Extensive Biotransformation Profiling of AZD8205, an Anti-B7-H4 Antibody-Drug Conjugate, Elucidates Pathways Underlying its Stability In Vivo

What happens to macromolecules in vivo? What drives structure-activity relationship and in vivo stability for antibody-drug conjugates (ADCs)? These interrelated questions are increasingly relevant due to re-emerging importance of ADCs as an impactful therapeutic modality and the gaps that exist in our understanding of ADC structural determinants that underlie ADC in vivo stability. Complex macromolecules, such as ADCs, may undergo changes in vivo due to their intricate structure as biotransformations may occur on the linker, the payload and/or at the modified conjugation site. Dissection of ADC metabolism presents a substantial analytical challenge due to the difficulty in identification or quantification of minor changes on a large macromolecule. We employed immunocapture-LCMS methods to evaluate in vivo changes in DAR profile in four different lead ADCs. This resulted in selection of AZD8205, a B7-H4-directed cysteine-conjugated ADC bearing a topoisomerase I inhibitor payload, with durable DAR. The results showed that vast majority of the biotransformation species identified contained at least one payload. This comprehensive characterization revealed that critical structural determinant contributing to the design of AZD8205 was the PEG section of the linker, resulting in competition between linker deconjugation and maleimide hydrolysis reactions resulting in a durable high DAR in vivo. To our knowledge, this study is the most extensive characterization of ADC in vivo biotransformation to date, which also sheds light on determinants of ADC stability in vivo and informs the selection of optimal linker. These results highlight the relevance of studying macromolecule biotransformation in elucidating the ADC structure-in vivo stability relationship.