Soluble Production and Function of Vascular Endothelial Growth Factor/Basic Fibroblast Growth Factor Complex Peptide Qing Zhang The State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China

Xuejun Lao Dept. of Gastrointestinal Surgery, The First Clinical School in Jinan University, Guangzhou, China

Jianhua Huang The Clinical Laboratory of the Third Affiliated Hospital in Sun Yat-Sen University, Guangzhou, China

Zhongsong Zhu, Lei Pang, Yong Tang, Qifang Song, Jiangfang Huang, Jie Deng, and Ning Deng Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering, Biomedicine Translational Inst., Jinan University, Guangzhou, China

Qin Yang Guangzhou Ming Kang Bioengineering Limited Company, Guangzhou, China

Aditi M. Sengupta Harvard Medical School, Post Graduate Association, Boston, MA, USA

Likuan Xiong Central Laboratory, Baoan Maternal and Child Health Care Hospital, Shenzhen, China DOI 10.1002/btpr.1997 Published online October 10, 2014 in Wiley Online Library (wileyonlinelibrary.com)

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important proangiogenic factors in tumor procession. The autocrine and paracrine bFGF and the VEGF in tumor tissue can promote tumor angiogenesis, tumor growth, and metastasis. A VEGF/bFGF Complex Peptide (VBP3) was designed on the basis of epitope peptides from both VEGF and bFGF to elicit in vivo production of anti-bFGF and anti-VEGF antibodies. In this study, we reported on the production of recombinant VBP3 using high cell density fermentation. Fed-batch fermentation for recombinant VBP3 production was conducted, and the production procedure was optimized in a 10-L fermentor. The fraction of soluble VBP3 protein obtained reached 78% of total recombinant protein output under fedbatch fermentation. Purified recombinant VBP3 could inhibit tumor cell proliferation in vitro and stimulate C57BL/6 mice to produce high titer anti-VEGF and anti-bFGF antibodies in vivo. A melanoma-grafted mouse model and an immunohistochemistry assay showed that tumor growth and tumor angiogenesis were significantly inhibited in VBP3-vaccinated mice. These results demonstrated that soluble recombinant VBP3 could be produced by largescale fermentation, and the product, with good immunogenicity, elicited production of hightiter anti-bFGF and anti-VEGF antibodies, which could be used as a therapeutic tumor vacC 2014 American Institute of Chemicine to inhibit tumor angiogenesis and tumor growth. V cal Engineers Biotechnol. Prog., 31:194–203, 2015 Keywords: bFGF, VEGF, polypeptide, fermentation, vaccination, tumor, proangiogenic factor

Introduction Tumor growth and metastasis are critically dependent on the development and remodeling of the microvasculature.1 Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the key proangiogenic factors Xuejun Lao and Jianhua Huang contributed equally to this work. Correspondence concerning this article should be addressed to N. Deng at [email protected] 194

in tumor procession.2,3 In many different types of cancer, the expression of VEGF and bFGF is elevated and most tumor cells secrete VEGF and bFGF.4 In tumor’s occurrence and development, the autocrine and paracrine bFGF and VEGF in the tumor tissue can promote tumor angiogenesis, tumor growth, and metastasis.5,6 Furthermore, bFGF and VEGF synergistically promote angiogenesis and tumor growth by enhancing the endogenous platelet derived growth factor B and its receptor b (PDGF-B-PDGFRb) signaling.6–9 Therefore, inhibition of bFGF and VEGF by neutralizing C 2014 American Institute of Chemical Engineers V

