JP7644949B2 - Method for detecting PD-L1-positive exosomes - Google Patents
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Description
本発明は一般に、腫瘍診断の分野に関する。具体的には、本発明は、ヒト体液中のエクソソームを定量する方法、より具体的にはPD-L1陽性エクソソームを定量および定性する方法、それに使用する抗PD-L1モノクローナル抗体等に関する。 The present invention generally relates to the field of tumor diagnosis. Specifically, the present invention relates to a method for quantifying exosomes in human body fluids, more specifically, a method for quantifying and qualifying PD-L1-positive exosomes, and an anti-PD-L1 monoclonal antibody for use therein.
1992年に発見されたプログラム化死－１（Programmed death-1、PD-1）は、T細胞表面に発現するタンパク質受容体であり、細胞のアポトーシスに関与している。PD-1はCD28ファミリーに属し、細胞傷害性Tリンパ球抗原４（CTLA-4）とアミノ酸配列において23％の相同性を示す。しかし、PD-1は、CTLA4とは異なり、主に活性化T細胞、B細胞および骨髄系細胞に発現する。PD-1は２つのリガンド、それぞれPD-L1およびPD-L2を有する。PD-L1は、主にがん細胞に発現し、PD-L2の発現は、活性化マクロファージや樹状細胞などの抗原提示細胞に限定される。 Programmed death-1 (PD-1), discovered in 1992, is a protein receptor expressed on the surface of T cells and is involved in cellular apoptosis. PD-1 belongs to the CD28 family and shows 23% homology in amino acid sequence with cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, unlike CTLA4, PD-1 is mainly expressed on activated T cells, B cells, and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed on cancer cells, and PD-L2 expression is limited to antigen-presenting cells such as activated macrophages and dendritic cells.
腫瘍微小環境中の多くの腫瘍細胞および免疫細胞はプログラム化死－リガンド１（Programmed death-ligand 1、PD-L1）を発現する。新しい研究では、乳がん、肺がん、胃がん、腸がん、腎がん、メラノーマ、非小細胞肺がん、結腸がん、膀胱がん、卵巣がん、膵がん、肝がん等のヒト腫瘍組織でPD-L1タンパク質の高発現が検出されており、PD-L1の発現レベルは、患者の臨床症状および予後と密接に相関している。 Many tumor cells and immune cells in the tumor microenvironment express programmed death-ligand 1 (PD-L1). New studies have detected high expression of PD-L1 protein in human tumor tissues, including breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and liver cancer, and the expression level of PD-L1 is closely correlated with the clinical symptoms and prognosis of patients.
PD-L1は、第二のシグナル経路を使ってT細胞の増殖を阻害するため、PD-L1とPD-1との結合をブロックすることは、腫瘍免疫療法の分野において、非常に有望な標的となっている（例えば、特許文献１および２）。 PD-L1 inhibits T cell proliferation using a second signaling pathway, so blocking the binding of PD-L1 to PD-1 is a very promising target in the field of tumor immunotherapy (e.g., Patent Documents 1 and 2).
オプジーボやキートルーダなど、PD-1を標的とする医薬は免疫チェックポイント阻害剤として注目されているが（ Yarchoan M., Johnson B.A. III, Lutz E.R., Laheru D.A., and Jaffee E.M. 2017. Targeting neoantigens to augment antitumour immunity. Nat. Rev. Cancer. 17:569 10.1038/nrc.2017.74）、単独の医薬ではその薬効が見られる症例は20％余りと決して高くはない。これは、PD-1のリガンドであるPD-L1が、すべてのがん細胞において発現していないためであると考えられている。このため、PD-1を標的とするキートルーダを処方するためには、患者を層別化するためのコンパニオン診断薬として、PD-L1の組織染色が必須となっている（ Reck M., Rodriguez-Abreu D., Robinson A.G., Hui R., Csoszi T., Fulop A., Gottfried M., Peled N., Tafreshi A., Cuffe S., et al. KEYNOTE-024 Investigators. 2016. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N. Engl. J. Med. 375:1823-1833. 10.1056/NEJMoa1606774：日本肺癌学会編、「肺癌患者におけるPD-L1検査の手引き」）。内視鏡や針を使って腫瘍組織を採取する生検（biopsy）を前提とする組織染色は侵襲性が高い検査であり、患者の負担が問題である。そこで、生検に代え、血液などの体液使って診断や治療効果予測を行う、患者負担が軽減されるリキッドバイオプシーの開発が望まれている。 Drugs that target PD-1, such as Opdivo and Keytruda, have been attracting attention as immune checkpoint inhibitors (Yarchoan M., Johnson B.A. III, Lutz E.R., Laheru D.A., and Jaffee E.M. 2017. Targeting neoantigens to augment antitumour immunity. Nat. Rev. Cancer. 17:569 10.1038/nrc.2017.74), but the efficacy of these drugs alone is only seen in about 20% of cases, which is not very high. This is thought to be because PD-L1, the ligand for PD-1, is not expressed in all cancer cells. For this reason, in order to prescribe Keytruda, which targets PD-1, tissue staining for PD-L1 is essential as a companion diagnostic for patient stratification (Reck M., Rodriguez-Abreu D., Robinson A.G., Hui R., Csoszi T., Fulop A., Gottfried M., Peled N., Tafreshi A., Cuffe S., et al. KEYNOTE-024 Investigators. 2016. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N. Engl. J. Med. 375:1823-1833. 10.1056/NEJMoa1606774: "Guide to PD-L1 Testing in Lung Cancer Patients," edited by the Japan Lung Cancer Society). Tissue staining, which is based on a biopsy in which tumor tissue is taken using an endoscope or needle, is a highly invasive test, and the burden on patients is an issue. Therefore, there is a need for the development of liquid biopsies, which use bodily fluids such as blood to make diagnoses and predict treatment outcomes instead of biopsies, thereby reducing the burden on patients.
PD-L1陽性の腫瘍からは、PD-L1陽性のエクソソームが分泌されていることが知られている（Chen G., Huang A.C., Zhang W., Zhang G., Wu M., Xu W., Yu Z., Yang J., Wang B., Sun H., et al. 2018. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 560:382-386. 10.1038/s41586-018-0392-8）。本発明者らは、生物学的試料中のPD-L1陽性エクソソームを直接定量する目的から、PD-L1の細胞外領域に特異的な抗体を作製し、それを基本とする測定系の構築に成功した。 It is known that PD-L1-positive exosomes are secreted from PD-L1-positive tumors (Chen G., Huang A.C., Zhang W., Zhang G., Wu M., Xu W., Yu Z., Yang J., Wang B., Sun H., et al. 2018. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 560:382-386. 10.1038/s41586-018-0392-8). In order to directly quantify PD-L1-positive exosomes in biological samples, the present inventors have produced an antibody specific to the extracellular domain of PD-L1 and succeeded in constructing a measurement system based on the antibody.
すなわち、本発明は、以下の態様を含む。
＜PD-L1陽性腫瘍を検出＞
［１］
PD-L1をその表面に発現しているPD-L1陽性エクソソームを、生物学的試料から分離することなく直接検出することを特徴とする、PD-L1陽性腫瘍を検出する方法。
［２］
PD-L1陽性エクソソームの検出を、抗体を用いて行う［１］記載の方法。
［３］
抗体が、PD-L1を認識する抗体、およびエクソソームを認識する抗体である、［２］記載の方法。
［４］
検出を、免疫沈降によって行う、［１］から［３］のいずれか記載の方法。
［５］
［１］から［３］のいずれか記載の方法によって、PD-L1陽性腫瘍を有する患者を選別する方法。
［６］
［４］記載の方法によって、免疫チェックポイント阻害剤が奏功する患者を選別する方法。
＜抗体分子＞
［７］
(ｉ)配列番号３のVHCDR1アミノ酸配列、配列番号４のVHCDR2アミノ酸配列および配列番号５のVHCDR3アミノ酸配列を含む重鎖可変領域(VH)、ならびに
(ii)配列番号１１のVLCDR1アミノ酸配列、配列番号１２のVLCDR2アミノ酸配列および配列番号１３のVLCDR3アミノ酸配列を含む軽鎖可変領域(VL)
を含む、PD-L1に結合することができる単離された抗体分子、またはその誘導体。
［８］
配列番号１のVHアミノ酸配列、またはそれと少なくとも８０％同一であるアミノ酸配列、または配列番号１のVHアミノ酸配列と比較して１または数個のアミノ酸残基の置換、欠失、挿入もしくは付加を有するアミノ酸配列を含む少なくとも１個のフレームワーク(FW)領域を含む重鎖可変領域を有する、［７］記載の抗体分子、またはその誘導体。
［９］
配列番号９のVLアミノ酸配列、またはそれと少なくとも８０％同一であるアミノ酸配列、または９のVLアミノ酸配列と比較して１または数個のアミノ酸残基の置換、欠失、挿入もしくは付加を有するアミノ酸配列を含む少なくとも１個のフレームワーク領域を含む軽鎖可変領域を有する、［７］または［８］記載の抗体分子、またはその誘導体。
［１０］
Fab、Ｆ(ab')２、Fvまたは単鎖Fv断片(scFv)である、［７］から［９］のいずれか記載の抗体分子の誘導体。
＜核酸＞
［１１］
［７］から［１０］のいずれか］記載の抗体分子の抗体重鎖または軽鎖可変領域をコードする単離された核酸。
［１２］
重鎖CDR1から3をコードする単離された核酸であって、配列番号６、７または８のヌクレオチド配列を含む核酸。
［１３］
軽鎖CDR1から3をコードする単離された核酸であって、配列番号１４、１５または１６のヌクレオチド配列を含む核酸。
［１４］
［１１］から［１３］のいずれか記載の核酸を含む発現ベクター。
［１５］
［１４］記載のベクターを含む宿主細胞。
［１６］
遺伝子発現に適した条件下、［１４］記載の宿主細胞を培養することを含む、抗体分子またはその断片を産生する方法。
＜検出＞
［１７］
(ｉ) 被験者から生物学的試料を入手し、
(ii) 得られた生物学的試料と、［７］から［１０］のいずれか］記載の単離された抗体分子またはその誘導体とを接触させ、そして
(iii) PD-L1陽性エクソソームおよび抗体分子またはその誘導体の間の複合体の形成を検出すること
を含む、生物学的試料におけるPD-L1陽性エクソソームを検出する方法。
［１８］
［１７］記載の方法によって、PD-L1陽性エクソソームが検出される患者を選別することを特徴とする、免疫チェックポイント阻害剤が奏功する患者を選別する方法。
［１９］
［７］から［１０］のいずれか記載の単離された抗体分子またはその誘導体を含む、PD-L1陽性エクソソームを定量するためのキット。
［２０］
配列番号１９に示すアミノ酸配列を有するヒトPD-L1およびヒトCD9タンパクの融合タンパク質、または配列番号２１に示すアミノ酸配列を有するヒトPD-L1およびヒトCD63の融合タンパク質を標準試薬としてさらに含有する、［１９］記載のキット。
That is, the present invention includes the following aspects.
