JP6831112B2 - Biomarkers for predicting the efficacy of PD-1 pathway inhibitors - Google Patents

Description

The present invention relates to a method for determining drug susceptibility to a PD-1 pathway inhibitor, a biomarker for determining drug susceptibility to a PD-1 pathway inhibitor, and a kit for determining drug susceptibility to a PD-1 pathway inhibitor.

PD-1 (Programmed Cell Death -1) is a receptor for immunosuppressive auxiliary signals that suppress immunity to cancer cells expressed on T cells, and PD-L1 and PD-L2 are known as ligands. .. In recent years, new cancer treatments using anti-PD-1 antibody, anti-PD-L1 antibody, etc. that block this PD-1 pathway have been attracting attention, and biomarkers related to the therapeutic effect have been searched all over the world. It has been.

For example, Patent Document 1 reports that a human monoclonal antibody that specifically binds to PD-1 has been isolated. Among PD-1 pathway inhibitors, nivolumab and pembrolizumab are known as anti-PD-1 antibodies. It is considered that the immune response to cancer cells can be enhanced by inhibiting the PD-1 pathway by utilizing such an anti-PD-1 antibody or an anti-PD-L1 antibody that binds to a ligand.

Biomarkers relating to PD-1 pathway inhibitors have been reported, for example, in Non-Patent Documents 1 to 3.

According to Non-Patent Document 1, many clinical trials have reported that the expression of PD-L1 by tumors correlates to some extent with the therapeutic effect of PD-1 pathway inhibitors. There are still many problems such as inconsistency in timing, sampling method, analysis method and evaluation method.

Non-Patent Document 2 describes that a new antigen (neoantigen) generated by mutation in a tumor becomes a target of antitumor T cells, and that the neoantigen may cause a strong immune response in vivo. .. It is described that when anti-PD-1 antibody treatment was performed on a tumor that became resistant to immunity to the neoantigen, immunity to the tumor was restored. As described above, what is described in Non-Patent Document 2 is the relationship between neoantigen and PD-1 inhibition.

Non-Patent Document 3 reports that in patients to whom anti-PD-1 treatment is effective, the number of cells expressing PD-1 and PD-L1 in infiltrated tumors is large, and the repertoire of T cell antigen receptors is large. There is.

Japanese Patent No. 4361545

Patel SP, Kurzrock R, Mol Cancer Ther, 14 (4), 847-856. 2015 Apr Jedd D. Wolchok and Timothy A. Chan, Nature, Vol.515, 496-498, 17 November 2014 Tumeh, P.C. et al. Nature, Vol.515, 568-571, 27 November 2014

The present invention finds a gene whose expression is increased in ovarian cancer showing susceptibility to a PD-1 pathway inhibitor, and provides a method for determining susceptibility of ovarian cancer to a PD-1 pathway inhibitor using the gene. With the goal. Another object of the present invention is to provide a biomarker for determining drug susceptibility of ovarian cancer, which contains the gene, and a kit for determining drug susceptibility of ovarian cancer.

The present inventor conducted a phase II doctor-initiated clinical trial using an anti-PD-1 antibody drug in 20 patients with recurrent ovarian cancer at the Department of Obstetrics and Gynecology, Kyoto University Hospital, and found tumor shrinkage in 15% of the patients. A therapeutic effect (including unchanged) was observed in 45% of patients. Therefore, surgical specimens (tumor tissue) were obtained from these patients, and as a result of comprehensive gene expression analysis after extracting the RNA of the tumor, it was expressed in patients who confirmed the therapeutic effect of complete response with anti-PD-1 antibody drug. Genes and gene groups in which the number increases (or decreases) were statistically extracted. Then, from these genes, candidate genes that could effectively function as biomarkers for determining susceptibility to PD-1 pathway inhibitors were identified.

The present invention has been completed by further studies based on these findings, and is the following method for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer and determination of susceptibility to PD-1 pathway inhibitors for ovarian cancer. To provide a biomarker for ovarian cancer and a kit for determining susceptibility to a PD-1 pathway inhibitor for ovarian cancer.

