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Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

This study was aimed to investigate the effect of a single-chain fragment variable antibody of connective tissue growth factor (anti-CTGF scFv) against the differentiation of fibroblast into myofibroblast. The scFv antibody was firstly expressed in Escherichia coli cells and was then purified by affinity chromatography. The yield scFv protein reached a purity over 95% after purification. Immunoreactivity assay demonstrated that scFv possessed a special affinity toward CTGF. RT-PCR, western blot, and immunofluorescence experiments showed that increased expression of α-smooth muscle actin induced by TGF-β1 could be suppressed by this scFv antibody through inhibiting the phosphorylation of Akt.

Background and Objective: Connective tissue growth factor (CTGF) has been identified as playing critical roles in fibrosis and is a promising therapeutic target. In a previous study, we used a phage display library to develop a humanized single-chain variable fragment antibody (scFv) against CTGF. In the present study, the protective effect of anti-CTGF scFv against bleomycin (BL)-induced pulmonary fibrosis was investigated in mice., Methods: The expression of α-smooth muscle actin in human embryonic lung fibroblast (HELF) cells was analysed by western blotting. A mouse model of pulmonary fibrosis was established by tracheal injection of BL (5 mg/kg). Mice received anti-CTGF scFv (4 mg/kg, three times a week) by i.v. injection. The effects of anti-CTGF scFv were evaluated by leukocyte counts in BAL fluid, hydroxyproline measurements in lung tissue and pathological examination., Results: α-Smooth muscle actin expression was decreased in HELF cells treated with anti-CTGF scFv. Anti-CTGF scFv significantly reduced the numbers of inflammatory leukocytes (total and differential count) in BAL fluid, as well as the hydroxyproline content of lung tissue. The severity of alveolitis and fibrosis in the mouse model was markedly attenuated by treatment with anti-CTGF scFv., Conclusions: Anti-CTGF scFv may potentially be developed as a useful inhibitor of pulmonary fibrosis., (© 2011 Southeast University, P.R.China. Respirology © 2011 Asian Pacific Society of Respirology.)

Excessive expression of CTGF (connective tissue growth factor)/CCN2 has been observed in many fibrotic diseases. The inhibition of the CTGF/CCN2 by antibody has been shown to be clinically useful for the management of fibrosis. A phage display humanized single-chain Fv antibody library was screened using CTGF/C (CTGF/CCN2 C-terminal domain) as the target. A phage ELISA was performed after four rounds of biopanning, and ten positive clones were further evaluated by ELISA and were chosen for DNA sequencing. The DNA encoding scFv (single-chain variable fragment) containing a full-length variable domain fragment of heavy chain and light chain of human immunoglobulin was inserted into pET-32(a)+ vector, and the fusion protein (TrxA-scFv) containing a thrombin cleavage site was expressed mainly in soluble form. The scFv was obtained by purified fusion protein digested with thrombin and then separated from the fusion partner TrxA by gel-filtration chromatography. An immunological assay showed that the purified scFv reacted with CTGF/CCN2 in a concentration-dependent manner. The result of the cell migration assay demonstrated that the scFv at 100 ng/ml could effectively inhibit the migration of HUVEC (human umbilical-vein endothelial cells) caused by CTGF/C. The number of migratory cells was significantly decreased as compared with the negative control (1062+/-92 versus 3269+/-288, P<0.001) and the inhibition rate was 90.5%.