Mechano-bioconjugation Strategy Empowering Fusion Protein Therapeutics with Aggregation Resistance, Prolonged Circulation, and Enhanced Antitumor Efficacy
Yajie Liu, Xilin Bai, Chengliang Lyu, Jing Fang, Fan Zhang, Wenhao Wu, Wei Wei, Wenbin Zhang
Abstract
Bioconjugation is a powerful protein modification strategy to improve protein properties. Herein, we report mechano-bioconjugation as a novel approach to empower fusion protein therapeutics and demonstrate its utility by a protein heterocatenane (cat-IFN-ABD) containing interferon-α2b (IFN) mechanically interlocked with a consensus albumin-binding domain (ABD). The conjugate was selectively synthesized in cellulo following a cascade of post-translational events using a pair of heterodimerizing p53dim variants and two orthogonal split-intein reactions. The catenane topology was proven by combined techniques of LC–MS, SDS-PAGE, SEC, and controlled proteolytic digestion. Not only did cat-IFN-ABD retain activities comparable to those of the wild-type IFN and ABD, the conjugate also exhibited enhanced aggregation resistance and prolonged circulation time over the simple linear and cyclic fusions. Consequently, cat-IFN-ABD potently inhibited tumor growth in the mouse xenograft model. Therefore, mechano-bioconjugation by catenation accomplishes function integration with additional benefits, providing an alternative pathway for developing advanced protein therapeutics.