WO2019146759A1 - 受容体タンパク質を標的とする治療薬、検査薬、受容体タンパク質に結合する抗体、及び分子標的薬のスクリーニング方法 - Google Patents

受容体タンパク質を標的とする治療薬、検査薬、受容体タンパク質に結合する抗体、及び分子標的薬のスクリーニング方法 Download PDF

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WO2019146759A1
WO2019146759A1 PCT/JP2019/002504 JP2019002504W WO2019146759A1 WO 2019146759 A1 WO2019146759 A1 WO 2019146759A1 JP 2019002504 W JP2019002504 W JP 2019002504W WO 2019146759 A1 WO2019146759 A1 WO 2019146759A1
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nptxr
chrnb2
antibody
expression
cancer
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English (en)
French (fr)
Japanese (ja)
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光郎 神田
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Nagoya University NUC
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Nagoya University NUC
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Priority to US16/964,236 priority Critical patent/US11773169B2/en
Priority to JP2019567188A priority patent/JP7312451B2/ja
Priority to EP22196119.6A priority patent/EP4169532A1/en
Priority to CN202211167212.XA priority patent/CN116059349A/zh
Priority to EP19744055.5A priority patent/EP3744346A4/en
Priority to CN201980022443.1A priority patent/CN112739380A/zh
Application filed by Nagoya University NUC filed Critical Nagoya University NUC
Publication of WO2019146759A1 publication Critical patent/WO2019146759A1/ja
Anticipated expiration legal-status Critical
Priority to JP2022134818A priority patent/JP7621664B2/ja
Priority to US17/932,450 priority patent/US12129301B2/en
Priority to US18/364,644 priority patent/US12098205B2/en
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Definitions

  • the present invention relates to therapeutic agents that target receptor proteins, and test agents.
  • the present invention relates to a target drug that targets a receptor that is highly expressed on the surface of a cancer cell and a signal transduction system that carries a signal downstream of the receptor, in particular, an antibody drug against a receptor protein, a nucleic acid drug.
  • the present invention relates to a companion diagnostic agent when administering the above-mentioned molecule-targeted drug, or a test agent for predicting prognosis such as relapse.
  • the present invention relates to a method for screening a drug targeted for a receptor protein.
  • Gastric cancer is the fourth most affected and the second most cancer-related death worldwide in the world (2012 statistics). Gastric cancer is common in Asia and South America such as Japan, China, Korea, etc. According to the statistics of cancer patients in Japan, the number of newly acquired gastric cancer is 133,000 (third place) and the number of deaths is 49,400 ( 3) (2015 statistics), it is an important disease to be overcome with high prevalence and priority.
  • HER2-positive gastric cancer is about 20% or so of advanced and recurrent gastric cancer
  • trastuzumab is effective only for HER2-positive gastric cancer.
  • the response rate of nivolumab, a newly approved immune checkpoint inhibitor remains at about 20%, creating a therapeutic limit.
  • ramucirumab which has been approved for the treatment of advanced gastric cancer in the United States, is not very effective in prolonging the prognosis. Therefore, the creation of inhibitors that can control the development of gastric cancer from a completely different mechanism is essential for improving treatment results.
  • An object of the present invention is to find a molecule that can be a target by identifying a molecule involved in gastric cancer development, and to provide a targeted therapeutic agent.
  • cancer cells in which the target molecule is expressed are considered to exert effects by the same action mechanism, it is expected to have effects not only for gastric cancer but also for other cancers. Ru.
  • the present invention relates to the following molecular targeted drugs, antibodies, test agents, and methods of screening for molecular targeted drugs.
  • a molecule-targeted therapeutic agent containing, as an active ingredient, a substance that neutralizes or suppresses the expression of Cholinergic Receptor Nicotinic Beta 2 Subunit (CHRNB2).
  • CHRNB2 Cholinergic Receptor Nicotinic Beta 2 Subunit
  • Therapeutic agent containing, as an active ingredient, a substance that neutralizes or suppresses the expression of Cholinergic Receptor Nicotinic Beta 2 Subunit (CHRNB2).
  • CHRNB2 Cholinergic Receptor Nicot
  • a test kit comprising an antibody that recognizes CHRNB2 or a primer that quantitates CHRNB2 mRNA expression, and a reagent necessary for measurement.
  • the antibody that recognizes CHRNB2 is any one that recognizes WKPEEFDNMKKVRLPBKH (SEQ ID NO: 8) or TFLHSDHSAPSSK (SEQ ID NO: 9) of CHRNB2.
  • the test kit according to (10), wherein the antibody that recognizes CHRNB2 is produced by the hybridoma represented by Accession No. NITE P-02857.
  • the test kit according to (10), wherein the primer for quantifying the CHRNB2 mRNA is a primer represented by SEQ ID NO: 1 and 2.
