JP6771203B2 - Method for predicting the effect of peptide vaccine therapy - Google Patents

Description

The present invention comprises a method for predicting the effect of peptide vaccine therapy on colorectal cancer patients, which comprises measuring the expression level of microRNA in the body fluid of a subject, and a kit for predicting the effect of peptide vaccine therapy on colorectal cancer patients. And related to the effect improver of peptide vaccine therapy for colorectal cancer patients.

Cancer is the most prevalent disease in the world, and among them, colorectal cancer is a disease in which about 700,000 people die every year. Over the last decade, the prognosis of patients with metastatic colorectal cancer has been significantly improved by combination therapy with standard chemotherapy (FOLFOX) and monoclonal antibodies such as anti-VEGF and anti-EGFR antibodies. However, the current situation is that many colorectal cancer patients have lost their lives due to progress due to chemotherapy resistance.

Early detection of cancer is important for its treatment, and various cancer diagnostic methods have been developed for this purpose. Among them, in recent years, there is a method of diagnosing cancer by measuring microRNA. MicroRNA (hereinafter, also referred to as "miR") is an RNA having a length of 18 to 25 bases existing in a cell, and is a non-coding RNA that is considered to have a function of regulating the expression of other genes. It is a kind. So far, colorectal cancer test markers consisting of microRNA molecules (see Patent Document 1) and colon cancer cells have been cultured in a serum-free medium and the microRNA released into the culture medium has been measured. A method for evaluating the malignancy of cells (see Patent Document 2) has been proposed. It is also a method of diagnosing whether a subject has or is at risk of developing prostate cancer and measures the level of miR-125b-1 in a test sample from the subject. A method including the above (see Patent Document 3) and a method for quantifying the recurrence probability of cancer in a patient having prostate cancer, in a biological sample containing prostate tissue collected from the patient. A method including a step of measuring the expression level of miR-486-5p (see Patent Document 4), that tumor-derived miR-486-5p in serum is associated with lung cancer (see Non-Patent Document 1), and It has been reported that the expression of miR-147b was more than doubled between gastric cancer tissue and normal gastric tissue (see Non-Patent Document 2).

On the other hand, as a therapy for patients for whom standard cancer therapy is inadequate, immunotherapy with a different mechanism of action is being developed worldwide in order to be used in combination with standard chemotherapy. It was found that a simple tumor antigen peptide vaccine in the early stages of development was not sufficiently effective in immunotherapy. Therefore, the development of immunoregulatory technology that controls important points of the anti-tumor immune network is currently underway, and the development of combined immunotherapy that combines them is expected in the future. Tumor antigen epitope peptides are used for immunotherapy, for example, RNF43 (ring finger protein 43), TOMM34 (34kDa translocase of the outer mitochondrial membrane), KOC1 (IMP-3; IGF-II mRNA binding protein 3), etc. have been reported. (See Non-Patent Document 3). Since the effect of such immunotherapy varies from patient to patient and it is preferable to predict the therapeutic effect before the therapy is given, it is an important issue to develop a method that can appropriately predict the effect before the immunotherapy is given. It has become.

So far, it is selected from the group consisting of miR-660, miR-324-5p, miR-532-5p, and microRNAs belonging to the same family as these microRNAs contained in the samples obtained from the subjects. A method for predicting the effect of therapy for liver fibrosis in the subject, which includes a step of measuring one or more (see Patent Document 5), has been proposed.

Further, the present inventors are characterized in measuring the expression level of at least one microRNA selected from 13 types of microRNAs such as miR-147b and miR-486-5p expressed in cancer tissues. We proposed a method for predicting the effect of peptide vaccine therapy on cancer patients (see Patent Document 6). However, the above 13 types of microRNAs are microRNAs derived based on microRNA analysis in cancer tissues, and it may not be possible to collect cancer tissues, and even if possible, excessive invasion may occur. Often accompanied. In addition, the expression level of microRNA expressed in cancer tissue and the expression level of microRNA in plasma do not always correlate, and even if they do correlate, it can be said to be a rare case. In fact, when the expression level of microRNA in cancer tissue and the expression level of microRNA in plasma in Patent Document 6 were compared and examined, only one microRNA showed a correlation. Therefore, in order to accurately predict the effect of peptide vaccine therapy, it is necessary to excise the cancer tissue and analyze the expression level of microRNA derived based on the analysis of the cancer tissue, which is a less invasive peptide. A method for predicting the effect of vaccine therapy has been sought.

