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 mAb(2) format where a set of mutated amino acid residues in the C(H)3 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(H)1/C(L) domain pair with a pair of antigen-binding C(H)3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding C(H)3 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(H)3 domains offer an alternative solution in positioning and valency of antigen binding sites.