JP2018203632A - Monoclonal antibodies and assay kits - Google Patents
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Abstract
Description
本発明は、MERSコロナウイルスに対するモノクローナル抗体、及び、MERSコロナウイルスのウイルス活性を中和する中和抗体の存在を測定するための測定キットに関する。 The present invention relates to a measurement kit for measuring the presence of a monoclonal antibody against MERS coronavirus and a neutralizing antibody that neutralizes the viral activity of MERS coronavirus.
中東呼吸器症候群(以下、MERS(Middle East respiratory syndrome)と称することがある。)は、2012年にサウジアラビアで最初に報告された、MERSコロナウイルス(以下、MERS-CoVと略することがある。)による重症の呼吸器疾患である。2015年の韓国国内での院内感染によるMERS流行の際には、近隣諸国への感染拡大が懸念された。現在までに、日本国内で患者発生はないが、中東への渡航者が帰国後にMERSを発症する可能性もある。 The Middle East respiratory syndrome (hereinafter sometimes referred to as MERS (Middle East respiratory syndrome)) was first reported in 2012 in Saudi Arabia and may be abbreviated as MERS coronavirus (hereinafter MERS-CoV). ) Is a serious respiratory disease. During the MERS epidemic caused by nosocomial infections in Korea in 2015, there was concern about the spread of infection to neighboring countries. To date, no cases have occurred in Japan, but it is possible that travelers to the Middle East will develop MERS after returning to Japan.
MERS-CoVは、典型的なコロナウイルスの形態を持つ。他のコロナウイルスと同様、脂質二重膜のエンベロープに包まれた直径100nmの楕円体で、エンベロープ表面に王冠に似た突起であるスパイク（S）がある。 MERS-CoV has a typical coronavirus form. Like other coronaviruses, it is an ellipsoid with a diameter of 100 nm wrapped in a lipid bilayer envelope and has a spike (S) that is a crown-like projection on the envelope surface.
MERS-CoVは、冬季の上気道炎の原因ウイルスとして巷で蔓延しているヒトコロナウイルス(OC43)と同じベータコロナウイルスに属する。MERSコロナウイルスはOC43等、他のコロナウイルスと血清学的に交差するため、ウイルス感染細胞を用いた通常のELISA、蛍光抗体法ではMERSコロナウイルス特異的抗体の検出が困難である。 MERS-CoV belongs to the same beta coronavirus as the human coronavirus (OC43) that is prevalent in cocoons as a causative virus for upper respiratory tract inflammation in winter. Since MERS coronaviruses serologically cross with other coronaviruses such as OC43, it is difficult to detect MERS coronavirus-specific antibodies using conventional ELISA and fluorescent antibody methods using virus-infected cells.
非特許文献１及び２には、MERS-CoVのスパイク（S）蛋白が主要抗原であることに着目して、MERS-CoVのスパイク（S）蛋白と親和性の高いモノクローナル抗体の樹立が記載されている。このモノクローナル抗体は培養細胞レベルあるいは、動物モデルでの感染は効率良く阻害が可能であるが、ヒトへの感染をどの程度阻害するのかは不明である。 Non-Patent Documents 1 and 2 describe the establishment of a monoclonal antibody having high affinity with MERS-CoV spike (S) protein, focusing on the fact that MERS-CoV spike (S) protein is the main antigen. ing. Although this monoclonal antibody can efficiently inhibit infection at the level of cultured cells or animal models, it is unclear how much it inhibits human infection.
本発明はかかる問題点に鑑みてなされたものであって、MERS-CoVに対する新規なモノクローナル抗体を提供することを目的とする。また試験サンプル中においてMERSコロナウイルスのウイルス活性を中和する中和抗体の存在を測定するための測定キットを提供することを目的とする。 The present invention has been made in view of such problems, and an object thereof is to provide a novel monoclonal antibody against MERS-CoV. It is another object of the present invention to provide a measurement kit for measuring the presence of neutralizing antibodies that neutralize the viral activity of MERS coronavirus in a test sample.
本発明にかかるモノクローナル抗体は、MERSコロナウイルスに特異的に結合し、そのウイルス活性を中和するMERSコロナウイルスに対するモノクローナル抗体である。 The monoclonal antibody according to the present invention is a monoclonal antibody against MERS coronavirus that specifically binds to MERS coronavirus and neutralizes its viral activity.
本発明にかかる測定キットは、試験サンプル中においてMERSコロナウイルスのウイルス活性を中和する中和抗体の存在を測定するための測定キットであって、(i)固体支持体に結合させたMERSコロナウイルス抗原と、(ii)請求項１乃至４の何れか１項に記載のモノクローナル抗体と、及び(iii)該モノクローナル抗体を認識する標識二次抗体と、を有する測定キットである。 The measurement kit according to the present invention is a measurement kit for measuring the presence of a neutralizing antibody that neutralizes the viral activity of MERS coronavirus in a test sample, and comprises (i) a MERS corona bound to a solid support. A measurement kit comprising a viral antigen, (ii) the monoclonal antibody according to any one of claims 1 to 4, and (iii) a labeled secondary antibody that recognizes the monoclonal antibody.