Re-table 2011-040525 Re-table 2011-001906 Japanese Unexamined Patent Publication No. 2013-046612 Special Table 2013-532482 Japanese Unexamined Patent Publication No. 2012-125215 JP-A-2015-213492

Li Y, Liang L, Zhang CY. Anal Chem. 2013 Dec3; 85 (23): 11174-9 Yao Y, Suo AL, Li ZF, Liu LY, Tian T, Ni L, Zhang WG, Nan KJ, Song TS, Huang C. Mol Med Rep. 2009 Nov-Dec; 2 (6): 963-70 Shoichi 硲, Masao Oka Journal of the Japanese Surgery Association 2012 37 (1): 41-45

In cancer immunotherapy, it is important to establish a method that can predict its effect with minimal invasiveness, easily and quickly, and a biomarker for that purpose. Furthermore, by developing the immunocontrol technology necessary for constructing combined immunotherapy. , It is necessary to aim at the construction of a new combined immunotherapy treatment strategy. Therefore, the subject of the present invention is a method for predicting the effect of peptide vaccine therapy on colorectal cancer patients in a minimally invasive, simple and rapid manner by measuring the expression level of microRNA in body fluids, and peptides for colorectal cancer patients. The purpose is to provide a kit for predicting the effect of vaccine therapy and an agent for improving the effect of peptide vaccine therapy for cancer patients.

In order to search for biomarkers that can predict the effect of peptide vaccine therapy on colorectal cancer patients, we conducted an exploratory analysis of 93 cases of standard chemotherapy and peptide vaccine therapy as the first-line therapy for advanced colorectal cancer. As a result, they found that the effect of peptide vaccine therapy differs depending on the level of expression of microRNA in plasma, and completed the present invention.

That is, the present invention is as shown in the following [1] to [5].
[1] A method for predicting the effect of peptide vaccine therapy on a colorectal cancer patient, which comprises measuring the expression level of microRNA in the body fluid of a subject, wherein the microRNA is the following (1) or A method characterized by being any of the microRNAs shown in (2).
(1) MicroRNA-6826-5p (hsa-miR-6826-5p) shown in SEQ ID NO: 1;
(2) MicroRNA-6875-5p (hsa-miR-6875-5p) shown in SEQ ID NO: 2;
[2] The method according to the above [1], wherein the colorectal cancer patient is a patient carrying a human leukocyte antigen (HLA) -A * 2402.
[3] The method according to the above [1] or [2], wherein the epitope peptide used for peptide vaccine therapy is an epitope peptide having an ability to bind to human leukocyte antigen (HLA) -A * 2402.
[4] A kit for predicting the effect of peptide vaccine therapy for colorectal cancer patients, which is a probe that hybridizes with any of the microRNAs shown in (1) or (2) below under stringent conditions, or the following (1). ) Or a kit comprising a primer pair capable of amplifying either of the microRNAs shown in (2).
(1) MicroRNA-6826-5p (hsa-miR-6826-5p) shown in SEQ ID NO: 1;
(2) MicroRNA-6875-5p (hsa-miR-6875-5p) shown in SEQ ID NO: 2;
[5] An agent for improving the effect of peptide vaccine therapy for colorectal cancer patients, which comprises a microRNA inhibitor for any of the microRNAs shown in (1) or (2) below.
(1) MicroRNA-6826-5p (hsa-miR-6826-5p) shown in SEQ ID NO: 1;
(2) MicroRNA-6875-5p (hsa-miR-6875-5p) shown in SEQ ID NO: 2;

According to the present invention, it is possible to predict the effect of peptide vaccine therapy on colorectal cancer patients by analyzing microRNAs in body fluids. Therefore, it is possible to perform immunotherapy for colorectal cancer patients in a minimally invasive, simple and rapid manner.

