
TANOX, INC.
10301 Stella Link
Houston
TX
|
Family ID: |
26856568 |
Appl. No.: |
11/457765 |
Filed: |
July 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10101482 |
Mar 18, 2002 |
7108989 |
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11457765 |
Jul 14, 2006 |
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08470865 |
Jun 6, 1995 |
6586395 |
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10101482 |
Mar 18, 2002 |
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08320157 |
Oct 7, 1994 |
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08470865 |
Jun 6, 1995 |
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08160067 |
Nov 30, 1993 |
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08320157 |
Oct 7, 1994 |
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Current U.S.
Class: |
435/69.1 ;
514/18.9; 514/19.3 |
Current CPC
Class: |
A61P 1/12 20180101; A61P
35/00 20180101; A61K 38/00 20130101; A61P 3/04 20180101; A01K
2227/105 20130101; A61P 31/12 20180101; A61P 37/02 20180101; A61P
13/08 20180101; A61P 9/10 20180101; A61P 25/00 20180101; A61P 9/00
20180101; A61P 31/04 20180101; A01K 2267/025 20130101; C12N 15/8509
20130101; A61P 37/06 20180101; A61P 43/00 20180101; C12Q 1/6876
20130101; A61P 7/06 20180101; A01K 2267/03 20130101; A61P 3/10
20180101; A61P 1/00 20180101; A01K 2217/05 20130101; A61P 27/02
20180101; C12Q 2600/158 20130101; A61P 17/14 20180101; C07K 14/4747
20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/00 20060101
A61K038/00 |
Claims
1-58. (canceled)
59. An antisense molecule comprising an oligonucleotide of length
sufficient to modulate apoptosis, wherein said antisense
oligonucleotide is complementary to a polynucleotide encoding
CDN-1, CDN-2, CDN-3, CDN1.DELTA.1, CDN1.DELTA.2, or
CDN1.DELTA.3.
60. The antisense compound of claim 59, which is targeted to a
nucleic acid molecule encoding CDN-1 and which preferentially
inhibits the expression of CDN-1.
61. The antisense compound of claim 59, which promotes
apoptosis.
62. The antisense compound of claim 59, which inhibits
apoptosis.
63. A pharmaceutical composition comprising the antisense molecule
of claim 59 and a pharmaceutically acceptable carrier or
diluent.
64. A method of modulating apoptosis in a cell in a mammal
diagnosed as having a proliferative disease, comprising
administering to said mammal an antisense oligonucleotide of claim
59.
65. The method of claim 64, wherein said mammal is a human.
66. The method of claim 64, wherein said proliferative disease is
cancer.
67. A method to modulate apoptosis-induced cell death, comprising
contacting an endogenous polynucleotide encoding SEQ ID NO:7 in a
cell with an antisense polynucleotide that inhibits the expression
of the polynucleotide encoding SEQ ID NO:7.
68. The method of claim 67, wherein the antisense polynucleotide
hybridizes under highly stringent conditions to a gene encoding SEQ
ID NO:7.
69. The method of claim 67, wherein the antisense polynucleotide is
fully complementary to a polynucleotide encoding SEQ ID NO:7.
70. The method of claim 67, wherein the antisense polynucleotide is
fully complementary to a polynucleotide encoding a fragment of SEQ
ID NO:7 lacking up to about the first 70 amino acids of SEQ ID
NO:7, wherein the fragment modulates apoptosis in a cell.
71. The method of claim 67, wherein the antisense polynucleotide is
fully complementary to a polynucleotide encoding a fragment of SEQ
ID NO:7 truncated after about amino acid position 112, wherein the
fragment modulates apoptosis in a cell.
72. The method of claim 67, wherein the antisense polynucleotide is
fully complementary to a polynucleotide encoding an amino acid
sequence that that is greater than 97% identical to SEQ ID NO:7,
wherein the amino acid sequence modulates apoptosis in a cell.
73. The method of claim 67, wherein apoptosis-induced cell death is
increased in the cell in the presence of the polynucleotide as
compared to in the absence of the polynucleotide.
74. The method of claim 67, wherein the cell displays uncontrolled
cell growth.
75. The method of claim 74, wherein the cell is a cancer cell.
76. The method of claim 74, wherein the cell is from a B cell
lymphoma.
Description
[0001] This is a continuation-in-part of U.S. patent application
Ser. No. 08/160,067 filed Nov. 30, 1993.
FIELD OF THE INVENTION
[0002] This invention relates to novel proteins with
apoptosis-modulating activity, recombinant DNA encoding the
proteins, compositions containing the proteins and methods of use
thereof.
BACKGROUND OF THE INVENTION
[0003] Apoptosis is a normal physiologic process that leads to
individual cell death. This process of programmed cell death is
involved in a variety of normal and pathogenic biological events
and can be induced by a number of unrelated stimuli. Changes in the
biological regulation of apoptosis also occur during aging and are
responsible for many of the conditions and diseases related to
aging. Recent studies of apoptosis have implied that a common
metabolic pathway leading to cell death may be initiated by a wide
variety of signals, including hormones, serum growth factor
deprivation, chemotherapeutic agents, ionizing radiation and
infection by human immunodeficiency virus (HIV). Wyllie (1980)
Nature, 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res.
Commun. 118:392-399; Duke and Cohen -(1986) Lymphokine Res.
5:289-299; Tomei et al. (1988) Biochem. Biophys. Res. Commun.
155:324-331; Kruman et al. (1991) J. Cell. Physiol. 148:267-273;
Ameisen and Capron (1991) Immunology Today 12:102; and Sheppard and
Ascher (1992) J. AIDS 5:143. Agents that modulate the biological
control of apoptosis thus have therapeutic utility in a wide
variety of conditions.
[0004] Apoptotic cell death is characterized by cellular shrinkage,
chromatin condensation, cytoplasmic blebbing, increased membrane
permeability and interchromosomal DNA cleavage. Kerr et al. (1992)
FASEB J. 6:2450; and Cohen and Duke (1992) Ann. Rev. Immunol.