本発明によれば、MERS-CoVに対する新規なモノクローナル抗体が得られる。 According to the present invention, a novel monoclonal antibody against MERS-CoV can be obtained.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。
（モノクローナル抗体）
本実施形態にかかるモノクローナル抗体は、MERS-CoVに特異的に結合し、そのウイルス活性を中和する。
Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.
(Monoclonal antibody)
The monoclonal antibody according to this embodiment specifically binds to MERS-CoV and neutralizes its viral activity.
MERS-CoVは、典型的なコロナウイルスの形態であり、プラス鎖の1本鎖RNAをゲノムに持つ（図１）。MERS-CoVのスパイク（S）蛋白はレセプター結合後、宿主細胞のプロテアーゼに解裂を受けて活性化し、ウイルス膜を細胞膜と融合させてウイルス遺伝子を細胞内に送り込む。その後、細胞内で脂質二重膜に包まれた構造、double membrane vesicles（DMVs）を形成し、この中でウイルスの遺伝子を複製する。ウイルス遺伝子は全長のウイルスRNAに加え、複製起点の異なる様々な長さの7本のサブジェノミックRNAを合成し、それぞれのRNAから、ウイルス粒子を構成する蛋白、spike（S）、envelope（E）、membrane（M）、nucleocapsid（N）を合成する。 MERS-CoV is a typical coronavirus form, and has a plus-strand single-stranded RNA in its genome (FIG. 1). After binding to the receptor, the MERS-CoV spike (S) protein is cleaved by the host cell protease and activated, and the virus membrane is fused with the cell membrane to send the viral gene into the cell. Thereafter, double membrane vesicles (DMVs) are formed inside the cell, and the viral genes are replicated. In addition to the full-length viral RNA, the viral gene synthesizes seven subgenomic RNAs of various lengths with different origins of replication. From each RNA, the proteins that make up the viral particle, spike (S), envelope (E ), Membrane (M), and nucleocapsid (N).
本実施形態にかかるモノクローナル抗体(45E11)は、重鎖可変部領域がMAVLALLFCLVTFPSCILSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGFGVNWVRQPPGKGLEWLGMIWGDGGTDYNSTLKSRLSFSKDNSKSQVFLKMNSLQTDDTARYYCARAAGTGDYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAA(配列番号１)であり、軽鎖可変部領域がMSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPWTFGGGTKLEIKRADAAP(配列番号２)である（図２）。 Monoclonal antibodies according to the present embodiment (45E11) includes a heavy chain variable region is EmueibuierueierueruefushierubuitiefuPiesushiaieruesukyubuikyuerukeiiesuJipijierubuieiPiesukyuesueruesuaitishitibuiesujiefuesuerutijiefujibuienudaburyubuiarukyuPiPijikeijieruidaburyuerujiemuaidaburyujidijijitidiwaienuesutierukeiesuarueruesuefuesukeidienuesukeiesukyubuiefuerukeiemuenuesuerukyutididitieiaruwaiwaishieiarueieijitijidiwaiefudiwaidaburyujikyuGTTLTVSSAKTTPPSVYPLAPGSAA (SEQ ID NO: 1), the light chain variable region is EmuesubuiPitikyubuierujieruerueruerudaburyuerutijieiarushidiaikyuemutikyuesuPieiesueruesueiesubuijiitibuitiaitishiarueiesuienuaiwaiesuwaierueidaburyuwaikyukyukeikyujikeiesuPikyueruerubuiwaienueikeitierueiijibuiPiesuaruefuesujiesujiesujitikyuefuesuerukeiaienuesuerukyuPiidiefujiesuwaiwaishiQHHYGTPWTFGGGTKLEIKRADAAP (SEQ ID NO: 2) (Figure 2).
抗体分子は、２つの重（Ｈ）鎖可変領域（ＶＨ）及び２つの軽（Ｌ）鎖可変領域（ＶＬ）を有する。ＶＨ及びＶＬ領域は、相補的決定領域（ＣＤＲ）に細分することができ、フレームワーク領域（ＦＲ）が散在している。 An antibody molecule has two heavy (H) chain variable regions (VH) and two light (L) chain variable regions (VL). VH and VL regions can be subdivided into complementary determining regions (CDRs) interspersed with framework regions (FR).
モノクローナル抗体(45E11)の重鎖可変領域において、CDR1はGFSLTGFGであり、CDR2はIWGDGGTであり、CDR3はARAAGTGDYFDYである。モノクローナル抗体(45E11)の重鎖可変領域において、FR1はMAVLALLFCLVTFPSCILSQVQLKESGPGLVAPSQSLSITCTVSであり、FR2はVNWVRQPPGKGLEWLGMであり、FR3はDYNSTLKSRLSFSKDNSKSQVFLKMNSLQTDDTARYYCであり、FR4はWGQGTTLTVSSAKTTPPSVYPLAPGSAAである。 In the heavy chain variable region of the monoclonal antibody (45E11), CDR1 is GFSLTGFG, CDR2 is IWGDGGT, and CDR3 is ARAAGTGDYFDY. In the heavy chain variable region of the monoclonal antibody (45E11), FR1 is MAVLALLFCLVTFPSCILSQVQLKESGPGLVAPSQSLSITCTVS, FR2 is VNWVRQPPGKGLEWLGM, FR3 is DYNSTLKSRLSFSKDNSKSQVFLKMNSLQTDDTARYYC, and FR4 is WGQGTTLTVSSAKTTP.
