JP7242761B2 - キメラ抗原受容体及び他の受容体の発現に対するt細胞 - Google Patents
キメラ抗原受容体及び他の受容体の発現に対するt細胞 Download PDFInfo
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Description
様々な方法を用いて、ヒトTCM/SCM/N細胞の集団を生成することができる。例えば、TCM/SCM/N細胞の集団を、混合Tリンパ球集団から調製することができる。Tリンパ球の集団は、細胞を用いて最終的に処置された対象に対して同種異系又は自己由来であり得、さらに、白血球フェレーシス又は採血によって対象から得ることができる。
1)CD62L+セントラルメモリー(TCM)細胞、及び、2)セントラルメモリー(TCM)集団も幹細胞様メモリー(TSCM)集団も、ナイーブT細胞(TN)と共に含むCD62L+TCM/SCM/N細胞のT細胞集団のCliniMACS/AutoMACS(商標)による濃縮を受けたヒト末梢血単核細胞(PBMC)の収率を評価するために研究を行った。生細胞数のGuava分析及び表現型のフローサイトメトリー分析を用いて評価を行った。
TCM/SCM/N細胞集団は、関心のある1つ又は複数のタンパク質を発現するように遺伝子操作することができる。例えば、細胞を、関心のある1つ又は複数のタンパク質を発現する機能を有するレンチウイルスで形質導入することができる。以下は、硫酸プロタミン/サイトカイン溶液を用いてレンチウイルスベクターでT細胞の形質導入に用いうる1つの方法の例である。
CD62L+TCMの集団又はCD62L+TCM/SCM/Nの集団(ナイーブ、セントラルメモリー及び幹細胞様メモリーのT細胞を含む)のいずれかについて、CliniMACS/AutoMACS(商標)による濃縮を受け、その後、臨床使用のために提案された方法を用いてビーズ刺激、及び、CD19Rを標的とするCARを発現するレンチウイルスであるCD19R(EQ)28Z-T2A-EGFRt_epHIV7又はIL-13Ralplaを標的とするレンチウイルスベクターであるIL13(EQ)BBZ-T2A-CD19t_epHIV7でレンチウイルス形質導入を行ったヒト末梢血単核細胞(PBMC)のex vivoでの増殖及び細胞表面表現型を評価するために研究を行った(ベクター及び発現したCARの詳細については特許文献1及び特許文献3を参照)。生細胞数のモニタリングによって増殖を評価し、さらに、フローサイトメトリー分析によって細胞表面表現型を評価した。
臨床使用に適した方法を用いて、CD19を標的としたCARを発現するレンチウイルスベクター(CD19R(EQ)28Z-T2A-EGFRt_epHIV7レンチウイルスベクター)で形質導入し、かつ増殖させた選択されていないPBMC、CD62L+TCM細胞及びCD62L+TCM/SCM/N細胞のインビボでの有効性を評価するために、研究を行った。静脈内(iv)投与されたこれらの細胞のインビボでの抗腫瘍の有効性を、緑色蛍光タンパク質(GFP)及びホタルルシフェラーゼ(ffLuc)レポーター遺伝子を発現するように操作されたCD19発現SUP-B15ヒト急性リンパ球性白血病の細胞株を用いて、免疫不全のNSGマウスにおいて調べた(PMID:22407828)。
臨床使用に適すると予想される方法を用いて、CD19R(EQ)28Z-T2A-EGFRt_epHIV7レンチウイルスベクターで形質導入し、増殖させたCD62L+セントラルメモリー(TCM)細胞又はCD62L+TCM/SCM/N細胞のCD19特異的な細胞溶解活性、脱顆粒及びサイトカイン生成の評価の研究を行った。細胞溶解活性を、フローサイトメトリーに基づく長期の死滅アッセイ及び5時間の脱顆粒アッセイを用いて評価した。サイトカイン生成を、刺激細胞との5時間の共培養後に、T細胞の細胞内染色によって評価した。
