JP2023162781A - Diagnostic agent, drug efficacy prediction kit, drug efficacy prediction method, and marker - Google Patents
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Abstract
Description
本発明は、診断薬、薬効予測キット、薬効予測方法、及びマーカーに関する。 The present invention relates to a diagnostic agent, a drug efficacy prediction kit, a drug efficacy prediction method, and a marker.
免疫チェックポイント阻害剤である抗ＰＤ－１抗体は、一部のがん患者では著明な効果を示すものの、奏功率は、２０～３０％程度である。血清ｍｉｃｒｏＲＮＡ（以下、ｍｉＲＮＡともいう。）は、がん診断や腫瘍免疫応答を反映する非常に有用なマーカーとして考えられている。 Although anti-PD-1 antibodies, which are immune checkpoint inhibitors, show remarkable effects in some cancer patients, the success rate is only about 20-30%. Serum microRNA (hereinafter also referred to as miRNA) is considered to be a very useful marker that reflects cancer diagnosis and tumor immune response.
食道がんにおいては、特定のｍｉＲＮＡを用いてニボルマブの薬効を予測する方法が提案されている（特許文献１参照。）。 For esophageal cancer, a method has been proposed for predicting the efficacy of nivolumab using specific miRNA (see Patent Document 1).
しかしながら、肺がんにおいては、臨床現場では腫瘍ＰＤ－Ｌ１発現を測定しているものの、精度の観点から改良の余地がある。 However, in lung cancer, although tumor PD-L1 expression is measured in clinical practice, there is room for improvement from the viewpoint of accuracy.
本発明は、以下の態様を含む。
［１］抗ＰＤ－１抗体を使用する肺がん患者に用いられるコンパニオン診断薬であって、
ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、ｍｉＲ－４５３４、及びｍｉＲ－６７２９－５からなる群から選択される少なくとも１つのｍｉＲＮＡを増幅するためのプライマーセット、及び／又は前記ｍｉＲＮＡ若しくはその増幅産物に結合するプローブを含む、診断薬。
［２］［１］に記載の診断薬を含む、肺がん患者における抗ＰＤ－１抗体薬効予測キット。
［３］患者由来の検体中のｍｉＲ－６７２９－５を内在性コントロールとし、前記検体中のｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡの発現量をｉｎ ｖｉｔｒｏで測定し、前記ｍｉＲＮＡの発現量を用いて患者に対する抗ＰＤ－１抗体の薬効を評価することを含む、抗ＰＤ－１抗体薬効予測方法。
［４］患者由来の検体中のｍｉＲ－６７２９－５を内在性コントロールとし、前記検体中のｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡの発現量をｉｎ ｖｉｔｒｏで測定し、抗ＰＤ－１抗体に薬効のあった対照者由来の検体中の対応するｍｉＲＮＡの対照発現量と、抗ＰＤ－１抗体に薬効の無かった対照者由来の検体中の対応するｍｉＲＮＡの対照発現量とを教師データとして作成された、抗ＰＤ－１抗体の薬効の有無を判別する判別式に、前記患者由来の検体中の前記ｍｉＲＮＡの発現量を代入し、抗ＰＤ－１抗体の薬効を評価することを含む、抗ＰＤ－１抗体薬効予測方法。
［５］肺がん患者における抗ＰＤ－１抗体の薬効予測マーカーであって、生体から分離された被検試料中に含まれる、ｍｉＲ－６７２９－５を内在性コントロールとしｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡを含む、マーカー。
The present invention includes the following aspects.
[1] A companion diagnostic agent used for lung cancer patients using anti-PD-1 antibodies,
a primer set for amplifying at least one miRNA selected from the group consisting of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, miR-4534, and miR-6729-5; /or a diagnostic agent comprising a probe that binds to the miRNA or its amplification product.
[2] A kit for predicting efficacy of anti-PD-1 antibody in lung cancer patients, comprising the diagnostic agent according to [1].
[3] miR-6729-5 in a patient-derived specimen is used as an endogenous control, and consists of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534 in the specimen. Anti-PD-1 antibody drug efficacy prediction, comprising measuring the expression level of at least one miRNA selected from the group in vitro, and evaluating the drug efficacy of the anti-PD-1 antibody for a patient using the expression level of the miRNA. Method.
[4] miR-6729-5 in a patient-derived specimen is used as an endogenous control, and consists of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534 in the specimen. The expression level of at least one miRNA selected from the group was measured in vitro, and the control expression level of the corresponding miRNA in a sample from a control subject for whom anti-PD-1 antibody had a therapeutic effect and the anti-PD-1 antibody were measured. The discriminant formula for determining the presence or absence of drug efficacy of an anti-PD-1 antibody was created using the control expression level of the corresponding miRNA in a sample from a control subject who had no drug effect as training data. A method for predicting the efficacy of an anti-PD-1 antibody, the method comprising evaluating the efficacy of the anti-PD-1 antibody by substituting the expression level of the miRNA.
