Fig. 4: Structural modelling of mimotopes complexed with FMC63.

a, Ribbon diagram of FMC63^scFv (purple) in complex with human CD19 (green, PDB 7URV). Interactions involving key residues of FMC63 are shown. Interacting side chain atoms are shown and boxed. Interactions involving backbone atoms are labelled without box. Created in BioRender. Ma, L. (2025) https://BioRender.com/yxmbsum. b,c, Computational models generated using AlphaFold and Rosetta for FMC63^scFv bound to F12 (b) (created in BioRender. Ma, L. (2025) https://BioRender.com/9lsos0m) or F12-A1 (c) (created in BioRender. Ma, L. (2025) https://BioRender.com/yxmbsum), respectively. Interactions involving previously mentioned key residues of FMC63 are labelled using the same scheme as in a. For F12-A1, the orientation of mimotope is shown to best exhibit how it engages FMC63. The panel in the bottom left depicts the complex in the same orientation as F12-FMC63. d, Interface footprint of mimotopes on FMC63. On the left side, the CD19 interface is shown in green, the F12 interface is shown in orange, and overlapping residues are shown in red. On the right side, the CD19 interface is shown in green, the F12A1 interface is shown in blue, and overlapping residues are shown in red (https://BioRender.com/yxmbsum). e, Recognition of natural CD19 recognition by FMC63^scFv in the presence of mimotopes. Biotinylated FMC63^scFv was pre-incubated with various concentrations of F12 or F12-A1 before staining NALM6 cells and detection via phycoerythrin (PE)-conjugated streptavidin. For FMC63^IgG binding, NALM6 cells were stained with the same concentrations of mimotope and FMC63^IgG with no previous incubation, and binding was detected using PE-conjugated anti-mouse IgG. A non-competing IgG clone HIB19 is shown as a control. Created in BioRender. Ma, L. (2025) https://BioRender.com/miyfij7. f, Activation of mimotope-bound CD19 CAR-T cells by target cells. CD19 CAR Jurkat T cells were pre-incubated with F12 or F12-A1 at indicated concentrations, and then co-cultured with NALM6 cells at a 1:1 E:T ratio for 16 h, followed by flow cytometry analysis of CD69 expression. Results in e and f are representative of three independent experiments.