WO2023276869A1 - Method for predicting prognosis after treatment with angiogenesis inhibitor, and combination therapy for cancer - Google Patents

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

Prognostic prediction method after treatment with angiogenesis inhibitors and cancer combination therapy

This application claims priority from Japanese Patent Application No. 2021-106928, which is incorporated herein in its entirety by reference.
The present disclosure includes methods for predicting prognosis after treatment with angiogenesis inhibitors, cancer combination therapy, and the like.

Cancer, especially solid tumors, require angiogenesis for their development and growth. Recent advances in molecular biology have elucidated various molecules involved in the induction of angiogenesis by tumor cells, and drugs that target them, ie, angiogenesis inhibitors, are widely used in clinical practice. Although anti-angiogenic drugs have brought beneficial results to many cancer patients, their maximum contribution has not been achieved due to problems such as the lack of biomarkers that are effective in predicting therapeutic effects and drug resistance. is.

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.

The present disclosure, in one aspect, relates to biomarkers, including IGFBP-1, for predicting the prognosis of a subject with cancer following treatment with an angiogenesis inhibitor.

The present disclosure provides, in one aspect, a method for predicting the prognosis of a subject with cancer after treatment with an angiogenesis drug, comprising: IGFBP- after treatment with an angiogenesis drug in a sample obtained from said subject; 1 relates to a method comprising measuring a quantity.

The present disclosure, in one aspect, 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.

The present disclosure, in one aspect, relates to a kit comprising an IGFBP-1 detection reagent for predicting the prognosis of a subject with cancer following treatment with an angiogenesis inhibitor.

The present disclosure provides, in one aspect, a pharmaceutical composition for treating cancer comprising:
including angiogenesis inhibitors, combined with IGFBP-1 inhibitors,
including an IGFBP-1 inhibitor combined with an angiogenesis inhibitor, or an angiogenesis inhibitor and an IGFBP-1 inhibitor;
It relates to pharmaceutical compositions.

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.

The present disclosure, in one aspect, relates to a method for treating cancer comprising administering an angiogenesis inhibitor and an IGFBP-1 inhibitor to a subject in need of treatment.

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.

Figure 1 shows the CT values (top) and serum IGFBP-1 levels (ng/ml) (bottom) before and one month after lenvatinib treatment in 31 cases of advanced hepatocellular carcinoma. Pre LEN indicates results before lenvatinib treatment, and Post LEN indicates results after lenvatinib treatment. (Student's t-test, **: p<0.01). (Hereafter, comparisons between two groups were analyzed by 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. are shown respectively.) Figure 2 shows the correlation (Pearson correlation coefficient) between the rate of change in serum IGFBP-1 value and the rate of change in CT value in lenvatinib treatment. Figure 3 shows the correlation between the serum IGFBP-1 level before lenvatinib treatment (top), the serum IGFBP-1 level after treatment (middle), or the rate of change in IGFBP-1 before and after treatment (bottom) and survival time (Log -rank test). FIG. 4 shows changes in tumor volume over time (left), gross observations of tumors (middle), and tumor weights (right) in hepatocellular carcinoma model mice administered with lenvatinib or sorafenib (day 14 of administration). VT indicates vehicle treatment, LEN indicates lenvatinib, SORA indicates sorafenib (same in following figures). FIG. 5 shows serum IGFBP-1 levels (left) in hepatocellular carcinoma model mice administered with lenvatinib or sorafenib, and IGFBP-1 mRNA expression (middle) and protein expression (right) in tumors. FIG. 6 shows the expression of IGFBP-1, CD31, and CA9 in tumor tissues of hepatocellular carcinoma model mice administered with lenvatinib or sorafenib. The upper photograph shows the results of immunostaining, and the lower graph shows the IGFBP-1 positive signal (%) per field of view (left), the number of CD31 positive cells (middle), and the CA9 positive area (%) (right). FIG. 7 shows the expression of IGFBP-1 and CD31 in the liver of hepatocellular carcinoma model mice to which lenvatinib was administered. The photograph on the left shows the results of immunostaining, and the graph on the right shows IGFBP-1-positive signal (%) per field of view (top) and the number of CD31-positive cells (bottom). FIG. 8 shows the expression of IGFBP-1 mRNA in Hep-55.1C cells after administration of lenvatinib. FIG. 9 shows IGFBP-1 mRNA (top) and protein (bottom) expression in Hep-55.1C cells cultured under normoxia or hypoxia conditions. Figure 10 shows proliferation of HuH7 cells (left) or HepG2 cells (right) cultured for 48 hours in the presence of IGFBP-1 (0, 250, 500 ng/ml). FIG. 11 shows vascular endothelial cell proliferation (top) and tube formation (bottom) in the presence of IGFBP-1. FIG. 12 shows vascular 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 a FGFR inhibitor and IGFBP-1. FIG. 14 shows the expression of IGFBP-1, HIF-1α, and HIF-2α in Hep-55.1C cells under normoxia and hypoxia conditions, and the expression of the HIF inhibitor YC-1 against this. show action. FIG. 15 shows the expression of IGFBP-1, HIF-1α, and HIF-2α in HuH7 cells under normoxia and hypoxia conditions, and the effect of the HIF inhibitor YC-1 on this. . FIG. 16 shows the expression of IGFBP-1, HIF-1α, and HIF-2α in HepG2 cells under normoxia and hypoxia conditions, and the effect of the HIF inhibitor YC-1 on this. . Figure 17 shows phosphorylation of FAK and ERK-1/2 by IGFBP-1 in HUVEC cells. FIG. 18 shows HUVEC cell proliferation by IGFBP-1 in the presence of integrin α5β1 inhibitor (left) or FAK inhibitor (right). FIG. 19 shows tube formation by IGFBP-1 in the presence of integrin α5β1 inhibitors or FAK inhibitors. Figure 20 shows macroscopic tumor findings (upper) and tumor weight (lower left) after administration of lenvatinib (day 14) and changes in tumor volume over time (right) in hepatocellular carcinoma model mice implanted with IGFBP-1-deficient HuH7 cells. below). IGFBP-1-KD indicates IGFBP-1 deficient tumors. Figure 21 shows gross observations of tumors (upper) and tumor weights (lower left) after administration of lenvatinib (day 14) and changes in tumor volume over time (right) in hepatocellular carcinoma model mice implanted with IGFBP-1-deficient HepG2 cells. below). IGFBP-1-KD indicates IGFBP-1 deficient tumors. Figure 22 shows macroscopic findings (upper) and tumor weight (lower left) of tumors (day 14) and tumor volume over time after administration of lenvatinib, anti-IGFBP-1 antibody, or a combination thereof in hepatocellular carcinoma model mice. Changes (lower right) are shown. FIG. 23 shows changes in tumor volume over time after administration of lenvatinib, an integrin α5/β1 inhibitor, or a combination thereof in hepatocellular carcinoma model mice.

