Fig. 2

Reconciling the differential blocking effects of Ipilimumab. a–d Ipilimumab does not break up pre-formed B7-CTLA-4 complex. a, b Impact of anti-CTLA-4 mAbs on B7-complexed CTLA-4. The B7-CTLA-4 complexes were formed by adding biotinylated CTLA-4 to plates pre-coated with either B7-1 (a) or B7-2 (b). Graded doses of anti-CTLA-4 mAbs were added to plates with pre-existing B7-1-CTLA-4 complex (a) or B7-2-CTLA-4 complex (b). After 2 h, the unbound proteins were washed away and the amounts of B7-1 or B7-2-complexed CTLA-4 were detected using HRP-labeled Streptavidin. c Dissociation kinetics of B7 and CTLA-4 complex based on flow cytometric assays using B7-expressing CHO cells. Surface hB7-1 or mB7-2-expressing CHO cells (1 × 10⁵/test) were incubated with soluble biotinylated CTLA-4-Fc (200 ng/test) for 30 min at room temperature. After washing, cells were incubated in 100 μl DPBS buffer for the indicated minutes. The amounts of B7-bound CTLA-4-Fc were detected with PE-streptavidin by flow cytometry, and the mean fluorescence intensity (MFI) of PE was calculated from triplicated samples. Data shown are results from one of two independent experiments. d L3D10 but not Ipilimumab significantly disrupts the pre-established interaction between soluble CTLA-4 and hB7-1 expressed on CHO cells. Surface hB7-1-expressing CHO cells (1 × 10⁵/test) were incubated with soluble biotinylated CTLA-4-Fc (200 ng/test) for 30 min at room temperature. After washing, cells were incubated with given doses of antibodies in 100 μl DPBS buffer for 1 h. The amounts of B7-bound CTLA-4-Fc were detected with PE-streptavidin, and MFI of PE was calculated. The results represent one of three independent assays with similar patterns. e Ipilimumab does not relieve CTLA-4-Fc-mediated inhibition of CD28-Fc binding to B7-1-transfected J558 cells (J558-B7). J558-B7 cells were incubated with biotinylated CD28-Fc (20 μg/ml) in the presence of CTLA-4-Fc (5 μg/ml) and graded doses of anti-CTLA-4 mAbs or control IgG-Fc. Data shown are means and S.E.M. of MFI from triplicate samples and are representative of at least three independent experiments with similar results. f Kinetics of B7-1-CTLA-4 interaction when B7-1 was immobilized. g Kinetics of B7-1-CTLA-4 interaction when CTLA-4 is immobilized. Data shown in this figure have been repeated two to five times