JP2021052669A - Fusion protein of protein recognition substance and streptavidin mutant - Google Patents
Fusion protein of protein recognition substance and streptavidin mutant Download PDFInfo
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Abstract
Description
本発明は、タンパク質認識物質（例えば、がん細胞を認識する抗体）とストレプトアビジン変異体との融合タンパク質、並びにその利用に関する。 The present invention relates to a fusion protein of a protein recognizing substance (eg, an antibody that recognizes cancer cells) and a streptavidin mutant, and its utilization.
アビジンとビオチン、あるいはストレプトアビジンとビオチンの間の親和性は非常に高く（Kd=10-15 から10-14M）、生体二分子間の相互作用としては、最も強い相互作用の一つである。現在、アビジン／ストレプトアビジン-ビオチン相互作用は、生化学、分子生物学、あるいは医学の分野で広く応用されている。アビジン／ストレプトアビジンとビオチンの高い結合能と抗体分子とを組み合わせたドラッグデリバリーの方法およびプレターゲティング法が考案されている。これらの研究に関連し、特許文献１には、天然ビオチンに対する親和性を低減させたストレプトアビジン変異体、並びにこの天然ビオチンに対する低親和性のストレプトアビジン変異体に対して高い親和性を有するビオチン改変二量体が報告されている。
The affinity between avidin and biotin, or streptavidin and biotin is very high (Kd = 10 -15 to 10 -14 M), and it is one of the strongest interactions between two biological molecules. .. Currently, the avidin / streptavidin-biotin interaction is widely applied in the fields of biochemistry, molecular biology, or medicine. A drug delivery method and a pretargeting method have been devised that combine the high binding ability of avidin / streptavidin and biotin with an antibody molecule. In connection with these studies,
本発明は、がんの治療又は診断において使用するための、がん細胞を認識する抗体とストレプトアビジン変異体との融合タンパク質を提供することを解決すべき課題とした。さらに本発明は、上記した融合タンパク質を用いた、がんの治療手段又はがんの診断手段を提供することを解決すべき課題とした。 The present invention has been set to solve the problem of providing a fusion protein of an antibody that recognizes cancer cells and a streptavidin mutant for use in the treatment or diagnosis of cancer. Further, the present invention has made it a problem to be solved to provide a means for treating cancer or a means for diagnosing cancer using the above-mentioned fusion protein.
本発明者は上記課題を解決するために鋭意検討した結果、がん細胞を認識する抗体として抗ＣＥＡ抗体を選択し、抗ＣＥＡ抗体とストレプトアビジン変異体との融合タンパク質であって、さらにＣ末端のアミノ酸残基を欠失させた融合タンパク質を調製した。そして、上記の融合タンパク質が、ビオチン改変二量体と親和性を有することを見出し、本発明を完成するに至った。 As a result of diligent studies to solve the above problems, the present inventor has selected an anti-CEA antibody as an antibody that recognizes cancer cells, and is a fusion protein of the anti-CEA antibody and a streptavidin variant, which is further C-terminal. A fusion protein was prepared in which the amino acid residue of was deleted. Then, they found that the above fusion protein had an affinity for a biotin-modified dimer, and completed the present invention.
即ち、本発明によれば、以下の発明が提供される。
＜１＞ タンパク質認識物質と、前記タンパク質認識物質に融合した配列番号１９に記載のアミノ酸配列とを含む融合タンパク質。
＜２＞ タンパク質認識物質が、がん細胞を認識する物質である、＜１＞に記載の融合タンパク質。
＜３＞ がん細胞を認識する抗体が、抗ＣＥＡ抗体または抗ＨＥＲ２抗体である、＜２＞に記載の融合タンパク質。
＜４＞ 配列番号１８に記載のアミノ酸配列を有する、＜１＞から＜３＞の何れか一に記載の融合タンパク質。
＜５＞ 配列番号１８に記載のアミノ酸配列をコードする核酸。
＜６＞ ＜１＞から＜４＞の何れか一に記載の融合タンパク質を含む、がん治療剤またはがん診断剤。
＜７＞ （１）＜１＞から＜４＞の何れか一に記載の融合タンパク質、及び（２）下記式（１）で示される化合物又はその塩と、診断用物質又は治療用物質とのコンジュゲートを含む、がんの治療または診断キット。
Ｘ１ａ、Ｘ１ｂ、Ｘ２ａ及びＸ２ｂはそれぞれ独立にＯ又はＮＨを示し、
Ｙ１及びＹ２はそれぞれ独立にＣ又はＳを示し、
Ｚ１及びＺ２はそれぞれ独立にＯ、Ｓ又はＮＨを示し、
Ｖ１及びＶ２はそれぞれ独立にＳ又はＳ＋−Ｏ−を示し、ｎ１及びｎ２はそれぞれ独立に０又は１の整数を示し、
Ｌ１及びＬ２はそれぞれ独立に、２価の連結基を示し、
Ｌ３は、診断用物質又は治療用物質と結合できる官能基を末端に含む基であり、
Ｌ４は、３価の連結基を示す。）
＜８＞ 診断用物質又は治療用物質が、フタロシアニン染料である、＜７＞に記載のキット。
That is, according to the present invention, the following invention is provided.
<1> A fusion protein containing a protein recognizing substance and the amino acid sequence shown in SEQ ID NO: 19 fused to the protein recognizing substance.
<2> The fusion protein according to <1>, wherein the protein recognizing substance is a substance that recognizes cancer cells.
<3> The fusion protein according to <2>, wherein the antibody that recognizes cancer cells is an anti-CEA antibody or an anti-HER2 antibody.
<4> The fusion protein according to any one of <1> to <3>, which has the amino acid sequence shown in SEQ ID NO: 18.
<5> A nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 18.
<6> A cancer therapeutic agent or a cancer diagnostic agent containing the fusion protein according to any one of <1> to <4>.
<7> (1) The fusion protein according to any one of <1> to <4>, and (2) the compound represented by the following formula (1) or a salt thereof, and a diagnostic substance or a therapeutic substance. Cancer treatment or diagnostic kits, including conjugates.
X1a, X1b, X2a and X2b independently indicate O or NH, respectively.
Y 1 and Y 2 independently represent C or S, respectively.
Z 1 and Z 2 independently represent O, S or NH, respectively.
V 1 and V 2 independently represent S or S + −O − , and n1 and n2 independently represent an integer of 0 or 1, respectively.
L 1 and L 2 each independently represent a divalent linking group.
L 3 is a group having a functional group at the end capable of binding to a diagnostic substance or a therapeutic substance.
L 4 represents a trivalent linking group. )
<8> The kit according to <7>, wherein the diagnostic substance or the therapeutic substance is a phthalocyanine dye.
本発明によるがん細胞を認識する抗体とストレプトアビジン変異体との融合タンパク質を用いることによれば、がん細胞の増殖を抑制することができる。 By using the fusion protein of the antibody that recognizes cancer cells and the streptavidin mutant according to the present invention, the growth of cancer cells can be suppressed.
以下、本発明について更に詳細に説明する。
＜タンパク質認識物質とストレプトアビジン変異体との融合タンパク質＞
本発明の融合タンパク質は、タンパク質認識物質と、前記タンパク質認識物質に融合した配列番号１９に記載のアミノ酸配列とを含む融合タンパク質である。
タンパク質認識物質としては、がん細胞を認識する物質が好ましい。
Hereinafter, the present invention will be described in more detail.
<Fusion protein of protein recognizing substance and streptavidin mutant>
The fusion protein of the present invention is a fusion protein containing a protein recognizing substance and the amino acid sequence set forth in SEQ ID NO: 19 fused to the protein recognizing substance.
As the protein recognizing substance, a substance that recognizes cancer cells is preferable.
がん細胞を認識する物質としては、がんに特異的に発現する以下の抗原を標的とした抗体、ペプチド、核酸、アプタマー等を用いることができる。 As the substance for recognizing cancer cells, antibodies, peptides, nucleic acids, aptamers and the like targeting the following antigens specifically expressed in cancer can be used.
エピレギュリン、ROBO1,2,3,4、1-40-β-アミロイド, 4-1BB, 5AC, 5T4, ACVR2B, 腺がん抗原, α-フェトプロテイン, アンギオポエチン2, 炭疽毒素, AOC3 (VAP-1), B-リンパ腫細胞, B7-H3, BAFF, βアミロイド, C242抗原, C5, CA-125, カルボニックアンヒドラーゼ9 (CA-IX), 心臓ミオシン, CCL11 (eotaxin-1), CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin), CD147 (basigin), CD15, CD152, CD154 (CD40L), CD154, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE受容体), CD25(IL-2受容体のα鎖), CD28, CD3, CD30 (TNFRSF8), CD33, CD37, CD38(サイクリックADPリボースヒドロラーゼ), CD4, CD40, CD41(インテグリンα-IIb), CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CFD, ch4D5, CLDN18.2, クロストリジウム・ディフィシレ（Clostridium difficile）,クランピング因子A, CSF2, CTLA-4, サイトメガロウイルス, サイトメガロウイルス糖タンパク質B, DLL4, DR5, E. coli 志賀毒素１型, E. coli志賀毒素２型、EGFL7, EGFR, エンドトキシン, EpCAM, エピシアリン（episialin）, ERBB3, 大腸菌（Escherichia coli）, 呼吸器合胞体｛こきゅうき ごうほうたい｝ウイルス（respiratory syncytial virus）のFタンパク質, FAP, フィブリンIIβ鎖, フィブロネクチンエクストラドメイン-B, 葉酸受容体1, Frizzled 受容体, GD2, GD3ガングリオシド, GMCSF受容体α鎖, GPNMB, Ｂ型肝炎表面抗原, Ｂ型肝炎ウイルス、HER1, HER2/neu, HER3, HGF, HIV-1, HLA-DRβ, HNGF, Hsp90, ヒトβアミロイド,ヒト分散因子（scatter factor）受容体キナーゼ, ヒトTNF, ICAM-1 (CD54), IFN-α, IFN-γ, IgE, IgE Fc 領域, IGF-1受容体, IGF-I, IgG4, IGHE, IL-1β, IL-12, IL-13, IL-17, IL-17A, IL-22, IL-23, IL-4, IL-5, IL-6, IL-6受容体, IL-9, ILGF2, インフルエンザA ヘマグルチニン, インスリン様増殖因子I受容体, インテグリンα4, インテグリンα4β7, インテグリンα5β1, インテグリンα7 β7, インテグリンαIIbβ3, インテグリンαvβ3, インテグリンγ誘導タンパク質,インターフェロン受容体, インターフェロンα/β受容体, ITGA2, ITGB2 (CD18), KIR2D, L-セレクチン(CD62L), Lewis-Y抗原, LFA-1 (CD11a), リポタイコ酸, LOXL2, LTA, MCP-1, メソテリン、MS4A1, MUC1, ムチンCanAg, ミオスタチン, N-グリコリルノイラミン酸, NARP-1, NCA-90 (顆粒球抗原), NGF, NOGO-A, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-R α, フォスファチジルセリン,前立腺がん細胞、緑膿菌（Pseudomonas aeruginosa）,狂犬病｛きょうけんびょう｝ウイルス糖タンパク質、RANKL, 呼吸器合胞体｛こきゅうき ごうほうたい｝ウイルス, RHD, Ｒｈ（Rhesus）因子, RON, RTN4, スクレロスチン, SDC1, セレクチンP, SLAMF7, SOST, スフィンゴシン-1-ホスフェート, TAG-72, TEM1, テネイシンC, TFPI, TGFβ1, TGFβ2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, 腫瘍抗原CTAA16.88,MUC1の腫瘍特異的グリコシル化, TWEAK受容体, TYRP1(グリコプロテイン75), VEGF-A, VEGFR-1, VEGFR2, ビメンチン, VWF
Epiregulin, ROBO1,2,3,4, 1-40-β-amyloid, 4-1BB, 5AC, 5T4, ACVR2B, adenocarcinoma antigen, α-fetoprotein,
がん細胞を認識する抗体としては、特に好ましくは、抗ＣＥＡ抗体または抗ＨＥＲ２抗体である。
本発明の融合タンパク質の一例は、配列番号１８に記載のアミノ酸配列を有する、融合タンパク質である。
The antibody that recognizes cancer cells is particularly preferably an anti-CEA antibody or an anti-HER2 antibody.
