Fermentation, purification and immunogenicity of a recombinant tumor multi-epitope vaccine, VBP3

Highlights

• The large-scale fermentation process of a recombinant tumor multi-epitope vaccine VBP3.

• Lactose is an economical, safe and industrial inducer for VBP3 fermentation..

• The large scale industrial conditions for producing VBP3 were established.

Abstract

The recombinant multi-epitope vaccine called VBP3 is designed to suppress tumor growth and angiogenesis through targeting both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA). We are aiming to produce VBP3 vaccine in a large scale and provide sufficient protein for pre-clinical study. High cost and potential toxicity are severe limitations of IPTG and we investigated whether lactose can mediate VBP3 induction. Firstly, we identified the biological characteristics and established a culture bank of VBP3 strains. The best-performing strains were selected and the fermentation mode of medium, bacterial growth and protein expression were optimized in shake flasks. We scaled up the VBP3 production in 10 L bioreactor using lactose as inducer and the protein yield was comparable with IPTG induction. Next, the target protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, with a SDS-PAGE purity over 90%. Further, the purified VBP3 vaccine was subcutaneously injected in BALB/c mice and elicited high-titer anti-bFGF (1:32,000) and anti-VEGFA (1:4000) antibodies. Take together, lactose was an applicable inducer for VBP3 production and the eligible product of VBP3 was harvested in the large-scale fermentation, supporting the industrial production and pre-clinical study in the future. The VBP3 vaccine with superior immunogenicity might be used as a potential therapeutic vaccine for tumor treatment.

Introduction

As a hallmark of solid tumor, angiogenesis is an essential program that supplies oxygen and nutrients for tumor development [1]. Some angiogenic factors including bFGF and VEGFA play critical roles in this process in an autocrine or paracrine manner [2]. It has been demonstrated that the abnormally expressed bFGF and VEGFA in tumor microenvironments can drive vascular formation by recruiting and activating endothelial cells [3]. Additionally, bFGF and VEGFA can work synergistically to enhance platelet-derived growth factor (PDGF) pathway and promote tumor angiogenesis [4]. Thus, anti-angiogenic therapy has emerged as a promising strategy against various cancers. Anti-angiogenic drugs, such as bevacizumab, that target VEGFA have been approved by the Food and Drug Administration (FDA) of USA and applied to malignant patients [5]. However, current anti-angiogenic strategies are dependent on single pathway, leading to modest and transient benefits in clinical treatment [6,7]. Therefore, in our previous research, we had designed a multi-epitope peptide of VBP3 comprised of three antigenic epitopes from bFGF and three antigenic epitopes from VEGFA. These peptide candidates were inserted into pET-32a and expressed via BL21 (DE3) [8]. Here we combined bFGF and VEGFA pathways in order to elicit better immunogenicity and endurable effects to suppress tumor angiogenesis.

We try to produce VBP3 vaccine in a large scale and provide sufficient protein for pre-clinical study. As an industrial strain for heterologous protein production, the recombinant Escherichia coli (E. coli) can grow rapidly with cheaper carbon source and reach a high cell density [9]. IPTG is an efficient analogue of allolactose which fully strengthen the expression of recombinant plasmid with T7 promoter and lac operon [10]. However, in some countries, IPTG is not applied to the production of human recombinant protein for the reason of high cost, non-metabolization and potential toxicity [11]. With these limitations in industrial production, we focus on whether lactose, a natural disaccharide, can be used as inducer in VBP3 production [12]. Lactose can be transported into the cell by permease and work as allolactose via β-galactosidase [13]. These processes result in energy loss and reduce recombinant protein accumulation, but the protein that is produced has improved solubility relative to the production in the presence of IPTG. On the other hand, lactose is also utilized as a carbon source, increasing the final biomass [14]. However, it will suppress the lactose effects if glycerol, glucose and other sugar analogues exist all at once in the broth [15]. Further, the heterogeneity of lactose permease in the cell membrane is closely related to the lag phase and yield decline [16]. Therefore, the induction of lactose is complex and the culture conditions of bacterial growth and protein expression should be optimized to a comparable yield of IPTG.

In this study, we had to establish a culture bank of VBP3 strains and completely investigate the biological characteristics. Before large-scale production, the best-performing strains should be selected and optimized in shake flasks for culture conditions. The biggest hurdle was to identify whether lactose could induce VBP3 production and its induction mode. The fed-batch fermentation of VBP3 strains was performed and induced by either IPTG or lactose in 10 L bioreactor, followed by biomass and protein production detection. Ni-NTA affinity chromatography was applied to isolate the target protein and the purified product was subjected to immunogenicity test. We were aiming to improve the industrial conditions for VBP3 production and provide sufficient protein of high immunogenicity for pre-clinical study in tumor therapy.