Journal: International Journal of Molecular Sciences

Article Title: Novel Bi-Specific Immuno-Modulatory Tribodies Potentiate T Cell Activation and Increase Anti-Tumor Efficacy

doi: 10.3390/ijms23073466

Figure Lengend Snippet: Effects of the combinatorial treatments on secretion of cytokines by stimulated T cells. hPBMCs were incubated with SEB (50 ng/mL) in the absence or presence of LAG-3_1, PD-1_1 or PD-L1_1 antibodies, used alone or in combinations for 66 h at 37 °C. Cytokine secretion was measured in the supernatants by evaluating the levels of IL-2 and IFNγ by ELISA. An unrelated antibody was used in parallel assays as a negative control. Error bars depict means ± SD. p -value: *** p ≤ 0.001; ** p < 0.01.

Article Snippet: The following recombinant proteins were used: Human PD-L1/Fc, human PD-1/Fc and human LAG-3/Fc protein (all from Bio-Thecne R&D Systems, Inc., Minneapolis, MN, USA); Human LAG-3/His-GST and Human HLA class II histocompatibility antigen, DRA (from Cusabio Technology LLC, Houston, TX, USA); and Staphylococcal enterotoxin B (SEB), a toxin produced by the bacterium Staphylococcus aureus and used as stimuli for the activation of lymphocytes (Sigma, S4881 St. Louis, MO, USA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Novel Bi-Specific Immuno-Modulatory Tribodies Potentiate T Cell Activation and Increase Anti-Tumor Efficacy

doi: 10.3390/ijms23073466

Figure Lengend Snippet: Schematic representation and purification of bispecific tribodies targeting LAG-3 and PD-L1 or PD-1, derived from LAG-3_1, PD-L1_1 and PD-1_1 parental mAbs. ( A ) Each TR was obtained by genetically fusing the indicated Fab with two identical scFvs. ( B ) Analysis of purity of the tribodies. Coomassie Blue stained SDS-PAGE of His-tagged Tribodies under reducing (lanes 1 and 2) or nonreducing conditions (lanes 3 and 4) obtained after purification by Ni-affinity chromatography. ( C ) SEC analysis of the final products to confirm the purity.

Article Snippet: The following recombinant proteins were used: Human PD-L1/Fc, human PD-1/Fc and human LAG-3/Fc protein (all from Bio-Thecne R&D Systems, Inc., Minneapolis, MN, USA); Human LAG-3/His-GST and Human HLA class II histocompatibility antigen, DRA (from Cusabio Technology LLC, Houston, TX, USA); and Staphylococcal enterotoxin B (SEB), a toxin produced by the bacterium Staphylococcus aureus and used as stimuli for the activation of lymphocytes (Sigma, S4881 St. Louis, MO, USA).

Techniques: Purification, Derivative Assay, Staining, SDS Page, Affinity Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Novel Bi-Specific Immuno-Modulatory Tribodies Potentiate T Cell Activation and Increase Anti-Tumor Efficacy

doi: 10.3390/ijms23073466

Figure Lengend Snippet: Binding of the novel generated tribodies to the purified recombinant proteins. Binding curves by ELISA assays of the tribodies (0.001–1000 nM), or their parental mAbs, onto immobilized PD-L1, PD-1 or LAG-3-Fc chimeric proteins. An unrelated human IgG antibody was used (at a concentration of 200 nM) in parallel assays as a negative control. The binding values were reported as the mean of at least three determinations obtained in three independent experiments. Error bars depict means ± SD.

Article Snippet: The following recombinant proteins were used: Human PD-L1/Fc, human PD-1/Fc and human LAG-3/Fc protein (all from Bio-Thecne R&D Systems, Inc., Minneapolis, MN, USA); Human LAG-3/His-GST and Human HLA class II histocompatibility antigen, DRA (from Cusabio Technology LLC, Houston, TX, USA); and Staphylococcal enterotoxin B (SEB), a toxin produced by the bacterium Staphylococcus aureus and used as stimuli for the activation of lymphocytes (Sigma, S4881 St. Louis, MO, USA).

Techniques: Binding Assay, Generated, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Novel Bi-Specific Immuno-Modulatory Tribodies Potentiate T Cell Activation and Increase Anti-Tumor Efficacy

doi: 10.3390/ijms23073466

Figure Lengend Snippet: Binding affinity of the selected tribodies for activated lymphocytes in the absence or presence of LAG-3_1 mAb. ( A ) Cell ELISA assays were performed by using increasing concentrations (0.1–100 nM) of tribodies on activated hPBMCs. ( B ) Cell ELISA assays were performed by measuring the binding of the indicated tribodies to human-activated lymphocytes in the absence (empty bars) or presence (dark bars) of the parental anti-LAG-3 antibody used at saturating concentration. The binding values are reported as the mean of at least three determinations obtained in three independent experiments. Error bars depict means ± SD.

Article Snippet: The following recombinant proteins were used: Human PD-L1/Fc, human PD-1/Fc and human LAG-3/Fc protein (all from Bio-Thecne R&D Systems, Inc., Minneapolis, MN, USA); Human LAG-3/His-GST and Human HLA class II histocompatibility antigen, DRA (from Cusabio Technology LLC, Houston, TX, USA); and Staphylococcal enterotoxin B (SEB), a toxin produced by the bacterium Staphylococcus aureus and used as stimuli for the activation of lymphocytes (Sigma, S4881 St. Louis, MO, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Novel Bi-Specific Immuno-Modulatory Tribodies Potentiate T Cell Activation and Increase Anti-Tumor Efficacy

doi: 10.3390/ijms23073466

Figure Lengend Snippet: Binding specificity of the TRBs with target ICs and their antagonistic abilities. ( A ) Binding of the immunomodulatory tribodies, or LAG-3_1 parental mAb, tested at increasing concentrations on HuT78 cells by cell ELISA. Error bars depict means ± SD; these were calculated on the basis of results obtained by at least three independent experiments. ( B ) Secretion of IL-2 measured in the supernatants of HuT78 cells, treated as indicated, using a DuoSet ELISA kit. An unrelated antibody was used in parallel as a negative control. ( C , D ) Competitive ELISA to test the interference of the tribodies in LAG-3/MHCII (HLA-DRA) and PD-1/PD-L1 interactions. The binding of biotinylated MHCII or PD-L1 ligand to immobilized LAG-3 or PD-1 receptor, respectively, was measured in the absence or presence of a molar excess of tribodies (3:1 M/M for anti-LAG-3 and 5:1 M/M for anti-PD-L1 mAbs). Error bars depict means ± SD. p -value: *** p ≤ 0.001; ** p < 0.01.

Article Snippet: The following recombinant proteins were used: Human PD-L1/Fc, human PD-1/Fc and human LAG-3/Fc protein (all from Bio-Thecne R&D Systems, Inc., Minneapolis, MN, USA); Human LAG-3/His-GST and Human HLA class II histocompatibility antigen, DRA (from Cusabio Technology LLC, Houston, TX, USA); and Staphylococcal enterotoxin B (SEB), a toxin produced by the bacterium Staphylococcus aureus and used as stimuli for the activation of lymphocytes (Sigma, S4881 St. Louis, MO, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Competitive ELISA