モノクローナル抗体(45E11)の軽鎖可変領域において、CDR1はENIYSYであり、CDR2はNAKであり、CDR3はQHHYGTPWTである。モノクローナル抗体(45E11)の軽鎖可変領域において、FR1はMSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASであり、FR2はLAWYQQKQGKSPQLLVYであり、FR3はTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCであり、FR4はFGGGTKLEIKRADAAPである。 In the light chain variable region of the monoclonal antibody (45E11), CDR1 is ENIYSY, CDR2 is NAK, and CDR3 is QHHYGTPWT. In the light chain variable region of the monoclonal antibody (45E11), FR1 is MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRAS, FR2 is LAWYQQKQGKSPQLLVY, FR3 is TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC, and FR4 is FGGGTK.
また、本実施形態にかかるモノクローナル抗体(45C2)は、重鎖可変部領域がMAVLALLFCLVTFPSCILSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGGTDYNSALKSRLSITKDNSKSQVFLKMNSLLPDDTARYYCARAAGTGDYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAA(配列番号３)であり、軽鎖可変部領域がMSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIDSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFGGSGSGTQFSLKIYSLQPEDFGNYYCQHHYGTPWTFGGGTKLEIKRADAAP(配列番号４)である（図３）。 Monoclonal antibodies according to the present embodiment (45C2) includes a heavy chain variable region is EmueibuierueierueruefushierubuitiefuPiesushiaieruesukyubuikyuerukeiiesuJipijierubuieiPiesukyuesueruesuaitishitibuiesujiefuesuerutijiwaijibuienudaburyubuiarukyuPiPijikeijieruidaburyuerujiemuaidaburyujidijijitidiwaienuesueierukeiesuarueruesuaitikeidienuesukeiesukyubuiefuerukeiemuenuesuerueruPididitieiaruwaiwaishieiarueieijitijidiwaiefudiwaidaburyujikyuGTTLTVSSAKTTPPSVYPLAPGSAA (SEQ ID NO: 3), light chain variable region is EmuesubuiPitikyubuierujieruerueruerudaburyuerutijieiarushidiaikyuemutikyuesuPieiesueruesueiesubuijiitibuitiaitishiarueiesuienuaidiesuwaierueidaburyuwaikyukyukeikyujikeiesuPikyueruerubuiwaienueikeitierueiijibuiPiesuaruefujijiesujiesujitikyuefuesuerukeiaiwaiesuerukyuPiidiefujienuwaiwaishiQHHYGTPWTFGGGTKLEIKRADAAP (SEQ ID NO: 4) (Fig. 3).
モノクローナル抗体(45C2)の重鎖可変領域において、CDR1はGFSLTGYGであり、CDR2はIWGDGGTであり、CDR3はIWGDGGTである。モノクローナル抗体(45C2)の重鎖可変領域において、FR1はMAVLALLFCLVTFPSCILSQVQLKESGPGLVAPSQSLSITCTVSであり、FR2はVNWVRQPPGKGLEWLGMであり、FR3はDYNSALKSRLSITKDNSKSQVFLKMNSLLPDDTARYYCであり、FR4はWGQGTTLTVSSAKTTPPSVYPLAPGSAAである。 In the heavy chain variable region of the monoclonal antibody (45C2), CDR1 is GFSLTGYG, CDR2 is IWGDGGT, and CDR3 is IWGDGGT. In the heavy chain variable region of the monoclonal antibody (45C2), FR1 is MAVLALLFCLVTFPSCILSQVQLKESGPGLVAPSQSLSITCTVS, FR2 is VNWVRQPPGKGLEWLGM, FR3 is DYNSALKSRLSITKDNSKSQVFLKMNSLLPDDTARYYC, and FR4 is WGQGTTLTVSSAKTTP.
モノクローナル抗体(45C2)の軽鎖可変領域において、CDR1はENIDSYであり、CDR2はNAKであり、CDR3はQHHYGTPWTである。モノクローナル抗体(45C2)の軽鎖可変領域において、FR1はMSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASであり、FR2はLAWYQQKQGKSPQLLVYであり、FR3はTLAEGVPSRFGGSGSGTQFSLKIYSLQPEDFGNYYCであり、FR4はFGGGTKLEIKRADAAPである。 In the light chain variable region of the monoclonal antibody (45C2), CDR1 is ENIDSY, CDR2 is NAK, and CDR3 is QHHYGTPWT. In the light chain variable region of the monoclonal antibody (45C2), FR1 is MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRAS, FR2 is LAWYQQKQGKSPQLLVY, FR3 is TLAEGVPSRFGGSGSGTQFSLKIYSLQPEDFGNYYC, and LE4 is FG.
