Structural highlights
Publication Abstract from PubMedCatalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11 A deep pocket that permits site-specific conjugation of beta-diketone-, beta-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. Sculpting a Uniquely Reactive Cysteine Residue for Site-Specific Antibody Conjugation.,Hwang D, Nilchan N, Park H, Roy RN, Roush WR, Rader C Bioconjug Chem. 2022 Jun 15;33(6):1192-1200. doi: , 10.1021/acs.bioconjchem.2c00146. Epub 2022 May 18. PMID:35584359[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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