Fig. 3.

Ipilimumab is ineffective in blocking B7/CTLA-4-mediated cell–cell interactions. a Profiles of B7-1-GFP or B7-2-GFP-transfected CHO cells or CTLA-4^Y201V-transfected 293T cells or mixture of B7-2 and CTLA-4 transfectants without co-incubation. b SDS-PAGE analysis for purify of Fabs used for the study. c, d Representative FACS profiles (c, Fabs used at 10 μg/ml) and dose responses (d) showing comparable binding by L3D10 and Ipilimumab Fabs to CTLA-4-OFP-transfected CHO cells. Alex Fluor 488-conjugated goat anti-human IgG (H+L) was used as the secondary antibody for the binding assay. Dose responses show similar binding activity of Ipilimumab and L3D10 Fabs. AF488-MFI, mean fluorescence intensity of Alex Fluor 488 dye. e Inhibition of B7-1-CTLA-4^Y201V-mediated cell–cell interaction by anti-CTLA-4 mAb Fabs. B7-1-GFP-transfected CHO cells and CTLA-4^Y201V-transfected 293T cells were co-incubated at 4 °C for 2 h in the presence of 10 μg/ml Fab or control proteins. Data shown are representative FACS profiles. f Quantitative comparison between L3D10 and Ipilimumab for their blocking of cell–cell interaction mediated by B7-1 and CTLA-4 expressed on opposing cells. As in e, except that graded doses of antibodies were added. g Inhibition of B7-2-CTLA-4^Y201V-mediated cell–cell interaction by anti-CTLA-4 mAb Fabs. As in e, except that B7-2-GFP transfectants were used. h Quantitative comparison between L3D10 and Ipilimumab for their blocking of cell–cell interaction mediated by B7-2 and CTLA-4 expressed on opposing cells. As in f, except that B7-2-GFP-transfected CHO cells were used. All assays have been repeated at least two times