Antibody RING-Mediated Destruction of Endogenous Proteins.

To understand gene function, the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. We therefore developed a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A construct combining the RING domain of ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates target destruction by the ubiquitin proteasome system, a process we describe as antibody RING-mediated destruction (ARMeD). The technique is highly specific because we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With increasing availability of protein-specific nanobodies, this method will allow rapid and specific degradation of a wide range of endogenous proteins.

Regulation of cancer-related pathways by protein NEDDylation and strategies for the use of NEDD8 inhibitors in the clinic.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) controls a vast if not every biological process in the cell. It is not surprising that deregulation in ubiquitin and UBL signalling has been implicated in the pathogenesis of many diseases and that these pathways are considered as major targets for therapeutic intervention. In this review, we summarise recent advances in our understanding of the role of the UBL neural precursor cell expressed developmentally downregulated-8 (NEDD8) in cancer-related processes and potential strategies for the use of NEDD8 inhibitors as chemotherapeutics.

Development of Cys38 knock-out and humanized version of NbAahII10 nanobody with improved neutralization of AahII scorpion toxin.

During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim, causing serious medical problems. Nanobodies (Nbs), the recombinant single-domain antigen-binding fragments of camel-specific heavy-chain only antibodies, offer special advantages in therapy over classic antibody fragments due to their robustness and smaller size, matching the size of the scorpion toxins. Recently, a potent AahII scorpion toxin-neutralizing Nb was identified. However, this NbAahII10 contains a single Cys in its first antigen-binding loop, leading to Nb dimerization upon prolonged storage. In this work, we first investigate the efficacy of NbAahII10 variants in which this Cys was substituted by Ala, Ser or Thr. Second, the NbAahII10 Cys/Ser mutant displaying the best functional properties is subsequently humanized. It is demonstrated that the maximally humanized version of NbAahII10 Cys/Ser maintains its high affinity for the antigen without conceding much on expression yield and stability. More importantly, its neutralizing capacity is preserved as all mice survive injections of seven LD(50) and 50% of mice survived nine LD(50) of the scorpion toxin. Thus, this humanized Nb is the best candidate to develop a therapy in human against the most toxic venom compound of one of the most dangerous scorpions.

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.

A bispecific nanobody to provide full protection against lethal scorpion envenoming.

Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was to ameliorate the current 100-kDa horse plasma antivenom serum (PAS)-derived Fab'(2) to more quickly reach the highly diffusible scorpion toxins (7 kDa). We immunized dromedaries with toxins from Androctonus australis hector (Aah) scorpions and cloned the single-domain antibody fragments or nanobodies (15 kDa) from their B cells. Nanobodies against AahI' toxin (with AahII the most toxic compound of the venom) were retrieved from the libraries, and their AahI'-toxin neutralization was monitored in mice. Remarkably, the NbAahI'F12 fully protected mice against 100 LD(50) of AahI' administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD(50) of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI'/AahII toxins succeeded in neutralizing 5 LD(50). Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 microg of NbF12-10 protected all mice, even if the whole procedure was repeated 3 times. Furthermore, the NbF12-10 remained fully protective when mice with severe signs of envenoming were treated a few minutes before the untreated mice died.