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- JPWO2019173693A5 JPWO2019173693A5 JP2020571330A JP2020571330A JPWO2019173693A5 JP WO2019173693 A5 JPWO2019173693 A5 JP WO2019173693A5 JP 2020571330 A JP2020571330 A JP 2020571330A JP 2020571330 A JP2020571330 A JP 2020571330A JP WO2019173693 A5 JPWO2019173693 A5 JP WO2019173693A5
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Description
本発明を、以下の実験実施例を参照してさらに詳細に説明する。これら実施例は、例示説明のみを目的として提供するものであり、特に指定のない限り、限定的であることを意図しない。したがって、本発明は、いかなる場合も以下の実施例に限定されると解釈されるべきではなく、むしろ、本明細書に示す教示の結果として明らかにされるすべてのいかなる変更をも包含すると解釈されるべきである。さらなる説明がなくても、当業者は、前述の説明及び以下の例示的な実施例を用いて、本発明の化合物を作製して利用し、請求される方法を実施することができると考えられる。以下の実施例は、本発明の種々の態様を具体的に示すものであり、いかなる場合においても本開示のそれ以外の部分を限定するものとみなされるべきではない。
本願は以下の発明を包含する。
［項目１］ 組換え核酸であって、
（ａ）Ｔ細胞受容体（ＴＣＲ）融合タンパク質（ＴＦＰ）をコードする配列であって、
（ｉ）以下：
（１）ＴＣＲ細胞外ドメインの少なくとも一部、
（２）膜貫通ドメイン、及び
（３）ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、ＴＣＲα、又はＴＣＲβの細胞内シグナル伝達ドメインからの刺激ドメインを含む細胞内ドメイン
を含むＴＣＲサブユニットと
（ｉｉ）抗原結合ドメインを含むヒト抗体又はヒト化抗体と
を含む前記配列、並びに
（ｂ）ＴＣＲ定常ドメインをコードする配列であって、前記ＴＣＲ定常ドメインが、
ＴＣＲα定常ドメイン、ＴＣＲβ定常ドメイン、又はＴＣＲα定常ドメインとＴＣＲβ定常ドメインである、前記配列
を含み、
前記ＴＣＲサブユニット及び前記抗体が機能的に連結されており、かつ
内因性ＴＣＲの機能的破壊を含む改変Ｔ細胞で発現すると前記ＴＦＰがＴＣＲ複合体に機能的に組み込まれる、組換え核酸。
［項目２］ 組換え核酸であって、
（ａ）Ｔ細胞受容体（ＴＣＲ）融合タンパク質（ＴＦＰ）をコードする配列であって、
（ｉ）以下：
（１）ＴＣＲ細胞外ドメインの少なくとも一部、
（２）膜貫通ドメイン、及び
（３）ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、ＴＣＲα、又はＴＣＲβの細胞内シグナル伝達ドメインからの刺激ドメインを含む細胞内ドメイン
を含むＴＣＲサブユニットと
（ｉｉ）抗体又はその断片に結合できる結合リガンド又はその断片と
を含む前記配列、並びに
（ｂ）ＴＣＲ定常ドメインをコードする配列であって、前記ＴＣＲ定常ドメインが、ＴＣＲα定常ドメイン、ＴＣＲβ定常ドメイン、又はＴＣＲα定常ドメインとＴＣＲβ定常ドメインである、前記配列
を含み、
前記ＴＣＲサブユニット及び結合リガンド又はその断片は機能的に連結されており、かつ
内因性ＴＣＲの機能的破壊を含む改変Ｔ細胞で発現すると前記ＴＦＰがＴＣＲ複合体に機能的に組み込まれる、組換え核酸。
［項目３］ 前記結合リガンドが、前記抗体のＦｃドメインと結合できる、項目２に記載の組換え核酸。
［項目４］ 前記結合リガンドが、ＩｇＧ１抗体と選択的に結合できる、項目２に記載の組換え核酸。
［項目５］ 前記結合リガンドが、ＩｇＧ４抗体と特異的に結合できる、項目２に記載の組換え核酸。
［項目６］ 前記抗体又はその断片が細胞表面抗原に結合する、項目２に記載の組換え核酸。
［項目７］ 前記抗体又はその断片が腫瘍細胞の表面にある細胞表面抗原に結合する、項目２に記載の組換え核酸。
［項目８］ 前記結合リガンドが、単量体、二量体、三量体、四量体、五量体、六量体、七量体、八量体、九量体、又は十量体を含む、項目２に記載の組換え核酸。
［項目９］ 前記結合リガンドが抗体又はその断片を含まない、項目２に記載の組換え核酸。
［項目１０］ 前記結合リガンドがＣＤ１６ポリペプチド又はその断片を含む、項目９に記載の組換え核酸。
［項目１１］ 前記結合リガンドがＣＤ１６結合ポリペプチドを含む、項目１０に記載の組換え核酸。
［項目１２］ 前記結合リガンドがヒトのものであるか、又はヒト化されている、項目２に記載の組換え核酸。
［項目１３］ 前記結合リガンドが結合できる抗体又はその断片をコードする核酸配列をさらに含む、項目２に記載の組換え核酸。
［項目１４］ 前記抗体又はその断片が細胞から分泌できる、項目１３に記載の組換え核酸。
［項目１５］ 組換え核酸であって、
（ａ）Ｔ細胞受容体（ＴＣＲ）融合タンパク質（ＴＦＰ）をコードする配列であって、
（ｉ）以下：
（１）ＴＣＲ細胞外ドメインの少なくとも一部、
（２）膜貫通ドメイン、及び
（３）ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、ＴＣＲα、又はＴＣＲβの細胞内シグナル伝達ドメインからの刺激ドメインを含む細胞内ドメインを含むＴＣＲサブユニットと
（ｉｉ）細胞の表面に発現する受容体又はポリペプチドに結合するリガンド又はその断片を含む抗原ドメインと
を含む前記配列、並びに
（ｂ）ＴＣＲ定常ドメインをコードする配列であって、前記ＴＣＲ定常ドメインが、ＴＣＲα定常ドメイン、ＴＣＲβ定常ドメイン、又はＴＣＲα定常ドメインとＴＣＲβ定常ドメインである、前記配列
を含み、
前記ＴＣＲサブユニット及び前記抗原ドメインは機能的に連結されており、かつ
内因性ＴＣＲの機能的破壊を含む改変Ｔ細胞で発現すると前記ＴＦＰがＴＣＲ複合体に機能的に組み込まれる、組換え核酸。
［項目１６］ 前記抗原ドメインがリガンドを含む、項目１５に記載の組換え核酸。
［項目１７］ 前記リガンドが細胞の受容体に結合する、項目１５に記載の組換え核酸。
［項目１８］ 前記リガンドが細胞の表面に発現するポリペプチドに結合する、項目１５に記載の組換え核酸。
［項目１９］ 細胞の表面に発現する前記受容体又はポリペプチドが、ストレス応答受容体又はポリペプチドを含む、項目１５に記載の組換え核酸。
［項目２０］ 細胞の表面に発現する前記受容体又はポリペプチドが、ＭＨＣクラスＩ関連糖タンパク質である、項目１５に記載の組換え核酸。
［項目２１］ 前記ＭＨＣクラスＩ関連糖タンパク質が、ＭＩＣＡ、ＭＩＣＢ、ＲＡＥＴ１Ｅ、ＲＡＥＴ１Ｇ、ＵＬＢＰ１、ＵＬＢＰ２、ＵＬＢＰ３、ＵＬＢＰ４、及びそれらの組合せからなる群から選択される、項目２０に記載の組換え核酸。
［項目２２］ 前記抗原ドメインが、単量体、二量体、三量体、四量体、五量体、六量体、七量体、八量体、九量体、又は十量体を含む、項目１５に記載の組換え核酸。
