Note: Descriptions are shown in the official language in which they were submitted.
CA 02773151 2012-03-28
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CANCER DIAGNOSTIC KIT TO MONITOR THE ACTpitY OF PROTEIN KINASE
CK2, THE METHODS AND COMPOSITIONS THEREOF
Field of the Invention
[0001] The present invention relates generally to cancer diagnosis, and more
particularly, to the
use of phospho-specific antibodies, peptides and proteins to monitor Protein
Kinase CK2 in
biopsies.
Background of the Invention
[0002] Apoptosis is an essential biological process required for normal cell
turnover, proper
embryonic development, as well as chemically induced cell death. Furthermore,
perturbations in
the regulation of apoptotic signaling pathways have been associated with many
human diseases,
including cancer. The progression of apoptosis is dependent on the tightly-
regulated proteolytic
cleavage of a variety of proteins required for cell survival by a group of
cysteine-dependent
aspartic-directed proteases called caspases. Studies investigating the
sequence requirements for
caspase substrate recognition identified a stringent specificity for an
aspartic acid residue within
the cleavage. Notably, the presence of charged or bulky residues adjacent to
the Asp residue of
position P1' are poorly tolerated in caspase catalysis, where the smaller
residues Gly, Ala, Thr,
Ser or Asn are preferred (Duncan, J.S., Turowec, J.P., Duncan, K.E., Vilk, G.,
Wu, C., Luscher,
B., Li, S.S-C., Gloor, G.B., and Litchfield, D.W. (2011) A peptide-based
target screen of
kinase/caspase overlapping substrates identifies pro-casnase-3 as a
nhysiological target of CK2.
Sci. Signal 4(172): 1-13).
[0003] Recently, a role for protein kinases in the regulation of caspase
signaling pathways has
emerged, where the phosphorylation of caspases and/or caspase substrates has
been shown to
negatively influence caspase-mediated cleavage. For example, the
phosphorylation of caspase-9
by ERK, PKCc, c-Abl, CK2 and Akt has been shown to influence caspase-9
activity, illustrating
the convergence of multiple protein kinases in the regulation of caspase
signaling pathways.
Interestingly, phosphorylation of caspase substrates by protein kinases at or
near the caspase
cleavage site has been shown to prevent caspase-mediated cleavage, signifying
a potential
mechanism for the regulation of caspase activity. Phosphorylation of Nogo-B
within a caspase
CA 02773151 2012-03-28
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recognition motif by CDK1 and 2 prevented its caspase-7-mediated cleavage,
while caspase-3-
mediated cleavage of Calsenilin was shown to be regulated by CK1
phosphorylation (Duncan,
J.S., Turowec, J.P., Duncan, K.E., Vilk, G., Wu, C., Luscher, B., Li, S.S-C.,
Gloor, G.B., and
Litchfield, D.W. (2011) A peptide-based target screen of kinase/caspase
overlapping substrates
identifies pro-caspase-3 as a physiological target of CK2. Sci. Signal 4(172):
1-13).
[0004] Using a large scale peptide screening technique, the constitutively
active and oncogenic
protein kinase CK2 was identified as the most prominent kinase to possess an
overlapping
consensus with caspases. Protein Kinase CK2 (formally known as, Casein ICinase
II) is a
serine/threonine protein kinase composed of three subunits (catalytic CK2a,
catalytic CK2a',
and regulatory CK243) that has been implicated in the regulation of key tumor
suppressors and
oncogenes within cell survival pathways, promoting tumorigenesis (Litchfield,
D.W. (2003)
Protein kinase CK2: structure, regulation and role in cellular decisions of
life and death. Biochem
J 369: 1-15). Mounting evidence suggests an anti-apoptotic role for CK2 via
the protection of
pro-survival proteins from caspase-mediated cleavage. Specifically, CK2
phosphorylation of
Bid, Max, HS1, PSN-2, Connexin 45.6, Caspase-9 and PTEN at residues at or near
the caspase
cleavage site has been shown to prevent caspase cleavage leading to the
protection of cells from
apoptosis. CK2 is a constitutively active, ubiquitously distributed protein
kinase, implicated in
tumorigenesis and transformation with a unique consensus sequence for
phosphorylation
Ser/Thr-X-X-Acidic, where the acidic residue is one of Asp, Glu, pSer or pTyr.
The requirement
for acidic residues for both CK2 phosphorylation and caspase protease activity
points towards a
potentially widespread mechanism for the regulation of apoptosis, via the
protection of caspase
targets by CK2 phosphorylation (Duncan, J.S., Turowec, J.P., Duncan, K.E.,
Vilk, G., Wu, C.,
Luscher, B., Li, S.S-C., Gloor, G.B., and Litchfield, D.W. (2011) A peptide-
based target screen
of kinase/caspase overlapping substrates identifies pro-caspase-3 as a
physiological target of
CK2. Sci. Signal 4(172): 1-13).
[0005] In the analysis, pro-caspase-3 was identified as a target of
phosphorylation in vitro and in
cells by CK2. Specifically, CK2 phosphorylated at least one residue on pro-
caspase-3 and its
phosphorylation prevented its cleavage in vitro and in cells thus preventing
the progression of
apoptosis. Threonine-174 on pro-caspase-3 was positively identified by mass
spectrometry as
the target of Protein kinase CK2. Validation of the phosphorylation-dependent
protection of
CA 02773151 2012-03-28
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procaspase-3 in cells, as well as identification of numerous other candidate
CK2/caspase targets
support a role for phosphorylation as a global mechanism of regulation of
caspase signaling
pathways (Duncan, J.S., Turowec, J.P., Duncan, K.E., Vilk, G., Wu, C.,
Luscher, B., Li, S.S-C.,
Gloor, G.B., and Litchfield, D.W. (2011) A peptide-based target screen of
kinase/caspase
overlapping substrates identifies pro-caspase-3 as a physiological target of
CK2. Sci. Signal
4(172): 1-13).
[0006] Previous literature suggests that CK2 exists in different flavours
within cells existing as
catalytic or regulatory monomers (CK2a, CK2a', CK2) or together as a holozyme
tetramer
(CK2a2CK2P2, CK2cC2CK2P2, or as CK2aCK2a'CK2132). In vitro evidence suggests
that the
different flavours may act differently on their protein targets. In other
words, the presence of
CK213 may regulate the activity of CK2 towards selected targets. For example,
the translation
initiation factor eif23 can only be phosphorylated by the CK2 holoenzyme
(CK2a2CK2132)
whereas the calcium-binding protein Calmodulin can only be phosphorylated by
the CK2a
monomer (Poletto et al (2008) The regulatory P subunit of Protein Kinase CK2
contributes to the
recognition of the substrate consensus sequence. A study with an eIF2b-derived
peptide.
Biochemistry 47:8317-8325; Arrigoni et al (2004) Phosphorylation of Calmodulin
fragments by
Protein Kinase CK2. Mechanistic aspects and structural consequences.
Biochemistry 43:12788-
12798). Notably,the CDC25p phosphatase was regulated through phosphorylation
in cells and
that the CK213 regulatory subunit of CK2 was important in bringing CDC250 into
close
proximity (Theis-Febvre et al (2003) Protein Kinase CK2 regulates CDC2513
phosphatase
activity. Oncogene 22(2):220-232). Thus, it seems reasonable to assume that
CK2 may regulate
life and death processes by a complex mechanism by which its different
flavours possess some
non-overlapping functions (Duncan, J.S., Turowec, J.P., Duncan, K.E., Vilk,
G., Wu, C.,
Luscher, B., Li, S.S-C., Gloor, G.B., and Litchfield, D.W. (2011) A peptide-
based target screen
of kinase/caspase overlapping substrates identifies pro-caspase-3 as a
physiological target of
CK2. Sci. Signal 4(172): 1-13).
[0007] Due to the role CK2 could thus play in the regulation of pathogenic
diseases such as
cancer by regulating essential processes such as apoptosis, much attention has
been garnered in
developing molecular therapeutics to regulate components of this kinase in
clinical applications.
