Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains

Abstract

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb² format where a set of mutated amino acid residues in the C_H3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab C_H1/C_L domain pair with a pair of antigen-binding C_H3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding C_H3 domains. Such bispecific molecules were produced in a "Fab-like" format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding C_H3 domains offer an alternative solution in positioning and valency of antigen binding sites.

Keywords: Ab, antibody; BLI, biolayer interferometry; BSA, bovine serum albumin; Bispecific antibody; CDR, complementarity determining region; DSC, differential scanning calorimetry; Domain-exchanged antibody; EC50, half-maximal effective concentration; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; Fab constant domain exchange; Fab, fragment antigen binding; Fc, fragment crystallizable; Fcab, Fc with antigen binding properties; HPLC-SEC, high pressure liquid chromatography-size exclusion chromatography; Her2 internalization; IgG, immunoglobulin G; LC-ESI-MS, liquid chromatography-electrospray ionization-mass spectrometry; PBS, phosphate buffered saline; PE, phycoerythrin; PEI, polyethylenimine; PNGase F, Peptide:N-glycosidase F; RMSD, root mean square deviation; TRA, trastuzumab; Tm, melting temperature; VEGF, vascular endothelial growth factor; “Knobs-into-holes” heterodimerization.