The production of a recombinant tandem single chain fragment variable capable of binding prolamins triggering celiac disease

Research article Open Access The production of a recombinant tandem single chain fragment variable capable of binding prolamins triggering celiac disease Britta Eggenreich1, Elke Scholz2, David Johannes Wurm1, Florian Forster2Email author and Oliver Spadiut1Email authorView ORCID ID profile BMC Biotechnology201818:30 https://doi.org/10.1186/s12896-018-0443-0 ©  The Author(s). 2018 Received: 13 October 2017Accepted: 9 May 2018Published: 29 May 2018 Abstract Background Celiac disease (CD) is one of the most common food-related chronic disorders. It is mediated by the dietary consumption of prolamins, which are storage proteins of different grains. So far, no therapy exists and patients are bound to maintain a lifelong diet to avoid symptoms and long-term complications. To support those patients we developed a tandem single chain Fragment variable (tscFv) acting as a neutralizing agent against prolamins. We recombinantly produced this molecule in E. coli, but mainly obtained misfolded product aggregates, so-called inclusion bodies, independent of the cultivation strategy we applied. Results In this study, we introduce this novel tscFv against CD and present our strategy of obtaining active product from inclusion bodies. The refolded tscFv shows binding capabilities towards all tested CD-triggering grains. Compared to a standard polyclonal anti-PT-gliadin-IgY, the tscFv displays a slightly reduced affinity towards digested gliadin, but an additional affinity towards prolamins of barley. Conclusion The high binding specificity of tscFv towards prolamin-containing grains makes this novel molecule a valuable candidate to support patients suffering from CD in the future. Keywords Celiac diseaseSingle chain fragment variable E. coli Inclusion bodyELISA Background Celiac disease (CD) is one of the most common food-related chronic disorders with a prevalence of 1–2% in Western nations [1, 2]. It is triggered by the dietary consumption of storage proteins (prolamin, alcohol soluble fraction of gluten) of wheat, barley, rye and others [3, 4]. Up to date it is still not completely clear which factors lead to the manifestation of CD. Genetically, patients carry genes for the human leukocyte antigens HLA-DQ2 and HLA-DQ8, but also environmental factors, like early exposure to dietary gluten, infection and/or change in the bacterial flora of the intestine contribute to this disorder [1, 3–5]. In patients with CD the uptake of gluten leads to the secretion of autoantibodies and tissue transglutaminase (TG2), as well as proinflammatory cytokines, such as Interleukin (IL) 15, IL 21, Tumor Necrosis Factor (TNF) alpha and Interferon (IFN) gamma (Fig. 1) [1, 3]. Thus, inflammations of the small bowel occur, ranging from intraepithelial lymphocytosis up to total villous atrophy combined with crypt hyperplasia [1, 3]. Hence, symptoms vary between asymptomatic, extra-intestinal manifestations, various abdominal complications, up to global malabsorption [3, 6]. Long-term complications include malignancy, such as intestinal lymphomas and adenocarcinoma [3, 7, 8]. Fig. 1 Adapted simplified pathogenesis of celiac disease [3, 5, 9]. Prolamin overcomes the epithelial barrier via a transcellular transport as a soluble IgA-prolamin complex bound to an epithelial receptor (CD71). The interaction of prolamin with a chemokine receptor CXCR3 leads to the release of Zonulin, a protein that increases the permeability of the epithelium, due to opening of Tight-junctions and hence allows paracellular transport of prolamin. CD71, CXCR3 and Zonulin are upregulated in patients with celiac disease. Prolamin that reaches the lamina propria gets deamidated by transglutaminase 2 (TG2) and hence binds more strongly to human leukocyte antigens (HLA)-DQ2 and DQ8 molecules on antigen-presenting cells. These presented prolamins activate CD4+T-cells, which then secrete proinflammatory cytokines. Furthermore, T-cells induce the expression of Interleukin (IL) 15 and autoantibodies against TG2 by innate immune cells. IL 15 has a very important role regarding the remodeling process of the intestinal surface. It leads to an upregulation of nonconventional HLA molecules, MICA on enterocytes, and activates NKG2D receptors on intraepithelial lymphocytes (IELs). The interaction of MICA and NKG2D promotes the downstream effect of IEL-mediated epithelial damage. Another source of IL 15 are epithelial and dendritic cells after contact with prolamin. To sum up, the contact of prolamin with the epithelial layer activates the innate and humoral immune system, which induces the destruction of the surface of the small intestine To reduce symptoms and avoid long-term complications, a strict gluten free diet (GFD) is the only effective treatment of CD so far [3]. Due to the high prevalence, severe symptoms, long-term complications and limited treatment possibilities, it is self-explanatory that patients are in pressing need of additional and alternative therapies. Many novel drugs are in development and the results of the respective clinical trials are impatiently anticipated. As shown in Table 1 various novel therapies are under development, however none of these has reached clinical phase 3 investigations yet. Hence, unfortunately no novel therapy will be introduced to the market in the near future. Next to this lack of therapeutic options, a high social burden lies upon patients with CD, because a lifelong GFD is difficult to maintain. Even in “gluten-free” dietary products traces of prolamins are found, which have a severe impact on the well-being [10]. To support those patients we recently developed a novel single chain Fragment variable (scFv) against prolamins [11]. This scFv works as a “neutralizing agent”, meaning that a complex between prolamin and the scFv is formed in the gut and no systemic interactions are expected, as the formed complex does not cross the epithelial barrier and is finally excreted. Thus, the scFv can be applied as a medical device. To obtain this novel scFv, we immunized chicken with peptic tryptic digested gliadin (PT-gliadin). Those immunized chicken were used as source for RNA, carrying the sequence for the recombinant scFv [11]. Since no effector function of the antibody (AB) is relevant for the neutralizing effect, but only the variable light and heavy chain are required, we generated a single chain Fragment variable (scFv). Since two antigen binding regions increase binding affinity, we joined two scFv with a peptide linker and constructed a tandem single chain Fragment variable (tscFv) [12, 13]. A block flow diagram of this process is presented in Additional file 1: Figure S1. Table 1 Potential therapies/supplementations for patients with celiac disease Site of action Target Principle of effect Information/Drug Phase of clinical trial ClinicalTrials.gov Identifier Ref. Intra-luminal Flours Pretreatment with lactobacilli, transamidation of gl (...truncated)