The programmed death-ligand 1 (PD-L1) is a major checkpoint protein that helps cancer cells evade the immune system. A non-invasive imaging agent with rapid clearance rate would be an ideal tool to predict and monitor the efficacy of anti-PD-L1 therapy. The aim of this research was to engineer a subnanomolar, high-affinity fibronectin type 3 domain (FN3)-based small binder targeted against human PD-L1 (hPD-L1) present on tumor cells. A naive yeast G4 library containing the FN3 gene with three binding loop sequences was used to isolate high-affinity binders targeted to purified full-length hPD-L1. The selected binder clones displayed several mutations in the loop regions of the FN3 domain. One unique clone (FN3hPD-L1-01) with a 6x His-tag at the C-terminus had a protein yield of >5 mg/L and a protein mass of 12 kDa. In vitro binding assays on six different human cancer cell lines (MDA-MB-231, DLD1, U87, 293 T, Raji and Jurkat) and murine CT26 colon carcinoma cells stably expressing hPD-L1 showed that CT26/hPD-L1 cells had the highest expression of hPD-L1 in both basal and IFN-γ-induced states, with a binding affinity of 2.38 ± 0.26 nM for FN3hPD-L1-01. The binding ability of FN3hPD-L1-01 was further confirmed by immunofluorescence staining on ex vivo CT26/hPD-L1 tumors sections. The FN3hPD-L1-01 binder represents a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells and would aid in earlier imaging of tumors. Future clinical validation studies of the labeled FN3hPD-L1 binder(s) have the potential to monitor immune checkpoint inhibitors therapy and predict responders.

中文翻译：

---

针对人类程序性死亡配体 1 的新型亚纳摩尔亲和力纤连蛋白 III 结构域结合剂的工程设计。


程序性死亡配体 1 (PD-L1) 是一种主要的检查点蛋白，可帮助癌细胞逃避免疫系统。具有快速清除率的非侵入性显像​​剂将是预测和监测抗 PD-L1 疗法疗效的理想工具。本研究的目的是设计一种亚纳摩尔、高亲和力的纤连蛋白 3 型结构域 (FN3) 小型结合物，靶向肿瘤细胞上存在的人 PD-L1 (hPD-L1)。使用含有具有三个结合环序列的 FN3 基因的天然酵母 G4 文库来分离靶向纯化全长 hPD-L1 的高亲和力结合物。选定的结合物克隆在 FN3 结构域的环区域中显示出多个突变。一种在 C 末端带有 6x His 标签的独特克隆 (FN3hPD-L1-01) 的蛋白质产量为 >5 mg/L，蛋白质质量为 12 kDa。对六种不同的人癌细胞系（MDA-MB-231、DLD1、U87、293 T、Raji 和 Jurkat）和稳定表达 hPD-L1 的鼠 CT26 结肠癌细胞进行体外结合测定，结果表明 CT26/hPD-L1 细胞具有hPD-L1 在基础状态和 IFN-γ 诱导状态下表达最高，与 FN3hPD-L1-01 的结合亲和力为 2.38 ± 0.26 nM。通过离体 CT26/hPD-L1 肿瘤切片的免疫荧光染色进一步证实了 FN3hPD-L1-01 的结合能力。 FN3hPD-L1-01 结合物代表了一种新型、小型、高亲和力的结合物，用于对肿瘤细胞上的 hPD-L1 表达进行成像，并有助于肿瘤的早期成像。标记的 FN3hPD-L1 结合剂的未来临床验证研究有可能监测免疫检查点抑制剂治疗并预测反应者。