Unless otherwise specified, the terms used in this specification have the meanings commonly understood by those skilled in the art in fields such as organic chemistry, medicine, pharmacy, molecular biology, and microbiology. Listed below are definitions for some terms used herein, which definitions supersede general understanding herein.

The present disclosure provides, in one aspect, a method for predicting the prognosis of a subject with cancer after treatment with an angiogenesis drug, comprising: IGFBP- after treatment with an angiogenesis drug in a sample obtained from said subject; 1 relates to a method comprising measuring a quantity.

Insulin-like Growth Factor Binding Protein 1 (IGFBP1) binds to insulin-like growth factors (IGFs) (particularly IGF-I and IGF-II) and regulates their activity and distribution. It is a secretory protein known to 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 of human IGFBP1 is 3484, the amino acid sequence is disclosed in Genbank Accession No. NP_000587, and the mRNA sequence is disclosed in Genbank Accession No. NM_000596, respectively.

The sample is, but is not limited to, for example, a blood sample or a cancer cell or cancer tissue sample. Blood samples include whole blood, plasma and serum. In the present disclosure, the term “sample obtained from a subject” is used in the sense of including a sample obtained from a subject that has undergone processing necessary for measurement, such as separation or concentration of proteins or nucleic acids, freezing, or fixation. Collection and subsequent processing of samples from subjects can be carried out as appropriate by those skilled in the art. In certain embodiments, the sample is serum.

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 human.

The sample is obtained from the subject after treatment with an anti-angiogenic drug. The sample may be, for example, 1-6 months, 1-3 months (eg, 1, 2, or 3 months), 1-5 weeks (eg, 1, 2, 3, 4, or 5 weeks), or 1 Obtained after ˜7 days (eg, 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, the sample is obtained after one month of anti-angiogenic drug treatment. Additionally, a pre-treatment sample with an anti-angiogenic drug may also be obtained. In one embodiment, the sample is obtained prior to anti-angiogenic drug treatment and after one month of anti-angiogenic drug treatment.

A person skilled in the art can appropriately measure the amount of IGFBP-1 in a sample, 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 determined by using a substance that specifically binds to IGFBP-1 (e.g., antibody, aptamer (e.g., DNA aptamer, RNA aptamer, peptide aptamer)), or by using the nucleic acid sequence of IGFBP-1. IGFBP-1-specific primers or probes (e.g., primers that specifically amplify IGFBP-1 mRNA or cDNA, probes that specifically bind to IGFBP-1 mRNA or cDNA) prepared according to can be measured by In one embodiment, the amount of IGFBP-1 is measured using an anti-IGFBP-1 antibody. A person skilled in the art can appropriately prepare the aforementioned antibodies, aptamers, primers and probes based on the amino acid and nucleic acid sequences of IGFBP-1. Measurement methods include ELISA (Enzyme-Linked Immunosorbent Assay), EIA (Enzyme Immuno Assay), RIA (Radioimmunoassay), immunological assays such as Western blot and dot blot, quantitative PCR, and mass spectrometry. A person skilled in the art can appropriately process the sample obtained from the subject according to the measurement method.