<Detection of PD-L1 positive tumors>
[1]
A method for detecting PD-L1-positive tumors, comprising directly detecting PD-L1-positive exosomes that express PD-L1 on their surface, without isolating them from a biological sample.
[2]
The method described in [1], wherein the detection of PD-L1-positive exosomes is performed using an antibody.
[3]
The method described in [2], wherein the antibody is an antibody that recognizes PD-L1 and an antibody that recognizes exosomes.
[4]
The method according to any one of [1] to [3], wherein the detection is carried out by immunoprecipitation.
[5]
A method for selecting a patient having a PD-L1-positive tumor by the method according to any one of [1] to [3].
[6]
[4] A method for selecting patients for whom an immune checkpoint inhibitor is effective, using the method described above.
<Antibody molecule>
[7]
(i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and
(ii) a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 11, the VLCDR2 amino acid sequence of SEQ ID NO: 12, and the VLCDR3 amino acid sequence of SEQ ID NO: 13
5. An isolated antibody molecule, or derivative thereof, capable of binding to PD-L1, comprising:
[8]
The antibody molecule of [7], or a derivative thereof, having a heavy chain variable region including at least one framework (FW) region comprising the VH amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical thereto, or an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the VH amino acid sequence of SEQ ID NO: 1.
[9]
An antibody molecule or derivative thereof according to [7] or [8], having a light chain variable region comprising at least one framework region comprising the VL amino acid sequence of SEQ ID NO: 9, or an amino acid sequence which is at least 80% identical thereto, or an amino acid sequence which has one or several amino acid residue substitutions, deletions, insertions or additions compared to the VL amino acid sequence of SEQ ID NO: 9.
[10]
A derivative of the antibody molecule according to any one of [7] to [9], which is a Fab, F(ab') 2 , Fv or single-chain Fv fragment (scFv).
<Nucleic Acid>
[11]
An isolated nucleic acid encoding an antibody heavy or light chain variable region of the antibody molecule described in any one of [7] to [10].
[12]
An isolated nucleic acid encoding heavy chain CDR1 to 3, said nucleic acid comprising the nucleotide sequence of SEQ ID NO:6, 7 or 8.
[13]
An isolated nucleic acid encoding light chain CDR1 to 3, said nucleic acid comprising the nucleotide sequence of SEQ ID NO: 14, 15 or 16.
[14]
An expression vector comprising the nucleic acid according to any one of [11] to [13].
[15]
[14] A host cell comprising the vector described above.
[16]
A method for producing an antibody molecule or a fragment thereof, comprising culturing the host cell according to [14] under conditions suitable for gene expression.
<Detection>
[17]
(i) Obtaining a biological sample from the subject;
(ii) contacting the obtained biological sample with an isolated antibody molecule or a derivative thereof according to any one of [7] to [10], and
(iii) A method for detecting PD-L1-positive exosomes in a biological sample, comprising detecting the formation of a complex between PD-L1-positive exosomes and an antibody molecule or a derivative thereof.
[18]
[17] A method for selecting a patient for whom an immune checkpoint inhibitor is effective, comprising selecting a patient in which PD-L1-positive exosomes are detected by the method described in the above.
[19]
A kit for quantifying PD-L1-positive exosomes, comprising the isolated antibody molecule or derivative thereof according to any one of [7] to [10].
[20]
The kit of [19], further comprising a fusion protein of human PD-L1 and human CD9 protein having the amino acid sequence set forth in SEQ ID NO: 19, or a fusion protein of human PD-L1 and human CD63 having the amino acid sequence set forth in SEQ ID NO: 21 as a standard reagent.
＜PD-L1陽性腫瘍を検出＞
本発明は、ひとつの態様として、PD-L1をその表面に発現しているPD-L1陽性エクソソームを、生物学的試料から分離することなく直接検出することを特徴とする、PD-L1陽性腫瘍を検出する方法に関する。
本明細書において、「PD-L1陽性エクソソーム」とは、PD-L1をその表面に発現しているエクソソームを意味する。PD-L1陽性エクソソームは主として、PD-L1陽性腫瘍から放出される。その存在は、Chen G., et al. 2018. Nature. 560:382-386. 10.1038/s41586-018-0392-8に記載されている。
<Detection of PD-L1 positive tumors>
In one aspect, the present invention relates to a method for detecting PD-L1-positive tumors, which is characterized by directly detecting PD-L1-positive exosomes that express PD-L1 on their surface without isolating them from a biological sample.
As used herein, "PD-L1-positive exosomes" refers to exosomes expressing PD-L1 on their surface. PD-L1-positive exosomes are primarily released from PD-L1-positive tumors. Their existence has been described in Chen G., et al. 2018. Nature. 560:382-386. 10.1038/s41586-018-0392-8.
エクソソームは30～120nmサイズの微小胞であり、通常、エキソサイトーシス経路を介して能動的に、血液などの体液中に分泌される。エクソソームは、主要組織適合性複合体タンパク質MHCI、MHCII）や抗原提示に関与するタンパク質に加え、多くの細胞機能に関与する膜および細胞質タンパク質を担持する。エクソソームは特定の生理的条件下で、樹状細胞、リンパ球、マスト細胞および上皮細胞のような多様な細胞タイプから分泌される。 Exosomes are microvesicles with a size of 30-120 nm that are usually actively secreted into body fluids such as blood via the exocytosis pathway. Exosomes carry major histocompatibility complex proteins (MHCI, MHCII) and proteins involved in antigen presentation, as well as membrane and cytoplasmic proteins involved in many cellular functions. Exosomes are secreted by a variety of cell types, such as dendritic cells, lymphocytes, mast cells, and epithelial cells, under certain physiological conditions.
エクソソームは細胞培養物の上清および体液を複数段階の遠心分離を行って調製される。しかし、エクソソームについての有用なインビボデータを得るための問題は、ヒト体液、特に血液検体からエクソソームを調製するための、エクソソームを得る現在利用可能な方法の効率が低レベルであることである。これらの現在直面している課題の解決法を提供するため、本発明は、体液、特にヒト血液検体からエクソソームを検出し、定量する単純で信頼のできる方法を提供する。 Exosomes are prepared from cell culture supernatants and body fluids using multiple centrifugation steps. However, a problem in obtaining useful in vivo data on exosomes is the low level of efficiency of currently available methods for obtaining exosomes for the preparation of exosomes from human body fluids, particularly blood samples. To provide a solution to these currently faced challenges, the present invention provides a simple and reliable method for detecting and quantifying exosomes from body fluids, particularly human blood samples.
かかる態様では、本発明は、PD-L1陽性エクソソームの検出を抗体、好ましくはPD-L1を認識する抗体およびエクソソームを認識する抗体を利用する。さらに好ましくは、本発明の抗体を利用する方法である。具体的には、PD-L1陽性エクソソームを検出する抗体として、以下に詳述するA1-7C11、A1-7E1、A1-9F7、A1-13A11、A1-13G7、A1-14A5およびA1-14E9の中から選ばれる少なくとも１つの抗ヒトPD-L1モノクローナル抗体、およびエクソソームを認識する抗体として、抗CD9モノクローナル抗体（コスモ・バイオ社製）または抗CD63モノクローナル抗体（コスモ・バイオ社製）、抗CD81モノクローナル抗体（コスモ・バイオ社製）を用いる方法である。 In this embodiment, the present invention uses antibodies to detect PD-L1-positive exosomes, preferably an antibody that recognizes PD-L1 and an antibody that recognizes exosomes. More preferably, the method uses the antibodies of the present invention. Specifically, the method uses at least one anti-human PD-L1 monoclonal antibody selected from A1-7C11, A1-7E1, A1-9F7, A1-13A11, A1-13G7, A1-14A5, and A1-14E9, which are described in detail below, as the antibody that detects PD-L1-positive exosomes, and an anti-CD9 monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.), an anti-CD63 monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.), or an anti-CD81 monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.) as the antibody that recognizes exosomes.
本発明において、「PD-L1陽性腫瘍」とは、PD-L1をその細胞表面に発現している腫瘍を意味する。PD-L1は、PD-1およびB7-1に結合し、免疫系を阻害するが、腫瘍微小環境中の多くの腫瘍細胞および免疫細胞はPD-L1を発現する。新しい研究では、乳がん、肺がん、胃がん、腸がん、腎がん、メラノーマ、非小細胞肺がん、結腸がん、膀胱がん、卵巣がん、膵がん、肝がん等のヒト腫瘍組織でPD-L1タンパク質の高発現が検出されており、PD-L1の発現レベルは、患者の臨床症状および予後と密接に相関している。また、PD-L1発現は、ヒト肺、卵巣および結腸癌ならびに様々な骨髄腫を含む多くのがんにおいて見出されており、予後不良に関連することが多いと報告されている（Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53）。PD-L1は、抗原特異的Ｔ細胞クローンのアポトーシスを増加させることにより、腫瘍免疫における役割を果たすことが示唆され（Dong et al. (2002) Nat Med 8:793-800）、また腸管粘膜炎症に関与し得ること、そして、PD-L1の阻害は大腸炎に関連する消耗性疾患を抑制することも示唆されている（Kanai et al. (2003) J Immunol 171:4156-63）。 In the present invention, "PD-L1 positive tumor" means a tumor expressing PD-L1 on its cell surface. PD-L1 binds to PD-1 and B7-1 and inhibits the immune system, but many tumor cells and immune cells in the tumor microenvironment express PD-L1. New studies have detected high expression of PD-L1 protein in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and liver cancer, and the expression level of PD-L1 is closely correlated with the clinical symptoms and prognosis of patients. In addition, PD-L1 expression has been found in many cancers, including human lung, ovarian and colon cancers and various myelomas, and has been reported to be often associated with poor prognosis (Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53). PD-L1 has been suggested to play a role in tumor immunity by increasing apoptosis of antigen-specific T cell clones (Dong et al. (2002) Nat Med 8:793-800), and may also be involved in intestinal mucosal inflammation, and inhibition of PD-L1 has been suggested to suppress debilitating disease associated with colitis (Kanai et al. (2003) J Immunol 171:4156-63).
本発明のPD-L1陽性腫瘍を検出する方法は、PD-L1陽性エクソソームを、生物学的試料から分離することなく直接検出することを特徴とする。生物学的試料とは、体液、特に血液、血漿、血清などの血液検体を意味し、さらには腹水、脳液、骨髄、尿、糞便または気管支肺胞洗浄液であってもよい。 The method for detecting PD-L1-positive tumors of the present invention is characterized in that PD-L1-positive exosomes are directly detected without being separated from a biological sample. The biological sample refers to a body fluid, particularly a blood sample such as blood, plasma, or serum, and may also be ascites, cerebral fluid, bone marrow, urine, feces, or bronchoalveolar lavage fluid.
