Fig. 2
Affinity maturation of TCRL antibody P1C1. a CDR1 and 3 of the heavy chain and CDR1 of the light chain of the original P1C1gl clone and the affinity-matured clones are shown. Mutants 2E3, 1E11 and 1G7 were identified by off-rate comparison for improved binding from the heavy-chain CDR1, CDR3 and light-chain CDR1 libraries, respectively. Mutations were sequenced and combined in a single triple mutant, P1C1TM. b Comparison of the binding affinities of the germline sequence converted P1C1 (P1C1gl), individual mutants and triple mutant (P1C1TM) was done by ELISA. Soluble biotinylated recombinant p53125–134/A24 pMHCs were incubated with immobilized antibodies and detected with HRP-conjugated streptavidin. c Binding kinetics of P1C1TM was analysed by surface plasmon resonance. Soluble recombinant p53125–134/A24 pMHCs was flowed over P1C1TM antibodies captured on an anti-human IgG-coated sensor chip at a range of concentrations between 200 and 2 nM. Binding specificity and avidity of P1C1TM to p53125–134/A24 pMHCs on cells were evaluated with SaoS2 cells pulsed with either a panel of six known A24 peptides, including p53125–134 (d) or a range of p53125–134 peptide concentrations (e). Data are representative of two or more experiments.
