Journal: OncoImmunology

Article Title: Anti-CD137 enhances anti-CD20 therapy of systemic B-cell lymphoma with altered immune homeostasis but negligible toxicity

doi: 10.1080/2162402x.2016.1192740

Figure Lengend Snippet: Figure 1. Treatment with aCD20 and aCD137 enhances survival against B lymphoma. (A) Tumor injection and treatment design. 1 £ 105 BL3750 cells were injected intra- venously at day (D) 0. Control Ig (2A3, 250 mg i.p.) or anti-CD20 (clone 5D2, 250 mg i.p.) was administered on day 4, and anti-CD137 (clone 3H3, 100 mg i.p.) either alone or in combination on day 5 in C57BL6 wild-type (WT) and various gene-targeted mice (FcRIII¡/¡, FcRIV¡/¡, and FcgR¡/¡). (B) Results are pooled from three independent experiments (n (2A3) D 24; n (5D2) D 31; n (3H3) D 12; n (5D2C3H3) D 34). Statistical analysis was calculated using Mantel–Cox test. p < 0.01 and p < 0.001 as indi- cated. (C) Results representative from one experiment, n D 6 each group. p < 0.05 and p < 0.01 as indicated. (D) Results representative from one experiment, n D 6 each group. (E) Results are pooled from two independent experiments (n (2A3) D 14; n (5D2) D 15; n (3H3) D 6; n (5D2C3H3) D 16). Statistical analysis was calculated using Mantel–Cox test. p < 0.01 as indicated. (F) Tumor inoculation (1 £ 105 BL3750 cells, i.v.) on D0 was followed by administration of anti-CD20 (clone 5D2, 250 mg i. p.) on day 4, and anti-CD137 (clone 3H3, 100 mg i.p.) at day 5, either alone or in combination in C57BL6 WT mice. Antibody treatment was then applied weekly until sacri- fice. Data shown is pooled from two independent experiments (n (2A3) D 16, n (5D2) D 6, n (3H3) D 6, n (5D2C3H3) D 16). p < 0.001, and p < 0.001 as indicated. (G) WT mice survival after single treatment with anti-CD20 (clone 5D2, 250 mg i.p. 20 d after BL3750 inoculation) and anti-CD137 (clone 3H3, 100 mg i.p. 21 d after BL3750 inoculation). Results are representative of one experiment, n D 6, each group. p < 0.01 as indicated.

Article Snippet: On day 5 post BL3750 lymphoma inoculation, anti-CD137 mAb (3H3, Bio X Cell) was administered (100 mg i.p. per injection).

Techniques: Injection, Control

Journal: OncoImmunology

Article Title: Anti-CD137 enhances anti-CD20 therapy of systemic B-cell lymphoma with altered immune homeostasis but negligible toxicity

doi: 10.1080/2162402x.2016.1192740

Figure Lengend Snippet: Figure 2. Treatment with aCD20 and aCD137 reduces blood B-cell numbers in mice bearing B-cell lymphoma. Blood samples were collected for hemogram assessment according to the experimental design shown in (A). 1 £ 105 BL3750 cells were injected i.v. at D0, and followed by treatment with control Ig (2A3, 250 mg i.p.) or anti- CD20 (clone 5D2, 250 mg i.p.) on day 4, and anti-CD137 (clone 3H3, 100 mg i.p.) either alone or in combination on day 5 in C57BL6 WT or various FcR-deficient mice, as indicated. Peripheral blood lymphocyte (B), and B cell numbers (C). Results are representative of one experiment n D 6 per group. Statistical analysis was calculated using Multiple T tests using the Holm–Sidak Method for multiple comparisons, where p < 0.05 was considered statistically significant between respective anti-CD137 (clone 3H3), anti-CD20 (clone 5D2), 5D2 C 3H3 groups versus (2A3) isotype control-treated mice.

Article Snippet: On day 5 post BL3750 lymphoma inoculation, anti-CD137 mAb (3H3, Bio X Cell) was administered (100 mg i.p. per injection).

