(191cg) Production of Homogeneous Antibody-Drug Conjugates Using the Nucleotide Binding Site

Antibody-drug conjugates (ADCs) are a recent pinnacle of immunotherapeutic agents that enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent drugs. To obtain an efficient ADC, a potent drug should be conjugated to an antibody while preserving antibody activity and high potency of the drug with an optimized drug-antibody ratio (DAR). Conventional ADCs are typically produced by conjugating a drug to an antibody through the side chains of either surface-exposed lysines or free cysteines generated through reduction of inter-chain disulfide bond. Many obstacles have been associated with the conventional drug conjugation methods to antibodies, such as aggregation of antibodies, high DAR yielding rapid in vivo clearance, heterogenous modifications causing weak performance of the ADC, and requirement of harsh chemical modification resulting in damaged antibodies. In this study, we describe a site-specific method for conjugation of drugs to monoclonal therapeutic antibodies using the nucleotide-binding site (NBS). We used a previously identified NBS ligand that has moderate binding affinity to the NBS located between heavy and light chains on the variable region of Fab fragment to conjugate a drug with a PEG linker to the antibody. We used the UV-NBS method that uses UV-energy to initiate a photo-cross-linking reaction between the NBS and the NBS-ligand, to obtain controlled site-specific conjugation of the drug to the antibody. We have assessed the effectiveness of the ADCs generated with the UV-NBS method utilizing a chemotherapeutic agent in combination with various pharmaceutical antibodies. We further characterized in vitro efficacy, potency, and the DAR of the ADCs as well as activity and sensitivity of the antibodies post-conjugation with the drugs.