Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

doi: 10.1007/s00262-012-1237-1

Figure Lengend Snippet: Saturation binding of PF-05082566 to human and cynomolgus monkey 4-1BB. Dose-dependent binding of AlexaFluor647-labeled PF-05082566 to a 300-19 cells transduced to express human 4-1BB, b 300-19 cells transduced to express cynomolgus monkey 4-1BB, c 72-h PHA-stimulated primary human PBMC, and d 72-h PHA-stimulated primary cynomolgus monkey PBMC. The data are expressed as fold increase in geometric mean fluorescence intensity (Geo MFI) of cells stained with PF-05082566 relative to cells stained with the corresponding concentration of human IgG2 isotype control as measured by FACS. a, b display data from representative experiments with samples stained in duplicate. Error bars represent SEM. c, d represent data of binding to 72-h PHA-stimulated CD3-positive primary PBMC. Each line represents the staining result of cells isolated from individual donors. Representative examples of flow cytometry data are demonstrated in Supplementary Figure 1 for 300-19 cells and Supplementary Figure 2 for PBMC

Article Snippet: Generation of fully human anti-4-1BB antibodies PF-05082566 was derived from a Fab which was selected from the MorphoSys AG HuCAL GOLD phage display library on the basis of binding to 4-1BB during two alternating rounds of panning on human 4-1BB-expressing 293T cells and recombinant human 4-1BB-IgG1Fc fusion protein followed by affinity maturation using MorphoSys AG RapMAT technology.

Techniques: Binding Assay, Labeling, Fluorescence, Staining, Concentration Assay, Isolation, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

doi: 10.1007/s00262-012-1237-1

Figure Lengend Snippet: PF-05082566 induces NF-κB pathway activation through both human and cynomolgus monkey 4-1BB. Dose-dependent activation of an NF-κB luciferase reporter in a 293T-cells-expressing human 4-1BB or b 293T-cells-expressing cynomolgus monkey 4-1BB. The data are representative of three separate experiments and shows fold increase in luciferase activity over unstimulated control cells by goat anti-huIgG Fc-specific F(ab′)2 cross-linking antibody plus PF-05082566 (black circles) or cross-linked human IgG2 isotype control (black triangles). Additionally, PF-05082566 treatment induces phospho-NF-κB p65 in anti-CD3-stimulated primary c human and d cynomolgus monkey CD3+ cells. The graphs demonstrate percentage of phospho-NF-κB p65+, 4-1BB+ T cells as measured by FACS. Cells were stimulated with CD3 alone (white bars), CD3 plus cross-linked 0.5 μg/ml PF-05082566 (gray bars), or CD3 plus cross-linked 5 μg/ml PF-05082566 (black bars). Significance relative to the control group was determined via two-tailed Student’s t test (*p < 0.05)

Article Snippet: Generation of fully human anti-4-1BB antibodies PF-05082566 was derived from a Fab which was selected from the MorphoSys AG HuCAL GOLD phage display library on the basis of binding to 4-1BB during two alternating rounds of panning on human 4-1BB-expressing 293T cells and recombinant human 4-1BB-IgG1Fc fusion protein followed by affinity maturation using MorphoSys AG RapMAT technology.

Techniques: Activation Assay, Luciferase, Expressing, Activity Assay, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

doi: 10.1007/s00262-012-1237-1

Figure Lengend Snippet: PF-05082566 enhances IL-2 production by primary human T cells and expansion of antigen-specific CD8+ T cells in vitro. a Dose-dependent enhancement of IL-2 production as measured by ELISA from purified T cells stimulated with plate bound CD3 plus increasing concentrations of plate bound PF-05082566. Each line represents stimulation of purified T cells from one of seven individual donors tested in duplicate. b PBMC isolated from four separate HLA-A2+ donors whom had received an influenza vaccine within the prior season were stimulated in vitro with influenza matrix peptide (58–66, GILGFVFTL) for 8 days in the presence of either huIgG2 control antibody (white bars) or PF-05082566 (black bars). The data are represented as the percent viable CD8+, MHC HLA-A2-GILGFVFTL pentamer+ cells as measured by FACS. Significance was determined via 2-way ANOVA comparison with the control IgG2-treated group (**p < 0.01, *p < 0.05). Results are representative of two separate experiments using four different donors

