Journal: Frontiers in Oncology

Article Title: Tackling Immune Targets for Breast Cancer: Beyond PD-1/PD-L1 Axis

doi: 10.3389/fonc.2021.628138

Figure Lengend Snippet: Examples of clinical trials of Immune targets in breast cancer immunotherapy.

Article Snippet: 4-1BB agonist , PF-05082566 (Utolimumab) , Pfizer , + Trastuzumab – Emtansine , I , NCT03364348.

Techniques:

Journal: Journal for Immunotherapy of Cancer

Article Title: 4-1BB co-stimulation further enhances anti-PD-1-mediated reinvigoration of exhausted CD39 + CD8 T cells from primary and metastatic sites of epithelial ovarian cancers

doi: 10.1136/jitc-2020-001650

Figure Lengend Snippet: 4-1BB agonistic antibodies enhanced the function of exhausted CD8 tumor-infiltrating lymphocytes (TILs). (A) Efficacy of 4-1BB co-stimulation with anti-CD3 stimulation in terms of CD8 TIL proliferation. In the presence of anti-CD3 antibodies, we analyzed the frequency of CTV low CD8 TILs (proliferated CD8 TILs) in the presence of isotype or anti-4-1BB agonistic antibodies. Representative flow cytometry plots are shown on the left, and data are presented as the stimulation index. (B–C) Effects of 4-1BB co-stimulation were also assessed in terms of cytokine production, representing functional capacity. Interferon (IFN)-γ and tumor necrosis factor (TNF)-α production in CD8 TILs was measured by intracellular staining. Representative flow cytometry plots are shown on the left, and data are presented as the relative ratio to the isotype-treated group, separately for cells from the ovary (B) and metastatic sites (C). *P<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Then those labeled cells were stimulated with 1 ng/mL anti-CD3 antibody (OKT-3; eBioscience), and with or without 5 µg/mL anti-PD-1 blocking antibody (EH12.2H7; BioLegend, San Diego, California, USA) and 10 µg/mL anti-4-1BB agonistic antibody (provided by ABL Bio, Seongnam, Korea).

Techniques: Flow Cytometry, Functional Assay, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: 4-1BB co-stimulation further enhances anti-PD-1-mediated reinvigoration of exhausted CD39 + CD8 T cells from primary and metastatic sites of epithelial ovarian cancers

doi: 10.1136/jitc-2020-001650

Figure Lengend Snippet: 4-1BB co-stimulation further enhances antiprogrammed cell death protein 1 (anti-PD-1)-mediated reinvigoration of exhausted CD8 tumor-infiltrating lymphocytes (TILs) from the ovary and metastatic sites. (A) In the presence of anti-CD3 antibodies, we analyzed the frequency of CTV low CD8 TILs (proliferated CD8 TILs) in the presence of isotype or anti-PD-1 blocking antibodies, or a combination of anti-PD-1 blocking antibodies and anti-4-1BB agonistic antibodies. Representative flow cytometry plots are shown to the left, and data are presented as the stimulation index. (B–C) We evaluated changes of the functional capacities of CD8 TILs after stimulation with antibodies by intracellular staining of cytokines (interferon (IFN)-γ and tumor necrosis factor (TNF)-α). We compared three different treatment groups: isotype-treated, anti-PD-1 blocking antibodies-treated and combined treatment with anti-PD-1 blocking antibodies and anti-4-1BB agonistic antibodies. Representative flow cytometry plots are shown to the left, and data are presented as the relative ratio to the isotype-treated group, separately for the ovary (B) and metastatic sites (C). *P<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Then those labeled cells were stimulated with 1 ng/mL anti-CD3 antibody (OKT-3; eBioscience), and with or without 5 µg/mL anti-PD-1 blocking antibody (EH12.2H7; BioLegend, San Diego, California, USA) and 10 µg/mL anti-4-1BB agonistic antibody (provided by ABL Bio, Seongnam, Korea).

Techniques: Blocking Assay, Flow Cytometry, Functional Assay, Staining

Journal: Frontiers in Immunology

Article Title: FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab

doi: 10.3389/fimmu.2023.1208631

Figure Lengend Snippet: Evaluation of 41BB, CD27, OX40, and GITR signaling in a Jurkat-based triple parameter reporter system. (A) Schematic of the Jurkat reporter – T cell stimulator cell system. (B) Flow cytometry staining of Jurkat reporter cells. (C) Flow cytometry staining of T cell stimulator cells (TCS). Upper panel: expression of the membrane-bound anti-human CD3 single chain fragment (mb-αCD3) on the indicated TCS; the paternal Bw cell line was used as control. Lower panels: expression of TNFR-ligands on TCS. Filled histogram: expression level on the indicated TCS; open histograms: staining of control TCS. (D) Jurkat-TPR expressing the indicated TNF receptor were stimulated with TCS control or with TCS expressing the corresponding ligand or left unstimulated (us). Reporter gene expression (NFκB::eCFP, NFAT::eGFP, and AP-1::mCherry) was assessed via flow cytometry. Left panel: Histograms show data from one representative experiment. Right panel: summarized data are shown (n=18 for CD27, n=16 for GITR, n=20 for OX40 and 41BB), each dot represents the mean of triplicate measurement, red line shows median; geometric mean fluorescence intensity (gMFI). The statistics were calculated using the Friedman test followed by Dunn’s multiple comparison test. *p ≤ 0.05; **p ≤ 0.01.

Article Snippet: Agonistic 41BB antibodies - Urelumab (BMS-663513), Utomilumab (PF-05082566), and the CD27 agonist mAb Varlilumab (CDX-1127) were purchased from Creative Biolabs (NY, USA).