It is a figure which shows the result of having investigated the relationship between the expression level of hsa-miR-6826-5p and the total survival time in Example 2, A is a study therapy group (HLA-A * 2402+), B is a control group (HLA). -A * 2402-). Low is a patient whose relative expression level for miR191 is less than the median, High is a patient whose relative expression level for miR191 is or more, the horizontal axis indicates the number of months since the start of administration, and the vertical axis indicates the overall survival rate. It is a figure which shows the result of having investigated the relationship between the expression level of hsa-miR-6875-5p and the total survival time in Example 2, A is a study therapy group (HLA-A * 2402+), B is a control group (HLA). -A * 2402-). Low is a patient whose expression level is less than the median relative to miR191, High is a patient whose expression level is greater than or equal to the median relative expression level to miR191, and the horizontal axis and the vertical axis are the same as in FIG. It is a figure which shows the result of having investigated the relationship between the expression level of miR-135a-3p and the total survival time in Example 2, A is a study therapy group (HLA-A * 2402+), and B is a control group (HLA-A). * 2402-). Low is a patient whose expression level is less than the median relative to miR191, High is a patient whose expression level is greater than or equal to the median relative expression level to miR191, and the horizontal axis and the vertical axis are the same as in FIG.

The method for predicting the effect of peptide vaccine therapy on colorectal cancer patients of the present invention is a method for predicting the effect of peptide vaccine therapy on colorectal cancer patients, which comprises measuring the expression level of microRNA in the body fluid of a subject. The method is not particularly limited as long as the method is characterized in that the microRNA is any of the microRNAs shown in (1) or (2) below, but humans are preferably used as the subject. Can be mentioned. The method for predicting the effect of the peptide vaccine therapy of the present invention does not include a diagnostic act by a doctor.
(1) MicroRNA-6826-5p (hsa-miR-6826-5p) shown in SEQ ID NO: 1;
(2) MicroRNA-6875-5p (hsa-miR-6875-5p) shown in SEQ ID NO: 2;

Examples of the body fluid of the subject include plasma, serum, stool, urine, saliva and the like of the subject.

The microRNA in the present invention includes microRNA-6826-5p (miRBase ID: hsa-miR-6826-5p, Accession NO: MIMAT0027552) shown in SEQ ID NO: 1 (5'-ucaauaggaaagaggugggaccu-3') and SEQ ID NO: 2 MicroRNA-6875-5p (miRBase ID: hsa-miR-6875-5p, Accession NO: MIMAT0027650) (hereinafter collectively referred to as "the MicroRNA") shown in (5'-ugagggacccaggacaggaga-3'). Either.

The method for extracting microRNA from the body fluid of the subject is not particularly limited as long as it is a method for extracting RNA containing microRNA from the body fluid of the subject, for example, miRNeay Mini Kit (manufactured by Kiagen). By using according to the attached protocol, a method for extracting total RNA including microRNA can be preferably exemplified.

In the present invention, the method for measuring the expression level of microRNA is not particularly limited, and examples thereof include a quantitative PCR method and a microarray method.

The quantitative PCR method is a method using a primer pair capable of amplifying the sequence of the MicroRNA, and is not particularly limited as long as the expression level of the MicroRNA can be measured, and is an agarose gel electrophoresis method. , SYBR green method, fluorescent probe method and other ordinary quantitative PCR methods can be used, but the fluorescent probe method is preferable in terms of quantification accuracy and reliability.

In the above microarray method, RNA extracted from body fluid is labeled with a label such as fluorescence, and the RNA is brought into contact with a microarray on which a probe that hybridizes with the microRNA under stringent conditions is fixed. A method of washing the microarray after hybridization and measuring the expression level of microRNA remaining on the microarray can be exemplified.

In the present invention, the peptide vaccine therapy is a method of treating a cancer by administering to a patient an epitope peptide capable of binding to HLA possessed by the subject to be administered to generate specific acquired immunity to the cancer. Means.