Article Snippet: Generation of fully human anti-4-1BB antibodies PF-05082566 was derived from a Fab which was selected from the MorphoSys AG HuCAL GOLD phage display library on the basis of binding to 4-1BB during two alternating rounds of panning on human 4-1BB-expressing 293T cells and recombinant human 4-1BB-IgG1Fc fusion protein followed by affinity maturation using MorphoSys AG RapMAT technology.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Purification, Isolation, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

doi: 10.1007/s00262-012-1237-1

Figure Lengend Snippet: PF-05082566 induces expansion of human CD45+ PBMC in vivo. Engraftment of human PBMC adoptively transferred via intraperitoneal injection to NSG host mice was analyzed by FACS for human CD45 and Ki-67 staining 28 days following injection. On day 7 following PBMC injection, mice were treated with a single injection of human IgG2 isotype control or the indicated concentration of PF-05082566. a Mean percentage of human CD45+ lymphocytes assessed at several time points over the course of the study b percentage of human CD45+ lymphocytes at Study Day 28, 21 days following mAb injection and c percentage of human CD45+ Ki-67+ present in the whole blood samples of each treatment group at Study Day 28, 21 days following mAb injection. Data are representative of n = 7 mice per group and significance relative to the control group on the final day of the study was determined via two-tailed Student’s t test (*p < 0.05, **p < 0.005)

Article Snippet: Generation of fully human anti-4-1BB antibodies PF-05082566 was derived from a Fab which was selected from the MorphoSys AG HuCAL GOLD phage display library on the basis of binding to 4-1BB during two alternating rounds of panning on human 4-1BB-expressing 293T cells and recombinant human 4-1BB-IgG1Fc fusion protein followed by affinity maturation using MorphoSys AG RapMAT technology.

Techniques: In Vivo, Injection, Staining, Concentration Assay, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

doi: 10.1007/s00262-012-1237-1

Figure Lengend Snippet: Intravenous administration of PF-05082566 to cynomolgus monkeys induces proliferation of CD8+ memory T cells. a, b Multiple dose administration. Cynomolgus monkeys were injected intravenously with the indicated doses of PF-05082566 on days 1 and 8 of the study (n = 10 animals per group). On day 13, blood was collected, and cells were analyzed by flow cytometry. a The far left plot shows the gating strategy for identifying CD8+ TCM (CD95+ CD28+) and CD8+ TEM (CD95+ CD28−). The remainder of the plots shows representative staining for Ki-67 versus CD28 for CD8+ TCM (top row) and CD8+ TEM (bottom row) at the indicated dose levels. b Multiple dose administration of the indicated dose levels of PF-05082566 on day 1 and day 8 demonstrates significant increases in Ki-67+ CD8+ TCM cells (graph on left) and Ki-67+ CD8+ TEM (graph on right) as measured on study day 13. c Single dose administration of 0.05 mg/kg (white bars), 1 mg/kg (gray bars), or 10 mg/kg (black bars) PF-05082566 to 2 individual animals per group demonstrates increases in Ki-67+ CD8+ TCM cells measured by FACS on study days 7 and 23 post dosing. For both b and c, fold changes were calculated relative to baseline pre-study samples collected prior to the start of dosing. Statistical significance was calculated using 1-way ANOVA with Dunnett’s Multiple Comparison test (*p < 0.05, ***p < 0.001)

Article Snippet: Generation of fully human anti-4-1BB antibodies PF-05082566 was derived from a Fab which was selected from the MorphoSys AG HuCAL GOLD phage display library on the basis of binding to 4-1BB during two alternating rounds of panning on human 4-1BB-expressing 293T cells and recombinant human 4-1BB-IgG1Fc fusion protein followed by affinity maturation using MorphoSys AG RapMAT technology.

Techniques: Injection, Flow Cytometry, Staining, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

doi: 10.1007/s00262-012-1237-1

Figure Lengend Snippet: PF-05082566 inhibits the growth of PC3 prostate carcinoma in vivo. a Mean tumor volume at time points following subcutaneous administration of 3 million PC3 prostate carcinoma cells plus 1.5 million human PBMC in the right flank of SCID Bg mice (n = 8 animals/group). b The volume of each individual tumor on the final study day (day 21). Statistical significance was determined using 1-way ANOVA with Tukey’s post-test (*p < 0.05, **p < 0.005). Data are representative of three independent experiments

Article Snippet: Generation of fully human anti-4-1BB antibodies PF-05082566 was derived from a Fab which was selected from the MorphoSys AG HuCAL GOLD phage display library on the basis of binding to 4-1BB during two alternating rounds of panning on human 4-1BB-expressing 293T cells and recombinant human 4-1BB-IgG1Fc fusion protein followed by affinity maturation using MorphoSys AG RapMAT technology.

