Objectives:Reduced expression of adhesion molecules in tumor vasculature can limit infiltration of effector T cells. To improve T cell adhesion to tumor endothelial cell (EC) antigens and enhance transendothelial migration, we developed bispecific, T-cell engaging antibodies (bsAb) that activate T cells after cross-linking with EC cell surface antigens.Methods:Recombinant T-cell stimulatory anti-VEGFR2–anti-CD3 and costimulatory anti-TIE2–anti-CD28 or anti-PD-L1–anti-CD28 bsAb were engineered and expressed. Primary lines of human umbilical vein endothelial cells (HUVEC) that constitutively express VEGFR2 and TIE2 growth factor receptors and PD-L1, but very low levels of adhesion molecules, served as models for anergic tumor EC.Results:In cocultures with HUVEC, anti-VEGFR2–anti-CD3 bsAb increased T cell binding and elicited rapid T cell activation. The release of proinflammatory cytokines TNF-α, IFN-γ, and IL-6 was greatly augmented by the addition of anti-TIE2–anti-CD28 or anti-PD-L1–anti-CD28 costimulatory bsAb. Concomitantly, T cell-released cytokines upregulated E-selectin, ICAM1, and VCAM1 adhesion molecules on HUVEC. HUVEC cultured in breast cancer cell-conditioned medium to mimic the influence of tumor-secreted factors were similarly activated by T cell-engaging bsAb. Migration of T cells in transwell assays was significantly increased by anti-VEGFR2–anti-CD3 bsAb. The combination with costimulatory anti-TIE2–anti-CD28 bsAb augmented activation and proliferation of migrated T cells and their cytotoxic capacity against spheroids of the MCF-7 breast cancer cell line seeded in the lower transwell chamber.Conclusions:T cells activated by anti-VEGFR2–anti-CD3 and costimulatory EC-targeting bsAb can reverse the energy of quiescent EC in vitro, resulting in improved T cell migration through an EC layer.
目的：肿瘤血管系统中黏附分子表达降低会限制效应T细胞的浸润。为增强T细胞对肿瘤内皮细胞表面抗原的黏附并促进跨内皮迁移，本研究开发了双特异性T细胞衔接抗体，该抗体通过与内皮细胞表面抗原交联激活T细胞。 方法：设计并表达重组T细胞刺激型抗VEGFR2-抗CD3双抗及共刺激型抗TIE2-抗CD28或抗PD-L1-抗CD28双抗。采用组成性表达VEGFR2、TIE2生长因子受体及PD-L1，但黏附分子表达水平极低的人脐静脉内皮细胞株作为无反应性肿瘤内皮细胞模型。 结果：在与HUVEC共培养体系中，抗VEGFR2-抗CD3双抗能增强T细胞结合并快速激活T细胞。联合使用共刺激型抗TIE2-抗CD28或抗PD-L1-抗CD28双抗可显著提升促炎细胞因子TNF-α、IFN-γ和IL-6的释放水平。同时，T细胞释放的细胞因子上调了HUVEC表面E-选择素、ICAM1和VCAM1黏附分子的表达。在模拟肿瘤分泌因子影响的乳腺癌细胞条件培养基中培养的HUVEC，同样可被T细胞衔接双抗激活。Transwell实验显示抗VEGFR2-抗CD3双抗显著促进T细胞迁移，联合共刺激型抗TIE2-抗CD28双抗可增强迁移T细胞的活化增殖能力及其对下层Transwell小室中MCF-7乳腺癌细胞球体的杀伤效能。 结论：抗VEGFR2-抗CD3双抗联合靶向内皮细胞的共刺激双抗激活的T细胞，可在体外逆转静息内皮细胞的无反应状态，从而提升T细胞穿越内皮细胞层的能力。
Reversal of Endothelial Cell Anergy by T Cell-Engaging Bispecific Antibodies
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