CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, United Kingdom.
Department of Medicine, Division of Infectious Diseases, University of Pittsburgh, Pittsburgh, USA.
Nucl Med Biol. 2019 May-Jun;72-73:49-54. doi: 10.1016/j.nucmedbio.2019.07.007. Epub 2019 Jul 18.
Increased activity of matrix metalloproteases (MMPs) is associated with reduced survival in several cancer subtypes. Aiming to produce an MMP tumour cell-selective cytotoxin, we genetically modified both components of the AB-type lethal toxin from Bacillus anthracis. Component A, Protective Antigen (PA-WT), was re-engineered to form an oligomeric pore in cell membranes only when cleaved by MMPs (PA-L1). The pore-translocation domain (LFn - N-terminal, 30 kDa) of the Lethal Factor (LF), component B, was fused to the catalytic domain of Pseudomonas exotoxin-A to increase its cytotoxic effect when delivered to cancerous cells. Here, we develop radiolabelled forms of LFn for MMP activity imaging by SPECT using the LFn/PA-L1 system.
DOTA-GA-maleimide was conjugated to LFn to allow radiolabelling with In via two different routes: (1) LFn was conjugated with maleimide-DOTA-GA under mild conditions, and then radiolabelled in acidic conditions at 95°C, or (2) In was coordinated to maleimide-DOTA-GA first and then conjugated via maleimide chemistry to LFn. Circular Dichroism Spectroscopy of LFn was performed to evaluate changes in its secondary structure. Cell uptake assays using the differently labelled forms of [In]In-DOTA-GA-LFn in the presence or not of PA-WT or PA-L1 were performed.
LFn was successfully radiolabelled by either strategy. Comparison of the secondary structure content of LFn exposed to 37°C or 95°C, showed a loss of alpha helix content at higher temperatures. Cell uptake of both forms of [In]In-DOTA-GA-LFn, labelled directly or indirectly, was significantly higher in MMP-positive cells, in the presence of PA-L1, compared to controls. Notably, despite being exposed to high temperatures, uptake of directly labelled [In]In-DOTA-GA-LFn was higher than indirectly labelled [In]In-DOTA-GA-LFn.
In-radiolabelling of LFn results in a functional molecule that targets MMP-activity in cells when combined with PA-L1. [In]In-LFn/PA-L1 is a promising MMP activity imaging agent for SPECT imaging.
基质金属蛋白酶（MMPs）活性增加与几种癌症亚型的生存率降低有关。为了产生 MMP 肿瘤细胞选择性细胞毒素，我们对炭疽芽孢杆菌 AB 型致死毒素的两个成分进行了基因修饰。A 成分保护性抗原（PA-WT）被重新设计成仅在被 MMP 切割时在细胞膜上形成寡聚孔（PA-L1）。B 成分致死因子（LF）的孔转运结构域（LFn-N 端，30 kDa）与假单胞菌外毒素-A 的催化结构域融合，以增加其输送至癌细胞时的细胞毒性。在这里，我们使用 LFn/PA-L1 系统通过 SPECT 发展放射性标记的 LFn 形式进行 MMP 活性成像。
DOTA-GA-马来酰亚胺与 LFn 缀合，以便通过两种不同的途径用 In 进行放射性标记：（1）在温和条件下，LFn 与马来酰亚胺-DOTA-GA 缀合，然后在 95°C 的酸性条件下进行放射性标记，或（2）首先将 In 与马来酰亚胺-DOTA-GA 配位，然后通过马来酰亚胺化学与 LFn 缀合。进行 LFn 的圆二色性光谱分析，以评估其二级结构的变化。在存在或不存在 PA-WT 或 PA-L1 的情况下，进行使用不同标记形式的 [In]In-DOTA-GA-LFn 的细胞摄取实验。
两种策略均成功地对 LFn 进行了放射性标记。与在 37°C 或 95°C 下暴露的 LFn 的二级结构含量进行比较，在较高温度下，α螺旋含量丧失。在存在 PA-L1 的情况下，与对照相比，MMP 阳性细胞中直接或间接标记的两种形式的 [In]In-DOTA-GA-LFn 的细胞摄取率均显著更高。值得注意的是，尽管暴露于高温下，直接标记的 [In]In-DOTA-GA-LFn 的摄取率高于间接标记的 [In]In-DOTA-GA-LFn。
当与 PA-L1 结合时，LFn 的放射性标记产生一种靶向细胞 MMP 活性的功能性分子。[In]In-LFn/PA-L1 是一种有前途的 MMP 活性 SPECT 成像剂。