また、本実施形態にかかるモノクローナル抗体(43A9)は、重鎖可変部領域がMECNWILPFILSVTSGVYSQVQLQQSGAELARPGASVKLSCNASGYIFSSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYSQKFRGKATVTADKSSSTAYMQLSSLASADSAVYYCARFGDGYYYPMDYWGQGTSVTVSSAKTTAPSV(配列番号５)であり、軽鎖可変部領域がMRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAP(配列番号６)である（図４）。 Monoclonal antibodies according to the present embodiment (43A9) includes a heavy chain variable region is EmuishienudaburyuaieruPiefuaieruesubuitiesujibuiwaiesukyubuikyuerukyukyuesujieiierueiaruPijieiesubuikeieruesushienueiesujiwaiaiefuesuesuwaidaburyuemukyudaburyubuikeikyuaruPijikyujieruidaburyuaijieiaiwaiPijidijiditiaruwaiesukyukeiefuarujikeieitibuitieidikeiesuesuesutieiwaiemukyueruesuesuerueiesueidiesueibuiwaiwaishieiaruefujidijiwaiYYPMDYWGQGTSVTVSSAKTTAPSV (SEQ ID NO: 5), the light chain variable region is EmuarushierueiiefuerujieruerubuierudaburyuaiPijieiaijidiaibuiemutikyueieiPiesubuiPibuitiPijiiesubuiesuaiesushiaruesuesukeiesueruerueichiesuenujienutiwaieruwaidaburyuefuerukyuaruPijikyuesuPikyuerueruaiwaiaruemuesuenuerueiesujibuiPidiaruefuesujiesujiesujitieiefutieruaruaiesuarubuiieiidibuijibuiwaiwaishiMQHLEYPFTFGSGTKLEIKRADAAP (SEQ ID NO: 6) (Fig. 4).
モノクローナル抗体(43A9)の重鎖可変領域において、CDR1はGYIFSSYWであり、CDR2はIYPGDGDTであり、CDR3はARFGDGYYYPMDYである。モノクローナル抗体(43A9)の重鎖可変領域において、FR1はMECNWILPFILSVTSGVYSQVQLQQSGAELARPGASVKLSCNASであり、FR2はMQWVKQRPGQGLEWIGAであり、FR3はRYSQKFRGKATVTADKSSSTAYMQLSSLASADSAVYYCであり、FR4はWGQGTSVTVSSAKTTAPSVである。 In the heavy chain variable region of the monoclonal antibody (43A9), CDR1 is GYIFSSYW, CDR2 is IYPGDGDT, and CDR3 is ARFGDGYYPMDY. In the heavy chain variable region of the monoclonal antibody (43A9), FR1 is MECNWILPFILSVTSGVYSQVQLQQSGAELARPGASVKLSCNAS, FR2 is MQWVKQRPGQGLEWIGA, FR3 is RYSQKFRGKATVTADKSSSTAYMQLSSLASADSAVYSA, KT is TS
モノクローナル抗体(43A9)の軽鎖可変領域において、CDR1はKSLLHSNGNTYであり、CDR2はRMSであり、CDR3はMQHLEYPFTである。モノクローナル抗体(43A9)の軽鎖可変領域において、FR1はMRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSSであり、FR2はLYWFLQRPGQSPQLLIYであり、FR3はNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCであり、FR4はFGSGTKLEIKRADAAPである。 In the light chain variable region of the monoclonal antibody (43A9), CDR1 is KSLLHSNGNTY, CDR2 is RMS, and CDR3 is MQHLEYPFT. In the light chain variable region of the monoclonal antibody (43A9), FR1 is MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSS, FR2 is LYWFLQRPGQSPQLLIY, FR3 is NLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC, and FR4 is FGSGTKLEIK.
上述のモノクローナル抗体の重鎖又は軽鎖可変部領域としてペプチドの誘導体も含まれる。ペプチドの誘導体の例としては、(1)上記アミノ酸配列中の１又は２個以上のアミノ酸が欠失したもの、(2)上記アミノ酸配列に１又は２個以上のアミノ酸が付加したもの、(3)上記アミノ酸配列に１又は２個以上のアミノ酸が挿入されたもの、又は(4)上記アミノ酸配列中の１又は２個以上のアミノ酸が他のアミノ酸で置換されたもの、又は(5)上記の組合せが挙げられる。 Derivatives of peptides are also included as the heavy chain or light chain variable region of the monoclonal antibody described above. Examples of peptide derivatives include: (1) one or more amino acids deleted in the amino acid sequence, (2) one or two or more amino acids added to the amino acid sequence, (3 1) one or more amino acids inserted into the amino acid sequence, or (4) one or more amino acids in the amino acid sequence substituted with other amino acids, or (5) the above Combinations are mentioned.
上述のモノクローナル抗体には、モノクローナル抗体分子そのもの、またモノクローナル抗体分子の断片が含まれる。モノクローナル抗体の断片とは、全長抗体の一部を指し、一般に、抗原結合領域又は可変領域を含む部分のことである。例えば、抗体断片にはＦａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、及びＦｖ断片が含まれる。 The above-mentioned monoclonal antibodies include monoclonal antibody molecules themselves and fragments of monoclonal antibody molecules. A monoclonal antibody fragment refers to a portion of a full-length antibody, and generally refers to a portion including an antigen-binding region or a variable region. For example, antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments.
モノクローナル抗体は、任意のアイソタイプであることができる。モノクローナル抗体は、例えば、マウスIgM、IgG1、IgG2a、IgG2b、IgG3、IgA、IgD、又はIgEであってもよい。また、モノクローナル抗体は、例えば、ヒトIgM、IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgD、又はIgEであってもよい。 The monoclonal antibody can be of any isotype. The monoclonal antibody may be, for example, mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, IgD, or IgE. The monoclonal antibody may be, for example, human IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, or IgE.
本実施形態にかかる抗体は、任意の種類の分子と抗体との共有結合により修飾又は複合化された、抗体誘導体を包含する。このような抗体誘導体として、例えば、アセチル化、グリコシル化、アミド化、ＰＥＧ化、リン酸化、既知の保護基／ブロック基による誘導体化、タンパク質分解的開裂、又は細胞内配位子又は他のタンパク質への結合により修飾されている抗体が挙げられる。 The antibody according to this embodiment includes an antibody derivative modified or conjugated by any type of molecule and the covalent bond of the antibody. Such antibody derivatives include, for example, acetylation, glycosylation, amidation, PEGylation, phosphorylation, derivatization with known protecting / blocking groups, proteolytic cleavage, or intracellular ligands or other proteins And antibodies that have been modified by binding to.
本実施形態にかかるモノクローナル抗体は、中東呼吸器症候群の予防剤及び治療剤等の医薬として使用することができる。医薬として使用する場合には、中東呼吸器症候群の治療又は予防が必要なヒト又は動物種に対し、適合性を有するモノクローナル抗体が用いられる。 The monoclonal antibody concerning this embodiment can be used as pharmaceuticals, such as a preventive agent and a therapeutic agent of Middle East respiratory syndrome. When used as a medicament, monoclonal antibodies that are compatible with human or animal species in need of treatment or prevention of the Middle East respiratory syndrome are used.
本実施形態にかかるモノクローナル抗体を医薬として用いる場合には、ヒトに投与した場合に抗原性を示す危険性が低減された抗体、具体的には、完全ヒト抗体、ヒト化抗体、マウス−ヒトキメラ抗体等のキメラ抗体が好ましく、特に好ましくは完全ヒト抗体である。 When the monoclonal antibody according to the present embodiment is used as a pharmaceutical, an antibody with reduced risk of showing antigenicity when administered to a human, specifically, a fully human antibody, a humanized antibody, a mouse-human chimeric antibody Chimeric antibodies such as are preferred, and particularly preferred are fully human antibodies.
本実施形態にかかるモノクローナル抗体を取得する方法としては、取得したい抗体を産生するハイブリドーマを培養し、得られた培養上清から常法によって抗体を精製して取得する。本実施形態においてはMERS-CoVのスパイク（S）蛋白を主要抗原とするのではなく、MERS-CoVそのものを免疫用ウイルス抗原とする。取得したハイブリドーマから抗体を採取する方法は、特に限定されるものではないが、例えば通常の腹水形成法や細胞培養法等を用いることが可能である。腹水形成法においては、骨髄腫細胞由来の哺乳動物と同種の動物の腹腔内にプリスタン等の鉱物油を投与し、その後ハイブリドーマ１×１０６〜１×１０９個、好ましくは１×１０７〜１×１０８個を腹腔内に投与し、ハイブリドーマを大量に増殖させる。そして、１〜４週間、好適には２〜３週間後に腹水又は血清を採集する。細胞培養法においては、ハイブリドーマを１０〜２０％仔ウシ血清含有IMDM、RPMI-1640、MEM、E-RDF又は無血清培地等の動物細胞培養培地中で、通常の培養条件（例えば37℃、５％ＣＯ２濃度）で３〜１４日間培養し、その培養上清から抗体を取得することができる。抗体の精製は、硫安塩析法、DEAEセルロース等の陰イオン交換体を利用するイオン交換クロマトグラフィー、プロテインＡセファロース等を用いるアフィニティークロマトグラフィー、分子量や構造によってふるい分ける分子ふるいクロマトグラフィー等の公知の方法を適宜に選択して精製することが可能である。
（測定キット）
本実施にかかる測定キットは、試験サンプル中においてMERS-CoVのウイルス活性を中和する中和抗体の存在を測定するための測定キットであって、(i)固体支持体に結合させたMERS-CoV抗原と、(ii)本実施形態にかかるモノクローナル抗体と、及び(iii)該モノクローナル抗体を認識する標識二次抗体と、を有する測定キットである。この測定キットにより競合ELISA法にて、試験サンプル中のMERS-CoVのウイルス活性を中和する中和抗体の存在を測定できる。
As a method for obtaining a monoclonal antibody according to this embodiment, a hybridoma that produces the antibody to be obtained is cultured, and the antibody is purified and obtained from the obtained culture supernatant by a conventional method. In this embodiment, the MERS-CoV spike (S) protein is not used as the main antigen, but MERS-CoV itself is used as the virus antigen for immunization. The method for collecting the antibody from the obtained hybridoma is not particularly limited, and for example, a normal ascites formation method, a cell culture method, or the like can be used. In the ascites formation method, mineral oil such as pristane is administered into the abdominal cavity of an animal of the same kind as a mammal derived from myeloma cells, and then 1 × 10 6 to 1 × 10 9 hybridomas, preferably 1 × 10 7 to 1 × 10 8 cells are administered intraperitoneally, and hybridomas are proliferated in large quantities. And ascites or serum is collected after 1 to 4 weeks, preferably 2 to 3 weeks. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as IMDM, RPMI-1640, MEM, E-RDF or serum-free medium containing 10-20% calf serum under normal culture conditions (for example, 37 ° C., 5 % CO 2 concentration) for 3 to 14 days, and antibodies can be obtained from the culture supernatant. For antibody purification, known methods such as ammonium sulfate salting-out, ion exchange chromatography using an anion exchanger such as DEAE cellulose, affinity chromatography using protein A sepharose, molecular sieve chromatography that screens according to molecular weight and structure, etc. It is possible to purify by selecting an appropriate method.
