Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 μM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16–F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.本研究设计并合成了一系列双功能PD-L1/CD73（分化簇73，cluster of differentiation 73）小分子抑制剂。其中，化合物CC-5展现出最强的PD-L1抑制活性，其半最大抑制浓度（IC50）为6 nM，同时具备高效的抗CD73活性，IC50值为0.773 μM。CC-5对PD-L1/CD73的高抑制活性进一步通过表面等离子体共振实验（surface plasmon resonance assays, SPR）得到验证，其与人PD-L1的解离常数（KD）为182 nM，与CD73的解离常数为101 nM。尤为重要的是，CC-5在CT26及B16-F10肿瘤模型中可显著抑制肿瘤生长，肿瘤生长抑制率（TGI）分别为64.3%与39.6%。对肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）的免疫组化（IHC）及流式细胞术分析结果显示，CC-5通过激活肿瘤免疫微环境发挥抗癌作用。综上，CC-5是首款双靶点PD-L1/CD73抑制剂，具备作为双功能免疫治疗药物开展后续研究的价值。