During the past two decades, tumor therapy based on monoclonal antibody has been found as a confident therapeutic approach in solid tumors and hematologic malignancies. Nanobodies are the smallest fragment of an antigen-binding domain in heavy chain-only antibody originated from the Camelidae family. Accordingly, they are being recently developed rapidly as diagnostic and therapeutic agents. In this regard, targeting of angiogenic factors like Placenta growth factor (PLGF) via nanobodies show a high effectiveness. In the current study, we developed a recombinant anti-PLGF bivalent nanobody based on the affinity enhancement mutant form of anti-PLGF nanobody to suppress the angiogenesis progression. Thereafter, the bivalent nanobody (bi-Nb) was cloned and then expressed into a bacterial system. Afterward, the purity was authorized using western blot assay and the affinity was assessed using ELISA. In this regard, proliferation, 3D capillary tube formation, and migration assays were employed as functional assays. The obtained data were analyzed using t-test and P < 0.05 was considered as statistically significant. The results indicate that the bivalent nanobody could inhibit proliferation, mobility, and formation of endothelial cell capillary-like structure. Moreover, the EC50 was estimated for endothelial cell’s proliferation and capillary tube’s formation to be about 100 ng/ml and 65 ng/ml, respectively. Migration of MCF-7 was inhibited as about 69%, rather than the control. Accumulation of data have shown that targeting of angiogenic factors like VEGF via monoclonal antibodies or nanobodies can be useful in the suppression of tumor progression. Also, the inhibition of PLGF with monoclonal antibody indicated that it is significant in angiogenesis suppression. However, due to intrinsic properties of nanobodies, they are suggested to be used. Since the small size is rapidly removed through liver or kidney system, so it is important to use bivalent or polymeric forms for extending the half-life. Our findings indicated that the inhibition of PLGF can prevent growth and proliferation of endothelial cells and tumor cells through the bivalent nanobody. So, it is suggested as a novel therapeutic agent for angiogenesis suppression.

在过去的二十年中，已经发现基于单克隆抗体的肿瘤疗法是治疗实体瘤和血液系统恶性肿瘤的可靠方法。纳米抗体是源自骆驼科的仅重链抗体中抗原结合结构域的最小片段。因此，它们最近被快速开发为诊断和治疗剂。在这方面，通过纳米抗体靶向血管生成因子（如胎盘生长因子（PLGF））显示出很高的效力。在当前的研究中，我们基于抗PLGF纳米抗体的亲和力增强突变体形式开发了重组抗PLGF二价纳米抗体，以抑制血管新生进程。此后，将二价纳米抗体（bi-Nb）克隆，然后表达到细菌系统中。之后，使用western印迹法确定纯度，并使用ELISA评估亲和力。在这方面，将增殖，3D毛细管形成和迁移测定用作功能测定。使用以下方法分析获得的数据t检验和P <0.05被认为具有统计学意义。结果表明，二价纳米抗体可以抑制内皮细胞的毛细血管样结构的增殖，迁移和形成。此外，据估计EC50的内皮细胞增殖和毛细管形成分别约为100 ng / ml和65 ng / ml。MCF-7的迁移被抑制了约69％，而不是对照。积累的数据表明，通过单克隆抗体或纳米抗体靶向血管生成因子（如VEGF）可用于抑制肿瘤进展。另外，用单克隆抗体抑制PLGF表明其在血管生成抑制中是重要的。然而，由于纳米抗体的固有性质，建议使用它们。由于小尺寸可以通过肝脏或肾脏系统迅速清除，因此使用二价或聚合形式延长半衰期很重要。我们的发现表明，PLGF的抑制可以通过二价纳米抗体阻止内皮细胞和肿瘤细胞的生长和增殖。因此，被建议作为抑制血管生成的新型治疗剂。