IL13Rα2を標的とするCARを発現するレンチウイルスベクターで形質導入したCD62L+TCM細胞及びCD62L+TCM/SCM/N細胞のインビボでの有効性を評価する研究を行った。CARは、ヒトIL-13変異体を含み、レンチウイルスベクター(IL13(EQ)BBZ-T2A-CD19t_epHIV7)によって発現される。このベクターを発現する細胞集団を実施例4において先に記載したように調製し、ホタルルシフェラーゼ(ffLuc)レポーター遺伝子を発現するように操作されたIL13Rα2+初代低継代GBM腫瘍様塊の株PBT030-2を用いた免疫不全NSGマウスにおける神経膠芽腫のマウスモデルにおいて評価した。
Claims (17)
- T細胞を含むヒト細胞の単離された集団であって、前記T細胞は、セントラルメモリーT細胞;メモリー幹T細胞、及びナイーブT細胞を含み、かつ、前記T細胞のうち、50%を超える前記T細胞はCD45RA+であり、90%を超える前記T細胞はCD62L+であり、前記T細胞の15%以下がCD14+であり、前記T細胞の5%以下がCD25+であり、かつ、前記T細胞のうち25%を超える前記T細胞はキメラ抗原受容体を発現する、単離されたヒト細胞の集団。
- 前記T細胞のうち少なくとも70%は、CD4+及びCD62L+、又はCD8+及びCD62L+である、請求項1に記載の単離されたヒト細胞の集団。
- 前記T細胞のうちの60%未満は、CD45RO+である、請求項1に記載の単離されたヒト細胞の集団。
- 前記キメラ抗原受容体がCD19を標的としている、請求項1~3のいずれかに記載の単離されたヒト細胞の集団。
- キメラ抗原受容体を発現するT細胞を含むヒト細胞の集団を調製する方法であって、以下の:
a)T細胞を含むヒト細胞の集団を提供する工程であって、ここで、前記T細胞は:セントラルメモリーT細胞;メモリー幹T細胞、及びナイーブT細胞を含み、ここで、前記T細胞の50%以上がCD45RA+であり、前記T細胞の90%以上がCD62L+であり;
b)前記T細胞を含むヒト細胞の集団を活性化する工程;かつ、
c)キメラ抗原受容体をコードする組換え核酸分子で、前記T細胞を含むヒト細胞の集団における細胞を導入又はトランスフェクションして、キメラ抗原受容体を発現するT細胞を含むヒト細胞の集団を提供する工程;
を含む、方法であって、
ここで、前記方法は、CD45RAを発現する細胞を枯渇させる工程を含まない、
方法。 - 前記組換え核酸分子は、ウイルスベクターである、請求項5に記載の方法。
- 前記ウイルスベクターは、レンチウイルスベクターである、請求項6に記載の方法。
- さらに、組換え核酸分子を保有するT細胞を含む前記ヒト細胞の集団を培養する工程を含む、請求項5に記載の方法。
- 前記培養する工程は、外因性IL-2及び外因性IL-15の添加を含む、請求項8に記載の方法。
- 活性化工程は、細胞を抗CD3抗体及び抗CD28抗体にさらす工程を含む、請求項5に記載の方法。
- 前記キメラ抗原受容体はCD19を標的とする、請求項5に記載の方法。
- 請求項1~4のいずれかに記載のT細胞を含む単離されたヒト細胞の集団を含む、がん、自己免疫又は感染症の治療用医薬組成物。
- 前記単離されたヒト細胞の単離された集団は、それが必要な患者に対して自己由来である、請求項12に記載の医薬組成物。
- 前記単離されたヒト細胞の単離された集団が、それが必要な患者に対して同種異系である、請求項12に記載の医薬組成物。
- 請求項5~11のいずれかに記載の方法で調製されたT細胞を含む単離されたヒト細胞の集団を含む、がん、自己免疫又は感染症の治療用医薬組成物。
- 前記単離されたヒト細胞の単離された集団が、それが必要な患者に対して自己由来である、請求項15に記載の医薬組成物。
- 前記単離されたヒト細胞の単離された集団が、それが必要な患者に対して同種異系である、請求項15に記載の医薬組成物。
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