本発明の抗体を用いたELISAによりPD-L1陽性精製エクソソームを定量したところ、いずれも濃度依存的な測定値が得られ、これらのELISAがPD-L1陽性エクソソームの定量に適していることが示された（図４）。また、固相抗体がCD9、CD63いずれの場合においても、市販抗体よりも本発明のA1-9F7抗体を用いたほうが有意に高感度にPD-L1陽性エクソソームを検出できるELISAとなることが判明した（図６）。さらに、抗CD9抗体を固相抗体としてPD-L1陽性精製エクソソームを定量するエクソソームELISAにおいて、血清あるいは血漿の存在下における抗体とエクソソームの結合特異性を評価するために添加回収試験を行った結果、回収率が100％前後となり、本発明では、生物学的試料からPD-L1陽性エクソソームを分離することなく直接検出できることが示された（図９Ａ、図９Ｂ）。直接検出できる系としては、生物学的試料、特に血液検体はその量が25μL以下、さらに好ましくは20μL以下、特に好ましくは10μL以下であれば、信頼できる測定値を得ることができる。特に、CD9/PD-L1 ELISAでは、検体の量が10μL以下で得られる測定値が信頼できる。当然ながら、検体は、検出レベル以上を与える量以上である必要がある。 When PD-L1-positive purified exosomes were quantified by ELISA using the antibodies of the present invention, concentration-dependent measurements were obtained, demonstrating that these ELISAs are suitable for quantifying PD-L1-positive exosomes (Figure 4). In addition, whether the solid-phase antibody was CD9 or CD63, it was found that the ELISA using the A1-9F7 antibody of the present invention was able to detect PD-L1-positive exosomes with significantly higher sensitivity than the commercially available antibody (Figure 6). Furthermore, in an exosome ELISA in which anti-CD9 antibody was used as the solid-phase antibody to quantify PD-L1-positive purified exosomes, an additive recovery test was performed to evaluate the binding specificity of the antibody and exosomes in the presence of serum or plasma, and the recovery rate was approximately 100%, demonstrating that the present invention can directly detect PD-L1-positive exosomes without isolating them from biological samples (Figures 9A and 9B). As for systems that can directly detect exosomes, reliable measurements can be obtained as long as the biological sample, particularly blood samples, are 25 μL or less in volume, more preferably 20 μL or less, and particularly preferably 10 μL or less. In particular, CD9/PD-L1 ELISA allows reliable measurements to be obtained with a sample volume of 10 μL or less. Of course, the sample volume must be at least large enough to provide a detection level or higher.
本発明の好ましい態様において、PD-L1陽性エクソソームは、免疫沈降法によって検出できる。免疫沈降法では、可溶性の抗原と抗体が特異的に反応して不溶化することを利用し、比較的穏和な条件で抗原を検出・分離・精製することが可能である。例えば、基質と抗体を多数架橋させることによって、大きな構造体として不溶化させる。一般に、セファロースビーズなどの担体に抗体を結合させるが、磁気ビーズを使用する方法もよく行われる。モノクローナル抗体よりもポリクローナル抗体の方が免疫沈降を行いやすい。免疫沈降法では、遠心分離によって非特異的に吸着する成分を取り除いてもよい。磁気ビーズを用いる場合は、遠心分離の代わりに磁気ビーズによる分離を行う。 In a preferred embodiment of the present invention, PD-L1-positive exosomes can be detected by immunoprecipitation. In immunoprecipitation, soluble antigens react specifically with antibodies to insolubilize them, making it possible to detect, separate, and purify antigens under relatively mild conditions. For example, a large structure is insolubilized by crosslinking a large number of substrates with antibodies. Generally, antibodies are bound to a carrier such as Sepharose beads, but a method using magnetic beads is also often used. Polyclonal antibodies are easier to immunoprecipitate than monoclonal antibodies. In immunoprecipitation, nonspecifically adsorbed components may be removed by centrifugation. When magnetic beads are used, separation using magnetic beads is performed instead of centrifugation.
詳細には、PD-L1陽性エクソソームをエクソソーム特異的な抗体に接触させ、免疫沈降した複合体を形成する。そのとき、PD-L1陽性エクソソームが固定された特異的な抗体と接触すると、抗体は通常、免疫沈降した複合体を形成するために固体支持体に結合している。固体支持体は、抗体の付着または会合のための適切な部位を含むように改変できる材料である。固体支持体の材料としては、アクリル樹脂、ポリスチレン、およびスチレンと他物質とのコポリマー、ポリプロピレン、ポリエチレン、ポリブチレン、ポリウレタン、またはテフロンＪ、ナイロン、ニトロセルロース、ポリサッカライドを含むプラスチック；シリカ、またはケイ素；および金属、無機ガラス等の物質が挙げられる。固体支持体のサイズおよび形状は、PD-L1陽性エクソソームを結合できるのであれば制限はない。固体支持体は、平面であってもよく、ウェル（穴）、すなわち３６４穴を有するプレートであってもよく、または固体支持体は、ビーズであってもよい。いくつかの実施形態では、固体支持体は、複数壁を有するプレートのウェルである。他の実施形態では、固体支持体は、好ましくはビーズであり、さらに好ましくは磁気ビーズである。 In detail, PD-L1-positive exosomes are contacted with an exosome-specific antibody to form an immunoprecipitated complex. When the PD-L1-positive exosomes are contacted with the immobilized specific antibody, the antibody is usually bound to a solid support to form an immunoprecipitated complex. The solid support is a material that can be modified to include suitable sites for antibody attachment or association. Materials for the solid support include plastics including acrylic resins, polystyrene and copolymers of styrene with other substances, polypropylene, polyethylene, polybutylene, polyurethane, or Teflon J, nylon, nitrocellulose, polysaccharides; silica, or silicon; and metals, inorganic glass, and other materials. The size and shape of the solid support are not limited as long as it can bind PD-L1-positive exosomes. The solid support may be a flat surface, a well (hole), i.e., a plate with 364 holes, or the solid support may be beads. In some embodiments, the solid support is a well of a plate with multiple walls. In other embodiments, the solid support is preferably a bead, and more preferably a magnetic bead.
以下の実施例にて示しているように、本発明の抗体は、免疫沈降に利用することができ、PD-L1陽性エクソソームを、生物学的試料から分離することなく直接検出することが可能であることが実証された。また、ELISAの場合、プレートのウェルの大きさに制限があり、処理できる検体量も限定されるところ、免疫沈降では検体量に制限がなく、比較的多い検体量においてエクソソームを濃縮できることが実証された。このように、検体自身の総量が比較的多い場合であっても、その検体中のPD-L1陽性エクソソームの存在量が少なく、すなわち濃度が低い場合においても、免疫沈降を用いることで定量が可能となる利点を提供し得る。 As shown in the following examples, it has been demonstrated that the antibodies of the present invention can be used for immunoprecipitation and that PD-L1-positive exosomes can be directly detected without isolating them from a biological sample. Furthermore, in the case of ELISA, there is a limit to the size of the plate wells, and the amount of sample that can be processed is also limited, whereas in immunoprecipitation, there is no limit to the amount of sample, and it has been demonstrated that exosomes can be concentrated in a relatively large amount of sample. Thus, even when the total amount of the sample itself is relatively large, or even when the amount of PD-L1-positive exosomes present in the sample is small, i.e., the concentration is low, immunoprecipitation can provide the advantage of enabling quantification.
本発明のPD-L1陽性腫瘍を検出する方法は、PD-L1陽性腫瘍を有する患者を選別する方法に応用することができ、かかる態様も本発明の範囲に入る。PD-L1陽性腫瘍を有する患者を選別する本発明の方法は、免疫チェックポイント阻害剤が奏功する患者を選別する方法に応用でき、かかる態様も本発明の範囲に入る。「免疫チェックポイント阻害剤」とは、新しいがん免疫療法に利用される抗体医薬、例えば抗PD-1抗体、抗PD-L1抗体および抗CTLA-4抗体を意味し、特に、本発明においては、「免疫チェックポイント阻害剤」としては抗PD-1抗体および抗PD-L1抗体が関連する。腫瘍細胞は、免疫細胞からの攻撃を逃れるためにPD-L1を発現し、これが免疫細胞のPD-1に結合すると、免疫細胞の働きが抑制される。抗PD-1抗体は免疫細胞のPD-1に結合し、PD-L1との結合を阻害し、抗PD-L1抗体はがん細胞が発現するPD-L1に結合し、PD-1との結合を阻害する。「免疫チェックポイント阻害剤」の市販薬の具体的には、抗PD-1抗体として小野薬品工業/米ブリストル・マイヤーズスクイブの「オプジーボ」（ニボルマブ）や米メルク（MSD）の「キートルーダ」（ペムブロリズマブ）、抗PD-L1抗体として独メルク/米ファイザーの「バベンチオ」（アベルマブ）、スイス・ロシュ/中外製薬の「テセントリク」（アテゾリズマブ）、英アストラゼネカの「イミフィンジ」（デュルバルマブ）が挙げられる。 The method of detecting PD-L1-positive tumors of the present invention can be applied to a method for selecting patients with PD-L1-positive tumors, and such an embodiment is also within the scope of the present invention. The method of selecting patients with PD-L1-positive tumors of the present invention can be applied to a method for selecting patients for whom immune checkpoint inhibitors are effective, and such an embodiment is also within the scope of the present invention. "Immune checkpoint inhibitors" refer to antibody drugs used in new cancer immunotherapy, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and in particular, in the present invention, anti-PD-1 antibodies and anti-PD-L1 antibodies are relevant as "immune checkpoint inhibitors." Tumor cells express PD-L1 to escape attacks from immune cells, and when this binds to PD-1 on immune cells, the function of the immune cells is suppressed. Anti-PD-1 antibodies bind to PD-1 on immune cells and inhibit binding to PD-L1, and anti-PD-L1 antibodies bind to PD-L1 expressed by cancer cells and inhibit binding to PD-1. Specific examples of commercially available "immune checkpoint inhibitor" drugs include anti-PD-1 antibodies such as "Opdivo" (nivolumab) from Ono Pharmaceutical Co., Ltd./Bristol-Myers Squibb Co., Ltd. (USA) and "Keytruda" (pembrolizumab) from Merck & Co., Inc. (MSD) (USA), and anti-PD-L1 antibodies such as "Bavencio" (avelumab) from Merck & Co., Ltd./Pfizer Co., Ltd. (USA), "Tecentriq" (atezolizumab) from Roche/Chugai Pharmaceutical Co., Ltd. (Switzerland), and "Imfinzi" (durvalumab) from AstraZeneca Co., Ltd. (UK).