10:267. The blebs, small, membrane-encapsulated spheres that pinch
off of the surface of apoptotic cells, may continue to produce
superoxide radicals which damage surrounding cell tissue and may be
involved in inflammatory processes.
[0005] Bcl-2 was discovered at the common chromosomal translocation
site t(14:18) in follicular lymphomas and results in aberrant
over-expression of bcl-2. Tsujimoto et al. (1984) Science
226:1097-1099; and Cleary et al. (1986) Cell 47:19-28. The normal
function of bcl-2 is the prevention of apoptosis; unregulated
expression of bcl-2 in B cells is thought to lead to increased
numbers of proliferating B cells which may be a critical factor in
the development of lymphoma. McDonnell and Korsmeyer (1991) Nature
349:254-256; and, for review see, Edgington (1993) Bio/Tech.
11:787-792. Bcl-2 is also capable of blocking of .gamma.
irradiation-induced cell death. Sentman et al. (1991) Cell
67:879-888; and Strassen (1991) Cell 67:889-899. It is now known
that bcl-2 inhibits most types of apoptotic cell death and is
thought to function by regulating an antioxidant pathway at sites
of free radical generation. Hockenbery et al. (1993) Cell
75:241-251.
[0006] While apoptosis is a normal cellular event, it can also be
induced by pathological conditions and a variety of injuries.
Apoptosis is involved in a wide variety of conditions including but
not limited to, cardiovascular disease, cancer regression,
immunoregulation, viral diseases, anemia, neurological disorders,
gastrointestinal disorders, including but not limited to, diarrhea
and dysentery, diabetes, hair loss, rejection of organ transplants,
prostate hypertrophy, obesity, ocular disorders, stress and
aging.
[0007] Bcl-2 belongs to a family of proteins some of which have
been cloned and sequenced. Williams and Smith (1993) Cell
74:777-779. All references cited herein, both supra and infra, are
hereby incorporated by reference herein.
SUMMARY OF THE INVENTION
[0008] Substantially purified DNA encoding novel bcl-2 homologs,
termed cdn-1, cdn-2 and cdn-3, as well as recombinant cells and
transgenic animals expressing the cdn-1 and cdn-2 genes are
provided. The substantially purified CDN-1 and CDN-2 proteins and
compositions thereof are also provided. Diagnostic and therapeutic
methods utilizing the DNA and proteins are also provided. Methods
of screening for pharmaceutical agents that stimulate, as well as
pharmaceutical agents that inhibit cdn-1 and cdn-2 activity levels
are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 depicts the PCR primers used to isolate the cdn-1
probes.
[0010] FIG. 2 depicts the cdn-1 clones obtained by the methods
described in Example 1.
[0011] FIG. 3 depicts the nucleotide sequence of cdn-1.
[0012] FIG. 4 depicts the results of a Northern blot analysis of
multiple tissues with probes specific for both bcl-2 and cdn-1.
[0013] FIG. 5 shows the sequence of the cdn-2 cDNA and flanking
sequences and the corresponding predicted amino acid sequence of
the cdn-2 protein.
[0014] FIG. 6 shows a comparison of N-terminal amino acid sequences
of cdn-1, cdn-2 and known bcl-2 family members.
[0015] FIG. 7 shows the nucleotide sequence of cdn-3.
[0016] FIG. 8 shows the anti-apoptotic effects of cdn-1 and some of
its derivatives in serum-deprivation induced apoptosis of WIL-2
cells.
[0017] FIG. 9 shows anti-apoptotic effects of cdn-1 and some of its
derivatives in FAS-induced apoptosis of WIL-2 cells.
[0018] FIG. 10 shows modulation of apoptosis by cdn-1 and cdn-2 in
FL5.12 cells.
[0019] FIG. 11 depicts the cdn-1 derivative proteins .DELTA.1,
.DELTA.2 and .DELTA.3. The N-terminal residues are indicated by the
arrows. The remainder of the derivative proteins is the same as
full-length cdn-1.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention encompasses substantially purified
nucleotide sequences encoding the novel bcl-2 homologs, cdn-1 and
cdn-2; and the proteins encoded thereby; compositions comprising
cdn-1 and cdn-2 genes and proteins and methods of use of thereof.
Note that in copending U.S. patent application Ser. No. 08/160,067,
cdn-1 was termed cdi-1; although the name has been changed, the
nucleotide sequence remains identical. The invention further
includes recombinant cells and transgenic animals expressing the
cloned cdn-1 or cdn-2 genes. The nucleotide and predicted amino
acid residue sequences of cdn-1 are shown in FIG. 3; and those of
cdn-2 are shown in FIG. 5. It has now been found that the proteins
encoded by the cdn genes are capable of modulating apoptosis. In a
lymphoblastoid cell line, cdn-1 was shown to decrease Fas-mediated
apoptosis. In a mouse progenitor B cell line, FL5.12, cdn-2 and a
derivative of cdn-1 decrease IL-3-induced apoptosis whereas cdn-1
slightly increased apoptosis. Thus, depending on the cell type, the
derivative of cdn and the method of induction of apoptosis,
apoptosis can be modulated in a highly specific manner by
controlling the concentration of cdns.
[0021] As used herein, "cdns" or "cdn" refers to the nucleic acid
molecules described herein (cdn-1, cdn-2, cdn-3 and derivatives
thereof), "the CDNs" or "CDN" refers to the proteins encoded
thereby (CDN-1, CDN-2, CDN-3 and derivatives thereof). The present
invention encompasses cdn-1 and cdn-2 nucleotide sequences. The cdn
nucleotides include, but are not limited to, the cDNA,
genome-derived DNA and synthetic or semi-synthetic DNA or RNA. The
nucleotide sequence of the cdn-1 cDNA with the location of
restriction endonuclease sites is shown in FIG. 2. As described in
the examples herein, cdn-1 mRNA has been detected in a variety of
human organs and tissues by Northern blot analysis. These organs
include liver; heart; skeletal muscle; lung; kidney; and pancreas
as shown in FIG. 3.
