Screening and construction of nanobodies against human CD93 using phage libraries and study of their antiangiogenic effects

Hui Miao; Yiling Wu; Hao Ouyang; Peiwen Zhang; Wenyun Zheng; Xingyuan Ma

doi:10.3389/fbioe.2024.1372245

Screening and construction of nanobodies against human CD93 using phage libraries and study of their antiangiogenic effects

Front Bioeng Biotechnol. 2024 May 1:12:1372245. doi: 10.3389/fbioe.2024.1372245. eCollection 2024.

Authors

Hui Miao¹, Yiling Wu¹, Hao Ouyang², Peiwen Zhang³, Wenyun Zheng³, Xingyuan Ma¹

Affiliations

¹ State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
² Department of Hepatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
³ Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China.

Abstract

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°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.

Keywords: CD93; angiogenesis; nanobody; phage display; vascular permeability.

Grants and funding

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).