The Role of MegaMolecule Antibody Structure in Internalization and Signaling

This manuscript describes the synthesis of 26 megamolecule-based antibody scaffolds that target the receptor tyrosine kinase HER2 (ERBB2). The scaffolds include mono-, bi-, and trivalent structures that present high- or low-affinity Fab or nanobody domains. Cell binding, internalization, and cytotoxicity were compared with those of the parent monoclonal antibody trastuzumab. Increasing scaffold valency from two to three domains only modestly increased binding efficiency and did not increase the internalization rate. Further, inhibition of cell proliferation was not impacted by scaffold valency. Targeting multiple epitopes on HER2 with a biparatopic scaffold significantly increased the internalization rate (approximately 3-fold) over trastuzumab but could either promote or inhibit cell proliferation. This work is significant both for demonstrating how the megamolecule approach can generate large numbers of diverse and structurally defined antibody mimics and revealing the critical influence of structural characteristics of the molecules are to their biological activities.