[2S03a] 07. Biocatalysis Engineering for Sustainable Bioprocessing - Part 2

3:30 PM - 3:45 PM JST(6:30 AM - 6:45 AM UTC)

[2Biocat-09] Enzymatic Domain Ligation: A Building Block Approach to High-Molecular-Weight Biopharmaceuticals and Targeted siRNA Delivery

○hikaru nakazawa¹, yu ando¹, maho ohtake¹, daisuke miura¹, mitsuo umetsu¹ (1. tohoku university (Japan))

Keywords:

protein engineering,antibody,transglutaminase,cell-penetrating peptide,delivery

The primary objective of this study was to establish a robust and versatile platform for the production of high molecular weight biopharmaceuticals, which are often challenging to manufacture using conventional methods. While molecular targeted therapies have revolutionized the treatment of complex diseases, their production faces significant hurdles. Many high performance proteins possess high molecular weights and require intricate folding, frequently leading to expression failure in Escherichia coli the most cost effective and scalable production host. Furthermore, we addressed challenges in the delivery of nucleic acid therapeutics. Fusion proteins comprising targeting antibodies and cationic peptides are promising candidates for the specific delivery of siRNA and the mitigation of systemic side effects. However, the direct expression of such fusion molecules in bacterial hosts is often unsuccessful, hindering research progress. To overcome these limitations, we applied a "building block" approach, wherein individual structural domains are synthesized separately and assembled site-specifically in vitro.In this study, we focused on the hierarchical structure of protein folding, treating the final molecule as an assembly of distinct structural units. We aimed to construct antiEGFR nanobody fused CD3 scFv and antiEGFR nanobody fused R9 complexes for Tcell recruitment and gene silencing applications, respectively.For the Tcell recruiting antibody, two functional components were prepared separately: the antiEGFR nanobody was modified with an Nterminal Ktag (MRHKGS), while the CD3 binding scFv was modified with a Cterminal Qtag (LLQGS). For nucleic acid delivery, the antiEGFR nanobody was expressed in E. coli with a Cterminal Ktag, while the R9 peptide was synthesized with an Nterminal Qtag.These molecules were enzymatically conjugated using microbial transglutaminase. This enzyme catalyzes the formation of a covalent bond between the glutamine side chain of the Qtag and the lysine/primary amine of the Ktag. This in vitro enzymatic assembly allowed us to bypass the cellular stress and misfolding issues typically associated with the direct expression of long chain fusion polypeptides.The VHH fused scFv was obtained with a reaction efficiency of 60%. Cytotoxicity assays revealed that this construct maintained higher cytotoxic activity than the diabody type Tcell recruiting antibody a conventional format comprising cancer-specific scFv and Tcell specific scFv fragments that can be prepared in E. coli. Regarding nucleic acid therapeutics, the nanobody R9 complex was successfully synthesized with a yield of 85%. Notably, due to the site specific nature of the MTGase mediated ligation, the binding affinity of the nanobody for the EGFR antigen remained entirely unaffected compared to its native state. In conclusion, this study demonstrates that enzymatic domain ligation is a powerful strategy for creating complex, multifunctional biopharmaceuticals. These findings provide a clear roadmap for the development of next-generation targeted therapies.