An example of a fusion protein of the present invention is a fusion protein having the amino acid sequence set forth in SEQ ID NO: 18.
本発明によればさらに、本発明の融合タンパク質をコードする核酸（例えば、ＤＮＡ）が提供される。
本発明の融合タンパク質をコードする核酸（例えば、ＤＮＡ）は、ベクターに組み込んで使用することができる。本発明の融合タンパク質を製造するためには、本発明の融合タンパク質をコードする核酸を発現ベクターに組み込み、この発現ベクターを宿主に形質転換することによって、本発明の融合タンパク質を発現させることができる。
Further according to the present invention, nucleic acids (eg, DNA) encoding the fusion proteins of the present invention are further provided.
The nucleic acid encoding the fusion protein of the present invention (eg, DNA) can be used by incorporating it into a vector. In order to produce the fusion protein of the present invention, the fusion protein of the present invention can be expressed by incorporating a nucleic acid encoding the fusion protein of the present invention into an expression vector and transforming this expression vector into a host. ..
大腸菌を宿主とする場合には、ベクターとしては、複製起点（ori）を有し、さらに形質転換された宿主を選択するための遺伝子（例えば、アンピシリン、テトラサイクリン、カナマイシン又はクロラムフェニコールなどの薬剤に対する薬剤耐性遺伝子など）を有していることが好ましい。また、発現ベクターの場合には、宿主において本発明のストレプトアビジン変異体を効率よく発現させることができるようなプロモーター、例えば、lacZプロモーターまたはT7プロモーターなどを持っていることが好ましい。このようなベクターとしては、ベクターの例としては、M13系ベクター、pUC系ベクター、pBR322、pBluescript、pCR-Script、pGEX-5X-1（ファルマシア）、「QIAexpress system」（キアゲン）、pEGFP、またはpET(この場合、宿主はT7 RNAポリメラーゼを発現しているBL21を使用することが好ましい)などが挙げられる。 When Escherichia coli is used as a host, the vector has an origin of replication (ori) and a gene for selecting a transformed host (for example, a drug such as ampicillin, tetracycline, kanamycin or chloramphenicol). It is preferable to have a drug resistance gene against Escherichia coli. Further, in the case of an expression vector, it is preferable to have a promoter capable of efficiently expressing the streptavidin mutant of the present invention in the host, for example, a lacZ promoter or a T7 promoter. Examples of such vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (Kiagen), pEGFP, or pET. (In this case, it is preferable to use BL21 expressing T7 RNA polymerase as the host).
宿主細胞へのベクターの導入は、例えば塩化カルシウム法、エレクトロポレーション法を用いて行うことができる。また、可溶性を向上させるためのタグ、例えばグルタチオンーS−トランスフェラーゼやチオレドキシン、マルトース結合蛋白質をコードする配列が付加されていてもよい。また、精製を容易にすることを目的にした設計されたタグ、例えばポリヒスチジンタグ、Ｍｙｃエピトープ、ヘマグルチニン（ＨＡ）エピトープ、Ｔ７エピトープ、ＸｐｒｅｓｓタグやＦＬＡＧペプチドタグ、その他の既知のタグ配列をコードする配列が付加されていてもよい。 The vector can be introduced into the host cell by using, for example, a calcium chloride method or an electroporation method. In addition, a tag for improving solubility, for example, a sequence encoding glutathione-S-transferase, thioredoxin, or maltose-binding protein may be added. It also encodes tags designed to facilitate purification, such as polyhistidine tags, Myc epitopes, hemagglutinin (HA) epitopes, T7 epitopes, Xpress and FLAG peptide tags, and other known tag sequences. An array may be added.
大腸菌以外にも、哺乳動物由来の発現ベクター（例えば、pcDNA3(インビトロゲン社製）や、pEGF-BOS(Nucleic Acids. Res.1990, 18(17),p5322)、pEF、pCDM8）、昆虫細胞由来の発現ベクター（例えば「Bac-to-BAC baculovairus expression system」（ギブコBRL社製）、pBacPAK8）、植物由来の発現ベクター（例えばpMH1、pMH2）、動物ウィルス由来の発現ベクター（例えば、pHSV、pMV、pAdexLcw）、レトロウィルス由来の発現ベクター（例えば、pZIPneo）、酵母由来の発現ベクター（例えば、「Pichia Expression Kit」（インビトロゲン社製）、pNV11 、SP-Q01）、枯草菌由来の発現ベクター（例えば、pPL608、pKTH50）が挙げられる。 In addition to Escherichia coli, expression vectors derived from mammals (for example, pcDNA3 (manufactured by In vitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8), derived from insect cells Expression vectors (eg, "Bac-to-BAC baculovairus expression system" (Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vector (eg, pZIPneo), yeast-derived expression vector (eg, "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), Bacillus subtilis-derived expression vector (eg, pAdexLcw) , PPL608, pKTH50).
CHO細胞、COS細胞、NIH3T3細胞等の動物細胞での発現を目的とした場合には、細胞内で発現させるために必要なプロモーター、例えばSV40プロモーター（Mulliganら, Nature (1979) 277, 108）、MMLV-LTRプロモーター、EF1αプロモーター（Mizushimaら, Nucleic Acids Res. (1990) 18, 5322）、CMVプロモーターなどを持っていることが不可欠であり、細胞への形質転換を選抜するための遺伝子（例えば、薬剤（ネオマイシン、G418など）により判別できるような薬剤耐性遺伝子）を有すればさらに好ましい。このような特性を有するベクターとしては、例えば、pMAM、pDR2、pBK-RSV、pBK-CMV、pOPRSV、pOP13などが挙げられる。 For expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, promoters required for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have the MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and genes for selecting transformation into cells (eg, for example) It is more preferable to have a drug resistance gene that can be identified by a drug (neomycin, G418, etc.). Examples of the vector having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13 and the like.
ベクターが導入される宿主細胞としては特に制限はなく、原核生物および真核生物のいずれでもよい。例えば、大腸菌や種々の動物細胞などを用いることが可能である。 The host cell into which the vector is introduced is not particularly limited, and may be either a prokaryote or a eukaryote. For example, Escherichia coli and various animal cells can be used.
真核細胞を使用する場合、例えば、動物細胞、植物細胞、真菌細胞を宿主に用いることができる。動物細胞としては、哺乳類細胞、例えば、CHO細胞、COS細胞、3T3細胞、HeLa細胞、Vero細胞、あるいは昆虫細胞、例えば、Sf9、Sf21、Tn5などを用いることができる。動物細胞において、大量発現を目的とする場合には特にCHO細胞が好ましい。宿主細胞へのベクターの導入は、例えば、リン酸カルシウム法、DEAEデキストラン法、カチオニックリボソームDOTAP（ベーリンガーマンハイム社製）を用いた方法、エレクトロポーレーション法、リポフェクションなどの方法で行うことが可能である。 When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts. As animal cells, mammalian cells such as CHO cells, COS cells, 3T3 cells, HeLa cells, Vero cells, or insect cells such as Sf9, Sf21, and Tn5 can be used. In animal cells, CHO cells are particularly preferable for the purpose of large-scale expression. The vector can be introduced into a host cell by, for example, a calcium phosphate method, a DEAE dextran method, a method using a cationic ribosome DOTAP (manufactured by Boehringer Manheim), an electroporation method, or a lipofection method.
植物細胞としては、例えば、ニコチアナ・タバカム（Nicotiana tabacum）由来の細胞が蛋白質生産系として知られており、これをカルス培養すればよい。真菌細胞としては、酵母、例えば、サッカロミセス（Saccharomyces）属、例えば、サッカロミセス・セレビシエ（Saccharomyces cerevisiae）、糸状菌、例えば、アスペルギルス（Aspergillus）属、例えば、アスペルギルス・ニガー（Aspergillus niger）が知られている。 As plant cells, for example, cells derived from Nicotiana tabacum are known as protein-producing systems, and callus culture may be performed. Known fungal cells include yeasts, such as the genus Saccharomyces, such as Saccharomyces cerevisiae, filamentous fungi, such as the genus Aspergillus, such as Aspergillus niger. ..
原核細胞を使用する場合は、大腸菌（E. coli）、例えば、JM109、DH5α、HB101等が挙げられ、その他、枯草菌が知られている。 When prokaryotic cells are used, E. coli, for example, JM109, DH5α, HB101, etc. are mentioned, and Bacillus subtilis is also known.
これらの細胞を、本発明の核酸により形質転換し、形質転換された細胞をin vitroで培養することにより本発明の融合タンパク質が得られる。培養は、公知の方法に従い行うことができる。例えば、動物細胞の培養液として、例えば、DMEM、MEM、RPMI1640、IMDMを使用することができる。その際、牛胎児血清（FCS）等の血清補液を併用することもできるし、無血清培養してもよい。培養時のpHは、約6〜8であるのが好ましい。培養は、通常、約30〜40℃で約15〜200時間行い、必要に応じて培地の交換、通気、攪拌を加える。また、細胞の増殖を促進するための成長因子の添加を行ってもよい。 The fusion protein of the present invention can be obtained by transforming these cells with the nucleic acid of the present invention and culturing the transformed cells in vitro. Culturing can be carried out according to a known method. For example, DMEM, MEM, RPMI1640, IMDM can be used as a culture medium for animal cells. At that time, a serum replacement fluid such as fetal bovine serum (FCS) can be used in combination, or serum-free culture may be used. The pH at the time of culturing is preferably about 6 to 8. Culturing is usually carried out at about 30-40 ° C. for about 15-200 hours, with medium replacement, aeration and stirring as needed. In addition, a growth factor for promoting cell proliferation may be added.
