KR100959020B1 - TMAP/CKAP2 specific monoclonal antibody - Google Patents

Abstract

The present invention relates to monoclonal antibodies or fragments thereof that specifically bind to Tumor associated microtubule associated protein (TMAP) / cytoskeleton associated protein 2 (CKAP2), and methods for identifying the presence of mitosis using these and methods for diagnosing cancer. It is about.

Monoclonal antibodies according to the present invention or fragments comprising the antigenic sites thereof are specifically bound to the non-phosphorylated form of TMAP / CKAP2 expressed in the interphase, according to changes in the cell cycle and TMAP / In addition to studies on the role of CKAP2, it may provide an important means for the study of the development and diagnosis of various cancers, including gastric cancer.

TMAP / CKAP2, Phosphorylated, Monoclonal Antibodies, Interstitial

Description

Monoclonal Antibody Specific to TPMAP / CGPAP2 {TMAP / CKAP2 specific monoclonal antibody}

The present invention relates to monoclonal antibodies or fragments thereof that specifically bind to Tumor associated microtubule associated protein (TMAP) / cytoskeleton associated protein 2 (CKAP2), and methods for identifying the presence of mitosis using these and methods for diagnosing cancer. It is about.

Tumor associated microtubule associated protein (TMAP) or cytoskeleton associated protein 2 (CKAP2), also known as LB1 and se20-10, is known as gastric cancer, diffuse B-cell lymphoma lymphoma, and cutaneous T-cell lymphoma have been cloned into genes that are overexpressed in several cancer tissues (Bae et al., 2003; Maouche-Chretien et al., 1998; Eichmuller et. al., 2001), and also increased expression of these proteins has been observed in various cancer cell lines (Bae et al., 2003; Jin et al., 2004).

However, there is a lack of information on the role of TMAP / CKAP2 in cancer development / development and the physiological role in cells in addition to the basic properties of TMAP / CKAP2. Interestingly, there have been reports of increased expression of TMAP / CKAP2 as the cancer progresses in stages of gastric cancer (Bae et al. 2003), suggesting that TMAP / CKAP2 may be a new marker in the diagnosis of several types of cancer, including gastric cancer. Suggests that you can. Recently published papers have shown that the expression of TMAP / CKAP2 is cell cycle-changing and an essential factor for normal cell growth (Jeon et al. 2006). It has been reported that it is involved in the formation and maintenance of phosphate (Hong et al. 2007; Jin et al. 2004).

TMAP / CKAP2 monoclonal antibody is expected to be able to study the formation and maintenance of mitotic spindles in the process of elucidating cell cycle control mechanisms, and to assist in the diagnosis or screening of cancers including gastric cancer.

U.S. Patent Publications US 2004/0116670 and US 2005/003447, International Application WO 0073450, et al., Relate to human cytoskeleton associated proteins and disclose human cytoskeleton associated proteins and genes encoding them as well as antagonists of the proteins. As an example of these antagonists, monoclonal antibodies of the protein are disclosed. However, this document does not mention any monoclonal antibodies that bind to interphase non-phosphorylated TMAP / CKAP2 proteins.

Under this background, the inventors produced monoclonal antibodies of TMAP / CKAP2 and hybridomas secreting them, which, unlike the polyclonal antibodies to the above proteins, bind to the medium phosphorylated form of TMAP / CKAP2. However, it was confirmed that the binding to the non-phosphorylated TMAP / CKAP2 of the interphase completed the present invention.

An object of the present invention is to provide a fragment comprising a monoclonal antibody or antigen-binding site thereof that specifically binds to TMAP / CKAP2 (Tumor associated microtubule associated protein / cytoskeleton associated protein 2) of interphase.

Still another object of the present invention is to provide a method for preparing a fragment comprising the monoclonal antibody or antigen-binding site thereof.

Still another object of the present invention is to provide a hybridoma which produces the monoclonal antibody.

Still another object of the present invention is to provide a method for determining whether cells are divided using a fragment comprising the monoclonal antibody or antigen-binding site thereof.

Still another object of the present invention is to provide a method for diagnosing cancer using the fragment comprising the monoclonal antibody or antigen-binding site thereof.

As one embodiment for achieving the above object, the present invention provides a monoclonal antibody or antigen-binding portion thereof that specifically binds to TMAP / CKAP2 (Tumor associated microtubule associated protein / cytoskeleton associated protein 2) of interphase. It relates to a fragment comprising a.

