淋巴细胞活化基因3蛋白（LAG3）是一种在肿瘤中枯竭T细胞上调的抑制性受体。LAG3是癌症免疫治疗的主要靶点，许多抗LAG3抗体正在临床试验中。然而，目前尚没有关于这些抗体所识别的表位的结构信息。我们通过单颗粒冷冻电子显微镜技术（cryoEM）确定了治疗性抗体（favezelimab）与LAG3之间的结合结构，分辨率为3.5 Å，揭示出favezelimab针对LAG3与二价性MHC-II结合位点的专一性配体。由于favezelimab定常区部分（单价性）Fab与LAG3形成的复合物很小（约100 kDa），这对cryoEM来说是一个挑战。因此，我们构建了二价性Fab favezelimab的版本，使Fab-LAG3复合物的大小翻倍，并赋予了复合物明显可辨识的形状，有利于粒子选择和图像处理的定位。本研究证明，二价性Fab可以作为冷冻电子显微镜分析小蛋白的新的基准标记物。版权所有 © 2023 Elsevier Ltd. 保留所有权利。

Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted T cells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 antibodies in clinical trials. However, there is no structural information on the epitopes recognized by these antibodies. We determined the single-particle cryoEM structure of a therapeutic antibody (favezelimab) bound to LAG3 to 3.5 Å resolution, revealing that favezelimab targets the LAG3-binding site for MHC class II, its canonical ligand. The small size of the complex between the conventional (monovalent) Fab of favezelimab and LAG3 (∼100 kDa) presented a challenge for cryoEM. Accordingly, we engineered a bivalent version of Fab favezelimab that doubled the size of the Fab-LAG3 complex and conferred a highly identifiable shape to the complex that facilitated particle selection and orientation for image processing. This study establishes bivalent Fabs as new fiducial markers for cryoEM analysis of small proteins.Copyright © 2023 Elsevier Ltd. All rights reserved.