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antibodies could be an important strategy for inhibiting tumor angiogenesis and tumor growth. Some epitope peptides mimicking peptides of bFGF selected from a peptide library using phage display technology, and a peptide vaccine based on bFGF have been reported.10–13 The bFGF epitope peptides corresponding to the bFGF heparin binding domain and the bFGF receptor binding domain were used to design vaccines to elicit specific anti-bFGF antibodies to block tumor angiogenesis and inhibit tumor growth and metastasis.10,11 For VEGF inhibition, the immunotherapy strategies of peptides, vaccines, and elicitation of antibodies have been reported.4,14–16 The antigenic B cell and T cell epitope peptides of VEGF were used as a vaccine to elicit high titer specific anti-VEGF antibodies to inhibit tumor angiogenesis, tumor cellular migration, proliferation, and invasion, for use as an immunotherapy.17–19 The clinical achievements with anti-VEGF therapies, such as bevacizumab, a humanized monoclonal antibody, constitute a milestone event for the field of anti-angiogenic research, eliciting survival benefits in many aggressive tumors. However, anti-VEGF treatments are failing to produce enduring clinical responses in most patients, in part due to the upregulation of other angiogenic factors such as bFGF and Platelet Derived Grwoth Factor-bb (PDGF-bb).20,21 The classical types of peptide vaccines only contain one to several epitope peptides, which are recognized by CD81 cytotoxic T lymphocyte (CTLs) or helper T cells. In contrast, the long peptide vaccines usually contain several restricted epitopes that are recognized by both CTLs and helper T cells. Synthetic long peptide vaccines are predominantly taken up by antigen presenting cells (APCs), where they are processed for presentation by both major histocompatibility complex (MHC) class I and II molecules.22 A synthetic long peptide vaccine targeted for p53, consisting of 10 synthetic 25–30 amino acids long overlapping peptides, spanning amino acids 70–248 of the wild type p53 protein, was used for metastatic colorectal cancer therapy.23 A bivalent polypeptide vaccine was designed with HER-2 peptide and VEGF peptide mimics, which combined HER-2 and VEGF-targeting therapy into active immunotherapy with conformational HER-2 B cell epitope vaccines and antiangiogenic therapy with peptides structured to mimic VEGF.24 Here, we designed a long polypeptide targeting both VEGF and bFGF (named VBP3), which was composed of three epitope peptides based on bFGF and three epitope peptides based on VEGF. The peptide fragments were combined with linkers and constructed into expression vector pET32a to encode recombinant VBP3 in E. coli.25 This combination strategy targeting both VEGF and bFGF signaling pathways could elicit the body to produce high titer specific antibodies to inhibit tumor angiogenesis, which could effectively inhibit tumor growth and prevent drug resistance in cancer therapy. In this study, we reported the soluble production of recombinant VBP3 with high density fermentation in a 10-L fermentor and a characterization of its products.

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units/mL penicillin/streptomycin in a 37 C, 5% CO2 incubator with 95% relative humidity. C57BL/6J mice (female, six and seven weeks old) were purchased from the Experimental Animal Center of the Southern Medical University in Guangzhou, China. All the animals used in the experiments were treated humanely in accordance with Institutional Animal Care and Use Committee guidelines. Gene cloning for the VBP3 complex peptide and the construction of the expression vector The peptide candidates of three VEGF epitope peptides and three bFGF epitope peptides were selected and combined together with linkers (GGGS) to construct the VBP3 complex peptide. The bFGF and VEGF epitope peptides were obtained from a phage peptide library scanning13 and prediction analysis according to the methods by Saha et al. and El-Manzalawy et al.26,27 The oligonucleotide fragments of VBP3 complex peptide were synthesized according to predetermined sequences from Sangon Biotech (Shanghai) Co. The synthesized oligonucleotide fragments were inserted into the pET32a vector to construct the expression vector pET32a-VBP3 by using restriction enzymes BamHI and HindIII.25 Expression of recombinant VBP3 in shake flasks Recombinant plasmid pET32a-VBP3 was transformed into competent cells of E. coli ER2566 to obtain recombinant cells. The transformants were selected from LB plates with ampicillin and identified by PCR and restriction enzyme analysis. The transformants were induced to express product by isopropyl b-D-1-thiogalactopyranoside (IPTG), and the cell lysates were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The high expression transformants of VBP3 were selected and cultured in LB medium in a shaking incubator at 37 C overnight, and seed cell aliquots were stored at 280 C. Conditions for inducing expression of product, such as inducing temperatures, media, IPTG concentration, and inoculum concentration were optimized in shake flask cultures. When the optical density (OD600) reached approximately 1.0, induction of the culture was conducted by adding IPTG to a final concentration of 0.2–1.0 mmol/L, followed by shaking for an additional 4–5 h at different inducing temperatures (26–37 C). The broth was collected and centrifuged at 3,000g for 10 min at 4 C. The pellet was re-suspended in phosphate buffered saline (PBS) buffer (10 mM phosphate, 2.7 mM potassium chloride, and 137 mM sodium chloride, pH 7.4) and ultrasonication. The cell lysis solution was centrifuged at 8,000g for 20 min at 4 C, and the resulting supernatant was used for purification or stored in aliquots at 280 C. Fed-batch fermentation of VBP3

Materials and Methods Cell lines and animals The tumor cell lines of melanoma cells (B16) and lung cancer cells (A549) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100

A 10-L fermentor (Biostat B-5, BRAUN, Germany) with pH, temperature, agitation, dissolved oxygen (DO), and air flow control was utilized. The original seed cells were activated and inoculated at 1:100 into 3 L of M9 fermentation media. The aeration rate was initially maintained at 1.0 vvm and increased to 1.5 vvm when required. DO was kept above 30% saturation. The pH of the medium was auto-controlled at 7.2 by adding 25% ammonia solution according to the pH

