JP2020115791A - Marker that determines effectiveness of application of cancer therapy including cancer immune therapy to cancer patient, and use thereof - Google Patents

Abstract

To provide a new technique that determines the effectiveness of application of cancer therapy including cancer immune therapy to a cancer patient.SOLUTION: Use of Cholesterol 25-hydroxylase(Ch25H) gene, Ch25H protein or 25-hydroxylation cholesterol (25OHC) as a marker that determines the effectiveness of application of cancer therapy including cancer immune therapy to a cancer patient.SELECTED DRAWING: None

Description

The present invention relates to a marker for determining the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient and use thereof. More specifically, the present invention provides a marker for determining the effectiveness of application of a cancer therapeutic method including cancer immunotherapy to a cancer patient, and efficacy evaluation of application of a cancer therapeutic method including cancer immunotherapy to a cancer patient. , A kit for determining the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient, an agent for improving the effectiveness of a cancer treatment method including cancer immunotherapy, and cancer treatment Kit and a method for screening an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy.

Recently, it has been shown that cancer immunotherapy that induces T cells that recognize cancer cells can cure even advanced stage solid tumors. One of cancer immunotherapy is a treatment method using an immune checkpoint inhibitor such as an anti-PD-1 antibody, which shows a clear clinical effect in many cancers including malignant melanoma and lung cancer.

Treatment with immune checkpoint inhibitors has a response rate of only about 20% with monotherapy. In many of the refractory cases, an immunosuppressive cancer microenvironment is constructed that inhibits the initiation of T cell responses to cancer. Therefore, it is necessary to develop a therapeutic method that releases this immunosuppression. There is also a demand for development of a method for determining the effectiveness of application of cancer immunotherapy to cancer patients (see, for example, Patent Document 1). Cancer patients who initially have a poor immune status even after general anticancer drug treatment and surgery may have a poor prognosis for these treatment methods.

By the way, Cholester 25-hydroxylase (hereinafter sometimes referred to as “Ch25H”) is an enzyme that metabolizes cholesterol into 25-hydroxylated cholesterol (hereinafter sometimes referred to as “25OHC”).

International Publication No. 2016/088791

However, the method for testing the therapeutic effect of an anticancer agent described in Patent Document 1 involves examining the expression of a predetermined cell surface molecule on T cells using a sample derived from a patient to whom the anticancer agent has been administered. .. Therefore, it is not possible to determine the effectiveness of applying cancer immunotherapy to cancer patients before treatment. It is an object of the present invention to provide a new technique for determining the effectiveness of applying a cancer treatment method including cancer immunotherapy to a cancer patient.

The present invention includes the following aspects.
[1] Use of the Cholesterol 25-hydroxylase (Ch25H) gene, Ch25H protein or 25-hydroxycholesterol (25OHC) as a marker for determining the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient.
[2] measuring the abundance of mRNA of Ch25H gene, Ch25H protein or 25OHC in a biological sample derived from a cancer patient, and measuring the abundance of said mRNA, said protein or said 25OHC in a biological sample of control. The amount of the amount compared to the abundance is data for evaluating the effectiveness of the application of the cancer treatment method including the cancer immunotherapy to the cancer patient, the cancer treatment method including the cancer immunotherapy to the cancer patient. How to obtain data for applicability assessment.
[3] The abundance of the mRNA, the protein, or the 25OHC is higher than the abundance in a control biological sample, so that the effectiveness of application of a cancer treatment method including cancer immunotherapy to the cancer patient is low. The method according to [2], which indicates that
[4] The abundance of the mRNA, the protein, or the 25OHC is higher than the abundance in a control biological sample, which is highly effective in applying a cancer treatment method including cancer immunotherapy to the cancer patient. The method according to [2], which indicates that
[5] The method according to any one of [2] to [4], wherein the biological sample is blood or tumor tissue.
[6] Cancer treatment method including cancer immunotherapy for cancer patients, which comprises a primer set for amplifying the cDNA of Ch25H gene, a probe hybridizing to mRNA of Ch25H gene, a specific binding substance for Ch25H protein, or a reagent for detecting 25OHC A kit for determining the effectiveness of application of.
[7] An agent for improving the effectiveness of a cancer treatment method including cancer immunotherapy, which comprises an inhibitor of Ch25H protein as an active ingredient.
[8] The agent for improving the efficacy of a cancer treatment method including the cancer immunotherapy according to [7], wherein the inhibitor of Ch25H protein is a substance that suppresses the expression of Ch25H gene.
[9] The agent for improving the efficacy of a cancer treatment method including the cancer immunotherapy according to [8], wherein the substance that suppresses the expression of Ch25H gene is siRNA or shRNA of Ch25H gene.
[10] A kit for treating cancer, which comprises an agent for improving the efficacy of the cancer treatment method including the immunotherapy for cancer according to any of [7] to [9], and an anticancer agent.
[11] A method for screening an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy, which comprises contacting cholesterol and Ch25H protein in the presence of a test substance to produce 25OHC, When the production amount of C. is reduced or increased compared to the production amount of 25OHC in the absence of the test substance, the test substance is determined to be an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy. Including doing.
[12] When the production amount of 25OHC is decreased as compared with the production amount of 25OHC in the absence of the test substance, the test substance is an agent for improving the effectiveness of a cancer treatment method including cancer immunotherapy. The method according to [11], which determines that there is.
[13] A method for screening an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy, comprising culturing a cancer cell in the presence of a test substance, and expressing the Ch25H gene or Ch25H protein in the cancer cell Or measuring the production amount of 25OHC, and the expression amount of the Ch25H gene or the Ch25H protein or the production amount of the 25OHC is decreased or increased as compared with the absence of the test substance, the test substance Determining that the agent is an efficacy enhancer of a cancer treatment method including cancer immunotherapy.
[14] When the expression level of the Ch25H gene or the Ch25H protein or the production level of the 25OHC is decreased as compared with the absence of the test substance, the test substance is a cancer treatment method including cancer immunotherapy. The method according to [13], wherein the method determines that the agent is an efficacy enhancer.

