WO2016136372A1 - Ckap4を標的分子とした抗腫瘍剤 - Google Patents
Ckap4を標的分子とした抗腫瘍剤 Download PDFInfo
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- WO2016136372A1 WO2016136372A1 PCT/JP2016/052485 JP2016052485W WO2016136372A1 WO 2016136372 A1 WO2016136372 A1 WO 2016136372A1 JP 2016052485 W JP2016052485 W JP 2016052485W WO 2016136372 A1 WO2016136372 A1 WO 2016136372A1
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- ckap4
- dkk1
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Definitions
- the present invention relates to an antitumor agent. More specifically, the present invention suppresses its expression or function using Cytoskeleton-associated protein 4 (CKAP4, 63-KDa Cytoskeleton-Linking Membrane Protein (CLIMP-63), P63, ERGIC-63)) as a target molecule.
- CKAP4 Cytoskeleton-associated protein 4
- CLIMP-63 Cytoskeleton-Linking Membrane Protein
- P63 ERGIC-63
- the present invention relates to an antitumor agent that suppresses the growth of tumor cells.
- the present invention relates to a method for examining postoperative prognosis of cancer patients and a method for examining cancer.
- Wnt signal transduction pathway when Wnt, an extracellular secreted glycoprotein, binds to a receptor, it is responsible for cell functions via LDL receptor-related protein 5 or 6 (LRP5 / 6) and 7-transmembrane receptor Frizzled. Transmit signals. Wnt signals are responsible for various cell function controls. Abnormalities in the Wnt signaling pathway are known to cause cancer, bone disease, inflammation, infection and the like.
- Dkk1 (Dickkopf1) is expressed by Wnt signal and is known to be a factor that suppresses Wnt signaling pathway by inhibiting the binding of Wnt ligand and LDL receptor-related protein 6 (LRP6).
- LRP6 LDL receptor-related protein 6
- Dkk1 has been reported to enhance the growth of tumor cells.
- Dkk1 is overexpressed in multiple myelopathy, hepatoblastoma, Wims tumor, prostate cancer, kidney cancer, breast cancer, esophageal cancer, lung cancer, etc.
- Dkk1 expression suppression And anti-Dkk1 antibody has been reported to be effective in suppressing the growth of tumor cells.
- Dkk1 has a function to enhance cell proliferation in spite of negatively controlling cell proliferation in the Wnt signaling pathway, so Dkk1 has a signaling pathway independent of the Wnt signaling pathway. It is assumed that cell proliferation is enhanced. However, the signal pathway that enhances cell proliferation by Dkk1 has not been elucidated.
- CKAP4 is a transmembrane receptor, and its ligands are known as anti-proliferative factor (APF), surfactant-protein A (SPA), tissue-plasminogen-activator (TPA), etc. There has been no report on the effect of CKAP4-mediated cell proliferation.
- APF anti-proliferative factor
- SPA surfactant-protein A
- TPA tissue-plasminogen-activator
- the present inventors established a Dkk1 expression strain (MDCK / Dkk1-FLAG cell) using canine tubule-derived MDCK cells, and Dkk1 enhances the proliferation ability of MDCK cells, as shown in Examples described later. It was confirmed. This suggests that Dkk1 enhances cell proliferation through a signal pathway independent of the Wnt signaling pathway.
- an object of the present invention is to specify a target molecule involved in enhancement of tumor cell proliferation by Dkk1, and to provide a new antitumor agent. Furthermore, another object of the present invention is to provide a method for screening an antitumor agent using the target molecule.
- Still another object of the present invention is to provide a test method for predicting the postoperative prognosis of cancer patients and a test method for predicting the presence or absence of cancer.
- the present inventors have intensively studied to elucidate the signal pathway that enhances the proliferation of tumor cells by Dkk1, and found that CKAP4 functions as a receptor for Dkk1 in a signal pathway independent of the Wnt signal pathway. I found it.
- the present inventors have found that Dkk1 promotes the growth of cancer cells through the association of CKAP4 and PI3K, and CKAP4 specifically regulates the cell proliferation promoting effect of Dkk1, It has also been found that it plays a part in an unregulated cell growth regulation mechanism.
- the present inventors have also found that tumor cell growth can be suppressed by suppressing the expression or function of CKAP4 in tumor cells.
- the present invention has been completed by further studies based on such knowledge.
- Item 1 An antitumor agent comprising a substance that suppresses the expression or function of CKAP4 as an active ingredient.
- Item 2. Item 2. The antitumor agent according to Item 1, wherein the substance is at least one nucleic acid drug selected from the group consisting of siRNA against CKAP4, shRNA, dsRNA, miRNA, and antisense nucleic acid.
- Item 3. Item 2. The antitumor agent according to Item 1, wherein the substance is an antibody or a fragment thereof that specifically binds to CKAP4.
- Item 4. Item 4. The antitumor agent according to any one of Items 1 to 3, which is used for cancer treatment, prevention or progression prevention.
- Item 5. Item 5.
- Item 6. The antitumor agent according to Item 5, wherein the cancer further expresses Dkk1.
- Item 7. Item 7.
- Item 8. Use of a substance that suppresses the expression or function of CKAP4 for the production of an antitumor agent.
- Item 9. A substance that suppresses the expression or function of CKAP4, which is used for the treatment of cancer.
- Item 10. A method for treating cancer comprising the step of administering to a cancer patient a substance that suppresses the expression or function of a therapeutically effective amount of CKAP4.
- a method for screening an active ingredient of an antitumor agent The first step of measuring the ability of CKAP4 to suppress the expression of CKAP4 or its ability to bind to CKAP4 on the cell membrane, and the test substance that was found to have the ability to suppress the expression of CKAP4 or bind to CKAP4 on the cell membrane
- a second step of selecting as a candidate for an active ingredient of a tumor agent A screening method comprising: Item 12.
- the screening method according to Item 11 comprising a step 1-a for contacting a test substance with a cell expressing CKAP4, and a step 1-b for measuring the expression level of CKAP4 in the cell after the step 1-a. .
- Item 15. The test method according to Item 14, wherein the expression of Dkk1 in the cancer tissue is measured.
- a prognostic agent for postoperative prognosis of cancer patients including a reagent for detecting CKAP4.
- Item 18. The test agent according to Item 17, further comprising a reagent for detecting Dkk1.
- Item 19. The test agent according to Item 17 or 18, wherein the cancer patient is a lung cancer patient or a pancreatic cancer patient.
- Item 20. Use of a reagent for detecting CKAP4 for the manufacture of a prognostic agent for postoperative prognosis in cancer patients.
- a test method for cancer comprising a step of measuring CKAP4 in exosomes collected from a subject.
- Item 23 A diagnostic agent for cancer comprising a reagent for detecting CKAP4.
- Item 24 Use of a reagent for detecting CKAP4 for the manufacture of a reagent for detecting CKAP4.
- Item 25 Reagent for detecting CKAP4, used for cancer testing.
- the antitumor agent of the present invention is based on the new finding that CKAP4 functions as a receptor for Dkk1, and Dkk1 transmits a signal for enhancing the growth of tumor cells via CKAP4. According to the above, by using CKAP4 as a target molecule and suppressing its expression or function, it becomes possible to effectively suppress the growth of tumor cells. In fact, the in vivo tumor suppressive effect of anti-CKAP4 antibody on various cancer cells has been confirmed.
- the antitumor agent of the present invention since Dkk1 and CKAP4 are not expressed in normal epithelial cells, the antitumor agent of the present invention has high tumor specificity and is excellent in safety.
- an excellent antineoplastic drug can be accelerated by screening a substance that suppresses its expression or function using CKAP4 as a target molecule. Can also bring
- the prognosis after surgery can be predicted with high accuracy, which can be used to determine the optimal treatment method after surgery for cancer patients.
- the presence or absence of cancer can be predicted by using exosomes collected from the body fluid of a subject, so that cancer diagnosis can be performed minimally or non-invasively. Is possible.
- (C) shows the result of paraffin embedding a kidney tissue section of embryonic day 13 (day E13) mice and immunostaining with anti-Dkk1 antibody (green) and anti-aPKC antibody (red).
- D shows the results of measuring MDCK / Dkk1-FLAG cells three-dimensionally on Matrigel for 5 days and measuring the cysts formed and the outer diameter (indicating the size of the cell mass).
- e shows the results of staining of anti-Ki67 antibody (green), anti-aPKC antibody (red), and DRAQ5 DNA Dye (blue) on MDCK / Dkk1-FLAG cells cultured three-dimensionally on Matrigel Indicates.
- MDCK cells cultured on Transwell polycarbonate filter were cultured for 24 hours after adding Dkk1 to the apical or basal side, followed by anti-Ki67 antibody (red) and DRAQ5 DNA Dye ( The result of staining with (blue) is shown.
- NT No processing.
- MDCK / Dkk1-FLAG cells were cultured two-dimensionally for 3 days, and the results of counting the number of cells are shown.
- (a) shows a schematic diagram of an experimental procedure for screening Dkk1-binding protein on the apical surface.
- B shows the result of purification of Dkk1 binding protein and silver staining (left figure) and the identified binding protein group (right figure).
- (c) shows a control (MDCK cell), MDCK / Dkk1-FLAG cell, or MDCK / Dkk1-FLAG-GPI cell (Dkk1-FLAG-GPI) lysate (Input) and its immunoprecipitate by anti-FLAG antibody
- the results of detecting CKAP4, LRP6, and Dkk1 for (IP) are shown.
- the upper figure of (d) shows a schematic diagram of the domain structure of Dkk1 used in the test, and the lower figure of (d) shows the control (MDCK cells) and wild type (wild type) Dkk1-FLAG (WT).
- CKAP4, LRP6, and Dkk1 against the lysate (Input) of MDCK cells expressing the deletion mutant Dkk1-FLAG ( ⁇ CRD-1, ⁇ CRD-2) and the immunoprecipitate (IP) of the anti-FLAG antibody The result of having been detected.
- the upper diagram of (e) shows a schematic diagram of the domain structure of CKAP4 used in the test
- the lower left diagram of (e) shows wild-type CKAP4 (WT) -HA or the deletion mutant CKAP4-HA (ECD , ⁇ C1, ⁇ C2, ⁇ C3)
- WT wild-type CKAP4
- ECD deletion mutant CKAP4-HA
- the lower right figure of (e) shows X293T / Dkk1-FLAG cell lysate transiently expressing wild-type CKAP4-HA (WT) or deletion mutant CKAP4-HA ( ⁇ LZ) and its input.
- WT wild-type CKAP4-HA
- ⁇ LZ deletion mutant CKAP4-HA
- a polypeptide in which glutathione-S-transferase (GST) is linked to the N-terminus of the extracellular domain of CKAP4 (CKAP4-ECD) or GST together with Dkk1 Incubation, Dkk1 bound to GST-CKAP4-ECD or GST was detected, and the measurement results are shown.
- GST glutathione-S-transferase
- (B) shows the result of detecting pAKT, AKT, and clathrin in the lysate of MDCK cells incubated with a preparation containing Dkk1.
- (C) shows the results of detection of various proteins in the lysate of MDCK cells (Control) or MDCK / Dkk1-FLAG cells transduced with control siRNA or CKAP4 siRNA.
- (D) shows the results of cell proliferation assay for MDCK cells (Control) or MDCK / Dkk1-FLAG cells (Dkk1-FLAG) transduced with control siRNA or CKAP4AP siRNA.
- (a) shows the control (MDCK cells) and MDCK cells (MDCK / Dkk1-FLAG) overexpressing wild-type Dkk1-FLAG (WT) as input (immunoprecipitate (IP) with anti-CKAP4 antibody). Shows the results of detecting p85 ⁇ , CKAP4 and Dkk1.
- (b) shows a lysate (input) of control (MDCK cells) and MDCK cells (MDCK / Dkk1-FLAG) overexpressing wild-type Dkk1-FLAG (WT) as an immunoprecipitate (IP) with anti-CKAP4 antibody. Shows the results of detecting p110 ⁇ , CKAP4 and Dkk1.
- (c) shows the p85 ⁇ against the lysate (Input) of X293T / Dkk1-FLAG cells expressing CKAP4-HA or CKAP4-ECD-HA together with p85 ⁇ and the immunoprecipitate (IP) of the anti-HA antibody. And the result of having detected HA (CKAP4) is shown.
