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遺伝暗号の拡張を利用した抗体λ軽鎖における化学コンジュゲーション高反応部位の同定

加藤, 明文 KATO, Akifumi カトウ, アキフミ 九州大学

2021.03.24

概要

抗体医薬は標的分子のみを認識して狙い撃ちするため薬効が高く副作用が少ないという利点があ り、これまでに 50 剤以上が世界で上市され、その数は年々増加している。特に最近では、一つの抗 体で二つの標的分子に結合する「二重特異性抗体」や、抗体に薬剤を結合した「ADC(Antibody Drug Conjugate)」など、タンパク工学や化学修飾技術を駆使して抗体を高機能化する研究が盛んになっ ている。タンパクを部位特異的に効率良く化学修飾する方法として、天然アミノ酸にはない機能を 持つ非天然アミノ酸を用いる方法が注目されている。しかし抗体への応用については、これまで高 効率で化学修飾可能な非天然アミノ酸の適切な導入部位を見出すことが難しく、抗体を高機能化す る上での障害となっていた。我々は先行研究において、理化学研究所(以下、理研)で開発された 遺伝暗号拡張技術を応用することで、抗体の定常領域への高効率での非天然アミノ酸の導入を可能 とし、重鎖定常領域 CH1 および、軽鎖定常領域 CLκへの化学修飾に適した複数の部位を見出し、 実際に薬剤付加による「武装化」や、抗体の 2 量体化による「高機能化」が可能であることを確認 していた。本研究では、この方法論を近年利用が進みつつある軽鎖定常領域 CLλに応用し、非天 然アミノ酸の導入と化学修飾に適した部位の網羅的な探索を実施した。

1. 非天然アミノ酸技術の抗体工学への応用と抗体軽鎖定常領域
CLλにおける非天然アミノ酸の好適導入部位の同定 抗体医薬に最も利用される IgG 抗体には、軽鎖定常領域がそれぞれ CLκと CLλである IgGκと IgGλの 2 種類が存在する。IgGκと IgGλの存在量は動物種によって異なり、マウスではおよそ 19:1 であるが、ヒトではおよそ 2:1 であることが知られている。これまでの抗体医薬は主としてげっ歯 類より取得されてきたため多くが IgGκであり、ADC 作製のための抗体の化学修飾法も軽鎖定常領 域については CLκに注目して研究されて、CLλに対しては検証されていなかった。そこで本研究 では、CLλ軽鎖を有する抗 IGF1R 抗体 Cixutumumab(Cix)をモデル抗体として用いて、非天然ア ミノ酸の導入に適した部位を網羅的に探索した。抗体の二つの腕の部分に相当する抗体フラグメン ト(Fab)調製に適した大腸菌 W3110 株を宿主として終止コドンのひとつ UAG コドンを非天然ア ミノ酸に 100 % 対応させた RF-1 knockout 株を活用し、非天然アミノ酸「o-Az-Z-Lys」の導入を試 みた。o-Az-Z-Lys は、天然アミノ酸のリジン(Lys)の側鎖に長いスペーサーが付加された構造をし ており、側鎖の長さが約 2 倍になっているため化学修飾における反応性に優れている。軽鎖定常領 域 CLλ 20 ケ所への導入を試みたところ、16 ケ所で o-Az-Z-Lys が導入された Fab を調製可能であ った。さらにクリック反応と呼ばれる化学反応を利用して蛍光基を結合させ、蛍光強度から導入し た人工アミノ酸の修飾反応効率が優れる部位を網羅的に探索した結果、反応効率が 80%以上となる 部位を多数同定することができた。さらに o-Az-Z-Lys の導入は、一部の例外を除き抗原結合活性お よび安定性に影響を与えないことを確認した。

2. Fab への薬剤付加
o-Az-Z-Lys 導入された Fab に薬剤を付加し、 「武装化」が可能か確認するため、特に反応性の高 い4ケ所(CLλ-125, 161, 165, 191)に o-Az-Z-Lys を導入し、クリック反応で抗癌剤 DM1 を結合 させた。これら武装化 Cix-Fab(Cix-Fab DM1)が標的となる IGF1R を高発現する乳がん細胞株 MCF-7 に対して細胞傷害活性を発揮するか解析した。IGF1R は二量体価することで細胞内への内 在化を示すことが知られている。本検討でも、二量体価を促進するクロスリンカーを併用した場 合のみで高い細胞傷害活性が認められ、クロスリンカーを併用せず Cix-Fab DM1 を単独添加した 場合では傷害活性が認められなかった。よって、Cix-Fab DM1 の細胞内への内在化選択的に高い 傷害活性を発揮することが示された。以上より、抗体への o-Az-Z-Lys 導入により簡便に ADC を 調製可能であることを確認した。

3. 2 量体化
Fab の調製と高機能化(アンタゴニスト化) 代表部位として選定した 4 部位について、それぞれ 2 種類の o-Az-Z-Lys 導入抗 HER2 抗体 Trastuzumab-Fab とリンカーで連結し、二重特異性 2 量体化 Fab (Bispecific Fab-dimer)を 8 種類調 製した。この Bispecific Fab-dimer の機能を IGF1R および HER2 を高発現する乳がん株 BT-474 細 胞を用いて解析した結果、いずれも細胞増殖を阻害するアンタゴニスト活性が認められた。興味 深いことに、それぞれの Fab 単独でのアンタゴニスト活性は非常に弱いものであった。がん細胞 膜表面で IGF1R と HER2 はヘテロダイマーを形成し、増殖シグナルを入力していると考えられて おり、Bispecific Fab-dimer はこのヘテロダイマーに結合し、効率的に増殖シグナルを遮断したと 考えられる。

抗体の軽鎖定常領域 CLλにおいて化学修飾に適した非天然アミノ酸の導入部位を多数同定した ことより、IgGλ型の ADC をデザインすることが容易となると考えられる。また、この部位を利 用して IgGκ、IgGλ問わず 2 分子の Fab を連結させることで、様々な受容体に対するアンタゴニ ストやアゴニストを創製することが可能となり、新たな抗体医薬の創薬展開が期待される。

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