Journal: Cell Reports Medicine

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

doi: 10.1016/j.xcrm.2022.100660

Figure Lengend Snippet: 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 mm 2 (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.

Article Snippet: Antibodies used include anti-human panCK (Genetech, GM351507 ), anti-human CD8 (Biolynx, BX50036), anti-human FoxP3 (Abcam, ab20034), anti-human PD-1 (Sinobiological, 10377-MM23), anti-human GITR (Cell Signaling, 68014), anti-human CD4 (Abcam, ab133616 ) and anti-human DAPI (Thermo Fisher, D1306).

Techniques: Expressing, Diffusion-based Assay, Multiplex Assay, Immunohistochemistry, Marker, Quantitation Assay, Software

Journal: Cell Reports Medicine

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

doi: 10.1016/j.xcrm.2022.100660

Figure Lengend Snippet: 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.

Article Snippet: Antibodies used include anti-human panCK (Genetech, GM351507 ), anti-human CD8 (Biolynx, BX50036), anti-human FoxP3 (Abcam, ab20034), anti-human PD-1 (Sinobiological, 10377-MM23), anti-human GITR (Cell Signaling, 68014), anti-human CD4 (Abcam, ab133616 ) and anti-human DAPI (Thermo Fisher, D1306).

Techniques: Binding Assay, Fluorescence, FACS, SPR Assay, Reporter Assay, Blocking Assay, Comparison

Journal: Cell Reports Medicine

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

doi: 10.1016/j.xcrm.2022.100660

Figure Lengend Snippet: 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.

Article Snippet: Antibodies used include anti-human panCK (Genetech, GM351507 ), anti-human CD8 (Biolynx, BX50036), anti-human FoxP3 (Abcam, ab20034), anti-human PD-1 (Sinobiological, 10377-MM23), anti-human GITR (Cell Signaling, 68014), anti-human CD4 (Abcam, ab133616 ) and anti-human DAPI (Thermo Fisher, D1306).

Techniques: Activity Assay, Expressing, Mutagenesis

Journal: Cell Reports Medicine

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

doi: 10.1016/j.xcrm.2022.100660

Figure Lengend Snippet: 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.

Article Snippet: Antibodies used include anti-human panCK (Genetech, GM351507 ), anti-human CD8 (Biolynx, BX50036), anti-human FoxP3 (Abcam, ab20034), anti-human PD-1 (Sinobiological, 10377-MM23), anti-human GITR (Cell Signaling, 68014), anti-human CD4 (Abcam, ab133616 ) and anti-human DAPI (Thermo Fisher, D1306).

Techniques: In Vitro, Incubation, Phospho-proteomics, Flow Cytometry, Activation Assay, Functional Assay

Journal: Cell Reports Medicine

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

doi: 10.1016/j.xcrm.2022.100660

Figure Lengend Snippet: 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.

Article Snippet: Antibodies used include anti-human panCK (Genetech, GM351507 ), anti-human CD8 (Biolynx, BX50036), anti-human FoxP3 (Abcam, ab20034), anti-human PD-1 (Sinobiological, 10377-MM23), anti-human GITR (Cell Signaling, 68014), anti-human CD4 (Abcam, ab133616 ) and anti-human DAPI (Thermo Fisher, D1306).

Techniques: In Vivo, Activity Assay, Drug discovery, Knock-In, Sandwich ELISA, Concentration Assay, In Vitro, Inhibition

Journal: Cell Reports Medicine

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

doi: 10.1016/j.xcrm.2022.100660

Figure Lengend Snippet: 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).

Article Snippet: Antibodies used include anti-human panCK (Genetech, GM351507 ), anti-human CD8 (Biolynx, BX50036), anti-human FoxP3 (Abcam, ab20034), anti-human PD-1 (Sinobiological, 10377-MM23), anti-human GITR (Cell Signaling, 68014), anti-human CD4 (Abcam, ab133616 ) and anti-human DAPI (Thermo Fisher, D1306).

Techniques: Membrane, Transduction, Activity Assay

Journal: Cell Reports Medicine

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

doi: 10.1016/j.xcrm.2022.100660

Figure Lengend Snippet:

Article Snippet: Antibodies used include anti-human panCK (Genetech, GM351507 ), anti-human CD8 (Biolynx, BX50036), anti-human FoxP3 (Abcam, ab20034), anti-human PD-1 (Sinobiological, 10377-MM23), anti-human GITR (Cell Signaling, 68014), anti-human CD4 (Abcam, ab133616 ) and anti-human DAPI (Thermo Fisher, D1306).

