Journal: Scientific Reports

Article Title: Pyrrothiogatain acts as an inhibitor of GATA family proteins and inhibits Th2 cell differentiation in vitro

doi: 10.1038/s41598-019-53856-1

Figure Lengend Snippet: Pyrrothiogatain inhibits Th2 cell differentiation and production of Th2 cytokines. ( A ) Intracellular staining of IL-5/IL-13, IL-4/IFN-γ, and IL-2/IFN-γ in naive CD4 + T cells cultured under Th2 conditions in the presence or absence of pyrrothiogatain (30 and 80 µM) for five days. ( B ) Cytokine production induced in the pyrrothiogatain treated Th2 cells shown in panel (A) was determined by ELISA. ( C ) Quantitative RT-PCR analysis of the pyrrothiogatain-treated Th2 cells shown in panel (A). ( D ) Immunoblot analysis of GATA3 in naive CD4 + T cells cultured under Th2 conditions in the presence or absence of pyrrothiogatain (80 µM) for 5 days. ( E ) Cytokine production from Th2 cells stimulated with immobilized anti-TCRβ mAb for 16 h in the presence or absence of pyrrothiogatain (0 to 100 µM). The amounts of IL-4, IL-5 and IL-13 in the culture supernatants were determined by ELSA. In ( B ), ( C ) and ( E ), all data are expressed as individual points of three independent experiments with error bars indicating standard deviation.

Article Snippet: The amount of cytokines in the recovered supernatants was determined with ELISA using the following antibodies and reagents: anti-IL-4 mAb (Cat#554387, BD Biosciences), biotin-anti-IL-4 mAb (Cat#554390, BD Biosciences), anti-IL-5 mAb (Cat#554393, BD Biosciences), biotin-anti-IL-5 mAb (Cat#554397, BD Biosciences), anti-IFN-γ mAb (Cat#551216, BD Biosciences), biotin-anti-IFN-γ mAb (Cat#554410, BD Biosciences), anti-IL-2 mAb (Cat#554424, BD Biosciences), biotin-anti-IL-2 mAb (Cat#554426, BD Biosciences), streptavidin horseradish peroxidase (Cat#434323, Invitrogen), and TMB peroxidase EIA substrate kit (Cat#1721066, BioRad).

Techniques: Cell Differentiation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Cancer immunology research

Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

doi: 10.1158/2326-6066.CIR-17-0549

Figure Lengend Snippet: Sustained IL2 stimulation is responsible for the adjuvant effect of IL2Cx. A, TgTR1 cells were incubated with Trp1 peptide (1 µg/ml) and IL2Cx122 or IL2Cx25 (100 ng IL2/ml) in the presence of increasing concentrations of homologue IL2 mAb (JES6-5H4 or JES6-1A12, respectively). Seven days later T-cell expansion was evaluated. B, CD45.1 mice were adoptively transferred with 1 × 105 TgTR1 cells followed by BiVax prime. Five days later the mice were boosted with BiVax, BiVax/IL2Cx25(10 µg) (2 µg IL2 + 10 µg IL2 mAb) or BiVax/IL2Cx25(300 µg) (2 µg IL2 + 300 µg IL2 mAb). On day 12 the total numbers of TgTR1 cells were evaluated in spleens. C, Representative flow dot plots from the experiment in panel B, showing the percentage of TgTR1 cells (MHCII-CD45.2+) in spleen gating in total live cells. (n = 3 mice/group). Representative results are shown from at least 3 independent experiments.

Article Snippet: IL2Cx were administered i.p. Human IL2Cx (huIL2Cx) was made from recombinant human IL2 (2 μg/mouse, Peprotech, cat. # 200-02) and human IL2 mAb (10 μg/ml, R&D Systems, cat. # MAB602).

Techniques: Adjuvant, Incubation

Journal: Cancer immunology research

Article Title: Sustained persistence of IL2 signaling enhances the antitumor effect of peptide vaccines through T-cell expansion and preventing PD-1 inhibition

doi: 10.1158/2326-6066.CIR-17-0549

Figure Lengend Snippet: PEG-IL2 is effective in expanding BiVax generated antigen-specific T cells. A, WT mice (3/group) were vaccinated with BiVax prime. Five days later the mice received boosters with BiVax, BiVax/IL2, BiVax/IL2Cx122 or BiVax/PEG-IL2. Seven days later the numbers of Trp1 tetramer+ T cells were evaluated in spleens. B, WT mice were inoculated s.c. with B16F10 cells and 7 days later, received a BiVax prime followed 5 days later by booster vaccines with BiVax, BiVax/IL2 or BiVax/PEG-IL2. Tumor size growth curves showing means with SD for each group. (n = 10 mice/group). C, purified CD8+ T cells from WT mice receiving BiVax prime-boost (5 days apart) and treated or not with PEG-IL2 were incubated with either B16F10 (PD-L1 low) cells, IFNγ-treated (PD-L1 high) B16F10 cells (pulsed with 1 µg Trp1) with or without PD-L1 mAb (10 µg/ml) and the numbers of IFNγ-producing cells were evaluated by EliSpot. Representative results are shown from at least 3 independent experiments.

