WO2018025869A1 - Biomarqueur pour prédiction de l'effet thérapeutique d'un inhibiteur de fstl1 chez un patient cancéreux - Google Patents

Biomarqueur pour prédiction de l'effet thérapeutique d'un inhibiteur de fstl1 chez un patient cancéreux Download PDF

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WO2018025869A1
WO2018025869A1 PCT/JP2017/027916 JP2017027916W WO2018025869A1 WO 2018025869 A1 WO2018025869 A1 WO 2018025869A1 JP 2017027916 W JP2017027916 W JP 2017027916W WO 2018025869 A1 WO2018025869 A1 WO 2018025869A1
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fstl1
cancer
dip2a
patient
expression level
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Japanese (ja)
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千恵 工藤
雅義 豊浦
有希子 石田
雄二 庄屋
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株式会社ファーマフーズ
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to a method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient, a method for determining the start of administration of an FSTL1 inhibitor to a cancer patient, and a method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient. It relates to the kit.
  • companion diagnosis is to assist the optimal medication by examining biomarkers related to the efficacy and side effects of the drug before the drug is administered and predicting the patient's individual responsiveness to the drug before the treatment. .
  • biomarkers related to the efficacy and side effects of the drug There are no limitations on the testing method, and genetic abnormality testing, gene expression testing, tissue testing of proteins and metabolites, blood component testing, and the like are used.
  • Pharmaceutical companies around the world are developing companion diagnostics using biomarkers at the same time as developing new drugs. In the oncology field, the development of innovative therapies such as cancer immunotherapy and cancer vaccine therapy using immune checkpoint inhibitors is progressing, and the effectiveness of specific drugs and the individual differences in the occurrence of side effects are ascertained.
  • the establishment of an appropriate companion diagnostic method that assists in determining drug validity and dosage will continue to be an increasingly important issue for research and development of new anticancer drugs.
  • FSTL1 Follistatin-like protein 1
  • MSC cancer-related mesenchymal stem cells
  • FSTL1 is secreted mainly in the arthritic joint matrix, and it has been reported that FSTL1 secretion is stimulated by IL-1 ⁇ and that FSTL1 can be a useful biomarker for systemic juvenile rheumatoid arthritis (non- Patent Document 2) and FSTL1 as a biomarker for inflammatory diseases such as juvenile rheumatoid arthritis and Kawasaki disease have been filed (Patent Documents 2 and 3).
  • FSTL1 binds to various receptors through the cell membrane (FSTL1 receptor) and affects various physiological actions (Non-patent Document 3).
  • DIP2A disco-interacting protein 2 homolog A
  • FSTL1 receptor present in the endothelial cell membranes of various tissues (Non-patent Document 4), and according to the human protein expression information database (Human Protein Inatlas), In normal tissues, expression is observed in some tissues such as smooth muscle, female genital organs, gastrointestinal tract, and in cancer tissues, it is detected in colorectal cancer, breast cancer, prostate cancer, lung cancer, liver cancer and the like.
  • One of the challenges of cancer treatment is to identify whether the tumor is a malignant tumor with high metastasis by biopsy, and to determine the appropriate treatment policy based not only on the primary lesion but also on the presence or possibility of metastasis. Is to decide.
  • An anti-FSTL1 antibody that is an FSTL1 inhibitor not only inhibits MSC that induces immune disruption, but also inhibits metastasis of cancer cells, and thus may be an effective anticancer agent against malignant tumors that are prone to metastasis.
  • the development of biomarkers useful for companion diagnosis such as the effectiveness of anti-FSTL1 antibody treatment, is strongly desired.
  • the present invention finds a biomarker useful for companion diagnosis for selecting a cancer patient that can be expected to have a therapeutic effect by an FSTL1 inhibitor, a method for predicting the therapeutic effect by an FSTL1 inhibitor in a cancer patient, It is an object of the present invention to provide a method for determining the start of administration of an FSTL1 inhibitor and a kit for predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients.
  • the present invention includes the following inventions in order to solve the above problems.
  • a method for predicting a therapeutic effect of an FSTL1 inhibitor in a cancer patient (1) measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient; (2) a step of comparing the measured value with a reference value, and (3) a step of determining that a therapeutic effect can be expected when the measured value is higher than the reference value,
  • a method comprising the steps of: [2] The method according to [1], wherein in the step (1), the expression levels of both FSTL1 and DIP2A are measured.
