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Developing and optimizing chimeric antigen receptor T cell approaches for the treatment of canine cancers

吉本, 翔 東京大学 DOI:10.15083/0002006920

2023.03.24

概要





















吉本



申請者は本学研究科に入学以降、イヌの悪性腫瘍に対する有効な治療法の
模索の中で、キメラ抗原受容体(CAR)発現 T 細胞(CAR-T 細胞)療法をテー
マとして、その臨床応用に向けた基礎的研究を重ねてきた。CAR 遺伝子導入法、
最適な共刺激分子の検索、標的抗原および適応癌腫の探索という CAR-T 細胞療
法の臨床応用に向けて克服すべき課題についての研究成果をまとめ、学位論文
として申請している。腫瘍免疫療法の 1 つである養子免疫療法は、患者の体内
から免疫細胞を採取し体外で活性化・増殖後、再び体内に戻す治療法であり、中
でも CAR-T 細胞療法が効果的治療法になりうると期待されている。CAR は、
抗体の抗原結合部位(scFv)と T 細胞受容体の CD3ζ鎖をキメラ化した受容体
であり、抗原が CAR に結合すると CD3ζ鎖から T 細胞に活性化シグナルが送
られ腫瘍細胞を特異的に攻撃する。ヒトでは CD19 抗原を標的とする CAR-T 細
胞が劇的な治療効果を示し、さらに様々な標的抗原や腫瘍種での臨床応用が進
められているが、イヌにおいてはその検討は始まったばかりであり臨床応用に
は至っていない。
そこで、イヌの悪性腫瘍に対する CAR-T 細胞療法の臨床応用を目指し、イ
ヌ CAR-T 細胞の開発と最適化の検討を行った。まず第 1 章では、イヌの T 細胞
に CAR を持続的に発現させるために、レトロウイルスベクターによる CAR 遺
伝子の導入を試み、作製したイヌ CAR-T 細胞の機能を評価している。2 種のレ
トロウイルスベクター(MigR1、pMND)を用いてイヌ T 細胞への CAR 遺伝子
の導入を行ったところ、pMND では約 40%、MigR1 では約 55%の CAR 発現が
確認された。さらに標的抗原発現細胞との共培養から、MigR1 により作製した
CAR-T 細胞における抗原特異的な細胞傷害活性、脱顆粒マーカーの発現および
IFNγ、TNFα、IL-17 産生を明らかにした。さらに 8 日目後には pMND では
約 80%、MigR1 では約 95%の CAR 発現が確認され、レトロウイルスベクター
による CAR の持続的発現が成功した。また、MigR1 により作製された CAR-T
細胞が、抗原特異的に腫瘍細胞を攻撃することも明らかにした。
第 2 章では、イヌ CAR-T 細胞の増殖などの機能を向上させる共刺激ドメイ

ンの検索と評価を行っている。3 種の共刺激ドメイン(CD28、4-1BB、ICOS)
を挿入した CAR を設計し、作製した CAR-T 細胞の細胞増殖能および、細胞傷
害能、脱顆粒マーカーの発現、サイトカイン産生能を評価した。細胞増殖能は
CD28、次いで 4-1BB を挿入した CAR-T 細胞で高く、細胞傷害能と脱顆粒マー
カーの発現は共刺激ドメインを含む各種 CAR-T 細胞間で明らかな差は認めら
れなかった。また IFNγ、TNFα、IL-17A 産生能では ICOS 挿入 CAR-T 細胞
は他に劣ることが示された。これら 3 つの共刺激分子の中では CD28、4-1BB が
有用であることが示された。
第 3 章では、イヌ CAR-T 細胞療法の標的抗原および適応腫瘍種の探索を
行っている。候補となりうるヒト上皮成長因子受容体 2(HER2)およびポドプ
ラニン(PDPN)について、様々な腫瘍組織における陽性率は免疫組織化学染色
法(IHC)で、細胞表面の抗原検出はフローサイトメトリーで検討した。その結
果、HER2 については既報の乳腺癌、移行上皮癌、骨肉腫、悪性黒色腫以外に適
応癌腫として肛門嚢腺癌、原発性肺癌、甲状腺癌を新たに見出し、フローサイト
メトリーによる解析から抗ヒト HER2 抗体(トラスツズマブ)由来の scFv がイ
ヌ HER2 標的 CAR-T 療法に有用であることを明らかにした。PDPN について
は、腫瘍特異的糖鎖修飾 PDPN(csgPDPN)が扁平上皮癌、肺癌、肛門嚢腺癌、
移行上皮癌などに有用である可能性を示した。
本論文において、レトロウイルスベクターによりイヌ T 細胞上に持続的な
CAR の発現を得ることができ、CAR の構造内に共刺激ドメイン(CD28 および
4-1BB)を挿入することによりイヌ CAR-T 細胞の増殖能を増強することを明ら
かにした。また、イヌ CAR-T 細胞療法の適応拡大のためには、HER2 と csgPDPN
を標的分子とすることが有用であることを示しており、イヌ CAR-T 細胞療法の
臨床応用につながる学術的に、臨床的に意義ある内容と考えられる。
審査会においては、これら研究の科学的意義、実験デザインの妥当性、解析
方法、研究結果のなどについて専門的な質疑が行われた。申請者は研究全体の背
景から実験手技、科学的解釈に至るまで、詳細かつ明解な説明を行い、博士学位
取得に足る科学的論理性や技術的背景を有していると考えられた。
以上これらの研究成果は、学術上応用上寄与するところが少なくない。よっ
て、審査委員一同は本論文が博士(獣医学)の学位論文として価値あるものと認
めた。

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