Fig. 9

Therapeutic effect of Ipilumumab is not achieved by blocking CTLA-4-B7 negative signaling. a Confirmation of the blocking activities of anti-B7 mAbs. CHO cells expressing mouse B7-1 or B7-2 were incubated with a mixture of antibodies (20 μg/ml) and biotinylated human CTLA-4-Fc (2 μg/ml) for 1 h. After washing away unbound proteins, the cell surface CTLA-4-Fc was detected by PE-conjugated streptavidin and measured by flow cytometry. Data shown are representative FACS profiles and have been repeated two times. b Diagram of experimental design. MC38 tumor-bearing Ctla4^h/m mice received anti-B7-1 and anti-B7-2 antibodies (300 μg/mouse/injection, once every 3 days for a total of three injections) in conjunction with either control Ig or Ipilimumab, mice that received Ipilimumab without anti-B7-1 and anti-B7-2 were used as positive control for tumor rejection. c, d Saturation of B7-1 and B7-2 by antibody treatments as diagrammed in b. The PBL were stained with FITC-conjugated anti-B7-1 and anti-B7-2 mAbs at 24 h after the last anti-B7 treatment on day 13. PBL from Cd80^−/−Cd86^−/− mice were used as negative control. e Complete blocking of B7-2 in vivo. As in c and d, except that CD45⁺ leukocytes were gated from single-cell suspensions of draining lymph nodes in mice-bearing MC38 were used. The top panel depicts profiles of B7-2 staining, while the lower panel shows the mean fluorescence intensities. This study has been repeated three times. f Ablation of antibody responses confirmed the functional blockade of B7 by anti-B7-1 and anti-B7-2 mAbs. Sera were collected at day 22 after tumor challenge to evaluate anti-human IgG antibody response. g Saturating blocking by anti-B7-1 and anti-B7-2 mAbs does not affect immunotherapeutic effect of Ipilimumab. Data shown in g are tumor volumes over time and have been repeated twice with similar results. Data in d–g represent mean ± S.E.M.; n.s., not significant