Large-scale production, purification, and function of a tumor multi-epitope vaccine: Peptibody with bFGF/VEGFA

Abstract

In tumor tissue, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) promote tumorigenesis by activating angiogenesis, but targeting single factor may produce drug resistance and compensatory angiogenesis. The Peptibody with bFGF/VEGFA was designed to simultaneously blockade these two factors. We were aiming to produce this Fc fusion protein in a large scale. The biological characterizations of Peptibody strains were identified as Escherichia coli and the fermentation mode was optimized in the shake flasks and 10-L bioreactor. The fermentation was scaled up to 100 L, with wet cell weight (WCW) 126 g/L, production 1.41 g/L, and productivity 0.35 g/(L·h) of IPTG induction. The target protein was isolated by cation-exchange, hydrophobic and Protein A chromatography, with total recovery of 60.28% and HPLC purity of 86.71%. The host cells protein, DNA, and endotoxin residues were within the threshold. In mouse model, immunization of Peptibody vaccine could significantly suppressed the tumor growth and angiogenesis, with inhibition rate of 57.73 and 39.34%. The Peptibody vaccine could elicit high-titer anti-bFGF and anti-VEGFA antibodies, which inhibited the proliferation and migration of Lewis lung cancer cell cells by decreasing the Akt/MAPK signal pathways. Therefore, the Peptibody with bFGF/VEGFA might be used as a therapeutic tumor vaccine. The large-scale process we developed could support its industrial production and pre-clinical study in the future.

Keywords: Peptibody; bFGF/VEGFA; fermentation; purification; tumor angiogenesis.

FIGURE 1

The biological characterizations of Peptibody strains. (A) The expression levels of Peptibody strains were analyzed by SDS‐PAGE and Western blotting assays. Lanes 1–5, cell lysate at the storage of 1, 3, 6, 9 and 12 months; Lane 6, cell lysate before induction; Lane 7, cell lysate of the empty vector; Lane M, protein molecular weight marker. The target protein was 37.4 kDa in which the red arrow pointed at. (B) The morphology of Peptibody strains was detected by Gram staining and scanning electron microscope assays. Scale bars: (a) 10 µm; (b) 2 µm; (c) 1 µm; (d) 200 nm. (C) The biochemical properties of Peptibody strains were detected by API 20E assay. (D) The species identification of Peptibody strains was conducted by 16S rDNA sequencing. Lane 1, the total bacterial DNA; Lane 2, PCR product of 16S rDNA V4 region; Lane M, nucleic acid marker

FIGURE 2

Optimization of Peptibody production from flask to 10‐L bioreactor fermentation. (A) SDS‐PAGE analysis of flask fermentation optimization. The Peptibody strains were cultured and induced in different conditions. Lane M, protein molecular weight marker; Lane NC‐1, cell lysate before induction; Lane NC‐2, cell lysate of the empty vector. For expression temperature, lanes from left to right, cell lysate at the temperature of 20, 24, 28, 32, and 36℃; For induction concentration, lanes from left to right, cell lysate at the concentration of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mM IPTG; For induction time, lanes from left to right, cell lysate at the induction of 1, 2, 3, 4, 5 and 6 h; For inoculation density, lanes from left to right, cell lysate at the density of 1, 2, 4, and 8%; For DO, lanes from left to right, cell lysate at the dissolved oxygen (DO) above 30, 40, 50, 60 and 70%; For inducer added time, lanes from left to right, cell lysate at the timing of 2, 4, 6, and 8 h. (B) The growth curve and SDS‐PAGE analysis of Peptibody production in 10‐L bioreactor. The fermentation mode were controlled at 37℃, pH 7.0 ± 0.2 and DO 30%. The inoculation density was 2% and the feeding speed was set at 10.0 mL/min. Lane M, protein molecular weight marker; Lanes from left to right, IPTG induction from 6 to 11 h. The target protein was 37.4 kDa in which the red arrow pointed at

FIGURE 3

Large‐scale fermentation of Peptibody production in 100‐L bioreactor. (A) The time‐course profile of Peptibody production in 100‐L bioreactor. The fermentation mode was controlled at 37℃, pH 7.0 ± 0.2 and dissolved oxygen (DO) 30%. The inoculation density was 2% and the feeding speed was set at 100.0 mL/min. The denoted signals were temperature (blue), DO (black), pH (red) and stirr (purple) and the denoted phases were biomass production, feed and expression. (B) The growth and expression curves of Peptibody strains during fermentation. (C) SDS‐PAGE analysis of Peptibody production during fermentation. Lane M, protein molecular weight marker; Lanes from left to right, IPTG induction from 6 to 10 h. The bands corresponding to the target protein were indicated by red arrows, 37.4 kDa

