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The formylglycine-generating enzyme (FGE) is utilized for the regioselective conjugation of proteins. In an oxygen-dependent reaction it catalyzes the formation of the non-canonical amino acid Cα-formylglycine (FGly) from the cysteine residue of a CxPxR-type core recognition se-quence. In addition to cysteines, the prokaryotic, anaerobic iron-sulfur protein AtsB can also convert serine residues to FGly. For protein conjugation, recombinant proteins with aldehyde tags, consisting of a core motif (C/SxP/AxR) and flanking auxiliary sequences, were converted enzymatically, both in vivo or in vitro. The resulting aldehyde function of the FGly was chemi-cally conjugated. Antibody-drug conjugates (ADCs), directed against the epidermal growth fac-tor receptor (EGFR), were generated by the FGly conjugation of the model protein DARPin E01 (designed ankyrin repeat protein) or the single-chain antibody scFv425-Fc. First, the protein was functionalized with an azide by the aldehyde-specific HIPS ligation (Hydrazino-iso-Pictet-Spengler) and then loaded with a toxin by click reaction. The consecutive use of enzymes with different substrate specificity enables bioorthogonality of the conjugation, whereby for the first time dual ADCs were generated using FGly technology.

In this thesis the use of FGE for selective and bioorthogonal dual conjugation as well as the cop-per-dependency of the enzyme was considered. As a cofactor of the aerobic enzymes, copper has a great influence on the substrate specificity of the prokaryotic FGE variants. In high enzyme-to-substrate ratios and in the presence of copper the acceptance of the proline-free motif increased. This effect was observed regardless whether the addition of copper was performed during the reaction or by previous enzyme reconstitution. However, even in the presence of the cofactor the human FGE (hFGE) has the closest substrate specificity for proline-containing cysteine sequences. To investigate the intracellular systems which enable the ER-permanent human FGE to incorporate copper ions, indirect activity assays based on in vivo sulfatase activation were per-formed. In FGE knock-out cells (ARPE19 sumf1-/-) the addition of copper sulfate significantly increased the activity of transiently expressed hFGE. In contrast, the treatment with the copper chelator tetraethylene pentamine (TEPA) resulted in an inhibiting effect. The relationship be-tween enzymatic activity and intracellular copper homeostasis was initially analyzed using a copper transporter ATP7B-deficient hepatocyte cell line (HepG2 ATP7B-KO). It has been hy-pothesized that the secretory route of the hFGE, its N-terminal domain with still unknown func-tion, and an interaction with the Golgi-permanent copper transporter are involved in the genera-tion of the holoenzyme.

The attachment of the C-terminal amino acid sequences KDEL or PGEL successfully increased both, the ER retention of hFGE and the FGly conversion rate of in situ antibody production in CHO cells, resulting in an FGly content of up to 91.7%. Using an extensive model protein library for the establishment of new FGly conjugation strategies, single and double conjugates were created in this cooperation project between the disciplines of biochemistry, organic chemistry, and biotechnology. Factors such as protein stability, conjugate storage, and biological functional-ity were analyzed. The single-chain antibody scFv425-Fc was loaded with monomethyl auri-statin E (MMAE) and carboxyfluorescein. In the cell-based methods live cell imaging and ala-marBlue® assay a direct correlation between the presentation of the EGFR on the cell surface and the ADC-dependent fluorescence or cytotoxicity was demonstrated, respectively. According-ly, the C-terminal conjugation of the single-chain antibody via the non-canonical amino acid FGly did not interfere with successful antigen binding, which led to ADC internalization and lysosomal release of the toxin. A targeted administration of the drug could be identified with an IC50 value for the MMAE-ADC in the picomolar range.