The amount of IGFBP-1 after treatment with angiogenesis inhibitors or the rate of change in the amount of IGFBP-1 before and after treatment correlates with the prognosis of the subject, and the higher the amount of IGFBP-1 after treatment or the amount of IGFBP-1 before treatment The greater the post-treatment increase in IGFBP-1 levels, the worse the prognosis. Therefore, prognosis can be predicted by measuring the amount of IGFBP-1 after treatment with angiogenesis inhibitors.

As used herein, prognosis means the medical outlook for the course of a subject with cancer after treatment with an angiogenesis inhibitor or the subject's life expectancy. In one embodiment, the prognostic prediction is prediction of overall survival or progression-free survival. Poor prognosis can mean short overall or progression-free survival, and good prognosis can mean long overall or progression-free survival.

In one embodiment, the method of the present disclosure includes comparing the amount of IGFBP-1 after treatment with an anti-angiogenic drug to a reference value. In this embodiment, the reference value separates a group of subjects with cancer after treatment with an angiogenesis inhibitor into a group with a good prognosis after treatment and a group with a poor prognosis with a statistically significant difference. can be the amount of IGFBP-1 that can Statistical significance may be analyzed by any test method. Test methods include, for example, log-rank test. The reference value should be a percentage (e.g., 10%, 20%, 30%, 40%, or 50%) that separates the top IGFBP-1 levels for each subject in the subject group after treatment with an angiogenesis inhibitor. can be any value. In one embodiment, the subject is predicted to have a poor prognosis after treatment with an anti-angiogenic drug if the amount of IGFBP-1 after treatment with an anti-angiogenic drug is greater than or equal to the reference value or is higher than the reference value. be. In one embodiment, the subject is predicted to have a favorable prognosis after treatment with an anti-angiogenic drug if the amount of IGFBP-1 following treatment with an anti-angiogenic drug is less than or equal to the reference value or is lower than the reference value. be done. In one embodiment, the reference value is a value within the range of 30000 ng/mL to 60000 ng/ml or 40000 ng/mL to 50000 ng/ml.

In one embodiment, the methods of the present disclosure comprise comparing the percent change in IGFBP-1 amount after anti-angiogenic drug treatment to IGFBP-1 amount before treatment with a reference value. The rate of change can be obtained by the following formula.

Percent change in IGFBP-1 amount = IGFBP-1 amount after treatment with antiangiogenic drug/IGFBP-1 amount before treatment with antiangiogenic drug

In this embodiment, the reference value separates a group of subjects with cancer after treatment with an angiogenesis inhibitor into a group with a good prognosis after treatment and a group with a poor prognosis with a statistically significant difference. can be the rate of change in the amount of IGFBP-1 that can be Statistical significance may be analyzed by any test method. Test methods include, for example, log-rank test. Alternatively, the reference value is a constant percentage (e.g., 10%, 20%, 30%, 40%, or 50%) of the top percent change in IGFBP-1 levels for each subject in a group of subjects after treatment with an angiogenesis inhibitor. %) may be used. In one embodiment, the reference value is a value within the range of 1.5-3 or 2-2.5. In one embodiment, the subject is predicted to have a poor prognosis after treatment with an anti-angiogenic drug if the percent change is greater than or equal to the reference value or higher than the reference value. In one embodiment, the subject is predicted to have a favorable prognosis after treatment with an anti-angiogenic drug if the percent change is less than or equal to the reference value or is lower than the reference value.

The amount of IGFBP-1 after treatment with angiogenesis inhibitors may be compared with multiple reference values. For example, the prognosis of each subject in the subject group after treatment with an angiogenesis inhibitor is ranked into 3 or more stages, and 2 or more IGFBP-1 amounts that can separate each ranked group with a statistically significant difference Alternatively, its rate of change may be used as a reference value. This can predict the prognostic rank of a subject after treatment with an angiogenesis inhibitor. Statistical significance may be analyzed by any test method. Test methods include, for example, log-rank test. Alternatively, the reference value is a constant percentage from the top of the IGFBP-1 amount or its rate of change for each subject in the subject group after treatment with an angiogenesis inhibitor (e.g., 10%, 20%, 30%, 40%, or 50%) can be separated from each other.

IGFBP-1 is expressed in tumors and secreted into the blood by hypoxic stimulation, and IGFBP-1 has been shown to enhance the proliferation and tube formation of vascular endothelial cells. It is suggested to be involved in resistance to drugs that induce ischemia or hypoxia. Therefore, the disclosed methods can be used for prognosis after treatment with angiogenesis inhibitors that induce tumor ischemia or hypoxia.

Cancers include liver cancer (including hepatocellular carcinoma and intrahepatic cholangiocarcinoma), esophageal cancer, gastric cancer, small bowel cancer, colon cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer), breast cancer, germ cell cancer, and 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, myelodysplasia syndrome, multiple myeloma, and malignant lymphoma. In one embodiment, the cancer is hepatocellular carcinoma.

