Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells

doi: 10.4049/jimmunol.1800907

Figure Lengend Snippet: Characterization of IL-2/CD25. (A) Diagram of mIL-2/mCD25 and hybrid mIL-2/hCD25 FPs. (B, C) Bioactivity of mIL-2/mCD25 with varied linker lengths. COS7 cells were transfected with cDNAs encoding mIL-2/mCD25 with different gly/ser linkers. 3 days after transfections supernatants were tested for IL-2 activity using anti-CD3 pre-activated T cells blasts in the B) absence or C) presence of anti-IL-2 (S4B6, 10 μg/ml) (D) SDS-PAGE of purified mIL-2/mCD25 and mIL-2/hCD25 (5 μg each) under reducing and non-reducing conditions and identified by Blue BANDit™ stain (AMRESCO). (E) Western blot analysis of purified mIL-2/mCD25 and mIL-2/hCD25 under reducing conditions and detection with a primary mAb to 6×-His. (F) SDS-PAGE under reducing conditions of purified mIL-2/mCD25 after de-glycosylation using Protein Deglycosylation Mix II). IL-2/CD25s were detected using BlueBANBitTM stain.

Article Snippet: The binding of the FP analytes were tested in 10 mM NaPO 4 , 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.1) on surfaces consisting of a low density (~300RU) of biot-mCD25(22–186)-BioP-TVMV-His which had been His cleaved (mCD25) and captured on a streptavidin sensor chip (GE Healthcare) or mCD122(27–241)-hFc-D/mCD132(23–263)-hFc-K heterodimeric Fc fusion (mIL-2-Rb/g) that had been captured via Protein A immobilized CM5 sensorchip surface using standard ethyl(dimethylaminopropyl) carbodiimide/ N -hydroxysuccinimide chemistry, with ethanolamine blocking (GE Healthcare).

Techniques: Transfection, Activity Assay, SDS Page, Purification, Staining, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells

doi: 10.4049/jimmunol.1800907

Figure Lengend Snippet: Epitope mapping and bioactivity of mIL-2/mCD25 and mIL-2/hCD25. (A,B) For epitope mapping, the indicated fluorescent-labeled mAbs were pre-incubated with various concentrations of purified mIL-2/mCD25 or soluble mCD25 (A) or mIL-2/hCD25 or soluble hCD25 (B) prior to incubation with mCD25 transfected EL4 cells (EL4J3.4) (A) or hCD25 transfected CHO cells( B) Data are represented as the percent inhibition of cellular staining with fluorescent anti-CD25 mAbs alone. Results (mean ± SEM) represent 3 independent determinations for FPs and 2 for soluble CD25. (C) Bioactivity of mIL-2/mCD25 and mIL-2/hCD25 relative to mouse and human IL-2 using the IL-2-dependent CTLL mouse cell line. The right bar graph depicts the activity of the indicated molecules to mIL-2, where the number of replicates are represents within each bar. Results represent the mean ± SEM. A two-sided one sample t-test was performed where the activity of mIL-2 was considered 1. The p value is listed above each bar. In some cases for the data in Fig. 2A and ​and2B,2B, the error bars were too small to show relative to the symbols.

Article Snippet: The binding of the FP analytes were tested in 10 mM NaPO 4 , 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.1) on surfaces consisting of a low density (~300RU) of biot-mCD25(22–186)-BioP-TVMV-His which had been His cleaved (mCD25) and captured on a streptavidin sensor chip (GE Healthcare) or mCD122(27–241)-hFc-D/mCD132(23–263)-hFc-K heterodimeric Fc fusion (mIL-2-Rb/g) that had been captured via Protein A immobilized CM5 sensorchip surface using standard ethyl(dimethylaminopropyl) carbodiimide/ N -hydroxysuccinimide chemistry, with ethanolamine blocking (GE Healthcare).

Techniques: Labeling, Incubation, Purification, Transfection, Inhibition, Staining, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells

doi: 10.4049/jimmunol.1800907

Figure Lengend Snippet: Molecular characteristics of IL-2/CD25. (A) Size determination of IL-2/CD25 by HPLC. The indicated purified proteins were subjected to HPLC using Sephacryl S-200 HR. The elution profile (black line) by O.D. (280 nm) and bioactivity (blue lines) using CTLL cells are shown. The highest values for the O.D. or cpm for 3H-thymidine proliferation were normalized to 1. (B) UV tracing of elution profiles of SEC-MALS of mIL-2/mCD25 and mIL-2/hCD25. The inset plots the molar mass vs. time light scattering signal of the main peak. The predicted MW of the protein backbone and observed MW of the FPs (right), where Carb reflect the amount of glycosylation. (C) Dynamic light scattering histograms of mIL-2/mCD25 and mIL-2/hCD25, with recombinant mIL-2 and mCD25, lysosozyme, and antibody, serving as reference proteins. Rh refers to the hydrodynamic radius. D. Models of FP dimers.

