Inhibition of VEGF-dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC14a

Abstract

The C-type lectin-like domain of CLEC14a (CLEC14a-C-type lectin-like domain [CTLD]) is a key domain that mediates endothelial cell-cell contacts in angiogenesis. However, the role of CLEC14a-CTLD in pathological angiogenesis has not yet been clearly elucidated. In this study, through complementarity-determining region grafting, consecutive deglycosylation, and functional isolation, we generated a novel anti-angiogenic human monoclonal antibody that specifically targets CLEC14a-CTLD and that shows improved stability and homogeneity relative to the parental antibody. We found that this antibody directly inhibits CLEC14a-CTLD-mediated endothelial cell-cell contact and simultaneously downregulates expression of CLEC14a on the surface of endothelial cells. Using various in vitro and in vivo functional assays, we demonstrated that this antibody effectively suppresses vascular endothelial growth factor (VEGF)-dependent angiogenesis and tumor angiogenesis of SNU182 human hepatocellular carcinoma, CFPAC-1 human pancreatic cancer, and U87 human glioma cells. Furthermore, we also found that this antibody significantly inhibits tumor angiogenesis of HCT116 and bevacizumab-adapted HCT116 human colorectal cancer cells. These findings suggest that antibody targeting of CLEC14a-CTLD has the potential to suppress VEGF-dependent angiogenesis and tumor angiogenesis and that CLEC14a-CTLD may be a novel anti-angiogenic target for VEGF-dependent angiogenesis and tumor angiogenesis.

Keywords: CTLD; VEGF; CLEC14a; angiogenesis; tumor angiogenesis.

Figure 1

Generation of an optimized antibody with improved stability via CDR grafting. (A) Schematic illustration of the CDR grafting of the parental IgG into omalizumab, trastuzumab, adalimumab, or bevacizumab. (B) Visual observation of antibody aggregation in preparations of the parental IgG and Clone 1 IgG. (C) The aggregation indices of parental IgG and Clone 1 IgG were measured spectrophotometrically. (D) Quantification of parental IgG and Clone 1 IgG antibodies before and after antibody precipitation. All of the values represent the mean ± SEM of triplicate measurements from two independent experiments. *P < 0.05 and ***P < 0.001.

Figure 2

Selection of an optimized lead antibody via consecutive deglycosylation and functional isolation. (A) Schematic illustration of antibody deglycosylation and the phage display technique used to select four deglycosylated IgG clones (deglyco C1‐C4 IgGs). (B) The binding specificities of the four deglycosylated IgG clones to human CTLD‐Fc, mouse CTLD‐Fc, and Fc alone were measured by ELISA. (C) HUVEC tube formation was assessed, and an optimized lead antibody that inhibited CLEC14a‐mediated angiogenesis was selected. Clone 1 IgG was used as the positive control. (D) The total number of tube branches formed was expressed as a percent of tube formation by the control (MOCK). (E) A wound‐healing assay was performed to measure the effect of deglyco C1 IgG on endothelial cell migration. (F) The extent of closure at the wound margins was expressed as a percent of the migration of the control (MOCK). (F) Motility analyses of Clone 1 and deglyco C1 IgG were performed using one‐dimensional gel electrophoresis under reducing conditions. All of the values represent the mean ± SEM of triplicate measurements from two independent experiments. **P < 0.01 and ***P < 0.001.

Figure 3

Elucidation of the mechanism of action of the optimized lead antibody. (A) Images depicting tube formation by HUVECs in the absence (MOCK) or presence of 20 μg·mL⁻¹ deglyco C1 IgG or bevacizumab as the positive control. Images were captured at 18 h. (B) The number of total branches in the absence (MOCK) or presence of 20 μg·mL⁻¹ deglyco C1 IgG or bevacizumab was expressed as a percent of tube formation in the control (MOCK). (C) HEK293F cells transfected with wild‐type CLEC14a were incubated in the absence (MOCK) or presence of deglyco C1 IgG for 6 h. Cell aggregates (aggregate mass > 4 cells; arrowheads) were counted under a light microscope. (D) The number of aggregates per field was expressed as a percent of the control (MOCK). (E) HUVECs were coated onto the wells of a microtiter plate, and binding of hCLEC14a‐(CTLD)‐Fc‐HRP to the HUVECs in the presence or absence of increasing concentrations of deglyco C1 IgG was measured by ELISA. (F) hCLEC14a‐ECD was coated onto the wells of a microtiter plate and binding of hCLEC14a‐CTLD‐Fc‐HRP to hCLEC14a‐ECD in the presence or absence of increasing concentrations of deglyco C1 IgG was measured by ELISA. (G) HUVECs that were incubated in the presence or absence of deglyco C1 IgG were fixed, stained with anti‐CLEC14a antibody, and analyzed by flow cytometry. All of the values represent the mean ± SEM of triplicate measurements from three independent experiments. ***P < 0.001.

