Method for predicting prognosis after treatment with angiogenesis inhibitor, and combination therapy for cancer

Abstract

The present disclosure includes: a biomarker for predicting the prognosis of a subject having cancer containing IGFBP-1 after a treatment of the subject with an angiogenesis inhibitor; a method for predicting the prognosis of a subject having cancer after a treatment of the subject with an angiogenesis inhibitor, the method including measuring the amount of IGFBP-1 in a specimen obtained from the subject after the treatment with the angiogenesis inhibitor; a composition and a kit, each of which contains an IGFBP-1 detection reagent and is used for the prediction of the prognosis of a subject having cancer after a treatment of the subject with an angiogenesis inhibitor; a pharmaceutical composition, a kit and a method, each of which is used for the treatment of cancer, and in each of which an angiogenesis inhibitor and an IGFBP-1 inhibitor are used in combination; and others.

Description

Method for predicting prognosis after treatment with angiogenesis inhibitors and combination cancer therapy

[0001] This application claims priority to Japanese Patent Application No. 2021-106928, the entire contents of which are incorporated herein by reference. The present disclosure includes a method for predicting prognosis after treatment with an angiogenesis inhibitor and a combination cancer therapy.

[0002] Angiogenesis is essential for the progression and growth of cancer, especially solid tumors. Recent advances in molecular biology have revealed various molecules involved in the induction of angiogenesis by tumor cells, and drugs targeting these molecules, i.e., angiogenesis inhibitors, are widely used in clinical practice. Although angiogenesis inhibitors have provided beneficial results to many cancer patients, their full potential has not been realized due to issues such as the lack of effective biomarkers to predict therapeutic efficacy and drug resistance.

[0003] One object of the present disclosure is to provide biomarkers, methods, compositions, and kits for predicting prognosis after treatment with angiogenesis inhibitors.A further object of the present disclosure is to provide pharmaceutical compositions, kits, and methods for treating cancer.

[0004] In one aspect, the present disclosure relates to biomarkers for predicting the prognosis of a subject with cancer following treatment with an anti-angiogenic drug, including IGFBP-1.

[0005] In one aspect, the present disclosure relates to a method for predicting the prognosis of a subject having cancer after treatment with an angiogenesis inhibitor, the method comprising measuring the amount of IGFBP-1 in a sample obtained from the subject after treatment with the angiogenesis inhibitor.

[0006] In one aspect, the present disclosure relates to a composition comprising an IGFBP-1 detection reagent for predicting the prognosis of a subject with cancer after treatment with an angiogenesis inhibitor.

[0007] In one aspect, the present disclosure relates to a kit comprising an IGFBP-1 detection reagent for predicting the prognosis of a subject with cancer after treatment with an angiogenesis inhibitor.

[0008] In one aspect, the present disclosure relates to a pharmaceutical composition for treating cancer, comprising an angiogenesis inhibitor in combination with an IGFBP-1 inhibitor, an IGFBP-1 inhibitor in combination with an angiogenesis inhibitor, or an angiogenesis inhibitor and an IGFBP-1 inhibitor.

[0009] In one aspect, the present disclosure relates to a kit for treating cancer, the kit comprising a composition comprising an angiogenesis inhibitor and a composition comprising an IGFBP-1 inhibitor.

[0010] In one aspect, the present disclosure relates to a method for treating cancer, comprising administering to a subject in need thereof an angiogenesis inhibitor and an IGFBP-1 inhibitor.

[0011] The present disclosure provides biomarkers, methods, compositions, and kits for predicting prognosis after treatment with angiogenesis inhibitors. Additionally, the present disclosure provides pharmaceutical compositions, kits, and methods for treating cancer.