［項目２３］ 前記抗原ドメインが、前記リガンド又はその断片の単量体又は二量体を含む、項目２２に記載の組換え核酸。
［項目２４］ 前記リガンド又はその断片が、単量体、二量体、三量体、四量体、五量体、六量体、七量体、八量体、九量体、又は十量体である、項目１５に記載の組換え核酸。
［項目２５］ 前記リガンド又はその断片が単量体又は二量体である、項目２４に記載の組換え核酸。
［項目２６］ 前記抗原ドメインが抗体又はその断片を含まない、項目１５に記載の組換え核酸。
［項目２７］ 前記抗原ドメインが可変領域を含まない、項目１５に記載の組換え核酸。
［項目２８］ 前記抗原ドメインがＣＤＲを含まない、項目１５に記載の組換え核酸。
［項目２９］ 前記リガンド又はその断片が、ナチュラルキラーグループ２Ｄ（ＮＫＧ２Ｄ）リガンド又はその断片である、項目１５に記載の組換え核酸。
［項目３０］ Ｔ細胞で発現すると、前記ＴＣＲ定常ドメインが機能的ＴＣＲ複合体に組み込まれる、項目１～２９のいずれか１項に記載の組換え核酸。
［項目３１］ 前記ＴＣＲ定常ドメインが、Ｔ細胞で発現すると、前記ＴＦＰを組み込む前記機能的ＴＣＲ複合体と同じ機能的ＴＣＲ複合体に組み込まれる、項目１～３０のいずれか１項に記載の組換え核酸。
［項目３２］ 前記ＴＦＰをコードする配列と前記ＴＣＲ定常ドメインをコードする配列が、同じ核酸分子内に含まれている、項目１～３１のいずれか１項に記載の組換え核酸。
［項目３３］ 前記ＴＦＰをコードする配列と前記ＴＣＲ定常ドメインをコードする配列が、異なる核酸分子内に含まれている、項目１～３１のいずれか１項に記載の組換え核酸。
［項目３４］ 前記ＴＣＲサブユニットと、前記抗体ドメイン、前記抗原ドメイン、もしくは前記結合リガンド、又はそれらの断片とが、リンカー配列によって機能的に連結されている、項目１～３３に記載の組換え核酸。
［項目３５］ 前記リンカー配列が（Ｇ
４
Ｓ）
ｎ
（式中、ｎ＝１～４）を含む、項目３４に記載の組換え核酸。
［項目３６］ 前記膜貫通ドメインが、ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、ＴＣＲα、又はＴＣＲβからのＴＣＲ膜貫通ドメインである、項目１～３５のいずれか１項に記載の組換え核酸。
［項目３７］ 前記細胞内ドメインが、ＣＤ３εのみ、ＣＤ３γのみ、ＣＤ３δのみ、ＴＣＲαのみ、又はＴＣＲβのみに由来する、項目１～３６のいずれか１項に記載の組換え核酸。
［項目３８］ 前記ＴＣＲサブユニットが、（ｉ）ＴＣＲ細胞外ドメインの少なくとも一部、（ｉｉ）ＴＣＲ膜貫通ドメイン、及び（ｉｉｉ）ＴＣＲ細胞内ドメインを含み、（ｉ）、（ｉｉ）、及び（ｉｉｉ）のうち少なくとも２つは同じＴＣＲサブユニット由来である、項目１～３７のいずれか１項に記載の組換え核酸。
［項目３９］ 前記ＴＣＲ細胞外ドメインが、ＴＣＲα鎖、ＴＣＲβ鎖、ＣＤ３ε ＴＣＲサブユニット、ＣＤ３γ ＴＣＲサブユニット、ＣＤ３δ ＴＣＲサブユニット、それらの機能的断片、及び少なくとも１つだが２０以下の修飾を有するそのアミノ酸配列からなる群から選択される、タンパク質の細胞外ドメイン又はその一部を含む、項目１～３８のいずれか１項に記載の組換え核酸。
［項目４０］ 前記ＴＣＲサブユニットが、ＴＣＲα鎖、ＴＣＲβ鎖、ＴＣＲゼータ鎖、ＣＤ３ε ＴＣＲサブユニット、ＣＤ３γ ＴＣＲサブユニット、ＣＤ３δ ＴＣＲサブユニット、ＣＤ４５、ＣＤ４、ＣＤ５、ＣＤ８、ＣＤ９、ＣＤ１６、ＣＤ２２、ＣＤ３３、ＣＤ２８、ＣＤ３７、ＣＤ６４、ＣＤ８０、ＣＤ８６、ＣＤ１３４、ＣＤ１３７、ＣＤ１５４、それらの機能的断片、及び少なくとも１つだが２０以下の修飾を有するそのアミノ酸配列からなる群から選択される、タンパク質の膜貫通ドメインを含む膜貫通ドメインを含む、項目１～３９のいずれか１項に記載の組換え核酸。
［項目４１］ 前記ＴＣＲサブユニットが、ＣＤ３ε、ＣＤ３γ、又はＣＤ３δの細胞内シグナル伝達ドメインから選択されるタンパク質の刺激ドメイン、又はそれらに対して少なくとも１つの修飾を有するアミノ酸配列を含むＴＣＲ細胞内ドメインを含む、項目１～４０のいずれか１項に記載の組換え核酸。
［項目４２］ 前記ＴＣＲサブユニットが、４－１ＢＢの機能的シグナル伝達ドメイン及び／又はＣＤ３ζの機能的シグナル伝達ドメインから選択されるタンパク質の刺激ドメイン、又はそれらに対して少なくとも１つの修飾を有するアミノ酸配列を含む細胞内ドメインを含む、項目１～４１のいずれか１項に記載の組換え核酸。
［項目４３］ 共刺激ドメインをコードする配列をさらに含む、項目１～４２のいずれか１項に記載の組換え核酸。
［項目４４］ 前記共刺激ドメインが、ＯＸ４０、ＣＤ２、ＣＤ２７、ＣＤ２８、ＣＤＳ、ＩＣＡＭ－１、ＬＦＡ－１（ＣＤ１１ａ／ＣＤ１８）、ＩＣＯＳ（ＣＤ２７８）、及び４－１ＢＢ（ＣＤ１３７）、ならびに少なくとも１つだが２０以下の修飾を有するそのアミノ酸配列からなる群から選択される、タンパク質の機能的シグナル伝達ドメインを含む、項目４３に記載の組換え核酸。
［項目４５］ 前記ＴＣＲサブユニットが、ＣＤ３ζ ＴＣＲサブユニット、ＣＤ３ε ＴＣＲサブユニット、ＣＤ３γ ＴＣＲサブユニット、ＣＤ３δ ＴＣＲサブユニット、ＴＣＲζ鎖、Ｆｃε受容体１鎖、Ｆｃε受容体２鎖、Ｆｃγ受容体１鎖、Ｆｃγ受容体２ａ鎖、Ｆｃγ受容体２ｂ１鎖、Ｆｃγ受容体２ｂ２鎖、Ｆｃγ受容体３ａ鎖、Ｆｃγ受容体３ｂ鎖、Ｆｃβ受容体１鎖、ＴＹＲＯＢＰ（ＤＡＰ１２）、ＣＤ５、ＣＤ１６ａ、ＣＤ１６ｂ、ＣＤ２２、ＣＤ２３、ＣＤ３２、ＣＤ６４、ＣＤ７９ａ、ＣＤ７９ｂ、ＣＤ８９、ＣＤ２７８、ＣＤ６６ｄ、それらの機能的断片、及び少なくとも１つだが２０以下の修飾を有するそのアミノ酸配列からなる群から選択される、タンパク質の免疫受容体チロシン活性化モチーフ（ＩＴＡＭ）又はその一部を含む、ＴＣＲサブユニットＩＴＡＭを含む、項目１～４４のいずれか１項に記載の組換え核酸。
［項目４６］ 前記ＩＴＡＭが、ＣＤ３γ、ＣＤ３δ、又はＣＤ３εのＩＴＡＭを置換する、項目４５に記載の組換え核酸。
［項目４７］ 前記ＩＴＡＭが、ＣＤ３ζ ＴＣＲサブユニット、ＣＤ３ε ＴＣＲサブユニット、ＣＤ３γ ＴＣＲサブユニット、及びＣＤ３δ ＴＣＲサブユニットからなる群から選択され、ＣＤ３ζ ＴＣＲサブユニット、ＣＤ３ε ＴＣＲサブユニット、ＣＤ３γ ＴＣＲサブユニット、及びＣＤ３δ ＴＣＲサブユニットからなる群から選択される別のＩＴＡＭを置換する、項目４５に記載の組換え核酸。
［項目４８］ 前記ＴＦＰ、前記ＴＣＲα定常ドメイン、前記ＴＣＲβ定常ドメイン、及びそれらの任意の組合せが、内因性ＴＣＲ複合体及び／又は少なくとも１つの内因性ＴＣＲポリペプチドと機能的に相互作用できる、項目１～４７のいずれか１項に記載の組換え核酸。
［項目４９］ （ａ）前記ＴＣＲ定常ドメインはＴＣＲα定常ドメインであり、前記ＴＦＰがＴＣＲβ、ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、又はそれらの組合せの内因性サブユニットを含むＴＣＲ複合体に機能的に統合されるか、
