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

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.

FIGURE 1

Anti-CD93 Nbs screening and phage ELISA. (A) The antigen-coated was CD93, and the negative control was coated with BSA. OD450 nm values were measured three times. (B–G) Analysis of CD93-binding phage by phage ELISA. The coated antigen was CD93, and the negative control was skimmed milk powder. The ratio of absorbance was calculated to obtain the positive monoclonal (H) Sequence of different positive monoclonals Nbs. Data are expressed as mean ± SEM.

FIGURE 2

Anti-CD93 Nbs construction and expression purification. (A) PCR analysis of phagemid insert size. M, molecular weight marker. From top to bottom, 1,000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp; lane 1-5, NC81; lane 6-10, NC89. There was a predicted 540/564 bp band in each lane. (B) SDS-PAGE analysis of purified anti-CD93 Nbs NC81 and NC89. Lane M: protein marker. (C) The model structures of anti-CD93 Nbs NC81 were obtained by I-Tasser. (D) The model structures of anti-CD93 Nbs NC89 were obtained by I-Tasser. Protein structures are shown in PYMOL.

FIGURE 3

Anti-CD93 Nbs affinity and stability assays. (A) NC81 affinity assay by ELISA. The logistic curve fitting was analyzed by GraphPad (n = 3). (B) NC89 affinity assay by ELISA. The logistic curve fitting was analyzed by GraphPad (n = 3). (C) CD93 expression levels on the surface of HUVECs and HEK293 (n = 3). (D) Binding capacity of anti-CD93 Nbs to HUVECs and HEK293 (n = 3). (E) The binding capacity of NC81 to CD93 under different temperature treatments was determined by ELISA (n = 3). (F) The binding capacity of NC89 to CD93 under different temperature treatments was determined by ELISA (n = 3). (G) Secondary structure of NC81 under different temperature treatments. (H) Secondary structure of NC89 under different temperature treatments. Data are expressed as mean ± SEM.

FIGURE 4

Anti-CD93 Nbs inhibit HUVECs proliferation and migration (A). MTT assay. NC81 specifically inhibits HUVEC proliferation (n = 3). (B) NC89 specifically inhibits HUVEC proliferation (n = 3). (C) Quantitative analysis of NC81 inhibition of HUVEC proliferation (n = 3). (D) Quantitative analysis of NC89 inhibition of HUVEC proliferation (n = 3). (E) Migration assay. NC81 dose-dependently reduced the migration of HUVECs. Quantitative analysis on the right. (Original magnification × 100). (F) NC89 dose-dependently reduced the migration of HUVECs. Quantitative analysis on the right. (Original magnification × 100). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus Control.

FIGURE 5

Inhibitory effects of the anti-CD93 Nbs on tube formation. (A) HUVECs were incubated with PBS and VEGF as negative and positive controls, respectively, and various concentrations (1 μM and 10 μM) of the Nbs were used (n = 3). (Original magnification × 100). (B) Quantitative analysis of tube length (n = 3). (C) Quantitative analysis of tubes formed (n = 3). Data are expressed as mean ± SEM. ***p < 0.001 versus Control; ^### p < 0.001 versus VEGF Group.

FIGURE 6

Anti-CD93 Nbs increase vascular permeability (A). Schematic representation of intercellular tight junctions and adhesion junctions. (B) The expression of CD93, and VE-cadherin was detected by Western blot, and β-actin was used as control. The results represent at least three independent experiments. (C) Quantitative analysis of CD93 protein expression. The protein expression of CD93 was normalized to basal β-actin (n = 3). (D) Quantitative analysis of VE-cadherin protein expression. The protein expression of VE-Cadherin was normalized to basal β-actin (n = 3). Data are expressed as mean ± SEM. *p < 0.05 versus Control; ^& p < 0.05 versus 293T.

FIGURE 7

Three-dimensional structure simulation and docking analysis of anti-CD93 Nbs (A). Three-dimensional structure of the CD93-NC81 complex. (B) Three-dimensional structure of the CD93-NC89 complex. (C) Interacting residues of the CD93-NC81 complex. (D) Interacting residues of the CD93-NC89 complex.