Site-Specific Conjugation of a Drug to an Antibody Via Nucleotide Binding Site

Antibody-drug conjugates (ADCs) are 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, the drug should be conjugated to the antibody with a linker 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 number of the DAR yielding rapid in vivo clearance, heterogenous modifications causing weak performance of the ADC, and requirement of harsh chemical modification resulting with damaged antibodies. In this study, we describe a site-specific method for conjugation of drugs to monoclonal therapeutic antibodies utilizing nucleotide-binding site (NBS). We used an NBS ligand –ring structured small molecule– 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 have used previously described UV-NBS method that utilizes 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 ADC generated with the UV-NBS method utilizing various drugs such as paclitaxel and bortezomib in combination with various antibodies including Rituximab, anti-CD133 and anti-CD38. We further characterized in vitro efficacy, toxicity, and the DAR of the ADCs as well as activity and sensitivity of the antibodies post-conjugation with the drugs.