  • a method of screening for a molecule-targeted drug which uses as an index the binding ability of a test substance and a cell surface of CHRNB2 to the cell surface.
  • a method of screening a molecule-targeted drug by culturing cells in the presence of a test substance and using CHRNB2 expression as an index.
  • a molecule-targeted therapeutic agent which contains, as an active ingredient, a substance which neutralizes or suppresses the expression of Neuronal pentraxin receptor (NPTXR).
  • NPTXR Neuronal pentraxin receptor
  • Therapeutic agent (19) The molecule-targeted therapeutic agent according to (18), wherein the active ingredient is an antibody.
  • the test kit according to (27), wherein the antibody that recognizes the NPTXR is one that recognizes either CESGLPRGLQGAGPRRDT (SEQ ID NO: 10) or KERVALSHSSRRQRQEVE (SEQ ID NO: 11).
  • the test kit according to (27), wherein the antibody that recognizes NPTXR is produced by the hybridoma represented by Accession No. NITE P-02856.
  • the primers for quantifying the mRNA expression of NPTXR are SEQ ID NOs: 3 and 4.
  • (31) A method of screening for a molecularly targeted drug using as an index the binding of a test substance to an area of the cell surface of NPTXR.
  • a therapeutic method comprising using the molecule-targeted therapeutic agent as described in (1) to (7).
  • a therapeutic method comprising using the molecule-targeted therapeutic agent according to (16) to (22).
  • the therapeutic method according to (37) which comprises examining NPTXR expression using the test kit according to (27) to (30).
  • the anticancer agent currently used for treatment is a therapeutic agent having a different mechanism of action, it is expected to have an effect on patients who have not been successfully treated with conventional treatment.
  • the figure which shows the analysis result of the influence which NPTXR expression gives to an apoptosis pathway shows mitochondrial depolarization and (B) shows analysis results of caspase activity.
  • (C) shows the analysis results of the activity of representative caspase family molecules.
  • the figure which analyzed the influence on the cell cycle of NPTXR expression is a figure which shows the analysis result by microscope observation by (B) by a cell sorter.
  • (A) is a figure which shows the analysis result of PI3K pathway which influences proliferation of a cancer cell.
  • (B) is a figure which shows the analysis result of the sensitivity to anticancer drug 5-FU.
  • A Analysis results of tumorigenicity of CHRNB2 knockout cells in mouse subcutaneous tumor model.
  • B Analysis results of tumorigenicity of NPTXR knockout cells in a mouse subcutaneous tumor model.
  • A) is a figure which shows the analysis result using MKN1 cell.
  • B) shows the results of analysis using cancer cell lines derived from various organs in addition to MIKN1 cells.
  • FIG. 1 shows the analysis results of the effect of CHRNB2 and (B) the cell proliferation of siRNA of NPTXR.
  • (D) gastric cancer tissue The figure which shows the analysis result of protein expression in the inside.
  • the results of analysis of expression distribution in various organs and normal tissues are shown.
  • (A) shows CHRNB2 expression and (B) shows NPTXR expression distribution.
  • (A) shows CHRNB2 expression and (B) shows NPTXR expression distribution.
  • (A) shows CHRNB2 expression and (B) shows NPTXR expression distribution.
  • (A) shows CHRNB2 expression and (B) shows NPTXR expression distribution.
  • the inventor classified cases after gastrectomy according to the progress, and used a next-generation sequencer to target mRNAs obtained from gastric cancer primary lesion tissues. Expression analysis was performed. As a result, we revealed that the receptor molecules CHRNB2 and NPTXR are molecules associated with the development of gastric cancer.
  • CHRNB2 and NPTXR are both genes whose expression is high in organs of nervous system such as brain.
  • the expression of CHRNB2 is not very high in other organs including the stomach, and there is no difference between the organs.
  • NPTXR is uniformly expressed in organs other than the nervous system, there is no difference in expression among organs.
  • CHRNB2 is associated with tumors such as gastric cancer and tumors The role of in Japan has never been reported and is not clear.
  • NPTXR although neuroblastoma has been reported as described above, there are no reports on other cancer types, and the role in epithelial tumors is also unknown.
  • antibodies and siRNA are used as substances that neutralize or inhibit the expression of CHRNB2 and NPTXR.
  • any substance that neutralizes or inhibits the expression of CHRNB2 and NPTXR as well as these antibodies and siRNA may be used.
  • the binding property of the CHRNB2 or NPTXR to the region exposed to the cell surface may be evaluated.
  • the region exposed on the cell surface of CHRNB2 or NPTXR can be predicted by a program for analyzing the three-dimensional structure of a protein. For example, since the peptides represented by SEQ ID NOS: 8 to 11 are considered to be exposed on the cell surface, screening can be performed by evaluating their binding to these peptides.
  • CHRNB2 or NPTXR expression can be screened using CHRNB2 or NPTXR expression as an index.