(I) Method for determining susceptibility of ovarian cancer to PD-1 pathway inhibitors
(I-1) ATP7B, BAIAP2L2, DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, GRAMD3, LOC100422737, MMP24, NHSL1, PTHLH, RHOD in biological samples collected from test subjects. , SAA2, SLCO4A1, UNC5A, and VNN1 as an index of the expression level of at least one gene selected from the group.
A method for determining the susceptibility of ovarian cancer to a PD-1 pathway inhibitor in the test subject.
(I-2) The expression level of at least one gene selected from the group consisting of DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, MMP24, SLCO4A1, UNC5A, and VNN1 is used as an index. , (I-1).
(I-3) The method according to (I-1) or (I-2), wherein the expression level of at least one gene selected from the group consisting of EEF1A2, HNF1B, and VNN1 is used as an index.
(I-4) Detection of the expression level of the gene,
The method according to any one of (I-1) to (I-3), which is carried out by detecting the mRNA of the gene or the protein encoded by the gene.
(I-5) The method according to any one of (I-1) to (I-4), wherein the PD-1 pathway inhibitor is an anti-PD-1 antibody.
(I-6) In any one of (I-1) to (I-5), which determines that ovarian cancer is sensitive to a PD-1 pathway inhibitor when the expression level is elevated. The method described.
(I-7) PD for test subjects whose ovarian cancer was determined to be sensitive to PD-1 pathway inhibitors by the method according to any one of (I-1) to (I-6). -1 A method of treating ovarian cancer, including the step of administering an effective amount of a pathway inhibitor.

(II) Biomarker for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer
(II-1) ATP7B, BAIAP2L2, DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, GRAMD3, LOC100422737, MMP24, NHSL1, PTHLH, RHOD, RNF182, SAA2, SLCO4 Contains at least one gene selected from the group
A biomarker for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer.
(II-2) Contains at least one gene selected from the group consisting of DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, MMP24, SLCO4A1, UNC5A, and VNN1 (II-1) ). The biomarker.
(II-3) The biomarker according to (II-1) or (II-2), which comprises at least one gene selected from the group consisting of EEF1A2, HNF1B, and VNN1.
(II-4) The biomarker according to any one of (II-1) to (II-3), wherein the PD-1 pathway inhibitor is an anti-PD-1 antibody.

(III) Kit for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer
(III-1) ATP7B, BAIAP2L2, DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, GRAMD3, LOC100422737, MMP24, NHSL1, PTHLH, RHOD, RNF182, SAA2, SLCO4 A nucleic acid that binds to at least one gene selected from the group, or an antibody or antibody fragment that binds to a protein encoded by the gene.
A kit for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer.
(III-2) Nucleic acid that binds to at least one gene selected from the group consisting of DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, MMP24, SLCO4A1, UNC5A, and VNN1, or the said The kit according to (III-1), which comprises an antibody or antibody fragment that binds to a protein encoded by a gene.
(III-3) A nucleic acid that binds to at least one gene selected from the group consisting of EEF1A2, HNF1B, and VNN1, or an antibody or antibody fragment that binds to a protein encoded by the gene (III-1). ) Or the kit according to (III-2).
(III-4) The kit according to any one of (III-1) to (III-3), wherein the PD-1 pathway inhibitor is an anti-PD-1 antibody.

INDUSTRIAL APPLICABILITY According to the present invention, a gene that serves as a biomarker for susceptibility to a PD-1 pathway inhibitor of ovarian cancer can be found, and by using the expression level of the gene as an index, an ovary that exhibits susceptibility to a PD-1 pathway inhibitor. It is possible to determine whether or not it is cancer.

According to the present invention, it is possible to efficiently select patients who are effective against the drug.

It is a figure which shows the result of having performed the hierarchical cluster analysis with CR as a complete response case and PR, SD and PD as other cases in a test example. It is a graph which shows the expression level of the EEF1A2 gene of each sample in a test example. The vertical axis represents the gene expression intensity log2 expression signal intensity (relative amount of gene expression normalized and log2 converted in 17 samples). It is a graph which shows the expression level of the VNN1 gene of each sample in a test example. The vertical axis represents the gene expression intensity log2 expression signal intensity. It is a graph which shows the expression level of the HNF1B gene of each sample in a test example. The vertical axis represents the gene expression intensity log2 expression signal intensity.

Hereinafter, the present invention will be described in detail.

In addition, in this specification, "comprise" also includes the meaning of "essentially consist of" and the meaning of "consist of".

Unless otherwise specified, the term "gene" in the present invention includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof. Further, in the present invention, the term "gene" shall be used without distinguishing between a regulatory region, a coding region, an exon, and an intron unless otherwise specified.

Further, in the present invention, "nucleic acid", "nucleotide" and "polynucleotide" are synonymous, and these include both DNA and RNA, and may be double-stranded or single-stranded.