（ｂ）前記ＴＣＲ定常ドメインはＴＣＲβ定常ドメインであり、前記ＴＦＰがＴＣＲα、ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、又はそれらの組合せの内因性サブユニットを含むＴＣＲ複合体に機能的に統合されるか、あるいは
（ｃ）前記ＴＣＲ定常ドメインはＴＣＲα定常ドメイン及びＴＣＲβ定常ドメインであり、前記ＴＦＰが、ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、又はそれらの組合せの内因性サブユニットを含むＴＣＲ複合体に機能的に統合される、項目１～４８のいずれか１項に記載の組換え核酸。
［項目５０］ 前記少なくとも１つだが２０以下の修飾が、細胞シグナル伝達を媒介するアミノ酸の修飾、又はＴＦＰへのリガンド結合に応答してリン酸化されるアミノ酸の修飾を含む、項目１～４９のいずれか１項に記載の組換え核酸。
［項目５１］ 前記ヒト抗体又はヒト化抗体が抗体断片である、項目１及び３４～５０のいずれか１項に記載の組換え核酸。
［項目５２］ 前記抗体断片が、ｓｃＦｖ、単一ドメイン抗体ドメイン、Ｖ
Ｈ
ドメイン、又はＶ
Ｌ
ドメインである、項目５１に記載の組換え核酸。
［項目５３］ 抗原結合ドメインが、抗ＣＤ１９結合ドメイン、抗Ｂ細胞成熟抗原（ＢＣＭＡ）結合ドメイン、抗メソテリン（ＭＳＬＮ）結合ドメイン、抗ＩＬ１３Ｒα２結合ドメイン、抗ＭＵＣ１６結合ドメイン、抗ＣＤ２２結合ドメイン、抗ＰＤ－１結合ドメイン、抗ＢＡＦＦ又はＢＡＦＦ受容体結合ドメイン、及び抗ＲＯＲ－１結合ドメインからなる群から選択される、項目１及び３４～５２のいずれか１項に記載の組換え核酸。
［項目５４］ 前記核酸が、ＤＮＡ及びＲＮＡからなる群から選択される、項目１～５３のいずれか１項に記載の組換え核酸。
［項目５５］ 前記核酸がｍＲＮＡである、項目１～５４のいずれか１項に記載の組換え核酸。
［項目５６］ 前記組換え核酸が核酸類似体を含み、前記核酸類似体は前記組換え核酸のコード配列にはない、項目１～５５のいずれか１項に記載の組換え核酸。
［項目５７］ 前記核酸類似体が、２’－Ｏ－メチル、２’－Ｏ－メトキシエチル（２’－Ｏ－ＭＯＥ）、２’－Ｏ－アミノプロピル、２’－デオキシ、Ｔ－デオキシ－２’－フルオロ、２’－Ｏ－アミノプロピル（２’－Ｏ－ＡＰ）、２’－Ｏ－ジメチルアミノエチル（２’－Ｏ－ＤＭＡＯＥ）、２’－Ｏ－ジメチルアミノプロピル（２’－Ｏ－ＤＭＡＰ）、Ｔ－Ｏ－ジメチルアミノエチルオキシエチル（２’－Ｏ－ＤＭＡＥＯＥ）、２’－Ｏ－Ｎ－メチルアセトアミド（２’－Ｏ－ＮＭＡ）修飾、ロックド核酸（ＬＮＡ）、エチレン核酸（ＥＮＡ）、ペプチド核酸（ＰＮＡ）、１’，５’－アンヒドロヘキシトール核酸（ＨＮＡ）、モルホリノ、メチルホスホン酸ヌクレオチド、チオールホスホン酸ヌクレオチド、及び２’－フルオロＮ３－Ｐ５’－ホスホロアミダイトからなる群から選択される、項目５６に記載の組換え核酸。
［項目５８］ リーダー配列をさらに含む、項目１～５７のいずれか１項に記載の組換え核酸。
［項目５９］ プロモーター配列をさらに含む、項目１～５８のいずれか１項に記載の組換え核酸。
［項目６０］ ポリ（Ａ）尾部をコードする配列をさらに含む、項目１～５９のいずれか１項に記載の組換え核酸。
［項目６１］ ３’ＵＴＲ配列をさらに含む、項目１～６０のいずれか１項に記載の組換え核酸。
［項目６２］ 前記核酸が、単離された核酸又は天然に存在しない核酸である、項目１～６１のいずれか１項に記載の組換え核酸。
［項目６３］ 前記核酸がｉｎ ｖｉｔｒｏ転写した核酸である、項目１～６２のいずれか１項に記載の組換え核酸分子。
［項目６４］ ＴＣＲα膜貫通ドメインをコードする配列をさらに含む、項目１～６３のいずれか１項に記載の組換え核酸分子。
［項目６５］ ＴＣＲβ膜貫通ドメインをコードする配列をさらに含む、項目１～６３のいずれか１項に記載の組換え核酸分子。
［項目６６］ ＴＣＲα膜貫通ドメインをコードする配列及びＴＣＲβ膜貫通ドメインをコードする配列をさらに含む、項目１～６３のいずれか１項に記載の組換え核酸。
［項目６７］ 項目１～６６のいずれか１項に記載の組換え核酸を含むベクター。
［項目６８］ ＤＮＡ、ＲＮＡ、プラスミド、レンチウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター（ＡＡＶ）、ラウス肉腫ウイルス（ＲＳＶ）ベクター、又はレトロウイルスベクターからなる群から選択される、項目６７に記載のベクター。
［項目６９］ ＡＡＶ６ベクターである、項目６７又は６８に記載のベクター。
［項目７０］ プロモーターをさらに含む、項目６７～６９のいずれか１項に記載のベクター。
［項目７１］ ｉｎ ｖｉｔｒｏ転写したベクターである、項目６７～７０のいずれか１項に記載のベクター。
［項目７２］ 項目１～６６のいずれか１項に記載の組換え核酸又は項目６７～７１のいずれか１項に記載のベクターを含む改変Ｔ細胞であって、内因性ＴＣＲの機能的破壊を含む、改変Ｔ細胞。
［項目７３］ 項目１～６６のいずれか１項に記載の核酸のＴＦＰをコードする配列、又はＴＦＰをコードする項目１～６６のいずれか１項に記載の核酸の配列によってコードされるＴＦＰを含む改変Ｔ細胞であって、内因性ＴＣＲの機能破壊を含む、改変Ｔ細胞。
［項目７４］ 項目１～６６のいずれか１項に記載のＴＦＰをコードする配列、又はＴＦＰをコードする項目１～６６のいずれか１項に記載の核酸の配列によってコードされるＴＦＰを含む、改変同種Ｔ細胞。
［項目７５］ ＴＣＲ定常ドメインをコードする異種配列をさらに含み、前記ＴＣＲ定常ドメインは、ＴＣＲα定常ドメイン、ＴＣＲβ定常ドメイン、又はＴＣＲα定常ドメインとＴＣＲβ定常ドメインである、項目７２～７４のいずれか１項に記載の改変Ｔ細胞。
［項目７６］ 前記機能的に破壊される内因性ＴＣＲが、内因性ＴＣＲα鎖、内因性ＴＣＲβ鎖、又は内因性ＴＣＲα鎖と内因性ＴＣＲβ鎖である、項目７２～７５のいずれか１項に記載の改変Ｔ細胞。
［項目７７］ 前記機能的に破壊された内因性ＴＣＲが、未改変の対照Ｔ細胞のものと比較して、ＭＨＣペプチド複合体への結合が減少する、項目７２～７６のいずれか１項に記載の改変Ｔ細胞。
［項目７８］ 前記機能的破壊が、前記内因性ＴＣＲをコードする遺伝子の破壊である、項目７２～７７のいずれか１項に記載の改変Ｔ細胞。
［項目７９］ 前記内因性ＴＣＲをコードする遺伝子の前記破壊が、Ｔ細胞のゲノム由来の前記内因性ＴＣＲをコードする前記遺伝子の配列の除去である、項目７８に記載の改変Ｔ細胞。
［項目８０］ ＣＤ４細胞、ＣＤ８細胞、ナイーブＴ細胞、メモリー幹Ｔ細胞、セントラルメモリーＴ細胞、二重陰性Ｔ細胞、エフェクターメモリーＴ細胞、エフェクターＴ細胞、Ｔｈ０細胞、Ｔｃ０細胞、Ｔｈ１細胞、Ｔｃ１細胞、Ｔｈ２細胞、Ｔｃ２細胞、Ｔｈ１７細胞、Ｔｈ２２細胞、γ／δＴ細胞、ナチュラルキラー（ＮＫ）細胞、ナチュラルキラーＴ（ＮＫＴ）細胞、造血幹細胞、及び多能性幹細胞から選択されるヒトＴ細胞である、項目７２～７９のいずれか１項に記載の改変Ｔ細胞。
［項目８１］ ＣＤ８
＋
Ｔ細胞又はＣＤ４
＋
Ｔ細胞である、項目７２～８０のいずれか１項に記載の改変Ｔ細胞。
［項目８２］ 同種Ｔ細胞である、項目７２～８１のいずれか１項に記載の改変Ｔ細胞。
