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WO2025004683A1 - Procédé d'examen cancéreux et agent de traitement cancéreux - Google Patents

Procédé d'examen cancéreux et agent de traitement cancéreux Download PDF

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Publication number
WO2025004683A1
WO2025004683A1 PCT/JP2024/019760 JP2024019760W WO2025004683A1 WO 2025004683 A1 WO2025004683 A1 WO 2025004683A1 JP 2024019760 W JP2024019760 W JP 2024019760W WO 2025004683 A1 WO2025004683 A1 WO 2025004683A1
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Prior art keywords
cancer
protein
membrane surface
specific
expressed
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English (en)
Japanese (ja)
Inventor
和丈 辻川
健太郎 神宮司
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University of Osaka NUC
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Osaka University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a cancer testing method, a cancer treatment agent, and a reagent for cancer research.
  • Biomarkers are substances in the body, such as proteins and genes, that serve as physiological indicators of the presence or absence of a disease and its progression.
  • Biomarkers include a variety of things, such as so-called clinical test values such as biochemical tests, blood tests, and tumor markers, as well as diagnostic imaging data such as CT (Computed Tomography) and MRI (Magnetic Resonance Imaging). Examples include indicators that quantify and convert biological information into numerical values in order to quantitatively grasp biological changes within the body.
  • biomarkers that indicate the presence or progression of a disease as indicators of disease are indispensable in current medical treatment, such as for the detection, diagnosis, and prognosis prediction of diseases. Biomarkers are also used in the selection and evaluation of development compounds in new drug development.
  • Biopsies are extremely important for cancer patients, especially for making decisions about diagnosis, prognosis, and treatment.
  • NSCLC non-small cell lung cancer
  • liquid biopsies in which bodily fluids, secretions, or excretions such as blood, urine, saliva, cerebrospinal fluid, pericardial effusion, pleural effusion, ascites, and stool are collected as test samples, have been attracting attention as an alternative sample acquisition method to complement or overcome the barriers to obtaining tumor tissue samples.
  • Liquid biopsies are less invasive than tissue biopsies and can be performed repeatedly.
  • Targets for liquid biopsy analysis include circulating tumor cells in the blood (Circulating Tumor Cells: CTCs), DNA released by cell death (cell free DNA: cfDNA), and DNA derived from cancer cells (Circulating Tumor DNA: ctDNA).
  • CTCs and ctDNA are expected to be detected when cancer cells metastasize, they are suitable for predicting cancer prognosis but cannot be used for early diagnosis.
  • Patent Document 1 A method for testing for lung cancer has been disclosed (Patent Document 1), which includes a step of analyzing the complex between CHL1 (cell adhesion molecule L1 like) and a CHL1 partner protein present in the membrane of extracellular vesicles present in a blood sample from a subject. Patent Document 1 also discloses various CHL1 partner proteins.
  • CHL1 cell adhesion molecule L1 like
  • Patent Document 2 There is a disclosure of isolating tissue-exuded extracellular vesicles (te-EVs) from diseased tissues and analyzing biomarkers (Patent Document 2).
  • Patent Document 2 reports that te-EVs derived from cancer tissues and normal tissues of cancer patients were used to identify tetraspanin molecules such as CD63, CD81, and CD9, which are known as exosome markers, and their expression levels were analyzed by liquid chromatography mass spectrometry (LC/MS).
  • LC/MS liquid chromatography mass spectrometry
  • EVs cancer-related extracellular vesicles
  • ccRCC viable renal cell carcinoma
  • n520 normal kidney tissues
  • Te-EV tissue-exuded EV
  • Patent Publication No. 2021-188970 International Publication No. WO2018/079689 (Patent Publication No. 6712737) International Publication No. WO2020/071489
  • the present invention comprises the following. 1.
  • a method for testing for cancer comprising detecting a protein that is expressed in a cancer-specific manner on the membrane surface of extracellular vesicles contained in a collected body fluid sample, thereby detecting cancer-specific extracellular vesicles.
  • the protein expressed in a cancer-specific manner on the membrane surface of the extracellular vesicle is one or more proteins selected from ABCE1 (ATP binding cassette subfamily E member 1), STAT1 (Signal transducer and activator of transcription 1), and ZA2G (Alpha-2-glycoprotein 1, zinc-binding). 3.