Angiogenesis inhibitors include VEGF (vascular endothelial growth factor) inhibitors, VEGFR (vascular endothelial growth factor receptor) inhibitors (inhibitors of one or more of VEGFR-1, VEGFR-2, and VEGFR-3). ), soluble VEGFR, kinase inhibitors, fibroblast growth factor receptor (FGFR) inhibitors (including inhibitors of one or more of FGFR-1, FGFR-2, FGFR-3 and FGFR-4), mTOR ( mammalian target of rapamycin) inhibitors. VEGF inhibitors include anti-VEGF antibodies (eg, bevacizumab, ranibizumab). VEGFR inhibitors include fluquintinib, brivanib and anti-VEGFR antibodies (eg, ramucirumab). Soluble VEGFRs include aflibercept. Kinase inhibitors include lenvatinib, sorafenib, axitinib, sunitinib, pazopanib, regorafenib, cabozantinib. FGFR inhibitors include AZD4547, erdafitinib, pemigatinib, futivatinib, anti-FGFR antibodies (eg, bofatamab). mTOR inhibitors include sirolimus, everolimus, and temsirolimus. In one embodiment, the angiogenesis inhibitor is a kinase inhibitor, VEGFR inhibitor, or FGFR inhibitor. In one embodiment, the angiogenesis inhibitor is lenvatinib, sorafenib, fluquintinib, or AZD4547. In the present specification, the name of each drug is used in the sense of including its pharmaceutically acceptable salts. For example, lenvatinib includes lenvatinib mesylate and sorafenib includes sorafenib tosylate. In the present disclosure, the term antibody refers to a molecule comprising an immunoglobulin or a portion thereof that has the ability to bind to an antigen, and includes not only molecules in the form of naturally occurring immunoglobulins, but also chimeric antibodies, humanized antibodies, multispecific It is used in the sense of including molecules with various structures such as antibodies and antibody fragments.

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

The present disclosure, in one aspect, provides compositions and kits comprising IGFBP-1 detection reagents for predicting the prognosis of a subject with cancer after treatment with an angiogenesis inhibitor. Compositions and kits can be used to practice the disclosed methods. IGFBP-1 detection reagents include substances that specifically bind to IGFBP-1 (e.g., antibodies, aptamers (e.g., DNA, RNA, or peptide aptamers)), IGFBP-1-specific primers or probes. . In one embodiment, the IGFBP-1 detection reagent is an anti-IGFBP-1 antibody. The compositions and kits can be compositions and kits for measurements by immunological assays such as ELISA, EIA, RIA, western blot, dot blot, quantitative PCR, mass spectrometry and the like. In addition to the IGFBP-1 detection reagent, the kit may contain necessary reagents, controls, buffer solutions, containers, and the like depending on the measurement method.

It was shown that the combined use of an angiogenesis inhibitor and an IGFBP-1 inhibitor resulted in a synergistic antitumor effect. The disclosure provides, in one aspect, combination therapy with an angiogenesis inhibitor and an IGFBP-1 inhibitor in the treatment of cancer.

In the present disclosure, an IGFBP-1 inhibitor means a substance that inhibits signal transduction from IGFBP-1, and can be a low-molecular-weight compound, protein, peptide, nucleic acid, or the like. An IGFBP-1 inhibitor can be a substance that binds to IGFBP-1 or a substance that suppresses the expression of IGFBP-1. Also, the IGFBP-1 inhibitor can 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 aptamer (eg, 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 binding of IGFBP-1 to integrin α5/β1. An anti-IGFBP-1 antibody can be appropriately produced by a person skilled in the art by a conventional method. In another embodiment, the IGFBP-1 inhibitor is a peptide comprising the amino acid sequence RGD (referred to as the RGD peptide) that inhibits binding to integrin α5/β1 through the RGD domain. RGD peptides include GRGDNP (CAS: 114681-65-1) (SEQ ID NO: 1) and RGD (CAS: 99896-85-2). As an angiogenesis inhibitor, the angiogenesis inhibitors exemplified herein can be used.

A pharmaceutical composition can be formulated by a conventional method. Pharmaceutical compositions contain, in addition to the active ingredient, pharmaceutically acceptable agents such as sterile water, saline, stabilizers, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, and the like. carriers or additives used. Dosage forms include tablets, powders, granules, capsules, pills, liquids, syrups, suspensions, emulsions, suppositories, injections and the like.

The dosage, administration schedule and administration method of angiogenesis inhibitors and IGFBP-1 inhibitors can be appropriately determined by those skilled in the art based on factors such as the characteristics of the inhibitors, the target cancer type, age, body weight and condition. can. For example, when the angiogenesis inhibitor is sorafenib, 200 to 400 mg once to several times a day (e.g., 1, 2, 3, or 4 times) for consecutive days, or for several days (2, 3, or every 4 days). If the angiogenesis inhibitor is lenvatinib, 4 to 24 mg (e.g., 8, 12, or 24 mg) once to several times a day (e.g., 1, 2, 3, or 4 times) on consecutive days , or every few days (2, 3, or 4 days). If the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody, 10 μg/kg to 100 mg/kg, 100 μg/kg to 10 mg/kg, or 1 mg/kg to 10 mg/kg daily , or every few days (2, 3, or 4 days). Administration methods include oral administration and parenteral administration, and parenteral administration includes subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, and the like.