Techniques: Injection, Control

Journal: OncoImmunology

Article Title: Anti-CD137 enhances anti-CD20 therapy of systemic B-cell lymphoma with altered immune homeostasis but negligible toxicity

doi: 10.1080/2162402x.2016.1192740

Figure Lengend Snippet: Figure 3. Treatment with aCD20 and aCD137 induces BAFF and cytokines in plasma with or without B lymphoma inoculation. Control Ig (2A3, 250 mg i.p.) or anti-CD20 (clone 5D2, 250 mg i.p.) was administered on day (D) 0, and anti-CD137 (clone 3H3, 100 mg i.p.) either alone or in combination on D1 in C57BL6 wild-type (WT) or gene- targeted mice. In some experiments (B, D), 1 £ 105 BL3750 cells were injected intravenously on day (D) 0, and control Ig (2A3, 250 mg i.p.) or anti-CD20 (clone 5D2, 250 mg i.p.) was administered on day (D) 4, and anti-CD137 (clone 3H3, 100 mg i.p.) either alone or in combination on D5 in WT or gene-targeted mice. (A) and (B) BAFF assessment from plasma of mice at D2, D5, D8 and D15 post anti-CD20 treatment. Data is representative of two experiment, n D 5 each group (n D 10 total).

Article Snippet: On day 5 post BL3750 lymphoma inoculation, anti-CD137 mAb (3H3, Bio X Cell) was administered (100 mg i.p. per injection).

Techniques: Clinical Proteomics, Control, Injection

Journal: Clinical Cancer Research

Article Title: FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models

doi: 10.1158/1078-0432.ccr-19-2958

Figure Lengend Snippet: Figure 1. mAb2 structure and concurrent high-affinity binding of CD137/PD-L1 mAb2 equivalent to individual component antibodies. A, Representation of the bispecific CD137/ PD-L1 mAb2, FS222, on a human IgG1 backbone with FcgR binding removed by L234A and L235A (LALA) mutations highlighted in green. The PD-L1 complementarity- determining regions (CDR) of heavy and light chains are highlighted in orange. The CD137 CH3 domain AB- and EF-binding loops are highlighted in cyan. B, FS222 simultaneous binding to both human PD-L1 and human CD137 as determined by SPR. C, FS222 binding to DO11.10 T cells expressing human CD137 as determined by flow cytometry. D, FS222 binding to HEK cells expressing human PD-L1 as determined by flow cytometry. E, FS222 binding to in vitro–activated human primary CD4þ and CD8þ T cells as determined by flow cytometry.

Article Snippet: No dose-limiting toxicity has been observed with CD137 mAb utomilumab (PF-05082566, Pfizer), a human IgG2 isotype antibody, in dose-escalation phase I clinical trials dosing up to 10 mg/kg in phase I clinical trials of advanced cancer (6, 8).

Techniques: Binding Assay, Expressing, Cytometry, In Vitro

Journal: Clinical Cancer Research

Article Title: FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models

doi: 10.1158/1078-0432.ccr-19-2958

Figure Lengend Snippet: Figure 2. CD137 agonism via FS222 is dependent upon cross-linking via PD-L1 in a human primary T-cell assay and has activity superior to mAb combinations in an MLR. A, FS222 activity in a human primary CD8þ T-cell activation assay with varying ratios of HEK cells that are positive for PD-L1 to HEK cells that are negative for PD-L1. Significance determined by extra sum-of-squares F test. , P < 0.001. B, FS222 activity in MLR against monospecific component parts that make up the complete FS222 mAb2 either alone or in combination with each other. Significance determined by extra sum-of-squares F test. , P < 0.001.

Article Snippet: No dose-limiting toxicity has been observed with CD137 mAb utomilumab (PF-05082566, Pfizer), a human IgG2 isotype antibody, in dose-escalation phase I clinical trials dosing up to 10 mg/kg in phase I clinical trials of advanced cancer (6, 8).

Techniques: Activity Assay, Activation Assay

Journal: Clinical Cancer Research

Article Title: CB307: A Dual Targeting Costimulatory Humabody V H Therapeutic for Treating PSMA-Positive Tumors

doi: 10.1158/1078-0432.CCR-23-3052

Figure Lengend Snippet: CB307 design and induced T-cell phenotypes. A, Illustration of CB307 format: three V H domains separated by peptide linkers. B, PSMA expression on cell lines. DU145-PSMA is isogenic to DU145-Parent other than for constitutive stable PSMA expression. C, Schematic of a Jurkat-based CD137 agonism reporter system whereby CB307 confers the clustering status of PSMA upon CD137 to generate a CD137 agonist signal. CD137 signaling is measured by luciferase expression from an NFκB-based reporter gene. D, Comparison of the reporter gene signal in the presence (DU145-PSMA) and absence (DU145-parent) of PSMA expression. E, Schematic of a primary cell CD137 activity assay whereby PBMCs stimulated by plate-bound αCD3 are co-incubated with tumor cells in the presence of CB307, leading to ( F ) PSMA-dependent secretion of IFNγ, ( G ) PSMA-dependent production of granzyme B, and ( H ) PSMA-dependent production of Bcl.xL. I, PSMA-dependent proliferation indicated by Ki67 expression. J, CB307 pharmacodynamic effects on NK cells. Statistical significance of CB307 treated versus untreated outcomes with PSMA-expressing tumor cells is indicated: **, P < 0.01; ****, P < 0.0001.

