JP6506691B2 - Fab領域結合性ペプチド - Google Patents
Fab領域結合性ペプチド Download PDFInfo
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- JP6506691B2 JP6506691B2 JP2015534279A JP2015534279A JP6506691B2 JP 6506691 B2 JP6506691 B2 JP 6506691B2 JP 2015534279 A JP2015534279 A JP 2015534279A JP 2015534279 A JP2015534279 A JP 2015534279A JP 6506691 B2 JP6506691 B2 JP 6506691B2
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- amino acid
- fab region
- peptide
- fab
- acid sequence
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
(2) 上記(1)に規定されるアミノ酸配列において、上記第13位、第19位、第30位および第33位を除く領域中で1個以上、20個以下のアミノ酸残基が欠損、置換および/または付加されたアミノ酸配列を有し、且つ、免疫グロブリンのFab領域への結合力が配列番号1のアミノ酸配列を有するペプチドよりも高いFab領域結合性ペプチド;または
(3) 上記(1)に規定されるアミノ酸配列に対して80%以上の配列同一性を有するアミノ酸配列を有し、且つ、免疫グロブリンのFab領域への結合力が配列番号1のアミノ酸配列を有するペプチドよりも高いFab領域結合性ペプチド(但し、上記(1)に規定されるアミノ酸配列における第13位、第19位、第30位および第33位から選択される1以上の位置のアミノ酸残基の置換は、(3)においてさらに変異しないものとする)。
上記[11]に記載のアフィニティー分離マトリックスと、Fab領域を含むタンパク質を含む液体試料とを接触させる工程と、
アフィニティー分離マトリックスに結合したFab領域を含むタンパク質を、アフィニティー分離マトリックスから分離する工程を含むことを特徴とする方法。
(1) 各種SpG−β1変異体の発現プラスミド調製
発現プラスミドの調製方法に関し、野生型SpG−β1を例に示す。野生型SpG−β1(配列番号1)のアミノ酸配列から逆翻訳を行い、当該ペプチドをコードする塩基配列(配列番号3)を設計した。次に、発現プラスミドの作製方法を図1に示す。野生型SpG−β1をコードするDNAは、同じ制限酵素サイトを有する2種の二本鎖DNA(f1とf2)を連結する形で調製し、発現ベクターのマルチクローニングサイトに組み込む。実際には、2種の二本鎖DNAと発現ベクターの3種の二本鎖DNAを連結する3断片ライゲーションによって、コードDNA調製とベクター組込みを同時に実施した。2種の二本鎖DNAの調製方法は、互いに30塩基程度の相補領域を含む2種の一本鎖オリゴDNA(f1−1/f1−2、または、f2−1/f2−2)を、オーバーラップPCRによって伸長し、目的の二本鎖DNAを調製した。具体的な実験操作については、次の通りとなる。一本鎖オリゴDNAf1−1(配列番号4)/f1−2(配列番号5)を外注によって合成し(シグマジェノシス社)、ポリメラーゼとしてPyrobest(タカラバイオ社)を用い、オーバーラップPCR反応を行った。PCR反応生成物をアガロース電気泳動にかけ、目的のバンドを切り出すことで抽出した二本鎖DNAを、制限酵素BamHIとEco52I(いずれもタカラバイオ社)により切断した。同様に、一本鎖オリゴDNAf2−1(配列番号6)/f2−2(配列番号7)を外注によって合成し、オーバーラップPCR反応を経て、合成・抽出した二本鎖DNAを、制限酵素Eco52IとEcoRI(いずれもタカラバイオ社)により切断した。次に、プラスミドベクターpGEX−6P−1(GEヘルスケア・バイオサイエンス社)のマルチクローニングサイト中のBamHI/EcoRIサイトに上記2種の二本鎖DNAをサブクローニングした。サブクローニングにおけるライゲーション反応は、Ligation high(TOYOBO社)を用いて、製品に添付のプロトコルに準ずる形で実施した。
上記(1)で得られた、各種SpG−β1変異体遺伝子を導入した各形質転換細胞を、アンピシリン含有2×YT培地にて、37℃で終夜培養した。これらの培養液を、100倍量程度のアンピシリン含有2×YT培地に接種し、37℃で約2時間培養した後で、終濃度0.1mMになるようIPTG(イソプロピル1−チオ−β−D−ガラクシド)を添加し、さらに、37℃にて18時間培養した。
(1) IgG由来Fabフラグメント(IgG−Fab)の調製