[0022] Similarly, cdn-2, cdn cDNA, genomic DNA and synthetic or
semi-synthetic DNAs and RNAs are additional embodiments of the
present invention. The nucleotide sequence of cdn-2 cDNA, along
with the predicted amino acid sequence of cdn-2 protein and the
locations of restriction endonuclease recognition sites, is given
in FIG. 5. The examples presented herein indicate that cdn-1 is on
human chromosome 6 and that cdn-2 is on human chromosome 20. There
is also a member of the family cdn-3 which is on human chromosome
11. Fluorescence in situ hybridization (FISH) indicated an
approximate location of cdn-1 to be at 6p21-23. Within this region
resides the gene for spinocerebellar ataxia type 1. Interestingly,
apoptosis has been proposed recently to be involved in the related
genetic disorder ataxia telangiectasia. Taken together with the
chromosomal localization and the expression of cdn-1 in brain
tissue, this suggests the possibility that cdn-1/cdn-2 might
represent the SCA1 gene locus. It is possible that cdn-2 and cdn-3
are pseudogenes. While these may not be expressed endogenously,
they are capable of expression from a recombinant vector providing
the appropriate promoter sequences. Thus, both cdn-2 and cdn-3
genes are encompassed by the present invention as are recombinant
constructs thereof and proteins encoded thereby.
[0023] Derivatives of the genes and proteins include any portion of
the protein, or gene encoding the protein, which retains apoptosis
modulating activity. FIG. 10 depicts three such derivatives of
cdn-1 which have been shown to retain apoptosis-modulating
activity. These derivatives, cdn1-.DELTA.1, cdn1-.DELTA.2 and
cdn1-.DELTA.3, are encompassed by the present invention.
[0024] The invention includes modifications to cdn DNA sequences
such as deletions, substitutions and additions particularly in the
non-coding regions of genomic DNA. Such changes are useful to
facilitate cloning and modify gene expression.
[0025] Various substitutions can be made within the coding region
that either do not alter the amino acid residues encoded or result
in conservatively substituted amino acid residues. Nucleotide
substitutions that do not alter the amino acid residues encoded are
useful for optimizing gene expression in different systems.
Suitable substitutions are known to those of skill in the art and
are made, for instance, to reflect preferred codon usage in the
particular expression systems.
[0026] The invention encompasses functionally equivalent variants
and derivatives of cdns which may enhance, decrease or not
significantly affect the properties of CDNs. For instance, changes
in the DNA sequence that do not change the encoded amino acid
sequence, as well as those that result in conservative
substitutions of amino acid residues, one or a few amino acid
deletions or additions, and substitution of amino acid residues by
amino acid analogs are those which will not significantly affect
its properties.
[0027] Amino acid residues which can be conservatively substituted
for one another include but are not limited to: glycine/alanine;
valine/isoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine; lysine/arginine; and
phenylalanine/tyrosine. Any conservative amino acid substitution
which does not significantly affect the properties of CDNs is
encompassed by the present invention.
[0028] Techniques for nucleic acid manipulation useful for the
practice of the present invention are described in a variety of
references, including but not limited to, Molecular Cloning: A
Laboratory Manual, 2nd ed., Vol. 1-3, eds. Sambrook et al. Cold
Spring Harbor Laboratory Press (1989); and Current Protocols in
Molecular Biology, eds. Ausubel et al., Greene Publishing and
Wiley-Interscience: New York (1987) and periodic updates.
[0029] The invention further embodies a variety of DNA vectors
having cloned therein the cdn nucleotide sequences encoding.
Suitable vectors include any known in the art including, but not
limited to, those for use in bacterial, mammalian, yeast and insect
expression systems. Specific vectors are known in the art and need
not be described in detail herein.
[0030] The vectors may also provide inducible promoters for
expression of the cdns. Inducible promoters are those which do not
allow constitutive expression of the gene but rather, permit
expression only under certain circumstances. Such promoters may be
induced by a variety of stimuli including, but not limited to,
exposure of a cell containing the vector to a ligand, metal ion,
other chemical or change in temperature.
[0031] These promoters may also be cell-specific, that is,
inducible only in a particular cell type and often only during a
specific period of time. The promoter may further be cell cycle
specific, that is, induced or inducible only during a particular
stage in the cell cycle. The promoter may be both cell type
specific and cell cycle specific. Any inducible promoter known in
the art is suitable for use in the present invention.
[0032] The invention further includes a variety of expression
systems transfected with the vectors. Suitable expression systems
include but are not limited to bacterial, mammalian, yeast and
insect. Specific expression systems and the use thereof are known
in the art and are not described in detail herein.
[0033] The invention encompasses ex vivo transfection with cdns, in
which cells removed from animals including man are transfected with
vectors encoding CDNs and reintroduced into animals. Suitable
transfected cells include individual cells or cells contained
within whole tissues. In addition, ex vivo transfection can include
the transfection of cells derived from an animal other than the
animal or human subject into which the cells are ultimately
introduced. Such grafts include, but are not limited to,
allografts, xenografts, and fetal tissue transplantation.
[0034] Essentially any cell or tissue type can be treated in this
manner. Suitable cells include, but are not limited to,
cardiomyocytes and lymphocytes. For instance, lymphocytes, removed,
transfected with the recombinant DNA and reintroduced into an
HIV-positive patient may increase the half-life of the reintroduced
T cells.
[0035] As an example, in treatment of HIV-infected patients by the
above-described method, the white blood cells are removed from the
patient and sorted to yield the CD4.sup.+ cells. The CD4.sup.+
cells are then transfected with a vector encoding CDNs and
reintroduced into the patient. Alternatively, the unsorted
lymphocytes can be transfected with a recombinant vector having at
least one cdn under the control of a cell-specific promoter such
that only CD4.sup.+ cells express the cdn genes. In this case, an
ideal promoter would be the CD4 promoter; however, any suitable
CD4.sup.+ T cell-specific promoter can be used.
