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CEAを標的抗原とするキメラ抗原受容体T細胞による膵臓癌への抗腫瘍効果の研究

佐藤, 理 北海道大学

2023.09.25

概要

【背景と目的】
膵臓癌 (以下膵癌) は消化器癌の中で最も悪性度が高い癌種の 1 つであり、未だに外
科的治療が長期生存を期待しうる唯一の治療法である。しかしながら、初診断時に手
術適応のある患者は約 20% に過ぎず、また根治切除ができても術後の再発率は高く
術後の 5 年生存率は 20- 40% と不良である。化学療法として様々な薬剤が開発されて
いるが、その効果は限定的で重篤な副作用も懸念されており、全 5 年生存率は 12.5%
程度と極めて予後不良な悪性疾患である。そのため、抗腫瘍効果が高く副作用の少な
い新規治療法の開発が期待され数多くの研究が行われているが、いずれにおいても決
して良好な成績とは言えず、アンメットメディカルニーズのある代表的疾患の 1 つで
ある。
癌治療において手術、化学療法、放射線療法の 3 大療法に続く第 4 の治療法として
免疫療法が長らく期待され、癌ワクチンや養子免疫療法など多くの臨床試験が行われ
てきた。近年、キメラ抗原受容体 T 細胞 (chimeric antigen receptor engineered T cells: CAR-T)
療法が血液腫瘍に対して顕著な治療効果を示して以来、関心が高まっている。しかし、
固形腫瘍においては未だ有効な治療効果の報告が少なく、臨床応用へは至っていない。
本研究では、難治性癌である膵癌に対する CAR-T 療法の有効性を検証する事とし
た。標的抗原としては、腫瘍特異性が高くかつオフターゲット効果が低い抗原として、
膵癌で高頻度に発現を認める carcinoembryonic antigen (CEA) を有効な標的抗原と考えた。
今回、抗 CEA-CAR-T を作製し in vitro、in vivo で膵癌に対する抗腫瘍効果の検証を行い、
また、膵癌切除症例の臨床データと病理学的所見から、将来的な CAR-T 療法の臨床
応用に向けてバイオマーカーの探索も行った。
【材料と方法】
ヒト膵癌細胞株として MIA PaCa-2、PANC-1、Capan-1、AsPC-1、PK-9、BxPC-3、
SUIT-2、KP1N、PCI-66 の 9 種類、対照として CEA 発現陽性が既知である胃癌細胞株
MKN45 を使用した。まず、各種細胞株において免疫蛍光染色、western blot analysis (WB)、flowcytometry (FCM) を試行して CEA 発現の評価を行った。また、FCM による
CEA に対する mean fluorescence intensity (MFI) から細胞あたりの CEA 分子数 (sites/ cell) を
導き、高発現・中発現・低発現群に分類した。続いて、各細胞株に対して抗 CEACAR-T の抗腫瘍効果を確認するため、まず in vitro で抗 CEA-CAR-T の機能アッセイを
行った。抗 CEA-CAR-T と細胞株の共培養液中の IFN-γ 分泌を ELISA 法で解析し、さ
らに、細胞毒性アッセイとして共培養後の標的細胞の生細胞/死細胞率を FCM で解
析した。また、可溶性 CEA (sCEA) が抗 CEA-CAR-T の抗腫瘍効果に競合する可能性を
考え、機能アッセイを高濃度 sCEA の存在下でも行った。 ...

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