[5] A predictive marker for the drug efficacy of anti-PD-1 antibodies in lung cancer patients, using miR-452-3p, miR-6729-5, which is contained in a test sample isolated from a living body, as an endogenous control. A marker comprising at least one miRNA selected from the group consisting of 3129-3p, miR-4304, miR-4492, and miR-4534.
本発明によれば、肺がん患者における抗ＰＤ－１抗体の治療効果の予測に寄与する。 According to the present invention, it contributes to predicting the therapeutic effect of anti-PD-1 antibodies in lung cancer patients.
≪薬効予測マーカー≫
本実施形態は、肺がん患者における抗ＰＤ－１抗体の薬効予測マーカーであって、ｍｉＲ－６７２９－５を内在性コントロールとし、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡ、又は前記ｍｉＲＮＡの相補鎖とストリンジェントな条件下でハイブリダイズすることができる核酸を含む、マーカーを提供する。
実施例において後述するように、本発明者は、ニボルマブに対する薬効の有無によって、高い汎用性で発現量が変動するｍｉＲＮＡ、及びその組み合わせを見出した。
≪Drug efficacy prediction marker≫
This embodiment is a marker for predicting the drug efficacy of anti-PD-1 antibodies in lung cancer patients, using miR-6729-5 as an endogenous control, miR-452-3p, miR-3129-3p, miR-4304, miR- 4492, and miR-4534, or a nucleic acid capable of hybridizing under stringent conditions with the complementary strand of said miRNA.
As described later in Examples, the present inventors have discovered miRNAs and combinations thereof whose expression levels vary with high versatility depending on the presence or absence of drug efficacy against nivolumab.
本実施形態において、「ストリンジェントな条件下」とは、例えば、５×ＳＳＣ（２０×ＳＳＣの組成：３Ｍ 塩化ナトリウム，０．３Ｍ クエン酸溶液，ｐＨ７．０）、０．１重量％ Ｎ－ラウロイルサルコシン、０．０２重量％のＳＤＳ、２重量％の核酸ハイブルダイゼーション用ブロッキング試薬、及び５０％フォルムアミドから成るハイブリダイゼーションバッファー中で、５５～７０℃で数時間から一晩インキュベーションを行うことによりハイブリダイズさせる条件を挙げることができる。なお、インキュベーション後の洗浄の際に用いる洗浄バッファーとしては、好ましくは０．１重量％ＳＤＳ含有１×ＳＳＣ溶液、より好ましくは０．１重量％ＳＤＳ含有０．１×ＳＳＣ溶液である。 In the present embodiment, "stringent conditions" refers to, for example, 5×SSC (composition of 20×SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 0.1% by weight N- Incubation at 55-70°C for several hours to overnight in a hybridization buffer consisting of lauroylsarcosine, 0.02% by weight SDS, 2% by weight blocking reagent for nucleic acid hybridization, and 50% formamide. The conditions for hybridization can be listed as follows. The washing buffer used for washing after incubation is preferably a 1×SSC solution containing 0.1% by weight SDS, more preferably a 0.1×SSC solution containing 0.1% by weight SDS.
本実施形態において、「薬効がある」とは、完全奏功又は部分奏功が認められることを意味し、「薬効が無い」とは、完全奏功及び部分奏功のいずれも認められないことを意味する。 In this embodiment, "medicinally effective" means that a complete response or partial response is observed, and "no medicinal efficacy" means that neither a complete response nor a partial response is observed.
ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、ｍｉＲ－４５３４、及びｍｉＲ－６７２９－５の塩基配列は、それぞれ順に配列番号１～６で表される。
本実施形態の薬効予測マーカーは、配列番号１～６のいずれかで表される塩基配列と９５％以上の同一性を有する塩基配列を含む核酸であることが好ましい。同一性としては、９５％以上が好ましく、９７％以上がより好ましく、９８％以上が特に好ましく、９９％以上が最も好ましい。
The base sequences of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, miR-4534, and miR-6729-5 are represented by SEQ ID NOs: 1 to 6, respectively.
The drug efficacy predictive marker of this embodiment is preferably a nucleic acid containing a base sequence having 95% or more identity with the base sequence represented by any of SEQ ID NOS: 1 to 6. The identity is preferably 95% or more, more preferably 97% or more, particularly preferably 98% or more, and most preferably 99% or more.
