JP7414714B2 - 抗腫瘍および抗ウイルスt細胞の生存および機能性を増強する方法および組成物 - Google Patents
抗腫瘍および抗ウイルスt細胞の生存および機能性を増強する方法および組成物 Download PDFInfo
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Description
本出願は2017年9月29日に出願された米国仮特許出願第62/565,820号の優先権を主張し、その全内容は、参照により全体が本明細書に組み込まれる。
1つの実施形態において、本発明はミリストイル化Akt分子を細胞膜上に固定することができ、またこれらがリン酸化され得ることを実証する。レシピエントマウスに養子移植された後、Akt2のCTL集団は肝臓および脾臓で活発に増殖する。これは、Aktの過剰発現が抗原刺激への応答としての肝内生存またはCTLの二次的増殖に関係することを示す。
別の実施形態において、本発明はAktシグナル伝達が免疫チェックポイント、特にHBV(B型肝炎ウイルス)特異的CTL上のLAG-3およびTIGITの発現を妨げることを実証した。
いくつかの実施形態において、本発明はAkt2発現CTLが2つの異なるモデルにおいて効率的に肝内のウイルス感染を排除し、回復した個体において持続し、防御記憶免疫を提供することを実証する。
以下、図中の“liver”は「肝臓」、“spleen”は「脾臓」、“ctrl”は「コントロール(対照実験)」、“adoptive transfer”は「養子移植」、“irrelevant”は「無関係の」、“cytokine-secreting cells”は「サイトカイン産生細胞」、“infection”は「感染」をそれぞれ意味する。
HBVゲノム(AdHBV)を保有するアデノウイルスに感染させたコンジェニック(類遺伝子)系統のC57BL/6マウスに、in vitro活性化CD45.1O+Hc93-100O特異的CD8ODT細胞を養子移植し、これらのマウスにおけるHBeAgの血清レベルの変化を検出する。ほとんどのマウスは42日以内に持続性HBV感染を排除できなかったことが見出された(図1A)。
マウス幹細胞レトロウイルス(MSCV)系は、造血細胞系統を形質導入する効率が高いため、Tリンパ球への遺伝子送達のために選択される。pMSCV-CD90.1プラスミドは、CD90.1遺伝子の3’非翻訳領域におけるウッドチャック肝炎ウイルス転写後調節因子(WPRE)によるP2Aペプチド配列およびマウスCD90.1オープンリーディングフレーム(ORF)によるヒグロマイシン耐性遺伝子の置換から生成され、導入遺伝子の発現を増強する。CD90.1遺伝子およびWPRE配列を、pLKO_TRC024プラスミド(RNAi core lab、Taipei、Taiwan)から増幅する。マウスAKT1(配列番号1)、AKT2(配列番号3)、およびAKT3(配列番号5)の各遺伝子を、AKT遺伝子の上流にPCRプライマーによるsrcミリストイル化配列を添加したマウス4T1乳がん細胞からのcDNAを用いるPCRを通してそれぞれクローニングし、膜標的化およびAkt分子の構成活性を確保する。ミリストイル化配列およびAKT遺伝子はそれぞれ、pMSCV-CD90.1におけるP2Aペプチド配列によってマウスCD90.1遺伝子に連結され、pMSCV-mAkt1-CD90.1、pMSCV-mAkt2-CD90.1およびpMSCV-mAkt3-CD90.1をもたらす。発現カセットは、5’および3’MSCV長末端反復(LTR)に隣接する。4つのプラスミドを用いて、それぞれマウスAKT1、AKT2、AKT3または対照実験CD90.1遺伝子を担持する組換えレトロウイルスを作製する(図2A)。
卵白アルブミン(OVA)とルシフェラーゼ発現は、アルブミンプロモーター(pENTRY‐Albp‐OL)の制御下で、OVAとルシフェラーゼをコードするプラスミドの流体力学的注入(HDI)によってレシピエントマウスの肝臓で誘発される。レシピエントマウスに養子移植された後、Akt1およびAkt2は肝臓および脾臓で活発に増殖したが、Akt3遺伝子組み換えCTLまたはCD90.1遺伝子組み換え(対照実験)集団は増殖しなかった。
