WO2011096698A2 - Composition for cancer prognosis prediction comprising anti-tmap/ckap2 antibodies - Google Patents

Abstract

The present invention relates to an antibody which specifically binds to TMAP (tumour associated microtubule associated protein)/CKAP2 (cytoskeleton associated protein 2) or a fragment of the same, and to a method for confirming the presence or absence of mitosis and a method for predicting cancer prognosis by using the same. More specifically, the present invention relates to a composition for cancer prognosis prediction comprising anti-TMAP/CKAP2 antibodies or an antigen-binding site thereof, a method for detecting TMAP/CKAP2 by using the composition, an anti-TMAP/CKAP2 antibody for cancer prognosis prediction, a method for providing information for cancer prognosis prediction using the composition, a cancer-treatment agent screening method comprising the measurement of changes in the level of TMAP/CKAP2 antigen-antibody reaction by the treatment of a candidate substance, and a composition for measuring cell-division cycles using the composition.

Description

Composition for diagnosing prognosis of cancer containing anti-TPM / CPAAP2 antibody

The present invention relates to an antibody or fragment thereof and a fragment thereof that specifically binds to a Tumor associated microtubule associated protein (TMAP) / cytoskeleton associated protein 2 (CKAP2) and a method for identifying the presence of mitosis and a method for diagnosing the prognosis of cancer. It is about. More specifically, the present invention provides a composition for prognostic diagnosis of cancer comprising an anti-TMAP / CKAP2 antibody or antigen-binding site thereof, a method for detecting TMAP / CKAP2 using the composition, an anti-TMAP / CKAP2 antibody for prognostic diagnosis of cancer, Method for providing information for prognostic diagnosis of cancer using the composition, a method for screening a cancer therapeutic agent comprising measuring a change in antigen-antibody response level of TMAP / CKAP2 by treating a candidate substance, and for measuring cell division cycle using the composition It relates to a composition.

The number of cancer (malignant neoplasm) deaths in Korea was 62,887, which was 25.5% (29.6% of male deaths and 20.5% of female deaths) out of 246,515 deaths in Korea (512 deaths per 100,000 population). Death (130.7 deaths per 100,000 population) is the number one cause of death. The cancer death rankings are lung cancer, stomach cancer, liver cancer, colon cancer, and pancreatic cancer, with the deaths of these five cancers accounting for about 70% of all cancer deaths. The main cause of cancer deaths in men is lung cancer, stomach cancer, liver cancer, and colon cancer. The number of deaths from these four cancers (28,147) accounts for 70% of the total number of deaths from men (40,177). The main causes of cancer deaths are gastric cancer, lung cancer, liver cancer, colon cancer and pancreatic cancer. The number of deaths from these five cancers (13,630) accounted for 60% of all female cancer deaths (22,710).

There are dozens of these types of cancer, which have been identified to date, and are mainly classified by the location of the diseased tissue. Cancer grows very fast and metastasis occurs as it invades surrounding tissues, threatening life. Types of cancer include cerebral spinal cord tumor, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, cancer, colon cancer, liver cancer, pancreatic cancer and biliary tract cancer. It may also be classified by other classification systems, depending on the mechanism or form of the cancer.

Breast cancer is a common cancer among women along with skin cancer. Despite improved detection methods, mass screening, and treatment methods over the past decade, the prospect of women diagnosed with breast cancer is improving. Still, a large number of women suffer from breast cancer. About 20% of women diagnosed with early stage breast cancer have a poor prognosis after 10 years, relapse, metastasize or die within this period. In contrast, the remaining 80% of breast cancer patients diagnosed at an early stage have a good prognosis after 10 years. As such, even if breast cancer is found in the same stage of progression, the prognosis of early stage breast cancer patients is different from each other, and there is an urgent need for an evaluation method to distinguish them.

Prognostic indicators provide some information about the prognosis as well as tumor size, lymph node status, and histological grade, and may suggest the likelihood of response to a particular therapeutic agent. For example, estrogen (ER) and progesterone (PR) steroid hormone receptor status measurements have become common methods for evaluating breast cancer patients. In addition, if diagnosed as a tumor that is hormone receptor positive, the prognosis is better in patients with ER + / PR + tumors because they will respond to hormone therapy and show relatively slow proliferation.

On the other hand, overexpression of human epidermal growth factor receptor 2 (HER-2), transmembrane tyrosine kinase receptor protein is associated with poor breast cancer prognosis (Ross et al. (2003) The Oncologist: 307-325). Currently, Her2 / neu expression levels in breast tumors are used to predict response to the anti-Her-2 / neu antibody therapeutic trastuzumab (Herceptin; Genentech). In addition, Ki-67 is a non-histone nuclear protein expressed in the G1 phase of the cell cycle and goes to the M phase. By checking whether Ki-67 is overexpressed, Ki-67 can be diagnosed as correlated with poor prognosis of breast cancer. Way.

As such, prognostic criteria and molecular markers provide some guidelines for predicting patient fate and selecting appropriate treatment routes, but there are still limitations in accurately diagnosing the prognosis of breast cancer. There is an urgent need to study markers that can be diagnosed.

In relation to other cancers, lung cancer was a rare disease until the 19th century. However, smoking has started to increase rapidly in the twentieth century, and the incidence of lung cancer is increasing rapidly in Korea. In addition, lung cancer is poorly treated compared to other cancers, so the incidence is not the first, but deaths are known to be the most cancer patients.

It is not yet clear how and how cancer cells are produced. However, it is a general opinion that cancer occurs due to the generation of cells that are not controlled for proliferation by modification of a gene having a function of normal cell proliferation control. . These cancers are classified as early cancers in which cancer cells are confined in the mucous membrane according to the degree of progression. In various cancers, the prognosis of treatment of patients found in early cancer states appears to be relatively good. Therefore, early diagnosis and treatment of cancer are expected to contribute to lower cancer mortality rate and lower cancer treatment cost. However, in many cases, most of the early symptoms are asymptomatic, and even if they are mild enough to feel a slight indigestion or discomfort in the upper abdomen, most people overlook this, causing the cancer death rate.

Until now, most cancer testing means are physical. Examples include gastrointestinal X-rays, dual imaging, compression, or mucosal imaging. By using an endoscope to visually identify internal organs, you can find very small lesions that do not appear on X-rays. Biopsy can be performed directly at this suspicious place, increasing the diagnosis rate. However, this method has the disadvantages of hygiene problems and patient suffering during the examination.

In addition, the treatment of cancer that has been advanced so far is to relieve the lesion by surgery, in particular, surgical resection is the only way to target the cure. In this surgical resection, the principle of resection is to include the widest possible range of surgery aimed at cure, but the extent of resection may be defined in consideration of the sequelae after extensive resection. Even in this case, when the cancer has spread to other organs, radical surgery is impossible. Therefore, other methods such as chemotherapy may be taken. The anti-cancer drugs currently available on the market are relieved and survived after temporary relief or resection. There is only a temporary effect of prolonging the duration, there is a limit to the treatment of the underlying cancer, and the patient suffers a double pain due to the side effects and economic burden of the anticancer drug.

Therefore, in order to treat cancer, it is important to develop a diagnosis method of cancer with high sensitivity and specificity in the pre-treatment stage, and such a diagnosis should be found early in the cancer. Furthermore, customized diagnosis and treatment are required to predict the prognosis for specific cancers. However, to date, the molecular diagnosis technology that detects lesions by detecting specific lesions in the early stage of cancer diagnosis is insignificant, and there is no method yet for a specific cancer.

On the other hand, cancer progression is essential for the proliferation and growth of cells, most of which undergo abnormal cell division. Normal cell division, called the cell cycle, is a series of processes in which one cell divides and divides into two cells. The S (interphase) phase, where DNA replication occurs through the G1 phase, is a preparatory stage for cell proliferation, and DNA After the synthesis, the G2 phase undergoes M (Mitotic phase) phase of cell division. In the cell cycle, there are two check points to check the growth of cells between G1 and S, G2 and M, so that normal cells proceed to the next step and abnormal cells induce self-killing. do. In the normal cell it is picked stop if growth factors and sufficient nutrients are not supplied to the cell cycle groups G1 and the turn of the growth of resting G ₀ state. In particular, the M phase, the dividing phase where the actual division of the cells occurs, is the stage in which the cloned genome is separated into the cell's anode and the two daughter cells are formed in the shortest and most dramatic phase in time. This series of processes is a very important process for maintaining the life of cells because all cells go through in order for one cell to grow and divide into two cells. When the cell divides in phase M, it has to go through the growth phase in G1 to the next. Cancer cells lose their self-killing function due to DNA damage and cannot continue to the next step at the point of confirmation. Because of this, the number of cells grows and grows rapidly. In other words, cancer breaks the normal division of cells and proliferates through endless repetition of phase M. Therefore, it is expected that the development of markers for detecting such abnormal cell cycles can be effectively used not only for the diagnosis of cancer but also for the development of therapeutics. .

