Generation and validation of therapeutically relevant, novel TRAIL-fusion proteins
In cancer drug development programs immuno therapy concepts based on pro-apoptotic as well as on immuno-regulatory cytokines of the Tumor Necrosos factor (TNF) family are considered as being very promising, in particular in combination with established (chemo-) therapeutics. As for pro-apoptotic ligands, TRAIL had raised considerable hopes due its potentially tumor selective action, but the first generation TRAIL reagent showed little clinical efficacy. Basic research on the mechanism of ligand–receptor interaction and signal initiation has meanwhile provided a rational basis for construction of highly active and stable ligands with potential of clinical use. The basic principle rests on a conversion of a rather labile, homotrimeric assembly of a soluble fragment of the naturally occuring membrane TRAIL into a genetically engineered single stranded version, named scTRAIL, comprising the three TRAIL monomers covalently linked by short peptides. The subproject here exploits this principle, which allows the construction of bi- or multifunctional molecules by simples genetic fusion to other modular protein components, e. g. allowing targeting to tumor markers or generation of oligomers to increase activity. The subproject focusses on scTRAIL molecules in a dimeric, i.e. hexavalent format, which has been shown by the PIs to be the minimum essential structure of a bioactive TRAIL. An important aspect is to increase plasma half-life of the product, which can be achieved by fusion to Fc parts of immunoglobulin, responsible for the long plasma half life of antibodies. The Fc part also serves as the covalent dimerization module, constituting e.g. an scTRAIL-Fc molecule. Prototypes of this reagent are already available and will be distributed to partners for use in the different melanoma treatment models.
Based on this prototype, optimization of format with respect to bioactivity, stability as well as pharmcokinetic properties will be performed by genetic engineering. Analyses include detailed biochemical/biophysical properties, in vitro apoptosis assays, and PK studies. Together with partners, analyses on tissue penetration of the various scTRAIl formats will be performed by immunohistochemistry of scTRAIL treated 3D-cultures and of ex vivo melanoma biopsies. It is the overall aim of this subproject to identify a hexavalent scTRAIL molecule that serves as a lead candidate for clinical development together with a partner from biotech or pharmaceutic industry.