Linker and\nConjugation Site Synergy in Antibody–Drug\nConjugates: Impacts on Biological Activity

Antibody–drug conjugates (ADCs) produced using\ngeneral conjugation\nmethods yield heterogeneous products containing mixtures of species\nwith different numbers of payloads per antibody (drug–antibody\nratios) conjugated at multiple sites. This heterogeneity affects the\nstability, efficacy, and safety of ADCs. Thus, various site-specific\nconjugation methods have been developed to achieve homogeneity in\nADCs. It was reported that linker structures and conjugation sites\ngenerally affected the characteristics of site-specific ADCs such\nas stability, efficacy, and safety. However, the combined effects\nof conjugation sites and linker structures on the physicochemical\nand biological characteristics of site-specific ADCs have remained\nunclear. In this study, we generated 30 homogeneous site-specific\nADCs with a combination of six conjugation sites and five linker structures\nusing THIOMAB technology and evaluated the characteristics of these\nhomogeneous ADCs. We found that both conjugation sites and linker\nstructures affected characteristics unique to ADCs (linker stability\nas well as target-dependent and target-independent cytotoxicity) in\nsite-specific ADCs. Especially, conjugation to the constant regions\nof the light chain and the presence of polyethylene glycol structures\nin the linker are important for those ADC-specific characteristics.\nInterestingly, we also found that the effects of linker structures\non the target-independent cytotoxicity of homogeneous ADCs at certain\nconjugation sites differed from those seen in conventional heterogeneous\nADCs. Our results suggest that optimizing linker structures based\non the conjugation site may be necessary for site-specific ADCs.