Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 binds to human TNFR2 and cynomolgus TNFR2 selectively. (A) Binding assay of AN3025 to human TNFR2 on plate by ELISA (EC 50 = 0.052nM). (B) Binding assay of AN3025 to human TNFR1 on plate by ELISA. (C) Binding assay of AN3025 to cynomolgus TNFR2 on plate by ELISA (EC 50 = 0.053nM). (D) Binding assay of AN3025 to mouse TNFR2 on plate by ELISA. (E) Binding assay of AN3025 to rat TNFR2 on plate by ELISA. The ELISA assays were tested in duplicates. Values were expressed as Mean ± SEM.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 inhibits TNFα-TNFR2 mediated cell death of hTNFR2 overexpressing Jurkat cell and enhances T effector cell function in Tregs/Teff co-culture assay. (A) Expression of human TNFR2 on wildtype Jurkat cells. (B) Viability of wildtype Jurkat cells after stimulation with titrated human TNFα. (C) Expression of human TNFR2 on established hTNFR2 overexpressing Jurkat single cell clone. (D) Viability of hTNFR2 overexpressing Jurkat cells after stimulation with titrated human TNFα. (E) Viability of hTNFR2 overexpressing Jurkat cells after stimulation with constant 0.5ng/mL human TNFα in the presence of titrated AN3025 or titrated human IgG 1 , κ. (F) Competitive ELISA binding assay of AN3025 with biotinylated human TNFα to human TNFR2 on plate. (G, H) Human iTregs were co-cultured with CFSE-labeled autologous CD4 + Teff cells for 4 days. (G) CFSE lo T effector cell percentage was quantified by flow cytometry. (H) IFNγ in the supernatants was detected by ELISA assay. Values were expressed as Mean ± SEM. **P < 0.01; ***P < 0.001.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Cell Function Assay, Co-culture Assay, Expressing, Competitive ELISA, Binding Assay, Cell Culture, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 significantly inhibits MC38 tumor growth as a monotherapy in hTNFR2 mouse model. (A–E) Murine colon cancer MC38 cells (5E5) were implanted subcutaneously into homozygous TNFR2 humanized mice (female). Mice were grouped when tumor volume reached approximately 100 mm 3 . (A, B) MC38 tumor bearing TNFR2 humanized mice were treated with AN3025 at the dosage of 10mg/kg, 3mg/kg or 1mg/kg every 4 days intraperitoneally for 5 doses in total. (A) Tumor volume measurement during the treatment (n=8 each group). (B) Body weight record during the treatment (n=8 each group). (C) MC38 tumor bearing TNFR2 humanized mice were treated with AN3025 at the dosage of 10mg/kg, 3mg/kg every 3 days intraperitoneally for 3 doses in total. Tregs (CD45 + CD3 + CD4 + Foxp3 + ) frequency in total CD4 + T cells in the MC38 tumors was quantified by flow cytometry (n=6 each group). (D, E) MC38 tumor bearing TNFR2 humanized mice were treated intraperitoneally with 10mg/kg AN3025 alone or combination of 10mg/kg AN3025, 0.3mg/mouse anti-mouse CD4 depletion antibody and 0.3mg/mouse anti-mouse CD8 depletion antibody every 4 days for 5 doses in total. (D) Tumor volume measurement during the treatment (n=8 each group). (E) Body weight record during the treatment (n=8 each group). Values were expressed as Mean ± SEM. **P < 0.01; ***P < 0.001.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 enhances anti-tumor efficacy of mouse PD-1 antibody in combination study. (A–H) Murine colon cancer MC38 cells (5E5) were implanted subcutaneously into homozygous TNFR2 humanized mice (female). Mice were grouped when tumor volume reached approximately 100 mm 3 . (A–F) MC38 tumor bearing TNFR2 humanized mice were treated with 10 mg/kg AN3025 or 10mg/kg mouse PD-1 antibody (mPD-1 Ab) every 3 days intraperitoneally for 7 doses in total. (A) Tumor volume measurement during the treatment (n=8 each group). (B) Body weight record during the treatment (n=8 each group). (C) Representative immunohistochemistry images of CD8 staining on MC38 tumors. (D) Quantifications of CD8 + T cells per mm 2 by immunohistochemistry (n=3 each group). (E) Representative immunohistochemistry images of CD4 staining on MC38 tumors. (F) Quantifications of CD4 + T cells per mm 2 by immunohistochemistry (n=3 each group). (G, H) MC38 tumor bearing TNFR2 humanized mice were treated with 3mg/kg AN3025 or 3mg/kg mPD-1 Ab or combination of AN3025 and mPD-1 Ab every 3 days intraperitoneally for 7 doses in total. (G) Tumor volume measurement during the treatment (n=8 each group). (H) Body weight record during the treatment (n=8 each group). Values were expressed as Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Immunohistochemistry, Staining

Journal: Journal of immunological methods

Article Title: Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

doi: 10.1016/j.jim.2021.113173

Figure Lengend Snippet: Fig. 1. Characterization of anti-mouse TNFR2 antibodies in vitro. (A and B) mTNFR2 stable transfected CHO-K1 cells were incubated with 3-fold increasing con centrations of each rat IgG2a mAbs, and binding was detected by flow cytometry assessing TNFR2 + population percentage (A) and gMFI (B). (C) TNFα ligand competition with generated antibodies assessed by FACS. Data represented as a three-parameter gMFI dose-response curve fit of the blocker antibodies with appropriate controls incubations with 3-fold increasing concentrations. Two benchmark hamster-anti-mTNFR2 antibodies with known blocking activity were added as controls. All data based on mean and SEM is representative of three independent experiments.