  • the method for detecting cancer according to the preceding item 1 or 2 characterized in that the cancer-specific extracellular vesicles are detected using an immunosandwich method or a flow cytometry method using an antibody against a protein that is expressed in a cancer-specific manner on the membrane surface of the extracellular vesicles and an antibody against an extracellular vesicle-specific membrane surface marker protein or an antibody against a protein that is expressed in a cancer-specific manner on the membrane surface of the extracellular vesicles. 6.
  • extracellular vesicle-specific membrane surface marker protein is one or more selected from CD9, CD63, CD81, integrin, selectin, and CD40, and the protein that binds to the extracellular vesicle membrane is Tim4.
  • a cancer testing device comprising a substrate having immobilized thereon an antibody against one or more proteins selected from ABCE1, STAT1 and ZA2G. 10.
  • a cancer testing kit for detecting cancer-specific extracellular vesicles from extracellular vesicles contained in a collected body fluid sample comprising at least one of the following configurations selected from 1) to 3): 1) A substrate on which an antibody against a protein that is specifically expressed on the membrane surface of extracellular vesicles is immobilized, and a labeling reagent capable of specifically detecting extracellular vesicles; 2) a substrate on which an antibody against an extracellular vesicle-specific membrane surface marker protein or a protein that binds to an extracellular vesicle membrane is immobilized, and a labeling reagent capable of detecting a protein that is expressed specifically in a cancer on the membrane surface of an extracellular vesicle; 3) A substrate on which an antibody against a protein that is expressed specifically in cancer on the membrane surface of extracellular vesicles is immobilized, and a labeling reagent capable of detecting the protein that is expressed specifically in cancer on the membrane surface of extracellular vesicles.
  • the extracellular vesicle-specific membrane surface marker protein is one or more selected from CD9, CD63, CD81, integrin, selectin, and CD40, and the protein that binds to the extracellular vesicle membrane is Tim4.
  • a cancer therapeutic agent comprising, as an active ingredient, an antibody against a protein expressed on the membrane surface of cancer-specific extracellular vesicles. 14.
  • cancer-specific EVs are present in bodily fluid samples, specifically blood samples and saliva, and that cancer-specific proteins are expressed on the membrane surface of EVs. It was confirmed that the level of these proteins decreased after surgery. Therefore, it is expected that detecting cancer-specific proteins on the membrane surface of EVs as markers can be used as indicators for early cancer diagnosis, cancer stage determination, and treatment.
  • Immune cells infiltrate into cancer tissue to attack cancer cells. Among immune cells, NK cells are cells that detect and attack cancer cells early.
  • Example 2 The reactivity of anti-ABCE1 antibodies in colorectal cancer patients was confirmed by ELISA using plasma from colorectal cancer patients before and after surgery and plasma from healthy subjects (Example 3). The results of confirming the effect of EVs collected from non-cancerous and cancerous areas of colon cancer patients on the cancer cell cytotoxicity of NK cells are shown below (Reference Example 1). The results of confirming the effect of anti-ABCE1 antibody on the inhibitory effect of colon cancer-derived EVs on the cytotoxic activity of NK cells are shown.
  • Figure 5a shows the protocol for treating HT29 cell-derived EVs with anti-ABCE1 antibody or control antibody
  • Figure 5b shows the results of Western blotting using anti-ABCE1 antibody on HT29 cell-derived SEV lysate
  • Figure 5c shows the results of confirming the effect of EVs on the cytotoxic activity of NK cells with or without anti-ABCE1 antibody treatment, based on the survival rate of HT29 cells.
  • Example 4 The results of confirming the expression of STAT1 in large EVs and small EVs derived from cancer tissues (N, Stage I-IV) and non-cancerous tissues (N) of colorectal cancer patients using an anti-STAT1 antibody (Santacruz, sc-417) are shown below (Reference Example 2).
  • Example 6 shows the results of confirming the reactivity of anti-ABCE1 antibody, anti-STAT1 antibody, and anti-ZA2G antibody in ovarian cancer patients by ELISA using plasma from ovarian cancer patients before and after surgery and plasma from healthy subjects.
  • extracellular vesicles refer to tiny vesicles with a membrane structure secreted from various types of cells, including, for example, exosomes, ectosomes, microvesicles, microvesicles, and apoptotic bodies.
