Journal:

Article Title: CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell

doi: 10.1111/j.1365-2567.2007.02537.x

Figure Lengend Snippet: Effects of in vitro CTLA-4 blockade on GATA-3 protein level/cell and frequency of IL-4-producing cells. Naive CD4+ T cells purified from the spleens of normal mice were induced to differentiate with anti-CD3 mAb, a recombinant form of CD80-Fc, IL-4 and anti-IFN-γ mAb for 3 days. Anti-CTLA-4 mAb was added in soluble form at increasing concentrations (0·1–10 μg/ml). (a) Mean fluorescence intensity for GATA-3 staining, (b) percentage of IL-4+ cells.

Article Snippet: 18 CD4 cell culture For in vitro experiments, splenic naive CD4 cells were purified from non-immunized BALB/c mice by immuno-magnetic cell sorting as previously described 19 and cultured in 24-well plates precoated with anti-CD3ε mAb (clone 145-2C11; 10 μg/ml) and/or recombinant mouse B7-1(CD80)/Fc chimeric protein (R & D Systems, cat. 740-B1; 0·33 μg/ml).

Techniques: In Vitro, Purification, Recombinant, Fluorescence, Staining

Journal: bioRxiv

Article Title: Lack of blocking activity in anti-CTLA-4 antibodies reduces toxicity, but not anti-tumor efficacy

doi: 10.1101/2021.07.12.452090

Figure Lengend Snippet: A. The ability of CTLA-4 mAbs to block binding of CD80 or CD86 was measured using a plate-based ELISA method. CTLA-4 was used to coat the plate, then after the antibody samples were incubated, His-tagged CD80 or CD86 was added, and the amount of ligand able to bind to CTLA-4 was measured. Blocking mAbs that prevent CD80 or CD86 from binding to CTLA-4 reduce the absorbance signal due to lack of CD80 or CD86 binding to CTLA-4. Weak-blocking mAbs still allow CD80 or CD86 to bind, preventing the loss of all absorbance signal. B. Plots show the ability of GIGA-564 to block the interaction between CTLA-4 and the B7 ligands CD80 and CD86 compared to ipilimumab and CTLA-4.28, as assessed by ELISA as described in (A). Absorbance values were normalized to an anti-PD-1 control (pembrolizumab) and displayed as the average of two technical replicates. C-D. The key residues mediating CTLA-4 binding were identified for GIGA-564 and ipilimumab by shotgun mutagenesis of CTLA-4, followed by staining and flow cytometry assessment of binding. C. Shown is the crystal structure (Protein Database [PDB] 1I8L) of the complex between CD80 (blue) and CTLA-4 (gray) on which the CTLA-4 epitope residues shared between ipilimumab and GIGA-564 were colored orange and the key differentiating residue R70 was colored red (visualized with Pymol). Additionally, G142 was identified as a secondary residue for the epitope of ipilimumab but not GIGA-564. D. Table showing key amino acids on CTLA-4 of interest for these epitopes; those found by mutational analysis to be important for binding of CTLA-4 to CD80 or CD86 in a cell-based assay are marked in gray to indicate the epitope residues for those proteins .

Article Snippet: Recombinant human His-tagged CD80 (R&D Systems 9050-B1, Minneapolis, MN, USA) or CD86 (R&D Systems 9090-B2, Minneapolis, MN, USA) was then added to the plates at 1 μg/mL.

Techniques: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control, Mutagenesis, Staining, Flow Cytometry, Residue, Cell Based Assay

Journal: bioRxiv

Article Title: Lack of blocking activity in anti-CTLA-4 antibodies reduces toxicity, but not anti-tumor efficacy

doi: 10.1101/2021.07.12.452090

Figure Lengend Snippet: Top panel: Ipilimumab blocks CTLA-4 interaction with CD80/CD86, which allows antigen presenting cells (APCs) to co-stimulate peripheral Tregs enhancing their proliferation. GIGA-564 weakly blocks CTLA-4 interaction with CD80/CD86 and thus induces less Treg proliferation. Bottom panel: Ipilimumab and GIGA-564 bind CTLA-4 on intratumoral Tregs to induce Treg killing via interactions with Fc receptor (FcR) on effector cells. GIGA-564 induces stronger FcR signaling and thus more efficiently depletes intratumoral Tregs than ipilimumab.

Article Snippet: Recombinant human His-tagged CD80 (R&D Systems 9050-B1, Minneapolis, MN, USA) or CD86 (R&D Systems 9090-B2, Minneapolis, MN, USA) was then added to the plates at 1 μg/mL.