Techniques: Flow Cytometry, Staining, Expressing, Membrane, Control, Gene Expression, Fluorescence, Comparison

Journal: Frontiers in Immunology

Article Title: FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab

doi: 10.3389/fimmu.2023.1208631

Figure Lengend Snippet: Influence of human Fcγ receptors on the agonistic activity of Urelumab and Utomilumab. (A) Schematic of the experimental set up. Jurkat-NFκB::eGFP expressing 41BB were stimulated either with TCS control, TCS expressing 41BBL (TCS-41BBL) or TCS expressing one of the indicated Fcγ receptors in the presence of different concentrations of 41BB agonistic antibodies. (B) Jurkat-41BB reporters were stimulated with TCS control (TCS-ctrl) or TCS-41BBL or left unstimulated. NFκB::eGFP reporter gene activation was analyzed by flow cytometry. (C, D) Jurkat-41BB reporters were stimulated with TCS control (TCS-ctrl) or TCS expressing the indicated Fcγ receptors (TCS-CD32A, TCS-CD32B, TCS-CD64) in the presence of different concentrations (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, or 3.16 μg/ml) of Urelumab (C) or Utomilumab (D) . Reporter gene induction was analyzed by flow cytometry. Upper panels: histograms show the results of one representative experiment. Middle: Summarized data +/- SD are shown (n=3, each performed in triplicates), dotted line indicates reporter gene expression upon stimulation via TCS-41BBL. Lower panels: stimulation curves and half-maximum effective concentration (EC50) were calculated as described in material and methods (n=3, performed in duplicates).

Article Snippet: Agonistic 41BB antibodies - Urelumab (BMS-663513), Utomilumab (PF-05082566), and the CD27 agonist mAb Varlilumab (CDX-1127) were purchased from Creative Biolabs (NY, USA).

Techniques: Activity Assay, Expressing, Control, Activation Assay, Flow Cytometry, Gene Expression, Concentration Assay

Journal: Frontiers in Immunology

Article Title: FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab

doi: 10.3389/fimmu.2023.1208631

Figure Lengend Snippet: EC50 values and CI intervals for Urelumab, Utomilumab and Varlilumab.

Article Snippet: Agonistic 41BB antibodies - Urelumab (BMS-663513), Utomilumab (PF-05082566), and the CD27 agonist mAb Varlilumab (CDX-1127) were purchased from Creative Biolabs (NY, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab

doi: 10.3389/fimmu.2023.1208631

Figure Lengend Snippet: Effects of Urelumab, Utomilumab, and Varlilumab on the proliferation and cytokine production of primary human T cells. (A) Gating strategy used. (B, C) CFSE labeled human PBMCs were stimulated with CD3 antibodies (final 30 ng/ml) in the presence or absence of Urelumab, Utomilumab, or Varlilumab (soluble, all used at a final concentration of 1 μg/ml) for 5 days. (B) CFSE dilution was analyzed in gated CD4 and CD8 T cell populations. Left panel: Histogram overlay shows CFSE dilution in CD4 and CD8 T cells of one representative donor; right panels: box plots show summarized data from all donors. (C) Cytokine content (IFN-γ, GM-CSF, TNF-α and IL-13) of stimulation cultures was assessed using a Luminex-based assay. (B, C) Summarized data are shown. n=7, each performed in triplicates. For statistical analysis, the Friedman test followed by Dunn’s multiple comparison correction were used. ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. (D) CFSE-labeled human PBMCs were stimulated with plate-bound CD3 antibodies in the presence or absence of Urelumab, Utomilumab, or Varlilumab (soluble, used at 0.03, 0.1, 0.3, or 1 μg/ml) for 5 days. Counts of CFSE low cells were analyzed in gated CD4 and CD8 T cell populations. Flow cytometry analysis was performed using constant cell volumes, flow rates, and acquisition time for all samples. Counts of CFSE low CD4 or CD8 cells are depicted normalized to control stimulated cells (CD3 antibody alone). Summarized data of 3 donors are shown (n=3, each performed in triplicates).

Article Snippet: Agonistic 41BB antibodies - Urelumab (BMS-663513), Utomilumab (PF-05082566), and the CD27 agonist mAb Varlilumab (CDX-1127) were purchased from Creative Biolabs (NY, USA).

Techniques: Labeling, Concentration Assay, Luminex, Comparison, Flow Cytometry, Control

Journal: Frontiers in Immunology

Article Title: FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab

doi: 10.3389/fimmu.2023.1208631

Figure Lengend Snippet: Varlilumab-induced apoptosis in CD4 and CD8 T cells. Human PBMCs were stimulated with plate-bound CD3 antibodies in the presence or absence of Urelumab, Utomilumab, or Varlilumab (1 μg/ml) for 48h. (A) Annexin V expression was analyzed in gated CD4 and CD8 T cells. Flow cytometry analysis was performed using constant cell volumes, flow rates, and acquisition time for all samples. (B) Summarized data of annexin V staining and cell counts of all donors are shown (n=5, each performed in triplicates). (C) Cytokine content (IL-2, GM-CSF, IL-13) of stimulation cultures was assessed using a Luminex-based assay. B-C) For statistical analysis, the Friedman test followed by Dunn’s multiple comparison correction were used. Median and +/- 95% CI is shown. ns, not significant; ns > 0.05, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Article Snippet: Agonistic 41BB antibodies - Urelumab (BMS-663513), Utomilumab (PF-05082566), and the CD27 agonist mAb Varlilumab (CDX-1127) were purchased from Creative Biolabs (NY, USA).

Techniques: Expressing, Flow Cytometry, Staining, Luminex, Comparison