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[Construction and function of Glypican-3-targeted fourth-generation chimeric antigen receptor T cells (secreting IL-7
Wanli Huang1, Yu Liu1, Yaodi Hu1
1School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China.
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.
Insights
The fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19) show enhanced proliferation and memory stem T cell (Tscm) generation compared to earlier versions. These novel CAR-T cells effectively eliminate GPC3-positive hepatocellular carcinoma (HCC) xenografts in preclinical models.
Area of Science:
- Immunotherapy
- Oncology
- Biotechnology
Background:
- Adoptive immunotherapy using chimeric antigen receptor-T cells (CAR-T) shows promise for cancer treatment but faces limitations in solid tumors.
- Glypican-3 (GPC3) is a validated biomarker for hepatocellular carcinoma (HCC), making it a target for immunotherapy.
- Existing GPC3-targeted CAR-T cells offer therapeutic potential, but enhancements are needed for improved efficacy.
Purpose of the Study:
- To develop and evaluate a fourth-generation GPC3-targeted CAR-T cell therapy (GPC3-BBZ-7×19) for hepatocellular carcinoma (HCC).
- To enhance CAR-T cell function by incorporating interleukin-7 (IL-7) and CCL19 into a lentiviral vector.
- To compare the preclinical efficacy of the fourth-generation CAR-T cells against second-generation CAR-T cells in vitro and in vivo.
Main Methods:
- Constructed a fourth-generation lentiviral vector encoding GPC3 CAR, IL-7, and CCL19.
- Generated GPC3-BBZ-7×19 CAR-T cells by transducing human T lymphocytes.
- Assessed CAR-T cell proliferation, chemotaxis, cytotoxicity, and Tscm populations in vitro.
- Evaluated tumor elimination in a GPC3-positive HCC xenograft mouse model using bioluminescence imaging.
Main Results:
- GPC3-BBZ-7×19 CAR-T cells demonstrated significantly enhanced proliferation and chemotactic ability compared to GPC3-BBZ CAR-T cells.
- A higher proportion of memory stem T cells (Tscm) was observed in the GPC3-BBZ-7×19 CAR-T cell group.
- No significant differences were found in cytotoxicity or cytokine secretion between the CAR-T cell generations.
- GPC3-BBZ-7×19 CAR-T cells effectively suppressed tumor growth in GPC3-positive HCC xenografts.
Conclusions:
- The fourth-generation GPC3-targeted CAR-T cells secreting IL-7 and CCL19 exhibit improved preclinical anti-tumor activity against HCC.
- These enhanced CAR-T cells show potential for more durable and effective treatment of HCC by promoting tumor-specific memory.
- The study provides a preclinical foundation for future clinical trials of this advanced CAR-T therapy for HCC.