FIGURE 4

Large‐scale purification of Peptibody fusion protein. (A) The purification procedures and SDS‐PAGE analysis of 15.7 mL cation‐exchange chromatography. The supernatant of cell lysate was mixed with PB balance buffer and loaded into the cation‐exchange column. Peaks 1–10, the gradients of NaCl‐PB from 0.1 to 1.0 M. Lanes from left to right, supernatant of cell lysate (load), flow through (FT), elution of 0.1 to 1.0 M NaCl‐PB. (B) The purification procedures and SDS‐PAGE analysis of 465.1 mL cation‐exchange chromatography. Peaks 1–3, 0.5, 0.7, and 1.0 M NaCl‐PB. Lanes from left to right, load, FT and elution of 0.5, 0.7, 1.0 M NaCl‐PB. (C) The purification procedures and SDS‐PAGE analysis of hydrophobic chromatography. The eluted samples of (B) were mixed with 2 M NaCl‐PB balance buffer and loaded into the hydrophobic column. Peaks 1–5, 1.5, 1, 0.5, 0 M NaCl‐PB and H₂O. Lanes from left to right, the eluted sample of (B), the eluted sample of (B) with 2 M NaCl‐PB (load), FT, 1.5 M NaCl‐PB, 1.0 M NaCl‐PB, 0.5 M NaCl‐PB, 20 mM PB, H₂O. (D) The purification procedures and SDS‐PAGE analysis of Protein A affinity chromatography. The eluted samples of (C) were mixed with 20 mM PB balance buffer and loaded into the Protein A column. Peaks 1–2, 10 mM NaCl‐PB and 20 mM CB‐PB. Lanes from left to right, the eluted sample of (C) with 20 mM PB (load), 10 mM NaCl‐PB, 20 mM CB‐PB, the final product concentrated in 0.015 M PBS (final). Lane M, protein molecular weight marker. The lines in blue, red, green, coffee, and purple were UV, condition, concentration, pressure, and pH in the columns, respectively. The bands corresponding to the target protein were indicated by red arrows, 37.4 kDa

FIGURE 5

Purity analysis and the host cells protein (HCP), DNA and endotoxin residues. (A) HPLC analysis of the purified Peptibody. The purified samples were subjected to Ultimate XB‐C4 column and analyzed by Agilent 1260 Infinity system. (B) The residual HCP was detected by ds‐ELISA assay. (a) Standard curve. (b) Detection of three replicates. (C) The residual DNA was detected by qRT‐PCR assay. (a) Total DNA extraction. Lane M, nucleic acid marker. Lane 1, host bacteria; Lanes 2–6, five sample replicates. (b–d) The amplification, melt and standard curves of host DNA. (e, f) The amplification and melt curves of five sample replicates. (g) DNA concentration of five sample replicates. (D) The residual DNA was detected by fluorescent staining assay. (a) Standard curve. (b) Detection of three replicates

FIGURE 6

Inhibitory effects of Peptibody vaccine on tumor growth and angiogenesis in mice. (A) The C57BL/6 mice (n = 4/group) were subcutaneously immunized with PBS and Peptibody vaccine (100 µg/mouse). Ten days after the last vaccination, the mice were inoculated with LL‐2 cells (1 × 10⁶/mouse) and the tumor volume was measured every 3 days. (a) Tumor images. (b) Tumor growth curves. (c) Tumor weight. (B) Representative images and quantitation of microvessels and lymph vessels by immunohistochemistry assay. (a, b, e), tumor section and the quantitation of microvessels. (c, d, f), tumor section and the quantitation of lymph vessels. Scale bars, 100 µm. Data were shown as mean ± SD of three independent experiments (*p < 0.05 for the Peptibody‐vaccinated mice vs. the negative control. p Values were analyzed by two‐way ANOVA test, using SPSS 19.0 software)

FIGURE 7

Inhibitory effects of anti‐Peptibody antibody on the proliferation, migration and Akt/MAPK signal pathways of LL‐2 cells. (A) Immunogenicity of the Peptibody vaccine in mice. The BALB/c mice were immunized with Peptibody vaccine and PBS for three times, respectively. Ten days after the last vaccination, the blood samples were collected. The anti‐Peptibody antibody was extracted and the titers were detected by ELISA assay. (B) The proliferation inhibition assay of LL‐2 cells. The cells (1 × 10³/well) were treated with anti‐Peptibody antibody at the concentration from 12.5 to 400 µg/mL. (C) Western blotting analysis of the phosphorylation of Akt and MAPK in LL‐2 cells. The cells (3 × 10⁵/well) were activated with bFGF and treated with anti‐Peptibody antibody at the concentration from 25 to 400 µg/mL. GAPDH served as the loading control. (D) Transwell chamber migration assay of LL‐2 cells. The cells (3 × 10⁵/well) were incubated with 200 µg/mL anti‐Peptibody antibody in the upper chamber for 24 h. PBS and irrelevant IgG served as the negative control. Scale bars, 50 µm. Data were shown as mean ± SD of three independent experiments (**p < 0.01 for the anti‐Peptibody antibody vs. the control. p Values were analyzed by one‐way ANOVA test, using SPSS 19.0 software)