The immune checkpoint receptor LAG3: Structure, function, and target for cancer immunotherapy.

Lymphocyte activation gene 3 protein (LAG3) is an immune checkpoint receptor that is highly upregulated on exhausted T cells in the tumor microenvironment. LAG3 transmits inhibitory signals to T cells upon binding to MHC class II and other ligands, rendering T cells dysfunctional. Consequently, LAG3 is a major target for cancer immunotherapy with many anti-LAG3 monoclonal antibodies (mAbs) that block LAG3 inhibitory activity in clinical trials. In this review, we examine the molecular basis for LAG3 function in light of recently determined crystal and cryoEM structures of this inhibitory receptor. We review what is known about LAG3 interactions with MHC class II, its canonical ligand, and the newly discovered ligands FGL1 and the T cell receptor (TCR)-CD3 complex, including current controversies over the relative importance of these ligands. We then address the development and mechanisms of action of anti-LAG3 mAbs in clinical trials for cancer immunotherapy. We discuss new strategies to therapeutically target LAG3 using mAbs that not only block the LAG3-MHC class II interaction, but also LAG3 interactions with FGL1 or TCR-CD3, or that disrupt LAG3 dimerization. Finally, we assess the possibility of developing mAbs that enhance, rather than block, LAG3 inhibitory activity as treatments for autoimmune diseases.

CryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3 determined using a bivalent Fab as fiducial marker.

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.

Structure-Based Design of Potent Iminosugar Inhibitors of Endoplasmic Reticulum α-Glucosidase I with Anti-SARS-CoV-2 Activity.

Enveloped viruses depend on the host endoplasmic reticulum (ER) quality control (QC) machinery for proper glycoprotein folding. The endoplasmic reticulum quality control (ERQC) enzyme α-glucosidase I (α-GluI) is an attractive target for developing broad-spectrum antivirals. We synthesized 28 inhibitors designed to interact with all four subsites of the α-GluI active site. These inhibitors are derivatives of the iminosugars 1-deoxynojirimycin (1-DNJ) and valiolamine. Crystal structures of ER α-GluI bound to 25 1-DNJ and three valiolamine derivatives revealed the basis for inhibitory potency. We established the structure-activity relationship (SAR) and used the Site Identification by Ligand Competitive Saturation (SILCS) method to develop a model for predicting α-GluI inhibition. We screened the compounds against SARS-CoV-2 in vitro to identify those with greater antiviral activity than the benchmark α-glucosidase inhibitor UV-4. These host-targeting compounds are candidates for investigation in animal models of SARS-CoV-2 and for testing against other viruses that rely on ERQC for correct glycoprotein folding.

Identification of Endoplasmic Reticulum α-Glucosidase I from a Thermophilic Fungus as a Platform for Structure-Guided Antiviral Drug Design.

All viruses depend on host cell proteins for replication. Denying viruses' access to the function of critical host proteins can result in antiviral activity against multiple virus families. In particular, small-molecule drug candidates which inhibit the α-glucosidase enzymes of the endoplasmic reticulum (ER) translation quality control (QC) pathway have demonstrated broad-spectrum antiviral activities and low risk for development of viral resistance. However, antiviral drug discovery focused on the ERQC enzyme α-glucosidase I (α-GluI) has been hampered by difficulties in obtaining crystal structures of complexes with inhibitors. We report here the identification of an orthologous enzyme from a thermophilic fungus, Chaetomium thermophilum (Ct), as a robust surrogate for mammalian ER α-GluI and a platform for inhibitor design. Previously annotated only as a hypothetical protein, the Ct protein was validated as a bona fide α-glucosidase by comparing its crystal structure to that of mammalian α-GluI, by demonstrating enzymatic activity on the unusual α-d-Glcp-(1 → 2)-α-d-Glcp-(1 → 3) substrate glycan, and by showing that well-known inhibitors of mammalian α-GluI (1-DNJ, UV-4, UV-5) also inhibit Ct α-GluI. Crystal structures of Ct α-GluI in complex with three such inhibitors (UV-4, UV-5, EB-0159) revealed extensive interactions with all four enzyme subsites and provided insights into the catalytic mechanism. Identification of ER Ct α-GluI as a surrogate for mammalian α-GluI will accelerate the structure-guided discovery of broad-spectrum antivirals. This study also highlights Ct as a source of thermostable eukaryotic proteins, such as ER α-Glu I, that lack orthologs in bacterial or archaeal thermophiles.

N-Substituted Valiolamine Derivatives as Potent Inhibitors of Endoplasmic Reticulum α-Glucosidases I and II with Antiviral Activity.

Most enveloped viruses rely on the host cell endoplasmic reticulum (ER) quality control (QC) machinery for proper folding of glycoproteins. The key ER α-glucosidases (α-Glu) I and II of the ERQC machinery are attractive targets for developing broad-spectrum antivirals. Iminosugars based on deoxynojirimycin have been extensively studied as ER α-glucosidase inhibitors; however, other glycomimetic compounds are less established. Accordingly, we synthesized a series of N-substituted derivatives of valiolamine, the iminosugar scaffold of type 2 diabetes drug voglibose. To understand the basis for up to 100,000-fold improved inhibitory potency, we determined high-resolution crystal structures of mouse ER α-GluII in complex with valiolamine and 10 derivatives. The structures revealed extensive interactions with all four α-GluII subsites. We further showed that N-substituted valiolamines were active against dengue virus and SARS-CoV-2 in vitro. This study introduces valiolamine-based inhibitors of the ERQC machinery as candidates for developing potential broad-spectrum therapeutics against the existing and emerging viruses.

How Natural Killer Cell Receptors Stick to Cell-Cell Adhesion Proteins.

The cytolytic activity of NK cells is regulated by activating and inhibitory receptors, including ones that recognize cell-cell adhesion molecules. In this issue of Structure, Deuss et al. (2019) report the structure of nectin-like protein-5 bound to the NK inhibitory receptor CD96, a promising new target for cancer immunotherapy.