According to the present invention, it is possible to provide a new technique for determining the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient.

3 is a graph showing the change over time in tumor volume of mice in each group in Experimental Example 1. 5 is a graph showing the change over time in tumor volume of mice in each group in Experimental Example 2. (A) And (b) is a graph which shows the result of having quantified the production amount of IFN-(gamma) in Experimental example 3. (A) and (b) are optical micrographs of tissue sections immunostained in Experimental Example 4. 9 is a graph showing measured values of 25OHC in serum in Experimental Example 5. 9 is a graph showing the change over time in tumor volume of mice in each group in Experimental Example 6. (A) And (b) is a graph which shows the result of having quantified the production amount of IFN-(gamma) in Experimental example 7. 9 is a graph showing the expression level of Ch25H gene mRNA in various human cancer tissues examined in Experimental Example 8.

[Marker for judging the effectiveness of application of cancer therapy including cancer immunotherapy to cancer patients]
In one embodiment, the present invention provides the Cholesterol 25-hydroxylase (Ch25H) gene, Ch25H protein or 25-hydroxylated cholesterol (marked as a marker for determining the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient. 25OHC).

Here, the Ch25H protein is synonymous with Cholesterol 25-hydroxylase, and is an enzyme that introduces a hydroxyl group at the 25-position of cholesterol to produce 25OHC.

As will be described later in Examples, the inventors have revealed that in mice in which the expression of Ch25H in cancer tissues is high, the effectiveness of application of cancer treatment methods including cancer immunotherapy is reduced. Further, it was revealed that the amount of 25OHC present in serum was increased in mice having cancer tissues with high expression of Ch25H. In addition, depending on the type of cancer therapeutic method including cancer immunotherapy, conversely, when the expression of Ch25H in the cancer tissue is high, the effectiveness of application of the cancer therapeutic method including cancer immunotherapy may be improved. .. The expression of Ch25H or blood 25OHC is considered to reflect the immune status of the patient.

Therefore, the Ch25H gene, Ch25H protein, or 25OHC can be used as a marker for determining the effectiveness of application of cancer treatment methods including cancer immunotherapy to cancer patients.

The NCBI accession number of the human Ch25H gene is NM_003956.3, and the NCBI accession number of the human Ch25H protein is NP_003947.1. The NCBI accession number of mouse Ch25H gene is NM_009890.1, and the NCBI accession number of mouse Ch25H protein is NP_034020.1.

[Method of determining effectiveness of application of cancer treatment method including cancer immunotherapy to cancer patient]
In one embodiment, the present invention provides a method for determining the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient, wherein the Ch25H gene mRNA, Ch25H in a biological sample derived from the cancer patient is used. Measuring the abundance of protein or 25OHC, wherein the abundance of said mRNA, said protein or said 25OHC is higher or lower than the abundance in a control biological sample. Methods related to the effectiveness of application of cancer therapies, including therapy, are provided. It can be said that the method of the present embodiment is a method of acquiring data for evaluating the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient.

As will be described later in Examples, the inventors have revealed that in mice in which the expression of Ch25H in cancer tissues is high, the effectiveness of application of cancer treatment methods including cancer immunotherapy is reduced. Further, it was revealed that the amount of 25OHC present in serum was increased in mice having cancer tissues with high expression of Ch25H. In addition, depending on the type of cancer therapeutic method including cancer immunotherapy, conversely, when the expression of Ch25H in the cancer tissue is high, the effectiveness of application of the cancer therapeutic method including cancer immunotherapy may be improved. ..

Therefore, when the abundance of mRNA of Ch25H gene, Ch25H protein or 25OHC in a biological sample derived from a cancer patient is higher or lower than that in a control biological sample, cancer immunotherapy for said cancer patient. It can be determined that the effectiveness of applying the cancer treatment method including is low. Further, according to the method of the present embodiment, it is possible to determine the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient before treatment.

The cancer is not particularly limited, but in particular, cancers such as malignant melanoma, lung cancer, renal cancer, and gastric cancer whose anti-PD-1 antibody is covered by insurance are mentioned.

In the method of the present embodiment, the mRNA or Ch25 protein of the Ch25 gene to be detected is preferably an mRNA or protein that is the same species as the cancer patient (or animal), and the cancer including cancer immunotherapy for human cancer patients. When determining the effectiveness of application of a therapeutic method, it is preferable to detect human Ch25 gene mRNA or human Ch25 protein.

In the method of the present embodiment, examples of the biological sample include blood and tumor tissue. In addition, as a control biological sample, a biological sample derived from a healthy subject or a patient to whom application of a cancer treatment method including cancer immunotherapy is effective can be used. Examples of blood samples include serum and plasma. Further, when the biological sample is a tumor tissue, as a control biological sample, for example, a tumor tissue derived from a patient confirmed to be effective to apply a cancer treatment method including cancer immunotherapy, in a healthy subject A sample derived from a tissue corresponding to the tumor tissue can be used.

Further, it is not necessary to prepare a control biological sample every time when the method of the present embodiment is carried out, and the amount of Ch25H gene mRNA, Ch25H protein or 25OHC present in the control biological sample is measured in advance, The measured value may be compared with the abundance of Ch25H gene mRNA, Ch25H protein or 25OHC in a biological sample derived from a cancer patient.

The method for measuring the abundance of mRNA of Ch25H gene is not particularly limited, and for example, RT-PCR, quantitative RT-PCT, DNA microarray analysis and the like can be performed.

The method for measuring the abundance of Ch25H protein is not particularly limited, and for example, ELISA, Western blotting, flow strip method, protein chip and the like can be used.

The method for measuring the abundance of 25OHC is not particularly limited, and it can be measured by, for example, mass spectrometry, ELISA, various chromatography and the like.

