Protein-antibody conjugates represent major advancements in targeted therapeutics. However, platforms enabling 'off-the-shelf' antibody conjugation are seldom reported. The SpyTag/SpyCatcher system, known for its stable isopeptide bond formation, is widely used to engineer protein architectures and study protein folding. This work introduces the fusion of SpyCatcher with native antibodies using cysteine-reactive tetra-divinylpyrimidine (TetraDVP)-SpyTag linkers. This platform allows for the rapid and stable conjugation of a native antibody with SpyCatcher proteins. As a proof of concept, the HER2-targeting antibody trastuzumab was conjugated to different SpyCatcher proteins using a TetraDVP-SpyTag linker, producing robust conjugates that retained specific binding to HER2-positive cells with excellent conversion rates. To demonstrate the platform's broader applicability, the TetraDVP-SpyTag linker was successfully conjugated to additional native IgG1 and IgG4 antibodies (durvalumab, brentuximab, cetuximab, and gemtuzumab) with similarly high efficiency as trastuzumab. Moreover, a scalable solid-phase synthesis of TetraDVP linkers has been developed, achieving high yields and purity. This innovative platform enables precise, single-step antibody bioconjugation, offering strong potential for protein-antibody conjugate synthesis. With applications across therapeutics and diagnostics, this method advances antibody-based drug development.

Department of Biochemistry University of Oxford South Parks Road OX1 3QU Oxford UK

Department of Organic Chemistry Palacky University Olomouc Tr 17 Listopadu 12 77900 Olomouc Czech Republic

Department of Pharmacology University of Cambridge Tennis Court Road CB2 1PD Cambridge UK

Yusuf Hamied Department of Chemistry University of Cambridge Lensfield Road CB2 1EW Cambridge UK

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