Novel lectin-based chimeric antigen receptors target Gb3-positive tumour cells

Abstract

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.

A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death

“…This result corroborates previously described studies which illustrated the abundance and variety of Gb3 isoforms present on HT-29 cells [101]. In contrast, the LS-174 cell line presented very low or no amounts of Gb3 [101], which we confirmed by our assay employing 2.6 nM Stx1B. Nevertheless, for higher concentrations of Stx1B (26 and 65 nM), a slight shift in fluorescence intensity was visible, indicating a basal presence of the Gb3 antigen at the surface of these cells.…”

“…By flow cytometry analysis, HT-29 were found to express a high amount of Gb3, as incubation with 2.6 nM Stx1B-Cy5 revealed a remarkable shift in fluorescence intensity. This result corroborates previously described studies which illustrated the abundance and variety of Gb3 isoforms present on HT-29 cells [101]. In contrast, the LS-174 cell line presented very low or no amounts of Gb3 [101], which we confirmed by our assay employing 2.6 nM Stx1B.…”

“…Moreover, Meléndez et al developed a panel of lectin-based chimeric antigen receptors (CARs) T cells, which demonstrated high therapeutical potential towards a variety of hematological malignancies and solid tumors expressing Gb3. In their studies, the Gb3-binding lectins StxB from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered Mitsuba from Mytilus galloprovincialis were employed to recognize the TACA Gb3 and fused to a second-generation CAR, achieving excellent target-specific cytotoxicity against Burkitt's lymphomaderived cell lines as well as colorectal and triple-negative breast-cancer [101]. Overall, these studies together with our current study support the potential of lectins as tools in many therapeutical applications where the glycome plays a crucial role in the development and sustainment of pathological conditions.…”

Dimeric Lectin Chimeras as Novel Candidates for Gb3-Mediated Transcytotic Drug Delivery through Cellular Barriers

“…The B-subunit of Shiga toxin (StxB), which binds to the glycosphingolipid Gb3 as a host cell receptor, has been studied as a drug carrier for a long time as well, specifically for targeting colorectal diseases [ 48 , 49 ]. Remarkably, Gb3-binding lectin-CAR T cells have demonstrated target-specific cytotoxicity against several cancer cell lines in vitro [ 50 ]. In addition, a bispecific construct composed of a Gb3-binding lectin linked to an antibody fragment (anti-CD3), termed ‘lectibody’, has been reported to kill Gb3-positive carcinoma cells in a highly specific and efficient manner by redirecting cytotoxic T lymphocytes in vitro [ 51 ].…”

Discrimination between protein glycoforms using lectin-functionalised gold nanoparticles as signal enhancers

“…19 Engineered chimeric antigen receptor (CAR) T cells decorated with lectins were able to target tumour-associated glycosphingolipid globotriaosylceramide (Gb3 -a glycan), demonstrating how glycoform targeting has real biomedical application. 20 The importance of studying aberrant glycosylation in cancer 21 is highlighted by the case of the prostate specific antigen (PSA). PSA is a clinical biomarker for prostate cancer, a disease estimated to have led to 375 000 deaths and 1.4 million new cases worldwide in 2020.…”