In the method of the present embodiment, examples of cancer treatment methods including cancer immunotherapy include administration of immune checkpoint inhibitors, cytokine therapy, adoptive immunotherapy, cancer vaccine therapy, and the like. As adoptive immunotherapy, a treatment method in which tumor-infiltrating lymphocytes (TIL) cultured in vitro are administered, a treatment method in which a T cell receptor (TCR) gene-transferred T cell (TCR-T) against a cancer antigen is administered, and a chimeric antigen The treatment method etc. which administer a receptor gene transfer T cell (CAR-T) are mentioned.

Examples of the immune checkpoint inhibitor include anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody and the like. Examples of the anti-PD-1 antibody include nivolumab, pembrolizumab and the like. Examples of the anti-CTLA-4 antibody include ipilimumab and the like. Examples of the anti-PD-L1 antibody include avelumab, atezolizumab and the like.

[Kit for Determining Effectiveness of Application of Cancer Treatment Method including Cancer Immunotherapy to Cancer Patient]
In one embodiment, the present invention provides a cancer immunity to a cancer patient, which comprises a primer set for amplifying the cDNA of the Ch25H gene, a probe that hybridizes with mRNA of the Ch25H gene, a specific binding substance for the Ch25H protein, or a reagent for detecting 25OHC. A kit for determining the effectiveness of application of a cancer treatment method including therapy.

With the kit of the present embodiment, it is possible to carry out the above-described method for determining the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient.

The primer set is not particularly limited as long as it can amplify the cDNA of Ch25H gene.

The probe is not particularly limited as long as it specifically hybridizes with the mRNA of Ch25H gene. The probe may be immobilized on a carrier to form a DNA microarray or the like.

Examples of specific binding substances include antibodies, antibody fragments, nucleic acid aptamers, peptide aptamers and the like. The antibody fragment includes F(ab')2, Fab', Fab, Fv, scFv and the like. The specific binding substance is not particularly limited as long as it can specifically bind to the Ch25H protein, and may be commercially available. The specific binding substance may be immobilized on a carrier to form a protein chip or the like.

[Efficacy improver for cancer treatment including cancer immunotherapy]
In one embodiment, the present invention provides an agent for improving the effectiveness of a cancer treatment method including cancer immunotherapy, which comprises an inhibitor of Ch25H protein as an active ingredient.

As described below in Examples, by suppressing the expression of the Ch25H gene or protein on the host side or the cancer cell side, the effectiveness of cancer treatment methods including cancer immunotherapy can be improved. Therefore, the inhibitor of Ch25H protein can be used as an agent for improving the efficacy of cancer treatment methods including cancer immunotherapy.

In the improving agent of the present embodiment, the inhibitor of Ch25H protein is not particularly limited as long as it is a substance that inhibits the function of Ch25H protein. More specifically, for example, a substance that inhibits or suppresses the expression (transcription or translation) of the Ch25H gene, an inhibitor of the Ch25H protein, and the like can be mentioned. The inhibitor of Ch25H protein may be a low molecular weight compound.

Specific examples of the substance that inhibits the transcription or translation of the Ch25H gene include an inhibitory nucleic acid for the mRNA of the Ch25H gene. Examples of the inhibitory nucleic acid include siRNA and shRNA. SiRNA or shRNA for the Ch25H gene can be prepared by a well-known method.

Examples of the inhibitor of Ch25H protein include a substance that introduces a hydroxyl group at the 25-position of cholesterol of Ch25H protein and inhibits the activity of producing 25OHC.

[Cancer treatment kit]
In one embodiment, the present invention provides a cancer treatment kit comprising an agent for improving the efficacy of a cancer treatment method including the above-mentioned cancer immunotherapy, and an anticancer agent.

To improve the effectiveness (therapeutic effect) of a cancer treatment method including cancer immunotherapy by administering to a cancer patient a combination of an agent for improving the effectiveness of the above-described cancer treatment method including cancer immunotherapy and an anti-cancer agent. You can In the kit of this embodiment, the agent for improving the efficacy of a cancer treatment method including cancer immunotherapy and the anti-cancer agent may be administered to the patient separately or may be mixed and administered to the patient.

Examples of the anti-cancer agent include drugs used in cancer treatment methods including cancer immunotherapy, for example, immune checkpoint inhibitors, cytokines used in cytokine therapy (eg, IFN-α, IFN-β, IFN-2, IFN). -Γ etc.), CAT-T cells used for chimeric antigen receptor gene transfer T cell (CAR-T) therapy, TCR-T cells used for T cell receptor (TCR) gene transfer T cell therapy for cancer antigens, Examples include TIL used in a therapeutic method of administering tumor-infiltrating lymphocytes (TIL) cultured in vitro, a peptide vaccine used in cancer vaccine therapy, and the like. The immune checkpoint inhibitors are the same as those mentioned above, and examples thereof include nivolumab, pembrolizumab, ipilimumab, avelumab, atezolizumab, and other commonly used anticancer agents.

[Method of screening for agent for improving efficacy of cancer treatment method including cancer immunotherapy]
(First embodiment)
A method for screening an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy according to the first embodiment, wherein cholesterol and Ch25H protein are contacted with each other in the presence of a test substance to produce 25OHC, When the production amount of 25OHC is decreased or increased as compared with the production amount of 25OHC in the absence of the test substance, the test substance is an agent for improving the effectiveness of cancer treatment methods including cancer immunotherapy. It is a method including determining.

As will be described later in Examples, the inventors have revealed that in mice in which the expression of Ch25H in cancer tissues is high, the effectiveness of application of cancer treatment methods including cancer immunotherapy is reduced. Further, it was revealed that the amount of 25OHC present in serum was increased in mice having cancer tissues with high expression of Ch25H.

Therefore, when the production amount of 25OHC is decreased as compared with the production amount of 25OHC in the absence of the test substance, the test substance is determined to be an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy. can do.

