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

  • Bioconjug Chem. 2025 Dec 17;36(12):2655-2664. doi: 10.1021/acs.bioconjchem.5c00487.
Sangwoo Lee  1 ,  Jisoo Park  1 ,  Byoung Jun Lee  1 ,  Jeonghyun Lee  1 ,  Byoung Joon Ko  2 ,  Eunha Kim  1  3 ,  Tae Hyeon Yoo  1  3
Affiliations
  • 1. Department of Molecular Science and Technology, Ajou University, 206 World cup-ro, Yeongtong-gu, Suwon 16499, Korea.
  • 2. School of Biopharmaceutical and Medical Science, Sungshin Women's University, Gangbuk-gu, Seoul 01133, Republic of Korea.
  • 3. Advanced College of Bio-Convergence Engineering, Ajou University, 206 World cup-ro, Yeongtong-gu, Suwon 16499, Korea.
PMID: 41292069 DOI: 10.1021/acs.bioconjchem.5c00487
Abstract

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.

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