JP7268059B2 - アグリコシル化抗体生産用形質転換マウスおよびこれから生産されたアグリコシル化抗体の用途 - Google Patents
アグリコシル化抗体生産用形質転換マウスおよびこれから生産されたアグリコシル化抗体の用途 Download PDFInfo
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Description
1-1.動物の用意
動物の使用および管理手続きはKRIBBの動物実験倫理委員会(IACUC)で検討および承認された。接合子(zygote)はC57BL/6Jマウスから得ており、ICR雌マウスを代理母(recipient)として用いた。遺伝子編集されたC57BL/6JマウスはBalb/cマウスと逆交配(back-cross)された。すべての動物は12時間の明暗周期で24℃の恒温および40%の湿度の無菌飼育場で飼育された。
pCMV-ABE7.10(addgene、#102919)およびxCas9(3.7)-ABE(7.10)(addgene、#108382)プラスミドベクターはAddgeneから購入した。プラスミドをAgeI(NEB)で37℃で2時間分解し、線形化されたベクターをPCR精製キット(QIAGEN)を用いて精製した。精製されたベクター1μgをmMESSAGE mMACHINE T7 Ultraキット(Thermo Fisher Scientific)を用いてmRNA合成のための鋳型として用いた。mRNAはMEGAclearキット(Thermo Fisher Scientific)を用いて分離し、凍結チューブバイアルに小分けした後、液体窒素に保存した。使用したABEの塩基配列は表1の通りである。
PMSGホルモン(5IU、Merck)の注入後、48時間の間隔でC57BL/6J雌マウス(5週齢)の腹腔内にhCGホルモン(5IU)を注入した。雌マウスを9週齢のC57BL/6J雄マウスと交配させ、1細胞接合子を雌マウスの卵管膨大部(ampulla of oviduct)から得た。卵丘細胞(cumulus cell)は3mg/mlのhyaluronidase(Merck)が含有されたM2培地で培養して除去された。実施例1-2の表1の塩基配列を有するABE mRNA3μg/μlと、標的DNA結合のためのsgRNA(Toolgen、韓国)3μg/μlとを含む混合物を、LEICA DMIRB遠隔操縦機(Leica-microsystems)が装着されたFemtojet微量注入器(Eppendorf、ドイツ)を用いて接合子の細胞質に微量注入した。
ゲノムDNAを分離するために、1週齢のpupから足指を切断した。
TiterMax Gold補助剤(Merck)100μlにヒトAFP(Mybiosource)50μgを溶解させて抗原性溶液(antigenic solution)を製造し、ゲノム編集されたマウス(6週齢)の足裏に1週間隔で4回注入した。最終注入後、膝窩リンパ節(popliteal lymph node)を解剖してB細胞を得た後、既存の方法によりFO骨髓腫(myeloma)細胞と融合させた。単一融合細胞を培養皿に入れて、20%FBS、1xHAT(100μM hypoxanthine、0.4μM aminopterinおよび16μM thymidine)(Merck)、1x抗生剤-抗菌溶液(100units/mlペニシリン、100μg/ml streptomycin sulfateおよび0.25μg/ml amphotericin B)(Welgene、Korea)を添加したまま、2週間培養した。hAFPに対する抗体を生産する陽性クローンをindirect ELISA検査を用いて選別した。ヒトAFP溶液は1μg/mlの濃度でhAFPをPBSに溶解させて製造した。96-ウェルプレート(Thermo Fisher scientific)の表面をコーティングするために抗原溶液(100μl)を使用した。ブロッキング後、培養培地を100倍希釈しコーティングされたプレートのウェルに添加した後、インキュベーションした。TBS-Tween20(0.02%)で3回洗浄した後、HRP(Cell Signaling Technology、希釈比率:1:2000)がコンジュゲートされた抗-マウス二次抗体を処理し、TMB-ultra solution(Thermo Fisher Scientific)を処理した。0.2N H2SO4 100μlを添加して化学反応を停止させた後、VERSA maxマイクロプレートリーダー(Molecular Devices)を用いて450nmでの吸光度を測定した。アグリコシル化(aglycosylated)単クローン抗体を生産するために、陽性クローンを無血清培地(Gibco)で培養した。