In addition, depending on the type of cancer therapeutic method including cancer immunotherapy, conversely, when the expression of Ch25H in the cancer tissue is high, the effectiveness of application of the cancer therapeutic method including cancer immunotherapy may be improved. .. In such a case, when the production amount of 25OHC is increased as compared with the production amount of 25OHC in the absence of the test substance, the test substance improves the efficacy of cancer treatment methods including cancer immunotherapy. It can be determined to be an agent.

The test substance is not particularly limited, and examples thereof include a natural compound library, a synthetic compound library, an existing drug library, and a metabolite library.

In the screening method of the first embodiment, first, cholesterol and Ch25H protein are brought into contact with each other in the presence of a test substance to produce 25OHC. This step may be performed in vitro using the purified Ch25H protein, or may be performed using cells in which the Ch25H gene has been introduced.

Subsequently, the amount of 25OHC produced is quantified. The method for quantifying 25OHC is not particularly limited, and examples thereof include mass spectrometry, ELISA, various chromatography and the like. As a result, when the production amount of 25OHC in the presence of the test substance decreases or increases as compared with the production amount of 25OHC in the absence of the test substance, the test substance is effective for cancer treatment including cancer immunotherapy. It can be determined to be a sex improving agent or a candidate thereof.

(Second embodiment)
A method for screening an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy according to the second embodiment comprises culturing a cancer cell in the presence of a test substance, and measuring the Ch25H gene or the Ch25H protein in the cancer cell. Measuring the expression level or the production amount of 25OHC, and the expression level of the Ch25H gene or the Ch25H protein or the production amount of the 25OHC is decreased or increased as compared with the absence of the test substance, The test substance is a method including determining that the test substance is an agent for improving the effectiveness of a cancer treatment method including cancer immunotherapy.

As will be described later in Examples, the inventors have revealed that in mice in which the expression of Ch25H in cancer tissues is high, the effectiveness of application of cancer treatment methods including cancer immunotherapy is reduced. Further, it was revealed that the amount of 25OHC present in serum was increased in mice having cancer tissues with high expression of Ch25H.

Therefore, when the expression level of the Ch25H gene or Ch25H protein or the production amount of 25OHC by the cancer cells in the presence of the test substance is decreased as compared with the absence of the test substance, the test substance includes cancer immunotherapy. It can be determined that it is an agent for improving the efficacy of cancer treatment.

Further, as described above, conversely, depending on the type of cancer treatment method including cancer immunotherapy, the effectiveness of application of the cancer treatment method including cancer immunotherapy is improved when the expression of Ch25H in the cancer tissue is high. It is also possible to do. In such a case, the expression level of the Ch25H gene or Ch25H protein or the production amount of 25OHC by the cancer cells in the presence of the test substance is the expression amount of the Ch25H gene or the Ch25H protein or the production of 25OHC in the absence of the test substance. When the amount is increased as compared with, it can be determined that the test substance is an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy.

In the screening method of the second embodiment, first, cancer cells are cultured in the presence of a test substance. The test substance is the same as in the screening method of the first embodiment. The cancer cell may be a cancer cell derived from a cancer patient or an established cancer cell.

Then, the expression level of the Ch25H gene or Ch25H protein or the production amount of 25OHC in the cancer cells is measured. The method for measuring the abundance of mRNA of Ch25H gene, the method for measuring the abundance of Ch25H protein, and the method for measuring the abundance of 25OHC are the same as those described above.

As a result, the expression level of the Ch25H gene or the Ch25H protein or the production amount of 25OHC in the presence of the test substance is decreased as compared with the expression amount of the Ch25H gene or the Ch25H protein or the production amount of 25OHC in the absence of the test substance, or When the amount increases, the test substance can be determined to be an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy or a candidate thereof.

[Other Embodiments]
In one embodiment, the present invention provides the measurement of the abundance of Ch25H gene mRNA, Ch25H protein or 25OHC in a biological sample derived from a cancer patient, and the abundance of said mRNA, said protein or said 25OHC is a control. The present invention provides a method for treating cancer, which comprises applying a cancer treatment method including cancer immunotherapy to the cancer patient when the amount is equal to or less than the amount present in the biological sample.

In one embodiment, the present invention provides the measurement of the abundance of Ch25H gene mRNA, Ch25H protein or 25OHC in a biological sample derived from a cancer patient, and the abundance of said mRNA, said protein or said 25OHC is a control. The method for treating cancer, which comprises applying a cancer treatment method including cancer immunotherapy to the cancer patient when the amount thereof is higher than that in the biological sample.

In one embodiment, the present invention comprises measuring the abundance of Ch25H gene mRNA, Ch25H protein or 25OHC in a biological sample derived from a cancer patient, and measuring the abundance of said mRNA, said protein or residue 25OHC, The present invention provides a method for treating cancer, which comprises administering an inhibitor of Ch25H protein and an anti-cancer agent to the cancer patient when the amount thereof is higher than that in a control biological sample.

In one embodiment, the invention provides an inhibitor of Ch25H protein for the treatment of cancer.

In one embodiment, the present invention provides a Ch25H gene expression inhibitor for the treatment of cancer.

In one embodiment, the present invention provides use of an inhibitor of Ch25H protein or an inhibitor of expression of Ch25H gene for producing a therapeutic agent for cancer.

In each of these embodiments, a biological sample, a control, a cancer treatment method including cancer immunotherapy, an anticancer agent, and an inhibitor of Ch25H protein are the same as those described above.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

<I. Examination in a model in which cancer cells forcibly expressing Ch25H were transplanted to wild-type mice>
[Experimental Example 1]
(Examination of efficacy of anti-PD-1 antibody therapy 1)
A mouse Ch25H gene expression vector was introduced into the mouse colon cancer cell line CT26 to obtain a stable overexpression cell line.