Under these circumstances, the present inventors have made intensive efforts to develop markers for measuring the cell cycle for the diagnosis of cancer.As a result of detecting whether the expression and amount of TMAP / CKAP2 are expressed, not only specific division but also similar cell division are necessary. In addition to confirming that it can be used for diagnostics, and confirmed that it can be used as a marker for predicting the survival rate and disease-free survival rate of breast cancer patients, the present invention was completed.

An object of the present invention is to provide a composition for diagnosing prognosis of cancer comprising an anti-TMAP / CKAP2 (Tumor associated microtubule associated protein / cytoskeleton associated protein 2) antibody or an antigen-binding site thereof.

Another object of the present invention is to provide an antibody for prognostic diagnosis of cancer that specifically binds TMAP / CKAP2.

It is another object of the present invention to provide a kit for prognostic diagnosis of cancer comprising an anti-TMAP / CKAP2 antibody or an antigen-binding site thereof.

Still another object of the present invention is to provide a method of detecting TMAP / CKAP2 in a subject having cancer using the composition comprising the antibody or binding site thereof.

Another object of the present invention is to (a) treating an antibody of the anti-TMAP / CKAP2 or an antigen-binding site thereof to a control sample isolated from an individual known to have a good prognosis and a sample isolated from an individual suspected of having cancer; (b) comparing the antigen-antibody response level of step (a); and (c) the antigen-antibody response level of the sample isolated from the individual suspected of cancer in step (b) compared to the control sample. In a high case, the present invention provides a method of providing information for prognostic diagnosis of cancer, which includes determining the subject as a cancer patient with a poor prognosis.

Another object of the present invention is to measure the antigen-antibody response level of TMAP / CKAP2 in cancer cells using a composition comprising an anti-TMAP / CKAP2 antibody or antigen-binding site thereof; (b) treating the cell with a candidate substance; And (c) confirming that the antigen-antibody response level is reduced after the candidate material treatment of step (b) compared to step (a).

Still another object of the present invention is to provide a composition for measuring cell division cycle comprising an anti-TMAP / CKAP2 antibody or an antigen-binding site thereof.

Antibodies or fragments comprising the antigenic sites of the present invention specifically bind to TMAP / CKAP2 in phosphorylated or non-phosphorylated form, which is expressed in the metaphase and anaphase of the mitotic phase. As well as studies on the role of TMAP / CKAP2 and changes in cell cycle, detection of cell division cycles such as breast cancer and GIST can provide an important means for the diagnosis and treatment of cancers that can predict the prognosis. In addition, it is possible to accurately predict the disease-free survival and survival rate of breast cancer patients, it can provide important information in the diagnosis of breast cancer, the discovery of treatments, as well as the selection of future treatment.

1 is a diagram showing the separation of PCR-reacted solution using 2% agarose gel electrophoresis after performing PCR using an immunoglobulin-specific primer combination.

Figure 2 is a diagram showing the DNA sequence analysis results and amino acid sequence of the heavy chain region of the human TMAP / CKAP2 antibody.

Figure 3 is a diagram showing the DNA sequence analysis results and amino acid sequence of the light chain region of the human TMAP / CKAP2 antibody.

Figure 4 shows the results of tissue immunohistochemical staining (x100) using anti-TMAP / CKAP2 antibody in liver cancer tissue. Brown or black stained cells show that the chromosomal region is stained by the antibody, and it can be confirmed that most of the dividing cells are stained.

Figure 5 shows the results of tissue immunohistochemical staining (x200) using anti-TMAP / CKAP2 antibody in liver cancer tissue. As a result of tissue immunochemical staining observed at a higher magnification than in FIG. 4, the cells stained in the cytoplasm were not stained in the nucleus, and cells that would soon enter the division stage could be observed.

FIG. 6 shows the results of tissue immunochemical staining using anti-TMAP / CKAP2 antibody in various types of lung cancer tissues. The expression of squamous cell carcinoma was higher than that of adenocarcinoma, and the expression of small cell lung cancer was much higher than that of the two non-small cell cancers.

Figure 7 shows the results of tissue immunohistochemical staining using TMAP / CKAP2 antibody in primary lung cancer and metastatic lung cancer tissue. It can be seen that the expression of TMAP / CKAP2 is significantly increased in metastatic lung cancer tissues compared to primary lung cancer. This increased expression was seen in squamous cell carcinoma in non-small cell lung cancer, but in adenocarcinoma, the increase in expression in metastatic lung cancer tissues was not observed.

Figure 8 shows the immunohistochemical staining results using anti-TMAP / CKAP2 antibody and anti-Ki67 antibody in normal tissue, anti-TMAP / CKAP2 antibody (A) and anti-Ki67 antibody in cervical tissue (B) Tissue immunochemical staining was performed. Both antibodies were stained only at the base of cervical epidermal tissue known to be dividing, and the number of cells stained by anti-TMAP / CKAP2 antibody was less than that of anti-Ki67 antibody. This is believed to be related to the expression of TMAP / CKAP2 during the narrower cell division stage. In addition, tissue immunohistochemical staining using anti-TMAP / CKAP2 antibody (C) and anti-Ki67 antibody (D) in LF (lymphoid follicle) tissues in which cell division occurs actively, compared to anti-Ki67 antibody, was also performed. A small number of cells stained with anti-TMAP / CKAP2 antibodies could be observed.

9 shows immunohistochemical staining results using anti-TMAP / CKAP2 antibodies in gastric and colonic tissues. Cells stained with anti-TMAP / CKAP2 antibodies in gastric and colonic tissues are stained in cells of the crypt region known to be dividing. 9A (x100) and 9B (x200) are stomach tissues, and FIGS. 9C (x100) and 9D (x200) are colon tissues.

10 is a diagram showing the results of immunohistochemical staining using anti-TMAP / CKAP2 antibody in breast cancer tissues. In breast cancer tissues, there are many tissues stained with anti-TMAP / CKAP2 antibody (A) and cancer tissues with little staining (B), which is closely related to the degree of division of cancer cells. Among the cells stained with the anti-TMAP / CKAP2 antibody, the cells stained on the chromosome or the chromosomal structures can be easily identified as the cells in the mitotic phase. And cells that stain relatively poorly in the cytoplasm are considered to be entering or preparing to enter the M phase into cells of the G2 phase. Cells that are stained on chromosomes or perchromosomal structures are thought to be important markers for mitotic activity index, and the ratio of cells in phase G2 to those in phase M is determined by the phase G2-M checkpoint ( It may be related to anticancer reactivity because it reflects the state of the checkpoint.

FIG. 11 is a Kaplan-Meier plot of overall survival and disease free survival according to TMAP / CKAP2 expression or Ki-67 expression in breast cancer patients. Kaplan-Meier plots showed higher TMAP / CKAP2 positive cell chromosome fractions in groups 2, 3, and 4, and survival (A) and disease-free survival (B) were worse than those in group 1 with low TMAP / CKAP2 expression. In (C), the survival rates are lower in groups 2, 3, and 4 having relatively higher percentages of nuclear Ki-67 positive cells than in group 1 having a low percentage of positive cells. (D) is a diagram showing the survival rate according to N stages by Kaplan-Meier plot. The prediction of survival rate using the expression level of TMAP / CKAP2 is better than the prediction of survival rate of patients using N stage, which is known to have the highest predictive value for predicting the prognosis of breast cancer. X axis; Survival date after surgery, Y-axis; viability

12 is a Kaplan-Meier plot of survival (OS) and disease-free survival (DFS) of breast cancer patients according to TMAP / CKAP2 expression. Survival rates of Groups 2, 3, and 4 compared to Group 1 with the lowest expression by the respective evaluation methods based on field chromosome counts (A and D), field total counts (B and E), or total fractions (C and F) Kaplan-Meier plots (AC) and disease free survival (DF) were performed. In this figure, higher TMAP / CKAP2 expression indicates that breast cancer patients have lower survival and disease free survival.

13 is a Kaplan-Meier plot of the survival of patients who died from breast cancer. Survival was significantly shorter in Group 4 patients than in Group 3 patients. (A) Analysis based on chromosome fraction (p = 0.026) (B) Analysis based on total fraction (p = 0.013)

As one aspect for achieving the above object, the present invention relates to a composition for prognostic diagnosis of cancer comprising an anti-TMAP / CKAP2 (Tumor associated microtubule associated protein / cytoskeleton associated protein 2) antibody or antigen-binding site thereof.