Hereinafter, each gene used in the present invention will be described. The RefSeq ID and UniGene ID shown below are registered on the NCBI website. In addition, some proteins indicated by the following RefSeq ID include a signal sequence, but in that case, the meaning of a mature protein is also included.

The nucleotide sequence of the ATP7B (ATPase, Cu ²⁺ transporting, beta polypeptide) gene is registered as RefSeq Accession No.NM_000053 (SEQ ID NO: 1), NM_001005918, NM_0012431821, and the amino acid sequence is also RefSeq Accession No.NP_000044 (SEQ ID NO: 1). 2), NP_001005918, NP_001230111 are registered. In addition, ATP7B is registered as UniGene ID: Hs.492280.

The nucleotide sequence of the BAIAP2L2 (BAI1-associated protein 2-like 2) gene is registered as RefSeq Accession No.NM_025045 (SEQ ID NO: 3), and the amino acid sequence is also registered as RefSeq Accession No.NP_079321 (SEQ ID NO: 4). ing. BAIAP2L2 is registered as UniGene ID: Hs.474822.

The base sequence of the DNAH5 (Dynein, axonemal, heavy chain 5) gene is registered as RefSeq Accession No.NM_001369 (SEQ ID NO: 5), and the amino acid sequence is also registered as RefSeq Accession No.NP_001360 (SEQ ID NO: 6). There is. DNAH5 is also registered as UniGene ID: Hs.212360.

The nucleotide sequence of the DUX2 (Double homeobox 2) gene is registered as RefSeq Accession No.NM_012147 (SEQ ID NO: 7), and the amino acid sequence is also registered as RefSeq Accession No.NP_036279 (SEQ ID NO: 8). In addition, DUX2 is registered as UniGene ID: Hs.729867.

The nucleotide sequence of the DUX4 (Double homeobox 4) gene is registered as RefSeq Accession No.NM_033178 (SEQ ID NO: 9), and the amino acid sequence is also registered as RefSeq Accession No.NP_149418 (SEQ ID NO: 10). In addition, DUX4 is registered as UniGene ID: Hs.553518.

The nucleotide sequence of the DUX4L2 (Double homeobox 4 like 2) gene is registered as RefSeq Accession No.NM_001127386 (SEQ ID NO: 11), and the amino acid sequence is also registered as RefSeq Accession No.NP_001120858 (SEQ ID NO: 12). In addition, DUX4L2 is registered as UniGene ID: Hs.714689.

The nucleotide sequence of the DUX4L4 (Double homeobox 4 like 4) gene is registered as RefSeq Accession No.NM_001177376 (SEQ ID NO: 13), and the amino acid sequence is also registered as RefSeq Accession No.NP_001170847 (SEQ ID NO: 14). In addition, DUX4L4 is registered as UniGene ID: Hs.725918.

The nucleotide sequence of the EEF1A2 (Eukaryotic translation elongation factor 1 alpha 2) gene is registered as RefSeq Accession No.NM_001958 (SEQ ID NO: 15), and the amino acid sequence is also registered as RefSeq Accession No.NP_001949 (SEQ ID NO: 16). There is. EEF1A2 is registered as UniGene ID: Hs.433839.

The nucleotide sequence of the EPHA7 (EPH receptor A7) gene is registered as RefSeq Accession No.NM_004440 (SEQ ID NO: 17), and the amino acid sequence is also registered as RefSeq Accession No.NP_004431 (SEQ ID NO: 18). EPHA7 is registered as UniGene ID: Hs.73962.

The nucleotide sequence of the HNF1B (HNF1 homeobox B) gene is registered as RefSeq Accession No.NM_000458 (SEQ ID NO: 19) and NM_001165923, and the amino acid sequence is also registered as RefSeq Accession No.NP_000449 (SEQ ID NO: 20) and NP_001159395. There is. HNF1B is registered as UniGene ID: Hs.191144.

The nucleotide sequence of the IL12RB2 (Interleukin 12 receptor, beta 2) gene is registered as RefSeq Accession No. NM_001559 (SEQ ID NO: 21), NM_001258214, NM_001258215, NM_001258216, and the amino acid sequence is also RefSeq Accession No. NP_001550 (SEQ ID NO: 22). ), NP_001245143, NP_001245144, NP_001245145. IL12RB2 is also registered as UniGene ID: Hs.479347.

