GoKawiilEthical compliance
Animal studies were performed at the Washington University School of Medicine in compliance with institutional guidelines and protocols approved by the Institutional Animal Care and Use Committee (animal protocol 21-0087, 24-0274; IBC protocol 13652, principal investigator: P.M.R.P.). Radiolabelling and PET–CT imaging experiments were performed using appropriately shielded equipment within a licensed radiation-safety facility by trained personnel under an active licence.
Preparation of antibody–TCO and antibody–tetrazine conjugates (random and site-specific)
Random conjugation
Antibodies were conjugated at a molar ratio of 15 TCO-PEG 4 -NHS-ester (TCO; BroadPharm, BP-22418) or tetrazine-PEG 5 -NHS-ester (tetrazine: BroadPharm, BP-22681) per antibody. Conjugations were performed in PBS (pH 8.8–9) at 37 °C, 500 rpm for 1 h. Conjugates were purified using a desalting column (PD-10, GE Healthcare) and concentrated using Amicon filters with a 50 kDa molecular weight cutoff (Millipore, UFC8050) in 1× PBS (pH 7.4).
For indocyanine green-Sulfo-Osu (ICG) conjugation, trastuzumab–tetrazine was conjugated with ICG (Fisher Scientific, 501952790) at a molar ratio of 3 ICG per antibody in PBS (pH 8.8) at 37 °C, 500 rpm for 1 h. The fluorescently labelled antibody conjugate was purified using a desalting column (PD-10) and concentrated using 50 kDa molecular weight cutoff Amicon filters in 1× PBS (pH 7.4).
Site-specific conjugation
Antibodies were modified with azide groups through the glycosylation sites in the Fc region (SiteClick Antibody Azido Modification kit, ThermoFisher, S10901). In brief, antibodies (5 mg) were incubated with 100 µl β-galactosidase at 37 °C, 450 rpm for 6 h, followed by overnight incubation at 30 °C with UDP-GalNAz containing GalT enzyme. Azide-modified antibody was purified and concentrated using Amicon filters with a 50 kDa molecular weight cutoff in 1× Tris buffer (pH 7.0). Then, DBCO-PEG 12 -TCO (TCO; BroadPharm, BP-22423) or DBCO-methyl-tetrazine (tetrazine; Vector Laboratories, CCT-1022) at a molar ratio of 15 was conjugated with the azide-modified antibody overnight at room temperature. Site-specific conjugates were purified using a desalting column (PD-10) and concentrated with Amicon filters with a 50 kDa molecular weight cutoff in 1× PBS (pH 7.4).
The concentration of the antibody conjugates was determined using a UV-visible spectrophotometer or a Pierce 660 assay (Thermo Fisher Scientific, 22660).
Cell culture
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