- (d) shows, together with FLAG-p110 ⁇ , lysis solution (Input) of X293T / Dkk1 cells expressing CKAP4-HA or CKAP4-ECD-HA and immunoprecipitate (IP) of the anti-HA antibody against FLAG
- the results of detecting (p110 ⁇ ) and HA (CKAP4) are shown.
- (e) shows a lysis solution (Input) of S2-CP8 cells expressing control shRNA (Control) or Dkk1 shRNA (Dkk1) and its immunoprecipitate (IP) with anti-CKAP4 antibody, p85 ⁇ , CKAP4 and The result of having detected Dkk1 is shown.
- (f) shows a lysis solution (Input) of S2-CP8 cells expressing control shRNA (Control) or Dkk1 shRNA (Dkk1) and its immunoprecipitate (IP) with anti-CKAP4 antibody, p110 ⁇ , CKAP4 and The result of having detected Dkk1 is shown.
- the upper diagram of (g) shows the amino acid sequence of the proline-rich region of CKAP4.
- FIG. 1 The figure below shows a lysate of X293T / Dkk1-FLAG cells transfected with a gene encoding CKAP4-HA (PA mutant CKAP4-HA) in which wild-type CKAP4-HA or proline is substituted with alanine (Input) And the result of having detected p85 (alpha) and HA (CKAP4) with respect to the immunoprecipitate (IP) by the anti-HA antibody is shown.
- the upper diagram shows a schematic diagram of the domain structure of p85 ⁇ .
- the lower figure of (h) shows wild-type p85 ⁇ (WT) (GFP-WT), ⁇ SH3 lacking the SH3 domain (GFP- ⁇ SH3), ⁇ SH2 lacking the SH2 and iSH2 domains (GFP- Results of detection of GFP (p85 ⁇ ) and CKAP4 against the lysate (Input) of X293T / Dkk1-FLAG cells transfected with the gene encoding ⁇ SH2) and its immunoprecipitate (IP) with anti-CKAP4 antibody Show.
- the results of detection of Dkk1, CKAP4, and HSP90 in the lysates of lung cancer cells, pancreatic cancer cells, cervical cancer cells, gastric cancer cells, esophageal squamous cell carcinoma cells, liver cancer cells, and fetal kidney cells are shown.
- the left figure of (a) shows the result of immunostaining pancreatic cancer tissue excised from a pancreatic cancer patient with anti-Dkk1 antibody or anti-CKAP4 antibody and hematoxylin.
- the left figure of (a) shows the result of immunostaining the lung adenocarcinoma tissue extracted from the lung adenocarcinoma patient with anti-Dkk1 antibody or anti-CKAP4 antibody and hematoxylin.
- the left figure of (c) shows the result of immunostaining of a lung squamous cell carcinoma tissue removed from a lung squamous cell carcinoma patient with anti-Dkk1 antibody or anti-CKAP4 antibody and hematoxylin.
- the left figure of (e) shows the result of immunostaining with anti-Dkk1 antibody or anti-CKAP4 antibody and hematoxylin on lung tissue showing lung atypical adenomatous hyperplasia.
- Human pancreatic cancer (a, b), human lung adenocarcinoma (c), and human lung squamous cell carcinoma (d) cancer tissues were immunostained with anti-Dkk1, anti-CKAP4 antibody, or anti-pAKT antibody and hematoxylin Results are shown.
- the upper left figure of (a) shows the results of detecting various proteins in the lysis solution of S2-CP8 cells transduced with control shRNA, Dkk1 shRNA, or Dkk1 shRNA and Dkk1-FLAG, and (a) The upper right figure shows the results of detecting various proteins in the lysate of S2-CP8 cells transduced with control shRNA, CKAP4 shRNA, or CKAP4 shRNA and CKAP4-HA.
- the lower figure of (a) shows the results of cell proliferation assay for each of the cells.
- the upper left figure of (b) shows the results of detecting various proteins in the lysate of A549 cells transduced with control shRNA, Dkk1 shRNA, or Dkk1 shRNA and Dkk1-FLAG, and the upper right diagram of (b) Shows the results of detecting various proteins in the lysate of A549 cells transduced with control shRNA, CKAP4 shRNA, or CKAP4 shRNA and CKAP4-HA.
- the lower figure of (b) shows the results of cell proliferation assay for each of the cells.
- S2-CP8 cells expressing control shRNA, Dkk1kkshRNA, CKAP4 shRNA, Dkk1 shRNA and Dkk1-FLAG, or CKAP4 shRNA and CKAP4-HA are three-dimensionally cultured on Matrigel, and one field of view The result of having measured the number of aggregates per hit is shown.
- A549 cells expressing control shRNA, Dkk1 shRNA, CKAP4 shRNA, Dkk1 shRNA and Dkk1-FLAG, or CKAP4 shRNA and CKAP4-HA are three-dimensionally cultured on Matrigel, and per field of view. The result of having measured the number of aggregates is shown.
- (a) shows tumor volume change and final weight of xenogeneic tumor tissue pieces between 21 after injection in nude mice subcutaneously injected with control shRNA, CKAP4 shRNA, or S2-CP8 cells expressing CKAP4 shRNA and CKAP4-HA The result of analyzing is shown.
- (b) Analyzes tumor volume change and final weight of heterogeneous tumor tissue pieces in nude mice subcutaneously injected with control shRNA, CKAP4 shRNA, or A549 cells expressing CKAP4 shRNA and CKAP4-HA The results are shown.
- (C) shows the results of analyzing the tumor volume change and the final weight of the heterogeneous tumor tissue pieces for 21 days after injection in nude mice subcutaneously injected with S2-CP8 cells expressing control shRNA, or Dkk1 shRNA.
- (D) shows the results of analyzing the tumor volume change and the final weight of the heterogeneous tumor tissue pieces 28 days after injection in nude mice subcutaneously injected with A549 cells expressing control shRNA, or Dkk1 shRNA.
- the left figure of (a) shows a schematic diagram of the epitope of the polyclonal anti-CKAP4 antibody, and the right figure of (a) shows S2-CP8 cells (Control), or S2-CP8 expressing CKAP4 cDNA or CKAP4 shRNA.
- (d) shows the results of detection of various proteins in the lysate of MDCK cells incubated with Dkk1 or insulin in the presence of anti-CKAP4 antibody or anti-GST antibody after treatment with nocodazole.
- (E) shows the results of detecting various proteins in the lysate of S2-CP8 cells and A549 cells incubated with anti-CKAP4 antibody or anti-GST antibody added.
- (f) shows the results of three-dimensional culture of S2-CP8 cells and A549 cells in Matrigel in the presence of anti-GST antibody or anti-CKAP4 antibody, and counting the number of cell aggregates in one field of view. .
- (g) shows the result of measuring the number of aggregates of S2-CP8 cells in one field by culturing S2-CP8 cells three-dimensionally in Matrigel in the presence of anti-GST antibody or anti-CKAP4 antibody.
- (H) shows the results of immunostaining of cancer-issue-originated spheroids (CTOS) of specimens LC189 and LB95 with anti-Dkk1 antibody or anti-CKAP4 antibody and hematoxylin.
- CTOS cancer-issue-originated spheroids
- I shows the results of proliferation assay of CTOS of specimens LC189 and LB95 in the presence of anti-CKAP4 antibody or anti-GST antibody.
- the left figure of (a) shows the results of observing nude mice subcutaneously injected with S2-CP8 cells from the top and the results of observing heterogeneous tumor tissue pieces excised from the nude mice.
- the right figure of (a) shows the results of measuring the tumor volume change and the final weight of the heterogeneous tumor tissue pieces for 14 days after antibody injection.
- the left figure of (b) shows the result of observing a nude mouse subcutaneously injected with A549 cells from the top and the result of observing a heterogeneous tumor tissue piece excised from the nude mouse.
- the right figure of (b) shows the results of measuring the tumor volume change and the final weight of the xenogeneic tumor tissue pieces 25 days after antibody injection.
- the results of detection of CKAP4 and TSG101 in exosomes recovered from cell culture solutions of various cancer cells and normal cell lines and CKAP4 and clathrin in cell lysate (Input) are shown.
- the antitumor agent of the present invention comprises a substance that suppresses the expression or function of CKAP4 as an active ingredient.
- CKAP4 as an active ingredient
- CKAP4 is involved in the growth regulation mechanism of tumor cells as a Dkk1 receptor, and has the effect of enhancing the growth of tumor cells. In the antitumor agent of the present invention, it is possible to effectively suppress the growth of tumor cells by suppressing the expression or function of CKAP4.
- CKAP4 is known as a transmembrane protein, and its amino acid sequence and base sequence are also known.
- amino acid sequence of human CKAP4 is known as SEQ ID NO: 1
- base sequence of the gene encoding human CKAP4 is known as SEQ ID NO: 2.
- the “substance that suppresses the expression of CKAP4” is not particularly limited as long as it is pharmaceutically acceptable and can suppress the expression of CKAP4 from DNA encoding CKAP4 (CKAP4 gene).
- the substance that suppresses the expression of CKAP4 may exhibit an inhibitory action on the expression of CKAP4 at any stage such as transcription, post-transcriptional regulation, translation, and post-translational modification of the CKAP4 gene.
- a nucleic acid molecule that suppresses transcription of CKAP4 gene such as decoy nucleic acid
- an RNA molecule having RNA interference action on CKAP4 mRNA such as siRNA, shRNA, dsRNA or the like
- These nucleic acid drugs may be used alone or in combination of two or more.
- nucleic acid drugs can be appropriately designed by those skilled in the art based on the information on the base sequence of the CKAP4 gene by a known method.
- siRNA, shRNA, dsRNA, miRNA, antisense nucleic acid, and more preferably siRNA and shRNA are preferable from the viewpoint of ease of clinical application.
- the nucleic acid molecule when it is an RNA molecule, it may be designed so that it can be generated in vivo.
- a DNA encoding the RNA molecule may be inserted into an expression vector for mammalian cells.
- expression vectors include virus vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai viruses, and animal cell expression plasmids.
- the “substance that suppresses the function of CKAP4” is not particularly limited as long as it is pharmaceutically acceptable and can suppress the function of CKAP4 present in the cell membrane of tumor cells.
- the substance that suppresses the function of CKAP4 include an antibody or a fragment thereof that specifically binds to CKAP4, an aptamer that specifically binds to CKAP4, a low molecular compound that suppresses the function of CKAP4, and the like. These substances may be used alone or in combination of two or more. Among these substances, an antibody or a fragment thereof that binds specifically to CKAP4 is preferable.
- the antibody that specifically binds to CKAP4 may be either a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
- the isotype of these antibodies is not particularly limited and may be any of IgG, IgM, IgA, etc., preferably IgG.
- the antibody is an antibody having reduced antigenicity in the human body, specifically, a fully human antibody, a humanized antibody, a mouse-human chimera.
- a fully human antibody a humanized antibody
- a mouse-human chimera a humanized antibody
- Preferred are antibodies, chicken-human chimeric antibodies and the like. Among these, fully human antibodies and humanized antibodies are more preferable.
- the binding site of the antibody to CKAP4 is a site exposed to the outside of CKAP4 (for example, in the case of human CKAP4, positions 128 to 602 in SEQ ID NO: 1). It is desirable that the region is a region within the region (preferably a region within the region of positions 128 to 503 of SEQ ID NO: 1).
- the leucine zipper region of CKAP4 positions 468 to 503 in SEQ ID NO: 1 is required for the binding between CKAP4 and Dkk1, so that the binding to Dkk1 in the leucine zipper region can be suppressed, A binding site for CKAP4 of the antibody may be set.
- the antibody fragment may be any fragment that has at least a complementarity determining region (CDR) for specifically recognizing and binding to a target antigen.
- CDR complementarity determining region
- the antibody and fragments thereof can be prepared by genetic engineering according to a conventional method.
- the antitumor agent of the present invention can suppress the growth or growth of tumor cells by suppressing the expression or function of CKAP4. . Therefore, the antitumor agent of the present invention can be used for cancer treatment.
- the cancer to which the antitumor agent of the present invention is applied is not particularly limited, and specifically, lung cancer, pancreatic cancer, colon cancer, colon cancer, stomach cancer, rectal cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, Solid cancers such as cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer, tongue cancer, pharyngeal cancer, laryngeal cancer, brain tumor, glioma, glioblastoma, glioblastoma multiforme
- a blood cancer such as leukemia or malignant lymphoma;
- the antitumor agent of the present invention effectively exerts a tumor cell growth inhibitory effect on tumor cells expressing CKAP4, particularly tumor cells expressing both CKAP4 and Dkk1, so that CKAP4 is expressed.
