Fig. 2.

Functional properties of scFv-OX40L, scFv-LIGHT and scFv-TNC-GITRL. a Antibody binding by flow cytometry. HT1080-FAP (EDG⁺) and HEK293 (EDG⁻) cells were incubated with 300 nM scFv-ligand. Bound construct were detected by an anti-hexahistidyl-tag-PE conjugated antibody. Gray filled: cells only; gray line: detection antibody only; black line: scFv-ligand followed by detection antibody. b Ligand activity of the fusion proteins in coated and soluble form. scDbFAPCD3 was coated and incubated with CFSE-labled PBMC together with scFv-ligand (50nM) presented either coated or in solution. Proliferation was determined after 4 days by flow cytometry. [n = 3. Mean ± SD, One-way ANOVA, Tukey post test, *p < 0.05, **p < 0.01, ***p < 0.0001]. c Costimulatory properties of the fusion proteins in a cellular assay. Target cells (HT1080-FAP) were incubated with scDb in combination with the scFv-ligands for 1 h before the addition of CFSE-labeled PBMC. Proliferation of PBMC was measured after 4 days by flow cytometry