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feedback. In the fed-batch cultivation phase, the temperature was kept at 37 C. The complementary fresh medium was fed for about 5 h. The fermentation temperature was adjusted to 28 C, and IPTG was fed to keep the final concentration of 0.5 mM. During the procedure of fermentation, timed sampling was performed every hour for biomass and expression level analyses. Biomass analyses The total cell concentrations were spectrophotometrically determined by measuring the absorbance of the broth at 600 nm (OD600). To determine the biomass in terms of the cell weight (one unit of OD600 was found to be equivalent to 0.45 mg dry cell weight), the samples of culture broth (100 mL) were taken in duplicate, centrifuged at 6,000 rpm and washed twice with deionized water. The precipitate was dried to a constant weight at 90 C for 48 h. The cells on the samples acquired at various times were lysed by ultrasonication in PBS and centrifuged at 12,000g. The recombinant soluble target protein in the lysate supernatant and the inclusion body protein in the lysate sediment were analyzed with 12% SDS-PAGE and stained with Coomassie brilliant blue (CBB) according to a standard protocol.28 The total protein in the lysate was detected with bicinchoninic acid assay (BCA) using Protein Assay Reagent (Thermo Co.), and the quality of the target protein was estimated by a densitometry analysis of the SDSPAGE gel by using Quantity One software (Bio-Rad Co.). The specific VBP3 protein expression was detected by Western-blot assay. The SDS-PAGE-separated proteins of cell lysates were transferred to a polyvinyl difluoride (PVDF) membrane. The membrane was incubated with rabbit anti-human bFGF antibodies (1:1,000 dilution in PBS, Sigma-Aldrich) for 1 h at room temperature and washed three times with PBST (0.2% Tween-20, in PBS), followed by incubation of horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies (1:5,000 dilution in PBS, SigmaAldrich) for 30 min at room temperature and then washed six times with PBST. The signal was then detected with an ECL Kit (Amersham Bioscience).

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buffer at 4 C. The target protein components were collected according to the absorbance value and identified by SDSPAGE. The purified VBP3 peptide was used immediately or stored in aliquots at 280 C. Tumor cell proliferation and signal pathway assay Lung cancer A549 cells were transferred to 96-well plates with 1,000 cells per well in the medium of RPMI1640 with 0.5% FBS and 10 ng/mL bFGF. The recombinant VBP3 peptides were added to the 96-well plates in different concentrations of 12.8, 6.4, 3.2, 1.6, and 0.8 lg/ mL. An Internalin A polypeptide (an irrelevant negative control peptide) expressed in E. coli in our lab 29 was used as a control at a concentration of 6.4 lg/mL. The 96-well plates were incubated for 48 h in a 37  C, 5% CO 2 incubator and cytotoxicity of the treatments was assessed with a CCK8 cell counting kit (Sigma-Aldrich). The OD 450 value was obtained in an enzyme linked immunosorbent assay (ELISA) plate reader (MK3, Thermo Lab Systems, USA). The effects of the VBP3 peptide on the pathway assay of Akt and ERK were assayed by Western blot. The lung cancer cells A549 were transferred to 12-well plates with the 3,000 cells per well in the medium of RPMI-1640 with 0.5% FBS and 10 ng/mL bFGF. The recombinant VBP3 peptides were added in different concentrations of 6.4, 3.2, 1.6, and 0.8 lg/mL. The Internalin A polypeptide was the control. The plates were incubated for 5 h at 37 C in a 5% CO2 incubator. The cells were collected and lysed with cell lysis buffer (RIPA buffer, Sigma-Aldrich). The proteins from the cell lysate were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk and incubated with the primary antibodies of anti-phospho-Erk1/2 rabbit mAb and anti-phospho-Akt rabbit mAb (1:1,000 dilution in PBS, Sigma-Aldrich) respectively for 1 h at room temperature, followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution in PBS, Sigma-Aldrich) for 30 min at room temperature. The signals were detected with an ECL Kit (Amersham Bioscience). Nonphosphorylated Erk1/2, Akt, and b-actin were used as reference controls.

Purification of recombinant VBP3 Recombinant VBP3 proteins were purified from the bacterial lysates with affinity chromatography and gel filtration. The expressive recombinant cells were collected by centrifugation at 5,000g, and the cell pellet was lysed by sonication in PBS buffer on ice with four sequential, 30 s periods with 10 s cooling breaks in between. The lysate solution was centrifuged at 15,000g (Beckman J2-21 centrifuge) and the lysate supernatant was collected (the soluble target protein was in the lysate supernatant and the inclusion bodies and other insoluble materials were separated from the soluble components). The supernatant of the cell lysate was mixed with 80 mM imidazole-PBS balance buffer and loaded in a nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography column (Qiagen Co.), equilibrated with 3–4 volumes of 80 mM imidazole-PBS. The other proteins were eluted with 3 volumes of 130 mM imidazole-PBS. The target protein was eluted with 2 volumes of 500 mM imidazole-PBS. The eluted target protein solution was concentrated with ultrafiltration (ultrafilter purchased from Millipore Co.) and loaded in a gel filtration column (GE Co.) and eluted with PBS

