Abstract

Neuropilin-1 (NRP-1) is a non-tyrosine kinase receptor and when overexpressed, leads to angiogenesis. High expression of NRP-1 has been observed in various cancers. Unique characteristic of nanobodies (small size, high affinity and stability, and ease production) make them potential therapeutic tools. Oligoclonal nanobodies which detect multiple functional epitopes on the target antigen could be potential tools for inhibition of cancer resistance problems due to escape variant of tumor cells. In this study, oligoclonal anti-NRP-1 nanobodies were selected from camel immune library and their binding activities as well as in vitro functionality were evaluated. Anti-NRP-1 nanobodies were expressed in an Escherichia coli host, and purified using nickel affinity chromatography. The effect of each individual and oligoclonal nanobodies on human endothelial cells were evaluated by MTT, Tube formation, and migration assay as well. Results showed that oligoclonal anti-NRP-1 nanobodies detected different epitopes of NRP-1 antigen and inhibited in vitro angiogenesis of human endothelial cells better than each individual nanobody. Results indicate promising oligoclonal anti-NRP-1 nanobodies for inhibition of angiogenesis.

摘要

Neuropilin-1 (NRP-1) 是一种非酪氨酸激酶受体，当过度表达时会导致血管生成。已在多种癌症中观察到 NRP-1 的高表达。纳米抗体的独特特性（体积小、高亲和力和稳定性以及易于生产）使其成为潜在的治疗工具。检测靶抗原上多个功能性表位的寡克隆纳米抗体可能是抑制由于肿瘤细胞逃逸变体引起的癌症耐药性问题的潜在工具。在这项研究中，寡克隆抗 NRP-1 纳米抗体选自骆驼免疫文库，并评估了它们的结合活性和体外功能。抗 NRP-1 纳米抗体在大肠杆菌中表达主机，并使用镍亲和层析纯化。每个个体和寡克隆纳米抗体对人内皮细胞的影响也通过 MTT、管形成和迁移测定进行评估。结果表明，寡克隆抗 NRP-1 纳米抗体检测到 NRP-1 抗原的不同表位，并且比单个纳米抗体更好地抑制人内皮细胞的体外血管生成。结果表明有前途的寡克隆抗 NRP-1 纳米抗体可抑制血管生成。