実施例1-5で製造したハイブリドーマ細胞を5%CO2が補充されたチャンバで72時間100RPMで撹拌しながら、無血清培地(Gibco)で培養した。条件培地(conditioned media)を得ており、13,000gで30分間遠心分離して細胞破片を除去し、残留破片を0.22μmの注射器フィルタ(Millipore)で濾過して除去した。FPLCシステム(AKTA浄水器、GE healthcare Life Sciences)を用いたHiTrapTM Protein G HPcolumn(GE healthcare Life Sciences)で濾過液を精製した。PBS(phosphate-buffered saline)バッファーで平衡を合わせたカラムに1ml/minの流速で濾過液を通過させ、結合タンパク質を3ml/minの流速で50mMグリシン-HCl(pH2.5)で溶出させた。溶出した分画を1M Tris-HCl(pH8.0)で中和させた後、タンパク質安定化カクテル溶液(Thermo Fisher Scientific)と混合し、-20℃で保管した。
混成化されたオリゴヌクレオチド対をpSpCas9(BB)-2A-Puro(PX459)(Addgene plasmid #48139)ベクターにクローニングすることにより、FUT8標的化ガイドRNA構造物(表5)を生成した。
自動分析器(automatic analyzer)(mTAS Wako i30、Wako Pure Chemical Industries、Osaka、Japan)でマイクロチップ毛細管電気泳動(microchip capillary electrophoresis)および液相結合分析(liquid-phase binding assay)を行って、残留血清検体でアルファ胎児蛋白(alpha-fetoprotein)であるAFP-L3を測定した。AFPに対して0.3~2000ng/mLの範囲で測定し、AFPの濃度が0.3ng/mlを超える血清でAFP-L3の濃度を計算した。試料のAFPの濃度が2,000ng/mL以上の場合、試料を製造会社の指針に従い、既存の結果に基づいて手動で希釈した。すべての検査は釜山(プサン)大学の梁山病院で行われ、検査前に被検体に関するいかなる情報も提供されなかった。
編集されたHEK293-T細胞(2×104)をDEME培地の18mm×18mmのカバーガラス上で1日間成長させた。細胞をBD cytofix/cytoperm溶液(BD bioscience)で12時間固定した。その後、細胞を2時間室温で9ng/μlのPhoSL-alexa488の存在下で培養した。PBSで3回洗浄した後、1.5μg/mlのDAPIを含有するVectashield封入剤(mounting medium)(VECTOR Laboratories)でカバーガラスを覆った。Zeiss LSM510 Meta顕微鏡(Carl Zeiss MicroImaging)で蛍光をモニタリングした。
緩衝液(pH7.4)0.1M重炭酸ナトリウム(sodium-bicarbonate)に溶解した抗-hAFPマウス単クローン抗体(#ab54745、Abcam)または抗-hAFPアグリコシル化抗体0.5μgでELISAウェルプレートを4℃で一晩コーティングした。プレートをTBS-0.1% Tween20で2回洗浄し、無蛋白ブロッキング緩衝液(Thermo Fisher Scientific)または0.5%ポリビニルアルコール+0.1% Tween 20を用いて室温で1時間ブロッキングした。それぞれのウェルに臨床試料または培養培地(100μl)を分注した。それぞれのウェルをRIPAバッファー(25mM Tris-HCl7.6、150mM NaCl、1%NP40、1%sodium deoxycholate、0.1%SDS)で5回以上洗浄し、再びTBS-0.1% Tween20で繰り返した。1:2,000で希釈した抗-hAFPウサギ多クローン抗体(#ab8201、Abcam)または1:1,000で希釈したビオチン-標識されたAAL(aleuria aurantia lectin)(#B-1395、VECTOR Laboratories)のうちの1つを室温で1時間処理した。十分に洗浄した後、それぞれHRPとコンジュゲートされた抗ウサギ二次抗体(Cell Signaling)またはストレプトアビジンを1:2,000で希釈し、室温で1時間処理した。TBS-0.1% Tween20で簡単に洗浄した後、TMP基質溶液(Thermo fisher Scientific)100μlを各ウェルに添加し、2N硫酸で反応を停止させた後、450nmでの吸光度を測定した。
脱グリコシル化された(deglycosylated)抗体は、50℃で15分間Rapid PNGase F非-還元型バッファーでPNGase-F(NEB)100unitsと市販中の抗-hAFP抗体(#MIA1305、Thermo fisher Scientific)10μgをインキュベーションして用意された。時間依存的および温度依存的タンパク質安定性は、PBS緩衝液でグリコシル化、脱グリコシル化またはアグリコシル化抗体を4℃または37℃で最大14日間インキュベーションして測定した。インキュベーションされた抗体を4~20%のMini-Protein TGXTMゲル(BioRad)で電気泳動させ、クマシーブルー染色によって視覚化した。タンパク質安定性は、pH3.0、7.0または10.0に調整された0.1Mリン酸塩バッファーで抗体を0~14日間インキュベーションしてモニタリングした。また、各抗体を0~3%のH2O2を含有したPBSバッファーで5時間、37℃の培養器でインキュベーションした。抗体の完全性(integrity)は前記記載のELISA分析によって測定された。