As used herein, the term "diagnosis" refers to identifying the presence or characteristics of a pathological condition, and for the purposes of the present invention, not only confirms whether cancer develops, but also recurs, metastases, drug reactivity, It is to judge whether or not such as tolerance. Preferably, when using the anti-TMAP / CKAP2 antibody of the present invention, by confirming the expression level of TMAP / CKAP2 from the sample of the subject, not only whether the cancer of the subject, but also whether the future prognosis of the subject is good predicted This is possible. The term "prognosis" in the present invention also refers to the progress and cure of diseases such as cancer-causing death or progression, including, for example, relapse, metastatic spread and drug resistance of neoplastic diseases such as cancer. For the purposes of the present invention may be to predict the prognosis of breast cancer, gastrointestinal basal tumor, liver cancer, squamous cell carcinoma, non-small cell cancer or small cell cancer, preferably to predict disease free survival or survival of breast cancer patients.

In the present invention, the term "disease free survival rate" means the possibility of survival without cancer recurrence after surgery of cancer, and "survival rate" means the possibility of survival regardless of recurrence of cancer.

As used herein, the term "diagnostic marker, diagnostic marker, or diagnostic marker" is a substance that can diagnose cancer cells by distinguishing them from normal cells, and has a poor prognosis compared to normal cells or cancer cells with a good prognosis. Polypeptides or nucleic acids (eg, mRNAs, etc.), lipids, glycolipids, glycoproteins or organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like that exhibit increased or decreased in cells with cancer. Preferably, the cancer diagnostic marker of the present invention is TMAP / CKAP2.

As used herein, the term "TMAP / CKAP2" is an abbreviation for Tumor associated microtubule associated protein / cytoskeleton associated protein 2, also known as LB1 and se 20-10. Preferably, the composition of the present invention can detect the cancer in the subject and the prognosis of the cancer by detecting the TMAP / CKAP2. Preferably, the composition of the present invention is liver cancer, lung cancer, breast cancer, thyroid cancer, testicular cancer, myelodysplasia, oral cancer, mycelial sarcoma, acute myeloid leukemia, acute lymphoblastic, basal cell cancer, ovarian epithelial cancer, ovarian germ cell tumor, brain tumor, pituitary adenoma , Multiple myeloma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, chronic myelogenous leukemia, retinoblastoma, choroidal melanoma, bladder cancer, parathyroid cancer, non-small cell lung cancer, small cell lung cancer, childhood brain tumor, childhood lymphoma, vulvar cancer, primary cancer Gastric lymphoma, gastric cancer, gastric carcinoma, gastrointestinal stromal tumor, gastrointestinal stromal tumor, wale tumor, breast cancer, penile cancer, pharyngeal cancer, pregnancy chorionic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic brain tumor, rectal carcinoma, Can be used to diagnose vaginal cancer, spinal cord cancer, auditory schwannoma, pancreatic cancer, salivary gland cancer, tonsil cancer, squamous cell carcinoma, adenocarcinoma, large cell carcinoma, skin cancer and laryngeal cancer. Preferably for the diagnosis and prognostic diagnosis of metastatic cancer to the cancer active cancer cell division, more preferably for the diagnosis or prognosis of liver or lung cancer, even more preferably depending on the activity of the mitosis of the cell It can be used for prognostic diagnosis of cancers such as breast cancer or gastrointestinal basal cancer (GIST) whose prognosis is determined. Most preferably, it can be used for prognostic diagnosis for predicting disease-free survival or survival in breast cancer patients.

The present inventors performed immunohistochemical staining in human cancer tissues and various normal tissues using antibodies against TMAP / CKAP2 produced by the present inventors to confirm the diagnostic and prognostic effects of the composition of the present invention. It was confirmed that the number of stained cells in cancer tissues was significantly higher than that in the tissues, and almost no expression of TMAP / CKAP2 was observed in most cells of normal tissues. In addition, we found for the first time that expression of TMAP / CKAP2 is strongly associated with survival and disease-free survival in breast cancer patients. This confirmation was analyzed by four different analytical methods: chromosome permillage analysis, total permillage analysis, field chromosome count analysis, and field total count analysis. As a result, it was confirmed that the higher the TMAP / CKAP2 expression group, the lower the disease-free survival rate and survival rate. In particular, the composition of the present invention can be identified by binding to the chromosome and spindle of the mitotic cell, the specific position where the cancer cell proliferation due to abnormal cell division is actively detected by detecting the staining position. That is, the composition of the present invention can distinguish between the region in which mitosis occurs and the surrounding substrate due to the occurrence and progression of cancer, and thus it is possible to determine the specific site and the time of division.

Although TMAP / CKAP2 is partially known about its relationship with B-cell lymphoma, cutaneous T-cell lymphoma, and breast cancer cell line, it is not known that the antibody can be used to diagnose or treat cancer. It is not disclosed at all that the diagnosis is possible. In addition, there is no known possibility of diagnosing liver cancer and lung cancer, and it has not been reported that it can be used as a means for diagnosing small cell lung cancer and distinguishing it from non-small cell lung cancer. Particularly with respect to cancers such as breast cancer or gastrointestinal basal tumors, the inventors have found that the prognosis of cancer patients with such cancers can be diagnosed due to their proliferation by cancer cell division. Accordingly, the present inventors first confirmed that the antibody against TMAP / CKAP2 was directly prepared, and that the diagnosis of cancer, and further the diagnosis and prognosis of the cancer, were possible using these. In addition, it was first identified that it can be used as a diagnostic marker for predicting disease-free survival or survival of breast cancer patients. More preferably, the composition of the present invention may be used for the diagnosis and prognostic diagnosis of liver and lung cancer, and more preferably breast cancer or gastrointestinal stromal tumor (GIST, gastrointestinal stromal) which can predict the prognosis by confirming the mitosis of cells. It can be used for prognostic diagnosis of cancer such as tumor). Most preferably, it can be used for prognostic diagnosis of cancer predicting disease-free survival or survival. In a preferred embodiment of the present invention, as a result of staining the breast cancer tissue using the composition of the present invention, it was confirmed that the specific staining of cells that actively divide breast cancer cells. On the other hand, as compared with Ki-67 antibody known to predict the prognosis of breast cancer as a marker of cell division, it was confirmed that the composition of the present invention can be stained tissue-specific staining is occurring compared to Ki-67 (FIG. 8). In addition, in a preferred embodiment of the present invention by analyzing the survival and disease-free survival rate of breast cancer patients undergoing cancer resection using the composition of the present invention, it was confirmed that the relatively low survival rate and disease-free survival rate in the group with high TMAP / CKAP2 expression (FIGS. 11-13).

Breast cancer is a mass of cancerous cells in the breast and generally refers to cancers occurring in the milk ducts and lobules. The causative factors have not been elucidated, but are presumed to be due to factors such as the effects of estrogen, age, birth experience, drinking and family history. Survival rate of breast cancer is reported to be close to 100% for stage 0 cancer, but less than 20% for stage 4 cancer. Surgery transfer, chemotherapy, radiation therapy, anti-hormonal therapy, etc. have. The prognosis of breast cancer is reported to vary depending on the stage of the cancer, but even if the patient has substantially the same stage, a lot of cases have been reported completely different, it is necessary to set specific markers for the prognosis of the breast cancer.

Meanwhile, gastrointestinal stromal tumor (GIST) is one of the mesoderm tumors of the gastrointestinal tract, and is known to be caused by mutations in the Kit gene or PDGFRA gene. Patients with GIST have a dietary eating disorder, gastrointestinal hemorrhage, and are known to metastasize mainly to the liver. GIST is also important to predict the prognosis of cancer. Although the proliferation of cancer cells is known to have a decisive effect on the prognosis, it is difficult to predict the prognosis because the specific markers that can be effectively predicted are not known.

Even in patients diagnosed with early stage breast cancer, 20% of patients treated with the same procedure have a poor prognosis and either relapse or metastasize or die within 10 years. Therefore, an effective diagnostic method is required for accurate prediction of such prognosis. Prognostic diagnosis in cancer can be achieved by detecting whether cell division is active. However, there is little means to provide information on whether cancer cell proliferation is actively progressing or cell division is actively occurring apart from cancer progression. In the above case, the composition of the present invention provides a highly accurate prognostic means for breast cancer or a GIST patient, and specifically, the composition of the present invention is administered to a cell to be diagnosed to determine whether the cell is divided and the degree of activity. This can be done by checking.