  • cells expressing CHRNB2 or NPTXR may be cultured in the presence of the candidate substance, CHRNB2 or NPTXR mRNA or protein may be quantified, and the effect of the candidate substance may be examined.
  • gene expression and protein expression known methods can be used. For example, gene expression can be measured by RT-PCR, and protein expression can be measured by ELISA or the like.
  • the anti-CHRNB2 antibody and the anti-NPTXR antibody refer to an immunoglobulin molecule that binds to CHRNB2 or NPTXR. Furthermore, as shown below, the inventor has obtained not only polyclonal antibodies against CHRNB2 and NPTXR, but also has already obtained monoclonal antibodies. Humanized antibodies in which these monoclonal antibodies are made into human chimeric antibodies, humanized CDR-grafted antibodies using human recombinant technology, and human antibodies using genetically modified mice are also included in the antibodies of the present invention. When administered to humans, humanized antibodies and human antibodies have fewer side effects than antibodies of non-human animals, and their therapeutic effects last for a long time. In addition, antibody fragments such as Fab, Fab ′, (Fab ′) 2 , and Fv fragments, linear antibodies, single chain Fv (scFv), bispecific antibodies, and the like are also included in the scope of the present invention.
  • anti-NPTXR monoclonal antibodies are also performing epitope analysis.
  • An "epitope" refers to a portion of an antigen that is recognized by an antibody, and means a site on the antigen to which a domain including the antibody variable region disclosed herein binds.
  • an antibody may recognize a linear amino acid sequence or a three-dimensional three-dimensional structure, but an epitope may refer to the amino acid sequence or the structure of an antigen. It can be defined by Since antibodies that bind to the same epitope are considered to have similar properties, anti-NPTXR monoclonal antibodies can also be defined by epitope sequences.
  • the nucleic acid drug is not limited to the siRNAs shown in the following examples, but may be any one that inhibits the expression of the CHRNB2 or NPTXR gene, and is not limited to siRNAs, and like miRNAs and antisense oligos, mRNA expression Including those that inhibit It also includes nucleic acids that bind to CHRNB2 or NPTXR proteins, such as aptamers, decoys, etc., and neutralize the activity.
  • an agent that abolishes the signal from these receptors by inhibiting the signal transduction system located downstream of the CHRNB2 or NPTXR receptor may be used.
  • the presence of PI3K / Akt signaling pathway was confirmed as a signal downstream of NPTXR. Therefore, in treating NPTXR high-expressing tumors, the effect is enhanced by using PI3K / Akt inhibitor alone or in combination with a molecule-targeted therapeutic agent that neutralizes or suppresses the expression of NPTXR there is a possibility.
  • CHRNB2 or NPTXR expression needs to be checked in advance to prevent unnecessary treatment.
  • the expression of CHRNB2 or NPTXR can be examined by PCR or immunohistochemical staining with an antibody as shown below. Primer sets used for PCR to measure expression, or antibodies, and reagents required for measurement can be combined to form a test kit. Since the expression of CHRNB2 and NPTXR is closely correlated with the prognosis, it is also possible to perform prognosis using these test kits.
  • CHRNB2 is equal to or better than gastric cancer, acute myeloid leukemia, breast cancer, cholangiocarcinoma, glioblastoma, glioma, lung cancer, malignant melanoma, during malignant It is highly expressed in skin cancer, pancreatic cancer, uterine cancer, uterine sarcoma, uveal melanoma, and renal cell carcinoma. Since the expression of CHRNB2 is high even in normal tissues in the brain and nervous system, high expression of CHRNB2 in glioblastoma and glioma is considered to reflect the expression of the original tissue.
  • CHRNB2 chronic myeloid leukemia, breast cancer, cholangiocarcinoma, lung cancer, malignant melanoma, malignant mesothelioma, pancreatic cancer, uterine cancer, uterine sarcoma, renal cell carcinoma, expression of CHRNB2 in the original organ is equivalent to that of stomach, Or less than that.
  • the high expression of CHRNB2 in these cancer types is likely to be correlated with carcinogenesis.
  • drugs targeting CHRNB2 may be effective in these cancer types as well. Sex is high.
  • NPTXR is equivalent to or better than gastric cancer, bladder cancer, breast cancer, esophageal cancer, glioblastoma, glioma, lung cancer, malignant melanoma, ovarian cancer, malignant pheochromocytoma, prostate cancer, testicular germ cell tumor, It is highly expressed in thyroid cancer, pancreatic cancer, uterine cancer and renal cell carcinoma. Therefore, in these carcinomas as well as gastric cancer, drugs targeting NPTXR are likely to be effective.
  • antibodies targeting CHRNB2 and NPTXR or nucleic acids are used alone, but they can be used in combination.
