A platform for SpyCatcher conjugation to native antibodies

Abstract

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.

Fig. 1. TetraDVP linkers and SpyTag/SpyCatcher system – then and now (size of the cartoons is purely schematic).

Scheme 1. Synthesis of TetraDVP-SpyTag conjugates 15–18. Full structures can be found in the ESI. Black ball indicates 2-chlorotrityl chloride polystyrene resin. Reaction conditions: (i) glycine, Bu₂SnCl₂, PhSiH₃, THF, reflux, 16 h (98%, 2); (ii) H-Gly-2CT resin, key branching amine 2, HOBt, DIC, r.t., 16 h; (iii) DBU/CH₂Cl₂ 5 : 95, r.t., 15 min; (iv) sarcosine DVP S1, HOBt, DIC, DMF, r.t., 16 h; (v) {2-[2-(Fmoc-amino)ethoxy]ethoxy}acetic acid (FAEEAA), HOBt, DIC, DMF, r.t., 16 h; (vi) HFIP/CH₂Cl₂ 1 : 4, r.t., 3 h; (vii) for 8–10: TetraDVP acids 5–7 (respectively), azido spacer S4, HOBt, DIC, DMSO, r.t., 16 h; for 11: TetraDVP acid 6, azido spacer S6, HOBt, DIC, DMSO, r.t., 16 h. (viii) DMSO, r.t., 1 h (94%, 14).

Fig. 2. Formation of SpyCatcher-trastuzumab conjugates. The SpyTag-trastuzumab conjugates Tras-15 to Tras-18 originate from the conjugation of reduced trastuzumab with TetraDVP-SpyTag conjugate 15 for Tras-15, 16 for Tras-16, 17 for Tras-17, and 18 for Tras-18 (the full structures of the TetraDVP-SpyTag conjugates are available in the ESI Section 1.3†). Conjugations run in TBS (1×, 2.5 mg per mL trastuzumab, 100 μL) with 10 eq. TCEP for 1 h then 5 eq. of TetraDVP conjugates 15, 16, 17, or 18 were added and incubated at 37 °C for 23 h; repeated daily for 3 days. (A) Hydrophobic interaction chromatography (HIC) analysis of trastuzumab-TetraDVP-SpyTag conjugates. Percentage shows conversion from native trastuzumab (*). (B) Deconvoluted MS data of the most successful conjugate Tras-17; intensity vs. deconvoluted mass. (C) Conjugation of Tras-17 to different SpyCatcher constructs. 1 μM Tras-17 was incubated with 5 μM of either SpyCatcher003, SpyCatcher002 linked to maltose-binding protein (SpyCatcher002-MBP), or DoubleCatcher, in PBS pH 7.4 at 37 °C for the time indicated. Reactivity of Tras-17 with each Catcher was monitored by SDS-PAGE analysis followed by Coomassie staining (first two lanes are broad range unstained protein standard, molecular weight ladders). Cartoons represent protein conjugation partners, with SpyCatcher003 in light blue, MBP in lilac, SpyTag003 in dark blue with D117A indicated in light blue in DoubleCatcher, TEV site in orange. (D) Tras-17 pre-conjugated to SpyCatcher003-sfGFP (2 μM) was incubated with HER2-positive SKBR3 cells, or (E) HER2-negative MDA-MB-468 cells in HBS pH 7.2 + 10% (v/v) FBS for 30 min at 37 °C with 5% (v/v) CO₂ before detection of sfGFP fluorescence by flow cytometry. As a positive control for HER2 binding, the anti-HER2 nanobody nanoHER2 was used instead of Tras-17. Cells incubated with either HER2-binder alone, SpyCatcher003-sfGFP alone, or buffer only were used to detect background signal. Abbreviations: sfGFP = superfolder Green Fluorescent Protein; HER2 = Human Epidermal Growth Factor Receptor 2; TCEP = Tris(2-carboxyethyl)phosphine; Tras = Trastuzumab (marked with asterisk *).

Fig. 3. Formation of antibody-SpyTag conjugates Dur-17, Bren-17, Cet-17 (IgG1), and Gem-17 (IgG4). Bioconjugation of IgG1 antibodies: conjugations run in TBS (1×, 2.5 mg per mL trastuzumab, 100 μL) with 10 eq. TCEP for 1 h then 5 eq. of TetraDVP conjugate 17 was added and incubated at 37 °C for 23 h. Conjugation of gemtuzumab (IgG4) with 17 required three daily reagent additions to reach completion. (A) Hydrophobic interaction chromatography (HIC) analysis of Dur-17, Bren-17, Cet-17, and Gem-17. Percentage shows conversion from native antibody. (B) SDS-PAGE gel of Dur-17, Bren-17, Cet-17 and Gem-17 with Coomassie blue staining. +/− indicates with or without reducing stain. TCEP = Tris(2-carboxyethyl)phosphine, TBS = Tris Buffered Saline.