Optimal target saturation of ligand-blocking anti-GITR antibody IBI37G5 dictates FcγR-independent GITR agonism and antitumor activity

Abstract

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a co-stimulatory receptor and an important target for cancer immunotherapy. We herein present a potent FcγR-independent GITR agonist IBI37G5 that can effectively activate effector T cells and synergize with anti-programmed death 1 (PD1) antibody to eradicate established tumors. IBI37G5 depends on both antibody bivalency and GITR homo-dimerization for efficient receptor cross-linking. Functional analyses reveal bell-shaped dose responses due to the unique 2:2 antibody-receptor stoichiometry required for GITR activation. Antibody self-competition is observed after concentration exceeded that of 100% receptor occupancy (RO), which leads to antibody monovalent binding and loss of activity. Retrospective pharmacokinetics/pharmacodynamics analysis demonstrates that the maximal efficacy is achieved at medium doses with drug exposure near saturating GITR occupancy during the dosing cycle. Finally, we propose an alternative dose-finding strategy that does not rely on the traditional maximal tolerated dose (MTD)-based paradigm but instead on utilizing the RO-function relations as biomarker to guide the clinical translation of GITR and similar co-stimulatory agonists.

Keywords: GITR; agonist antibody; cancer immunotherapy; costimulatory receptor; receptor occupancy.

Graphical abstract

Figure 1

Dynamic regulations of GITR and PD1 expression on TILs from human CRC (A) Experimental design. (B–G) Transcriptomic (B–E) and histological analyses (F–G) on tumor-infiltrating lymphocytes (TILs) from human CRC samples. (B) Uniform manifold approximation and projection (UMAP) representation of CD3⁺ TIL clusters from 7 CRC patients (top). Fraction of cells in each CD3⁺ TIL clusters per sample (bottom). p, patient; core, neoplasm core; border, neoplasm border. (C) Heatmap of differentially expressed genes in CD3⁺ clusters from (B). (D) Diffusion map of CD3⁺ clusters using the first two diffusion components (top). Clusters are colored according to identities in (B). Pseudotime (bottom left) and feature plots for CD8 and CD4 (bottom right) are depicted on the same scale. (E) Sliding windows (N = 200) of average expression of genes of interest in CD8⁺ T cells are quantified along the pseudotime cell order. (F) A representative image showing the localization of PD1⁺GITR⁺CD8⁺ T cells (arrow heads) in CRC tissues using multiplex immunohistochemistry (left, scale bar: 100 μm). The inset illustrates higher resolution images (right, scale bar: 25 μm). PanCK (orange) is tumor cell marker, and white depicts merged green (PD1) and purple (GITR). (G) Quantitation of PD1⁺, GITR⁺, and GITR⁺PD1⁺ expression in tumor-infiltrated CD8⁺ T cells. Each dot represents one tumor sample. Regression analysis showing PD1⁺ and GITR⁺CD8⁺ T cells numbers per mm² (top). p value was calculated automatically by GraphPad software in linear regression module. Box and whiskers plot represents the frequency of expression of PD1 and GITR in CD8⁺ TILs (bottom). p values were calculated using one-way ANOVA, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 2

Characterization of IBI37G5, a ligand-mimetic anti-GITR agonist antibody (A) Binding of IBI37G5 to CHOS-hGITR cells using fluorescence-activated cell sorting (FACS) analysis. (B) Kinetic analysis of IBI37G5 binding to hGITR using surface plasmon resonance. (C) Agonistic activities of different IBI37G5 formats in Jurkat-hGITR NF-κB reporter assay. Graph shows representative results of at least 3 replicate experiments. (D and E) IBI37G5 competes GITRL binding to GITR in FACS and bio-layer interferometry (BLI) analysis. (D) Competitive binding of IBI37G5 with hGITRL-mFc on Jurkat-hGITR cells. Mean ± SD is presented. (E) Sandwich ligand-blocking assay showing hGITR/hGITRL interaction blocked by IBI37G5. Representative sensorgrams from duplicate measurements are shown. (F) Alanine scanning on GITR shows residues required for IBI37B5 or GITRL binding (red) and residues only required for IBI37G5 binding (purple). (G) Modeled structure of hGITR and Fv (IBI37G5) complex shown in cartoon. hGITR, VH, and VL are colored in yellow, marine, and blue, respectively. Interface residues included in the epitope and CDR3 regions are shown as sticks. (H) hGITRL, Fv, and overlapped binding regions on hGITR. (I) Superimposed structures of hGITR/Fv (IBI37G5) and hGITR/hGITRL complexes (left). Schematic diagram elucidated the significant interactions between hGITR (gray) and IBI37G5-VL (magenta) and IBI37G5-VH (yellow) and hGITRL (cyan). Hydrogen bonds, salt bridges, and van der Waals interactions are indicated in orange dashed lines, purple lines, and green lines, respectively (right). The table listed the information of interactions (bottom). (J) Comparison of modeled receptor-antibody (left) and receptor-ligand (right) complexes shown in surface representation. hGITR/IBI37G5 was modeled based on the most probable conformation of hIgG1. One GITR receptor dimer was masked from hGITR/hGITRL complex to show receptor-ligand interaction. The distance was measured between the C termini of modeled hGITR.