  • the present inventors have found regions that can be targets in two places for the anti-CHRNB2 antibody and the anti-NPTXR antibody, respectively. Therefore, these two types of antibodies against CHRNB2 or NPTXR may be used simultaneously, or CHRNB2 and NPTXR, or antibodies against two molecules may be used in combination. It can also be used in combination with already approved chemotherapeutic agents and targeted medicines such as trastuzumab, ramsirumab and nivolumab.
  • the recurrence and metastasis group is divided into peritoneal metastasis recurrence group, liver metastasis recurrence group and lymph node recurrence group, and it is long-term for more than 5 years.
  • Transcriptome analysis was performed for expression profiling of RNA obtained from each of four cases of gastric cancer primary tissue in 4 groups in combination with the recurrence free group.
  • the surgical sample extracted RNA using RNeasy kit (made by QIAGEN).
  • the extracted total RNA was subjected to preparation of a library for sequencing according to a standard protocol using TruSeq RNA Sample Prep Kit (manufactured by illumina).
  • next-generation sequencer Hiseq manufactured by illumina
  • transcriptome analysis was performed. Data was acquired at a read base length of 100 bases / lead, a reference acquisition lead count of 100 million read pairs (200 million reads) / lane, and a reference acquisition data volume of 20 Gb / lane.
  • CHRNB2 and NPTXR are significantly elevated in any recurrence group as compared to the control long-term recurrence-free group of 5 years or more.
  • CHRNB2 and NPTXR molecules have unknown roles in malignancy but are receptor proteins, they may be targets for drug discovery that have completely different mechanisms from existing gastric cancer molecular target drugs, so we will investigate further.
  • RNA expression levels of CHRNB2 and NPTXR were quantified by quantitative PCR using 14 types of gastric cancer cell lines and FHS74 which is a non-neoplastic ductal epithelial cell line (FIG. 1).
  • RNA was extracted and purified by the RNeasy kit (manufactured by QIAGEN), and quantitative PCR was carried out by heating at 95 ° C. for 10 minutes using an ABI STEPOne Plus Real-Time PCR System (manufactured by Applied Biosystems) for 60 minutes at 95 ° C. for 60 seconds.
  • glycerolaldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control.
  • the sequences of the PCR primers used are as follows.
  • [CHRNB2] Forward: AGCGAGGACGATGACCAG (SEQ ID NO: 1) Reverse: GGTGCCAAAGACACACAGACAA (SEQ ID NO: 2)
  • [NPTXR] Forward: TCATTCTGGAGCTGGAGGAC (SEQ ID NO: 3) Reverse: GGCAGCTGAGAGGTTCACA (SEQ ID NO: 4)
  • [Control GAPDH] Forward: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 5) Reverse: GAAGATGGTGATGGGATTTC (SEQ ID NO: 6)
  • CHRNB2 and NPTXR were increased in expression in gastric cancer tissues and gastric cancer cell lines of patients in the relapsed / metastatic group.
  • the proliferation ability of the cells was analyzed by the following method.
  • the cells were seeded in 96-well plates at 1 ⁇ 10 4 cells each, and cell growth was measured over time until day 5 with DMEM medium supplemented with 2% fetal bovine serum.
  • 10 ⁇ L of Cell Counting Kit-8 (manufactured by Dojindo Molecular Technologies) was added to measure the absorbance, and the value at the start of the measurement was displayed as 1 (FIG. 2A).
  • MKN1 cells (KO-CHRNB2 cells) in which CHRNB2 was knocked out, the cell proliferation rate decreased, and a significant difference was observed in the number of cells from 1 day after seeding (indicated by * in the figure. The following significant differences were observed) Things are displayed similarly.).
  • KO-CHRNB2 cells showed almost no growth even 5 days after seeding. The significant difference is analyzed by the Mann-Whitney test.
  • the apoptotic cell ratio was analyzed as follows. Annexin V staining was performed using Annexin V Alexa Fluor 568 conjugate (manufactured by Thermo Fisher Scientific). Add 10 ⁇ L of Annexin V reagent to 1 ⁇ 10 6 cells, allow to stand at room temperature for 15 minutes, then place on a slide and use the fluorescence microscope (FSX100, manufactured by Olympus) for cell number and Annexin V The number of positive cells was counted.
  • Annexin V staining was performed using Annexin V Alexa Fluor 568 conjugate (manufactured by Thermo Fisher Scientific). Add 10 ⁇ L of Annexin V reagent to 1 ⁇ 10 6 cells, allow to stand at room temperature for 15 minutes, then place on a slide and use the fluorescence microscope (FSX100, manufactured by Olympus) for cell number and Annexin V The number of positive cells was counted.
  • FSX100 fluorescence microscope
  • the apoptosis ratio was significantly increased in KO-CHRNB2 cells (FIG. 2B).
