Mobile website
Basic Research
Research on the in vitro cytotoxic effects of CAR-T cells targeting CD117 on acute myeloid leukemia Kasumi-1 cells
- Article
- Figures
- Metrics
- Preview PDF
- Reference
- Related
- Cited by
- Materials
Abstract:
[Abstract] Objective: To generate low-affinity CD117-targeting CAR-T cells and investigate their in vitro cytotoxic effects on acute myeloid leukemia (AML) Kasumi-1 cells. Methods: Low-affinity CD117-targeting monoclonal antibody (barzolvolimab) and Fab-79D VH and VL sequences were retrieved for the design of single-chain variable fragments (scFvs) with a VH-(G4S)?-VL configuration. These scFvs were then integrated individually into canonical second-generation chimeric antigen receptor (CAR) constructs harboring the 4-1BB costimulatory domain. Subsequent to gene synthesis, the resultant CAR sequences were subcloned into the pMFG retroviral vectors, yielding the CD117-79D CAR and CD117-0159 CAR plasmids, respectively. The two CAR plasmids were subjected to separate retroviral packaging and production procedures. After validation of viral titers, the recombinant retroviruses were transduced into activated T cells to generate CD117-79D CAR-T and CD117-0159 CAR-T cell products. The CAR-positive fractions of the two CAR-T cell populations were quantified utilizing flow cytometry. Untransduced T cells and the two engineered CAR-T cells were co-cultured independently with CD117 ? Kasumi-1 cells. Thereafter, the apoptotic rates of Kasumi-1 cells were detected via flow cytometry for the comparative evaluation of the anti-tumor activity of the two CAR-T cell products. Results: CD117-79D CAR-T and CD117-0159 CAR-T cells were successfully constructed, with positive rates of (59.4 ± 2.6)% and (62.5 ± 1.2)% , respectively. Untransduced T cells, CD117-79D CAR-T cells, and CD117-0159 CAR-T cells all exhibited stable proliferation in in vitro cultivation, and no statistically significant difference was observed in their proliferative abilities (all P > 0.05). Results of the in vitro cytotoxicity assay against Kasumi-1 cells demonstrated that, in varying effector-to-target ratios, both CD117-79D CAR-T and CD117-0159 CAR-T cells exerted significantly enhanced cytotoxicity compared with untransduced T cells (P < 0.05 or P < 0.01). However, no statistically significant difference was detected in the killing efficiency between the two CAR-T cell products (P > 0.05). Conclusion: CD117-79D CAR-T and CD117-0159 CAR-T cells with low-affinity were successfully generated. In vitro experiments verified that these CAR-T cells could effectively eliminate CD117? Kasumi-1 cells, thereby providing experimental evidence for the targeted therapy of AML.
Keywords:
Project Supported:
Clc Number:
