The team led by Wen-Bin Zhang at the College of Chemistry and Molecular Engineering, Peking University, and Wei Wei at the Institute of Process Engineering, Chinese Academy of Sciences, has developed mechano-bioconjugation as a novel approach to empower fusion protein therapeutics, and demonstrated its utility by a protein heterocatenane (cat-IFN-ABD) containing interferon-α2b (IFN) mechanically interlocked with a consensus albumin-binding domain (ABD). The research results were published online by the journal J. Am. Chem. Soc. on September 30, 2022 (https://pubs.acs.org/doi/full/10.1021/jacs.2c06532), with the title "Mechano-bioconjugation Strategy Empowering Fusion Protein Therapeutics with Aggregation Resistance, Prolonged Circulation, and Enhanced Antitumor Efficacy".
Protein therapeutics, such as antibodies, enzymes and cytokines, have expanded rapidly over the past decades and have become one of the most important classes in modern biopharmaceuticals. Compared to small molecule drugs, protein therapeutics have the advantages of high activity, high specificity, low toxicity and unambiguous biological functions. However, due to their inherent instability, protein drugs often tend to aggregate/fibrillize during storage and exhibit poor in vivo pharmacokinetics. Extra efforts are usually required to enhance their stability and overall therapeutic efficacy while reducing their side effects. Currently, bioconjugation of protein drugs with various functional moieties, such as polyethylene glycol (PEG) or human serum albumin (HSA), has proven to be an effective strategy for extending the half-life of protein drugs in vivo. Meanwhile, this method also suffers from drawbacks such as decreased protein activity, complex manufacturing process, and strong immunogenicity. Therefore, new bioconjugation strategies are strongly demanded to make protein therapeutics less aggregation-prone, less frequently dosed, more stable and efficacious.
Mechanical bond is a unique type of bonding that involves no gain or loss of electrons, but results from an entanglement in space between two or more molecular entities. In supramolecular chemistry, mechanical bonds are commonly encountered in the making of molecular machines. In Nature, proteins containing mechanical bonds have also been identified and found to exhibit enhanced stabilities. Wen-Bin Zhang group has made considerable progresses in cultivating artificial mechanical bonds in proteins, such as protein catenanes, lasso proteins, and protein pretzelanes, in the past few years. Indeed, they found that protein catenation could bring in enhanced stability without compromising activity, which represents a novel mode of bioconjugation. By enabling simultaneous function integration and stability enhancement, it shall open a new avenue for protein conjugation beyond simple fusion.
Mechano-bioconjugation refers to conjugation of two cyclic motifs by mechanical bonds to form a heterocatenane structure. Wen-Bin Zhang group has previously shown that protein heterocatenanes could be biosynthesized upon expression of a single gene precursor containing p53dim domains for intramolecular entwining and orthogonal split inteins for spontaneous chain cleavage and cyclization via trans-splicing (Angew. Chem., Int. Ed. 2020, 59, 16122). Based on this, they chose interferon (IFN, an antiviral and antitumor drug) as a model protein drug and albumin-binding domain (ABD) as a model functional motif and prepared a novel protein mechano-bioconjugate: the heterocatenane cat-IFN-ABD. Notably, by replacing the original p53dim homodimer domains with a pair of their recently reported, engineered p53dim heterodimers (J. Am. Chem. Soc. 2021, 143, 18029), highly selective biosynthesis of protein heterocatenane with decent yields has been achieved, providing a facile method for preparing protein mechano-bioconjugates.
To further explore the structure-property relationship of topological proteins, they also constructed and synthesized linear and cyclic IFN-ABD fusion proteins (l/c-IFN-ABD) as controls together with the wild-type interferon (wt-IFN). The results showed that cat-IFN-ABD not only retained activities comparable to that of the wt-IFN and ABD, but also exhibited a significantly improved resistance to thermal aggregation. As ABD prolongs the in vivo half-life of the protein drug by binding to human serum albumin, all IFN-ABD fusion proteins have a longer in vivo half-life than wt-IFN, with cat-IFN-ABD in particular having the longest in vivo half-life of 11.8 hours. Biodistribution and intratumoral accumulation experiments further showed that cat-IFN-ABD was significantly enriched in the tumor sites compared to the control samples. Consequently, cat-IFN-ABD showed the most superior antitumor activity in the mouse xenograft tumor model, fully demonstrating the functional advantages of mechano-bioconjugation. This work extends the linear backbone of the protein into a catenane structure by mechano-bioconjugation, which is not only a simple and effective method for multi-function integration, but also offers additional advantages including enhanced stability and improved efficacy. As this approach is also compatible with other existing engineering techniques, it has great potential for the development of new-generation protein therapeutics.
Fig. 1 The mechano-bioconjugation strategy leads to fully retained affinity and activity upon multi-function integration, and also empowers fusion protein therapeutics with aggregation resistance, prolonged circulation, and enhanced anti-tumor efficacy.
Yajie Liu, a postdoc at Peking University, is the first author of this paper. Prof. Wen-Bin Zhang from Peking University and Prof. Wei Wei from the Institute of Process Engineering, Chinese Academy of Sciences are the co-corresponding authors. This research was jointly supported by the National Natural Science Foundation of China, the National Key R&D Program of China, Beijing National Laboratory for Molecular Sciences, Clinical Medicine Plus X project of Peking University, and Open Funding Project of the State Key Laboratory of Bio-chemical Engineering. This work was also supported by the fellowship of China Postdoctoral Science Foundation and the National Center for Protein Sciences at Peking University.
Original link for the paper: https://pubs.acs.org/doi/full/10.1021/jacs.2c06532.