  • cancer-specific expression means cancer tissue-specific expression, but also includes cases where the expression is selectively expressed in cancer tissue compared to normal tissue, or where the expression is significantly higher due to cancer tissue compared to normal tissue and is clearly distinguishable from normal tissue.
  • examples of “proteins that are cancer-specifically expressed on the membrane surface of EVs” include one or more proteins selected from ABCE1, STAT1, and ZA2G.
  • examples of “EV-specific membrane surface marker proteins” include one or more proteins selected from CD9, CD63, CD81, integrin, selectin, and CD40, and an example of "a protein that binds to the EV membrane” is Tim4.
  • an immunological detection method can be applied as a method capable of detecting a protein that is expressed in a cancer-specific manner on the membrane surface of EVs.
  • the immunological detection method any method known per se or any method to be developed in the future can be applied, for example, an immunosandwich method can be applied, and specific examples of suitable methods include ELISA, sandwich ELISA, EIA, RIA, and Western blot methods.
  • an antibody against a protein expressed in a cancer-specific manner on the membrane surface of an EV is reacted with a test sample, and detection can be performed using a detection reagent that reacts with the test sample that has reacted with the antibody against the protein.
  • a substrate is used to which an antibody against a protein expressed in a cancer-specific manner on the membrane surface of an EV is immobilized, and the test sample that has reacted with the antibody captured on the substrate can be detected using a labeled reagent that can be specifically detected in an EV, specifically a labeled antibody against an EV-specific membrane surface marker protein or a labeled protein that binds to the EV membrane, as a detection reagent.
  • an antibody against an EV-specific membrane surface marker protein or a protein that binds to the EV membrane can be reacted with a test sample, and the test sample that has reacted with the antibody against the EV-specific membrane surface marker protein or the protein that binds to the EV membrane can be detected using a labeled antibody against a protein that is expressed in a cancer-specific manner on the membrane surface of the EV as a detection reagent.
  • the labeled antibody may be a labeled anti-IgG antibody or a labeled anti-Fc antibody, as long as it can detect the desired target.
  • the peptides constituting the target biomarker can be separated and purified by liquid chromatography (LC) or HPLC to prepare a test sample.
  • LC liquid chromatography
  • HPLC HPLC
  • the detection method of the present invention is characterized by its ease of testing, in that the above-mentioned immunological detection method can be applied.
  • the present invention also extends to a testing device used in the cancer testing method of the present invention, i.e., a testing device capable of detecting a cancer-specific protein expressed on the membrane surface of an EV.
  • a testing device is a substrate on which an antibody against a cancer-specific protein expressed on the membrane surface of an EV is immobilized.
  • a substrate on which an antibody against an EV marker protein or a protein that binds to the EV membrane is immobilized can also be used.
  • a detection reagent capable of detecting a cancer-specific protein expressed on the membrane surface of an EV is required.
  • the present invention also includes a test kit capable of detecting a protein that is specifically expressed on the membrane surface of EV.
  • the test kit includes at least the test device and may further include a detection reagent and the like.
  • the test kit includes at least one of the following configurations 1) to 3).
  • the beads were washed three times with PBS-T, and 20 ⁇ l of 1 ⁇ Laemmli SDS sample buffer was added to the beads and treated at 70 ° C for 10 minutes.
  • the supernatant (EV lysate) was used for Western blotting as described below in 2).
  • anti-ABCE1 antibody-bound beads or control antibody-bound beads were reacted with SEV, and the reactants were removed using a magnet to obtain the supernatant.
  • the SEV treated with anti-ABCE1 antibody-bound beads and control antibody-bound beads were designated as colon cancer SEV low and colon cancer SEV high, respectively (Fig. 5a).
  • Example 5 Confirmation of ZA2G expression in EVs derived from cancerous parts of ovarian cancer postoperative specimens EVs from ovarian cancer clinical specimens (non-cancerous part EVs: Normal (1), (2); cancerous part EVs: Stage I (3), (4), Stage II (5), (6), Stage III (7), (8), Stage IV (9), (10); Small EVs: 3.0 ⁇ 10 8 particles, Large EVs: 3.0 ⁇ 10 7 particles) were added with 1 ⁇ Laemmli SDS sample buffer to prepare samples, which were developed by SDS-PAGE (12% gel) and transferred to a PVDF membrane.
