Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, 35205, USA.
Department of Radiology, University of Alabama at Birmingham, 1824 6th Ave S, WTI 310F, Birmingham, AL, 35294, USA.
Mol Imaging Biol. 2024 Oct;26(5):858-868. doi: 10.1007/s11307-024-01948-4. Epub 2024 Aug 27.
Due to their long circulation time in the blood, monoclonal antibodies (mAbs) such as trastuzumab, are usually radiolabeled with long-lived positron emitters for the development of agents for Positron Emission Tomography (PET) imaging. Manganese-52 (Mn, t = 5.6 d, β = 29.6%, E(β) = 242 keV) is suitable for imaging at longer time points providing a complementary technique to Zirconium-89 (Zr, t = 3.3 d, β = 22.7%, E(β) = 396 keV)) because of its long half-life and low positron energy. To exploit these properties, we aimed to investigate suitable bifunctional chelators that could be readily conjugated to antibodies and labeled with Mn under mild conditions using trastuzumab as a proof-of-concept.
Trastuzumab was incubated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 1-Oxa-4,7,10-tetraazacyclododecane-5-S-(4-isothiocyantobenzyl)-4,7,10-triacetic acid (p-SCN-Bn-Oxo-DO3A), and 3,6,9,15-tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA) at a tenfold molar excess. The immunoconjugates were purified, combined with [Mn]MnCl at different ratios, and the labeling efficiency was assessed by iTLC. The immunoreactive fraction of the radiocomplex was determined through a Lindmo assay. Cell studies were conducted in HER2 + (BT474) and HER2- (MDA-MB-468) cell lines followed by in vivo studies.
Trastuzumab-Oxo-DO3A was labeled within 30 min at 37 °C with a radiochemical yield (RCY) of 90 ± 1.5% and with the highest specific activity of the chelators investigated of 16.64 MBq/nmol. The labeled compound was purified with a resulting radiochemical purity of > 98% and retained a 67 ± 1.2% immunoreactivity. DOTA and PCTA immunoconjugates resulted in < 50 ± 2.5% (RCY) with similar specific activity. Mouse serum stability studies of [Mn]Mn-Oxo-DO3A-trastuzumab showed 95% intact complex for over 5 days. Cell uptake studies showed higher uptake in HER2 + (12.51 ± 0.83% /mg) cells compared to HER2- (0.85 ± 0.10%/mg) cells. PET images of mice bearing BT474 tumors showed high tumor uptake that was consistent with the biodistribution (42.02 ± 2.16%ID/g, 14 d) compared to MDA-MB-468 tumors (2.20 ± 0.80%ID/g, 14 d). Additionally, both models exhibited low bone uptake of < 1% ID/g.
The bifunctional chelator p-SCN-Bn-Oxo-DO3A is promising for the development of Mn radiopharmaceuticals as it was easily conjugated, radiolabeled at mild conditions, and illustrated stability for a prolonged duration both in vitro and in vivo. High-quality PET/CT images of [Mn]Mn-Oxo-DO3A-trastuzumab were obtained 14 d post-injection. This study illustrates the potential of [Mn]Mn-Oxo-DO3A for the evaluation of antibodies using PET imaging.
由于其在血液中的循环时间较长，曲妥珠单抗等单克隆抗体（mAbs）通常使用长寿命的正电子发射体进行放射性标记，以开发正电子发射断层扫描（PET）成像的试剂。锰-52（Mn，t=5.6 d，β=29.6%，E（β）=242 keV）适合在较长时间点进行成像，为锆-89（Zr，t=3.3 d，β=22.7%，E（β）=396 keV）提供了一种互补技术，因为它的半衰期长，正电子能量低。为了利用这些特性，我们旨在研究合适的双功能螯合剂，这些螯合剂可以在温和条件下与抗体结合，并使用曲妥珠单抗作为概念验证进行 Mn 标记。
将曲妥珠单抗与 S-2-（4-异硫氰酸苄基）-1,4,7,10-四氮杂环十二烷四乙酸（p-SCN-Bn-DOTA）、1-氧代-4,7,10-四氮杂环十二烷-5-S-（4-异硫氰酸苄基）-4,7,10-三乙酸（p-SCN-Bn-Oxo-DO3A）和 3,6,9,15-四氮杂双环[9.3.1]十五烷-1（15），11，13-三烯-4-S-（4-异硫氰酸苄基）-3,6,9-三乙酸（p-SCN-Bn-PCTA）以十倍摩尔过量孵育。将免疫偶联物纯化，与[Mn]MnCl 以不同的比例结合，并通过 iTLC 评估标记效率。通过 Lindmo 测定法确定放射性复合物的免疫反应性分数。在 HER2+（BT474）和 HER2-（MDA-MB-468）细胞系中进行细胞研究，然后进行体内研究。
曲妥珠单抗-Oxo-DO3A 在 37°C 下 30 分钟内标记，放射性化学产率（RCY）为 90±1.5%，所研究的螯合剂中具有最高的比活度 16.64 MBq/nmol。标记化合物用放射性纯度>98%纯化，保留了 67±1.2%的免疫反应性。DOTA 和 PCTA 免疫偶联物的 RCY 均<50±2.5%，具有相似的比活度。[Mn]Mn-Oxo-DO3A-曲妥珠单抗在小鼠血清中的稳定性研究表明，在超过 5 天的时间内，有 95%的完整复合物。细胞摄取研究表明，HER2+（12.51±0.83%/mg）细胞的摄取量高于 HER2-（0.85±0.10%/mg）细胞。携带 BT474 肿瘤的小鼠的 PET 图像显示，与 MDA-MB-468 肿瘤（2.20±0.80%ID/g，14 d）相比，肿瘤摄取较高，与生物分布一致（42.02±2.16%ID/g，14 d）。此外，两种模型的骨摄取均<1%ID/g。
双功能螯合剂 p-SCN-Bn-Oxo-DO3A 具有开发 Mn 放射性药物的潜力，因为它易于结合，在温和条件下进行放射性标记，并在体外和体内均表现出长时间的稳定性。在注射后 14 天获得了[Mn]Mn-Oxo-DO3A-曲妥珠单抗的高质量 PET/CT 图像。本研究说明了[Mn]Mn-Oxo-DO3A 用于使用 PET 成像评估抗体的潜力。
