Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions

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.

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins

“…Specifically, we first investigated the binding epitopes and neutralizing mechanisms for a selected group of VHHs. Based on these structural and functional information, we further enhanced the potency of these VHHs by rational design of heterodimeric VNAs, which use custom-designed peptide linkers to achieve simultaneous binding of two parental VHHs that have non-overlapping yet closely apposed epitopes (Beirnaert et al, 2017;Desmyter et al, 2017).…”

A New Venue of TNF Targeting

“…As this drug has a different mode of binding, i.e. it can bind a single trimeric TNF and block two of the three receptor binding sites of TNF, it distinguishes itself from other TNF neutralizing drugs [257]. The ease of cloning and production allows Nbs to be locally secreted by the genetically modified probiotic indications such as sepsis, RA and MS.…”

AAV mediated delivery of a novel anti-BACE1 VHH reduces Abeta in an Alzheimer’s disease mouse model

“…However, if required, modifications to the system can easily be made by taking advantage of the relative ease with which VHH and AAV can be engineered. For example, VHH affinities in the low picomolar range can be achieved using standard affinity maturation techniques (Mahajan et al, 2018), or via production of bivalent VHH constructs (Beirnaert et al, 2017) that bind distinct epitopes on a given target. Expression levels of a given VHH from a vector-based system can be improved by use of a self-complementary genome configuration, containing multiple copies of the VHH coding sequence (Verhelle et al, 2017).…”