Abstract
The activity of tumor necrosis factor (TNF), a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™) were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3) were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.
Highlights
Tumor necrosis factor (TNF) is a pleiotropic cytokine with beneficial functions in immune regulation and host defense, but deleterious pro-inflammatory and cytotoxic functions during inflammation
For isolation of NanobodiesTM that act as antagonists of tumor necrosis factor (TNF), two llamas were immunized with human TNF, and phage display libraries were generated using RNA derived from peripheral blood lymphocytes
The action of TNF can be inhibited by antibodies or trap molecules targeting the cytokine [32]
Summary
Tumor necrosis factor (TNF) is a pleiotropic cytokine with beneficial functions in immune regulation and host defense, but deleterious pro-inflammatory and cytotoxic functions during inflammation. TNF signaling is mediated by binding to two cellsurface receptors: TNF receptor type 1 (TNFR1 or p55), expressed in most tissues, or TNF receptor type 2 (TNFR2 or p75), which is inducible and typically found in cells of the immune system [2]. TNFR1 induces pro-inflammatory cascades and apoptosis, while TNFR2 has a role in cell survival, proliferation, and immune regulation [3, 4]. The structure of the related lymphotoxin α (LTα, previously called TNFβ) in complex with TNFR1 has shown that the trimeric cytokine binds three receptor molecules in a symmetrical way [5]. TNF has a similar structure as LTα and both compete for binding to the same receptors. It was concluded that the structural insights provided by the analysis of the complex of LTα with p55 should apply for TNF
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