[0012] Figure 1 shows CT values ​​(top) and serum IGFBP-1 levels (ng / ml) (bottom) before and one month after lenvatinib treatment in 31 patients with advanced hepatocellular carcinoma. Pre-LEN indicates the results before lenvatinib treatment, and Post-LEN indicates the results after lenvatinib treatment. (Student's t-test, **: p<0.01) (Comparisons between two groups were analyzed using Student's t-test, and comparisons between three or more groups were analyzed using one-way ANOVA. In the figure, * indicates p<0.05, and ** indicates p<0.01.) Figure 2 shows the correlation (Pearson correlation coefficient) between the rate of change in serum IGFBP-1 levels and the rate of change in CT values ​​during lenvatinib treatment. Figure 3 shows the correlation (Log-rank test) between serum IGFBP-1 levels before lenvatinib treatment (top), serum IGFBP-1 levels after treatment (middle), or the percentage change in IGFBP-1 levels before and after treatment (bottom) and survival time. Figure 4 shows the time course of tumor volume (left), as well as the macroscopic appearance of tumors (middle) and tumor weight (right) (day 14 of treatment) in hepatocellular carcinoma model mice treated with lenvatinib or sorafenib. VT indicates vehicle treatment, LEN indicates lenvatinib, and SORA indicates sorafenib (the same applies in the following figures). Figure 5 shows serum IGFBP-1 levels (left), as well as IGFBP-1 mRNA expression (middle) and protein expression (right) in tumors in hepatocellular carcinoma model mice treated with lenvatinib or sorafenib. Figure 6 shows the expression of IGFBP-1, CD31, and CA9 in tumor tissue from hepatocellular carcinoma model mice treated with lenvatinib or sorafenib. The top photograph shows the results of immunostaining, and the bottom graphs show the IGFBP-1 positive signal (%) per field (left), the number of CD31-positive cells (center), and the CA9-positive area (%) (right). Figure 7 shows the expression of IGFBP-1 and CD31 in the liver of hepatocellular carcinoma model mice treated with lenvatinib. The left photograph shows the results of immunostaining, and the right graphs show the IGFBP-1 positive signal (%) per field (top) and the number of CD31-positive cells (bottom). Figure 8 shows the expression of IGFBP-1 mRNA in Hep-55.1C cells after lenvatinib treatment.Figure 9 shows IGFBP-1 mRNA (top) and protein (bottom) expression in Hep-55.1C cells cultured under normoxia or hypoxia conditions. Figure 10 shows the proliferation of HuH7 cells (left) or HepG2 cells (right) cultured for 48 hours in the presence of IGFBP-1 (0, 250, or 500 ng / ml). Figure 11 shows endothelial cell proliferation (top) and tube formation (bottom) in the presence of IGFBP-1. Figure 12 shows endothelial cell proliferation (top) and tube formation (bottom) in the presence of lenvatinib and IGFBP-1. Figure 13 shows tube formation in the presence of an angiogenesis inhibitor selected from lenvatinib, sorafenib, a VEGFR inhibitor, or an FGFR inhibitor, and IGFBP-1. Figure 14 shows the expression of IGFBP-1, HIF-1α, and HIF-2α in Hep-55.1C cells under normoxia and hypoxia, as well as the effect of the HIF inhibitor YC-1 on these levels. Figure 15 shows the expression of IGFBP-1, HIF-1α, and HIF-2α in HuH7 cells under normoxia and hypoxia, as well as the effect of the HIF inhibitor YC-1 on these levels. Figure 16 shows the expression of IGFBP-1, HIF-1α, and HIF-2α in HepG2 cells under normoxia and hypoxia, as well as the effect of the HIF inhibitor YC-1 on these levels. Figure 17 shows the phosphorylation of FAK and ERK-1 / 2 by IGFBP-1 in HUVEC cells. Figure 18 shows IGFBP-1-induced HUVEC cell proliferation in the presence of an integrin α5β1 inhibitor (left) or a FAK inhibitor (right). Figure 19 shows IGFBP-1-induced tube formation in the presence of an integrin α5β1 inhibitor or a FAK inhibitor. Figure 20 shows the macroscopic appearance of tumors (top), tumor weight (bottom left) (day 14), and changes in tumor volume over time (bottom right) after administration of lenvatinib in a hepatocellular carcinoma model mouse transplanted with IGFBP-1-deficient HuH7 cells. IGFBP-1-KD indicates IGFBP-1-deficient tumors.Figure 21 shows the macroscopic appearance of tumors (top) and tumor weight (bottom left) (day 14) and the change in tumor volume over time (bottom right) after administration of lenvatinib in a mouse model of hepatocellular carcinoma transplanted with IGFBP-1-deficient HepG2 cells. IGFBP-1-KD indicates an IGFBP-1-deficient tumor. Figure 22 shows the macroscopic appearance of tumors (top) and tumor weight (bottom left) (day 14) and the change in tumor volume over time (bottom right) after administration of lenvatinib, an anti-IGFBP-1 antibody, or a combination thereof in a mouse model of hepatocellular carcinoma. Figure 23 shows the change in tumor volume over time after administration of lenvatinib, an integrin α5 / β1 inhibitor, or a combination thereof in a mouse model of hepatocellular carcinoma.

[0013] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0014] In one aspect, the present disclosure relates to a method for predicting the prognosis of a subject having cancer after treatment with an angiogenesis inhibitor, the method comprising measuring the amount of IGFBP-1 in a sample obtained from the subject after treatment with the angiogenesis inhibitor.

[0015] Insulin-like growth factor binding protein 1 (IGFBP1) is a secreted protein known to bind to insulin-like growth factors (IGFs), particularly IGF-I and IGF-II, and regulate their activity and distribution. IGFBP1 is also known to bind to integrin α5 / β1 on the cell membrane via its RGD (Arg-Gly-Asp) domain. The Genbank Gene ID for human IGFBP1 is 3484, and its amino acid sequence is available under Genbank Accession No. NP_000587, while its mRNA sequence is available under Genbank Accession No. NM_000596.

[0016] The sample is, but is not limited to, a blood sample or a cancer cell or cancer tissue sample. Blood samples include whole blood, plasma, and serum. In the present disclosure, a sample obtained from a subject is used to mean a sample collected from a subject and subjected to processing necessary for measurement, such as protein or nucleic acid separation or concentration, freezing, or fixation. Collection of a sample from a subject and subsequent processing can be performed appropriately by those skilled in the art. In one embodiment, the sample is serum.

[0017] As used herein, the subject is a mammal. Mammals include, for example, mice, rats, rabbits, cats, dogs, sheep, pigs, horses, cows, monkeys, and humans. In one embodiment, the subject is a human.

[0018] A sample is obtained from a subject after treatment with an angiogenesis inhibitor. The sample is obtained, for example, after 1 to 6 months, 1 to 3 months (e.g., 1, 2, or 3 months), 1 to 5 weeks (e.g., 1, 2, 3, 4, or 5 weeks), or 1 to 7 days (e.g., 1, 2, 3, 4, 5, 6, or 7 days) of treatment with an angiogenesis inhibitor. Samples may be obtained at multiple time points after initiation of treatment. In one embodiment, a sample is obtained after 1 month of treatment with an angiogenesis inhibitor. Additionally, a sample may be obtained before treatment with an angiogenesis inhibitor. In one embodiment, samples are obtained before treatment with an angiogenesis inhibitor and after 1 month of treatment with an angiogenesis inhibitor.

