Michael John Scott Saunders

Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5. The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve “best-in-class” and broader neutralization capacity.

Neutralizing Nanobodies Targeting Diverse Chemokines Effectively Inhibit Chemokine Function

Background: Chemokines play a prominent role in inflammatory diseases. Results: Nanobodies targeting chemokines display high affinity and potently neutralize chemokine-induced receptor binding and signaling. Conclusion: Neutralizing Nanobodies targeting chemokines effectively inhibit chemokine function. Significance: Nanobodies directed against inflammatory and homeostatic chemokines form a promising new class of potent and specific inhibitors of chemokine function, to be used for research and therapeutic purposes. Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.

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.

Generation of Immune Globulin Single Variable Domains by Display Technologies

Rabies virus-specific, cross-neutralizing, llama-derived heavy-chain antibody fragments (VHH) can be generated rapidly from immune llamas using phage display technology. The selection procedure can be tailor-made depending on the epitope to be targeted, whereas the single-chain nature of the VHH allows for facile genetic fusion of different VHH to create multivalent VHH. These VHH can target one or several different epitopes, thereby increasing the chance of neutralization of different strains of virus.

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. Anti-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. Contrary 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. Our 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.