Development of Nanobodies Against Hemorrhagic and Myotoxic Components of Bothrops atrox Snake Venom.

Snake envenoming is a globally neglected public health problem. Antivenoms produced using animal hyperimmune plasma remain the standard therapy for snakebites. Although effective against systemic effects, conventional antivenoms have limited efficacy against local tissue damage. In addition, potential hypersensitivity reactions, high costs for animal maintenance, and difficulties in obtaining batch-to-batch homogeneity are some of the factors that have motivated the search for innovative and improved therapeutic products against such envenoming. In this study, we have developed a set of nanobodies (recombinant single-domain antigen-binding fragments from camelid heavy chain-only antibodies) against Bothrops atrox snake venom hemorrhagic and myotoxic components. An immune library was constructed after immunizing a Lama glama with whole venom of B. atrox, from which nanobodies were selected by phage display using partially purified hemorrhagic and myotoxic proteins. Biopanning selections retrieved 18 and eight different nanobodies against the hemorrhagic and the myotoxic proteins, respectively. In vivo assays in mice showed that five nanobodies inhibited the hemorrhagic activity of the proteins; three neutralized the hemorrhagic activity of whole B. atrox venom, while four nanobodies inhibited the myotoxic protein. A mixture of the anti-hemorrhagic and anti-myotoxic nanobodies neutralized the local tissue hemorrhage and myonecrosis induced by the whole venom, although the nanobody mixture failed to prevent the venom lethality. Nevertheless, our results demonstrate the efficacy and usefulness of these nanobodies to neutralize important pathologies of the venom, highlighting their potential as innovative therapeutic agents against envenoming by B. atrox, a viperid species causing many casualties in South America.

Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition.

Monoclonal antibodies are playing an increasing role in both human and animal health. Different strategies of protein and chemical engineering, including humanization techniques of non-human antibodies were applied successfully to optimize clinical performances of antibodies. Despite the emergence of techniques allowing the development of fully human antibodies such as transgenic Xeno-mice, antibody humanization remains a standard procedure for therapeutic antibodies. An important prerequisite for antibody humanization requires standardized numbering methods to define precisely complementary determining regions (CDR), frameworks and residues from the light and heavy chains that affect the binding affinity and/or specificity of the antibody-antigen interaction. The recently generated deep-sequencing data and the increasing number of solved three-dimensional structures of antibodies from human and non-human origins have led to the emergence of numerous databases. However, these different databases use different numbering conventions and CDR definitions. In addition, the large fluctuation of the variable chain lengths, especially in CDR3 of heavy chains (CDRH3), hardly complicates the comparison and analysis of antibody sequences and the identification of the antigen binding residues. This review compares and discusses the different numbering schemes and "CDR" definition that were established up to date. Furthermore, it summarizes concepts and strategies used for numbering residues of antibodies and CDR residues identification. Finally, it discusses the importance of specific sets of residues in the binding affinity and/or specificity of immunoglobulins.

Distinct antibody species: structural differences creating therapeutic opportunities.

Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies.

Single-domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge.

UNLABELLED: Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity. IMPORTANCE: Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses. Here we show that single-domain antibody fragments that are specific for NA can bind and inhibit H5N1 viruses in vitro and can protect laboratory mice against a challenge with an H5N1 virus, including an oseltamivir-resistant virus. In addition, plant-produced VHH fused to a conventional Fc domain can protect in vivo even in the absence of NA-inhibitory activity. Thus, NA of influenza virus can be effectively targeted by single-domain antibody fragments, which are amenable to further engineering.

Development of 177Lu-nanobodies for radioimmunotherapy of HER2-positive breast cancer: evaluation of different bifunctional chelators.

