Structure and analysis of scFvMTBHsp70 fusion protein. A, anti-MSLN VH and VL are linked with a (G4S)3 linker and fused to full length MTBHsp70 with a (G4S)3 linker. B, RAPIDstain based on Coomassie dye following purification and hIgG-Fc tag removal of MTBHsp70 and scFvMTBHsp70. C, BR5FVB1 ovarian cancer cells and 40L mesothelioma cells were incubated with 40 μg/ml scFvMTBHsp70 or 26 μg/ml MTBHsp70 (blue line), or without either protein (solid), followed by anti-MTBHsp70 (IgG2a), biotinylated anti-IgG2a, and Streptavidin-APC, and then analyzed by flow cytometry. To confirm that the scFv portion of the fusion protein binds to MSLN on the surface of tumor cells, scFvMTBHsp70 or MTBHsp70 was preincubated with 12 μg/ml recombinant human MSLN for 30 min (red line) before being added to the cells. Data are representative of three independent experiments in duplicate tubes. D, Median fluorescence intensity (MFI) values of cells stained with scFvMTBHsp70 or MTBHsp70 normalized to cells stained without either protein. Data are expressed as means ± SEM in arbitrary units. P values were determined using One-Way ANOVA followed by Turkey’s multiple comparison tests. *,p < 0.05; **,p < 0.01;ns, non-significant. E, scFvMTBHsp70 binds with peritoneal mesothelial cells at a low level compared to ovarian cancer and mesothelioma cells. Binding of the fusion protein is at very low or undetectable levels on PBLs and splenocytes. Thick line, with incubation of scFvMTBHsp70; solid, without incubation of scFvMTBHsp70. Data are representative of three independent experiments.