Fig. 1.
a Modular schema of the bispecific antibody and antibody–ligand fusion proteins. V H/L variable region of the heavy/light antibody chain, FAP fibroblast activation protein, EDG endoglin, scDb single-chain diabody, Db diabody, ECD extracellular domain, H Histidine tag, TNF tenascin, L n linker of n amino acids. Schema of the combinatorial settings: b targeting scDb/B7-Db to FAP and the scFv-ligand to endoglin; c Targeting scDb to FAP and scFv-4-1BBL plus a second scFv-ligand to EDG. CR costimulatory receptor. d SDS-PAGE analysis of the scFv–ligand fusion proteins (3 μg/lane) under reducing (R) and non-reducing (NR) conditions on a 12 % SDS-PAGE and 4–12 % Bis–Tris Gel, respectively; Coomassie staining. e HPLC analysis on a TSK-GEL G3000SWXL column (Tosoh Bioscience) of scFv-OX40L (2 μg), scFv-LIGHT (15 μg) and scFv-TNC-GITRL (15 μg). Mobile phase was PBS at a flow rate of 0.5 ml/min