[0036] Further, the invention encompasses cells transfected in vivo
by the vectors. Suitable methods of in vivo transfection are known
in the art and include, but are not limited to, that described by
Zhu et al. (1993) Science 261:209-211. In vivo transfection by cdns
may be particularly useful as a prophylactic treatment for patients
suffering from atherosclerosis. Elevated modulation of the levels
of CDN could serve as a prophylaxis for the apoptosis-associated
reperfusion damage that results from cerebral and myocardial
infarctions. In these patients with a high risk of stroke and heart
attack, the apoptosis and reperfusion damage associated with
arterial obstruction could be prevented or at least mitigated.
[0037] Infarctions are caused by a sudden insufficiency of arterial
or venous blood supply due to emboli, thrombi, or pressure that
produces a macroscopic area of necrosis; the heart, brain, spleen,
kidney, intestine, lung and testes are likely to be affected.
Apoptosis occurs to tissues surrounding the infarct upon
reperfusion of blood to the area; thus, modulation of CDN levels,
achieved by a biological modifier-induced change in endogenous
production or by in vivo transfection, could be effective at
reducing the severity of damage caused by heart attacks and
stroke.
[0038] Transgenic animals containing the recombinant DNA vectors
are also encompassed by the invention. Methods of making transgenic
animals are known in the art and need not be described in detail
herein. For a review of methods used to make transgenic animals,
see, e.g. PCT publication No. WO 93/04169. Preferably, such animals
express recombinant cdns under control of a cell-specific and, even
more preferably, a cell cycle specific promoter.
[0039] In another embodiment, diagnostic methods are provided to
detect the expression of cdns either at the protein level or the
mRNA level. Any antibody that specifically recognizes CDNs is
suitable for use in CDN diagnostics. Abnormal levels of CDNs are
likely to be found in the tissues of patients with diseases
associated with inappropriate apoptosis; diagnostic methods are
therefore useful for detecting and monitoring biological conditions
associated with such apoptosis defects. Detection methods are also
useful for monitoring the success of CDN-related therapies.
[0040] Purification or isolation of CDNs expressed either by the
recombinant DNA or from biological sources such as tissues can be
accomplished by any method known in the art. Protein purification
methods are known in the art. Generally, substantially purified
proteins are those which are free of other, contaminating cellular
substances, particularly proteins. Preferably, the purified CDNs
are more than eighty percent pure and most preferably more than
ninety-five percent pure. For clinical use as described below, the
CDNs are preferably highly purified, at least about ninety-nine
percent pure, and free of pyrogens and other contaminants.
[0041] Suitable methods of protein purification are known in the
art and include, but are not limited to, affinity chromatography,
immunoaffinity chromatography, size exclusion chromatography, HPLC
and FPLC. Any purification scheme that does not result in
substantial degradation of the protein is suitable for use in the
present invention.
[0042] The invention also includes the substantially purified CDNs
having the amino acid residue sequences depicted in FIGS. 3 and 5,
respectively. The invention encompasses functionally equivalent
variants of CDNs which do not significantly affect their properties
and variants which retain the same overall amino acid sequence but
which have enhanced or decreased activity. For instance,
conservative substitutions of amino acid residues, one or a few
amino acid deletions or additions, and substitution of amino acid
residues by amino acid analogs are within the scope of the
invention.
[0043] Amino acid residues which can be conservatively substituted
for one another include but are not limited to: glycine/alanine;
valine/isoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine; lysine/arginine; and
phenylalanine/tyrosine. Any conservative amino acid substitution
which does not significantly affect the properties of CDNs is
encompassed by the present invention.
[0044] Suitable antibodies are generated by using the CDNs as an
antigen or, preferably, peptides encompassing the CDN regions that
lack substantial homology to the other gene products of the bcl
family. Methods of detecting proteins using antibodies and of
generating antibodies using proteins or synthetic peptides are
known in the art and are not be described in detail herein.
[0045] CDN protein expression can also be monitored by measuring
the level of cdn MRNA. Any method for detecting specific mRNA
species is suitable for use in this method. This is easily
accomplished using the polymerase chain reaction (PCR). Preferably,
the primers chosen for PCR correspond to the regions of the cdn
genes which lack substantial homology to other members of the bcl
gene family. Alternatively, Northern blots can be utilized to
detect cdn mRNA by using probes specific to cdns. Methods of
utilizing PCR and Northern blots are known in the art and are not
described in detail herein.
[0046] Methods of treatment with cdns also include modulating
cellular expression of cdns by increasing or decreasing levels of
cdn mRNA or protein. Suitable methods of increasing cellular
expression of cdn include, but are not limited to, increasing
endogenous expression and transfecting the cells with vectors
encoding cdns. Cellular transfection is discussed above and is
known in the art. Suitable indications for increasing endogenous
levels of cdn include, but are not limited to, malignancies and
cardiac-specific over-expression. Cardiac specific over-expression
is particularly suitable for use in indications including, but not
limited to, patients susceptible to heart disease and in advance of
cardiotoxic therapies including, but not limited to, chemotherapies
such as adriamycin, so as to offer cardioprotection.
[0047] In addition, increasing endogenous expression of cdns can be
accomplished by exposing the cells to biological modifiers that
directly or indirectly increase levels of CDNs either by increasing
expression or by decreasing degradation of cdn mRNA. Suitable
biological modifiers include, but are not limited to, molecules and
other cells. Suitable molecules include, but are not limited to,
drugs, cytokines, small molecules, hormones, combinations of
interleukins, lectins and other stimulating agents e.g. PMA, LPS,
bispecific antibodies and other agents which modify cellular
functions or protein expression. Cells are exposed to such
biological modifiers at physiologically effective concentrations,
and the expression of cdns is measured relative to a control not
exposed to the biological modifiers. Those biological modifiers
which increase expression of cdns relative to the control are
selected for further study.
[0048] The invention further encompasses a method of decreasing
endogenous levels of cdns. The methods of decreasing endogenous
levels of cdns include, but are not limited to, antisense
nucleotide therapy and down-regulation of expression by biological
modifiers. Antisense therapy is known in the art and its
application will be apparent to one of skill in the art.
[0049] Screening for therapeutically effective biological modifiers
is done by exposing the cells to biological modifiers which may
directly or indirectly decrease levels of CDNs either by decreasing
expression or by increasing the half-life of cdn mRNA or CDNs.
