Use of peptide asparginyl ligases (PALs) in generating alternative non-Ig protein-drug conjugates for cancer therapy

Abstract

Past research on antibody–drug conjugates (ADCs) focused on protein site-specific modification as chemical conjugation lacks site-selectivity. And despite monoclonal antibodies being the most common immunotherapeutic agent, its applicability & functionality are limited by several restrictions. Thus, site-selective conjugation focused in utilizing optimized peptidyl asparaginyl ligases (PALs) and novel therapeutics targeting a promising antibody alternatives: non-Ig (non-immunoglobulin) protein (Affibody, Nanobody, etc.); but this field remains small due to challenges in engineering them for clinical success. This project aims to further optimize Oldenlandict affinis asparaginyl endopeptidase (OaAEP1b) ligation of Affibody and DARPin conjugates with minimal hydrolysis. Results presented successful OaAEP1b-C247A expression, purification & activation and anti-HER2 non-Ig proteins expression & purification; though troubleshooting required in purifying non-Ig protein as proteins were not completely eluted. We verified pH-controlled OaAEP1b orthogonal ligation between Affibody & DARPin with 5(6)-Carboxyfluorescein (FAM), generating satisfactory yield. But further improvement necessary for OaAEP1b-C247A ligase activity optimization and reducing DARPin protein precipitation during pH rebuffering. Due to challenges regarding limited time constraints and unforeseen power outage, future investigation to assess Affibody & DARPin binding affinity (Kd) and half-maximal inhibitory concentration (IC50) to unravel further insights into non-Ig proteins with enzymatic ligation for therapeutic applications.