Clone C CAR T cells recognize the ALNEQIARL peptide with high specificity. (A) Alanine screen: peptides used for T2 pulsing (left), mIgG1 binding (middle), and CAR T-cell killing (right). (B) Western blot of NDC80 knockdown using small interfering RNA (siRNA). (C) CAR T-cell killing against JMN NDC80 knockdown cells corresponding to panel B. (D) HLA ligands identified via MS after immunoprecipitation with either HLA-A*02–specific BB7.2 antibody (black) or mIgG1 clone C (red). Peptides are ranked by predicted binding to HLA-A*02 using netMHCpan 4.0 in EL mode. Peptides in green were identified in 2/2 MS runs; peptide in black was identified in 1/2 runs. (E) Flow cytometry of clone C or mIgG1 isotype binding of T2 cells pulsed with either the ALNEQIARL peptide or the potential off-targets from panel D. Two NDC80-derived peptides (GLNEEIARV and HLEEQIAKV) were used as internal negative controls. Error bars denote standard deviation in panels A and C. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; gMFI, geometric mean fluorescence intensity. PE-A, phycoerythrin.