W.M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA; Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA; University of Maryland Marlene and Stewart Greenebaum Cancer Center, University of Maryland Medical Center, Baltimore, MD 21201, USA; Center for Biomolecular Therapeutics, University of Maryland School of Medicine, Rockville, MD 20850, USA.
Structure. 2023 Oct 5;31(10):1149-1157.e3. doi: 10.1016/j.str.2023.07.013. Epub 2023 Aug 23.
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.
淋巴细胞激活基因 3 蛋白（LAG3）是一种抑制性受体，在肿瘤中耗竭的 T 细胞上上调。LAG3 是癌症免疫治疗的一个主要靶点，许多抗 LAG3 抗体正在临床试验中进行研究。然而，这些抗体所识别的表位还没有结构信息。我们测定了与 LAG3 结合的治疗性抗体（favezelimab）的单颗粒 cryoEM 结构，分辨率为 3.5Å，揭示了 favezelimab 靶向 MHC 类 II 的 LAG3 结合位点，这是其典型配体。favezelimab 的常规（单价）Fab 与 LAG3（~100kDa）之间的复合物体积较小，这对 cryoEM 提出了挑战。因此，我们设计了一种双价 Fab favezelimab，使 Fab-LAG3 复合物的大小增加了一倍，并赋予复合物一个高度可识别的形状，这有助于颗粒的选择和定向，便于图像处理。这项研究确立了双价 Fab 作为 cryoEM 分析小蛋白的新基准标记物。
