biostudies-literatureUnknown36(8)Baniahmad SFNatural Sciences and Engineering Research Council of CanadaAntibody-drug conjugates are revolutionizing cancer treatment. However, their manufacturing still requires improvements in conjugation technology, especially for the control of the drug-to-antibody ratio (DAR). Here, we investigate the use of the de novo designed coiled-coil heterodimer, composed of the Ecoil and Kcoil peptides, as a new strategy for generating antibody conjugates with high homogeneity and a controllable DAR. More precisely, we investigated the assembly, stability, and tumor targeting of two conjugated antibodies made of (1) trastuzumab with C-terminal Ecoils (TZM-Ecoil) noncovalently paired with Kcoil peptides fused to the monomeric red fluorescent protein (Kcoil-mRFP), yielding TZM-E/K-mRFP or (2) TZM-Ecoil noncovalently paired to Kcoil peptide covalently linked to the fluorescent dye CF750 (Kcoil-CF750), yielding TZM-E/K-CF750. Results from the <i>in vitro</i> stability assessment of these complexes in blood serum revealed that their integrity was maintained. Furthermore, <i>in vivo</i> biodistribution and tumor localization data using a HER2-expressing SKOV3 xenograft mouse model indicated efficient tumor targeting and retention for up to 10 days postinjection of the TZM-E/K-CF750 conjugate.Bioconjugate chemistry1670-1682https://www.ebi.ac.uk/biostudies/studies/S-EPMC12371696biostudies-literatureUse of Coiled-Coil Affinity Peptides to Manufacture Antibody Conjugates.PMC12371696Burlacu ADurocher YLing BDe Crescenzo GAcchione MBaniahmad SFIqbal USimmons MDelafosse LMoreno MJfalseUse of Coiled-Coil Affinity Peptides to Manufacture Antibody Conjugates.Antibody-drug conjugates are revolutionizing cancer treatment. However, their manufacturing still requires improvements in conjugation technology, especially for the control of the drug-to-antibody ratio (DAR). Here, we investigate the use of the de novo designed coiled-coil heterodimer, composed of the Ecoil and Kcoil peptides, as a new strategy for generating antibody conjugates with high homogeneity and a controllable DAR. More precisely, we investigated the assembly, stability, and tumor targeting of two conjugated antibodies made of (1) trastuzumab with C-terminal Ecoils (TZM-Ecoil) noncovalently paired with Kcoil peptides fused to the monomeric red fluorescent protein (Kcoil-mRFP), yielding TZM-E/K-mRFP or (2) TZM-Ecoil noncovalently paired to Kcoil peptide covalently linked to the fluorescent dye CF750 (Kcoil-CF750), yielding TZM-E/K-CF750. Results from the <i>in vitro</i> stability assessment of these complexes in blood serum revealed that their integrity was maintained. Furthermore, <i>in vivo</i> biodistribution and tumor localization data using a HER2-expressing SKOV3 xenograft mouse model indicated efficient tumor targeting and retention for up to 10 days postinjection of the TZM-E/K-CF750 conjugate.2025-01-01T00:00:00Z2025 Aug2026-05-08T10:53:32.326Z2026-05-02T03:07:20.196ZS-EPMC123716964072816710.1021/acs.bioconjchem.5c00178