Large-Scale Screening Campaign of a ROR1-CAR Library with scFv Mutations
(A) Schematic representation of the WT ROR1-specific R11 scFv with nucleotide (NT) and amino acid (AA) sequence of the VH CDR3 region. Nucleotides targeted by mutagenesis are underlined. SP, signal peptide; (G4S)3, linker. (B) EGFRt expression and ROR1-specific CAR expression of ROR1-CAR scFv library and ROR1-CAR WT reporter cells after nucleofection and subsequent EGFRt-based magnetic bead enrichment (first step) and flow cytometry-based sorting with AF647-labeled ROR1 protein (second step). (C) ECFP and EGFP reporter signal after stimulation of ROR1-CAR scFv library and ROR1-CAR WT reporter cells with BW-ROR1 cells at a 2.5:1 ratio for 24 hr. (D) NF-κB and NFAT activation in percent ± SD of BW-ROR1-stimulated ROR1-CAR scFv library clones normalized to the positive control (stimulation with BW-OKT3) and compared to the ROR1-CAR WT (NF-κB, black line; NFAT, gray line). Statistical significance (n = 3) in comparison to the ROR1-CAR WT was determined using two-way ANOVA with Holm-Sidak post hoc test. ↑, higher; ↓, lower; ns, not significant; *p < 0.05.