Suitable biological modifiers include, but are not limited to,
molecules and other cells. Suitable molecules include, but are not
limited to, drugs, cytokines, small molecules, hormones,
combinations of interleukins, lectins and other stimulating agents
e.g. PMA, LPS, bispecific antibodies and other agents which modify
cellular functions or protein expression. Cells are grown under
conditions known to elicit expression of at least one cdn
(preferably cdn-1), exposed to such biological modifiers at
physiologically effective concentrations, and the expression of
cdns is measured relative to a control not exposed to biological
modifiers. Those biological modifiers which decrease the expression
of cdns relative to a control are selected for further study. Cell
viability is also monitored to ensure that decreased cdn expression
is not due to cell death.
[0050] In determining the ability of biological modifiers to
modulate (increase or decrease) cdn expression, the levels of
endogenous expression may be measured or the levels of recombinant
fusion proteins under control of cdn-specific promoter sequences
may be measured. The fusion proteins are encoded by reporter
genes.
[0051] Reporter genes are known in the art and include, but are not
limited to chloramphenicol acetyl transferase (CAT) and
.beta.-galactosidase. Expression of cdn-1 and -2 can be monitored
as described above either by protein or mRNA levels. Expression of
the reporter genes can be monitored by enzymatic assays, or
antibody-based assays, like ELISAs and RIAs, also known in the art.
Potential pharmaceutical agents can be any therapeutic agent or
chemical known to the art, or any uncharacterized compounds derived
from natural sources such as fungal broths and plant extracts.
Preferably, suitable pharmaceutical agents are those lacking
substantial cytotoxicity and carcinogenicity.
[0052] Suitable indications for modulating endogenous levels of
cdns are any in which cdn-mediated apoptosis is involved. These
include, but are not limited to, various types of malignancies and
other disorders resulting in uncontrolled cell growth such as
eczema, or deficiencies in normal programmed cell death such as
malignancies, including, but not limited to, B cell lymphomas.
[0053] The invention also encompasses therapeutic methods and
compositions involving treatment of patients with biological
modifiers to increase or decreast expression of cdns. Effective
concentrations and dosage regimens may be empirically derived. Such
derivations are within the skill of those in the art and depend on,
for instance, age, weight and gender of the patient and severity of
the disease. Alternatively, patients may be directly treated with
either native or recombinant CDNs. The CDNs should be substantially
pure and free of pyrogens. It is preferred that the recombinant
CDNs be produced in a mammalian cell line so as to ensure proper
glycosylation. CDNs may also be produced in an insect cell line and
will be glycosylated.
[0054] For therapeutic compositions, a therapeutically effective
amount of substantially pure CDN is suspended in a physiologically
accepted buffer including, but not limited to, saline and phosphate
buffered saline (PBS) and administered to the patient. Preferably
administration is intravenous. Other methods of administration
include but are not limited to, subcutaneous, intraperitoneal,
gastrointestinal and directly to a specific organ, such as
intracardiac, for instance, to treat cell death related to
myocardial infarction.
[0055] Suitable buffers and methods of administration are known in
the art. The effective concentration of a CDN will need to be
determined empirically and will depend on the type and severity of
the disease, disease progression and health of the patient. Such
determinations are within the skill of one in the art.
[0056] Bcl-2 is thought to function in an antioxidant pathway. Veis
et al. (1993) Cell 75:229-240. Therefore, therapy involving CDNs is
suitable for use in conditions in which superoxide is involved.
Administration of CDNs results in an increased extracellular
concentration of CDNs, which is thought to provide a method of
directly inhibiting superoxide accumulation that may be produced by
the blebs associated with apoptosis. The therapeutic method thus
includes, but is not limited to, inhibiting superoxide mediated
cell injury.
[0057] Suitable indications for therapeutic use of CDNs are those
involving free radical mediated cell death and include, but are not
limited to, conditions previously thought to be treatable by
superoxide dismutase. Such indications include but are not limited
to HIV infection, autoimmune diseases, cardiomyopathies, neuronal
disorders, hepatitis and other liver diseases, osteoporosis, and
shock syndromes, including, but not limited to, septicemia.
[0058] Hybridization of cloned cdn DNA to messenger mRNA from
various regions of the brain indicated high levels of expression of
cdn-1 in each of the regions studied (FIG. 8). Therefore,
neurological disorders are another area in which therapeutic
applications of CDNs may be indicated.
[0059] The following examples are provided to illustrate but not
limit the present invention. Unless otherwise specified, all
cloning techniques were essentially as described by Sambrook et al.
(1989) and all reagents were used according to the manufacturer's
instructions.
EXAMPLE 1
Identification and Cloning of cdn-1 cDNA
[0060] An amino acid sequence comparison of the six known bcl-2
family members (FIG. 6) revealed two regions with considerable
sequence identity, namely amino acids 144-150 and 191-199. In an
attempt to identify new bcl-2 family members, degenerate PCR
primers based on sequences in these regions were designed (FIG. 1)
and PCR was performed using human heart cDNA and human B
lymphoblastoid cell line (WIL-2) cDNA. PCR was performed using the
Hot Start/Ampliwax technique (Perkin Elmer Cetus). The final
concentration of the PCR primers and the template cDNA were 4 .mu.M
and 0.1-0.2 ng/ml, respectively. The conditions for cDNA synthesis
were identical to those for first strand cDNA synthesis of the cDNA
library as described below. PCR was performed in a Perkin Elmer
Cetus DNA Thermal Cycler according to the method described by
Kiefer et al. (1991) Biochem. Biophys. Res. Commun. 176:219-225,
except that the annealing and extension temperatures during the
first 10 cycles were 36.degree. C. Following PCR, samples were
treated with 5 units of DNA polymerase I, Klenow fragment for 30
min at 37.degree. C. and then fractionated by electrophoresis on a
7% polyacrylamide, 1 X TBE (Tris/borate/EDTA) gel. DNA migrating
between 170-210 base pars was excised from the gel, passively
eluted for 16 hours with gentle shaking in 10 mM Tris-HCl pH 7.5, 1
mM EDTA (TE), purified by passage over an Elutip-D column
(Schleicher and Schuell), ligated to the pCR-Script vector
(Stratagene) and transformed into Escherichia coli strain XL1-Blue
MRF (Stratagene). Plasmid DNA from transformants (white colonies)
containing both the heart and WIL-2 PCR products was isolated using
the Magic Miniprep DNA Purification System (Promega), and the DNA
inserts were sequenced by the dideoxy chain termination method
according to Sanger et al. (1977) Proc. Natl. Acad. Sci. USA
74:5463-5467 (USB, Sequenase version 2.0). DNA sequence analysis of
the eleven heart PCR products revealed two sequences identical to
bcl-x (Boise et al. (1993) Cell 74:597-608) and ten other sequences
unrelated to the bcl-2 family.