＜がん治療剤またはがん診断剤＞
本発明の融合タンパク質は、がん治療剤またはがん診断剤として有用である。
本発明によれば、（１）本発明の融合タンパク質、及び（２）後記する式（１）で示される化合物又はその塩と、診断用物質又は治療用物質とのコンジュゲートを含む、がんの治療または診断キットが提供される。
<Cancer therapeutic agent or cancer diagnostic agent>
The fusion protein of the present invention is useful as a cancer therapeutic agent or a cancer diagnostic agent.
According to the present invention, a cancer comprising (1) a fusion protein of the present invention and (2) a compound represented by the formula (1) described later or a salt thereof and a diagnostic substance or a therapeutic substance. Treatment or diagnostic kits are provided.
本発明の融合タンパク質を患者に投与することで、がん細胞に特異的にストレプトアビジン変異体を集積できることができる。次に、ストレプトアビジン変異体に親和性を有するビオチン改変二量体と診断用物質又は治療用物質とのコンジュゲートを患者に投与することによって、癌細胞へ的確に診断用物質又は治療用物質を集積させることが可能になる。 By administering the fusion protein of the present invention to a patient, a streptavidin mutant can be specifically accumulated in cancer cells. Next, by administering to the patient a conjugate of a biotin-modified dimer having an affinity for the streptavidin mutant and a diagnostic substance or a therapeutic substance, the diagnostic substance or the therapeutic substance can be accurately delivered to the cancer cells. It becomes possible to accumulate.
あるいは、「本発明の融合タンパク質」と、「ストレプトアビジン変異体に親和性を有するビオチン改変二量体と診断用物質又は治療用物質とのコンジュゲート」とを、結合させた複合体を調製し、この複合体を患者に投与することもできる。 Alternatively, a complex in which the "fusion protein of the present invention" and "a conjugate of a biotin-modified dimer having an affinity for a streptavidin mutant and a diagnostic substance or a therapeutic substance" are bound is prepared. , This complex can also be administered to the patient.
＜ビオチン改変二量体＞
ビオチン改変二量体は、下記式（１）で示される化合物又はその塩であり、好ましくは下記式（２）で示される化合物又はその塩である。ビオチン改変二量体は、国際公開ＷＯ２０１５／１２５８２０号に記載されている化合物を使用することができる。
<Biotin-modified dimer>
The biotin-modified dimer is a compound represented by the following formula (1) or a salt thereof, and preferably a compound represented by the following formula (2) or a salt thereof. As the biotin-modified dimer, the compounds described in International Publication WO2015 / 125820 can be used.
（式中、
Ｘ１ａ、Ｘ１ｂ、Ｘ２ａ及びＸ２ｂはそれぞれ独立にＯ又はＮＨを示し、
Ｙ１及びＹ２はそれぞれ独立にＣ又はＳを示し、
Ｚ１及びＺ２はそれぞれ独立にＯ、Ｓ又はＮＨを示し、
Ｖ１及びＶ２はそれぞれ独立にＳ又はＳ＋−Ｏ−を示し、ｎ１及びｎ２はそれぞれ独立に０又は１の整数を示し、
Ｌ１及びＬ２はそれぞれ独立に、２価の連結基を示し、
Ｌ３は、診断用物質又は治療用物質（例えば、フタロシアニン染料）と結合できる官能基を末端に含む基であり、
Ｌ４は、３価の連結基を示す。）
(During the ceremony,
X1a, X1b, X2a and X2b independently indicate O or NH, respectively.
Y 1 and Y 2 independently represent C or S, respectively.
Z 1 and Z 2 independently represent O, S or NH, respectively.
V 1 and V 2 independently represent S or S + −O − , and n1 and n2 independently represent an integer of 0 or 1, respectively.
L 1 and L 2 each independently represent a divalent linking group.
L 3 is a group having a functional group at the end capable of binding to a diagnostic substance or a therapeutic substance (for example, a phthalocyanine dye).
L 4 represents a trivalent linking group. )
式（１）及び式（２）において、下記構造：
Ｘ１ａ、Ｘ１ｂ、Ｘ２ａ及びＸ２ｂがＮＨを示すことが好ましく、Ｙ１及びＹ２がＣを示すことが好ましく、Ｚ１及びＺ２がＮＨを示すことが好ましく、Ｖ１及びＶ２がＳを示すことが好ましい。 Shown X1a, X1b, it is preferable that X2a and X2b represents a NH, it is preferred that Y 1 and Y 2 represents a C, Z 1 and Z 2 preferably exhibit NH, V 1 and V 2 is the S Is preferable.
Ｌ１及びＬ２はそれぞれ独立に、−ＣＯＮＨ−、−ＮＨＣＯ−、−ＣＯＯ−、−ＯＣＯ―、−ＣＯ−、−Ｏ−、及び炭素数１から１０のアルキレン基から選択される基の組み合わせからなる２価の連結基であることが好ましい。
Ｌ１及びＬ２はそれぞれ独立に、−ＣＯＮＨ−、−ＮＨＣＯ−、−Ｏ−、及び炭素数１から１０のアルキレン基から選択される基の組み合わせからなる２価の連結基であることが好ましい。
Ｌ１、及びＬ２はそれぞれ独立に、−ＣＯＮＨ−、−ＮＨＣＯ−、及び炭素数１から１０のアルキレン基から選択される基の組み合わせからなる２価の連結基であることが好ましい。
L 1 and L 2 are independently a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, and alkylene groups having 1 to 10 carbon atoms. It is preferably a divalent linking group consisting of.
It is preferable that L 1 and L 2 are divalent linking groups each independently consisting of a combination of groups selected from -CONH-, -NHCO-, -O-, and an alkylene group having 1 to 10 carbon atoms. ..
It is preferable that L 1 and L 2 are divalent linking groups each independently consisting of a combination of groups selected from -CONH-, -NHCO-, and an alkylene group having 1 to 10 carbon atoms.
Ｌ４は、３価の連結基を示し、好ましくは、
Ｌ３は、好ましくは、−ＣＯＮＨ−、−ＮＨＣＯ−、−ＣＯＯ−、−ＯＣＯ―、−ＣＯ−、−Ｏ−、及び炭素数１から１０のアルキレン基から選択される基の組み合わせからなる基であって、さらにアミノ基を末端に含む基である。 L 3 is preferably a group consisting of a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, and alkylene groups having 1 to 10 carbon atoms. It is a group further containing an amino group at the end.
＜ビオチン改変二量体と診断用物質又は治療用物質とのコンジュゲート＞
ビオチン改変二量体に、診断用物質又は治療用物質を結合させることにより、ビオチン改変二量体と診断用物質又は治療用物質とのコンジュゲートを調製することができる。診断用物質又は治療用物質としては、蛍光色素、化学発光剤、放射性同位元素、金属化合物等からなる増感剤、金属化合物等からなる中性子捕捉剤、フタロシアニン染料、低分子化合物、マイクロあるいはナノバブル、タンパク質などを挙げることができる。好ましくは、フタロシアニン染料を使用することができる。
<Combination of biotin-modified dimer with diagnostic or therapeutic substance>
By binding a diagnostic substance or a therapeutic substance to the biotin-modified dimer, a conjugate of the biotin-modified dimer and the diagnostic substance or the therapeutic substance can be prepared. Diagnostic or therapeutic substances include fluorescent dyes, chemiluminescent agents, radioactive isotopes, sensitizers made of metal compounds, neutron trapping agents made of metal compounds, phthalocyanine dyes, low molecular weight compounds, micro or nanobubbles, etc. Examples include proteins. Preferably, a phthalocyanine dye can be used.
＜フタロシアニン染料＞
フタロシアニン染料は、好ましくはシリコンフタロシアニン染料である。ＩＲＤｙｅ（登録商標）７００ＤＸのようなフタロシアニン染料の具体例は、例えば、米国特許第７，００５，５１８号に記載されている。
<Parthalocyanine dye>
The phthalocyanine dye is preferably a silicone phthalocyanine dye. Specific examples of phthalocyanine dyes such as IRDye® 700DX are described, for example, in US Pat. No. 7,005,518.
フタロシアニン染料としては、下記式（２１）で示される染料を使用することができ、一例としては、下記式（２２）で示される染料を使用することができ As the phthalocyanine dye, a dye represented by the following formula (21) can be used, and as an example, a dye represented by the following formula (22) can be used.
式中、Ｌ２１は２価の連結基を示し、Ｒ２１は、式（１）で示される化合物又はその塩と結合できる官能基を示す。 In the formula, L 21 represents a divalent linking group, and R 21 represents a functional group capable of binding to the compound represented by the formula (1) or a salt thereof.
Ｘ及びＹはそれぞれ独立に親水性基、−ＯＨ、水素原子又は置換基である。ここで言う置換基としては、ハロゲン原子（フッ素原子）、炭素原子を含む置換基（炭化水素基など）、窒素原子を含む置換基（アミノ基など）などを挙げることができるが、特に限定されない。
Ｘ及びＹの具体例としては、
（ｉ）Ｘ及びＹがともに親水性基である場合、
（ｉｉ）Ｘ及びＹの一方が親水性基であり、Ｘ及びＹの他方が−ＯＨ、又は水素原子である場合、
（ｉｉｉ）Ｘ及びＹが、−ＯＨ、又は水素原子である場合、
を挙げることができる。
X and Y are independently hydrophilic groups, -OH, hydrogen atoms or substituents, respectively. Examples of the substituent referred to here include a halogen atom (fluorine atom), a substituent containing a carbon atom (such as a hydrocarbon group), and a substituent containing a nitrogen atom (such as an amino group), but are not particularly limited. ..
Specific examples of X and Y include
(I) When both X and Y are hydrophilic groups
(Ii) When one of X and Y is a hydrophilic group and the other of X and Y is -OH or a hydrogen atom
(Iii) When X and Y are -OH or a hydrogen atom,
Can be mentioned.
Ｘ及び／又はＹで示される親水性基は特に限定されないが、一例を下記に示す。
フタロシアニン染料としては、ＩＲＤｙｅ（登録商標）７００ＤＸなどの市販品を使用することができる。本発明においては、 ＩＲＤｙｅ（登録商標）７００ＤＸのＮＨＳエステルを使用して、これを、アミノ基を有するビオチン改変二量体と反応させることによりコンジュゲートを製造することができる。ＩＲＤｙｅ（登録商標）７００ＤＸの他のバリエーションは、米国特許第７，００５，５１８号に記載されており、それらを使用することもできる。 As the phthalocyanine dye, a commercially available product such as IRDye (registered trademark) 700DX can be used. In the present invention, a conjugate can be produced by using an NHS ester of IRDye® 700DX and reacting it with a biotin-modified dimer having an amino group. Other variations of IRDye® 700DX are described in US Pat. No. 7,005,518, and they can also be used.
Ｒ２１は、式（１）で示される化合物又はその塩と結合できる官能基を示す。Ｒ２１は、好ましくは、ビオチン改変二量体上のカルボキシル基、アミン、又はチオール基と反応して結合できる官能基である。Ｒ２１は、好ましくは、活性化エステル、ハロゲン化アシル、ハロゲン化アルキル、適宜置換されたアミン、無水物、カルボン酸、カルボジイミド、ヒドロキシル、ヨードアセトアミド、イソシアネート、イソチオシアネート、マレイミド、ＮＨＳエステル、ホスホロアミダイト、スルホン酸エステル、チオール、又はチオシアネートなどが挙げられるが特に限定されない。 R 21 represents a functional group capable of binding to the compound represented by the formula (1) or a salt thereof. R 21 is preferably a functional group capable of reacting and binding to a carboxyl group, amine, or thiol group on the biotin-modified dimer. R 21 is preferably an activated ester, an acyl halide, an alkyl halide, an appropriately substituted amine, an anhydride, a carboxylic acid, a carbodiimide, a hydroxyl, an iodoacetamide, an isocyanate, an isothiocyanate, a maleimide, an NHS ester, a phosphoro. Amidite, sulfonic acid ester, thiol, thiocyanate and the like can be mentioned, but are not particularly limited.
Ｌ２１は２価の連結基を示し、例えば、エーテル、チオエーテル、アミン、エステル、カルバマート、ウレア、チオウレア、オキシ又はアミド結合の任意の組み合わせ、又は一重、二重、三重若しくは芳香族炭素−炭素結合；又はリン−酸素、リン−硫黄、窒素−窒素、窒素−酸素、若しくは窒素−プラチナ結合；又は芳香族若しくは複素芳香族結合を含んでいてもよい。Ｌ２１は、好ましくは、−ＣＯＮＨ−、−ＮＨＣＯ−、−ＣＯＯ−、−ＯＣＯ―、−ＣＯ−、−Ｏ−、及び炭素数１から１０のアルキレン基から選択される基の組み合わせからなる基である。 L 21 represents a divalent linking group, eg, any combination of ether, thioether, amine, ester, carbamate, urea, thiourea, oxy or amide bond, or single, double, triple or aromatic carbon-carbon bond. Alternatively, it may contain a phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bond; or an aromatic or heteroaromatic bond. L 21 is preferably a group consisting of a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, and alkylene groups having 1 to 10 carbon atoms. Is.
−Ｌ２１−Ｒ２１は、ホスホロアミダイト基、ＮＨＳエステル、活性カルボン酸、チオシアネート、イソチオシアネート、マレイミド、及びヨードアセトアミドを含んでいてもよい。 -L 21- R 21 may contain a phosphoramidite group, an NHS ester, an active carboxylic acid, a thiocyanate, an isothiocyanate, a maleimide, and an iodoacetamide.
Ｌ２１は、−（ＣＨ２）ｎ−基を含み、式中、ｎは１〜１０の整数であり、好ましくは、ｎは１〜４の整数である。一例としては、−Ｌ２１−Ｒ２１は、−Ｏ−（ＣＨ２）３−ＯＣ（Ｏ）−ＮＨ−（ＣＨ２）５−Ｃ（Ｏ）Ｏ−Ｎ−スクシンイミジルである。 L 21 contains a − (CH 2 ) n − group, in which n is an integer of 1 to 10, preferably n is an integer of 1 to 4. As an example, -L 21- R 21 is -O- (CH 2 ) 3- OC (O) -NH- (CH 2 ) 5- C (O) ON-succinimidyl.
＜光免疫療法＞
光免疫療法とは、体内で特定の細胞を破壊するために光増感剤及び照射光を使用する治療法である。光増感剤は、特異的な波長の光に晒されるとき、付近の細胞のアポトーシス、ネクローシス、及び／又は自食作用を誘発できる細胞傷害性の活性酸素種を生じる。例えば、特許６１２７０４５号公報には、細胞を死滅させる方法であって、 細胞表面タンパク質を含む細胞を、治療有効量の１または複数の抗体−ＩＲ７００分子と接触させるステップであって、該抗体が該細胞表面タンパク質に特異的に結合するステップと；該細胞に、６６０〜７４０ｎｍの波長で、かつ少なくとも１Ｊｃｍ−２の線量で照射するステップと；該細胞に照射した約０〜８時間後に、該細胞を１または複数の治療剤と接触させ、これにより、該細胞を死滅させるステップとを含む方法が記載されている。特表２０１７−５２４６５９号公報には、疾患又は病態を患っている対象において細胞毒性を誘発する方法であって、（ａ）対象の細胞に特異的に結合するプローブにコンジュゲートしたＩＲＤｙｅ（登録商標）７００ＤＸなどのフタロシアニン染料を包含する治療的に有効な薬剤を該対象に投与し；そして、（ｂ）細胞死を誘発するのに有効な量で適切な励起光を前記細胞に照射することを含む、方法が記載されている。
<Photoimmunotherapy>
Photoimmunotherapy is a treatment method that uses a photosensitizer and irradiation light to destroy specific cells in the body. Photosensitizers produce cytotoxic reactive oxygen species that can induce apoptosis, necrosis, and / or autophagy of nearby cells when exposed to light of specific wavelengths. For example, Japanese Patent No. 6127045 describes a method of killing a cell, the step of contacting a cell containing a cell surface protein with a therapeutically effective amount of one or more antibody-IR700 molecules, wherein the antibody is said to be A step of specifically binding to a cell surface protein; a step of irradiating the cell with a wavelength of 660 to 740 nm and a dose of at least 1 Jcm-2 ; about 0-8 hours after irradiation of the cell, the cell. Is described as a method comprising contacting the cells with one or more therapeutic agents, thereby killing the cells. Japanese Patent Application Laid-Open No. 2017-524659 describes a method for inducing cytotoxicity in a subject suffering from a disease or pathological condition, wherein (a) IRDye® conjugated to a probe that specifically binds to the target cell. ) A therapeutically effective agent, including a phthalocyanine dye, such as 700DX, is administered to the subject; and (b) the cells are irradiated with an appropriate amount of excitation light in an amount effective to induce cell death. Including, methods are described.
本発明の融合タンパク質と、ビオチン改変二量体とフタロシアニン染料とのコンジュゲートとを対象に投与し、細胞増殖の抑制または細胞死を誘発するのに有効な量で励起光を細胞に照射することによって、細胞増殖の抑制または細胞死を誘発し、対象を治療することができる。 To administer the fusion protein of the present invention and a conjugate of a biotin modified dimer and a phthalocyanine dye to a subject, and irradiate the cells with excitation light in an amount effective for suppressing cell proliferation or inducing cell death. Can suppress cell proliferation or induce cell death and treat a subject.
好ましくは、本発明の融合タンパク質と、ビオチン改変二量体とフタロシアニン染料とのコンジュゲートとを対象に投与し、細胞増殖の抑制または細胞死を誘発するのに有効な量で励起光を細胞に照射することによって、細胞増殖の抑制または細胞死を誘発し、対象を治療することができる。 Preferably, the fusion protein of the present invention and a conjugate of a biotin-modified dimer and a phthalocyanine dye are administered to the cells, and excitation light is applied to the cells in an amount effective for suppressing cell proliferation or inducing cell death. Irradiation can suppress cell proliferation or induce cell death and treat the subject.
対象としては、ヒトおよび非ヒト哺乳動物を包含し、ヒト、またはマウスなどの実験動物が挙げられる。対象としては、細胞増殖の抑制または細胞死の誘発が望まれる疾患を患っている対象が好ましく、例えば、がん又は固形腫瘍を有する対象を挙げることができる。 Subjects include humans and non-human mammals and include laboratory animals such as humans or mice. The subject is preferably a subject suffering from a disease in which suppression of cell proliferation or induction of cell death is desired, and examples thereof include a subject having cancer or solid tumor.
「がん」とは、癌腫、リンパ腫、芽細胞腫、肉腫、及び白血病又は悪性リンパ腫が挙げられる。がんの具体例としては、扁平上皮癌（例えば、上皮性扁平細胞癌）、小細胞肺癌を含めた肺癌、非小細胞肺癌（「ＮＳＣＬＣ」）、肺腺癌及び肺の扁平上皮癌、腹膜の癌、肝細胞性癌、消化器癌を含めた胃体又は胃癌、膵臓癌、神経膠芽腫、子宮頚癌、卵巣癌、肝臓癌、膀胱癌、肝細胞癌、乳癌、結腸癌、直腸癌、結腸直腸癌、子宮内膜又は子宮癌腫、唾液腺癌腫、腎臓又は腎臓部癌、前立腺癌、外陰部癌、甲状腺癌、肝細胞癌腫、肛門癌腫、陰茎癌腫、並びに頭頚部癌が挙げられる。 "Cancer" includes carcinomas, lymphomas, blastocytes, sarcomas, and leukemias or malignant lymphomas. Specific examples of cancer include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer (“NSCLC”), lung adenocarcinoma and lung squamous cell carcinoma, and peritoneum. Cancer, hepatocellular carcinoma, gastric or gastric cancer including gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectum Examples include cancer, colorectal cancer, endometrial or uterine cancer, salivary adenocarcinoma, kidney or kidney cancer, prostate cancer, pudendal cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, and head and neck cancer.
固形腫瘍とは、良性でも悪性でも、通常包嚢を含まない細胞の異常な塊を指す。固形腫瘍としては、神経膠腫、星状細胞腫、髄芽腫、頭蓋咽頭腫、上衣腫、松果体腫、血管芽腫、聴神経腫、希突起神経膠腫、髄膜腫、黒色腫、神経芽細胞腫、及び網膜芽細胞腫が挙げられる。 A solid tumor is an abnormal mass of cells, benign or malignant, that usually does not contain a capsule. Solid tumors include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pine fruit tumor, hemangioblastoma, acoustic neuroma, rare glioma, meningioma, melanoma, Examples include neuroblastoma and retinoblastoma.
対象への投与方法としては、局所経路、注射（皮下注射、筋肉内注射、皮内注射、腹腔内注射、腫瘍内注射、および静脈内注射など）、経口経路、眼経路、舌下経路、直腸経路、経皮経路、鼻腔内経路、膣経路、および吸入経路などが挙げられるが、これらに限定されない。 Subjects can be administered by local route, injection (subcutaneous injection, intramuscular injection, intradermal injection, intraperitoneal injection, intratumoral injection, and intravenous injection, etc.), oral route, ocular route, sublingual route, rectal route. Routes, percutaneous routes, intranasal routes, vaginal routes, and inhalation routes include, but are not limited to.
