TNFRSF receptor-specific antibody fusion proteins with targeting controlled Fc\u03b3R-independent agonistic activity

Juliane Medler,Thomas Hünig,Moritz Rothaug,Andreas Beilhack,Johannes Nelke,Tim Steinfatt,Harald Wajant,Daniela Weisenberger,Susanne Berr

doi:10.1038/s41419-019-1456-x

Juliane Medler, Thomas Hünig + Show 7 more

Open Access

https://doi.org/10.1038/s41419-019-1456-x

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Abstract

Antibodies specific for TNFRSF receptors that bind soluble ligands without getting properly activated generally act as strong agonists upon FcγR binding. Systematic analyses revealed that the FcγR dependency of such antibodies to act as potent agonists is largely independent from isotype, FcγR type, and of the epitope recognized. This suggests that the sole cellular attachment, achieved by Fc domain-FcγR interaction, dominantly determines the agonistic activity of antibodies recognizing TNFRSF receptors poorly responsive to soluble ligands. In accordance with this hypothesis, we demonstrated that antibody fusion proteins harboring domains allowing FcγR-independent cell surface anchoring also act as strong agonist provided they have access to their target. This finding defines a general possibility to generate anti-TNFRSF receptor antibodies with FcγR-independent agonism. Moreover, anti-TNFRSF receptor antibody fusion proteins with an anchoring domain promise superior applicability to conventional systemically active agonists when an anchoring target with localized disease associated expression can be addressed.

Highlights

Receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are naturally activated by ligands of the TNF superfamily (TNFSF)[1,2]
We analyzed at first a panel of ~30 murine antibodies recognizing more than 10 members of the TNFRSF in coculture assays of TNFRSF receptorresponsive target cells and empty vector (EV) or murine
Since we were interested in the potential agonistic activity of the antibodies, we excluded, antibodies recognizing the intracellular part of TNFRSF receptors or antibodies, which do not work in ELISA or FACS applications and which are obviously not able to recognize the native receptor molecules

Summary

Introduction

Receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are naturally activated by ligands of the TNF superfamily (TNFSF)[1,2]. TNFSF ligands, with exception of LTα, are type II transmembrane proteins and are characterized by a C-terminal TNF homology domain (THD), which promotes assembly of homotrimeric molecules and binding to TNFRSF receptors[1,2]. Soluble TNFSF ligands occur naturally due to proteolytic processing in the stalk region separating the THD from the transmembrane domain but can be generated by recombinant DNA technology[1,3]. In the case of trimeric receptor complexes formed in response to soluble ligand binding, signalingpromoting secondary interaction may largely depend on a receptor type-specific intrinsic oligomerization capacity. The poor response of TNFRSF receptors to soluble ligand trimers can be overcome by oligomerization (e.g., via antibody cross-linking or genetic fusion with oligomerization domains) or cell surface anchoring of the soluble ligand molecules[3,4]

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