CA2773151A1 - Cancer diagnostic kit to monitor the activity of protein kinase ck2, the methods and compositions thereof - Google Patents

Abstract

A diagnostic screening kit to monitor the kinase activity of protein kinase CK2 in cancer biopsies is provided. The compositions of the kit include a phospho-specific anti-caspase-3 antibody, a caspase-3 peptide fragment, and a human pro-caspase-3 protein.

Description

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 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 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.

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.

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.

[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 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.

[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.

[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.

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 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 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.

[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 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, 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 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., 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 =

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, 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 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*).

[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

(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 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 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.

[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 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 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.

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 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 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 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.

Claims (19)

1. An In vitro method for diagnosing cancer in a subject, said method comprising quantifying the activity of the human signaling protein CK2.alpha. and the human signaling protein holoenzyme tetramer CK2.alpha.2CK2.beta.2 in a cell obtained from a biological sample of said subject.

2. The method according to claim 1, wherein the activity of CK2.alpha. and CK2.alpha.2CK2.beta.2 is quantified using an antibody or a fragment thereof which binds to an amino acid sequence comprising a phosphorylation site identified in Table 2 and set forth in SEQ ID
NO: 2, and wherein the antibody does not bind to said amino acid sequence when the serine or threonine is not phosphorylated.

3. The method according to claim 1, wherein the activity of CK2.alpha. and CK2.alpha.2CK2.beta.2 is quantified using an antibody or a fragment thereof which binds to an amino acid sequence comprising a phosphorylation site identified in Table 2 and set forth in SEQ ID
NO: 3, and wherein the antibody does not bind to said amino acid sequence when the serine or threonine is not phosphorylated.

4. The method according to claim 1, wherein the activity of CK2.alpha. and CK2.alpha.2CK2.beta.2 is quantified using an antibody or a fragment thereof which binds to an amino acid sequence comprising a phosphorylation site identified in Table 2 and set forth in SEQ ID
NO: 6, and wherein the antibody does not bind to said amino acid sequence when the serine or threonine is not phosphorylated.

5. The method according to claims 1 to 4, where said biological sample is a cancer biopsy.

6. The method according to claim 5, where said biological sample contains human signal proteins CK2.alpha. and CK2.alpha.2CK2.beta.2.

7. An antibody or fragment thereof which binds specifically to an amino acid sequence comprising a phosphorylation site identified in Table 2 and set forth in SEQ
ID NO: 2, and wherein the antibody does not bind to said amino acid sequence when the serine or threonine is not phosphorylated.

8. An antibody or fragment thereof which binds specifically to an amino acid sequence comprising a phosphorylation site identified in Table 2 and set forth in SEQ
ID NO: 3, and wherein the antibody does not bind to said amino acid sequence when the serine or threonine is not phosphorylated.

9. An antibody or fragment thereof which binds specifically to an amino acid sequence comprising a phosphorylation site identified in Table 2 and set forth in SEQ
ID NO: 6, and wherein the antibody does not bind to said amino acid sequence when the serine or threonine is not phosphorylated.

10. An antibody in claims 7, 8 or 9, wherein the antibody is monoclonal or polyclonal.

11. An antibody or a fragment according to claims 7, 8 or 9, wherein said antibody or fragment is labelled with a detectable molecule or substance.

12. A nucleic acid comprising a sequence encoding an antibody or fragment according to claims 7, 8 or 9.

13. A vector comprising a nucleic acid according to claim 10.

14. A host cell, which has been transformed by a nucleic acid according to claim 10 and/or a vector according to claim 13.

15. A method of producing an antibody or fragment according to claims 7, 8 and 9, wherein the method comprises the steps of: (i) culturing a transformed host cell according to claim 11 under conditions suitable to allow expression of said antibody or fragment;
and (ii) recovering the expressed antibody or fragment.

16. A method for detecting CK2.alpha., said method comprising using an antibody or a fragment according to any of claims 7 to 11.

17. A method for detecting CK2.alpha.2CK2.beta.2, said method comprising using an antibody or a fragment according to any of claims 7 to 11.

18. Use of an antibody or a fragment according to any of claims 7 to 11 for diagnosing cancer.

19. A kit for diagnosing cancer, comprising an amino acid sequence according to claims 2 and 3, an amino acid sequence according to claim 4 and an antibody or a fragment thereof according to any of claims 7 to 11.