PUB - Publikationen an der Universität Bielefeld

Antibody-drug conjugates (ADCs) combine the ability of an antibody to target tumor-specific antigens with the cell-killing potential of a cytotoxic payload after cellular internalization. The epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. This thesis examined the development of bivalent anti-EGFR protein-drug conjugates, analyzed _in vitro_ and _in vivo_, and provided insights into novel concepts of enzyme-based bioconjugation strategies.
All FDA-approved ADCs are currently produced by stochastical conjugation of payloads resulting in a heterogeneous product. Site-specific conjugation of the toxic drug to the protein vehicle allows for defined drug-to-antibody ratios (DAR) improving the pharmacokinetics and therapeutic index. The formylglycine-generating enzyme (FGE) can be used as a tool for the site-specific oxidation of a cysteine into a Cα-formylglycine (FGly) within the recognition motif CxPxR. A mammalian cell surface display system was evaluated for the site-directed evolution of human FGE towards alternative recognition motifs. The biocompatible hydrazine-_iso_-Pictet-Spengler (HIPS) and tandem Knoevenagel ligations allowed for the bioconjugation of aldehyde moieties on the surface of live cells, which were introduced by sodium periodate oxidation. However, labeling of _in situ_ formed FGly in transiently expressed cell surface constructs was not detected and the chemical approaches resulted in high background fluorescence.
Moreover, new enzymatic strategies were explored for the generation of ADCs. A putative MibH-like peptidyl halogenase was identified by phylogenetic relationship analysis. Also, expression of the human lysyl oxidase-like 2 (hLOXL2) and the amine oxidase from _Escherichia coli_ (ECAO) was established, which can be used for the scanning of possible recognition motifs that would allow the site-specific incorporation of allysine into protein-tags.
Bivalent anti-EGFR DARPin E01 fusion constructs were expressed as a single-chain dimer (__DD1__) or N-terminally fused to the human IgG1 Fc domain (__DFc__). Monomethyl auristatin E (MMAE)-conjugates of __DD1__ (DAR = 2.0) and __DFc__ (DAR = 4.0) revealed sub-nanomolar cytotoxicity against EGFR-overexpressing A431 cells. Confocal fluorescence microscopy of corresponding fluorescent dye derivatives showed receptor-mediated endocytosis upon specific EGFR-binding. Despite excellent _in vitro_ performance, anti-tumor activity of the MMAE-conjugates was not satisfactory in an SCC model _in vivo_. Tweaking of binding kinetics might play a key role for anti-EGFR protein-conjugates as dye-conjugates suggested excessive unspecific binding in xenografted mice during _in vivo_ imaging experiments.
Furthermore, genetically encoded pH sensors were designed, fused to the ligand epidermal growth factor (EGF) and characterized in detail. Ratiometric analysis of flow cytometry of A431 cells yielded an internalization rate for the EGF fusion protein. This innovative highthroughput assay combined with off-target analysis might facilitate ADC development projects for specific cancer therapy in the future. Overall, these studies provide important information to unravel the significance of protein-drug conjugates on therapeutic application.