Figure 4

Evaluation of the in vitro and in vivo toxicity of the optimized lead antibody. (A) HUVECs were incubated in the absence (MOCK) or presence of deglyco C1 IgG or 5‐FU (positive control) for 2 days. Cell viability was assessed by measuring absorbance at 450 nm. Values represent the mean ± SEM of triplicate measurements from two independent experiments. ***P < 0.001. (B) HUVECs cultured in the presence or absence of deglyco C1 IgG were stained with rhodamine–phalloidin and 4′, 6‐diamidino‐2‐phenylindole, dihydrochloride, and cell morphologies were examined by confocal microscopy. Scale bars = 20 μm. (C) HUVECs were cultured in the presence or absence of hTNFα or deglyco C1 IgG, stained with anti‐ICAM‐1 (upper panel) or VCAM‐1 (lower panel) polyclonal antibody, and analyzed by flow cytometry. hTNFα served as the positive control for endothelial cell activation. Results are representative of three independent experiments. (D) In vivo toxicity was detected based on changes in the serum concentrations of GOT, GPT, blood urea nitrogen, creatinine, and TBIL measured 30 days after antibody injection. (E) In vivo toxicity was also detected based on changes in the body weights of mice between day 1 and day 28 after antibody injection. (F) The apoptotic status of kidney and liver tissues 30 days after antibody injection was analyzed by TUNEL assay. All of the data represent the mean ± SD from four independent experiments.

Figure 5

Effect of the optimized lead antibody on VEGF‐dependent angiogenesis. (A) rhVEGF‐dependent tube formation was assessed in the absence (MOCK) or presence of deglyco C1 IgG or bevacizumab. (B) Total branch numbers were expressed as a percent of tube formation in the control (MOCK). (C) Images of rhVEGF‐dependent vessel sprouting were obtained from sectioned aortic rings cultured in the absence (MOCK) or presence of deglyco C1 IgG or bevacizumab. (D) The numbers of sprouting vessels were counted manually. (E) Nude mice were implanted with Matrigel plugs in the absence (MOCK) or presence of deglyco C1 IgG or bevacizumab, and images of rhVEGF‐dependent microvessel formation were obtained. (F) The extent of microvessel formation was determined by measuring the hemoglobin content, which was expressed as a percent of the hemoglobin content in the control (MOCK). All of the values represent the mean ± SEM of triplicate measurements from two independent experiments. ***P < 0.001. (G) Immunoblot analysis was performed to assess rhVEGF‐dependent phosphorylation of VEGFR, Akt, and ERK in HUVECs in the absence (MOCK) or presence of VEGF or VEGF plus deglyco C1 IgG.

Figure 6

Effect of the optimized lead antibody on tumor angiogenesis. (A) Microvessel formation by SNU182, CFPAC‐1, and U87 cell‐derived tumors was measured in the absence (MOCK) or presence of deglyco C1 IgG or bevacizumab. (B) Hemoglobin content was expressed as a percent of the hemoglobin content in the control (MOCK). (C) Microvessel formation by HCT116 and HCT116/Beva cell‐derived tumors in the absence (MOCK) or presence of deglyco C1 IgG or bevacizumab was determined by immunohistochemistry with anti‐CD31 antibody. Scale bars = 100 μm. (D) CD31 positivity per field was expressed as a percent of the CD31‐positive microvessel density in the control (MOCK). All of the values represent the mean ± SEM of quadruplicate measurements from two independent experiments. BALB/c male nude mice were subcutaneously implanted with U87 glioma cell‐loaded Matrigel plugs, with or without 10 mg·kg⁻¹ deglyco C1 IgG or bevacizumab. Tumor volume (E) and total body weight (F) were measured once a week for 1 month. Tumor images at 1 month after injection are also shown (inset). All of the values represent mean ± SEM. (G) Apoptotic tumor cells were determined by TUNEL staining in control (MOCK) group and deglyco C1 IgG‐ or bevacizumab‐treated groups. Scale bars = 50 μm. (H) TUNEL positivity per field in each group was expressed as a graph bar. All of the values represent the mean ± SEM of quadruplicate measurements. *P < 0.05 and ***P < 0.001.