Interconversion of Unexpected Thiol States Affects the Stability, Structure, and Dynamics of Antibody Engineered for Site-Specific Conjugation

Abstract

Antibody–drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody–drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterize an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge during manufacture. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody intermediate. This quantitative and structurally resolved model of the local and global changes in structure and dynamics associated with the engineering and subsequent disulfide-bonded variant can assist future engineering strategies.

Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation

“…Initial characterization across four antibody intermediates revealed that the inserted cysteine at position C239 could adopt several, distinct thiol states during manufacture: free thiol (2xSH), cysteinylated (1x/2x Cys), and an additional disulfide bond between both C239i residues (iDSB) (Figure A, C). These findings correlate with those of Orozco et al and Cao et al (2xSH and iDSB variants discussed only). Orozco et al used high resolution tandem mass spectrometry and the ensuing unique peptide fragmentation patterns to confirm that the additional disulfide bond was configured similarly to the canonical mAb interchain disulfide bridgea heavy–heavy link at equivalent sites (C239) of each chain.…”

“…Rapid formation of iDSB within 24 h at 37 °C after deglycosylation further supports the hypothesis that a steric constraint must be overcome to form the iDSB and is consistent with previous findings (Figure S6). 26 Why the level of iDSB remains relatively constant (ca. ∼ 20%) during the fed-batch process remains unclear.…”

“…The insertion of a cysteine near the hinge region of an antibody IgG1 scaffold (C239i) has been of particular interest, exhibiting favorable properties such as consistent DAR ≈ 2, payload stability over time, decreased FcγR binding, and retaining wildtype half-life by not reducing neonatal Fc receptor (FcRn) affinity . Recently, we reported changes to the stability, structure, and dynamics resulting from the formation of an unexpected disulfide bond that can occur during antibody manufacture and the subsequent conjugation . While it does not preclude this format from successful preclinical and clinical development, it highlights that characterizing pCQAs of engineered scaffolds and monitoring these through development is important.…”

“…25 Recently, we reported changes to the stability, structure, and dynamics resulting from the formation of an unexpected disulfide bond that can occur during antibody manufacture and the subsequent conjugation. 26 While it does not preclude this format from successful preclinical and clinical development, it highlights that characterizing pCQAs of engineered scaffolds and monitoring these through development is important.…”

All-in-one disulfide bridging enables the generation of antibody conjugates with modular cargo loading

“…These data corroborate the findings of the DSC analysis and indicate that TetraDVP conjugates may have increased stability compared to conjugates made via rebridging of two cysteine residues. Overall, it should be noted that the modest destabilising effects caused by both DVP and TetraDVP conjugation compare favourably to those caused by other clinically successful site-selective antibody modification approaches or mutations in the C H 2 domain, 53–55 showcasing the merits of this form of cysteine modification.…”