- Cancers, particularly those expressing both CKAP4 and Dkk1, are suitable applications.
- lung cancer and pancreatic cancer have high expression of CKAP4 and Dkk1, and are particularly suitable as cancers to which the antitumor agent of the present invention is applied.
- CKAP4 in cancer can be confirmed by tissue immunization against the collected cancer tissue. Specifically, when the collected cancer tissue is immunostained with an anti-CKAP4 antibody and there is 5% or more of the region where CKAP4 expression is observed in the tumor region, CKAP4 is expressed. To be judged.
- a cancer in which the CKAP4 expression region is present in the tumor region is 5% or more, more preferably, the CKAP4 expression region is present in the tumor region is 20% or more.
- a cancer, particularly preferably a cancer in which 50% or more of the expression region of CKAP4 is present in the tumor region is mentioned.
- the expression of CKAP4 in cancer can be measured by collecting RNA from the collected cancer tissue and quantitative PCR.
- the determination of the presence or absence of CKAP4 expression may be performed using a non-cancerous tissue of the same case as an index. Specifically, when the amount of CKAP4 in the cell lysate of cancer tissue is greater than the amount of CKAP4 in the cell lysate of non-cancerous tissue of the same case, it is determined that CKAP4 is expressed in the cancer.
- Dkk1 when the collected cancer tissue is immunostained with an anti-Dkk1 antibody, Dkk1 is expressed if there is 5% or more of the region where Dkk1 expression is observed in the tumor region. It is judged that As a suitable example of the cancer to which the antitumor agent of the present invention is applied, a cancer in which the Dkk1 expression region is present in the tumor region is 5% or more, more preferably, the Dkk1 expression region is present in the tumor region is 20% or more. A cancer, particularly preferably a cancer in which 50% or more of the expression region of Dkk1 is present in the tumor region.
- the amount of Dkk1 in the cell lysate of cancer tissue is greater than the amount of Dkk1 in the cell lysate of non-cancerous tissue of the same case. It is determined that Dkk1 is expressed in the cancer.
- the antitumor agent of the present invention can be used not only for humans but also for mammals such as cows, pigs, dogs, cats, goats, rats, mice, rabbits, etc., but is preferably used as a pharmaceutical for humans.
- mammals such as cows, pigs, dogs, cats, goats, rats, mice, rabbits, etc., but is preferably used as a pharmaceutical for humans.
- the dosage form of the antitumor agent of the present invention may be either oral administration or parenteral administration as long as the antitumor effect is obtained, and may be appropriately set according to the type of active ingredient to be used. Good. Specific administration forms of the antitumor agent of the present invention include oral administration; parenteral administration such as injection administration (intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, local injection into the affected area, etc.) and suppository administration. Can be mentioned.
- the dosage of the antitumor agent of the present invention may be appropriately set according to the type of active ingredient used, the mode of administration, the type of cancer to be applied, the degree of symptoms of the patient, and the like.
- a nucleic acid molecule is used as an active ingredient
- a single dose of the nucleic acid molecule is usually about 0.01 to 1000 ⁇ g / kg body weight, preferably about 0.1 to 100 ⁇ g / kg body weight.
- an antibody, a fragment thereof, or a low molecular weight compound is used as an active ingredient, a dose of these substances is usually about 0.1 to 20 mg / kg body weight every 1 to 3 weeks. May be administered at a frequency of about once.
- the antitumor agent of the present invention may be used alone, or may be used in combination with one or more other drugs having antitumor activity and / or radiation therapy.
- the antitumor agent of the present invention is prepared in a preparation form according to the type of active ingredient and the administration form.
- Examples of the dosage form of the antitumor agent of the present invention include solid preparations such as tablets, capsules, pills, powders, granules and suppositories; liquid preparations such as liquids, suspensions, emulsions and injections. Can be mentioned.
- the antitumor agent of the present invention is formulated by adding a pharmaceutically acceptable carrier or additive depending on the formulation form.
- a pharmaceutically acceptable carrier or additive depending on the formulation form.
- a solid preparation it can be formulated using an excipient, a binder, a disintegrant, a lubricant and the like.
- a liquid preparation it can be formulated using physiological saline, a buffer solution, or the like.
- nucleic acid molecule when used as an active ingredient, it is desirable that the nucleic acid molecule is formulated together with a nucleic acid introduction aid so that the nucleic acid molecule is easily transferred into tumor cells.
- nucleic acid introduction aid include lipofectamine, oligofectamine, RNAifect, liposome, polyamine, DEAE dextran, calcium phosphate, dendrimer and the like.
- the screening method of the present invention is a method for screening an active ingredient of an antitumor agent, the first step of detecting the ability to suppress the expression of CKAP4 or the ability to bind to CKAP4 on a cell membrane for a test substance, and The method includes a second step of selecting, as a candidate for an active ingredient of an antitumor agent, a test substance in which the ability to suppress the expression of CKAP4 or the ability to bind to CKAP4 on a cell membrane is recognized.
- the screening method of the present invention will be described in detail.
- the test substance is not particularly limited as long as it can be a candidate substance for an active ingredient of an antitumor agent.
- the test substance is a natural substance such as a biological substance. Alternatively, it may be a chemically synthesized material.
- examples of the test substance include low molecular weight compounds; proteins such as antibodies; nucleic acid molecules such as siRNA, shRNA, dsRNA, miRNA, antisense DNA, antisense RNA, and aptamer.
- First step In the screening method of the present invention, first, as a first step, the ability to suppress the expression of CKAP4 or the ability to bind to CKAP4 on the cell membrane is detected.
- a specific technique for detecting the ability of the test substance to suppress CKAP4 expression is not particularly limited, and examples thereof include a method of performing the following Steps 1-a and 1-b.
- Step 1-a A step of bringing a test substance into contact with a cell expressing CKAP4.
- Step 1-b A step of measuring the expression level of CKAP4 in the cells after Step 1-a.
- the cells expressing CKAP4 used in the step 1-a are not particularly limited as long as they express CKAP4.
- cells that endogenously express CKAP4 such as tumor cells, normal cells
- Cells transfected with CKAP4 gene can be used by transfecting with CKAP4 gene.
- Transfection of normal cells with the CKAP4 gene can be performed using known genetic engineering techniques.
- the cell and the test substance in order to bring the test substance into contact with the cell expressing CKAP4, the cell and the test substance can be added to a medium in which the cell can grow to grow the cell. What is necessary is just to culture
- nucleic acid molecules such as siRNA, shRNA, dsRNA, miRNA, antisense DNA, and antisense RNA are used as test substances, nucleic acid introduction is performed so that these nucleic acid molecules can be easily introduced into the cells. It is preferable that an adjuvant is also present.
- the type of such a nucleic acid introduction auxiliary agent is as exemplified in the column of “1. Antitumor agent”.
- the CKAP4 expression level in the cells can be measured by measuring the intracellular CKAP4 mRNA level and / or CKAP4 level (protein level) according to a known method.
- Step 1-i A step of bringing a test substance into contact with a cell expressing CKAP4.
- Step 1-ii Step of confirming whether or not the test substance is bound to CKAP4 on the cell membrane after Step 1-i.
- the step 1-i can be performed by the same method as the step 1-a.
- the specific method for confirming whether or not the test substance is bound to CKAP4 on the cell membrane is not particularly limited, but the amount of the test substance is determined using a known immunostaining method. It can be determined by measuring.
- CKAP4 can be recovered from the cells after Step 1-i and the amount of the test substance bound to CKAP4 can be determined.
- test substance in which the ability to suppress the expression of CKAP4 or the binding ability to CKAP4 on the cell membrane in the first step is recognized is selected as a candidate for an active ingredient of an antitumor agent.
- the expression level of CKAP4 is smaller than the expression level of CKAP4 when the first step is performed without adding the test substance.
- the test substance is determined to be capable of suppressing the expression of CKAP4.
- a test substance that has been found to bind to CKAP4 on the cell membrane is determined to have the ability to bind to CKAP4 on the cell membrane.
- the test substance in which the ability to suppress the expression of CKAP4 or the ability to suppress the binding of CKAP4 to Dkk1 is selected as a candidate for an active ingredient of an antitumor agent because it has a tumor cell growth inhibitory effect.
- the test substance thus selected may actually be tested for tumor cell growth inhibitory action and evaluated for its clinical application as an antitumor agent.
- the postoperative prognostic examination method for cancer patients according to the present invention includes a step of measuring the expression of CKAP4 and Dkk1 in cancer tissues collected from cancer patients.
- the postoperative prognosis of a cancer patient can be estimated by using as an index the expression of both CKAP4 and Dkk1 in cancer tissue collected from the cancer patient.
- the type of cancer of the patient who is the subject of the prognosis examination method is not particularly limited, and specifically, lung cancer, pancreatic cancer, colon cancer, colon cancer, stomach cancer, rectal cancer, liver cancer, Breast cancer, bladder cancer, prostate cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer, tongue cancer, pharyngeal cancer, laryngeal cancer, brain tumor, glioma, glioblastoma, polymorphism
- Examples include solid cancers such as glioblastoma; blood cancers such as leukemia and malignant lymphoma.
- lung cancer and pancreatic cancer are mentioned as a suitable example of the test object of the test
- the expression of CKAP4 in cancer tissue collected from a cancer patient can be measured using a reagent capable of detecting CKAP4.
- Examples of the method for measuring the expression of CKAP4 include a method of immunostaining the collected cancer tissue using an anti-CKAP4 antibody. Specifically, when the collected cancer tissue is immunostained with an anti-CKAP4 antibody and there is 5% or more of the region where CKAP4 expression is observed in the tumor region, CKAP4 is expressed. To be judged.
- the expression of CKAP4 can be measured, for example, by collecting RNA from the collected cancer tissue and performing quantitative PCR.
- the determination of the presence or absence of CKAP4 expression may be performed using a non-cancerous tissue of the same case as an index. Specifically, when the amount of CKAP4 in the cell lysate of cancer tissue is greater than the amount of CKAP4 in the cell lysate of non-cancerous tissue of the same case, it is determined that CKAP4 is expressed in the cancer.
- test method of the present invention it is desirable to measure the expression of Dkk1 in a cancer tissue collected from a cancer patient from the viewpoint of further improving the accuracy of predicting the prognosis after surgery of the patient.
- Measurement of Dkk1 expression in cancer tissues collected from cancer patients can be performed using a reagent capable of detecting Dkk1.
- the method for measuring the expression of Dkk1 should be confirmed by the method of tissue immunization against the collected cancer tissue, the method of collecting RNA from the collected cancer tissue, and measuring by quantitative PCR, etc. Can do.
- Dkk1 when the collected cancer tissue is immunostained with an anti-Dkk1 antibody, if there is 5% or more of the region where Dkk1 expression is observed in the tumor region, Dkk1 is not expressed in the cancer. It is judged that it is expressed.
- RNA is collected from collected cancer tissue and Dkk1 is measured, the amount of Dkk1 in the cell lysate of cancer tissue is greater than the amount of Dkk1 in the cell lysate of non-cancerous tissue of the same case. It is determined that Dkk1 is expressed in the cancer.
- cancer patients in which both CKAP4 and Dkk1 are expressed are estimated to have a poor prognosis after surgery, such as a short overall survival and a short relapse-free survival.
- the present invention provides a reagent for detecting CKAP4 in cancer tissue as a test kit for performing the test method.
- the reagent for detecting CKAP4 is not particularly limited, and examples thereof include an anti-CKAP4 antibody and a fragment thereof.
- the test kit of the present invention may contain a reagent for detecting Dkk1 in cancer tissue. These antibodies may be labeled with biotin, fluorescent labels, magnetic beads, or the like as necessary.
- the method for examining cancer according to the present invention is characterized by including a step of measuring CKAP4 in exosomes collected from a subject. According to the present invention, it is possible to diagnose whether or not a subject suffers from cancer by using the presence or absence of CKAP4 in exosomes collected from the subject as an index.
- the type of cancer to be examined is not particularly limited, but specifically, lung cancer, pancreatic cancer, colon cancer, colon cancer, stomach cancer, rectal cancer, liver cancer, breast cancer, bladder cancer, prostate Cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer, tongue cancer, pharyngeal cancer, laryngeal cancer, brain tumor, glioma, glioblastoma, polymorphic glioblastoma, etc.
