FRβ CAR construction and expression in primary human T cells. (A) Schematic of lentiviral CAR expression vectors containing the anti-human FRβ scFv m909 linked to either intracellular signaling domains from CD3-ζ alone (m909-Z) or CD28 and CD3-ζ in tandem (m909-28Z). Both constructs also encode GFP separated by a viral T2A (2A) ribosomal skipping peptide. (B) CAR expression in primary human T cells. Expression of m909 CAR in primary human T cells was confirmed by GFP, and surface expression was confirmed by labeling with a rabbit anti-human IgG antibody that binds the human m909 scFv portion of the CAR. Upper and lower rows show results from 2 representative donors. (C) After 13 days of expansion, m909 CAR–transduced T-cell populations comprise ∼70% CD8⁺ and 30% CD4⁺. Upper and lower rows show results from 2 representative donors. L, linker; TM, transmembrane domain; UN, untransduced T cells; VH, variable heavy chain; VL, variable light chain.