Winfried Römer

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.

The present report assesses the capability of a soluble glycosyltransferase to modify glycolipids organized in two synthetic membrane systems that are attractive models to mimic cell membranes: giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs). The objective was to synthesize the Gb3 antigen (Galα1,4Galβ1,4Glcβ-Cer), a cancer biomarker, at the surface of these membrane models. A soluble form of LgtC that adds a galactose residue from UDP-Gal to lactose-containing acceptors was selected. Although less efficient than with lactose, the ability of LgtC to utilize lactosyl–ceramide as an acceptor was demonstrated on GUVs and SLBs. The reaction was monitored using the B-subunit of Shiga toxin as Gb3-binding lectin. Quartz crystal microbalance with dissipation analysis showed that transient binding of LgtC at the membrane surface was sufficient for a productive conversion of LacCer to Gb3. Molecular dynamics simulations provided structural elements to help rationalize experimental data.