ビオチン改変二量体とフタロシアニン染料とのコンジュゲートと、本発明の融合タンパク質はそれぞれ、治療有効量で投与することが好ましい。
治療有効量は、上記のコンジュゲートと融合タンパク質のそれぞれについて、６０キログラム当たり少なくとも０．５ミリグラム（ｍｇ／６０ｋｇ）、少なくとも５ｍｇ／６０ｋｇ、少なくとも１０ｍｇ／６０ｋｇ、少なくとも２０ｍｇ／６０ｋｇ、少なくとも３０ｍｇ／６０ｋｇ、少なくとも５０ｍｇ／６０ｋｇである。例えば、静脈内投与される場合、１ｍｇ／６０ｋｇ、２ｍｇ／６０ｋｇ、５ｍｇ／６０ｋｇ、２０ｍｇ／６０ｋｇ、または５０ｍｇ／６０ｋｇの用量など、例えば、０．５〜５０ｍｇ／６０ｋｇである。別の例では、治療有効量は、少なくとも１００μｇ／ｋｇ、少なくとも５００μｇ／ｋｇまたは少なくとも５００μｇ／ｋｇなど、少なくとも１０μｇ／ｋｇ、例えば、腫瘍内投与または腹腔内投与される場合、１００μｇ／ｋｇ、２５０μｇ／ｋｇ、約５００μｇ／ｋｇ、７５０μｇ／ｋｇ、または１０００μｇ／ｋｇの用量など、例えば、１０μｇ／ｋｇ〜１０００μｇ／ｋｇである。一例では、治療有効量は、局所用溶液で投与される１０μｇ／ｍｌ、２０μｇ／ｍｌ、３０μｇ／ｍｌ、４０μｇ／ｍｌ、５０μｇ／ｍｌ、６０μｇ／ｍｌ、７０μｇ／ｍｌ、８０μｇ／ｍｌ、９０μｇ／ｍｌ、または１００μｇ／ｍｌなど、２０μｇ／ｍｌ〜１００μｇ／ｍｌの間など、少なくとも５００μｇ／ｍｌなど、少なくとも１μｇ／ｍｌである。
It is preferable that the conjugate of the biotin-modified dimer and the phthalocyanine dye and the fusion protein of the present invention are each administered in a therapeutically effective amount.
Therapeutically effective amounts are at least 0.5 milligrams (mg / 60 kg), at least 5 mg / 60 kg, at least 10 mg / 60 kg, at least 20 mg / 60 kg, at least 30 mg / 60 kg, per 60 kilograms, for each of the above conjugates and fusion proteins. At least 50 mg / 60 kg. For example, when administered intravenously, the dose is 1 mg / 60 kg, 2 mg / 60 kg, 5 mg / 60 kg, 20 mg / 60 kg, or 50 mg / 60 kg, for example, 0.5 to 50 mg / 60 kg. In another example, the therapeutically effective amount is at least 10 μg / kg, such as at least 100 μg / kg, at least 500 μg / kg or at least 500 μg / kg, eg, 100 μg / kg, 250 μg / kg when administered intratumorally or intraperitoneally. Doses such as kg, about 500 μg / kg, 750 μg / kg, or 1000 μg / kg, for example, 10 μg / kg to 1000 μg / kg. In one example, therapeutically effective amounts are 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml administered in topical solution. , Or at least 1 μg / ml, such as at least 500 μg / ml, such as between 20 μg / ml and 100 μg / ml, such as 100 μg / ml.
上記した投与量を、１回もしくは複数回にわたる分割用量（２、３、または４回の用量など）または単一の製剤で投与することができる。 The above doses can be administered in single or multiple divided doses (such as 2, 3, or 4 doses) or in a single formulation.
ビオチン改変二量体とフタロシアニン染料とのコンジュゲートと、本発明の融合タンパク質はそれぞれ、単独で投与することもでき、薬学的に許容されるキャリアの存在下で投与することもでき、他の治療剤（他の抗がん剤など）の存在下で投与することもできる。 The conjugate of the biotin-modified dimer with the phthalocyanine dye and the fusion protein of the invention can each be administered alone or in the presence of a pharmaceutically acceptable carrier, and other therapies. It can also be administered in the presence of agents (such as other anti-cancer agents).
ビオチン改変二量体とフタロシアニン染料とのコンジュゲートと、本発明の融合タンパク質は、循環している腫瘍細胞又は固形腫瘍の細胞などの標的細胞又は標的組織に結合することができる。その後に、光を照射すると、上記コンジュゲート又は複合体は、光を吸収し、標的細胞又は組織を損傷又は破壊することができる。 The conjugate of the biotin-modified dimer and the phthalocyanine dye and the fusion protein of the present invention can bind to a target cell or tissue such as a circulating tumor cell or a solid tumor cell. Subsequent irradiation with light allows the conjugate or complex to absorb the light and damage or destroy the target cell or tissue.
光免疫療法において照射光の波長は、好ましくは６６０〜７４０ｎｍであり、例えば６６０ｎｍ、６７０ｎｍ、６８０ｎｍ、６９０ｎｍ、７００ｎｍ、７１０ｎｍ、７２０ｎｍ、７３０ｎｍ、又は７４０ｎｍの波長を有する。光の照射は、近赤外（ＮＩＲ）発光ダイオードを備えたデバイスを使用することによって行ってもよい。 In photoimmunotherapy, the wavelength of the irradiation light is preferably 660 to 740 nm, and has a wavelength of, for example, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, or 740 nm. Light irradiation may be performed by using a device equipped with a near infrared (NIR) light emitting diode.
光の照射量は、少なくとも１Ｊ／ｃｍ２、例えば、少なくとも４Ｊ／ｃｍ２、少なくとも１０Ｊ／ｃｍ２、少なくとも１５Ｊ／ｃｍ２、少なくとも２０Ｊ／ｃｍ２、少なくとも５０Ｊ／ｃｍ２、または少なくとも１００Ｊ／ｃｍ２、例えば、１〜５００Ｊ／ｃｍ２である。光照射は、複数回（例えば、２、３、４、５、６、７、８、９、または１０回）実施してもよい。 The dose of light is at least 1 J / cm 2 , for example at least 4 J / cm 2 , at least 10 J / cm 2 , at least 15 J / cm 2 , at least 20 J / cm 2 , at least 50 J / cm 2 , or at least 100 J / cm 2. For example, 1 to 500 J / cm 2 . Light irradiation may be performed a plurality of times (for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).
以下の実施例により本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.
合成例１：
2-（（4-（5-（（3aS、4S、6aR）-2-イミニオヘキサヒドロ-1H-チエノ[3,4-d]イミダゾール-4-イル）ペンタンアミド）ブチル）アミノ）-N-（2- （（4-（5-（（3aS、4S、6aR）-2-イミニオヘキサヒドロ-1H-チエノ[3,4-d]イミダゾール-4-イル）ペンタンアミド）ブチル）アミノ）-2-オキソエチル）-N- （2-（2-（2-（3-ヨードベンズアミド）エトキシ）エトキシ）エチル）-2-オキソエタン-1-アミニウム）2,2,2-トリフルオロアセテート：2-((4-(5-((3aS,4S,6aR)-2-Iminiohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)butyl)amino)-N-(2-((4-(5-((3aS,4S,6aR)-2-iminiohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)butyl)amino)-2-oxoethyl)-N-(2-(2-(2-(3-iodobenzamido)ethoxy)ethoxy)ethyl)-2-oxoethan-1-aminium) 2,2,2-trifluoroacetate
Synthesis example 1:
2-((4- (5-((3aS, 4S, 6aR) -2-iminiohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamide) butyl) amino) -N -(2-((4- (5-((3aS, 4S, 6aR) -2-iminiohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamide) butyl) amino) -2-oxoethyl) -N- (2- (2- (2- (3-iodobenzamide) ethoxy) ethoxy) ethyl) -2-oxoethane-1-aminium) 2,2,2-trifluoroacetate: 2- ((4- (5-((3aS, 4S, 6aR) -2-Iminiohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamido) butyl) amino) -N- (2-((( 4- (5-((3aS, 4S, 6aR) -2-iminiohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamido) butyl) amino) -2-oxoethyl) -N- (2 -(2-(2- (3-iodobenzamido) ethoxy) ethoxy) ethyl) -2-oxoethan-1-aminium) 2,2,2-trifluoroacetate
ビスイミノビオチン1 (2.7 mg, 2.06 μmol)のDMF (100 μl)溶液に、室温でDMF (100 μl)に溶解した化合物2 (0.7 mg, 2.06 μmol)とトリエチルアミン (4.3 μl, 30.9 μmol)を加えた。アルゴン雰囲気下、室温5時間攪拌し、溶媒を減圧除去した。得られた粗生成物を逆相HPLC（グラジエント：0% for 5 min; 0-100% for 90 min CH3CN in 0.1% CF3COOH水溶液, 保持時間：41.5 min, YMC-Triart C18, flow rate = 3.5 ml/min）で精製し、標題化合物3 (1.5 mg, 収率 51％)を得た。LRMS (ESI): m/z 362 [M+3H]3+.
標題化合物3のことをPsycheＪ-ヨウ素体とも称する。
To a solution of bisiminobiotin 1 (2.7 mg, 2.06 μmol) in DMF (100 μl), compound 2 (0.7 mg, 2.06 μmol) dissolved in DMF (100 μl) at room temperature and triethylamine (4.3 μl, 30.9 μmol) were added. It was. The solvent was removed under reduced pressure by stirring at room temperature for 5 hours under an argon atmosphere. The obtained crude product was subjected to reverse phase HPLC (gradient: 0% for 5 min; 0-100% for 90 min CH 3 CN in 0.1% CF 3 COOH aqueous solution, holding time: 41.5 min, YMC-Triart C18, flow rate. Purification at (= 3.5 ml / min) gave the title compound 3 (1.5 mg, yield 51%). LRMS (ESI): m / z 362 [M + 3H] 3+ .
The title compound 3 is also referred to as PsycheJ-iodine.