In the present disclosure, using an angiogenesis inhibitor and an IGFBP-1 inhibitor in combination means using them to treat the same subject at the same time. The dosing schedules of the angiogenesis inhibitor and 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 may be contained in different compositions, and the composition containing the angiogenesis inhibitor and the IGFBP-1 inhibitor are combined. may be provided as a kit in combination with the composition comprising.

In one aspect, the disclosure provides a method for treating cancer comprising administering an angiogenesis inhibitor and an IGFBP-1 inhibitor to a subject in need thereof.
In one aspect, the present disclosure provides an angiogenesis inhibitor for use in treating cancer, wherein the angiogenesis inhibitor is used in combination with an IGFBP-1 inhibitor; An IGFBP-1 inhibitor in combination with an angiogenesis inhibitor; and a combination of an angiogenesis inhibitor and an IGFBP-1 inhibitor for use in treating cancer.
In one aspect, the present disclosure is the use of an angiogenesis inhibitor for the manufacture of a medicament for use in treating cancer, wherein said angiogenesis inhibitor is combined with an IGFBP-1 inhibitor; use of an IGFBP-1 inhibitor for the manufacture of a medicament for use in the treatment of use of a combination of an angiogenesis inhibitor and an IGFBP-1 inhibitor for the manufacture of
These aspects can be carried out according to the description regarding the pharmaceutical composition of the present disclosure.

An exemplary embodiment of the present disclosure is shown below.

[1]
A method for predicting the prognosis of a subject with cancer following treatment with an anti-angiogenic drug, comprising measuring the amount of IGFBP-1 after treatment with an anti-angiogenic drug in a sample obtained from said subject. ,Method.
[2]
2. The method of 1, wherein the sample is a blood sample.
[3]
3. The method according to 2 above, wherein the blood sample is serum.
[4]
4. The method according to any one of 1 to 3 above, wherein the cancer is hepatocellular carcinoma.
[5]
5. The method of any one of 1 to 4, further comprising comparing said post-treatment IGFBP-1 amount to a reference value.
[6]
6. The method of 5, wherein the subject is predicted to have a poor prognosis after treatment with the angiogenesis inhibitor if the post-treatment IGFBP-1 level is higher than the reference value.
[7]
5. The method according to any one of 1 to 4 above, further comprising measuring the amount of IGFBP-1 in the sample obtained from the subject before treatment with the angiogenesis inhibitor.
[8]
8. The method of 7 above, further comprising comparing the rate of change in the amount of IGFBP-1 after treatment relative to the amount of IGFBP-1 before treatment with a reference value.
[9]
9. The method of 8, wherein the subject is predicted to have a poor prognosis after treatment with the anti-angiogenic drug if the rate of change is higher than the reference value.
[10]
10. The method according to any one of 1 to 9 above, wherein the angiogenesis inhibitor is a VEGF inhibitor, VEGFR inhibitor, soluble VEGFR, kinase inhibitor, FGFR inhibitor, or mTOR inhibitor.
[11]
11. The method according to any one of 1 to 10 above, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fluquintinib, or AZD4547.

[12]
Biomarkers to predict prognosis after treatment with anti-angiogenic drugs in subjects with cancer, including IGFBP-1.

[13]
A composition comprising an IGFBP-1 detection reagent for predicting the prognosis of a subject with cancer after treatment with an angiogenesis inhibitor.
[14]
14. The composition according to 13 above, wherein the IGFBP-1 detection reagent is an antibody, aptamer, primer or probe.
[15]
14. The composition according to 13 above, wherein the IGFBP-1 detection reagent is an anti-IGFBP-1 antibody.

[16]
A kit comprising an IGFBP-1 detection reagent for predicting the prognosis of a subject with cancer after treatment with an anti-angiogenic drug.
[17]
17. The kit according to 16 above, wherein the IGFBP-1 detection reagent is an antibody, aptamer, primer or probe.
[18]
17. The kit according to 16 above, wherein the IGFBP-1 detection reagent is an anti-IGFBP-1 antibody.

