Fig. 2: Characterization of iAb induced TNFRSF agonism.

a Cartoons and corresponding representative negative stain electron microscopy 2D classification images for each antibody format. All of the formats in the images contain the variable region of the 3C8 anti-OX40 antibody as representative. b OX40 agonism activity from a panel of ten anti-OX40 antibodies for the indicated antibody format. Each symbol represents a unique clone. Data is shown as fold change over an untreated control (n = 2 independent wells). c Comparison of OX40 agonism activity by the anti-OX40 iAb_aff1 clone, 3C8, to that of the native ligand of OX40, OX40L. Data is shown as fold change over an untreated control (n = 3 independent wells) and presented as mean values±SEM. d Surface plasmon resonance (SPR) affinity data comparing K_D values for each anti-OX40 clone as either an iAb_aff1 or WT IgG. The dotted gray line has a slope of 1 and indicates no change between the two formats. e Cell surface binding to OX40+ Jurkat cells for each anti-OX40 clone comparing the EC₅₀ values of the iAb_aff1 and WT IgG formats. The dotted gray line has a slope of 1 and indicates no change between the two formats. f Effect of antibody fragmentation on OX40 agonism activity with and without iAb_aff1 mutation set engraftment for a single anti-OX40 clone, 3C8 (n = 2 independent wells). g–j TNFRSF agonism activity of various formats of clones against CD40 (g, 4 nM), 4-1BB (h, 22.2 nM), DR4 (i, 100 nM), and DR5 (j, 100 nM). Data are shown at a single concentration (indicated above in paretheses) taken from titration curves in Fig. S9 (n = 2 independent wells). CD40 and 4-1BB agonism are shown as fold change over an untreated control, while DR4 and DR5 agonism are shown as % killing relative to an untreated control. Each CD40 clone was produced as a WT IgG1, IgG2 C131S, and iAb_aff1, while all clones against other targets were only produced as a WT IgG1 and iAb_aff1. Source data are provided as a Source Data file.