Vaccination and tumor challenge C57BL/6 female mice were vaccinated with the VBP3 vaccine and challenged with melanoma B16 cells as reported previously.17,30 The mice (five per group) were vaccinated three times at 10-day intervals with recombinant VBP3 peptide (at a dose of 5 mg/kg) emulsified completely with an equal volume of Freund’s adjuvant (SigmaAldrich). In the control group, the mice (n 5 5) were vaccinated with a mixture of Internalin A (an irrelevant polypeptide, with the dose of 5 mg/kg) and adjuvant. Ten days after the last vaccination, the blood samples were collected from the caudal veins of the mice for antibody titer analysis, and the mice were injected subcutaneously with 4 3 105 B16 tumor cells in the left shoulder flank. The tumors were assessed by palpating the injection site, and the mice were killed upon the development of 10–15 mm tumors in the control group. The tumor size was measured every day. The tumor volume was determined by the following formula: Tumor volume (mm3) V5 p/6(a 3 b2). a 5 length, b 5 width.

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Figure 1. The construction and expression of recombinant VBP3. A: Combination schematic diagram of peptide candidates and linkers and construction of vector expression. B: Identification of expression vector with restriction enzyme assays and PCR. Lane 1, plasmid; Lane 2, enzyme digestion analysis with BamHI and HindIII; and lane 3, PCR analysis. C: SDS-PAGE and Western-blot analysis of recombinant VBP3 expression. 1, SDS-PAGE results for the empty vector; 2, SDS-PAGE results from samples taken before inducing; 3. SDS-PAGE results of samples after inducting; 4 Western-blot results of samples from empty vector; 5, Western-blot results of samples from inducing VBP3 expression (Rabbit anti-human bFGF antibodies were used as the primary antibody and HRP-conjugated goat anti-rabbit antibodies were used as the secondary antibody).

Immunogenicity analyses Blood samples were collected from the caudal veins of all vaccinated mice before the tumor challenge and after the mice were sacrificed. The specific anti-bFGF and anti-VEGF antibodies were detected by indirect ELISA. The recombinant bFGF (Sigma-Aldrich) and VEGF (Invitrogen) were dispensed at 50 ng/ well into 96-well plates. The 96-well plates were blocked with 5% nonfat milk and then incubated with a serial dilution of sera from VBP3 and PBS-vaccinated mice for 2 h at 37 C, washed with PBS-T and incubated with HRP-conjugated goat anti-mouse IgG (dilution of 1:6,000 in PBS, Sigma-Aldrich). The OD405 values were detected in an ELISA plate reader. The antibody titers were determined as previously described31 and defined as the reciprocal of the serum dilution with an absorbance of two standard deviations above the mean OD value of normal mouse serum.

Immunohistochemistry analyses Tumor samples were collected from the mice and fixed with formalin. The paraffin-embedded and formalin-fixed samples were cut into 5 mm sections, which were subsequently processed for immunohistochemistry according to

the standard experimental protocol according to the Immunohistochemistry Kit (Santa Cruz Biotechnology). The sections were incubated with the primary antibody (rabbit anti-mouse CD31 IgG, 1:500 diluted in PBS) for 1 h at 37 C, the secondary antibody (HRP-conjugated goat anti-rabbit IgG, 1:5,000 dilution in PBS) for 30 min at 37 C, and then stained with 3, 30 -diaminobenzidine (DAB) and hematoxylin (H.E.). The vessel density was determined by counting the number of microvessels per high power field within the sections, as described.32 Statistical analysis Statistical significance of compiled data from individual experiments was determined using ANOVA test with the software of Sigma-Plot 10.0

Results Construction strategies for complex peptide VBP3 in E. coli Six peptide candidates were used to construct the VBP3 complex peptide. Three VEGF epitope peptides were used,

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Figure 3.

