T cells are the most predominant effector cells in immune-mediated elimination of cancer and circumventing tumor progression. Among various approaches, T cells activation by specific antibodies independently of their TCR specificity, is considered as an effective approach to circumvent tumor progression. The most common surface marker for all T cells which is crucial for T cell activation is regarded as CD3. Therefore, the goal of our study was to evaluate the preclinical efficacy of recombinant anti-CD3 nanobody. To this end, anti-CD3 sequence, was PCR amplified, following cloning and expression in E.coli and purification, the purified nanobody with a molecular weight of ∼17 kDa was confirmed by western blot. Furthermore, flow cytometry analysis demonstrated that purified nanobody could bind to CD3 on Jurkat cell line. Subsequently, results from inoculation of 3 μg/g of nanobody to tumor bearing balb/c mice indicate inhibition of tumor growth. Furthermore, circulating levels of tumoricidal cytokines such as IL-2 and IFNγ were raised whereas tolerogenic cytokines such as IL-4, 6 and 10 were decreased at the end of the treatment. Moreover, IHC analysis confirmed the presence and also the percentage of TILs in tumor sites in response to anti-CD3 therapy. Hence, our results suggest that the purified anti-CD3 nanobody may become a promising candidate for targeting and activating CTLs to induce anti-tumor responses and may provide groundwork for future studies involving other kind of cancers.

中文翻译：

---

评价重组抗CD3纳米抗体对免疫调节功能的潜力。

在免疫介导的消除癌症和规避肿瘤进展中，T细胞是最主要的效应细胞。在各种方法中，独立于其TCR特异性而被特异性抗体激活的T细胞被认为是规避肿瘤进展的有效方法。对于T细胞活化至关重要的所有T细胞最常见的表面标记被视为CD3。因此，我们研究的目的是评估重组抗CD3纳米抗体的临床前疗效。为此，在克隆并在大肠杆菌中表达并纯化后，PCR扩增了抗CD3序列，通过蛋白质印迹证实了分子量约为17kDa的纯化的纳米抗体。此外，流式细胞仪分析表明纯化的纳米抗体可以结合Jurkat细胞系上的CD3。随后，荷瘤的balb / c小鼠接种3μg/ g纳米抗体的结果表明抑制了肿瘤的生长。此外，在治疗结束时，肿瘤细胞因子如IL-2和IFNγ的循环水平升高，而耐受性细胞因子如IL-4、6和10降低。此外，IHC分析证实了响应抗CD3治疗的肿瘤部位中TIL的存在以及百分比。因此，我们的结果表明，纯化的抗CD3纳米抗体可能成为靶向和激活CTL以诱导抗肿瘤反应的有希望的候选者，并可能为涉及其他类型癌症的未来研究提供基础。在治疗结束时，肿瘤细胞因子如IL-2和IFNγ的循环水平升高，而致耐受性细胞因子如IL-4、6和10降低。此外，IHC分析证实了响应抗CD3治疗的肿瘤部位中TIL的存在以及百分比。因此，我们的结果表明，纯化的抗CD3纳米抗体可能成为靶向和激活CTL以诱导抗肿瘤反应的有希望的候选者，并可能为涉及其他类型癌症的未来研究提供基础。在治疗结束时，肿瘤细胞因子如IL-2和IFNγ的循环水平升高，而致耐受性细胞因子如IL-4、6和10降低。此外，IHC分析证实了响应抗CD3治疗的肿瘤部位中TIL的存在以及百分比。因此，我们的结果表明，纯化的抗CD3纳米抗体可能成为靶向和激活CTL以诱导抗肿瘤反应的有希望的候选者，并可能为涉及其他类型癌症的未来研究提供基础。