嵌合抗原受体修饰的T细胞（CAR-T）在浆膜腔内的抗肿瘤效应及其机制

Chimeric antigen receptor-modified T cells (CAR-T) have powerful killing activity for tumor cells in an antigen dependent manner. Tumor metastases in serous cavity frequently occurs in patients with lung cancer, ovarian cancer, and so on, accompanying with severe clinical symptom and worse prognosis. Cancer cells are prone to initiate epithelial–mesenchymal transition (EMT) and generate more cancer stem cells in serous cavity, modulate antigens expression. Moreover, both cancer and monocytes also unregulated the level of PD-L1 expression. These changes can induce immune escape. The project aims to regional immunotherapy using CAR-T for cancer metastases in serous cavity. We will monitor the modulation of surface targeting antigen profile of CAR-T therapy on cancer cells when exposed in serosa cavity. Previously, we found that VEGFR1 expressed on cancer cells from malignant ascites and malignant ascites treated cancer cell lines. Therefore, we developed a split signal CAR to target VEGFR1 and PD-L1, trying to switch inhibitory signal of PD-L1 to activating signal, 4-1BB, for promoting CAR-T killing and survival. We also found that IL-2 enhanced killing activity within malignant ascites. Based on our previous studies, we are going to further investigate the effects on prevention and treatment for cancer metastases in serous cavity, confirm the immune mechanism, and optimize the therapeutic strategy. The project will be helpful to promote clinical application through providing an effective war to prevent and treat cancer metastases in serous cavity.

嵌合抗原受体修饰T细胞（CAR-T）具有抗原依赖的强大杀肿瘤活性。肿瘤浆膜腔转移伴恶性积液临床疗效差、危害大。癌细胞在浆膜腔易于上皮间质转化并产生更多肿瘤干细胞，发生抗原调变，同时癌细胞和免疫细胞PD-L1上调，促进免疫逃逸。本项目致力于浆膜腔转移的CAR-T局部治疗，拟监测癌细胞浆膜腔暴露后CAR-T靶向抗原的变化，已在前期证实VEGFR1在腹水癌细胞及腹水处理细胞系稳定表达。本项目设计了双靶向VEGFR1和PD-L1的拼接信号CAR，将PD-L1抑制转化为激活信号4-1BB，增强CAR-T的杀伤和存活。项目前期还发现IL-2有助增强CAR-T在腹水环境下的杀伤活性。在前期基础上，本项目拟研究CAR-T，特别是双靶向CAR-T对肿瘤浆膜腔转移的预防和治疗作用，验证免疫效应的分子机制，优化CAR-T治疗策略。项目有望推动CAR-T在实体瘤中的临床应用，为浆膜腔癌转移提供有效预防和治疗手段。

CAR-T具有膜抗原依赖的细胞毒性，是肿瘤治疗新手段。肿瘤浆膜腔转移伴积液临床疗效及预后差，本项目立足局部输注CAR-T来治疗浆膜腔转移。我们前期发现浆膜腔转移肿瘤仍然高表达VEGFR1（JBC, 2016），此外，本项目前期发现恶性积液可诱导肿瘤细胞高表达PD-L1。基于此，我们设计了双靶向CAR-T，一个靶向肿瘤抗原作为杀伤信号进行肿瘤杀伤，一个靶向PD-L1并将PD-L1抑制信号转化为4-1BB激活信号促进CAR-T的活性和存活。本项目以VEGFR1和HER-2作为肿瘤杀伤靶抗原，设计了VEGFR1/PD-L1和HER2/PD-L1双靶向CAR-T。体外研究发现，VEGFR1或HER-2单靶向CAR-T与联合PD-L1双靶CAR-T相比，双靶向CAR-T细胞因子IFN-γ、IL-2、TNF-α分泌水平更高，靶抗原刺激后增殖能力更强。在腹腔荷瘤模型等剂量对比研究中，双靶向CAR-T展现出更快速、更持久的肿瘤抑制效果。进一步机制研究发现，PD-L1转化为 4-1BB激活信号增强了双CAR-T细胞效应功能，CAR-T在恶性胸腹水中和肿瘤细胞共培养后RNA-Seq测序显示，双CAR-T细胞因子如IL-2、IFN-γ、TNF-α基因、归巢和迁移相关基因的转录水平更高，而在促凋亡和免疫抑制相关基因的转录水平更低。我们的研究有望为浆膜腔癌转移提供有效预防和治疗手段，目前华西医院已批准进一步临床研究（NCT04684459）。.此外，在本项目支持下，我们还发现在EGFR突变的NSCLC细胞系体外TKI靶向治疗耐药后，肿瘤细胞PD-L1表达显著升高（Sci Rep, 2019, IF=3.998）；部分NSCLC患者靶向治疗后出现浆膜腔积液增加，但肿瘤细胞生长仍然依赖EGFR信号（STTT, 2020, IF=13.493），这些发现提示我们的双靶向设计可能进一步应用于肺癌。但我们的另一项研究针发现对肿瘤相关抗原采用全身治疗需关注肺毒性（OncoImmunology, 2020, IF=5.869）。