［項目８３］ 抑制分子の少なくとも一部を含む第１のポリペプチドを含む抑制分子をコードする核酸を、細胞内シグナル伝達ドメインからの陽性シグナルを含む第２のポリペプチドと会合してさらに含む、項目７２～８２のいずれか１項に記載の改変Ｔ細胞。
［項目８４］ 前記抑制分子が、ＰＤ１の少なくとも一部を含む第１のポリペプチドと、共刺激ドメイン及び一次シグナル伝達ドメインを含む第２のポリペプチドとを含む、項目８３に記載の改変Ｔ細胞。
［項目８５］ （ａ）項目７２～８４のいずれか１項に記載の改変Ｔ細胞と、
（ｂ）薬学的に許容される担体と、を含む、医薬組成物。
［項目８６］ 項目７２～８４のいずれか１項に記載の改変Ｔ細胞を作製する方法であって、
（ａ）ＴＣＲα鎖、ＴＣＲβ鎖、又はＴＣＲα鎖とＴＣＲβ鎖をコードする内因性ＴＣＲ遺伝子を破壊し、それにより内因性ＴＣＲ遺伝子の機能的破壊を含んだＴ細胞を作製することと、
（ｂ）内因性ＴＣＲ遺伝子の機能的破壊を含んだＴ細胞に、項目１～６３のいずれか１項に記載の組換え核酸又は項目６７～７１のいずれか１項に記載のベクターを形質導入することを含む、方法。
［項目８７］ 前記破壊が、ＴＣＲα鎖、ＴＣＲβ鎖、又はＴＣＲα鎖とＴＣＲβ鎖をコードする内因性遺伝子を標的とするヌクレアーゼタンパク質、又はヌクレアーゼタンパク質をコードする核酸配列をＴ細胞に形質導入することを含む、項目８６に記載の方法。
［項目８８］ 項目７２～８４のいずれか１項に記載の改変Ｔ細胞を作製する方法であって、内因性ＴＣＲ遺伝子の機能的破壊を含んだＴ細胞に、項目１～６３のいずれか１項に記載の組換え核酸又は項目６７～７１のいずれか１項に記載のベクターを形質導入することを含む、方法。
［項目８９］ 内因性ＴＣＲ遺伝子の機能的破壊を含んだ前記Ｔ細胞が、ＴＣＲα鎖、ＴＣＲβ鎖、又はＴＣＲα鎖とＴＣＲβ鎖をコードする内因性ＴＣＲ遺伝子の機能的破壊を含んだＴ細胞である、項目８８に記載の方法。
［項目９０］ 前記Ｔ細胞がヒトＴ細胞である、項目８６～８９のいずれか１項に記載の方法。
［項目９１］ 内因性ＴＣＲ遺伝子の機能的破壊を含んだ前記Ｔ細胞が、未改変の対照Ｔ細胞のものと比較して、ＭＨＣ－ペプチド複合体への結合が減少する、項目８６～９０のいずれか１項に記載の方法。
［項目９２］ 前記ヌクレアーゼが、メガヌクレアーゼ、ジンクフィンガーヌクレアーゼ（ＺＦＮ）、転写活性化因子様エフェクターヌクレアーゼ（ＴＡＬＥＮ）、ＣＲＩＳＰＲ／Ｃａｓヌクレアーゼ、又はｍｅｇａＴＡＬヌクレアーゼである、項目８６～９１のいずれか１項に記載の方法。
［項目９３］ 前記組換え核酸又は前記ベクターに含まれる前記配列が、切断部位で前記内因性ＴＣＲサブユニット遺伝子に挿入され、前記内因性ＴＣＲサブユニット遺伝子への前記配列の前記挿入により前記内因性ＴＣＲサブユニットが機能的に破壊される、項目８６～９２のいずれか１項に記載の方法。
［項目９４］ 前記ヌクレアーゼがメガヌクレアーゼである、項目８６～９３のいずれか１項に記載の方法。
［項目９５］ 前記メガヌクレアーゼが第１のサブユニット及び第２のサブユニットを含み、前記第１のサブユニットは前記認識配列の第１の認識半部位に結合し、前記第２のサブユニットは前記認識配列の第２の認識半部位に結合する、項目９４に記載の方法。
［項目９６］ 前記メガヌクレアーゼが、リンカーを含む一本鎖メガヌクレアーゼであり、前記リンカーは前記第１のサブユニットと前記第２のサブユニットとを共有結合する、項目９５に記載の方法。
［項目９７］ 治療を必要とする対象におけるがんの治療方法であって、項目８５に記載の治療有効量の医薬組成物を前記対象に投与することを含む、方法。
［項目９８］ 治療を必要とする対象におけるがんの治療方法であって、（ａ）項目８６～９６のいずれか１項に記載の方法に従って作製される改変Ｔ細胞と、（ｂ）薬学的に許容される担体とを含む医薬組成物を前記対象に投与することを含む、方法。
［項目９９］ 治療を必要とする対象におけるがんの治療方法であって、（ａ）項目８８～９６のいずれか１項に記載の方法に従って作製される改変Ｔ細胞と、（ｂ）薬学的に許容される担体とを含む医薬組成物を前記対象に投与することを含む、方法。
［項目１００］ 前記改変Ｔ細胞が同種異系Ｔ細胞である、項目９７～９９のいずれか１項に記載の方法。
［項目１０１］ 有効量の未改変の対照Ｔ細胞を投与した対象と比較して、前記対象において放出されるサイトカインが少ない、項目９７～１００のいずれか１項に記載の方法。
［項目１０２］ 項目１～６６のいずれか１項に記載の組換え核酸又は項目６７～７１のいずれか１項に記載のベクターを含む有効量の改変Ｔ細胞を投与した対象と比較して、前記対象において放出されるサイトカインが少ない、項目９７～１０１のいずれか１項に記載の方法。
［項目１０３］ 前記方法が、前記医薬組成物の有効性を増加させる薬剤と組み合わせて前記医薬組成物を投与することを含む、項目９７～１０２のいずれか１項に記載の方法。
［項目１０４］ 前記方法が、前記医薬組成物に付随する１つ以上の副作用を改善する薬剤と組み合わせて前記医薬組成物を投与することを含む、項目９７～１０３のいずれか１項に記載の方法。
［項目１０５］ 前記がんが固形癌、リンパ腫、又は白血病である、項目９７～１０４のいずれか１項に記載の方法。
［項目１０６］ 前記がんが、腎細胞癌、乳癌、肺癌、卵巣癌、前立腺癌、結腸癌、子宮頸癌、脳癌、肝臓癌、膵臓癌、腎臓癌、及び胃癌からなる群から選択される、項目９７～１０５のいずれか１項に記載の方法。
［項目１０７］ 医薬品として又は医薬品の調製に使用するための、項目１～６６のいずれか１項に記載の組換え核酸、項目６７～７１のいずれか１項に記載のベクター、項目７２～８４のいずれか１項に記載の改変Ｔ細胞、又は項目８５に記載の医薬組成物。
The present invention will be described in more detail with reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limited unless otherwise specified. Accordingly, the invention should not be construed to be confined to the following examples in any case, but rather to embrace any modifications manifested as a result of the teachings presented herein. Should be. Without further description, one of ordinary skill in the art will be able to formulate, utilize and implement the claimed method using the above description and exemplary examples below. .. The following examples illustrate the various aspects of the invention and should not be considered in any way to limit the rest of the disclosure.