「免疫チェックポイント阻害剤」を利用する免疫療法はがんに対する標準療法の一つとして認められる時代となっており、これらの薬剤は既存の標準療法に不応性となった難治性がんに対して長期的な治療効果を発揮し，全生存期間の延長をもたらす優れた治療法であると認められている。しかし，免疫チェックポイント阻害剤には、例えば免疫関連有害事象の発症、最適な治療レジメンの同定など、克服すべき課題も多く存在している。特に、現在の免疫チェックポイント阻害剤は単剤での奏効率が10-30％程度と不十分であるため、その治療効果をさらに向上させることを目指した複合的がん免疫療法の開発が世界中で積極的に進められている。複合的がん免疫療法は抗PD-1/PD-L1抗体との組み合わせを中心として実施されており、複数の免疫チェックポイント分子を同時に阻害する治療法、免疫チェックポイント阻害剤と免疫刺激性分子を活性化する薬剤の併用などが検討されている。また、非免疫療法との組み合わせとして、殺細胞性抗がん剤や分子標的薬、血管新生阻害剤のみならず、放射線治療と抗PD-1/PD-L1抗体の併用による臨床試験が多く実施されている。いずれにせよ、免疫チェックポイント阻害剤は単剤での奏効率が10-30％程度と不十分である理由は、PD-L1が、すべてのがん細胞において発現していないためであると考えられる。このため、患者の負担も考慮し、生検に代え、血液などの体液を使って診断や治療効果予測を行う、患者負担が軽減されるリキッドバイオプシーの開発が望まれ、本発明方法はこの要請に適う発明である。 Immunotherapy using immune checkpoint inhibitors has become recognized as one of the standard therapies for cancer. These drugs are recognized as an excellent treatment that exerts long-term therapeutic effects on intractable cancers that are refractory to existing standard therapies and extends overall survival. However, there are many challenges to overcome with immune checkpoint inhibitors, such as the occurrence of immune-related adverse events and the identification of optimal treatment regimens. In particular, the response rate of current immune checkpoint inhibitors as a single agent is insufficient at about 10-30%, so the development of composite cancer immunotherapy aimed at further improving the therapeutic effect is being actively promoted around the world. Composite cancer immunotherapy is mainly performed in combination with anti-PD-1/PD-L1 antibodies, and therapies that simultaneously inhibit multiple immune checkpoint molecules and the combination of immune checkpoint inhibitors and drugs that activate immune stimulatory molecules are being considered. In addition, as a combination with non-immunotherapy, many clinical trials are being conducted using not only cytotoxic anticancer drugs, molecular targeted drugs, and angiogenesis inhibitors, but also radiation therapy in combination with anti-PD-1/PD-L1 antibodies. In any case, the reason why the efficacy rate of immune checkpoint inhibitors as a single agent is insufficient at about 10-30% is thought to be because PD-L1 is not expressed in all cancer cells. For this reason, it is desirable to develop a liquid biopsy that reduces the burden on patients by using body fluids such as blood to perform diagnosis and predict treatment effects instead of biopsies, taking into consideration the burden on patients, and the method of the present invention meets this need.
＜抗体分子＞
本発明は、別の態様として、
(ｉ)配列番号３のVHCDR1アミノ酸配列、配列番号４のVHCDR2アミノ酸配列および配列番号５のVHCDR3アミノ酸配列を含む重鎖可変領域(VH)、ならびに
(ii)配列番号１１のVLCDR1アミノ酸配列、配列番号１２のVLCDR2アミノ酸配列および配列番号１３のVLCDR3アミノ酸配列を含む軽鎖可変領域(VL)
を含む、PD-L1に結合することができる単離された抗体分子、またはその誘導体に関する。本発明において、「抗体分子」は、2本の同一の重鎖と2本の同一の軽鎖との間でジスルフィド結合により相互に結合されたテトラペプチド鎖構造の免疫グロブリン分子を意味する。抗体重鎖および軽鎖のN末端に隣接する約110個のアミノ酸配列は、高度に変化しやすく、可変領域（Fv領域）として知られている。C末端に近い残りの部分のアミノ酸配列は、相対的に安定であり、定常領域として知られている。可変領域は、３つの超可変領域（HVR）と相対的に保存された４つのフレームワーク領域（FR）を備える。３つの超可変領域は、抗体の特異性を決定し、相補性決定領域（CDR）としても知られている。各軽鎖可変領域（LCVR）および各重鎖可変領域（HCVR）は、アミノ末端からカルボキシル末端へ、FR1、CDR1、FR2、CDR2、FR3、CDR3およびFR4の連続した順番で、３つのCDR領域および４つのFR領域からなる。３つの軽鎖CDRは、LCDR1、LCDR2およびLCDR3を指し、また３つの重鎖CDRは、HCDR1、HCDR2およびHCDR3を指す。
<Antibody molecule>
In another aspect, the present invention provides a method for producing a composition comprising:
(i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and
(ii) a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 11, the VLCDR2 amino acid sequence of SEQ ID NO: 12, and the VLCDR3 amino acid sequence of SEQ ID NO: 13
The present invention relates to an isolated antibody molecule, or derivative thereof, capable of binding to PD-L1, comprising: a) a tetrapeptide chain structure of two identical heavy chains and two identical light chains linked together by disulfide bonds. The approximately 110 amino acid sequences adjacent to the N-terminus of the antibody heavy and light chains are highly variable and known as the variable region (Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and known as the constant region. The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody and are also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) consists of three CDR regions and four FR regions in the following consecutive order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three light chain CDRs are referred to as LCDR1, LCDR2 and LCDR3, and the three heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3.
本発明において、「単離された」とは、その本来の天然環境から単離された試料を意味する。例えば、生きている動物に存在する天然起源のポリヌクレオチドまたはポリペプチドは、単離されていないが、天然の系において共存する試料の一部または全部から人為的に分離された同じポリヌクレオチドまたはポリペプチドは、単離されている。 In the present invention, "isolated" refers to a sample that is isolated from its original natural environment. For example, a polynucleotide or polypeptide of natural origin present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been artificially separated from some or all of the sample with which it coexists in the natural system is isolated.
本発明において、「抗体分子の誘導体」とは、ダイアボディおよび単鎖分子、ならびに「抗原結合断片」を意味する。「抗原結合断片」は、抗原結合活性を有するFab断片、Fab'断片、F(ab')2断片、およびヒトPD-L1と結合するFv断片、scFv断片を指し、本発明で規定されている抗体における１つ以上のCDR領域を含むことができる。Fv断片は、重鎖可変領域、軽鎖可変領域、および定常領域を持たないすべての抗原結合部位を含む最小の抗体断片である。一般に、Fv抗体は、VHおよびVLドメイン間にポリペプチドリンカーをさらに含み、抗原結合に必要な構造を形成することができる。また、異なるリンカーが、２つの抗体の可変領域を連結するのに用いられ、単鎖抗体または単鎖Fv (scFv)と名付けられたポリペプチドを形成する。本明細書では、「PD-L1と結合する」という用語は、ヒトPD-L1と相互作用する能力があることを意味する。本明細書では、本発明の「抗原結合部位」は、本発明の抗体または抗原結合断片により認識される、抗原上の不連続の３次元部位を指す。 In the present invention, the term "derivative of an antibody molecule" refers to diabodies and single chain molecules, as well as "antigen-binding fragments". The term "antigen-binding fragments" refers to Fab fragments, Fab' fragments, F(ab')2 fragments having antigen-binding activity, and Fv fragments and scFv fragments that bind to human PD-L1, and may include one or more CDR regions in the antibody defined in the present invention. The Fv fragment is the smallest antibody fragment that contains all of the antigen-binding site without the heavy chain variable region, the light chain variable region, and the constant region. In general, the Fv antibody further contains a polypeptide linker between the VH and VL domains, which can form the structure required for antigen binding. Also, different linkers are used to link the variable regions of two antibodies to form a polypeptide named a single chain antibody or single chain Fv (scFv). In the present specification, the term "binds to PD-L1" means that it has the ability to interact with human PD-L1. In the present specification, the term "antigen-binding site" refers to a discrete three-dimensional site on the antigen that is recognized by the antibody or antigen-binding fragment of the present invention.
本発明は別の態様として、配列番号１のVHアミノ酸配列、またはそれと少なくとも８０％同一であるアミノ酸配列、または配列番号１のVHアミノ酸配列と比較して１または数個のアミノ酸残基の置換、欠失、挿入もしくは付加を有するアミノ酸配列を含む少なくとも１個のフレームワーク(FW)領域を含む重鎖可変領域を有する、本発明の抗体分子、またはその誘導体、ならびに、配列番号９のLHアミノ酸配列、またはそれと少なくとも８０％同一であるアミノ酸配列、または９のLHアミノ酸配列と比較して１または数個のアミノ酸残基の置換、欠失、挿入もしくは付加を有するアミノ酸配列を含む少なくとも１個のフレームワーク領域を含む軽鎖可変領域を有する、本発明の抗体分子、またはその誘導体に関する。VHおよびVL領域は、「フレームワーク領域」(FRまたはFW)と命名されるより保存された領域が散りばめられた、「相補性決定領域」(CDR)と命名される高頻度可変性の領域へと細分することができる。 In another aspect, the present invention relates to an antibody molecule of the present invention, or a derivative thereof, having a heavy chain variable region comprising at least one framework (FW) region comprising the VH amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 80% identical thereto, or an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the VH amino acid sequence of SEQ ID NO: 1, and an antibody molecule of the present invention, or a derivative thereof, having a light chain variable region comprising at least one framework region comprising the LH amino acid sequence of SEQ ID NO: 9, or an amino acid sequence at least 80% identical thereto, or an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the LH amino acid sequence of SEQ ID NO: 9. The VH and VL regions can be subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions, termed "framework regions" (FR or FW).
ある態様では、抗PD-L1抗体分子の軽鎖または重鎖可変フレームワークはヒトコンセンサス配列に由来する軽鎖もしくは重鎖可変フレームワーク残基の少なくとも８０％、８５％、８７％、９０％、９３％、９５％、９７％、９９％を含む軽鎖または重鎖可変フレームワークを含むことができる。 In some embodiments, the light or heavy chain variable framework of the anti-PD-L1 antibody molecule may comprise a light or heavy chain variable framework that comprises at least 80%, 85%, 87%, 90%, 93%, 95%, 97%, 99% of the light or heavy chain variable framework residues derived from a human consensus sequence.
＜核酸＞
本発明は別の態様として、本発明の抗体分子の抗体重鎖または軽鎖可変領域をコードする単離された核酸、具体的には重鎖CDR1から3をコードする単離された核酸であって、配列番号６、７または８のヌクレオチド配列を含む核酸、または軽鎖CDR1から3をコードする単離された核酸であって、配列番号１４、１５または１６のヌクレオチド配列を含む核酸に関する。
本発明において、用語「核酸」、「核酸配列」、「ヌクレオチド配列」または「ポリヌクレオチド」は、互換的に使用される。これらは、デオキシリボヌクレオチドもしくはリボヌクレオチドまたはこれらのアナログのいずれかである、いずれかの長さのヌクレオチドのポリマー型を意味する。
別の態様として、本発明は、本発明の核酸を含む発現ベクター、および本発明のベクターを含む宿主細胞に関する。
<Nucleic Acid>
In another aspect, the present invention relates to an isolated nucleic acid encoding an antibody heavy or light chain variable region of an antibody molecule of the present invention, specifically an isolated nucleic acid encoding heavy chain CDR1 to 3 comprising the nucleotide sequence of SEQ ID NO: 6, 7 or 8, or an isolated nucleic acid encoding light chain CDR1 to 3 comprising the nucleotide sequence of SEQ ID NO: 14, 15 or 16.