Figure 2. Soluble expression of VBP3 at different inducing temperatures. A: Soluble expression of VBP3 at different inducing temperatures in shaking flasks. Soluble expression of the target protein was decreased with the increased inducing temperature while the total expression level was increased. Up to 28 C, the expression level of the total target protein was maintained in a high expression level while the soluble expression got decreased. The illustrated values represent the mean number (6SEM) of three separate experiments. B: SDS-PAGE analysis of VBP3 expression in different temperatures. Lane M, protein molecular weight marker; Lane 1, cell lysate before induction; Lanes 2–6, total recombinant protein of cell lysate at the temperatures of 22 C, 25 C, 28 C, 32 C, and 37 C, respectively; Lanes 7–11, protein in the cell lysate sediments of cell lystae at the temperatures of 22 C, 25 C, 28 C, 32 C, and 37 C, respectively; Lanes 12–16, the protein in the supernatant of the cell lysate at the temperatures of 22 C, 25 C, 28 C, 32 C, and 37 C, respectively.

namely V1, QKRKRKKSRYKS (125–136); V2, VASAVFYSALVE (105–117); and V3, IHPDGRVDGVREKS (which mimics peptides scanned from the phage peptide library.13 Three bFGF peptides were used, named B1, FAMKEDGRLLASK (75–86); B2, RPKKDRARQEKKS (34–48); and B3, RLESNNYNTYRSRKYTS (97–113). The peptide candidates were combined together with the soft linker (GGGGS) according to Figure 1A. The expression vector plasmid pET32a-VBP3 was transformed to E. coli strain of ER2566. Highly expressing recombinant cells were selected and the recombinant VBP3 peptide was highly expressed in E. coli cells (Figure 1C). The soluble expression of recombinant VBP3 The further expression assay showed that the induction temperature could influence the soluble expression of

Growth curve and expression analyses of VBP3 in fed-batch fermentation. A: A growth curve and expression analysis of VBP3 in fedbatch fermentation. The growth curve and the expression of VBP3 in two independent fed-batch fermentations in a 10-L fermentor. In the cell culture phase, the fresh media was fed and the conditions were controlled with the pH 7.2, DO 30%, and the temperature at 37 C. During the inducing phase, the temperature was adjusted to 28 C. The feeding speed of fermentation I was 3.5 mL/min. By adjusting the feeding speed to 4.5 mL/min for fermentation II, the growth speed and expression level got significantly improved. The data were the mean number (6 SEM) of groups consisting of three observations. B: SDS-PAGE analysis of the time samples of fermentation II. The results showed that the target protein was expressed in 0.5–5 h, and the expression level increased quickly when the induction time was prolonged. The highest expression level was reached in 3–4.5 h in inducing cultivation at 28 C. C: SDS-PAGE analysis of the timing samples of fermentation I. The target protein was increased a bit more slowly than that of fermentation II. D: SDS-PAGE analysis of purification of recombinant VBP3. The Ni-NTA affinity chromatography and gel filtration chromatography were used to purify the recombinant VBP3. The cell lysate supernatant of the fermentation products was mixed with PBS balance buffer and loaded into the Ni-NTA affinity chromatography column. The target proteins were eluted with 150 mM imidazole-PBS. The eluted samples were concentrated by ultrafiltration and loaded into the gel filtration column. The target protein component was eluted with PBS buffer.M: Protein molecular weight marker. Lanes 1–9, expression analysis of timing samples during the fermentation. Lane 10, supernatant of cell lysate. Lane 11, purified VBP3 peptide with Ni-NTA affinity chromatography. Lane 12, purified VBP3 peptide with gel filtration chromatography.

recombinant VBP3 in shaking flasks. The formation of inclusion bodies occurred heavily when the inducing temperature was 37 C. More than 75% of the target proteins were expressed in inclusion bodies. By decreasing the inducing temperature, the soluble expression increased. When induced at 22 C, the percentage of soluble target protein expressed was more than 89% of the total target protein, but the absolute amount of total target protein was much decreased. Figure 2 shows that the expression level of the total target

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Figure 4. VBP3 peptide inhibits tumor cell proliferation, and ERK and Akt activation assay. A: The proliferation inhibition assay of tumor cells by recombinant VBP3 peptide. A549 cells were treated with serially diluted VBP3 peptides in 96-well plates. (The negative control group was treated with the irrelevant peptide Internalin A). The proliferation inhibition rates were assayed with CCK8 Kit after 48-h incubation at 37 C, 5% CO2 incubator. The percentage (%) of proliferation inhibition of tumor cells under different concentration of recombinant VBP3 peptide is shown. Compared with negative control, tumor cell proliferation was inhibited significantly by recombinant VBP3 peptide when the concentration was up to 0.8 lg/mL. The data were represented as the mean6 S.D. Asterisks indicated significant differences (n 5 3 replicates; *P < 0.05; **P < 0.01). P values were calculated using one-way ANOVA test with the software of SigmaPlot 10.0). B: The Western-blot assay of phosphorylation of ERK and Akt. bFGF-induced A549 cells were treated with serially diluted recombinant VBP3 peptides in 12-well plates, and the cell lysates were collected and the phosphorylated and total levels of Erk1/2 and Akt were detected by Western blot analysis. b-actin served as the loading control. Lane 1, negative control (A549 cells treated with 10 ng/mL bFGF in PBS). Lane 2, A549 cells treated with the irrelevant peptide as a negative control at the concentration of 6.4 lg/mL. Lanes 3–6, A549 cells treated with VBP3 peptide in the concentration of 0.8, 1.6, 3.2, and 6.4 lg/mL respectively.