The present application includes the following inventions.
[Item 1] Recombinant nucleic acid
(A) A sequence encoding a T cell receptor (TCR) fusion protein (TFP).
(I) Below:
(1) At least part of the TCR extracellular domain,
(2) Transmembrane domain and
(3) An intracellular domain containing a stimulating domain from the intracellular signaling domain of CD3ε, CD3γ, CD3δ, TCRα, or TCRβ.
With TCR subunits including
(Ii) With a human antibody or humanized antibody containing an antigen-binding domain
The sequence containing, as well as
(B) A sequence encoding a TCR constant domain, wherein the TCR constant domain is
TCRα constant domain, TCRβ constant domain, or TCRα constant domain and TCRβ constant domain, said sequence.
Including
The TCR subunit and the antibody are functionally linked and
A recombinant nucleic acid in which the TFP is functionally integrated into the TCR complex when expressed in a modified T cell comprising functional disruption of the endogenous TCR.
[Item 2] Recombinant nucleic acid
(A) A sequence encoding a T cell receptor (TCR) fusion protein (TFP).
(I) Below:
(1) At least part of the TCR extracellular domain,
(2) Transmembrane domain and
(3) An intracellular domain containing a stimulating domain from the intracellular signaling domain of CD3ε, CD3γ, CD3δ, TCRα, or TCRβ.
With TCR subunits including
(Ii) With a binding ligand or fragment thereof that can bind to the antibody or fragment thereof
The sequence containing, as well as
(B) A sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRα constant domain, a TCRβ constant domain, or a TCRα constant domain and a TCRβ constant domain.
Including
The TCR subunit and binding ligand or fragment thereof are functionally linked and
A recombinant nucleic acid in which the TFP is functionally integrated into the TCR complex when expressed in a modified T cell comprising functional disruption of the endogenous TCR.
[Item 3] The recombinant nucleic acid according to item 2, wherein the binding ligand can bind to the Fc domain of the antibody.
[Item 4] The recombinant nucleic acid according to item 2, wherein the binding ligand can selectively bind to an IgG1 antibody.
[Item 5] The recombinant nucleic acid according to item 2, wherein the binding ligand can specifically bind to an IgG4 antibody.
[Item 6] The recombinant nucleic acid according to item 2, wherein the antibody or a fragment thereof binds to a cell surface antigen.
[Item 7] The recombinant nucleic acid according to item 2, wherein the antibody or a fragment thereof binds to a cell surface antigen on the surface of a tumor cell.
[Item 8] The binding ligand is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptameric, an octamer, a nonamer, or a decamer. 2. The recombinant nucleic acid according to item 2.
[Item 9] The recombinant nucleic acid according to item 2, wherein the binding ligand does not contain an antibody or a fragment thereof.
[Item 10] The recombinant nucleic acid according to item 9, wherein the binding ligand comprises a CD16 polypeptide or a fragment thereof.
[Item 11] The recombinant nucleic acid according to item 10, wherein the binding ligand comprises a CD16-binding polypeptide.
[Item 12] The recombinant nucleic acid according to item 2, wherein the binding ligand is human or humanized.
[Item 13] The recombinant nucleic acid according to item 2, further comprising a nucleic acid sequence encoding an antibody or a fragment thereof to which the binding ligand can bind.
[Item 14] The recombinant nucleic acid according to item 13, wherein the antibody or fragment thereof can be secreted from cells.
[Item 15] Recombinant nucleic acid.
(A) A sequence encoding a T cell receptor (TCR) fusion protein (TFP).
(I) Below:
(1) At least part of the TCR extracellular domain,
(2) Transmembrane domain and
(3) With a TCR subunit containing an intracellular domain containing a stimulating domain from the intracellular signaling domain of CD3ε, CD3γ, CD3δ, TCRα, or TCRβ.
(Ii) With an antigen domain containing a ligand or a fragment thereof that binds to a receptor or polypeptide expressed on the surface of a cell.
The sequence containing, as well as
(B) A sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRα constant domain, a TCRβ constant domain, or a TCRα constant domain and a TCRβ constant domain.
Including
The TCR subunit and the antigen domain are functionally linked and
A recombinant nucleic acid in which the TFP is functionally integrated into the TCR complex when expressed in a modified T cell comprising functional disruption of the endogenous TCR.
[Item 16] The recombinant nucleic acid according to item 15, wherein the antigen domain contains a ligand.
[Item 17] The recombinant nucleic acid according to item 15, wherein the ligand binds to a cell receptor.
[Item 18] The recombinant nucleic acid according to item 15, wherein the ligand binds to a polypeptide expressed on the surface of a cell.
[Item 19] The recombinant nucleic acid according to item 15, wherein the receptor or polypeptide expressed on the surface of a cell comprises a stress response receptor or polypeptide.
[Item 20] The recombinant nucleic acid according to item 15, wherein the receptor or polypeptide expressed on the surface of a cell is an MHC class I-related glycoprotein.
[Item 21] The recombinant nucleic acid according to item 20, wherein the MHC class I-related glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof.
[Item 22] The antigen domain is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptameric, an octamer, a nonamer, or a decamer. The recombinant nucleic acid of item 15.
[Item 23] The recombinant nucleic acid according to item 22, wherein the antigen domain comprises a monomer or a dimer of the ligand or a fragment thereof.
[Item 24] The ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a hepatic, an octamer, a nonamer, or a decamer. The recombinant nucleic acid according to item 15, which is a body.
[Item 25] The recombinant nucleic acid according to item 24, wherein the ligand or a fragment thereof is a monomer or a dimer.
[Item 26] The recombinant nucleic acid according to item 15, wherein the antigen domain does not contain an antibody or a fragment thereof.
[Item 27] The recombinant nucleic acid according to item 15, wherein the antigen domain does not contain a variable region.
[Item 28] The recombinant nucleic acid according to item 15, wherein the antigen domain does not contain a CDR.
[Item 29] The recombinant nucleic acid according to item 15, wherein the ligand or a fragment thereof is a natural killer group 2D (NKG2D) ligand or a fragment thereof.
[Item 30] The recombinant nucleic acid according to any one of items 1 to 29, wherein the TCR constant domain is integrated into a functional TCR complex when expressed in T cells.
[Item 31] The set according to any one of items 1 to 30, wherein when the TCR constant domain is expressed in T cells, it is incorporated into the same functional TCR complex as the functional TCR complex that incorporates the TFP. Replacement nucleic acid.
[Item 32] The recombinant nucleic acid according to any one of items 1 to 31, wherein the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained in the same nucleic acid molecule.
[Item 33] The recombinant nucleic acid according to any one of items 1 to 31, wherein the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained in different nucleic acid molecules.
[Item 34] The recombination according to item 1-33, wherein the TCR subunit and the antibody domain, the antigen domain, or the binding ligand, or a fragment thereof are functionally linked by a linker sequence. Nucleic acid.
[Item 35] The recombinant nucleic acid according to item 34, wherein the linker sequence comprises (G4S ) n (n = 1-4 in the formula).
Item 36. The recombinant nucleic acid according to any one of items 1 to 35, wherein the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, or TCRβ.
[Item 37] The recombinant nucleic acid according to any one of Items 1 to 36, wherein the intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, or TCRβ only.
[Item 38] The TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, (i), (ii), and. The recombinant nucleic acid according to any one of items 1 to 37, wherein at least two of (iii) are derived from the same TCR subunit.
[Item 39] The TCR extracellular domain has a TCRα chain, a TCRβ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and at least one but 20 or less modifications thereof. The recombinant nucleic acid according to any one of items 1 to 38, which comprises an extracellular domain of a protein or a part thereof, which is selected from the group consisting of amino acid sequences.
[Item 40] The TCR subunit is a TCRα chain, a TCRβ chain, a TCR zeta chain, a CD3ε TCR subsystem, a CD3γ TCR subsystem, a CD3δ TCR subsystem, a CD45, a CD4, a CD5, a CD8, a CD9, a CD16, a CD22, or a CD33. , CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and the transmembrane domain of the protein selected from the group consisting of at least one of the amino acid sequences having a modification of 20 or less. The recombinant nucleic acid according to any one of items 1 to 39, which comprises a transmembrane domain comprising.