The nucleotide sequence of the GRAMD3 (GRAM domain containing 3) gene is registered as RefSeq Accession No.NM_001146319 (SEQ ID NO: 23), NM_001143620, NM_001146321, NM_001146322, NM_023927, and the amino acid sequence is also RefSeq Accession No.NP_001139791 (SEQ ID NO: 24). ), NP_001139792, NP_001139793, NP_001139794, NP_0076416. In addition, GRAMD3 is registered as UniGene ID: Hs.363558.

The base sequence (long non-coding RNA) of the LOC100422737 gene is registered as RefSeq Accession No.NR_033557 (SEQ ID NO: 25). In addition, LOC100422737 is registered as UniGene ID: Hs.634681.

The nucleotide sequence of the MMP24 (Matrix metallopeptidase 24 (membrane-inserted)) gene is registered as RefSeq Accession No.NM_006690 (SEQ ID NO: 26), and the amino acid sequence is also registered as RefSeq Accession No.NP_006681 (SEQ ID NO: 27). ing. In addition, MMP24 is registered as UniGene ID: Hs.715494.

The nucleotide sequence of the NHSL1 (NHS-like 1) gene is registered as RefSeq Accession No.NM_020464 (SEQ ID NO: 28) and NM_001144060, and the amino acid sequence is also registered as RefSeq Accession No.NP_065197 (SEQ ID NO: 29) and NP_001137532. ing. NHSL1 is also registered as UniGene ID: Hs.652741.

The nucleotide sequence of the PTHLH (Parathyroid hormone-like hormone) gene is registered as RefSeq Accession No. NM_198965 (SEQ ID NO: 30), NM_002820, NM_198964, NM_198966, and the amino acid sequence is also RefSeq Accession No. NP_945316 (SEQ ID NO: 31). , NP_002811, NP_945315, NP_945317. In addition, PTHLH is registered as UniGene ID: Hs.591159.

The nucleotide sequence of the RHOD (Ras homolog family member D) gene is registered as RefSeq Accession No.NM_014578 (SEQ ID NO: 32), and the amino acid sequence is also registered as RefSeq Accession No.NP_055393 (SEQ ID NO: 33). RHOD is registered as UniGene ID: Hs.15114.

The nucleotide sequence of the RNF182 (Ring finger protein 182) gene is registered as RefSeq Accession No. NM_152737 (SEQ ID NO: 34), NM_001165034, NM_001165033, NM_001165032, and the amino acid sequence is also RefSeq Accession No. NP_689950 (SEQ ID NO: 35), It is registered as NP_001158506, NP_001158505, NP_001158504. In addition, RNF182 is registered as UniGene ID: Hs.111164.

The nucleotide sequence of the SAA2 (Serm amyloid A2) gene is registered as RefSeq Accession No.NM_030754 (SEQ ID NO: 36) and NM_001127380, and the amino acid sequence is also registered as RefSeq Accession No.NP_110381 (SEQ ID NO: 37) and NP_001120852. There is. SAA2 is also registered as UniGene ID: Hs.731376.

The nucleotide sequence of the SLCO4A1 (Solute carrier organic anion transporter family, member 4A1) gene is registered as RefSeq Accession No.NM_016354 (SEQ ID NO: 38), and the amino acid sequence is also registered as RefSeq Accession No.NP_057438 (SEQ ID NO: 39). Has been done. SLCO4A1 is also registered as UniGene ID: Hs.235782.

The nucleotide sequence of the UNC5A (Unc-5 netrin receptor A) gene is registered as RefSeq Accession No.NM_133369 (SEQ ID NO: 40), and the amino acid sequence is also registered as RefSeq Accession No.NP_588610 (SEQ ID NO: 41). ing. In addition, UNC5A is registered as UniGene ID: Hs.33191.

The nucleotide sequence of the VNN1 (Vanin 1) gene is registered as RefSeq Accession No.NM_004666 (SEQ ID NO: 42), and the amino acid sequence is also registered as RefSeq Accession No.NP_004657 (SEQ ID NO: 43). In addition, VNN1 is registered as UniGene ID: Hs.12114.

In the present invention, the above-mentioned gene includes a reduced product and a mutant thereof even if the gene does not have the base sequence registered in the database as described above, and the reduced product and the mutant include the same. It is desirable to encode a protein having the same biological activity as the protein consisting of the above amino acid sequence. Proteins with equivalent biological activity include, for example, proteins from other organisms.

Examples of the mutant include (a) 1 or 2 or more, for example, 1 to 50, 1 to 25, 1 to 12, 1 to 9 in the amino acid sequence registered in the database as described above. , A gene encoding a protein consisting of an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, (b) 70% or more, 80% or more of the base sequence registered in the database as described above, Examples thereof include genes consisting of base sequences having 90% or more and 95% or more identity.