  • FIG. 2A by knocking out CHRNB2, the cell proliferation ability was significantly reduced compared to the parent gastric cancer cell MKN1 cell. From this result, it became clear that one of the mechanisms by which the growth of KO-CHRNB2 cells is suppressed is apoptosis induction. That is, by overexpression of CHRNB, apoptosis is less likely to be induced, and it is considered that malignancy is therefore high.
  • the migration ability of the cells was evaluated by a wound healing assay (Wound-healing assay).
  • Ibidi Culture insert method manufactured by ibid was seeded in a 12-well plate at a predetermined width to form 2 ⁇ 10 4 cells of each cell, and the cells were seeded with wound gaps and cultured in a serum-free medium. The inserts were removed 24 hours after seeding, and the wound width was measured at 200 ⁇ m intervals every 6 hours. The measurement was performed using a microscope at 40 ⁇ magnification, and 10 wells were measured to determine the mean and standard deviation (FIG. 2C). By knocking out CHRNB2, it was revealed that the migration ability was also significantly reduced.
  • the infiltration ability of cells was evaluated by control cells, KO-CHRNB2 cells by Matrigel invasion assay.
  • the assay was performed according to the protocol using BioCoat Matrigel invasion Chambers (manufactured by BD Biosciences). Specifically, each cell is seeded at 2.5 ⁇ 10 4 cells / well and cultured in serum-free DMEM medium for 24 hours, and then cells on the bottom of the membrane are fixed and DiffQuick (Sysmex Corporation) And stained under a microscope to count the number of cells. Microscopy was performed at 200 ⁇ magnification to determine the mean and standard deviation of 5 randomly selected fields. By knocking out CHRNB2, invasive ability was also significantly reduced (FIG. 2D).
  • the cell adhesion ability was analyzed using CytoSelect 48-Well Cell Adhesion Assay (manufactured by Cell Biolabs) as follows. Each cell was seeded at 7.5 ⁇ 10 4 cells / well on an adhesion factor coated plate, and each absorbance was measured after 1 hour of culture in serum-free DMEM medium. Analysis of the adhesion ability to five types of cell adhesion factors revealed that the adhesion ability was also reduced in KO-CHRNB2 cells (FIG. 2E).
  • MKN1 cells have significantly changed properties related to cancer development such as proliferation ability, apoptosis ratio, migration ability, invasion ability, and adhesion ability by losing the expression of CHRNB2. Therefore, by inhibiting the action of CHRNB2, it is possible to suppress the cancer progression of gastric cancer cells.
  • the proliferation ability, the migration ability, the infiltration ability, and the adhesion ability are all significantly the parent strain MKN1 cells Decreased, and the apoptosis rate was significantly increased.
  • the growth ability, the migration ability, the infiltration ability, and the adhesion ability of each strain were significantly reduced with respect to the parent strain MKN1 cells.
  • the results showed that the apoptosis rate was significantly increased. Therefore, it became clear that the loss of NPTXR, like CHRNB2, significantly impairs the property of gastric cancer cell MKN1 involved in cancer progression.
  • NPTXR expression affects apoptosis and enhances cell proliferation. Therefore, the NPTXR knockout cell line was used to analyze the properties associated with apoptosis. Mitochondria are known to be deeply involved in apoptosis. It is known that the loss of mitochondrial membrane potential promotes the release of cytochrome c, which activates the caspase cascade and induces apoptosis. Therefore, it was analyzed whether NPTXR expression is involved in apoptosis via mitochondria (FIG. 4).
  • the mitochondrial membrane potential was visualized to visualize live cells in which mitochondrial depolarization has occurred, that is, cells in which apoptosis is induced (FIG. 4 (A)).
  • the NPTXR knockout cells NPTXR-KO-1 and NPTXR-KO-2 and the parent MNK1 cells as a control are each stained with 1 ⁇ 10 5 cells with Muse MitoPotential kit (Merck Millipore) and analyzed with a cell sorter Did.
  • caspase activity which is a protease that promotes apoptosis
  • caspase activity was analyzed.
  • Each 1 ⁇ 10 5 cell lines of each cell line were stained with Muse Multi-Caspase kit (manufactured by Merck Millipore) and analyzed by a cell sorter.
  • Caspase positive cells Caspase + / Dead and Caspase + / Live fractions
  • Caspase Colorimetric Assay Kit manufactured by BioVision
  • caspase 3 is an effector caspase
  • caspases 8 and 9 are initiator caspases.
  • Caspase 12 is a caspase found only in mice and is considered to act as an initiator to induce apoptosis due to endoplasmic reticulum stress.
  • NPTXR expression suppresses the induction of apoptosis in the mitochondrial pathway and consequently also suppresses the activation of caspases.
  • Cancer cells are characterized by having high proliferation ability, resistance to apoptosis, and invasion / metastatic ability as described above. However, not all cancer cells have similar properties, and a cancer stem cell hypothesis has been proposed in which some of the cancer cells have stem cell properties and cancer develops.
