High-Throughput Screening of Photo-Cross-Linking Peptide Libraries for Site-Specific Conjugation of IgG

An affinity-guided photo-cross-linking reaction based on Fc-binding peptide harboring p-benzoyl-l-phenylalanine (PEptide-DIrected Photo-cross-linking; PEDIP) enables site-specific modification of native antibodies but suffers from issues coming from long UV exposure and high peptide concentrations. In this study, we report a bacterial surface-display system of the photo-cross-linking peptide and high-throughput screening of its libraries with FACS for higher photo-cross-linking efficiency. The lead peptide (B1) exhibited a higher conjugation yield than the original peptide (95.5% vs 78.4%) while preserving site fidelity at heavy chain Met252, confirmed by LC-MS/MS. A cyclooctyne group introduced to the N-terminus of B1 enabled conjugation of IgG with payloads via strain-promoted azide-alkyne cycloaddition. The conjugate of trastuzumab (antihuman HER2 IgG) and monomethyl auristatin retained antigen selectivity and exhibited potent cytotoxicity in HER2⁺ HCC1954 cells with minimal activity in HER2^- MDA-MB-231 cells.