title: Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format.
abstract: Co-stimulation 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 co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory 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 co-stimulatory 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 co-stimulatory 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 co-stimulatory activity. Hence, this format exhibits favorable properties that make it a promising choice for further therapeutic fusion protein development.
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart , Stuttgart, Germany.
authors.forename: Sina
authors.initials: S
authors.lastname: Fellermeier
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart , Stuttgart, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Beha
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart , Stuttgart, Germany.
authors.forename: Jan-Erik
authors.initials: JE
authors.lastname: Meyer
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart , Stuttgart, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Ring
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart , Stuttgart, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bader
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart , Stuttgart, Germany.
authors.forename: Roland E
authors.initials: RE
authors.lastname: Kontermann
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart , Stuttgart, Germany.
authors.forename: Dafne
authors.initials: D
authors.lastname: Müller
keywords: Antibody-fusion proteins; TNFSF ligands; cancer immunotherapy; co-stimulation