Techniques:

Journal: Journal for immunotherapy of cancer

Article Title: XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, demonstrates tumor-growth inhibition and tumor-selective pharmacodynamics in mouse models of cancer.

doi: 10.1136/jitc-2023-007785

Figure Lengend Snippet: Figure 4 In vitro function of XTX101. (A,B) In vitro inhibition of human CTLA-4 binding to CD80 (A) and CD86 (B) by XTX100 (black), XTX101 (red), and activated XTX101 (blue), as assessed by ELISA. (C) In vitro activity of intact (red) and activated XTX101 (blue) compared with XTX100 (black) in antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay. (D) IL-2 production of SEB-stimulated human peripheral blood mononuclear cells (PBMCs) incubated with XTX100 (black), XTX101 (red), and activated XTX101 (blue), as measured by ELISA. Fold-change from isotype at highest mAb concentration is reported. ND, not determined.

Article Snippet: Serial dilutions of test articles were added to the washed ELISA plates followed by addition of a 2.6 μg/ mL solution of recombinant human CD80 or CD86 (9050- B1- 100 or 9090- B2- 100, R&D Systems).

Techniques: In Vitro, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Bioassay, Incubation, Concentration Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 1 Pre-engagement of Ipilimumab and Tremelimumab with soluble CTLA-4-Ig prevents CTLA4 binding to cell bound CD80 or CD86 ligand. (A) Schematic of experimental set-up. A fixed dose (2µg/ml) of soluble APC conjugated Abatacept was incubated on ice with a titration of soluble anti- CTLA-4 Abs to pre-engage CTLA-4 with anti-CTLA-4. This was added to DG75 B cells expressing either CD80 or CD86-GFP and incubated on ice for 30 minutes. Cells were washed and analysed for Abatacept-ligand interaction by flow cytometry. All reagents were chilled on ice before use. (B) Representative concatenated FACS plots show impact of Abatacept-ligand engagement as the dose of anti-CTLA-4 Abs increased. (C) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab(Ipi) (Black line) and tremelimumab (Treme) (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates and data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Binding Assay, Incubation, Titration, Expressing, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 2 Ipilimumab and Tremelimumab outcompete cell bound CD80 or CD86 for engagement with soluble CTLA-4-Ig. (A) Schematic of experimental set-up. DG75 B cells expressing either CD80 or CD86-GFP were mixed with a titration of soluble anti-CTLA-4 Abs. A fixed dose (2µg/ml) of soluble APC conjugated Abatacept was added to assess direct competition between soluble anti-CTLA-4 and cell bound ligand for Abatacept binding. Cells were incubated on ice, washed and Abatacept-ligand engagement analysed by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced Abatacept-ligand engagement as the dose of anti-CTLA-4 Abs increased. (C) Anti- CTLA-4 doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates and data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Titration, Binding Assay, Incubation, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 3 Ipilimumab and Tremelimumab are unable to displace pre-engaged CTLA-4-Ig from cell bound CD80 or CD86. (A) Schematic of experimental set-up. DG75 B cells expressing either CD80 or CD86-GFP were pre-incubated on ice with a fixed dose (2µg/ml) of soluble APC conjugated Abatacept to pre-engage soluble CTLA-4 with ligand. Cells were washed, treated with a titration of soluble anti-CTLA-4 Abs and incubated on ice or at 37°C. Cells were washed and analysed for Abatacept-ligand engagement by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show Abatacept-ligand binding at different doses of anti-CTLA-4 Abs when incubated on ice or when incubated at 37°C. (C) Dose response curves for ipilimumab (Black line) and tremelimumab (Blue line) presented as MFI CTLA-4-Ig binding from data shown in (B) Data presented are mean +/- SD from 3-6 independent experiments. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3-6).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Incubation, Titration, Cytometry, Ligand Binding Assay, Binding Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 4 Pre-engagement of cell bound CTLA-4 with soluble CD80 and CD86Ig inhibits Ipilimumab and Tremelimumab from binding. (A) Schematic of experimental set-up. Jurkat cells expressing CTLA-4 were pre-incubated on ice with either soluble CD80 or CD86Ig to pre-engage CTLA-4 with ligand. Cells were washed, incubated with a fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA4 Abs and incubated on ice. Cells were washed and analysed for anti-CTLA-4-CTLA-4 binding by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced anti-CTLA-4 engagement as the dose of CD80 or