[Item 41] The TCR subunit contains a stimulating domain of a protein selected from the intracellular signaling domain of CD3ε, CD3γ, or CD3δ, or an amino acid sequence having at least one modification to them. The recombinant nucleic acid according to any one of items 1 to 40.
[Item 42] The TCR subunit is a stimulating domain of a protein selected from the functional signaling domain of 4-1BB and / or the functional signaling domain of CD3ζ, or an amino acid having at least one modification to them. The recombinant nucleic acid according to any one of items 1-41, comprising an intracellular domain comprising a sequence.
[Item 43] The recombinant nucleic acid according to any one of items 1-42, further comprising a sequence encoding a co-stimulation domain.
[Item 44] The co-stimulation domain is OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and at least one. However, the recombinant nucleic acid of item 43, comprising the functional signaling domain of the protein, selected from the group consisting of its amino acid sequence with a modification of 20 or less.
[Item 45] The TCR subunit is a CD3ζ TCR subunit, a CD3ε TCR subsystem, a CD3γ TCR subsystem, a CD3δ TCR subunit, a TCRζ chain, an Fcε receptor 1 chain, an Fcε receptor 2 chain, and an Fcγ receptor 1 chain. , Fcγ receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, The immunoreceptor tyrosine of a protein selected from the group consisting of CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and at least one of the amino acid sequences having a modification of 20 or less. The recombinant nucleic acid according to any one of items 1-44, comprising the TCR subunit ITAM, comprising the activation motif (ITAM) or a portion thereof.
[Item 46] The recombinant nucleic acid according to item 45, wherein the ITAM replaces the ITAM of CD3γ, CD3δ, or CD3ε.
[Item 47] The ITAM is selected from the group consisting of a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, and a CD3δ TCR subunit, and a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, 45. The recombinant nucleic acid of item 45, which replaces another ITAM selected from the group consisting of the CD3δ TCR subunit.
Item 48, wherein the TFP, the TCRα constant domain, the TCRβ constant domain, and any combination thereof can functionally interact with an endogenous TCR complex and / or at least one endogenous TCR polypeptide. The recombinant nucleic acid according to any one of 1 to 47.
[Item 49] (a) The TCR constant domain is a TCRα constant domain, and the TFP is functionally integrated into a TCR complex containing an endogenous subunit of TCRβ, CD3ε, CD3γ, CD3δ, or a combination thereof. mosquito,
(B) The TCR constant domain is a TCRβ constant domain, and the TFP is functionally integrated or integrated into a TCR complex containing an endogenous subunit of TCRα, CD3ε, CD3γ, CD3δ, or a combination thereof.
(C) The TCR constant domain is a TCRα constant domain and a TCRβ constant domain, and the TFP is functionally integrated into a TCR complex containing an endogenous subunit of CD3ε, CD3γ, CD3δ, or a combination thereof. The recombinant nucleic acid according to any one of items 1 to 48.
[Item 50] Items 1-49, wherein the at least one but 20 or less modifications include modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to TFP. The recombinant nucleic acid according to any one item.
[Item 51] The recombinant nucleic acid according to any one of Items 1 and 34 to 50, wherein the human antibody or humanized antibody is an antibody fragment.
[Item 52] The recombinant nucleic acid according to item 51, wherein the antibody fragment is a scFv, a single domain antibody domain, a VE domain , or a VL domain.
[Item 53] The antigen-binding domain is an anti-CD19-binding domain, an anti-B cell maturation antigen (BCMA) -binding domain, an anti-mesotelin (MSLN) -binding domain, an anti-IL13Rα2-binding domain, an anti-MUC16-binding domain, an anti-CD22-binding domain, and an anti-PD. The recombinant nucleic acid according to any one of items 1 and 34 to 52, selected from the group consisting of a -1 binding domain, an anti-BAFF or BAFF receptor binding domain, and an anti-ROR-1 binding domain.
[Item 54] The recombinant nucleic acid according to any one of items 1 to 53, wherein the nucleic acid is selected from the group consisting of DNA and RNA.
[Item 55] The recombinant nucleic acid according to any one of items 1 to 54, wherein the nucleic acid is mRNA.
[Item 56] The recombinant nucleic acid according to any one of Items 1 to 55, wherein the recombinant nucleic acid comprises a nucleic acid analog, and the nucleic acid analog is not in the coding sequence of the recombinant nucleic acid.
[Item 57] The nucleic acid analog is 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-. 2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'- O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA) modification, locked nucleic acid (LNA), ethylene nucleic acid (ENA), Peptide Nucleic Acid (PNA), 1', 5'-Anhydrohexitol Nucleic Acid (HNA), Morphorino, Methylphosphonic Acid Nucleotide, Thiolphosphonate Nucleic Acid, and 2'-Fluoro N3-P5'-Phophoroamideite Item 5. The recombinant nucleic acid according to item 56, which is selected from the group consisting of.
[Item 58] The recombinant nucleic acid according to any one of items 1 to 57, further comprising a leader sequence.
[Item 59] The recombinant nucleic acid according to any one of items 1 to 58, further comprising a promoter sequence.
[Item 60] The recombinant nucleic acid according to any one of items 1 to 59, further comprising a sequence encoding a poly (A) tail.
[Item 61] The recombinant nucleic acid according to any one of items 1 to 60, further comprising a 3'UTR sequence.
[Item 62] The recombinant nucleic acid according to any one of items 1 to 61, wherein the nucleic acid is an isolated nucleic acid or a nucleic acid that does not exist in nature.
[Item 63] The recombinant nucleic acid molecule according to any one of items 1 to 62, wherein the nucleic acid is an in vitro transcribed nucleic acid.
[Item 64] The recombinant nucleic acid molecule according to any one of items 1 to 63, further comprising a sequence encoding a TCRα transmembrane domain.
[Item 65] The recombinant nucleic acid molecule according to any one of items 1 to 63, further comprising a sequence encoding a TCRβ transmembrane domain.
[Item 66] The recombinant nucleic acid according to any one of items 1 to 63, further comprising a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain.
[Item 67] A vector containing the recombinant nucleic acid according to any one of items 1 to 66.
Item 68. Item 67, which is selected from the group consisting of DNA, RNA, plasmids, lentivirus vectors, adenoviral vectors, adeno-associated virus vectors (AAV), Rous sarcoma virus (RSV) vectors, or retroviral vectors. Vector.
[Item 69] The vector according to item 67 or 68, which is an AAV6 vector.
[Item 70] The vector according to any one of items 67 to 69, further comprising a promoter.
[Item 71] The vector according to any one of items 67 to 70, which is an in vitro transcribed vector.
[Item 72] A modified T cell containing the recombinant nucleic acid according to any one of items 1 to 66 or the vector according to any one of items 67 to 71, which causes functional disruption of endogenous TCR. Including modified T cells.
[Item 73] A TFP encoded by the sequence encoding the TFP of the nucleic acid according to any one of items 1 to 66, or the sequence of the nucleic acid according to any one of items 1 to 66 encoding TFP. Modified T cells comprising, comprising disrupting the function of an endogenous TCR.
[Item 74] The TFP comprises the sequence encoding the TFP according to any one of items 1 to 66, or the TFP encoded by the sequence of the nucleic acid according to any one of items 1 to 66 encoding TFP. Modified allogeneic T cells.
Item 75, any one of items 72-74, further comprising a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRα constant domain, a TCRβ constant domain, or a TCRα constant domain and a TCRβ constant domain. The modified T cells described in.
[Item 76] The item according to any one of items 72 to 75, wherein the functionally disrupted endogenous TCR is an endogenous TCRα chain, an endogenous TCRβ chain, or an endogenous TCRα chain and an endogenous TCRβ chain. Modified T cells.
[Item 77] In any one of items 72-76, the functionally disrupted endogenous TCR has reduced binding to the MHC peptide complex as compared to that of unmodified control T cells. Described modified T cells.
[Item 78] The modified T cell according to any one of items 72 to 77, wherein the functional disruption is disruption of the gene encoding the endogenous TCR.
[Item 79] The modified T cell of item 78, wherein the disruption of the gene encoding the endogenous TCR is the removal of the sequence of the gene encoding the endogenous TCR from the genome of the T cell.
[Item 80] CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells. , Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ / δT cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells, and pluripotent stem cells. , The modified T cell according to any one of items 72 to 79.
