C4BP: An Alternative Scaffold to Antibody Drug Conjugates with Improved Target Binding Strength and Cytotoxicity

摘要

Multimeric scaffold complexes hold great potential for oligomerization of therapeutic molecules in order to achieve avidity by multivalent interactions. We chose the C-terminal oligomerization domain of human C4b binding protein (C4BP) as an oligomerization scaffold due to its heptameric structure and high stability [1]. Previous structural analyses have shown that N- and C-termini are easily accessible for modification. The aim of this work is the N- and C-terminal functionalization of C4BP to generate a multivalent bi-specific molecule by genetic fusion and enzymatic conjugation. A target-binding peptide is genetically fused to the N-terminus and a SortaseA recognition sequence (LPETG) to the C-terminus of C4BP. The transpeptidase activity of SortaseA from Staphylococcus aureus allows covalent attachment of various oligoglycine substrates as for example toxins and fluorophores, which are not feasible for genetic fusion [2]. Final constructs are able to bind their target with high avidity and are internalized upon cell binding. This finding shows that a toxic payload fused to C4BP can be transported into cells expressing the targeted receptor. Furthermore it has to be shown that the modified C4BP scaffold can specifically kill targeted cells.