CA 02773151 2012-03-28
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However, a reliable and specific diagnostic assay is lacking in which one
could also monitor the
activity of CK2 in vivo using clinical biopsies or other bioactive tissues.
[0008] In view of the foregoing, it would then be desirable to develop such a
diagnostic assay to
prognosticate the aggressiveness of CK2-related cancers.
Summary of the Invention
[0009] Surprisingly, by developing a polyclonal phospho-specific antibody to
Threonine-174 on
human pro-caspase-3, we can monitor the CK2 activities of its different forms
in vitro and in
vivo using pro-caspase-3 protein and a caspase-3 peptide fragment.
[0010] In one aspect of the invention, the CK2a monomer phosphorylates human
pro-caspase-3
recombinant protein in vitro but is unable to phosphorylate the human pro-
caspase-3 peptide
fragment in vitro.
[0011] In another aspect of the invention, the CK213 regulatory subunit
regulates the activity of
CK2a in that the holoenzyme tetramer consisting of CK2a2CK2f32 can
phosphorylate the pro-
caspase-3 fragment in vitro but is unable to phosphorylate the pro-caspase-3
recombinant protein
in vitro.
[0012] In further aspect, the ratio of CK2a/CK2a2CK2132 activity in vitro and
in vivo using
separately the polyclonal phospho-specific antibody to pro-caspase-3, the pro-
caspase-3
fragment and the pro-caspase-3 recombinant protein could be used as a
diagnostic biomarker kit
for cancer in a mammal.
[0013] In even further aspect, an article of manufacture is provided
comprising packaging and a
composition comprising the polyclonal phospho-specific antibody to pro-caspase-
3, the pro-
caspase-3 fragment, the pro-caspase-3 recombinant protein and a suitable
support to permit
binding of the peptide and protein for the diagnostic analysis using the
specific antibody. The
packaging is labeled to indicate that the composition is suitable as a cancer
diagnostic assay.
[0014] These and other aspects, features and advantages of the invention will
become apparent
from the following detailed descriptions, claims and drawings.
CA 02773151 2012-03-28
Brief Description of the Drawings
[0015] Table 1 illustrates the phospho-specific antibody against pro-caspase-3
that was raised in
rabbits and the peptide sequence epitope that was used to immunize these
animals. Column 3
shows which amino acids in the epitope were modified prior to immunization.
Lower case
letters highlight the amino acids that are phosphorylated.
[0016] Table 2 illustrates the sequence of peptides that are used in the
cancer diagnostic assay.
The lower case letters highlight the amino acids that can be modified through
phosphorylation.
[0017] Figure 1 graphically illustrates the specificity of the polyclonal
phospho-antibody,
known herein as aC3T174, towards wild-type (wt) pro-caspase-3, pro-caspase-3
(T174A)
mutant, pro-caspase-3 (Si 76A) mutant and pro-caspase-3 (T174A/S176A) mutant
that had been
phosphorylated with GST-CK2a monomer kinase using 32P-y-ATP.
[0018] Figure 2A graphically illustrates the unique specificity of CK2a or
holoenzyme tetramer
CK2a2CK2(32 towards the pro-caspase-3 recombinant protein (C3) using
radioactive kinase
assays in vitro. We also note the specificity of phosphorylation of Protein
kinase CK2 towards
other proteins such as Cahnodulin (CaM), a-casein (CAS), and pro-caspase-8
(C8).
[0019] Figure 2B graphically illustrates the unique specificity of CK2a or
holoenzyme tetramer
CK2a2CK2132 towards the pro-caspase-3 peptide fragment (RC3) using radioactive
kinase assays
in vitro. We also note the specificity of phosphorylation of Protein kinase
CK2 towards pro-
caspase-8 peptide fragment (RC8), eif243 peptide fragment (eif213), RRRDDDSDDD
peptide
(DSD), and no peptide control. Below shows the fold of induction by dividing
the activity
obtained using CK2a by the activity obtained using holoenzyme tetramer
CK2a2CK2132.
[0020] Figure 3A is a figure denoting the ability of the phospho-specific
antibody to recognize
the pro-caspase-3 peptide fragment (RC3) or pro-caspase-3 recombinant protein
(C3) that had
been pre-phosphorylated with holoenzyme tetramer CK2a2CK2f32. The results were
quantified
in relative light units.
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[0021] Figure 3B is a figure denoting the ability of the phospho-specific
antibody to recognize
the pro-caspase-3 peptide fragment (RC3) or pro-caspase-3 recombinant protein
(C3) that had
been pre-phosphorylated with either CK2a or holoenzyme tetramer CK2a2CK2f32
and adhered
to a 96-well plate. The results are presented in relative light units.
[0022] Figure 4A is an illustration that shows a coronal magnetic resonance
image (MRI) view
of growing neoplastic tissue in nude mice. PC-3 carcinoma cells were injected
into nude mice to
induce a neoplastic carcinoma and MRI images at day 9 and day 13 post-
injection are shown.
The symbol a represents the growing neoplastic tissue of the prostata. The
symbol b represents
the vesica urinaria. The symbol c represents the non-cancerous mouse prostata
organ. Cranial,
Caudal, Left, and Right emphasize the directionality of the MRI image.
[0023] Figure 4B graphically shows the utility of the cancer diagnostic kit
when bio-active
mouse tissue (cancerous tumour or normal) is used to monitor the activity of
Protein kinase CK2
in vivo. The RC3 peptide fragment (RC3) and pro-caspase-3 recombinant protein
(C3) were first
adhered in separate wells to 96-well plates. Tumour or control tissue extracts
were then diluted
in CK2 kinase assay buffer and incubated in the wells for 2 hours at 37 C. The
CK2 inhibitor,
4,5,6,7-tetrabromobenzotriazole (TBB), was included in some of the wells as an
additional
control. After extensive washing, detection of Protein kinase CK2 activity in
the tumour or
control tissue extracts (amounts indicated in iig) was monitored using the
ocC3T174 polyclonal
antibody towards the bound phosphorylated RC3 peptide and the pro-caspase-3
recombinant
protein. The inset figure is a western blot showing the expression of CK2a,
CK2a', and CK213
in the same control (non-cancerous (C)), and tumour (T) extracted mouse
prostate tissues. The
results are presented in relative light units.
Detailed Description
[0024] A method to diagnose cancer using a diagnostic kit is provided. The
method comprises
the use of a phospho-specific antibody recognizing separately phosphorylated
pro-caspase-3
recombinant protein and pro-caspase-3 peptide fragment. By monitoring CK2a and
holoenzyme
tetramer CK2a2CK2132 kinase activity in cancerous and non-cancerous tissues or
cells using the
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antibody targeted towards these particular substrates, we can therefore
reliably predict a
neoplastic condition in mammals.
[0025] The term "mammal" is used herein to encompass both human and non-human
mammals.
[0026] A "coding sequence" or a sequence "encoding" an expression product,
such as a RNA,
polypeptide, protein, or enzyme, is a nucleotide sequence that, when
expressed, results in the
production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide
sequence encodes
an amino acid sequence for that polypeptide, protein or enzyme. A coding
sequence for a protein
may include a start codon (usually ATG) and a stop codon. As used herein,
references to specific
proteins (e.g., antibodies or pro-caspase-3) can include a polypeptide having
a native amino acid
sequence, as well as variants and modified forms regardless of their origin or
mode of
preparation. A protein which has a native amino acid sequence is a protein
having the same
amino acid sequence as obtained from nature (e.g., a naturally occurring CK2)
Such native
sequence proteins can be isolated from nature or can be prepared using
standard recombinant
and/or synthetic methods. Native sequence proteins specifically encompass
naturally occurring
truncated or soluble forms, naturally occurring variant forms (e.g.,
alternatively spliced forms),
naturally occurring allelic variants and forms including post-translational
modifications. A native
sequence protein includes proteins following post-translational modifications
such as
glycosylation, or phosphorylation, or other modifications of some amino acid
residues.
