ox 40 150nd  (fluidigm)

Journal: Frontiers in Immunology

Article Title: Generation of novel human anti-OX-40 mAbs endowed with different biological properties as tools for cancer therapy

doi: 10.3389/fimmu.2025.1644391

Figure Lengend Snippet: Binding of the novel anti-OX-40 mAbs on OX-40 recombinant protein and human lymphocytes. (A) Binding of the novel human mAbs was tested at increasing concentrations (0.3–100 nM) on purified rhOX-40 and the signal was detected by using the secondary HRP-conjugated anti-Fab antibody. OX-40_8 mAb was used as a negative control. (B) The expression level of OX-40 protein was determined on untreated or activated hPBMCs by using a commercial polyclonal anti-OX-40 Ab. (C) Binding of mAbs to untreated (grey curves) and activated (black curves) hPBMCs tested by cell ELISA at increasing concentrations (0.3–200 nM). Concentration values were reported as the mean of at least three determinations and error bars depicted means ± SD. The binding curves were obtained by using Prism (GraphPad Prism 5) tool. The results were obtained by at least three independent experiments.

Article Snippet: The clinically validated anti-PD-L1 Atezolizumab mAb (N298A, InvivoGen) and anti-OX-40 Rocatinlimab (HY-P99955, MedChemExpress) were also used.

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

Journal: Frontiers in Immunology

Article Title: Generation of novel human anti-OX-40 mAbs endowed with different biological properties as tools for cancer therapy

doi: 10.3389/fimmu.2025.1644391

Figure Lengend Snippet: Binding kinetics of the novel anti-OX-40 mAbs on immobilized OX-40 protein via BLI analyses. The sensorgrams report the binding of novel converted mAbs to OX-40 obtained via BLI analyses. The recombinant human OX-40/Fc protein was used as a ligand and immobilized on ProA sensor (3 µg/mL), whereas OX-40_1 (A) , OX-40_2 (B) , OX-40_3 (C) and OX-40_5 (D) were used as analytes and tested at increasing concentrations (10–100 nM), after saturation with Fc protein by two subsequent injections (6 µg/mL). The sensorgrams show association and dissociation rates of the analytes.

Article Snippet: The clinically validated anti-PD-L1 Atezolizumab mAb (N298A, InvivoGen) and anti-OX-40 Rocatinlimab (HY-P99955, MedChemExpress) were also used.

Techniques: Binding Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Generation of novel human anti-OX-40 mAbs endowed with different biological properties as tools for cancer therapy

doi: 10.3389/fimmu.2025.1644391

Figure Lengend Snippet: Assessment of competitive binding and interference of novel anti-OX-40 mAbs and OX-40-L. To test the ability of the novel anti-OX-40 mAbs, (A) for OX-40_1 and OX-40_5 and (B) for OX-40_2 and OX-40_3, to interfere in the OX-40/OX-40L interaction, a BLI analysis was performed. OX40-L/His was used as ligand and immobilized on HIS1K sensor at the concentration of 5 µg/mL and then OX40/Fc receptor was tested at the concentration of 10 nM, before or after preincubation with each indicated anti-OX-40 mAbs (used at 10-fold molar excess). A human unrelated IgG was used as a negative control.

Article Snippet: The clinically validated anti-PD-L1 Atezolizumab mAb (N298A, InvivoGen) and anti-OX-40 Rocatinlimab (HY-P99955, MedChemExpress) were also used.

Techniques: Binding Assay, Concentration Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: Generation of novel human anti-OX-40 mAbs endowed with different biological properties as tools for cancer therapy

doi: 10.3389/fimmu.2025.1644391

Figure Lengend Snippet: Assessment of the effects of novel anti-OX-40 mAbs on OX-40 intracellular signalling. The ability of the indicated antibodies to activate OX-40 receptor was tested by using OX-40 Bioassay. Genetically engineered Jurkat T cells expressing luciferase reporter gene under the control of OX-40 signalling were used. The cells were treated with the indicated antibodies in a dose escalation range (1.5–200 nM). After treatment, the bioluminescent signal was quantified. OX-40_8 mAb was used as a negative control. Error bars depicted means ± SD and the P values reported is: ***P < 0.001, calculated by Sidak’s multiple comparisons test performed by 2way ANOVA analysis. RLU values at all the doses of the antibody OX-40_1, OX-40_2, and OX-40_3 were compared to those of OX-40_5.

Article Snippet: The clinically validated anti-PD-L1 Atezolizumab mAb (N298A, InvivoGen) and anti-OX-40 Rocatinlimab (HY-P99955, MedChemExpress) were also used.

