LAG3 ectodomain structure reveals functional interfaces for ligand and antibody recognition

Abstract

The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible 'loop 2' region in LAG3 domain 1. We also defined the LAG3-FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.

Extended Data Figure 1.. Yeast display selections and protein engineering of hLAG3.

a, Yeast display selection strategy used to isolate high-affinity FGL1^FD binders from the hLAG3^D12 mutant library. Flow cytometry dot plots depict the first round (upper panel) and final round of selection (lower panel) stained with 100 nM FGL1^FD tetramers and 20 nM FGL1^FD monomers, respectively. The gating strategies for yeast and single cells were shown in the bottom. b, Mutation-frequency map generated from sequencing of the five clones isolated from the final round of yeast selection. The recurring mutation, M171I, is highlighted in blue. c, Table of clones containing the M171I mutation. d, Dose-response titrations of yeast expressing hLAG3^D12 or LAG3^D12 variants with biotinylated FGL1^FD. e, SDS-PAGE analysis of LAG3 protein expression and secretion. Two replicated transfections were performed for hLAG3, hLAG3*, hLAG3^D12, hLAG3*^D12 (hLAG3^D12 with M171I mutation). High-Five cells were inoculated with consistent titer of virus. After 48 hours, proteins were retrieved using Ni-NTA. Quantifications of the relative intensity of the bands (right panel) were based on triplicates inoculation.

Extended Data Figure 2.. Analysis of LAG3 structures.

a, Electron density maps of hLAG3*:F7 complex structure at 3.71 Å. Upper panel: the composite omit map; lower panel: the 2Fo-Fc map from the final round of refinement. The maps were contoured at 0.8 σ. b, Alignment of D3 and D4 domains from the two protomers in the hLAG3 dimer reveals a rotation angle of 161° about the D2-D3 hinge. The two protomers are colored in cyan and grey. c, the M171I mutation and its surrounding residues in hLAG3* structure. M167 in mLAG3 is the equivalent site to M171I. f, Structural comparison of D1 domains from LAG3 (mLAG3) and CD4 (PDB ID: 3T0E). MHCII-binding residues of CD4 D1 are shown as sticks and are clustered in an analogous region to LAG3 Loop2. CD4: pink; mLAG3: green; Loop2: blue.

Extended Data Figure 3.. LAG3 dimer interface and LAG3:MHCII binding analyses with SPR.

a, The 2Fo-Fc electron density map, contoured at 1.0 σ, surrounding the mLAG3 dimer interface residue Trp¹⁸⁰. b, SPR analyses of hLAG3* binding to HLA-DR4^HIV. Biotinylated HLA-DR4^HIV was immobilized on a SA chip. hLAG3* protein dilutions starting from 20,000 nM were injected in turn. c-d, SPR sensorgram of recombinant mLAG3 flowed over an SA-chip immobilized with I-A(b)^MOG (c) or I-A(b)^CLIP (d). e, SPR analyses of LAG3 binding affinity binding to human MHCII allomorphs. MHCII proteins bound to CLIP peptides were biotinylated and immobilized on an SA chip and hLAG3* proteins were flowed over the chip. The curves were then fitted to determine the K_D. Mean K_D and S.D was from two independent replicates. f, Sequence alignment of the D2 dimer interface region from multiple LAG3 orthologs. Residues forming dimer contacts are outlined in red, conserved residues are highlighted in green, and biochemically similar residues are colored in green.

Extended Data Figure 4.. LAG3 and MHCII protein colocalization on cell surface.

a-b, Untreated HuT 78 cells or HuT 78 cells treated with anti-CD3 (1 ug/ml), anti-CD3 /anti-CD28 (1 ug/ml) or PMA (50 ng/ml) for 48h and LAG3 expression was analyzed by flow cytometry. c, Flow cytometry analyses of HLA-DR expression on unstimulated or stimulated Hut-78 cells. The cells were stained with anti-HLA-DR (clone L243, BioLegend, 1:100). d, Subcellular localization of LAG-3 and HLA-DR on HuT-78 cells. Confocal fluorescence images displaying nuclei (blue), LAG-3 (green) and HLA-DR (red) in HuT-78 cells, either untreated or treated with anti-CD3/anti-CD28. Representative confocal images for the individual channels and merged images are shown in the zoomed panel. One of three representative experiments is shown. Horizontal bars in (b) represent the mean and statistics was determined by one-way ANOVA with P values noted in the figure.

