Cytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. However, itsefficacy isrestricted by the immunosuppression, which is mediated by the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) pathway. In order to overcome the negative co-stimulation from these T cells,we screened a nanobody targeted for CTLA-4 (Nb36) and blocked the CTLA-4 signaling with Nb36. Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors to beused to induce CIK cells in vitro, after which they were co-cultured with DC cells that had received tumor antigens. In addition, wetested whether blocking CTLA-4 signaling with Nb36 could promote in vitro DC-CIK cells proliferation, pro-inflammatory cytokine production and cytotoxicity,or not. For the in vivo experiments, we constructed a subcutaneously transplanted tumor model and placed it in NOD/SCID mice to verify the anti-tumor effect of this therapy. After stimulation with Nb36, the DC-CIK cells presented enhanced proliferation and production of IFN-γ in vitro, which strengthened the killing effect on the tumor cells. For the in vivo experiments, it was found that Nb36-treated DC-CIK cells significantly inhibited the growth of subcutaneously transplanted livercancer tumors, as well as reduced the tumor weight and prolonged the survival of tumor-bearing NOD/SCID mice. Ourfindings demonstrated that in response to CTLA-4 specific nanobody stimulation, DC-CIK cells exhibited a better anti-tumor effect. In fact, this Nb-based CTLA-4 blocking strategy achieved an anti-tumor efficacy close to that of monoclonal antibodies. Our findings suggest that DC-CIK cells + Nb36 have the potential totreatmalignant tumors through in vivo adoptive therapy.

中文翻译：

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基于 Nanoboby 的 CTLA-4 阻断策略在树突状细胞刺激的细胞因子诱导的杀伤细胞中增强其抗肿瘤作用

用肿瘤抗原脉冲树突状细胞 (DC-CIK) 免疫疗法诱导的细胞因子诱导杀伤细胞是治疗恶性肿瘤的一种有前途的策略。然而，其功效受到免疫抑制的限制，免疫抑制由细胞毒性 T 淋巴细胞相关抗原 4 (CTLA-4) 途径介导。为了克服这些 T 细胞的负共刺激，我们筛选了靶向 CTLA-4 (Nb36) 的纳米抗体，并用 Nb36 阻断 CTLA-4 信号传导。外周血单个核细胞（PBMCs）从健康供者身上采集，用于体外诱导CIK细胞，然后与接受肿瘤抗原的DC细胞共培养。此外，我们测试了用 Nb36 阻断 CTLA-4 信号是否可以促进体外 DC-CIK 细胞增殖、促炎细胞因子的产生和细胞毒性。在体内实验中，我们构建了皮下移植的肿瘤模型并将其置于NOD/SCID小鼠体内以验证该疗法的抗肿瘤作用。用Nb36刺激后，DC-CIK细胞在体外表现出增强的增殖和IFN-γ的产生，从而加强了对肿瘤细胞的杀伤作用。在体内实验中，发现Nb36处理的DC-CIK细胞显着抑制了皮下移植肝癌肿瘤的生长，并降低了肿瘤重量并延长了荷瘤NOD/SCID小鼠的存活期。我们的研究结果表明，响应 CTLA-4 特异性纳米抗体刺激，DC-CIK 细胞表现出更好的抗肿瘤作用。事实上，这种基于 Nb 的 CTLA-4 阻断策略实现了接近单克隆抗体的抗肿瘤功效。