  • [3] The method according to [1] or [2], wherein the sample is a sample containing cancer cells of a patient or blood of the patient.
  • [7] A method for determining the start of administration of an FSTL1 inhibitor to a cancer patient, (1) measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient; (2) comparing the measured value with a reference value, and (3 ′) determining the start of administration of the FSTL1 inhibitor when the measured value is higher than the reference value, A method comprising the steps of: [8] The method according to [7], wherein in the step (1), the expression levels of both FSTL1 and DIP2A are measured. [9] The method according to [7] or [8] above, wherein the sample is a sample containing cancer cells of the patient or the blood of the patient.
  • the sample containing cancer cells of the patient is a biopsy cancer tissue or a cancer tissue obtained by excision surgery.
  • the expression level of FSTL1 and / or DIP2A and the blood FSTL1 concentration in a biopsy cancer tissue or a cancer tissue obtained by excision are measured, and the blood FSTL1 concentration is measured [7] ] Method.
  • the expression level of FSTL1 and / or DIP2A and the blood DIP2A soluble type concentration in a biopsy cancer tissue or a cancer tissue obtained by excision are measured. The method according to [7] above.
  • kits for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient comprising a reagent for measuring the expression level of FSTL1 and / or DIP2A in a sample collected from the patient.
  • the reagent contains one or more selected from anti-FSTL1 antibody, anti-DIP2A antibody, FSTL1 RT-PCR primer set, and DIP2A RT-PCR primer set Kit.
  • the present invention also includes the following aspects.
  • a method for providing information on the therapeutic effect of an FSTL1 inhibitor against cancer comprising measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a cancer patient.
  • a method for providing the cancer patient with information on when to start administration of an FSTL1 inhibitor which comprises measuring the expression level of FSTL1 and / or DIP2A in a sample collected from the cancer patient.
  • a method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient a method for determining the start of administration of an FSTL1 inhibitor to a cancer patient, and a method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient Kits can be provided.
  • FIG. It is a figure which shows the result of having administered anti-FSTL1 antibody to the mouse
  • both FSTL1 and DIP2A are strongly expressed in mouse cancer cell lines (B16-F10, CT26 and 3LL) in which anti-FSTL1 antibody has been confirmed to be successful in a tumor-bearing mouse model. confirmed.
  • the present invention provides a method for predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients.
  • the therapeutic effect prediction method of the present invention only needs to include the following steps (1) to (3).
  • (1) A step of measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient (2)
  • a therapeutic effect can be expected when the measured value is higher than the reference value
  • the therapeutic effect prediction method of the present invention may include steps other than the above steps (1) to (3) as long as the object of the present invention can be achieved, and the content thereof is not limited.
  • cancer there are no particular limitations on the type of cancer of the cancer patient for which the therapeutic effect is predicted, including skin cancer, colon cancer, breast cancer, lymphoma, lung cancer, prostate cancer, kidney cancer, head and neck cancer, and esophagus.
  • Examples of the substance that inhibits the binding between FSTL1 and its receptor include an antibody (anti-FSTL1 antibody) or peptide that specifically binds to FSTL1, or an antibody or peptide that specifically binds to the FSTL1 receptor.
  • a substance that inhibits the binding between FSTL1 and its receptor is preferable, and an anti-FSTL1 antibody is more preferable.
  • step (1) the expression level of FSTL1 and / or DIP2A in a sample collected from a patient is measured.
  • the sample collected from the patient is not particularly limited as long as it can measure the expression level of FSTL1 or the expression level of DIP2A.
  • tissues obtained by biopsy or surgery blood, tissue fluid, lymph fluid, bone marrow, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, feces, ascites, pleural effusion, etc .; nasal cavity, bronchial, skin, Examples of the washing liquid after washing various organs and bones;
  • a sample containing cancer cells or blood is preferred.
  • the sample containing cancer cells is preferably a biopsy cancer tissue or a cancer tissue obtained by excision.
  • the sample for measuring the expression level of FSTL1 and the sample for measuring the expression level of DIP2A may be the same or different. If it is a sample which can measure an expression level, it will not specifically limit.
  • the sample for measuring the expression level of FSTL1 is blood containing cancer cells
  • the sample for measuring the expression level of DIP2A is a biopsy cancer tissue or a cancer tissue obtained by excision surgery. It is done.