protein was increased, and soluble VBP3 decreased with increasing temperature. When the induction temperature was at 28 C, the soluble target protein could reach 78% and the total output could be maintained at a relatively high level. Fed-batch fermentation of recombinant VBP3 engineering cells Key fermentation factors were sought from among media compositions and cultivation conditions for efficient VBP3 production using recombinant E. coli. The standard fed-batch fermentation was performed for two stages of cell growth, and the inducing culture in a fermentor. It was important to maintain high cell-density fermentation and high level of soluble expression for recombinant VBP3. During the initial fermentation phase for logarithmic growth, it was important to maintain 30% DO, constant feeding, and pH feedback. The feeding speed of fresh supplemental media could affect the cell’s growth. When the feeding speed was increased from 3.5 mL/min in fermentation I to 4.5 mL/min in fermentation II, the cell growth rate exponentially increased to maintain a quasi-steady specific growth rate. The output of the recombinant VBP3 also improved significantly (Figure 3A). During the inducing phase, the key was to obtain soluble expression by controlling the induction temperature and fermentation conditions. The high-density fermentation was established and a high level of soluble target protein expression of the target protein was obtained in fermentation II. About 560 mg/L total target proteins were obtained with 78% soluble expression by fed-batch fermentation at a feeding speed of 4.5 mL/min and induction at 28 C. Tumor cell proliferation and signal pathway assay The effects of recombinant VBP3 peptide on tumor cell proliferation were assayed with CCK8 reagents. A549 cells were treated with serially diluted recombinant VBP3

peptides in 96-well plates. The negative control group was treated with the irrelevant peptide Internalin A. The results showed that recombinant VBP3 could inhibit tumor cell proliferation effectively (Figure 4A). The proliferation of A549 cells was inhibited by recombinant VBP3 peptide on a dosedependent manner. Compared with negative control, tumor cell proliferation was inhibited significantly by recombinant VBP3 peptide when the concentration was up to 0.8 lg/mL (statistical analysis was conducted using one-way ANOVA). For assays of phosphorylation of ERK and Akt, bFGFinduced A549 cells were treated with serially diluted recombinant VBP3 peptides in 12-well plates. The cell lysates were collected and the phosphorylated and total levels of Erk1/2 and Akt were detected by Western blot analysis. The results of a Western-blot assay showed that the phosphorylation level of ERK and AKT in A549 cells was inhibited by recombinant VBP3 peptide. Akt and ERK are the main signal pathway molecules activated by bFGF and VEGF to promote cell proliferation. The phosphorylation of Akt and ERK were reduced gradually by recombinant VBP3 peptide in a dose-dependent manner (Figure 4B). Immunogenicity analyses of recombinant VBP3 In order to evaluate the immunogenicity of VBP3 produced by fermentation, mice were subcutaneously vaccinated with the purified VBP3 using different doses. After the third vaccination, blood samples were collected from the mice and the specific anti-bFGF and anti-VEGF antibodies were detected. The results showed that the VBP3 complex peptide could effectively elicit high titer anti-bFGF and anti-VEGF antibodies in response to a peptide dose of 5 mg/kg in mice. The antibodies reacted against both bFGF and VEGF with endpoint titers of 1:64,000 and 1:32,000, respectively (the antibody titer was defined as the serum dilution that gave an OD value of two standard deviations above the mean OD value for normal mouse serum). In contrast, the serum

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Figure 6. Tumor growth curves in mice vaccinated with recombinant VBP3 vaccine. Figure 5. Immunogenicity assays of recombinant VBP3 in vivo. The mice (five per group) were vaccinated three times at 10day intervals with the recombinant VBP3 vaccine (5 mg/kg), or they were vaccinated with irrelevant polypeptide (as a negative control, 5 mg/kg). 10 days after the last immunization, the blood samples were collected and the anti-bFGF and antiVEGF antibodies were analyzed by indirect ELISA. Specific anti-bFGF and anti-VEGF antibodies could be detected in the serum of VBP3-vaccinated mice, and there’s no specific immunoreactivity to bFGF and VEGF in the serum of the control group (the irrelevant peptide-vaccinated mice). The data were represented as the mean 6 S.D. (n 5 5 mice/group).

