Related Experiment Video
Updated: Nov 27, 2025
Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level
Published on: March 14, 2025
Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line
Chung Hyo Kang1,2, Yeongrin Kim1,3, Heung Kyoung Lee1
1Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 34114, Korea.
Abstract:
CD19 is the most promising target for developing chimeric-antigen receptor (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.
Insights
Researchers screened CD19 single-chain variable fragments (scFvs) for chimeric antigen receptor (CAR) T-cell therapy. The 4G7 scFv demonstrated comparable efficacy to the established FMC63 scFv in preclinical models of B-cell leukemia.
Area of Science:
- Immunology
- Oncology
- Cell Therapy
Background:
- CD19 is a key target for chimeric antigen receptor (CAR) T-cell therapy in B-cell leukemias.
- Existing CAR T-cell products like Kymriah and Yescarta utilize CD19-targeting CARs.
- The FMC63 single-chain variable fragment (scFv) is commonly used in anti-CD19 CAR T-cells.
Purpose of the Study:
- To screen various CD19 scFvs for developing novel anti-CD19 CAR T-cell therapies.
- To evaluate the efficacy of CAR T-cells engineered with different CD19 scFvs.
- To identify alternative scFvs to FMC63 for potential use in CAR T-cell treatments.
Main Methods:
- Screening of multiple CD19 scFvs using the KHYG-1 NK/T-cell line.
- Construction and expression of anti-CD19 CARs with different scFvs in KHYG-1 and Jurkat cell lines.
- In vitro cytotoxicity assays and IL-2 secretion assays using CD19-positive target cells.
- In vivo efficacy studies in NSG mice models with B-cell leukemia xenografts.
Main Results:
- Only FMC63 CAR and 4G7 CAR KHYG-1 cells efficiently lysed CD19-positive cell lines.
- FMC63 CAR and 4G7 CAR Jurkat cells secreted IL-2 upon co-culture with NALM-6 cells.
- In vitro and in vivo studies showed 4G7 CAR T-cells were as potent as FMC63 CAR T-cells in eradicating B-cell leukemia.
- 4G7 CAR T-cells demonstrated comparable lysis of CD19-positive cell lines to FMC63 CAR T-cells.
Conclusions:
- The 4G7 scFv is a promising alternative to FMC63 for developing effective anti-CD19 CAR T-cell therapies.
- 4G7 CAR T-cells exhibit potent anti-leukemic activity comparable to established FMC63 CAR T-cells.
- These findings suggest 4G7 CAR T-cells could be a viable option for treating B-cell leukemia patients.
