Advancing targeted costimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format
Sina Fellermeier, Nadine Beha, Jan-Erik Meyer, Sarah Ring, Stefan Bader, Roland E. Kontermann & Dafne Müller
Costimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a costimulatory ligand of the TNFSF constitute an attractive option to focus the costimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging. Aiming for molecules of simple and stable configuration, we used TNFSF ligands in a single-chain format (scTNFSF), i.e three units of the ectodomain connected by polypeptide linkers, folding into an intramolecular trimer. By fusing tumor-directed scFv antibody fragments directed against EpCAM or FAP to costimulatory scTNFSF molecules (sc4-1BBL, scOX40L, scGITRL or scLIGHT) a set of monomeric scFv-scTNFSF fusion proteins was generated. In comparison to the scFv-TNFSF format, defined by intermolecular homotrimerization via the TNFSF part, scFv-scTNFSF showed equal or enhanced costimulatory activity despite reduced avidity in antibody binding. In addition, enhanced serum stability and improved bioavailability in mice were observed. We show that the scFv-scTNFSF format can be applied to various members of the TNFSF, presenting targeting-dependent costimulatory activity. Hence, this format exhibits favorable properties that make it a promising choice for further therapeutic fusion protein development.