As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
In further aspects, the invention relates to an expression vector comprising a nucleic acid of the invention, and a host cell comprising a vector of the invention.
ある態様では、ベクターは、本明細書に記載の抗体分子をコードするヌクレオチド配列を含む。ある態様では、ベクターは、本明細書に記載のヌクレオチド配列を含む。ベクターとしては、限定されないが、ウイルス、プラスミド、コスミド、ラムダファージ、または酵母人工染色体(YAC)が挙げられる。さらに、DNAが染色体に安定的に統合された細胞を、トランスフェクトされた宿主細胞の選択を可能にする１つまたは複数のマーカーを導入することにより選択してもよい。マーカーは、栄養要求性宿主に、例えば原栄養性、殺生物剤耐性(例えば、抗生物質)、または銅などの重金属への耐性を付与し得る。選択性マーカー遺伝子は、発現されるＤＮＡ配列に直接連結させてもよく、または同じ細胞に共形質転換により導入してもよい。さらに追加的なエレメントが、ｍＲＮＡの最適な合成のために必要であり得る。これらのエレメントは、スプライスシグナル、転写プロモーター、エンハンサー、および終止シグナルを含んでもよい。 In some embodiments, the vector comprises a nucleotide sequence encoding an antibody molecule as described herein. In some embodiments, the vector comprises a nucleotide sequence as described herein. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). Additionally, cells in which the DNA has stably integrated into the chromosome may be selected by introducing one or more markers that allow for selection of transfected host cells. The markers may confer, for example, prototrophy, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper, to an auxotrophic host. The selectable marker gene may be directly linked to the DNA sequence to be expressed or may be introduced by co-transformation into the same cell. Additionally, additional elements may be necessary for optimal synthesis of mRNA. These elements may include splice signals, transcription promoters, enhancers, and termination signals.
ある態様では、宿主細胞は抗体分子をコードする核酸を含むように遺伝子的に改変されている。ある態様では、宿主細胞は、発現カセットの使用によって遺伝子的に改変されている。表現「発現カセット」は、配列が適合する宿主において遺伝子の発現に影響を与えることのできるヌクレオチド配列を指す。そのようなカセットは、プロモーター、イントロン有りまたは無しのオープンリーディングフレーム、および終止シグナルを含んでもよい。発現を有効にするために必要なさらなる因子、例えば誘導性プロモーターを使用してもよい。細胞は、真核細胞、細菌細胞、昆虫細胞またはヒト細胞であり得る。適切な真核細胞には、限定されないが、Vero細胞、HeLa細胞、CHO細胞等が含まれる。適切な昆虫細胞としては、限定されないが、Sf9細胞が挙げられる。別の態様として、本発明は、遺伝子発現に適した条件下、本発明の宿主細胞を培養することを含む、抗体分子またはその断片を産生する方法に関する。 In one embodiment, the host cell is genetically modified to contain a nucleic acid encoding an antibody molecule. In one embodiment, the host cell is genetically modified by the use of an expression cassette. The expression "expression cassette" refers to a nucleotide sequence capable of affecting expression of a gene in a host with which the sequence is compatible. Such a cassette may include a promoter, an open reading frame with or without an intron, and a termination signal. Additional elements required to effect expression may be used, such as an inducible promoter. The cell may be a eukaryotic cell, a bacterial cell, an insect cell or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, CHO cells, etc. Suitable insect cells include, but are not limited to, Sf9 cells. In another embodiment, the invention relates to a method of producing an antibody molecule or a fragment thereof, comprising culturing a host cell of the invention under conditions suitable for gene expression.
＜検出＞
本発明はさらなる態様として、
(ｉ) 被験者から生物学的試料を入手し、
(ii) 得られた生物学的試料と、本発明の単離された抗体分子またはその誘導体とを接触させ、そして
(iii) PD-L1陽性エクソソームおよび抗体分子またはその誘導体の間の複合体の形成を検出すること
を含む、生物学的試料におけるPD-L1陽性エクソソームを検出する方法に関する。
本発明のPD-L1陽性腫瘍を検出する方法と同様、本発明のPD-L1陽性エクソソームを検出する方法は、PD-L1陽性エクソソームを、生物学的試料から分離することなく直接検出することを特徴とする。生物学的試料とは、体液、特に血液、血漿、血清を意味し、さらには腹水、脳液、骨髄、尿、糞便または気管支肺胞洗浄液であってもよい。本発明のPD-L1陽性エクソソームを検出する方法は、PD-L1陽性腫瘍を有する患者を選別する方法に応用することができ、かかる態様も本発明の範囲に入る。PD-L1陽性腫瘍を有する患者を選別する本発明の方法は、免疫チェックポイント阻害剤が奏功する患者を選別する方法に応用でき、かかる態様も本発明の範囲に入る。
<Detection>
The present invention relates to a further aspect,
(i) Obtaining a biological sample from the subject;
(ii) contacting the obtained biological sample with an isolated antibody molecule or derivative thereof of the present invention; and
(iii) A method for detecting PD-L1-positive exosomes in a biological sample, comprising detecting the formation of a complex between PD-L1-positive exosomes and an antibody molecule or a derivative thereof.
Similar to the method for detecting PD-L1-positive tumors of the present invention, the method for detecting PD-L1-positive exosomes of the present invention is characterized in that PD-L1-positive exosomes are directly detected without being separated from a biological sample. The biological sample refers to a body fluid, particularly blood, plasma, or serum, and may also be ascites, cerebral fluid, bone marrow, urine, feces, or bronchoalveolar lavage fluid. The method for detecting PD-L1-positive exosomes of the present invention can be applied to a method for selecting patients with PD-L1-positive tumors, and such an embodiment is also within the scope of the present invention. The method for selecting patients with PD-L1-positive tumors of the present invention can be applied to a method for selecting patients who respond to immune checkpoint inhibitors, and such an embodiment is also within the scope of the present invention.
PD-L1陽性エクソソームおよび抗体分子またはその誘導体の間の複合体の形成を検出する手法は、プラスチック・プレートやマグネティック・ビーズなどの担体に抗CD9抗体などのエクソソームを認識する抗体を結合させ、そこにエクソソームを含む生物学的試料を接触させることによりエクソソームを担体上の抗体に結合させ、さらにそのエクソソームにホースラディッシュペルオキシダーゼなどの標識体で標識した抗PD-L1抗体を結合させた複合体に対して発光基質を作用させて、プレートリーダなどで発光強度を検出することにより、複合体を定量するものである。 The method for detecting the formation of a complex between PD-L1-positive exosomes and antibody molecules or their derivatives involves binding an antibody that recognizes exosomes, such as an anti-CD9 antibody, to a carrier such as a plastic plate or magnetic beads, contacting a biological sample containing exosomes with the carrier to bind the exosomes to the antibody on the carrier, and then reacting a luminescent substrate with the complex formed by binding the exosomes to an anti-PD-L1 antibody labeled with a label such as horseradish peroxidase, and quantifying the complex by detecting the luminescence intensity using a plate reader or the like.
本発明は別の態様として、本発明の単離された抗体分子またはその誘導体を含む、PD-L1陽性エクソソームを定量するためのキットに関する。
本発明のキットは、次に挙げる１つまたは複数の他の要素を含むことができる：
使用説明書；他の試薬、例えば、標識、または抗体を標識するための薬剤。
本発明のキットは好ましくは、標準試薬を含有する。標準試薬としては、例えば、配列番号１９に示すアミノ酸配列を有するヒトPD-L1およびヒトCD9タンパクの融合タンパク質、および配列番号２１に示すアミノ酸配列を有するヒトPD-L1およびヒトCD63の融合タンパク質を挙げることができる。
In another aspect, the present invention relates to a kit for quantifying PD-L1-positive exosomes, comprising the isolated antibody molecule or derivative thereof of the present invention.
The kits of the invention may contain one or more of the following other components:
Instructions for use; other reagents, such as labels or agents for labeling the antibody.
The kit of the present invention preferably contains a standard reagent, such as a fusion protein of human PD-L1 and human CD9 protein having the amino acid sequence as set forth in SEQ ID NO:19, and a fusion protein of human PD-L1 and human CD63 protein having the amino acid sequence as set forth in SEQ ID NO:21.
以下、本発明を実施例により、詳細に説明するが、これらは本発明の範囲を限定するものでなく、単なる例示であることに留意すべきである。 The present invention will be described in detail below with reference to examples. However, it should be noted that these examples are merely illustrative and do not limit the scope of the present invention.
実施例１
抗PD-L1モノクローナル抗体の調製
１－１：抗原の調製
米国立生物工学情報センターのデータベースにアクセッション番号BC069381で登録されたヒトPD-L1タンパクの細胞外領域のMet1-Thr239のカルボキシル末端側にウサギ免疫グロブリンのFc領域を接続したcDNA（配列番号１７）を合成し、発現ベクターpD603（ATUM社製）におけるサイトメガロウイルスのプロモーター下流に組み込んだ。FreeStyle 293 Expression System（サーモフィッシャー・サイエンティフィック社製）によりこのプラスミドを293-6E細胞に導入して一過的に発現させた後、Monofinity A Resin Prepacked Column（ジェンスクリプト社製）を用いて純度約95%まで精製することにより、PD-L1のFc融合タンパク質としてPD-L1抗原を調製した。
参考までに、PD-L1における細胞外ドメインのほか、膜貫通ドメインと細胞内ドメインも含まれるPD-L1の全長DNA配列を、配列番号１８に示す。
Example 1
Preparation of anti-PD-L1 monoclonal antibody 1-1: Preparation of antigen cDNA (SEQ ID NO: 17) was synthesized by connecting the Fc region of rabbit immunoglobulin to the carboxyl terminal side of Met1-Thr239 of the extracellular domain of human PD-L1 protein registered in the National Center for Biotechnology Information database under accession number BC069381, and was integrated downstream of the cytomegalovirus promoter in expression vector pD603 (ATUM). This plasmid was introduced into 293-6E cells using the FreeStyle 293 Expression System (Thermo Fisher Scientific) for transient expression, and then purified to a purity of approximately 95% using a Monofinity A Resin Prepacked Column (GenScript) to prepare the PD-L1 antigen as an Fc fusion protein of PD-L1.
For reference, the full-length DNA sequence of PD-L1, including the extracellular domain as well as the transmembrane and intracellular domains, is shown in SEQ ID NO:18.