Nucleotide sequence identity can be calculated using commercially available or analysis tools available through telecommunication lines (Internet). Nucleotide sequence identity (%) can be determined using a program commonly used in the art (eg, BLAST, FASTA, etc.) by default.

Method for determining susceptibility of ovarian cancer to PD-1 pathway inhibitor The method for determining susceptibility of ovarian cancer to PD-1 pathway inhibitor in the subject of the present invention is ATP7B, in a biological sample collected from the subject. BAIAP2L2, DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, GRAMD3, LOC100422737, MMP24, NHSL1, PTHLH, RHOD, RNF182, SAA2, SLCO4A1, UNC5A, and NN It is characterized by using the expression level of one gene as an index.

The above gene is preferably at least one gene selected from the group consisting of DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, MMP24, SLCO4A1, UNC5A, and VNN1, more preferably. Is at least one gene selected from the group consisting of EEF1A2, HNF1B, and VNN1.

In addition, in the method of the present invention, the use of the expression level of a gene other than the above in addition to the above gene is not excluded, and the expression level of the gene other than the above is used to determine the susceptibility to the PD-1 pathway inhibitor. Can be used for.

The test subject in the present invention is a mammal (human, mouse, rat, monkey, dog, cat, rabbit, pig, horse, cow, etc.) suffering from ovarian cancer, preferably a human, particularly a human female. is there. Biological samples include cells, tissues (biopsy samples, surgically removed specimens, tissue specimens, etc.), body fluids (blood, plasma, serum, lymph, saliva, urine, spinal fluid, etc.) isolated from the test subject, and stool. Etc., preferably tumor cells or tissues, especially ovarian cancer cells or tissues.

In the present invention, the type of ovarian cancer is not particularly limited, and any histological type such as mucinous adenocarcinoma, clear cell adenocarcinoma, endometrioid adenocarcinoma, and serous adenocarcinoma can be applied.

As the PD-1 pathway inhibitor, any drug capable of inhibiting the PD-1 pathway can be used without particular limitation. As the PD-1 pathway inhibitor in the present invention, those used especially for humans are desirable. It is considered that PD-1 pathway inhibitors can inhibit immunosuppression against cancer cells and enhance the immune response against cancer cells by inhibiting the PD-1 pathway.

Examples of the PD-1 pathway inhibitor include agents that inhibit the binding of PD-L1 and / or PD-L2 to PD-1. Examples of such agents include compounds that bind to PD-L1, PD-L2 or PD-1 (eg, antibodies). The PD-1 pathway inhibitor in the present invention is preferably an anti-PD-1 antibody. Nivolumab and pembrolizumab are known as such anti-PD-1 antibodies. The antibody may be either a monoclonal antibody or a polyclonal antibody, or may be a humanized chimeric antibody. The antibody isotype includes IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and the like, but any of them may be used without limitation. Antibodies may also be antibody fragments, such as antibody fragments include Fab, Fab', F (ab') ₂ , Fv, scFv and the like.

Examples of the method for detecting the expression level of a gene include detecting the mRNA of the above gene or the protein encoded by the gene. Extraction of mRNA or protein from a biological sample can be performed according to a conventional method.

The method for detecting mRNA is not particularly limited as long as it can quantify mRNA. Examples of the detection method include a DNA chip (or microarray) method, a realtime PCR method, a Northern blotting method, and the like. The method for detecting mRNA here also includes detecting the cDNA corresponding to the mRNA.

The method for detecting a protein is not particularly limited as long as it can quantify the protein. Examples of the detection method include immunohistochemical staining, immune cell staining, flow cytometry, protein chip, ELISA method, analysis method by Western blotting, and the like.

In the method of the present invention, it is determined whether or not the ovarian cancer to be tested has susceptibility to a PD-1 pathway inhibitor by using the expression level of the above gene as an index. In this case, for example, when the expression level of the gene is higher than the preset cutoff value, it is determined that the ovarian cancer to be tested has susceptibility to the PD-1 pathway inhibitor. The above cutoff value can be appropriately set by those skilled in the art, and is, for example, the average value or the median value of the expression level of the above gene in ovarian cancer cells or tissues that are not sensitive to PD-1 pathway inhibitors. It can be a significantly higher value than the above. In addition, the above cutoff value can be standardized from the data obtained in the past.