As used herein, the term “antibody” includes immunoglobulin molecules that are immunologically reactive with specific antigens, and include both polyclonal antibodies and monoclonal antibodies. The term "monoclonal antibody" is also a term known in the art and refers to highly specific antibodies directed against a single antigenic site (epitope) as antibody molecules of monomolecular composition obtained from substantially the same antibody population. In addition, such monoclonal antibodies are typically directed against a single epitope on an antigen, unlike polyclonal antibodies that include different antibodies directed against different epitopes. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and are also not contaminated by other immunoglobulins because they are produced by methods such as culturing hybridomas. Has an advantage.

In one specific embodiment, the monoclonal antibodies of the invention bind to the interphase, non-phosphorylated form of TMAP / CKAP2 protein, preferably the C-terminus of TMAP / CKAP2, ie TMAP / It binds to the 458th-682th amino acid sequence of the CKAP2 protein, and more preferably the 569th to 625th amino acid sequence of the region.

Those skilled in the art will readily appreciate that the monoclonal antibodies according to the present invention can be converted to chimeric antibodies, humanized antibodies and human monoclonal antibodies having reduced immunogenicity for application in the human body. Such chimeric antibody is a recombinant region of the monoclonal antibody of the present invention with the constant region of the human antibody, the humanized antibody complementarity determining region (complementarity determining directly binding to the antigen in the variable region of the monoclonal antibody of the invention Regions (CDRs) or only specificity determining residues (SDRs), which are involved in antigen binding specificity, are implanted in human antibodies.

The human antibody replaces the heavy or light chain variable region of the variable region of the monoclonal antibody of the present invention instead of the heavy or light chain variable region of the human antibody, and as a result, a hybrid (mouse heavy / human light chain) showing antigen binding ability Alternatively, the mouse light chain / human heavy chain) antibody is replaced with a human heavy or light chain variable region, and then a human human variable region showing antigen-binding ability is selected and linked to the human antibody constant region. It can be easily prepared from the monoclonal antibody of the present invention using a known method such as such, it is natural that such a variant is within the scope of the present invention. Chimeric antibodies, humanized antibodies and human monoclonal antibodies prepared as described above can be produced in animal cells using known methods.

In addition, the monoclonal antibodies of the present invention can be used for the treatment and diagnosis of cancer as functional fragments of antibody molecules, as well as complete forms having the full length of two heavy and two light chains, as long as they have the properties of binding as described above. Can be. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and can include Fab, F (ab '), F (ab') ₂ , Fv, etc.

The monoclonal antibodies of the present invention can be easily prepared by known monoclonal antibody production techniques. For example, it may be made by a fusion method well known in the art (Kohler et al., European Journal of Immunology 6; 511-519). In addition, a monoclonal antibody according to the present invention may be prepared by using an antibody library using phage display technology in which an antibody gene is directly obtained from B lymphocytes without expressing a hybridoma and expressing an antibody on a phage surface. However, it is not limited to these.

In a preferred embodiment, the monoclonal antibodies of the present invention produce hybridomas and are produced by the hybridomas. In general, hybridoma cells secreting monoclonal antibodies are made by fusing cancer cell lines with immune cells from immunologically suitable host animals such as mice injected with antigenic proteins. These two populations of cell fusion are fused using methods known in the art, such as polyethylene glycol, and the antibody producing cells are propagated by standard culture methods. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antibody specific for the antigen are cultured in large quantities in vitro or in vivo. In one specific embodiment, the inventors amplify the mouse TMAP / CKAP2 clone to the expression vector and overexpressed in Bacteria to immunize the fusion protein obtained in Balb / c mice to fuse the splenocytes and SP2 myeloma cells of the mouse The monoclonal antibody of the present invention was prepared by the method.

Thus, as another aspect, the present invention

a) immunizing TMAP / CKAP2 to a non-human animal; And

b) providing a method for producing a monoclonal antibody according to the present invention comprising the step of preparing a hybridoma using cells of the immunized animal and myeloma cells in step a).

The monoclonal antibodies of the present invention can be used without purification, and also in high purity using various conventional methods such as dialysis, salt precipitation, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and the like. It can be used by purification.

