Inhibition activity of a disulfide-stabilized diabody against basic fibroblast growth factor in lung cancer

Abstract

The over-expression of basic fibroblast growth factor (bFGF) plays a crucial role in the development, invasion and metastasis of lung cancer. Therefore, neutralizing antibodies against bFGF may inhibit the growth of lung cancer. In this study, a Disulfide-stabilized diabody (ds-Diabody) against bFGF was constructed by site-directed mutation and overlap extension PCR (SOE-PCR) at the position of VH44 and VL100 in the scFv. The ds-Diabody was constructed and expressed in Pichia pastoris. We found that the ds-Diabody against bFGF could efficiently suppress the proliferation, migration and invasion of human lung cancer A549 cells in vitro. Moreover, in A549 cells, the ds-Diabody against bFGF could inhibit bFGF-induced activation of downstream signaling regulators, such as phospho-Akt and phospho-MAPK. In the nude mouse xenograft model of lung cancer, the ds-Diabody against bFGF could significantly inhibit tumor growth and decrease the densities of micro-vessels and lymphatic vessels in tumor tissue. Our data indicate that the ds-Diabody against bFGF could effectively suppress the lung cancer growth through blockade of bFGF signaling pathway and inhibition of tumor angiogenesis, which may make it a potential therapeutic candidate antibody drug for human lung cancer therapy.

Keywords: angiogenesis; bFGF; ds-diabody; lung cancer; lymphangiogenesis.

Figure 1. Construction of the ds-Diabody against bFGF

a. The ds-Diabody against bFGF was constructed by introducing disulfide bonds between VL and VH. b. Schematic representation of the construction of ds-Diabody against bFGF

Figure 2. Purification and identification of the ds-Diabody against bFGF by SDS-PAGE and western-blot

a. SDS-PAGE of ds-Diabody against bFGF. Lane M: Protein molecular weight marker; Lane 1: Proteins from culture supernatant; Lane 2: Other proteins; Lane 3: Fractions obtained by Ni Sepharose affinity chromatography and anion-exchange chromatography; Lane 4: Other proteins. b. Western-blot assay of ds-Diabody against bFGF. Lane 1: Western blot assay of the ds-Diabody against bFGF under reducing condition; Lane 2: Western blot assay of the ds-Diabody against bFGF under non-reducing condition

Figure 3. Antigen binding activity of the ds-Diabody and full-length human antibody against bFGF were assayed by indirect ELISA

Figure 4. Proliferation inhibition effects of the ds-Diabody against bFGF on lung cancer cells

The lung cancer A549 cells (2×10³ cells/well) were transferred to 96-well plates and treated with 15 ng/mL bFGF plus ds-Diabody at serially diluted concentrations. The results of CCK-8 showed that ds-Diabody against bFGF could inhibit the proliferation of lung cancer cells in dose dependent. The data were represented as the mean±SD of three independent experiments performed in triplicate

Figure 5. Western-blot assays of Akt and MAPK phosphorylation in lung cancer cells treated with the ds-Diabody against bFGF

The A549 cells (2×105 cells/well) transferred in 6-well plates were serum-starved cultured and treated with 15 ng/mL bFGF and serially concentrations of the ds-Diabody and incubated for 30 min. The cell lysates were collected and transferred in PVDF membrane for western-blot assay. The primary antibodies were anti-MAPK, anti-p-MAPK, anti-Akt and anti-p-Akt. The β-actin was served as the reference control. a. Akt and MAPK phosphorylation of human lung cancer A549 cells treated by various concentrations of ds-Diabody against bFGF (1-100 μg/mL) for 30 min. b. Quantitative analysis of phosphorylated/total Akt. c. Quantitative analysis of phosphorylated/total MAPK

Figure 6. The migration inhibition of A549 cells by ds-Diabody against bFGF

a. The migration of A549 cells in different conditions at different times. b. The quantitative analysis of the migration rate of different groups. The results indicated that the ds-Diabody against bFGF could significantly inhibit the migration of A549 cells when compared with the Irrelevant IgG group (*P < 0.05, **P < 0.01)

Figure 7. The invasion inhibitory of A549 cells by the ds-Diabody against bFGF

The cells were cultured in serum-free medium containing 15 ng/mL bFGF in the upper chamber and were chemo-attracted by the 10% serum in the lower chamber. The invasion cells migrated into the lower side were stained by crystal violet and imaged with a computerized imaging system. a. A549 cells were treated with DMEM serum-free medium. b. A549 cells were treated with the irrelevant IgG. c. A549 cells were treated with the ds-Diabody against bFGF. d. A549 cells were treated with the full-length human antibody against bFGF. e. The quantitative analysis of A549 invasion. The number of cells observed from serum-free DMEM medium group was set as 100. The data were represented as the means ± SD. *P<0.05; **P<0.01

Figure 8. The inhibition of tumor growth by the ds-Diabody against bFGF in mice model

The lung cancer cells (A549, 1×106 cells) were injected in the shoulders of BALB/c nude mice (n=6). After the tumor palpable, the mice were intravenously injected with the ds-Diabody against bFGF (10 mg/kg) six times with 3 days interval and the tumor volume was measured at different time-points after treatment. a. Tumor growth curve in different groups. b. Stripped tumors in different groups. c. Quantitative analysis of the tumor weight. d. The growth inhibition rate of tumors in different groups. The data were represented as the means ± SD (error bars) from 6 animals. *P<0.05; **P<0.01

Figure 9. Immunohistochemical analyses of tumor tissues

a. Paraffin sections of A549 tumors were stained for vascular endothelial cells with anti-CD31 antibody and lymphatic endothelial cells with anti-LYVE1 antibody respectively. b. Quantitative analysis of microvessel density and lymphatic vessels density. The number of blood vessels and lymphatic vessels at 5 high-power fields (x400) per section were counted. The data were represented as the means ± SD (error bars). *P<0.05; **P<0.01