In the present invention, the epitope peptide means a peptide used for immunotherapy consisting of 8 to 10 amino acids derived from a protein specifically present in cancer cells, and the epitope peptide is a subject to be administered. It is preferably an epitope peptide capable of binding to HLA possessed by HLA. For example, when a subject possesses HLA-A * 2402, an epitope peptide having an ability to bind to HLA-A * 2402 (HLA-A *) It is preferably an epitope peptide (2402 binding epitope peptide), and when the subject possesses HLA-0201, it is preferably an epitope peptide (HLA-0201 binding epitope peptide) having an ability to bind to HLA-0201. When the subject carries HLA-3101, it is preferably an epitope peptide having an ability to bind to HLA-3101 (HLA-3101 binding epitope peptide), and the subject carries HLA-1101. In some cases, it is preferably an epitope peptide having an ability to bind to HLA-1101 (HLA-1101 binding epitope peptide), and when the subject has HLA-2601, the ability to bind to HLA-2601 is determined. It is preferably an epitope peptide having (HLA-2601 binding epitope peptide), and when the subject has HLA-3303, an epitope peptide having a binding ability to HLA-3303 (HLA-3303 binding epitope peptide). ), And when the subject possesses HLA-0206, it is preferably an epitope peptide (HLA-0206 binding epitope peptide) having an ability to bind to HLA-0206, and the subject When HLA-0207 is possessed, it is preferably an epitope peptide having an ability to bind to HLA-0207 (HLA-0207-binding epitope peptide). Specific examples of the HLA-A * 2402 binding epitope peptide include tumor antigen-derived RNF43-721 (SEQ ID NO: 3: NSQPVWLCL), TOMM34-299 (SEQ ID NO: 4: KLRQEVKQNL), and KOC1 (IMP-3)-. 508 (SEQ ID NO: 5: KTVNELQNLAS) and VEGFR (vascular endothelial)
Growth factor receptor) 1, VEGFR1-1084 (SEQ ID NO: 6: SYGVLLWEI), VEGFR2-169 (SEQ ID NO: 7: RFVPDGNRI), which are angiogenesis-suppressing cancer vaccines targeting VEGFR2, can be mentioned. Only one of them may be used, two or more may be mixed, three or more may be mixed, four or more may be mixed, or all five may be mixed. Since the epitope peptides on the surface vary depending on the colorectal cancer cells, the number of epitope peptides to be mixed is 3, or more, preferably 4 or more, more preferably 5, from the viewpoint of further enhancing the effect of peptide vaccine therapy. More than one.

In the present invention, the cancer patient is not particularly limited as long as it is a patient having an HLA to which an epitope peptide used for peptide vaccine therapy can be bound. For example, when an HLA-A * 2402 binding epitope peptide is used. , HLA-A * 2402 is preferred, and when the HLA-0201 binding epitope peptide is used, the patient is preferably HLA-0201-bearing, and the HLA-3101 binding epitope peptide is used. When used, it is preferably a patient carrying HLA-3101, and when using an HLA-1101 binding epitope peptide, it is preferably a patient carrying HLA-1101, preferably an HLA-2601 binding epitope. When using a peptide, it is preferable to be a patient carrying HLA-2601, and when using an HLA-3303-binding epitope peptide, it is preferable to be a patient carrying HLA-3303, and HLA-0206 restraint. When a sex epitope peptide is used, it is preferable that the patient has HLA-0206, and when an HLA-0206 binding epitope peptide is used, it is preferable that the patient has HLA-0206. Whether or not a cancer patient has HLA type can be confirmed by the HLA genotyping method.

In the present invention, predicting the effect of peptide vaccine therapy means predicting whether the effect of peptide vaccine therapy is high or low in patients who have undergone peptide vaccine therapy. Specifically, peptide vaccine therapy is performed. It can be mentioned to predict whether the effect of improving the overall survival time or the overall survival rate of the patient is high or low. The overall survival rate means the proportion of patients who have been treated with peptide vaccine therapy and who are alive after a certain period of time has passed since the treatment.

The effect of peptide vaccine therapy on the above cancer patients can be predicted by measuring the expression level of the MicroRNA in the body fluid of the subject. For example, the expression level of the MicroRNA in the body fluids of 2 or more, preferably 4 or more, more preferably 5 or more cancer patients with and without the effect of peptide vaccine therapy was measured in advance. Then, the median or average value of the expression level is calculated, and the cutoff value is determined based on the median or average value. Next, by measuring the expression level of the MicroRNA in the body fluid of the subject and comparing the expression level of the microRNA of the subject with the cutoff value, the effect of peptide vaccine therapy on the cancer patient can be obtained. It is possible to predict.

As cancer patients with and without the effect of peptide vaccine therapy, cancer patients with the effect of peptide vaccine therapy are defined as "patients who have survived for 3 years or more after the start of peptide vaccine therapy". A method can be mentioned in which a cancer patient who has no effect of the above is regarded as a "patient who died less than two years after the start of peptide vaccine therapy".