  • step (1) only the expression level of FSTL1 may be measured or only the expression level of DIP2A may be measured, but it is preferable to measure the expression levels of both FSTL1 and DIP2A. It has been confirmed that the accuracy of predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients is improved by judging the combination of both expression levels. Moreover, since FSTL1 is a secreted protein, the expression level of FSTL1 can be evaluated based on the blood FSTL1 concentration. Therefore, as an aspect of measuring the expression level of FSTL1, only the expression level of FSTL1 in a sample containing cancer cells (cancer tissue) may be measured, or only the FSTL1 concentration in blood may be measured. May be measured.
  • an aspect of measuring only the expression level of FSTL1 an aspect of measuring only the blood FSTL1 concentration, an aspect of measuring only the expression level of DIP2A; the expression level of FSTL1 and the blood FSTL1 concentration
  • An embodiment for measuring both FSTL1 expression level and DIP2A expression level an embodiment for measuring blood FSTL1 concentration and DIP2A expression level; an FSTL1 expression level, DIP2A expression level and blood FSTL1 concentration
  • concentration are mentioned.
  • a method for measuring the expression level in a sample containing cancer cells is not particularly limited, a method for measuring the amount of target protein in the sample, and a method for measuring the amount of mRNA (target mRNA) of a gene encoding the target protein in the sample.
  • a method for measuring the expression level using an immunohistochemical method can be preferably used.
  • a sample containing cancer cells it is preferable to use a biopsy cancer tissue or a cancer tissue obtained by excision surgery.
  • the amount of target protein can be measured by extracting a protein from a sample by a known method and using a known protein measurement method such as Western blotting, EIA, ELISA, or RIA.
  • the target mRNA amount is measured by extracting RNA from a sample by a known method and using a known mRNA amount measuring method such as Northern blotting, RT-PCR, quantitative RT-PCR, or RNase protection assay. be able to.
  • a known mRNA amount measuring method such as Northern blotting, RT-PCR, quantitative RT-PCR, or RNase protection assay.
  • the RT-PCR method or the quantitative RT-PCR method is preferable, and the quantitative RT-PCR method is more preferable.
  • a method of preparing a tissue specimen according to a conventional method and performing immunostaining using an antibody that specifically binds to the target protein can be suitably used.
  • the sample is preferably a biopsy cancer tissue or a cancer tissue obtained by excision surgery.
  • any of the fluorescent antibody method, the enzyme antibody method, and the metal-labeled antibody method may be used, but the fluorescent antibody method is preferred.
  • the fluorescent antibody method may be performed by a direct method or an indirect method.
  • the fluorescence intensity of the antibody on the specimen indicates the expression level of the target protein.
  • the fluorescence intensity can be measured using a commercially available pixel counter or the like.
  • the expression level of FSTL1 can be measured by immunostaining using an anti-FSTL1 antibody
  • the expression level of DIP2A can be measured by immunostaining using an anti-DIP2A antibody.
  • FSTL1 and DIP2A can also be immunofluorescently stained using antibodies labeled with different fluorescent substances (double staining).
  • the in situ hybidization method can be used to measure the amount of target mRNA in the tissue specimen.
  • the blood FSTL1 concentration can be measured, for example, by separating serum from blood collected from a patient and quantifying the serum FSTL1 concentration using a known method such as ELISA. It has been confirmed that the blood FSTL1 concentration is high in patients suffering from highly metastatic cancer and advanced in degree of progression. Therefore, it can be expected that the therapeutic effect of the FSTL1 inhibitor in the metastatic lesion is predicted by performing the determination by combining the measured values of the blood FSTL1 concentration. Specifically, for example, in a cancer patient whose primary lesion has been completely removed, when the FSTL1 and / or DIP2A expression level of the resected cancer tissue is high and the blood FSTL1 concentration is also high, the primary lesion has already been removed.
  • the amino acid sequences of human FSTL1 and human DIP2A and the base sequence of the encoding gene can be obtained from a known database such as GenBank.
  • the NCBI accession number of the base sequence of the gene encoding human FSTL1 is NM_007085, and the NCBI accession number of the amino acid sequence of human FSTL1 is NP_009016.
  • the NCBI accession number of the nucleotide sequence of the gene encoding human DIP2A is NM_015151
  • the NCBI accession number of the amino acid sequence of human DIP2A is NP_055966.