ヒト化モノクローナルIgG製剤を原料として、これをパパインによって、FabフラグメントとFcフラグメントに断片化し、Fabフラグメントのみを分離精製することで調製した。ここでは、抗Her2モノクローナル抗体(一般名「トラスツズマブ」)由来のIgG−Fabの調製方法を示すが、基本的には他のIgG−Fab、例えば、抗TNFαモノクローナル抗体(一般名「アダリムマブ」)由来のIgG−Fabも同様の方法で調製した。
表面プラズモン共鳴を利用したバイオセンサーBiacore3000(GEヘルスケア・バイオサイエンス社)を用いて、実施例1(2)で取得した各種SpG−β1変異体のIgG−Fabとの親和性を解析した。本実施例では、実施例2(1)で取得したIgG−Fabをセンサーチップに固定化し、各種ペプチドをチップ上に流して、両者の相互作用を検出した。IgG−FabのセンサーチップCM5への固定化は、N−ヒドロキシスクシンイミド(NHS)、および、N−エチル−N’−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)を用いたアミンカップリング法にて行い、ブロッキングにはエタノールアミンを用いた(センサーチップや固定化用試薬は、全てGEヘルスケアバイオサイエンス社製)。IgG−Fab溶液は、固定化用緩衝液(10mM CH3COOH−CH3COONa,pH4.5)を用いて10倍程度に希釈し、Biacore 3000付属のプロトコルに従い、センサーチップへ固定した。また、チップ上の別のフローセルに対して、EDC/NHSにより活性化した後にエタノールアミンを固定化する処理を行うことで、ネガティブ・コントロールとなるリファレンスセルも用意した。各種SpG−β1変異体は、ランニング緩衝液(20mM NaH2PO4−Na2HPO4,150mM NaCl,0.005% P−20,pH7.4)を用いて、0.1〜100μMの範囲で適宜調製し、各々のペプチド溶液を、流速40μL/minで60秒間センサーチップに添加した。測定温度25℃にて、添加時(結合相,60秒間)、および、添加終了後(解離相,60秒間)の結合反応曲線を順次観測した。各々の観測終了後に、20mM NaOH(30秒間)を添加してセンサーチップを再生した。この操作は、センサーチップ上に残った添加ペプチドの除去が目的であり、固定化したヒトIgGの結合活性がほぼ完全に戻ることを確認した。得られた結合反応曲線(リファレンスセルの結合反応曲線を差し引いた結合反応曲線)に対して、システム付属ソフトBIA evaluationを用いた1:1の結合モデルによるフィッティング解析を行い、ヒトIgGに対する親和定数(KA=kon/koff)を算出した。結果を表1に示す。
上記実施例2の実験と同様にして、SpG−β1変異体のIgG−Fabに対する親和性を測定した。IgG−Fabについては、上記実施例2の実験において、1種類のIgG−Fabで見た結果について、他の種類のIgG−Fabでも概ね同様の傾向が見られることを確認したので、1種類のみについて実験を行った。結果を表2に示す。
上記実施例3の実験と同様にして、SpG−β1変異体のIgG−Fabに対する親和性を測定した。この実験では、GSTを切断したGST切断型(Pep−)で測定を実施した。また、1ドメイン型(Pep.1d)だけでなく、C末端にCysが結合した1ドメイン型(Pep.1dC)とC末端にCysが結合した2ドメイン型(Pep.2dC)を用いて実験を行った。その結果を表3に示す。
Claims (10)
- 下記(1)〜(3)の何れかのFab領域結合性ペプチド。
(1) プロテインGのβ1ドメイン由来のアミノ酸配列(配列番号1)において、第13位の位置のアミノ酸残基がThrまたはSerに置換されているアミノ酸配列を有し、
前記アミノ酸配列の第19位、第30位および第33位から選択される1以上の位置のアミノ酸残基が更に置換されていても良く、
且つ、免疫グロブリンのFab領域への結合力が置換導入前よりも高いFab領域結合性ペプチドであって、
前記第19位のアミノ酸残基がVal、LeuまたはIleに置換されており、
前記第30位のアミノ酸残基がVal、LeuまたはIleに置換されており、
前記第33位のアミノ酸残基がPheに置換されているFab領域結合性ペプチド;
(2) 上記(1)に規定されるアミノ酸配列において、上記第13位、第19位、第30位および第33位を除く領域中で1個以上、5個以下のアミノ酸残基が欠損、置換および/または付加されたアミノ酸配列を有し、且つ、免疫グロブリンのFab領域への結合力が配列番号1のアミノ酸配列を有するペプチドよりも高いFab領域結合性ペプチド;または