- lung cancer and pancreatic cancer are mentioned as a suitable example of the test object of the test
- the origin of exosome is not particularly limited, and examples thereof include body fluids such as serum and urine. Among these, serum is preferable.
- the method for collecting exosomes from a subject is not particularly limited as long as a sample that can be used as a sample for detecting CKAP4 is obtained, and can be appropriately selected from conventionally known methods.
- Exosomes can be separated by ultracentrifugation, but more conveniently, exosomes can be separated using commercially available kits. Specific examples of commercially available exosome separation kits include ExoQuick TM Exosome Precipitation Solution, Exoquick-TC, etc. (both manufactured by System Biosciences).
- CKAP4 in exosome can be detected by eluting the protein from exosome and using a reagent for detecting Dkk1 such as anti-CKAP4 antibody.
- the present invention provides a reagent for detecting CKAP4 in exosomes as a test kit for performing the test method.
- the reagent for detecting CKAP4 is not particularly limited, and examples thereof include an anti-CKAP4 antibody and a fragment thereof.
- the anti-CKAP4 antibody may be labeled with biotin, a fluorescent label, a magnetic bead or the like, if necessary.
- the test kit of the present invention may contain a reagent for separating exosomes from body fluids.
- the transfected cells may be referred to as “X / Y cells” (for example, MDCK / Dkk1-FLAG-GPI cells, etc.), but these cells encode protein Y in the X cells.
- a protein may be expressed in the form of “A-B” (for example, Dkk1-FLAG), and the protein means a protein in which peptide A (peptide etc.) is fused to peptide A.
- Transfected cells were prepared according to the following production method or according to a known genetic engineering technique, except for those described below for the production method.
- Test method 1-1 Separation of protein binding to Dkk1
- Dkk1-FLAG-GPI Dkk1 gene in which a FLAG epitope and a glycosylphosphatidylinositol (GPI) anchor signal sequence were inserted in series on the C-terminal side was constructed.
- MDCK cells which are normal cells derived from canine tubules, were transduced with the above vector, and human Dkk1 gene (Dkk1-FLAG-GPI; human Dkk1) in Dulbecco's modified Eagle's medium (DMEM) medium containing 10 ⁇ g / ml blasticidin S.
- DMEM Dulbecco's modified Eagle's medium
- the collected beads were eluted by three incubations at 30 ° C. for 30 minutes in TBS (50 ⁇ l) containing 0.2 ⁇ mg / ml FLAG peptide.
- TBS 50 ⁇ l
- a 50% slurry of neutravidin beads was added 40 ⁇ l of a 50% slurry of neutravidin beads and incubated at 4 ° C. with shaking for 2 hours.
- the beads were then washed twice with 1 ml NP40 buffer and once with 1 ml 10 mM Tris-HCl (pH 7.5), and the Dkk1-bound complex was then washed with 20 ⁇ l Laemmli sample buffer. Elute. Thereafter, the protein bound to Dkk1 was detected by protein silver staining. Seventeen bands (arrows in FIG. 2 (b)) were cut from the gel and analyzed by mass spectrometry.
- MDCK cells (2 ⁇ 10 5 cells) expressing Dkk1 polar secretion Dkk1 were seeded on a Transwell dye filter (Corning Costar Quality Biological, Gaithersburg, MD, USA). After 48 hours, the medium was changed and further cultured for 24 hours. In order to detect Dkk1 secreted in the culture solution, Blue Sepharose was added to the culture solution obtained from the apical side and the basal side and incubated at 4 ° C. for 2 hours, followed by centrifugation. Next, the precipitate was collected, and Dkk1 was detected using an anti-Dkk1 antibody.
- MDCK I Cell lines and cell culture renal tubular cells MDCK type I (MDCK I) were provided by Dr. S. Tsukita (Osaka University), and human lung adenocarcinoma cells A549 and NCI-H1579 were provided by Dr. Y. Shintani (Osaka) From the University), human cervical cancer cell HeLaS3 from Dr. K. Matsumoto (Nagoya University), human gastric cancer cell AGS from Dr. M. Hatakeyama (University of Tokyo), KKLS was provided by Dr. W. Yasui (Hiroshima University), human hepatoblastoma cell HepG2 was provided by Dr. Y.
- DMEM Dulbecco's modified Eagle's medium
- FBS fetal bovine serum
- RPMI-1640 fetal bovine serum
- Antibodies The antibodies used in this test are as shown in Table 1 below.
- MDCK I cells, S2-CP8 cells, and A549 cells were seeded in 35 mm dishes at 1 ⁇ 10 5 cells / ml.
- MDCK I cells were cultured in DMEM containing 10% FBS.
- S2-CP8 cells and A549 cells were cultured in DMEM containing 1% FBS. The cell number was counted after a predetermined number of days.
- MDCK I cells, S2-CP8 cells and A549 cells were tested by three-dimensional culture and three-dimensional culture according to previously reported contents ((Matsumoto et al., EMBO J, 2014).
- Matrigel BD Biosciences, To analyze MDCK I cell cyst formation, S2-CP8 and A549 cell proliferation in San Jose, CA, USA), first place 40 ⁇ l Matrigel on a round glass slide and incubate at 37 ° C for 30 minutes The gel was then solidified and then suspended in MDCK I cells (3 ⁇ 10 4 cells) suspended in DMEM containing 10% FBS and 2% Matrigel, and DMEM containing 0.1% bovine serum albumin and 2% Matrigel. Turbid S2-CP8 cells or A549 cells (2 ⁇ 10 4 cells) were added onto the solidified Matrigel and cultured.
- Tumor Graft analysis was performed using a modified technique of previously reported content (Fujii et al., Oncogene, 2014). After anesthetizing 6-week-old male BALB / cAnNCrj-nu nude mice (Charles River Laboratory Japan Inc, Osaka, Japan) with medetomidine (0.3 mg / kg body weight) and midazolam (4 mg / kg body weight), 100 ⁇ l S2-CP8 cells (5 ⁇ 10 6 cells) or A549 cells (5 ⁇ 10 6 cells) suspended in a phosphate buffer (PBS) were injected subcutaneously. Next, nude mice 21 days after transplantation (S2-CP8 cells) and 28 days after transplantation (A549 cells) were subjected to analysis.
- nude mice were divided into two groups when the average tumor size reached 50 mm 3 .
- Anti-pCKAP4 antibody 150 ⁇ g / body
- anti-glutathione-S-transferase (GST) antibody 150 ⁇ g / body twice a week for 2 weeks (at days 0, 4, 8, 12) (S2-CP8 cells) and 3.5 weeks (at days 0, 4, 9, 12, 16, 18, 22) (A549 cells) were injected intraperitoneally.
- Nude mice were subjected to analysis on day 14 (S2-CP8 cells) and day 25 (A549 cells) after antibody administration.
- the size and weight of the xenogeneic tumor tissue piece formed at the transplant site was measured and further subjected to immunohistochemical analysis.
- the size of the tumor was calculated by the formula: major axis ⁇ minor axis ⁇ minor axis ⁇ 0.5. All animal experiments in this study were conducted under the approval of the Osaka University Animal Experiment Committee (No. 21-048-1).
- Knockdown of protein expression by siRNA Table 2 shows the types of proteins targeted for knockdown and their target sequences in this test.
- Cells were transduced with a mixture of siRNA (20 nM each) against the target protein using RNAiMAX (Invitrogen, Carlsbad, CA, USA). The test was performed using cells 36 to 48 hours after transduction.
- Plasmids FLAG-p110 ⁇ , FLAG-p110 ⁇ in pcDNA3mycTEVflag and p85 ⁇ in pCAGGS used were those provided by Dr. T. Asano (Hiroshima University).
- CKAP4 cDNA was amplified from the A549 cDNA library, and all mutants were prepared by a general method.
- a lentiviral vector was constructed by inserting cDNA into CSII-CMV-MCS-IRES2-Bsd provided by Dr. H. Miyoshi (RIKEN).
- each lentivirus was introduced into a parent cell (5 ⁇ 10 4 cells / well in a 12-well plate) in the presence of a conditioned medium and 10 ⁇ g / mL polybrene.
- MDCK I cells S2-CP8 cells that stably express Dkk1-Flag, CKAP4-HA, Dkk1 shRNA, or CKAP4 shRNA by collecting cells by centrifugation at 1200 xg for 1 hour and further incubating for 24 hours And A549 cells were prepared.
- Pancreatic cancer patients 59 patients
- lung adenocarcinoma patients 67 patients
- squamous cell lung cancer patients 61 patients
- tumor tissue pieces 61 patients
- the age of patients with pancreatic cancer ranged from 47 to 83 years, with an average age of 70 years.
- the age of lung adenocarcinoma patients was 39-85 years, with an average age of 68 years.
- the age of patients with squamous cell carcinoma of the lung was 38-82 years, with an average age of 71 years.
- the age of lung atypical adenomatous hyperplasia was 58-78 years, with an average age of 67 years.
- antigen activation was first performed using a Pascal pressure chamber (manufactured by Dako), and Dako REAL Peroxidase-Blocking Solution was reacted for 5 minutes to block nonspecific binding sites.
- tissue sections were treated with anti-Dkk1 antibody (1: 100), anti-CKAP4 antibody (1: 100), or pAKT antibody (1:50) at 4 ° C. for 16 hours.
- HRP horseradish peroxidase
- DAB diaminobenzidine
- Test results 2-1 Effect of Dkk1 on cell growth ability
- MDCK cells expressing the Dkk1 gene Dkk1-FLAG
- the results of confirming that Dkk1 with a FLAG tag on the C-terminal side is expressed are shown in Fig. 1 (a). Shown above.
- the control shown in (a) of FIG. 1 is a result of using MDCK cells as parent cells. HSP90 was used as a loading control.
- MDCK / Dkk1-FLAG cells cultured on a transwell filter are cultured, and after apical polarity is established, the expression of Dkk1 is analyzed separately for apical and basolateral sides. It is the result.
- Fig. 1 (a) MDCK / Dkk1-FLAG cells cultured on a transwell filter are cultured, and after apical polarity is established, the expression of Dkk1 is analyzed separately for apical and basolateral sides. It is the result.
- Fig. 1 (a) MDCK /
- MDCK / Dkk1-FLAG cells were fixed on a Transwell polycarbonate filter, and the cells were stained with anti-Dkk1 antibody (green), anti-E-cadherin antibody (red), and DRAQ5 DNA Dye (blue). Shown in (b). E-cadherin was used as a basolateral membrane marker.
- Ap indicates the top end side
- Bl indicates the side bottom side
- the scale bar is 20 ⁇ m. From this result, it was confirmed that Dkk1-FLAG was localized in the apical membrane in MDCK / Dkk1-FLAG cells.
- FIG. 1 (c) shows the result of paraffin embedding of kidney tissue sections of embryonic day 13 (day E13) mice and immunostaining with anti-Dkk1 antibody.
- the upper right diagram in FIG. 1C is an enlarged view in the frame of the upper left diagram.
- the scale bar shown in the upper right diagram of FIG. 1 (c) is 100 ⁇ m.
- the lower figure of (c) of FIG. 1 shows the result of immunostaining the tissue piece with anti-Dkk1 antibody (green) and anti-aPKC antibody (red).
- aPKC is a marker of the apical membrane.
- the white arrow in FIG. 1 (c) indicates Dkk1 localized in the apical membrane.
- the scale bar shown in the lower part of FIG. 1 (c) is 20 ⁇ m. From this result, it was revealed that Dkk1 is expressed on the apical side in the ureter of fetal kidney.
- MDCK / Dkk1-FLAG cells were three-dimensionally cultured on Matrigel for 5 days.
- wild type MDCK cells were used as a control.
- the result is shown in FIG.
- cysts that were three-dimensionally cultured on Matrigel were immobilized, and then stained with anti-Ki67 antibody (green), anti-aPKC antibody (red), and DRAQ5 DNA Dye (blue).
- MDCK cells were cultured two-dimensionally on a Transwell-polycarbonate filter for 5 days, and then 250 ⁇ ng / ml Dkk1 was added to the apical side or the basal side and cultured for 24 hours. Next, after fixing the cells, the cells were stained with anti-Ki67 antibody (red) and DRAQ5 DNA Dye (blue). The obtained result is shown in FIG.