One of the most important prognostic factors of cancer patients such as breast cancer or GIST is the degree of cell division, and thus a mitotic activity index can be used as a measure of this. Among them, as a mitotic activity marker, conventionally, H & E staining of cancer tissue was used to count the number of cells in the mitotic phase under a microscope. This method takes a lot of time and is subjective to the subjective judgment of a pathologist. Judging by this, there is a disadvantage that is severe deviation for each individual. Under these backgrounds, the present inventors have tried to develop a method for identifying cancer cells in the dividing phase, and as a result, tissue immunochemical staining was observed in various tissues and human tissues using anti-TMAP / CKAP2 antibodies against humans. For the first time, we have shown that anti-TMAP / CKAP2 antibodies can easily distinguish cancer cells in the division as well as normal cells in the division of the cancer tissue.

Anti-TMAP / CKAP2 antibodies have the advantage of being able to easily detect mitotic cells by staining the chromosome or perichromosome structures in the dividing phase, making it an objective tool for diagnosing the prognosis of cancers such as breast cancer or GIST, where the information is important. And can provide a quick way. Many efforts and various attempts have been made using anti-Ki-67 antibodies in the conventional effort to easily measure cells in the dividing stage. The expression of Ki-67 is not limited to the dividing stage but is later G1, S, G2 and M. There is a limit to being used as a mitotic activity marker because the number of cells expressed and stained during many cell cycles during the period is excessive. However, the anti-TMAP / CKAP2 antibody of the present invention has an advantage of being able to easily identify mitotic cells by expressing specifically on a chromosome or a chromosomal structure in the dividing phase. In addition, the composition comprising the anti-TMAP / CKAP2 antibody of the present invention is often found in the cytoplasm, these cells are presumed to be G2 phase cells before the dividing stage. The ratio of cells in phase G2 to cells in phase M reflects the state of the G2 / M phase checkpoint, which can provide important information about the prognosis or anticancer resistance and responsiveness of cancer patients. As described above, the composition of the present invention is the first composition capable of analyzing the distribution of both G2 and M groups, and may be used as important data for research in the field of prognostic diagnosis of anticancer and cancer.

In addition, the TMAP / CKAP2 of the present invention has a high correlation with the disease-free survival and survival rate of breast cancer patients, it can be predicted that the higher the expression of TMAP / CKAP2, the lower the disease-free survival rate and survival rate, specific therapeutic agents of breast cancer patients, And / or predict the survival and / or likelihood of survival after surgical removal of cancer, and / or treatment with chemotherapy for a certain period of time without cancer recurrence. The compositions of the present invention can be used clinically to make treatment decisions by selecting the most appropriate mode of treatment for any particular patient. Predictive compositions of the present invention determine whether a patient responds favorably to a treatment regimen, such as a prescribed regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc. Predict whether long-term survival is possible. In a preferred embodiment of the present invention, as a result of comparing the correlation between Ki-67 expression and survival rate, which was used as a marker for measuring cell division and proliferation, compared with TMAP / CKAP2, the case of TMAP / CKAP2 is related to the survival rate. It was much larger (FIGS. 11A-11C) and these results suggest that TMAP / CKAP2 can be strongly used as a predictive prognostic marker for disease-free survival and survival in breast cancer patients.

On the other hand, the composition of the present invention shows sensitivity and specificity for breast cancer or GIST, in particular small cell lung cancer, it was confirmed that it can be used for diagnosing small cell lung cancer and further distinguishing small cell lung cancer from other cancers.

Lung cancer is largely classified into small cell lung cancer and non-small cell lung cancer, and non-small cell lung cancer is divided into adenocarcinoma, squamous cell carcinoma and large cell cancer. Although small cell lung cancer is classified as a part of lung cancer by the location of the onset tissue, it is distinguished from other lung cancers because it has distinct characteristics in clinical course, treatment, and prognosis.

Specifically, small cell lung cancer, unlike non-small cell lung cancer, is most often undergoing surgical resection at the time of diagnosis, and it is rapidly growing, and is well known to respond to chemotherapy or radiation therapy. Small cell lung cancer has a strong malignancy and is often found in metastases to other organs, opposite lungs, and mediastinum through lymphatic vessels or blood circulation. It is known for the first time mainly in the airways (bronchi or bronchioles). In general, the mass is large, grayish white and proliferates along the bronchial wall. The major metastases are known to be in the order of brain, liver, bone, lung, adrenal gland and kidney.

Meanwhile, non-small cell lung cancer is divided into adenocarcinoma, squamous cell acrcinoma, adenocarcinoma, and large-cell carcinoma as described above, and squamous cell carcinoma is mainly found in the center of lung. It is common in men and is known to be associated with smoking. As a clinical symptom, squamous cell carcinoma is mainly caused by the bronchial lumen growing and blocking the bronchus. Adenocarcinoma, on the other hand, occurs mainly in the peripheral part of the lungs, and occurs well in women or non-smokers, even if the size is often metastatic. Adenocarcinoma has recently been increasing in frequency. Finally, large cell carcinoma is a type of cancer that occurs in about 4 to 10% of lung cancers, and occurs mainly near the lung surface (peripheral lung), and about half occurs in large bronchus. Cell size is generally large, and some of them tend to rapidly proliferate and metastasize, and are known to have a poor prognosis compared to other non-small cell lung cancers.

Cancer diagnostic markers comprising the antibody or antigen-binding site thereof against TMAP / CKAP2 of the present invention showed a high expression of the lung cancer as a whole, it was confirmed that the diagnosis in most lung cancers. In addition, it was confirmed that the lung cancer, in particular small cell lung cancer can be specifically detected, according to the characteristics of the present invention, as well as the diagnosis of cancer, including lung cancer and liver cancer, can be diagnosed for small cell lung cancer, and by cancer cell proliferation Progress can be diagnosed and used as a way of predicting prognosis. In addition, the present inventors confirmed that the composition of the present invention can be effectively used to distinguish between small cell lung cancer and non-small cell lung cancer, which are difficult to distinguish by conventional methods known in the art. Specifically, the inventors of the present invention conducted tissue immunochemistry using lung cancer tissues in order to confirm the diagnosis and detection ability of the present invention, and observed that 1) there was a difference in expression of TMAP / CKAP2 according to the type of lung cancer. 2) In particular, it was confirmed that the expression of squamous cell carcinoma was increased rather than adenocarcinoma. 3) In particular, the composition of the present invention was confirmed that the response rate is high specifically for small cell lung cancer, and in fact, it was confirmed that TMAP / CKAP2 staining was increased in small cell lung cancer cells. As described above, small cell lung cancer may be very useful as a composition for diagnosing small cell lung cancer, unlike other non-small cell lung cancers, in terms of clinical course, prognosis, and treatment method. When it is difficult to distinguish non-small cell lung cancer, it means that the composition of the present invention can be substituted for these methods or used as an additional diagnostic method.

The composition of the present invention comprises an antibody against TMAP / CKAP2 and may also comprise a fragment of said antibody. Antibodies of the invention include immunoglobulin molecules that are immunologically reactive with specific antigens, and include both polyclonal antibodies and monoclonal antibodies. "Monoclonal antibody" is also a known term and refers to a highly specific antibody directed against a single antigenic site (epitope) as an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such a monoclonal antibody Typically, unlike polyclonal antibodies that include different antibodies directed against different epitopes, monoclonal antibodies are directed against a single epitope on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic light 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.

Those skilled in the art will also readily understand 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 present invention Regions (CDRs) or specificity determining residues (SDRs), which are involved in antigen binding specificity, are inserted into human antibodies.

The humanized antibody replaces the heavy chain variable region or the light chain variable region of the variable region of the monoclonal antibody of the present invention with the heavy chain variable region or the light chain variable region of the human antibody, and as a result, a hybrid (hybrid: heavy / human) Light chain or mouse light chain / human heavy chain) antibody can be easily prepared from the monoclonal antibody of the present invention using a known method such as selecting a mouse heavy chain variable region or a light chain variable region and linking it with a human antibody constant region, It is natural that such variants fall 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.

The monoclonal antibody of the present invention may be a fragment thereof as long as it has the above-described binding characteristics. That is, the antibody of the present invention can be used for diagnosis of cancer and whether or not as a functional fragment of an antibody molecule, as well as a complete form having the full length of two heavy chains and two light chains. 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). This method is similar to the traditional hybridoma production method, but can be obtained by inactivating the mouse's immunoglobulin gene and preparing a transgenic mouse transplanted with the human immunoglobulin locus.

