Site-specifically89 Zr-labeled monoclonal antibodies for ImmunoPET

Abstract Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled89 Zr-Df-thio-trastuzumab conjugates were investigated. Methods The amino group of desferrioxamine B was acylated by bromoacetyl bromide, N -hydroxysuccinimidyl iodoacetate, or N -hydroxysuccinimidyl 4-[ N -maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with89 Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model. Results The chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with89 Zr in yields exceeding 75%.89 Zr-Df-Ac-thio-trastuzumab and89 Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20–25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1–7.1. Conclusions The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with89 Zr. The site-specifically89 Zr-labeled thio-antibodies were stable in serum and showed PET imaging properties comparable to lysine conjugates.