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

Abstract

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.

Figure 1

Validation of the L2A5 Fab specificity. (A) Interaction of L2A5 Fab and L2A5 IgG in a focused glycan microarray containing α2–3 and α2–6 sialylated glycans. The full list of probes and their sequences is detailed in Table S1. The chart position assigned to each probe is also referenced in Table S1. (B) Cell binding assays using flow cytometry. Left panel: EC50 MDA-MB231 vs STn expressing cells using L2A5 Fab; Center panel: EC50 MDA-MB231 vs STn expressing cells using L2A5 IgG; Righ panel: EC50 MDA-MB231 vs STn expressing cells using L2A5 Fab vs L2A5 IgG.

Figure 2

Molecular recognition of sTn–Thr and sTn–Ser by L2A5 Fab. (A) Off-resonance and STD-NMR spectrum of sTn–Thr and sTn–Ser (800 μM) in the presence of L2A5 Fab (20 μM) (40:1 molar ratio) at 310 K and 600 MHz (* ethanol contamination; # Tris). STD-NMR derived epitope map of sTn–Thr and sTn–Ser in the presence of L2A5 Fab is also depicted. The relative STD response is coded according to the legend. (B) TR-ROESY spectrum of sTn–Thr and sTn–Ser (600 μM) in the presence of L2A5 Fab (40 μM) (15:1 molar ratio) at 310 K and 800 MHz. Exchange cross-peaks between the free and bound states are circled and identified with the respective assignment and chemical shift change.

Figure 3

Crystal structure of L2A5 Fab in complex with sTn–Ser or sTn–Thr. (A) Cartoon and surface representation of the side and top views of the crystal structure of the L2A5 Fab. (B) Buried surface area (BSA) values on the L2A5 Fab crystal structures in complex with sTn–Ser or sTn–Thr. (C) Interactions between L2A5 Fab and sTn–Ser and sTn–Thr. H-bonds are depicted with black dashes. Water molecules are shown as red spheres. The composite omit map for sTn–Ser and sTn–Thr, contoured at 1σ, is shown. (D) Schematic representation of the interactions between L2A5 Fab and sTn–Ser and sTn–Thr, respectively.

Figure 4

Evaluation of binding of L2A5 Fab WT and W91^L mutants to sTn–Thr and sTn–Ser by ITC and NMR. (A) Dissociation constant and thermodynamic parameters obtained from binding of sTn–Ser or sTn–Thr with L2A5 Fab. (B) Representative ITC binding isotherms for binding to sTn–Thr of L2A5 WT, W91A^L, and W91R^L. (C) Off-resonance and STD-NMR spectra of sTn–Thr (800 μM) in the presence of L2A5 Fab WT, W91A^L, and W91R^L.

Figure 5

Binding of L2A5 to MUC1 and MUC4 derived glycopeptides. (A) Binding of L2A5 Fab to glycopeptide microarrays of MUC1 derived glycopeptides. (B) Binding of L2A5 Fab to glycopeptide microarrays of MUC4 derived glycopeptides. (C) 3D Model of the L2A5 Fab/APGST*APPA (* = sTn) complex obtained through molecular dynamic simulations. (D) 3D Model of the L2A5 Fab/TS*SAST (* = sTn) complex obtained through molecular dynamic simulations. In (C, D), L2A5 Fab is in cartoon representation (VH in light gray and VL in dark gray), with some residues of the binding site identified and represented in sticks. The glycopeptides are represented in sticks (Neu5Ac, purple; Gal, yellow; peptide, black). Polar interactions and CH–π interactions are depicted with black dashed lines.