Article Snippet: The binding of the FP analytes were tested in 10 mM NaPO 4 , 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.1) on surfaces consisting of a low density (~300RU) of biot-mCD25(22–186)-BioP-TVMV-His which had been His cleaved (mCD25) and captured on a streptavidin sensor chip (GE Healthcare) or mCD122(27–241)-hFc-D/mCD132(23–263)-hFc-K heterodimeric Fc fusion (mIL-2-Rb/g) that had been captured via Protein A immobilized CM5 sensorchip surface using standard ethyl(dimethylaminopropyl) carbodiimide/ N -hydroxysuccinimide chemistry, with ethanolamine blocking (GE Healthcare).

Techniques: Purification, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells

doi: 10.4049/jimmunol.1800907

Figure Lengend Snippet: Binding properties of IL-2/CD25 FPs to captured IL-2R by Surface Plasmon Resonance (SPR). (A) Binding of mIL-2, mIL-2/mCD25, and mIL-2/hCD25 to captured mouse and human CD25 or mouse IL-2Rβ/γc heterodimers by SPR. The left shows representative binding sensograms and the right indicates the association and dissociation constants and the Kd for the binding of each protein to CD25 or IL-2Rβ/γc. The top concentrations for IL-2 was 1000 nM for binding to captured CD25 or IL-2Rβ/γc, except for mIL-2 to mIL-2Rβ/γc, which was 111 nM and 4000nM for the FPs. (B) Direct comparison of the binding levels of the indicated proteins to captured mCD25, where the ligand concentrations are show in the graph.

Article Snippet: The binding of the FP analytes were tested in 10 mM NaPO 4 , 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.1) on surfaces consisting of a low density (~300RU) of biot-mCD25(22–186)-BioP-TVMV-His which had been His cleaved (mCD25) and captured on a streptavidin sensor chip (GE Healthcare) or mCD122(27–241)-hFc-D/mCD132(23–263)-hFc-K heterodimeric Fc fusion (mIL-2-Rb/g) that had been captured via Protein A immobilized CM5 sensorchip surface using standard ethyl(dimethylaminopropyl) carbodiimide/ N -hydroxysuccinimide chemistry, with ethanolamine blocking (GE Healthcare).

Techniques: Binding Assay, SPR Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells

doi: 10.4049/jimmunol.1800907

Figure Lengend Snippet: Activation of STAT5 by IL-2/CD25. (A) Representative FACS analysis of induction of pSTAT5 after incubation of spleen cells from female C57BL/6 mice with mIL-2 or mIL-2/mCD25 for 15 min. (B) Dose-response curves of in vitro STAT5 activation (mean ± SEM) of the indicated cell populations in the spleen after incubation with IL-2 (n=3), mIL-2/mCD25 (n=2), or mIL-2/hCD25 (n=2) for 15 min. (C) Representative normalized dose-responses curves (left) for CD4+ Foxp3+ Tregs; the EC50s (right) of each dose-response (n=5) were determined by non-linear regression analysis. Shown is the mean ± SEM, where the number in the graph represents the p values of an unpaired two-sided t-test. (D-F) Time-course of in vivo responsiveness of the indicated cells after injection of female C57BL/6 mice with IL-2 (5 μg), mIL-2/mCD25 (20 μg), or mIL-2/hCD25 (20 μg). At the indicated time, spleen cells were harvested and immediately processed for ex vivo pSTAT5 staining or (D) FACS analysis for Foxp3 and CD25. Shown is the mean ± SEM. The number in each graph represents the p value for the analysis of mIL-2/mCD25 vs mIL-2 (D, E) or mIL-2/mCD25 vs.mIL-2/hCD25 (F) by two-way ANOVA using Tukey”s multiple comparison test. (D) Responses by Tregs to mIL-2 and mIL-2/mCD25. (E) Responsiveness by various cells populations to mIL-2 and mIL-2/mCD25. (F) Comparison of the responses by Tregs to mIL-2/mCD25 or mIL-2/hCD25. In some cases for the data in Fig. 4, the error bars were too small to show relative to the symbols.

Article Snippet: The binding of the FP analytes were tested in 10 mM NaPO 4 , 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.1) on surfaces consisting of a low density (~300RU) of biot-mCD25(22–186)-BioP-TVMV-His which had been His cleaved (mCD25) and captured on a streptavidin sensor chip (GE Healthcare) or mCD122(27–241)-hFc-D/mCD132(23–263)-hFc-K heterodimeric Fc fusion (mIL-2-Rb/g) that had been captured via Protein A immobilized CM5 sensorchip surface using standard ethyl(dimethylaminopropyl) carbodiimide/ N -hydroxysuccinimide chemistry, with ethanolamine blocking (GE Healthcare).

