CEA, INRAE, Medicines and Healthcare Technologies Department, Université Paris-Saclay, SIMoS, France.
Sanofi, Large Molecule Research, Vitry-sur-Seine, France.
MAbs. 2023 Jan-Dec;15(1):2175311. doi: 10.1080/19420862.2023.2175311.
Delineating the precise regions on an antigen that are targeted by antibodies has become a key step for the development of antibody therapeutics. X-ray crystallography and cryogenic electron microscopy are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, they are labor-intensive and a successful outcome is not guaranteed. We used deep mutational scanning (DMS) of the human LAMP-1 antigen displayed on yeast surface and leveraged next-generation sequencing to observe the effect of individual mutants on the binding of two LAMP-1 antibodies and to determine their functional epitopes on LAMP-1. Fine-tuned epitope mapping by DMS approaches is augmented by knowledge of experimental antigen structure. As human LAMP-1 structure has not yet been solved, we used the AlphaFold predicted structure of the full-length protein to combine with DMS data and ultimately finely map antibody epitopes. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one of the two antibodies with a LAMP-1 luminal domain. Finally, we used AlphaFold models of non-human LAMP-1 to understand the lack of mAb cross-reactivity. While both epitopes in the murine form exhibit multiple mutations in comparison to human LAMP-1, only one and two mutations in the Macaca form suffice to hinder the recognition by mAb B and A, respectively. Altogether, this study promotes a new application of AlphaFold to speed up precision mapping of antibody-antigen interactions and consequently accelerate antibody engineering for optimization.
阐明抗体所针对的抗原的精确区域已成为开发抗体治疗药物的关键步骤。X 射线晶体学和低温电子显微镜被认为是提供这些结合位点在原子分辨率下的精确信息的金标准。然而，它们非常耗费人力，并且不能保证取得成功的结果。我们使用在酵母表面展示的人 LAMP-1 抗原的深度突变扫描（DMS），并利用下一代测序来观察单个突变对两种 LAMP-1 抗体结合的影响，并确定它们在 LAMP-1 上的功能表位。通过 DMS 方法进行微调的表位作图，通过对实验抗原结构的了解得到增强。由于人类 LAMP-1 的结构尚未解决，我们使用全长蛋白的 AlphaFold 预测结构与 DMS 数据结合，最终精细地绘制了抗体表位。通过将结果与两种抗体之一与 LAMP-1 腔域的共晶结构进行比较，证实了该方法的准确性。最后，我们使用非人类 LAMP-1 的 AlphaFold 模型来了解 mAb 交叉反应性缺失的原因。虽然与人类 LAMP-1 相比，鼠形式的两个表位都表现出多个突变，但只有一个和两个突变足以分别阻止 mAb B 和 A 的识别。总的来说，这项研究促进了 AlphaFold 的新应用，以加速抗体-抗原相互作用的精确作图，从而加速抗体工程的优化。