Article Snippet: First, a single estimation screening of KD value was performed with an expected saturating concentration of 100 nM His tagged mTNFR2 (R&D Systems, 426-R2/CF) 100 nM diluted in 10× Kinetics Buffer (KB) followed by a dissociation step.

Techniques: In Vitro, Transfection, Incubation, Binding Assay, Flow Cytometry, Generated, Blocking Assay, Activity Assay

Journal: Journal of immunological methods

Article Title: Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

doi: 10.1016/j.jim.2021.113173

Figure Lengend Snippet: Fig. 2. Characterization of anti-mTNFR2 mAbs targeting CRDs 1–4. (A) Schematic representation of the 6 mouse-human TNF2 chimeras CRD1-CRD4 (Cystein Rich Domain). (B) The targeting CRD of each mAb were determined by cell ELISA with mouse-human TNFR2 domain swap mutants. Data represented as a three-parameter OD450–620 detection based on mean and SD of three independent experiments. (C) The domain epitopes of the 13 mAbs are indicated on a hTNFR2-hTNFα trimer structure (PDB: 3ALQ), 74% similar to mouse TNFR2. The CRDs for one TNFR2 receptor are shown in indicated colors.

Article Snippet: First, a single estimation screening of KD value was performed with an expected saturating concentration of 100 nM His tagged mTNFR2 (R&D Systems, 426-R2/CF) 100 nM diluted in 10× Kinetics Buffer (KB) followed by a dissociation step.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunological methods

Article Title: Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

doi: 10.1016/j.jim.2021.113173

Figure Lengend Snippet: Fig. 3. Characterization of anti-mouse TNFR2 antibodies with ex vivo material. (A) TNFR2 expression upon binding of anti-mTNFR2 antibodies to Treg cell pop ulation. Detection by commercial hamster anti-TNFR2 direct labeled with PE with the respective hamster-isotype control labeled with PE (left). Generated rat anti- mTNFR2 antibodies and a rat isotype control were detected by a secondary antibody anti-rat AF647 label (right). Gating strategy shown in (Sup. Fig. 2 A) The isotype control has been overlaid in each anti-mTNFR2 antibody histogram represented with % of max. Data representative of two independent experiments. (B) TNFR2 expression upon binding of anti-mTNFR2 antibodies to activated CD8+ cells. Detection by commercial hamster anti-TNFR2 direct labeled with PE with the respective hamster-isotype control labeled with PE (left). Generated rat anti-mTNFR2 antibodies and a rat isotype control were detected by a secondary antibody anti-rat PE label (right). Gating strategy not shown. Gating strategy for CD8+ population was done on unstained OT1 activated cells. First, OT1 cells were gated based on FSC-A / SSC-A properties. Next, single cells were gated based FSC-A / FSC-H. CD8+ population were gated as CD8-PerCP-Vio700 positive. Next to the CD8+ population, a mouse TNFR2+ gate was set with a rat isotype control via histogram. The isotype control has been overlaid in each anti-mTNFR2 antibody histogram represented with % of max. Data representative of single experiment out of two independent experiments. (C)TNFR2 expressing Treg cells co-staining, representation of can didates 18 and 25 with a benchmark antibody, clone TR75–89. Data representative of two independent experiments. (D) Costimulation of CD8+ T-cells with anti- TNFR2 antibodies. Assessment of in vitro CD8+ T-cell costimulation for different anti-TNFR2 antibodies (plate bound anti-CD3 at 0.5 μg/mL). Anti-TNFR2 antibodies were plate bound in 2-fold decreasing dilution starting at 50 μg/mL. Data representative of three independent experiments with n = 3 biological replicates on the read out of IFNγ in supernatant at 50 μg/mL per each candidate and mean of independent experiment. Blank was subtracted, IFNγ was calculated based on the standard curve and normalized based on single incubation of anti-CD3 antibodies as 0% costimulation and double incubation of anti-CD3 + anti-CD28 antibodies as a 100% costimulation.

Article Snippet: First, a single estimation screening of KD value was performed with an expected saturating concentration of 100 nM His tagged mTNFR2 (R&D Systems, 426-R2/CF) 100 nM diluted in 10× Kinetics Buffer (KB) followed by a dissociation step.

Techniques: Ex Vivo, Expressing, Binding Assay, Labeling, Control, Generated, Staining, In Vitro, Incubation

Journal: Immunity

Article Title: Six distinct NFκB signaling codons convey discrete information to distinguish stimuli and enable appropriate macrophage responses

doi: 10.1016/j.immuni.2021.04.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant Mouse sTNFRII/TNFRSF1B , R & D Systems , 426-R2–050.

Techniques: Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Software, Cell Tracking Assay, Flow Cytometry

Journal: Cell systems

Article Title: Measuring Signaling and RNA-Seq in the Same Cell Links Gene Expression to Dynamic Patterns of NF-κB Activation

doi: 10.1016/j.cels.2017.03.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Reagents used were: LPS (Enzo Life Sciences, ALX-581–010-L001), recombinant mouse sTNFRII/TNFRSF1B (Fisher Scientific, 426-R2–050), brefeldin A (BD Biosciences, 555029, Golgi Plug), Leptomycin B (Cell Signaling Technology, 9676).

Techniques: Recombinant, Control, Picogreen Assay, Sample Prep, Software, Imaging