in vitro活性化および形質導入後、活性化後3日目のHBc93-100特異的CD8+T細胞を、種々の免疫チェックポイントのそれらの表面発現について分析する。構成的に活性なAkt1/2の過剰発現はPD‐1およびTIGITの表面発現を変化させない(図4A、4Bおよび4D、図5A、5Bおよび5D)。しかしそれはAkt1‐およびAkt2‐CTLの表面上のLAG‐3の発現を有意に減少させる(図4Cおよび4D、図5Cおよび5D)。
HBVキャリアマウスの肝臓および脾臓における養子移植された対照実験またはAkt1過剰発現HBc93-100特異的CTLの細胞数を測定すると、養子移植後6日目および19日目の両方で肝臓から回収された対照実験‐CTLよりも多くのAkt1‐CTLが存在する(図7A~C)。
さらにレシピエントマウスの肝臓における生物発光の測定により、肝臓から抗原を排除するための発現CTLの能力を調べた。生物発光の損失は肝臓からの抗原の除去を示した。本発明者らは、Akt1‐OT‐I CTLが対照実験OT‐I CTLよりも効率的であり、肝臓からOVAを排除することを見出した(図10A)。それらは7日以内に抗原を除去し、またこれは肝臓における細胞集団の増殖のピークでもあった(図10Bおよび10C)。これらのAkt1-OT-I CTLは対照実験CTLよりもOVA発現肝細胞に対して細胞毒性を実行する能力が高い。これは、養子移植後7日目にAkt1-CTLを投与されたマウスの血清ALTレベルの上昇によって明らかになった(図10D)。
ウイルス感染肝細胞はCTL誘発性細胞毒性に対して高い感受性を示すことが示されている。HDI後の肝臓微小環境はウイルス感染中の微小環境と完全には類似していない可能性がある。そこで肝内持続性ウイルス感染状況下でのCTLにおけるAktの機能を研究するため、アルブミンプロモーターの転写制御の下、肝臓のみでOVAとルシフェラーゼを持続的に表現するアデノウイルス(Ad-Albp-OL)ベースの肝臓感染モデルを確立した。著者らは最初に菌の投与量を標定し、それぞれAd-Albp-OLの2×108および4×108iuによる免疫が2か月以上にわたってルシフェラーゼの安定した発現を誘発し得ることを見出した(図11)。次にAd-Albp-OLの4×108iuをマウスに感染させ、それぞれAkt-および対照実験CTLをマウスに養子移植し、図12Aに示した実験計画に従っていくつかの分析を行った。
肝細胞がん(HCC)殺傷に関するAkt導入CTLの能力をさらに試験し、腫瘍抗原特異的Akt2移植CD8+CTLがHCCを有するマウスへの養子移植後10日目に腫瘍部位および肝臓に蓄積し得ることを実証する(図14A)。これらのAkt2‐CTLは腫瘍微小環境を変化させ、腫瘍部位における周囲のF4/80+マクロファージを誘引または活性化する(図14B)。
がん免疫療法に対するAkt分子の潜在的適用をさらに探索するため、ヒトまたはマウスAkt1またはAkt2遺伝子を有するプラスミドを構築し、抗CEAキメラ抗原レセプタ(CAR)をコードするORFを構築する(図16A)。本発明において使用される組換え抗CEAキメラ抗原レセプタの構築は、Hombachらの論文(Hombach, A.; Wieczarkoweicz, A.; Marquardt, T.; Heuser, C.; Usai, L.; Pohl, C.; Seliger, B.; Abken, H.)および論文「組換え免疫レセプタによる腫瘍特異的T細胞活性化:CD3ζシグナル伝達およびCD28共刺激は効率的なIL-2分泌のために同時に必要とされ、そして1つの組み合わされたCD28/CD3ζシグナル伝達レセプタ分子に組み込まれ得る」(J Immunol 2001, 167(11), 6123‐31)に記載されたものである。マウスAKT1遺伝子、抗CEA CAR ORFまたはその両方を保有する組換えレトロウイルスによって活性化マウスCD3+T細胞を改変し、次にそれらの増殖能力、サイトカイン産生および細胞傷害性についてモニターする。
本発明はさらに、Akt分子の発現が抗ウイルスおよび抗腫瘍CTL応答(例えば増殖、サイトカイン産生および細胞毒性)を促進し得ることを示す。これはさらにMDSCによって誘発される増殖停止に対するCTL耐性の可能性を示す。