(3) 上記(1)に規定されるアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列を有し、且つ、免疫グロブリンのFab領域への結合力が配列番号1のアミノ酸配列を有するペプチドよりも高いFab領域結合性ペプチド(但し、上記(1)に規定されるアミノ酸配列における第13位、並びに第19位、第30位および第33位から選択される1以上の位置のアミノ酸残基の置換は、(3)においてさらに変異しないものとする)。 - 上記(2)に規定されるアミノ酸配列において、上記欠損、置換および/または付加されたアミノ酸残基の位置が、第2位、第10位、第15位、第18位、第21位、第22位、第23位、第24位、第25位、第27位、第28位、第31位、第32位、第35位、第36位、第39位、第40位、第42位、第45位、第47位および第48位から選択される1以上である請求項1に記載のFab領域結合性ペプチド。
- 上記(2)に規定されるアミノ酸配列において、上記欠損、置換および/または付加されたアミノ酸残基の位置がN末端および/またはC末端である請求項1または2に記載のFab領域結合性ペプチド。
- 上記(3)に規定されるアミノ酸配列において、上記配列同一性が95%以上である請求項1〜3のいずれかに記載のFab領域結合性ペプチド。
- 請求項1〜4のいずれかに記載のFab領域結合性ペプチドを2個以上連結した複数ドメインを有することを特徴とするFab領域結合性ペプチド多量体。
- 請求項1〜4のいずれかに記載のFab領域結合性ペプチド、または請求項5に記載のFab領域結合性ペプチド多量体がリガンドとして水不溶性担体に固定化されたものであることを特徴とするアフィニティー分離マトリックス。
- Fab領域を含むタンパク質を製造する方法であって、
請求項6に記載のアフィニティー分離マトリックスと、Fab領域を含むタンパク質を含む液体試料とを接触させる工程と、
アフィニティー分離マトリックスに結合したFab領域を含むタンパク質を、アフィニティー分離マトリックスから分離する工程を含むことを特徴とする方法。 - 請求項1〜4のいずれかに記載のFab領域結合性ペプチドをコードすることを特徴とするDNA。
- 請求項8に記載のDNAを含むことを特徴とするベクター。
- 請求項9に記載のベクターにより形質転換されたものであることを特徴とする形質転換体。
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WO2016031902A1 (ja) * | 2014-08-28 | 2016-03-03 | 株式会社カネカ | Fab領域含有ペプチド用アフィニティー分離マトリックス |
JP6731345B2 (ja) | 2014-08-28 | 2020-07-29 | 株式会社カネカ | 免疫グロブリンg結合性ペプチド |
US20170305965A1 (en) * | 2014-08-28 | 2017-10-26 | Kaneka Corporation | Fab REGION-BINDING PEPTIDE |
WO2016136910A1 (ja) * | 2015-02-26 | 2016-09-01 | 株式会社カネカ | 改変型Fab領域結合性ペプチド |
KR102468989B1 (ko) * | 2016-11-18 | 2022-11-22 | 가고시마 유니버시티 | IgG 결합 펩타이드를 포함하는 고상 담체 및 IgG의 분리 방법 |
JP7118949B2 (ja) | 2017-03-31 | 2022-08-16 | 株式会社カネカ | 安定性改良型免疫グロブリン結合性ペプチド |
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WO2022010806A1 (en) | 2020-07-06 | 2022-01-13 | ALX Oncology Inc. | Methods for reducing the interference of drugs that bind therapeutic targets expressed on blood cells in serological assays |
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CN112778426B (zh) * | 2021-01-06 | 2023-12-12 | 深圳伯生生物传感技术有限公司 | 一种精准抗体核酸定向连接方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4977247A (en) | 1986-02-14 | 1990-12-11 | Genex Corporation | Immobilized protein G variants and the use thereof |