実施例１：タンパク質の発現と精製
V2122は、国際公開ＷＯ２０１５／１２５８２０の実施例３（国際公開ＷＯ２０１５／１２５８２０の配列番号４）に記載されているストレプトアビジン変異体である。V2122のアミノ酸配列（Ｃ末端に６×Ｈｉｓタグを有する配列）を配列表の配列番号１に記載する。
Example 1: Protein expression and purification
V2122 is a streptavidin variant described in Example 3 of WO2015 / 125820 (SEQ ID NO: 4 of WO2015 / 125820). The amino acid sequence of V2122 (a sequence having a 6 × His tag at the C-terminal) is shown in SEQ ID NO: 1 of the sequence listing.
scFv-V2122は、上記のV2122に、CEACAM5に対する一本鎖抗体（scFv）を結合したものである。このscFv型の抗CEACAM5抗体は特許文献US7626011B2に記載されているscFv配列である。scFv型の抗CEACAM5抗体のアミノ酸配列を配列表の配列番号２に記載する。また、scFv型の抗CEACAM5抗体とV2122とをアミノ酸リンカー(GGGGSGGGG)（配列番号６）で結合したCEA-V2122のアミノ酸配列を配列表の配列番号３に記載する。 scFv-V2122 is a single-chain antibody (scFv) against CEACAM5 bound to the above V2122. This scFv-type anti-CEACAM5 antibody is the scFv sequence described in Patent Document US7626011B2. The amino acid sequence of the scFv type anti-CEACAM5 antibody is shown in SEQ ID NO: 2 of the sequence listing. In addition, the amino acid sequence of CEA-V2122 in which scFv-type anti-CEACAM5 antibody and V2122 are bound with an amino acid linker (GGGGSGGGG) (SEQ ID NO: 6) is shown in SEQ ID NO: 3 of the sequence listing.
CEA-V2122融合タンパク質の発現のために、大腸菌にて分泌発現するためのpelBシグナルをN末端に、また 6xHis-Tag配列をC末端に組み込んだCEA-V2122遺伝子配列のDNAコドンを大腸菌に最適化し人工遺伝子合成を行なった。このアミノ酸配列を配列表の配列番号４、DNA配列を配列表の配列番号５に記載する。 For the expression of the CEA-V2122 fusion protein, the DNA codon of the CEA-V2122 gene sequence with the pelB signal secreted and expressed in E. coli at the N-terminus and the 6xHis-Tag sequence at the C-terminus was optimized for E. coli. Artificial gene synthesis was performed. This amino acid sequence is shown in SEQ ID NO: 4 of the sequence listing, and the DNA sequence is shown in SEQ ID NO: 5 of the sequence listing.
具体的なタンパク質発現ベクターは、pETDuet1ベクターのMCS2にシャペロンskp遺伝子が組み込まれたベクターを使用した。skp遺伝子は配列表の配列番号６に記載されたアミノ酸配列を元にコドンを大腸菌に最適化したDNAの人工遺伝子合成を行った。合成されたskp遺伝子はプライマー（AAGGAGATATACATATGGATAAAATTGCCATTGTTAATAT（配列番号７）, TTGAGATCTGCCATATGTTATTTCACTTGTTTCAGAACG（配列番号８））を使用しPCRで増幅し、制限酵素NdeIで直鎖化されたpETDue1ベクターのMCS2にIn-Fusion HD Cloning Kitをもちいてクローニングを行いpETDuet_skp とした。次に、 pETDuet_skp のMCS1にCEA-V2122遺伝子を組み込んだ。具体的には、人工合成されたCEA-V2122遺伝子をプライマー（AGAAGGAGATATACCATGAAATATCTGCTGCCGAC（配列番号９）、CGCCGAGCTCGAATTTTAATGATGGTGATGATGATG（配列番号１０））を用いPCRにて増幅した。また、pETDuet_skp をプライマー（GGTATATCTCCTTCTTAAAGTTAAAC（配列番号１１）、AATTCGAGCTCGGCGCGCCTGCAG（配列番号１２））を使用しPCRにて直鎖化した。PCRにより増幅された CEA-V2122と直鎖化された pETDuet_skp を In-Fusion HD Cloning Kit をもちいてクローニングを行った。クローニングされたベクターはシーケンスにより組み込まれた遺伝子配列の確認を行い pETDuet_CEA-V2122_skp とした。 As a specific protein expression vector, a vector in which the chaperone skp gene was integrated into MCS2 of the pETDuet1 vector was used. For the skp gene, artificial gene synthesis of DNA with codons optimized for Escherichia coli was performed based on the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing. The synthesized skp gene was amplified by PCR using primers (AAGGAGATATACATATGGATAAAATTGCCATTGTTAATAT (SEQ ID NO: 7), TTGAGATCTGCCATATGTTATTTCACTTGTTTCAGAACG (SEQ ID NO: 8)) and in-Fusion HD Cloning into MCS2 of the pETDue1 vector linearized with the restriction enzyme NdeI. It was cloned using and used as pETDuet_skp. Next, the CEA-V2122 gene was integrated into MCS1 of pETDuet_skp. Specifically, the artificially synthesized CEA-V2122 gene was amplified by PCR using primers (AGAAGGAGATATACCATGAAATATCTGCTGCCGAC (SEQ ID NO: 9), CGCCGAGCTCGAATTTTAATGATGGTGATGATGATG (SEQ ID NO: 10)). In addition, pETDuet_skp was linearized by PCR using primers (GGTATATCTCCTTCTTAAAGTTAAAC (SEQ ID NO: 11), AATTCGAGCTCGGCGCGCCTGCAG (SEQ ID NO: 12)). CEA-V2122 amplified by PCR and pETDuet_skp linearized with PCR were cloned using the In-Fusion HD Cloning Kit. The cloned vector was designated as pETDuet_CEA-V2122_skp by confirming the integrated gene sequence by sequencing.
CEA-V2122 del5はpETDuet_CEA-V2122_skpをテンプレートとし、PCRにてC末端から１１番目のアミノ酸をコードするコドンをストップコドンに変え6xHis tagを含む１１アミノ酸を除去したアミノ酸配列となる。具体的にはプライマー（AGTTAAATAACGAGCTCGGCGCGCCTG（配列番号１３）,GCTCGTTATTTAACTTTGGTGAATGTATC（配列番号１４））を使用してpETDuet_CEA-V2122_skp をテンプレートにしPCRにてC末端から１１番目のアミノ酸をコードするコドンをストップコドンに変異を導入した。PCR産物は定法に従い、制限酵素Dnp I で処理し、その後コンピテントセル DH5a (TOYOBO)にトランスフォーメンションした。形質転換した大腸菌はLBプレートに播種し、翌日に2mLのLB培地にコロニーを植菌し培養を行い、miniprep kit (QIAGEN)にてプラスミドを調製しシーケンスを行い変異の導入を確認した。ペリプラズム発現のためのpelBシグナル配列を含む変異導入前の遺伝子配列を配列番号１５、アミノ酸配列を配列番号１６、また変異導入後の遺伝子配列を配列番号１７、アミノ酸配列を配列番号１８で示す。また、変異を導入したプラスミドベクターをpETDuet_CEA-V2122-del5_skpとした。配列番号１８においてV2122からＣ末端の6xHis tagを含む１１アミノ酸を除去した部分に対応するアミノ酸配列を配列番号１９に示す。 CEA-V2122 del5 uses pETDuet_CEA-V2122_skp as a template, and the codon encoding the 11th amino acid from the C-terminal is changed to a stop codon by PCR, and 11 amino acids including 6xHis tag are removed. Specifically, using primers (AGTTAAATAACGAGCTCGGCGCGCCTG (SEQ ID NO: 13), GCTCGTTATTTAACTTTGGTGAATGTATC (SEQ ID NO: 14)), pETDuet_CEA-V2122_skp is used as a template, and the codon encoding the 11th amino acid from the C-terminal is mutated to a stop codon by PCR. Introduced. The PCR product was treated with the restriction enzyme Dnp I according to a conventional method, and then transformed into competent cell DH5a (TOYOBO). The transformed Escherichia coli was inoculated on an LB plate, and the next day, colonies were inoculated into 2 mL of LB medium and cultured, a plasmid was prepared using a miniprep kit (QIAGEN), and sequencing was performed to confirm the introduction of mutations. The gene sequence before the introduction of the mutation containing the pelB signal sequence for periplasmic expression is shown by SEQ ID NO: 15, the amino acid sequence is shown by SEQ ID NO: 16, the gene sequence after the introduction of the mutation is shown by SEQ ID NO: 17, and the amino acid sequence is shown by SEQ ID NO: 18. The plasmid vector into which the mutation was introduced was pETDuet_CEA-V2122-del5_skp. The amino acid sequence corresponding to the portion of SEQ ID NO: 18 from which 11 amino acids including the C-terminal 6xHis tag have been removed from V2122 is shown in SEQ ID NO: 19.
タンパク質発現のために、pETDuet_CEA-V2122-del5_skpをBL21(DE3)(ニッポン・ジーン社)に形質転換し 2xYT培地(SIGMA-ADLRICH社)、37°Cにて一晩、前培養を行った。前培養を行った培地を 新しい培地に100倍希釈になるように添加し、OD(600nm) = 0.5〜2.0になるまで37℃で培 養を行った。次に最終濃度0.5mM IPTGを添加し37℃で4時間培養し培養上清を回収した後、4℃で保存した。 For protein expression, pETDuet_CEA-V2122-del5_skp was transformed into BL21 (DE3) (Nippon Gene) and precultured overnight in 2xYT medium (SIGMA-ADLRICH) at 37 ° C. The pre-cultured medium was added to a new medium at 100-fold dilution and cultivated at 37 ° C until OD (600 nm) = 0.5-2.0. Next, the final concentration of 0.5 mM IPTG was added, and the cells were cultured at 37 ° C. for 4 hours to collect the culture supernatant, which was then stored at 4 ° C.
回収した培養上清を用いてProtein L カラムによる精製を行った。具体的にはCapto L (GE Healthcare Life Sciences) 1mLをPD-10カラムに充填し、10カラムボリュームのPBSで平衡化後、上述の粗精製物をアプライし、10カラムボリュームのPBSで洗浄後、10mM グリシン塩酸pH2.0で溶出しVivaspin Turbo 15 (MWCO 100,000) による遠心濃縮を行なった。さらに PD-10 (GE Healthcare Life Sciences社) を用いてPBSへバッファー置換を行い、さらに Vivaspin Turbo 4 (MWCO 100,000) による遠心濃縮を行い最終精製物とした。同様に発現精製を行ったCEA-V2122とCEA-V2122-del5をSDS-PAGE電気泳動後、CBB染色を行なった結果を図1に示す。SDS-PAGEゲルは Mini-PROTEAN TGX 4-15% (Bio-Rad社) を使用し CBB染色液は Bullet CBB Stain One(Ready To Use) (ナカライテスク社）を使用した。 The collected culture supernatant was used for purification by a Protein L column. Specifically, 1 mL of Capto L (GE Healthcare Life Sciences) was filled in a PD-10 column, equilibrated with 10-column volume of PBS, applied with the above crude product, washed with 10-column volume of PBS, and then washed. It was eluted with 10 mM glycine hydrochloride at pH 2.0 and centrifuged with Vivaspin Turbo 15 (MWCO 100,000). Furthermore, buffer was substituted into PBS using PD-10 (GE Healthcare Life Sciences), and centrifugal concentration was performed with Vivaspin Turbo 4 (MWCO 100,000) to obtain the final purified product. Figure 1 shows the results of CBB staining of CEA-V2122 and CEA-V2122-del5, which had been similarly expressed and purified, after SDS-PAGE electrophoresis. Mini-PROTEAN TGX 4-15% (Bio-Rad) was used for the SDS-PAGE gel, and Bullet CBB Stain One (Ready To Use) (Nacalai Tesque) was used for the CBB stain.
