Fig. 5

HERA-GITRL demonstrates significantly enhanced agonistic activity compared to a clinical benchmark antibody. a ELISA showing binding of hexavalent HERA-GITRL and bivalent anti-GITR antibody (TRX518) to immobilized human GITR-Fc. TRX518 bound to human GITR-Fc was detected by goat anti-human IgG-Peroxidase. Values are mean OD (n = 3) ± S.D. Representative data from at least three independent experiments are shown. The p value represents a comparison between samples using a two-way ANOVA. ****p < 0.0001. b, c NFκB-luc2/GITR-expressing Jurkat cells were incubated with the indicated concentrations of HERA-GITRL, trimeric GITRL or anti-GITR antibody, without (b) or with (c) crosslinking, at 37 °C for six hours. Data are shown as mean values (± S.D.) from three independent experiments. b The p values represent a comparison between samples using a two-way ANOVA. ****p < 0.0001. c The p values represent a comparison between soluble (b) and crosslinked (c) samples using a two-way ANOVA. ****p < 0.0001. n.s. not significant. d Naïve CD4+ T cells  were isolated from the peripheral blood of healthy volunteers and stimulated with anti-CD3 antibody (1 μg/mL) in the presence of HERA-GITRL or anti-GITR antibody (both 1000 ng/mL). On day five, T cells were harvested and examined by FCM. Due to high donor variability observed when comparing PBS to anti-CD3 antibody treatment, all values have been normalized to the anti-CD3 antibody alone group. The dashed line indicates the 1.0 value. The p values represent a comparison between samples using a one-way ANOVA. *p < 0.05, **p < 0.01. e NFκB-luc2/GITR-expressing Jurkat cells were incubated with the indicated concentrations of trimeric GITRL and/or anti-GITR antibody at 37 °C for six hours. A constant ratio by mass (1:1) was maintained in the combination group. Data are shown as mean values (± S.D.) from three independent experiments. The p values represent a comparison between samples using a two-way ANOVA. ****p < 0.0001. n.s. not significant