Fig 1.
The neutralizing activity of TNFα antagonists on sTNFα-induced cytotoxicity in mouse L929 cells.
1.5×104 L929 cells treated with TNFα antagonists in presence of 1 μg/ml actinomycin D and 20 IU/ml sTNFα for 18 h at 37°C, followed by MTS test. The cell viability expressed as the percentage of the control group as described in “Materials and methods”.
Fig 2.
Competitive binding of TNFα antagonists to mTNFα on transfected cells.
5×105 mTNF expressing CHO cells were incubated with TNFα antagonists competed with 13.3 nM biotin-T0001 for 0.5 h at 4°C, followed by incubation with R-phycoerythrin-conjugated avidin as a secondary antibody for 0.5 h at 4°C. The binding of TNFα antagonists to mTNFα expressed as the percentage competition as described in “Materials and methods”.
Fig 3.
Competitive binding of TNFα antagonists to FcγRIIIa on transfected cells.
2×105 Jurkat cells transfected with FcγRIIIa(158V) were incubated with TNFα antagonists competed with 6.7 nM biotin-IgG1 alone or in the presence of 1:1 molar excess sTNFα for 0.5 h at 4°C, followed by staining with Streptavidin R-PE Conjugate as a secondary antibody for 0.5 h at 4°C. The binding of TNFα antagonists to FcγRIIIa expressed as the percentage competition as described in “Materials and methods”.
Fig 4.
Ability of TNFα antagonists to mediate ADCC.
Jurkat-NFAT luciferase reporter cells (A) or PBMCs (B) were used as effector cells and mTNF-expressing CHO cells were used as targets (E:T ratio 20:1) in ADCC assays. TNFα antagonists were serially diluted and incubated with the CHO cells, followed by incubation with the effector cells for 6 h at 37°C. In report gene assay, ADCC activity was measured for luciferase production using a luminescent substrate. ADCC expressed as the fold of induction (FI). In PBMC-based assay, ADCC activity was measured for LDH release and expressed as percentage of lysis.
Fig 5.
Competitive binding of TNFα antagonists to C1q.
96-well plates were coated with 5 μg/mL goat anti-human C1q antibody for 16 h at 4°C, and 3 μ/mL C1q was added to each well and incubated for 2 h at RT. 133.3 nM biotin-IgG1 and varying concentrations of TNFα antagonists were added to the plates alone or in the presence of a 0.8-fold molar excess of sTNFα, incubated at RT for 15 h and then followed by staining with streptavidin-HRP as a secondary antibody for 1 h at RT. The binding of TNFα antagonists to C1q expressed as the percentage competition as described in “Materials and methods”.
Fig 6.
Ability of TNFα antagonists to mediate CDC.
5×104 mTNF-expressing CHO cells were incubated with TNFα antagonists for 1 h at 4°C, followed by incubation with 8% human complement serum for 2 h at 37°C. At the end of incubation, AlamaBlue dye was added into each well and the plate was incubated for additional 17 h before read at 530 nm excitation/590 nm emission on a fluorescence plate reader. CDC expressed as the percentage of cell death as described in “Materials and methods”.
Fig 7.
Apoptosis induced by TNFα antagonists in mTNF-transfected Jurkat T cells.
(A) mTNF-transfected Jurkat T cells were untreated or treated with TNFα antagonists or rituximab, at 100 nM and cultured for 24 hours. The stimulated cells were stained with FITC-conjugated Annexin V and PI and were then analyzed by flow cytometry. Ten thousand cells were measured and plotted. The proportion of cells residing in each quadrant is expressed as a percentage. (B) The proportions of Annexin V-positive cells in each sample after stimulation are indicated as solid columns.
Fig 8.
Schematic illustrations summarizing the different actions between TNFR-Fc fusion proteins and monoclonal antibodies.
TNFR-Fc fusion protein (T0001 or etanercept) binds TNFα in a 1:1 stoichiometry, while up to three molecules of mAb (adalimumab or infliximab) can bind to each sTNFα trimer. (A) When blocking sTNFα to recruit TNFR trimer, one TNFR-Fc fusion protein molecule is sufficient, but at least two mAb molecules may be needed. (B) When inducing reverse signal though mTNFα, mAb formed more stable complexes with mTNFα and exhibited higher avidity than TNFR-Fc fusion protein. (C) When activating ADCC, TNFR-Fc fusion protein binds both mTNFα trimer and FcγRIIIa in a 1:1:1 stoichiometry, while three mAb molecules can bind to each mTNFα trimer, consequently recruiting three FcγRIIIa. (D) When activating CDC, TNFR-Fc fusion protein binds both mTNFα trimer and C1q in a 1:1:1 stoichiometry, while three mAb molecules can bind to each mTNFα trimer, consequently recruiting three C1q.