A new enzymatic proximity-based labeling method for native IgG Fab was developed.

The resulting fluorescent-modified Fab retained specific antigen binding activity.

The proposed approach is promising for various applications where Fabs are used.

The fragment antigen-binding (Fab) fragment of IgG has been studied widely as a delivery vehicle for tumor-targeting drugs and dyes due to its high specificity and enhanced tumor penetration, which is attributed to its small size. Functionalizing Fab with chemical entities requires site-specific modification to preserve the binding ninity and ensure product homogeneity. In this study, we report a tag-free, site-specific labeling approach targeting a Lys residue in Fab using the recently developed engineered zymogen of microbial transglutaminase fused with an antibody-binding protein G. Fab of trastuzumab, prepared via papain digestion, was selectively modified at Lys 65 in the heavy chain with a glutamine-donor fluorescent substrate, achieving a high labeling efficiency (∼96 %). Bio-layer interferometry experiments confirmed that the modified Fab retained antigen-binding affinity (KD = 5.71 ± 3.89 nM) comparable to its native counterpart (4.72 ± 3.19 nM). Confocal microscopy analysis demonstrated selective binding of the fluorescent-modified Fab to human epidermal growth factor receptor type2 (HER2)-positive SK-BR-3 cells, with negligible binding to HER2-negative MDA-MB-231 cells. The proposed strategy enables site-specific Fab modification without genetic engineering, offering a streamlined approach to producing homogeneous Fab conjugates for diagnostic imaging and therapeutic antibody engineering applications.

Antibody-drug conjugate

Antibody fragment

Antibody-binding protein

Crosslinking

Enzymatic modification

Microbial transglutaminase

© 2025 The Authors. Published by Elsevier B.V.