  • the proportion of stem cells was analyzed using aldehyde dehydrogenase (ALDH) activity expressed in stem cells as an indicator.
  • ALDH aldehyde dehydrogenase
  • the percentage of ALDH positive cells was measured by ALDEFLOUR fluorescent reagent system (manufactured by Stem Cell Technologies) (FIG. 6).
  • the parent strains MKN1 cells and NPTXR knockout cells were each stained at 1 ⁇ 10 5 cells and analyzed.
  • PI3 kinase is an intracellular signal transduction system that regulates various functions such as Akt-mediated survival, proliferation, protein synthesis, cell movement via Rac, etc. It is known to be highly expressed.
  • the activity of PI3K in NPTXR knockout cells was measured using Muse PI3K Activation Dual Detection Kit (manufactured by Millipore) (FIG. 7 (A)).
  • Muse PI3K Activation Dual Detection Kit manufactured by Millipore
  • PI3K was activated in most of the cells, whereas in NPTXR knockout cells, a decrease in the proportion of PI3K positive cells was observed. It was shown that the abolition of NPTXR expression suppresses PI3K activity.
  • NPTXR expression affects the sensitivity to 5-FU, which is a commonly used anticancer agent in gastric cancer (FIG. 7 (B)).
  • the parental control MKN1, NPTXR-KO-1, and NPTXR-KO-2 cells are seeded at 5 ⁇ 10 3 cells into wells, and 0, 0.01, 0.1, 1, 10, 100 mg / L.
  • the cells were exposed to 5-FU, and the number of cells after 72 hours was measured by Cell Counting Kit-8.
  • tumors When mouse subcutaneous tumor models were prepared and tumorigenicity was compared, the tumors (Tat. MKN1) increased with time, but the CHRNB2 knockout cells were significant from the fourth week after transplantation (FIG. 8 *). A difference in the tumor volume was observed in (1), and knockout of CHRNB2 revealed that tumorigenicity was reduced (FIG. 8 (A)). In addition, in mice transplanted with NPTXR-knock out cells, a significant difference in tumor volume was observed from the third week after transplantation, which revealed that tumorigenicity was reduced (FIG. 8 (B)). .
  • CHRNB2 antibody-1 WKPEEFDNMKKVRLPSKH (SEQ ID NO: 8)
  • CHRNB2 antibody-2 TFLHSDHSAPSSK (SEQ ID NO: 9)
  • MKN1 cells were seeded at 1 ⁇ 10 4 in a 96-well plate, each antibody was added to a concentration of 0.7 ⁇ g / mL, and cell proliferation was measured by WST-1 assay over time (Fig. 9). Prebleed serum was used as a control.
  • CHRNB2 antibodies were added to gastric cancer cell line MKN1
  • Example 2 ⁇ Preparation of NPTXR Neutralizing Antibody (Polyclonal Antibody)>
  • estimation of hydrophilicity, secondary structure, antigenicity and optimum antigen are similarly performed using an epitope search tool We performed part search.
  • the following two sequences were selected as target sequences, and rabbits were immunized according to a conventional method, and two polyclonal antibodies against NPTXR (Accession number: NP_055108) were produced.
  • the NPTXR antibody-1 is an antibody to amino acid sequences at positions 161-178 (target sequence 1, SEQ ID NO: 10) and the NPTXR antibody-2 (target sequence 2, SEQ ID NO: 11) is positions 251-268.
  • NPTXR antibody-1 CESGLPRGLQGAGPRRDT (SEQ ID NO: 10)
  • NPTXR antibody-2 KERVALSHSSRRQRQEVE (SEQ ID NO: 11)
  • Example 3 ⁇ Analysis of the effect of CHRNB2 neutralizing antibody using mouse peritoneal dissemination model> Next, the effect of CHRNB2 antibody that recognizes the above two types of targets was analyzed by a mouse peritoneal dissemination model. 1 ⁇ 10 6 MKN1 cells were transplanted into the abdominal cavity of 8-week-old nude mice (BALB cnu / nu, obtained from Japan SLC Co., Ltd.) to examine the therapeutic effect. In addition, CHRNB2 antibody (each CHRNB2 antibody-1 is 6.5 ⁇ g / 500 ⁇ L, CHRNB2 antibody-2 is 8.7 ⁇ g / 500 ⁇ L) as per the administration schedule shown in FIG. Intraperitoneal injection was performed intraperitoneally for 6 weeks, and gastric cancer peritoneal dissemination was compared with the control group. The control is a group to which no antibody has been administered.
  • the seeded nodules were collected from the open mice, and the total weight of the seeded nodules in each mouse was measured (FIG. 10 lower). In the antibody-administered group, the total tumor weight was significantly reduced (*) with respect to the control.
  • Example 4 ⁇ Analysis of the effect of NPTXR neutralizing antibody using mouse peritoneal dissemination model> Similar to Example 3, the effect of NPTXR antibody that recognizes two targets of NPTXR was analyzed by the mouse peritoneal dissemination model (FIG. 11).
