Fig. 3

Fine specificity of affinity matured TCRL antibody P1C1TM. a SaoS2 cells pulsed with p53_125–134 peptide analogues containing single amino acid mutations to alanine were stained with 10 µg mL⁻¹ of P1C1gl (left) or the affinity-matured P1C1TM (right) and detected with an Alexa Fluor 647-conjugated anti-human IgG secondary antibody. Data are representative of two independent experiments. b Potential off-target peptides were identified using the MOTIF search tool and analysed by NetMHC 3.0 for predicted binding affinity to HLA-A*24:02. Peptides with predicted binding affinity of 100 nM or less were used to determine the cross-reactivity of P1C1TM. The murine p53_118–127 that differs from the human p53_125–134 by only two amino acids at positions 5 and 9 was also included. c Soluble pMHCs presenting the panel peptides was produced by UV exchange and used to assess P1C1TM’s fine specificity by ELISA. No UV exchange pMHC and no peptide exchange controls were used as negative controls, while pMHC UV-exchanged with p53_125–134 peptide was used as a positive control. Data are means of triplicates ± SEM. d The panel of peptides were pulsed on SaoS2 cells and stained with 10 µg mL⁻¹ P1C1TM. Unpulsed SaoS2 cells were used as negative control, while SaoS2 cells pulsed with p53_125–134 peptide was used as a positive control. Data are representative of two independent experiments.