最近，针对程序性细胞死亡1（PD-1）及其配体（PD-L1）的治疗性抗体在多种肿瘤中表现出强效的抗癌作用。然而，单独阻断PD-1/PD-L1通路并不足以恢复正常的免疫应答。TGF-β和VEGFA等其他负调控抗肿瘤免疫应答的因子也参与了肿瘤细胞的免疫逃逸并诱导免疫治疗抵抗性。我们基于Nano-YBODY™技术平台开发了一种新型的抗TGF-β/VEGF双特异性抗体Y332D。采用CCK-8、流式细胞术、SBE4荧光素酶报告基因检测、免疫印迹和Transwell实验来测量抗TGF-β结构域的生物活性。采用NFAT荧光素酶报告基因检测、发光细胞活性检测和管形成实验来测量抗VEGF结构域的生物活性。在H22、EMT-6、4T1和AKT/Ras驱动的小鼠肝细胞癌肿瘤模型中评估了Y332D单独或与PD-1阻断联合治疗的活体抗癌效果。采用免疫荧光染色、流式细胞术、RNA-测序和定量RT-PCR分析肿瘤微环境的变化。Y332D能够保持对TGF-β和VEGFA的特异性结合亲和力。Y332D几乎完全对抗了TGF-β和VEGFA的体外生物学功能，包括免疫抑制、激活TGF-β信号传导、上皮间质转化（EMT）、激活VEGF/VEGFR信号传导、HUVEC增殖和管形成。体内实验数据表明，与抗TGF-β和抗VEGF单一治疗相比，Y332D在抑制肿瘤生长和转移方面更为有效。在联合治疗中，Y332D加上PD-1阻断显示出最强大且持久的抗癌效果。机制上，Y332D加上PD-1阻断上调了肿瘤浸润淋巴细胞的密度和功能，并表现出重新激活的抗肿瘤免疫。Y332D能同时阻断TGF-β和VEGF信号传导。与单一治疗相比，Y332D联合PD-1阻断通过改善免疫微环境，展现出卓越的抗肿瘤效果。© 2023. BioMed Central有限公司，Springer Nature的一部分。

Recently, therapeutic antibodies against programmed cell death 1 (PD-1) and its ligand (PD-L1) have exerted potent anticancer effect in a variety of tumors. However, blocking the PD-1/PD-L1 axis alone is not sufficient to restore normal immune response. Other negative regulators of antitumor immunity, like TGF-β and VEGFA, are also involved in immune escape of tumor cells and induce immunotherapy resistance.We developed a novel anti-TGF-β/VEGF bispecific antibody Y332D based on the Nano-YBODY™ technology platform. The CCK-8, flow cytometry, SBE4 luciferase reporter assay, western blotting and transwell assays were used to measure the biological activities of the anti-TGF-β moiety. The NFAT luciferase reporter assay, luminescent cell viability assay and tube formation assay were used to measure the biological activities of the anti-VEGF moiety. The in vivo anticancer efficacy of Y332D alone or in combination with PD-1 blockade was evaluated in H22, EMT-6, 4T1, and AKT/Ras-driven murine hepatocellular carcinoma tumor models. Immunofluorescent staining, flow cytometry, RNA-seq and quantitative RT-PCR were adopted to analyze the alterations in the tumor microenvironment.Y332D could maintain specific binding affinities for TGF-β and VEGFA. Y332D almost entirely counteracted the in vitro biological functions of TGF-β and VEGFA, including immunosuppression, activated TGF-β signaling, epithelial-mesenchymal transition (EMT), activated VEGF/VEGFR signaling, HUVEC proliferation and tube formation. The in vivo experiment data demonstrated that Y332D was more effective in inhibiting tumor growth and metastasis than anti-TGF-β and anti-VEGF monotherapies. In combination therapies, Y332D plus PD-1 blockade exhibited the most potent and durable anticancer effect. Mechanistically, Y332D plus PD-1 blockade upregulated the density and function of tumor-infiltrating lymphocytes and exerted reinvigorated antitumor immunity.Y332D could simultaneously block TGF-β and VEGF signalings. In comparison with the monotherapies, Y332D combined with PD-1 blockade exerts superior antitumor effect through improving immune microenvironment.© 2023. BioMed Central Ltd., part of Springer Nature.