Extended Data Figure 5.. Epitope mapping and functional characterization of LAG3 antagonist antibodies.

a-c, SPR sensograms measuring hLAG3 binding to MHCII in the presence of scFv-Fc fusion proteins. d, SPR was used to determine scFv:LAG3 binding affinities. e, Flow cytometry analyses of binding of recombinant HLA-DR4^HIV(4 μM) to yeast-displayed CD4 or hLAG3 and its mutants. f-g, A luciferase reporter assay was used to assess the ability of 15011-Fc to inhibit LAG3 in the presence of FGL1. To activate TCR signaling, LAG3⁺ Jurkat cells were either co-cultured with MHCII-expressing Raji cells (f) or stimulated with anti-CD3 (α-CD3) (g). In f and g, the cells were supplemented with 10 nM FGL1 and an isotype control antibody, 15011-Fc, or F7-Fc (300 nM). The luminescence was measured after incubating the cells overnight. In f and g, the graph represents mean ± SD of three replicates from representative of two independent experiments. All statistics was determined by one-way ANOVA, with P values noted in the figure.

Extended Data Figure 6.. Mapping of FGL1 binding sites on hLAG3.

a, Flow chart depicting the selection strategy used to isolate mutations that decreased LAG3 binding to FGL1. In the first two rounds, the hLAG3^D12 library was negatively selected against Alexa Fluor 647-labeled streptavidin to remove non-specific binders. In rounds 3-5, the indicated concentrations of biotinylated FGL1^FD were incubated with the library and negative selections were performed to remove FGL1 binders. b, Yeast expressing LAG3 mutants were stained with MHCII tetramers (200 nM) to determine whether the FGL1 loss-of-binding mutations affected LAG3 binding to MHCII. c, SPR-sensograms recorded following the injection of samples containing fixed concentrations of hLAG3 (800 nM) and varying concentrations 15011.scFv over a chip coated with FGL1^FD.

Extended Data Figure 7.. Mapping the binding interface between FGL1FD and hLAG3.

a, Structural alignment of the three copies of FGL1^FD (FD-a, FD-b and FD-c) in the crystal asymmetric unit. Different P subdomain loop conformations differences are depicted in the zoom panel. b, Structural alignment of fibrinogen-like domains from FGL1, Ang1 (RMSD=1.12 Å) and FIBCD1 (RMSD=1.21 Å). The FD-b protomer from the FGL1^FD structure was used in this alignment. FGL1, blue; Ang1, yellow; FICD1, gray. c, Grouped residues mutated in the alanine scanning assay. Residues that were mutated in a single construct are colored accordingly. d, Flow cytometry histogram plots depicting increased binding of FGL1^FD variant populations following iterative rounds of selection against hLAG3 (left) and a heat map showing the mutation frequency of amino acid substitutions after sequencing six clones (right). e, Yeast expressing hLAG3 were stained with biotinylated FGL1^FD and FGL1^FD containing the affinity-enhancing G290E mutation (FD^G290E).

Extended Data Figure 8.. FGL1-induced LAG3 clustering correlates with FGL1 suppression mechanism.

a, LAG3-NFAT Jurkat T cells were treated with 1, 10 or 100 nM of FGL1 protein and incubated for 30 min. Binding was detected using an anti-LAG3 antibody. Data reflect the mean ± SD of n=3 technical replicates, with representative of two experiments is shown. b, In the presence of the blocking antibody, 15011Fc, the FGL1-LAG3 clustering effect was diminished. Stacked confocal microscopy images displaying LAG3 (green) and DAPI (blue) in Jurkat T cells expressing LAG3, either untreated or treated with the indicated reagents: 10 nM FGL1, 15011Fc, isotype control, 10 nM FGL1 and 300 nM 15011Fc, or 10 nM FGL1 and 300 nM isotype control. c, The number of LAG3 clusters per cell was quantified and plotted as the average per field of view (FOV) with analyses of n=7 FOV from one of three representative experiments. d, NFAT luciferase reporter assay performed to assess the effect of FGL1 on LAG3-mediated suppression of T cell activation mediated by anti-CD3. The relative activation was obtained by normalizing baseline-subtracted luciferase signals of tested concentrations to the buffer control group. The graph represents mean ± SD of three independent replicates from one of two representative experiments. All significance was determined by one-way ANOVA, with P values noted in the figure.

