Table 1.
Experimentally observed enhancement of cytokine signaling by cytokine-neutralizing antibodies (CNAs).
Fig 1.
Schematics of main biochemical reactions in a cytokine-receptor model.
1 –binding of cytokine with antibody, 2 –binding of cytokine with receptor on the cell membrane, 3 –internalization of cytokine/receptor complex (relatively fast), 4 –degradation of cytokine in endosome (lysosome), 5 –recycling of receptor from endosome to the cell surface and internalization (relatively slow) of free receptor.
Fig 2.
The cytokine receptor consists of two subunits, the α-chain (IL-4Rα) and the common γ-chain (CD132). IL-4Rα subunits are internalized and recycled, while common γ-chain, antibody and cytokine are degraded after internalization.
Fig 3.
Kinetics of free interleukin, signaling, proliferation rate and cell count.
In the model, T cells were incubated with IL-4 250 pM and different concentrations of mAb (5 pM—red lines, 250 pM–blue lines, 10000 pM–green lines; mAb KD set at 20 pM) during 6 days. (a, e) Time course of free IL-4 (100% represents the initial IL-4 concentration of 250 pM); (b, f) Time course of number of the signaling complexes per cell; (c, g) Proliferation rates, expressed as an Emax function of the number of signaling complexes, with EC50 = 20 signaling complexes per cell; (d, h) Time course of cellular proliferation, expressed as T-cell count. The initial number of T cells is 1 x 106 per ml.
Fig 4.
The agonistic effect in the IL-4/T cell model.
Blue lines are for the γ-chain blocking antibody, red lines are for the α-chain blocking antibody. (a) Reproduction of the sIL-4Rα agonistic effect from [5]. IL-4 bioactivity measurements (red squares) were normalized to baseline (incubation with 250 pM IL-4, without sIL-4Rα). For sIL-4Rα /IL-4 binding, a KD of 600 pM was used [29]; the initial number of T cells (2e+05 per ml) was estimated from the experimental description. (b,c,d) Dependencies of the agonistic effect upon parameter values in the IL-4/T cell model (sensitivity analysis), (b) Effect of varying IL-4/mAb binding affinities (solid, dashed and dashed-dotted lines are for Kd values of, respectively, 20, 100 and 500 pM); (c) Effect of varying EC50 values in the proliferation rate Emax function (solid, dashed, and dashed-&-dotted lines are for, respectively, 20, 50 and 100 signaling complexes per cell); (d) effect of varying incubation times (solid, dashed and dashed-dotted lines are for, respectively, cell counts measured after 2, 3, and 5 days). In all simulations, reference parameters were set as follows: initial IL-4 concentration was 250 pM and the initial number of T cells was 1e+06 per ml, other parameter values: IL-4/mAb binding KD = 20 pM; EC50 = 20 signaling complexes per cell; incubation time = 3 days. 100% of the effect corresponds to the calculated number of cells for model parameters (KD, incubation time, EC50) set in the absence of any mAb. With an antibody that blocks the binding to the α-chain (red lines), the agonistic effect may occur. With an antibody that blocks the binding to the γ-chain, a monotonic decrease in the cytokine effect is observed (blue lines).
Fig 5.
Agonistic effect of an IL-4 neutralizing mAb: model simulations for an in vivo case vs. an in vitro case.
Red lines are for the α-chain blocking mAb, blue lines are for the γ-chain blocking mAb. (a) simulations for the in vitro case, (b) simulations for the in vivo case. The following experimental conditions were used in the model simulations: incubation time = 3 days; initial concentration of IL-4 = 250 pM; initial cells density = 4e+06 cells/ml; mAb/IL-4 binding KD = 20 pM; EC50 = 20 signaling complexes per cell. For simulations of the in vivo conditions, systemic elimination rates of 1.26 1/h and 0.004 1/h were added, respectively, for IL-4 and the mAb.