A simple and broadly applicable nanobody-based approach to generate potent TNFR agonists.

The receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are of overwhelming scientific and clinical relevance and stand at the center of intensive basic and translational research efforts. TNFRSF receptors (TNFRs) are engaged by membrane-bound ligands of the TNF superfamily (TNFSF) and, in some cases, by soluble ligand molecules released from the membrane-bound TNFSF ligand (TNFL) molecules. The development of recombinant TNFL-based TNFR agonists for research and especially therapeutic purposes is highly "individualized", as ligand type-specific hurdles must be overcome in terms of stability, manufacturability, TNFR-specificity and need for oligomerization. TNFR-specific antibodies can also show agonistic activity, but this agonism typically requires FcγR-binding, resulting in a reciprocal conditional bispecific FcγR/TNFR agonism not useful for the study or exploitation of pure TNFR agonism. Some antibodies trigger intrinsic TNFR agonism independent from FcγR-binding, but the rational development of such antibodies is poorly predictable and furthermore challenging due to isotype- and epitope-requirements and poor specific activity when benchmarked with FcγR-bound anti-TNFR antibodies.Using a series of nanobodies (or single-domain antibodies (sdAbs) or variable heavy domain of heavy chains (VHHs)) specific for the TNFRSF members 41BB, BCMA, CD40, CD95, TRAILR2/DR5, GITR, OX40, TNFR1 and TNFR2, we show here that genetic fusion of single-chain encoded triplets of these nanobodies with oligomerizing protein scaffolds regularly results in potent hexa-, nona- and dodecavalent agonists inducing TNFR signaling with EC50-values in the sub-nanomolar range. The oligovalent nanobody formats described exhibit superior CMC properties and enable the simple generation of highly active TNFR agonists from virtually any TNFR-specific nanobody.