In addition, a monoclonal antibody may be prepared by phage display technology, which is a technology for selecting antibody clones for a specific antigen by expressing a human antibody library on the surface of bacteriophage in the form of manufactured antibody fragments (Fab, ScRv). The method for producing the antibody of the present invention is not limited.

Typically, immunoglobulins have heavy and light chains, each heavy and light chain comprising a constant region and a variable region (the site is also known as a "domain"). The variable regions of the light and heavy chains comprise three variable regions and four "framework regions" called "complementarity determining resions" (hereinafter referred to as "CDRs"). The CDRs mainly serve to bind epitopes of antigens. The CDRs of each chain are typically called CDR1, CDR2, CDR3, starting sequentially from the N-terminus and also identified by the chain in which the particular CDR is located.

The antibody of the present invention includes, without limitation, an antibody capable of specifically binding to TMAP / CKAP2 for diagnosing the prognosis of cancer, preferably, heavy chain CDR1 as set forth in SEQ ID NO: 38; Heavy chain CDR2 set forth in SEQ ID NO: 39; And a heavy chain variable region comprising a heavy chain CDR3 as set out in SEQ ID NO: 40 and a light chain CDR1 as set out in SEQ ID NO: 42; Light chain CDR2 set forth in SEQ ID NO: 43; It may be an antibody comprising a light chain variable region comprising the light chain CDR3 set forth in SEQ ID NO: 44, more preferably an antibody comprising a heavy chain amino acid sequence set forth in SEQ ID NO: 45 and a light chain amino acid sequence set forth in SEQ ID NO: 46.

As another aspect, the composition of the present invention relates to an antibody for prognostic diagnosis of cancer that specifically binds TMAP / CKAP2. Antibodies of the invention include immunoglobulin molecules that are immunologically reactive with specific antigens, and include both polyclonal antibodies and monoclonal antibodies.

The description of the antibody is the same as described above.

The antibody of the present invention includes, without limitation, an antibody capable of specifically binding to TMAP / CKAP2 for diagnosing the prognosis of cancer, preferably, heavy chain CDR1 as set forth in SEQ ID NO: 38; Heavy chain CDR2 set forth in SEQ ID NO: 39; And a heavy chain variable region comprising a heavy chain CDR3 as set out in SEQ ID NO: 40 and a light chain CDR1 as set out in SEQ ID NO: 42; Light chain CDR2 set forth in SEQ ID NO: 43; It may be an antibody comprising a light chain variable region comprising the light chain CDR3 set forth in SEQ ID NO: 44, more preferably an antibody comprising a heavy chain amino acid sequence set forth in SEQ ID NO: 45 and a light chain amino acid sequence set forth in SEQ ID NO: 46.

In a preferred embodiment of the present invention, by producing a human TMAP / CKAP2 monoclonal antibody hybridoma, the analysis of the sequence of the enclosing was confirmed to have a heavy and light chain region having the DNA sequence of SEQ ID NO: 37 and 41 ( 2 and 3).

In another aspect, the present invention relates to a kit for prognostic diagnosis of cancer comprising an anti-TMAP / CKAP2 antibody or antigen-binding site thereof. Fragments comprising TMAP / CKAP2 and available antibodies or antigen-binding sites thereof are as described above and can be prepared in the form of kits commonly used in the art, including such compositions of the present invention as components. Is self explanatory. Cancers capable of diagnosing the prognosis of cancers by anti-TMAP / CKAP2 antibodies or antigen-binding sites thereof for the purposes of the present invention include, without limitation, the cancers described above, but preferably predict disease free survival or survival of breast cancer patients. It is a kit for diagnosing breast cancer prognosis, characterized in that.

In another aspect, the present invention relates to a method for detecting TMAP / CKAP2 in a subject with cancer using a composition comprising an anti-TMAP / CKAP2 antibody or antigen-binding site thereof.

Using the compositions of the present invention as described below, it is possible to detect TMAP / CKAP2 expressed in the mitotic phase of cells, and furthermore, such compositions can be used for cancer patients, preferably individuals with lung or liver cancer, more preferably It can be used to detect TMAP / CKAP2 from individuals with small cell lung cancer, more preferably from cancer cell division such as breast cancer or GIST to diagnose prognosis, most preferably disease free survival or survival rate of breast cancer patients. It can be used to detect TMAP / CKAP2 from a subject for diagnosing prognosis.

 In another embodiment, the present invention provides a method of treating an anti-TMAP / CKAP2 antibody or antigen-binding site thereof in (A) a control sample isolated from an individual with a known prognosis and a sample isolated from a suspected cancer. ; (b) comparing the antigen-antibody response level of step (a), and (c) the antigen-antibody response level of the sample isolated from the individual suspected of cancer in step (b) is higher than the control sample. If so, the present invention relates to a method for providing information for prognostic diagnosis of cancer, comprising determining the subject as a cancer patient with a poor prognosis.

Preferably, in the present invention, by treating a sample with an antibody against TMAP / CKAP2, and comparing the degree of response, it is possible to confirm the prognosis for cancer of the individual having the target sample. This method can also be accomplished by comparing the response levels of antigen-antibodies with individuals from a control group that is known to have a good prognosis. Preferably it means to confirm the disease-free survival or survival of breast cancer patients.

In the present invention, an individual known to have a good prognosis means an individual having no history of metastasis, recurrence, and death after the onset of cancer. A sample of a suspected cancer is a sample of a subject or tissue in which a cancer or a tumor has already occurred or is expected to occur, and means a sample to be diagnosed with a prognosis.

As used herein, the term "sample" includes samples such as whole blood, serum, blood, plasma, saliva, urine, sputum, lymphatic fluid, cerebrospinal fluid, and intercellular fluid with different expression levels of TMAP / CKAP2 in cancer tissues of an individual. The sample is not limited as long as it is a sample containing cells capable of detecting cell division.

The information providing method for prognostic diagnosis of cancer of the present invention can compare the reaction level of antigen-antibody after treatment with anti-TMAP / CKAP2 antibody in the control group and the experimental group as described above. TMAP / CKAP2 protein antigen in the mean amount and the amount of the antigen-antibody complex bound to the antibody or antigen-binding portion thereof of the present invention that recognizes it. The response levels of these antigen-antibodies can be compared using, without limitation, measurement methods commonly used in the art. Examples of antigen-antibody response levels include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and Ouchterlony immunodiffusion. , Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, aggregation and protein chip, but are not limited thereto.

The ELISA method, which is one of the most representative methods for determining the antigen-antibody reaction level, is one of the most widely used antigen-antibody assays because of its simplicity, cost-effectiveness, and large-scale analysis. In particular, this method is increasingly used because it is a very sensitive reaction such as radioimmunoassay (RIA) and does not use radioactivity as in RIA.

Agglutination means that antigens and antibodies meet and particles form agglomerates with each other. In general, agglutinates in which granular antigens, such as cells, that are insoluble in water do not precipitate and form entangled with the antibody. These agglutination reactions occur when the antibody acts as agglutinin (agglutin). It is a reaction that can be identified with the naked eye by connecting antigens).

As another method for analyzing the response level of antigen-antibody, radio-immuno assay (RIA) can be used. This method radiolabels the standard antigen, and immunoprecipitates with the antibody. When the antigen (sample) not labeled with radioactivity is added, the precipitation of the radiolabeled standard antigen is reduced, and the radioactivity in the precipitation is reduced. This principle is used to measure the amount of antigen in a sample.

On the other hand, immunohistochemical staining (immunohistochemistry) or immunohistochemical staining method, there is an immunofluorescence method and immunoenzyme method, the immunofluorescence method is a direct method, an indirect method, the immunoenzyme method includes a direct method, an indirect method, PAP method, ABC method and LSAB method. Immunohistochemical staining is a method of staining to prove the presence of a specific antigen in tissue sections using affinity of the antibody for the antigen. This immunohistochemical staining confirms the origin of undifferentiated cells, predicts the presence or absence of enzymes, hormones, tumor markers and prognostic factors, distinguishes carcinomas and sarcomas, differentiates benign and malignant tumors, and estimates the origin of metastatic cancer. It is used. Information providing method for cancer diagnosis of the present invention can be applied to the above-described method to measure the antigen-antibody response level, preferably the present inventors through tissue immunostaining the expression level and expression of TMAP / CKAP2 in cells The location was confirmed.

In addition, various labels can be used to measure the antigen-antibody reaction level of the present invention. 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-lactase, and the like. In addition, the light emitting material includes acridinium ester, isoluminol derivatives, and the like, and the microparticles include colloid, gold, colored latex, and the like, and the radioisotopes include ³⁷ Co, ³ H, ¹²⁵ I, ¹²⁵ I. Bolton-Hunter reagents and the like.

