Site-Specific Conjugation Strategy for Dual Antibody–Drug Conjugates Using Aerobic Formylglycine-Generating Enzymes

Multiple, site-specific protein conjugation is increasingly attractive for the generation of antibody–drug conjugates (ADCs). As it is important to control the number and position of cargoes in an ADC, position-selective generation of reactive sites in the protein of interest is required. Formylglyc...

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Published inBioconjugate chemistry Vol. 32; no. 6; pp. 1167 - 1174
Main Authors Boschanski, Mareile, Krüger, Tobias, Karsten, Lennard, Falck, Georg, Alam, Sarfaraz, Gerlach, Marcus, Müller, Benjamin, Müller, Kristian M, Sewald, Norbert, Dierks, Thomas
Format Journal Article
LanguageEnglish
Published Washington American Chemical Society 16.06.2021
Subjects
Amino acid sequence
Amino acids
Ankyrins
Antibodies
Cell culture
Chains
Conjugates
Conjugation
Conversion
Copper converters
Cytotoxicity
Enzymatic activity
Enzyme activity
Enzymes
Epidermal growth factor
Growth factors
Internalization
Proteins
Receptors
Residues
Toxicity
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Summary:Multiple, site-specific protein conjugation is increasingly attractive for the generation of antibody–drug conjugates (ADCs). As it is important to control the number and position of cargoes in an ADC, position-selective generation of reactive sites in the protein of interest is required. Formylglycine (FGly) residues are generated by enzymatic conversion of cysteine residues embedded in a certain amino acid sequence motif with a formylglycine-generating enzyme (FGE). The addition of copper ions increases FGE activity leading to the conversion of cysteines within less readily accepted sequences. With this tuned enzyme activity, it is possible to address two different recognition sequences using two aerobic formylglycine-generating enzymes. We demonstrate an improved and facile strategy for the functionalization of a DARPin (designed ankyrin repeat protein) and the single-chain antibody scFv425-Fc, both directed against the epidermal growth factor receptor (EGFR). The single-chain antibody was conjugated with monomethyl auristatin E (MMAE) and carboxyfluorescein (CF) and successfully tested for receptor binding, internalization, and cytotoxicity in cell culture, respectively.
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ISSN:1043-1802
1520-4812
DOI:10.1021/acs.bioconjchem.1c00246