[19]
A pharmaceutical composition for treating cancer, comprising:
including angiogenesis inhibitors, combined with IGFBP-1 inhibitors,
including an IGFBP-1 inhibitor combined with an angiogenesis inhibitor, or an angiogenesis inhibitor and an IGFBP-1 inhibitor;
pharmaceutical composition.
[20]
20. The pharmaceutical composition according to 19 above, which comprises an angiogenesis inhibitor and is used in combination with an IGFBP-1 inhibitor.
[21]
20. The pharmaceutical composition according to 19 above, which comprises an IGFBP-1 inhibitor and is used in combination with an angiogenesis inhibitor.
[22]
20. The pharmaceutical composition according to 19 above, comprising an angiogenesis inhibitor and an IGFBP-1 inhibitor.
[23]
23. The pharmaceutical composition according to any one of 19 to 22 above, wherein the angiogenesis inhibitor is a VEGF inhibitor, VEGFR inhibitor, soluble VEGFR, kinase inhibitor, FGFR inhibitor, or mTOR inhibitor.
[24]
24. The pharmaceutical composition according to any one of 19 to 23 above, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fluquintinib, or AZD4547.
[25]
25. The pharmaceutical composition according to any one of 19 to 24 above, wherein the IGFBP-1 inhibitor is a substance that inhibits the binding of IGFBP-1 to integrin α5/β1.
[26]
26. The pharmaceutical composition according to any one of 19 to 25 above, wherein the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody.
[27]
27. The pharmaceutical composition according to any one of 19 to 26 above, wherein the cancer is hepatocellular carcinoma.

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

[29]
A method for treating cancer comprising administering an angiogenesis inhibitor and an IGFBP-1 inhibitor to a subject in need thereof.
[30]
30. The method of 29, wherein the angiogenesis inhibitor is a VEGF inhibitor, VEGFR inhibitor, soluble VEGFR, kinase inhibitor, FGFR inhibitor, or mTOR inhibitor.
[31]
31. The method according to 29 or 30 above, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fluquintinib, or AZD4547.
[32]
32. The method according to any one of 29 to 31 above, wherein the IGFBP-1 inhibitor is a substance that inhibits the binding of IGFBP-1 to integrin α5/β1.
[33]
33. The method according to any one of 29 to 32 above, wherein the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody.
[34]
34. The method according to any one of 29 to 33 above, wherein the cancer is hepatocellular carcinoma.

Specific examples will be described below, but these examples do not limit the invention described in the claims in any way.

Of the 251 cases of advanced hepatocellular carcinoma who underwent lenvatinib treatment (body weight 60 kg or more: 12 mg/day, body weight less than 60 kg: 8 mg/day, daily), case 31 had contrast-enhanced CT taken 1 month before and after treatment. Selected examples. Using the stored serum before and after lenvatinib treatment, changes in 55 angiogenesis-related proteins were comprehensively searched using the Angiogenesis Assay Kit (R&D Systems). As a result, IGFBP-1 was identified as a protein with high expression intensity and a large rate of change before and after lenvatinib treatment.

The CT value of the tumor before and after treatment was obtained, and the rate of change in CT value was calculated as follows. CT values were average values of three different regions.

Pre-CT value = a/b (Tumor/Aorta)
a: CT value of tumor before treatment b: CT value of aorta before treatment Post CT value = c/d (Tumor/Aorta)
c: CT value of tumor after treatment d: CT value of aorta after treatment

Rate of change in CT value = post-CT value / pre-CT value

Also, the rate of change in serum IGFBP-1 levels before and after treatment was calculated by the following formula.

Rate of change in serum IGFBP-1 level = serum IGFBP-1 level after treatment / serum IGFBP-1 level before treatment

The CT value decreased after lenvatinib treatment, and the serum IGFBP-1 value increased (Fig. 1). Focusing on each case, a correlation was observed between the degree of decrease in CT value and the degree of increase in IGFBP-1 value after lenvatinib treatment (Fig. 2). It was shown that the higher the IGFBP-1 level after treatment, the more ischemia (lower CT value) in the tumor was obtained.

The survival times of the 31 cases were stratified by the serum IGFBP-1 level before lenvatinib treatment, the serum IGFBP-1 level after treatment, or the rate of change in IGFBP-1 before and after treatment. Both were divided into two groups by median value (before treatment: 22006 ng/mL, after treatment: 44081 ng/mL, rate of change: 2.05). It was found that cases with high IGFBP-1 levels after lenvatinib treatment and cases with large increases in IGFBP-1 levels after lenvatinib treatment had a worse prognosis (Fig. 3).

For in vivo experiments, a hepatocellular carcinoma cell line (Hep-55.1C) (5×10 ⁶ cells) was subcutaneously inoculated into the dorsal side of C57BL/6 mice to prepare hepatocellular carcinoma model mice. After administering lenvatinib (10 mg/kg/day) or sorafenib (30 mg/kg/day) to this model mouse for 14 days, tumors were excised and measured. Serum IGFBP-1 was assessed by ELISA, and IGFBP-1 mRNA and protein expression in tumors by qPCR and Western blotting, respectively.

It was shown that lenvatinib and sorafenib reduced tumor volume and weight and inhibited tumor growth (Fig. 4). It was also shown that administration of lenvatinib and sorafenib increased serum IGFBP-1, and furthermore, IGFBP-1 expression in tumors was increased at the gene and protein levels (Fig. 5).