Techniques: Purification, Recombinant, Antibody Labeling, Luciferase, Staining, Gene Expression, Software, Imaging

Journal: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology

Article Title: Combination of local radiotherapy and anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) therapy augments PD-L1 blockade-mediated anti-tumor effects in murine breast cancer model.

doi: 10.1016/j.radonc.2023.109981

Figure Lengend Snippet: Fig. 2. Antitumor effects of triple-combination therapy of RT, anti-GITR agonist and PD-L1 blockade using an in vivo syngenic murine triple negative breast cancer model. (A) Treatment schedule for mice treated with RT, anti-GITR agonist and PD-L1 blockade. (B) Mean tumor volume of subcutaneous implants at hind limb (left) and flank (right) in mice of each treatment group: Control, agonistic anti-GITR antibody, PD-L1 blockade, RT, agonist anti-GITR antibody + PD-L1 blockade, agonist anti-GITR antibody + RT, PD-L1 blockade + RT and triple-combination therapy. (n = 5 mice per group) (C) The representative samples of lung with metastatic nodules and lung metastasis nodule count by each treatment group. (D) The representative bioluminescence images of subcutaneous nodules before and after each treatment, after subcutaneous injection of 4T1-luc tumor cells. The relative tumor burden of each treatment group is quantified by measuring the luminous intensity of photons emitted from each tumor in the images. (E) Representative images of lung samples with metastatic nodules indicated with an arrow and counts grouped by treatment. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; RT, radiation therapy; αGITR, agonist anti-GITR antibody; αPD-L1., PD-L1 blockade.

Article Snippet: The anti-PD-L1 blocking antibody (5 mg/kg; BioXCell, BE0101) and the anti-GITR agonistic antibody (5 mg/kg; BioXCell, BE0063) were intraperitoneally injected on days 10, 12, 14, 17, and on days 11, 13, 15, 17, 19, and 21, respectively.

Techniques: In Vivo, Control, Injection

Journal: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology

Article Title: Combination of local radiotherapy and anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) therapy augments PD-L1 blockade-mediated anti-tumor effects in murine breast cancer model.

doi: 10.1016/j.radonc.2023.109981

Figure Lengend Snippet: Fig. 3. Profile of CD8+ cytotoxic T-cells in the tumor microenvironment according to treatment groups. (A) Flow cytometry analysis and immunohistochemistry results for CD8+ cytotoxic T-cells (CD3+ CD8+) (B) in the whole tumor microenvironment, (C) intratumoral region, (D) tumor periphery, and (E) in the stromal region are presented, respectively. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. RT, radiation therapy; αGITR, agonist anti-GITR antibody; αPD-L1, PD- L1 blockade.

Article Snippet: The anti-PD-L1 blocking antibody (5 mg/kg; BioXCell, BE0101) and the anti-GITR agonistic antibody (5 mg/kg; BioXCell, BE0063) were intraperitoneally injected on days 10, 12, 14, 17, and on days 11, 13, 15, 17, 19, and 21, respectively.

Techniques: Flow Cytometry, Immunohistochemistry

Journal: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology

Article Title: Combination of local radiotherapy and anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) therapy augments PD-L1 blockade-mediated anti-tumor effects in murine breast cancer model.

doi: 10.1016/j.radonc.2023.109981

Figure Lengend Snippet: Fig. 4. Profile of regulatory T-cells (Treg) in the tumor microenvironment according to treatment groups. (A) Flow cytometry analysis and (B) immunohisto chemistry results for Tregs (CD25+ Foxp3+) are presented, respectively. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. RT, radiation therapy; αGITR, agonist anti-GITR antibody; αPD-L1, PD-L1 blockade; Treg, regulatory T-cells.

Article Snippet: The anti-PD-L1 blocking antibody (5 mg/kg; BioXCell, BE0101) and the anti-GITR agonistic antibody (5 mg/kg; BioXCell, BE0063) were intraperitoneally injected on days 10, 12, 14, 17, and on days 11, 13, 15, 17, 19, and 21, respectively.

Techniques: Flow Cytometry, Immunohistochemistry

Journal: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology

Article Title: Combination of local radiotherapy and anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) therapy augments PD-L1 blockade-mediated anti-tumor effects in murine breast cancer model.

doi: 10.1016/j.radonc.2023.109981

Figure Lengend Snippet: Fig. 5. Immune cell profiles in the spleen according to treatment groups analyzed by flow cytometry. (A,B,C) Flow cytometric analysis results for (A) CD8+ cytotoxic T-cells, and (B) Effector memory T-cells (CD44high CD62Llow) and (C) Tregs (CD25+ Foxp3+) of the spleen are presented, respectively. * P < 0.05; ** P < 0.01; *** P<0.001; **** P < 0.0001. RT, radiation therapy; αGITR, agonist anti-GITR antibody; αPD-L1, PD-L1 blockade; Treg, regulatory T-cells.