- the Ki67 positive rate was calculated as the ratio of the number of cells stained with anti-Ki67 antibody to the number of cells stained with nuclear staining (5 visual fields). The results are shown as the mean value ⁇ s.d. Of three independent tests. In (f) of FIG.
- NT shows the case where Dkk1 is not added
- AP shows the case where Dkk1 is added to the apical side
- Bl shows the case where Dkk1 is added to the basal side
- the scale bar is 20 ⁇ m.
- MDCK / Dkk1-FLAG cells were cultured two-dimensionally for 3 days, and the number of cells was counted. The obtained results are shown in FIG. MDCK cells were used as a control. From this result, it was confirmed that MDCK / Dkk1-FLAG cells had higher proliferation ability than control, and Dkk1 promoted cell proliferation.
- FIG. 2 (b) A schematic diagram of the experimental procedure for screening Dkk1 binding protein on the apical surface is shown in FIG.
- the result of purifying the Dkk1-binding protein and performing silver staining is shown in FIG. 2 (b).
- FIG. 2 (b) When the 17 bands eluted as proteins binding to Dkk1-FLAG-GPI were subjected to mass spectrometry, the proteins shown in the table on the right side of FIG. 2 (b) were detected. Among them, CKAP4 was band 10 As detected.
- the upper diagram of (d) in Fig. 2 shows a schematic diagram of the domain structure of Dkk1 used in this test.
- S indicates a signal peptide region.
- control MDCK cells
- lysate of MDCK cells expressing wild type Dkk1-FLAG (WT) or deletion mutant Dkk1-FLAG ( ⁇ CRD-1, ⁇ CRD-2) with an anti-FLAG antibody Immunoprecipitation.
- CKAP4, LRP6, or Dkk1 was detected from the cell lysate (Input) and immunoprecipitate (IP) using an anti-CKAP4 antibody, an anti-LRP6 antibody, or an anti-Dkk1 antibody.
- IP immunoprecipitate
- WT -FLAG and ⁇ CRD-2-FLAG formed a complex with CKAP4, but did not form a complex with ⁇ CRD-1 -FLAG. Consistent with previous reports, WT-FLAG and ⁇ CRD-2-FLAG formed a complex with LRP6, but ⁇ CRD-2-FLAG and LRP6 did not form a complex. Therefore, it was revealed that Dkk1 forms a complex with CKAP4 and LRP6 through different regions.
- FIG. 2 (e) Schematic diagram of the domain structure of CKAP4 used in this study is shown in the upper part of Fig. 2 (e).
- ICD is the intracellular domain
- ECD is the extracellular domain
- E is the endoplasmic reticulum anchor domain
- MB is the microtubule binding domain
- M is the transmembrane domain
- T is the tyrosine sulfated region
- LZ represents a leucine zipper region
- ⁇ represents an alpha helix region.
- a lysate (Input) of X293T / Dkk1-FLAG cells in which wild-type CKAP4-HA (WT) or deletion mutant CKAP4-HA ( ⁇ LZ) was temporarily expressed was input with an anti-FLAG antibody.
- HA and Dkk1 were detected using anti-HA antibody or anti-Dkk1 antibody against cell lysate (Input) and immunoprecipitate (IP).
- IP immunoprecipitate
- GST-CKAP4-ECD Polypeptide in which glutathione-S-transferase (GST) is linked to the N-terminus of the extracellular domain (ECD) of CKAP4 (positions 128 to 602 of the amino acid sequence shown in SEQ ID NO: 1) (intracellular A variant having a deleted domain) was prepared.
- NP40 buffer 0.5 nM GST-CKAP4-ECD or 0.5 nM GST was reacted with 0 to 16 nM Dkk1 (R & D® Systems) at 4 ° C. for 2 hours.
- GST-CKAP4-ECD and GST were collected by centrifugation and washed 3 times with NP40 buffer.
- Dkk1 was detected for the obtained precipitate using an anti-Dkk1 antibody. The results are shown in (a) and (b) of FIG. From this result, it was confirmed that Dkk1 directly binds to the extracellular domain of CKAP4.
- the MDCK cells were treated with 1 nM nocodazole for 24 hours, the MDCK cells were stimulated for 60 minutes with an adjustment solution containing 250 ng / ml Dkk1 or an adjustment solution not containing Dkk1.
- Cells were collected at 0, 10, 30 and 60 minutes after the start of stimulation to prepare cell lysates.
- pAKT, AKT and clathrin were detected from the cell lysate using anti-pAKT antibody, anti-AKT antibody and anti-clathrin antibody. Clathrin was used as a loading control.
- the obtained result is shown in FIG. This result showed that in the presence of Dkk1, phosphorylation of AKT in MDCK cells was promoted, suggesting that Dkk1 activates AKT in a time-dependent manner.
- MDCK cells control or MDCK / Dkk1-FLAG cells (Dkk1-FLAG) were transduced with control siRNA or CKAP4 siRNA.
- Cell proliferation assays were performed on the resulting cells. The obtained result is shown in FIG. From these results, when the expression of CKAP4 was suppressed in cells expressing Dkk1, a decrease in cell proliferation ability was observed, and it was confirmed that Dkk1 promoted cell proliferation via CKAP4.
- MDCK cells (control) or MDCK / Dkk1-FLAG cells were transduced with control siRNA or CKAP4 siRNA.
- the resulting cell lysate was prepared, and the lysate was reacted with antibodies against various proteins shown in FIG. 4 (d) to detect the various proteins.
- the obtained result is shown in FIG.
- phosphorylation of AKT was significantly reduced by suppressing the expression of CKAP4 when Dkk1 was expressed. Therefore, it was suggested that Dkk1 and CKAP4 are required for the activation of AKT in MDCK cells.
- CKAP4 a lysate of MDCK cells (MDCK / Dkk1-FLAG) overexpressing control (MDCK cells) and wild-type Dkk1-FLAG (WT) (Input ) was immunoprecipitated with anti-CKAP4 antibody.
- detection of p85 ⁇ , p110 ⁇ , CKAP4 and Dkk1 is performed on the cell lysate (Input) and immunoprecipitate (IP) using anti-p85 ⁇ antibody, anti-p110 ⁇ antibody, anti-CKAP4 antibody, and anti-Dkk1 antibody. went. The results are shown in (a) and (b) of FIG.
- CKAP4-HA or CKAP4-ECD-HA was expressed together with p85 ⁇ in X293T / Dkk1-FLAG cells.
- the obtained cell lysate (Input) was prepared.
- the obtained lysate was immunoprecipitated with an anti-HA antibody.
- p85 ⁇ and CKAP4 were detected from the cell lysate (Input) and immunoprecipitate (IP) using anti-p85 ⁇ antibody or anti-HA antibody.
- IP immunoprecipitate
- CKAP4-HA or CKAP4-HA ECD was expressed together with FLAG-p110 ⁇ in X293T / Dkk1-FLAG cells.
- the obtained cell lysate (Input) was prepared.
- the obtained lysate was immunoprecipitated with an anti-HA antibody.
- p110 ⁇ and CKAP4 were detected from the cell lysate (Input) and immunoprecipitate (IP) using anti-p110 ⁇ antibody or anti-HA antibody.
- IP immunoprecipitate
- a lysate (Input) of S2-CP8 cells stably expressing control shRNA or Dkk1kkshRNA (Dkk1) was prepared. Further, the obtained lysate was immunoprecipitated with an anti-CKAP4 antibody. Next, detection of p85 ⁇ , p110 ⁇ , CKAP4 and Dkk1 is performed on the cell lysate (Input) and immunoprecipitate (IP) using anti-p85 ⁇ antibody, anti-p110 ⁇ antibody, anti-CKAP4 antibody and anti-Dkk1 antibody. went. The results are shown in (e) and (f) of FIG. From this result, it was revealed that CKAP4, p85 ⁇ and p110 ⁇ also form a complex in S2-CP8 cells in a Dkk1-dependent manner.
- Fig. 5 (g) shows the amino acid sequence of the proline-rich region of CKAP4.
- PA mutant-CKAP4-HA
- a gene encoding a mutant of CKAP4 (PA (mutant-CKAP4-HA) in which a part of proline in the proline-rich region of CKAP4 was substituted with alanine was prepared.
- the lysate of X293T / Dkk1-FLAG cells transfected with a gene encoding CKAP4-HA or PA mutant-CKAP4-HA was immunoprecipitated with anti-HA antibody or non-immune antibody.
- Fig. 5 (h) shows a schematic diagram of the domain structure of p85 ⁇ .
- wild type p85 ⁇ (WT) (GFP-WT)
- ⁇ SH3 lacking the SH3 domain (GFP- ⁇ SH3)
- SH2 and iSH2 domains are deleted.
- a gene encoding ⁇ SH2 (GFP- ⁇ SH) is prepared.
- the lysate of X293T / Dkk1-FLAG cells transfected with a gene encoding GFP-WT, GFP- ⁇ SH3 or GFP- ⁇ SH was immunoprecipitated with anti-CKAP4 antibody or non-immune antibody.
- GFP (p85 ⁇ ) and CKAP4 were detected from the cell lysate (Input) and immunoprecipitate (IP) using anti-GFP (p85 ⁇ ) antibody and anti-CKAP4 antibody. The result is shown in the lower diagram of FIG. This result revealed that the SH3 domain of p85 ⁇ (PI3K) is involved in binding to CKAP4.
- the region where Dkk1 or CKAP4 is expressed is 5% or more and less than 20%, low, 20% or more and less than 50%, medium, 50% These cases were considered high. Expression of 5% or less was negative. From these results, it was confirmed that Dkk1 was expressed at a high frequency of 76.3% and CKAP4 at 66.1% in pancreatic cancer cases. Furthermore, (b) and (c) of FIG. 7 classify each specimen based on the presence or absence of expression of Dkk1 and CKAP4, and show the results of analyzing the relationship between the number of postoperative days and overall survival rate and recurrence-free survival rate. . From these results, it became clear that pancreatic cancer patients expressing both Dkk1 and CKAP4 have reduced overall survival and relapse-free survival after surgery.
- the area surrounded by a frame in the left diagram of FIG. 8A is an enlarged image of a tumor area and a non-tumor area.
- the scale bar in the left figure of FIG. 8A is 100 ⁇ m.
- the region where Dkk1 or CKAP4 is expressed is 5% or more and less than 20%, low, 20% or more and less than 50%, medium, 50% These cases were considered high. Expression of 5% or less was negative.
- FIG. 8 (b) shows the results of classifying each specimen based on the presence or absence of expression of Dkk1 and CKAP4 and analyzing the relationship between the number of postoperative days and the recurrence-free survival rate.
- the area surrounded by a frame in the left figure of FIG. 8C is an enlarged image of the tumor area and the non-tumor area.
- the scale bar in the left figure of FIG. 8 (c) is 100 ⁇ m.
- the region in which Dkk1 or CKAP4 is expressed is 5% or more and less than 20%, low, 20% or more and less than 50%, medium, 50% These cases were considered high.
- FIG. 8 (d) shows the results of classifying each specimen based on the presence or absence of expression of Dkk1 and CKAP4 and analyzing the relationship between the number of postoperative days and the recurrence-free survival rate.
- the region enclosed by a frame in the left diagram of FIG. 8 (e) is an enlarged image of a tumor region and a non-tumor region.
- the scale bar in the left figure of FIG. 8 (e) is 100 ⁇ m.
- the case where the region where Dkk1 or CKAP4 is expressed in AAH is 5% or more and less than 20% is low, 20% or more and less than 50% is middle, 50% or more The case was high.
- CKAP4 expression may be associated with the transition from precancerous lesions to lung cancer, considering that AAH is Dkk1-positive but CKAP4 negative, and that AAH is a precancerous lesion. It was.
- A549 cells were transduced with control shRNA, Dkk1 shRNA, Dkk1 shRNA and Dkk1-FLAG, CKAP4 shRNA, or CKAP4 shRNA and CKAP4-HA.
- a lysate of the obtained cells was prepared, and each protein was detected using antibodies against various proteins shown in the upper diagram of FIG. 10 (b).
- a cell proliferation assay was performed on the obtained cells. The obtained results are shown in the lower diagram of FIG. From this result, by suppressing the expression of Dkk1 or CKAP4 in A549 cells, the activity of AKT is reduced by inhibiting the activity of AKT and reducing the cell proliferation ability. Has been confirmed to be recoverable.