In addition, the expression of IGFBP-1, CD31 and CA9 (Carbonic Anhydrase IX) in tumor tissue after administration of lenvatinib in hepatocellular carcinoma model mice was evaluated by immunostaining. CD31 is an angiogenic marker and CA9 is a hypoxic marker for hepatocellular carcinoma. Administration of lenvatinib or sorafenib decreased the expression of CD31 and increased the expression of CA9, indicating that the administration of lenvatinib decreased blood vessels in the tumor and created a hypoxic environment (Fig. 6). On the other hand, in the liver, which is the main production site of IGFBP-1, no increase in IGFBP-1 was observed after administration of lenvatinib (Fig. 7). It was suggested that IGFBP-1, which is elevated by intratumoral hypoxia, may be involved in resistance to lenvatinib or sorafenib.

For in vitro experiments, Hep-55.1C cells were cultured for 24 hours in the presence of lenvatinib (0-10 μM) (normal oxygen). Expression of IGFBP-1 was then assessed by qPCR. Expression of mRNA was normalized by expression of GAPDH. No increase in IGFBP-1 was observed in Hep-55.1C cells due to lenvatinib administration (Fig. 8).

Hep-55.1C cells were cultured under normoxic (20% O ₂ ) or hypoxic (2% O ₂ ) conditions for 24 hours and the expression of IGFBP-1 was assessed by qPCR and Western blotting. mRNA and protein expression were normalized by expression of β-actin (ATCB) or GAPDH. Hypoxic stimulation increased the expression of IGFBP-1 at the gene and protein levels in Hep-55.1C cells (Fig. 9).

To evaluate the effect of IGFBP-1 on cancer cells, IGFBP- ¹ (0, 250, 500 ng/ml ) for 48 h and the growth rate was assessed. Cell proliferation was assessed using the MTT assay. Direct application of IGFBP-1 to cancer cells did not increase cell proliferation (Fig. 10).

To evaluate the effect of IGFBP-1 on vascular endothelial cells, human vascular endothelial cells (HUVEC) 1×10 ³ cells were cultured in the presence of IGFBP-1 (0, 100, 250 ng/ml) for 48 hours and then proliferated. Evaluated speed. In the presence of IGFBP-1 (0, 100, 250 ng/ml), tube formation ability of HUVEC was similarly evaluated using tube formation assay.

　IGFBP-1 was shown to enhance vascular endothelial cell proliferation and tube formation (Fig. 11). In addition, IGFBP-1 was shown to enhance vascular endothelial cell proliferation and tube formation even in the presence of lenvatinib (Fig. 12).

We evaluated the effects of IGFBP-1 on vascular endothelial cell proliferation and tube formation in the presence of angiogenesis inhibitors other than lenvatinib. selected from lenvatinib (3 μM), sorafenib (3 μM), VEGFR-1,2,3 inhibitor (fluquintinib) (1 μM), and FGFR-1,2,3 inhibitor (AZD4547) (1 μM) Tube formation assays were performed as described above in the presence of angiogenesis inhibitors and IGFBP-1 (250 ng/ml). IGFBP-1 was shown to enhance angiogenesis in the presence of any angiogenesis inhibitor (Fig. 13).

We investigated the signaling pathways involved in the upregulation of IGFBP-1 by hypoxic stimulation. Hep-55.1C cells, HuH7 cells, or HepG2 cells (1 × 10 ⁵ cells) were treated under normoxic (20% O ₂ ) or hypoxic (2% O ₂ ) conditions with the HIF inhibitor YC-1 (Lificiguat ) (5 μM) for 24 h. Expression of IGFBP-1, HIF-1α, HIF-2α was assessed by Western blot. The expression of each protein was normalized by the expression of tubulin.

The expression of IGFBP-1 was increased by hypoxic stimulation, and this increase was suppressed by the addition of YC-1 (Figs. 14-16). This suggests that the expression of IGFBP-1 is increased by hypoxic stimuli, particularly by signaling pathways mediated by HIF-1α and HIF-2α.

Furthermore, we investigated the signaling pathways through which IGFBP-1 expression promotes vascular endothelial cell proliferation and tube formation. After serum-free culture of HUVEC (1×10 ⁵ cells) for 12 hours, IGFBP-1 (250 ng/ml) was added, and phosphorylation of FAK and ERK-1/2 was evaluated after 5 and 15 minutes. did. Expression of each protein was normalized by expression of GAPDH. In addition, HUVEC (5×10 ³ cells) were treated with integrin α5/β1 inhibitor (ATN-161) at various concentrations (0, 1, 3, 10 μM) in the presence of IGFBP-1 (250 ng/ml) ( CAS No: 262438-43-7) or FAK inhibitor 14 (CAS No: 4506-66-5), cultured for 48 hours in the presence or absence, and performed a proliferation assay using the MTT assay. did. Furthermore, HUVEC (1 × 10 ³ cells) were cultured in the presence of IGFBP-1 (250 ng/ml) and integrin α5/β1 inhibitor (3 μM) or FAK inhibitor (10 μM) to promote tube formation. An assay was performed.

IGFBP-1 enhanced the phosphorylation of FAK and ERK-1/2 (Fig. 17). In addition, integrin α5β1 inhibitors and FAK inhibitors suppressed IGFBP-1-induced enhancement of vascular endothelial cell proliferation and tube formation (FIGS. 18 and 19). IGFBP-1 was suggested to promote vascular endothelial cell proliferation and angiogenesis through signaling through integrin α5/β1 - FAK - ERK.