The present invention can determine the suspected cancer as a cancer pathogen by measuring the response level of the antigen-antibody using the method as described above, and further can predict and diagnose the prognosis of the cancer patient. Specifically, TMAP / CKAP2 of the present invention is a cancer cell, in particular, a cell in mitosis, which is characterized by an increase in its expression in metaphase and anaphase even during mitosis. When this occurs, the response level of the antigen-antibody is increased compared to the control sample. By confirming this, the subject can be determined to be a cancer patient, and in the case of a patient with a history of cancer disease, the proliferation can be predicted to have a poor prognosis. Will be. Preferably, the judgment was more pronounced in patients with suspected liver cancer or lung cancer, and in the case of small cell lung cancer, the contrast was stronger, and it was confirmed that these cancers were easily diagnosed. In addition, it was confirmed that accurate prediction of the prognosis was possible in the case of cancer where verification of cancer cell proliferation such as breast cancer and GIST is closely related to the prognosis. Most preferably, the disease-free survival rate and survival rate of breast cancer patients can be accurately predicted, and compared with Ki-67, which is used as a proliferative marker, the TMAP / CKAP2 of the present invention more clearly shows the disease-free survival rate and survival rate. As a result, it was confirmed that it could provide clinical information to determine future treatment regimens of breast cancer patients.

In still another aspect, the present invention provides a method for treating cancer, comprising the steps of: (a) measuring the antigen-antibody response level of TMAP / CKAP2 in cancer cells using a fragment comprising an anti-TMAP / CKAP2 antibody or antigen-binding site thereof; (b) treating the cell with the candidate in step (a), and (c) confirming that the antigen-antibody response level is reduced after processing the candidate in step (b) compared to step (a). It relates to a screening method of a cancer therapeutic agent comprising a.

Determining the expression level of the anti-TMAP / CKAP2 antibody may be used without limitation, conventional expression level measurement methods used in the art as described above, such as western blot, ELISA, radiation Immunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS or protein chip method.

In addition, the term "candidate" of the present invention is a substance that is expected to be able to treat cancer or a substance that is expected to improve the prognosis, and a substance that is expected to improve or improve cancer directly or indirectly. It is not limited. Examples of such candidates include all therapeutically predictable substances such as compounds, genes or proteins. The screening method of the present invention confirms the expression level of the anti-TMAP / CKAP2 antibody before and after administration of the candidate material, and when the expression level is decreased compared with the candidate material, the candidate material is used as a predictive treatment for cancer. You can decide.

In another aspect, the present invention relates to a composition for measuring cell division cycle comprising an anti-TMAP / CKAP2 antibody or antigen-binding site thereof.

Substantially, cell dividers express many proteins, enzymes, and kinases that do not appear in other cycles, but not all of them can be used as specific markers for cell division. Therefore, many researchers continue to make efforts to find effective markers.

The antibody of the present invention is expressed in a mitotic phase in which cell division is active and can specifically recognize and bind TMAP / CKAP2, which exhibits the greatest expression in the G2 / M phase of the cell cycle. Although TMAP / CKAP2 has been known to be expressed in the dividing phase, it has not been reported yet whether it can be easily used as a marker for mitotic division through tissue immunochemical staining. The present inventors confirmed that cells with high expression rate of TMAP / CKAP2 have a high rate of cell division and growth, and that cells with low expression rate of TMAP / CKAP2 have a low correlation with cell division and growth rate. .

Currently, Ki-67 is used as a marker to measure cell mitosis and proliferation, but since this marker is mainly stained in the nucleus, the cells in mitosis enter the division because the nucleus disappears. There is a disadvantage that it cannot be observed for cells, and in general, the percentage of cells stained by Ki-67 is too high to be used as an index, and thus it is difficult to be used as an effective detection marker. There was. However, in the case of using the composition of the present invention, it is possible to specifically observe the cells in the spine that reacts specifically with or without the nucleus, and has specificity with respect to the cells just before entering the dividing cell and the cells being dividing. Cells to be proliferated and proliferating cells can be detected at one time. In more detail, the present inventors first identified that a specific cell division time can be detected using the composition of the present invention, and in order to confirm the treatment of the antibody of the present invention to the cells, the present invention is directed to chromosomes and spindles of the dividing cells. Specific staining was observed.

More preferably, the composition of the present invention can bind to TMAP / CKAP2, which is expressed independently of the phosphorylation of TMAP / CKAP2, and is not detected only instantaneously in the cleavage phase, but is continuously identified throughout the middle and late stages of cleavage. It is possible. In other words, if the detectability of the protein changes depending on the phosphorylation involved in the activity of TMAP / CKAP2, it is possible to confirm the expression level of the entire TMAP / CKAP2 regardless of phosphorylation only in a very short time. There are disadvantages. However, the composition of the present application can confirm the expression level regardless of whether or not phosphorylation of the protein, it is possible to confirm whether more accurate cell division.

In another aspect, the present invention relates to the use of the anti-TMAP / CKAP2 antibody or antigen-binding site thereof for the prognostic diagnosis of cancer. The description of the anti-TMAP / CKAP2 antibody or antigen-binding site, cancer, and prognosis thereof is as described above.

In another aspect, the present invention relates to a use for measuring a cell division cycle comprising an anti-TMAP / CKAP2 antibody or antigen-binding site thereof. The description of the anti-TMAP / CKAP2 antibody or antigen-binding site and cell division cycle thereof is as described above.

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 Production of Monoclonal Antibody Hybridomas

To prepare a human TMAP / CKAP2 monoclonal antibody, the mouse (m) TMAP / CKAP2 was first amplified and cloned into an expression vector pET-28a (+) vector designed to express His tag at the N-terminus. After overexpression in bacteria, a single band on SDS-PAGE is obtained through His-bind affinity chromatography, gel filtration chromatography, and gel elution after SDS-PAGE. Purified to yield. ELISA plate coated with His-hTMAP / CKAP2 by fusing spleen and SP2 myeloma cells obtained by immunizing recombinant His-TMAP / CKAP2 fusion protein obtained by the above method three times in Balb / c mice Screening was performed using to obtain positive clones.

Example 2. Amino Acid Sequence and DNA Sequence Analysis of Produced Anti-human TMAP / CKAP2 Antibodies Using PCR

Total RNA was isolated from the monoclonal antibody-producing hybridoma cells produced in Example 1, cDNA was synthesized therefrom, and then PCR was performed using immunoglobulin-specific primer combinations to analyze their sequences.

More specifically, for immunoglobulin PCR analysis, total RNA was isolated from human TMAP / CKAP2 monoclonal antibody-producing hybridoma cells using TRIzol reagent (Invitrogen, catalog number: 15596). CDNA synthesis was carried out using the SuperScript III First-strand Synthesis System for RT-PCR (Invitrogen, catalog number: 18080-051). 5 μg of each sample of the isolated total RNA was used for cDNA synthesis. Primers used for cDNA synthesis were carried out using immunoglobulin 3′-conserved site sequence primers instead of oligo-d (T) primers. Immunoglobulin 3'-conservation site sequence primers were used according to sub-types of antibodies produced by each hybridoma cell. That is, MuIgMV _H 3'-1 was used when the heavy-chain subtype of the producing antibody was immunoglobulin-M, and MuIgGV _H 3'-2 was used when the subtype was immunoglobulin-G. . In addition, the subtype of the light chain (Light-chain) kappa (k) - When the chain is was used MuIgkV _L 3'-1, subtype a lambda (λ) - were used, the MuIgλV _L 3'-1 when the chain . And, the MuIgMV _H 3'-1, MuIgGV _H 3'-2, MuIgkV _L 3'-1, MuIgλV _L 3'-1 primers used at this time are components of the mouse Ig-Primer Set (Novagen, catalog number 69831-3). Were used, and the sequence information of each primer is shown in Table 1.

Table 1

Immunoglobulin-PCR responses were derived from the cDNA synthesized sequentially from anti-human TMAP / CKAP2 monoclonal antibody-producing hybridoma cells, using mouse Ig-Primer Set (Novagen, catalog number 69831-3) and 2X PCR pre-Mix (SolGent , catalog number: STD01-M50h). The PCR procedure was performed by the method recommended by the manufacturer of the mouse Ig-Primer Set (Novagen, catalog number 69831-3), which is a 5'-primer used for heavy and light chain PCR. The configuration was different.