本実施形態の薬効予測マーカーにおける標的核酸としては、ｍｉＲ－６７２９－５を内在性コントロールとし、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される１つのｍｉＲＮＡが好ましく、２つがより好ましく、３つが更に好ましく、４つが更に好ましく、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、ｍｉＲ－４５３４、及びｍｉＲ－６７２９－５の５つの組み合わせが最も好ましい。 The target nucleic acids in the drug efficacy predictive marker of this embodiment include miR-6729-5 as an endogenous control, miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534. One miRNA selected from the group is preferred, two are more preferred, three are even more preferred, four are even more preferred, miR-452-3p, miR-3129-3p, miR-4304, miR-4492, miR-4534, and miR-6729-5 are most preferred.
本実施形態において、抗ＰＤ－１抗体としては、ニボルマブ、ペムブロリズマブ等が挙げられ、ニボルマブが好ましい。 In this embodiment, anti-PD-1 antibodies include nivolumab, pembrolizumab, etc., with nivolumab being preferred.
≪コンパニオン診断薬≫
本実施形態は、抗ＰＤ－１抗体を使用する肺がん患者に用いられるコンパニオン診断薬であって、ｍｉＲ－６７２９－５を内在性コントロールとし、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡを増幅するためのプライマーセット、及び／又は前記ｍｉＲＮＡ若しくはその増幅産物に結合するプローブを含む、診断薬を提供する。
≪Companion diagnostic agent≫
This embodiment is a companion diagnostic agent used for lung cancer patients using anti-PD-1 antibodies, with miR-6729-5 as an endogenous control, miR-452-3p, miR-3129-3p, miR- 4304, miR-4492, and miR-4534, and/or a probe that binds to the miRNA or its amplification product. .
実施例において後述するように、本発明者は、上記マーカーは、抗ＰＤ－１抗体が、薬効があった患者と薬効が無かった患者との間において、検体中の発現量に大きな差が生じることを見出した。よって、本発明の診断薬は、抗ＰＤ－１抗体の薬効を予測するために有効に使用できる。 As will be described later in the Examples, the present inventors have determined that there is a large difference in the expression levels of the above markers in samples between patients for whom the anti-PD-1 antibody was effective and patients for whom the drug was ineffective. I discovered that. Therefore, the diagnostic agent of the present invention can be effectively used to predict the efficacy of anti-PD-1 antibodies.
≪薬効予測キット≫
本実施形態は、上記本発明の診断薬を含む、肺がん患者における抗ＰＤ－１抗体薬効予測用キットを提供する。
≪Drug efficacy prediction kit≫
The present embodiment provides a kit for predicting the efficacy of anti-PD-1 antibodies in lung cancer patients, which includes the diagnostic agent of the present invention.
本実施形態のキットは、体液、細胞、組織等から核酸（例えば、ｔｏｔａｌ ＲＮＡ）を抽出するためのキット、標識用蛍光物質、核酸増幅用試薬等を含むことが好ましい。 The kit of this embodiment preferably includes a kit for extracting nucleic acids (eg, total RNA) from body fluids, cells, tissues, etc., a fluorescent substance for labeling, a reagent for nucleic acid amplification, and the like.
≪薬効予測デバイス≫
本実施形態は、肺がん患者における抗ＰＤ－１抗体の薬効予測デバイスであって、固相と、前記固相に結合したｍｉＲ－６７２９－５を内在性コントロールとし、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡとハイブリダイズし得る核酸を含む、薬効予測デバイスを提供する。
≪Drug efficacy prediction device≫
This embodiment is a device for predicting the drug efficacy of anti-PD-1 antibodies in lung cancer patients, in which a solid phase and miR-6729-5 bound to the solid phase are used as endogenous controls, miR-452-3p, miR- The present invention provides a drug efficacy prediction device comprising a nucleic acid capable of hybridizing with at least one miRNA selected from the group consisting of 3129-3p, miR-4304, miR-4492, and miR-4534.
本実施形態のデバイスは、上記核酸が固相に結合したものである。固相としては、ガラス基板、シリコン基板、プラスチック基板、金属基板等が挙げられる。上記核酸が固相に結合したものとして、ＤＮＡアレイ、ＲＮＡアレイ等の核酸アレイが挙げられる。核酸アレイとしては、Ｌ－リジンやアミノ基、カルボキシル基等の官能基でコートされた固相表面に核酸が固定されたものが挙げられる。 The device of this embodiment is one in which the above nucleic acid is bound to a solid phase. Examples of the solid phase include glass substrates, silicon substrates, plastic substrates, metal substrates, and the like. Examples of the nucleic acids bound to a solid phase include nucleic acid arrays such as DNA arrays and RNA arrays. Examples of nucleic acid arrays include those in which nucleic acids are immobilized on a solid phase surface coated with functional groups such as L-lysine, amino groups, and carboxyl groups.