[0027] A "biological sample" encompasses a variety of sample types obtained
from a subject and
can be used in a diagnostic or monitoring assay. Biological samples include
but are not limited to
solid tissue samples such as a biopsy specimen or tissue cultures or cells
derived therefrom, and
the progeny thereof. For example, biological samples include cells obtained
from a tissue sample
collected from an individual suspected of having a prostate cancer. Therefore,
biological samples
encompass clinical samples, cells in culture, cell supernatants, cell lysates,
and tissue samples. In
a preferred embodiment, said biological sample is a prostate biopsy.
[0028] The term "detection" as used herein includes qualitative and/or
quantitative detection
(measuring levels) with or without reference to a control.
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[0029] According to the present invention, "antibody" or "inununoglobulin"
have the same
meaning, and will be used equally in the present invention. In natural
antibodies, two heavy
chains are linked to each other by disulfide bonds and each heavy chain is
linked to a light chain
by a disulfide bond. There are two types of light chain, lambda (I) and kappa
(k). There are five
main heavy chain classes (or isotypes) which determine the functional activity
of an antibody
molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence
domains. The light
chain includes two domains, a variable domain (VL) and a constant domain (CL).
The heavy
chain includes four domains, a variable domain (VH) and three constant domains
(CH1, CH2
and CH3, collectively referred to as CH). The variable regions of both light
(VL) and heavy
(VH) chains determine binding recognition and specificity to the antigen. The
constant region
domains of the light (CL) and heavy (CH) chains confer important biological
properties such as
antibody chain association, secretion, trans-placental mobility, complement
binding, and binding
to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab
fragment of an
immunoglobulin and consists of the variable portions of one light chain and
one heavy chain.
The specificity of the antibody resides in the structural complementarity
between the antibody
combining site and the antigenic determinant. Antibody combining sites are
made up of residues
that are primarily from the hypervariable or complementarity determining
regions (CDRs).
Occasionally, residues from nonhypervariable or framework regions (FR)
influence the overall
domain structure and hence the combining site. The light and heavy chains of
an
immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-
CDR1 ,
H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six
CDRs,
comprising the CDR set from each of a heavy and a light chain V region.
Framework Regions
(FRs) refer to amino acid sequences interposed between CDRs.
[0030] According to the present invention, the term "diagnosis" encompasses
the identification
of a disease and the determination of the future course and outcome of a
disease.
[0031] The term "monoclonal antibody" or "mAb" as used herein refers to an
antibody of a
single amino acid composition, that is directed against a specific antigen and
that is produced by
a single clone of B cells or hybridoma.
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[0032] The term "polyclonal antibody" as used herein refers to an antibody
that is directed
against a specific antigen that is derived from different B-cell lines.
[0033] The term "Fab" denotes an antibody fragment having a molecular weight
of about 50,000
Da and antigen binding activity, in which about a half of the N-terminal side
of H chain and the
entire L chain, among fragments obtained by treating IgG with a protease,
papaine, are bound
together through a disulfide bond.
[0034] The term "F(ab')2" refers to an antibody fragment having a molecular
weight of about
100,000 Da and antigen binding activity, which is slightly larger than the Fab
bound via a
disulfide bond of the hinge region, among fragments obtained by treating IgG
with a protease,
pepsin.
[0035] The term "Fab' " refers to an antibody fragment having a molecular
weight of about
50,000 Da and antigen binding activity, which is obtained by cutting a
disulfide bond of the
hinge region of the F(ab')2. A single chain Fv ("scFv") polypeptide is a
covalently linked
VH::VL heterodimer which is usually expressed from a gene fusion including VH
and VL
encoding genes linked by a peptide-encoding linker. The human scFv fragment of
the invention
includes CDRs that are held in appropriate conformation, preferably by using
gene
recombination techniques.
[0036] The term "hybridoma" denotes a cell, which is obtained by subjecting a
B cell prepared
by immunizing a non-human mammal with an antigen to cell fusion with a myeloma
cell derived
from a mouse or the like which produces a desired monoclonal antibody having
an antigen
specificity.
[0037] The term "chimeric antibody" refers to a monoclonal antibody which
comprises a VH
domain and a VL domain of an antibody derived from a non-human animal, a CH
domain and a
CL domain of a human antibody. As the non-human animal, any animal such as
mouse, rat,
hamster, rabbit or the like can be used.
[0038] The term "humanized antibody" refers to antibodies in which the
framework or
"complementarity determining regions" (CDR) have been modified to comprise the
CDR from a
donor immunoglobulin of different specificity as compared to that of the
parent immunoglobulin.
CA 02773151 2012-03-28
In a preferred embodiment, a mouse CDR is grafted into the framework region of
a human
antibody to prepare the "humanized antibody".
[0039] By "purified" and "isolated" it is meant, when referring to a
polypeptide (i.e. the antibody
fragment of the invention) or a nucleotide sequence, that the indicated
molecule is present in the
substantial absence of other biological macromolecules of the same type. The
term "purified" as
used herein preferably means at least 75% by weight, more preferably at least
85% by weight,
more preferably still at least 95% by weight, and most preferably at least 98%
by weight, of
biological macromolecules of the same type are present. An "isolated" nucleic
acid molecule
which encodes a particular polypeptide refers to a nucleic acid molecule which
is substantially
free of other nucleic acid molecules that do not encode the subject
polypeptide; however, the
molecule may include some additional bases or moieties which do not
deleteriously affect the
basic characteristics of the composition.
[0040] The term CK2 denotes the casein kinase 2 or II protein, in particular
the Human CK2.
Protein kinase CK2 is a highly conserved and ubiquitous serine/threonine
kinase. Accordingly
the term "CK2a" denotes the CK2a subunit of CK2, the term "CK213" denotes the
CK213 subunit
of CK2 and "CK2a2CK2132" denotes the holoenzyme tetramer consisting of 2 CK2a
and 2 CK213
subunits in a tetramer complex of CK2. The polypeptide sequence for human CK2a
is deposited
in the database under accession number NM 001895. The polypeptide sequence for
human
CK2 13 is deposited in the database under accession number NM_001320.5.
[0041] The term C3 denotes the pro-caspase-3 protein, in particular human
Caspase3. Caspase3
is a member of a family of cysteine proteases that are key mediators of
programmed cell death or
apoptosis. The precursor form of all caspases is composed of a prodomain, and
large and small
catalytic subunits. The polypeptide sequence for pro-caspase-3 is deposited in
the database
= under the accession number AAA65015.1. Caspase3 is also known as Apopain,
CPP32 or
Yama.
[0042] The term C8 denotes the pro-caspase-8 protein, in particular human
Caspase8. Caspase8
is a member of a family of cysteine proteases that are key mediators of
programmed cell death or
apoptosis. The precursor form of all caspases is composed of a prodomain, and
large and small
catalytic subunits. The polypeptide sequence for pro-caspase-8 is deposited in
the database
CA 02773151 2012-03-28
11
under the accession number CAA66853.1. Caspase8 is also known as Casp-8,
FLICE, MACH,
or MCH5.
[0043] The term single mutant refers to the replacement of one amino acid in a
polypeptide
chain with a different amino acid. In preferred embodiments, threonine or
serine was replaced at
the identical position of the polypeptide with the amino acid alanine.
[0044] The term double mutant refers to the replacement of two amino acids in
a polypeptide
chain with two amino acids. In preferred embodiments, both threonine and
serine was replaced at
the identical positions of the same polypeptide chain with the amino acid
alanine.
[0045] As used herein, the term "subject" denotes a mammal, such as a rodent,
a feline, a
canine, and a primate. Preferably a subject according to the invention is a
human.
[0046] An object of the invention relates to an in vitro method for diagnosing
cancer in a
subject, said method comprising measuring the activity of CK2a and CK2a2CK2132
in a cell
obtained from a biological sample of said subject.
[0047] The term "activity" relating to Protein kinase CK2 refers to the degree
of phosphorylation
of SEQ ID NO: 2 and SEQ ID NO: 6. In preferred embodiments, we measure the
activity to
CK2a and CK2a2CK202.
[0048] Diagnostic Methods: Typically the activity of CK2a and CK2a2CK2132 can
be measured
by using an antibody or a fragment thereof which specifically binds to
phosphorylated caspase-3
polypeptide fragments or proteins.
