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.

C‐type lectin domain group 14 proteins in vascular biology, cancer and inflammation

“…tested a human colorectal cancer cell line HCT116 as well as a bevacizumab‐resistant version of this line. Both cell lines showed significant reductions in in vivo angiogenesis following treatment with CLEC14A antibodies when these cells were embedded in Matrigel and injected subcutaneously . This suggests a possible use for targeting of CLEC14A in patients that have acquired resistance to VEGF blockade.…”

“…Finally, these antibodies could reduce HUVEC cell migration and tube formation based on in vitro assays. Further studies optimised the solubility and stability of the CLEC14A CTLD‐targeting antibodies and showed that they could block angiogenesis in mouse models utilising Matrigel plugs injected with recombinant VEGF or human tumour cells . Collectively, these results suggest that the CTLD of CLEC14A has functional roles in angiogenesis.…”

Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin-binding protein

“…The C-type lectin domain has been shown to engage other matrix proteins, such as multimerin 2 (MMRN2) and heat-shock protein 70-1A (61,62). Antibodies blocking these interactions or targeting the C-type lectin domain have been shown to decrease cell migration and tumor angiogenesis in a manner dependent on MMRN2 (42,62) or VEGFA (68). Whether these antibodies block the GAG-binding site is not known.…”

C‑type lectin family XIV members and angiogenesis: A review

“…CLEC14A has also been reported to regulate pro-angiogenic phenotypes such as filopodia formation, cell migration and tube formation in HUVECs (31,32); it localizes to the inter-cellular boundary and regulates cell adhesion through its CTLD (31). Targeted neutralization of CLEC14A using an anti-CTLD antibody was shown to inhibit endothelial cell migration, cell-cell contact and tube formation by blocking CTLD-CTLD interactions, and downregulating CLEC14A expression at the endothelial cell surface (36,37). A Stable Isotope Labelling with Amino Acids in Cell Culture-based proteomics study showed the upregulation of CLEC14A expression during tubule morphogenesis, and the analysis of post-translational modifications of CLEC14A identified a phosphorylation site at Ser483, near the PDZ-binding domain in the cytoplasmic tail (38).…”