  • NPTXR antibody-1 and NPTXR antibody-2 administration groups are shown (FIG. 11 upper).
  • NPTXR antibody-administered group only a few peritoneal disseminated nodules were observed with either antibody, indicating its therapeutic effect.
  • the disseminated nodules were collected from the open mice, and the total weight of the disseminated nodules in each mouse was measured (FIG. 11 lower). In the antibody-administered group, the total tumor weight was significantly reduced (*) with respect to the control.
  • CHRNB2 and NPTXR are targeted and their expression is neutralized, thereby significantly proliferating cancer cells. Can be suppressed. Therefore, relapsed / metastatic gastric cancer can be treated by using an antibody that recognizes CHRNB2 or NPTXR as a drug.
  • CHRNB2 and NPTXR are upregulated in many cancers, not only in gastric cancer. There is a high probability that these cancers can be treated similarly using CHRNB2 and NPTXR neutralizing antibodies.
  • Example 5 ⁇ Establishment of anti-CHRNB2 monoclonal antibody> Using the peptide represented by SEQ ID NO: 9 as an antigen, a monoclonal antibody against CHRNB2 was established by a conventional method. Three clones were selected from the obtained hybridomas to obtain monoclonal antibodies. In the same manner as in Example 1, MKN1 cells were seeded to 1 ⁇ 10 4 in a 96-well plate, each antibody was added to a concentration of 0.7 ⁇ g / mL, and cell proliferation was WST over time. -1 measured by the assay (FIG. 12). Antibodies obtained from any of the clones showed similar inhibitory effects on cell proliferation.
  • CH-01 clone 3
  • NITE National Institute of Technology and Evaluation
  • NPMD Patent Microorganisms Depositary Center
  • Example 6 Establishment of anti-NPTXR monoclonal antibody A monoclonal antibody against NPTXR was established using the peptide represented by SEQ ID NO: 10 as an antigen. In the same manner as in Example 1, MKN1 cells were seeded to 1 ⁇ 10 4 in a 96-well plate, each antibody was added to a concentration of 0.7 ⁇ g / mL, and cell proliferation was WST over time. -1 assay (FIG. 14 (A)). All clones showed similar inhibitory effects on cell proliferation.
  • anti-NPTXR monoclonal antibody clone 1 was analyzed on cancer cell lines derived from various organs. The antibody was added to each cancer cell line in the same manner as described above, and the proliferation ratio to the control of the antibody administration group on day 5 was calculated (FIG. 14 (B)). In all cell lines, it was shown that antibody addition suppresses cell growth. In particular, a strong cell growth inhibitory effect of the antibody was observed against MKN1 and NUGC4, which are cell lines established from gastric cancer.
  • NP-01 Clone 1
  • NITE BP-02856 accession number NITE BP-02856
  • NITE National Institute of Technology and Evaluation
  • NPMD Patent Microorganisms Depositary Center
  • the left graph shows the result of quantitative PCR analysis of the expression level of CHRNB2 mRNA, and the right shows the result of cell proliferation analyzed by Cell Counting Kit-8.
  • the growth potential of gastric cancer cells was significantly reduced (*) by a specific siRNA against CHRNB2.
  • Example 8 ⁇ Study of nucleic acid medicine for NPTXR> Next, using a specific siRNA against NPTXR, in the same manner as in Example 7, the effect on proliferation was analyzed in vitro. After the siRNA of NPTXR was introduced into MKN1 cells and cultured in serum-free DMEM medium for 72 hours, the proliferation ability was evaluated (FIG. 16 (B)).
  • the left graph shows the results of quantitative PCR analysis of the expression level of NPTXR mRNA, and the right shows the results of cell proliferation analyzed by Cell Counting Kit-8.
  • the growth potential of gastric cancer cells was significantly reduced (*) by specific siRNA against NPTXR.
  • nucleic acid drugs can suppress the development of cancer by suppressing the expression of these molecules.
  • Example 9 ⁇ A quantitative analysis of CHRNB2 expression level in gastric cancer tissue>
  • the mRNA expression level of CHRNB2 in gastric cancer tissues obtained from 300 cases of gastric cancer was examined by a quantitative PCR method.
  • CHRNB2 expression was measured by quantitative PCR in tissues obtained from normal gastric mucosa, 121 cases of gastric cancer of stages I and II, and 179 cases of gastric cancer of stages III and IV (FIG. 17 (A)).
  • CHRNB2 was higher in gastric cancer tissues than in normal gastric mucosa, and was significantly increased in stages III and IV gastric cancer, as compared with stages I and II. From these data, it is recognized that expression of CHRNB2 is enhanced as the stage progresses.
  • CHRNB2 protein in gastric tissue was detected by immunohistochemical staining (FIG. 17 (D)).
