Extended Data Fig. 11: Saturation mutagenesis of A7 construct resulted in single-antigen-specific population.
From: RETRACTED ARTICLE: Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs
a-b. Cross-reactive binding to mismatched HLA-A24 peptides. Flow cytometry of Jurkat cells transduced with A7 CAR stained with PHOX2B dextramer on x-axis and mismatched PBK peptide on HLA-A*24:02 on y-axis. c. Saturation mutagenesis was performed on CDR loops 1-3 of the heavy chain. Each pool of mutants was stained with target pMHC and counter-stained with mismatched pMHC. Contribution of each CDR binding loop (mutagens labeled CDR loop # - position #) to binding HLA-A*24:02 is shown (green: no contribution to binding; red: significant HLA binding at specific amino acid). Contribution of each position to HLA binding was calculated as follows: (MFItarget(mut)/MFImismatch(mut))/((MFItarget(WT)/MFImismatch(WT). d. Mutations of A7 CAR at CDR3 positions 2 and 3 result in 12.4x and 4.7x shifts towards single specificity, respectively. Double mutation of D3A and R2A resulted in modified A7 CAR with no MHC cross-reactivity and reduced binding to target. Created with BioRender.com.