  • Anti-ABCE1 antibody (Abcam, Cat No. ab185548, 0.1 ⁇ g), anti-STAT1 antibody (Santa Cruz Biotechnology, Cat No. sc-417, used at 1/1000 dilution, 0.1 ⁇ g) or anti-ZA2G antibody (Abcam, ab-180574, 0.05 ⁇ g) were each added to a 96-well plate and coated at 4°C O/N. After washing three times with PBS-T (PBS containing 0.05% Tween-20), the plate was blocked at room temperature for 1 hour at 37°C using PBS containing 1% BSA.
  • PBS-T PBS containing 0.05% Tween-20
  • bodily fluid samples such as blood and saliva can be used as samples for testing, and testing can be performed without invasively obtaining biological samples from the subject, which is useful with a low burden on the subject.
  • NK cells are cells that detect and attack cancer cells at an early stage.
  • cancer cells release EVs as a means of evading such attacks, and that the various RNAs and proteins contained in these EVs allow them to avoid attacks from NK cells.
  • the antibodies against the proteins that are specifically expressed in the membrane surface of EVs discovered in this invention can remove cancer-specific EVs. This can restore the function of NK cells that were suppressed by cancer-specific EVs, and are expected to suppress the survival of cancer cells.

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Abstract

L'invention concerne un biomarqueur utile pour le diagnostic d'un cancer et le développement de nouveaux médicaments. L'invention concerne en outre un procédé d'examen cancéreux, un agent de traitement et un réactif pour des études sur le cancer, dans chacun desquels le biomarqueur est utilisé. Un biomarqueur utile est fourni par l'identification d'une protéine qui est exprimée sur la surface de la membrane d'une vésicule extracellulaire (EV) d'une manière spécifique d'un cancer. Le procédé d'examen du cancer consiste à détecter une protéine (un biomarqueur) qui est exprimée, d'une manière spécifique d'un cancer, sur la surface de la membrane d'une vésicule extracellulaire (EV) contenue dans un échantillon de fluide corporel prélevé pour détecter une vésicule extracellulaire (EV) spécifique d'un cancer. Un anticorps dirigé contre une protéine exprimée sur la surface de la membrane d'une vésicule extracellulaire (EV) d'une manière spécifique d'un cancer peut être utilisé dans un procédé d'examen cancéreux, un agent de traitement cancéreux et un réactif pour des études sur le cancer.
PCT/JP2024/019760 2023-06-30 2024-05-29 Procédé d'examen cancéreux et agent de traitement cancéreux Pending WO2025004683A1 (fr)

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GAO YING, XU HUI-HUI, WANG RUI, FANG HUI: " Expression of a New Tumor Metastasis-related Gene ABCE1 in Non-small Cell Lung Cancer and Its Significance", JOURNAL OF CHINA MEDICAL UNIVERSITY, CHINA MEDICAL UNIVERSITY, SHENYANG,, CN, vol. 40, no. 10, 1 October 2011 (2011-10-01), CN , pages 911 - 914, XP093254938, ISSN: 0258-4646 *
GLAVINAS HRISTOS; KRAJCSI PETER; CSEREPES JUDIT; SARKADI BALAZS: "The role of ABC transporters in drug resistance, metabolism and toxicity", CURRENT DRUG DELIVERY, BENTHAM SCIENCE PUBLISHERS, HILVERSUM, NL, vol. 1, no. 1, 1 January 2004 (2004-01-01), NL , pages 27 - 42, XP008120176, ISSN: 1567-2018 *
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HAYASHI, TETSUTARO: "P-3015: STAT1 inhibition restored chemotherapy sensitivity in bladder cancer", ABSTRACTS OF THE 75TH ANNUAL MEETING OF THE JAPANESE CANCER ASSOCIATION, JAPANESE CANCER ASSOCIATION, JP, vol. 75, 1 January 2016 (2016-01-01) - 8 October 2016 (2016-10-08), JP, pages 1247, XP009560382 *
ISHIZUYA, YU: "AOP-112: Search and identification of novel therapeutic targets by comprehensive protein analysis of serum exosomes from patients with castration-resistant prostate cancer", THE 107TH ANNUAL MEETING OF THE JAPANESE UROLOGICAL ASSOCIATION; APRIL 18-21, 2019, JAPANESE UROLOGICAL ASSOCIATION, JP, vol. 107, 1 January 2019 (2019-01-01) - 21 April 2019 (2019-04-21), JP, pages 631, XP009559803 *
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