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.
Highlights
T cells genetically modified to express chimeric-antigen receptor (CAR) specific to a tumor have shown dramatic results in preclinical and clinical tests [1]
Two CD19 CAR T cell products are approved by the US FDA, and numerous clinical trials using CD19 CAR T cells are still ongoing by various institutions [12]
The NK-92 cell line is more popular than the KHYG-1 cell line among NK cell researchers, we found that KHYG-1 has several advantages over the NK-92 cell line
Summary
T cells genetically modified to express chimeric-antigen receptor (CAR) specific to a tumor have shown dramatic results in preclinical and clinical tests [1]. Two CD19 CAR T cell products are approved by the US FDA, and numerous clinical trials using CD19 CAR T cells are still ongoing by various institutions [12]. The National Cancer Institute (NCI) adopted γ-retrovirus for gene transduction in T cells, while the University of Pennsylvania (UPenn) adopted lentivirus for this purpose [16]. They have quite different strategies to manufacture CD19 CAR T cells, there is one thing which is common in most institutions—CD19 scFv. Nearly 90% of the worldwide clinical anti-CD19 CAR T cell products use FMC63 as a CD19 scFv [17]. We postulated that it is meaningful to test these CD19 scFvs for developing potent CD19 CAR T cells
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