Genetic Code Expansion-Driven in Situ Click Labeling Enables Rapid Imaging-Based Selection of Functional Nanobody-Dye...

Area of Science:

• Bioconjugation Chemistry
• Protein Engineering
• Molecular Imaging

Background:

• Antigen-binding proteins like nanobodies are valuable for imaging and drug delivery.
• Traditional labeling methods yield heterogeneous conjugates; site-specific methods are often limited.
• Genetic code expansion allows precise labeling but requires extensive screening for optimal sites.

Purpose of the Study:

• To develop a convenient, cell-based screening platform for evaluating site-specifically labeled nanobodies.
• To enable rapid, purification-free assessment of labeling efficiency and antigen-binding activity.
• To streamline the identification of suitable labeling sites for engineered antigen-binder conjugates.

Main Methods:

• A mammalian cell-based system was used to screen nanobody variants.
• Nanobodies were fused to BFP, secreted, and labeled in situ with a tetrazine-fluorescein probe.
• Supernatant containing labeled nanobodies was applied to HER2-expressing cells, and binding was assessed via fluorescence imaging.

Main Results:

• The platform allows simultaneous assessment of labeling efficiency and antigen-binding retention.
• It efficiently identifies labeling sites that support click conjugation while preserving nanobody function.
• The method proved effective using an anti-HER2 nanobody and HER2 as a model system.

Conclusions:

• The developed platform offers a convenient and rapid method for screening site-specifically labeled nanobodies.
• This approach significantly reduces the time and cost associated with identifying optimal labeling sites.
• The method is broadly applicable to various antigen-binder pairs, facilitating the development of engineered conjugates.