Article Snippet: Briefly, a rabbit monoclonal anti-CD137 antibody (clone E6Z7F, #19541; Cell Signaling Technology) was diluted 1:100 in Bond primary antibody diluent (AR9352; Leica Biosystems), applied to formalin-fixed paraffin-embedded (FFPE) tissue following Bond Epitope Retrieval Solution 1 (ER1; AR9961; Leica Biosystems) antigen retrieval for 30 minutes and detected using the Bond Polymer Refine Detection Kit (DS9800; Leica Biosystems).

Techniques: Expressing, Luciferase, Comparison, Activity Assay, Incubation

Journal: Clinical Cancer Research

Article Title: CB307: A Dual Targeting Costimulatory Humabody V H Therapeutic for Treating PSMA-Positive Tumors

doi: 10.1158/1078-0432.CCR-23-3052

Figure Lengend Snippet: CB307 activity in combination with immune checkpoint inhibitors. A, Schematic of a 2D primary cell-based experimental system assessing CB307 combination with immune checkpoint inhibitors. SEB is used to induce CD137 expression on primary T cells. B, IL2 secretion following drug treatment in the presence of PSMA-expressing tumor cells. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001. C, Isobologram of CB307-pembrolizumab additivity as assessed in 2D cocultures. D, Comparison of IL2 production between PD-L1–expressing versus PD-L1 CRISPR gene deleted nonexpressing tumor cells. In both cases, PSMA expression is similar. E, Schematic of a 3D tumor spheroid experimental system, whereby tumor cells fluoresce red whilst immune cells appear black. F, Single-agent versus combination drug activity measured as reduction in tumor cell count and ( G ) increasing CD8 + T-cell count after 10 days in culture. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001. H, Changing red fluorescent tumor spheroid and surrounding black immune cell “cloud” over time.

Article Snippet: Briefly, a rabbit monoclonal anti-CD137 antibody (clone E6Z7F, #19541; Cell Signaling Technology) was diluted 1:100 in Bond primary antibody diluent (AR9352; Leica Biosystems), applied to formalin-fixed paraffin-embedded (FFPE) tissue following Bond Epitope Retrieval Solution 1 (ER1; AR9961; Leica Biosystems) antigen retrieval for 30 minutes and detected using the Bond Polymer Refine Detection Kit (DS9800; Leica Biosystems).

Techniques: Activity Assay, Expressing, Comparison, CRISPR, Cell Counting

Journal: Clinical Cancer Research

Article Title: CB307: A Dual Targeting Costimulatory Humabody V H Therapeutic for Treating PSMA-Positive Tumors

doi: 10.1158/1078-0432.CCR-23-3052

Figure Lengend Snippet: PSMA and CD137 expression measured by IHC. A, Primary human prostate cancer tumor co-stained for PSMA (green) and CD137 (brown). Arrows indicate patches of CD137-positive cells. B, PSMA positivity across a selection of human multitumor tissue microarrays. Shown are percent of cells in a core showing any level of PSMA positivity by IHC as quantified by image analysis. Where duplicate cores have been stained and scored, results are presented as the average. C, Density of CD137-positive cells per mm 2 of viable tissue. Cells with any level of CD137 expression as determined by image analysis of IHC staining on sample cores are shown. Where duplicate cores have been stained and scored, results are presented as the average. D, PSMA expression in sites of primary and metastatic human prostate cancer are shown—any level of PSMA expression in a given cell is counted as positive. The median value for each anatomical site is indicated. Boxes represent inner quartiles. Each dot represents a sample from an individual patient. E, CD137-positive cell density in regions determined by image analysis to be tumor. F, The density of CD137-positive cells in the tumor-adjacent nontumor stromal tissue.

