Site-specifically 89Zr-labeled monoclonal antibodies for ImmunoPET

Tinianow, Jeff N.; Gill, Herman S.; Ogasawara, Annie; Flores, Judith E.; Vanderbilt, Alexander N.; Luis, Elizabeth; Vandlen, Richard; Darwish, Martine; Junutula, Jagath R.; Williams, Simon-P.; Marik, Jan

doi:10.1016/j.nucmedbio.2009.11.010

« Previous Next »Nuclear Medicine and Biology
Volume 37, Issue 3 , Pages 289-297, April 2010

Site-specifically ⁸⁹Zr-labeled monoclonal antibodies for ImmunoPET

Genentech Research and Early Development, Genentech Inc., South San Francisco, CA 94080, USA

Received 10 October 2009; received in revised form 25 November 2009; accepted 27 November 2009. published online 11 February 2010.

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 radiolabeled ⁸⁹Zr-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 with ⁸⁹Zr 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 with ⁸⁹Zr in yields exceeding 75%. ⁸⁹Zr-Df-Ac-thio-trastuzumab and ⁸⁹Zr-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 with ⁸⁹Zr. The site-specifically ⁸⁹Zr-labeled thio-antibodies were stable in serum and showed PET imaging properties comparable to lysine conjugates.

Keywords: ImmunoPET, Zirconium-89, Site-specifically labeled antibodies, Thiol specific desferrioxamine reagent, Iodoacetate, Bromoacetate, Maleimide, Imaging, Trastuzumab, PHESELECTOR, THIOMAB