１－２：モノクローナル抗体の調製
PD-L1のFc融合タンパク質を、初回免疫には完全フロイント・アジュバント（ベクトン・ディッキンソン社製）と、２回目以降は不完全フロイント・アジュバント（ベクトン・ディッキンソン社製）と等量混和することにより、免疫原としての乳剤を調製した。
モノクローナル抗体は、K.Watanabe et al., Vasohibin as an endothelium-drived negative feedback regulator of angiogenesis, J. Clin. Inves. 114(2004), 898-907に記載されている方法で作製した。即ち、５週齢の雌A/J系マウスに、投与１回につき、一匹あたり100μgのFc融合タンパク質を皮下と腹腔内に等量に分けて投与した。その後、6回目の免疫後に、最終のブースター（細胞融合の４日前）を行ったマウスより脾臓を摘出し、脾細胞を調製した。脾細胞とミエローマ細胞（P3U1）との細胞融合は、K.Watanabe et al., Vasohibin as an endothelium-drived negative feedback regulator of angiogenesis, J. Clin. Inves. 114(2004), 898-907に記載されている方法で行った。
1-2: Preparation of monoclonal antibodies
An emulsion was prepared as an immunogen by mixing the PD-L1 Fc fusion protein with equal amounts of complete Freund's adjuvant (Becton Dickinson) for the first immunization and incomplete Freund's adjuvant (Becton Dickinson) for the second and subsequent immunizations.
The monoclonal antibody was prepared by the method described in K. Watanabe et al., Vasohibin as an endothelium-driven negative feedback regulator of angiogenesis, J. Clin. Inves. 114(2004), 898-907. Specifically, 100 μg of Fc fusion protein per mouse was administered subcutaneously and intraperitoneally in equal amounts to 5-week-old female A/J mice. After the sixth immunization, the spleen was removed from the mice that had received the final booster (4 days before cell fusion), and splenocytes were prepared. Cell fusion between splenocytes and myeloma cells (P3U1) was performed by the method described in K. Watanabe et al., Vasohibin as an endothelium-driven negative feedback regulator of angiogenesis, J. Clin. Inves. 114(2004), 898-907.
１－３：モノクローナル抗体の評価
抗血清とハイブリドーマ上清の抗体価の評価（１次評価）は下記に記述するELISA法にて行った。即ち、ヤギ抗マウスIgG抗体を固相した96穴マイクロプレートに、抗血清またはハイブリドーマ上清を添加し、さらにビオチン・ラベリング・キット（Dojindo社製）によりビオチン化したPD-L1タンパク質とユーロピウム標識ストレプトアビジン（パーキンエルマー社製）を混合攪拌後、室温で2時間反応させた。反応後、洗浄液（0.01%Tween20と0.1%ProClin150を含む生理食塩水）にて３回洗浄し、100μl増強試薬（Delfia Enhancement Solution, パーキンエルマー社製）を加え、5分間撹拌した。時間分解蛍光強度をNivo (Perkin Elmer社製)にて測定し、抗血清やハイブリドーマを未添加の場合に得られるシグナルの３倍を超えるシグナル強度を示す場合を陽性と判断し、７クローンの抗体を選択した（表１）。
1-3: Evaluation of monoclonal antibodies Antiserum and hybridoma supernatants were evaluated for antibody titers (primary evaluation) by the ELISA method described below. Specifically, antiserum or hybridoma supernatant was added to a 96-well microplate with goat anti-mouse IgG antibody immobilized thereon, and then PD-L1 protein biotinylated with a biotin labeling kit (Dojindo) and europium-labeled streptavidin (PerkinElmer) were mixed and stirred, and reacted at room temperature for 2 hours. After the reaction, the plate was washed three times with a washing solution (physiological saline containing 0.01% Tween 20 and 0.1% ProClin 150), and 100 μl of enhancement reagent (Delfia Enhancement Solution, PerkinElmer) was added and stirred for 5 minutes. Time-resolved fluorescence intensity was measured using Nivo (Perkin Elmer), and a signal intensity more than three times that obtained when no antiserum or hybridoma was added was judged to be positive, and seven antibody clones were selected (Table 1).
表１：選択した抗ヒトPD-L1モノクローナル抗体
実施例２
抗PD-L1モノクローナル抗体のPD-L1陽性エクソソームとの反応性の確認：２次評価（エクソソームELISA）
２－１：PD-L1陽性エクソソームの調製
ヒトPD-L1の全領域をコードしHisタグを付加したcDNAを全合成し（ユーロフィンジェノミクス社に委託）、発現ベクターpD603（ATUM社製）に挿入したものをHEK293細胞株に GenePORTER Gold トランスフェクション試薬（コスモ・バイオ社製）により導入し、1mg/mL G418存在下で複数の安定導入株を選択した。これらの細胞株の抽出液を調製し、抗Hisタグ抗体（プロテインテック社製）を用いたウェスタンブロット法により、安定導入株の中から導入遺伝子の発現量が高い細胞株を選択し（図１Ａ）、その細胞株を150mm径の培養皿10枚まで拡大培養して無血清培地（Advanced DMEM,サーモフィッシャー・サイエンティフィック社製）による培養液から超遠心法（4℃で100,000 ×gにて70分遠心）によりエクソソームを調製し、エクソソーム上にPD-L1を多く発現している細胞株（クローン番号８）を確認した（図１Ｂ）。この細胞株より調製したPD-L1陽性エクソソームを以下のELISA構築に供した。
Example 2
Confirmation of reactivity of anti-PD-L1 monoclonal antibodies with PD-L1-positive exosomes: Secondary evaluation (exosome ELISA)
2-1: Preparation of PD-L1-positive exosomes A His-tagged cDNA encoding the entire region of human PD-L1 was synthesized (outsourced to Eurofins Genomics), inserted into the expression vector pD603 (ATUM), and transfected into HEK293 cells using GenePORTER Gold transfection reagent (Cosmo Bio). Several stable transfectants were selected in the presence of 1 mg/mL G418. Extracts from these cell lines were prepared, and cell lines with high expression of the introduced gene were selected from the stable transfectants by Western blotting using an anti-His tag antibody (Proteintech) (Figure 1A). These cell lines were then expanded to ten 150 mm culture dishes, and exosomes were prepared from the culture medium in serum-free medium (Advanced DMEM, Thermo Fisher Scientific) by ultracentrifugation (100,000 × g for 70 minutes at 4°C), and a cell line (clone number 8) that expressed high levels of PD-L1 on exosomes was identified (Figure 1B). The PD-L1-positive exosomes prepared from this cell line were used to establish the following ELISA.
２－２：サンドイッチELISAの構築
実施例１－３で調製した抗PD-L1モノクローナル抗体7クローンと抗CD9モノクローナル抗体（コスモ・バイオ社製）、および抗CD63モノクローナル抗体（コスモ・バイオ社製）を用い、PD-L1を発現しているエクソソームを定量するためのサンドイッチELISA測定系を構築した（図２）。
詳細には、まず、抗CD9モノクローナル抗体および抗CD63モノクローナル抗体を固相した96穴マイクロプレートを調製した。トリス塩酸緩衝液にて5μg/mLに調製した各抗体溶液を、96穴マイクロプレートに100μL／ウェルずつ添加し、終夜反応させた。その後、反応液を除き、洗浄液（0.01%Tween20と0.1%ProClin150を含むリン酸緩衝生理食塩水）で1回洗浄後、2%ブロックエース（大日本住友製薬社製）を200μL／ウェルずつ添加し、４℃で終夜放置することによりブロッキングを行った。一方、表１に示した全ての抗体について、20倍モル量のSulfo-NHS-LC-Biotin(サーモフィッシャー社製)と室温で2時間反応し、PD-10カラム（GEヘルスケア社製）でゲル濾過することにより、標識抗体を調製した。
2-2: Construction of sandwich ELISA Using the 7 anti-PD-L1 monoclonal antibody clones prepared in Examples 1-3, an anti-CD9 monoclonal antibody (Cosmo Bio), and an anti-CD63 monoclonal antibody (Cosmo Bio), a sandwich ELISA measurement system for quantifying exosomes expressing PD-L1 was constructed (Figure 2).
In detail, first, a 96-well microplate was prepared with anti-CD9 monoclonal antibody and anti-CD63 monoclonal antibody immobilized. Each antibody solution, prepared at 5 μg/mL with Tris-HCl buffer, was added to the 96-well microplate at 100 μL/well and reacted overnight. After that, the reaction solution was removed, and the plate was washed once with a washing solution (phosphate-buffered saline containing 0.01% Tween 20 and 0.1% ProClin 150), and then 200 μL/well of 2% Block Ace (Sumitomo Dainippon Pharma) was added and left at 4°C overnight for blocking. Meanwhile, all the antibodies shown in Table 1 were reacted with 20 times the molar amount of Sulfo-NHS-LC-Biotin (Thermo Fisher) at room temperature for 2 hours, and gel filtration was performed with a PD-10 column (GE Healthcare) to prepare labeled antibodies.
抗体が固相されたプレートにPD-L1陽性エクソソーム（10μg/mL）100μLを添加し撹拌後、室温で2時間反応した。その後、反応液を除き、上記洗浄液で３回洗浄後、1μg/mLの上記で調製した標識抗体100μLを添加し、攪拌後室温で2時間反応させた。３回洗浄後、HRP(Horseradish Peroxidase)標識ストレプトアビジン(R&D社製)を100μL添加し、室温で20分間反応させた。同様に3回洗浄後、TMB溶液（Colorburst Blue, ALerCHEK社製）を100μL添加し、室温で20分間反応させた。そこに、2N硫酸を50μL添加して反応を停止し、各ウェルの450nmにおける吸光度を測定した（図３）。その結果、７種類の抗PD-L1抗体のうちクローンA1-9F7を標識抗体として用いると、固相抗体が抗CD9、抗CD63いずれの抗体の場合でももっとも強いシグナルが検出されることが判明した。 100 μL of PD-L1 positive exosomes (10 μg/mL) was added to the plate on which the antibody was immobilized, and after stirring, the plate was reacted at room temperature for 2 hours. After that, the reaction solution was removed, and the plate was washed three times with the above washing solution, after which 100 μL of the labeled antibody prepared above at 1 μg/mL was added, and after stirring, the plate was reacted at room temperature for 2 hours. After washing three times, 100 μL of HRP (Horseradish Peroxidase)-labeled streptavidin (R&D) was added, and the plate was reacted at room temperature for 20 minutes. After washing three times in the same manner, 100 μL of TMB solution (Colorburst Blue, ALerCHEK) was added, and the plate was reacted at room temperature for 20 minutes. The reaction was stopped by adding 50 μL of 2N sulfuric acid, and the absorbance at 450 nm of each well was measured (Figure 3). As a result, it was found that when clone A1-9F7 of the seven anti-PD-L1 antibodies was used as the labeled antibody, the strongest signal was detected regardless of whether the solid-phase antibody was anti-CD9 or anti-CD63.
