Fig. 2

Screening and characterization of mature mesothelin (matMSLN)-specific scFvs. a Schematic representation of matMSLN (residues 296–606) and its soluble counterpart (solMSLN, residues 296–592). The top bar depicts the full-length matMSLN ending in a GPI anchor at residue 606; the bottom bar represents solMSLN, which lacks a C-terminal GPI-anchoring region. The corresponding amino acid sequence is aligned with the annotated residue positions. b Heatmap showing the levels and antigen-binding affinities of individual clones. Yeast-displayed clones were analyzed by flow cytometry using an anti-FLAG antibody (expression) and 10 nM biotinylated matMSLN or solMSLN (binding). SS1 and 15B6 were included as positive controls, and an isotype antibody served as a negative control. Clones are ranked by mean fluorescence intensity (MFI). ELISA-based binding of recombinant anti-MSLN scFvs to matMSLN (c) and solMSLN (d). Binding in immobilized scFvs incubated with serial dilutions of biotinylated antigen and detected via streptavidin-HRP. Note that the yeast-display binding values in (b) represent semiquantitative MFI-based screening at a fixed antigen concentration, whereas the ELISA results in (c, d) reflect the quantitative binding of the purified scFvs. Apparent differences between these assays arise from their distinct experimental formats, with ELISA providing a more accurate measure of solMSLN binding. Flow cytometry of cell surface binding by scFvs to MSLN⁺ (e) and MSLN^– K562 cells (f). Serially diluted antibodies were incubated with the cells, and the bound scFvs were detected via an anti-His-FITC secondary antibody. g Epitope binning of anti-MSLN scFvs (CLMS10, CLMS32, CLMS36, CLMS76, CLMS95, SS1, and 15B6) by competitive ELISA. The immobilized scFvs were challenged with biotinylated matMSLN preincubated with competing antibodies. The heatmap intensity reflects the degree of competitive binding, indicating epitope overlap. h Predicted epitope mapping of anti-MSLN scFvs on the basis of binning data. According to UniProt annotations, the extracellular domain of MSLN is divided into Region I (residues 296–398), Region II (399–494), and Region III (495–606). Three independent experiments were performed; the data are presented as the means ± standard deviations (s.d.)