230 / 2024-07-30 10:52:04

Precision Engineering of Single Domain Antibody for Biomedical Imaging

medical optic,single domain antibody,nanobody,biomedical imaging,fluorescence imaging

Single domain antibody (sdAb), also called nanobody (Nb), is a small antibody fragment first discovered in Camelidae. It exhibits several encouraging properties not regularly found in full-sized antibodies, such as a very small size (~15 kDa), high stability, deep tissue penetration, renal clearance and low immunogenicity. These unique features make nanobody an appropriate and promising tool for labeling subcellular structures with high density and minimal linkage error in advanced cell imaging, and also facilitate targeted, real-time and high-specific imaging in vivo. However, design of antibody molecular probes frequently necessitates the incorporation of additional groups, such as dyes, chelators, drugs, and other labels. Because of the small size and limited active sites of nanobodies, the effects of conjugation sites, labeling methods, and properties of labels on them are significantly more pronounced than those on conventional antibodies. Although random labeling methods that rely on endogenous cysteine or lysine residues in proteins are simple and widely used, they may lead to heterogeneous products with variable functionalization ratios, and sometimes a loss of targeting ability. Site-specific engineering strategies, such as enzyme-mediated methods and click chemistry, have gained huge interest in the industry and the research field. Herein, we describe a universal and facile strategy to produce homogenous nanobody molecular probes with ideal properties for biomedical imaging. Specifically, we first developed a kind of zwitterionic magnetic beads (MB) for the microbial transglutaminase (mTGase) immobilization. The immobilized enzyme (MB-mTGase) shows high catalytic activity and reusability, effectively simplifies the protein separation steps and reduces the loss of nanobody during purification. Using MB-mTGase and “click” strategies, dye, linker, photosensitizers, polyethylene glycol and other functional molecules can be easily and precisely conjugated to the specific site of nanobody. We demonstrated the fluorescent nanobody probes that enable targeted immunofluorescence imaging and PEGylated nanobody probes with improved circulation dynamics in vivo. In addition, glucose-incorporated nanobody probes with enhanced blood brain barrier (BBB) penetration and tumor targeting were yielded for brain tumor diagnostics and image-guided surgery. Furthermore, we synthesized a series of multifunctional linkers for the precision engineering of nanobody, which may contribute to the development of nanobody-associated multimodal imaging systems and antibody drug conjugates (ADCs). This study provides viable approach for the development of molecular probes in nanobiophotonics.