Anti-Human TNFRSF9 Recombinant Antibody (Utomilumab) (CAT#: TAB-457CQ)

Figure 1 Sandwich ligand-blocking assay by SPR.

Full sensorgrams for a utomilumab Fab was captured onto anti-human lambda surface or b urelumab Fab was captured onto anti-human kappa surface to determine blocking of the interaction of h4-1BB receptor with h4-1BBL. The legend shows the first analyte sample name followed by the second analyte. Triplicate measurements are consistent with one another; therefore, sensorgrams from third replicates are shown. Utomilumab Fab blocks the h4-1BB receptor/h4-1BBL interaction while urelumab Fab does not. Anti-Avi tag antibody was used to demonstrate binding to h4-1BB receptor by its Avi-tag when receptor is bound to utomilumab Fab or urelumab Fab.

Chin, S. M., Kimberlin, C. R., Roe-Zurz, Z., Zhang, P., Xu, A., Liao-Chan, S., ... & Wang, F. (2018). Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab. Nature communications, 9(1), 4679.

Figure 2 Urelumab activates 4-1BB signaling more strongly than utomilumab.

Human 4-1BB NF-κB luciferase reporter HEK293 cells were incubated with fivefold increasing concentrations (max 250 nM) of a utomilumab or b urelumab antibody at 37 °C for 6 h before measuring luciferase activity and comparing to unstimulated cells. Mean fold induction was plotted against concentrations of test antibodies. Error bars represent the propagated standard deviation (SD) of three measurements. Solid lines are fits with dose-response curves with maximal fold induction of 1.38 ± 0.051 and 1.84 ± 0.053 for utomilumab and 1.99 ± 0.046 and 2.24 ± 0.033. c, d Purified human CD8 T cells were stimulated with plate bound anti-CD3 (50 ng/ml) plus each plate bound antibody with fourfold increasing concentrations (max 333.33 nM) at 37° C for 72 h and human cytokine production was measured by ELISA. Error bars represent the standard deviation of two measurements. All cytokines are measured from the same donor.

Chin, S. M., Kimberlin, C. R., Roe-Zurz, Z., Zhang, P., Xu, A., Liao-Chan, S., ... & Wang, F. (2018). Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab. Nature communications, 9(1), 4679.

Figure 3 Differential clustering of 4-1BB receptors on Jurkat cells induced by urelumab and utomilumab.

A Confocal images of 4-1BB receptor co-clusters with Urelumab-Alexa Fluor 488 conjugate (URE, green), Urelumab-Fab Alexa Fluor 488 Conjugate (URE-Fab), Utomilumab Alexa Fluor 594 conjugate (UTO, red), or with a combination of URE and UTO in the presence (top row) or absence (bottom row) of 4-1BB His-Avi ligand. Nuclei are stained with DAPI (blue). Scale bar = 10 µm. b Numbers of observed clusters per 100 µm2 of cellular area. Data shown as mean + S.E.M., N = at least 10 cells from three independent experiments. *p-value ≤ 0.05; **p-value ≤ 0.01; ***p-value ≤ 0.001; n.s., not significant

Chin, S. M., Kimberlin, C. R., Roe-Zurz, Z., Zhang, P., Xu, A., Liao-Chan, S., ... & Wang, F. (2018). Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab. Nature communications, 9(1), 4679.

Figure 1 Anti-Human TNFRSF9 Recombinant Antibody (Utomilumab) (TAB-457CQ) in WB

Western blot analysis of TAB-457CQ was performed with Recombinant Human TNFRSF9 Protein onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with TAB-457CQ and HRP Goat Anti-Human IgG as a secondary antibody. Chemiluminescent detection was performed.

Figure 2 Anti-Human TNFRSF9 Recombinant Antibody (Utomilumab) (TAB-457CQ) in ELISA

ELISA analysis of TAB-457CQ was performed by coating with Recombinant Human TNFRSF9 Protein. Then blocked with BSA, and incubated with Anti-Human TNFRSF9 Antibody (TAB-457CQ). The HRP-conjugated goat anti-human IgG as a secondary antibody. Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 3 Anti-Human TNFRSF9 Recombinant Antibody (TAB-457CQ)-Low endotoxin in SDS-PAGE

SDS-PAGE analysis of TAB-457CQ-LowE in β-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 1) conditions.

Figure 4 Anti-Human TNFRSF9 Recombinant Antibody (TAB-457CQ)-Low endotoxin in WB

Western blot analysis of TAB-457CQ-LowE was performed by loading human TNFRSF9 protein, His tag.

Figure 5 Anti-Human TNFRSF9 Recombinant Antibody (TAB-457CQ)-Low endotoxin in ELISA

ELISA analysis of TAB-457CQ-LowE was performed by coating with human TNFRSF9 protein (His tag). Then blocked with BSA and incubated with TAB-457CQ-LowE. The HRP conjugated Anti-human IgG as a secondary antibody (1: 2000). Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.