Background
The mucin O-glycan sialylTn antigen (sTn, Neu5Acα2-6GalNAcα1-O-Ser/Thr) is an antigen associated with diverse cancer types and is generally associated with a higher risk of metastasis and poor prognosis. Despite efforts to develop highly specific anti-s-Tn antibodies for diagnostic and immunotherapy applications, challenges in eliciting high-affinity antibodies against the carbohydrate structure have limited their effectiveness, resulting in low titers and short-lived protection. The lack of experimental structural insights into the specificity of anti-s-Tn antibodies has hindered the optimization of their tumor cell recognition.
On December 16, 2024, Cátia O. Soares' team published a study titled "Decoding the Molecular Basis of the Specificity of an Anti-sTn Antibody" in JACS Au. The study used a comprehensive structural approach, combining X-ray crystallography, nuclear magnetic resonance spectroscopy, computational methods, sugar/glycopeptide microarrays, and biophysical techniques, to delve into the molecular basis of sTn recognition by the novel preclinical anti-sTn monoclonal antibody L2A5. The data demonstrated that the L2A5 antigen-binding fragment (Fab) specifically binds to the sTn core. The Neu5Ac group establishes a key interaction with the receptor, while the GalNAc group provides additional contacts. L2A5 exhibits excellent specificity for sTn glycopeptides derived from the tumor-associated MUC1 and MUC4 mucins, which may contribute to its selective targeting of tumor cells. This novel finding offers hope for the rational improvement and potential application of this anti-sTn antibody in the diagnosis and targeted therapy of tumors such as breast, colorectal, and bladder cancers.
L2A5 IgG and Fab formats retain specificity for sTn antigen
Glycan binding specificity was assessed using a neoglycolipid (NGL) microarray, and both antibody formats (Fab and IgG) retained primary binding to the short mucin o-glycan core sTn disaccharide NeuAcα2-6GalNAc-linked serine. Flow cytometry analysis revealed that both L2A5 Fab and IgG retained binding to sTn-expressing triple-negative breast cancer cells (MDA-sTn), whereas they failed to bind to the non-sTn-expressing parental cells (MDA-WT).
Molecular insights into sialoglycan recognition by L2A5 via NMR spectroscopy
The molecular details of L2A5 Fab recognition of two sTn antigen presenters, sTn-Thr and sTn-Ser, were further analyzed using (STD) NMR experiments. STD-derived epitope maps obtained for sTn–Thr and sTn–Ser revealed that the Neu5Ac unit is closest to the L2A5 Fab binding site, with the NHAc group receiving the highest STD response. This suggests that the Neu5Ac moiety is the core region of L2A5 Fab recognition of sTn antigens, with its NHAc group strongly interacting with the L2A5 Fab binding site. The STD intensity of the adjacent GalNAc group is weak, indicating that this monosaccharide is less involved in the binding event.
The molecular basis of the interaction between L2A5 and sTn determined by X-ray crystallography
L2A5 Fab was co-crystallized with sTn–Ser and sTn–Thr antigens to obtain crystal structures with resolutions of 2.3 Å and 2.6 Å. In the crystals, the bound conformation of sTn–Thr/Ser to L2A5 Fab showed that the α(2,6)Neu5Ac-GalNAc linkage adopted the −g (−60°) and gt (60°) configurations around the φ and ω dihedral angles, respectively.
The L2A5 Fab recognizes the Neu5Ac moiety, while the GalNAc moiety makes few contacts with the Fab. The attached Ser/Thr amino acid residues are solvent-exposed and contribute minimal additional non-covalent contacts. The key Neu5Ac group interacts with the W91L residue through a CH–π interaction, while hydrogen bonds are present between the GalNAc unit and residues D50L, Q89L, and R46L. Accordingly, H4 Neu5Ac points toward the center of the W91L residue and the N-acetyl group. This is consistent with the chemical shifts observed in the bound state in the TR-ROESY NMR spectrum. The α2–6-sialic acid geometry is crucial for L2A5 recognition, favoring the presence of consecutive GalNAc/Gal groups.
The key role of W91L in the recognition of sTn by L2A5 monoclonal antibody
Isothermal titration thermodynamic (ITC) analysis of L2A5 Fab with either sTn-Ser or sTn-Thr revealed a 1:1 complex with dissociation constants (KD's) of 1.76 and 4.47 μM, respectively. Mutation of W91L to a smaller (W91AL) or positively charged amino acid (W91RL) resulted in a complete loss of binding to sTn antigens (sTn-Ser and sTn-Thr), highlighting the critical relevance of this W residue in the interaction.
Interaction of L2A5 with sTn in MUC1 and MUC4 glycopeptides
L2A5 Fab only recognized peptides glycosylated with the sTn antigen, highlighting L2A5's specific binding to the sTn antigen without any recognition of Tn-, T-, or sT– antigens. In glycopeptide array presentation, the anti-sTn L2A5 Fab recognized peptide sequences from MUC1 and MUC4 in a glycosylation-site-specific manner. L2A5 Fab preferentially bound to the sTn-Thr antigen on the GST*AP sequence of MUC1 and the sTn-Ser antigen on the TS*SAST sequence of MUC4. In the MUC1 glycopeptide, the binding pattern for the sTn-Thr antigen showed the sTn group perpendicular to the peptide; in the MUC4 glycopeptide, the binding pattern for the sTn-Ser antigen showed the sTn group parallel to the peptide.
Glycopeptide array and MD data suggest that the unique peptide backbone and amino acid composition of the cancer-associated MUC1 and MUC4 glycoproteins may mediate the optimal binding interaction between L2A5 Fab and the sTn antigen. Specific recognition of MUC1 and MUC4, as suggested by microarray analysis, likely contributes to the selective targeting of tumor cells over healthy cells.
Summarize
This article reveals the mechanism of monoclonal antibody recognition of the sTn antigen. The data show that anti-sTn mAbs have potential in the diagnosis and treatment of sTn-expressing cancers (such as breast cancer, colorectal cancer, and bladder cancer). The structural approach reported in this article is robust and can be extended to accelerate the humanization process of different anti-glycan antibodies and improve their specificity and affinity, making a significant contribution to the research field of antibodies.
