Phage Display Reveals VLRB-Mediated Recognition of Minimal Tumor Glycan Antigen Sialyl-Tn

TL;DR

This study shows that VLRB proteins can be used to detect a cancer-related sugar called sTn, offering a new alternative to traditional antibodies.

Contribution

The study introduces VLRB as a novel scaffold for high-affinity recognition of the tumor glycan sialyl-Tn.

Findings

Two VLRB binders, A8 and B11, were isolated with high specificity for sTn and related O-glycans.

Both ccombodies bound to sTn-positive cell lines and glycoproteins, showing potential for diagnostic and therapeutic use.

The Vicia villosa lectin inhibited ccombody binding, suggesting shared epitope recognition.

Abstract

Sialyl-Tn (sTn) is a tumor-associated carbohydrate antigen (TACA) abundantly expressed by various types of carcinomas. While conventional antibody-based platforms have traditionally been used for the detection and targeting of sTn, alternative binding scaffolds may offer distinct advantages. Variable lymphocyte receptor B (VLRB), the immunoglobulin-like molecule of jawless vertebrates, offers a promising alternative for glycan recognition. In this study, a phage-displayed VLRB library was utilized to identify sTn-specific binders. Two candidates, designated as ccombodies A8 and B11, were isolated after four rounds of biopanning. Both were expressed and purified using Ni-affinity and FPLC, yielding proteins with apparent molecular weights of ~27 kDa in SDS-PAGE. Sequence analysis revealed a preference for glycan-binding residues in randomized hypervariable regions, with A8 exhibiting an increased aliphatic content. ELISA confirmed selective binding to sTn and other O-glycans containing the core α-GalNAc, with EC50 values of 18.2 and 14.2 nM for A8 and B11, respectively. Vicia villosa lectin inhibited ccombody binding to sTn, indicating shared epitope recognition. Additionally, both ccombodies bound to sTn-positive glycoproteins and carcinoma cell lines HeLa and LS174T. These findings demonstrate that phage display of VLRBs enables the identification of high-affinity, glycan-specific binders, offering a compelling alternative to immunoglobulin-based platforms for future diagnostic and therapeutic applications targeting tumor-associated glycans.

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