インデル(indel)効率に対する統計的テストは、two-tailed Student’s t-testを用いてSigma Plotで行われ、p value<0.05は有意なものと見なした。
免疫グロブリンG(IgG)はN-連結された糖タンパク質で、一般的な糖タンパク質と同じく、グリカン構造に異質性(heterogeneity)を示す。このような特性のため、レクチンを含むグリカン特異的プローブを用いて疾病特異的グリコール-バイオマーカーを測定するにあたり、ELISAまたはCLIAのような日常的に使用される免疫測定法(immunoassay)の使用が制限された。一方、レクチンは被分析物が存在しなくても捕獲抗体に結合可能であり(図1)、グリカン構造は予測しにくく、batch-to-batch変形が容易なため、統制できない大きな空試験値(blank value)を生成する。このような問題は、適切なレクチンおよびアグリコシル化抗体を用いる免疫分析プラットフォームであるALIQUAT(Aglycosylated antibody-Lectin coupled Immunoassay for the QUAntification of Tumor marker)による特定のグリコフォーム(glycoform)分析によって解決できる(図2)。したがって、本実施例では、ゲノム編集によりアグリコシル化抗体を生産するマウスを確立した。
HDR効率性を向上させるために様々なアプローチ方法が開発されたが、これらの方法は依然として非相同末端連結(Non-homologous end joining、NHEJ)ベースのノックアウト(knock-out)効率に比べて非常に低い。最近、二重鎖DNAの切断なしに、シトシンまたはアデニン塩基ベースの編集により高い効率でそれぞれ「C-G to T-A」または「A-T to G-C」転換が可能な塩基編集システム(base editing system)が開発された。C57BL/6のIgG遺伝子の配列分析の結果、塩基編集ウィンドウ(図3)内でIgG2b、IgG2cおよびIgG3がN-グリコシル化アスパラギンコーディング配列(AAC)に編集可能なアデニンを共有することが明らかになった。1つまたは2つとものアデニンをグアニンに転換させると、非-アスパラギンコーディング配列が生成され、IgGからN-グリカンが無くなる。
実施例2-1で選別した9匹のpupのうち、IgG1遺伝子座でゲノム編集を行うのに用いるために、他のpupに対比して、IgG2c(図8)、IgG2b(図9)およびIgG3(図10)遺伝子で二対立因子性(biallelic)「A to G」転換率が高いpup(#11)を選択した。このpupはIgG2bで同型接合突然変異(homozygous mutation)を示し、IgG2cとIgG3でキメラ突然変異を示した(図11)。
IgG1の標的部位はcanonical(NGG)を含まないが、標的の二本鎖ともNG PAM配列のみを含む。したがって、可能な隣接サイトを標的として供与DNAを用いてHDR媒介遺伝子編集を行ったが、IgG1遺伝子が編集されたpupを生成することができなかった。したがって、IgG1の遺伝子編集のために、NG配列に対する遺伝子標的化活性を示す実施例1-2のxCas9-ABEを用いて、実施例1-3によりIgG1遺伝子を編集した。
3-1.免疫グロブリンのすべてのサブクラス生産能力の確認
免疫グロブリンG(IgG)のN-グリコシル化はマウス、ウサギおよびヒトを含む高等真核生物の間で高度に保存されている。IgG N-グリコシル化が安定性を付与したり免疫反応調節に関与するという報告があるが、進化論的圧力によって高等生物体がIgGのN-グリコシル化を採用するようになったのかは明確ではない。
ゲノム編集されたマウスから生産される抗体がアグリコシル化抗体であるか否かを確認した。
抗-hAFP抗体は、ヒト血清アルブミン(human serum albumin、HAS)に存在するエピトープに対して交差反応を示し得るため、HASに対する反応性を確認して反応性クローンを除いた。また、時間の経過により抗体の生産が減少したクローンは除き、抗体の生産が持続するクローンを選別して、1E5、2A2および3A5と指定された3個のクローンを選別した。3個のクローンはいずれもIgG1サブクラスおよびカッパ(kappa)軽鎖の単クローン抗体を分泌することが確認された(図19)。また、MS分析により、新たに形成された突然変異領域にO-linkedグリコシル化のような翻訳後変形(post-translational modification)は存在しないことが確認された。
生成された抗体が完全に無糖化されたことを確認するために、Concanavalin A(Con-A)を用いたレクチンブロット分析および抗-マウスIgG抗体を用いた免疫ブロット分析を行った。