≪抗ＰＤ－１抗体薬効予測方法≫
患者由来の検体中のｍｉＲ－６７２９－５を内在性コントロールとし、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡの発現量をｉｎ ｖｉｔｒｏで測定し、前記ｍｉＲＮＡの発現量を用いて患者に対する抗ＰＤ－１抗体の薬効を評価することを含む、抗ＰＤ－１抗体薬効予測方法を提供する。
例えば、患者由来の検体中の上記ｍｉＲＮＡの発現量と、抗ＰＤ－１抗体に薬効の無いことが分かっている対照者の対照発現量とを用いて両発現量を比較して、検体中の上記ｍｉＲＮＡの発現量が、抗ＰＤ－１抗体に薬効の無い対照者の対照発現量より有意に差がある場合、患者が、抗ＰＤ－１抗体に薬効があると予測できる。
また、例えば、患者由来の検体中の上記ｍｉＲＮＡの発現量と、抗ＰＤ－１抗体に薬効の有ることが分かっている対照者の対照発現量とを用いて両発現量を比較して、検体中の上記ｍｉＲＮＡの発現量が、抗ＰＤ－１抗体に薬効の有る対照者の対照発現量より有意に差がある場合、患者が、抗ＰＤ－１抗体に薬効が無いと予測できる。
≪Method for predicting anti-PD-1 antibody efficacy≫
miR-6729-5 in a patient-derived specimen is used as an endogenous control, and at least one selected from the group consisting of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534 is used as an endogenous control. The present invention provides a method for predicting the efficacy of an anti-PD-1 antibody, which comprises measuring the expression level of two miRNAs in vitro, and evaluating the efficacy of the anti-PD-1 antibody for a patient using the expression level of the miRNA.
For example, by comparing the expression level of the above miRNA in a patient-derived sample with the expression level of a control person for whom anti-PD-1 antibodies are known to have no drug effect, If the expression level of the miRNA is significantly different from the expression level of a control person for whom anti-PD-1 antibodies do not have a therapeutic effect, it can be predicted that the anti-PD-1 antibody will have a therapeutic effect on the patient.
In addition, for example, the expression level of the above-mentioned miRNA in a patient-derived sample is compared with the expression level of a control person for whom anti-PD-1 antibodies are known to have a therapeutic effect, and the expression level of both miRNAs is compared. If the expression level of the above-mentioned miRNA in the patient is significantly different from the expression level of a control subject for whom anti-PD-1 antibody has a therapeutic effect, it can be predicted that the anti-PD-1 antibody has no therapeutic effect on the patient.
本実施形態において、検体としては、血液、尿、唾液、汗、組織浸出液等が挙げられ、血液が好ましい。血液の中でも、血清、血しょう等が挙げられ、血清が好ましい。検体からｍｉＲＮＡを抽出する方法としては、酸性フェノールを含むＲＮＡ抽出用試薬を用いた方法等が挙げられ、定法に従う。 In this embodiment, examples of the specimen include blood, urine, saliva, sweat, tissue exudate, and the like, with blood being preferred. Among blood, serum, plasma, etc. can be mentioned, and serum is preferable. A method for extracting miRNA from a specimen includes a method using an RNA extraction reagent containing acidic phenol, and a conventional method is followed.
患者由来の検体中のｍｉＲＮＡの検出方法としては、プライマーを用いてＰＣＲにより、特定のｍｉＲＮＡの断片を増幅し、その増幅産物を解析してもよく、特定のｍｉＲＮＡに相補的なプローブを用いて、ハイブリダイゼーションを用いた方法により、解析してもよい。
定量性の観点から、ＰＣＲにより特定のｍｉＲＮＡの断片を増幅し、その増幅産物を解析することが好ましい。具体的な定量方法としては、次世代シークエンサー（ＮＧＳ）法やリアルタイムＰＣＲ（ＲＴ－ＰＣＲ）法が挙げられる。
As a method for detecting miRNA in patient-derived samples, a specific miRNA fragment may be amplified by PCR using primers and the amplified product may be analyzed, or a probe complementary to the specific miRNA may be used. The analysis may be performed by a method using hybridization.
From the viewpoint of quantitative performance, it is preferable to amplify a specific miRNA fragment by PCR and analyze the amplified product. Specific quantitative methods include the next generation sequencer (NGS) method and the real-time PCR (RT-PCR) method.
