Fig 1: Structure and characterization of the bispecific anti-EGFR/EPHA2 antibody. A, An asymmetric monovalent bispecific anti-EphA2/EGFR antibody, BMX-661, composed of Fab fragment of BMX-066 and scFv derived from the anti-EGFR antibody cetuximab, fused to the human IgG1 Fc domain. Fc fragments of the antibody are heterodimerized by knob-in-hole mutations in the CH3 domains of the heavy chains. His5 amino acid tag is incorporated into the linker between scFv and the Fc region for purification purposes. B, Binding of BMX-661 and cetuximab to soluble immobilized rhEGFR (extracellular portion, top) or BMX-661 and BMX-066 to immobilized rhEPHA2 (bottom) in the ELISA assay. Soluble antigen proteins (2 µg/mL) were immobilized onto ELISA plates, and after incubation with the indicated concentrations of BMX-661, BMX-066, or cetuximab, bound antibodies were detected with goat anti-human IgG(Fc)-HRP. C, The surface plasmon resonance–binding curves for BMX-066 and BMX-661. Soluble rhEPHA2 was immobilized onto the ProteOn GLH chip through amino groups in the 150 mmol/L NaCl, 10 mmol/L HEPES pH 7.4, 0.05% Tween-20 buffer. After washing out the unbound protein, BMX-066 and BMX-661 were applied at 500 nmol/L (IgG) and the unbound antibodies were removed with the buffer. Following this, rhEGFR was introduced, as indicated (sEGFR), and the unbound ligand was removed after binding saturation. The measurements were done using Bio-Rad ProteOn XPR36, at 25°C. Bispecific BMX-661 bound both immobilized rhEPHA2 and soluble rhEGFR, whereas, as expected, monospecific anti–EPHA2 BMX-066 did not interact with rhEGFR. D, Dependence of integral volume of particles, V, from their sizes, d, for BMX-661 preparation measured by dynamic light scattering. BMX-661 (0.16 mg/mL) was analyzed in the 20 mmol/L l-histidine, 30 mmol/L citric acid, 32 mmol/L Na2HPO4, 1% trehalose, 0.05% Tween-20; pH = 6.0 buffer. The content of multimeric forms of the MABs was estimated using spectrometer ZS Zetasizer Nano (Malvern Instruments) at 25°C. E, HPLC analysis of BMX-661 solution, size exclusion chromatography. F, Stability of BMX-661 estimated by SDS-PAGE under reducing conditions. Freshly isolated antibodies BMX-066 (lanes 1 and 2) and BMX-661 (lanes 4 and 5); BMX-661 stored for 200 days at 4°C (lanes 6 and 7) or incubated at 37°C for 3 days (lanes 8 and 9) in the formulation buffer were loaded at 11 (lanes 1, 4, 6, and 8) and 4 (lanes 2, 5, 7, and 9) micrograms per well. Lanes 3 and 10 molecular weight markers. The top band in the heavy chain region of BMX-661 represents the EGFR-binding scFV-Fc portion of the antibody, whereas the second band and the lower band represent heavy and light chains of the EPHA2-binding portion of bispecific antibody. G, Blood serum stability of BMX-661. ELISA titration curves before (yellow) and after (red) incubation of 2 mg/mL of BMX-661 in human blood serum (Sigma-Aldrich, Cat # H4522) for 7 days at 37°C. ELISA plates were coated with rhEPHA2 (left panel) or sEGFR (right), and blood serum samples with BMX-661 were applied to titration. Staining was performed with goat anti-human IgG(Fc)-HRP. H, Flow cytometry analysis of BMX-661 with HEK-EPHA2 and HEK-pcDNA3 cells. Anti-hIgG-PE was used as a secondary antibody. Staining with nonspecific hIgG was used as a specificity control. I, EGFR levels in HEK-293 cells stably transfected with the EGFR-encoding pCW45 expression vector (HEK-EGFR) or mock-transfected HEK-293 (HEK-pCW45). J, Flow cytometry analysis of BMX-661 with HEK-EGFR and HEK-pCW45 cells. Western blot images were optimized using PowerPoint software where required.