The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody’s activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb.

肠上皮受体鸟苷酸环化酶 C (GUCY2C) 是一种在胃肠道恶性肿瘤中表达的肿瘤相关细胞表面抗原，可作为结直肠癌免疫治疗的有效靶点。在这里，我们描述了一种酵母表面展示方法与基于正交肽的作图策略相结合，以识别一种新型抗 GUCY2CxCD3 双特异性抗体 (BsAb) 的 GUCY2C 结合表位，该抗体最近已进入临床用于治疗癌症。目标表位位于人 GUCY2C 的 N 端螺旋 H2，这使得能够确定与抗 GUCY2C 抗体结构域复合的最小 GUCY2C 表位的晶体结构。为了了解这个最小表位是否覆盖整个抗体结合区并研究表位位置对抗体活性的影响，我们进一步确定了 GUCY2C 全长胞外结构域 (ECD) 背景下这种相互作用的结构。我们发现该表位位于 GUCY2C 的突出膜远端螺旋区域上，并且其在 GUCY2C 表面上的特定位置决定了双抗体排列中两个抗原臂的紧密空间接近度，这对于 GUCY2CxCD3 BsAb 的肿瘤杀伤活性至关重要。