- Control shRNA, CKAP4 shRNA, or S2-CP8 cells (5 ⁇ 10 6 cells) expressing CKAP4 shRNA and CKAP4-HA were subcutaneously injected into the back of nude mice. Twenty-one days later, xenogeneic tumor tissue pieces at the transplant site of nude mice were analyzed. The obtained result is shown in FIG.
- the left figure of (a) of FIG. 11 shows the result of observing nude mice 21 days later from the top and the result of observing the excised tumor tissue pieces.
- the dotted line shows the outline of the heterogeneous tumor tissue piece, and the scale bar is 10 mm.
- the scale bar is 10 mm.
- the right figure shows the result of measuring the weight of the heterogeneous tumor tissue piece.
- the median is a thick line
- the box is in the 25 th to 75 th percentile range
- the error bar is in the 5 th to 95 th percentile range
- * is P ⁇ 0.05
- ** is P ⁇ . 0.01.
- Control shRNA, CKAP4 shRNA, or A549 cells (5 ⁇ 10 6 cells) expressing CKAP4 shRNA and CKAP4-HA were subcutaneously injected into the back of nude mice. Twenty-eight days later, xenogeneic tumor tissue pieces at the transplant site of nude mice were analyzed. The obtained result is shown in FIG.
- the left figure of (b) of FIG. 11 shows the result of observing nude mice after 28 days from the top and the result of observing excised tumor tissue pieces.
- the dotted line shows the outline of the heterogeneous tumor tissue piece, and the scale bar is 10 mm.
- the scale bar is 10 mm.
- the right figure shows the result of measuring the weight of the heterogeneous tumor tissue piece.
- the median is a bold line
- the box is in the 25 th to 75 th percentile range
- the error bar is in the 5 th to 95 th percentile range
- * is P ⁇ 0.05
- ** is P ⁇ 0.01.
- S2-CP8 cells (5 ⁇ 10 6 cells) expressing control shRNA or Dkk1 shRNA were subcutaneously injected into the back of nude mice. Twenty-one days later, xenogeneic tumor tissue pieces at the transplant site of nude mice were analyzed. The obtained results are shown in FIG.
- the left figure of FIG. 11 (c) shows the result of observing nude mice 21 days later from the top and the result of observing excised tumor tissue pieces.
- the dotted line shows the outline of the heterogeneous tumor tissue piece, and the scale bar is 10 mm.
- the right figure shows the result of measuring the weight of the heterogeneous tumor tissue piece.
- the median is a thick line
- the box is in the 25 th to 75 th percentile range
- the error bar is in the 5 th to 95 th percentile range
- * is P ⁇ 0.05
- ** is P ⁇ . 0.01. From this result, it was confirmed that the ability to form tumors by S2-CP8 cells can be reduced by suppressing the expression of Dkk1.
- A549 cells (5 ⁇ 10 6 cells) expressing control shRNA or Dkk1 shRNA were subcutaneously injected into the back of nude mice. Twenty-eight days later, xenogeneic tumor tissue pieces at the transplant site of nude mice were analyzed. The obtained result is shown in FIG.
- the left figure of FIG. 11 (d) shows the result of observing nude mice 28 days later from the top and the result of observing the excised tumor tissue pieces.
- the dotted line shows the outline of the heterogeneous tumor tissue piece, and the scale bar is 10 mm.
- the right figure shows the result of measuring the weight of the heterogeneous tumor tissue piece.
- the median is a bold line
- the box is in the 25 th to 75 th percentile range
- the error bar is in the 5 th to 95 th percentile range
- * is P ⁇ 0.05
- ** is P ⁇ . 0.01. From this result, it was confirmed that the ability to form tumors by A549 cells can be reduced by suppressing the expression of Dkk1.
- FIG. 12 (a) shows the antigenic site of polyclonal anti-CKAP4 antibody (CKAP4 pAb) Indicates.
- a lysate of S2-CP8 cells (control) or S2-CP8 cells expressing CKAP4 cDNA or CKAP4 shRNA was prepared, and CKAP4 was detected using an anti-CKAP4 antibody in the lysate.
- the obtained results are shown in the right figure of FIG. From this result, the expression level of CKAP4 increased in S2-CP8 cells expressing CKAP4 cDNA compared to the control, and the expression level of CKAP4 could be suppressed in S2-CP8 cells expressing CKAP4 ⁇ ⁇ shRNA compared to the control. It was confirmed that Therefore, it was shown that the prepared CKAP4 antibody was acting correctly.
- S2-CP8 cells grown on a glass-coated slide glass were unfixed and stained with an anti-CKAP4 antibody (green).
- the actin cytoskeleton and nucleus were stained with phalloidin (red) and DRAQ5ADNA Dye (blue).
- the obtained result is shown in FIG. From this result, it was confirmed that CKAP4 was strongly expressed in the cell membrane of S2-CP8 cells.
- MDCK cells were treated with 1 nM nocodazole for 24 hours and then incubated with 25 ⁇ g / ml anti-CKAP4 antibody or anti-GST antibody for 4 hours. Cells were then stimulated with 250 ng / ml Dkk1 or 1 nM insulin for 30 minutes in the presence of anti-CKAP4 antibody or anti-GST antibody. Thereafter, the cells were collected to prepare a cell lysate, and pAKT, AKT and clathrin were detected from the lysate using anti-pAKT antibody, anti-AKT antibody and anti-clathrin antibody. Anti-GST antibody was used as a control and clathrin was used as a loading control. The obtained result is shown in FIG. From these results, it was confirmed that the anti-CKAP4 antibody suppresses the activation of Dkk1-dependent AKT, but does not affect the activity of insulin-dependent AKT.
- S2-CP8 cells or A549 cells were incubated with 5 ⁇ g / ml anti-CKAP4 antibody or anti-GST antibody for 4 hours. Thereafter, the cells were collected to prepare a cell lysate, and pAKT, AKT and clathrin were detected from the lysate using anti-pAKT antibody, anti-AKT antibody and anti-clathrin antibody.
- Anti-GST antibody was used as a control and clathrin was used as a loading control. The obtained result is shown in FIG. From this result, it was confirmed that the anti-CKAP4 antibody suppresses the activation of AKT in cancer cells.
- S2-CP8 cells (2 ⁇ 10 4 cells) or A549 cells (2 ⁇ 10 4 cells) are three-dimensionally on Matrigel.
- the cells were cultured for 5 days, and the number of cell aggregates per field was counted (5 fields).
- Anti-GST antibody was used as a control.
- the obtained result is shown in FIG. ** is P ⁇ 0.01.
- S2-CP8 cells (2 ⁇ 10 4 cells) are three-dimensionally displayed on Matrigel. After culturing for 7 days, the number of cell aggregates per 1 mm 2 was counted. The obtained result is shown in FIG.
- the scale bar in the upper diagram of FIG. 12 (g) is 200 ⁇ m.
- the average value is a bold line, the box is in the 25 th to 75 th percentile range, the error bar is in the 5 th to 95 th percentile range, and ** is P ⁇ 0.01.
- Surgical specimens or pleural effusions were provided by two cancer patients for whom consent was obtained at the Osaka Medical Center for Adult Diseases. Disperse two specimens (LC189 and LB95) mechanically and enzymatically, collect 40-250 ⁇ m tissue pieces, and culture in suspension on StemPro hESC (Invitrogen, Carlsbad, CA), cancer tissue-originated spheroid (CTOS) Got.
- CTOS cancer tissue-originated spheroid
- the obtained CTOS was transplanted to a NOD / scid mouse and reared to form a transplanted tumor at the transplantation site of the mouse. Thereafter, sections prepared from the transplanted tumor were immunostained with anti-Dkk1 antibody or anti-CKAP4 antibody and hematoxylin.
- the obtained result is shown in FIG. As a result, it was confirmed that the expression level of Dkk1 and CKAP4 was high in the transplanted tumor of specimen LC189, and the expression of Dkk1 and
- CTOS was isolated and cryopreserved again from the transplanted tumor obtained above and then thawed, and then subjected to the following proliferation assay. Specifically, CTOS was embedded in matrigel® Growth® Factor® Reduced® (GFR) ® (BD® Biosciences, Bedford, MA) and 7 ° C. in StemPro® hESC medium containing anti-CKAP4 antibody or anti-GST antibody at each concentration. Cultured for days. During the culture period, the area of CTOS was measured with Image J.
- GFR matrigel® Growth® Factor® Reduced®
- StemPro® hESC medium containing anti-CKAP4 antibody or anti-GST antibody at each concentration. Cultured for days. During the culture period, the area of CTOS was measured with Image J.
- the middle figure of (a) and (b) of FIG. 13 shows the result of measuring the volume of the heterogeneous tumor tissue piece, and the right figure of FIGS. 13 (a) and (b).
- the results are shown as mean values ⁇ sd. * Is P ⁇ 0.05, ** is P ⁇ 0.01. From these results, it was confirmed that administration of anti-CKAP4 antibody can reduce the tumor-forming ability of pancreatic cancer cells (S2-CP8) and lung cancer cells (A549).
- the exosome pellet was washed with 1.2 ml of PBS and again subjected to ultracentrifugation under conditions of 4 ° C., 100,000 ⁇ g, 2 hours. Thereafter, the supernatant was discarded to recover the exosome, and the exosome was dissolved in the Laemmli sample buffer.
- CKAP4, TSG101, and clathrin were detected using the anti-CKAP4 antibody, the anti-TSG101 antibody, and the anti-clathrin antibody with respect to the exosome and each cell lysate.
