[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-494] Proximity-driven enzymatic crosslinking for site-specific antibody fragment-drug conjugate generation

○Koki Murozono¹, Riko Nishioka¹, Yoshirou Kawaguchi¹, Michio Kimura¹, Noriho Kamiya^1,2 (1. Graduate School of Engineering, Kyushu University (Japan), 2. Center for Future Chemistry, Kyushu University (Japan))

Keywords:

Enzyme,Antibody,Protein engineering,Antibody-drug conjugate,Biopharmaceutical

Fab, a fragment antibody consisting only of the antigen-binding region of an antibody, exhibits high permeability into tumor tissues, rapid systemic distribution, and clearance due to its small molecular weight compared to whole antibodies. Therefore, the application of Fabs modified with functional molecules such as drugs and diagnostic agents as next-generation antibody drugs is expected. Conventionally, chemical modification methods targeting Lys residues on the molecular surface have been widely used for the modification of Fab, but control of the drug-conjugate number and modification positions is difficult, and the heterogeneity of the products and the impact on antigen-binding ability are major issues. While the use of cross-linking enzymes is a powerful means for site-specific modification of Fab, the introduction of enzyme-reactive tags into Fab by genetic recombination has generally been required. However, the introduction of tags requires optimization of insertion sites and linker sequences, which complicates the development process.
In this study, we aimed to develop a new technology that enables tag-free site-specific modification by using Fab obtained by fragmenting off-the-shelf antibodies as is. Specifically, we designed fusion proteins EzMTG-pG and pG-EzMTG, each an engineered zymogen of microbial transglutaminase (EzMTG) fused to a Fab-binding protein G (pG), which cross-links the side chains of Gln and Lys residues. We then investigated the establishment and the potential of a site-specific modification method without genetic modification, targeting Fab derived from an off-the-shelf antibody (trastuzumab).
First, labeling reactions of Fab by pG-fused EzMTG were performed using a fluorescent-dye-modified Gln substrate as a drug model, and their cross-linking catalytic properties were evaluated. As a result, a significant improvement in the modification efficiency was confirmed compared to EzMTG lacking the pG moiety. This is considered to be because the cross-linking efficiency between specific Lys residues in Fab and the fluorescent-dye-modified Gln substrate improved due to proximity, by positioning EzMTG near the Fab via pG. As a result of identifying the modification sites, we found that EzMTG-pG selectively modifies only Lys65 of Fab, and pG-EzMTG selectively modifies Lys225 of Fab. When computational chemical analysis was performed regarding the factors of modification selectivity, it was suggested that the orientation of EzMTG was controlled by the difference in the fusion direction of pG binding to Fab, making it easier to reach the vicinity of Lys65 in EzMTG-pG and the vicinity of Lys225 in pG-EzMTG. Furthermore, Lys65 and Lys225 were located in the largest positive residue patch area among all Lys residues of trastuzumab. It is considered that the electrostatic interaction between the negatively charged-rich region of the EzMTG active center and the positive charge patch formed around the target Lys residues promoted association with the enzyme active site, becoming advantageous for the initiation of the reaction.
Finally, since all Fabs modified with either fluorescent dyes or drugs maintained antigen-binding capacity and showed antigen-specific cytotoxic activity, it was confirmed that this method is useful as a means for highly efficient production of Fabs in which various functional molecules are modified at specific Lys residues, using off-the-shelf antibody drugs as starting materials.