[0019] Measurement of the amount of IGFBP-1 in a sample can be performed by those skilled in the art as appropriate, and the measurement method is not particularly limited. The amount of IGFBP-1 can be the amount of IGFBP-1 protein or mRNA. For example, the amount of IGFBP-1 can be measured by using a substance that specifically binds to IGFBP-1 (e.g., an antibody, an aptamer (e.g., a DNA aptamer, an RNA aptamer, or a peptide aptamer)), or by using IGFBP-1-specific primers or probes (e.g., primers that specifically amplify IGFBP-1 mRNA or cDNA, or probes that specifically bind to IGFBP-1 mRNA or cDNA) prepared based on the nucleic acid sequence of IGFBP-1. In one embodiment, the amount of IGFBP-1 is measured using an anti-IGFBP-1 antibody. Those skilled in the art can appropriately prepare the antibody, aptamer, primer, and probe based on the amino acid sequence and nucleic acid sequence of IGFBP-1. Measurement methods include immunological assays such as ELISA (Enzyme-Linked Immunosorbent Assay), EIA (Enzyme Immuno Assay), RIA (Radioimmunoassay), Western blot, dot blot, quantitative PCR, mass spectrometry, etc. Those skilled in the art can appropriately process samples obtained from subjects depending on the measurement method.

[0020] The amount of IGFBP-1 after treatment with an angiogenesis inhibitor or the rate of change in the amount of IGFBP-1 before and after treatment correlates with the prognosis of the subject, and it has been shown that the higher the amount of IGFBP-1 after treatment or the greater the increase in the amount of IGFBP-1 after treatment relative to the amount before treatment, the worse the prognosis. Therefore, measuring the amount of IGFBP-1 after treatment with an angiogenesis inhibitor can predict the prognosis.

[0021] As used herein, prognosis refers to the medical outlook for the course of cancer subject after treatment with anti-angiogenic drugs or the subject's remaining life expectancy.In one embodiment, prognosis prediction is prediction of overall survival or progression-free survival.Poor prognosis can mean that overall survival or progression-free survival is short, and good prognosis can mean that overall survival or progression-free survival is long.

[0022] In one embodiment, the method of the present disclosure includes comparing the amount of IGFBP-1 after treatment with an angiogenesis inhibitor with a reference value. In this embodiment, the reference value may be an IGFBP-1 amount that can statistically significantly separate a group of subjects with cancer after treatment with an angiogenesis inhibitor into those with a good prognosis and those with a poor prognosis. The statistical significance may be analyzed by any test method. Examples of such test methods include the log-rank test. The reference value may be a value that can separate a certain percentage (e.g., 10%, 20%, 30%, 40%, or 50%) of subjects with the highest IGFBP-1 amount in a group of subjects after treatment with an angiogenesis inhibitor. In one embodiment, if the amount of IGFBP-1 after treatment with an angiogenesis inhibitor is equal to or greater than the reference value, the subject is predicted to have a poor prognosis after treatment with an angiogenesis inhibitor. In one embodiment, when the amount of IGFBP-1 after treatment with an angiogenesis inhibitor is equal to or lower than the reference value, the subject is predicted to have a good prognosis after treatment with an angiogenesis inhibitor. In one embodiment, the reference value is a value within the range of 30,000 ng / mL to 60,000 ng / ml or 40,000 ng / mL to 50,000 ng / ml.

[0023] In one embodiment, the method of the present disclosure includes comparing the rate of change in IGFBP-1 levels after angiogenesis inhibitor treatment relative to the IGFBP-1 levels before treatment with a reference value. The rate of change can be calculated using the following formula: Rate of change in IGFBP-1 levels = IGFBP-1 levels after angiogenesis inhibitor treatment / IGFBP-1 levels before angiogenesis inhibitor treatment. In this embodiment, the reference value may be a rate of change in IGFBP-1 levels that can statistically significantly separate a group of subjects with good prognosis from a group with poor prognosis after treatment with angiogenesis inhibitors. Statistical significance may be analyzed using any test method, such as the log-rank test. Alternatively, the reference value may be a value that separates a certain percentage (e.g., 10%, 20%, 30%, 40%, or 50%) of the highest rate of change in IGFBP-1 levels for each subject in a group of subjects after angiogenesis inhibitor treatment. In one embodiment, the reference value is a value within the range of 1.5 to 3 or 2 to 2.5. In one embodiment, when the rate of change is equal to or greater than the reference value or is higher than the reference value, the subject is predicted to have a poor prognosis after treatment with an angiogenesis inhibitor. In one embodiment, when the rate of change is equal to or less than the reference value or is lower than the reference value, the subject is predicted to have a good prognosis after treatment with an angiogenesis inhibitor.

[0024] The IGFBP-1 level after treatment with an angiogenesis inhibitor may be compared with multiple reference values. For example, the prognosis of each subject in a group of subjects after treatment with an angiogenesis inhibitor may be ranked into three or more levels, and two or more IGFBP-1 levels or their change rates that can separate the groups with statistical significance may be used as reference values. This allows the prognosis rank of a subject after treatment with an angiogenesis inhibitor to be predicted. Statistical significance may be analyzed by any test method. Examples of test methods include the log-rank test. Alternatively, the reference values ​​may be multiple values ​​that can separate a certain percentage (e.g., 10%, 20%, 30%, 40%, or 50%) from the top of the IGFBP-1 levels or their change rates for each subject in the group of subjects after treatment with an angiogenesis inhibitor.