  • step (2) the measured value of the expression level of FSTL1 and / or DIP2A obtained in step (1) is compared with each reference value.
  • the reference value the measured value of the expression level of FSTL1 and / or DIP2A in the healthy subject sample measured simultaneously in the step (1) can be used.
  • you may set arbitrary reference values based on the accumulated measurement value (health person accumulation data) of the expression level of FSTL1 and / or DIP2A in a healthy person sample. Therefore, the reference value in the present specification may be a measurement value obtained by simultaneously measuring the expression level of FSTL1 and / or DIP2A in a healthy subject sample or an accumulated measurement value, and the blood FSTL1 concentration of a healthy subject may be measured simultaneously.
  • step (3) when the measured value of the expression level of FSTL1 and / or DIP2A obtained in step (1) is higher than the reference value, it is determined that the cancer patient can expect a therapeutic effect by the FSTL1 inhibitor. .
  • the therapeutic effect can be expected when the measured value is five times higher than the reference value, and the therapeutic effect is expected when the measured value is ten times higher than the reference value. It is more preferable to determine that it is possible.
  • a therapeutic effect can be expected when the measured value is 1.5 times higher than the reference value, It is more preferable to determine that a therapeutic effect can be expected when the measured value is twice or more higher than the reference value.
  • the therapeutic effect prediction method of the present invention is based on the determination that the therapeutic effect in step (3) can be expected.
  • the sample provider is a patient whose primary lesion has been excised
  • the expression level of FSTL1 and / or DIP2A in the provided sample early detection of metastasis and early treatment with an FSTL1 inhibitor Information can be provided.
  • the expression of FSTL1 and / or DIP2A is significantly enhanced in the early stage cancer, it is possible to select an early stage cancer patient who can expect a therapeutic effect by the FSTL1 inhibitor with high accuracy.
  • FSTL1 in a sample containing cancer cells cancer tissue
  • DIP2A in a sample containing cancer cells cancer tissue
  • blood FSTL1 concentration blood FSTL1 concentration
  • this invention also provides the parameter
  • the present invention provides a method for determining the start of administration of an FSTL1 inhibitor to a cancer patient.
  • the administration start determination method of the present invention only needs to include the following steps (1), (2) and (3 ′).
  • (1) A step of measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient
  • (2) A step of comparing the measured value with a reference value (3 ′) When the measured value is higher than the reference value, the FSTL1 inhibitor Step of determining the start of administration
  • the present invention provides a kit for predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients. Since the kit of the present invention measures the expression level of FSTL1 and / or DIP2A, it contains either or both of a reagent for measuring the expression level of FSTL1 and a reagent for measuring the expression level of DIP2A. What is necessary is just to include the reagent for measuring the expression level of DIP2A preferably. Examples of the reagent for measuring the expression level of DIP2A include an anti-DIP2A antibody and a primer set for RT-PCR of DIP2A. The kit of the present invention preferably contains one or both of these. The anti-DIP2A antibody can be used for immunohistochemical staining, and the primer set can be used for quantification of DIP2A mRNA.
  • kit of the present invention contains an anti-DIP2A antibody
  • a secondary antibody a blocking reagent, a washing buffer, an instruction manual, etc.
  • a primer set for DIP2A RT-PCR in addition to this, a DNA synthase, a nucleotide serving as a substrate for the DNA synthase, an amplification reaction solution, a reaction tube, a sample preparation reagent, It is preferable to include an instruction manual and the like.
  • the kit of the present invention can also be suitably used for carrying out the method for determining the start of administration of the FSTL1 inhibitor to the cancer patient of the present invention. Therefore, the kit of the present invention can be rephrased as a kit for determining the start of administration of an FSTL1 inhibitor to cancer patients.
  • Example 1 Expression analysis of FSTL1 and DIP2A in high metastasis human cancer cell line and low metastasis human cancer cell line
  • FIG. 1 shows double-stained images of FSTL1 and DIP2A excluding DAPI nuclear staining.
  • Panc1-mock (upper left) contained a small amount of DIP2A positive cells, but FSTL1 showed only faint staining in the nuclei of some cells.
  • Panc1-Snail + (upper right), both FSTL1 and DIP2A were strongly positive in almost all cells.
  • DIP2A was basically expressed on the cell membrane, but accumulated with FSTL1 in the periphery of the nucleus, and this was also observed in the nucleus in some cells.