CD86Ig was increased. CD80 or CD86Ig doses were Log(x) transformed and dose response curves fit using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (C) CD80 or CD86Ig doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Binding Assay, Expressing, Incubation, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 5 Soluble CD80 and CD86Ig outcompete Ipilimumab and Tremelimumab for engagement with cell bound CTLA4. (A) Schematic of experimental set-up. A fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA4 Abs was pre-incubated with a titration of either soluble CD80 or CD86Ig on ice. Jurkat expressing CTLA-4 cells were added to facilitate direct competition between anti-CTLA4 Abs and CD80 or CD86Ig for cell bound CTLA-4 occupancy. Cells were incubated on ice, washed and anti-CTLA4-CTLA4 engagement analysed by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced anti-CTLA-4-CTLA-4 engagement as the dose of CD80 or CD86Ig was increased. (C) CD80 or CD86Ig doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Incubation, Titration, Expressing, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 6 Soluble CD80 and CD86Ig are unable to displace pre-engaged Ipilimumab and Tremelimumab from cell bound CTLA-4. (A) Schematic of experimental set-up. Jurkat cells expressing CTLA-4 were pre-treated with a fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA-4 Abs on ice to pre-engage anti-CTLA-4-CTLA-4. Cells were washed and a titration of either soluble CD80 or CD86Ig was added. Cells expressing WT CTLA-4 were incubated on ice and cells expressing a non-internalising CTLA-4 mutant (Del 36) were incubated at either on ice or at 37°C. Cells were washed and analysed for anti-CTLA-4-CTLA-4 staining by flow cytometry. (B) Concatenated FACS plots showinganti-CTLA-4 binding at various doses of CD80 or CD86Ig (C) Dose response curves for ipilimumab (Black line) and tremelimumab (Blue line) presented as MFI anti- CTLA-4 binding from data in (B) Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3-6).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Titration, Incubation, Mutagenesis, Staining, Cytometry, Binding Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 7 Ipilimumab and Tremelimumab block CD80 and CD86 ligand loss for APC by transendocytosis. (A) CTV stained CHO cells expressing either CD80 or CD86-GFP were cultured in the presence of CHO cells expressing CTLA-4 at a 1:1 ratio for 5 hours at 37°C. A titration of soluble ipilimumab (Black line) or tremelimumab (Blue line) were added at 0h. Percentage of CD80 or CD86-GFP ligand loss from CTV+ CHO cells by transendocytosis was determined by making the GFP MFI of CTV+ CHO cells relative to a negative control where no CTLA-4 was present. (B) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fit using Prism v6 to obtain Log EC50 for Iipilimumab (Black line) and tremelimumab (Blue line). (C) Graphs show the mean EC50 with 95% confidence interval calculated using Prism v6. (D-F) As in A, B & C except with CTLA-4 expressing Jurkat and CTV stained DG75 B cells expressing CD80 or CD86-GFP. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates, data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Blocking Assay, Staining, Expressing, Cell Culture, Titration, Negative Control, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 8 Ipilimumab and Tremelimumab interfere with pre-established transendocytosis of CD80 and CD86 to prevent further ligand loss from APC. (A) CTV stained CHO cells expressing either CD80 or CD86-GFP were cultured in the presence of CHO cells expressing CTLA-4 at a 1:1 ratio for 0, 3 or 6 hours at 37°C. Transendocytosis was established for 3 hours in the absence of anti-CTLA-4 Abs at which point soluble ipilimumab (dotted black line) or tremelimumab (dotted blue line) were spiked into culture. Transendocytosis was continued for a further 3 hours to observe the effect of anti-CTLA4 spike. Soluble anti-CTLA-4 antibodies were also added at timepoint 0h to block transendocytosis from the start of the assay (solid black – ipilimumab or blue – tremelimumab lines). Transendocytosis was also performed in the absence of anti-CTLA-4 treatment (Purple line). (B) % of CD80 or CD86-GFP ligand loss from CTV+ CHO cells by transendocytosis was determined by making the GFP MFI CTV+ CHO cells relative to a negative control where CTLA4 was not expressed (n=3). (C, D) As in A & B except with CTLA-4 expressing Jurkat and CTV stained DG75 B cells expressing CD80 or CD86-GFP cultured at a Jurkat:DG75 ratio of 2:1. Also an additional spike was performed at 6h and the assay was run for a total of 20h. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=2).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Staining, Expressing, Cell Culture, Blocking Assay, Negative Control