Fig. 1

Structure, production and activity of HERA-GITRL. a Schematic depiction of the structure of the hexavalent HERA-GITRL. Three copies of a GITRL protomer sub-sequence (called the GITRL-receptor binding domain or GITRL-RBD) were combined into one single chain (sc) polypeptide. This generated a trivalent scGITRL-RBD, which was fused to the Fc part of a human IgG1-mutein to create a hexavalent scGITRL-RBD dimer. b, c Purification was accomplished by a two-step process combining AFC followed by preparative SEC. b For analytical SEC; purified HERA-GITRL was detected by online measurement of absorption at 280 nm. Content of monomer and aggregates was calculated as the AUC from the elution profile of the SEC. HERA-GITRL eluted as a single peak and showed no detectable aggregates. c Purity and aggregation status of purified hexavalent HERA-GITRL was also assessed by non-reducing and reducing SDS-PAGE. d ELISA showing binding of hexavalent HERA-GITRL to immobilized human, mouse and cynomolgus monkey GITR-Fc. Plate-bound GITR-Fc was probed with the indicated formats and concentrations. Receptor-bound HERA-GITRL was detected via a Strep-Tag II-specific antibody conjugated to horseradish peroxidase. Values are mean OD (n = 3) ± S.D. at a wavelength of 450 nm (with a 630 nm correction). Representative data from three independent experiments are shown. e NFκB-luc2/GITR-expressing Jurkat cells were incubated with the indicated concentrations of HERA-GITRL or trimeric GITRL at 37 °C. After six hours, luminescence was measured and the fold induction in luciferase activity was calculated in order to compare multiple experiments. Data are shown as mean values (± S.D.) from three independent experiments. A two-way ANOVA plus post hoc Bonferroni multiple comparisons analysis was conducted to compare the effects of treatment and concentration on luminescence. ****p < 0.0001