Article Snippet: IL2Cx were administered i.p. Human IL2Cx (huIL2Cx) was made from recombinant human IL2 (2 μg/mouse, Peprotech, cat. # 200-02) and human IL2 mAb (10 μg/ml, R&D Systems, cat. # MAB602).

Techniques: Generated, Vaccines, Purification, Incubation, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Pyrrothiogatain acts as an inhibitor of GATA family proteins and inhibits Th2 cell differentiation in vitro

doi: 10.1038/s41598-019-53856-1

Figure Lengend Snippet: Pyrrothiogatain inhibits Th2 cell differentiation and production of Th2 cytokines. ( A ) Intracellular staining of IL-5/IL-13, IL-4/IFN-γ, and IL-2/IFN-γ in naive CD4 + T cells cultured under Th2 conditions in the presence or absence of pyrrothiogatain (30 and 80 µM) for five days. ( B ) Cytokine production induced in the pyrrothiogatain treated Th2 cells shown in panel (A) was determined by ELISA. ( C ) Quantitative RT-PCR analysis of the pyrrothiogatain-treated Th2 cells shown in panel (A). ( D ) Immunoblot analysis of GATA3 in naive CD4 + T cells cultured under Th2 conditions in the presence or absence of pyrrothiogatain (80 µM) for 5 days. ( E ) Cytokine production from Th2 cells stimulated with immobilized anti-TCRβ mAb for 16 h in the presence or absence of pyrrothiogatain (0 to 100 µM). The amounts of IL-4, IL-5 and IL-13 in the culture supernatants were determined by ELSA. In ( B ), ( C ) and ( E ), all data are expressed as individual points of three independent experiments with error bars indicating standard deviation.

Article Snippet: The amount of cytokines in the recovered supernatants was determined with ELISA using the following antibodies and reagents: anti-IL-4 mAb (Cat#554387, BD Biosciences), biotin-anti-IL-4 mAb (Cat#554390, BD Biosciences), anti-IL-5 mAb (Cat#554393, BD Biosciences), biotin-anti-IL-5 mAb (Cat#554397, BD Biosciences), anti-IFN-γ mAb (Cat#551216, BD Biosciences), biotin-anti-IFN-γ mAb (Cat#554410, BD Biosciences), anti-IL-2 mAb (Cat#554424, BD Biosciences), biotin-anti-IL-2 mAb (Cat#554426, BD Biosciences), streptavidin horseradish peroxidase (Cat#434323, Invitrogen), and TMB peroxidase EIA substrate kit (Cat#1721066, BioRad).

Techniques: Cell Differentiation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Protein engineering, design & selection : PEDS

Article Title: A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adaptable cytokine activity.

doi: 10.1093/protein/gzt045

Figure Lengend Snippet: Fig. 1. Expression and purification of ICs with IL2 fused to the L- or H-chain C-termini. IC34 and IC35 were transiently expressed in HEK 293F cells in suspension and the secreted proteins were bound to and eluted from protein A using low pH. Fractions were analyzed by SDS–PAGE using non-reducing or reducing conditions as indicated. Fractions in lanes 4, 5 and 6 of the non-reduced gel correspond to lanes 6, 7 and 8 of the reduced gel. The intact ICs are indicated with an arrow. The faster running species in the non-reduced samples are consistent with the molecular weights of the H-chain dimer (IC35) or the H-IL2 dimer (IC34).

Article Snippet: After centrifugation, the amount of intact IC was measured in plasma using an enzyme-linked immunosorbent assay (ELISA), in which an anti-human IgG anti-serum was used for capture (PierceThermo, Cat. #PA1-29961), and a biotinylated monoclonal anti-human IL2 antibody (R&D Systems, Cat. #BAF 202) and a streptavidin–horseradish peroxidase conjugate (Thermo Fisher, Cat. #N100) were used for detection.

Techniques: Expressing, Suspension, SDS Page

Journal: Protein engineering, design & selection : PEDS

Article Title: A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adaptable cytokine activity.

doi: 10.1093/protein/gzt045

Figure Lengend Snippet: Fig. 2. In vitro biological activities of ICs fused to either the H- or L-chain. (A) ADCC activity against labeled M21 melanoma target cells was tested using three different effector (resting human PBMC) to target ratios of 12.5 : 1 (filled triangle), 25 : 1 (filled square) or 50 : 1(filled rhombus) and varying concentrations of the ICs. (B) CDC activity was also tested against M21 melanoma cells using a 1 : 8 dilution of pooled human plasma as a source of complement and varying concentrations of the indicated anti-GD2 IC or chimeric antibody. (C) A standard CTLL-2 bioassay based on 3H-thymidine incorporation, shown as counts per minute (CPM), was used to compare the two ICs with the same molar amounts of recombinant IL2. The activity of the ICs was assumed to be 3000 IU/mg based on the fraction (1/6) that is IL2 and the specific activity of the recombinant IL2 preparation (18 000 IU/mg).