In addition, as a method for predicting the effect of peptide vaccine therapy on cancer patients by measuring the expression level of the MicroRNA and comparing the expression level of the subject with the cutoff value, a specific method is used. When the microRNA of the present case is measured, if the expression level is less than the cutoff value, it can be predicted that the effect of the peptide vaccine therapy is high.

The kit for predicting the effect of peptide vaccine therapy on cancer patients of the present invention is particularly limited as long as it is a kit provided with a probe that hybridizes with the MicroRNA under stringent conditions or a primer pair capable of amplifying the MicroRNA. However, in addition to the probe or the primer pair, the kit may include reagents used for labeling reactions of buffers, dNTPs, RNA, reagents used for hybridization reactions, instructions, and the like.

The probe or primer pair can be obtained by chemical synthesis or the like using a method well known in the art. By performing a quantitative PCR method or a microarray method using the probe or the primer pair, it is possible to measure the expression level of microRNA.

The stringent condition refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Specifically, 80% or more, preferably 90% or more, more preferably 95% or more. More preferably, DNAs having 100% identity hybridize with each other, and DNAs with lower identity do not hybridize with each other, or 65 ° C., 1 × SSC, which is a condition for normal Southern hybridization washing. Conditions for hybridizing with a solution (the composition of a 1-fold SSC solution is 150 mM sodium chloride, 15 mM sodium citrate), 0.1% SDS, or 0.1 × SSC, a salt concentration corresponding to 0.1% SDS. Can be mentioned.

Examples of the probe include a polynucleotide consisting of the base sequence of the microRNA of the present microRNA or a base sequence complementary to a part thereof. If such a polynucleotide is fixed to a microarray as a probe and used, a microarray method can be used. It can be performed.

Further, the primer pair may be any primer pair capable of amplifying the MicroRNA, and a polynucleotide (forward primer) consisting of a part of the base sequence on the 5'side of the base sequence of the MicroRNA and the Micro. A primer consisting of a polynucleotide (reverse primer) complementary to a part of the 3'side of a polynucleotide (reverse transcription primer) complementary to a part of the base sequence on the 3'side of the RNA base sequence You can also list a pair. By using such a primer pair, a quantitative PCR method can be performed, and specifically, a commercially available kit such as TaqMan (registered trademark) MicroRNA Assay (manufactured by Thermo Fisher Scientific) can be used.

The agent for improving the effect of peptide vaccine therapy for cancer patients of the present invention is not particularly limited as long as it contains a microRNA inhibitor for the MicroRNA, and the microRNA inhibitor has an antagonistic effect on the MicroRNA. Means a compound having.

The above-mentioned effect enhancer of peptide vaccine therapy includes excipients, binders, lubricants, disintegrants, preservatives, isotonic agents, stabilizers, which are usually used for formulation and are pharmaceutically acceptable. It may contain additives such as dispersants, antioxidants, colorants, flavors and buffers. The dosage form of the preparation may be a solid preparation such as a powder or a granule, but from the viewpoint of improving the effect of the excellent peptide vaccine therapy, a liquid preparation such as a solution, an emulsion or a suspension may be used. preferable.

The method of administering the effect-enhancing agent of the peptide vaccine therapy is not particularly limited as long as the desired effect of the peptide vaccine therapy can be improved, and is intravenous administration, oral administration, intramuscular administration, subcutaneous administration, transdermal administration, and nasal administration. Nasal administration, transpulmonary administration and the like can be mentioned. Further, the effect improving agent of the peptide vaccine therapy may be administered to the patient at the same time as a drug such as an epitope peptide used in the peptide vaccine therapy, or either one may be administered to the patient first.

[Exploratory analysis of cases in which a phase II study (PII) was performed]
(Treatment for cancer patients)
Combination therapy (II) in which peptide vaccine therapy is added to FOLFOX therapy (Schmoll HJ, et all, J Clin Oncol (2012) 30: 3588-3595), which is standard chemotherapy, as first-line therapy for patients with unresectable advanced colorectal cancer. Phase study (PII): FXV) was performed on a total of 96 patients. This study complies with the Declaration of Helsinki and was approved by the Ethics Review Board of Yamaguchi University. Informed consent was also obtained from the patient.