The fragment containing the monoclonal antibody or antigen-binding site thereof of the present invention prepared by the above-described production method, the conventional TMAP / CKAP2 polyclonal antibody in the midphase of the cell (centrosome) and microtubules ( Unlike the reaction with phosphorylated TMAP / CKAP2 present in the microtubule, it does not bind to them, but specifically binds to the non-phosphorylated TMAP / CKAP2 of the interphase. Accordingly, the monoclonal antibody or fragment thereof according to the present invention provides an important means for the study of the development of cancer, including gastric cancer, as well as the study of the role of TMAP / CKAP2 and the study of cell cycle, in particular cancer cell changes in the cell cycle. can do.

The present invention also relates to hybridomas that produce and secrete monoclonal antibodies that specifically recognize surface antigens of cancer cells. In a preferred embodiment, the hybridoma of the present invention is a hybridoma of Accession No. KCLRF-BP-00176, and the present inventors deposited the hybridoma with the Korea Cell Line Bank on November 28, 2007.

As used herein, the term "hybridoma" refers to a cell obtained by fusing an antibody-producing B lymphocyte and myeloma cancer cells. Myeloma cells are transformed cells, which have the advantage of being cultured for a long time in a culture dish. For example, the isolation of myeloma hybridomas that produce only one antibody means that a large amount of monoclonal antibodies can be easily obtained.

The hybridoma of the present invention for producing a monoclonal antibody according to the present invention is cultured in a large amount of cells expressing TMAP / CKAP2 as described in the method for producing a monoclonal antibody, non-human animals, Preferably, the mice are immunized, and cells obtained from the immunized mice may be prepared by fusing with myeloma cancer cells.

Myeloma cells used for cell fusion include p3 / x63-Ag8 from mouse, p3-U1, NS-1, MPC-11, SP-2 / 0, F0, P3x63Ag8, V653, S194 and R210 from rat. Cell lines can be used. The cell line used in the specific examples of the invention is myeloma cell SP2.

In addition, the method for producing a hybridoma, may further comprise the step of selecting a hybridoma to produce a monoclonal antibody that specifically binds to biliary cancer cells.

In still another aspect, the present invention relates to a method for identifying cell division by using a monoclonal antibody specific for TMAP / CKAP2 or a fragment comprising an antigen-binding site thereof.

For example, by reacting a monoclonal antibody of the present invention with a biological sample and detecting the formation of an antigen-antibody complex, it is possible to determine whether the cell is divided, that is, whether the cell is in a dividing phase including the intermediate phase or in an interstitial state.

As used herein, the term “biological sample” includes tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. It is possible, but not limited to. These biological samples can be reacted with or without manipulation of the antibody of the present invention to determine whether the cells are in a dividing or interphase state, i.e., whether phosphorylation of TMAP / CKAP2 has occurred.

As used herein, the term “antigen-antibody complex” refers to the combination of a TMAP / CKAP2 protein antigen in a sample with a monoclonal antibody according to the invention that recognizes it, and the formation of such antigen-antibody complexes is carried out using a colormetric method. ), Electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and scintillation counting method. It can be detected by any method. However, it is not necessarily limited to these and various applications and applications are possible.

In the present invention, various labels can be used for detecting the antigen-antibody complex. Specific examples may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and radioisotopes, but are not limited thereto.

Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, β-latamase, and the like. Phosphorus, Eu³ +, Eu³ + chelate or cryptate and the like, and ligands include biotin derivatives and the like, luminescent materials include acridinium esters, isoluminol derivatives and the like, and microparticles include colloidal gold, Pigmented latexes, and the like, and radioisotopes include ⁵⁷ Co, ³ H, ¹²⁵ I, ¹²⁵ I-Bolton Hunter reagent, and the like.

Hereinafter, the present invention will be described in detail through examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example

1. Secreting Monoclonal Antibodies Hybridoma  production

To prepare a mouse TMAP / CKAP2 monoclonal antibody, a mouse (m) TMAP / CKAP2 was first amplified and cloned into an expression vector (pET-28 (a) vector) designed to express histidine tags at the N-terminus. After overexpression in BL21 (DE3) bacteria, His-bind affinity chromatography, gel filtration chromatography and SDS-PAGE were performed, followed by gel elution, followed by SDS-PAGE. Purified to yield a single band. Splenocytes and sp2 myeloma cells obtained by immunizing the recombinant His-TMAP / CKAP2 fusion protein thus obtained were fused with Balb / c mice and screened using His-mTMAP / CKAP2 coated ELISA plate to obtain positive clones. .