Article Snippet: After centrifugation, the amount of intact IC was measured in plasma using an enzyme-linked immunosorbent assay (ELISA), in which an anti-human IgG anti-serum was used for capture (PierceThermo, Cat. #PA1-29961), and a biotinylated monoclonal anti-human IL2 antibody (R&D Systems, Cat. #BAF 202) and a streptavidin–horseradish peroxidase conjugate (Thermo Fisher, Cat. #N100) were used for detection.

Techniques: In Vitro, Activity Assay, Labeling, Clinical Proteomics, Bioassay, Recombinant

Journal: Protein engineering, design & selection : PEDS

Article Title: A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adaptable cytokine activity.

doi: 10.1093/protein/gzt045

Figure Lengend Snippet: Fig. 4. IL2R specificity and CDC activities of L-chain IL2 variants. Proliferation assays based on mitochondrial dye reduction were performed using the TF-1b cell line expressing only the bg IL2R (A) or the CTLL-2 cell line expressing the abg IL2R (B). The results are representative of at least three individual experiments for each protein. The ranges of ED50 values for the individual ICs in different assays are shown in Table II. (C) The L-IL2 based IC45 (with a five residue deletion in the N-terminus of IL2) was compared with an H-IL2 based IC at two concentrations for binding to TF-1b cells expressing only bg IL2R. (D) GD2-expressing M21 melanoma cells were mixed with dilutions of the indicated ICs and diluted human serum as a source of complement. Cell killing was measured by loss of mitochondrial dye reduction.

Article Snippet: After centrifugation, the amount of intact IC was measured in plasma using an enzyme-linked immunosorbent assay (ELISA), in which an anti-human IgG anti-serum was used for capture (PierceThermo, Cat. #PA1-29961), and a biotinylated monoclonal anti-human IL2 antibody (R&D Systems, Cat. #BAF 202) and a streptavidin–horseradish peroxidase conjugate (Thermo Fisher, Cat. #N100) were used for detection.

Techniques: Expressing, Residue, Binding Assay

Journal: Protein engineering, design & selection : PEDS

Article Title: A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adaptable cytokine activity.

doi: 10.1093/protein/gzt045

Figure Lengend Snippet: Fig. 3. PK analysis of ICs administered IV or SC in mice. The anti-GD2 ICs with IL2 fused to either the H- or L-chain were compared in terms of their clearance rate from the circulation after IV (A) or SC (B) dosing. Intact IC was measured using an ELISA based on capturing from serum with an anti-human IgG anti-serum and detection with anti-human IL2 detection. Since the initial concentrations after IV dosing of the two ICs were not the same, due to differences in ELISA reactivity, the kinetics were reported as percentages of different starting concentrations.

Article Snippet: After centrifugation, the amount of intact IC was measured in plasma using an enzyme-linked immunosorbent assay (ELISA), in which an anti-human IgG anti-serum was used for capture (PierceThermo, Cat. #PA1-29961), and a biotinylated monoclonal anti-human IL2 antibody (R&D Systems, Cat. #BAF 202) and a streptavidin–horseradish peroxidase conjugate (Thermo Fisher, Cat. #N100) were used for detection.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Protein engineering, design & selection : PEDS

Article Title: A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adaptable cytokine activity.

doi: 10.1093/protein/gzt045

Figure Lengend Snippet: Fig. 6. IL2 bioactivity and selectivity of ICs bound to antigen-idiotype antibody-coated beads. (A) The ability of ICs to present IL2 to TF-1b cells after binding to magnetic beads coated with 1A7 anti-idiotype antibody was first compared using two H-IL2 molecules reactive with either GD2 (and 1A7 antibody—IC34) or CD20 (non-specific binding control). Dilutions of beads were combined with a fixed number of responder cells and incubated for 48 h before measuring proliferation by dye reduction. (B) The relative ability of serial dilutions of bead-bound L-IL2 ICs, IC35 and IC45 to activate TF-1b cells were compared with IC34 (H-IL2 format). (C) and (D) Four different L-IL2 ICs were compared for the ability to stimulate the proliferation of CTLL-2 cells (C) or TF-1b cells (D) using the same preparation of coated beads, although a 10-fold dilution was used for the CTLL-2 assay. IC35, IC45 and IC64 all bind GD2 (and the 1A7 anti-idiotype antibody). IC65 binds human EpCAM (negative control) but otherwise has the L-IL2 structure of IC35.

Article Snippet: After centrifugation, the amount of intact IC was measured in plasma using an enzyme-linked immunosorbent assay (ELISA), in which an anti-human IgG anti-serum was used for capture (PierceThermo, Cat. #PA1-29961), and a biotinylated monoclonal anti-human IL2 antibody (R&D Systems, Cat. #BAF 202) and a streptavidin–horseradish peroxidase conjugate (Thermo Fisher, Cat. #N100) were used for detection.

Techniques: Binding Assay, Magnetic Beads, Control, Incubation, Negative Control