次世代シークエンサーでは、解析されたＤＮＡ断片をリードと呼び、リード数と１リード当たりに決定される塩基数（リード長）の積が出力データとなる。本実施形態において、ｍｉＲ－６７２９－５を内在性コントロールとし、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つをｓｉｎｇｌｅｐｌｅｘ ＰＣＲまたはｍｕｌｔｉｐｌｅｘ ＰＣＲで増幅し、次世代シークエンサー（ＮＧＳ）でその増幅産物の塩基配列を解析し、当該領域のリード数を計数することにより定量することが好ましい。ｍｕｌｔｉｐｌｅｘ ＰＣＲは、一つのＰＣＲ反応系に複数のプライマー対を同時に使用することで、複数の遺伝子領域を同時に増幅する方法である。上述した特定のｍｉＲＮＡにアニールするプライマーの組み合わせの少なくとも一部を、一つのＰＣＲ反応系で使用してもよい。 In next-generation sequencers, the analyzed DNA fragments are called reads, and the product of the number of reads and the number of bases determined per read (read length) is output data. In this embodiment, miR-6729-5 is used as an endogenous control, and at least one selected from the group consisting of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534 It is preferable to amplify it by singleplex PCR or multiplex PCR, analyze the base sequence of the amplified product with a next-generation sequencer (NGS), and quantify it by counting the number of reads in the region. Multiplex PCR is a method of simultaneously amplifying multiple gene regions by simultaneously using multiple primer pairs in one PCR reaction system. At least some of the above-described combinations of primers that anneal to specific miRNAs may be used in one PCR reaction system.
リアルタイムＰＣＲでは、インターカレータ－法、プローブ法、サイクリングプローブ法等を用いて、蛍光強度を検出することにより、増幅産物の生成量をモニターすることができる。本実施形態において、ｍｉＲ－６７２９－５を内在性コントロールとし、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つをｓｉｎｇｌｅｐｌｅｘまたはｍｕｌｔｉｐｌｅｘのリアルタイムＰＣＲ法により定量することが好ましい。ｍｕｌｔｉｐｌｅｘ ＰＣＲにおいては、上述した特定のｍｉＲＮＡにアニールする様々なプライマーの組み合わせの少なくとも一部を、一つのＰＣＲ反応系で使用してもよい。 In real-time PCR, the amount of amplified product produced can be monitored by detecting fluorescence intensity using an intercalator method, a probe method, a cycling probe method, or the like. In this embodiment, miR-6729-5 is used as an endogenous control, and at least one selected from the group consisting of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534 It is preferable to quantify by singleplex or multiplex real-time PCR method. In multiplex PCR, at least some of the various primer combinations that anneal to the specific miRNA described above may be used in one PCR reaction system.
また、リアルタイムＰＣＲにおいては、特定のｍｉＲＮＡに相補的な蛍光色素標識プローブを用いることが好ましい。
定量方法としては、ターゲットの実際のコピー数を決定する絶対定量法とサンプル間の相対値を決定する比較定量法が挙げられ、取得したいデータの質に応じて使い分けられる。
Furthermore, in real-time PCR, it is preferable to use a fluorescent dye-labeled probe complementary to a specific miRNA.
Quantitation methods include absolute quantification, which determines the actual copy number of the target, and comparative quantification, which determines the relative value between samples, and these methods can be used depending on the quality of the data to be obtained.
本実施形態は、患者由来の検体中のｍｉＲ－６７２９－５を内在性コントロールとし、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡの発現量をｉｎ ｖｉｔｒｏで測定し、抗ＰＤ－１抗体に薬効のあった対照者由来の検体中の対応するｍｉＲＮＡの対照発現量と、抗ＰＤ－１抗体に薬効の無かった対照者由来の検体中の対応するｍｉＲＮＡの対照発現量とを教師データとして作成された、抗ＰＤ－１抗体の薬効の有無を判別する判別式に、前記患者由来の検体中の前記ｍｉＲＮＡの発現量を代入し、抗ＰＤ－１抗体の薬効を評価することを含む、抗ＰＤ－１抗体薬効予測方法を提供する。 In this embodiment, miR-6729-5 in a patient-derived sample is used as an endogenous control, and miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534 are selected as an endogenous control. The expression level of at least one selected miRNA was measured in vitro, and the control expression level of the corresponding miRNA in a sample from a control subject for whom the anti-PD-1 antibody had a therapeutic effect was compared with the control expression level of the corresponding miRNA in a sample from a control subject for whom the anti-PD-1 antibody had a therapeutic effect. The expression level of the corresponding miRNA in the sample from the patient who did not have the above expression was used as training data to determine the presence or absence of the drug efficacy of the anti-PD-1 antibody. Provided is a method for predicting the efficacy of an anti-PD-1 antibody, which includes substituting the expression level of miRNA and evaluating the efficacy of the anti-PD-1 antibody.