(Measurement kit)
The measurement kit according to this embodiment is a measurement kit for measuring the presence of a neutralizing antibody that neutralizes the virus activity of MERS-CoV in a test sample, and (i) MERS- bound to a solid support. A measurement kit comprising a CoV antigen, (ii) a monoclonal antibody according to the present embodiment, and (iii) a labeled secondary antibody that recognizes the monoclonal antibody. With this assay kit, the presence of neutralizing antibodies that neutralize the viral activity of MERS-CoV in the test sample can be measured by competitive ELISA.
マイクロプレートにMERS-CoV抗原を固相化し、試験サンプル中の被検対象の抗体と、本実施形態にかかるモノクローナル抗体とを、同一マイクロプレート内で同時に反応させる。試料サンプル中にMERS-CoVのウイルス活性を中和する中和抗体が多い場合は、MERS-CoV抗原と結合できる本実施形態にかかるモノクローナル抗体が減少し、発色が弱くなる。逆に試料サンプル中のMERS-CoVのウイルス活性を中和する中和抗体が少ない場合は、MERS-CoV抗原と結合できる実施形態にかかるモノクローナル抗体が増加し、発色が強くなる。 The MERS-CoV antigen is immobilized on a microplate, and the antibody to be tested in the test sample and the monoclonal antibody according to this embodiment are reacted simultaneously in the same microplate. When there are many neutralizing antibodies in the sample sample that neutralize the viral activity of MERS-CoV, the monoclonal antibodies according to this embodiment that can bind to the MERS-CoV antigen are decreased, and the color development is weakened. Conversely, when there are few neutralizing antibodies that neutralize the viral activity of MERS-CoV in the sample sample, the monoclonal antibody according to the embodiment capable of binding to the MERS-CoV antigen increases and the color development becomes stronger.
具体的には競合ELISAは下記手順により行われる。
1.抗原の調製
MERSコロナウイルスを感染させたVero細胞に1%NP40を加え、8,000g 20分遠心し、上清から感染細胞ライセートを回収する。UV照射により完全にウイルスを不活化し競合ELISA用抗原とする。
2.抗原のコーティング
MERSコロナウイルス感染細胞ライセートをPBSで適当な濃度に（例えば800倍）希釈し、100μLをELISAプレートの各ウェルに分注する。プレートにシールをして4℃で一晩静置する。
3.ELISAプレートのブロッキング
抗原をコーティングしたELISAプレートの各ウェルを0.05% tween 20入りPBS(PBS-T) 300μLで2回洗浄後、2%ウシ血清アルブミン(BSA)入りPBS-T (2BPBS-T)200μLを加え、プレートにシールをして37℃で2時間インキュベートする。
4.モノクローナル抗体と被験血清の混合液の調製
ビオチン標識したモノクローナル抗体45C2、45E11、43A9のいずれか、あるいはこれらの混合液50μLと、段階希釈（例えば、4倍希釈から2倍ずつ段階希釈）した被検血清50μLとを混合する。
5.モノクローナル抗体と被験血清の混合液の反応
ブロッキングしたELISAプレートの各ウェルを300μLのPBS-Tで2回洗浄後、モノクローナル抗体と被験血清の混合液100μLを各ウェルに加える。プレートにシールをして37℃で1時間インキュベートする。
6.標識
ELISAプレートの各ウェルを300μLのPBS-Tで3回洗浄後、適当な濃度に（例えば8,000倍）希釈したHorseradish peroxidase (HRP)標識アビジン100μLを各ウェルに加える。プレートにシールをして37℃で1時間インキュベートする。なお、4.でHRP標識されたモノクローナル抗体を使用した場合はこのステップを省略できる。
7.吸光度測定
ELISAプレートの各ウェルを300μLのPBS-Tで3回洗浄後、ペルオキシダーゼ用発色基質(ABTS)溶液100μLを加える。室温で15-20分インキュベート後、ELISAプレートリーダーで各ウェルの405nm波長の吸光度を測定する。
8.判定
被検血清による、モノクローナル抗体と抗原の結合阻害の程度（% inhibition）を下記の式で表す。
Specifically, competitive ELISA is performed according to the following procedure.
1. Preparation of antigen
1% NP40 is added to Vero cells infected with MERS coronavirus, centrifuged at 8,000g for 20 minutes, and the infected cell lysate is recovered from the supernatant. The virus is completely inactivated by UV irradiation and used as a competitive ELISA antigen.
2. Antigen coating
The MERS coronavirus-infected cell lysate is diluted to an appropriate concentration (eg, 800 times) with PBS, and 100 μL is dispensed into each well of the ELISA plate. Seal the plate and let stand at 4 ° C overnight.
