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WO2011096698A9 - Composition pour le pronostic du cancer comprenant des anticorps anti-tmap/ckap2 - Google Patents

Composition pour le pronostic du cancer comprenant des anticorps anti-tmap/ckap2 Download PDF

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Publication number
WO2011096698A9
WO2011096698A9 PCT/KR2011/000691 KR2011000691W WO2011096698A9 WO 2011096698 A9 WO2011096698 A9 WO 2011096698A9 KR 2011000691 W KR2011000691 W KR 2011000691W WO 2011096698 A9 WO2011096698 A9 WO 2011096698A9
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Prior art keywords
cancer
tmap
ckap2
antibody
composition
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Ceased
Application number
PCT/KR2011/000691
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English (en)
Korean (ko)
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WO2011096698A2 (fr
WO2011096698A3 (fr
Inventor
신창호
홍경만
배창대
박주배
최용복
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Cancer Center Japan
National Cancer Center Korea
Sungkyunkwan University Foundation for Corporate Collaboration
Original Assignee
National Cancer Center Japan
National Cancer Center Korea
Sungkyunkwan University Foundation for Corporate Collaboration
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Filing date
Publication date
Priority claimed from KR1020100081081A external-priority patent/KR20110091423A/ko
Application filed by National Cancer Center Japan, National Cancer Center Korea, Sungkyunkwan University Foundation for Corporate Collaboration filed Critical National Cancer Center Japan
Priority to US13/577,126 priority Critical patent/US8980570B2/en
Publication of WO2011096698A2 publication Critical patent/WO2011096698A2/fr
Publication of WO2011096698A3 publication Critical patent/WO2011096698A3/fr
Publication of WO2011096698A9 publication Critical patent/WO2011096698A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • 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.
  • cancer 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.
  • cancer testing means are physical. Examples include gastrointestinal X-rays, dual imaging, compression, or mucosal imaging.
  • endoscope 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.
  • this method has the disadvantages of hygiene problems and patient suffering during the examination.
  • 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.
  • 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.
  • the cancer breaks the normal division of cells and proliferates through the endless repetition of the M phase. Therefore, the development of markers for detecting such abnormal cell cycles is expected to be effectively used not only for the diagnosis of cancer but also for the development of therapeutics. .
  • 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.
  • an anti-TMAP / CKAP2 Tumor associated microtubule associated protein / cytoskeleton associated protein 2
  • 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 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).
  • 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.
  • TMAP / CKAP2 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 rate and survival rate of breast cancer patients, it can provide important information in the diagnosis of breast cancer, discovery of therapeutic agents, as well as selection of future treatment methods.
  • 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.
  • tissue immunohistochemical staining observed at a higher magnification than in FIG. 4, the cells stained in the cytoplasm were not stained in the nucleus, and cells which 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 carcinomas.
  • FIG. 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.
  • FIG. 10 is a diagram showing the results of immunohistochemical staining using anti-TMAP / CKAP2 antibody in breast cancer tissues.
  • 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.
  • A anti-TMAP / CKAP2 antibody
  • B cancer tissues with little staining
  • the cells stained on the chromosome or the chromosomal structures can be easily identified as the cells in the mitotic phase.
  • 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.
  • FIG. 12 is a Kaplan-Meier plot of survival (OS) and disease-free survival (DFS) of breast cancer patients according to TMAP / CKAP2 expression.
  • OS survival
  • DFS disease-free survival
  • 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.
  • higher TMAP / CKAP2 expression indicates that breast cancer patients have lower survival and disease free survival.
  • 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.
  • an anti-TMAP / CKAP2 Tumor associated microtubule associated protein / cytoskeleton associated protein 2
  • 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.
  • 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.
  • 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.
  • 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.
  • TMAP / CKAP2 is an abbreviation for Tumor associated microtubule associated protein / cytoskeleton associated protein 2, also known as LB1 and se 20-10.
  • the composition of the present invention can detect the cancer in the subject and the prognosis of the cancer by detecting the TMAP / CKAP2.
  • 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.
  • GIST gastrointestinal basal cancer
  • it can be used for prognostic diagnosis for predicting disease-free survival or survival in breast cancer patients.
  • 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.
  • GIST gastrointestinal stromal tumor
  • Kit gene or PDGFRA gene 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • phage display technology 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.
  • immunoglobulins typically 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").
  • 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.
  • 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.
  • 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.
  • 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 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.
  • RIA radioimmunoassay
  • Agglutination means that antigens and antibodies meet and particles form agglomerates with each other.
  • agglutinates in which granular antigens, such as cells, that are insoluble in water do not precipitate and form entangled with the antibody.
  • 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).
  • 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.
  • 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.
  • 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.
  • the response level of the antigen-antibody is increased compared to the control sample.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un anticorps qui se lie spécifiquement à TMAP (protéine associée aux microtubules associés aux tumeurs)/CKAP2 (protéine associée au cytosquelette 2) ou un fragment de celle-ci, un procédé permettant de confirmer la présence ou l'absence de mitose, et un procédé de pronostic du cancer utilisant cet anticorps. Plus spécifiquement, l'invention concerne une composition pour le pronostic du cancer comprenant des anticorps anti-TMAP/CKAP2 ou un site de liaison à l'antigène de ceux-ci, un procédé de détection de TMAP/CKAP2 utilisant la composition, un anticorps anti-TMAP/CKAP2 pour le pronostic du cancer, un procédé permettant d'obtenir des informations pour le pronostic du cancer utilisant la composition, un procédé de criblage d'agents anticancéreux consistant à mesurer les changements de niveau de réaction antigène de TMAP/CKAP2-anticorps par le traitement d'une substance candidate, et un procédé de mesure des cycles de division cellulaire utilisant la composition.
PCT/KR2011/000691 2010-02-05 2011-02-01 Composition pour le pronostic du cancer comprenant des anticorps anti-tmap/ckap2 Ceased WO2011096698A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/577,126 US8980570B2 (en) 2010-02-05 2011-02-01 Composition for cancer prognosis prediction comprising anti-TMAP/CKAP2 antibodies

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2010-0011126 2010-02-05
KR20100011126 2010-02-05
KR10-2010-0081081 2010-08-20
KR1020100081081A KR20110091423A (ko) 2010-02-05 2010-08-20 항―ｔｍａｐ／ｃｋａｐ２ 항체를 포함하는 암의 예후 진단용 조성물

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WO2011096698A2 WO2011096698A2 (fr) 2011-08-11
WO2011096698A3 WO2011096698A3 (fr) 2011-12-29
WO2011096698A9 true WO2011096698A9 (fr) 2012-03-01

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