The mice (five per group) were vaccinated with recombinant VBP3 vaccine. 10 days after the last immunization, the mice were challenged with melanoma B16 cells (4 3 105) and the tumor sizes were measured every day. The tumor growth in the VBP3 vaccination group was significantly slower than the negative control (irrelevant polypeptide-vaccinated mice, n 5 5). The data were presented as the mean tumor volume and S.D. (**P < 0.01 for the VBP3 vaccination group vs. the negative control, P values were calculated using two-way ANOVA test with the software of SigmaPlot 10.0).

high titer anti-VEGF and anti-bFGF antibodies, but could also inhibit tumor angiogenesis and tumor growth.

collected from mice treated with PBS did not exhibit any immunoreactivity (Figure 5).

Discussion

Tumor challenge assays in the vaccinated mice To evaluate the effects of protective anti-tumor immunity, the mice vaccinated with the recombinant VBP3 were grafted with melanoma in the left shoulder flank. Melanoma cells are a tumor cell type with high expression of VEGF and bFGF proangiogenic factors. Tumors were assessed by palpation of the injection site, and the mice were killed upon development of 10–15 mm tumors. In the course of tumor growth in vaccinated mice, the tumor size was inhibited and progressed steadily, while in the negative control group, tumor kept progressing fast. Statistical analysis of tumor volume in recombinant VBP3 vaccination group relative to the control group was conducted using two-way ANOVA. The tumor growth in recombinant VBP3 vaccination group was significantly slower than that of the negative control group (Figure 6). Tumor microvessels were analyzed with immunohistochemistry using anti-CD31 antibodies. The microvessels in the tumor sections were visualized based on a standard assay wherein the blood vessel endothelial cells were stained with an anti-CD31 antibody. Figure 7 shows that the tumor microvessels were stained brown. There was a high density of CD31 immunopositive microvessels in the tumor sections of the PBS group. In the tumor sections of VBP3 vaccination group, there were few microvessels. The number of microvessels visible in five high power fields was counted. The tumor microvessels were decreased significantly in the VBP3 vaccination group relative to the control group (statistical analysis was conducted using two-way ANOVA). These results demonstrated that the recombinant VBP3 produced by fermentation could not only induce the mice to produce

VEGF and bFGF are the key proangiogenic factors promoting tumor angiogenesis synergistically in tumor progression.2,3 Complex peptide VBP3 was designed with the six epitope peptides based on VEGF and bFGF. We hypothesized that the complex peptide VBP3 could not only inhibit tumor cell’s proliferation by competitively binding with receptors, but also elicit the body to produce high titers of both anti-bFGF and anti-VEGF antibodies to inhibit tumor angiogenesis, tumor growth, and metastasis. The complex peptide VBP3 was constructed into an E. coli expression system, and the high level expressed transformants were selected. The main purpose of recombinant protein expression is to obtain a great quantity of soluble products from the bacterial cells. In this expression system, it is not always successful. The availability of high-yield soluble recombinant protein in this expression system is reportedly affected by many factors, including bacterial strains, vectors, medium composition, inducer, induction temperature, and fermentation parameter controls.33,34 First, the target protein may be aggregated into insoluble inclusion bodies in the bacterial cells. The inclusion body proteins are, in generally, misfolded and biologically inactive. Aggregation is favored at higher temperatures due to the strong temperature dependence of hydrophobic interactions that determined the aggregation reaction.35 However, a sudden decrease in the cultivation temperature could inhibit replication, transcription, and translation. The promoters used in vectors for recombinant protein expression are also strongly affected. Heat shock protease may be partially eliminated at low temperatures, which might reduce the formation of inclusion body and increase peptide stability and potential for correct folding.36 Reducing the temperature could limit the

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Figure 7. Tumor microvessel assays by immunohistochemistry. A: Tumor section from the negative control group. B: Tumor section from recombinant VBP3 vaccination group. C: Tumor section with no-primary antibody control. D: Quantification of the microvessels. The tumor sections were incubated with primary antibody of rabbit anti-mouse CD31 IgG for 1 h at 37 C, and incubated with secondary antibodies of HRP-conjugated goat anti-rabbit IgG for 30 min at 37 C. The number of microvessels was counted in five high power fields (the arrows show the microvessels). The number of microvessels in the tumor section of the recombinant VBP3 vaccination group was significantly lower than that in control group. The data were represented as the means 6 S.D. (n 5 5; **P < 0.01, P values were calculated using two-way ANOVA test with the software of SigmaPlot 10.0).