For bacterial expression, clones of mTMAP / CKAP2 were PCR amplified using two primers, 5'-TCC GAA TTC ATG GCA GAG TCC AGG-3 'and 5'-TTC GTC GAC GCT ATT CCT CTT CCG C-3'. The amplified product was cut with restriction enzymes EcoRI and SalI and cloned into pET-28a (+) vector.

2. In mouse cell lines  Manifested mTMAP Of CKAP2 Specific reaction test with

C2C12 (mouse myoblast) cells were transfected with control luciferase siRNA (Ctrl siRNA), mCKAP2 siRNA, pCAGGS vector (Vector) and pCAGGS-mCKAP2 (overexpression), respectively, and then cultured for 2 days to obtain whole cell lysate. Thereafter, 50 μg of protein was separated from SDS-PAGE, and Western blot was performed using monoclonal antibody (MAb). The results are shown in FIG. 1. Monoclonal antibodies recognized two protein bands, 75-kDa and 85-kDa, in each cell. Both bands responded in lanes treated with mCKAP2 siRNA (second lane) to specifically reduce mTMAP / CKAP2 expression. All of the responses were increased in the pCAGGS-mCKAP2 lane (lane 4), which resulted in a specific increase in expression of TMAP / CKAP2. These results suggest that both bands are TMAP / CKAP2 proteins, and that these two bands are thought to be caused by splicing variants of mRNA.

Therefore, it was found that the monoclonal antibody specifically reacts with the mTMAP / CKAP2 protein inherent in cells.

In this experiment, the sequence of the mCKAP2 siRNA used to suppress the expression of mTMAP / CKAP2 in the mouse cell line is 5'-GAUACUGACCAGCGCAGA-3 ', and the pCAGGS-mCKAP2 used to overexpress mTMAP / CKAP2 is the coding sequence for mTMAP / CKAP2 ( NM_001004140) was PCR amplified using the primers described above and subcloned into EcoR I and Bgl II sites of the pCAGGS vector.

3. Immunofluorescence staining experiment  through mTMAP Of CKAP2 Intracellular positioning of

C2C12 cells were cultured on glass cover slides, fixed with 3.7% formaldehyde, and then permeabilized in 0.25% Triton X-100 solution. After standing in 5% bovine serum albumin (BSA) solution for 30 minutes, a monoclonal antibody was added and reacted at room temperature for 1 hour. Cy3 fluorescently labeled goat anti-mouse IgG was added and reacted to fluoresce the position of TMAP / CKAP2. Further, DAPI staining was performed to observe the nucleus.

Monoclonal antibodies reacted with mTMAP / CKAP2 located in microtubules and centrosomes in interphase cells. However, unlike previously reported polyclonal antibodies, expression could not be observed in metaphase cells (FIG. 2). This suggests that monoclonal antibodies do not react with mTMAP / CKAP2, which has undergone post-translational modification unlike polyclonal antibodies (Hong et al., 2007) that have already been reported. In particular, the presence of phosphorylation sites in mTMAP / CKAP2 may cause monoclonal antibodies to react with unphosphorylated mTMAP / CKAP2, suggesting the possibility of phosphorylation of mTMAP / CKAP2 in the medium term.

4. Human TMAP Of CKAP2 Reactivity with

Infected with baculovirus expressing human TMAP / CKAP2 (hCKAP2 baculovirus), or harvesting uninfected HighFive insect cells to obtain whole cell lysate, 5 μg of each nitrocellulose membrane The dot blot was directly prepared and analyzed for reactivity using a monoclonal antibody, wherein a rabbit polyclonal antibody (hCKAP2 PolyAb) against human TMAP / CKAP2 was used as a positive control (FIG. 3A).

It was confirmed that the monoclonal antibody also reacts with human TMAP / CKAP2.

Monoclonal antibody also detected a single protein of about 80-kDa, unlike TMAP / CKAP2 in mice.