US5082773A (en) | 1986-02-14 | 1992-01-21 | Pharmacia Lkb Biotechnology Ab | Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G |
US5229492A (en) | 1986-02-14 | 1993-07-20 | Pharmacia Lkb Biotechnology Ab | Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G |
US5312901A (en) | 1986-02-14 | 1994-05-17 | Pharmacia Lkb Biotechnology Ab | Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G |
US4956296A (en) | 1987-06-19 | 1990-09-11 | Genex Corporation | Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G |
US4954618A (en) | 1986-02-14 | 1990-09-04 | Genex Corporation | Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G |
SE459662B (sv) | 1986-03-21 | 1989-07-24 | Pharmacia Ab | Hybrid-dna-molekyl som kodar foer ett protein med samma igg specificitet som protein g, plasmid innehaallande densamma samt foerfarande foer framstaellning av proteinet |
DE69733467T2 (de) | 1996-01-25 | 2006-03-23 | Kaneka Corp. | Adsorbtionsvorrichtung und absorbens für immunuglobine und deren komplexe |
GB9614033D0 (en) | 1996-07-04 | 1996-09-04 | Univ Manchester | Modified protein G and fragments thereof |
US6663862B1 (en) * | 1999-06-04 | 2003-12-16 | Duke University | Reagents for detection and purification of antibody fragments |
EP1830947A4 (en) | 2004-12-14 | 2012-04-18 | Ge Healthcare Bio Sciences Ab | CLEANING OF IMMUNOGLOBULINS |
US8728828B2 (en) | 2004-12-22 | 2014-05-20 | Ge Healthcare Bio-Sciences Ab | Purification of immunoglobulins |
WO2007019376A2 (en) * | 2005-08-03 | 2007-02-15 | Rq Bioscience, Inc. | Methods and compositions for diagnosis of iga-and igm-mediated kidney diseases |
JP5004165B2 (ja) | 2006-10-10 | 2012-08-22 | 独立行政法人産業技術総合研究所 | タンパク質の配向制御固定化に適したタンパク質 |
JP5236311B2 (ja) * | 2008-02-22 | 2013-07-17 | 株式会社カネカ | IgG−Fab断片抗体結合性ペプチド |
JP5550109B2 (ja) | 2010-04-26 | 2014-07-16 | 独立行政法人産業技術総合研究所 | 溶液中のイムノグロブリン量の測定方法 |
US20140221613A1 (en) * | 2011-08-04 | 2014-08-07 | Daicel Corporation | Novel modified protein comprising tandem-type multimer of mutant extracellular domain of protein g |
JP2014529997A (ja) * | 2011-09-23 | 2014-11-17 | ウニヴェルズィテート シュトゥットガルト | 免疫グロブリン結合ドメインを使った血清中半減期延長 |
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