Fig. 1
ATOR-1015 binds to CTLA-4 and OX40 and blocks binding to the natural ligands. (a) Design of ATOR-1015. The Fab domains bind to OX40. The CTLA-4 binding domains, which are fused to the light chain via a S (GGGGS)2 linker, consists of 111 amino acids from CD86 with 5 mutations for enhanced CTLA-4 affinity. (b) Binding of ATOR-1015 to CTLA-4-expressing CHO cells. Cells were stained with serially diluted ATOR-1015 or IgG1 control, followed by a PE-conjugated anti-human IgG. Mean fluorescence intensity (MFI) was determined by flow cytometry (n = 3). (c) ATOR-1015 completely blocks CTLA-4 from interacting with CD80 and CD86 in a competitive ELISA. Plates were coated with CD80-Fc or CD86-Fc. Serially diluted ATOR-1015 was mixed with a fixed concentration of biotinylated CTLA-4-mFc and added to the plates. Streptavidin-HRP and substrate was added, and luminescence was measured. Percent inhibition calculated based on the maximal signal in the absence of ATOR-1015 is shown (n = 2). (d) Binding of ATOR-1015 to OX40 expressing CHO cells. Cells were stained as described in (B) (n = 2). (e) OX40 expressing CHO cells were pre-incubated with or without ATOR-1015 followed by the addition of OX40L and a fluorescently labelled detection antibody. Percent OX40L inhibition was assessed by flow cytometry (n = 3). Statistical analysis was performed using the Mann Whitney, two-tailed test (****, p < 0.0001). All data presented as mean ± SEM. n equals the number of independent experiments