7TUS

Sculpting a uniquely reactive cysteine residue for site-specific antibody conjugation

Summary for 7TUS

• Entry DOI: 10.2210/pdb7tus/pdb
• Descriptor: Antibody Heavy Chain, Antibody Light Chain, DI(HYDROXYETHYL)ETHER, ... (6 entities in total)
• Functional Keywords: antibody therapeutics, antibody-drug conjugate, catalytic antibody, protein modification, engineered cysteine, immune system
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 8
• Total formula weight: 192963.24

Authors

Park, H.,Rader, C. (deposition date: 2022-02-03, release date: 2022-06-08, Last modification date: 2024-10-16)

Primary citation

Hwang, D.,Nilchan, N.,Park, H.,Roy, R.N.,Roush, W.R.,Rader, C.
Sculpting a Uniquely Reactive Cysteine Residue for Site-Specific Antibody Conjugation.
Bioconjug.Chem., 33:1192-1200, 2022

PubMed Abstract: Catalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11 Å deep pocket that permits site-specific conjugation of β-diketone-, β-lactam-, and heteroaryl methylsulfonyl-functionalized small and large molecules. Various dual variable domain formats pair a tumor-targeting antibody with h38C2 to enable precise, fast, and stable assembly of antibody-drug conjugates (ADCs). Here, we expand the scope of this ADC assembly strategy by mutating h38C2's reactive lysine to a cysteine. X-ray crystallography of this point mutant, h38C2_K99C, confirmed a deeply buried unpaired cysteine. Probing h38C2_K99C with maleimide, monobromomaleimide, and dibromomaleimide derivatives of a fluorophore revealed highly disparate conjugation efficiencies and stabilities. Dibromomaleimide emerged as a suitable electrophile for the precise, fast, efficient, and stable assembly of ADCs with the h38C2_K99C module. Mass spectrometry indicated the presence of a thio-monobromomaleimide linkage which was further supported by in silico docking studies. Using a dibromomaleimide derivative of the highly potent tubulin polymerization inhibitor monomethyl auristatin F, h38C2_K99C-based ADCs were found to be as potent as h38C2-based ADCs and afford a new assembly route for ADCs with single and dual payloads.

PubMed: 35584359
DOI: 10.1021/acs.bioconjchem.2c00146

Experimental method

X-RAY DIFFRACTION (2.4 Å)