T细胞免疫疗法的成功取决于肿瘤微环境（TME），异常肿瘤血管是大多数实体肿瘤的标志，与免疫逃逸有关。T细胞结合双特异性抗体（BsAb）治疗的功效依赖于T细胞在实体肿瘤中的成功转运和细胞溶解活性。使用抑制血管内皮生长因子（VEGF）的方法规范化肿瘤血管，可提高基于BsAb的T细胞免疫疗法的有效性。使用抗人VEGF（bevacizumab，BVZ）或抗小鼠VEGFR2抗体（DC101）作为VEGF阻滞剂，使用携带抗-GD2、抗-HER2或抗-glypican3（GPC3）IgG-(L)-scFv平台的EATs进行外体武装。使用癌细胞系导出的异种移植瘤（CDXs）或患者导出的异种移植瘤（PDXs）在BALB-Rag2-/-IL-2R-γc-KO（BRG）小鼠中进行评估 BsAb驱动的肿瘤内T细胞浸润和体内抗肿瘤反应。使用流式细胞术分析人癌细胞系中的VEGF表达，并使用VEGF Quantikine ELISA Kit测量小鼠血清中的VEGF水平。使用流式细胞术和生物荧光技术评估肿瘤浸润淋巴细胞（TILs），使用免疫组织化学方法研究TIL和肿瘤血管。人癌细胞系中的VEGF表达随着体外播种密度的增加而增加。BVZ显着降低了小鼠血清中的VEGF水平。BVZ或DC101增加了TME中的高内皮静脉（HEVs），并大大增强了神经母细胞瘤和骨肉瘤异种移植瘤中（2.1-8.1倍）BsAb驱动的T细胞浸润，这种浸润对CD8（+）TIL与CD4（+）TIL更有利，从而在多种CDX和PDX肿瘤模型中带来了更好的抗肿瘤效果，而没有额外的毒性。使用针对VEGF或VEGFR2的特异抗体的VEGF阻滞可以增加TME中的HEVs和细胞毒性CD8（+）TILs，显著提高EAT策略在临床前模型中的治疗效果，支持进一步研究VEGF阻滞以进一步增强基于BsAb的T细胞免疫疗法。©作者（或他们的雇主）2023年。根据CC BY-NC许可重新使用。不得进行商业再利用。由BMJ出版。

Success of T cell immunotherapy hinges on the tumor microenvironment (TME), and abnormal tumor vasculature is a hallmark of most solid tumors and associated with immune evasion. The efficacy of T cell engaging bispecific antibody (BsAb) treatment relies on the successful trafficking and cytolytic activity of T cells in solid tumors. Normalization of tumor vasculature using vascular endothelial growth factor (VEGF) blockades could improve efficacy of BsAb-based T cell immunotherapy.Anti-human VEGF (bevacizumab, BVZ) or anti-mouse VEGFR2 antibody (DC101) was used as VEGF blockade, and ex vivo armed T cells (EATs) carrying anti-GD2, anti-HER2, or anti-glypican3 (GPC3) IgG-(L)-scFv platformed BsAb were used. BsAb-driven intratumoral T cell infiltration and in vivo antitumor response were evaluated using cancer cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) carried out in BALB-Rag2 -/-IL-2R-γc-KO (BRG) mice. VEGF expression on human cancer cell lines was analyzed by flow cytometry, and VEGF levels in mouse serum were measured using VEGF Quantikine ELISA Kit. Tumor infiltrating lymphocytes (TILs) were evaluated using flow cytometry and by bioluminescence; both TILs and tumor vasculature were studied using immunohistochemistry.VEGF expression on cancer cell lines increased with seeding density in vitro. BVZ significantly reduced serum VEGF levels in mice. BVZ or DC101 increased high endothelial venules (HEVs) in the TME and substantially enhanced (2.1-8.1 fold) BsAb-driven T cell infiltration into neuroblastoma and osteosarcoma xenografts, which was preferential for CD8(+) TILs versus CD4(+) TILs, leading to superior antitumor effects in multiple CDX and PDX tumor models without added toxicities.VEGF blockade using specific antibodies against VEGF or VEGFR2 increased HEVs in the TME and cytotoxic CD8(+) TILs, significantly improving the therapeutic efficacy of EAT strategies in preclinical models, supporting the clinical investigation of VEGF blockades to further enhance BsAb-based T cell immunotherapies.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.