IGFBP-1 was deficient in HuH7 cells or HepG2 cells, and these IGFBP-1 deficient cells (5 × 10 ⁶ cells) or GFP-expressing cells as a control were subcutaneously inoculated into the dorsal side of Balb/c mice to form a hepatocellular carcinoma model. I made a mouse. Tumors were excised and measured after 14 days of administration of lenvatinib (10 mg/kg/day).

IGFBP-1-deficient tumors showed stronger growth suppression by lenvatinib than control tumors (Figs. 20 and 21). Deletion of IGFBP-1 expression was shown to increase sensitivity to lenvatinib.

HepG2 cells were transplanted into Balb/c mice as described above, and lenvatinib (10 mg/kg/day) alone, anti-IGFBP-1 antibody (IGFBP1 Monoclonal Antibody; ThermoFisher Catalog # MA5-23727) (0.03 mg/ kg/day) alone or in combination for 14 days, tumors were excised and measured.

In this experiment, lenvatinib or anti-IGFBP-1 antibody alone did not significantly suppress tumor growth, but when used in combination, tumor growth was significantly suppressed (Fig. 22). It was shown that the combined use of lenvatinib and anti-IGFBP-1 antibody enhanced the antitumor effect.

Similarly, in model mice in which HepG2 cells were transplanted into Balb/c mice, lenvatinib (10 mg/kg/day) alone, integrin α5/β1 inhibitor (ATN-161) (CAS No: 262438-43-7) ( 1 mg/kg/day) alone or in combination for 14 days, tumors were excised and measured.

In this experiment, lenvatinib or integrin α5/β1 inhibitor alone did not significantly suppress tumor growth, but their combined use significantly suppressed tumor growth (Fig. 23). Combined use of lenvatinib and an integrin α5/β1 inhibitor was shown to enhance the antitumor effect. 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 binding of IGFBP-1 to integrin α5/β1 enhances the antitumor effect. It was suggested that inhibition is important.

Claims (20)

1. A method for predicting the prognosis of a subject with cancer following treatment with an anti-angiogenic drug, comprising measuring the amount of IGFBP-1 after treatment with an anti-angiogenic drug in a sample obtained from said subject. ,Method.
2. The method according to claim 1, wherein the sample is a blood sample.
3. The method according to claim 2, wherein the blood sample is serum.
4. The method according to any one of claims 1 to 3, wherein the cancer is hepatocellular carcinoma.
5. The method according to any one of claims 1 to 4, further comprising comparing the post-treatment IGFBP-1 amount 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 post-treatment IGFBP-1 level is higher than the reference value.
7. The method according to any one of claims 1 to 4, further comprising measuring the amount of IGFBP-1 before treatment with said angiogenesis inhibitor in a sample obtained from said subject.
8. 8. The method of claim 7, further comprising comparing the rate of change in the amount of IGFBP-1 after treatment relative to the amount of IGFBP-1 before treatment with a reference value.
9. 9. The method of claim 8, wherein the subject is predicted to have a poor prognosis after treatment with the anti-angiogenic drug if 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, VEGFR inhibitor, soluble VEGFR, kinase inhibitor, FGFR inhibitor, or mTOR inhibitor.
11. The method according to any one of claims 1 to 10, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fluquintinib, or AZD4547.
12. A composition comprising an IGFBP-1 detection reagent for predicting the prognosis of a subject with cancer after treatment with an angiogenesis inhibitor.
13. A kit containing an IGFBP-1 detection reagent for predicting the prognosis of cancer-bearing subjects after treatment with angiogenesis inhibitors.
14. 　Biomarkers, including IGFBP-1, to predict prognosis after treatment with anti-angiogenic drugs in subjects with cancer.
15. A pharmaceutical composition for treating cancer, comprising:
    including angiogenesis inhibitors, combined with IGFBP-1 inhibitors,
    including an IGFBP-1 inhibitor combined with an angiogenesis inhibitor, or an angiogenesis inhibitor and an IGFBP-1 inhibitor;
    pharmaceutical composition.
16. The pharmaceutical composition according to claim 15, wherein the angiogenesis inhibitor is a VEGF inhibitor, VEGFR inhibitor, soluble VEGFR, kinase inhibitor, FGFR inhibitor, or mTOR inhibitor.
17. The pharmaceutical composition according to any one of claims 15 or 16, wherein the angiogenesis inhibitor is sorafenib, lenvatinib, fluquintinib, or AZD4547.
18. The pharmaceutical composition according to any one of claims 15 to 17, wherein the IGFBP-1 inhibitor is an anti-IGFBP-1 antibody.
19. The pharmaceutical composition according to any one of claims 15 to 18, wherein the cancer is hepatocellular carcinoma.
20. A kit for treating cancer, comprising a composition containing an angiogenesis inhibitor and a composition containing an IGFBP-1 inhibitor.

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