Fig. 3: Affinity maturation of CD19 mimotopes.

a, Schematic workflow for identifying affinity-enhanced mimotope variants of P1 from a motif-fixed mimotope library V2. Created in BioRender. Ma, L. (2025) https://BioRender.com/xd8q66t. b, Flow cytometry plots of 20 nM FMC63^IgG binding to yeast cells expressing the V2 mimotope library. The yeast clone P1 was included as a control. The V2 mimotope library was pre-enriched using FMC63^IgG-coated magnetic beads (Methods). c, Weblogo showing the consensus mimotope sequence with high-affinity binding to FMC63^IgG. d, Binding of FMC63^IgG to select yeast clones assessed by flow cytometry. Amino acid sequences and apparent binding affinity are shown for each yeast clone. Median fluorescence intensities (MFI) at each concentration were fit for the calculation of apparent K_d values. e, Impact of individual AA positions on FMC63^IgG binding. Yeast clones bearing F12 variants with 1-2 alanine (red) insertions or individual AA mutated to alanine (red) were assessed for FMC63^IgG binding by flow cytometry. f, ELISA showing the effect of flanking residues on mimotope^F12 binding to FMC63^IgG. The apparent binding affinity was determined as in d. g, Schematic workflow for identifying affinity-enhanced mimotopes from mimotope library V3 using kinetic sorting. Yeast library V3 was first stained with monovalent biotin-FMC63scFv, followed by competition with non-biotinylated bivalent FMC63^IgG. Created in BioRender. Ma, L. (2025) https://BioRender.com/xd8q66t. h, Binding of FMC63^scFv to select yeast clones bearing various evolved mimotopes. The parental mimotope^F12 sequence was underlined. The apparent binding affinity was determined as in d and specified after the mimotope sequence. i, Representative histograms showing retention of pre-bound FMC63^scFv on yeast clone F12-A1 in the presence of FMC63^IgG. Yeast cells were pre-stained with 50 nM biotin-FMC63^scFv followed by incubation with 1 μM FMC63^IgG. Yeast cells were sampled at indicated times for PE-streptavidin staining as an indicator for the retention of biotin-FMC63^scFv. j, CD19⁻ K562 cells were labelled by incubation with 500 nM of the indicated amph-mimotopes, washed and then incubated with various concentrations of fluorescent FMC63^scFv followed by flow cytometry analysis of scFv binding; apparent binding affinities were determined as in h. Data are mean ± s.d. with triplicate samples.

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