  • Example 2 Expression analysis of FSTL1 and DIP2A of a mouse cancer cell line in which anti-FSTL1 antibody is successful in a tumor-bearing model
  • Snail forced expression cell line of mouse melanoma B16-F10 (F10-snail +) 3.
  • Mouse colon cancer cell line CT26 4 Mouse colon cancer cell line CT26 4).
  • Mouse lung cancer cell line 3LL Using the same PE-labeled FSTL1 antibody and DIP2A antibody (Santa Cruz; combined with FITC-labeled secondary antibody) as in Example 1, immunostaining and observation were performed in the same manner as in Example 1.
  • Anti-FSTL1 Clone # 8-1 (1-2) Experimental procedure When Snail-positive tumor cells were transplanted subcutaneously and intravenously in mice, they metastasized not only to various organs but also to the bone marrow, which increased mesenchymal stem cells (MSC) starting from the bone marrow. It is known that the induction of anti-tumor immunity is strongly suppressed systemically (Cancer Research 73: 6185, 2013). Therefore, 5 ⁇ 10 5 GFP positive Snail positive B16-F10 tumor cells introduced by forced introduction of GFP gene and mouse Snail gene were subcutaneously injected into C57BL / 6N mice, and 1 ⁇ 10 5 cells were injected into the tail vein.
  • MSC mesenchymal stem cells
  • anti-FSTL1 antibody (# 6-55, # 7-34, # 8-1) prepared to 1 mg / mL with physiological saline or the Mouse IgG (anti-DNP antibody), which is an isotype, was intraperitoneally administered at a dose of 10 mg / kg as a control antibody. Saline was intraperitoneally administered to the untreated group.
  • Various immunological assays were performed 14 days after cell transplantation (Day 14). Subcutaneous tumor diameters were measured 7 days, 11 days, and 14 days after cell transplantation to calculate the tumor volume, and the inhibitory effect on subcutaneous tumor growth was evaluated. As an assay, the number of GFP positive Snail positive B16-F10 tumor cells in bone marrow cells, the number of CD45 negative cells in bone marrow cells, and body weight were measured.
  • FIG. 3 shows the results of the antitumor effect of the anti-FSTL1 antibody. All three anti-FSTL1 antibodies used significantly suppressed the growth of subcutaneous tumors relative to the control antibody.
  • the numerical value in parentheses is the P value when compared with the untreated group
  • the numerical value shown in the horizontal line is the cell transplantation day 14 between the group with one end of the horizontal line and the group with the other end. P value when compared later is shown.
  • FIG. 4 shows the effect on bone metastasis (left: GFP-positive Snail-positive B16-F10 tumor cells in the bone marrow), the effect on MSC increase (middle: the number of CD45-negative cells in the bone marrow), the effect on weight loss ( Right).
  • Anti-FSTL1 antibodies # 6-55 and # 8-1 showed an effect of suppressing weight loss.
  • all of the three types of anti-FSTL1 antibodies decreased MSC and showed bone metastasis inhibitory effect.
  • FIG. 5 shows the results of the antitumor effect
  • FIG. 6 shows the results of the antimetastatic effect.
  • Anti-FSTL1 antibody (# 6-55) suppressed CT26 subcutaneous tumor growth and lung metastasis very strongly. Specifically, the solid tumor disappeared in 3 out of 5 mice, and the number of lung metastases was very small (the average number of nodules in the control antibody group was 14 and the number of nodules in the anti-FSTL1 antibody group was 0). ⁇ 3).
  • the numerical value shown in the place of a horizontal line in FIG. 5 shows P value at the time of a cell transplant 14 days after a cell transplantation between the group with the one end of a horizontal line, and the group with the other end.
  • FIG. 7 shows the results of the antitumor effect
  • FIG. 8 shows the results of the effect on weight loss.
  • Anti-FSTL1 antibody (# 6-55) strongly suppressed the growth of 3LL subcutaneous tumors. Specifically, solid tumors disappeared in 2 out of 5 mice. In addition, no metastasis was observed in any organ. In the anti-FSTL1 antibody group, weight loss, thinning, and fluffing were not observed at all, and all mice were healthy.