実施例２：CEA-V2122-del5とビオチン改変体との相互作用解析
CEA-V2122-del5とビオチン改変体との相互作用解析を Biacore T200 を用いて実施した。使用したビオチン改変体は、製造例１に記載のPsycheＪ-ヨウ素体である。
Example 2: Interaction analysis between CEA-V2122-del5 and biotin variant
The interaction analysis between CEA-V2122-del5 and the biotin variant was performed using Biacore T200. The biotin variant used is the PsycheJ-iodine compound described in Production Example 1.
具体体的な解析方法は以下の通りである。アミンカップリングキットを使用し、Sensor Chip CM5 に目標値 3000RUとなるように設定し、精製された CEA-V2122-del5 の固定化を行なった。アナライトの濃度は 2.5E-09 Mを２倍希釈し、2.5E-09Mを含む５種類の希釈系列を調製し測定に使用した。相互作用解析は Single-Cycle Kinetics 解析にてデータの取得を行った。得られたデータを Biacore T200 Evaluation Software, version 2.0 にて Bivalent Analyze モードによるカーブフィッティングを実施しka1=6.3E+03, kd1=8.0E-06の値を得た。また、Bivalent AnalyzeではKD＝kd1/ka1で評価できることからKD＝kd1/ka1= 6.3E+03/8.0E-06=1.3E-09の評価値を得た。それらの結果を図２に示す。
図２に示されるセンサーグラム、KD値よりCEA-V2122 del5とビオチン改変体は強く結合することが確認された。
The specific analysis method is as follows. Using an amine coupling kit, the Sensor Chip CM5 was set to a target value of 3000 RU, and purified CEA-V2122-del5 was immobilized. As for the concentration of allite, 2.5E-09 M was diluted 2-fold, and 5 kinds of dilution series including 2.5E-09M were prepared and used for the measurement. For the interaction analysis, data was acquired by Single-Cycle Kinetics analysis. The obtained data were subjected to curve fitting in Bivalent Analyze mode with Biacore T200 Evaluation Software, version 2.0, and the values of ka1 = 6.3E + 03 and kd1 = 8.0E-06 were obtained. Moreover, to obtain an evaluation value of the K D = kd1 / ka1 = 6.3E + 03 / 8.0E-06 = 1.3E-09 because it can be evaluated by K D = kd1 / ka1 in Bivalent Analyze. The results are shown in FIG.
Sensorgram shown in Figure 2, it was confirmed that from the K D values CEA-V2122 del5 and biotin variants bind strongly.
配列番号１
AEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH
SEQ ID NO: 1
AEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH
配列番号２(sm3E-scFv配列)
QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIK
SEQ ID NO: 2 (sm3E-scFv sequence)
QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACS
配列番号３
QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH
SEQ ID NO: 3
QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH
配列番号４
MKYLLPTAAAGLLLLAAQPAMAQVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH
SEQ ID NO: 4
MKYLLPTAAAGLLLLAAQPAMAQVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH
配列番号５
ATGAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCAATGGCACAGGTTAAACTGGAACAGAGCGGTGCCGAAGTTGTTAAACCGGGTGCAAGCGTTAAACTGAGCTGTAAAGCAAGCGGCTTTAACATCAAAGATAGCTATATGCATTGGCTGCGTCAGGGTCCGGGTCAGCGTCTGGAATGGATTGGTTGGATTGATCCGGAAAATGGTGATACCGAATATGCACCGAAATTTCAGGGTAAAGCAACCTTTACCACCGATACCAGCGCAAATACCGCATATCTGGGTCTGAGCAGCCTGCGTCCGGAAGATACCGCAGTGTATTATTGTAATGAAGGCACCCCGACCGGTCCGTATTATTTCGATTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGGTGGTGGTGGTAGTGGTGGCGGTGGTTCAGGCGGTGGCGGTAGCGAAAATGTTCTGACCCAGAGCCCGAGCAGCATGAGCGTTAGCGTTGGTGATCGTGTTAATATTGCATGTAGCGCAAGCAGCAGCGTTCCGTACATGCACTGGCTGCAGCAGAAACCGGGTAAAAGCCCGAAACTGCTGATTTATCTGACCAGCAATCTGGCAAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAGTGGCACCGATTATAGCCTGACCATTAGCAGCGTGCAGCCTGAAGATGCAGCAACCTATTATTGTCAGCAGCGTAGCAGTTATCCGCTGACCTTTGGTGGTGGCACCAAACTGGAAATTAAAGGGGGTGGTGGCTCAGGTGGCGGAGGTGCAGAAGCAGGTATTACCGGTACATGGTCAGATCAGCTGGGTGATACCTTTATTGTTACCGCAGGCGCAGATGGTGCACTGACCGGCACCTATGAAAATGCAGTTGGTGGTGCAGAAAGCCGTTATGTGCTGACCGGTCGTTATGATAGCGCACCGGCAACCGATGGTAGCGGCACCGCACTGGGTTGGACCGTTGCATGGAAAAATAACAGCAAAAATGCACATAGCGCAACCACCTGGTCAGGTCAGTATGTGGGTGGTGCCGATGCCAAAATTAACACCCAGTGGCTGCTGACCAGCGGTACAACCAATGCAAATGCCTGGAAAAGTACCCTGGTTGGTCATGATACATTCACCAAAGTTAAACCGAGCGCAGCAAGCCATCATCATCACCATCATTAA
SEQ ID NO: 5
ATGAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCAATGGCACAGGTTAAACTGGAACAGAGCGGTGCCGAAGTTGTTAAACCGGGTGCAAGCGTTAAACTGAGCTGTAAAGCAAGCGGCTTTAACATCAAAGATAGCTATATGCATTGGCTGCGTCAGGGTCCGGGTCAGCGTCTGGAATGGATTGGTTGGATTGATCCGGAAAATGGTGATACCGAATATGCACCGAAATTTCAGGGTAAAGCAACCTTTACCACCGATACCAGCGCAAATACCGCATATCTGGGTCTGAGCAGCCTGCGTCCGGAAGATACCGCAGTGTATTATTGTAATGAAGGCACCCCGACCGGTCCGTATTATTTCGATTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGGTGGTGGTGGTAGTGGTGGCGGTGGTTCAGGCGGTGGCGGTAGCGAAAATGTTCTGACCCAGAGCCCGAGCAGCATGAGCGTTAGCGTTGGTGATCGTGTTAATATTGCATGTAGCGCAAGCAGCAGCGTTCCGTACATGCACTGGCTGCAGCAGAAACCGGGTAAAAGCCCGAAACTGCTGATTTATCTGACCAGCAATCTGGCAAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAGTGGCACCGATTATAGCCTGACCATTAGCAGCGTGCAGCCTGAAGATGCAGCAACCTATTATTGTCAGCAGCGTAGCAGTTATCCGCTGACCTTTGGTGGTGGCACCAAACTGGAAATTAAAGGGGGTGGTGGCTCAGGTGGCGGAGGTGCAGAAGCAGGTATTACCGGTACATGGTCAGATCAGCTGGGTGATACCTTTATTGTTACCGCAGGCGCAGATGGTGCACTGACCGGCACCTATGAAAATGCAGTTGGTGGTGCAGAAAGCCGTTATGTGCTGACCGGTCGTTATGATAGCGCACCGGCAACCGATGGTAGCGGCACCGCACTGG GTTGGACCGTTGCATGGAAAAATAACAGCAAAAATGCACATAGCGCAACCACCTGGTCAGGTCAGTATGTGGGTGGTGCCGATGCCAAAATTAACACCCAGTGGCTGCTGACCAGCGGTACAACCAATGCAAATGCCTGGAAAAGTACCCTGGTTGGTCATGATACATTCACCAAAG
配列番号１５
ATGAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCAATGGCACAGGTTAAACTGGAACAGAGCGGTGCCGAAGTTGTTAAACCGGGTGCAAGCGTTAAACTGAGCTGTAAAGCAAGCGGCTTTAACATCAAAGATAGCTATATGCATTGGCTGCGTCAGGGTCCGGGTCAGCGTCTGGAATGGATTGGTTGGATTGATCCGGAAAATGGTGATACCGAATATGCACCGAAATTTCAGGGTAAAGCAACCTTTACCACCGATACCAGCGCAAATACCGCATATCTGGGTCTGAGCAGCCTGCGTCCGGAAGATACCGCAGTGTATTATTGTAATGAAGGCACCCCGACCGGTCCGTATTATTTCGATTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGGTGGTGGTGGTAGTGGTGGCGGTGGTTCAGGCGGTGGCGGTAGCGAAAATGTTCTGACCCAGAGCCCGAGCAGCATGAGCGTTAGCGTTGGTGATCGTGTTAATATTGCATGTAGCGCAAGCAGCAGCGTTCCGTACATGCACTGGCTGCAGCAGAAACCGGGTAAAAGCCCGAAACTGCTGATTTATCTGACCAGCAATCTGGCAAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAGTGGCACCGATTATAGCCTGACCATTAGCAGCGTGCAGCCTGAAGATGCAGCAACCTATTATTGTCAGCAGCGTAGCAGTTATCCGCTGACCTTTGGTGGTGGCACCAAACTGGAAATTAAAGGGGGTGGTGGCTCAGGTGGCGGAGGTGCAGAAGCAGGTATTACCGGTACATGGTCAGATCAGCTGGGTGATACCTTTATTGTTACCGCAGGCGCAGATGGTGCACTGACCGGCACCTATGAAAATGCAGTTGGTGGTGCAGAAAGCCGTTATGTGCTGACCGGTCGTTATGATAGCGCACCGGCAACCGATGGTAGCGGCACCGCACTGGGTTGGACCGTTGCATGGAAAAATAACAGCAAAAATGCACATAGCGCAACCACCTGGTCAGGTCAGTATGTGGGTGGTGCCGATGCCAAAATTAACACCCAGTGGCTGCTGACCAGCGGTACAACCAATGCAAATGCCTGGAAAAGTACCCTGGTTGGTCATGATACATTCACCAAAGTTAAACCGAGCGCAGCAAGCCATCATCATCACCATCATTAA
SEQ ID NO: 15
ATGAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCAATGGCACAGGTTAAACTGGAACAGAGCGGTGCCGAAGTTGTTAAACCGGGTGCAAGCGTTAAACTGAGCTGTAAAGCAAGCGGCTTTAACATCAAAGATAGCTATATGCATTGGCTGCGTCAGGGTCCGGGTCAGCGTCTGGAATGGATTGGTTGGATTGATCCGGAAAATGGTGATACCGAATATGCACCGAAATTTCAGGGTAAAGCAACCTTTACCACCGATACCAGCGCAAATACCGCATATCTGGGTCTGAGCAGCCTGCGTCCGGAAGATACCGCAGTGTATTATTGTAATGAAGGCACCCCGACCGGTCCGTATTATTTCGATTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGGTGGTGGTGGTAGTGGTGGCGGTGGTTCAGGCGGTGGCGGTAGCGAAAATGTTCTGACCCAGAGCCCGAGCAGCATGAGCGTTAGCGTTGGTGATCGTGTTAATATTGCATGTAGCGCAAGCAGCAGCGTTCCGTACATGCACTGGCTGCAGCAGAAACCGGGTAAAAGCCCGAAACTGCTGATTTATCTGACCAGCAATCTGGCAAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAGTGGCACCGATTATAGCCTGACCATTAGCAGCGTGCAGCCTGAAGATGCAGCAACCTATTATTGTCAGCAGCGTAGCAGTTATCCGCTGACCTTTGGTGGTGGCACCAAACTGGAAATTAAAGGGGGTGGTGGCTCAGGTGGCGGAGGTGCAGAAGCAGGTATTACCGGTACATGGTCAGATCAGCTGGGTGATACCTTTATTGTTACCGCAGGCGCAGATGGTGCACTGACCGGCACCTATGAAAATGCAGTTGGTGGTGCAGAAAGCCGTTATGTGCTGACCGGTCGTTATGATAGCGCACCGGCAACCGATGGTAGCGGCACCGCACTGG GTTGGACCGTTGCATGGAAAAATAACAGCAAAAATGCACATAGCGCAACCACCTGGTCAGGTCAGTATGTGGGTGGTGCCGATGCCAAAATTAACACCCAGTGGCTGCTGACCAGCGGTACAACCAATGCAAATGCCTGGAAAAGTACCCTGGTTGGTCATGATACATTCACCAAAG
