CD19- and ROR1-specific CAR constructs have been previously described (49, 59). For work in this study, a single STII sequence and two G4S linkers were inserted between the FMC63 or R12 scFv and immunoglobulin G4 (IgG4) hinge (25). These were linked to the 27–amino acid transmembrane domain of human CD28 (UniProt: P10747) and to a signaling module comprising either (i) the 41–amino acid cytoplasmic domain of human CD28 with an LL→GG substitution located at positions 186 to 187 of the native CD28 protein (60) or (ii) the 42–amino acid cytoplasmic domain of human 4-1BB (UniProt: Q07011), each of which was linked to the 112–amino acid cytoplasmic domain of isoform 3 of human CD3ζ (UniProt: P20963-3). Mutant CD28/CD3ζ CARs with tyrosine to phenylalanine substitutions at CD28 UniProt positions 206, 209, and 218 and/or with proline to alanine substitutions at CD28 UniProt positions 208 and 211 were generated by site-directed mutagenesis. All CAR constructs were linked by T2A sequence to EGFRt, codon-optimized, and cloned into an HIV7 lentiviral vector. For fluorescence microscopy, the CD3ζ endodomain was directly fused to enhanced green fluorescent protein (eGFP). To make CAR antigen–expressing K562 cells, amino acids 1 to 325 of human CD19 (UniProt: P15391) were cloned into an HIV7 lentiviral vector, and amino acids 1 to 937 of human ROR1 (UniProt: Q01973) were cloned into an mp71 retroviral vector, which was a gift of W. Uckert (Max Delbruck Center for Molecular Medicine). All cloning was performed by PCR, enzyme digest, and/or Gibson assembly. Plasmids were verified by capillary sequencing and restriction digest.

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