Decoding the Molecular Basis of the Specificity of an Anti-sTn Antibody

The mucin O-glycan sialyl Tn antigen (sTn, Neu5Acα2-6GalNAcα1-O-Ser/Thr) is an antigen associated with different types of cancers, often linked with a higher risk of metastasis and poor prognosis. Despite efforts to develop anti-sTn antibodies with high specificity for diagnostics and immunotherapy, challenges in eliciting high-affinity antibodies for glycan structures have limited their effectiveness, leading to low titers and short protection durations. Experimental structural insights into anti-sTn antibody specificity are lacking, hindering their optimization for cancer cell recognition. In this study, we used a comprehensive structural approach, combining X-ray crystallography, NMR spectroscopy, computational methods, glycan/glycopeptide microarrays, and biophysical techniques, to thoroughly investigate the molecular basis of sTn recognition by L2A5, a novel preclinical anti-sTn monoclonal antibody (mAb). Our data unequivocally show that the L2A5 fragment antigen-binding (Fab) specifically binds to core sTn moieties. NMR and X-ray structural data suggest a similar binding mode for the complexes formed by the sTn moiety linked to Ser or Thr and the L2A5 Fab. The sugar moieties are similarly oriented in the paratope of mAb, with the Neu5Ac moiety establishing key interactions with the receptor and the GalNAc moiety providing additional contacts. Furthermore, L2A5 exhibits fine specificity toward cancer-related MUC1 and MUC4 mucin-derived sTn glycopeptides, which might contribute to its selective targeting against tumor cells. This newfound knowledge holds promise for the rational improvement and potential application of this anti-sTn antibody in diagnosis and targeted therapy against sTn expressing cancers such as breast, colorectal, and bladder cancer, improving patient care.

Descrição

Funding Information: This work is funded by the European Union under Horizon Europe (project 101079417 \u2500 GLYCOTwinning).The authors also thank Agencia Estatal de Investigacio\u0301n of Spain (PID2021-127662OB-I00 to F.C. and PID2019-107770RA-I00 to J.E.-O), the Juan de la Cierva Formacio\u0301n 2021 (FJC2021-046465-I to M.E.L). J.J.-B, J.E.-O, and M.E.L. also thank the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain). The NMR spectrometers are part of the National NMR Network (PTNMR) and were partially supported by Infrastructure Project No 22161 (cofinanced by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). X-ray diffraction experiments described in this paper were performed using beamline i24 at Diamond Light Source (Oxfordshire, UK). Publisher Copyright: © 2024 The Authors. Published by American Chemical Society.

Palavras-chave

antibody glycan NMR sialic acid X-ray crystallography Analytical Chemistry Chemistry (miscellaneous) Physical and Theoretical Chemistry Organic Chemistry SDG 3 - Good Health and Well-being