Programmed cell death ligand 1 (PD-L1) plays a critical role in the tumor microenvironment and overexpression in several solid cancers has been reported. This was associated with a downregulation of the local immune response, specifically of T-cells. Immune checkpoint inhibitors showed a potential to break this localized immune paralysis, but only 30% of patients are considered responders. New diagnostic approaches are therefore needed to determine patient eligibility. Small molecule radiotracers targeting PD-L1, may serve as such diagnostic tools, addressing the heterogeneous PD-L1 expression between and within tumor lesions, thus aiding in therapy decisions. Four biphenyl-based small-molecule PD-L1 ligands were synthesized using a convergent synthetic route with a linear sequence of up to eleven steps. As a chelator NODA-GA, CB-TE2A or DiAmSar was used to allow radiolabeling with copper-64 ([64Cu]Cu-14–[64Cu]Cu-16). In addition, a dimeric structure based on DiAmSar was synthesized ([64Cu]Cu-17). All four radioligands exhibited high proteolytic stability (> 95%) up to 48 h post-radiolabeling. Saturation binding yielded moderate affinities toward PD-L1, ranging from 100 to 265 nM. Real-time radioligand binding provided more promising KD values around 20 nM for [64Cu]Cu-14 and [64Cu]Cu-15. In vivo PET imaging in mice bearing both PC3 PD-L1 overexpressing and PD-L1-mock tumors was performed at 0–2, 4–5 and 24–25 h post injection (p.i.). This revealed considerably different pharmacokinetic profiles, depending on the substituted chelator. [64Cu]Cu-14, substituted with NODA-GA, showed renal clearance with low liver uptake, whereas substitution with the cross-bridged cyclam chelator CB-TE2A resulted in a primarily hepatobiliary clearance. Notably, the monomeric DiAmSar radioligand [64Cu]Cu-16 demonstrated a higher liver uptake than [64Cu]Cu-15, but was still renally cleared as evidenced by the lack of uptake in gall bladder and intestines. The dimeric structure [64Cu]Cu-17 showed extensive accumulation and trapping in the liver but was also cleared via the renal pathway. Of all tracer candidates and across all timepoints, [64Cu]Cu-17 showed the highest accumulation at 24 h p.i. in the PD-L1-overexpressing tumor of all timepoints and all radiotracers, indicating drastically increased circulation time upon dimerization of two PD-L1 binding motifs. This study shows that chelator choice significantly influences the pharmacokinetic profile of biphenyl-based small molecule PD-L1 radioligands. The NODA-GA-conjugated radioligand [64Cu]Cu-14 exhibited favorable renal clearance; however, the limited uptake in tumors suggests the need for structural modifications to the binding motif for future PD-L1 radiotracers.

中文翻译：

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螯合剂影响：研究铜 64 标记的 PD-L1 放射性配体的药代动力学行为

程序性细胞死亡配体 1 (PD-L1) 在肿瘤微环境中发挥着关键作用，并且已报道在几种实体癌中过度表达。这与局部免疫反应（特别是 T 细胞）的下调有关。免疫检查点抑制剂显示出打破这种局部免疫麻痹的潜力，但只有 30% 的患者被认为是有反应者。因此需要新的诊断方法来确定患者的资格。针对 PD-L1 的小分子放射性示踪剂可以作为此类诊断工具，解决肿瘤病灶之间和肿瘤病灶内的异质性 PD-L1 表达，从而有助于治疗决策。采用收敛合成路线合成了四种基于联苯的小分子 PD-L1 配体，线性序列最多有 11 个步骤。作为螯合剂 NODA-GA、CB-TE2A 或 DiAmSar 用于使用铜 64 ([64Cu]Cu-14–[64Cu]Cu-16) 进行放射性标记。此外，还合成了基于DiAmSar的二聚结构([64Cu]Cu-17)。所有四种放射性配体在放射性标记后长达 48 小时内均表现出高蛋白水解稳定性 (> 95%)。饱和结合对 PD-L1 产生中等亲和力，范围为 100 至 265 nM。实时放射性配体结合为 [64Cu]Cu-14 和 [64Cu]Cu-15 提供了更有前景的 KD 值，约为 20 nM。在注射后 0-2、4-5 和 24-25 小时 (pi) 对携带 PC3 PD-L1 过表达和 PD-L1 模拟肿瘤的小鼠进行体内 PET 成像。这揭示了显着不同的药代动力学特征，具体取决于取代的螯合剂。用 NODA-GA 取代的 [64Cu]Cu-14 显示出肾脏清除率和较低的肝脏摄取率，而用交桥 cyclam 螯合剂 CB-TE2A 取代则主要通过肝胆清除。值得注意的是，单体 DiAmSar 放射性配体 [64Cu]Cu-16 表现出比 [64Cu]Cu-15 更高的肝脏摄取，但仍被肾脏清除，胆囊和肠道缺乏摄取证明了这一点。二聚体结构[64Cu]Cu-17在肝脏中显示出广泛的积累和捕获，但也通过肾途径被清除。在所有候选示踪剂和所有时间点中，[64Cu]Cu-17 在所有时间点和所有放射性示踪剂的 PD-L1 过表达肿瘤中在注射后 24 小时显示出最高积累，表明两个 PD-L1 二聚化后的循环时间急剧增加结合图案。这项研究表明，螯合剂的选择显着影响基于联苯的小分子 PD-L1 放射性配体的药代动力学特征。NODA-GA 结合的放射性配体 [64Cu]Cu-14 表现出良好的肾脏清除率；然而，肿瘤中的有限摄取表明未来 PD-L1 放射性示踪剂的结合基序需要进行结构修饰。