Figure 1.. Structures of human and murine LAG3 ECDs.

a, Cartoon schematic of the hLAG3^D12 yeast display construct used to generate a mutant library. Biotinylated FGL1^FD and an anti-Myc antibody were used to select for binders with increased ligand binding affinity and surface expression, respectively. b, Steady state SPR binding isotherms were fitted to determine the binding affinities between FGL1^FD and hLAG3 or the hLAG3* variant. The average K_D and standard deviation of two replicates are presented. c, Cell surface expression level of hLAG3 or hLAG3* as determined by flow cytometry. Data represent the mean MFI ± SD from n=3 independent biological replicates and statistics determined by using one-way analysis of variance (ANOVA) with P values noted in the figure. d, Crystal structure of hLAG3* bound to the scFv of F7. Unmodeled residues are shown as dashed lines. hLAG3: cyan; F7: yellow; M171I: red. mLAG3: green. Loop 1 of hLAG3: magenta. e, Crystal structure of mLAG3^D12. f, Crystal structure of hLAG3^D34 bound to F7. g, Structural alignment of mLAG3 D1, and hLAG3 D1. The Loop 2 region between the C’ and D strands adopts distinct conformations in each domain. h, Topology diagram of D1 from the mLAG3 structure.

Figure 2.. Structural and biochemical characterization of human and murine LAG3 dimers.

a-b, Comparison of mLAG3 and hLAG3 dimer conformations (top). Different D2-D2 packing geometries were observed in mLAG3 (a) and hLAG3 (b). The dihedral angles between D2 beta strands are highlighted in red. The lower panels depict residue networks at the dimer interfaces of mLAG3 (a) and hLAG3 (b). c, SEC chromatograms from hLAG3 and mLAG3 purification. d, SEC-MALS analyses of mLAG3, hLAG3:pk1, and hLAG3:pk2. Calculated and predicted molecular weights are listed in the table. e, Analyses of surface hydrophobicity for D2 domains of mLAG3 (left) and hLAG3 (right); lighter coloring indicates increased surface hydrophobicity. f, SEC-MALS analyses indicate that mLAG3^D12 is a homodimer in solution while the mLAG3^D12EE mutant is predominantly monomeric. g, SPR was used to determine the binding affinities of hLAG3* and mLAG3 for HLA-DR4^HIV and I-A(b)^MOG, respectively. h, SPR analyses of the binding affinities of mLAG3 binding to MHCII allomorphs I-A(b), I-A(d) and I-A(g7) loaded with CLIP peptide. The K_D and standard deviation were determined by averaging the values from two independent experiments.

Figure 3.. Epitope mapping and functional characterization of LAG3 antagonist scFvs.

a, Zoom window depicting F7 epitope on the hLAG3^D34:F7 structure. The F7-bound residues in hLAG3 (depicted below the zoom panel) are not conserved in mLAG3. b, The Arg¹⁰⁶ residue and Loop 2 epitope recognized by 15011 are highlighted on the structure of the mLAG3 D1 domain. A sequence alignment shows the conservation of mLAG3 and hLAG3 Loop 2 residues. c, Flow cytometry histogram plots depicting the binding of yeast-displayed CD4, hLAG3, hLAG3^R110G, and hLAG3^ΔLoop2 to 15011. d, Cartoon representations of F7 and 15011 scFvs binding to hLAG3. e, An SPR-based competition assay was performed to determine whether the 15011 or F7 inhibits hLAG3:MHCII interactions. Fixed concentrations of hLAG3 (800 nM) mixed with various concentrations of 15011-Fc or F7-Fc fusion proteins were injected over a sensor chip coated with HLA-DR4. ΔRU was calculated by subtracting the R_max of hLAG3 alone from the R_max of hLAG3:scFv complexes. The curves were plotted from 1 of 2 representative experiments. f, NFAT reporter assay comparing potency of F7 and 15011 as LAG3 antagonists. Net signals were normalized to a PBS control for analyzing RLU. The graph represents the mean RLU ± SD of n=3 replicates from representative of two independent experiments and statistics in comparison with PBS control group determined by using one-way ANOVA with P values noted in the figure.

