Tang Jie, Liu Nan, Zhu Yongjie, Li Yi, Zhao Xudong
Department of Targeting Therapy & Immunology and Laboratory of Animal Tumor Models, Cancer Center and Department of Respiratory and Critical care Medicine and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
Core Facilities, West China Hospital, Sichuan University, Chengdu, Sichuan, PR China.
Immunol Invest. 2023 Nov;52(8):985-996. doi: 10.1080/08820139.2023.2264332. Epub 2023 Nov 24.
CAR-T cell immunotherapy has achieved remarkable success in malignant B-cell malignancies, but progress in solid tumors is slow, and one of the key reasons is the lack of ideal targets. Cancer-specific extra domain B of fibronectin (EDB-FN) is widely upregulated in solid tumors and expressed at low levels in normal tissues. Many imaging and targeted cancer therapies based on EDB-FN targets have been developed and tested in clinical trials, making EDB-FN an ideal target for immunotherapy.
We constructed two EDB-FN-targeted CAR-Ts based on the peptide APT0 and the single-chain antibody CGS2 in a lentiviral infection manner for the first time. Luciferase cytotoxicity assay to assess CAR-T killing of tumor cells. An enzyme-linked immunosorbent assay was used to detect the release of the cytokine IFN-γ. Fluorescence imaging to evaluate the dynamics of CAR-T cell and tumor cell coculture. Knockdown assays were used to validate the target specificity of CAR-T cells.
In this research, two CAR-Ts targeting EDB-FN, APT0 CAR-T, and CGS2 CAR-T, were constructed. In vitro, both CAR-T cells produced broad-spectrum killing of multiple EDB-FN-positive solid tumor cell lines and were accompanied by cytokine IFN-γ release. Regarding safety, the two CAR-T cells did not affect T cells' normal growth and proliferation and were not toxic to HEK-293T human embryonic kidney epithelial cells.
APT0 CAR-T and CGS2 CAR-T cells are two new CAR-Ts targeting EDB-FN. Both CAR-T cells can successfully identify and specifically kill various EDB-FN-positive solid tumor cells with potential clinical applications.
嵌合抗原受体T细胞（CAR-T）免疫疗法在恶性B细胞肿瘤中取得了显著成功，但在实体瘤中的进展缓慢，关键原因之一是缺乏理想的靶点。纤连蛋白的癌症特异性额外结构域B（EDB-FN）在实体瘤中广泛上调，在正常组织中低表达。许多基于EDB-FN靶点的成像和靶向癌症治疗方法已被开发并在临床试验中进行测试，使EDB-FN成为免疫治疗的理想靶点。
我们首次以慢病毒感染的方式构建了两种基于肽APT0和单链抗体CGS2的靶向EDB-FN 的CAR-T。采用荧光素酶细胞毒性试验评估CAR-T对肿瘤细胞的杀伤作用。使用酶联免疫吸附试验检测细胞因子IFN-γ 的释放情况。通过荧光成像评估CAR-T细胞与肿瘤细胞共培养的动态过程。采用敲低试验验证CAR-T细胞的靶点特异性。
在本研究中构建了两种靶向EDB-FN的CAR-T细胞，即APT0 CAR-T和CGS2 CAR-T。在体外，两种CAR-T细胞均对多种EDB-FN阳性实体瘤细胞系产生广谱杀伤作用，并伴有细胞因子IFN-γ 的释放。在安全性方面,两种CAR-T细胞不影响T细胞的正常生长和增殖,对人胚肾上皮细胞HEK-293T无毒性。
APT0 CAR-T和CGS2 CAR-T细胞是两种新型的靶向EDB-FN的CAR-T细胞。两种CAR-T细胞均能成功识别并特异性杀伤各种EDB-FN阳性实体瘤细胞，具有潜在的临床应用价值。
