Francesca Rosato

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments.

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (K_d = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell.

A chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation—a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions.