Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: LAG3 , REA351 , 25 , 130-120-470 , PE , Miltenyi Biotec.

Techniques: Imaging

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

Journal: International journal of molecular sciences

Article Title: Immune Checkpoints in Recurrent Pregnancy Loss: New Insights into a Detrimental and Elusive Disorder.

doi: 10.3390/ijms241713071

Figure Lengend Snippet: Figure 3. LAG3 gene (A) and protein (B) expression levels in endometrial biopsies. (A) Fold change of LAG3 mRNA in endometrium tissue samples from the RPL and the control group. (B) Fold change of LAG3 protein in endometrium tissue samples from the RPL and the control group. Data are presented as mean ± SE. Significance was calculated using Student’s t-test (* p < 0.05). RPL denotes recurrent pregnancy loss; CTRL-END: endometrial tissue from the control group; RPL-END endometrial tissue from the RPL group.

Article Snippet: Once normalized the total protein concentrations of each sample to the lowest one, the quantitative measurement of CD276 (Cusabio, Houston, TX, USA, Catalog Number CSB-E14285h), LAG3 (Cusabio, CSB-EL012719HU) and CTLA-4 (Cusabio, Catalog Number CSB-E09171h) endometrial levels were determined using commercially available competitive ELISA kits according to the manufacturer’s instructions.

Techniques: Expressing, Control