文献类型：期刊文献

英文题名：Screening and construction of nanobodies against human CD93 using phage libraries and study of their antiangiogenic effects

作者：Miao, Hui[1];Wu, Yiling[1];Ouyang, Hao[2];Zhang, Peiwen[3];Zheng, Wenyun[3];Ma, Xingyuan[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[2]Shanghai Univ Tradit Chinese Med, Yueyang Hosp Integrated Tradit Chinese & Western M, Dept Hepatol, Shanghai, Peoples R China;[3]East China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, Shanghai, Peoples R China

年份：2024

卷号：12

外文期刊名：FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY

收录：;EI(收录号：20242016102011);WOS:【SCI-EXPANDED(收录号:WOS:001222459700001)】；

基金：The FP7 WeNMR (project# 261572), H2020 West-Life (project# 675858), the EOSC-hub (project# 777536) and the EGI-ACE (project# 101017567) European e-Infrastructure projects are acknowledged for the use of their web portals, which make use of the EGI infrastructure with the dedicated support of CESNET-MCC, INFN-PADOVA-STACK, INFN-LNL-2, NCG-INGRID-PT, TW-NCHC, CESGA, IFCA-LCG2, UA-BITP, SURFsara and NIKHEF, and the additional support of the national GRID Initiatives of Belgium, France, Italy, Germany, the Netherlands, Poland, Portugal, Spain, UK, Taiwan and the US Open Science Grid.r The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Key Research and Development Project of China (2018YFA0902804), and the National Natural Science Foundation (31670944 and 81673345).

语种：英文

外文关键词：CD93; nanobody; phage display; angiogenesis; vascular permeability

摘要：Background Cluster of Differentiation 93 (CD93) plays an important role in angiogenesis and is considered an important target for inhibiting tumor angiogenesis, but there are currently no therapeutic antibodies against CD93 in the clinic. Thus, we describe the screening of novel nanobodies (Nbs) targeting human CD93 from a phage library of shark-derived Nbs. Methods Screening and enrichment of phage libraries by enzyme-linked immunosorbent assay (ELISA). Anti-CD93 Nbs were purified by expression in E. coli. The binding affinity of anti-CD93 Nbs NC81/NC89 for CD93 was examined by flow cytometry (FC) and ELISA. The thermal stability of NC81/NC89 was examined by ELISA and CD spectroscopy. Afterward, the anti-angiogenic ability of NC81/NC89 was examined by MTT, wound healing assay, and tube formation assay. The expression level of VE-cadherin (VE-Ca) and CD93 was detected by Western Blot (WB). The binding sites and binding forms of NC81/NC89 to CD93 were analyzed by molecular docking. Results The anti-CD93 Nbs were screened in a phage library, expressed in E. coli, and purified to >95% purity. The results of FC and ELISA showed that NC81/NC89 have binding ability to human umbilical vein endothelial cells (HUVECs). The results of ELISA and CD spectroscopy showed that NC81/NC89 retained the ability to bind CD93 at 80 degrees C and that the secondary structure remained stable. In vitro, the results showed that NC81 and NC89 significantly inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs) as well as tube formation on Matrigel. Western Blot showed that NC81 and NC89 also inhibited the expression of VE-Ca thereby increasing vascular permeability. It was found during molecular docking that the CDR regions of NC81 and NC89 could be attached to CD93 by strong hydrogen bonds and salt bridges, and the binding sites were different. Conclusion We have successfully isolated NC81 and NC89, which bind CD93, and both Nbs significantly inhibit angiogenesis and increase vascular permeability. These results suggest that NC81 and NC89 have potential clinical applications in angiogenesis-related therapies.