Nanobodies show favourable pharmacokinetic characteristics for tumor targeting, including high tumor-to-background-ratios. Labelled with a therapeutic radionuclide, nanobodies could be used as an adjuvant treatment option for HER2-overexpressing minimal residual disease. The therapeutic radionuclide Lutetium-177 is linked to the nanobody using a bifunctional chelator. The choice of the bifunctional chelator could affect the in vivo behaviour of the radiolabeled nanobody. Consequently, we compared four different bifunctional chelators - p-SCN-Bn-DOTA, DOTA-NHS-ester, CHX-A"-DTPA or 1B4M-DTPA - in order to select the optimal chemical link between Lutetium-177 and a HER2 targeting nanobody. MS results revealed different degrees of chelator-conjugation. High stability in time was observed, together with nanomolar affinities on HER2-expressing tumor cells. Ex vivo biodistributions as well as SPECT/micro-CT analyses showed high activities in tumors expressing medium HER2 levels with low background activity except for the kidneys. The 1B4M-DTPA-coupled conjugate was further evaluated in a high HER2-expressing tumor model. Here, tumor uptake values of 5.99 ± 0.63, 5.12 ± 0.17, 2.83 ± 0.36 and 2.47 ± 0.38 %IA/g were obtained at 1, 3, 24 and 48h p.i., which coincided with exceptionally low background values, except for the kidneys, and unprecedented tumor-to-background ratios. No specific binding was observed in a HER2-negative model. In conclusion, the in-house developed anti-HER2 nanobody 2Rs15dHIS can be successfully labeled with (177) Lu using different bifunctional chelators. Both macrocyclic and acyclic chelators show high stability in time. High specific tumor uptake combined with the lowest background uptake was measured using the 1B4M-DTPA-based conjugate.

Immunological aspects of scorpion toxins: current status and perspectives.

Significant progress has been made in immunological studies of scorpion toxins and several formats of antibodies directed against scorpion toxins have been reported. Some of these are commonly used in a specific treatment against envenoming; others are primarily used for immuno-biochemical characterizations. The preparation protocol of the antibody or its fragments can be substantially different from one laboratory to another, which complicates a direct comparison of the potency of the antivenom. The use of immune sera, the total immunoglobulin fraction or Fab and Fab'2 fragments as the therapeutic agent is widespread. A number of monoclonal antibodies have also been reported and used for engineering of Fv, ScFv or Fab fragments. Recently, a novel antibody format - known as nanobodies - derived from HCAbs of camelids and selected after phage display shows great potential to provide a more efficient therapy against scorpion envenoming. Subsequent bispecific derivatives have been designed and their pharmacokinetics have been studied. Distinct advantages and disadvantages have been attributed to these equine, murine or camelid antibodies and their derived fragments. Some fragments are easily amenable into next generation therapeutics after proper manufacturing and provide an ensured availability of the product to the medical community. Through examples, we will show how the comparison of the serotherapeutic effectiveness is compromised due to the absence of standardization, on the preparation of immunogens, production processes and / or nature of the products. We will report on recent advances in the field.

Specific cell targeting with nanobody conjugated branched gold nanoparticles for photothermal therapy.

Branched gold nanoparticles are potential photothermal therapy agents because of their large absorption cross section in the near-infrared window. Upon laser irradiation they produce enough heat to destroy tumor cells. In this work, branched gold nanoparticles are biofunctionalized with nanobodies, the smallest fully functional antigen-binding fragments evolved from the variable domain, the VHH, of a camel heavy chain-only antibody. These nanobodies bind to the HER2 antigen which is highly expressed on breast and ovarian cancer cells. Flow cytometric analysis and dark field images of HER2 positive SKOV3 cells incubated with anti-HER2 conjugated branched gold nanoparticles show specific cell targeting. Laser irradiation studies reveal that HER2 positive SKOV3 cells exposed to the anti-HER2 targeted branched gold nanoparticles are destroyed after five minutes of laser treatment at 38 W/cm(2) using a 690 nm continuous wave laser. Starting from a nanoparticle optical density of 4, cell death is observed, whereas the control samples, nanoparticles with anti-PSA nanobodies, nanoparticles only, and laser only, do not show any cell death. These results suggest that this new type of bioconjugated branched gold nanoparticles are effective antigen-targeted photothermal therapeutic agents for cancer treatment.