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Novel monoclonal antibody L2A5 specifically targeting sialyl-Tn and short glycans terminated by alpha-2–6 sialic acids

By Loureiro L, Sousa D, Ferreira D, Chai W, Lima L, Pereira C, Lopes C, Correia V, Silva L, Li C, Santos L, Ferreira JA, Barbas A, Palma A, Novo C and Videira P

Abstract

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.We thank Dinora Ferreira, Vitor Domingos and Maria Rosário Tito for assistance with animals in the IHMT/ NOVA animal room. We acknowledge the contribution of Prof Ten Feizi and Dr Yan Liu in the glycan microarray analysis and their input in the MIRAGE document; Dr Yibing Zhang for preparation and analysis of the NGL probes; Pengtao Zhang for the preparation of the lipid reagent and the NGL probes and Prof Makoto Kiso and Prof Hideharu Ishida for the synthetic glycosylceramides (GSC) included in the microarray. We also thank Mylène Carrascal for valuable input and expertise.This work was supported by the Bluepharma innovation award “Trifunctional antibodies and dendritic cell-based technologies: a combined approach to cancer immunotherapy” 2013 and the Scientific merit prize Santander - Totta – Lisbon New University - “Antibody engineering for breast cancer therapy” 2013. LRL is supported by the grant PD/BD/52476/2013 from the Portuguese Foundation for Science and Technology (FCT). The work is partially supported by FCT-MCTES through the grants PD/ BD/105727/2014 (VC), IF/00033/2012 (ASP), SFRH/BPD/101827/2014 (LL) and SFRH/BPD/111048/2015 (JAF), the Wellcome Trust Biomedical Resource grant (WC) and the NSFC-Shandong Joint Fund (U1606403) (CL). The authors also acknowledge FCT funding for CI-IPOP research unit (PEst-OE/SAU/UI0776/201). The Applied Molecular Biosciences Unit-UCIBIO is financed by national funds from FCT-MCTES (UID/Multi/04378/2013) and co-financed by the European Regional Development Fund under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The funders did not play a role in the study design, data collection or decision to publish

Topics: Animals, Antibodies, Monoclonal / immunology, Antibodies, Monoclonal / isolation & purification, Antibodies, Monoclonal / metabolismo, Antibodies, Monoclonal / therapeutic use, Antigens, Tumor-Associated, Carbohydrate / immunology, Antigens, Tumor-Associated, Carbohydrate / physiology, Breast Neoplasms / pathology, Cell Line, Tumor, Female, Glycosylation, Humans, Hybridomas, Mice, Mice, Inbred BALB C, Neoplasm Proteins / metabolismo, Polysaccharides / chemistry, Polysaccharides / immunology, Sialic Acids / metabolismo, Urinary Bladder Neoplasms / pathology
Publisher: 'Springer Science and Business Media LLC'
Year: 2018
DOI identifier: 10.1038/s41598-018-30421-w
OAI identifier: oai:repositorio-aberto.up.pt:10216/127416
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