[0025] It has been shown that IGFBP-1 expression in tumors and secretion into the blood are increased by hypoxic stimulation, and that IGFBP-1 promotes proliferation and lumen formation in vascular endothelial cells, suggesting that increased IGFBP-1 is involved in resistance to drugs that induce ischemia or hypoxia. Therefore, the method of the present disclosure can be used to predict prognosis after treatment with angiogenesis inhibitors that induce ischemia or hypoxia in tumors.

[0026] Cancers include liver cancer (including hepatocellular carcinoma and intrahepatic cholangiocarcinoma), esophageal cancer, stomach cancer, small intestine cancer, colon cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer), breast cancer, germ cell cancer, gallbladder cancer, head and neck cancer, skin cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, ovarian cancer, thyroid cancer, gallbladder cancer, brain tumor, thymic cancer, malignant melanoma, leukemia, myelodysplastic syndrome, multiple myeloma, and malignant lymphoma. In one embodiment, the cancer is hepatocellular carcinoma.

[0027] Antiangiogenic agents include vascular endothelial growth factor (VEGF) inhibitors, vascular endothelial growth factor receptor (VEGFR) inhibitors (including inhibitors of one or more of VEGFR-1, VEGFR-2, and VEGFR-3), soluble VEGFRs, kinase inhibitors, fibroblast growth factor receptor (FGFR) inhibitors (including inhibitors of one or more of FGFR-1, FGFR-2, FGFR-3, and FGFR-4), and mammalian target of rapamycin (mTOR) inhibitors. VEGF inhibitors include anti-VEGF antibodies (e.g., bevacizumab, ranibizumab). VEGFR inhibitors include fruquintinib, brivanib, and anti-VEGFR antibodies (e.g., ramucirumab). Soluble VEGFRs include aflibercept. Kinase inhibitors include lenvatinib, sorafenib, axitinib, sunitinib, pazopanib, regorafenib, and cabozantinib. FGFR inhibitors include AZD4547, erdafitinib, pemigatinib, futibatinib, and anti-FGFR antibodies (e.g., bofatamab). mTOR inhibitors include sirolimus, everolimus, and temsirolimus. In one embodiment, the angiogenesis inhibitor is a kinase inhibitor, a VEGFR inhibitor, or an FGFR inhibitor. In one embodiment, the angiogenesis inhibitor is lenvatinib, sorafenib, fruquintinib, or AZD4547. In this specification, the names of each drug are used to include its pharmaceutically acceptable salts. For example, lenvatinib includes lenvatinib mesylate, and sorafenib includes sorafenib tosylate. In the present disclosure, the term "antibody" means a molecule comprising an immunoglobulin or a portion thereof that has antigen-binding ability, and is used to include not only molecules in the form of natural immunoglobulins, but also molecules of various structures such as chimeric antibodies, humanized antibodies, multispecific antibodies, and antibody fragments.

[0028] As described above, in one aspect, the present invention provides a biomarker, including IGFBP-1, for predicting the prognosis of a subject with cancer after treatment with an angiogenesis inhibitor.

[0029] In one aspect, the present disclosure provides compositions and kits containing an IGFBP-1 detection reagent for predicting the prognosis of a cancer subject after treatment with an angiogenesis inhibitor. The compositions and kits can be used to practice the methods of the present disclosure. The IGFBP-1 detection reagent can include a substance that specifically binds to IGFBP-1 (e.g., an antibody, an aptamer (e.g., a DNA, RNA, or peptide aptamer)), or a primer or probe specific to IGFBP-1. In one embodiment, the IGFBP-1 detection reagent is an anti-IGFBP-1 antibody. The compositions and kits can be used for measurement by immunological assays such as ELISA, EIA, RIA, Western blot, and dot blot, quantitative PCR, mass spectrometry, and the like. In addition to the IGFBP-1 detection reagent, the kits can also include reagents, controls, buffers, or containers required depending on the measurement method.

[0030] It has been shown that the combined use of an angiogenesis inhibitor and an IGFBP-1 inhibitor can produce a synergistic antitumor effect. In one aspect, the present disclosure provides a combination therapy of an angiogenesis inhibitor and an IGFBP-1 inhibitor in the treatment of cancer.

[0031] In the present disclosure, an IGFBP-1 inhibitor refers to a substance that inhibits signal transduction from IGFBP-1 and may be a small molecule compound, a protein, a peptide, a nucleic acid, or the like. The IGFBP-1 inhibitor may be a substance that binds to IGFBP-1 or a substance that suppresses IGFBP-1 expression. The IGFBP-1 inhibitor may also be a substance that inhibits the binding of IGFBP-1 to IGF or a substance that inhibits the binding of IGFBP-1 to integrin α5 / β1. In one embodiment, the IGFBP-1 inhibitor is an antibody or an aptamer (e.g., a DNA, RNA, or peptide aptamer). In one embodiment, the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody. In one embodiment, the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody that inhibits the binding of IGFBP-1 to integrin α5 / β1. Anti-IGFBP-1 antibodies can be appropriately prepared by those skilled in the art using standard methods. In another embodiment, the IGFBP-1 inhibitor is a peptide containing the amino acid sequence RGD (referred to as an RGD peptide) that inhibits binding to integrin α5 / β1 via the RGD domain. RGD peptides include GRGDNP (CAS: 114681-65-1) (SEQ ID NO: 1) and RGD (CAS: 99896-85-2). The angiogenesis inhibitor may be any of the angiogenesis inhibitors exemplified herein.

