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Bioconjug Chem. 2019 Sep 18;30(9):2452-2457. doi: 10.1021/acs.bioconjchem.9b00522. Epub 2019 Aug 21.

Substrate Design Enables Heterobifunctional, Dual "Click" Antibody Modification via Microbial Transglutaminase.

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Robert Frederick Smith School of Chemical and Biomolecular Engineering , Cornell University , 113 Ho Plaza , Ithaca , New York 14850 , United States.
Department of Chemical and Biomolecular Engineering , University of Illinois at Urbana-Champaign , 600 S Mathews Ave , Urbana , Illinois 61801 , United States.


Site-specific modification of native antibodies has proven advantageous, as it enhances the properties of antibody-based bioconjugates without the need to manipulate the genetic code. However, native antibody modification is typically limited to strategies that introduce a single functional handle. In this work, we addressed this limitation by designing heterobifunctional substrates for microbial transglutaminase (MTG) that contain both azide and methyltetrazine "click" handles. Structure-conjugation relationships for these substrates were evaluated using the Her2-targeted antibody trastuzumab. Förster resonance energy transfer (FRET) was used to demonstrate that these chemical handles are mutually orthogonal. This orthogonality was leveraged for the one-pot synthesis of a bifunctional antibody-drug conjugate (ADC). This ADC, containing a maytansine-derived payload and a hydrophobicity-masking polyethylene glycol (PEG) side chain, demonstrated potent in vitro activity in SKOV3 cells. These studies establish the dual "click" approach as a powerful technique in the toolbox for native antibody modification.

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