Techniques: Bioassay, Expressing, Luciferase, Control, Negative Control

Journal: Frontiers in Immunology

Article Title: Generation of novel human anti-OX-40 mAbs endowed with different biological properties as tools for cancer therapy

doi: 10.3389/fimmu.2025.1644391

Figure Lengend Snippet: Effects of anti-OX-40 mAbs on co-cultures of NK with Treg, CD4 + /CD25 - cells, or with MDA-MB-231 TNBC cells. (A) ADCC induced by anti-OX-40 mAbs at indicated concentration on co-cultures of NK with Treg or with CD4 + /CD25 - T cells, used as a negative control. INFγ secretion was measured by ELISA on supernatant in co-cultures of NK and Treg cells (B) , treated as mentioned above. (C) Binding of OX-40_5 on Treg and control CD4 + /CD25 - cells was assessed by Cell ELISA. (D) ADCC induced by anti-OX-40 mAbs at indicated concentration on co-cultures of NK with MDA-MB-231 tumor cells. (E) INFγ secretion was measured by ELISA from supernatant of NK and MDA-MB-231 tumor cells co-cultures. The values were obtained by at least three independent experiments and error bars depicted means ± SD. The P values reported are: ***P < 0.01; **P < 0.01; *P < 0.05, by student’s t test (two variables), calculated by comparing the treatment of the non-agonistic OX-40_5 mAb to each other agonistic mAbs (A, B) , or the combination of OX-40_3 with OX-40_5 respect to each one used as single agents (E) .

Article Snippet: The clinically validated anti-PD-L1 Atezolizumab mAb (N298A, InvivoGen) and anti-OX-40 Rocatinlimab (HY-P99955, MedChemExpress) were also used.

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

Journal: Frontiers in Immunology

Article Title: Generation of novel human anti-OX-40 mAbs endowed with different biological properties as tools for cancer therapy

doi: 10.3389/fimmu.2025.1644391

Figure Lengend Snippet: Expression of OX-40 on hPBMCs, NK, CD8 + and CD4 + T cells and effects of the novel mAbs on these subpopulations. Analyses by WB with a commercial anti-OX-40 antibody of cell extracts from stimulated subpopulations of lymphocytes. The protein levels are expressed as fold increase with respect to untreated cells and the intensity of the bands was normalized to α-tubulin by calculating the ratio of OX-40/tubulin for poorly glycosylated (white bars), medium glycosylated (grey bars) and highly glycosylated (black bars) signal intensities for each cell extract (A) . The effects of novel anti-OX-40 mAbs were tested on NK and CD8 + cells isolated or in co-cultures with monocytes. IFNγ secretion detected in supernatant of NK cells (B) CD8 + T cells (D) isolated or in co-cultures with monocytes ( C , E ) treated with anti-OX-40 mAbs at concentrations of 2 and 10 nM for 48 h at 37°C. Concentration values were obtained by at least three independent experiments and expressed in pg/mL. Error bars depicted means ± SD. The P values reported are: **P < 0.01; *P ≤ 0.05, by student’s t test (two variables), calculated by comparing the non-agonistic OX-40_5 mAb with the agonistic OX-40_3 mAb.

Article Snippet: The clinically validated anti-PD-L1 Atezolizumab mAb (N298A, InvivoGen) and anti-OX-40 Rocatinlimab (HY-P99955, MedChemExpress) were also used.

Techniques: Expressing, Isolation, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Generation of novel human anti-OX-40 mAbs endowed with different biological properties as tools for cancer therapy

doi: 10.3389/fimmu.2025.1644391

Figure Lengend Snippet: Effects of anti-OX-40 mAbs on co-cultures of lymphocytes and tumor cells used alone or in combination with anti-PD-L1 mAbs. (A) IL-2 secretion in supernatants of hPBMCs treated with anti-OX-40 mAbs alone or combined with anti-PD-L1 mAb. IL-2 levels were measured by following the manufacturer’s recommendations of cytokines secretion kit by R & D Systems. (B) Analysis of OX-40 and PD-L1 expression on different tumor cell lines by cell ELISA. (C) Tumor cell lysis induced by OX-40 mAbs alone or in combination with anti-PD-L1 mAbs on co-cultures of MDA-MB-231 tumor cells with unfractionated hPBMCs (left) or NK cells (right). The results were obtained by at least three independent experiments. Error bars depicted means ± SD and The P values reported are: ***P < 0.001; **P < 0.01; * ≤ 0.05, by student’s t test (two variables), calculated by comparing the combinations with each respective compound used as a single agent.

Article Snippet: The clinically validated anti-PD-L1 Atezolizumab mAb (N298A, InvivoGen) and anti-OX-40 Rocatinlimab (HY-P99955, MedChemExpress) were also used.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Lysis

Journal: eLife

Article Title: Tailoring T fh profiles enhances antibody persistence to a clade C HIV-1 vaccine in rhesus macaques

doi: 10.7554/eLife.89395

Figure Lengend Snippet: Flow cytometry antibodies.

Article Snippet: Mouse anti-human CD134 (OX-40) (Clone L106) , AIM assay , BD Biosciences , Cat#744746; RRID: AB_2742454.

Techniques: Flow Cytometry

Journal: Cell

Article Title: Enhanced safety and efficacy of protease-regulated CAR-T cell receptors

doi: 10.1016/j.cell.2022.03.041

Figure Lengend Snippet: bd

Article Snippet: Anti-OX-40 (clone ACT35) , Fluidigm , 3150023B.

Techniques: Purification, Virus, Recombinant, Membrane, Transfection, Enzyme-linked Immunosorbent Assay, Antibody Labeling, Flow Cytometry, Luciferase, Software, Imaging