(719a) Antibody-Enzyme Conjugate Megamolecules with Control of Domain Stoichiometry for Cancer Therapy

Antibody conjugates are crosslinked protein structures that use the cell-targeting properties of antibodies to deliver a molecular cargo to a desired cell type. We proposed to use an antibody-enzyme conjugate to enable tumor-specific enzymatic activation of a chemotherapeutic prodrug to improve the precision of anti-cancer chemotherapy. However, current conjugation strategies do not allow for precise crosslinking of antibody and enzyme, yielding a heterogeneous product with poorly defined properties and batch-to-batch variability. In this study, we applied megamolecules, a site-specific crosslinking strategy, to synthesize homogeneous nanobody-enzyme conjugates with precise covalent bonding. We varied the number of domains of nanobody and enzyme in the conjugate independently and measured their effect on cancer cell proliferation in both 2D and 3D culture. We found that increasing the number of enzyme domains increased drug activation and cytotoxicity, while increasing the number of nanobody domains had a weaker effect on cytotoxicity. This study provides evidence for the need to control antibody conjugate structure with molecular precision.