肿瘤细胞治疗研究
英文原题：Next-generation of mesothelin-targeted CAR-T cells secreting anti-PD-L1 scFv for potent immunotherapy against 3D patient-derived colorectal cancer organoids.
最后，我们的14天离体CRC-PDOs/CAR-T平台为实体瘤中的肿瘤相关抗原验证、简化的PDO分离、自体CAR-T细胞毒性测试以及个性化免疫治疗优化提供了一种有前景的快速转化工具。
背景：嵌合抗原受体（CAR）T细胞疗法已彻底改变癌症治疗格局，尤其是在血液系统恶性肿瘤领域。然而，其成功转化至实体瘤仍受多重障碍限制，包括免疫抑制性肿瘤微环境以及靶向肿瘤同时损伤正常组织的毒性。提高CAR-T细胞疗效的一种有前景策略，是合理选择肿瘤特异性抗原，并结合局部免疫调节工程设计，例如使CAR-T细胞分泌阻断免疫检查点的抗PD-L1单链可变片段（scFv）。 方法：为更好地模拟治疗应答，我们建立了动态实时自体共培养平台，整合结直肠癌（CRC）患者来源类器官（PDO）和CAR-T细胞，在体外评估其浸润、持久性和细胞毒性。尽管间皮素（MSLN）靶向CAR-T细胞的早期临床试验显示安全性较高，其抗肿瘤疗效仍有限，提示需要改进构建体。因此，我们构建了抗MSLN-CAR4-T细胞，其采用全人源抗MSLN scFv，连接至三重共刺激骨架（CD28、4-1BB和CD27），并融合CD3ζ；还构建了额外包含抗PD-L1 scFv的抗MSLN-CAR5-T细胞。 结果：CAR4-T和CAR5-T细胞对MSLN高表达/PD-L1高表达的自体PDO均表现出相近的细胞毒性。然而，在较低效靶比（5:1）下，只有抗MSLN-CAR5-T细胞能够对PD-L1高表达PDO维持强效杀伤，并产生较高水平的IFN-γ/细胞毒性细胞因子，显示出对PD-L1介导抑制的抵抗力增强。 结论：最终，我们建立的为期14天的体外CRC-PDO/CAR-T平台，为肿瘤相关抗原验证、简化PDO分离、自体CAR-T细胞毒性检测以及实体瘤个体化免疫治疗优化，提供了有前景且可快速转化的工具。
BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies have revolutionized the landscape of cancer treatment, particularly in hematological malignancies. However, their successful translation to solid tumors remains limited by several barriers, including immunosuppressive tumor microenvironment and on-target/off-tumor toxicity. One promising strategy to enhance efficacy of CAR-T cells is the rational selection of tumor-specific antigens coupled with engineering strategies that incorporate localized immune modulation, such as CAR-T cells secreting immune checkpoint-blocking anti-PD-L1 scFv. METHODS: To better model therapeutic responses, we established a dynamic real-time autologous co-culture platform integrating colorectal cancer (CRC) patient-derived organoids (PDOs) and CAR-T cells to assess infiltration, persistence, and cytotoxicity ex vivo. Although early clinical trials of mesothelin (MSLN)-directed CAR-T cells have demonstrated high safety, their anti-tumor efficacy remains modest, highlighting the need for improved constructs. Therefore, we engineered anti-MSLN-CAR4-T cells using fully human anti-MSLN scFv linked to a triple costimulatory backbone (CD28, 4-1BB, and CD27) fused to CD3 , and anti-MSLN-CAR5-T cells, incorporating an additional anti-PD-L1 scFv. RESULTS: Both CAR4- and CAR5-T cells exhibited comparable cytotoxic efficacy against MSLN High /PD-L1 High autologous PDOs. However, only anti-MSLN-CAR5-T cells were able to sustain potent killing activity against PD-L1 High PDOs with high IFN- /cytolytic cytokine production at a low effector-to-target ratio (5:1), reflecting improved resilience to PD-L1-mediated suppression. CONCLUSIONS: Finally, our 14-day ex vivo CRC-PDOs/CAR-T platform provides a promising rapid and translational tool for tumor-associated antigen validation, streamlined PDO isolation, autologous CAR-T cytotoxicity testing, and personalized immunotherapy optimization in solid tumors.
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