肠上皮受体鸟苷酸环化酶C（GUCY2C）是一种与肠道恶性肿瘤有关的细胞表面抗原，在结直肠癌免疫治疗中可以作为一个有效的靶点。在这里，我们描述了一种酵母表面展示方法，结合一个正交肽基映射策略，来确定最近开始用于癌症治疗的一种新型抗GUCY2CxCD3双特异性抗体（BsAb）的GUCY2C结合表位。目标表位被定位在人类GUCY2C的N-末端螺旋H2上，这使得我们能够确定GUCY2C表位和抗GUCY2C抗体域之间的最小结晶结构。为了了解这个最小表位是否涵盖了整个抗体结合区域，并且研究表位位置对抗体活性的影响，我们进一步确定了这个相互作用在GUCY2C全长细胞外区域（ECD）环境中的结构。我们发现这个表位位于突出的距离膜的螺旋区域，它在GUCY2C表面的特定位置决定了两个抗原臂在GUCY2CxCD3 BsAb的肿瘤杀伤活性中的紧密空间接近的diabody排列。© 2023. Springer Nature Limited.

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.© 2023. Springer Nature Limited.