[Item 81] The modified T cell according to any one of items 72 to 80, which is a CD8 + T cell or a CD4 + T cell.
[Item 82] The modified T cell according to any one of items 72 to 81, which is an allogeneic T cell.
[Item 83] A nucleic acid encoding an inhibitory molecule comprising a first polypeptide comprising at least a portion of the inhibitory molecule is further included associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. The modified T cell according to any one of items 72 to 82.
[Item 84] The modified T cell according to item 83, wherein the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a co-stimulation domain and a primary signaling domain. ..
[Item 85] (a) The modified T cells according to any one of items 72 to 84, and
(B) A pharmaceutical composition comprising a pharmaceutically acceptable carrier.
[Item 86] The method for producing a modified T cell according to any one of items 72 to 84.
(A) Disrupting the TCRα chain, TCRβ chain, or the endogenous TCR gene encoding the TCRα chain and the TCRβ chain, thereby producing T cells containing the functional disruption of the endogenous TCR gene.
(B) Transduction of the recombinant nucleic acid according to any one of items 1 to 63 or the vector according to any one of items 67 to 71 into T cells containing functional disruption of an endogenous TCR gene. Methods, including doing.
[Item 87] The disruption transduces a TCRα chain, a TCRβ chain, or a nuclease protein targeting an endogenous gene encoding a TCRα chain and a TCRβ chain, or a nucleic acid sequence encoding a nuclease protein into T cells. Included, the method of item 86.
[Item 88] The method for producing a modified T cell according to any one of items 72 to 84, wherein the T cell containing functional disruption of an endogenous TCR gene is one of items 1 to 63. A method comprising transducing a recombinant nucleic acid according to item or a vector according to any one of items 67 to 71.
[Item 89] The T cell containing the functional disruption of the endogenous TCR gene is a TCRα chain, a TCRβ chain, or a T cell containing a functional disruption of the endogenous TCR gene encoding the TCRα chain and the TCRβ chain. , Item 88.
[Item 90] The method according to any one of items 86 to 89, wherein the T cell is a human T cell.
[Item 91] Items 86-90, wherein the T cells containing functional disruption of the endogenous TCR gene have reduced binding to the MHC-peptide complex as compared to those of unmodified control T cells. The method according to any one.
[Item 92] In any one of items 86 to 91, wherein the nuclease is a meganuclease, a zinc finger nuclease (ZFN), a transcriptional activator-like effector nuclease (TALEN), a CRISPR / Casnuclease, or a megaTAL nuclease. The method described.
[Item 93] The sequence contained in the recombinant nucleic acid or vector is inserted into the endogenous TCR subunit gene at the cleavage site, and the sequence is inserted into the endogenous TCR subunit gene to cause the endogenous. The method of any one of items 86-92, wherein the TCR subunit is functionally disrupted.
[Item 94] The method according to any one of items 86 to 93, wherein the nuclease is a meganuclease.
[Item 95] The meganuclease contains a first subunit and a second subunit, the first subunit binds to the first recognition half site of the recognition sequence, and the second subunit is The method of item 94, which binds to the second recognition subunit of the recognition sequence.
[Item 96] The method of item 95, wherein the meganuclease is a single-stranded meganuclease containing a linker, wherein the linker covalently binds the first subunit to the second subunit.
[Item 97] A method for treating cancer in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 85.
[Item 98] A method for treating cancer in a subject in need of treatment, wherein (a) modified T cells produced according to the method according to any one of items 86 to 96, and (b) pharmaceutical. A method comprising administering to said subject a pharmaceutical composition comprising an acceptable carrier.
[Item 99] A method for treating cancer in a subject in need of treatment, wherein (a) modified T cells produced according to the method according to any one of items 88 to 96, and (b) pharmaceutical. A method comprising administering to said subject a pharmaceutical composition comprising an acceptable carrier.
[Item 100] The method according to any one of items 97 to 99, wherein the modified T cells are allogeneic T cells.
[Item 101] The method according to any one of items 97 to 100, wherein a subject to which an effective amount of unmodified control T cells has been administered has less cytokine released in the subject.
[Item 102] Compared with a subject to which an effective amount of modified T cells containing the recombinant nucleic acid according to any one of items 1 to 66 or the vector according to any one of items 67 to 71 was administered. Item 6. The method according to any one of Items 97 to 101, wherein the subject releases less cytokine.
[Item 103] The method according to any one of items 97 to 102, wherein the method comprises administering the pharmaceutical composition in combination with an agent that increases the efficacy of the pharmaceutical composition.
[Item 104] The item according to any one of items 97 to 103, wherein the method comprises administering the pharmaceutical composition in combination with an agent that improves one or more side effects associated with the pharmaceutical composition. Method.
[Item 105] The method according to any one of items 97 to 104, wherein the cancer is solid cancer, lymphoma, or leukemia.
[Item 106] The cancer is selected from the group consisting of renal cell cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, and gastric cancer. The method according to any one of items 97 to 105.
[Item 107] The recombinant nucleic acid according to any one of items 1 to 66, the vector according to any one of items 67 to 71, and items 72 to 84 for use as a drug or for preparation of a drug. The modified T cell according to any one of the above items, or the pharmaceutical composition according to item 85.
Claims (21)
（ａ）Ｔ細胞受容体（ＴＣＲ）融合タンパク質（ＴＦＰ）をコードする配列であって、
（ｉ）以下：
（１）ＴＣＲ細胞外ドメインの少なくとも一部、
（２）膜貫通ドメイン、及び
（３）ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、ＴＣＲα、又はＴＣＲβの細胞内シグナル伝達ドメインからの刺激ドメインを含む細胞内ドメイン
を含むＴＣＲサブユニットと
（ｉｉ）抗原結合ドメインを含む抗体と
を含む前記配列、並びに
（ｂ）ＴＣＲ定常ドメインをコードする配列であって、前記ＴＣＲ定常ドメインが、ＴＣＲα定常ドメイン、ＴＣＲβ定常ドメイン、又はＴＣＲα定常ドメインとＴＣＲβ定常ドメインである、前記配列
を含み、
前記ＴＣＲサブユニット及び前記抗体が機能的に連結されており、かつ内因性ＴＣＲの機能的破壊を含む改変Ｔ細胞で発現すると前記ＴＦＰがＴＣＲ複合体に機能的に組み込まれる、前記組換え核酸。 Recombinant nucleic acid
(A) A sequence encoding a T cell receptor (TCR) fusion protein (TFP).
(I) Below:
(1) At least part of the TCR extracellular domain,
Includes (2) transmembrane domain and (3) TCR subunit containing intracellular domain including stimulation domain from intracellular signaling domain of CD3ε, CD3γ, CD3δ, TCRα, or TCRβ and (ii) antigen binding domain. The sequence comprising the antibody and (b) the sequence encoding the TCR subunit, wherein the TCR subunit is a TCRα subunit, a TCRβ subunit, or a TCRα subunit and a TCRβ subunit. Including,
The recombinant nucleic acid in which the TCR subunit and the antibody are functionally linked and the TFP is functionally integrated into the TCR complex when expressed in a modified T cell containing functional disruption of the endogenous TCR.
前記細胞内ドメインが、ＣＤ３εのみ、ＣＤ３γのみ、ＣＤ３δのみ、ＴＣＲαのみ、又はＴＣＲβのみに由来し、及び／又は
前記ＴＣＲ細胞外ドメインが、ＴＣＲα鎖、ＴＣＲβ鎖、ＣＤ３ε ＴＣＲサブユニット、ＣＤ３γ ＴＣＲサブユニット、ＣＤ３δ ＴＣＲサブユニット、それらの機能的断片、及び少なくとも１つだが２０以下の修飾を有するそのアミノ酸配列からなる群から選択される、タンパク質の細胞外ドメイン又はその一部を含む、請求項１又は２に記載の組換え核酸。 The transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, or TCRβ .
The intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, or TCRβ only, and / or
The TCR extracellular domain consists of a TCRα chain, a TCRβ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, a functional fragment thereof, and an amino acid sequence having at least one modification of 20 or less. The recombinant nucleic acid according to claim 1 or 2 , comprising an extracellular domain of a protein or a portion thereof, selected from the group.