Article Snippet: Briefly, a rabbit monoclonal anti-CD137 antibody (clone E6Z7F, #19541; Cell Signaling Technology) was diluted 1:100 in Bond primary antibody diluent (AR9352; Leica Biosystems), applied to formalin-fixed paraffin-embedded (FFPE) tissue following Bond Epitope Retrieval Solution 1 (ER1; AR9961; Leica Biosystems) antigen retrieval for 30 minutes and detected using the Bond Polymer Refine Detection Kit (DS9800; Leica Biosystems).

Techniques: Expressing, Staining, Selection, Immunohistochemistry

Journal: Circulation

Article Title: CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

doi: 10.1161/circulationaha.107.699173

Figure Lengend Snippet: Figure 1. Semiquantitative real-time reverse-transcription poly- merase chain reaction of CD137 mRNA. A, In snap-frozen human arteries, CD137 was significantly increased in athero- sclerotic lesions vs normal arteries (2 leftmost bars; n8 normal and 11 atherosclerotic biopsies). Six hours of incubation with lipopolysaccharide (LPS) induced CD137 in organ cultures derived from normal and atherosclerotic arteries (4 rightmost bars; n5). B, CD137 was induced in HUVECs vs control (CON) in response to 8 hours of exposure to TNF- or a mix of the cytokines TNF-, IL-1, and IFN- (cytokine mix [CM]) (n4). C, CD137 was induced in AoSMCs vs control in response to the cytokine mix (n4). Results were normalized to the housekeep- ing gene cyclophilin A. *P0.05; **P0.01; ***P0.001.

Article Snippet: Acetone-fixed sections were incubated with 10% goat serum in 50 mmol/L NH4Cl followed by a mouse monoclonal anti-human CD137 antibody (Abcam, Cambridge, UK) or an isotype control (Dakopatts, Glostrup, Denmark) and a polyclonal rabbit anti–human von Willebrand factor (Dakopatts) or a polyclonal rabbit anti-human CD3 (Dakopatts) antibody.

Techniques: Reverse Transcription, Incubation, Derivative Assay, Control

Journal: Circulation

Article Title: CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

doi: 10.1161/circulationaha.107.699173

Figure Lengend Snippet: Figure 2. Immunostaining of frozen sec- tions of human arterial biopsies. CD137 and CD137L cells are stained red; cell- type markers are stained green. Colocal- ization of red and green results in yellow. CD137 was expressed on endothelial and T cells in lesions (A through C, E through G, respectively) but not in nor- mal arteries (D). A, anti-CD137; B, anti– von Willebrand factor; C, A and B over- laid; D, anti-CD137 in a normal artery; E, anti-CD137 and anti-CD3; F, anti-CD137 and anti-CD8; G, anti-CD137 and anti- CD4; and H, anti-CD137L and anti- CD68. Nuclei are stained blue with DAPI. White bars are 50 m.

Article Snippet: Acetone-fixed sections were incubated with 10% goat serum in 50 mmol/L NH4Cl followed by a mouse monoclonal anti-human CD137 antibody (Abcam, Cambridge, UK) or an isotype control (Dakopatts, Glostrup, Denmark) and a polyclonal rabbit anti–human von Willebrand factor (Dakopatts) or a polyclonal rabbit anti-human CD3 (Dakopatts) antibody.

Techniques: Immunostaining, Staining

Journal: Circulation

Article Title: CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

doi: 10.1161/circulationaha.107.699173

Figure Lengend Snippet: Figure 3. Fluorescence-activated cell sorter analysis of ECs and SMCs. A, CD137 in HUVECs after 24 hours of incubation with (black solid line) or with- out (gray solid line) TNF- at 10 ng/mL. B, CD137 in AoSMCs after 24 hours of incubation with (black) or without (gray) a mix of TNF-, IFN-, and IL-1, each at 10 ng/mL. In A and B, the dotted gray lines represent antibody isotype controls. C, VCAM-1 in HUVECs after 12 hours of incubation of unstimulated cells (light gray), with beads alone (dark gray), or with beads with crosslinked rCD137L (black). D, ICAM-1 in HUVECs after 12 hours of incubation of unstimulated cells (light gray), with beads alone (dark gray), or with beads with crosslinked rCD137L (black). Graphs are representative of 3 independent experiments.

Article Snippet: Acetone-fixed sections were incubated with 10% goat serum in 50 mmol/L NH4Cl followed by a mouse monoclonal anti-human CD137 antibody (Abcam, Cambridge, UK) or an isotype control (Dakopatts, Glostrup, Denmark) and a polyclonal rabbit anti–human von Willebrand factor (Dakopatts) or a polyclonal rabbit anti-human CD3 (Dakopatts) antibody.