配列番号１６
MKYLLPTAAAGLLLLAAQPAMAQVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH*
SEQ ID NO: 16
MKYLLPTAAAGLLLLAAQPAMAQVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH *
配列番号１７
ATGAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCAATGGCACAGGTTAAACTGGAACAGAGCGGTGCCGAAGTTGTTAAACCGGGTGCAAGCGTTAAACTGAGCTGTAAAGCAAGCGGCTTTAACATCAAAGATAGCTATATGCATTGGCTGCGTCAGGGTCCGGGTCAGCGTCTGGAATGGATTGGTTGGATTGATCCGGAAAATGGTGATACCGAATATGCACCGAAATTTCAGGGTAAAGCAACCTTTACCACCGATACCAGCGCAAATACCGCATATCTGGGTCTGAGCAGCCTGCGTCCGGAAGATACCGCAGTGTATTATTGTAATGAAGGCACCCCGACCGGTCCGTATTATTTCGATTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGGTGGTGGTGGTAGTGGTGGCGGTGGTTCAGGCGGTGGCGGTAGCGAAAATGTTCTGACCCAGAGCCCGAGCAGCATGAGCGTTAGCGTTGGTGATCGTGTTAATATTGCATGTAGCGCAAGCAGCAGCGTTCCGTACATGCACTGGCTGCAGCAGAAACCGGGTAAAAGCCCGAAACTGCTGATTTATCTGACCAGCAATCTGGCAAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAGTGGCACCGATTATAGCCTGACCATTAGCAGCGTGCAGCCTGAAGATGCAGCAACCTATTATTGTCAGCAGCGTAGCAGTTATCCGCTGACCTTTGGTGGTGGCACCAAACTGGAAATTAAAGGGGGTGGTGGCTCAGGTGGCGGAGGTGCAGAAGCAGGTATTACCGGTACATGGTCAGATCAGCTGGGTGATACCTTTATTGTTACCGCAGGCGCAGATGGTGCACTGACCGGCACCTATGAAAATGCAGTTGGTGGTGCAGAAAGCCGTTATGTGCTGACCGGTCGTTATGATAGCGCACCGGCAACCGATGGTAGCGGCACCGCACTGGGTTGGACCGTTGCATGGAAAAATAACAGCAAAAATGCACATAGCGCAACCACCTGGTCAGGTCAGTATGTGGGTGGTGCCGATGCCAAAATTAACACCCAGTGGCTGCTGACCAGCGGTACAACCAATGCAAATGCCTGGAAAAGTACCCTGGTTGGTCATGATACATTCACCAAAGTTAAATAA
SEQ ID NO: 17
ATGAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCAATGGCACAGGTTAAACTGGAACAGAGCGGTGCCGAAGTTGTTAAACCGGGTGCAAGCGTTAAACTGAGCTGTAAAGCAAGCGGCTTTAACATCAAAGATAGCTATATGCATTGGCTGCGTCAGGGTCCGGGTCAGCGTCTGGAATGGATTGGTTGGATTGATCCGGAAAATGGTGATACCGAATATGCACCGAAATTTCAGGGTAAAGCAACCTTTACCACCGATACCAGCGCAAATACCGCATATCTGGGTCTGAGCAGCCTGCGTCCGGAAGATACCGCAGTGTATTATTGTAATGAAGGCACCCCGACCGGTCCGTATTATTTCGATTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGGTGGTGGTGGTAGTGGTGGCGGTGGTTCAGGCGGTGGCGGTAGCGAAAATGTTCTGACCCAGAGCCCGAGCAGCATGAGCGTTAGCGTTGGTGATCGTGTTAATATTGCATGTAGCGCAAGCAGCAGCGTTCCGTACATGCACTGGCTGCAGCAGAAACCGGGTAAAAGCCCGAAACTGCTGATTTATCTGACCAGCAATCTGGCAAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAGTGGCACCGATTATAGCCTGACCATTAGCAGCGTGCAGCCTGAAGATGCAGCAACCTATTATTGTCAGCAGCGTAGCAGTTATCCGCTGACCTTTGGTGGTGGCACCAAACTGGAAATTAAAGGGGGTGGTGGCTCAGGTGGCGGAGGTGCAGAAGCAGGTATTACCGGTACATGGTCAGATCAGCTGGGTGATACCTTTATTGTTACCGCAGGCGCAGATGGTGCACTGACCGGCACCTATGAAAATGCAGTTGGTGGTGCAGAAAGCCGTTATGTGCTGACCGGTCGTTATGATAGCGCACCGGCAACCGATGGTAGCGGCACCGCACTGG GTTGGACCGTTGCATGGAAAAATAACAGCAAAAATGCATAGCGCAACCACCTGGTCAGGTCAGTATGTGGGTGGTGCCGATGCCAAAATTAACACCCAGTGGCTGCTGACCAGCGGTACAACCAATGCAAATGCCTGGAAAAGTACCGGTTGGTCATGATACATTCACCAAAGTTAAATAA
配列番号１８
MKYLLPTAAAGLLLLAAQPAMAQVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVK*
SEQ ID NO: 18
MKYLLPTAAAGLLLLAAQPAMAQVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVK *
配列番号１９
AEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTW
SGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVK*
SEQ ID NO: 19
AEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTW
SGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVK *
Claims (8)
Ｘ１ａ、Ｘ１ｂ、Ｘ２ａ及びＸ２ｂはそれぞれ独立にＯ又はＮＨを示し、
Ｙ１及びＹ２はそれぞれ独立にＣ又はＳを示し、
Ｚ１及びＺ２はそれぞれ独立にＯ、Ｓ又はＮＨを示し、
Ｖ１及びＶ２はそれぞれ独立にＳ又はＳ＋−Ｏ−を示し、ｎ１及びｎ２はそれぞれ独立に０又は１の整数を示し、
Ｌ１及びＬ２はそれぞれ独立に、２価の連結基を示し、
Ｌ３は、診断用物質又は治療用物質と結合できる官能基を末端に含む基であり、
Ｌ４は、３価の連結基を示す。） (1) The fusion protein according to any one of claims 1 to 4, and (2) a compound represented by the following formula (1) or a salt thereof, and a conjugate of a diagnostic substance or a therapeutic substance. , Cancer treatment or diagnostic kit.
X1a, X1b, X2a and X2b independently indicate O or NH, respectively.
Y 1 and Y 2 independently represent C or S, respectively.
Z 1 and Z 2 independently represent O, S or NH, respectively.
V 1 and V 2 independently represent S or S + −O − , and n1 and n2 independently represent an integer of 0 or 1, respectively.
L 1 and L 2 each independently represent a divalent linking group.
L 3 is a group having a functional group at the end capable of binding to a diagnostic substance or a therapeutic substance.
L 4 represents a trivalent linking group. )
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| JP2019179280A JP2021052669A (en) | 2019-09-30 | 2019-09-30 | Fusion protein of protein recognition substance and streptavidin mutant |
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| JP2019179280A JP2021052669A (en) | 2019-09-30 | 2019-09-30 | Fusion protein of protein recognition substance and streptavidin mutant |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116554300A (en) * | 2023-04-27 | 2023-08-08 | 湖北医药学院 | Polypeptide capable of interacting with clostridium difficile toxin TcdB and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080003646A1 (en) * | 2002-07-01 | 2008-01-03 | Cancer Research Technology Limited | Antibodies against tumor surface antigens |
| WO2015125820A1 (en) * | 2014-02-18 | 2015-08-27 | サヴィッド・セラピューティックス株式会社 | Biotin variant, streptavidin mutant, and uses thereof |
| JP2018528268A (en) * | 2015-08-18 | 2018-09-27 | アスピリアン セラピューティクス インコーポレイテッド | Compositions, combinations and related methods for photoimmunotherapy |
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2019
- 2019-09-30 JP JP2019179280A patent/JP2021052669A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080003646A1 (en) * | 2002-07-01 | 2008-01-03 | Cancer Research Technology Limited | Antibodies against tumor surface antigens |
| WO2015125820A1 (en) * | 2014-02-18 | 2015-08-27 | サヴィッド・セラピューティックス株式会社 | Biotin variant, streptavidin mutant, and uses thereof |
| JP2018528268A (en) * | 2015-08-18 | 2018-09-27 | アスピリアン セラピューティクス インコーポレイテッド | Compositions, combinations and related methods for photoimmunotherapy |
Non-Patent Citations (1)
| Title |
|---|
| JSMI REPORT, vol. 10, no. 1, JPN6019042507, 2016, pages 43 - 45, ISSN: 0005123672 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116554300A (en) * | 2023-04-27 | 2023-08-08 | 湖北医药学院 | Polypeptide capable of interacting with clostridium difficile toxin TcdB and application thereof |
| CN116554300B (en) * | 2023-04-27 | 2023-10-24 | 湖北医药学院 | Polypeptide capable of interacting with clostridium difficile toxin TcdB and application thereof |
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