[0032] Pharmaceutical compositions can be formulated by conventional methods. In addition to the active ingredient, pharmaceutical compositions may contain pharmaceutically acceptable carriers or additives such as sterilized water, physiological saline, stabilizers, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, etc. Dosage forms include tablets, powders, granules, capsules, pills, liquids, syrups, suspensions, emulsions, suppositories, injections, etc.

[0033] The dosage, administration schedule, and administration method of the angiogenesis inhibitor and IGFBP-1 inhibitor can be appropriately determined by one skilled in the art based on factors such as the characteristics of the inhibitor, the type of cancer, age, weight, and condition of the subject. For example, if the angiogenesis inhibitor is sorafenib, it can be administered at a dose of 200 to 400 mg, once to several times (e.g., 1, 2, 3, or 4 times) per day, every day or every few days (2, 3, or 4 days). If the angiogenesis inhibitor is lenvatinib, it can be administered at a dose of 4 to 24 mg (e.g., 8, 12, or 24 mg), once to several times (e.g., 1, 2, 3, or 4 times) per day, every day or every few days (2, 3, or 4 days). When the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody, it can be administered at a dose of 10 μg / kg to 100 mg / kg, 100 μg / kg to 10 mg / kg, or 1 mg / kg to 10 mg / kg daily, either daily or every few days (2, 3, or 4 days). Administration methods include oral and parenteral administration, and parenteral administration includes subcutaneous, intradermal, intramuscular, and intravenous administration.

[0034] In the present disclosure, the combined use of an angiogenesis inhibitor and an IGFBP-1 inhibitor means that they are used to treat the same subject at the same time. The administration schedules of the angiogenesis inhibitor and the IGFBP-1 inhibitor may be the same or different. The angiogenesis inhibitor and the IGFBP-1 inhibitor may be contained in the same composition or in different compositions, and a composition containing an angiogenesis inhibitor and a composition containing an IGFBP-1 inhibitor may be combined and provided as a kit.

[0035] In one embodiment, the present disclosure provides a method for treating cancer, comprising administering an angiogenesis inhibitor and an IGFBP-1 inhibitor to a subject in need of treatment.In one embodiment, the present disclosure provides an angiogenesis inhibitor for use in treating cancer, wherein the angiogenesis inhibitor is combined with an IGFBP-1 inhibitor; an IGFBP-1 inhibitor for use in treating cancer, wherein the IGFBP-1 inhibitor is combined with an angiogenesis inhibitor; and a combination of an angiogenesis inhibitor and an IGFBP-1 inhibitor for use in treating cancer.In one embodiment, the present disclosure provides the use of an angiogenesis inhibitor for the manufacture of a medicament for use in treating cancer, wherein the angiogenesis inhibitor is combined with an IGFBP-1 inhibitor; the use of an IGFBP-1 inhibitor for the manufacture of a medicament for use in treating cancer, wherein the IGFBP-1 inhibitor is combined with an angiogenesis inhibitor; and the use of a combination of an angiogenesis inhibitor and an IGFBP-1 inhibitor for the manufacture of a medicament for use in treating cancer. These aspects can be implemented in accordance with the description of the pharmaceutical compositions of the present disclosure.

[0036] Exemplary embodiments of the present disclosure are set forth below.

[0037] [1] A method for predicting the prognosis of a subject having cancer after treatment with an angiogenesis inhibitor, the method comprising measuring the amount of IGFBP-1 in a sample obtained from the subject after treatment with the angiogenesis inhibitor. [2] The method described in [1], wherein the sample is a blood sample. [3] The method described in [2], wherein the blood sample is serum. [4] The method described in any one of [1] to [3], wherein the cancer is hepatocellular carcinoma. [5] The method described in any one of [1] to [4], wherein the amount of IGFBP-1 after the treatment is compared with a reference value. [6] The method described in [5], wherein the subject is predicted to have a poor prognosis after treatment with the angiogenesis inhibitor if the amount of IGFBP-1 after the treatment is higher than the reference value. [7] The method described in any one of [1] to [4], further comprising measuring the amount of IGFBP-1 before treatment with the angiogenesis inhibitor in a sample obtained from the subject. [8] The method described in [7], further comprising comparing the rate of change in the amount of IGFBP-1 after the treatment relative to the amount of IGFBP-1 before the treatment with a reference value. [9] The method according to claim 8, wherein the subject is predicted to have a poor prognosis after treatment with the angiogenesis inhibitor when the rate of change is higher than the reference value.

[10] The method according to any one of claims 1 to 9, wherein the angiogenesis inhibitor is a VEGF inhibitor, a VEGFR inhibitor, a soluble VEGFR, a kinase inhibitor, an FGFR inhibitor, or an mTOR inhibitor.

[11] The method according to any one of claims 1 to 10, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fruquintinib, or AZD4547.

[0038]

[12] A biomarker for predicting the prognosis of a subject with cancer after treatment with an angiogenesis inhibitor, including IGFBP-1.

[0039]

[13] A composition comprising an IGFBP-1 detection reagent for predicting the prognosis of a cancer subject after treatment with an angiogenesis inhibitor.

[14] The composition according to 13, wherein the IGFBP-1 detection reagent is an antibody, an aptamer, a primer, or a probe.

[15] The composition according to 13, wherein the IGFBP-1 detection reagent is an anti-IGFBP-1 antibody.

[0040]

[16] A kit for predicting the prognosis of a cancer subject after treatment with an angiogenesis inhibitor, comprising an IGFBP-1 detection reagent.