4-1.L3-null AFP標準を作るためのFUT8 -/- 突然変異クローンの作製
HEK293-T細胞は、GDP-フコース(fucose)においてcore N-グリカンへのフコース転移を触媒するFUT8(alpha-(1,6)-fucosyltransferase)を発現する。FUT8遺伝子には3つのスプライシング変異が存在し、触媒の活性に関連があり、すべての変異型が共有するエクソン9および11はグリコシルトランスフェラーゼ(glycosyltransferase)ファミリー23に属する。
FUT8遺伝子の除去による機能的損失を調べるために、core-fucosylated N-glycanに特異的な結合性を示すことが知られたPhoSL(mushroom Pholiota squarrosa)由来レクチンを用いて、実施例1-9によりレクチン結合テストを行った。免疫蛍光(immunofluorescence)分析を行った結果、FUT8-/-HEK293-T細胞はPhoSL結合性がないことが確認された(図23)。
canonical sandwich ELISAおよびAALを用いるlectin-coupled ELISA分析により標準曲線を作成した。
μ-TASは、LCAが内蔵された毛細管におけるAFP-L3の移動を電気泳動で分析する体外診断用免疫分析器である。分析器の高感度および強固性とAFP-L3測定の臨床的有用性のため、μ-TASはHCC進行の危険性評価のための試験管内検査のFDA承認を受けた。分析的妥当性と臨床的有用性にもかかわらず、μ-TAS分析は臨床的用途にいくつかの限界がある。一般的に高費用を要する分析であるので、癌検診のための日常的な臨床的使用が制限される。また、μ-TASはAFP-L3の用途で開発され、他のグリコフォーム分析への使用が困難である。
タンパク質グリコシル化は、タンパク質の機能、タンパク質-タンパク質の相互作用、細胞-宿主認識など多様な効果を示す。また、タンパク質グリカンはin vivoおよびin vitroでタンパク質安定性を付与することが知られている(Bowden2012)。したがって、無糖質化によって抗体が保存および試験中にタンパク質安定性を失うか否かを、実施例1-11により確認した。
PBSバッファーで多様な形態の抗体を14日間、4℃および37℃の条件で放置した。脱グリコシル化抗体は矢印で表しているように2つの異なる分解断片(degradation fragment)を示すことが確認された(図30)。処理初期に断片が観察されたため、断片のうち、分子量が約35kDaの断片はPNGase-F処理によって発生したことが確認された。断片のうち、分子量が約50kDaの断片は37℃で時間により増加したことが確認された。しかし、本来のグリコシル化された形態は、調べられた条件下でいかなる分解生成物も現れていない。同様に、本発明者らはゲル上でアグリコシル化抗体に対する分解産物の跡を検出することができなかった。
抗体は多様な反応条件または精製過程でpHの変化に露出しうる。したがって、多様なpH条件で多様なグリコフォーム抗体の安定性を調べた。
酸化条件下で抗体の完全性について調べた結果、グリコシル化およびアグリコシル化抗体ともこのような条件に対する抵抗性を示すことが確認され、これは脱グリコシル化された形態の物理的特性と非常に対照的であった(図32)。アグリコシル化抗体は3.0%のH2O2を含有するPBS緩衝液で1時間恒温処理した場合、10%未満の完全性の損失を示した。しかし、脱グリコシル化された抗体はこのような条件下で約50%の結合活性(avidity)の損失が確認された。この結果により、グリコシル化された免疫グロブリンGに対比してアグリコシル化抗体の安定性が低下しないことが確認された。
Claims (3)
- IgG(immunoglobulin G)遺伝子が変形されたアグリコシル化抗体生産用のヒトを除く動物モデルであって、
前記変形は、
IgG1重鎖の297番~299番アミノ酸配列をコードする領域を含む塩基のうち、配列番号2に相当する配列の、配列番号40の配列への置換;
IgG2b重鎖の297番~299番アミノ酸配列をコードする領域を含む塩基のうち、配列番号3に相当する配列の、配列番号37の配列への置換;
IgG2c重鎖の297番~299番アミノ酸配列をコードする領域を含む塩基のうち、配列番号4に相当する配列の、配列番号38の配列への置換;および
IgG3重鎖の297番~299番アミノ酸配列をコードする領域を含む塩基のうち、配列番号5に相当する配列の、配列番号39の配列への置換
からなる群より選択される1つ以上の置換により発生したものである、アグリコシル化抗体生産用のヒトを除く動物モデル。 - 前記動物は、ウサギ、ヤギまたはマウスである、請求項1に記載のアグリコシル化抗体生産用のヒトを除く動物モデル。
- 請求項1に記載のヒトを除く動物モデルに診断しようとする抗原を投与するステップを含む抗原に対するアグリコシル化抗体の生産方法。
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