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Abstract
Description
項1. CKAP4の発現又は機能を抑制する物質を有効成分とすることを特徴とする、抗腫瘍剤。
項2. 前記物質が、CKAP4に対するsiRNA、shRNA、dsRNA、miRNA、及びアンチセンス核酸よりなる群から選択される少なくとも1種の核酸医薬である、項1に記載の抗腫瘍剤。
項3. 前記物質がCKAP4特異的に結合する抗体又はその断片である、項1に記載の抗腫瘍剤。
項4. 癌の治療、予防又は進行防止の用途に使用される、項1~3のいずれかに記載の抗腫瘍剤。
項5. 癌がCKAP4を発現している癌である、項4に記載の抗腫瘍剤。
項6. 癌が更にDkk1を発現している癌である、項5に記載の抗腫瘍剤。
項7. 癌が肺癌又は膵癌である、項4~6のいずれかに記載の抗腫瘍剤。
項8. CKAP4の発現又は機能を抑制する物質の、抗腫瘍剤の製造のための使用。
項9. 癌の処置に使用される、CKAP4の発現又は機能を抑制する物質。
項10. 癌患者に、治療有効量のCKAP4の発現又は機能を抑制する物質を投与する工程を含む、癌の治療方法。
項11. 抗腫瘍剤の有効成分をスクリーニングする方法であって、
被験物質について、CKAP4の発現抑制能、又は細胞膜上のCKAP4との結合能を測定する第1工程、及び
CKAP4の発現抑制能、又は細胞膜上のCKAP4との結合能が認められた被験物質を抗腫瘍剤の有効成分の候補として選択する第2工程、
を含むスクリーニング方法。
項12. 前記第1工程として、
被験物質を、CKAP4を発現する細胞に接触させる第1-a工程、及び
第1-a工程後に、前記細胞におけるCKAP4発現量を測定する第1-b工程
を含む、項11に記載のスクリーニング方法。
項13. 前記第1工程として、
被験物質を、CKAP4を発現する細胞に接触させる第1-i工程、及び
第1-i工程後に、被験物質が細胞膜上のCKAP4に結合しているか否かを確認する第1-ii工程
を含む、項11に記載のスクリーニング方法。
項14. 癌患者から採取した癌組織におけるCKAP4の発現を測定する工程を含む、癌患者の術後の予後の検査方法。
項15. 更に、前記癌組織におけるDkk1の発現を測定する、項14に記載の検査方法。
項16. 前記癌患者が肺癌患者又は膵癌患者である、項14又は15に記載の検査方法。
項17. CKAP4を検出する試薬を含む、癌患者の術後の予後の検査薬。
項18. 更にDkk1を検出するための試薬を含む、項17に記載の検査薬。
項19. 癌患者が肺癌患者又は膵癌患者である、項17又は18に記載の検査薬。
項20. CKAP4を検出するための試薬の、癌患者の術後の予後の検査薬の製造のための使用。
項21. 癌患者の術後の予後の検査に使用される、CKAP4を検出するための試薬。
項22. 被験者から採取されたエクソソーム中のCKAP4を測定する工程を含む、癌の検査方法。
項23. CKAP4を検出する試薬を含む、癌の検査薬。
項24. CKAP4を検出する試薬の、CKAP4を検出する試薬の製造のための使用。
項25. 癌の検査に使用される、CKAP4を検出する試薬。
本発明の抗腫瘍剤は、CKAP4の発現又は機能を抑制する物質を有効成分とすることを特徴とする。以下、本発明の抗腫瘍剤について詳述する。
本発明の抗腫瘍剤では、有効成分として、CKAP4の発現又は機能を抑制する物質を使用する。CKAP4は、Dkk1の受容体として腫瘍細胞の増殖調節機構に関与しており、腫瘍細胞の増殖を亢進させる作用がある。本発明の抗腫瘍剤では、CKAP4の発現又は機能を抑制することによって、腫瘍細胞の増殖を効果的に抑制させることが可能になる。
CKAP4は、腫瘍細胞において発現し、腫瘍細胞の増殖を亢進させているので、本発明の抗腫瘍剤では、CKAP4の発現又は機能を抑制することにより、腫瘍細胞の増殖抑制が可能になっている。従って、本発明の抗腫瘍剤は、癌の治療の用途に使用できる。本発明の抗腫瘍剤の適用対象となる癌としては、特に制限されないが、具体的には、肺癌、膵癌、大腸癌、結腸癌、胃癌、直腸癌、肝癌、乳癌、膀胱癌、前立腺癌、子宮頚癌、頭頚部癌、胆管癌、胆嚢癌、口腔癌、舌癌、咽頭癌、喉頭癌、脳腫瘍、神経膠腫(グリオーマ)、膠芽腫、多型性神経膠芽腫等の固形癌;白血病、悪性リンパ腫等の血液癌が挙げられる。
本発明の抗腫瘍剤の投与形態については、抗腫瘍効果が得られることを限度として、経口投与又は非経口投与のいずれであってもよく、使用する有効成分の種類に応じて適宜設定すればよい。本発明の抗腫瘍剤の投与形態として、具体的には、経口投与;注射投与(静脈注射、皮下注射、筋肉注射、腹腔注射、患部への局所注射等)、座薬投与等の非経口投与が挙げられる。
本発明の抗腫瘍剤は、有効成分の種類や投与形態に応じた製剤形態に調製される。本発明の抗腫瘍剤の製剤形態としては、例えば、錠剤、カプセル剤、丸剤、散剤、顆粒剤、坐剤等の固形製剤;液剤、懸濁剤、乳剤、注射剤等の液状製剤等が挙げられる。
本発明のスクリーニング方法は、抗腫瘍剤の有効成分をスクリーニングする方法であって、被験物質について、CKAP4の発現抑制能、又は細胞膜上のCKAP4との結合能を検出する第1工程、及びCKAP4の発現抑制能、又は細胞膜上のCKAP4との結合能が認められた被験物質を抗腫瘍剤の有効成分の候補として選択する第2工程を含むことを特徴とする。以下、本発明のスクリーニング方法について詳述する。
本発明のスクリーニング方法において、被験物質は、抗腫瘍剤の有効成分の候補物質となり得るものであればよく、その種類については、特に制限されず、例えば、生体由来物質等の天然物質であってもよく、また化学的に合成された合成物質であってもよい。具体的には、被験物質として、低分子化合物;抗体等のタンパク質;siRNA、shRNA、dsRNA、miRNA、アンチセンスDNA、アンチセンスRNA、アプタマー等の核酸分子等が挙げられる。
本発明のスクリーニング方法では、先ず、第1工程として、CKAP4の発現抑制能、又は細胞膜上のCKAP4との結合能を検出する。
第1-a工程:被験物質を、CKAP4を発現する細胞に接触させる工程。
第1-b工程:第1-a工程後に、前記細胞におけるCKAP4発現量を測定する工程。
第1-i工程:被験物質を、CKAP4を発現する細胞に接触させる工程。
第1-ii工程:第1-i工程後に、被験物質が細胞膜上のCKAP4に結合しているか否かを確認する工程。
第2工程では、前記第1工程において、CKAP4の発現抑制能、又は細胞膜上のCKAP4との結合能が認められた被験物質を抗腫瘍剤の有効成分の候補として選択する。
本発明の癌患者の術後の予後の検査方法は、癌患者から採取した癌組織におけるCKAP4およびDkk1の発現を測定する工程を含むことを特徴とする。本発明の検査方法によれば、癌患者から採取した癌組織におけるCKAP4およびDkk1双方の発現を指標とすることにより、癌患者の術後の予後を推定することが可能になる。
本発明の癌の検査方法は、被験者から採取されたエクソソーム中のCKAP4を測定する工程を含むことを特徴とする。本発明によれば、被験者から採取されたエクソソーム中のCKAP4の有無を指標とすることにより、被験者が癌に罹患しているか否かを診断することが可能になる。
1-1.Dkk1と結合するタンパク質の分離
FLAGエピトープ及びグリコシルフォスファチジルイノシトール(GPI)アンカーシグナル配列をC末端側に直列に挿入したDkk1遺伝子(Dkk1-FLAG-GPI)を有するレンチウイルスベクターを構築した。
Dkk1を発現するMDCK細胞(2×105 cells)をTranswell polycarbonate filter(Corning Costar Quality Biological, Gaithersburg, MD, USA)に播種した。48時間後に培地を交換し、更に24時間培養を行った。培養液中に分泌されたDkk1を検出するために、頂端側と基底側から得られた培養液にBlue Sepharoseを加えて、4℃で2時間インキュベートした後に、遠心分離を行った。次いで、沈殿物を回収し、抗Dkk1抗体を用いてDkk1の検出を行った。
腎尿細管細胞MDCK type I(MDCK I)はDr. S. Tsukita(大阪大学)から供与されたもの、ヒト肺腺癌細胞A549及びNCI-H1579はDr. Y. Shintani(大阪大学)から供与されたもの、ヒト子宮頸癌細胞HeLaS3はDr. K. Matsumoto (名古屋大学) から供与されたもの、ヒト胃癌細胞AGS はDr. M. Hatakeyama (東京大学) から供与されたもの、 KKLSは Dr. W. Yasui (広島大学) から供与されたもの、ヒト肝芽腫細胞HepG2 は Dr. Y. Matsuura (大阪大学) から供与されたもの、ヒト肺腺癌細胞A549、Calu-6とヒト食道扁平上皮癌KYSE-70およびTE-11 は塩野義製薬株式会社から供与されたものを使用した。膵癌細胞S2-CP8及びSUIT-2は東北大学加齢医学研究所医用細胞資源センターから購入した。X293T細胞はタカラバイオ株式会社から購入した。
MDCK I細胞、S2-CP8細胞、及びA549細胞を、1×105cell/mlとなるように35mmディッシュに播種した。MDCK I細胞については、10%FBSを含むDMEMで培養を行った。S2-CP8細胞及びA549細胞については、1%FBSを含むDMEMで培養を行った。所定日数経過後に細胞数を計測した。
3次元培養による試験は、従来報告されている内容((Matsumoto et al., EMBO J, 2014)に従って実施した。マトリゲル上(BD Biosciences, San Jose, CA, USA)でのMDCK I細胞の嚢胞形成、S2-CP8細胞及びA549細胞の増殖を分析するために、先ず、40μlのマトリゲルを円形のスライドグラスに載せ、37℃で30分間インキュベートしてゲルを固化させた。次いで、10%FBS及び2%マトリゲルを含むDMEMに懸濁したMDCK I細胞(3×104 cells)、及び0.1%ウシ血清アルブミン及び2%マトリゲルを含むDMEMに懸濁したS2-CP8細胞又はA549細胞(2×104 cells)を、固化させたマトリゲルの上に添加し、培養を行った。
腫瘍移植片分析は、従来報告されている内容(Fujii et al., Oncogene, 2014)を改変した手法を用いて行った。6週齢の雄BALB/cAnNCrj-nuヌードマウス(Charles River Laboratory Japan Inc, Osaka, Japan)をメデトミジン(0.3 mg/kg体重)及びミダゾラム(4 mg/kg体重)で麻酔を行った後に、100 μlのリン酸緩衝液(PBS)に懸濁したS2-CP8細胞(5×106 cells)又はA549細胞(5×106 cells)を皮下注入した。次いで、移植21日後(S2-CP8細胞)及び移植28日後(A549細胞)のヌードマウスを分析に供した。
本試験において、ノックダウンの標的としたタンパク質の種類とその標的配列を表2に示す。RNAiMAX(Invitrogen, Carlsbad, CA, USA)を用いて、標的タンパク質に対するsiRNA(各々20 nM)の混合物を細胞に形質導入した。形質導入から36~48時間後の細胞を用いて試験を行った。
pcDNA3mycTEVflagにおけるFLAG-p110α、FLAG-p110β及びpCAGGSにおけるp85αは、Dr. T. Asano(広島大学)から供与されたものを使用した。A549 cDNAライブラリーからCKAP4 cDNAを増幅し、全ての変異体は一般的な手法で作成した。Dr. H. Miyoshi(理化学研究所)から供与されたCSII-CMV-MCS-IRES2-BsdにcDNAを挿入することにより、レンチウイルスベクターを構築した。Gateway technology (Invitrogen)を用いて、H1プロモーターとshRNAを含むオリゴDNAフラグメントをCS-RfA-EVBsd中にクローン化し、shRNAを含むレンチウイルスベクターを構築した。本試験で使用したshRNAの標的タンパク質とその標的配列を表3に示す。
FuGENE HD トランスフェクション試薬(Roche Applied Science, Basel, Switzerland)を用いて、パッケージングベクター(pCAG-HIV-gp及びpCMV-VSV-G-RSV-Rev)及びレンチウイルスベクターをX293T細胞にトランスフェクトし、レンチウイルスを増幅させた。また、条件培地及び10 μg/mLのポリブレンの存在下で各レンチウイルスを親細胞(5×104 cells/well in a 12-well plate)に導入した。細胞を1200×gで1時間遠心分離して回収し、更に24時間インキュベートすることにより、Dkk1-Flag、CKAP4-HA、Dkk1 shRNA、又はCKAP4 shRNAを安定に発現するMDCK I細胞、S2-CP8細胞、及びA549細胞を作製した。
細胞をスライドガラス上に播種し、4%パラホルムアルデヒドを用いて室温で20分間固定した。次いで、細胞をPBSで3回洗浄した後、1% BSA及び0.05% Tween-20を含むPBSで20分間ブロッキングを行った。次いで、一次抗体を添加して、終夜インキュベートした。PBSで細胞を3回洗浄した後に、蛍光標識二次抗体を添加して室温で1時間インキュベートした。その後、PBSでスライドガラスを十分に洗浄し、50%グリセロールを含むPBSをにより封入した。本試験の操作及び測定は、LSM510 system (Carl Zeiss Microscopy Co., Ltd, Jena, Germany)を用いて行った。
大阪大学附属病院にて2001年4月~2015年4月に外科的処置を受けた膵癌患者(59名)、肺腺癌患者(67名)、肺扁平上皮癌患者(61名)、及び肺異型腺腫様過形成患者(11名)から切除された腫瘍組織片(標本)を用いて行った。何れも腫瘍病変に対して術前に化学療法及び放射線療法を施行されていない患者のみを対象とした。膵癌患者の年齢は47~83歳であり、平均年齢は70歳であった。肺腺癌患者の年齢は39~85歳であり、平均年齢は68歳であった。肺扁平上皮癌患者の年齢は38~82歳であり、平均年齢は71歳であった。肺異型腺腫様過形成患者の年齢は58~78歳であり、平均年齢は67歳であった。