  • Immunostaining of tissues was performed by a conventional method using a polyclonal antibody against CHRNB2. It is possible to determine cases in which the expression clearly appears in the tumor tissue and cases in which the expression does not occur. Therefore, expression of CHRNB2 protein can be confirmed by immunohistochemical staining.
  • Example 10 ⁇ Quantitative analysis of NPTXR expression level in gastric cancer tissue>
  • the mRNA expression level of NPTXR in gastric cancer tissues obtained from 300 gastric cancer cases was examined by a quantitative PCR method (FIG. 18 (A)).
  • the expression of NPTXR was significantly increased in gastric cancer tissues as compared to normal gastric mucosa regardless of the stage.
  • NPTXR NPTXR
  • the above 300 cases were divided into stages in detail and examined by quantitative PCR in the same manner as described above (FIG. 19).
  • the expression of NPTXR tended to increase as the stage progressed, and it was found that there was a significant difference in expression between normal tissues and stages I and II.
  • NPTXR protein in gastric tissue was detected by immunohistochemical staining (FIG. 18 (D)).
  • Immunostaining of tissues was performed by a conventional method using a polyclonal antibody against NPTXR. It is possible to determine cases in which the expression clearly appears in the tumor tissue and cases in which the expression does not occur. Therefore, expression of NPTXR protein can be confirmed by immunohistochemical staining.
  • the CHRNB2 antibody and the NPTXR antibody have been shown to be applicable to companion diagnostics that enable screening of CHRNB2 and NPTXR targeted patients targeted for molecular targeted therapy.
  • CHRNB2 and NPTXR compare proteins in the long-term recurrence-free group and recurrent metastasis group, and since they are proteins in which a difference in expression was observed, CHRNB2 and NPTXR expression can also be used as a prognostic marker. Since immunohistochemical staining can be determined even from a small amount of tissue sample, not only a surgical sample but also a biopsy sample at the time of endoscopic examination are targeted. In addition, since immunohistochemical staining is a routine examination in clinical practice, the ability of antibodies to confirm the expression of these proteins in a sample is of great clinical significance.
  • Example 11 ⁇ Expression distribution by tissue microarray> (1) Distribution of expression in various organs and normal tissues Expression of CHRNB2 and NPTXR in various organs and normal tissues as data for examining the safety of antibody drugs that bind to CHRNB2 and NPTXR or drugs that inhibit expression Analysis of distribution was performed (FIG. 20).
  • CHRNB2 and NPTXR were analyzed by immunohistochemical staining using a tissue microarray (manufactured by Provitro). As a result of tissue staining, the analyzed tissue is classified into no expression, low expression and high expression, and the ratio is shown in the graph. The graph shows the number of tissues analyzed as (n). In human normal tissues, both CHRNB2 expression and NPTXR expression are low, suggesting that the possibility of adverse events occurring to undesired organs is low.
  • CHRNB2 and NPTXR Expression analysis of CHRNB2 and NPTXR in breast cancer was performed (FIG. 22). CHRNB2 was expressed in about 40% of breast cancer and NPTXR in about 30%. Expression analysis of CHRNB2 and NPTXR in lung cancer was performed (FIG. 23). Both CHRNB2 and NPTXR were expressed in about 50% of lung cancers. The expression analysis of CHRNB2 and NPTXR in pancreatic cancer was performed (FIG. 24). CHRNB2 was expressed in about 40% of pancreatic cancer and NPTXR in about 50%. Expression analysis of CHRNB2 and NPTXR in esophageal cancer was performed (FIG. 25). Both CHRNB2 and NPTXR were expressed in about 50% of esophageal cancer.
  • CHRNB2 and NPTXR are low in normal tissues, and expression is observed not only in gastric cancer but also in various cancers such as breast cancer, lung cancer, pancreatic cancer and esophageal cancer. Therefore, it has been suggested that, in these cancers as well, an inhibitor that inhibits CHRNB2, NPTXR expression, or an antibody drug that binds to CHRNB2, NPTXR can be used to treat.
  • CHRNB2 and NPTXR high expression groups are found in the group of patients treated with S-1 postoperative adjuvant therapy, which is positioned as the standard treatment, means that these two molecules can not be controlled by the existing treatment It is a key molecule to overcome the situation.
  • Drugs that neutralize and suppress the expression of CHRNB2 and NPTXR are completely new therapeutic agents because they are completely different from existing targets for growth factor receptor-centered molecular targeted therapeutics. In addition to gastric cancer, it can be a drug having an effect on tumors with high expression of CHRNB2 and NPTXR.

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PCT/JP2019/002504 2018-01-26 2019-01-25 受容体タンパク質を標的とする治療薬、検査薬、受容体タンパク質に結合する抗体、及び分子標的薬のスクリーニング方法 Ceased WO2019146759A1 (ja)

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