Hans De Haard

Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies.

The development of a number of different solid tumours is associated with over-expression of ErbB1, or the epidermal growth factor receptor (EGFR), and this over-expression is often correlated with poor prognosis of patients. Therefore, this receptor tyrosine kinase is considered to be an attractive target for antibody-based therapy. Indeed, antibodies to the EGFR have already proven their value for the treatment of several solid tumours, especially in combination with chemotherapeutic treatment regimens. Variable domains of camelid heavy chain-only antibodies (called Nanobodies™) have superior properties compared with classical antibodies in that they are small, very stable, easy to produce in large quantities and easy to re-format into multi-valent or multi-specific proteins. Furthermore, they can specifically be selected for a desired function by phage antibody display. In this report, we describe the successful selection and the characterisation of antagonistic anti-EGFR Nanobodies. By using a functional selection strategy, Nanobodies that specifically competed for EGF binding to the EGFR were isolated from ‘immune’ phage Nanobody repertoires. The selected antibody fragments were found to efficiently inhibit EGF binding to the EGFR without acting as receptor agonists themselves. In addition, they blocked EGF-mediated signalling and EGF-induced cell proliferation. In an in vivo murine xenograft model, the Nanobodies were effective in delaying the outgrowth of A431-derived solid tumours. This is the first report describing the successful use of untagged Nanobodies for the in vivo treatment of solid tumours. The results show that functional phage antibody selection, coupled to the rational design of Nanobodies, permits the rapid development of novel anti-cancer antibody-based therapeutics.

Lactobacillli expressing llama VHH fragments neutralise Lactococcus phages

BackgroundBacteriophages infecting lactic acid bacteria (LAB) are widely acknowledged as the main cause of milk fermentation failures. In this study, we describe the surface-expression as well as the secretion of two functional llama heavy-chain antibody fragments, one binding to the major capsid protein (MCP) and the other to the receptor-binding proteins (RBP) of the lactococcal bacteriophage p2, by lactobacilli in order to neutralise lactococcal phages.ResultsThe antibody fragment VHH5 that is directed against the RBP, was fused to a c-myc tag and expressed in a secreted form by a Lactobacillus strain. The fragment VHH2 that is binding to the MCP, was fused to an E-tag and anchored on the surface of the lactobacilli. Surface expression of VHH2 was confirmed by flow cytometry using an anti-E-tag antibody. Efficient binding of both the VHH2 and the secreted VHH5 fragment to the phage antigens was shown in ELISA. Scanning electron microscopy showed that lactobacilli expressing VHH2 anchored at their surface were able to bind lactococcal phages. A neutralisation assay also confirmed that the secreted VHH5 and the anchored VHH2 fragments prevented the adsorption of lactococcal phages to their host cells.ConclusionLactobacilli were able to express functional VHH fragments in both a secreted and a cell surface form and reduced phage infection of lactococcal cells. Lactobacilli expressing llama heavy-chain antibody fragments represent a novel way to limit phage infection.

Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine–Nanobody Complex

The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (VHH) domains (Nanobodies®) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo, as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohns disease, rheumatoid arthritis, and psoriasis.

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

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.

VHH selected against the viral spike protein can protect mice against lethal rabies virus challenge

VHH are polypeptides (15 kDa) derived from the variable domain of single heavy-chain antibodies of Camelidae. They represent the smallest antigen-binding fragment of an antibody (human IgG = 150 kDA). VHH are currently being explored for a number of applications. n nAnti-rabies virus VHH were cloned from lymphocytes of vaccinated llamas and selected for their affinity with the viral spike glycoprotein G and neutralizing potency in a cellular infection assay. Linkage of two VHH allowed recognition of two identical or different epitopes and increased the neutralizing potency in cells more than a hundred-fold. Next, we examined the protection efficiency of these bimeric VHH in vivo. n nContrary to irrelevant control VHH, pre-incubation of the virus with anti-G VHH fully protected mice against disease and mortality upon inoculation of the virus-VHH mix in the nose, muscle or brain. Preventive administration of anti-G VHH in the nose, 24 hours prior to intranasal virus challenge, also almost completely prevented disease and lethal infection. This suggests that VHH remain sufficiently active for at least 24 hours at the site of administration in the nose to neutralize a significant part of the invading virus. Post exposure prophylaxis (PEP) by injection of anti-G VHH in the left quadriceps muscle 10 minutes after virus challenge in the right quadriceps muscle reduced mortality by 50%. Treatment 24 hours after virus challenge was however no longer effective, most likely because the virus had already reached the central nervous system and was no longer exposed to locally administered VHH. n nOur results show that anti-G VHH can neutralize rabies virus in an Fc-independent way. VHH probably hinder the recognition of cellular receptors or interfere with the fusion of viral and cellular membranes. The bimeric constructs proved protective in different challenge models, but that protection in the PEP model was weak. This might be due to the short half-life of the used VHH. Considering that prolongation of the half-life of VHH is feasible by different approaches, VHH technology may offer perspectives as an alternative to antibodies for PEP. VHH have a low production cost, not contaminated by blood-borne pathogenic agents and are less likely to evoke allergic or immunopathological reactions. They have good thermal stability, which is an advantage in developing countries, where the cold chain for distribution and preservation can not always be guaranteed.

P4-245: Nanobodies™ targeting amyloid beta as potential therapeutics for Alzheimer’s disease

discovery of a GSI possessing minimal inhibition of Notch processing and maximal inhibition of APP processing to reduce 40/42 levels. Utilizing HTS screening of the Wyeth and ArQule compound collections, we have identified the lead, 4-chloro-N-[(1S,2S)-1-(hydroxymethyl)-2-methylbutyl]-benzenesulfonamide, which not only inhibited -secretase cleavage of APP (EC50 40 and 42 2078 nM and 1938 nM, respectively) but also was sparing of Notch cleavage (EC50 20,000 nM). Structure-activity relationships in this new series and the discovery of more potent GSIs will be discussed.