Figure 3

IBI37G5 combined with PD1 blockade synergistically activates T cells in vitro and in vivo (A) Human T cell activations after indicated treatments were determined by measuring released IFN-γ in the supernatant. Biological replicates from 4 different donors were shown. (B and C) Individual tumor growth curves post treatments in MC38- (B) or CT26- (C) inoculated mice. Mice were randomized at day 7 when tumor volume reached around 60–80 mm³ before being treating with αPD1 (0.5 mg/kg) or IBI37G5 (1 mg/kg) twice weekly. The waterfall plots show tumor volume changes by the end of study. Tumor complete regression was defined as CR, and tumor volume reduced >30% from baseline was defined as PR. N = 5–7 mice in each group. (D–F) Tumor weight (D) and percentage of tumor-infiltrating CD8⁺ T cells (E) or CD4⁺ T cells (F). (D–I) Mice bearing MC38 tumors were treated with indicated antibodies and sacrificed 1 week post treatment. (G and H) Representative FACS plots and bar graphs showing intracellular staining of TNF-α and IFN-γ in CD8⁺ TILs (G) and CD4⁺ TILs (H) after ex vivo stimulation with MC38 cells. (I) Representative FACS plots of Treg cell populations in tumors and spleens of mice after treatments. Bar graphs showing Treg cell percentage in CD4⁺ T cells and ratio of CD8⁺ T/Treg in tumors and spleens. Each dot represents one mouse (N = 9 mice/group). 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.

Figure 4

GITR homodimerization and antibody bivalency are both required for the agonistic activity of IBI37G5 (A) The cartoon models of WT dimeric hGITR and mutants when mutating two critical interface residues F137 and F139 into alanines (AA), arginines (RR), or aspartic acids (DD). The mutated residues are shown as sticks. Overall stability and interface energy density of WT dimeric hGITR and mutants were calculated by ROSETTA relax application. (B–D) Activities of GITR agonists: IBI37G5 (B), GITRL trimer or GITRL hexamer MEDI1873 (C), or monovalent (mv) IBI37G5 (D) measured in Jurkat NF-κB reporter cells expressing WT or mutant GITRs. Mean ± SD is presented. (E) Confocal images showing GITR receptor clustering on Jurkat cells expressing WT or mutant GITRs upon indicated treatments at 10 nM. Scale bar: 10 μm. Quantification of GFP foci number and intensity (cell number = 20–50). Mean florescence intensity of foci or diffused cytoplasmic GFP signal was measured. Median (50%) and quartiles (25%, 75%) were shown in violin plots. Experiments were repeated at least twice. p values were calculated using one-way ANOVA, ∗∗∗∗p < 0.0001.

Figure 5

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-κB 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.

Figure 6

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.

Figure 7

Proposed working models (A) IBI37G5 binds two GITR dimers simultaneously to form the basic signaling unit-like complex on the cell membrane in a 2:2 stoichiometry, and free IBI37G5 antibody can engage and link two pre-arranged complexes to form higher-order cross-linking for signal transduction. (B) If GITR were not able to form homodimer (due to disrupted dimeric interface on CRD3), IBI37G5 can only engage two GITR monomers but is unable to induce GITR cross-linking. (C) mvIBI37G5 can only bind one GITR dimer and fails to form GITR cross-linking. (D) Hypothetical dose-dependent RO-activity relationship of IBI37G5 on GITR agonism (left). At optimal RO, IBI37G5 links GITR dimers to form a linear chained network to transduce robust downstream signaling (top right). At oversaturated RO, IBI37G5 binds to GITR in a monovalent pose and only induces weak GITR agonism due to failed receptor cross-linking (bottom right).