Genentech Research and Early Development, Genentech Inc., South San Francisco, CA 94080, USA.
Nucl Med Biol. 2010 Apr;37(3):289-97. doi: 10.1016/j.nucmedbio.2009.11.010. Epub 2010 Feb 10.
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.
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.
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.
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.
开发三种巯基反应试剂，用于通过工程化半胱氨酸残基（硫代曲妥珠单抗）将去铁胺（Df）化学选择性地连接到单克隆抗体上。研究了通过定点放射性标记的（89）Zr-Df-硫代曲妥珠单抗缀合物的体外稳定性和体内成像特性。
通过溴乙酰溴、N-羟基琥珀酰亚胺基碘乙酸酯或 N-羟基琥珀酰亚胺基 4-[N-马来酰亚胺甲基]环己烷-1-羧酸盐酰化去铁胺 B 的氨基，分别得到巯基反应试剂溴乙酰去铁胺（Df-Bac）、碘乙酰去铁胺（Df-Iac）和马来酰亚胺环己基去铁胺（Df-Chx-Mal）。Df-Bac 和 Df-Iac 通过亲核取代作用使硫代曲妥珠单抗的游离巯基烷基化，形成 Df-Ac-硫代曲妥珠单抗，而马来酰亚胺试剂 Df-Chx-Mal 通过迈克尔加成反应提供 Df-Chx-Mal-硫代曲妥珠单抗。用（89）Zr 标记缀合物，并评估其血清稳定性，并在 BT474M1（HER2 阳性）乳腺癌小鼠模型中研究其正电子发射断层扫描（PET）成像特性。
以 14%（Df-Bac）、53%（Df-Iac）和 45%（Df-Chx-Mal）的产率获得了化学选择性试剂。Df-Chx-Mal 与硫代曲妥珠单抗的定点缀合在 pH 7.5 下完全在 1 小时内完成，而 Df-Iac 和 Df-Bac 分别需要 2 小时和 5 小时。每个 Df 修饰的硫代曲妥珠单抗与（89）Zr 的螯合产率超过 75%。（89）Zr-Df-Ac-硫代曲妥珠单抗和（89）Zr-Df-Chx-Mal-硫代曲妥珠单抗在小鼠血清中稳定，在 BT474M1（HER2 阳性）乳腺癌模型中具有相当的 PET 成像能力，肿瘤摄取达到 20-25%ID/g，肿瘤与血液的比值为 6.1-7.1。
新试剂与曲妥珠单抗的工程化巯基基团表现出良好的反应性，并且与（89）Zr 具有很好的螯合特性。通过定点放射性标记的硫代抗体在血清中稳定，并且显示出与赖氨酸缀合物相当的 PET 成像特性。
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