[0061] DNA sequence analyses of the eleven WIL-2 PCR products
yielded one bcl-x sequence, five sequences identical to another
bcl-2 family member, bax (Oldvai et al. (1993) Cell 74:609-619),
four unrelated sequences and one novel bcl-2 related sequence,
termed cdn-1. The unique cdn-1 amino acid sequence encoded by the
PCR product is shown in FIG. 6 from amino acid 151-190 (top
row).
[0062] To isolate the cdn-1 cDNA, a human heart cDNA library
(Clontech) and a WIL-2 cDNA library, constructed as described by
Zapf et al. (1990) J. Biol. Chem. 265:14892-14898 were screened
using the cdn-1 PCR DNA insert as a probe. The DNA was
.sup.32P-labeled according to the method described by Feinberg and
Vogelstein (1984) Anal. Biochem. 137:266-267 and used to screen
150,000 recombinant clones from both libraries according to the
method described by Kiefer et al. (1991). Eight positive clones
from the WIL-2 cDNA library and two positive clones from the heart
cDNA library were identified. Four clones from the WIL-2 cDNA
library and two from the heart cDNA library were further purified
and plasmid DNA containing the cDNA inserts was excised from the
.lamda.ZAPII vector (Stratagene) (FIG. 2). The two longest clones,
W7 (2.1 kb) and W5 (2.0 kb) were sequenced and shown to contain the
cdn-1 probe sequence, thus confirming their authenticity. The heart
cDNAs also encoded cdn-1.
[0063] The W7 DNA sequence along with the deduced amino acid
residue sequence is shown in FIG. 2. The deduced amino acid
sequence of cdn-1 was also aligned for maximum sequence identity
with the other bcl-2 family members and is shown in FIG. 6. As can
be seen, there is considerable sequence identity between cdn-1 and
other family members between amino acids 100 and 200. Beyond this
central region, sequence conservation falls off sharply. Like
bcl-2, cdn-1 appears to be an intracellular protein in that it does
not contain a either a hydrophobic signal peptide or N-linked
glycosylation sites. Cdn-1 does contain a hydrophobic C-terminus
that is also observed with all bcl-2 family members except LMW5-HL,
suggesting its site of anti-apoptotic activity, like that of bcl-2,
is localized to a membrane bound organelle such as the
mitochondrial membrane, the endoplasmic reticulum or the nuclear
membrane. Hockenbery et al. (1990); Chen-Levy et al. (1989) Mol.
Cell. Biol. 9:701-710; Jacobsen et al. (1993) Nature 361:365-369;
and Monighan et al. (1992) J. Histochem. Cytochem.
40:1819-1825.
EXAMPLE 2
Northern Blot Analysis of cDNA Clones
[0064] Northern blot analysis was performed according to the method
described by Lehrach et al. (1977) Biochem. 16:4743-4651 and Thomas
(1980) Proc. Natl. Acad. Sci. USA 77:5201-5205. In addition, a
human multiple tissue Northern blot was purchased from Clontech.
The coding regions of bcl-2 and cdn-1 cDNAs were labeled by the
random priming method described by Feinberg and Vogelstein (1984)
Anal. Biochem. 137:266-267. Hybridization and washing conditions
were performed according to the methods described by Kiefer et al.
(1991).
[0065] The results, presented in FIG. 4 indicate that cdn-1 is
expressed in all organs tested (heart, brain, placenta, lung,
liver, skeletal muscle, kidney and pancreas) whereas bcl-2 is not
expressed or expressed at only low levels in heart, brain, lung,
and liver. Thus, cdn-1 appears to be more widely expressed
throughout human organs than bcl-2 and may be more important in
regulating apoptosis in these tissues.
EXAMPLE 3
Expression of Recombinant cdn-1
[0066] In order to express recombinant cdn-1 in the baculovirus
system, the cdn-1 cDNA generated in Example 1 was used to generate
a novel cdn-1 vector, by a PCR methodology as described in Example
1, using primers from the 3' and 5' flanking regions of the gene
which contain restriction sites to facilitate cloning. The plasmids
were sequenced by the dideoxy terminator method (Sanger et al.,
1977) using sequencing kits (USB, Sequenase version 2.0) and
internal primers. This was to confirm that no mutations resulted
from PCR.
[0067] A clone was used to generate recombinant viruses by in vivo
homologous recombination between the overlapping sequences of the
plasmid and AcNPV wild type baculovirus. After 48 hours
post-transfection in insect Spodoptera frugiperda clone 9 (SF9)
cells, the recombinant viruses were collected, identified by PCR
and further purified. Standard procedures for selection, screening
and propagation of recombinant baculovirus were performed
(Invitrogen). The molecular mass, on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), of the
protein produced in the baculovirus system was compared with the
predicted molecular mass of cdn-1 according to the amino-acid
sequence.
[0068] In addition, similar clones can be expressed preferably in a
yeast intracellular expression system by any method known in the
art, including the method described by Barr et al. (1992)
Transgenesis, ed. JAH Murray, (Wiley and Sons) pp. 55-79.