Techniques: In Vivo

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: Neuro-Oncology Advances

Article Title: Preclinical assessment of checkpoint blockade combined with DNA methyltransferase inhibition in high-risk pediatric brain tumors reveals limited therapeutic synergy

doi: 10.1093/noajnl/vdaf241

Figure Lengend Snippet: Immune composition of syngeneic TME at baseline versus DAC + 4H2 combination treatment. The infiltration of immune cells and effect of combination treatment was analyzed using CyTOF. (A) Phenograph clustering of all samples. A t-SNE plot representing all models at baseline with different cell populations as clusters indicated by color. (B) Heat map showing expression of specific immune markers in the various cluster groups. (C) t-SNE plots for each individual tumor model at both baseline (upper) and following DAC + 4H2 (lower) treatment ( n = 3 tumors per condition, per model).

Article Snippet: Isotype control and 4H2 (an anti-mouse PD-1 antibody kindly provided by Bristol Myers Squibb) were dosed IP at 200 μg per mouse on Monday and Friday of each week, beginning on the Friday of the first week of dosing.

Techniques: Expressing

Journal: Neuro-Oncology Advances

Article Title: Preclinical assessment of checkpoint blockade combined with DNA methyltransferase inhibition in high-risk pediatric brain tumors reveals limited therapeutic synergy

doi: 10.1093/noajnl/vdaf241

Figure Lengend Snippet: Cluster analysis of syngeneic tumors at baseline versus DAC + 4H2 treatment. (A) Relative proportions of leukocyte populations in each tumor type at baseline and following DAC+4H2 treatment. (B) Relative percentage of T cells, macrophages, MDSCs, and neutrophils in each tumor model from vehicle-treated mice and those treated with DAC + 4H2. Significant changes in individual cell clusters for (C) IUE-24-C5, (D) SU2C-54_i_5, (E) IUE-K27APP, and (F) SHH57835 models. P = .001 − .01**, .01 − .05*.

Article Snippet: Isotype control and 4H2 (an anti-mouse PD-1 antibody kindly provided by Bristol Myers Squibb) were dosed IP at 200 μg per mouse on Monday and Friday of each week, beginning on the Friday of the first week of dosing.

Techniques:

Journal: Neuro-Oncology Advances

Article Title: Preclinical assessment of checkpoint blockade combined with DNA methyltransferase inhibition in high-risk pediatric brain tumors reveals limited therapeutic synergy

doi: 10.1093/noajnl/vdaf241

Figure Lengend Snippet: Variable responses to DAC, DAC + 4H2, or 4H2 treatment in orthotopic syngeneic models. (A) 5-methylcytosine-specific dot blot assay was used to evaluate activity of DAC in mouse brain tumor tissue. gDNA was isolated from brain tumors established from different models and treated with DAC or a combination of DAC and 4H2. 100 ng of gDNA were loaded per dot and methylene blue staining was used as a loading control. (B) Evaluation of neoantigen gene expression and inflammation using quantitative PCR in tumor tissue harvested from IUE-24-C5 syngeneic mice treated with either vehicle (Veh), DAC, or DAC+4H2. (C) Schematic representation of treatment strategy of DAC and 4H2 in syngeneic models. Red arrows indicate DAC IP dosing (0.5 mg/kg) and blue arrows indicated anti-PD-L1 (4H2) IP dosing (200 µg). Figure created with BioRender.com. Kaplan–Meier curves representing survival data for (D) DIPG model IUE-24-C5 (Veh vs DAC P = .001, Veh vs 4H2 P = .6088, Veh vs DAC+4H2 P < .0170), (E) ATRT model SU2C_54_i_5 (Veh vs DAC p=0.0079, Veh vs 4H2 P = 0.0027, Veh vs DAC+4H2 P = .0176), (F) DMG model IUE-K27M-APP (Veh vs DAC P = .6030, Veh vs 4H2 P = .4658, Veh vs DAC+4H2 P = .7927), and (G) MB model SHH57835 (Veh vs DAC P = .9873, Veh vs 4H2 P = .9690, Veh versus DAC+4H2 P = .9831. Stars represent: P = 0 - 0.001 ***, 0.001 - 0.01 **, 0.01 - 0.05 *.).

Article Snippet: Isotype control and 4H2 (an anti-mouse PD-1 antibody kindly provided by Bristol Myers Squibb) were dosed IP at 200 μg per mouse on Monday and Friday of each week, beginning on the Friday of the first week of dosing.

Techniques: Dot Blot, Activity Assay, Isolation, Staining, Control, Gene Expression, Real-time Polymerase Chain Reaction