 That is, for the heavy chain, both immunoglobulin-G or M, the 5'-primer is MuIgV_H5'-A, MuIgV_H5'-B, MuIgV_H5'-C, MuIgV_H5'-D, MuIgV_H5'-E, MuIgV_H5'-F were used respectively, and for the light chain, when the kappa (k) -chain, the 5'-primer was MuIgkV_L5'-A, MuIgkV_L5'-B, MuIgkV_L5'-C, MuIgkV_L5'-D, MuIkkV_L5'-E, MuIgkV_L5'-F, MuIgkV_L5'-G, and in the lambda-chain, MuIgλV_L5'-A was used as the 5'-primer, respectively. Each primer sequence information is shown in Table 1 above. Indicated.

 In addition, the conditions under which PCR was performed were 94 ° C, 3-min-> 94 ° C, 1-min / 50 ° C, 1-min / 72 ° C, 2- when 5'-primers were used as A and B primers. min (35-cycles)-> 72 ℃, 6-min-> 4 ℃ was carried out under the condition, when using the 5'-primer as C, D, E, F, G primer 94 ℃, 3-min- PCR reactions were performed under conditions of> 94 ° C, 1-min / 60 ° C, 1-min / 72 ° C, 2-min (35-cycles)-> 72 ° C, 6-min-> 4 ° C. PCR reaction solution was separated using 2% agarose gel electrophoresis (Fig. 1).

The resulting PCR-band was cut out on UV, extracted with DNA according to the gel illusion method, and used for sequencing experiments.

The results are shown in FIGS. 2 and 3.

Example 3 Tissue Immunochemical Staining with Anti-TMAP / CKAP2 Antibodies or Anti-Ki-67 Antibodies in Liver Cancer, Lung Cancer, Cervical, Stomach, Colon, and Breast Cancer Tissues

Tissue immunochemical staining was performed using Ultravision LP Detection kit and DAB (Lab Vision Corporational Fremont, CA, USA). Arrays of normal and cancerous tissues of several people were purchased from Superbiochip company (www.Tissue-array.com). Tissue sections were deparafinized with xylene and heated for 15 minutes at 121 ° C. in 10 mM Tris buffer pH 9.0 containing 1 mM EDTA to retrive the antigen. After immobilization with 95% ethanol, the sections were digested with primary antigen (diluted culture of hybridoma producing anti-TMAP / CKAP2 monoclonal antibody or anti-Ki-67 monoclonal antibody) and TBST (Tris buffered saline) containing 20% FBS. with 0.05% Tween) solution at room temperature for 1 hour. After washing again with TBST, color reaction was performed using DAB and counterstaining was performed with Mayer's Hematoxylin (Daco Cytomation Denmark).

The results are shown in FIGS. 4 to 10.

Example 4 Preparation and Clinical Pathology of Breast Cancer Patients

The study was performed on 112 patients with invasive breast cancer who were diagnosed with invasive breast cancer from 1999 to 2003. The patients had a survival rate of at least 7 years. The detailed pathological characteristics of these patients are shown in Table 2 below. Most patients had invasive catheter cancer (84.8%) and luminal type A (45.5%).

TABLE 2

Example 5: Measurement of TMAP / CKAP2-positive cells and evaluation of expression level of TMAP / CKAP2 in breast cancer tissue

Mean chromosome TMAP / CKAP2 positive cells were counted under 200 magnification. The total number of cancer cells at 200 magnifications was calculated based on four times the number of cells at 400 magnifications. The measurement of cell number at 400 magnification was performed by comparing with 400 magnification photographs of 150, 200, 300, 350, 450, 500, 550, 900, and 1000 cancer cell numbers. The method further increases the measurement efficiency. Counting cancer cells was difficult to measure by simple comparisons and when irregular, all cancer cells in the microscopic field used to count TMAP / CKAP2 positive cells were counted. When two specific areas of cancer tissue were seen, ie, different levels of chromosome TMAP / CKAP2 staining, a high positive area was used to count cancer cell numbers if more than 20% were covered with cancer cells. TMAP / CKAP2 expression levels were measured using the following four assays.

1) Chromosome permillage analysis

Chromosome TMAP / CKAP2 positive cell fraction is the chromosome TMAP / CKAP2 positive cell number (A) divided by the total number of cancer cells (B) in the area used when counting TMAP / CKAP2 positive cells (A / B). This assay was also used to evaluate the expression level of TMAP / CKAP2, and three other measures were also used to assess the relationship between TMAP / CKAP2 expression and patient survival.

2) total permillage analysis

In addition to the chromosome, the cytoplasm is also stained with TMAP / CKAP2, and the cells stained only in the cytoplasm were determined to be G2 or early M phase cells of dividing cells. Therefore, in order to include these cells as proliferating cells, multiply the number of cytoplasmic positive cells (A) by 0.1, add this number and chromosome positive cells (B), and divide the total number of cancer cells (C) by the total. Thousand fraction [(A * 0.1 + B) / C].

3) field chromosome count analysis

The average number of TMAP / CKAP2 chromosome positive cells in the microscope 200x magnification was defined as the field chromosome count.

4) Field total count analysis

To account for cytoplasmic staining cells, multiply the number of cytoplasmic staining cells (A) within a microscope 200 magnification by 0.1 and add this number to the number of chromosome positive cells (B) plus the total number of TMAP / CKAP2 positive cells (A * 0.1 + B).

TMAP / CKAP2 expression was measured by the above four different assay methods.

Example 6: Analysis of association of TMAP / CKAP2 expression or Ki-67 expression with clinicopathological variation

The association between TMAP / CKAP2 expression or Ki-67 expression and clinicopathological variables was analyzed by Spear man's rank correlation test or Wilcoxon rank sum test.

As a result, as shown in Table 3, TMAP / CKAP2 expression is associated with the histopathological variables, histological grade, nuclear grade, T stage, N stage, ER and PR state, Ki-67 expression also The results were statistically correlated with histologic grade, nuclear grade, and status of ER and PR.

TABLE 3

Table 4 below shows that chromosome permillage is very closely associated with Ki-67 expression, and TMAP / CKAP2 expression is very closely associated with clinicopathological parameters in other methods of analyzing TMAP / CKAP2 expression. It was confirmed that it is related.

Table 4

Example 7 Analysis of Association of TMAP / CKAP2 Expression or Ki-67 Expression with Overall Survival (OS) or Disease Free Survival (DFS) in Breast Cancer Patients

According to the method of chromosomal permillage analysis of TMAP / CKAP2 positive cells, patients with different TMAP / CKAP2 or Ki-67 expression levels were divided into 4 different groups: 4 in group 1 (low expression). Group (high expression).

Kaplan-Meier plots show the differences between relative survival and disease free survival, indicating that groups 3 and 4 show higher chromosomal fractions in TMAP / CKAP2 expression than those in group 1 OS; FIG. 11A) and obscurity survival (DFS; FIG. 11B).

Several variables, such as age and nuclear grade, stage T and stage N, were all found to be significantly associated with patient survival (Table 5). The recurrence information of breast cancer is not complete and the recurrence time can be studied only about 20 cases, most of which are related to death (16 cases). In 17 cases (85%), the cancer had spread far and local recurrence was found in 1 case (5%). In addition, two cases (10%) were found to spread far and local recurrence occurred. Frequent metastasis occurred mainly to the lungs, bones, liver and brain. All 23 patients were associated with breast cancer.

Table 5

Table 5 is based on the analysis of chromosomal permillage (chromosomal permillage) analysis of patients with different TMAP / CKAP2 expression in the same ratio divided into four different groups, for the statistical treatment of one group and two groups for one treatment Groups 1 and 2 were compared with the remaining 3 and 4 groups, which were analyzed using univariate analysis and multivariate analysis.

As a result, based on the univariate analysis, Groups 3 and 4 showed lower survival rates than Groups 1 and 2. In addition, the results of multivariate analysis considering age, nuclear grade, HER, T, and N stages at the time of diagnosis showed that chromosome fraction was significantly related to survival (p = 0.016). In addition, the risk ratios were 12.9 and 24.7 in groups 3 and 4, respectively, which were the same as or better than the N stages and risks (4.8 in group 2 and 11.2 in group 3), which are known to be most relevant to survival. In view of the degree, the hazard ratio also suggests that TMAP / CKAP2 of the present invention is a good prognostic diagnostic marker, comparable to stage N.

Based on the Kaplan-Meier plot, the results showed different levels of Ki-67 expression, indicating that Ki-67 was higher in Groups 2, 3, and 4 than in Group 1, and that Ki-67 was higher. The lower the survival rate (Fig. 11C).

