# A Recognition Tag of Human Origin for Bioorthogonal Generation of Antibody‐Drug Conjugates using Microbial Biotin Ligase

## TL;DR

Researchers developed a new method to create antibody-drug conjugates using a human-derived tag and microbial enzymes, achieving high potency in cancer cell tests.

## Contribution

A human-derived recognition tag is introduced for site-specific conjugation using microbial biotin ligase, expanding enzymatic tools for antibody-drug conjugates.

## Key findings

- The p67 tag fused to trastuzumab enables efficient site-specific conjugation with high conversion rates.
- Conjugates show single-digit nanomolar potency in Her2-expressing cell lines.
- Trz-HC:p67-MMAE is more potent on high Fc-γIIIa receptor cells due to Fc-blocking by p67 on the light chain.

## Abstract

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.

A human‐derived recognition tag of Pyrococcus horikoshii biotin ligase is fused to the monoclonal antibody trastuzumab at the C‐termini of either light or heavy chain. These fusion antibodies allow for the PhBL‐mediated introduction of desthiobiotin azide and the subsequent attachment of DBCO‐AF488 and DBCO‐Val‐Cit‐PAB‐MMAE via strain‐promoted azide‐alkyne cycloaddition. The generated antibody‐drug conjugates exhibit single‐digit nanomolar potency on Her2‐expressing cell lines.© 2025 WILEY‐VCH GmbH