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Site-specifically 89Zr-labeled monoclonal antibodies for ImmunoPET.

Authors :: Tinianow JN
Gill HS
Ogasawara A
Flores JE
Vanderbilt AN
Luis E
Vandlen R
Darwish M
Junutula JR
Williams SP
Marik J
Source :: Nuclear medicine and biology [Nucl Med Biol] 2010 Apr; Vol. 37 (3), pp. 289-97. Date of Electronic Publication: 2010 Feb 10.
Publication Year :: 2010
Abstract: Unlabelled: 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 radiolabeled (89)Zr-Df-thio-trastuzumab conjugates were investigated.<br />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 with (89)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.<br />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 with (89)Zr in yields exceeding 75%. (89)Zr-Df-Ac-thio-trastuzumab and (89)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.<br />Conclusions: The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with (89)Zr. The site-specifically (89)Zr-labeled thio-antibodies were stable in serum and showed PET imaging properties comparable to lysine conjugates.<br /> (Copyright 2010 Elsevier Inc. All rights reserved.)