Flavors of biotin ligases: From research tools towards biopharmaceutical applications

Biotin ligases are enzymes commonly attaching biotin to biotin-dependent enzymes, which are fundamental for essential metabolic pathways like gluconeogenesis or fatty acids synthesis. However, in times of protein engineering and genome editing those enzymes can be utilized in versatile ways. In this work biotin ligases were deployed in different fields of research starting with a tool for fundamental understanding of cell biology and culminating in biopharmaceutical approaches. First, a novel enzyme called ultraID applicable for protein-protein interaction studies was engineered by directed evolution. This undertaking was carried out by error-prone PCR mutagenesis and yeast surface display in combination with fluorescence activated cell sorting. While based on a biotin ligase derived from Aquifex aeolicus the novel enzyme exhibited a massively improved catalytic turnover, which was tracked back to one single mutation in the active site. To date, ultraID is one of the smallest and most efficient enzymes for the proximity-dependent biotin identification method. Second, screening of the randomized biotin ligase library was conducted towards a modified biotin substrate. Hereby, a propargyl functionalized biotin derivative was examined for enzyme turnover. Even tough different yeast surface presentation and assay strategies were analyzed a propargyl biotin using enzymes was not identified. Third, fundamental experiments for the generation of an antibody-drug conjugate by biotin ligases were performed. A biotin ligase derived from Pyrococcus horikoshii was exploited to conjugate propargyl biotin and desthiobiotin azide to the therapeutic antibody trastuzumab, which was equipped with a biotin acceptor domain. It was successfully demonstrated that chemoenzymatic modification was feasible by conjugating a fluorophore to the antibody. However, the usage of desthiobiotin azide as a substrate renders the biotin ligase unspecific and consequently, optimizations must be done in future approaches. In parallel an antibody-drug conjugate was generated by a different approach. Lipoate-protein ligase A derived from Escherichia coli was used to conjugate a click chemistry moiety to a tagged trastuzumab. Subsequent attachment of a Monomethyl auristatin E toxin led to the generation of an antibody-drug conjugate with potency in the picomolar range. Additionally, it was shown that the applied conjugation strategy had no impact on the antibodies affinity and enzymatic conjugation was complete within a few minutes. Last, identification of internalizing single-chain variable fragments out of systemic lupus erythematosus phage display library was executed. Hereby different enrichment strategies were examined involving incubation on mammalian cells and biotinylation of phages upon successful cell penetration. Ultimately, discovery of an internalizing binder was not achieved but crucial knowledge on phage display-based enrichment of cell penetrating binders was obtained