MetCAR T Cells Transduction and Characterization

Example 3

To produce MetCAR T cells, peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were stimulated with anti-CD3/CD28 antibodies in the presence of IL-7 (10 ng/ml) and IL-15 (5 ng/ml) for 24 hr to expand CD3+ T cells for transduction with MetCARs. CD3+ T cells are regularly tested to reach >90% after stimulation (data not shown). Because all MetCAR constructs use CD19 as a tag, the expression of CD19 by flow cytometer is used as the transduction efficacy (FIGS. 2A, 2B). With NT cells as a negative control, MetCAR transduction efficiency ranged from 60-90% with no significant differences between MetCAR constructs (FIGS. 2A, 2B). MetCAR expression levels were determined using CD3ζ as a marker (FIG. 2C). Results showed that both MetCAR.CD28.ζ. and MetCAR.4-1BB.ζ.-transduced T cells had CD3ζ overexpression comparing with non-transduced or CAR.Δ-transduced T cells. p-CD3ζ expression was also observed in MetCAR.CD28.ζ- and MetCAR.4-1BB.ζ.-transduced T cells (FIG. 2C), indicating the activation of TCR in MetCAR-T cells.