Subsequently, a CT26 cell line overexpressing Ch25H was transplanted into the flank region of a wild-type Balb/c mouse, and the tumor volume was measured over time. For comparison, a CT26 cell line not introduced with Ch25H (CT26 cell line into which only an empty expression vector was introduced) was transplanted into the flank region of a wild-type Balb/c mouse, and the tumor volume was increased. It was measured over time.

On the 5th, 9th and 13th days after cell transplantation, 200 μg/mouse of anti-PD-1 antibody (BioXcell) was intraperitoneally administered to each mouse. Also, for comparison, a group was prepared in which 200 μg/mouse of an isotype control antibody (hamster IgG, BioXcell) was intraperitoneally administered instead of the anti-PD-1 antibody.

1(a) to 1(d) are graphs showing changes over time in tumor volume of mice in each group. 1(a) to 1(d), "MOCK" indicates the result of a mouse transplanted with a CT26 cell line into which Ch25H is not introduced, and "Ch25H" indicates a CT26 cell line overexpressing Ch25H. "Isotype" indicates the result of the transplanted mouse, "Isotype" indicates the result of the mouse administered with the isotype control antibody, and "Anti-PD-1 antibody" indicates the result of the mouse administered with the anti-PD-1 antibody. Is shown.

As a result, in the mouse (MOCK) transplanted with the CT26 cell line into which Ch25H was not introduced, the increase in tumor volume was significantly suppressed by the administration of the anti-PD-1 antibody. On the other hand, in the mouse (Ch25H) transplanted with the CT26 cell line overexpressing Ch25H, suppression of increase in tumor volume due to administration of the anti-PD-1 antibody was not observed, and the anti-PD-1 antibody therapy was It became clear that it became maladaptive.

[Experimental Example 2]
(Examination of efficacy of anti-PD-1 antibody therapy 2)
A mouse Ch25H gene expression vector was introduced into the mouse fibrosarcoma cell line MCA205 to obtain a stable overexpression cell line.

Subsequently, the MCA205 cell line overexpressing Ch25H was transplanted into the flank of wild-type C57BL/6 mice, and the tumor volume was measured over time. For comparison, a wild-type C57BL/6 mouse was transplanted into the flank region with a Ch25H-introduced MCA205 cell line (an MCA205 cell line into which only an empty expression vector was introduced) to reduce the tumor volume. It was measured over time.

On the fourth, eighth, twelfth, and fifteenth days after cell transplantation, 200 μg/mouse of anti-PD-1 antibody (BioXcell) was intraperitoneally administered to each mouse. Also, for comparison, a group was prepared in which 200 μg/mouse of an isotype control antibody (hamster IgG, BioXcell) was intraperitoneally administered instead of the anti-PD-1 antibody.

2(a) to 2(d) are graphs showing changes over time in tumor volume of mice in each group. 2(a) to (d), "MOCK" indicates the result of the mouse transplanted with the MCA205 cell line into which Ch25H was not introduced, and "Ch25H" indicates the MCA205 cell line overexpressing Ch25H. "Isotype antibody" indicates the result of the transplanted mouse, "Isotype antibody" indicates the result of the mouse administered with the isotype control antibody, and "Anti-PD-1 antibody" indicates that of the mouse administered with the anti-PD-1 antibody. Indicates the result.

As a result, in the mouse (MOCK) transplanted with the MCA205 cell line into which Ch25H was not introduced, the increase in tumor volume was significantly suppressed by the administration of the anti-PD-1 antibody, and 5 out of 6 mice were completely cured. On the other hand, in the mouse (Ch25H) transplanted with the MCA205 cell line overexpressing Ch25H, suppression of the increase in tumor volume due to administration of the anti-PD-1 antibody was not observed, and the anti-PD-1 antibody therapy was It became clear that it became maladaptive.

[Experimental Example 3]
(Study on cytotoxic T cells 1)
The effect of inducing tumor antigen-specific T cells in regional lymph nodes and tumor tissues in the tumor-bearing mice prepared in Experimental Example 2 was evaluated by an IFN-γ production assay.

Specifically, first, the cells to which the isotype control antibody-administered MCA205 cell line into which the Ch25H had not been introduced and transplanted with the MCA205 cell line that overexpressed Ch25H were transplanted were used as cells. It was euthanized 38 days after transplantation.

Subsequently, the tumor tissue was excised from each mouse, cut into pieces with scissors, placed in 20 mL of collagenase solution (2 mg collagenase+30U DNase/1 mL RPMI1640), and shaken at 37° C. for 30 minutes. Then, CD8 magnetic T beads were separated using CD8 magnetic beads (Miltenyi).

Regional lymph nodes (axillary and inguinal lymph nodes on the side of cancer cell transplantation) were removed from each mouse, and the lymph nodes were destroyed by grinding with a slide glass to collect lymph node cells. Then, CD8 magnetic T beads were separated using CD8 magnetic beads (Miltenyi).

Each of the obtained T cells (3×10 ^{5 to} 5×10 ⁵ ) derived from the tumor tissue and lymph nodes was collected from a wild-type healthy mouse, and 32 Gy of radiation-treated spleen cells (1×10 ⁷ cells) were collected. ), and a tumor antigen gp70 peptide (peptide consisting of amino acids 604 to 611 of mouse leukemia virus MuLV gp70 p15E: KSPWFTTL, SEQ ID NO: 1) (1.0 μg/mL), human interleukin (IL)-2. (20 U/mL) and mouse IL-7 (10 ng/mL) in 10% fetal bovine serum-containing RPMI1640 medium, 48-well plate (1 mL/well), tumor tissue-derived T cells for 2 days, lymph node-derived T cells The cells were cultured for 7 days.

