Extensive Biotransformation\nProfiling of AZD8205,\nan Anti-B7-H4 Antibody-Drug Conjugate, Elucidates Pathways Underlying\nIts Stability <i>In Vivo</i>

What happens to macromolecules <i>in vivo</i>? What drives\nthe structure–activity relationship and <i>in vivo</i> stability for antibody-drug conjugates (ADCs)? These interrelated\nquestions are increasingly relevant due to the re-emerging importance\nof ADCs as an impactful therapeutic modality and the gaps that exist\nin our understanding of ADC structural determinants that underlie\nADC <i>in vivo</i> stability. Complex macromolecules, such\nas ADCs, may undergo changes <i>in vivo</i> due to their\nintricate structure as biotransformations may occur on the linker,\nthe payload, and/or at the modified conjugation site. Furthermore,\nthe dissection of ADC metabolism presents a substantial analytical\nchallenge due to the difficulty in the identification or quantification\nof minor changes on a large macromolecule. We employed immunocapture-LCMS\nmethods to evaluate <i>in vivo</i> changes in the drug-antibody\nratio (DAR) profile in four different lead ADCs. This comprehensive\ncharacterization revealed that a critical structural determinant contributing\nto the ADC design was the linker, and competition of the thio-succinimide\nhydrolysis reaction over retro-Michael deconjugation can result in\nsuperb conjugation stability in vivo. These data, in conjunction with\nadditional factors, informed the selection of AZD8205, puxitatug samrotecan,\na B7-H4-directed cysteine-conjugated ADC bearing a novel topoisomerase\nI inhibitor payload, with durable DAR, currently being studied in\nthe clinic for the potential treatment of solid malignancies (NCT05123482).\nThese results highlight the relevance of studying macromolecule biotransformation\nand elucidating the ADC structure–<i>in vivo</i> stability\nrelationship. The comprehensive nature of this work increases our\nconfidence in the understanding of these processes. We hope this analytical\napproach can inform future development of bioconjugate drug candidates.