Further, in the present invention, the expression level of the above gene is preferably 10% or more, more preferably 50% or more, as compared with the expression level of a biological sample in a test subject in which ovarian cancer is not sensitive to a PD-1 pathway inhibitor. When it is increased by% or more, more preferably 100% or more, particularly preferably 200% or more, it can be determined that the ovarian cancer of the test subject is sensitive to the PD-1 pathway inhibitor.

In addition, even if it is determined that the ovarian cancer of the test subject has sensitivity to the PD-1 pathway inhibitor when the expression level of one of the above genes is increased as described above. It is good, but it is desirable that the expression level as described above is increased in two or more kinds of genes.

Further, in the present invention, the ovarian cancer to be tested is PD- even when the expression profile of the plurality of sets of the above genes includes the characteristics when the ovarian cancer is sensitive to the PD-1 pathway inhibitor. It can be determined to be sensitive to one pathway inhibitor.

In this case, for example, the expression level of the gene set is scored, and if the score is higher than the preset cutoff value, it is determined that the ovarian cancer to be tested has susceptibility to the PD-1 pathway inhibitor. As described above, a method for scoring the expression level of a gene set is well known, and for example, an ssGSEA (single sample gene set enrichment analysis) method is known. The above cutoff values can be appropriately set by those skilled in the art and are, for example, the mean or median score obtained from the expression level of ovarian cancer cells or tissues that are not sensitive to PD-1 pathway inhibitors. The value can be significantly higher than the value. In addition, the above cutoff value can be standardized from the data obtained in the past.

In the present invention, when ovarian cancer is determined to be sensitive to a PD-1 pathway inhibitor, it is predicted that ovarian cancer has a high therapeutic effect by the PD-1 pathway inhibitor. That is, the method for determining susceptibility of the present invention also includes the meaning of a method for predicting a therapeutic effect. The therapeutic effect expected when determined to be highly sensitive in the present invention may be complete response (CR), partial response (PR), or invariant (SD), but is preferably complete response (CR). ).

In the present invention, when ovarian cancer is determined to be sensitive to a PD-1 pathway inhibitor, it is considered that the therapeutic effect on ovarian cancer is high, and therefore the PD-1 pathway inhibitor is administered to the test subject. Therefore, it is desirable that ovarian cancer be treated.

The method of administering the PD-1 pathway inhibitor is not particularly limited, and for example, it can be administered by intra-arterial administration, intravenous administration, oral administration, rectal administration, enteral administration, transdermal administration, oral administration and the like.

PD-1 pathway inhibitors can be used as is or with non-toxic carriers, diluents or excipients allowed in pharmaceuticals, tablets (bare tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewables). Tablets, troches, etc.), capsules, pills, powders (powder), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, excipients (distilled water when used) Alternatively, it is prepared and used in the form of (including the case where it is mixed with an infusion solution such as an amino acid infusion solution or an electrolyte infusion solution and prepared as a liquid preparation).

The content of the active ingredient in the PD-1 pathway inhibitor administered to the test subject is appropriately in the range of 0.0001 to 100% by weight, preferably 0.001 to 99.9% by weight, more preferably 0.01 to 99% by weight in the total amount of the pharmaceutical preparation. It is possible to select.

The dose of the PD-1 pathway inhibitor can be appropriately determined according to various conditions such as the patient's weight, age, sex, and symptoms.

Biomarkers for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer The biomarkers for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer of the present invention are ATP7B, BAIAP2L2, DNAH5, DUX2, DUX4, DUX4L2, It is characterized by containing at least one gene selected from the group consisting of DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, GRAMD3, LOC100422737, MMP24, NHSL1, PTHLH, RHOD, RNF182, SAA2, SLCO4A1, UNC5A, and VNN1. To do.

By using the expression level of the gene as an index, it is possible to determine whether or not the ovarian cancer is susceptible to the PD-1 pathway inhibitor. Therefore, the gene is used for the PD-1 pathway inhibitor of ovarian cancer. It can be a biomarker for susceptibility determination.

The ovarian cancer, PD-1 pathway inhibitor, susceptibility determination method, etc. are the same as those described above.

Kit for determining susceptibility to PD-1 pathway inhibitor of ovarian cancer The kit for determining susceptibility to PD-1 pathway inhibitor for ovarian cancer of the present invention is ATP7B, BAIAP2L2, DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, Nucleic acid that binds to or encodes at least one gene selected from the group consisting of EPHA7, HNF1B, IL12RB2, GRAMD3, LOC100422737, MMP24, NHSL1, PTHLH, RHOD, RNF182, SAA2, SLCO4A1, UNC5A, and VNN1. It is characterized by containing an antibody or antibody fragment that binds to the protein to be produced.