Figure 4.. Identification of an FGL1-binding surface on the LAG3.

a, Flow cytometry histogram plots depicting the reduction in hLAG3 binding to FGL1^FD over subsequent rounds of negative selection. b, Yeast expressing various LAG3 mutants were stained with FGL1^FD (750 nM) or 15011 (100 nM) and binding was analyzed by flow cytometry. Relative binding indicates the MFI normalized to wildtype hLAG3. Data represent mean MFI ± SD of n=3 independent staining, and statistics determined by using one-way analysis of variance (ANOVA) with P values noted in the figure. Representative of two independent experiments is shown. c-d, Competition assays evaluating the ability of the scFv of 15011 to inhibit FGL1^FD binding to hLAG3. In c, yeast expressing hLAG3 were incubated with biotinylated FGL1^FD (500 nM) and the indicated concentrations of 15011. In d, samples containing hLAG3 (800 nM) and various concentrations of 15011 scFv were injected over an SPR sensor chip coated with FGL1^FD. In c and d, one representative of two independent experiments is shown. e, Mutations that diminished LAG3:FGL1 binding (red) are mapped on to the structure of hLAG3*.

Figure 5.. Structure and LAG3-interacting residues of FGL1^FD.

a, Crystal structure of FGL1^FD and structural comparison to Fibrinogen-like domains from FIBCD1 (PDB ID: 4M7F) and Ang1 (PDB ID: 4EPU). The A, B and P subdomains of FGL1^FD are colored in red, blue and yellow, respectively (left panel). The FIBCD1 ligand ManNAc and the Ang1 ligand Tie2 bind to similar positions on the P domains. b, Alanine scanning mutagenesis was used to identify LAG3-binding residues on FGL1. Yeast expressing alanine mutants of FGL1^FD were stained with biotinylated hLAG3 and binding was analyzed by flow cytometry. Relative binding was adjusted based on cell surface expression levels of each mutant. c, Cartoon schematic of the FGL1^FD yeast display construct used to generate a mutant library. Flow cytometry dot plots depict the binding of hLAG3 to yeast isolated from the initial and final rounds of selection. d, Table containing mutations identified in FGL1 variants following yeast display selections for enhanced LAG3-binding (top). A series of reversion mutants were expressed on yeast, stained with hLAG3, and analyzed by flow cytometry to identify which mutations conferred increased binding (bottom). MFI of LAG3 binding were adjusted to the surface expression level of each variant and normalized to that of wildtype FGL1^FD. e, Residues that positively or negatively affect FGL1 binding to hLAG3 were mapped on the structure of FGL1^FD. The affinity-enhancing mutations K181, G136 and G290 are colored in yellow, and pairs of alanine mutants are shown in matching colors. In b and d, data represent the mean relative binding ± SD of three independent replicates from representative of two independent experiments and statistics determined by using one-way analysis of variance (ANOVA) with P values noted in the figure.

Figure 6.. FGL1-induced LAG3 clustering correlates with FGL1 suppression mechanism.

a, A cartoon model of full-length FGL1 bound to LAG3 is shown as the interaction is predicted to occur on the cell surface. The left panel depicts stoichiometric amounts of FGL1 crosslinking LAG3 dimers to form higher-order oligomers, and the right panel depicts the loss of LAG3 clustering in the presence of excess FGL1. b, Stacked confocal microscopy images displaying LAG3 (green) and DAPI (blue) in Jurkat T cells expressing LAG3, either untreated or treated with 1, 10 or 100 nM of FGL1 protein for 30 min. c, The number of LAG3 clusters per cell was quantified and plotted as the average per field of view (FOV) from 1 of 3 representative experiments. Analysis was performed from n = 7 fields of view. d, An NFAT luciferase reporter assay was performed to assess the effect of FGL1 on LAG3-mediated suppression of T cell activation mediated by pMHCII. The relative activation was obtained by normalizing baseline-subtracted luciferase signals of tested concentrations to the buffer control group. In d, the graph represents n=3 replicates from representative of two independent experiments. All staistics was determined by one-way ANOVA with P values noted in the figure.