Article Snippet: The anti-PD-L1 blocking antibody (5 mg/kg; BioXCell, BE0101) and the anti-GITR agonistic antibody (5 mg/kg; BioXCell, BE0063) were intraperitoneally injected on days 10, 12, 14, 17, and on days 11, 13, 15, 17, 19, and 21, respectively.

Techniques: Flow Cytometry

Journal: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology

Article Title: Combination of local radiotherapy and anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) therapy augments PD-L1 blockade-mediated anti-tumor effects in murine breast cancer model.

doi: 10.1016/j.radonc.2023.109981

Figure Lengend Snippet: Fig. 6. Production of interferons in plasma. Serum levels of (A) interferon-beta and (B) interferon–gamma at 1 week after administration of RT. * P < 0.05; ** P < 0.01; *** P < 0.001. CON, contrast; RT, radiation therapy; αGITR, agonist anti-GITR antibody; αPD-L1, PD-L1 blockade.

Article Snippet: The anti-PD-L1 blocking antibody (5 mg/kg; BioXCell, BE0101) and the anti-GITR agonistic antibody (5 mg/kg; BioXCell, BE0063) were intraperitoneally injected on days 10, 12, 14, 17, and on days 11, 13, 15, 17, 19, and 21, respectively.

Techniques: Clinical Proteomics

Journal: Journal of neuroendocrinology

Article Title: GHR−/− Mice are Protected from Obesity-Related White Adipose Tissue Inflammation

doi: 10.1111/jne.12854

Figure Lengend Snippet: Flow Cytometry Antibodies (24 Months)

Article Snippet: Results were analyzed with FlowJo V10 software. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Target Conjugate Manufacturer Catalog # RRID CD206 AF488 BioRad MCA2235A488 AB_324891 Ly-6C PerCP-Cy5.5 Thermo Fisher 45–5932-80 AB_1518762 MHC-II Biotin Thermo Fisher 13–5321-81 AB_466661 F4/80 PE-Cy7 Thermo Fisher 25–4801-82 AB_469653 CD11c APC Thermo Fisher 117–0114-81 AB_469345 CD45 APC-eFluor 780 Thermo Fisher 47–0451-82 AB_1548781 CD11b Alexa Fluor 700 Thermo Fisher 56–0112-80 AB_657586 CCR2 Phycoerythrin R&D Systems FAB5538P AB_10718414 NK1.1 FITC Thermo Fisher 11–5941-81 AB_465317 NKT Phycoerythrin BD Biosciences 550082 AB_393552 CD44 Biotin Thermo Fisher 13–0441-81 AB_466441 CD4 PerCP-Cy5.5 Thermo Fisher 45–0042-80 AB_906231 CD3 PE-Cy7 Thermo Fisher 25–0031-81 AB_469571 CD25 APC Thermo Fisher 17–0251-81 AB_469365 CD62L Alexa Fluor 700 Thermo Fisher 56–0621-80 AB_494004 CD45 Alexa Fluor 700 Thermo Fisher 56–0451-80 AB_891456 CD16/CD32 Thermo Fisher 14–0161-82 AB_467133 Streptavidin eFluor 615 Thermo Fisher 42–4317-80 AB_11218079 Open in a separate window Flow Cytometry Antibodies (8 Months) table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Target Conjugate Manufacturer Catalog # RRID CD11b VioGreen Miltenyi Biotec 130–113-811 AB_2726328 CD11c APC-Vio770 Miltenyi Biotec 130–110-841 AB_2654715 CD206 APC BioLegend 141707 AB_10896057 CD25 PE Miltenyi Biotec 130–108-996 AB_2656655 CD3 APC-Vio770 Miltenyi Biotec 130–109-840 AB_2657087 CD38 PE-Vio770 Miltenyi Biotec 130–109-258 AB_2657842 CD4 VioGreen Miltenyi Biotec 130–109-413 AB_2657964 CD44 PE-Vio770 Miltenyi Biotec 130–110-085 AB_2658157 CD45 VioBlue Miltenyi Biotec 130–110-802 AB_2658222 CD62L PerCP-Vio700 Miltenyi Biotec 130–107-046 AB_2660523 CD80 PE Miltenyi Biotec 130–116-460 AB_2727557 CD8a FITC Miltenyi Biotec 130–102-490 AB_2659883 F4/80 PerCP-Vio700 Miltenyi Biotec 130–102-161 AB_2651711 GITR APC Miltenyi Biotec 130–116-427 AB_2727531 Open in a separate window Flow Cytometry Antibodies (24 Months) 2.5.

Techniques: Flow Cytometry