VEGF-targeted scFv inhibits corneal neovascularization via STAT3 pathway in alkali burn model

Abstract

We developed a novel anti-vascular endothelial growth factor (VEGF) single-chain variable fragment (scFv) and evaluated its anti-inflammatory and anti-corneal neovascularization (CoNV) effects in the alkali burn model. VEGF-scFv was prepared from the derived sequence of bevacizumab with a glycine and serine repetitive linker. After being evaluated using surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and cellular fluorescent staining, VEGF-scFv showed high specificity and good affinity with VEGF antigen and HUVEC cells. The KD value was 3.07 ± 0.37 nM and the Bmax value was 0.23 ± 0.01 pmol (n = 3). The corneal alkali burn model was built on male Sprague Dawley (SD) rats. ScFv was injected subconjunctivally immediately after the alkali burn. On 1, 3, 7, and 14 d after alkali burn, the area of CoNV in the VEGF-scFv group was similar to that of the dexamethasone group (2 mg/mL) and significantly smaller than the 0.9% NaCl and the bevacizumab control groups (2 mg/mL). Especially on 14 d, the CoNV area was 45.15% ± 8.46% in the VEGF-scFv group. Hematoxylin and eosin staining (HE) confirmed that few new blood vessels in the VEGF-scFv group. Both immunofluorescence staining and western blotting verified that the expression levels of VEGF, vascular endothelial growth factor receptor 2 (VEGFR2), and signal transducer and activator of transcription 3 (STAT3) in the burned corneal tissue in VEGF-scFv group were significantly lower than other groups (P < 0.05), suggesting this scFv inhibited CoNV by regulating the VEGF/VEGFR2/STAT3 pathway. Therefore, our study prepared an anti-VEGF scFv which may provide a potential treatment for corneal alkali burn for future clinical application.