(Peptide vaccine therapy)
For peptide vaccine therapy, 3 mg of each of the five HLA-A * 2402 restrictive epitope peptides is mixed with 1.5 ml of incomplete Freund's adjuvant (IFA) Montanide ISA51 (manufactured by Seppci) and administered subcutaneously once a week. Subcutaneous administration was performed for 13 weeks and then once every 2 weeks. The five types of epitope peptides include RNF43-721 (SEQ ID NO: 3: NSQPVWLCL), TOMM34-299 (SEQ ID NO: 4: KLRQEVKQNL), KOC1 (IMP-3) -508 (SEQ ID NO: 5: KTVNELQNL), which are derived from tumor antigens. VEGFR1-1084 (SEQ ID NO: 6: SYGVLLWEI), an angiogenesis-suppressing cancer vaccine that targets VEGFR1, and VEGFR2-169 (SEQ ID NO: 7: RFVPDGNRI) (American), an angiogenesis-suppressing cancer vaccine that targets VEGFR2. (Manufactured by Peptide Company) was used. The above peptides were synthesized by the standard solid phase method, purified, and used as GMP grade peptides.

(Presence or absence of HLA-A * 2402 in patients)
The therapy was a biological double-blind study without notifying the patient and the attending physician whether the patient had HLA-A * 2402. The HLA genome typing method was used to examine whether each patient had HLA-A * 2402, and 50 patients were in the study therapy group (HLA-A * 2402 patients with the effect of the epitope peptide used in this study). Group: HLA-A * 2402 +), 46 patients were in the control group (group of patients who did not have HLA-A * 2402, no effect of the epitope peptide used in this study: HLA-A * 2402-). It turned out at a later key opening. Peripheral blood obtained from 93 of the 96 patients mentioned above was collected in an EDTA tube, centrifuged at 400 g for 15 minutes at 4 ° C to prepare plasma, and stored at -80 ° C until use. It was used for extraction of total RNA in Example 1 and Example 2 described later.

(Extraction of total RNA)
Using total RNA extracted from the plasma of 13 of 93 patients, the relationship between the expression level of about 2600 types of microRNA and overall survival was comprehensively analyzed by microRNA microarray analysis. Total RNA was extracted using a 3D-Gene® RNA extraction reagent from liquid sample kit (manufactured by Toray Industries, Inc.) according to the protocol.

(MicroRNA microarray analysis)
MicroRNA microarray analysis was performed using a 3D-Gene miRNA Labeling kit and a 3D-Gene Human miRNA Oligo Chip (manufactured by Toray Industries, Inc.). The relative fluorescence intensity of each hybridization signal was evaluated by Microarray Data Analysis Tool Ver3.2 (manufactured by Filgen), and the expression level of microRNA was examined.

Next, of the above 13 patients, 5 were long-term survivors (patients who survived for 3 years or more after the start of peptide vaccine therapy), and 8 were short-term survivors (less than 2 years after the start of peptide vaccine therapy). (Dead patient), the expression level of microRNA expressed in the plasma of each group of patients (plasma miR expression level) was compared, and Log2 Radio and Fisher Radio were determined. The results are shown in Table 1.

According to the above microarray analysis, there are four microarrays that distinguish between the two groups: miR-6826-5p, miR-6875-5p, miR-135a-3p, and miR-6835-5p, which have an absolute value of Log2Ratio of 1.3 or more. MicroRNA was selected as a candidate.

Total RNA was extracted from the plasma of 93 cases obtained in Example 1 above, purified with miRNeasy serum / plasma kit (manufactured by QIAGEN), and then miR-6826-5p, miR-6875- by RT-PCR analysis shown below. The expression of 5p, miR-135a-3p, and miR-6835-5p was quantified. Extraction of total RNA was carried out in the same manner as in Example 1. Since miR-6835-p could not be detected by quantitative PCR and its expression level was extremely low, it was excluded from this analysis.