aggregation of recombinant proteins and improve the solubility of the recombinant proteins.37,38 In bioreactors, E. coli could be successfully cultivated at high cell densities by applying the fed-batch principle, which has been widely used since the late 1980s.39,40 The key to high cell-density cultivation is to achieve the lowest cost and largest-scale production of the recombinant protein.33,34,41 The production procedure could increase the concentration of the target protein in favor of extraction and purification, and decrease the volume of the reactor in the same production. Ripathi et al. (2009) reported fed-batch fermentation in a 5-L bioreactor with TY medium. They adjusted the cultivation time to change the cells yield. The cells were gown up to 8 h prior to inducing cultivation, and the dry cell weight was up to 14.35 g/L.42 Krause et al. (2010) used an enzymatic glucose release system together with a combination of mineral salts and complex medium that provided high cell densities, high protein yields, and a considerably improved proportion of soluble proteins in harvested cells.43 They controlled cell growth by glucose-limited fed-batch and slowed the growth period during which the recombinant protein was slowly synthesized and folded. Compared to standard cultures in LB, they achieved over 10-fold higher volumetric yields of soluble recombinant proteins. In this study, the standard fed-batch fermentation was used. During the early fermentation phase for logarithmic growth, DO was maintained 30%, pH was controlled with pH feedback, and the cells were supplemented with media feeding at 4.5 mL/min, which was exponentially varied over time in order to maintain a quasi-steady specific growth rate. The cell densities corresponding to 10–12 g/L cell dry weight could be achieved. The soluble expression of recombinant VBP3 was greatly influenced by the inducing temperature. Most target protein was expressed in the form of inclusion bodies when induced at 37 C. The consequence of temperature reduction was an increase in soluble target protein. On the other hand, the amount of biomass and the expression level of the total target protein decreased at a low induction temperature. In consideration of output of target protein and its solubility, the induction temperature was set at 28 C.The soluble recombinant VBP3 could reach 78% of total target protein, and the total target protein production could be maintained at a high level. In this fermentation,

high cell-density fermentation and a high level soluble expression of the recombinant VBP3 were realized. Lung cancer (A549) cells are cells with autocrine release of bFGF and VEGF,4 which were selected to assay the effects of VBP3 peptide on tumor cell proliferation. The results showed that the proliferation of A549 cells were effectively inhibited by recombinant VBP3 peptide, and the inhibition rate reached about 35% at a concentration of 12.8 lg/mL of recombinant VBP3 peptide. Akt and ERK are the main signaling pathways activated by bFGF and VEGF to promote cell proliferation.44 The results of Western-blot assay of the signal pathway showed that the phosphorylation of Akt and ERK as induced by bFGF could be inhibited by a recombinant VBP3 peptide in a dose-dependent manner. The recombinant VBP3 peptide could also elicit production of high titer anti-VEGF and anti-bFGF antibodies when introduced in vivo. A strategy vaccination in anti-angiogenic therapies targeting tumors is to induce a specific, strong, and persistent immune response leading to the eradication of cancer. Antiangiogenic vaccine approaches have shown promising results in reducing tumor growth and metastases. An endothelial cell vaccine was reported to demonstrate antitumor activity and vaccines targeting specific angiogenic targets have been reported.45 Peptide vaccines based on VEGF have been used to elicit the body to produce anti-VEGF antibodies to inhibit tumor angiogenesis reducing tumor growth and metastases.15,17,24,46 Moreraa et al. (2010) developed a therapeutic cancer vaccine (CIGB-247) with recombinant modified human VEGF expressed in E. coli as antigen, that when combined with an adjuvant of proteoliposomes (VSSP) could block tumor growth and increase animal’s survival.46 Wang et al. (2010) synthesized the B-cell epitopes of the VEGF (127–144) peptide. Using mice immunized with this peptide, the specific anti-VEGF antibody from the high titer antiserum could be separated. The separated anti-VEGF peptide antibodies inhibited cellular migration, proliferation, invasion, tube formation, and the growth of aortic ring cultures.17 Peptide vaccines of bFGF could also elicit high-titer antibFGF antibodies to inhibit tumor angiogenesis for tumor therapy. Plum et al. (2000) designed vaccines with epitope peptides from the heparin binding domain and receptor domain of bFGF to elicit specific anti-bFGF antibodies to

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block tumor angiogenesis and inhibit tumor growth and metastasis.10 In our study, recombinant complex peptide VBP3 vaccine elicited excellent immunoreactions in mice and elicited production of specific high-titer anti-VEGF and anti-bFGF antibodies at the same time, which could efficiently inhibit tumor growth and metastases in vivo. This combination strategy targeting both VEGF and bFGF signaling pathways to inhibit angiogenesis might effectively prevent drug resistance in cancer therapy. Our results indicated the potential for large-scale VBP3 production of VBP3 by E. coli fermentation to produce VBP3 complex peptide vaccine for tumor therapy.

Acknowledgments This work was supported by grants from the State Natural Science Foundation of China (81372281), the Shenzhen Science and Technique Plan (JCYJ20140416085544636), and the Fundamental Research Funds for the Central Universities (21612112). Authors thank Miss Mu G (Mingkang Bioengineering Inc, Guangzhou, China) for project management assistance and Dr. Ruan Y (Life Science school in Jinan University, Guangzhou, China) for technical support for the fermentation.

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