  • Example 2 From the results of Example 2 and Reference Examples 1, 2, and 3, it can be confirmed that the anti-FSTL1 antibody has an antitumor effect against a mouse model transplanted with mouse cancer cells highly expressing FSTL1 and DIP2A. It was. In Example 1, since it was confirmed that both FSTL1 and DIP2A are highly expressed in human cancer cells with high metastasis, such a cancer that highly expresses FSTL1 and DIP2A was developed. The anti-FSTL1 antibody can be expected to have a therapeutic effect on existing cancer patients.
  • FSTL1 / DIP2A co-positive small Snail-induced MSC sMSC
  • large numbers of FSTL1 / DIP2A co-positive large cancer stem cell (CSC) -like tumor cells were also increased.
  • the vertical axis is the FSTL1 expression level and the horizontal axis is the DIP2A expression level, and the correlation between the expression in each tumor tissue has been confirmed.
  • FIG. 11 shows the results of analyzing the expression levels of FSTL1 and DIP2A in the above 6 cancer types according to the progression stage (stage I-IV).
  • black circles ( ⁇ ) indicate average values of expression levels in each stage, and white circles ( ⁇ ) indicate individual values. From this analysis result, it was revealed that both FSTL1 and DIP2A were enhanced in expression in stages I to III, and FSTL1 was particularly enhanced in stage I.
  • stage I the co-expression of FSTL1 and DIP2A at the same time is significantly enhanced compared to the other stages, and both molecules regulate cancer metastasis, so cancer cells are exactly It is speculated that FSTL1 and DIP2A expression is enhanced in the primary lesion to be detached, and that cancer progression and metastasis are promoted. From these results, it was suggested at the clinical level that detecting the expression of FSTL1 and DIP2A, particularly the co-expression of both molecules and their correlation, may lead to early detection and early treatment of cancer metastasis.
  • Example 4 Expression analysis of FSTL1 and DIP2A in tumor tissue derived from patients with advanced stage melanoma
  • a tumor tissue derived from an advanced stage melanoma patient a section set (Human malignant melanoma, product number CK2) containing a primary tumor tissue and a metastatic tumor tissue was purchased from Super Bio Chip, and the same method as in Example 3 was used. Immunostaining of FSTL1 and DIP2A was performed, and the expression intensity was analyzed by measuring the pixel count.
  • FIG. 13 shows the analysis of the expression levels of FSTL1 and DIP2A in the primary lesion by various classifications, but no significant correlation was found in age, sex, patient survival month, and the like.
  • FIG. 13 shows the analysis of the expression levels of FSTL1 and DIP2A in the primary lesion by various classifications, but no significant correlation was found in age, sex, patient survival month, and the like.
  • DIP2A is higher than the average value.
  • Patients with high expression and high expression of FSTL1 were also observed to have
  • Example 5 Measurement of serum FSTL1 concentration in various cancer patients
  • Serum of various types skin cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, breast cancer
  • stage IV terminal cancer patients
  • FSTL1 concentration in the serum was measured using an R & D Systems DuoSet ELISA Human FSTL1 (R & D Systems, Cat # DY1694). The measurement was performed according to the product protocol.
  • Example 6 Efficacy evaluation of anti-FSTL1 antibody using mouse osteosarcoma cell transplantation model
  • FIG. 15 shows the results of the antitumor effect
  • FIG. 16 shows the results of measuring the FSTL1 concentration in serum.
  • the anti-FSTL1 antibody (# 6-55) suppressed the growth of subcutaneous tumors and decreased the serum FSTL1 concentration to support the antitumor effect. From this, it was suggested that anti-FSTL1 antibody may be effective for osteosarcoma.

Abstract

La présente invention concerne un procédé de prédiction de l'effet thérapeutique d'un inhibiteur de FSTL1 chez un patient cancéreux, ledit procédé étant caractérisé en ce qu'il comprend les étapes de : (1) mesure du taux d'expression de FSTL1 et/ou DIP2A dans un échantillon prélevé à partir du patient ; (2) comparaison d'une valeur de mesure à une valeur de référence ; et (3) détermination que l'effet thérapeutique est prédit lorsque la valeur de mesure est supérieure à la valeur de référence. Par exemple, selon la présente invention, un diagnostic compagnon pour prédire l'efficacité ou similaire d'un traitement avec un inhibiteur de FSTL1 peut être effectué de manière appropriée.
PCT/JP2017/027916 2016-08-01 2017-08-01 Biomarqueur pour prédiction de l'effet thérapeutique d'un inhibiteur de fstl1 chez un patient cancéreux WO2018025869A1 (fr)

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