Fig 2: Distribution of GITR protein in T cells from different tissues of GC patients(A) Total GITR (GITRTOT) expression in Treg cells from different tissues.(B) Comparison of surface GITR (GITRSUR) and GITRTOT expression in Treg cells among different tissues.(C) Composition ratio of GITRSUR and intracellular GITR (GITRIC) in Treg cells from different tissues.(D) Confocal images of GITR (clone: D919D) distributed on the surface membrane of Treg cells from GC tissues.(E) Confocal images of GITR (clone: D919D) distributed intracellularly in Treg cells from PBMC. The images showed in (D and E) are stained with DAPI (blue), CD4 (green), FOXP3 (magenta), and GITR (red). The rightmost panel is merged view of the four left images. The scale bar (10 μm) is placed at the left corner of each image.(F) Right: confocal image of GITR (clone: ERP20566) distributed intracellularly in iTreg cells and stained with DAPI (blue), CD4 (green), and GITR (red). Left: confocal image of isotype control in iTreg cells and stained with DAPI (blue), CD4 (green), and IgG (red). The scale bar (10 μm) is placed at the left corner of each image.(G) GITRTOT expression in Tconv cells from different tissues.(H) Comparison of GITRSUR and GITRTOT expression in Tconv cells among different tissues.(I) Composition ratio of GITRSUR and GITRIC in Tconv cells from different tissues.(J) GITRTOT expression in CD8 T cells from different tissues.(K) Comparison of GITRSUR and GITRTOT expression in CD8 T cells among different tissues.(L) Composition ratio of GITRSUR and GITRIC in CD8 T cells from different tissues.The clone number of GITR antibody used in (A–C and G–L) is 108–17. Data are represented as mean +/− SEM and were analyzed by unpaired Student’s t test in (A–C and G–L). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 3: IBI37G5 induces a bell-shaped dose response in vitro(A) Bell-shaped response of IBI37G5 in human CD4+ T cells. SEB-primed human CD4+ T cells were incubated with IBI37G5 for 5 min, and NF-?B p65 phosphorylation was detected by flow cytometry.(B) Detection of freely exposed Fabs of IBI37G5 at different levels of target saturation.(C) GITR receptor clustering upon IgG or IB37G5 treatment at different concentrations. Scale bar: 10 µm.(D) Quantification of foci number and intensity by GFP florescence (cell number = 23–66). Mean florescence intensity of foci (cells with foci formation) or diffused cytoplasmic GFP signal (cells without foci) was measured. Median (50%) and quartiles (25%, 75%) were shown in violin plots. Experiments were performed in duplicate.(E) Bell-shaped response induced by IBI37G5 in human CD4+ T cell activation and functional analyses. Experiments were performed in triplicate using T cells from 4 healthy donors. Mean ± SEM is presented, and p values were calculated using one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: The bell-shaped antitumor response of IBI37G5 is associated with the level of GITR receptor saturation in vivo(A–C) Antitumor activity of IBI37G5 as monotherapy or in combination with anti-PD1 antibody in MC38 tumor model. Mice (N = 6 mice/group) were treated with antibodies at indicated doses twice weekly for 5 times. Pooled data from two independent experiments were shown.(A) Individual tumor growth curves in mice from different treatment groups. Red curves highlight tumor regressions (CR or PR).(B) Waterfall plots showing tumor size changes at the end of study.(C) Individual, median (50%), and quartiles (25%, 75%) of tumor size change were shown in violin plots.(D and E) Anti-tumor activity of IBI37G5 in combination with anti-PD1 antibody in B16F10 tumor model. N = 7 mice/group, twice weekly dosing for 4 times.(F) Pharmacokinetics of IBI37G5 in mice. IBI37G5 at indicated doses were administered intravenously in hGITR knockin mice (N = 9 mice/group), and blood samples from indicated time points were collected and analyzed using sandwich ELISA. Dotted lines and blue areas in between depict the antibody concentration range matching the best in vitro activities.(G) PK/PD simulation of IBI37G5 in tumor models. Red (MC38) and blue (B16F10) curves show bell-shaped correlation between tumor-growth-inhibition percentage (TGI%) at different doses of IBI37G5 (left y axis). Regression analysis of IBI37G5 exposure levels (area under curve [AUC]) and dosages shows strong positive correlation (right y axis). Mean ± SEM is presented, and p values were calculated using one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Intratumoral GITR+ Treg cells exhibit stronger suppressive capacity(A) FOXP3 expression of GITR+ Treg cells and GITR− Treg cells from GC tissues.(B) CD25 expression of GITR+ Treg cells and GITR− Treg cells from GC tissues.(C–G) The immune checkpoints expression, including (C) CTLA-4, (D) PD-1, (E) OX40, (F) LAG3, and (G) CD39, of GITR+ Treg cells and GITR− Treg cells from GC tissues.(H) Suppressive capacity of GITR+ Treg cells and GITR− Treg cells isolated from GC tissues. CD8 T cells were co-stimulated with anti-CD3/CD28 beads. Left: histograms showing CTV dilution peaks indicating effector T (Teff) cell proliferation. Right: percentages of proliferative CD8 Teff cells co-cultured with GITR+ or GITR− Treg cells at ratios of 2:1, 4:1, 8:1, or no Treg. The schematic gating strategy for flow sorting of GITR+ Treg cells and GITR− Treg cells is shown in Figure S2A. Positive percentage is used to evaluate the level of CD39. MFI are used to evaluate the levels of FOXP3, CD25, CTLA-4, PD-1, OX40, and LAG3. Data are represented as mean +/− SEM and were analyzed by unpaired Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001; NS: not significant.