本実施形態において、ＲＯＣ曲線下面積（ＡＵＣ）のヒストグラムにおいて、中央値に焦点を当て、ＬＡＳＳＯ回帰を用いて２０回繰り返し１０分割交差により判別式を作成した。これにより、ｍｉＲ－６７２９－５を内在性コントロールとしｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４の組み合わせによる汎用性がｍｉＲＮＡの判別式を見出すことができた。 In this embodiment, in the histogram of the area under the ROC curve (AUC), a discriminant was created by repeating 20 times and 10-fold intersection using LASSO regression, focusing on the median value. This allows us to find a versatile miRNA discriminant using miR-6729-5 as an endogenous control and a combination of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534. was completed.
本実施形態により、低侵襲的に、感度及び特異度の高い、抗ＰＤ－１抗体の薬効予測を可能とし、早期の治療、及び予後の改善をもたらすことができる。 According to this embodiment, it is possible to predict the efficacy of an anti-PD-1 antibody with high sensitivity and specificity in a minimally invasive manner, leading to early treatment and improved prognosis.
以下、実施例により本発明を説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be explained with reference to examples, but the present invention is not limited to the following examples.
インフォームドコンセントを得たニボルマブを投与された肺がん患者１４３例（Ａ病院１１８例、Ｂ病院２５例）のうち、効果判定ができなかった５例を除いた１３８例の血清中のｍｉＲＮＡを解析に用いた。この１３８例を２群に分け、１１１例を学習データ（ｄｉｓｃｏｖｅｒｙ ｓｅｔ）に用い、２７例を検証データ（ｖａｌｉｄａｔｉｏｎ ｓｅｔ）に用いて解析を行った（図１参照）。
患者の背景を表１に示す。ｄｉｓｃｏｖｅｒｙ ｓｅｔ及びｖａｌｉｄａｔｉｏｎ ｓｅｔの患者の年齢は、共に平均６０代であり、両群で優位な差は認められなかった。
Of the 143 lung cancer patients who received nivolumab with informed consent (118 patients at Hospital A, 25 patients at Hospital B), we analyzed miRNA in the serum of 138 patients, excluding 5 patients for whom efficacy could not be determined. Using. These 138 cases were divided into two groups, and analysis was performed using 111 cases as learning data (discovery set) and 27 cases as validation data (validation set) (see FIG. 1).
The patient background is shown in Table 1. The average age of patients in the discovery set and validation set was 60 years old, and no significant difference was observed between the two groups.
解析方法を図２（Ａ）に示す。患者の血清４００μＬからｍｉＲＮｅａｓｙ Ｋｉｔｓ (ＱＩＡＧＥＮ社)を用いてＲＮＡを抽出し、ＱＩＡｓｅｑ ｍｉＲＮＡ Ｌｉｂｒａｒｙ Ｋｉｔ Ｋｉｔｓ (ＱＩＡＧＥＮ社)を用いてライブラリーを作成し，ｉｌｌｍｉｎａ ＮｅｘｔＳｅｑによる次世代シークエンサーを用いて、ニボルマブ奏功群と非奏功群の２２５０個のｍｉＲＮＡプロファイルを網羅的に解析した。検出したｍｉＲＮＡのリード数、及び設定した内在性コントロールの値を元に、ＬＡＳＳＯ回帰を用いて、２０回繰り返し１０分割交差検証を学習データと検証データで行い、汎用性能を評価した。 The analysis method is shown in FIG. 2(A). RNA was extracted from 400 μL of patient serum using miRNeasy Kits (QIAGEN), a library was created using QIAseq miRNA Library Kit Kits (QIAGEN), and a next-generation sequencer using illmina NextSeq was used to determine the success of nivolumab. group We comprehensively analyzed 2250 miRNA profiles in the non-response group and the non-response group. Based on the number of detected miRNA reads and the set endogenous control value, 10-fold cross-validation was performed 20 times using training data and validation data using LASSO regression to evaluate general-purpose performance.
学習データにおける２０回分のＲＯＣ曲線下面積（ＡＵＣ）のヒストグラムを図２（Ｂ）に示す。ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、ｍｉＲ－４５３４、及びｍｉＲ－６７２９－５の組み合わせを用いることにより、中央値は、０．７５であった。次いで、検証データにおける２０回分のＡＵＣのヒストグラムを図２（Ｃ）に示す。中央値は、０．６６であった。 A histogram of the area under the ROC curve (AUC) for 20 times in the learning data is shown in FIG. 2(B). By using the combination of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, miR-4534, and miR-6729-5, the median value was 0.75. Next, a histogram of AUC for 20 times in the verification data is shown in FIG. 2(C). The median value was 0.66.