免疫組織化学的分析は、従来報告されている内容(Fujii et al., Oncogene, 2014)を改変した手法を用いて行った。具体的手法の概要は以下の通りである。先ず、DakoRealTMEnVisionTM Detection System (Dako, Carpentaria, CA, USA)を用いて、その製造元の推奨する方法に従って、全ての組織切片を染色した。具体的には、先ず、Pascalプレッシャーチャンバー(Dako社製)を用いて抗原賦活化を行い、Dako REAL Peroxidase-Blocking Solutionを5分間反応させて非特異的結合サイトをブロックした。次いで、組織切片に対して、抗Dkk1抗体(1:100)、抗CKAP4抗体(1:100)、又はpAKT抗体(1:50)を用いて、4℃で16時間処理した。その後、組織切片に、ホースラディッシュペルオキシダーゼ(HRP)標識のヤギ抗マウスIgGを添加して1時間インキュベートした後に、ジアミノベンジジン(DAB)(Dako)を添加した。次いで、組織切片に0.1% (w/v)のヘマトキシリンで対比染色した。5%を超える領域が染色されていた腫瘍を、Dkk1陽性又はCKAP4陽性と分類した。
統計分析は、JMP software(SAS Institute. Inc., Cary NC, USA)を用いて行った。結果の差は、術後無再発生存期間および全生存期間の検討は、Kaplan-Meier 法にてlog-rank testを用いた。Dkk1、CKAP4、及びDkk1とCKAP4の発現との相関については、各検定間の相関をモンテカルロシミュレーションによって計算し、多重補正係数を検定間の相関を考慮に入れながら計算し、元のP値を多重補正係数でかけるという方法で多重性に対応した。その他の検定は、スチューデントのt検定、もしくはマン・ホイットニーのU検定を用いて、統計学的優位性を検定した。P値が0.05未満の場合には、統計学的に有意差があるとみなした。
2-1.Dkk1が細胞の増殖能に与える影響
Dkk1遺伝子(Dkk1-FLAG)を発現したMDCK細胞において、C末端側がFLAGタグ化されたDkk1が発現していることを確認した結果を図1の(a)の上図に示す。図1の(a)に示すコントロールは、親細胞であるMDCK細胞を使用した結果である。また、HSP90は、ローディングコントロールとして使用した。図1の(a)の下図には、Transwell polycarbonate filter上で培養したMDCK/Dkk1-FLAG細胞を培養し、頂底極性を確立した後に、頂端側と側底側に分けてDkk1の発現を分析した結果である。図1の(a)の下図において、Apは頂端側、Blは基底側を指している。この結果から、Dkk1遺伝子(Dkk1-FLAG)が形質導入されたMDCK細胞(MDCK/Dkk1-FLAG細胞)において、C末端側がFLAGタグ化されたDkk1が安定的に発現し、極性化したMDCK細胞において頂上側有意にDkk1-FLAGが分泌されることが確認された。
頂端側表面にあるDkk1結合タンパク質をスクリーニングした実験手順の概略図を図2の(a)に示す。Dkk1結合タンパク質を精製し、銀染色を行った結果を図2の(b)に示す。Dkk1-FLAG-GPIに結合するタンパク質として溶出された17個のバンドについて、質量分析を行ったところ、図2の(b)の右側の表に示すタンパク質が検出され、その内、CKAP4はバンド10として検出された。
MDCK細胞(WT)、MDCK/Dkk1-FLAG細胞(Dkk1-FLAG)、又はMDCK/Dkk1-GPI1-FLAG細胞(Dkk1-FLAG-GPI)の溶解液に対して、図4の(a)に示す各種タンパク質に対する抗体を反応させた。その結果、図4の(a)に示すように、MDCK細胞において、Dkk1を過剰発現することによりAKTのリン酸化が促進したが、ErK1/2、JNK、Srcのリン酸化の亢進は認められなかった。すなわち、Dkk1がAKTを活性化することが示唆された。
肺癌細胞(A549、Calu-6、及びNCl-H1579)、膵癌細胞(SUIT-2及びS2-CP8)、子宮頸管癌細胞(HeLaS3)、胃癌細胞(AGS及びKKLS)、食道扁平上皮癌細胞(KYSE-70及びTE-11)、肝芽腫細胞(HepG2)、及び胎児由来腎臓細胞(X239T)の溶解液に、抗Dkk1抗体、抗CKAP4抗体及び抗HSP90抗体を反応させ、Dkk1、CKAP4、及びHSP90(コントロール)量を測定した。得られた結果を図6に示す。この結果から、Dkk1は正常細胞では発現しておらず、癌細胞の一部で発現している一方、CKAP4はいずれの細胞においても発現していることが確認された。
膵癌患者から摘出した膵癌組織(n=59)に対して抗Dkk1抗体又は抗CKAP4抗体とヘマトキシリンで免疫染色した結果を図7の(a)の左図に示す。図7の(a)の左上図において枠で囲んだ領域は腫瘍領域の拡大像、右上図において枠で囲んだ領域は非腫瘍領域の拡大像である。図7の(a)の左図中のスケールバーは、50 μmである。また、図7の(a)の右図において、腫瘍領域においてDkk1又はCKAP4が発現している領域が5%以上20%未満の症例をlow、20%以上50%未満の症例をmedium、50%以上の症例をhighとした。5%以下の発現はnegativeとした。この結果から、膵癌症例においてDkk1が76.3%、CKAP4が66.1%という高頻度で発現していることが確認された。更に、図7の(b)および(c)に、各検体をDkk1とCKAP4の発現の有無に基づいて分類し、術後日数と全生存率および無再発生存率の関係を分析した結果を示す。これらの結果から、Dkk1とCKAP4の双方を発現している膵癌患者では、術後全生存期間および無再発生存期間の短縮が認められることが明らかとなった。
ヒト膵癌、ヒト肺腺癌、及びヒト肺扁平上皮癌の各癌組織に対して抗Dkk1抗体、抗CKAP4抗体、又は抗pAKT抗体とヘマトキシリンで免疫染色した。得られた結果を図9に示す。図9中、黒色のボックスは拡大像を示し、バーのスケールは200μmである。この結果から、試験に供したヒト膵癌、ヒト肺腺癌、及びヒト肺扁平上皮癌において、Dkk1及びCKAP4を発現している腫瘍領域では、AKTが活性化していることが確認された。
S2-CP8細胞に、コントロールshRNA、Dkk1 shRNA、Dkk1shRNA及びDkk1-FLAG、CKAP4 shRNA、又はCKAP4shRNA及びCKAP4-HAを形質導入した。得られた細胞の溶解液を作製し、当該溶解液に対して、図10の(a)の上図に示す各種タンパク質に対する抗体を用いて、当該各タンパク質の検出を行った。また、得られた細胞について、細胞増殖アッセイを行った。得られた結果を図10の(a)の下図に示す。この結果から、S2-CP8細胞においてDkk1又はCKAP4の発現を抑制することにより、AKTの活性が阻害され細胞増殖能を低減させ、各々に対してDkk1又はCKAP4の過剰発現することによって、AKTの活性と細胞増殖が回復し得ることが確認された。
図12の(a)の左図に、ポリクローナル抗CKAP4抗体(CKAP4 pAb)の抗原部位を示す。
膵癌細胞(SUIT-2及びS2-CP8)、肺癌細胞(A549及びCalu-6)、胎児由来正常腎臓細胞 (X293T)、及び犬尿細管由来正常細胞(MDCK)を10cmディッシュで80~90%コンフルエントな状態になるまで培養した後に、培養上清を回収した。培養上清を2,000×gで10分間の条件にて遠心分離した後に、更に10,000×gで10分間の条件で遠心分離を行い、細胞破砕物を分離した。回収した上清を4℃、100,000×g、3時間の条件で超遠心分離し、エクソソームペレットを得た。エクソソームペレットを1.2 mlのPBSで洗浄した後に、再度、4℃、100,000×g、2時間の条件で超遠心分離に供した。その後、上清を廃棄してエクソソームを回収し、エクソソームをLaemmliサンプルバッファーに溶解させた。エクソソームと各細胞の溶解液に対して、抗CKAP4抗体、抗TSG101抗体、及び抗クラスリン抗体を用いて、CKAP4、TSG101及びクラスリンの検出を行った。
Claims (15)
- CKAP4の発現又は機能を抑制する物質を有効成分とすることを特徴とする、抗腫瘍剤。
- 前記物質が、CKAP4に対するsiRNA、shRNA、dsRNA、miRNA、及びアンチセンス核酸よりなる群から選択される少なくとも1種の核酸医薬である、請求項1に記載の抗腫瘍剤。
- 前記物質がCKAP4特異的に結合する抗体又はその断片である、請求項1に記載の抗腫瘍剤。
- 癌の治療、予防又は進行防止の用途に使用される、請求項1~3のいずれかに記載の抗腫瘍剤。
- 癌がCKAP4を発現している癌である、請求項4に記載の抗腫瘍剤。
- 癌が更にDkk1を発現している癌である、請求項5に記載の抗腫瘍剤。
- 癌が肺癌又は膵癌である、請求項4~6のいずれかに記載の抗腫瘍剤。
- 抗腫瘍剤の有効成分をスクリーニングする方法であって、
被験物質について、CKAP4の発現抑制能、又は細胞膜上のCKAP4との結合能を測定する第1工程、及び
CKAP4の発現抑制能、又は細胞膜上のCKAP4との結合能が認められた被験物質を抗腫瘍剤の有効成分の候補として選択する第2工程、
を含むスクリーニング方法。 - 前記第1工程として、
被験物質を、CKAP4を発現する細胞に接触させる第1-a工程、及び
第1-a工程後に、前記細胞におけるCKAP4発現量を測定する第1-b工程
を含む、請求項8に記載のスクリーニング方法。 - 前記第1工程として、
被験物質を、CKAP4を発現する細胞に接触させる第1-i工程、及び
第1-i工程後に、被験物質が細胞膜上のCKAP4に結合しているか否かを確認する第1-ii工程
を含む、請求項8に記載のスクリーニング方法。 - 癌患者から採取した癌組織におけるCKAP4の発現を測定する工程を含む、癌患者の術後の予後の検査方法。
- 更に、前記癌組織におけるDkk1の発現を測定する、請求項11に記載の検査方法。
- CKAP4を検出する試薬を含む、癌患者の術後の予後の検査薬。
- 被験者から採取されたエクソソーム中のCKAP4を測定する工程を含む、癌の検査方法。
- CKAP4を検出する試薬を含む、癌の検査薬。
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CN201680012273.5A CN107249636B (zh) | 2015-02-27 | 2016-01-28 | 以ckap4作为靶分子的抗肿瘤剂 |
US15/553,941 US10618954B2 (en) | 2015-02-27 | 2016-01-28 | CKAP4-molecular-targeted antitumor agent |
CA2977917A CA2977917C (en) | 2015-02-27 | 2016-01-28 | Ckap4-molecular-targeted antitumor agent |
EP16755122.5A EP3287143A4 (en) | 2015-02-27 | 2016-01-28 | Ckap4-molecular-targeted antitumor agent |
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WO2019053274A1 (en) * | 2017-09-15 | 2019-03-21 | Jan Lotvall | METHOD AND SYSTEM FOR IDENTIFICATION OF MEMBRANE PROTEINS ON EXTRACELLULAR VESICLES |
WO2019065747A1 (ja) | 2017-09-26 | 2019-04-04 | 国立大学法人大阪大学 | 抗ckap4モノクローナル抗体 |
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CN110007071A (zh) * | 2019-04-01 | 2019-07-12 | 河南省生物工程技术研究中心有限公司 | 一种ckap4标记抗体-荧光微球复合物的制备方法及其应用 |
CN114720700A (zh) * | 2022-05-07 | 2022-07-08 | 浙江大学 | 检测抗细胞骨架相关蛋白4-IgG自身抗体的试剂在制备检测血管内皮损伤试剂盒的应用 |
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WO2019065747A1 (ja) | 2017-09-26 | 2019-04-04 | 国立大学法人大阪大学 | 抗ckap4モノクローナル抗体 |
CN111133108A (zh) * | 2017-09-26 | 2020-05-08 | 国立大学法人大阪大学 | 抗ckap4单克隆抗体 |
JPWO2019065747A1 (ja) * | 2017-09-26 | 2021-01-28 | 国立大学法人大阪大学 | 抗ckap4モノクローナル抗体 |
EP3690051A4 (en) * | 2017-09-26 | 2021-05-26 | Osaka University | ANTI-CKAP4 MONOCLONAL ANTIBODY |
US11807679B2 (en) | 2017-09-26 | 2023-11-07 | Osaka University | Anti-CKAP4 monoclonal antibody |
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CN107249636B (zh) | 2023-07-28 |
EP3287143A4 (en) | 2018-09-05 |
US20180037649A1 (en) | 2018-02-08 |
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