The development of antibodies that target specific glycan structures on cancer cells or human pathogens poses a significant challenge due to the immense complexity of naturally occurring glycans. Automated glycan assembly enables the production of structurally homogeneous glycans in amounts that are difficult to derive from natural sources. Nanobodies (Nbs) are the smallest antigen-binding domains of heavy-chain-only antibodies (hcAbs) found in camelids. To date, the development of glycan-specific Nbs using synthetic glycans has not been reported. Here, we use defined synthetic glycans for alpaca immunization to elicit glycan-specific hcAbs, and describe the identification, isolation, and production of a Nb specific for the tumor-associated carbohydrate antigen Globo-H. The Nb binds the terminal fucose of Globo-H and recognizes synthetic Globo-H in solution and native Globo-H on breast cancer cells with high specificity. These results demonstrate the potential of our approach for generating glycan-targeting Nbs to be used in biomedical and biotechnological applications.

由于天然存在的聚糖非常复杂，因此开发针对癌细胞或人类病原体上特定聚糖结构的抗体提出了重大挑战。自动聚糖组装能够以难以从天然来源获得的量生产结构均一的聚糖。纳米抗体 (Nbs) 是骆驼科动物中发现的仅重链抗体 (hcAbs) 的最小抗原结合结构域。迄今为止，尚未报道使用合成聚糖开发聚糖特异性 Nbs。在这里，我们使用定义的合成聚糖进行羊驼免疫以引发聚糖特异性 hcAb，并描述了对肿瘤相关碳水化合物抗原 Globo-H 特异性的 Nb 的鉴定、分离和生产。Nb 结合 Globo-H 的末端岩藻糖，并以高特异性识别溶液中的合成 Globo-H 和乳腺癌细胞上的天然 Globo-H。这些结果证明了我们的方法在生成聚糖靶向 Nbs 以用于生物医学和生物技术应用的潜力。