[17] The kit according to 16, wherein the IGFBP-1 detection reagent is an antibody, an aptamer, a primer, or a probe.

[18] The kit according to 16, wherein the IGFBP-1 detection reagent is an anti-IGFBP-1 antibody.

[0041]

[19] A pharmaceutical composition for treating cancer, comprising an angiogenesis inhibitor in combination with an IGFBP-1 inhibitor, an IGFBP-1 inhibitor in combination with an angiogenesis inhibitor, or an angiogenesis inhibitor and an IGFBP-1 inhibitor.

[20] The pharmaceutical composition according to 19, comprising an angiogenesis inhibitor in combination with an IGFBP-1 inhibitor.

[21] The pharmaceutical composition according to 19, comprising an IGFBP-1 inhibitor in combination with an angiogenesis inhibitor.

[22] The pharmaceutical composition according to 19, comprising an angiogenesis inhibitor and an IGFBP-1 inhibitor.

[23] The pharmaceutical composition according to any one of 19 to 22, wherein the angiogenesis inhibitor is a VEGF inhibitor, a VEGFR inhibitor, a soluble VEGFR, a kinase inhibitor, an FGFR inhibitor, or an mTOR inhibitor.

[24] The pharmaceutical composition according to any one of 19 to 23, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fruquintinib, or AZD4547.

[25] The pharmaceutical composition according to any one of 19 to 24, wherein the IGFBP-1 inhibitor is a substance that inhibits the binding of IGFBP-1 to integrin α5 / β1.

[26] The pharmaceutical composition according to any one of 19 to 25, wherein the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody.

[27] The pharmaceutical composition according to any one of 19 to 26, wherein the cancer is hepatocellular carcinoma.

[0042]

[28] A kit for treating cancer, comprising a composition containing an angiogenesis inhibitor and a composition containing an IGFBP-1 inhibitor.

[0043]

[29] A method for treating cancer, comprising administering an angiogenesis inhibitor and an IGFBP-1 inhibitor to a subject in need of treatment.

[30] The method according to the above item 29, wherein the angiogenesis inhibitor is a VEGF inhibitor, a VEGFR inhibitor, a soluble VEGFR, a kinase inhibitor, an FGFR inhibitor, or an mTOR inhibitor.

[31] The method according to the above item 29 or 30, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fruquintinib, or AZD4547.

[32] The method according to any one of the above items 29 to 31, wherein the IGFBP-1 inhibitor is a substance that inhibits the binding of IGFBP-1 to integrin α5 / β1.

[33] The method according to any one of the above items 29 to 32, wherein the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody.

[34] The method according to any one of the above items 29 to 33, wherein the cancer is hepatocellular carcinoma.

[0044] Specific examples will be described below, but these examples are not intended to limit the invention described in the claims in any way.

[0045] Of 251 patients with advanced hepatocellular carcinoma who underwent lenvatinib treatment (body weight 60 kg or more: 12 mg / day daily, body weight less than 60 kg: 8 mg / day daily), 31 patients who underwent contrast-enhanced CT scans one month before and after treatment were selected. Using serum samples stored before and after lenvatinib treatment, a comprehensive search for changes in 55 angiogenesis-related proteins was performed using the Angiogenesis Assay Kit (R&D Systems). IGFBP-1 was identified as a protein with a high expression level and a large percentage change before and after lenvatinib treatment.

[0046] The CT values ​​of the tumor were determined before and after treatment, and the rate of change in CT values ​​was calculated as follows. The average value of three different areas was used for the CT values. Pre-CT value = a / b (Tumor / Aorta) a: CT value of the tumor before treatment b: CT value of the aorta before treatment Post-CT value = c / d (Tumor / Aorta) c: CT value of the tumor after treatment d: CT value of the aorta after treatment Rate of change in CT values ​​= Post-CT value / Pre-CT value Furthermore, the rate of change in serum IGFBP-1 levels before and after treatment was calculated using the following formula. Rate of change in serum IGFBP-1 levels = Serum IGFBP-1 level after treatment / Serum IGFBP-1 level before treatment

[0047] After lenvatinib treatment, CT values ​​decreased and serum IGFBP-1 values ​​increased (Figure 1). Focusing on each individual case, a correlation was observed between the degree of decrease in CT values ​​and the degree of increase in IGFBP-1 values ​​after lenvatinib treatment (Figure 2). It was shown that cases with higher IGFBP-1 values ​​after treatment were more likely to have ischemia within the tumor (lower CT values).

[0048] The survival time of 31 patients was stratified by serum IGFBP-1 levels before and after lenvatinib treatment, and the rate of change in IGFBP-1 levels before and after treatment. Patients were divided into two groups based on the median (before treatment: 22,006 ng / mL, after treatment: 44,081 ng / mL, rate of change: 2.05). Patients with higher IGFBP-1 levels after lenvatinib treatment and patients with greater increases in IGFBP-1 levels due to lenvatinib treatment were found to have a worse prognosis (Figure 3).

[0049] For in vivo experiments, hepatocellular carcinoma cell line (Hep-55.1C) (5 x 10 6 C57BL / 6 mice were subcutaneously inoculated with lenvatinib (10 mg / kg / day) or sorafenib (30 mg / kg / day) for 14 days to create a mouse model of hepatocellular carcinoma. After treatment, tumors were excised and measured. Serum IGFBP-1 was assessed by ELISA, and tumor IGFBP-1 mRNA and protein expression were assessed by qPCR and Western blotting, respectively.