Techniques: Activation Assay, Incubation, In Vitro, In Vivo, Injection, Ex Vivo, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells

doi: 10.4049/jimmunol.1800907

Figure Lengend Snippet: Pharmacokinetics and pharmacodynamics of IL-2/CD25. (A) Female C57/B6 mice were injected i.p. with 20 μg of mIL-2/mCD25 or mIL-2/hCD25. Serum was collected at the indicated time points and the levels of the FPs were determined by ELISA developed to specifically detect these molecules. Shown are the mean ± SEM where an unpaired two-sided t-test was used to compare the half-life of the two FPs. (B, C) Male C57BL/6 mice received mIL-2/mCD25 (8 μg) and mIL-2 (1.0 μg)/anti-IL-2 (5 μg of JES6–12A1) (days 1, 3 and 5) and hIL-2 (25,000 U) or PBS (daily for 5 days); the indicated cell populations from the spleen was assessed 1, 3 and 7 days after the last injection. Shown is the mean ± SEM. The number in each graph represents the p value for the analysis of mIL-2/mCD25 (upper value) or mIL-2/anti-mIL-2 complex (lower values) to hIL-2 by two-way ANOVA using Tukey’s multiple comparison test. (B) Effect on the indicated parameter for Tregs and conventional CD4+ and CD8+ T cells. (C) The levels of CD25 and Foxp3 on Tregs. In some cases for the data in Fig. 5, the error bars were too small to show relative to the symbols.

Article Snippet: The binding of the FP analytes were tested in 10 mM NaPO 4 , 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.1) on surfaces consisting of a low density (~300RU) of biot-mCD25(22–186)-BioP-TVMV-His which had been His cleaved (mCD25) and captured on a streptavidin sensor chip (GE Healthcare) or mCD122(27–241)-hFc-D/mCD132(23–263)-hFc-K heterodimeric Fc fusion (mIL-2-Rb/g) that had been captured via Protein A immobilized CM5 sensorchip surface using standard ethyl(dimethylaminopropyl) carbodiimide/ N -hydroxysuccinimide chemistry, with ethanolamine blocking (GE Healthcare).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells

doi: 10.4049/jimmunol.1800907

Figure Lengend Snippet: Molecular consequences of mIL-2/mCD25 on Tregs. Male C57BL/6-Foxp3/RFP mice received a single injection (i.p.) of mIL-2/mCD25 (20 μg) or PBS. 3 days later, Tregs were sorted from the spleens and RNA was isolated for RNAseq. (A) Enriched canonical pathways grouped according to overlapped top differentially expressed (DE) genes. The canonical pathways were selected with a p value <0.05 and adjusted p value < 0.25. Red indicates up-regulated versus down-regulated (blue), and undetermined (white). Thickness of edges (line) indicates the number of DE genes overlapped between the pathways. (B) IL-2/CD25-dependent expression of selected immune related genes in Tregs. Error bars represent standard errors. Reference lines indicate fold-change of 0.5 and 2, respectively.

Article Snippet: The binding of the FP analytes were tested in 10 mM NaPO 4 , 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.1) on surfaces consisting of a low density (~300RU) of biot-mCD25(22–186)-BioP-TVMV-His which had been His cleaved (mCD25) and captured on a streptavidin sensor chip (GE Healthcare) or mCD122(27–241)-hFc-D/mCD132(23–263)-hFc-K heterodimeric Fc fusion (mIL-2-Rb/g) that had been captured via Protein A immobilized CM5 sensorchip surface using standard ethyl(dimethylaminopropyl) carbodiimide/ N -hydroxysuccinimide chemistry, with ethanolamine blocking (GE Healthcare).

Techniques: Injection, Isolation, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-2/CD25: A long-acting fusion protein that promotes immune tolerance by selectively targeting the IL-2R on regulatory T cells

