Fig 2: Surface plasmon resonance (SPR) results showing LAG-3, anti-LAG3 mAb, and Gal-3 binding kinetics. A SPR plot showing Gal-3 binding to LAG-3 in a dose-dependent way. B Fits to the experimental data were made with EVILFIT for the LAG-3/Gal-3 interaction. The distribution in ligand binding kinetics is shown in the two-dimensional grids. Each grid point represents a 1:1 interaction typified by KD and kd. A third coordinate, indicated with a color scale, represents the abundance of these interactions. C SPR plot and corresponding EVILFIT curve (D) showing Gal-3 binding to the antagonistic LAG-3 Ab (17B4) without the presence of LAG-3. Kinetics in a similar range as Gal-3/ LAG-3 interaction is seen. E SPR plot showing how Gal-3 interferes with the LAG-3/antagonistic LAG3 antibody binding in a dose-dependent way. Chip with immobilized antagonistic LAG-3-Ab (17B4). Flow containing a constant Fc:LAG3 amount and increasing concentrations of human recombinant Gal-3 were added. F The interaction between LAG-3 and LAG-3 mAb as a function of Gal-3 concentration is plotted with two-dimensional fits. To quantify the development in the distribution of binding kinetics, three bins (Bin 1, Bin 2, and Bin 3) were defined, guided by the major types of interactions observed for LAG-3, Gal-3, and LAG-3 mAb. G From bin 1, we see a typical signal from a mAb interaction (KD at 1 nM) at the lowest concentrations of Gal-3. This signal decreases with increasing Gal-3 concentrations and is lost above 1600 nM Gal-3. Bin 2 shows an intermediate state with complexes between LAG-3, Gal-3, and mAb that occurs with Gal-3 concentrations between 100 and 3200 nM Gal-3 (KD 10 µM). Bin 3 shows direct binding between Gal-3 and anti-LAG-3-mAb (KD 1 mM–1 µM)

Fig 3: Effects of rhLAG-3 and antagonistic LAG-3 mAb on cytokine production. Production of cytokines in PBMC and paired SFMC cultures from cRA patients (n = 10) with or without the addition of recombinant human LAG-3 (A) or antagonistic LAG-3 mAb (B). The level of significance is indicated by * < 0.05. Differences were analyzed using the Mann–Whitney test to compare the two groups

Fig 4: Cellular expression of LAG-3 in PBMCs and SFMCs. A Cellular expression of LAG-3 on CD3 + CD4 + T cells from PBMCs and SFMCs in HC (n = 6) and cRA (n = 9). B Representative FACS data from one of the cRA patients shown in the flow plot. C Distribution of LAG-3 + cells in relation to CD45R0 presented in the bar graph (n = 12) and representative FACS data from one of the cRA patients shown in the flow plot (D). E Percentages of LAG-3 + cells determined by PD-1 co-expression are presented in the bar graph (n = 4) and representative FACS data from one of the cRA patients are shown in the flow plot (F). G Percentages of LAG-3 + cells capable of IL-2 production or not (n = 4) with a representative flow plot in H. Bars indicate the median and whiskers standard deviation. The level of significance is indicated on the graph. Differences were analyzed using the paired t tests or Mann–Whitney test to compare the two groups

Fig 5: Bivalent LAG-3 binding by FS118 is required for maximal LAG-3 shedding in both CD4+ and CD8+ T cells. (a) A biologically similar antibody was generated, which was monovalent for LAG-3 (LAG-31) and bivalent for PD-L1 (PD-L12). Schematic representations of FS118 and LAG-31/PD-L12 mAb2 are shown. Orange, PD-L1-binding regions; blue, fragment crystallizable region; yellow, LAG-3-binding regions. (b) The sLAG-3 level from human SEB assays consisting of allogenic expanded CD4+ T cells and immature dendritic cells cocultured for 4 days, in response to either FS118 or LAG-31/PD-L12 mAb2. sLAG-3 levels in the supernatant are shown from three individuals. Assays performed in technical duplicate, and data shown as mean ± SD. (c) MHC I- or MHC II-restricted antigen recall assay. PBMCs were stimulated with MHC I- or MHC II-restricted peptides then exposed to FS118 or LAG-31/PD-L12 mAb2. The supernatant was collected, and sLAG-3 levels were measured by ELISA (data from two individual donors are shown). Assays performed in technical duplicate, and data shown as mean ± SD. LAG-3, lymphocyte-activation gene 3; MHC, major histocompatibility complex; PBMCs, peripheral blood mononuclear cells; PD-L1, programmed death ligand 1; SEB, Staphylococcal enterotoxin B; sLAG-3, soluble LAG-3.