Click-on Antibody Fragments for Customisable Targeted Nanomedicines – Site-specific Tetrazine and Azide Functionalisation through Non-canonical Amino Acid incorporation

Protein functionalisation for the development of imaging agents and antibody drug conjugates still often relies on statistical amidation of the protein through accessible lysine and cysteine residues, requiring protein to protein conjugation optimisation and can potentially impact the overall function. To combat this, focus has turned to developing proteins that have noncanonical amino acids incorporated into their structure, allowing for site-specific labelling and functionalisation. Herein we showcase the incorporation of non-canonical amino acids bearing a tetrazine or azide orthogonal coupling modality into biologics targeted to the prostate-specific membrane antigen and epidermal growth factor receptor respectively. The placement of these bioorthogonal residues into nanobody or single chain variable fragments (scFvs) is introduced by site-directed mutagenesis of the protein-coding DNA that allows for controlled insertion when these proteins are expressed. We show that bioorthogonal coupling of model compounds such as fluorophore or polymeric materials onto the protein does not significantly change the binding affinity, making these protein conjugation methods a powerful tool for development of simple customisable personalised targeted antibody-drug conjugates where affinity is retained.