6N9T

Structure of a peptide-based photo-affinity cross-linker with Herceptin Fc

Summary for 6N9T

• Entry DOI: 10.2210/pdb6n9t/pdb
• Descriptor: Immunoglobulin G1 FC, Photo-affinity peptide, 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[beta-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose, ... (5 entities in total)
• Functional Keywords: crosslinking, antibody, photo-reactive, immune system
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 4
• Total formula weight: 56371.25

Authors

Sadowsky, J.,Ultsch, M.,Vance, N.,Wang, W. (deposition date: 2018-12-04, release date: 2019-01-16, Last modification date: 2025-04-02)

Primary citation

Vance, N.,Zacharias, N.,Ultsch, M.,Li, G.,Fourie, A.,Liu, P.,LaFrance-Vanasse, J.,Ernst, J.A.,Sandoval, W.,Kozak, K.R.,Phillips, G.,Wang, W.,Sadowsky, J.
Development, Optimization, and Structural Characterization of an Efficient Peptide-Based Photoaffinity Cross-Linking Reaction for Generation of Homogeneous Conjugates from Wild-Type Antibodies.
Bioconjug. Chem., 30:148-160, 2019

PubMed Abstract: Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein we report the development and detailed characterization of a robust photoaffinity cross-linking method for site-specific conjugation to fully glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. After the initial discovery of an effective Bpa mutant peptide and optimization of the reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody, we assessed the scope of the photoconjugation reaction across different human and nonhuman antibodies and antibody mutants. Next, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and showed that the resulting conjugate is both stable in plasma and as potent as a conventional antibody-drug conjugate in cells, portending well for future biological applications.

PubMed: 30566343
DOI: 10.1021/acs.bioconjchem.8b00809

Experimental method

X-RAY DIFFRACTION (2.576 Å)