HEK 293A cells were transfected with pCMV6-myc vector (Vector), pCMV6-myc-hCKAP2, control luciferase siRNA (Control siRNA) and hCKAP2 siRNA, and then cultured for 2 days to obtain whole cell lysate. 50 μg of each protein was separated on 12% SDS-PAGE, and then subjected to Western blot analysis using monoclonal antibody to efficiently detect expressed myc-hCKAP2 fusion protein. Treatment of hCKAP2 siRNA with reduced expression of human TMAP / CKAP2 reduced the reactivity with the detected single protein band, indicating that monoclonal antibodies specifically reacted with human TMAP / CKAP2 (FIG. 3B). ). The sequence of hCKAP2 siRNA used at this time is 5'-GUUCUAUCUUGGCGCUAAA-3 '.

5. Against Monoclonal Antibodies Epitope Mapping  (Figure 4)

HeLa cells were transfected with pEGFP (Clontech) vector (Vector), GFP-fusion full-length (Full), and directed deletion mutant construct (1-263 aa, 264-457 aa, 458-682 aa), respectively. Incubate for 2 days and give whole cell lysate. Each 5 μg protein was applied directly to the nitrocellulose membrane to prepare a dot blot and analyzed using monoclonal antibody. The rabbit polyclonal antibody (hCKAP2 PolyAb) against human TMAP / CKAP2 was used as a positive control. The results are shown in Figure 4A.

Unlike rabbit polyclonal antibodies, monoclonal antibodies of the present invention selectively reacted with the full-length TMAP / CKAP2 to the C-terminus moiety (corresponding to the amino acid sequence 458 to 682). In contrast, rabbit polyclonal antibodies against human TMAP / CKAP2 did not react effectively with the C-terminal site.

To more accurately locate epitopes located at the C-terminus (458-682 aa), HeLa cells were transfected with deletion mutant constructs (GFP-fusion) in different ways in the same manner as in Figure 4A. After the infection, the whole cell lysate was obtained and dot blot analysis was performed using monoclonal antibodies. The results are shown in FIG. 4B.

It was confirmed that the epitope was located between 569 and 625 because the monoclonal antibody according to the present invention specifically reacted with an amino acid sequence of 569 to 625.

6. Humans and Mice TMAP Of CKAP2 Comparison of the amino acid sequence corresponding to the C-terminal part of (Fig. 5)

The ClustalW program was used to enumerate and compare the C-terminal (458-682 aa) region of human CKAP2 and the corresponding amino acid sequence of mouse CKAP2.

The 569-625 amino acids containing epitope regions for monoclonal antibodies showed high homology between human and mouse CKAP2. There were also three threonine residues (T578, T596, T622) that could be phosphorylated in connection with mitosis. As mentioned above, the monoclonal antibodies do not detect CKAP2 in the middle stage, suggesting that their phosphorylation is involved in the mitosis process.

7. Phosphorylated against Monoclonal Antibodies TMAP Of CKAP2 of Epitope Mapping  (Figure 6)

Mutant constructs in which T578 and T596 were substituted with glutamate (T578E and T596E) among the phosphorylated residues were prepared and transfected into HEK 293A cells, followed by dot blot analysis using monoclonal antibodies. Cell lysates transfected with GFP vectors alone were used as negative controls, and cell lysates transfected with a vector (GFP-WT hCKAP2) in which the fusion protein of GFP and wild type TMAP / CKAP2 were overexpressed were used as positive controls. The results are shown in Figure 6A.

Unlike the WT or T578E variants, the T596E variants did not react with monoclonal antibodies. This suggests that T596 residues in epitopes recognized by monoclonal antibodies are very important in antigen-antibody binding.

Dot blot analysis was performed by constructing non-phosphorylated (T578, T596) and phosphorylated (T578-P, T596-P) synthetic peptides to sites containing T578 or T596, respectively, and 1 μg peptide per dot was applied. The results are shown in Figure 6B.

Testing with non-phosphorylated or phosphorylated synthetic peptides confirmed that T596 non-phosphorylated peptides reacted with monoclonal antibodies, while T596 phosphorylated peptides (T596-P) did not. This means that if T596 is not phosphorylated, the monoclonal antibody binds, and the reason why the reaction between the monoclonal antibody and TMAP / CKAP2 disappeared in the middle of mitosis was due to phosphorylation of T596. In other words, phosphorylation occurs in T596 of TMAP / CKAP2 during mitosis, and phosphorylation is lost as mitosis progresses.