The nucleic acid that binds to the above gene is a polynucleotide that can hybridize to the gene. For example, mRNA can be detected by using it in a DNA chip (or microarray) method, realtime PCR method, Northern blot method, or the like. The expression level of the gene can be detected.

The antibody or antibody fragment that binds to the protein is mainly intended to be an antibody that specifically binds to the protein. The antibody or antibody fragment can be obtained according to a known method, and the antibody may be either a monoclonal antibody or a polyclonal antibody, or may be a humanized chimeric antibody. The antibody isotypes include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, etc., but any of them can be used without limitation. Examples of the antibody fragment include Fab, Fab', F (ab') ₂ , Fv, scFv and the like, and the antibody or antibody fragment may be modified with a fluorescent dye or the like. The antibody or antibody fragment is used for immunohistochemical staining, immunocell staining, flow cytometry, protein chip, ELISA method, Western blotting analysis method, etc. to detect protein and detect the expression level of the gene. be able to.

In the present invention, the expression level of the gene in the biological sample of the test control is detected by using the sensitivity determination kit in the same manner as described above, and the expression level is used as an index to detect PD- for ovarian cancer. 1 It is possible to determine whether or not the drug is sensitive to a pathway inhibitor.

In addition, the kit of the present invention may include items other than the above, if necessary. Examples of such a substance include a positive control, a negative control, and an instruction describing a method for detecting the gene using the kit of the present invention.

The ovarian cancer, PD-1 pathway inhibitor, susceptibility determination method, etc. are the same as those described above.

INDUSTRIAL APPLICABILITY According to the present invention, susceptibility to PD-1 pathway inhibitors is shown by using the expression level of a gene that serves as a biomarker for susceptibility to PD-1 pathway inhibitors (particularly anti-PD-1 antibody) of ovarian cancer as an index. It becomes possible to determine whether or not the patient has ovarian cancer. As a result, it becomes possible to efficiently select patients (patients showing remarkable efficacy) that are effective against PD-1 pathway inhibitors (particularly anti-PD-1 antibodies).

Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples and the like.

Test example <Target>
Of the 20 patients who underwent a study using anti-PD-1 antibody (nivolumab) for platinum-resistant recurrent / advanced ovarian cancer conducted at the Department of Obstetrics and Gynecology, Kyoto University Hospital, with the approval of the Ethics Committee. The subjects were 17 patients who could be evaluated by the following comprehensive gene expression analysis (microarray analysis) after obtaining the consent of the patients or their families.

<Expression analysis>
RNA was extracted from the surgical specimen (tumor part) of the patient who underwent the above clinical trial using the QIAGEN AllPrep DNA / RNA Kit (Cat. No.80234), and then the SensationPlus Amplification and WT Labeling Kit (Affymetrix, Part no.902312). ) Was used to amplify and label the RNA. Next, expression analysis was performed using the labeled DNA by GeneChip (registered trademark) Human Transcriptome Array 2.0 (Affymetrix, Part No. 902312), and GeneChip (registered trademark) Scanner 3000 system (Affymetrix, Part No. The microarray was scanned according to 00-0218) and the microarray data was acquired.

Signal Space Transformation-Robust multi-array Average (SST-RMA) normalization was performed on the obtained microarray data using Expression Console ver 1.4.1 (Affymetrix), and analysis was performed at the gene level.

<Statistical analysis>
Statistical analysis was performed by hierarchical cluster analysis (Average linkage). We extracted genes or gene groups that showed a significant difference in expression between cases with a complete response therapeutic effect and other cases (PR + SD + PD) (complete response (CR): 2 cases, partial). Response (PR): 1 case, unchanged (SD): 4 cases, exacerbation (PD): 10 cases)

For statistical analysis, FDR Q value <0.05 was defined as having a significant difference using Samroc from statistical software R3.1.1 and Bioconductor ‘SAGx’ package.

<Result>
As a result of hierarchical cluster analysis, there were 497 genes whose expression was significantly increased or decreased in CR. The results of the hierarchical cluster analysis are shown in FIG.

Among the genes whose expression was significantly increased or decreased by this CR, FDP Q value <0.0005, p <1 × 10 ^-6 , and 2 CR cases were twice as many as all non-CR cases (1 on log2 scale). The following 22 genes were listed as the genes whose expression was increased.