(RT-PCR analysis)
For each of miR-6826-5p, miR-6875-5p, miR-135a-3p, and miR-6835-5p, TaqMan® MicroRNA assay (Thermo Fisher Scientific) and LightCycler 480 Probe Master (Roche Diagnostics) ) Was used to perform RT-PCR with LightCycler® 480 System II (manufactured by Roche Diagnostics). As an intrinsic control, miR-191 was used. Using the Looped RT primer and template RNA included in the TaqMan MicroRNA assay, reverse transcription reaction was performed at 16 ° C. for 30 minutes, 42 ° C. for 30 minutes, and 85 ° C. for 5 minutes, followed by PCR reaction at 95 ° C. for 10 minutes. After ting, the test was performed at 95 ° C. for 15 seconds and 60 ° C. for 60 seconds in 40 cycles. The value of mean ± 3SD was excluded.

Furthermore, patients in the study therapy group (HLA-A * 2402+) and control group (HLA-A * 2402-) were divided into two groups according to the level of microRNA expression, and the overall survival rate was determined by the Kaplan-Meier method. The difference in overall survival between the two groups was compared and examined by the log-rank test. The median expression level of microRNA expressed in plasma in all cases (93 cases) was used as the reference cut-off value when dividing patients into two groups according to the expression level. Overall survival was calculated from the day the epitope peptide was first administered to the day the patient died. The relationship between the expression level of each microRNA and the overall survival time is shown in FIGS.

As shown in FIGS. 1 and 2, in the case where the expression levels of miR-6826-5p and miR-6875-5p were low (relative fluorescence intensity was less than the cutoff value) in the test therapy group (HLA-A * 2402+). Was found to have a longer overall survival time than cases with high expression levels (relative fluorescence intensity above the cutoff value). Regarding the above microRNAs, a difference in prognosis was confirmed only in the test therapy group (HLA-A * 2402 +) carrying HLA-A * 2402, and the expression level of each microRNA in plasma , Was shown to be associated with the effects of peptide vaccine therapy. On the other hand, as shown in FIG. 3, in the study therapy group (HLA-A * 2402+), there was no difference in overall survival between cases with high expression levels of miR-135a-3p and cases with low expression levels.

Therefore, miR-6826-5p and miR-6875-5p are biomarkers that predict the effect of peptide vaccine therapy, and by examining the expression level of the above microRNA in plasma, the effect of peptide vaccine therapy on cancer patients It became clear that it is possible to predict. In addition, it is considered that the effect of peptide vaccine therapy is enhanced by using a compound having an antagonistic effect on miR-6826-5p and miR-6875-5p.

INDUSTRIAL APPLICABILITY The present invention can predict the effect of peptide vaccine therapy on colorectal cancer patients, and can be used in the field of colorectal cancer treatment.

Claims (2)

A method for assisting prediction of the effect of peptide vaccine therapy on a patient with colorectal cancer, which comprises measuring the expression level of microRNA in plasma or serum of a subject, wherein the microRNA is described in (1) below. ) or (2) the Ri der either micro RNA shown, colon cancer patients, a patient carrying human leukocyte antigen (HLA) -A * 2402, epitope peptides used for the peptide vaccine therapy is human Expression of the microRNA in plasma or serum of a cancer patient having an effect of peptide vaccine therapy and a cancer patient having no effect in advance, which is an epitope peptide having a binding ability to leukocyte antigen (HLA) -A * 2402. defining a cut-off value based on the amount, the by comparing the expression level of a micro RNA and the cut-off value, you assist effect prediction of peptide vaccine therapy for subjects in plasma or serum of a subject A method characterized by that.
(1) MicroRNA-6826-5p (hsa-miR-6826-5p) shown in SEQ ID NO: 1;
(2) MicroRNA-6875-5p (hsa-miR-6875-5p) shown in SEQ ID NO: 2; It is an effect prediction kit for peptide vaccine therapy using an epitope peptide capable of binding to human leukocyte antigen (HLA) -A * 2402 for colon cancer patients carrying human leukocyte antigen (HLA) -A * 2402. Therefore, a probe that hybridizes with either of the microRNAs shown in (1) or (2) below under stringent conditions or a primer capable of amplifying either of the microRNAs shown in (1) or (2) below. Kit with pairs.
(1) MicroRNA-6826-5p (hsa-miR-6826-5p) shown in SEQ ID NO: 1;
(2) MicroRNA-6875-5p (hsa-miR-6875-5p) shown in SEQ ID NO: 2;