doi: 10.4049/jimmunol.1800907

Figure Lengend Snippet: The effect of IL-2/CD25 on diabetes in female NOD mice. (A) 6–7 week old female NOD mice received the indicate dose of mIL-2/mCD25 i.p. or PBS and diabetes instances were assessed until 30 weeks of age. Data were analyzed by Mantal-Cox log rank test. (B) Female NOD-Foxp3/RFP mice were injected (i.p.) twice per week 3.5 days apart with mIL-2/mCD25 or PBS for 2 weeks. 3 days after the last injection the proportion of Tregs within the CD4+ T cell compartment was determined for the indicated tissues, where PLN refers to the pancreatic lymph node. Shown is the mean ± SEM, where the number of replicates is shown within the bars of each graph. Data were analyzed by Kruskal-Wallis one-way ANOVA. (C-H) A distinct cohort of 6–7 week old female NOD mice were treated twice per week 3.5 days apart with PBS or mIL-2/mCD25 (4 μg) for 5 weeks. Two to 3 days after the last treatment, serum was collected, the mice were euthanized, the spleen and PLN were collected and lymphoid cells were isolated from the pancreas. (C) Levels of antibodies reactive to the FP (n=8). (D) In vivo pSTAT5 levels were assessed directly ex vivo for the indicated cell subsets by immediate fixation of the spleen cell suspension followed by staining and FACS analysis. (E-G) The cell number and phenotype of CD4+ Foxp3+ Tregs (E) and CD4+ Foxp3- (F) and CD8+ (G) T conventional cells were assessed for the indicated tissues. (H) The proportion of CD44hi CD62Llo activated T cells were determined for CD4+ Foxp3- and CD8+ conventional T cells, as indicated. Each time point (D-H) consisted of 5 mice/group. All data were assessed by a two-sided non-paired t-test and any p value <0.05 is shown in the graph when comparing FP vs the PBS control. (I) 12 week old female NOD mice were treated with PBS (n=11) or an initial 8 ug dose of mIL-2/mCD25 (n=9). Subsequently, the mice received PBS or mIL-2/mCD25 (4 μg) every 3.5 days for 5 weeks. Diabetes instances were assessed until 40 weeks of age. Data were analyzed by Mantel-Cox log rank test.

Article Snippet: The binding of the FP analytes were tested in 10 mM NaPO 4 , 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.1) on surfaces consisting of a low density (~300RU) of biot-mCD25(22–186)-BioP-TVMV-His which had been His cleaved (mCD25) and captured on a streptavidin sensor chip (GE Healthcare) or mCD122(27–241)-hFc-D/mCD132(23–263)-hFc-K heterodimeric Fc fusion (mIL-2-Rb/g) that had been captured via Protein A immobilized CM5 sensorchip surface using standard ethyl(dimethylaminopropyl) carbodiimide/ N -hydroxysuccinimide chemistry, with ethanolamine blocking (GE Healthcare).

Techniques: Injection, Isolation, In Vivo, Ex Vivo, Staining

Journal: International journal of cancer

Article Title: Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is induced by genistein through the expression of p53 in colorectal cancer cells.

doi: 10.1002/ijc.11173

Figure Lengend Snippet: FIGURE 3 – Expression of p53 and p21 in the presence of genistein. (a) HCT-116 cells were treated with genistein at various concentrations for 48 hr and total cell lysate was harvested for western analysis. Bands were quantified using Scion Image software (values for p53 and p21 were normalized to actin protein levels). (b) Time course of p53 levels. HCT-116 cells were treated with 50 M genistein for various time points and analyzed by western.

Article Snippet: The antibodies used were NAG-1,23 p53 (Santa Cruz, Santa Cruz, CA), p21 (Santa Cruz) and actin (Santa Cruz).

Techniques: Expressing, Western Blot, Software

Journal: International journal of cancer

Article Title: Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is induced by genistein through the expression of p53 in colorectal cancer cells.

doi: 10.1002/ijc.11173

Figure Lengend Snippet: FIGURE 4 – NAG-1 protein levels in the presence of genistein in HCT-116 and HCT-15 cells. HCT-116 (p53 wild type) and HCT-15 (p53 mutant) were treated with various concentrations of genistein for 48 hr and total cell lysate was harvested for western analysis. NAG-1 bands from HCT-116 cells were quantified using Scion Image soft- ware and normalized to actin values.

Article Snippet: The antibodies used were NAG-1,23 p53 (Santa Cruz, Santa Cruz, CA), p21 (Santa Cruz) and actin (Santa Cruz).

Techniques: Mutagenesis, Western Blot

Journal: International journal of cancer

Article Title: Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is induced by genistein through the expression of p53 in colorectal cancer cells.

doi: 10.1002/ijc.11173

Figure Lengend Snippet: FIGURE 6 – Activation of luciferase activity in p53-dependent manner. Each construct was transiently cotransfected with either empty vector or p53 expression vector into HCT-15 (p53-negative) cells. The cells were grown for 2 days and harvested for luciferase activity. The data show induction over relative luciferase activity of empty vector-transfected cells. Values are mean SD of 3 independent transfections. Statistical analysis was performed as described in Figure 1. *, The changes in the numbers of cells at genistein-treated cells are significant at p 0.001.

Article Snippet: The antibodies used were NAG-1,23 p53 (Santa Cruz, Santa Cruz, CA), p21 (Santa Cruz) and actin (Santa Cruz).

Techniques: Activation Assay, Luciferase, Activity Assay, Construct, Plasmid Preparation, Expressing, Transfection