EXAMPLE 4
Expression of cdn-1 in Mammalian Systems
[0069] The cdn-1 coding sequence was excised from a plasmid
generated in Example 1, and introduced into plasmids pCEP7, pREP7
and pcDNA3 (Invitrogen) at compatible restriction enzyme sites.
pCEP7 was generated by removing the RSV 3'-LTR of pREP7 with
XbaI/Asp718, and substituting the CMV promoter from pCEP4
(Invitrogen). 25 .mu.g of each cdn-1-containing plasmid was
electroporated into the B lymphoblastoid cell line WIL-2, and
stable hygromycin resistant transformants or G418 resistant
transformants (pcDNA3 constructs, FIG. 8) expressing cdn-1 were
selected.
[0070] The coding region of cdns can also ligated into expression
vectors capable of stably integrating into other cell types
including but not limited to cardiomyocytes, neural cell lines such
as GTI-7 and TNF sensitive cells such as the human colon
adenocarcinoma cell line HT29 so as to provide a variety of assay
systems to monitor the regulation of apoptosis by cdn-1.
EXAMPLE 5
Effect of the Anti-Apoptotic Activity of cdn-1 and its Derivatives
in the Wild Type B Lymphoblastoid Cell Line WIL2-729 HF2 and the
Transformed Cell Expressing Excess cdn-1
[0071] 2.times.10.sup.5 WIL-2, and WIL-2 cells transformed with a
vector encoding cdn-1 as described in Example 4 are grown in RPMI
supplemented with 10% fetal bovine serum (FBS) for the anti-fas
experiment or 0.1% FBS for serum deprivation experiments. In the
case of the anti-fas experiment, after washing with fresh medium,
the cells were suspended in RPMI supplemented with 10% FBS, exposed
to anti-fas antibodies and the kinetics of cell death in response
to an apoptosis inducing agent were analyzed by flow cytometry with
FACScan. In the case of the serum deprivation experiment, the WIL-2
cells were resuspended in RPMI supplemented with 0.1% FBS and
apoptosis was monitored according to the method described by
Henderson et al. (1993) Proc. Natl. Acad. Sci. USA 90:8479-8483.
Other methods of inducing apoptosis include, but are not limited
to, oxygen deprivation in primary cardiac myocytes, NGF withdrawal,
glutathione depletion in the neural cell line GTI-7 or TNF addition
to the HT29 cell line. Apoptosis was assessed by measuring cell
shrinkage and permeability to propidium iodide (PI) during their
death. In addition, any other method of assessing apoptotic cell
death may be used.
[0072] FIG. 8 shows the anti-apoptotic response of various WIL-2
transformants to anti-Fas treatment. FIG. 9 shows the
anti-apoptotic response of various WIL-2 transformants to serum
deprivation. In FIG. 8, duplicate wells containing 3.times.10.sup.5
cells were incubated with 50 ng/ml of the cytocidal anti-Fas
antibody for 24 hours. Cell death was then analyzed by flow
cytometry with FACScan. The proteins expressed from each construct
are shown beneath the columns. Since many of the constructs are
truncation or deletion variants, the exact amino acids expressed
are also indicated. As can be seen, all of the transformants had
some protective effect when compared to the control transformant
containing the pREP7 vector alone. The most apoptosis-resistant
transformant was the cdn-1.DELTA.2 expressing cell line, in which
over 90% of the cells survived anti-fas treatment. Significant
protection was also observed in transformants expressing full
length cdn-1 (1-211) and cdn-1.DELTA.1, followed by bcl-2.DELTA.
and bcl-2 expressing cell lines.
[0073] Cdn-1.DELTA.1 and cdn-1.DELTA.2 are lacking the N-terminal
59 and 70 amino acids of the full length cdn-1 molecule,
respectively. The observation that cdn-1.DELTA.2 is more effective
at blocking apoptosis than full length cdn-1 suggests that smaller,
truncated cdn-1 molecules may be potent therapeutics.
EXAMPLE 6
Determination of Other cdn Genes and Cloning of the cdn-2 Gene
[0074] Southern blot analyses of human genome DNA and a panel of
human/rodent somatic cell DNAs indicated that there were at least 3
cdn related genes and that they resided in chromosomes 6, 11 and
20. PCR/sequence analysis of the three hybrid DNAs showed that
cdn-1 was on chromosome 6 and that two closely related sequences
were on chromosome 20 (designated cdn-2) and chromosome 11
(designated cdn-3). We have cloned the cdn-2 and cdn-3 genes and
sequenced them. Interestingly, both cdn-2 and cdn-3 do not contain
introns and have all of the features of processed genes that have
returned to the genome. cdn-3 has a nucleotide deletion, causing a
frame shift and early termination and thus is probably a
pseudogene. Both, however, have promoter elements upstream of the
repeats CCAAT, TATAAA boxes but are probably not transcribed.
(Northern blot analysis with cdn-2 and cdn-3 specified probes.)
[0075] 900,000 clones from a human placenta genomic library in the
cosmid vector pWE15 (Stratagene, La Jolla, Calif.) were screened
with a 950 bp BgIII- HindIII cDNA probe containing the entire
coding region of Cdn-1. The probe was .sup.32P-labeled according to
the method of Feinberg and Vogelstein (1984) Anal. Biochem.
137:266-267. The library was processed and screened under high
stringency hybridization and washing conditions as described by
Sambrook et al. (1989) Molecular Cloning, 2nd edition, Cold Spring
Harbor Laboratory Press. Ten double positive clones were further
purified by replating and screening as above. Plasmid DNA was
purified using the Wizard Maxiprep DNA Purification System as
described by the supplier (Promega Corp., Madison, Wis.) and
analyzed by EcoRI restriction enzyme mapping and Southern blotting.
The probe used for Southern blotting and hybridization conditions
was the same as above.
[0076] The cosmid clones fell into two groups as judged by EcoRI
restriction analysis and Southern blotting. Cosmid clones (cos) 1-4
and 7 displayed one distinct pattern of EcoRI generated DNA
fragments and contained a single 6.5 kb hybridizing EcoRI DNA
fragment. Cos2 and Cos9 fell into the second group that was
characterized by a 5.5 kb hybridizing EcoRI DNA fragment. The 6.5
kb DNA fragment from cos2 and the 5.5 kb DNA fragment from cos9
were subcloned into pBluescript SK (Stratagene, La Jolla, Calif.)
using standard molecular biological techniques (Sambrook et al. as
above). Plasmid DNA was isolated and the DNA inserts from two
subclones, A4 (from cos2) and C5 (from cos9) were mapped with
BamHI, HindIII and EcoRI and analyzed by Southern blotting as
described above. Smaller restriction fragments from both clones
were subcloned into M13 sequencing vectors and the DNA sequence was
determined.