２－３：ELISA測定値のエクソソーム濃度依存性
抗CD9モノクローナル抗体（コスモ・バイオ社製）および抗CD63モノクローナル抗体（コスモ・バイオ社製）を固相抗体、実施例１－３で調製し実施例２－２で選別した抗PD-L1モノクローナル抗体（クローンA1-9F7）を標識抗体としたELISA測定系で、クローン番号８の細胞株の培養上清より超遠心法（4℃で100,000 ×gにて70分遠心）で調製した0、0.3125、0.625、1.25、2.5、5.0、10μg/mLのPD-L1陽性精製エクソソームを定量したところ、いずれも濃度依存的な測定値が得られ、これらのELISAがPD-L1陽性エクソソームの定量に適していることが示された（図４）。
2-3: Exosome concentration dependence of ELISA measurement values In an ELISA measurement system using anti-CD9 monoclonal antibody (Cosmo Bio) and anti-CD63 monoclonal antibody (Cosmo Bio) as the solid-phase antibody and the anti-PD-L1 monoclonal antibody (clone A1-9F7) prepared in Example 1-3 and selected in Example 2-2 as the labeled antibody, 0, 0.3125, 0.625, 1.25, 2.5, 5.0, and 10 μg/mL of PD-L1-positive purified exosomes prepared by ultracentrifugation (centrifugation at 100,000 × g for 70 minutes at 4°C) from the culture supernatant of the cell line of clone no. 8 were quantified. Concentration-dependent measurement values were obtained for all cases, demonstrating that these ELISAs are suitable for quantifying PD-L1-positive exosomes (Figure 4).
２－４：標準試薬
精製エクソソームは保存安定性が低いため、検量線を描く際に使用する標準試薬としてそのまま用いた場合、異なる試験で同一の標準曲線を得ることが困難であるため、キットのロット間差やテスト間差の補正に用いることはできない。そのため、検量線を描く際の標準試薬は保存安定性の高い精製組換えタンパク質であることが望ましい。
このような理由から、PD-L1陽性エクソソームELISAの標準試薬としてCD9あるいはCD63の細胞外領域とPD-L1の細胞外領域とを融合させた組換えタンパク質を作製した。即ち、ヒトPD-L1のMet1からArg258のカルボキシル末端側にヒトCD9タンパクのGly110からPhe193あるいはヒトCD63タンパクのAla103からLeu204までの配列Hisタグに接続させた融合タンパクをコードするcDNAを発現ベクターpD603に挿入したプラスミドを構築し、HEK293細胞に導入して一過的に強制発現させ、細胞から分泌されたPD-L1/CD9あるいはPD-L1/CD63融合タンパクをHis Mag Sepharose excel（GE Healthcare Life Sciences社製）により精製した。これらの融合タンパクを0、0.625、1.25、2.5、5.0、10および20ng/mLの濃度で、抗CD9モノクローナル抗体あるいは抗CD63モノクローナル抗体を固相抗体、抗PD-L1モノクローナル抗体（クローンA1-9F7）を標識抗体としたELISA測定系で測定したところ、いずれも濃度依存的な測定値が得られ、これらの融合タンパクが測定系の標準試薬として使用できることが示された（図５）。ヒトPD-L1のMet1からArg258のカルボキシル末端側にヒトCD9タンパクのGly110からPhe193を接続した融合タンパク質のアミノ酸配列およびDNA配列をそれぞれ、配列番号１９および２０に示す。また、ヒトPD-L1のMet1からArg258のカルボキシル末端側にヒトCD63タンパクのAla103からLeu204を接続した融合タンパク質のアミノ酸配列およびDNA配列をそれぞれ、配列番号２１および２２に示す。
2-4: Standard Reagents Purified exosomes have low storage stability, so if they are used as standard reagents to draw standard curves, it is difficult to obtain the same standard curve in different tests, and they cannot be used to correct differences between kit lots or between tests. Therefore, it is desirable to use purified recombinant proteins with high storage stability as standard reagents when drawing standard curves.
For this reason, we prepared recombinant proteins in which the extracellular domains of CD9 or CD63 were fused with the extracellular domain of PD-L1 as standard reagents for PD-L1-positive exosome ELISA. Specifically, we constructed a plasmid by inserting cDNA encoding a fusion protein in which the carboxyl-terminal side of Met1 to Arg258 of human PD-L1 was linked to a His tag sequence from Gly110 to Phe193 of human CD9 protein or Ala103 to Leu204 of human CD63 protein into the expression vector pD603, and introduced it into HEK293 cells for transient forced expression. The PD-L1/CD9 or PD-L1/CD63 fusion protein secreted from the cells was purified using His Mag Sepharose excel (GE Healthcare Life Sciences). These fusion proteins were measured at concentrations of 0, 0.625, 1.25, 2.5, 5.0, 10 and 20ng/mL in an ELISA system using anti-CD9 monoclonal antibody or anti-CD63 monoclonal antibody as the solid-phase antibody and anti-PD-L1 monoclonal antibody (clone A1-9F7) as the labeled antibody. The measured values were concentration-dependent, indicating that these fusion proteins can be used as standard reagents in the measurement system (Figure 5). The amino acid and DNA sequences of the fusion proteins in which Gly110 to Phe193 of human CD9 protein is linked to the carboxyl-terminal side of Met1 to Arg258 of human PD-L1 are shown in SEQ ID NOs: 19 and 20, respectively. The amino acid and DNA sequences of the fusion proteins in which Ala103 to Leu204 of human CD63 protein is linked to the carboxyl-terminal side of Met1 to Arg258 of human PD-L1 are shown in SEQ ID NOs: 21 and 22, respectively.
２－５：本発明抗体と市販の抗体との性能比較
実施例１－３に記載のようにして調製した抗PD-L1モノクローナル抗体（クローンA1-9F7）について、Chen G., Huang A.C., Zhang W., Zhang G., Wu M., Xu W., Yu Z., Yang J., Wang B., Sun H., et al. 2018. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 560:382-386. 10.1038/s41586-018-0392-8において標識抗体として使用されている、商業的に入手可能な抗PD-L1モノクローナル抗体（クローンMIH5、Invitrogen社製）との性能を比較した。すなわち、これら２種類のモノクローナル抗体をビオチンラベルしたものを標識抗体、抗CD9抗体（コスモ・バイオ社製）あるいは抗CD63抗体（コスモ・バイオ社製）を固相抗体としてPD-L1陽性精製エクソソームを定量するエクソソームELISAにおいて、得られた検量線の傾きからそれぞれの検出系の感度を比較した。その結果、固相抗体がCD9、CD63いずれの場合においても、市販抗体よりも本発明のA1-9F7抗体を用いたほうが有意に強いシグナルが得られ、2倍以上高感度にPD-L1陽性エクソソームを検出できるELISAとなることが判明した（図６）。
2-5: Performance comparison of the antibody of the present invention with commercially available antibodies The anti-PD-L1 monoclonal antibody (clone A1-9F7) prepared as described in Example 1-3 was compared in performance with the commercially available anti-PD-L1 monoclonal antibody (clone MIH5, Invitrogen) used as a labeled antibody in Chen G., Huang AC, Zhang W., Zhang G., Wu M., Xu W., Yu Z., Yang J., Wang B., Sun H., et al. 2018. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 560:382-386. 10.1038/s41586-018-0392-8. Specifically, these two types of monoclonal antibodies were labeled with biotin as the labeled antibody, and anti-CD9 antibody (Cosmo Bio) or anti-CD63 antibody (Cosmo Bio) was used as the solid-phase antibody to quantify PD-L1-positive purified exosomes. The sensitivity of each detection system was compared based on the slope of the obtained calibration curve in an exosome ELISA. As a result, it was found that, in both cases where the solid-phase antibody was CD9 or CD63, a significantly stronger signal was obtained when the A1-9F7 antibody of the present invention was used than when a commercially available antibody was used, resulting in an ELISA that can detect PD-L1-positive exosomes with more than twice the sensitivity (Figure 6).
実施例３
PD-L1陽性エクソソームの検出に用いるモノクローナル抗体A1-9F7の構造解析
PD-L1陽性エクソソームを検出するELISAにおいてもっとも高い反応性を示した抗PD-L1モノクローナル抗体（クローンA1-9F7）のVH鎖可変領域およびVL鎖可変領域の構造解析をRepertoire Genesis株式会社のバイオインフォマティクス解析システムにより行った。すなわち、クローンA1-9F7のモノクローナル抗体を産生するハイブリドーマから抽出したメッセンジャーRNAを鋳型にしてcDNAを合成し、次世代シーケンサーによりVH鎖可変領域およびVL鎖可変領域の遺伝子配列を決定し、それぞれの配列をアミノ酸配列に置換した（VH鎖可変領域：図７、配列番号１および配列番号２、VL鎖可変領域：図８、配列番号９および配列番号１０）。
Example 3
Structural analysis of monoclonal antibody A1-9F7 for detecting PD-L1-positive exosomes
Structural analysis of the VH and VL chain variable regions of the anti-PD-L1 monoclonal antibody (clone A1-9F7), which showed the highest reactivity in the ELISA detecting PD-L1-positive exosomes, was performed using the bioinformatics analysis system of Repertoire Genesis Co., Ltd. Specifically, cDNA was synthesized using messenger RNA extracted from a hybridoma producing the monoclonal antibody clone A1-9F7 as a template, and the gene sequences of the VH and VL chain variable regions were determined using a next-generation sequencer, and each sequence was replaced with an amino acid sequence (VH chain variable region: Figure 7, SEQ ID NO: 1 and SEQ ID NO: 2; VL chain variable region: Figure 8, SEQ ID NO: 9 and SEQ ID NO: 10).
実施例４
ELISAにおける添加回収試験
抗CD9抗体を固相抗体としてPD-L1陽性精製エクソソームを定量するエクソソームELISAにおいて、血清あるいは血漿の存在下における抗体とエクソソームの結合特異性を評価するために添加回収試験を行った。具体的には、抗CD9抗体（コスモ・バイオ社製）を固相したELISAにヒト由来プール血清（コスモ・バイオ社製）をウェルあたり0、2.5、5、10、25、50μLとPD-L1陽性エクソソームを濃度が2、6、16μg/mLになるように加えて測定し、血清0μL（無添加）時に得られたシグナルに対して各濃度の血清を加えた際に得られたシグナルの割合（回収率）を求めた。また、ヒトプール血漿（コスモ・バイオ社製）についても同様に割合を求めた。その結果、血清では添加量が10μL以下の場合に回収率が83.5～113.4%と良好な成績であった（図９Ａ）。血漿の場合でも添加量が10μL以下の場合に回収率が92.3～107.5%と良好であった（図９Ｂ）。
Example 4
Recovery test in ELISA In exosome ELISA, which quantifies PD-L1-positive purified exosomes using anti-CD9 antibody as a solid-phase antibody, a recovery test was performed to evaluate the binding specificity of the antibody and exosomes in the presence of serum or plasma. Specifically, 0, 2.5, 5, 10, 25, and 50 μL of human pooled serum (Cosmo Bio) and 2, 6, and 16 μg/mL of PD-L1-positive exosomes were added to an ELISA with anti-CD9 antibody (Cosmo Bio) as a solid phase, and measurements were performed. The ratio of the signal obtained when serum was added at each concentration to the signal obtained when 0 μL of serum (no addition) was calculated (recovery rate). The ratio was also calculated in the same way for human pooled plasma (Cosmo Bio). As a result, when the amount of serum added was 10 μL or less, the recovery rate was good, ranging from 83.5 to 113.4% (Figure 9A). Even in the case of plasma, when the amount added was 10 μL or less, the recovery rate was good, ranging from 92.3 to 107.5% (FIG. 9B).
