NeuroMab™ Anti-CD195 BBB Shuttle Antibody(NRZP-1022-ZP3699)

Figure 1 Binding of anti-CCR5 mAbs to CCR5+ cells.

Figure 2 CI values for different combinations of mAb and viral inhibitors.

Figure 3 Inhibition of CCR5 co-receptor function by anti-CCR5 mAbs. Inhibition of cell-cell fusion by anti-CCR5 mAbs was tested in the RET assay.

Add 0-250 μg/ml of PA8-PA12 or 0-25 μg/ml of PA14 or 2D7 to the mixture of HeLa-EnvJR-FL+ and PM1 cells labeled with F18 and R18, respectively. Fluorescent RET was measured after 4 hours of incubation. Results are the mean of three independent experiments and are expressed as percent fusion inhibition = [1 - (% RET in presence of mAb ÷ % RET in absence of mAb)] × 100%. Inhibition of HIV-1 entry by anti-CCR5 mAbs was tested in a single-round replicating luciferase-based entry assay.

Figure 4 Inhibition of CCR5 co-receptor function by anti-CCR5 mAbs. Inhibition of cell-cell fusion by anti-CCR5 mAbs was tested in the RET assay.

U87-CD4+CCR5+ cells were infected with NLluc+env-reporter virus carrying the JR-FL envelope in the presence of 0-250 μg/ml PA8-PA12 or 0-25 μg/ml PA14 or 2D7. Luciferase activity (relative light units, rlu) was measured in cell lysates 72 hours post infection. Results are from a representative experiment and expressed as % entry inhibition = [1 - (rlu in presence of mAb rlu in absence of mAb)] x 100%. Binding of biotinylated [b]gp120, sCD4 and b-gp120-CD4 complexes to L1.2-CCR5+ cells.

Figure 5 Inhibition of CCR5 co-receptor function by anti-CCR5 mAbs. Inhibition of cell-cell fusion by anti-CCR5 mAbs was tested in the RET assay.

Strong binding was observed when gp120 derived from the R5 virus HIV-1JR-FL was complexed with equimolar amounts of sCD4. No binding was observed in the absence of sCD4 or gp120 from the X4 virus HIV-1LAI. Background binding to CCR5-L1.2 cells has been subtracted from all curves. Inhibition of gp120/sCD4 binding to L1.2-CCR5+ cells was tested in the presence of different concentrations of each antibody.

Figure 6 Inhibition of CCR5 co-receptor function by anti-CCR5 mAbs. Inhibition of cell-cell fusion by anti-CCR5 mAbs was tested in the RET assay.

Cells were pre-incubated in 96-well plates with anti-CCR5 mAb and then incubated with saturating concentrations of biotinylated gp120/sCD4. Finally, the binding of PE-labeled streptavidin to the cells was measured using a fluorescent microplate reader. Results are from a representative experiment and expressed as percent inhibition of gp120/sCD4 binding = [1-(mfi in the presence of mAb ÷ mfi in the absence of mAb)] x 100%.

Figure 7 Synergistic inhibition of cell-cell fusion by PA12 and 2D7.

Dose response curves were obtained for the mAbs used alone and in combination. Add 0-50 μg/ml of PA12, 0-25 μg/ml 2D7, or both in a 2:1 ratio to the mixture of HeLa-EnvJR-FL+ and PM1 cells, with R18 and F18, respectively. Fluorescent RET was measured after 4 hours of incubation. Results are expressed as percent fusion inhibition and are the mean of values from three independent experiments.