靶向PD-L1小分子探针PET显像并指导NSCLC放疗联合免疫治疗研究

Recent breakthroughs in immune checkpoint blockade have led to a paradigm shift in the standard of care (SOC) for non-small-cell lung cancer (NSCLC). Despite unprecedented improvements in clinical outcomes, less than 20% of patients respond to anti-PD-1/PD-L1 agents. The ability of radiotherapy to induce an immunogenic response and neutralize the immune-suppressive effects of the tumor microenvironment, uniquely positions it as a synergistic tool at the center of emerging multimodal therapies utilizing immune checkpoint blockade. With the development of multimodal therapies, it becomes increasingly critical to identify predictive biomarkers to guide patients selection for treatment. . PET imaging providing noninvasively whole-body and real-time evaluation of PD-1/PD-L1 expression, has shown promise in assessment of immune checkpoints to select eligible candidates for anti-PD-1/PD-L1 immunotherapies. Recently, various immuno-PET tracers based on monoclonal antibodies (mAbs), engineered scaffold proteins and peptides were developed to target either PD-1 or PD-L1.. In our previous study, we developed a highly specific domain antibody-based probe (89Zr-Df-KN035) for PD-L1 immuno-PET imaging, showing efficient accumulation in PD-L1 expressed tumor xenografts (NSCLC and glioma) and favorable in vivo biodistribution in healthy non-human primates (NHPs). More importantly, the tracer demonstrated encouraging imaging performance at 24h post administration, much earlier than monoclonal antibody (mAb)-based probes. However, its circulation times remained long because of the relatively large size (79.6 kDa). . Small-molecule inhibitors with superior properties such as oral bioavailability, lower manufacture costs, higher tissues penetration, shorter half-lives, as well as less immune-related adverse events (irAEs), have been proven to be desirable candidates for clinical application. . In this study, two novel small-molecule inhibitors of PD-L1, BMS1001 and BMS1166, with crystal structure suitable for conjugation, will be labeled with 68Ga, a short half-life positron nuclide. Then the resulting probes, 68Ga-NOTA-BMS1001 and 68Ga-NOTA-BMS1166 will be sequentially evaluated by in vitro cell binding studies using PD-L1 positive human lung adenocarcinoma cell line A549. Next, serial PET imaging and biodistribution studies will be performed in humanized NSG mice bearing A549 xenografts to evaluate in vivo performance of the two probes. On this basis, the changes of tumor PD-L1 expression after radiotherapy will be further evaluated by PET imaging in humanized NSG mice bearing A549 xenografts. . We aim to develop a more appropriate PET tracer allowing standard clinical workflow of imaging within 1-2h post administration, in order to select eligible candidates for anti-PD-1/PD-L1 immunotherapy and guide the combination treatment of radiotherapy and immunotherapy in patients with NSCLC, extending the benefits of immune checkpoint blockade to a broader patient population.

靶向PD-1/PD-L1的肿瘤PET显像有助于筛选免疫治疗获益人群。前期工作构建的PET探针89Zr-Df-KN035在表达PD-L1的非小细胞肺癌（NSCLC）和胶质瘤移植瘤高度摄取，显像时间较抗体探针明显提前，但是血液清除仍较慢。小分子抑制剂具有成本低、口服给药、半衰期短、肿瘤穿透力强、免疫排斥反应少等优点。本项目拟利用新型非肽衍生物类小分子抑制剂BMS1001和BMS1166构建更适于临床显像的68Ga-NOTA-BMS1001和68Ga-NOTA-BMS1166正电子探针，通过体外研究、免疫人源化荷NSCLC小鼠体内生物学分布及PET显像评价其用于监测肿瘤PD-L1表达的可行性和效能。随后，基于放疗上调肿瘤PD-L1表达的报道，监测免疫人源化荷NSCLC小鼠放疗后PD-L1的变化，旨在决策放疗联合免疫治疗，增强抗肿瘤效应，扩大NSCLC免疫治疗获益患者。

靶向PD-1/PD-L1的肿瘤PET显像有助于筛选免疫治疗获益人群。近年来，研究人员基于单克隆抗体、抗体衍生物、基因工程蛋白（Affibody、Nanobody、Adnectin等）、多肽、以及小分子化合物等，开发出了多种靶向PD-L1的PET探针。基于小分子化合物的PET探针具有成本低、半衰期短、肿瘤穿透力强、免疫排斥反应少等优点。本项目根据百时美施贵宝公司报道的一类靶向PD-L1的小分子抑制剂化合物原型，在保留其药效基团的条件下，对其进行结构改造及修饰，采用药物化学全合成的方法，设计并合成一系列小分子探针的非放射性标准品及其标记前体。利用HTRF技术筛选出两种与PD-L1具有较高亲和力的非放射性标准品（IC50分别为154.7nM及545.5nM）。分别采用正电子核素18F及68Ga对探针前体进行标记，制备了两种小分子PET探针（18F-TJ-33和68Ga-TJ-34），放射化学纯度均>99%。68Ga-TJ-34体外120min血清稳定性＞95%，而18F-TJ-33＜50%。18F-TJ-33和68Ga-TJ-34的脂水分配系数分别为2.67±0.32和-1.13±0.21，表明前者亲脂性较强而后者亲水性较强。荷瘤小鼠模型PET显像显示，68Ga-TJ-34可明显区分不同PD-L1表达水平的肿瘤小鼠模型，生物分布结果与PET显像一致，而18F-TJ-33有待进一步优化以提高稳定性和靶向结合。两种探针单次大剂量注射后14天内均未对小鼠造成生化及组织学损伤。因此，本项目研发的小分子PET探针68Ga-TJ-34具有良好的PD-L1靶向特异性、显像效能和生物安全性，具有良好的研究及转化应用前景。