図２（Ｂ）で示した学習データ（ｄｉｓｃｏｖｅｒｙ ｓｅｔ）における中央値０．７５の内訳としては、感度が１００％、特異度が約９％であった（図３（Ａ）参照。）。図２（Ｃ）で示した検証データ（ｖａｌｉｄａｔｉｏｎ ｓｅｔ）における中央値０．６６の内訳としては、感度が１００％、特異度が約１３％であった（図３（Ｂ）参照。）。 As for the median value of 0.75 in the learning data (discovery set) shown in FIG. 2(B), the sensitivity was 100% and the specificity was about 9% (see FIG. 3(A)). The breakdown of the median value of 0.66 in the validation set shown in FIG. 2(C) was that the sensitivity was 100% and the specificity was approximately 13% (see FIG. 3(B)).
本明細書において、「感度」は、（真陽性の数）／（真陽性の数＋偽陽性の数）の値を意味する。感度が高ければニボルマブの薬効がある患者を予測することが可能となり、ニボルマブによる治療の選択につながる。 As used herein, "sensitivity" means the value of (number of true positives)/(number of true positives + number of false positives). If the sensitivity is high, it will be possible to predict which patients will benefit from nivolumab, which will lead to the selection of nivolumab treatment.
本明細書において、「特異度」は、（真陰性の数）／（真陰性の数＋偽陰性の数）の値を意味する。特異度が高ければニボルマブの薬効が無い患者を、ニボルマブの薬効が有る患者であると誤判別することによる無駄な治療を防ぎ、患者の負担の軽減や医療費の削減につながる。 As used herein, "specificity" means the value of (number of true negatives)/(number of true negatives + number of false negatives). If the specificity is high, it will prevent unnecessary treatment due to erroneously identifying patients for whom nivolumab is not effective as patients for whom nivolumab is effective, leading to a reduction in the burden on patients and medical costs.
次いで、学習データ（ｄｉｓｃｏｖｅｒｙ ｓｅｔ）及び検証データ（ｖａｌｉｄａｔｉｏｎ ｓｅｔ）それぞれにおける各症例のｍｉＲＮＡのｉｎｄｅｘ ｓｃｏｒｅをドットプロットで表した（図３（Ｃ）及び（Ｄ）参照。）。図３（Ｃ）及び（Ｄ）に示す様に、学習データ（ｄｉｓｃｏｖｅｒｙ ｓｅｔ）及び検証データ（ｖａｌｉｄａｔｉｏｎ ｓｅｔ）ともに、Ｎｏｎ－ｒｅｓｐｏｎｄｅｒとＲｅｓｐｏｎｄｅｒにおけるｉｎｄｅｘ ｓｃｏｒｅに差が確認された。 Next, the miRNA index score of each case in each of the learning data (discovery set) and validation data (validation set) was represented by a dot plot (see FIGS. 3(C) and (D)). As shown in FIGS. 3(C) and (D), a difference in index score between Non-responder and Responder was confirmed for both the learning data (discovery set) and the validation data (validation set).
次いで、Ｎｏｎ－ｒｅｓｐｏｎｄｅｒとＲｅｓｐｏｎｄｅｒにおけるｍｉＲＮＡのｉｎｄｅｘ ｓｃｏｒｅのカットオフ値を０とした際の学習データ（ｄｉｓｃｏｖｅｒｙ ｓｅｔ）と検証データ（ｖａｌｉｄａｔｉｏｎ ｓｅｔ）の比を図４（Ａ）及び（Ｂ）に示す。学習データ（ｄｉｓｃｏｖｅｒｙ ｓｅｔ）及び検証データ（ｖａｌｉｄａｔｉｏｎ ｓｅｔ）ともに、高い割合でＮｏｎ－ｒｅｓｐｏｎｄｅｒとＲｅｓｐｏｎｄｅｒを識別することができた。 Next, the ratio of learning data (discovery set) and validation data (validation set) when the cutoff value of miRNA index score in Non-responder and Responder is set to 0 is shown in FIGS. 4(A) and 4(B). Both the learning data (discovery set) and the validation data (validation set) were able to identify Non-responders and Responders at a high rate.
上記データセットから肺腺がんのみを抽出して分析した結果を図４（Ｃ）に示す。肺腺がんにおいても学習データ（ｄｉｓｃｏｖｅｒｙ ｓｅｔ）及び検証データ（ｖａｌｉｄａｔｉｏｎ ｓｅｔ）ともに、Ｎｏｎ－ｒｅｓｐｏｎｄｅｒとＲｅｓｐｏｎｄｅｒにおけるｉｎｄｅｘ ｓｃｏｒｅに差が確認された。 The results of extracting and analyzing only lung adenocarcinoma from the above data set are shown in FIG. 4(C). Also in lung adenocarcinoma, a difference in index score between non-responders and responders was confirmed in both the learning data (discovery set) and the validation data (validation set).
