Construction and function of Glypican-3-targeted fourth-generation chimeric antigen receptor T cells (secreting IL-7 and CCL19).
- Author:
Wanli HUANG
1
;
Yu LIU
1
;
Yaodi HU
1
;
Jimin GAO
1
Author Information
- Publication Type:Journal Article
- Keywords: chemotaxis; chimeric antigen receptor T cells; glypican-3; hepatocellular carcinoma; memory stem T cells; proliferation
- MeSH: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Chemokine CCL19; metabolism; Glypicans; metabolism; HEK293 Cells; Humans; Interleukin-7; metabolism; Lentivirus; genetics; Liver Neoplasms; Mice; Receptors, Chimeric Antigen; metabolism; T-Lymphocytes; metabolism; Xenograft Model Antitumor Assays
- From: Chinese Journal of Biotechnology 2020;36(5):979-991
- CountryChina
- Language:Chinese
- Abstract: Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.