[0049] In preferred embodiments, said antibody of fragment thereof binds to
phosphorylated
pro-caspase-3 epitopes as set forth in SEQ ID NOS: 2, 3, and SEQ ID NO: 6.
[0050] In preferred embodiments, the activity of CK2a and CK2a2CK2I32 is
measured in said
cell.
[0051] In order to monitor the outcome of the cancer, the method of the
invention may be
repeated at different intervals of time, in order to determine if the activity
of CK2a and
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CK2a2CI(2132 increases or decreases, whereby it is determined if the cancer
progresses or
regresses.
[0052] Antibodies and Antibody Fragments of the Invention: The inventor has
produced a
highly specific antibody against phosphorylated epitopes of caspase-3 or
fragments thereof. The
sequences are set forth in SEQ ID NOs: 2, 3, and SEQ ID NO: 6. Figure 1
highlights the
sensitivity and specificity of the antibody towards said sequences.
[0053] The antibodies of the present invention may be monoclonal or polyclonal
antibodies,
single chain or double chain, chimeric antibodies, or humanized antibodies.
Whereas polyclonal
antibodies may be used, monoclonal antibodies may be preferred. Said fragment
may be a Fab,
F(ab')2, Fab' or scFV fragment. Antibodies or fragments of the invention can
be used in an
isolated (e.g., purified) form or contained in a vector, such as a membrane or
lipid vesicle (e.g. a
lipo some) .
[0054] In a preferred embodiment, antibodies of the invention may be labelled
with a detectable
molecule or substance, such as a fluorescent molecule, a radioactive molecule
or any others
labels known in the art. Labels are known in the art that generally provide
(either directly or
indirectly) a signal. As used herein, the term "labeled", with regard to the
antibody, is intended to
encompass direct labeling of the antibody by coupling (i.e., (PE) or
lndocyanine (Cy5)) to the
antibody, as well as indirect labeling of the antibody by reactivity with a
detectable substance.
An antibody of the invention may be labelled with a radioactive molecule by
any method known
to the art. For example radioactive molecules include but are not limited
radioactive atom for
scintigraphic studies such as 1123, 1124, In111 , Re186, Re188. Antibodies of
the invention may
be also labelled with a spin label for nuclear magnetic resonance (NMR)
imaging (also known as
magnetic resonance imaging, mri), such an iodine-123, iodine-131 , indium-Ill,
fluorine-19,
carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Antibodies
of the invention
may be useful for staging of cancer (e.g., in radioimaging).
[0055] An object of the invention relates to a method for detecting the
activity of CK2a and
CK2a2C1(2132, said method comprising using an antibody or a fragment thereof
which binds to
pro-caspase-3 proteins or fragments thereof as set forth in SEQ ID NOs: 2, 3,
and SEQ ID NO:
6.
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[0056] A further object of the invention relates to a method to use the
antibody or a fragment
thereof which binds to pro-caspase-3 epitopes as set forth in SEQ ID NOs: 2,
3, and SEQ ID
NO: 6 for the diagnosis of cancer.
[0057] They may also be used alone or in combination with other means for
detecting cancer
markers, including but not limited to Prostate Specific Antigen (PSA).
[0058] Nucleic Acids, vectors and recombinant host cells: A further object of
the invention
relates to a nucleic acid sequence encoding an antibody of the invention or a
fragment thereof.
Typically, said nucleic acid is a DNA or RNA molecule, which may be included
in any suitable
vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a
viral vector.
[0059] The terms "vector", "cloning vector" and "expression vector" mean the
vehicle by which
a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host
cell, so as to
transform the host and promote expression (e.g. transcription and translation)
of the introduced
sequence. So, a further object of the invention relates to a vector comprising
a nucleic acid of the
invention. Such vectors may comprise regulatory elements, such as a promoter,
enhancer,
terminator and the like, to cause or direct expression of said polypeptide
upon administration to a
subject. Examples of promoters and enhancers used in the expression vector for
animal cell
include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR
promoter and
enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter
(Mason JO et al.
1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the
like. Any
expression vector for animal cell can be used, so long as a gene encoding the
human antibody C
region can be inserted and expressed. Examples of suitable vectors include
pAGE107 (Miyaji H
et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984),
pKCR
(O'Hare K et al. 1981), pSG1 beta d2-4-(Miyaji H et al. 1990) and the like.
Other examples of
plasmids include replicating plasmids comprising an origin of replication, or
integrative
plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples
of viral vector
include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant
viruses may be
produced by techniques known in the art, such as by transfecting packaging
cells or by transient
transfection with helper plasmids or viruses. Typical examples of virus
packaging cells include
PA317 cells, PsiCRIP cells, GPenv cells, 293 cells, etc. Detailed protocols
for producing such
CA 02773151 2012-03-28
14
replication defective recombinant viruses may be found for instance in WO
95/14785, WO
96/22378, US 5,882,877, US 6,013,516, US 4,861 ,719, US 5,278,056 and WO
94/19478.
[0060] A further object of the present invention relates to a cell which has
been transfected,
infected or transformed by a nucleic acid and/or a vector according to the
invention.
[0061] The term "transformation" means the introduction of a "foreign" (i.e.
extrinsic or
extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell
will express the
introduced gene or sequence to produce a desired substance, typically a
protein or enzyme coded
by the introduced gene or sequence. A host cell that receives and expresses
introduced DNA or
RNA bas been "transformed". The nucleic acids of the invention may be used to
produce a
recombinant antibody of the invention in a suitable expression system. The
term "expression
system" means a host cell and compatible vector under suitable conditions,
e.g. for the
expression of a protein coded for by foreign DNA carried by the vector and
introduced to the
host cell. Common expression systems include E. coli host cells and plasmid
vectors, insect host
cells and Baculovirus vectors, and mammalian host cells and vectors. Other
examples of host
cells include, without limitation, prokaryotic cells (such as bacteria) and
eukaryotic cells (such as
yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific
examples include E. coli,
Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells,
CHO cells,
3T3 cells, COS cells, HeLa, U2-0S, Saos-2 etc.) as well as primary or
established mammalian
cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells,
epithelial cells,
nervous cells, adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cell
(ATCC CRL1581
), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate
reductase
gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al;
1980), rat
YB2/3HLP2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as "YB2/0
cell"), and
the like.
[0062] Methods of producing antibodies of the invention: Antibodies and
fragments of the
invention may be produced by any technique known in the art, such as, without
limitation, any
biological, chemical, genetic or enzymatic technique, either alone or in
combination. Procedures
for raising polyclonal antibodies are well known. Polyclonal antibodies can be
obtained from
serum of an animal immunized against the pro-caspase-3 epitope as set forth in
SEQ ID NO: 1,
CA 02773151 2012-03-28
which may be produced by genetic engineering for example according to standard
methods well-
known by one skilled in the art. Typically, such antibodies can be raised by
administering the
epitope as set forth in SEQ ID NO: 1 subcutaneously to New Zealand white
rabbits which have
first been bled to obtain pre-immune serum. The antigens can be injected at a
total volume of 100
ttl per site at six different sites. Each injected material may contain
adjuvants with or without
pulverized acrylamide gel containing the protein or polypeptide after SDS-
polyacrylamide gel
electrophoresis. The rabbits are then bled two weeks after the first injection
and periodically
boosted with the same antigen three times every six weeks. A sample of serum
is then collected
10 days after each boost. Polyclonal antibodies are then recovered from the
serum by affinity
chromatography using the corresponding antigen to capture the antibody. This
and other
procedures for raising polyclonal antibodies are disclosed by Harlow et al.
(1988), which is
hereby incorporated in the references. The antibody-producing cells in the
immunized mammal
are isolated and fused with myeloma or heteromyeloma cells to produce hybrid
cells
(hybhdoma). The hybridoma cells producing the monoclonal antibodies are
utilized as a source
of the desired monoclonal antibody. This standard method of hybridoma culture
is described in
Kohler and Milstein (1975). Antibody generation techniques not involving
immunisation are also
contemplated such as for example using phage display technology to examine
naive libraries
(from non-immunised animals); see Barbas et al. (1992), and Waterhouse et al.
