Background

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7–19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects.

Methods

We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7–19-CAR-T cells in the clinic.

Results

GPC3-7–19-CAR-T cells had 1.5–2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7–19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm³ ± 1.17 vs. 0.34 cm³ ± 0.25. Of note, increased proportion of CD4⁺ T_EM and CD8⁺ T_CM cells was infiltrated in GPC3-7–19-CAR-T cells group. GPC3-7–19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7–19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion.

Conclusions

In conclusion, GPC3-7–19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4⁺ T_EM and CD8⁺ T_CM cell subsets. Most importantly, GPC3-7–19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic.

Graphical Abstract

► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3⁺HCCs.

►DC cells recruited by CCL19 could interact with CD4⁺ T cells and promote the differentiation of CD4⁺T_EFF cells into CD4⁺T_EM and CD8⁺T_CM subsets, leading a better anti-tumor effect on GPC3⁺HCCs.

►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

中文翻译：

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GPC3-IL7-CCL19-CAR-T 启动肝细胞癌治疗的免疫微环境重建

背景

嵌合抗原受体 (CAR)-T 细胞疗法是一种革命性的治疗方法，已成为晚期癌症治疗的支柱。传统的磷脂酰肌醇蛋白聚糖3（GPC3）-CAR-T细胞在晚期肝细胞癌（HCC）中尚未产生理想的临床效果，且机制尚不清楚。本研究旨在研究我们团队构建的新型GPC3-7-19-CAR-T细胞的临床实用性，并探讨其抗肿瘤作用的机制。

方法

我们设计了一种新型 GPC3 靶向 CAR，包括抗 GPC3 s​​cFv、CD3ze、CD28 和 4-1BB，可诱导中等水平 (500 pg/mL) 的 IL-7 和高水平 (15000 pg/mL) 的 CCL19 共表达。 /mL) 并将其转导至人类 T 细胞中。在体外，细胞杀伤功效通过 xCELLigence RTCA 系统、LDH 非放射性细胞毒性测定进行了验证，并在采用 3D 微流体芯片的原发性 HCC 类器官模型中得到了证实。在体内，在人源化 NSG 小鼠异种移植模型中评估了抗肿瘤能力。最后，我们启动了I期临床试验，以评估GPC3-7-19-CAR-T细胞在临床中的安全性和效果。

结果

GPC3-7-19-CAR-T细胞的杀伤效率比GPC3-CAR-T细胞高1.5-2倍。GPC3-7–19-CAR-T细胞治疗模型中的肿瘤形成率降低（3/5vs.5/5），平均肿瘤体积为0.74 cm 3 ^±  1.17 vs. 0.34 cm ³  ± 0.25。值得注意的是，GPC3-7-19-CAR-T 细胞组中 CD4 _{+ TEM}^和CD8 + ^T CM_细胞浸润的比例增加。GPC3-7-19-CAR-T细胞通过减少多形核（PMN）骨髓源性抑制细胞（MDSC）和调节性T（Treg）细胞浸润并招募更多树突状细胞（DC），明显逆转了免疫抑制肿瘤微环境（TME） HCC 异种移植肿瘤组织。在一名晚期 HCC 患者中，GPC3-7-19-CAR-T 细胞治疗在静脉输注 56 天后肿瘤缩小。

结论

^{总之，GPC3-7-19-CAR-T细胞通过重建占主导地位的CD4 +} TEM和CD8 ⁺ TCM细胞亚群诱导的TME _，取得了优于传统GPC3-CAR-T细胞的抗肿瘤效果_。最重要的是，GPC3-7-19-CAR-T细胞在临床上对HCC患者表现出良好的安全性和抗肿瘤功效。

图形概要

► 新型GPC3-7-19-CAR-T细胞设计具有中等水平的IL-7分泌和高水平的CCL19分泌，可以招募更多成熟的DC来协助杀死GPC3 ⁺ HCC。

►CCL19招募的DC细胞可以与CD4 + T细胞相互作用_{，促进CD4 + T EFF细胞分化为CD4 + TEM}^和CD8 + _T CM亚^群，从而^对GPC3 ₊ ^HCC具有更好的抗肿瘤作用。

►与传统GPC3-CAR-T相比，GPC3-7-CCL19-CAR-T细胞可以通过减少PMN-MDSC和Treg细胞浸润来逆转肿瘤免疫抑制微环境。