（ｂ）前記ＴＣＲ定常ドメインはＴＣＲβ定常ドメインであり、前記ＴＦＰがＴＣＲα、ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、又はそれらの組合せの内因性サブユニットを含むＴＣＲ複合体に機能的に統合されるか、あるいは
（ｃ）前記ＴＣＲ定常ドメインはＴＣＲα定常ドメイン及びＴＣＲβ定常ドメインであり、前記ＴＦＰが、ＣＤ３ε、ＣＤ３γ、ＣＤ３δ、又はそれらの組合せの内因性サブユニットを含むＴＣＲ複合体に機能的に統合される、請求項１～４のいずれか１項に記載の組換え核酸。 (A) The TCR constant domain is a TCRα constant domain, whether the TFP is functionally integrated into a TCR complex containing an endogenous subunit of TCRβ, CD3ε, CD3γ, CD3δ, or a combination thereof.
(B) The TCR constant domain is a TCRβ constant domain, and the TFP is functionally integrated into or (1) a TCR complex comprising an endogenous subunit of TCRα, CD3ε, CD3γ, CD3δ, or a combination thereof. c) The TCR constant domain is a TCRα constant domain and a TCRβ constant domain, wherein the TFP is functionally integrated into a TCR complex comprising an endogenous subunit of CD3ε, CD3γ, CD3δ, or a combination thereof. Item 2. The recombinant nucleic acid according to any one of Items 1 to 4 .
（ｂ）プロモーター配列、
（ｃ）ポリ（Ａ）尾部をコードする配列、
（ｄ）３’ＵＴＲ配列、
（ｅ）ＴＣＲα膜貫通ドメインをコードする配列、
（ｆ）ＴＣＲβ膜貫通ドメインをコードする配列、及び／又は
（ｇ）ＴＣＲα膜貫通ドメインをコードする配列及びＴＣＲβ膜貫通ドメインをコードする配列、
をさらに含む、請求項１～７のいずれか１項に記載の組換え核酸。 (A) Reader arrangement,
(B) Promoter sequence,
(C) Poly (A) Sequence encoding tail,
(D) 3'UTR array,
(E) A sequence encoding a TCRα transmembrane domain,
(F) A sequence encoding a TCRβ transmembrane domain and / or
(G) A sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain,
The recombinant nucleic acid according to any one of claims 1 to 7 , further comprising.
（ａ）Ｔ細胞受容体（ＴＣＲ）融合タンパク質（ＴＦＰ）をコードする配列であって、(A) A sequence encoding a T cell receptor (TCR) fusion protein (TFP).
（ｉ）以下：(I) Below:
（１）ＴＣＲ細胞外ドメインの少なくとも一部、及び(1) At least part of the TCR extracellular domain and
（２）膜貫通ドメイン、(2) Transmembrane domain,
を含むＴＣＲサブユニットと、With the TCR subunit, including
（ｉｉ）抗原結合ドメインを含むヒト抗体又はヒト化抗体、あるいは抗体又はその断片に結合できる結合リガンド又はその断片と、(Ii) A human antibody or humanized antibody containing an antigen-binding domain, or a binding ligand or fragment thereof capable of binding to the antibody or fragment thereof.
を含む前記配列、並びにThe sequence containing, as well as
（ｂ）ＴＣＲ定常ドメインをコードする配列、(B) A sequence encoding a TCR constant domain,
を含み、Including
前記ＴＣＲサブユニット及び前記抗体は機能的に連結されており、かつThe TCR subunit and the antibody are functionally linked and
内因性ＴＣＲの機能的破壊を含む改変Ｔ細胞で発現すると前記ＴＦＰがＴＣＲ複合体に機能的に組み込まれる、前記組換え核酸。The recombinant nucleic acid, wherein the TFP is functionally integrated into the TCR complex when expressed in a modified T cell comprising functional disruption of the endogenous TCR.
前記機能的に破壊された内因性ＴＣＲが、未改変の対照Ｔ細胞のものと比較して、ＭＨＣペプチド複合体への結合が減少し、及び／又は
前記機能的破壊が、前記内因性ＴＣＲをコードする遺伝子の破壊であり、
前記内因性ＴＣＲをコードする遺伝子の前記破壊が、Ｔ細胞のゲノム由来の前記内因性ＴＣＲをコードする前記遺伝子の配列の除去である、
請求項１２又は１３に記載の改変Ｔ細胞。 The functionally disrupted endogenous TCR is an endogenous TCRα chain, an endogenous TCRβ chain, or an endogenous TCRα chain and an endogenous TCRβ chain.
The functionally disrupted endogenous TCR has reduced binding to the MHC peptide complex and / or compared to that of unmodified control T cells.
The functional disruption is the disruption of the gene encoding the endogenous TCR.
The disruption of the gene encoding the endogenous TCR is the removal of the sequence of the gene encoding the endogenous TCR from the T cell genome.
The modified T cell according to claim 12 or 13 .
前記抑制分子が、ＰＤ１の少なくとも一部を含む第１のポリペプチドと、共刺激ドメイン及び一次シグナル伝達ドメインを含む第２のポリペプチドとを含んでもよい、
請求項１２～１４のいずれか１項に記載の改変Ｔ細胞。 Nucleic acid encoding a suppressor molecule, including a first polypeptide containing at least a portion of the suppressor molecule, is further included associated with a second polypeptide containing a positive signal from the intracellular signaling domain.
The inhibitory molecule may include a first polypeptide containing at least a portion of PD1 and a second polypeptide containing a co-stimulation domain and a primary signaling domain.
The modified T cell according to any one of claims 12 to 14 .
（ｂ）薬学的に許容される担体と、
を含む、医薬組成物。 (A) The modified T cell according to any one of claims 12 to 15 .
(B) Pharmaceutically acceptable carriers and
A pharmaceutical composition comprising.
（ａ）ＴＣＲα鎖、ＴＣＲβ鎖、又はＴＣＲα鎖とＴＣＲβ鎖をコードする内因性ＴＣＲ遺伝子を破壊し、それにより内因性ＴＣＲ遺伝子の機能的破壊を含んだＴ細胞を作製すること、及び
（ｂ）内因性ＴＣＲ遺伝子の機能的破壊を含んだＴ細胞に、請求項１～１０のいずれか１項に記載の組換え核酸又は請求項１１に記載のベクターを形質導入すること、
を含む、前記方法。 The method for producing a modified T cell according to any one of claims 12 to 15 .
(A) disrupting the TCRα chain, TCRβ chain, or endogenous TCR gene encoding the TCRα chain and TCRβ chain, thereby producing T cells containing the functional disruption of the endogenous TCR gene, and (b). Transducing the recombinant nucleic acid according to any one of claims 1 to 10 or the vector according to claim 11 into T cells containing functional disruption of an endogenous TCR gene.
The method described above.
前記ヌクレアーゼが、メガヌクレアーゼ、ジンクフィンガーヌクレアーゼ（ＺＦＮ）、転写活性化因子様エフェクターヌクレアーゼ（ＴＡＬＥＮ）、ＣＲＩＳＰＲ／Ｃａｓヌクレアーゼ、又はｍｅｇａＴＡＬヌクレアーゼでもよい、
請求項１９に記載の方法。 The disruption comprises transducing a TCRα chain, a TCRβ chain, or a nuclease protein targeting an endogenous gene encoding the TCRα chain and the TCRβ chain, or a nucleic acid sequence encoding the nuclease protein into T cells.
The nuclease may be a meganuclease, zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), CRISPR / Cas nuclease, or megaTAL nuclease.
19. The method of claim 19 .
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| ES2875959T3 (en) | 2016-10-07 | 2021-11-11 | Tcr2 Therapeutics Inc | Compositions and methods for T-cell receptor reprogramming using fusion proteins |
| WO2018098365A2 (en) | 2016-11-22 | 2018-05-31 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
| WO2021035170A1 (en) * | 2019-08-21 | 2021-02-25 | Precision Biosciences, Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
| KR20220078607A (en) * | 2019-09-12 | 2022-06-10 | 티씨알2 테라퓨틱스 인크. | Compositions and methods for TCR reprogramming using fusion proteins |
| WO2021133959A2 (en) * | 2019-12-24 | 2021-07-01 | TCR2 Therapeutics Inc. | Compositions and methods for gamma delta tcr reprogramming using fusion proteins |
| AU2021326035A1 (en) * | 2020-08-12 | 2023-03-09 | Migal Galilee Research Institute Ltd. | Alloreactive immune cell-distancing device and uses thereof for protecting donor-derived cells from allorejection |
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