要約すると、構成的に活性なAkt分子の発現により、T細胞が寛容原性の肝臓または腫瘍の微小環境で生存し、腫瘍細胞やウイルスを殺傷する能力を獲得することを可能にする。活性型Akt分子はTCRシグナル伝達と組み合わされる場合にのみCTLの大量増殖応答を誘発できるため、CTLのT細胞工学に安全に適用可能である。よって本発明者らは、抗腫瘍または抗ウイルス改変T細胞を含む組成物、ならびに慢性ウイルス感染および悪性腫瘍の処置のためのその使用方法について特許出願する。
Claims (16)
- Akt分子を過剰発現する改変細胞を含み、前記改変細胞は、
a.Aktアイソフォーム、および
b.前記Aktアイソフォームを細胞膜に固定する手段であるペプチド、
をコードするポリヌクレオチドで改変され、
前記Aktアイソフォームが、Akt2であり、前記改変細胞が、T細胞、ナチュラルキラー細胞、造血幹細胞、胚性幹細胞または多能性幹細胞である、免疫寛容を低下させるための組成物。 - 前記ペプチドが、配列番号7に記載のミリストイル化配列からなる、請求項1に記載の組成物。
- 前記ポリヌクレオチドが、キメラ抗原レセプタまたは組換えT細胞レセプタをコードするフラグメントをさらに含む、請求項1に記載の組成物。
- 前記ポリヌクレオチドが、前記Aktアイソフォームと前記キメラ抗原レセプタまたは組換えT細胞レセプタとの間のリンカーをコードするフラグメントをさらに含む、請求項3に記載の組成物。
- 前記リンカーが、配列番号9に記載の2Aペプチドである、請求項4に記載の組成物。
- 有効量の請求項1に記載の組成物を投与することを含む、ヒトを除く対象のウイルス感染症を治療するためのウイルス感染症治療方法。
- 前記ウイルス感染症が肝炎である、請求項6に記載のウイルス感染症治療方法。
- 有効量の請求項1に記載の組成物を投与することを含む、ヒトを除く対象のがんを治療するためのがん治療方法。
- 前記がんが肝がんである、請求項8に記載のがん治療方法。
- 前記がんが肝細胞がん、胆管がん、肝血管肉腫および類上皮血管内皮腫を含む、請求項9に記載のがん治療方法。
- 有効量の請求項3に記載の組成物を投与することを含む、ヒトを除く対象のがんを治療するためのがん治療方法。
- 請求項1に記載の組成物を製造する方法であって、請求項1に記載のポリヌクレオチドを含む組換えウイルスまたはトランスポゾンベクターを、請求項1に記載の改変細胞に導入し、前記改変細胞を増殖させることを含む、製造方法。
- 前記組換えウイルスまたはトランスポゾンベクターが、レトロウイルス、レンチウイルス、アデノウイルス、またはアデノ随伴ウイルスを含み、トランス遺伝子の形質導入または組込みに用いることができる、請求項12に記載の製造方法。
- 前記組換えウイルスまたは前記トランスポゾンベクターが、in vitro転写またはin vitro合成によって増幅され得、エレクトロポレーションまたはリポソームによって前記改変細胞にトランスフェクトされ得る、請求項12に記載の製造方法。
- 前記改変細胞が、ウイルスによる形質導入、DNAによる形質転換またはRNAによる形質転換によってさらに改変され得る、請求項12に記載の製造方法。
- 前記改変細胞の増殖を増強するために、可溶性・プレート結合性の抗CD3および抗CD28抗体、またはサイトカインの補充を伴う抗CD3および抗CD28ビーズで前記改変細胞を刺激することを含む、請求項12に記載の製造方法。
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WO2019068066A1 (en) | 2019-04-04 |
EP3687575A4 (en) | 2021-06-30 |
TWI731268B (zh) | 2021-06-21 |
US20200306303A1 (en) | 2020-10-01 |
EP3687575A1 (en) | 2020-08-05 |
TW201924698A (zh) | 2019-07-01 |
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