Techniques: Fluorescence, Incubation

Journal: Circulation

Article Title: CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

doi: 10.1161/circulationaha.107.699173

Figure Lengend Snippet: Figure 5. CD3 and I-Ab immunostaining in atherosclerotic lesions of Apoe/ mice. T-cell infiltration (A) and I-Ab expression (B) were significantly aug- mented on in vivo antibody 2A ligation of CD137 vs control rat IgG2a (n5). Immu- nostaining of I-Ab (C) and CD68 (D) in the aortic root from a control mouse. Immunostaining of I-Ab (E) and CD68 (F) in a 2A-treated mouse. Black bars indi- cate 0.2 mm. *P0.05; **P0.01.

Article Snippet: Acetone-fixed sections were incubated with 10% goat serum in 50 mmol/L NH4Cl followed by a mouse monoclonal anti-human CD137 antibody (Abcam, Cambridge, UK) or an isotype control (Dakopatts, Glostrup, Denmark) and a polyclonal rabbit anti–human von Willebrand factor (Dakopatts) or a polyclonal rabbit anti-human CD3 (Dakopatts) antibody.

Techniques: Immunostaining, Expressing, In Vivo, Ligation, Control

Journal: Circulation

Article Title: CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

doi: 10.1161/circulationaha.107.699173

Figure Lengend Snippet: Figure 6. Semiquantitative real-time reverse-transcription polymerase chain reaction of adhesion molecule and cytokine mRNA in Apoe/ aortas of CD137 agonist 2A–treated and control rat IgG2a-treated animals (n5 per group). ICAM-1, TNF-, IL-1, and IFN- were significantly induced in animals treated with 2A vs mice treated with control IgG2a. mRNA levels are expressed as arbitrary units normalized to the housekeeping gene hypoxantine-guanine phosphoribosyl transferase. *P0.05; **P0.01.

Article Snippet: Acetone-fixed sections were incubated with 10% goat serum in 50 mmol/L NH4Cl followed by a mouse monoclonal anti-human CD137 antibody (Abcam, Cambridge, UK) or an isotype control (Dakopatts, Glostrup, Denmark) and a polyclonal rabbit anti–human von Willebrand factor (Dakopatts) or a polyclonal rabbit anti-human CD3 (Dakopatts) antibody.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Control

Journal: Circulation

Article Title: CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

doi: 10.1161/circulationaha.107.699173

Figure Lengend Snippet: Figure 7. Atherosclerotic lesion size in 5 CD137 agonist–treated vs 5 control rat IgG2a-treated Apoe/ mice. Lesions were evalu- ated by microscopic morphometry of serial sections after stain- ing with Oil Red O at 100 to 800 m from the aortic root. Whis- kers indicate meanSE. *P0.05.

Article Snippet: Acetone-fixed sections were incubated with 10% goat serum in 50 mmol/L NH4Cl followed by a mouse monoclonal anti-human CD137 antibody (Abcam, Cambridge, UK) or an isotype control (Dakopatts, Glostrup, Denmark) and a polyclonal rabbit anti–human von Willebrand factor (Dakopatts) or a polyclonal rabbit anti-human CD3 (Dakopatts) antibody.

Techniques: Control, Staining

Journal: Circulation

Article Title: CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

doi: 10.1161/circulationaha.107.699173

Figure Lengend Snippet: Figure 8. Proposed model for CD137 in atherosclerosis. A, Cytokines produced by activated immune cells (macrophages, T cells) in the intima induce CD137 on vascu- lar ECs and SMCs. B, CD137L expressed by macrophages ligates CD137 on T cells and vascular cells, which results in endothe- lial expression of adhesion molecules (VCAM-1, ICAM-1), recruitment of T cells and monocytes, activation of T cells and monocyte-derived macrophages, and reduc- tion of smooth muscle proliferation (C). All of these responses contribute to plaque inflam- mation and disease aggravation.

Article Snippet: Acetone-fixed sections were incubated with 10% goat serum in 50 mmol/L NH4Cl followed by a mouse monoclonal anti-human CD137 antibody (Abcam, Cambridge, UK) or an isotype control (Dakopatts, Glostrup, Denmark) and a polyclonal rabbit anti–human von Willebrand factor (Dakopatts) or a polyclonal rabbit anti-human CD3 (Dakopatts) antibody.

Techniques: Produced, Expressing, Activation Assay, Derivative Assay