Center for Radiopharmaceutical Sciences ETH-PSI, Paul Scherrer Institut, 5232 Villigen-PSI, Switzerland; Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, 8093 Zurich, Switzerland.
Laboratory of Applied Mechanobiology, Institute of Translational Medicine, Department of Health Sciences and Technology, ETH Zurich, CH-8093 Zurich, Switzerland.
Bioorg Med Chem. 2022 Nov 1;73:117040. doi: 10.1016/j.bmc.2022.117040. Epub 2022 Sep 28.
Previously, we have exploited bacterial adhesins-derived fibronectin-binding peptides (FnBPs) for targeting mechanically altered fibronectin (Fn) fibrils within the cancer-associated extra-cellular matrix (ECM). However, despite the ability of FnBP probes to visualize pathological lesions, when labeled with metallic radionuclides and administered for targeted imaging, they exhibit high and persistent retention of radioactivity within the kidneys. Intending to overcome this issue towards a future translation of FnBPs to the clinic, the goal of the present study was to reduce the renal retention of In-labelled FnBPs employing dual renal brush border membrane (BBM) enzyme-sensitive Met-Val-Lys-based linkers, enabling a rapid washout of radioactivity from the kidneys.
Three maleimide-activated NOTA-conjugated brush border-enzyme cleavable linkers equipped with either single or dual consecutive MVK-based cleavable moieties were designed and synthesized. Their respective NOTA-MVK-based FnBPA5.1 conjugates were obtained by means of maleimide-thiol mediated conjugation at the N-terminus of the Fn-binding sequence, radiolabeled with indium-111, and further evaluated in vitro and in vivo in comparison to the control [In]In-FnBPA5.1.
The linker equipped with two MVK sites displayed a two-fold more effective cleavage rate than the single MVK featuring linker in vitro, as revealed by the quantification of the released Met-containing radiometabolites. SPECT/CT imaging and biodistribution studies of the series of FnBPA5.1 radioconjugates performed at 24 h post-injection (p.i.) confirmed the in vitro results, indicating that the renal retention of In-labelled FnBPs can be significantly lowered through the interposition of a single MVK-based sequence between the Fn-targeting moiety and the chelating unit (52.75 ± 9.79 vs 92.88 ± 4.85 % iA/g, P < 0.001), and even further reduced by the addition of a second one (down to 34.82 ± 6.04, P < 0.001), with minor influence on the biodistribution in other organs, such as tumors.
In summary, we report here promising In-labelled FnBP radiotracers equipped with dual MVK-based cleavable linkers leading to a more effective reduction of renal retention and improved tumor-to-kidney ratios compared to the single MVK-featuring derivative. Our dual MVK strategy is a crucial step towards the clinical translation of mechano-sensory FnBPs and might as well be adopted for other radiopharmaceuticals suffering from persistent renal retention of radioactivity.
此前，我们利用细菌黏附素衍生的纤连蛋白结合肽（FnBPs）来靶向癌症相关细胞外基质（ECM）中机械改变的纤连蛋白（Fn）纤维。然而，尽管 FnBP 探针能够可视化病理性病变，但当用放射性金属核素标记并用于靶向成像时，它们在肾脏中表现出高且持续的放射性保留。为了克服这一问题，以便将来将 FnBPs 转化为临床应用，本研究的目的是使用双肾刷状缘膜（BBM）酶敏感的 Met-Val-Lys 基连接物来减少 In 标记的 FnBPs 的肾脏保留，从而实现放射性从肾脏的快速清除。
设计并合成了三个马来酰亚胺活化的 NOTA 缀合的刷状缘酶可切割连接物，它们分别带有单个或双个连续的基于 MVK 的可切割部分。通过马来酰亚胺-巯基介导的连接，在 Fn 结合序列的 N 端将它们各自的 NOTA-MVK 基 FnBPA5.1 缀合物进行连接，并用铟-111 进行放射性标记，并与对照物 [In]In-FnBPA5.1 进行比较，在体外和体内进行进一步评估。
与具有单个 MVK 的连接物相比，体外具有两个 MVK 位点的连接物显示出两倍更有效的切割速率，这可以通过定量释放的含有 Met 的放射性代谢产物来证明。在注射后 24 小时进行的 FnBPA5.1 放射性缀合物的 SPECT/CT 成像和生物分布研究证实了体外结果，表明通过在 Fn 靶向部分和螯合单元之间插入单个基于 MVK 的序列，可以显著降低 In 标记的 FnBPs 的肾脏保留（52.75±9.79% vs 92.88±4.85% iA/g，P<0.001），甚至通过添加第二个序列进一步降低（降至 34.82±6.04，P<0.001），对肿瘤等其他器官的生物分布影响较小。
总之，我们在这里报告了带有双 MVK 基可切割连接物的有前途的 In 标记 FnBP 放射性示踪剂，与具有单个 MVK 的衍生物相比，其可更有效地降低肾脏保留并提高肿瘤与肾脏的比值。我们的双 MVK 策略是将机械感觉 FnBPs 转化为临床应用的重要步骤，并且对于其他放射性药物中持续存在的放射性肾保留也可能采用这种方法。