3.Blocking of ELISA plate Each well of antigen-coated ELISA plate was washed twice with 300 μL of PBS (PBS-T) containing 0.05% tween 20, and then PBS-T containing 2% bovine serum albumin (BSA) (2BPBS-T ) Add 200 μL, seal the plate and incubate at 37 ° C. for 2 hours.
4. Preparation of mixed solution of monoclonal antibody and test serum Dilute serially (for example, 4 times dilution to 2 times dilution) with biotinylated monoclonal antibody 45C2, 45E11, 43A9, or 50 μL of these mixtures. Mix with 50 μL of test serum.
5. Reaction of the mixture of monoclonal antibody and test serum Wash each well of the blocked ELISA plate twice with 300 μL of PBS-T, and then add 100 μL of the mixture of monoclonal antibody and test serum to each well. Seal the plate and incubate at 37 ° C for 1 hour.
6.Signs
Each well of the ELISA plate is washed 3 times with 300 μL of PBS-T, and then 100 μL of Horseradish peroxidase (HRP) -labeled avidin diluted to an appropriate concentration (eg, 8,000 times) is added to each well. Seal the plate and incubate at 37 ° C for 1 hour. Note that this step can be omitted when the HRP-labeled monoclonal antibody is used in 4.
7.Absorbance measurement
Each well of the ELISA plate is washed 3 times with 300 μL of PBS-T, and then 100 μL of peroxidase chromogenic substrate (ABTS) solution is added. After incubation at room temperature for 15-20 minutes, the absorbance at 405 nm wavelength of each well is measured with an ELISA plate reader.
8. Determination The degree of inhibition (% inhibition) between the monoclonal antibody and the antigen by the test serum is expressed by the following formula.
ELISAのカットオフ値は陰性コントロール血清による% inhibition値の平均+標準偏差の3倍値（3SD）とする。 The cut-off value of ELISA is the mean of the% inhibition value by the negative control serum + 3 times the standard deviation (3SD).
被検血清による% inhibition値がカットオフ値を上回る場合、被験血清はMERSコロナウイルス抗体陽性と判断される。 If the% inhibition value by the test serum exceeds the cut-off value, the test serum is determined to be MERS coronavirus antibody positive.
下記手順により本実施例にかかるモノクローナル抗体を作成した。
1.ウイルス抗原の調製
(a)MERSコロナウイルス(Human Beta-Coronavirus Lineage C-Novel/2012,参考文献： ZakiらN Engl J Med. 367(19):1814-1820, 2013)をアフリカミドリザル腎臓上皮由来の培養細胞（Vero細胞）に接種した。
(b)3-4日後、培養フラスコを-80℃に移し、凍結後に室温で融解することにより細胞破砕を行った。細胞浮遊液を高速遠心機で8,000g 30分遠心し、上清を集めた。
(c)(b)にポリエチレングリコール6,000と塩化ナトリウムをそれぞれ8%と0.5Mになるように加えてよく混ぜた後、 4℃で1晩静置してウイルスを沈殿させた。
(d)高速遠心機で8,000g 30分遠心し上清を捨て、沈殿をPBSに溶かして回収した。
(e)超遠心機ロータSW41用チューブに20%/60%のスクロースクッションを入れておき、その上から(d)でPBSに溶かしたウイルス液を重層した。30,000rpm, 2時間遠心後、中間層を集めてプールし、もう一度20%/60%のスクロースクッションで30,000rpm, 2時間の遠心を行い、中間層をウイルス液として回収した。回収したウイルス液を紫外線照射し、不活化したものを免疫用ウイルス抗原とした。
2.マウスへの免疫
上記のように精製したUV不活化MERSコロナウイルス抗原を、以下のようにBALB/cマウスに免疫した。初回免疫として、20マイクログラムのウイルス抗原をフロイント完全アジュバントと混合して皮下に注射した。２週間後に尾静脈より採血し、血清中の抗MERS-CoV抗体価をELISAで測定した。抗体価の上昇が確認できたマウスについて、初回免疫後１９日目に、7マイクログラムの精製ウイルス抗原をPBSに懸濁して尾静脈より静脈注射して追加免疫を行なった。追加免疫後３日目に、２匹のマウスより麻酔下全採血及び安楽死後の脾臓摘出を行い、マウスミエローマ細胞と細胞融合させて、HAT選択培地細胞培養系で抗体産生細胞（ハイブリドーマ）を作製した。
3.ハイブリドーマのスクリーニング
(a)MERSコロナウイルス抗原及び他のヒトコロナウイルス（ヒトコロナウイルス229E、ヒトコロナウイルスNL63、ヒトコロナウイルスOC43）を抗原としたELISA法で行った。
(b)ELISAでMERSコロナウイルス抗原にのみ反応し、他のヒトコロナウイルスに反応しないハイブリドーマを選択した。限界希釈法によりハイブリドーマを単クローン化した。
4.ハイブリドーマクローンの選別
(a)単クローン化したハイブリドーマ上清をMERSコロナウイルスのS蛋白質あるいは、N蛋白質を発現するハムスター腎細胞由来BHK細胞に反応させた。さらに蛍光標識された抗マウス二次抗体を反応させ、S蛋白質に反応するクローンをピックアップした。
(b)MERSコロナウイルスと単クローン化したハイブリドーマの上清を混合し、Vero細胞に接種した。MERSコロナウイルスの感染を阻止する（中和活性がある）ハイブリドーマクローン45C2, 45E11, 43A9を選別した。
5.ハイブリドーマクローンの抗体遺伝子解析
クローン45C2, 45E11, 43A9のハイブリドーマ細胞からRNAを抽出し、SMARTer RACE5’/3’Kit (Clonetech)を用いて各クローンの抗体遺伝子のH鎖、L鎖の5’末端側の可変領域の遺伝子配列を決定した。
6.モノクローナル抗体の精製
ハイブリドーマクローン45C2, 45E11, 43A9の培養上清からprotein G カラムあるいはprotein Aカラムを用いてモノクローナル抗体を精製した。
7.標識化
精製したモノクローナル抗体をビオチン標識した。
A monoclonal antibody according to this example was prepared according to the following procedure.
