Figure 1. Engineering and characterizing BaCa antibodies with superior cytotoxicity against ovarian cancer cells.
(A) Domain organization, and SDS-PAGE analyses of BaCa-1, BaCa-2, BaCa-3, and IgG1 antibodies in native and reducing conditions. Individual gel lanes for each antibody types are cropped from the same blot.
(B) Summary of BaCa-1, BaCa-2, BaCa-3 and parental IgG1 properties
(C) NIH-OVCAR-3 cells were treated with increasing concentrations of the indicated antibodies or cisplatin. The cell death was quantified using cell viability assays (n=3).
(D) rFOLR1 and rDR5 were coated on 96 well plates in 5:1 ratio. Relative avidity index of indicated antibodies was determined in presence of 6 M urea.
(E) The binding kinetics of immobilized biotinylated rDR5 against lexatumumab and BaCa or biotinylated rFOLR1 against farletuzumab and BaCa were measured using bio-layer interferometry (BLI) optical analytical technique.
(F) Domain organization of the non-anchoring BaCa (NBaCa) antibody.
(G) Cell viabilities of NIH-OVCAR-4 cells were analyzed in the presence of BaCa, lexatumumab, or NBaCa antibodies. IC50 values are shown at the bottom (n=3).
Abbreviation used in Figure 1A and B: * = 1-step protein-A purification, ** = Total % recovery after size exclusion purification (SEC), *** = Farletuzumab data is shown (Lexatumumab was comparable), Native = Antibody run on gel with non-reducing dye, Reducing = Antibody run on gel with reducing dye, HC = Heavy chain, LC = Light chain, Fab = Fragment antigen binding, Fv = Fragment variable, scFv = Single chain fragment variable, VL = Variable domain of light chain, VH = Variable domain of heavy chain, CK = Kappa chain
Error bars in C, D and G represent SEM. Unpaired Welch’s t-test was used to determine p values.
See also Figures S1 and S2