Enzymatic (Dual) Site-Specific Conjugation of Engineered and Native Antibodies

摘要

Site-specific drug attachment to antibodies has found increasing interest leading to homogenous, stoichiometrically well-defined antibody-drug conjugates (ADCs) which results in following key benefits: 1) facilitated characterization since the attachment sites are well-known, 2) a highly reliable and consistent batch-to-batch production and 3) a reported higher ADC tumor uptake with an increased applicable maximum-tolerated-dose (MTD) versus the conventionally (uncontrolled) modified ADCs. As a site-specific technology, enzymatic conjugation has generated considerable attraction since those conjugation reactions are typically fast and can be done under physiological conditions. Among the available enzymes, microbial transglutaminase (MTG) has recently been recognized as an interesting tool for bioconjugation applications since it catalyzes a transamidation reaction between a 'MTG-reactive' glutamine residue and a simple, low molecular weight primary amine. We have now stably solid-phase immobilized MTG for the efficient generation of site-specifically conjugated, engineered (deglycosylated) antibodies with various functional substrates at the glutamine residue Q295 and the lysine residues K288/290 and K340 (Spycher et al., ChemBioChem, 2017). We demonstrate that the combination of both approaches using immobilized MTG enables the generation of dual site-specifically modified antibody-drug conjugates that are suitable for imaging and therapy by conjugating a fluorescent dye and metal chelator for radiolabeling. Additionally, we introduce a novel proprietary enzymatic conjugation technology that enables site-specific payload attachment to native antibodies, thus generating well-defined ADCs that have a native IgG antibody structure.