(1993). While
mAbs can be produced by hybridoma culture the invention is not to be so
limited. For example,
knowing the amino acid sequence of the desired sequence, one skilled in the
art can readily
produce the antibodies, by standard techniques for production of polypeptides.
For instance, they
can be synthesized using well-known solid phase method, preferably using a
commercially
available peptide synthesis apparatus (such as that made by Applied
Biosystems, Foster City,
California) and following the manufacturer's instructions. Alternatively,
antibodies of the
invention can be synthesized by recombinant DNA techniques as is well-known in
the art. For
example, these fragments can be obtained as DNA expression products after
incorporation of
DNA sequences encoding the desired (poly)peptide into expression vectors and
introduction of
such vectors into suitable eukaryotic or prokaryotic hosts that will express
the desired
polypeptide, from which they can be later isolated using well-known
techniques. In particular,
the invention further relates to a method of producing an antibody or a
polypeptide of the
invention, which method comprises the steps consisting of: (i) culturing a
transformed host cell
CA 02773151 2012-03-28
16
according to the invention under conditions suitable to allow expression of
said antibody or
polypeptide; and (ii) recovering the expressed antibody or polypeptide.
Antibodies of the
invention are suitably separated from the culture medium by conventional
immunoglobulin
purification procedures such as, for example, protein A-Sepharose,
hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity chromatography. In
a particular
embodiment, the human chimeric antibody of the present invention can be
produced by obtaining
nucleic sequences encoding VL and VH domains as previously described,
constructing a human
chimeric antibody expression vector by inserting them into an expression
vector for animal cell
having genes encoding human antibody CH and human antibody CL, and expressing
the coding
sequence by introducing the expression vector into an animal cell. As the CH
domain of a human
chimeric antibody, it may be any region which belongs to human immunoglobulin,
but those of
IgG class are suitable and any one of subclasses belonging to IgG class, such
as IgGI , lgG2,
lgG3 and lgG4, can also be used. Also, as the CL of a human chimeric antibody,
it may be any
region which belongs to Ig, and those of kappa class or lambda class can be
used. Methods for
producing chimeric antibodies involve conventional recombinant DNA and gene
transfection
techniques are well known in the art (See Morrison SL. et al. (1984) and
patent documents
US5,202,238; and U55,204, 244). The humanized antibody of the present
invention may be
produced by obtaining nucleic acid sequences encoding CDR domains, as
previously described,
constructing a humanized antibody expression vector by inserting them into an
expression vector
for animal cell having genes encoding (i) a heavy chain constant region
identical to that of a
human antibody and (ii) a light chain constant region identical to that of a
human antibody, and
expressing the genes by introducing the expression vector into an animal cell.
The humanized
antibody expression vector may be either of a type in which a gene encoding an
antibody heavy
chain and a gene encoding an antibody light chain exists on separate vectors
or of a type in
which both genes exist on the same vector (tandem type). In respect of
easiness of construction
of a humanized antibody expression vector, easiness of introduction into
animal cells, and
balance between the expression levels of antibody H and L chains in animal
cells, humanized
antibody expression vector of the tandem type is preferred (Shitara K et al.
1994). Examples of
tandem type humanized antibody expression vector include pKANTEX93 (WO
97/10354),
pEE18 and the like. Methods for producing humanized antibodies based on
conventional
recombinant DNA and gene transfection techniques are well known in the art
(See, e. g.,
CA 02773151 2012-03-28
17
Riechrnann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be
humanized using a
variety of techniques known in the art including, for example, CDR-grafting
(EP 239,400; PCT
publication W091/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089),
veneering or
resurfacing (EP 592,106; EP 519,596; Padlan EA (1991 ); Studnicka GM et al.
(1994); Roguska
MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5,565,332). The general
recombinant DNA
technology for preparation of such antibodies is also known (see European
Patent Application
EP 125023 and International Patent Application WO 96/02576). The Fab of the
present invention
can be obtained by treating an antibody which specifically reacts against the
pro-caspase-3
epitopes as set forth in SEQ ID NO:1 with a protease, papaine. Also, the Fab
can be produced by
inserting DNA encoding Fab of the antibody into a vector for prokaryotic
expression system, or
for eukaryotic expression system, and introducing the vector into a procaryote
or eucaryote (as
appropriate) to express the Fab. The F(ab')2 of the present invention can be
obtained treating an
antibody which specifically reacts with the pro-caspase-3 epitope as set forth
in SEQ ID
NO:1 with a protease, pepsin. Also, the F(ab')2 can be produced by binding
Fab' described below
via a thioether bond or a disulfide bond. The Fab' of the present invention
can be obtained
treating F(ab')2 which specifically reacts with the pro-caspase-3 epitope as
set forth in SEQ ID
NO: 1 with a reducing agent, dithiothreitol. Also, the Fab' can be produced by
inserting DNA
encoding Fab' fragment of the antibody into an expression vector for
prokaryote, or an
expression vector for eukaryote, and introducing the vector into a prokaryote
or eukaryote (as
appropriate) to perform its expression. The scFv of the present invention can
be produced by
obtaining cDNA encoding the VH and VL domains as previously described,
constructing DNA
encoding scFv, inserting the DNA into an expression vector for prokaryote, or
an expression
vector for eukaryote, and then introducing the expression vector into a
prokaryote or eukaryote
(as appropriate) to express the scFv. To generate a humanized scFv fragment, a
well-known
technology called CDR grafting may be used, which involves selecting the
complementary
determining regions (CDRs) from a donor scFv fragment, and grafting them onto
a human scFv
fragment framework of known three dimensional structure (see, e. g.,
W098/45322; WO
87/02671 ; US5,859,205; US5,585,089; US4,816,567; EP0173494).
[0063] Phosphorylation Site-Specific Antibodies of the invention: In further
aspect of the
invention, the invention discloses phosphorylation site-specific binding
molecules that
specifically bind at a novel serine and/or threonine phosphorylation site of
the invention, and that
CA 02773151 2012-03-28
=
18
distinguish between the phosphorylated and unphosphorylated forms. In one
embodiment, the
binding molecule is an antibody or an antigen-binding fragment thereof. The
antibody may
specifically bind to an amino acid sequence comprising a phosphorylation site
identified in Table
1 and Table 2. In specific embodiments, the said antibody can bind the
phosphorylated forms of
SEQ ID NO: 1; SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 6.
[0064] In some embodiments, the antibody or antigen-binding fragment thereof
specifically
binds the phosphorylated site. An antibody or antigen-binding fragment thereof
specially binds
an amino acid sequence comprising a novel serine and/or threonine
phosphorylation site in Table
1 and Table 2 when it does not significantly bind any other site in the parent
protein and does not
significantly bind a protein other than the parent protein. An antibody of the
invention is
sometimes referred to herein as a "phospho-specific" antibody.
[0065] An antibody or antigen-binding fragment thereof specially binds an
antigen when the
dissociation constant is <1 mM, preferably <100 nM, and more preferably <10
nM.
[0066] In particularly preferred embodiments, an antibody or antigen-binding
fragment thereof
of the invention specially binds an amino acid sequence comprising a novel
serine and/or
threonine phosphorylation site shown as a lower case "s" or "t" (respectively)
in a sequence listed
in Table 1 and Table 2 selected from the group consisting of SEQ ID NOs: 1; 2;
3 ; and 6.
