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A Recognition Tag of Human Origin for Bioorthogonal Generation of Antibody‐Drug Conjugates using Microbial Biotin Ligase
Author(s) -
Bitsch Peter,
Bitsch Sebastian,
Murmann Noah,
Bork Ingo,
Becker Janine,
Kolmar Harald
Publication year - 2025
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202500261
The use of enzymes, such as microbial transglutaminase, lipoate protein ligase A, or sortase A, for the generation of antibody‐drug conjugates has proven to be a powerful tool for the site‐specific payload conjugation to tumor‐specific antibodies. Herein, the extension of this enzymatic toolbox by Pyrococcus horikoshii biotin ligase is reported. To this end, the therapeutic antibody trastuzumab is equipped with p67, the 67 amino acid carboxyl‐terminal domain of human propionyl‐CoA carboxylase α subunit, at the C‐terminus of either the light or heavy chain (Trz‐LC:p67 and Trz‐HC:p67). Upon incubation with PhBL, the azide‐bearing linker desthiobiotin azide is site‐specifically coupled to the p67 domains at the antibody. Subsequent strain‐promoted azide‐alkyne cycloaddition with DBCO‐AF488 and DBCO‐Val‐Cit‐PAB‐MMAE yielded conjugates near to full conversion. In cellular assays, these constructs exhibit single‐digit nanomolar EC50 values in cellular proliferation assays on SK‐BR‐3 and A431 cells, where no significant difference in the performance between the two variants Trz‐LC:p67‐MMAE and Trz‐HC:p67‐MMAE is observed. On high Fc‐γIIIa receptor expressing Jurkat cells, Trz‐HC:p67‐MMAE exhibits higher potency than Trz‐LC:p67‐MMAE, indicating an Fc‐blocking effect of p67 when fused to the light chain.