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. 2021 Feb 25:11:585457.
doi: 10.3389/fonc.2021.585457. eCollection 2021.

Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2

Simin Zhang  1 , Jiahui Huang  1 , Ligang Zhang  1 , Jiangtao Gu  1 , Qifang Song  1 , Yaxiong Cai  1 , Jiangchuan Zhong  1 , Hui Zhong  2 , Yanrui Deng  1 , Wenhui Zhu  1 , Jianfu Zhao  3 , Ning Deng  1
Affiliations

Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2

Simin Zhang et al. Front Oncol. .

Abstract

Angiogenesis is considered one of the hallmarks of cancer and plays a critical role in the development of tumor. Fibroblast growth factor 2 (FGF-2) is a member of the FGF family and participates in excessive cancer cell proliferation and tumor angiogenesis. Thus, targeting FGF-2 was considered to be a promising anti-tumor strategy. A disulfide-stabilized diabody (ds-Diabody) against FGF-2 was produced in Pichia pastoris (GS115) by fermentation and the anti-tumor activity was analyzed. The novel 10-L fed batch fermentation with newly designed media was established, and the maximum production of the ds-Diabody against FGF-2 reached 210.4 mg/L. The ds-Diabody against FGF-2 was purified by Ni2+ affinity chromatography and DEAE anion exchange chromatography. The recombinant ds-Diabody against FGF-2 could effectively inhibit proliferation, migration, and invasion of melanoma and glioma tumor cells stimulated by FGF-2. Furthermore, xenograft tumor model assays showed that the ds-Diabody against FGF-2 had potent antitumor activity in nude mice by inhibiting tumor growth and angiogenesis. The tumor growth inhibition rate of melanoma and glioma was about 70 and 45%, respectively. The tumor angiogenesis inhibition rate of melanoma and glioma was about 64 and 51%, respectively. The results revealed that the recombinant ds-Diabody against FGF-2 may be a promising anti-tumor drug for cancer therapy.

Keywords: angiogenesis; disulfide-stabilized diabody; fibroblast growth factor 2; glioma cancer; melanoma cancer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The fermentation, purification and binding activity studies of ds-Diabody against FGF-2 in Pichia pastoris. (A) Schematic illustration of the recombinant expression plasmid of pPICZαA-ds-Diabody. (B) The curve of cell wet weight and the production of ds-Diabody against FGF-2 in Pichia pastoris fermentation for strategy II. (C) The antigen combined activity of ds-Diabody against FGF-2 by ELISA. (D) The SDS-PAGE assays of ds-Diabody against FGF-2 in fermentation for strategy II. (E) The purification of ds-Diabody. Lane M, marker; Lane 1, the SDS-PAGE assay of the purified ds-Diabody in reducing condition; Lane 2: the Western blot assays of the purified ds-Diabody.
Figure 2
Figure 2
The tumor proliferation and migration inhibition of ds-Diabody against FGF-2. (A) The proliferation inhibition of ds-Diabody on A375 cells. (B) The proliferation inhibition of ds-Diabody on U87 cells. (C) The migration of A375 cells in different groups. (D) The migration of U87 cells in different groups. (E) The quantitative analysis of the migration rate of A375 cells in different groups. (F) The quantitative analysis of the migration rate of U87 cells in different groups. The results presented as the mean ± SD in triplicate. *p < 0.05 versus Irrelevant Group.
Figure 3
Figure 3
The invasion inhibition and phosphorylation assays of Akt and MAPK in tumor cells treated with ds-Diabody against FGF-2. (A) The invasion and the quantitative analysis of A375 cells in different groups. (B) The invasion and the quantitative analysis of U87 cells in different groups. (C) Western blot assays of Akt and MAPK phosphorylation in A375 cells treated by ds-Diabody against FGF-2. (D) Western blot assays of Akt and MAPK phosphorylation of U87 cells treated by ds-Diabody against FGF-2. The results presented as the mean ± SD in triplicate. *p < 0.05 versus Irrelevant Group.
Figure 4
Figure 4
The tumor inhibition of melanoma by ds-Diabody against FGF-2 in mice. The melanoma (A375) cells (2.0 × 106 cells in 100 μl medium) were subcutaneously injected on the shoulder of BALB/c nude mice (n = 7). When the tumors were palpable, the ds-Diabody against FGF-2 were i.v. injected (5 mg/kg in 100 μl PBS) six times at 3 days intervals. (A) The tumor growth curve in nude mice in different groups. (B) The stripped tumors from nude mice in different groups. (C) The scatter diagram of tumor weights in different groups. (D) The weight curves of nude mice in different groups. (E) Immunohistochemistry analysis of microvessels of tumor tissues. The results presented as the mean ± SD in triplicate. *p < 0.05 versus PBS Group.
Figure 5
Figure 5
The tumor inhibition of glioma by ds-Diabody against FGF-2 in mice. The glioma (U87) cells (5.0 ×106 cells in 100 μl medium) were subcutaneously injected on the shoulder of BALB/c nude mice (n = 7). When the tumors were palpable, the ds-Diabody against FGF-2 were i.v. injected (5 mg/kg in 100 μL PBS) six times at 3 days intervals. (A) The tumor growth curve in nude mice in different groups. (B) The stripped tumors from nude mice in different groups. (C) The scatter diagram of tumor weights in different groups. (D) The weight curves of nude mice in different groups. (E) Immunohistochemistry analysis of microvessels of tumor tissues. The results presented as the mean ± SD in triplicate. *p < 0.05 versus PBS Group.
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