[0050] Lenvatinib and sorafenib reduced tumor volume and weight, indicating that tumor growth was suppressed (Figure 4). Furthermore, administration of lenvatinib and sorafenib increased serum IGFBP-1 levels and increased IGFBP-1 expression in tumors at both the gene and protein levels (Figure 5).

[0051] We also evaluated the expression of IGFBP-1, CD31, and CA9 (carbonic anhydrase IX) in tumor tissue of a mouse model of hepatocellular carcinoma after lenvatinib administration by immunohistochemistry. CD31 is an angiogenesis marker, and CA9 is a hypoxia marker for hepatocellular carcinoma. Lenvatinib or sorafenib treatment reduced CD31 expression and increased CA9 expression, indicating that lenvatinib treatment reduced tumor blood vessels and created a hypoxic environment (Figure 6). However, in the liver, where IGFBP-1 is primarily produced, no increase in IGFBP-1 was observed after lenvatinib treatment (Figure 7). These findings suggest that IGFBP-1, which increases in response to hypoxia within the tumor, may contribute to resistance to lenvatinib or sorafenib.

[0052] For in vitro experiments, Hep-55.1C cells were cultured in the presence of lenvatinib (0-10 μM) under normoxia for 24 hours. IGFBP-1 expression was then assessed by qPCR. mRNA expression was normalized to GAPDH expression. Lenvatinib treatment did not significantly increase IGFBP-1 expression in Hep-55.1C cells (Figure 8).

[0053] Hep-55.1C cells were cultured under normoxic (20% O2) or hypoxic (2% O2) conditions for 24 hours, and IGFBP-1 expression was assessed by qPCR and Western blotting. mRNA and protein expression were normalized by the expression of β-actin (ATCB) or GAPDH. Hypoxia-induced increases in IGFBP-1 expression at both the gene and protein levels were observed in Hep-55.1C cells (Fig. 9).

[0054] To evaluate the effect of IGFBP-1 on cancer cells, hepatocellular carcinoma cell lines HuH7 cells or HepG2 cells (5 × 10 3 Cancer cells were cultured in the presence of IGFBP-1 (0, 250, or 500 ng / ml) for 48 hours, and the proliferation rate was assessed using the MTT assay. Direct application of IGFBP-1 to cancer cells did not result in an increase in cell proliferation (Figure 10).

[0055] To evaluate the effect of IGFBP-1 on vascular endothelial cells, 1 × 10 human vascular endothelial cells (HUVEC) were used. 3 The cells were cultured for 48 hours in the presence of IGFBP-1 (0, 100, 250 ng / ml), and the proliferation rate was evaluated.The tube formation ability of HUVECs was also evaluated in the presence of IGFBP-1 (0, 100, 250 ng / ml) using a tube formation assay.

[0056] IGFBP-1 was shown to enhance vascular endothelial cell proliferation and lumen formation (Figure 11), and IGFBP-1 was also shown to enhance vascular endothelial cell proliferation and lumen formation in the presence of lenvatinib (Figure 12).

[0057] The effect of IGFBP-1 on vascular endothelial cell proliferation and tube formation was evaluated in the presence of angiogenesis inhibitors other than lenvatinib. Tube formation assays were performed as described above in the presence of IGFBP-1 (250 ng / ml) and angiogenesis inhibitors selected from lenvatinib (3 μM), sorafenib (3 μM), the VEGFR-1, 2, 3 inhibitor (fruquintinib) (1 μM), and the FGFR-1, 2, 3 inhibitor (AZD4547) (1 μM). IGFBP-1 was shown to enhance angiogenesis in the presence of all angiogenesis inhibitors (Figure 13).

[0058] We investigated the signaling pathway involved in the increased expression of IGFBP-1 in response to hypoxia. Hep-55.1C cells, HuH7 cells, or HepG2 cells (1 × 10 5Cells were cultured for 24 hours under normoxic (20% O2) or hypoxic (2% O2) conditions in the presence or absence of the HIF inhibitor YC-1 (Lificiguat) (5 μM). Expression of IGFBP-1, HIF-1α, and HIF-2α was assessed by Western blot. Expression of each protein was normalized to that of tubulin.

[0059] IGFBP-1 expression was elevated by hypoxic stimulation, and this elevation was suppressed by the addition of YC-1 (Figs. 14-16). These results suggest that IGFBP-1 expression is elevated by hypoxic stimulation, particularly through signaling pathways mediated by HIF-1α and HIF-2α.

[0060] Furthermore, we investigated the signaling pathway by which IGFBP-1 expression promotes proliferation and tube formation in vascular endothelial cells (HUVECs) (1 × 10 5 After serum-free culture for 12 hours, IGFBP-1 (250 ng / ml) was added, and the phosphorylation of FAK and ERK-1 / 2 was evaluated 5 and 15 minutes later. The expression of each protein was normalized by the expression of GAPDH. HUVECs (5 × 10 3 HUVECs (1 × 10 cells) were cultured for 48 hours in the presence of IGFBP-1 (250 ng / ml) in the presence or absence of various concentrations (0, 1, 3, 10 μM) of an integrin α5 / β1 inhibitor (ATN-161) (CAS No.: 262438-43-7) or a FAK inhibitor (FAK inhibitor 14) (CAS No.: 4506-66-5), and proliferation assays were performed using the MTT assay. 3 cells) were cultured in the presence of IGFBP-1 (250 ng / ml) and an integrin α5 / β1 inhibitor (3 μM) or an FAK inhibitor (10 μM), and a tube formation assay was performed.

