Genetic Code Expansion-Driven <i>in Situ</i> Click Labeling Enables Rapid Imaging-Based Selection of Functional Nanobody–Dye Conjugates

Antigen-binding proteins, such as nanobodies, modified with functional small molecules hold great potential for applications including imaging probes, drug conjugates, and localized catalysts. However, traditional chemical labeling methods that randomly target lysine or cysteine residues often produce heterogeneous conjugates with limited reproducibility. Conventional site-specific conjugation approaches, which typically modify only the N- or C-terminus, may also be insufficient to achieve the desired functionalities. Genetic code expansion offers a powerful alternative by enabling the site-specific incorporation of noncanonical amino acids bearing reactive handles-such as trans-cyclooctene (TCO)-thereby allowing precise bioorthogonal conjugation via click chemistry. Nevertheless, identifying suitable incorporation sites that tolerate such modifications without disrupting antigen binding remains a time- and cost-intensive process, as this process typically requires labor-intensive screening involving the expression and purification of each candidate variant. Here, using HER2 and an anti-HER2 nanobody as a model antigen-binder pair, we present a convenient mammalian cell-based screening platform for rapid, purification-free evaluation of site-specifically labeled nanobodies. The nanobody is fused to blue fluorescent protein (BFP), secreted by HEK293T cells, and labeled in situ with a tetrazine-fluorescein probe. The resulting supernatant is then applied directly to HEK293T cells stably expressing HER2-mCherry. Labeling efficiency and retention of antigen-binding activity are simultaneously assessed by fluorescence imaging in the BFP, fluorescein, and mCherry channels. This approach enables efficient identification of labeling sites that support productive click conjugation while preserving binding function. It should be broadly applicable to other antigens and binders, streamlining early-stage screening of engineered antigen-binder conjugates for diverse applications.