Fig. 6: Therapeutic application of TCR-MAP to predict adverse cross-reactivities of clinical TCR.
a, Schematic of the human genome-wide TCR-MAP screen of the a3a TCR+ SrtA-Jurkats and peptide enrichment results. The 90-aa peptides tiling across the human proteome with 22-aa overlap were expressed in HLA-A*01:01+ G5-target cells. These cells were screened with the evolved MAGEA3 TCR. Each dot represents one peptide, with the y axis plotting the −log10 adjusted P values determined by Mageck and the x axis indicating the geometric mean of the enrichment of the peptide across three replicates. Fold enrichment is defined as the ratio of the abundance of the peptide in the sorted population relative to the input library. Peptides highlighted in red contain the epitope from MAGEA3. The remaining colored dots indicate additional cross-reactive fragments that were validated. Validated peptides that scored in the a3a TCR screen are listed with the antigenic epitope colored. b, Saturation mutagenesis screen heatmap representing the relative recognition of mutant epitopes from MAGEA3 relative to the original sequence. Each box in the heatmap shows the relative enrichment or depletion of a single mutant peptide, where the amino acid along the x axis was substituted to the amino acid indicated along the y axis. c, EpitopeID ranking of potentially cross-reactive peptides. Saturation mutagenesis heatmap values were used to rank 9-aa peptides tiling the entire human proteome. Scored peptides were then filtered for HLA-A*01:01 binders by NetMHC and listed in descending order of the calculated stimulation potential score. Peptides 6–10 were tested for reactivity by performing peptide pulsing experiments in HLA-A*01:01+ G5-target cells and the CD69 upregulation of a3a SrtA-Jurkats is reported. ****P < 0.0001 for each group relative to the no-antigen control, determined by a two-tailed t-test. Each dot represents a different biological replicate, where error bars indicate the mean and s.d. Peptide pulsing experiments are representative of n = 3 independent biological replicates.