[0061] IGFBP-1 enhanced the phosphorylation of FAK and ERK-1 / 2 (Fig. 17). Furthermore, integrin α5β1 and FAK inhibitors suppressed IGFBP-1-induced enhancement of endothelial cell proliferation and tube formation (Figs. 18 and 19). These findings suggest that IGFBP-1 promotes endothelial cell proliferation and angiogenesis through signal transduction via integrin α5 / β1, FAK, and ERK.

[0062] IGFBP-1 was deleted in HuH7 cells or HepG2 cells, and these IGFBP-1-deficient cells (5 × 10 6 Balb / c mice were subcutaneously inoculated with either lenvatinib (10 mg / kg / day) or GFP-expressing cells (control) to create hepatocellular carcinoma model mice. After 14 days of treatment with lenvatinib (10 mg / kg / day), tumors were excised and measured.

[0063] IGFBP-1-deficient tumors showed stronger growth inhibition by lenvatinib than control tumors (Figures 20 and 21), indicating that loss of IGFBP-1 expression increases sensitivity to lenvatinib.

[0064] Balb / c mice were implanted with HepG2 cells as described above, and then treated with lenvatinib (10 mg / kg / day) alone, anti-IGFBP-1 antibody (IGFBP1 Monoclonal Antibody; ThermoFisher Catalog # MA5-23727) (0.03 mg / kg / day) alone, or a combination of these for 14 days. Tumors were then excised and measured.

[0065] In this experiment, lenvatinib or an anti-IGFBP-1 antibody alone did not significantly inhibit tumor growth, but their combined use significantly inhibited tumor growth (Figure 22), demonstrating that the combined use of lenvatinib and an anti-IGFBP-1 antibody enhanced the antitumor effect.

[0066] Similarly, Balb / c mice were implanted with HepG2 cells and treated with lenvatinib (10 mg / kg / day) alone, the integrin α5 / β1 inhibitor (ATN-161) (CAS No: 262438-43-7) (1 mg / kg / day) alone, or a combination of these for 14 days. Tumors were then excised and measured.

[0067] In this experiment, lenvatinib or an integrin α5 / β1 inhibitor alone did not significantly inhibit tumor growth, but their combination significantly inhibited tumor growth (Figure 23). The combined use of lenvatinib and an integrin α5 / β1 inhibitor demonstrated enhanced antitumor effects. These results suggest that IGFBP-1 promotes angiogenesis by binding to integrin α5 / β1 expressed on the cell membrane of vascular endothelial cells, and that inhibition of the binding of IGFBP-1 to integrin α5 / β1 is important for enhancing the antitumor effect.

Claims

1. A method for predicting the prognosis of a subject having cancer after treatment with an angiogenesis inhibitor, the method comprising measuring the amount of IGFBP-1 in a sample obtained from the subject after treatment with the angiogenesis inhibitor.

2. The method of claim 1 , wherein the sample is a blood sample.

3. The method of claim 2 , wherein the blood sample is serum.

4. The method of claim 1 , wherein the cancer is hepatocellular carcinoma.

5. The method of claim 1, further comprising comparing the amount of IGFBP-1 after said treatment with a reference value.

6. The method of claim 5, wherein the subject is predicted to have a poor prognosis after treatment with the angiogenesis inhibitor if the amount of IGFBP-1 after treatment is higher than the reference value.

7. The method of claim 1, further comprising measuring the amount of IGFBP-1 in a sample obtained from the subject prior to treatment with the angiogenesis inhibitor.

8. The method of claim 7, further comprising comparing the rate of change in the amount of IGFBP-1 after the treatment relative to the amount of IGFBP-1 before the treatment with a reference value.

9. The method of claim 8, wherein the subject is predicted to have a poor prognosis after treatment with the angiogenesis inhibitor if the rate of change is higher than the reference value.

10. 2. The method of claim 1, wherein the angiogenesis inhibitor is a VEGF inhibitor, a VEGFR inhibitor, a soluble VEGFR, a kinase inhibitor, an FGFR inhibitor, or an mTOR inhibitor.

11. The method of claim 1, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fruquintinib, or AZD4547.

12. A composition comprising an IGFBP-1 detection reagent for predicting the prognosis of a subject having cancer after treatment with an angiogenesis inhibitor.

13. A kit for predicting the prognosis of a subject having cancer after treatment with an angiogenesis inhibitor, comprising an IGFBP-1 detection reagent.

14. A biomarker for predicting prognosis following treatment with an anti-angiogenic drug in a subject with cancer, comprising IGFBP-1.

15. A pharmaceutical composition for treating cancer, comprising: Contains angiogenesis inhibitors and is used in combination with IGFBP-1 inhibitors; Contains an IGFBP-1 inhibitor in combination with an antiangiogenic agent, or including angiogenesis inhibitors and IGFBP-1 inhibitors, Pharmaceutical compositions.

16. The pharmaceutical composition of claim 15, wherein the angiogenesis inhibitor is a VEGF inhibitor, a VEGFR inhibitor, a soluble VEGFR, a kinase inhibitor, an FGFR inhibitor, or an mTOR inhibitor.

17. The pharmaceutical composition of claim 15, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fruquintinib, or AZD4547.

18. The pharmaceutical composition of claim 15 , wherein the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody.

19. The pharmaceutical composition according to claim 15, wherein the cancer is hepatocellular carcinoma.

20. A kit for treating cancer, comprising a composition comprising an angiogenesis inhibitor and a composition comprising an IGFBP-1 inhibitor.