Kaplan-Meier plots the survival rate according to N stages, which are known to have a high predictive value in predicting the prognosis of breast cancer, according to the Kaplan-Meier plot.The survival rate using the expression level of TMAP / CKAP2 of the present invention is higher than predicting the survival rate of patients using the N stages. Suggested that the prediction may be better (FIG. 11D).

Table 5 below shows the high TMAP / CKAP2 as a result of analysis based on analytical methods such as field chromosome count, field total count, and total fraction, which are methods for evaluating TMAP / CKAP2 expression level other than chromosomal permillage analysis method. It was confirmed that expression was statistically significantly associated with low survival. In addition, Kaplan Meier plot of this relationship also confirms that TMAP / CKAP2 expression is associated with low survival rate as in chromosome millimeter analysis (FIG. 12).

Table 6

The expression of TMAP / CKAP2 was also highly related to disease free survival (DFS) (Tables 7 and 8 below). Based on the chromosomal permillage analysis method, patients with different TMAP / CKAP2 expression levels were divided into four different groups, and group 1 and 2 were grouped into one group for statistical treatment. The 3 groups and 4 groups were compared with the other groups. For this purpose, univariate analysis and multivariate analysis were used.

As a result, the disease free survival rate was significantly lower in group 3 or group 4 having higher TMAP / CKAP2 positive cell chromosome fraction compared to group 1 and group 2 (p = 0.014 in group 3 and p = 0.024 in group 4). Confirmed.

However, the association with the disease-free survival rate (DFS) has a limitation in obtaining patient information, and it is assumed that there is no recurrence until that time unless there is no recurrence until the final examination of the patient. All the patients who died died from breast cancer. It was not possible to determine whether the disease had relapsed, so the disease-free survival rate (DFS) was estimated based on the time of death. Therefore, the reliability is lower than the survival rate (OS).

In addition, as a result of analyzing the expression level and disease-free survival rate of TMAP / CKAP2 by analytical methods other than chromosome fraction (Table 8), all showed that the expression and disease-free survival rate of TMAP / CKAP2 were significantly related and the risk (harzard) ratio) is also similar to the result of chromosome fraction.

TABLE 7

Table 8

Example 8: Analysis of association between TMAP / CKAP2 expression and survival (OS) in 23 patients who died from breast cancer

If TMAP / CKAP2 expression is closely associated with prognostic factors such as N and T stages, TMAP / CKAP2 expression may also be associated with premature death.

As a result, based on the chromosome permillage (FIG. 13A) and the total fraction analysis method (FIG. 13B) in FIG. 13, the relationship between TMAP / CKAP2 expression and survival (OS) was analyzed and Kaplan-Meier plot was analyzed. The results are shown.

As a result, high TMAP / CKAP2 expression shows a low survival rate (OS), which means that high-risk patients are likely to die sooner, which means that more high-risk patients can use more aggressive treatments. Clinical significance was considered.

Example 9 Comparison of Ki-67 and TMAP / CKAP2 as Prognostic Markers

Currently, Ki-67 is used as a marker to measure cell mitosis and proliferation, but since this marker is mainly stained in the nucleus, cells in mitosis enter the division because the nucleus disappears. There is a disadvantage in that it cannot be observed in cells, and the relationship between the actual Ki-67 expression level and the survival rate of cancer patients has not been clearly demonstrated.

Comparing the results of the analysis of the correlation between Ki-67 expression and survival rate as shown in FIG. 11C, it was found that Ki-67 expression was less related to survival rate than TMAP / CKAP2 expression (FIGS. 11A and 11B). In addition, as shown in FIG. 11C, group 1, which has the lowest Ki-67 expression, had better survival than other groups, but showed no significant change between groups 2, 3, and 4. In contrast, TMAP / CKAP2 expression can be seen that there is a large difference in survival rate between the 2, 3 and 4 group in the analysis results of Figure 11A. These results suggest that Ki-67 may not be widely used as a good marker for survival.

Biologically, the chromosomal expression of TMAP / CKAP2 can be observed from the middle of the mitosis right after the cytoplasm, whereas Ki-67 is the nucleus for the entire period starting from the late G1 phase until the mitosis. Staining can be observed. Thus, it can be said that survival rate is more closely related to mitosis during the growth period. These results are in line with what is known to be better than using Ki-67 to directly count mitosis after H & E staining under a microscope.

These results support that expression of TMAP / CKAP2 of the present invention can be used as a powerful prognostic marker for predicting survival and disease-free survival in breast cancer patients.

Claims (23)

1. An anti-TMAP / CKAP2 (Tumor associated microtubule associated protein / cytoskeleton associated protein2) antibody or a composition for prognostic diagnosis of cancer comprising an antigen-binding site thereof.
2. The composition of claim 1, wherein the composition measures mitosis of cells.
3. The composition of claim 1, wherein the cancer is breast cancer or gastrointestinal basal tumor (GIST).
4. The composition of claim 1, wherein the cancer is liver cancer, squamous cell carcinoma, non-small cell lung cancer, or small cell lung cancer.
5. The composition of claim 1, wherein the composition predicts disease free survival or survival of a breast cancer patient.
6. The antibody of claim 1, wherein the antibody comprises a heavy chain CDR1 as set forth in SEQ ID NO: 38; Heavy chain CDR2 set forth in SEQ ID NO: 39; And a heavy chain variable region comprising a heavy chain CDR3 as set out in SEQ ID NO: 40 and a light chain CDR1 as set out in SEQ ID NO: 42; Light chain CDR2 set forth in SEQ ID NO: 43; A composition comprising an light chain variable region comprising the light chain CDR3 set forth in SEQ ID NO: 44.
7. 7. The composition of claim 6, wherein the antibody is an antibody comprising a heavy chain amino acid sequence as set out in SEQ ID NO: 45 and a light chain amino acid sequence as set out in SEQ ID NO: 46.
8. Antibody for prognostic diagnosis of cancer that specifically binds TMAP / CKAP2 (Tumor associated microtubule associated protein / cytoskeleton associated protein2).
9. The antibody of claim 8, wherein the antibody comprises a heavy chain CDR1 as set forth in SEQ ID NO: 38; Heavy chain CDR2 set forth in SEQ ID NO: 39; And a heavy chain variable region comprising a heavy chain CDR3 as set out in SEQ ID NO: 40 and a light chain CDR1 as set out in SEQ ID NO: 42; Light chain CDR2 set forth in SEQ ID NO: 43; An antibody comprising a light chain variable region comprising the light chain CDR3 set forth in SEQ ID NO: 44.
10. 10. The antibody of claim 9, wherein the antibody comprises a heavy chain amino acid sequence as set out in SEQ ID NO: 45 and a light chain amino acid sequence as set out in SEQ ID NO: 46.
11. Kit for diagnosing prognosis of cancer comprising anti-TMAP / CKAP2 antibody or antigen-binding site thereof.
12. The kit of claim 11, wherein the kit predicts disease free survival or survival of a breast cancer patient.
13. A method for detecting TMAP / CKAP2 in a subject with cancer using the composition of claim 1.
14. (a) treating an anti-TMAP / CKAP2 antibody or antigen binding site thereof to a control sample isolated from a subject with a known good prognosis and a sample isolated from a suspected cancer;

    (b) comparing the antigen-antibody response levels of step (a); And

    (c) if the antigen-antibody response level of the sample isolated from the suspected cancer is higher than that of the control sample in step (b), determining the subject as a cancer patient with a poor prognosis. How to provide information for prognostic diagnosis.
15. The method of claim 14, wherein the prognosis of the cancer is to determine disease free survival or survival of the breast cancer patient.
16. The method of claim 14, wherein the sample is any one or more samples selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph fluid, and cells isolated from an individual.
17. The method of claim 14, wherein the method of comparing antigen-antibody response levels is Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement Using a fixed assay, FACS or protein chip.
18. (a) measuring the antigen-antibody response level of TMAP / CKAP2 in cancer cells using the composition of claim 1;

    (b) treating the cells of step (a) with a candidate substance; And

    (c) confirming that the antigen-antibody response level is reduced after the candidate treatment of step (b) compared to step (a).
19. A composition for measuring cell division cycle comprising an anti-TMAP / CKAP2 antibody or antigen-binding site thereof.
20. 20. The composition of claim 19, wherein the dividing cycle detects mitosis of cells.
21. 21. The composition of claim 20, wherein the cleavage group is metaphase or anaphase.
22. Use for prognostic diagnosis of cancer of anti-TMAP / CKAP2 antibody or antigen-binding site thereof.
23. Use for measuring cell division cycles comprising an anti-TMAP / CKAP2 antibody or antigen binding site thereof.

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