[0067] It shall be understood that if a given sequence disclosed herein
comprises more than one
amino acid that can be modified, this invention includes sequences comprising
modifications at
one or more of the amino acids. In one non-limiting example, where the
sequence is:
RRRGIETDSGVDDDMA, and the * symbol indicates the preceding amino acid is
modified
(e.g., a T* or S* indicates a modified (e.g., phosphorylated) threonine or
serine residue, the
invention includes, without limitation, RRRGIET*DSGVDDDMA, RRRGIETDS*GVDDDMA,
HHHHHHMENTENSVDSKSIICNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNICN
FHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNICNDLTREEIVELMRDVSKEDHSICRS
SFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLIGKPKLFIIQAARGTELDCGIET
*DSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSICDGSWFIQSLCAMLKQYADKL
EFMHILTRVNRKVATEFESFSFDATFHAICKQIPCIVSMLTICELYFYH, as well as sequences
comprising more than one modified amino acid including GIET*DS*GVDDDMAC,
CA 02773151 2012-03-28
19
RRRGIET*DS*GVDDDMA and
HHHHHHMENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKN
FHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRS
SFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGIOKLFIIQAARGTELDCGIET
*DS *GVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQ SLCAMLKQYADKL
EFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH. Thus, an antibody of
the invention may specifically bind to RRRGIET*DSGVDDDMA, or may specifically
bind to
RRRGIETDS*GVDDDMA, or may specifically bind to RRRGIET*DS*GVDDDMA, and so
forth. In some embodiments, an antibody of the invention specifically binds
the sequence
comprising a modification at one amino acid residues in the sequence. In some
embodiments, an
antibody of the invention specifically binds the sequence comprising
modifications at two or
more amino acid residues in the sequence.
[0068] In some embodiments, an antibody or antigen-binding fragment thereof of
the invention
specifically binds an amino acid sequence comprising any one of the above
listed SEQ ID NOs.
In some embodiments, an antibody or antigen-binding fragment thereof of the
invention
especially binds an amino acid sequence comprises a fragment of one of said
SEQ ID NOs.,
wherein the fragment includes the phosphorylatable serine and/or threonine.
[0069] Antibodies of the invention may be advantageously conjugated to
fluorescent dyes (e.g.
A1exa488, PE) for use in diagnostic analyses. Also, antibodies of the
inventions may be used
with other antibodies, known in the art as secondary antibodies, that have
conjugated fluorescent
dyes (e.g. A1exa488, PE) to facilitate their detection in diagnostic analyses.
Examples of
secondary antibodies used in the invention may be but not limited to goat anti-
rabbit IgG
antibodies conjugated to Alexa488.
[0070] Peptides and Peptide Fragments of the invention: Polypeptide epitopes
used for the
invention may be synthesized using standard methods of peptide synthesis well
known in the art.
In preferred embodiments, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ
ID NO: 5 may be synthesized using these methods. Modifications of the said
epitopes may
include the attachment of molecules by chemical means. Molecules for
attachment may include
but not limited to amino acids, polypeptides, proteins or biotin.
Modifications may allow
CA 02773151 2012-03-28
binding of said epitopes to surfaces to facilitate the diagnosis. In preferred
embodiments, the
pro-caspase-3 epitopes may also be expressed on DNA plasmids or vectors in
host cells such as
but not limited to E. coli. The plasmids or vectors may be but not limited to
pET vectors well
known in the art. [0072] Peptides of the invention may be phosphorylated
according to Table 1
and Table 2. The phopho-specific antibodies of the invention or fragments
thereof bind to the
phosphorylated but not the non-phosphorylated forms of the peptides or
fragments thereof. In
specific but not limited examples, the phospho-specific antibodies of the
invention bind to the
phosphorylated form of SEQ ID NO: 2 (RRRGIET*DSGVDDDMA) where threonine is
phosphorylated as denoted by (T*).
[0071] Proteins of the invention: Pro-caspase-3 epitope as set forth in SEQ ID
NO: 6 may be
expressed in a host cell such as but not limited to E coli. In preferred
embodiments, the pro-
caspase-3 epitope as set forth in SEQ ID NO: 6 may be encoded in a DNA plasmid
or vector.
Modifications of the said epitopes may include the attachment of molecules by
chemical means.
Modifications may be but not limited to amino acids, polypeptides, proteins or
biotinylation.
Modifications may allow binding of said epitopes to surfaces to facilitate the
diagnosis. In
preferred embodiments, the pro-caspase-3 epitopes may also be expressed on DNA
plasmids or
vectors in host cells such as but not limited to E. coli. The plasmids of
vectors may be but not
limited to pET vectors known in the art.
[0072] Proteins of the invention may be phosphorylated according to Table 2.
The phopho-
specific antibodies of the invention or fragments thereof bind to the
phosphorylated but not the
non-phosphorylated forms of the protein or fragments thereof. In specific but
not limited
examples, the phospho-specific antibodies of the invention bind to the
phosphorylated form of
SEQ ID NO: 6
(HHHHHHMENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKN
FHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNICNDLTREEIVELMRDVSKEDHSKRS
SFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQAARGTELDCGIET
*DSGVDDDMACHKIP VEADFLYAYSTAPGYYSWRNSKDGSWFIQ SLCAMLKQYADKL
EFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH) where threonine is
phosphorylated as denoted by (T*).
CA 02773151 2012-03-28
21
[0073] Kits for diagnosing cancer: Finally, the invention also provides kits
comprising at least
one antibody or fragment of the invention. Kits find use in detecting the
activity of CK2a and
CK2a2CK21:32. Kits of the invention can contain an antibody or a fragment and
can be coupled to
a solid support, e.g., a tissue culture plate or beads (e.g., sepharose
beads). Kits of the inventions
can contain pro-caspase-3 epitopes such as SEQ ID NO: 2, 3, and 6 and can be
coupled to a solid
support, eg., a tissue culture plate or beads (e.g. Sepharose beads or avidin
membrane). Kits can
be provided which contain antibodies and pro-caspase-3 epitopes for detection
and quantification
of CK2a and CK2a2CK2f32, e.g. in an ELISA or a Western blot. Such antibody
useful for
detection may be provided with a label such as a fluorescent or radiolabel.
The kit according to
the invention is especially adapted for diagnosing, including but not limited
to, prostate cancer.
Kit may be used for diagnosing breast cancer. Kits can be labeled for
diagnostic use only and
components of the kit (e.g. antibody and pro-caspase-3 epitopes) are clearly
labeled.
[0074] Embodiments of the invention are described by reference to the
following specific
examples which are not to be construed as limiting.
Example 1 ¨ A Polyclonal phospho-specific antibody to T174 and S176 of Pro-
caspase-3
[0075] We chose to generate an antibody specific to phosphorylated pro-caspase-
3 at sites we
predicted to be targets of Protein kinase CK2 (Duncan, J.S., Turowec, J.P.,
Duncan, K.E., Vilk,
G., Wu, C., Luscher, B., Li, S.S-C., Gloor, G.B., and Litchfield, D.W. (2011)
A peptide-based
target screen of kinase/caspase overlapping substrates identifies pro-caspase-
3 as a physiological
target of CK2. Sci. Signal 4(172): 1-13). In order to generate antibodies
directed against
phosphorylated pro-caspase-3, we coupled SEQ ID NO: 1 to KLH (Keyhole Limpet
Hemocyanin) protein carrier and injected in two rabbits (Table 1).
[0076] Once the polyclonal antibody, aC3T174, was purified from the rabbit
serum, we
performed validation experiments to confirm its specificity to the sites on
pro-caspase-3.
Purified His-tagged Pro-caspase-3, His-tagged Pro-caspase-3 (Ti 74A), His-
tagged Pro-caspase-3
(Si 76A), and His-tagged Pro-caspase-3 (T174A/S176A) proteins were suspended
into CK2
kinase buffer (150 mM NaC1, 50 mM Tris-C1, 50 mM MgCl2, 1 mM DTT, 100 uM 32P-
y¨ATP
CA 02773151 2012-03-28
22
(1000 cpm/pmol specific activity) (herein referred to as CK2 kinase assay
buffer). To commence
the kinase assay, fifteen nanograms of GST-CK2a was introduced into the
reaction tubes and
placed in a 30 C water bath with shaking. At the appropriate reaction times up
to 64 minutes
(Figure 1), the kinase assay was stopped by the addition of 2 X laemmli buffer
and immediately
boiled at 95 C for 5 minutes.
