肿瘤细胞治疗研究
英文原题：A novel Chimeric Antigen Receptor (CAR) - strategy to target EGFR(VIII)-mutated glioblastoma cells via macrophages.
本工作所描述的模型适合用于向 iPSC 来源巨噬细胞的转化，以在表达突变变体 EGFR VIII 的实体瘤中产生未来可临床应用的细胞治疗方法。
自体嵌合抗原受体（CAR）表达 T 细胞（CAR-T）已有效用于血液系统恶性肿瘤，但对实体瘤的疗效仍有限。采用巨噬细胞的 CAR 疗法具有发展前景，因为巨噬细胞能够浸润肿瘤并启动吞噬作用。 方法：为建立 CAR 巨噬细胞（CAR-Mac）的临床前模型，研究者设计了一种新型 CAR 构建体，利用可分化为巨噬细胞的 THP-1 单核细胞系靶向表达 EGFRvIII 的胶质母细胞瘤细胞（DK-MG）。该 CAR 结构包括特异识别 EGFRvIII 的单链可变片段（scFv）和可增强靶细胞系留及吞噬活性的 MEGF10 胞内结构域。 结果：研究通过慢病毒转导生成表达 CAR 或对照构建体的 THP-1 细胞，随后诱导其分化为单核细胞及表达 CAR 的 M1 巨噬细胞（CAR-Mac）。研究将 THP-1 来源的野生型（WT）巨噬细胞或 CAR-Mac 与靶向 DK-MG 细胞共培养，以评估吞噬能力。共聚焦显微镜显示，CAR-Mac 对 DK-MG EGFRvIII 细胞的吞噬效率约为 60%，而 WT 细胞约为 15%。视频显微镜也观察到抗 EGFRvIII CAR-Mac 对胶质母细胞瘤细胞系的杀伤能力。对共培养上清液进行 ELISA 和乳酸脱氢酶（LDH）检测发现 IL-6 和 LDH 水平升高，提示 CAR-Mac 可有效介导细胞毒作用。纯化 CAR-Mac 的转录组分析发现了支持成功吞噬作用的分子特征。 结论：本文所述模型可用于向 iPSC 来源巨噬细胞转化，为表达突变型 EGFRvIII 的实体瘤开发未来具有临床应用潜力的细胞疗法。
BACKGROUND: Autologous chimeric antigen receptor (CAR) expressing T-Cells (CAR-T) have been efficiently used in hematological malignancies but their efficacy in solid tumors remains limited. CAR therapies via the use of macrophages, offer a promising avenue due to their unique ability to infiltrate tumors and to initiate phagocytosis. METHODS: To generate a preclinical model of CAR-Macs, we have designed a novel CAR-construct to target EGFR VIII -expressing glioblastoma cells (DK-MG) using THP-1 monocytic cell line able to differentiate towards macrophages. The CAR structure comprises a ScFv recognizing specifically EGFR VIII and MEGF10 intracellular domain which enhances tethering and phagocytosis activity. RESULTS: THP-1 cell line expressing CAR and control constructs were generated via lentiviral transduction followed by generation of monocytes and CAR-expressing M1 macrophages (CAR-Macs). To evaluate their ability of phagocytosis, we co-cultured THP-1 derived WT macrophages or CAR-Macs in the presence of target DK-MG cells. Confocal microscopy experiments revealed highly efficient phagocytosis of DK-MG EGFR VIII cells by CAR-Macs (~ 60%) as compared to WT cells (~ 15%). The killing potential of the anti-EGFR VIII CAR-Macs against the glioblastoma cell line has also been observed using video microscopy. ELISA and LDH assays performed in co-culture supernatants, showed an increase of IL-6 and LDH levels suggesting efficient cytotoxicity via CAR-Macs. Transcriptome analyses performed in purified CAR-Macs, revealed evidence of a molecular signature in favor of successful phagocytosis. CONCLUSIONS: The model described in this work is suitable for allowing translation to iPSC-derived macrophages to generate clinically applicable future cell therapy approaches in solid tumors expressing mutated variant EGFR VIII .
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