実施例５
免疫沈降によるエクソソームの濃縮
５－１：抗CD9抗体固相磁性ビーズの調製
抗CD9抗体（クローン12A12、コスモ・バイオ社製）100μgをアミノ基ビオチン標識試薬（サーモフィッシャー社製）と室温で2時間反応し、ビオチン化した。ストレプトアビジン固相磁性ビーズ（多摩川精機社製）1mgにビオチン化抗体を加え、PBS中、４℃で１時間結合反応を行い、10mM HEPES-NaOH (pH 7.9), 50mM KCl, 1mM EDTA, 10% glycerolで洗浄後、同液中で0.1 mg/20 μLの濃度で保存した。
Example 5
5. Concentration of exosomes by immunoprecipitation 5-1: Preparation of anti-CD9 antibody solid-phase magnetic beads 100 μg of anti-CD9 antibody (clone 12A12, Cosmo Bio) was reacted with amino group biotin labeling reagent (Thermo Fisher) at room temperature for 2 hours to be biotinylated. Biotinylated antibody was added to 1 mg of streptavidin solid-phase magnetic beads (Tamagawa Seiki), and binding reaction was carried out in PBS at 4°C for 1 hour. After washing with 10 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 1 mM EDTA, 10% glycerol, it was stored in the same solution at a concentration of 0.1 mg/20 μL.
５－２：肺がん細胞株PC9の培養上清中のエクソソームの免疫沈降
ヒト肺がん細胞株PC9（東京慈恵会医科大学より譲受）をサブコンフルエントな状態までダルベッコ変法イーグル培地に10%牛胎児血清を加えた培養液中で培養し、100nM ヒトγインターフェロンを加えてPD-L1の発現を誘導（Predictive Markers for the Efficacy of Anti-PD-1/PD-L1 Antibodies in Lung Cancer. J Thorac Oncol. 2016 Jul; 11(7): 976-88.）しながらさらに2日培養した培養液を回収し、対照として、γインターフェロンを加えない培地での培養液も回収した。これらの培養液１mLに0.1%ポリオキシエチレン10ノエルフェニルエーテル存在下、20mM HEPES-NaOH (pH 7.9)、150mM KCl、1mM MgCl２、0.2mM CaCl２、0.2mM EDTA、10% グリセロールで洗浄・懸濁した、実施例５－１で調製した抗CD9抗体固相磁性ビーズ50μg/10μLを加え、４℃にて終夜回転混和した。次いで、ビーズを磁気分離し、同液で3回洗浄、50μLに懸濁した。また、ブランク試料として細胞に加える前の培養液1mLにも同様の処理を行った。
5-2: Immunoprecipitation of exosomes in culture supernatant of lung cancer cell line PC9 Human lung cancer cell line PC9 (obtained from The Jikei University School of Medicine) was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum until subconfluent. PD-L1 expression was induced by adding 100 nM human gamma interferon (Predictive Markers for the Efficacy of Anti-PD-1/PD-L1 Antibodies in Lung Cancer. J Thorac Oncol. 2016 Jul; 11(7): 976-88.) and the culture medium was then collected after two more days of culture. As a control, culture medium without gamma interferon was also collected. To 1 mL of each of these culture solutions, 50 μg/10 μL of the anti-CD9 antibody solid-phase magnetic beads prepared in Example 5-1, which had been washed and suspended in 20 mM HEPES-NaOH (pH 7.9), 150 mM KCl, 1 mM MgCl 2 , 0.2 mM CaCl 2 , 0.2 mM EDTA, and 10% glycerol in the presence of 0.1% polyoxyethylene 10-phenyl ether, were added, and the mixture was rotated and mixed overnight at 4° C. The beads were then magnetically separated, washed three times with the same solution, and suspended in 50 μL. In addition, the same treatment was performed on 1 mL of the culture solution before adding it to the cells as a blank sample.
５－３：免疫沈降したエクソソーム上のPD-L1の検出
実施例１－３で調製し実施例２－２で選別した抗PD-L1抗体（クローンA1-9F7）200μgにペルオキシダーゼ標識キット（同仁化学社製）を用いてHRP標識し、標識抗体を調製した。免疫沈降と同一の液で2μg/mLに希釈した標識抗体50μLを等量の免疫沈降させたビーズに加え、室温で2時間反応後、ビーズを磁気分離し、同液で4回洗浄した。HRP基質液（モス社製）100μLの添加による発色を確認後、磁気分離でビーズを除去した液に2N硫酸50μLを添加し、波長450nmの吸光度を測定した。得られた測定値（B）からブランク試料の測定値（B(0)）を差し引いた値（B-B(0)）を求めたところ、γインターフェロンでPD-L1の発現を誘導した試料では、誘導していない試料に比べて有意に高い値が得られた（図１０左）。このことは、免疫沈降は、本実施例の検体について、PD-L1陽性エクソソームの存在・非存在を区別できることを示している。
5-3: Detection of PD-L1 on immunoprecipitated exosomes Anti-PD-L1 antibody (clone A1-9F7) (200 μg) prepared in Example 1-3 and selected in Example 2-2 was labeled with HRP using a peroxidase labeling kit (Dojindo Chemical Industries, Ltd.) to prepare a labeled antibody. 50 μL of the labeled antibody diluted to 2 μg/mL with the same solution as the immunoprecipitation was added to an equal amount of immunoprecipitated beads, and after 2 hours of reaction at room temperature, the beads were magnetically separated and washed four times with the same solution. After confirming color development by adding 100 μL of HRP substrate solution (Moss), 50 μL of 2N sulfuric acid was added to the solution from which the beads were removed by magnetic separation, and the absorbance at a wavelength of 450 nm was measured. The measured value (B) minus the measured value (B(0)) of the blank sample was calculated, and the value (BB(0)) was obtained. The sample in which PD-L1 expression was induced with gamma interferon was significantly higher than the sample in which PD-L1 expression was not induced (Figure 10, left). This indicates that immunoprecipitation can distinguish between the presence and absence of PD-L1-positive exosomes in the samples of this example.
同じ試料100μLについて、抗CD9抗体（クローン12A12、コスモ・バイオ社製）を固相、抗PD-L1抗体（クローンA1-9F7）をHRP標識抗体としたELISA（実施例２のサンドイッチELISA）で測定したところ、γインターフェロンの存在・非存在による測定値、すなわちPD-L1陽性エクソソームの存在・非存在を示す測定値にほとんど差が見られなかった（図１０右）。このことは、免疫沈降ではPD-L1陽性エクソソームの存在・非存在が区別できた試料について、ELISAは適用できないことを示している。
上記の通り、本実施例では、同一試料を用いて免疫沈降およびELISAの定量可能性を比較した。結果、免疫沈降を用いることで、PD-L1陽性エクソソームの発現量が少ない試料であっても、検出が可能であることが判明した。
When 100 μL of the same sample was measured by ELISA (sandwich ELISA in Example 2) using anti-CD9 antibody (clone 12A12, Cosmo Bio) as the solid phase and anti-PD-L1 antibody (clone A1-9F7) as the HRP-labeled antibody, almost no difference was observed in the measured values depending on the presence or absence of gamma interferon, i.e., the measured values indicating the presence or absence of PD-L1-positive exosomes (Figure 10, right). This indicates that ELISA cannot be applied to samples in which the presence or absence of PD-L1-positive exosomes could be distinguished by immunoprecipitation.
As described above, in this example, we compared the quantification feasibility of immunoprecipitation and ELISA using the same samples. As a result, we found that immunoprecipitation can detect PD-L1-positive exosomes even in samples with low expression levels.
Claims (12)
該PD-L1を認識する抗体が、
(ｉ)配列番号３のVHCDR1アミノ酸配列、配列番号４のVHCDR2アミノ酸配列および配列番号５のVHCDR3アミノ酸配列を含む重鎖可変領域(VH)、ならびに
(ii)配列番号１１のVLCDR1アミノ酸配列、配列番号１２のVLCDR2アミノ酸配列および配列番号１３のVLCDR3アミノ酸配列を含む軽鎖可変領域(VL)
を含む、PD-L1に結合することができる単離された抗体分子、またはPD-L1に結合することができる、単離されたその抗体分子のFab、F(ab')２、Fvまたは単鎖Fv断片(scFv)である、方法。 A method for detecting PD-L1-positive tumors, comprising directly detecting PD-L1-positive exosomes expressing PD-L1 on their surface using an antibody that recognizes PD-L1, without isolating the exosomes from a biological sample, comprising:
The antibody that recognizes PD-L1 is
(i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and
(ii) a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 11, the VLCDR2 amino acid sequence of SEQ ID NO: 12, and the VLCDR3 amino acid sequence of SEQ ID NO: 13
or an isolated antibody molecule capable of binding to PD-L1, or a Fab, F(ab') 2 , Fv or single-chain Fv fragment (scFv) of said isolated antibody molecule capable of binding to PD-L1.
(ii)配列番号１１のVLCDR1アミノ酸配列、配列番号１２のVLCDR2アミノ酸配列および配列番号１３のVLCDR3アミノ酸配列を含む軽鎖可変領域(VL)
を含む、PD-L1に結合することができる単離された抗体分子、またはPD-L1に結合することができる、単離されたその抗体分子のFab、F(ab')２、Fvまたは単鎖Fv断片(scFv)。 (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and
(ii) a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 11, the VLCDR2 amino acid sequence of SEQ ID NO: 12, and the VLCDR3 amino acid sequence of SEQ ID NO: 13
or an isolated antibody molecule capable of binding to PD-L1, comprising:
(ｉｉ) PD-L1陽性エクソソームおよび抗体分子またはPD-L1に結合することができる、単離されたその抗体分子のFab、F(ab')２、Fvまたは単鎖Fv断片(scFv)の間の複合体の形成を検出すること
を含む、生物学的試料におけるPD-L1陽性エクソソームを検出する方法。 (i ) contacting a biological sample obtained from a subject with an isolated antibody molecule of any one of claims 6 to 8 , or a Fab, F(ab') 2 , Fv or single-chain Fv fragment (scFv) of the isolated antibody molecule capable of binding to PD-L1; and
(ii) a method for detecting PD-L1-positive exosomes in a biological sample, comprising detecting the formation of complexes between PD-L1-positive exosomes and an antibody molecule, or an isolated Fab, F(ab') 2 , Fv or single-chain Fv fragment (scFv) of the antibody molecule capable of binding to PD-L1.
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