[0077] The radioactive samples were resolved on two 15% SDS-PAGE gels by
applying a
voltage of 200 Volts for 1 hour. One gel was fixed in a methanol-acetic acid
(50%/5%) solution
for 1 hour, stained using coomassie G-250 stain and then dried. The
incorporation of radioactive
phosphate was determined by exposing the gels to film and quantifiying using a
STORM 850
phosphoimager and expressed in pmol p32 incorporation (Figure 1,
autoradiography
quantification). The other gel was Western blotted onto 0.45 micron PVDF
membranes using
established procedures and immunoblotted using the aC3T174 antibody. Briefly,
the PVDF
membranes were blocked in 5% BSA/TBS-T (known herein as blocking solution) for
1 hour with
shaking at room temperature. Then, a 1:1000 dilution of rabbit aC3T174
polyclonal antibodies
in 1% BSA/TBS-T replaced the blocking solution and the PVDF membranes were
further
incubated overnight at 4 C with shaking. Next day, the antibody solution was
removed and the
membranes were extensively washed with TBS-T solution (3 X 15 minutes with
shaking at room
temperature). Finally the PVDF membranes were exposed for 1 hour at room
temperature to a
1:10 000 dilution of secondary goat anti-rabbit antibodies conjugated to a
fluorescent dye
(IRDye 680) (LiCOR Biosciences) in 1 % BSA/TBS-T. The immunoblots were washed
4 X 10
minutes with TBS-T and 1 X 5 minutes with TBS prior to exposure on a LiCOR
Odyssey near
infrared imager. Using Odyssey software, the western blot quantification was
expressed in
arbitrary units (Figure 1).
[0078] Results: We noted an increasing incorporation of phosphate into pro-
caspase-3 using
CK2a as the protein kinase (Figure 1). This phosphate incorporation increased
up to 15 pmol of
radioactive phosphate after 64 minutes of incubation time. This observation
was also seen when
using the aC3T174 antibody was used to probe the western blot. Specifically,
an increase of the
number of arbitrary units from 0 to approximately 300 was observed over the 64-
minute
incubation. We thus validated that pro-caspase-3 is a target for CK2 in vitro
using radioactive
ATP and that aC3T174 seems to react specifically to phosphorylated pro-caspase-
3.
CA 02773151 2012-03-28
23
[0079] We next proceeded to confirm that aC3T174 was targeting specific
phosphorylated
amino acids located in pro-caspase-3. We presumed that aC3T174 was targeting
phosphorylated
T174 and S176 in pro-caspase-3 since we used a similar peptide fragment (SEQ
ID NO: 1) to
immunize our rabbits. Thus, we generated mutants of pro-caspase-3 in which
T174, S176 or
both amino acids were replaced with alanine. We hypothesized that a change of
these amino
acids to alanine would prevent the incorporation of radioactive phosphate at
these specific sites.
We noted in both the Western blots and autoradiographs a reduction of
phosphate incorporation
when we mutated either T174 or S176 over the incubation period (Figure 1).
Interestingly, CK2
was unable to phosphorylate pro-caspase-3 (T174A/S176A) double mutant during
the incubation
period. With the observation that the aC3T174 antibody reacted less with the
single mutants or
not by any means with the pro-caspase-3 double mutant, we could conclude that
our antibody
was specific to phosphorylated T174 and S176.
Example 2 ¨ The Phosphorvlation of Pro-caspase-3 protein in vitro by different
forms of
Protein Kinase CK2
[0080] Plasmids encoding His-tagged recombinant pro-caspase-3 and pro-caspase-
8 were
introduced into E. coli BL21 (DE3) cells and grown in liquid Luria-Bertani
broth supplemented
with 100 mg/m1 ampicillin. The recombinant proteins were induced for
expression by the
addition of 350 p,M IPTG to Luria-Bertani liquid media. After 4 hours, the
cells were harvested
and the proteins were isolated using His-tagged Qiagen resins and standard
isolation techniques
using manufacturer's instructions. The proteins were suspended in an ice-cold
solution of 250
mM NaC1, 50 mM Tris-C1, pH 8, 0.5 mM DTT, 0.5 mM EDTA, 0.01 % Triton X-100, 50
%
glycerol and aliquoted and stored at -80 C.
[0081] Kinase assays were performed using either recombinant GST-tagged CIC2d
or GST-
CK2a2HIS-CK2132 holoenzyme tetramer. Briefly, various concentrations of pro-
caspase-3 (pro-
C3), pro-caspase-8 (pro-C8), a-casein (CAS), or calmodulin (CaM) were
suspended in an ice-
cold solution of CK2 kinase assay buffer. Kinase assays were initiated by
adding 10-15 ng of
CK2a or holoenzyme tetramer to the mixture and incubated for 10 minutes at 30
C. The
reactions were immediately stopped by the addition of 2 X laenunli buffer and
immediate
heating to 95 C for 10 minutes. Samples were loaded onto 12 or 15 % SDS-PAGE
gels to
CA 02773151 2012-03-28
24
resolve the phosphorylated proteins. The gels were fixed in a methanol -acetic
acid solution for
1 hour, stained using coomassie G-250 stain and then dried. The incorporation
of radioactive
phosphate was determined by exposing the gels to film and quantifiying using a
STORM 850
phosphoimager (Figure 2A). The results were performed at least in triplicate
and were plotted as
initial velocity versus substrate concentration. Note that the phosphorylation
of pro-caspase-3
and -8 in vitro is dependent upon the presence of CK213. In other words, the
activity of the CIC2
holoenzyme tetramer is significantly different from the CK2a monomer in vitro.
Example 3 ¨ The Phosphorvlation of specific peptides usin2 different forms of
Protein
kinase CK2 in vitro
[0082] RC3 peptide (RRRGIETDSGVDDDMA), DSD peptide (RRRDDDSDDD), and eif2I3
peptide (MSGDEMIFDPTMSKKICKKKKICP) were synthesized in-house to 95% purity
using
standard peptide synthesis procedures. The sequence masses were checked by
injection into a
Waters Triple-quad mass spectrometer and using MassLynx 4.0 software for
analysis.
[0083] Kinase assays were performed using either recombinant GST-tagged CIC2a
or GST-
CK2a2HIS-C1(2132 holoenzyme tetramer. Briefly, 100 M of RC3, DSD or eif2f3
peptides were
suspended in an ice-cold solution of kinase assay buffer consisting of CIC2
kinase assay buffer.
Kinase assays were initiated by adding 10-15 ng of CIC2a or holoenzyme
tetramer to the mixture
and incubated for 10 minutes at 30 C. The reactions were immediately stopped
by spotting the
mixture on P81 phosphocellulose paper and placing into 500 ml of 1% phosphoric
acid. The P81
papers were washed using 4 X 5 minutes with 1 % phosphoric acid and were
finally fixed by a
final wash of 95% ethanol. The P81 papers were dried under a heating lamp. The
incorporation
of radioactive phosphate was determined by exposing the P81 samples to a
phosphor-
intensifying screen and quantifiying using a STORM 850 phosphoimager (Figure
2B). The
results were performed at least in triplicate and were plotted as a histogram
as initial velocity
(Vi). Fold induction was compared by dividing the degree of phosphate
incorporation into the
peptide using the holoenzyme tetramer by the degree of phosphate incorporation
into the peptide
using the CIC2a monomer kinase. The assays were performed at least in
triplicate.
CA 02773151 2012-03-28
Example 4 ¨ Polvclonal antibody aC3T174 can reco2nize phosphorvlated RC3
peptide and
the C3 recombinant protein adhered to surfaces
[0084] Twenty IJNI of RC3 peptide (RRRGIETDSGVDDDMA) and recombinant pro-
caspase-3
was first buffered changed to remove residual Tris buffer using Zeba-Spin
desalting columns
(Thermo Scientific Inc.) and suspended into 150 mM Sodium phosphate, pH 7.4
containing 0.5
mM DTT. The peptides and proteins were then added to separate wells of a 96-
well Aminolink
plate (Thermo Scientific Inc.) and incubated overnight at 4 C to facilitate
biding to the plate.
After extensive washing to remove unbound peptide and protein, 10 mM glycine
was added to
each well to block unbound reactive sites.
