Incomplete O-glycosylation is a feature associated with malignancy resulting in the expression of truncated glycans such as the sialyl-Tn (STn) antigen. Despite all the progress in the development of potential anti-cancer antibodies, their application is frequently hindered by low specificities and cross-reactivity. In this study, a novel anti-STn monoclonal antibody named L2A5 was developed by hybridoma technology. Flow cytometry analysis showed that L2A5 specifically binds to sialylated structures on the cell surface of STn-expressing breast and bladder cancer cell lines. Moreover, immunoblotting assays demonstrated reactivity to tumour-associated O-glycosylated proteins, such as MUC1. Tumour recognition was further observed using immunohistochemistry assays, which demonstrated a high sensitivity and specificity of L2A5 mAb towards cancer tissue, using bladder and colorectal cancer tissues. L2A5 staining was exclusively tumoural, with a remarkable reactivity in invasive and metastasis sites, not detectable by other anti-STn mAbs. Additionally, it stained 20% of cases of triple-negative breast cancers, suggesting application in diseases with unmet clinical needs. Finally, the fine specificity was assessed using glycan microarrays, demonstrating a highly specific binding of L2A5 to core STn antigens and additional ability to bind 2–6-linked sialyl core-1 probes. In conclusion, this study describes a novel anti-STn antibody with a unique binding specificity that can be applied for cancer diagnostic and future development of new antibody-based therapeutic applications.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors, which plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-β. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-β secretion following co-culture of Siglec-15-expressing monocytic cell lines with tumor cells expressing sTn or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.

The structural diversity of the lipopolysaccharides (LPSs) from Helicobacter pylori poses a challenge to establish accurate and strain-specific structure-function relationships in interactions with the host. Here, LPS structural domains from five clinical isolates were obtained and compared with the reference strain 26695. This was achieved combining information from structural analysis (GC-MS and ESI-MSn) with binding data after interrogation of a LPS-derived carbohydrate microarray with sequence-specific proteins. All LPSs expressed Lewisx/y and N-acetyllactosamine determinants. Ribans were also detected in LPSs from all clinical isolates, allowing their distinction from the 26695 LPS. There was evidence for 1,3-d-galactans and blood group H-type 2 sequences in two of the clinical isolates, the latter not yet described for H. pylori LPS. Furthermore, carbohydrate microarray analyses showed a strain-associated LPS recognition by the immune lectins DC-SIGN and galectin-3 and revealed distinctive LPS binding patterns by IgG antibodies in the serum from H. pylori-infected patients.

The immune microenvironment of breast cancer (BC) is composed by high macrophage infiltrates, correlated with the most aggressive subtypes. Tumour-associated macrophages (TAM) within the BC microenvironment are key regulators of immune suppression and BC progression. Nevertheless, several key questions regarding TAM polarisation by BC are still not fully understood. Recently, the modulation of the immune microenvironment has been described via the recognition of abnormal glycosylation patterns at BC cell surface. These patterns rise as a resource to identify potential targets on TAM in the BC context, leading to the development of novel immunotherapies. Herein, we will summarize recent studies describing advances in identifying altered glycan structures in BC cells. We will focus on BC-specific glycosylation patterns known to modulate the phenotype and function of macrophages recruited to the tumour site, such as structures with sialylated or N-acetylgalactosamine epitopes. Moreover, the lectins present at the surface of macrophages reported to bind to such antigens, inducing tumour-prone TAM phenotypes, will also be highlighted. Finally, we will discuss and give our view on the potential and current challenges of targeting these glycan-lectin interactions to reshape the immunosuppressive landscape of BC.