[0077] The sequence of A4 contains an open reading frame that
displays 97% amino acid sequence identity with cdn-1. (FIG. 5) The
high degree of sequence identity of this gene with cdn-1 indicates
that it is a new cdn-1 related gene and therefore will be called
cdn-2. A sequence comparison of the encoded cdn-2 protein and the
other members of the bcl-2 family is shown in FIG. 5. Cdn-2
contains the conserved regions, BH1 and BH2, that are hallmarks of
the bcl-2 family, and displays a lower overall sequence identity
(.about.20-30%) to other members, which is also characteristic of
the bcl-2 family. cdn-3 has a frame shift and therefore does not
contain the structural features of cdn-1, cdn-2 or other bcl-2
family members.
EXAMPLE 7
Chromosomal Localization of the cdn-1 and cdn-2 Genes
[0078] Southern blot analysis of a panel of human/rodent somatic
cell hybrid DNAs (Panel #2 DNA from the NIGMS, Camden, N.J.) and
fluorescent in situ hybridization (FISH) of metaphase chromosomes
were used to map the cdn genes to human chromosomes. For Southern
blotting, 5.mu.g of hybrid panel DNA was digested with EcoRI or
BamHI/HindIII, fractionated on 0.8% or 1% agarose gels, transferred
to nitrocellulose and hybridized with the cdn-1 probe.
Hybridization and washing conditions were as described above. For
FISH, the cdn-2 subclone, A4, was biotinylated using the Bionick
Labeling System (Gibco BRL, Gaithersburg, Md.) and hybridized to
metaphase chromosomes from normal human fibroblasts according to
the method described by Viegas-Pequignot in In Situ Hybridization,
A Practical Approach, 1992, ed. D. G. Wilkinson, pp. 137-158, IRL
Press, Oxford. Probe detection using FITC-conjugated avidin and
biotinylated goat anti-avidin was according to the method described
by Pinkel et al. (1988) Proc. Natl. Acad. Sci. USA
85:9138-9142.
[0079] Southern blot analysis showed three hybridizing EcoRI bands
in the human DNA control that were approximately 12 kb, 11 kb and
5.5 kb in length. Analysis of the somatic cell hybrid DNA indicated
that the 12 kb band was in two different samples, NA10629, which
contained only human chromosome 6, and NA07299, which contained
both human chromosomes 1 and X and, importantly, a portion of
chromosome 6 telomeric to p21. The 11 kb band was in NA13140, which
contains human chromosome 20. The 5.5 kb hybridizing band was found
only in sample NA10927A, which contained human chromosome 11.
PCR/DNA sequencing analysis of these hybrid DNA samples using
primers for cdn-1 or cdn-2, showed cdn-1 sequences in NA10629 (the
chromosome 6-containing hybrid DNA) and NA07299 (the chromosome 1,
X and 6pter >p21-containing hybrid DNA), indicating that the
cdn-1 gene resides on chromosome 6, telomeric to p21. cdn-2
sequences were found in NA13140, indicating the cdn-2 gene resides
on chromosome 20, and cdn-3 sequences were found in NA10927A,
indicating the cdn-3 gene resides on chromosome 11.
EXAMPLE 8
Modulation of Apoptosis by cdn-1 and cdn-2 in FL5.12 Cells
[0080] FL5.12 is an IL-3-dependent lymphoid progenitor cell line
(McKearn et al. (1985) Proc. Natl. Acad. Sci USA 82:7414-7418) that
has been shown to undergo apoptosis following withdrawal of IL-3
but is protected from cell death by overexpression of bcl-2. Nunez
et al. (1990) J. Immunol. 144:3602-3610; and Hockenbery et al.
(1990) Nature 348:334-336. To assess the ability of cdn-1 and cdn-2
to modulate apoptosis, cDNAs encoding cdn-1, cdn-2, two truncated
forms of cdn-1 (described below) and bcl-2 were ligated into the
mammalian expression vector, pcDNA3 (Invitrogen, San Diego, Calif.)
and stably introduced into the mouse progenitor B lymphocyte cell
line FL5.12 by electroporation and selection in media containing
the antibiotic G418. Assays were then performed on bulk
transformants as described below.
[0081] The effects of the overexpressed genes on FL5.12 cell
viability were examined at various times following withdrawal of
IL-3 and are shown in FIG. 10. Cell viability was assessed by
propidium iodide (PI) exclusion on a flow cytometer (Becton
Dickinson FACScan). Bcl-2 expression protected the cells
significantly from cell death while cdn-1 appeared to enhance cell
death when compared to the vector control. Cdn-2 expression
conferred a low level of protection from cell death at earlier
times but was insignificant at later time points. Interestingly,
cdn-1.DELTA.2 gave a moderate level of protection against cell
death. Cdn-1-112, a molecule that contains the N-terminal 112 amino
acids of cdn-1, also appeared to partially protect the FL5.12 cells
although at lower levels than Bcl-2.
[0082] As shown in Example 7, expression of cdn-1 and cdn-1.DELTA.2
in WIL2 cells resulted in increased cell survival in response to
anti-Fas-mediated apoptosis and serum withdrawal. Taken together,
these data suggest that the various cdn molecules are capable of
modulating apoptosis in a positive or negative manner, depending on
the cell type and apoptotic stimuli. Thus, they are effective in
preventing cell death such as in the post-ischemic reperfusion
tissue damage in the heart or in inducing cell death in cells that
have escaped apoptotic control, as is the case in various
cancers.
[0083] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent to those skilled in the art
that certain changes and modifications may be practiced. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims.
Sequence CWU 1
1
* * * * *