２０回繰り返し１０分割交差検証を行った際の各モデルの判定式の詳細を表２に示す。また、各ｍｉＲＮＡ個別の精度を図５に示す。個別のｍｉＲＮＡよりこれら５つを組み合わせた方が、精度が高いことが確認された。 Table 2 shows details of the determination formula for each model when 10-fold cross validation was repeated 20 times. Furthermore, the accuracy of each miRNA individually is shown in FIG. It was confirmed that the combination of these five miRNAs was more accurate than the individual miRNAs.
本明細書において、「精度」は、（真陽性の数＋真陰性の数）／（全症例数）の値を意味する。精度は、全検体に対しての判別結果が正しかった割合を示しており、検出性能を評価する第一の指標となる。 As used herein, "accuracy" means the value of (number of true positives + number of true negatives)/(number of total cases). Accuracy indicates the percentage of correct discrimination results for all samples, and is the first index for evaluating detection performance.
更に、臨床で用いられている既存のＰＤ－Ｌ１マーカーと本発明における５つのｍｉＲＮＡの組み合わせとを比較した結果を図６（Ａ）、（Ｂ）に示す。既存のＰＤ－Ｌ１マーカーと比較して、本発明における５つのｍｉＲＮＡの組み合わせの方が、精度が高いことが確認された。 Furthermore, the results of a comparison between the existing PD-L1 marker used clinically and the combination of the five miRNAs of the present invention are shown in FIGS. 6(A) and (B). It was confirmed that the combination of the five miRNAs of the present invention has higher accuracy than the existing PD-L1 marker.
以上、網羅的な血清ｍｉＲＮＡプロファイルを用いることで、体液診断での免疫チェックポイント阻害剤の奏効予測を行えることが確認された。 As described above, it was confirmed that by using a comprehensive serum miRNA profile, it is possible to predict the effectiveness of immune checkpoint inhibitors in body fluid diagnosis.
本発明によれば、肺がん患者における抗ＰＤ－１抗体の治療効果の予測に寄与する。 According to the present invention, it contributes to predicting the therapeutic effect of anti-PD-1 antibodies in lung cancer patients.
Claims (5)
ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、ｍｉＲ－４５３４、及びｍｉＲ－６７２９－５からなる群から選択される少なくとも１つのｍｉＲＮＡを増幅するためのプライマーセット、及び／又は前記ｍｉＲＮＡ若しくはその増幅産物に結合するプローブを含む、診断薬。 A companion diagnostic agent used for lung cancer patients using anti-PD-1 antibodies,
a primer set for amplifying at least one miRNA selected from the group consisting of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, miR-4534, and miR-6729-5; /or a diagnostic agent comprising a probe that binds to the miRNA or its amplification product.
抗ＰＤ－１抗体に薬効のあった対照者由来の検体中の対応するｍｉＲＮＡの対照発現量と、抗ＰＤ－１抗体に薬効の無かった対照者由来の検体中の対応するｍｉＲＮＡの対照発現量とを教師データとして作成された、抗ＰＤ－１抗体の薬効の有無を判別する判別式に、前記患者由来の検体中の前記ｍｉＲＮＡの発現量を代入し、抗ＰＤ－１抗体の薬効を評価することを含む、抗ＰＤ－１抗体薬効予測方法。 miR-6729-5 in a patient-derived specimen is used as an endogenous control, and selected from the group consisting of miR-452-3p, miR-3129-3p, miR-4304, miR-4492, and miR-4534 in the specimen. measuring the expression level of at least one miRNA in vitro;
Control expression level of the corresponding miRNA in a sample from a control subject for whom the anti-PD-1 antibody had a therapeutic effect, and control expression level of the corresponding miRNA in a sample from a control subject for whom the anti-PD-1 antibody did not have a therapeutic effect. The expression level of the miRNA in the sample derived from the patient is substituted into the discriminant formula for determining the presence or absence of the drug efficacy of the anti-PD-1 antibody, which was created using the above as training data, to evaluate the drug efficacy of the anti-PD-1 antibody. A method for predicting anti-PD-1 antibody efficacy, comprising:
生体から分離された被検試料中に含まれる、ｍｉＲ－６７２９－５を内在性コントロールとし、前記被検試料中に含まれる、ｍｉＲ－４５２－３ｐ、ｍｉＲ－３１２９－３ｐ、ｍｉＲ－４３０４、ｍｉＲ－４４９２、及びｍｉＲ－４５３４からなる群から選択される少なくとも１つのｍｉＲＮＡを含む、マーカー。 A marker for predicting drug efficacy of anti-PD-1 antibody in lung cancer patients,
miR-6729-5 contained in a test sample isolated from a living body is used as an endogenous control, miR-452-3p, miR-3129-3p, miR-4304, miR contained in the test sample. -4492, and miR-4534.
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