Subsequently, T cells were collected using Lymphoprep (Alere Technologies AS). The collected tumor tissue-derived T cells (1×10 ⁵ cells) were transformed into mouse 96-well plates (1×10 ⁵ cells) in the presence of EL4 cells (1×10 ⁵ cells) and a final concentration of 1 μg/mL of the gp70 peptide ( 200 μL/well), 10% fetal bovine serum-containing RPMI1640 medium was mixed and cultured in 3 wells for each group, and IFN-γ in the culture supernatant after 24 hours of culture was measured by an ELISA method.

Similarly, the collected lymph node-derived T cells (5×10 ⁴ cells) were mixed with EL4 cells (1×10 ⁵ cells) at a final concentration of 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL, and 0. In the presence of 001 μg/mL and 0 μg/mL of gp70 peptide, 3 wells of each group were mixed-cultured in a 96-well plate (200 μL/well) with RPMI1640 medium containing 10% fetal bovine serum, and cultured for 24 hours. IFN-γ in Seiki was measured by the ELISA method.

For comparison, an irrelevant β-Gal peptide (peptide consisting of amino acids 96 to 103 of β-galactosidase: DAPIYTNV, SEQ ID NO: 2) (1.0 μg/mL) was contacted instead of the gp70 peptide. We also prepared a group.

As a result, when cancer antigen-specific cytotoxic T cells are present in the T cells, IFN-γ is produced by contact with the cancer antigen peptide.

3(a) and 3(b) are graphs showing the results of quantifying the amount of IFN-γ produced. FIG. 3( a) shows the result of cells derived from tumor tissue, and FIG. 3( b) shows the result of cells derived from lymph nodes. In FIGS. 3( a) and 3 (b ), “Tumor” indicates the result of cells derived from tumor tissue, “LN” indicates the result of cells derived from lymph nodes, and “MOCK” indicates Ch25H. It shows that it is the result of a tumor tissue or lymph node derived from a mouse transplanted with a non-transfected MCA205 cell line, and "Ch25H" is a tumor tissue or lymph derived from a mouse transplanted with an MCA205 cell line overexpressing Ch25H. It shows that it is the result of the section, "gp70" shows that it is the result of contacting the gp70 peptide, and "β-gal" shows that it is the result of contacting the β-gal peptide. Also, “*” indicates that there is a significant difference at p<0.05.

As a result, it was revealed that cancer antigen-specific cytotoxic T cells are present in tumor tissues and lymph nodes derived from mice transplanted with the MCA205 cell line into which Ch25H has not been introduced. Moreover, the number of cancer antigen-specific cytotoxic T cells in tumor tissues and lymph nodes derived from a mouse transplanted with an MCA205 cell line overexpressing Ch25H is the same as in a mouse transplanted with an MCA205 cell line into which Ch25H is not introduced. It was found to be significantly less than the number of cancer antigen-specific cytotoxic T cells in the tumor tissue and lymph nodes from which they were derived.

The above results indicate that when cancer cells overexpress Ch25H, the number of cancer antigen-specific cytotoxic T cells decreases in tumor tissues and lymph nodes.

[Experimental Example 4]
(Study of cytotoxic T cells 2)
A tumor tissue was excised from the tumor-bearing mouse (38 days after cell transplantation) prepared in Experimental Example 2 and fixed with formaldehyde to prepare a tissue section. Subsequently, the prepared tissue section was stained with an anti-CD8 antibody to detect CD8-positive T cells.

FIGS. 4A and 4B are optical micrographs of immunostained tissue sections. 4(a) and 4(b), "MCA205-MOCK" indicates the result of the tumor tissue derived from the mouse transplanted with the MCA205 cell line into which Ch25H was not introduced, and "MCA205-Ch25H" indicates Ch25H. It is a result of tumor tissue derived from a mouse transplanted with an overexpressed MCA205 cell line. All scale bars are 100 μm.

As a result, in the tumor tissue derived from the mouse transplanted with the MCA205 cell line overexpressing Ch25H, invasion of CD8-positive T cells was compared with the tumor tissue derived from the mouse transplanted with the MCA205 cell line into which Ch25H was not introduced. It was revealed that the value was significantly decreased.

[Experimental Example 5]
(Detection of 25-hydroxycholesterol (25OHC) in serum)
Blood was collected from the tumor-bearing mouse (38 days after cell transplantation) prepared in Experimental Example 2 to prepare serum. Subsequently, the amount of 25-hydroxylated cholesterol (25OHC) present in serum was quantified by mass spectrometry.

Specifically, first, phosphate-buffered saline (PBS) at 10 times the inside of 1μg serum 1mL diluted standard (deuterium to be 0.1 mg / mL in ethanol labeled _D 6 -25 hydroxide 10 μL of dissolved cholesterol) was added. Subsequently, 1 mL of the sample was applied to a diatomaceous earth column (trade name “SLE+”, Biotage) and eluted with 3 mL of n-hexane. Then, the total amount of the eluate was applied to a silica column (trade name “Sep-pak”, waters company), washed with 1 mL of n-hexane:ethyl acetate (9:1) to remove cholesterol, and further ethyl acetate was added. 1 mL was applied and the eluate was collected. All oxysterols were recovered in this fraction.

Subsequently, the eluate was heated to 40° C., dried under a nitrogen stream, dissolved in 10 μL of pyridine, and mixed with 50 μL of BSTFA:TMCS (99:1) to mix all OH groups. Trimethylsilylated. Subsequently, 2 μL of the derivatized sample was injected into GC-MS and analyzed. The quantitative value was calculated using the peak area ratio of the internal standard and 25OHC.

The analysis conditions of GC-MS were as follows.
<<GC-MS analysis conditions>>
Capillary column: Rtx-5MS (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm)
Oven temperature: 150° C., holding 1 minute−20° C./minute→250° C., 5° C./minute→280° C., holding 10 minutes−20° C./minute→330° C., holding 3 minutes.
Carrier gas: He, linear velocity 39.0 cm/sec Ion source temperature: 200°C
Interface temperature: 280℃
Vaporization chamber temperature: 250°C

FIG. 5 is a graph showing measured values of 25OHC in serum. In FIG. 5, “MCA205-MOCK” indicates a quantitative value of 25OHC in serum derived from a mouse transplanted with an MCA205 cell line into which Ch25H was not introduced, and “MCA205-Ch25H” indicates an MCA205 cell line in which Ch25H was overexpressed. The quantitative value of 25OHC in the serum from the mouse transplanted with is shown.