[0085] Kinase assays were performed using either recombinant GST-tagged CK2a
or GST-
CK2a2HIS-CK2132 holoenzyme tetramer. Briefly, kinase assays were initiated by
adding 10-15
ng of CK2a or holoenzyme tetramer to each well in a CK2 kinase assay buffer
solution (200 1)
and incubated for 2 hours at 37 C. In parallel, 20 uM of CIC2 inhibitor, TBB,
was added to the
kinase assay for control purposes. Also, selected wells were treated for 30
minutes at 30 C with
k-phosphatase after the kinase assays for control purposes following the
manufacturer's
recommendations. The reactions were immediately stopped by spiking each well
at the
appropriate time with 50 1 of 0.5 mM EDTA, pH 8Ø The wells were then
extensively washed
using 150 mM Sodium phosphate, pH 7.4 containing 0.5 mM DTT.
[0086] To monitor the degree of phosphorylation using the polyclonal aC3T174
antibody, the
wells were initially blocked using 5% BSA in 150 mM Tris-buffered Saline ¨
0.1% Tween-20
(TBS-T) (known herein as blocking solution) solution for 1 hour at room
temperature. Then, the
blocking solution was removed and replaced with a solution of 1:100 dilution
of polyclonal
rabbit aC3T174 antibody in 2% BSA/TBS-T and incubated for 1 hour at room
temperature. The
96-well plate was vigorously washed using TBS-T (at least 6 X 5 minutes), and
the secondary
antibody was then applied. The secondary antibody was an A1exa488-conjugate
goat anti-rabbit
antibody (Invitrogen) diluted to 1:1000 in 1% BSA/TBS-T. Incubation was 1 hour
at room
temperature followed again by extensive washing using TBS-T. A TBS without
Tween-20 was
used as the fmal wash prior to analysis on a Wallac 1420 VICTOR3 V Plate
reader. The plate
was read for 0.1 seconds in the green channel using the appropriate filters
supplied. The results
CA 02773151 2012-03-28
26
were graphed in relative light units and error bars are error of the mean
(Figure 3A). Note the
specificity of the aC3T174 to phosphorylated RC3 peptide and C3 protein. The
specificity of
the antibody is phosphorylation-dependent and CK2-specific since 1-phosphatase
or TBB either
removed the phosphate groups or inhibited the kinase assay, respectively. A
similar ELISA plate
assay was performed to denote the specificity of either CK2a or holoenzyme
tetramer
CK2a2CK2(32 to RC3 and C3 proteins adhered to 96-well plates. Detection was
monitored using
said antibody as described above. We noted that the holoenzyme tetramer
CK2a2CK2132
significantly phosphorylated RC3 peptide whereas it could not significantly
phosphorylate
recombinant pro-caspase-3 protein (Figure 3B). Thus, we conclude that CK2I3
regulates the
phosphorylation of C3 proteins and fragments by an unknown mechanism. Assays
were
performed at least in triplicate and additional controls were included such as
no peptide or
proteins in test wells or no primary antibody in the ELISA assay.
Example 5 ¨ A cancer dia2nostic kit assay that can detect different forms of
Protein kinase
CIC2 in mouse prostate tissue.
[0087] Cell culture: PC-3M cells (purchased from NIH/NCI Biorepository)
(Kozlowski 1984)
were cultured in RPMI-1640 (Gibco) plus 10% fetal bovine serum. Cells in log
phase were
harvested by trypsinization, washed once with HBSS (Gibco), and suspended in
HBSS for
injection (JM Kozlowski et al Cancer Res 44, 3522-3529, August 1984).
[0088] Animal model: Male nude mice (nu/nu, Charles River Laboratories) aged 6-
8 weeks
were housed in a specific pathogen free barrier facility. All animal
experiments were approved
by the Animal Use Subcommittee of the University Council on Animal Care at The
University of
Western Ontario following the guidelines of the Canadian Council on Animal
Care.
[0089] Tumour Induction: For tumour induction, mice were anaesthetized with
isoflurane, an
incision was made in the abdomen, and the bladder was retracted to expose the
prostate. PC-3M
cells (0.5 million cells in 30-40 tL HBSS) were injected into the left lateral
or dorsal lobes of the
prostate (K Rembrink et al The Prostate (1997) 31:168-174).
[0090] Tumour collection: On day 10 or 14 after tumour induction, mice were
sacrificed by
euthanyl injection and then perfused with 10% saline. The tumour was harvested
and snap frozen
CA 02773151 2012-03-28
27
in liquid nitrogen, then stored at -80 C until analysis. A magnetic resonance
image (MRI) was
taken to monitor the growth of the tumour in the nude mouse prostate (Figure
4A, coronal view).
[0091] Tissue sample preparation: Tissues were first suspended into a solution
composed of
0.5 % Nonidet P-40 (NP-40), 150 mM NaC1, 50 mM Tris-C1, pH 7.5,
protease/phosphatase
inhibitors. Then, the tissues were homogenized using a tissue homogenizer on
ice for up to 30
minutes. The homogenization process occurred in pulses to prevent over-
heating. After, the
samples were then spun at 20000 x g for 30 minutes to remove insolubles. The
supernatant was
collected and immediately used for analysis using the prescribed ELISA assay.
[0092] ELISA analysis:: Twenty !AM of RC3 peptide (RRRGIETDSGVDDDMA) and
recombinant pro-caspase-3 was first buffered changed to remove residual Tris
buffer using Zeba-
Spin desalting columns (Thermo Scientific Inc.) and suspended into 150 mM
Sodium phosphate,
pH 7.4 containing 0.5 mM DTT. The peptides and proteins were then added to
separate wells of
a 96-well Aminolink plate (Thermo Scientific Inc.) and incubated overnight at
4 C to facilitate
biding to the plate. After extensive washing to remove unbound peptide and
protein, 10 mM
glycine was added to each well to block unbound reactive sites.
[0093] Three or six micrograms of control or tumour tissues were suspended in
CK2 kinase
assay buffer (200u1) and was added to the 96-well plate that contained the
adhered RC3 and C3
molecules. The kinase assay was allowed to proceed for 2 hours at 37 C and
then immediately
stopped by the addition of 50 I of 0.5 mM EDTA, pH 8Ø Controls that were
included in the
assay were no antibody control, no peptide control, addition of TBB inhibitor,
and no protein
control. Following the 2-hour incubation, the wells were extensively washed
using TBS-T
solution and detection of phosphorylation was commenced using the polyclonal
aC3T174
antibody.
[00941 To monitor the degree of phosphorylation using the polyclonal aC3T174
antibody, the
wells were initially blocked using 5% BSA in 150 mM Tris-buffered Saline ¨
0.1% Tween-20
(TBS-T) (known herein as blocking solution) solution for 1 hour at room
temperature. Then, the
blocking solution was removed and replaced with a solution of 1:100 dilution
of polyclonal
rabbit aC3T174 antibody in 2% BSA/TBS-T and incubated for 1 hour at room
temperature. The
CA 02773151 2012-03-28
28
96-well plate was vigorously washed using TBS-T (at least 6 X 5 minutes), and
the secondary
antibody was then applied. The secondary antibody was an A1exa488-conjugate
goat anti-rabbit
antibody (Invitrogen) diluted to 1:1000 in 1% BSA/TBS-T. Incubation was 1 hour
at room
temperature followed again by extensive washing using TBS-T. A TBS without
Tween-20 was
used as the final wash prior to analysis on a Wallac 1420 VICTOR3 V Plate
reader. The plate
was read for 0.1 seconds in the green channel using the appropriate filters
supplied. The results
were graphed in relative light units and error bars are error of the mean
(Figure 4B). In parallel,
samples of the tumour (T) and control (C) tissues were lysed in 2 X laemmli
buffer and the
proteins were resolved on 12 % SDS-PAGE gels, western blotted to PVDF
membranes and
immuoblotted using CK2a, CK2a' and CK213 antibodies (Figure 4B, inset figure).
Note the
induction of expression of the three CK2 proteins in the tumour samples as
determined by
western blot analysis.