8. Mitosis  Of cells in the process TMAP Of CKAP2 Change (Figure 7)

The following experiment was carried out to determine if phosphorylation of endogenous TMAP / CKAP2 changes in mitosis during actual in vivo. HeLa cells are treated with 1 μM nocodazole for 20 hours to arrest G2 or mitosis. Gentle pipetting was used to isolate round mitotic cells from G2 cells attached to the bottom, and then obtain whole cell lysates for each of them. Western blot analysis was performed using monoclonal antibodies.

As a result, monoclonal antibodies, like rabbit polyclonal antibodies against human TMAP / CKAP2, react with TMAP / CKAP2 (non-phosphorylated) expressed on G2, while TMAP / CKAP2 (phosphorylated) expressed in cells in the middle stage. ) Did not react. These results confirm that the actual phosphorylation of the T596 residue of TMAP / CKAP2 during mitosis occurs during the medium term and then disappears.

Monoclonal antibodies according to the present invention or fragments comprising the antigenic sites thereof are specifically bound to the non-phosphorylated form of TMAP / CKAP2 expressed in the interphase, according to changes in the cell cycle and TMAP / In addition to studies on the role of CKAP2, it may provide an important means for the study of the development and diagnosis of various cancers, including gastric cancer.

Figure 1 shows the results of Western blot with mTMAP / CKAP2 expressed in the mouse cell line.

Figure 2 shows the results of immunofluorescence staining experiment using a monoclonal antibody.

Figure 3 shows the results of reactivity test with human TMAP / CKAP2 of the monoclonal antibody, A is for the whole cell of high-Five insect cells infected with or uninfected (hCKAP2 baculovirus) expressing human CKAP2 The results of the dot blot analysis using a monoclonal antibody, B is a HEK 293A cells with pCMV6-myc vector (Vector), pCMV6-myc-hCKAP2, control luciferase siRNA (Control siRNA) or hCKAP2 siRNA, respectively After transfection, the whole cell lysate was obtained, and each protein was separated on 12% SDS-PAGE, followed by Western blot using monoclonal antibody.

Figure 4 shows the epitope mapping results for monoclonal antibody, A is transformed HeLa cells with pEGFP (Clontech) vector (Vector), GFP-fusion full-length (Full) and the indicated deletion variant constructs, respectively The result of dot blot analysis using monoclonal antibody obtained by cell lysate, B is a different site of HeLa cells in order to more accurately locate the epitope located at the C-terminal (458-682 aa) site After transfection with the deletion mutant construct (GFP-fusion), the whole cell lysate was obtained, followed by dot blot analysis using monoclonal antibody.

5 is a result of comparing the amino acid sequence corresponding to the C-terminal region of human and mouse CKAP2. Two threonine residues (T578 and T596) that are likely to be phosphorylated are indicated by red boxes.

FIG. 6 shows the results of epitope mapping of phosphorylated TMAP / CKAP2 to monoclonal antibodies. After infection, dot blot analysis was performed using monoclonal antibody, and B was a non-phosphorylated (T578, T596) and phosphorylated (T578-P, T596-P) synthetic peptide for a site containing T578 or T596. This is the result of dot blot analysis by manufacturing each.

7 shows changes in TMAP / CKAP2 in cells in mitosis.

Claims (9)

A fragment comprising a monoclonal antibody or antigen-binding portion thereof that specifically binds to TMAP / CKAP2 (Tumor associated microtubule associated protein / cytoskeleton associated protein 2) of interphase. The monoclonal antibody or fragment thereof of claim 1, wherein the TMAP / CKAP2 is in non-phosphorylated form. The monoclonal antibody or fragment thereof of claim 1 or 2, wherein the monoclonal antibody binds to the C-terminus (458th to 682th amino acid sequence) of TMAP / CKAP2. The monoclonal antibody or fragment thereof of claim 3, wherein the monoclonal antibody binds to the 569th to 625th amino acid sequence of TMAP / CKAP2. The monoclonal antibody or fragment thereof according to claim 1 or 2, which is secreted by the hybridoma of Accession No. KCLRF-BP-00176. Claim 1 monoclonal antibody or a fragment comprising the antigen-binding site thereof, TMAP / CKAP2 detection composition for determining whether the cells divide. The composition of claim 6, wherein the cells are cancer cells. Hybridoma of Accession No. KCLRF-BP-00176. a) immunizing TMAP / CKAP2 to a non-human animal; And b) A method for producing the monoclonal antibody of claim 1, comprising preparing a hybridoma using cells of the immunized animal and myeloma cells in step a).

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