As a result, the amount of 25OHC in the serum derived from the mouse transplanted with the MCA205 cell line overexpressing Ch25H was higher than that in the serum derived from the mouse transplanted with the MCA205 cell line into which Ch25H was not introduced. It was also revealed that the number was significantly higher (p=0.0015). The results show that blood samples can be used to measure the abundance of 25OHC.

<II. Examination in a model in which mouse cancer cells were transplanted to Ch25H knockout mice>
[Experimental Example 6]
(Study of efficacy of anti-PD-L1 antibody therapy)
The mouse colon cancer cell line MC38 was transplanted into the flank region of a Ch25H gene knockout mouse, and the tumor volume was measured over time. For comparison, the MC38 cell line was transplanted into the flank of wild-type C57BL/6 mice, and the tumor volume was measured over time.

On the 4th, 7th, 10th, and 14th days after cell transplantation, 200 μg/mouse of anti-PD-L1 antibody (BioXcell) was intraperitoneally administered to each mouse. For comparison, a group was also prepared in which 200 μg/mouse of an isotype control antibody (rat IgG2b, BioXcell) was intraperitoneally administered instead of the anti-PD-L1 antibody.

FIGS. 6(a) to 6(d) are graphs showing changes over time in tumor volume of mice in each group. In FIG. 6(a) to (d), “WT” indicates the result of the wild-type mouse, “Ch25H KO” indicates the result of the Ch25H knockout mouse, and “isotype antibody” indicates the isotype control. The results are shown for the mice administered with the antibody, and the "anti-PD-L1 antibody" is the results for the mouse administered with the anti-PD-L1 antibody.

As a result, in wild-type mice, there was an individual whose increase in tumor volume was not suppressed by administration of anti-PD-L1 antibody, whereas in Ch25H knockout mouse (Ch25H-KO), administration of anti-PD-L1 antibody was performed. Caused complete tumor rejection in all individuals.

This result indicates that removal or inhibition of Ch25H on the host side improves the efficacy of anti-PD-L1 antibody therapy.

[Experiment 7]
(Study of cytotoxic T cells 3)
The effect of inducing tumor antigen-specific T cells in regional lymph nodes and tumor tissues in the tumor-bearing mice prepared in Experimental Example 6 was evaluated by an IFN-γ production assay.

Specifically, first, the wild-type mouse transplanted with the MC38 cell line, which was administered with the isotype control antibody in Experimental Example 6, and the Ch25H knockout mouse transplanted with the MC38 cell line were euthanized 22 days after the cell transplantation. It was

Subsequently, the tumor tissue was excised from each mouse, cut into pieces with scissors, placed in 20 mL of collagenase solution (2 mg collagenase+30U DNase/1 mL RPMI1640), and shaken at 37° C. for 30 minutes. Then, CD8 magnetic T beads were separated using CD8 magnetic beads (Miltenyi).

The obtained T cells derived from the tumor tissue were collected from wild-type healthy mice in RPMI1640 medium containing 10% fetal bovine serum and mixed with 32 Gy of radiation-treated spleen cells to prepare T cell spleen cell mixed suspension A. (4×10 ⁵ T cells+1×10 ⁷ spleen cells/mL) were prepared.

In addition, regional lymph nodes (axillary and inguinal lymph nodes on the side of cancer cell transplantation) were removed from each mouse, and the lymph nodes were destroyed by grinding with a slide glass, and lymph node cells were collected and 10% bovine A lymph node cell suspension B prepared at 7×10 ⁶ cells/mL in RPMI1640 medium containing fetal serum was prepared.

Subsequently, the T cell spleen cell mixed suspension A was treated with a tumor antigen gp70 peptide (peptide consisting of amino acids 604 to 611 of mouse leukemia virus MuLV gp70 p15E: KSPWFTTL, SEQ ID NO: 1) (1.0 μg/mL). , Human IL-2 (20 U/mL), mouse IL-7 (10 ng/mL) in RPMI1640 medium containing 10% fetal bovine serum, and cultured in a 48-well plate (1 mL/well) for 2 days.

In addition, lymph node cell suspension B contained 10% fetal bovine serum containing tumor antigen gp70 peptide (1.0 μg/mL), human IL-2 (20 U/mL), mouse IL-7 (10 ng/mL). The cells were cultured in a 48-well plate (1 mL/well) in RPMI1640 medium for 7 days.

Subsequently, lymphocytes were collected using Lymphoprep (Alere Technologies AS). The recovered T-cells (1×10 ⁵ cells) derived from the tumor tissue were mixed with 96 well plates (200 μL) in the presence of EL4 cells (1×10 ⁵ cells) and a final concentration of 1 μg/mL and 0.1 μg/mL of the gp70 peptide. /Well), 10% fetal bovine serum-containing RPMI1640 medium was mixed and cultured in 3 wells for each group, and IFN-γ in the culture supernatant after 24 hours of culture was measured by an ELISA method.

Similarly, the collected lymph node-derived cells (5×10 ⁴ cells) were mixed with EL4 cells (1×10 ⁵ cells) at a final concentration of 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL, 0.001 μg. /ML and 0 µg/mL of gp70 peptide, 96-well plate (200 µL/well), 10% fetal calf serum-containing RPMI1640 medium was mixed and cultured in each group with 3 wells, and the culture supernatant after 24 hours of culture IFN-γ therein was measured by the ELISA method.

For comparison, an irrelevant β-Gal peptide (peptide consisting of amino acids 96 to 103 of β-galactosidase: DAPIYTNV, SEQ ID NO: 2) (1.0 μg/mL) was contacted instead of the gp70 peptide. We also prepared a group.