From the above 22 genes, 13 types of genes (DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2) were shown based on the specific expression pattern in 2 CR cases and the characteristics of the genes. , MMP24, SLCO4A1, UNC5A, and VNN1) were extracted.

Furthermore, among the above 22 genes, the genes involved in gene mutation and cell proliferation and particularly contributing to susceptibility were EEF1A2, VNN1 and HNF1B (Figs. 2 to 4).

Claims (14)

At least one selected from the group consisting of D NAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2 , M MP24 , SLCO4A1, UNC5A, and VNN1 in biological samples collected from the test subject. The process of measuring the expression level of a gene , as well as
A step of determining that ovarian cancer in the test subject is sensitive to a PD-1 pathway inhibitor when the measured expression level or the score obtained by scoring the measured expression levels of multiple genes is higher than a preset cutoff value.
A method for determining the susceptibility of ovarian cancer to a PD-1 pathway inhibitor in the subject. The method of claim 1, wherein in the measurement step, the expression level of at least one gene selected from the group consisting of EEF1A2, HNF1B, and VNN1 is measured . Measurement of the expression level of the gene,
The method according to claim 1 or 2 , wherein the method is carried out by detecting the mRNA of the gene or the protein encoded by the gene. The method according to any one of claims 1 to 3 , wherein the PD-1 pathway inhibitor is an anti-PD-1 antibody. The method according to any one of claims 1 to 4, wherein the cutoff value is at least twice the expression level in a biological sample of a test subject who is not sensitive to a PD-1 pathway inhibitor. Claims 1 to 5 for determining that ovarian cancer in a test subject is sensitive to a PD-1 pathway inhibitor when the expression level measured in two or more genes is higher than the cutoff value in the determination step. The method according to any one of the above. At least one of the group consisting of D NAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2 , M MP24 , SLCO4A1, UNC5A, and VNN1 whose expression level is higher than the preset cutoff value . Use of the gene as a biomarker for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer. Use of at least one gene in the group consisting of EEF1A2, HNF1B, and VNN1 whose expression level is higher than the preset cutoff value as a biomarker for determining susceptibility to PD-1 pathway inhibitors of ovarian cancer . The use according to claim 7 or 8, wherein the PD-1 pathway inhibitor is an anti-PD-1 antibody. Nucleic acid that binds to at least one gene selected from the group consisting of D NAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2 , M MP24 , SLCO4A1, UNC5A, and VNN1, or to the gene. Contains an antibody or antibody fragment that binds to the encoded protein,
Kit for use in a method for determining the susceptibility to PD-1 pathway inhibitor of ovarian cancer according to claim 1. A nucleic acid that binds to at least one gene selected from the group consisting of EEF1A2, HNF1B, and VNN1, or an antibody or antibody fragment that binds to a protein encoded by the gene.
A kit for use in a method for determining susceptibility of ovarian cancer to a PD-1 pathway inhibitor according to claim 2 . The kit according to claim 10 or 11 , wherein the PD-1 pathway inhibitor is an anti-PD-1 antibody. ATP7B, BAIAP2L2, DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, GRAMD3, LOC100422737, MMP24, NHSL1, PTHLH, RHOD, RNF182SA Steps to measure the expression level of at least one gene selected from the group consisting of UNC5A and VNN1, and
A step of determining that ovarian cancer in the test subject is sensitive to a PD-1 pathway inhibitor when the measured expression level is higher than a preset cutoff value.
The susceptibility of ovarian cancer to PD-1 pathway inhibitors in the test subject is more than twice the expression level in the biological sample of the subject who is not sensitive to the PD-1 pathway inhibitor. How to judge. ATP7B, BAIAP2L2, DNAH5, DUX2, DUX4, DUX4L2, DUX4L4, EEF1A2, EPHA7, HNF1B, IL12RB2, GRAMD3, LOC100422737, MMP24, NHSL1, PTHLH, RHOD, RNF182SA Steps to measure the expression levels of at least two genes selected from the group consisting of UNC5A and VNN1, and
A step of determining that ovarian cancer in a test subject is sensitive to a PD-1 pathway inhibitor when the measured expression level is higher than a preset cutoff value.
In the step of determining the above, if the expression level measured in two or more genes is higher than the cutoff value, it is determined that the ovarian cancer in the test subject has susceptibility to the PD-1 pathway inhibitor. A method for determining the susceptibility of ovarian cancer to PD-1 pathway inhibitors in a subject.

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