1. Preparation of viral antigen
(a) MERS coronavirus (Human Beta-Coronavirus Lineage C-Novel / 2012, reference: Zaki et al. N Engl J Med. 367 (19): 1814-1820, 2013) was cultured in African green monkey kidney epithelium (Vero Cells).
(b) After 3-4 days, the culture flask was transferred to −80 ° C., and after freezing, the cells were disrupted by thawing at room temperature. The cell suspension was centrifuged at 8,000 g for 30 minutes with a high-speed centrifuge, and the supernatant was collected.
(c) Polyethylene glycol 6,000 and sodium chloride were added to (b) and 8% and 0.5M, respectively, and mixed well, and then allowed to stand at 4 ° C. overnight to precipitate the virus.
(d) The mixture was centrifuged at 8,000 g for 30 minutes with a high-speed centrifuge, the supernatant was discarded, and the precipitate was dissolved in PBS and collected.
(e) A 20% / 60% sucrose cushion was placed in the tube for the ultracentrifuge rotor SW41, and the virus solution dissolved in PBS in (d) was overlaid from above. After centrifugation at 30,000 rpm for 2 hours, the intermediate layer was collected and pooled, and centrifuged again at 30,000 rpm for 2 hours with a 20% / 60% sucrose cushion to recover the intermediate layer as a virus solution. The recovered virus solution was irradiated with ultraviolet rays and inactivated, and used as a virus antigen for immunization.
2. Immunization of mice BALB / c mice were immunized with the UV-inactivated MERS coronavirus antigen purified as described above as follows. As a first immunization, 20 micrograms of viral antigen was mixed with Freund's complete adjuvant and injected subcutaneously. Two weeks later, blood was collected from the tail vein, and the anti-MERS-CoV antibody titer in the serum was measured by ELISA. On the 19th day after the first immunization, 7 micrograms of purified viral antigen was suspended in PBS and injected intravenously from the tail vein of the mice whose antibody titer was confirmed to be boosted. On the third day after booster immunization, whole blood was collected from two mice under anesthesia and spleen was removed after euthanasia, and fused with mouse myeloma cells to produce antibody-producing cells (hybridoma) in a HAT selective medium cell culture system. did.
3. Hybridoma screening
(a) ELISA was performed using MERS coronavirus antigen and other human coronaviruses (human coronavirus 229E, human coronavirus NL63, human coronavirus OC43) as antigens.
(b) Hybridomas that reacted only with MERS coronavirus antigen by ELISA but did not react with other human coronaviruses were selected. The hybridoma was cloned by the limiting dilution method.
4. Selection of hybridoma clones
(a) Monoclonal hybridoma supernatant was reacted with hamster kidney cell-derived BHK cells expressing MRS coronavirus S protein or N protein. Further, a fluorescently labeled anti-mouse secondary antibody was reacted to pick up a clone that reacted with S protein.
(b) The supernatant of the hybridoma which had been cloned with MERS coronavirus was mixed and inoculated into Vero cells. Hybridoma clones 45C2, 45E11, and 43A9 that inhibit MERS coronavirus infection (having neutralizing activity) were selected.
5. Antibody gene analysis of hybridoma clones Extract RNA from clone 45C2, 45E11, 43A9 hybridoma cells and use SMARTer RACE5 '/ 3'Kit (Clonetech) for 5' of the H and L chains of the antibody gene of each clone The gene sequence of the terminal variable region was determined.
6. Purification of monoclonal antibody The monoclonal antibody was purified from the culture supernatant of the hybridoma clones 45C2, 45E11, and 43A9 using a protein G column or protein A column.
7. Labeling The purified monoclonal antibody was labeled with biotin.
中東呼吸器症候群に対する医薬として利用できる。 It can be used as a medicine for Middle East respiratory syndrome.
Claims (5)
(i)固体支持体に結合させたMERSコロナウイルス抗原と、
(ii)請求項１乃至４の何れか１項に記載のモノクローナル抗体と、及び
(iii)該モノクローナル抗体を認識する標識二次抗体と、
を有する測定キット。 A measurement kit for measuring the presence of neutralizing antibodies that neutralize the viral activity of MERS coronavirus in a test sample,
(i) a MERS coronavirus antigen bound to a solid support;
(ii) the monoclonal antibody according to any one of claims 1 to 4, and
(iii) a labeled secondary antibody that recognizes the monoclonal antibody;
A measurement kit having:
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