Department of Medical Oncology, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, P. R. China.
Key Laboratory of Tropical Disease Control of Ministry Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Institute of Human Virology, Sun Yat-sen University, Guangzhou, Guangdong, P. R. China.
Cancer Commun (Lond). 2023 Jul;43(7):788-807. doi: 10.1002/cac2.12452. Epub 2023 Jun 6.
Existing treatments for cholangiocarcinoma have poor efficacy. However, chimeric antigen receptor-T (CAR-T) cells are emerging as a potential therapeutic strategy. Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR-T cell infiltration and function. This study aimed to improve the function of CAR-T cells through knock down immune checkpoints and immunosuppressive molecular receptors.
We evaluated the expression of epidermal growth factor receptor (EGFR) and B7 homolog 3 protein (B7H3) antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry. Subsequently, we engineered CAR-T cells targeting EGFR and B7H3 antigens. We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR-T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR-T cells for antitumor activity both in vitro, using tumor cell lines and cholangiocarcinoma organoid models, and in vivo, using humanized mouse models.
We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues. EGFR-CAR-T and B7H3-CAR-T cells demonstrated specific anti-tumor activity. We found an abundance of programmed cell death protein 1 (PD-1), T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and T cell immunoglobulin and ITIM domain (Tigit) on infiltrated CD8 T cells in the cholangiocarcinoma microenvironment. We then decreased the expression of these 3 proteins on the surface of CAR-T cells, named PTG-scFV-CAR-T cells. Furthermore, we knocked-down the expression of transforming growth factor beta receptor (TGFβR), interleukin-10 receptor (IL-10R), and interleukin-6 receptor (IL-6R) of PTG-scFV-CAR-T cells. Those cells, named PTG-T16R-scFV-CAR-T cells, potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoid model. Finally, the PTG-T16R-scFv-CAR-T cells showed greater inhibitory effect on tumor growth in vivo, and were superior in prolonging the survival of mice.
Our results revealed that PTG-T16R-scFV-CAR-T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long-term efficacy both in vitro and in vivo. This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.
现有的胆管癌治疗方法疗效不佳。然而，嵌合抗原受体 T（CAR-T）细胞作为一种潜在的治疗策略正在出现。实体肿瘤在免疫抑制微环境中存在多种不良因素，这些因素会损害 CAR-T 细胞的浸润和功能。本研究旨在通过敲低免疫检查点和免疫抑制性分子受体来提高 CAR-T 细胞的功能。
我们使用免疫组织化学评估胆管癌组织中表皮生长因子受体（EGFR）和 B7 同源物 3 蛋白（B7H3）抗原的表达，并通过流式细胞术筛选胆管癌微环境中的特定免疫检查点。随后，我们设计了针对 EGFR 和 B7H3 抗原的 CAR-T 细胞。我们通过构建两个簇的短发夹 RNA 同时敲低 CAR-T 细胞中的免疫检查点和免疫抑制性分子受体，并在体外使用肿瘤细胞系和胆管癌类器官模型以及体内使用人源化小鼠模型评估工程化的 CAR-T 细胞的抗肿瘤活性。
我们观察到胆管癌组织中 EGFR 和 B7H3 抗原的高表达。EGFR-CAR-T 和 B7H3-CAR-T 细胞表现出特异性的抗肿瘤活性。我们发现胆管癌微环境中浸润的 CD8 T 细胞上存在大量程序性细胞死亡蛋白 1（PD-1）、T 细胞免疫球蛋白和粘蛋白结构域蛋白 3（Tim-3）和 T 细胞免疫球蛋白和 ITIM 结构域（Tigit）。然后，我们降低了 CAR-T 细胞表面这些 3 种蛋白的表达，命名为 PTG-scFV-CAR-T 细胞。此外，我们敲低了 PTG-scFV-CAR-T 细胞中转化生长因子β受体（TGFβR）、白细胞介素 10 受体（IL-10R）和白细胞介素 6 受体（IL-6R）的表达。这些细胞，命名为 PTG-T16R-scFV-CAR-T 细胞，在体外能有效杀伤肿瘤细胞，并在胆管癌类器官模型中促进肿瘤细胞凋亡。最后，PTG-T16R-scFv-CAR-T 细胞在体内对肿瘤生长的抑制作用更强，并且能延长小鼠的存活时间。
我们的结果表明，敲低六重抑制分子的 PTG-T16R-scFV-CAR-T 细胞对胆管癌具有强大的免疫作用，在体外和体内均具有长期疗效。该策略为胆管癌提供了一种有效的个性化免疫细胞治疗方法。
