[P006] Poster

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

[P03-350] Controlled antibody modification using engineered microbial transglutaminase fusion proteins via proximity labeling

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

Keywords:

Antibody-drug conjugate,Biopharmaceutics,Enzymatic conjugation

Antibody-drug conjugates (ADCs) are biopharmaceuticals that combine the target specificity of antibodies with the pharmacological potency of small-molecule drugs. By leveraging the high selectivity of antibodies for disease-associated antigens, potent cytotoxic payloads can be preferentially delivered to target cells, thereby enhancing therapeutic efficacy while minimizing systemic toxicity. ADCs have traditionally been synthesized through the stochastic modification of lysine or cysteine residues. However, this approach poses stability and efficacy issues, which has led to extensive research into achieving site-specific conjugation.
In this context, site-specific modification using microbial transglutaminase (MTG) has been widely explored. However, most existing methods require prior antibody engineering, such as deglycosylation or genetic modification. To enable the site-specific modification of native antibodies, we previously designed EzMTG-pG(Fab), a fusion protein consisting of an engineered zymogen of MTG (EzMTG) and protein G (pG(Fab)), which binds specifically to the Fab region of antibodies. While this fusion protein enabled selective modification at Lys65 of an IgG antibody (trastuzumab), Lys65 is located close to the antigen binding site, which limits the versatility of antibody types.
To address this challenge, we developed a novel pG-fused EzMTG construct. Since EzMTG retains its N-terminal pro-peptide to facilitate proper folding, it allows for the introduction of functional domains at the N-terminus—a modification that is challenging with conventional MTG. The resulting fusion protein, pG(Fab)-EzMTG, exhibited specific cross-linking activity toward Lys225 in the hinge site of trastuzumab when using a fluorescently labeled Gln donor substrate. These findings demonstrate that controlling the relative spatial orientation between EzMTG and IgG through molecular design is critical for directing the modification site.
Furthermore, optimization of the peptide linker between pG(Fab) and EzMTG significantly improved the conjugation efficiency, highlighting the pivotal role of linker design in fusion protein engineering. This Lys225-targeted modification was successfully extended to other IgG1 antibodies. Finally, ADCs prepared using the optimized fusion protein exhibited potent, antigen-specific cytotoxicity.