Interconversion of unexpected thiol states affects stability, structure and dynamics in engineered antibody for site-specific conjugation

Orozco, C. T.; Edgeworth, M. J.; Devine, P. W. A.; Hines, A. R.; Cornwell, O.; Wang, X.; Phillips, J. J.; Ravn, P.; Jackson, S. E.; Bond, N. J.

doi:10.1101/2020.09.28.317339

bioRxiv · 10.1101/2020.09.28.317339

Interconversion of unexpected thiol states affects stability, structure and dynamics in engineered antibody for site-specific conjugation

Orozco, C. T.·Edgeworth, M. J.·Devine, P. W. A.·Hines, A. R.·Cornwell, O.·Wang, X.·Phillips, J. J.·Ravn, P.·Jackson, S. E.·Bond, N. J.

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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 characterise an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge. 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. Insight is gained into the local and global destabilisation associated with the engineering and subsequent disulfide bonded variant that will inform future engineering strategies.

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Orozco, C. T., Edgeworth, M. J., Devine, P. W. A., Hines, A. R., Cornwell, O., Wang, X., Phillips, J. J., Ravn, P., Jackson, S. E., Bond, N. J.. 2020-09-29. Interconversion of unexpected thiol states affects stability, structure and dynamics in engineered antibody for site-specific conjugation. https://doi.org/10.1101/2020.09.28.317339

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