As a result, when cancer antigen-specific cytotoxic T cells are present in the cells, contact with a cancer antigen peptide produces IFN-γ.

FIGS. 7A and 7B are graphs showing the results of quantifying the amount of IFN-γ produced. FIG. 7( a) shows the result of cells derived from tumor tissue, and FIG. 7( b) shows the result of cells derived from lymph nodes. In FIGS. 7A and 7B, “Tumor” indicates the result of cells derived from tumor tissue, “LN” indicates the result of cells derived from lymph nodes, and “WT” indicates wild. "Ch25H KO" is the result of tumor tissue or lymph node derived from Ch25H knockout mouse, and "gp70" was contacted with gp70 peptide. The result is shown, and "β-gal" indicates the result of contacting the β-gal peptide. Also, “*” indicates that there is a significant difference at p<0.05.

As a result, the number of cancer antigen-specific cytotoxic T cells in the tumor tissue derived from the Ch25H knockout mouse is significantly higher than the number of cancer antigen-specific cytotoxic T cells in the tumor tissue derived from the wild-type mouse. It became clear. Moreover, it was revealed that cancer antigen-specific cytotoxic T cells were present in the lymph nodes derived from the Ch25H knockout mouse. In contrast, the presence of cancer antigen-specific cytotoxic T cells was not clearly recognized in the lymph nodes derived from wild-type mice.

The above results indicate that removal or inhibition of Ch25H on the host side increases the number of cancer antigen-specific cytotoxic T cells in tumor tissues and lymph nodes.

<III. Expression of Ch25H in various human cancer tissues>
[Experimental Example 8]
(Expression of Ch25H in various human cancer tissues)
Using RNA sequence data published in TCGA (The Cancer Genome Atlas) (http://cancergenome.nih.gov/), the expression level of Ch25H in various human cancer tissues can be calculated using The Human Protein Atlas(https:/). /Www.proteinatlas.org/) was used for analysis. FIG. 8 is a graph showing the expression level of mRNA of Ch25H gene in various human cancer tissues. As a result, disease cases in which the expression level of Ch25H gene was high were observed. In these disease cases, the effectiveness of cancer therapy including cancer immunotherapy may be low.

According to the present invention, it is possible to provide a new technique for determining the effectiveness of application of a cancer treatment method including cancer immunotherapy to a cancer patient.

Claims (14)

Use of the Cholesterol 25-hydroxylase (Ch25H) gene, Ch25H protein or 25-hydroxycholesterol (25OHC) as a marker for determining the effectiveness of application of cancer treatment methods including cancer immunotherapy to cancer patients. Measuring the abundance of Ch25H gene mRNA, Ch25H protein or 25OHC in a biological sample from a cancer patient,
The amount of the mRNA, the protein, or the 25OHC abundance is compared with the abundance in a control biological sample to determine the effectiveness of application of a cancer treatment method including cancer immunotherapy to the cancer patient. A method for obtaining data for evaluating the effectiveness of application of a cancer treatment method including cancer immunotherapy to the cancer patient. The abundance of the mRNA, the protein or the 25OHC is higher than the abundance in the control biological sample, which indicates that the application of the cancer treatment method including the cancer immunotherapy to the cancer patient is less effective. The method according to claim 2. The abundance of the mRNA, the protein, or the 25OHC is higher than the abundance in the control biological sample, which indicates that the cancer treatment method including cancer immunotherapy is highly effective for the application to the cancer patient. The method according to claim 2. The method according to any one of claims 2 to 4, wherein the biological sample is blood or tumor tissue. Application of a cancer treatment method including cancer immunotherapy to a cancer patient, which comprises a primer set for amplifying the cDNA of Ch25H gene, a probe that hybridizes with mRNA of Ch25H gene, a specific binding substance for Ch25H protein or a reagent for detecting 25OHC A kit for determining efficacy. An agent for improving the effectiveness of a cancer treatment method including cancer immunotherapy, which comprises an inhibitor of Ch25H protein as an active ingredient. The agent for improving the efficacy of a cancer treatment method including cancer immunotherapy according to claim 7, wherein the inhibitor of Ch25H protein is a substance that suppresses the expression of Ch25H gene. The agent for improving the efficacy of a cancer treatment method including cancer immunotherapy according to claim 8, wherein the substance that suppresses the expression of Ch25H gene is siRNA or shRNA of Ch25H gene. A cancer treatment kit comprising the agent for improving the efficacy of a cancer treatment method including the cancer immunotherapy according to any one of claims 7 to 9 and an anticancer agent. A method for screening an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy, comprising:
Contacting cholesterol and Ch25H protein in the presence of a test substance to produce 25OHC;
When the production amount of 25OHC is decreased or increased as compared with the production amount of 25OHC in the absence of the test substance, the test substance is an agent for improving the effectiveness of cancer treatment methods including cancer immunotherapy. Determining. When the amount of 25OHC produced is decreased as compared with the amount of 25OHC produced in the absence of the test substance, the test substance is determined to be an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy. The method of claim 11, wherein A method for screening an agent for improving the efficacy of a cancer treatment method including cancer immunotherapy, comprising:
Culturing the cancer cells in the presence of the test substance,
Measuring the expression level of the Ch25H gene or Ch25H protein or the production level of 25OHC in the cancer cell;
When the expression level of the Ch25H gene or the Ch25H protein or the production level of the 25OHC is decreased or increased as compared with the absence of the test substance, the test substance is effective for cancer treatment including cancer immunotherapy. Determining that it is a sex improving agent. When the expression level of the Ch25H gene or the Ch25H protein or the production level of the 25OHC is decreased as compared with the absence of the test substance, the test substance is effective for cancer treatment including cancer immunotherapy. The method according to claim 13, wherein the method is determined to be an enhancer.