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Twist1 promote the bypass of p53 response by interacting with p53 and facilitating its MDM2-mediated degradation. We reasoned that reagents able to interfere with the p53:Twist1 complex might alleviate Twist1 inhibitory effect over p53, thus representing potential therapeutic tools in p53 wild type tumors. From a pre-immune library of llama nanobodies (VHH), we isolated binders targeting the p53 C-terminal region (p53-CTD) involved in the interaction with Twist1 by using recombinant Twist1 as an epitope-specific competitor during elution. Positive hits were validated by proving their capacity to immunoprecipitate p53 and to inhibit Twist1:p53 binding in vitro. Molecular modeling confirmed a preferential docking of positive hits with p53-CTD. D11 VHH activity was validated in human cell models, succeeded in immunoprecipitating endogenous p53 and, similarly to Twist1 knock-down, interfered with p53 turnover, p53 phosphorylation at Serine 392 and affected cell viability. Despite the limited functional effect determined by D11 expression in target cells, our results provide the proof of principle that nanobodies ectopically expressed within a cell, have the capacity to target the assembly of the pro-tumorigenic Twist1:p53 complex. These results disclose novel tools for dissecting p53 biology and lay down the grounds for the development of innovative targeted therapeutic approaches.

Precise spatiotemporal organization and regulation of signal transduction networks are essential for cellular response to internal and external cues. To understand how this biochemical activity architecture impacts cellular function, many genetically encodable tools which regulate kinase activity at a subcellular level have been developed. In this review, we highlight various types of genetically encodable molecular tools, including tools to regulate endogenous kinase activity and biorthogonal techniques to perturb kinase activity. Finally, we emphasize the use of these tools alongside biosensors for kinase activity to measure and perturb kinase activity in real time for a better understanding of the cellular biochemical activity architecture.

Parkinson’s disease is a neurodegenerative disorder mainly characterized by the degeneration of dopaminergic neurons in the substantia nigra. Degenerating neurons contain abnormal aggregates called Lewy bodies, that are predominantly composed of the misfolded and/or mutated alpha-synuclein protein. Post-translational modifications, cellular stress, inflammation and gene mutations are thought to trigger its pathological misfolding and aggregation. With alpha-synuclein pathology being strongly associated with dopaminergic neuronal toxicity, strategies aimed to reduce its burden are expected to be beneficial in slowing disease progression. Moreover, multiple sources of evidence suggest a cell-to-cell transmission of pathological alpha-synuclein in a prion-like manner. Therefore, antibodies targeting extra- or intracellular alpha-synuclein could be efficient in limiting the aggregation and transmission. Several active and passive immunization strategies have been explored to target alpha-synuclein. Here, we summarize immunotherapeutic approaches that were tested in pre-clinical or clinical studies in the last two decades in an attempt to treat Parkinson’s disease.

Recombinant antibodies such as nanobodies are progressively demonstrating to be a valid alternative to conventional monoclonal antibodies also for clinical applications. Furthermore, they do not solely represent a substitute for monoclonal antibodies but their unique features allow expanding the applications of biotherapeutics and changes the pattern of disease treatment. Nanobodies possess the double advantage of being small and simple to engineer. This combination has promoted extremely diversified approaches to design nanobody-based constructs suitable for particular applications. Both the format geometry possibilities and the functionalization strategies have been widely explored to provide macromolecules with better efficacy with respect to single nanobodies or their combination. Nanobody multimers and nanobody-derived reagents were developed to image and contrast several cancer diseases and have shown their effectiveness in animal models. Their capacity to block more independent signaling pathways simultaneously is considered a critical advantage to avoid tumor resistance, whereas the mass of these multimeric compounds still remains significantly smaller than that of an IgG, enabling deeper penetration in solid tumors. When applied to CAR-T cell therapy, nanobodies can effectively improve the specificity by targeting multiple epitopes and consequently reduce the side effects. This represents a great potential in treating malignant lymphomas, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma and solid tumors. Apart from cancer treatment, multispecific drugs and imaging reagents built with nanobody blocks have demonstrated their value also for detecting and tackling neurodegenerative, autoimmune, metabolic, and infectious diseases and as antidotes for toxins. In particular, multi-paratopic nanobody-based constructs have been developed recently as drugs for passive immunization against SARS-CoV-2 with the goal of impairing variant survival due to resistance to antibodies targeting single epitopes. Given the enormous research activity in the field, it can be expected that more and more multimeric nanobody molecules will undergo late clinical trials in the next future.Systematic Review Registration

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) ‘2a’ consisting of the full-length 2a gene (839 amino acids, aa), (ii) ‘Motifs’ covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) ‘GDD’ located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the ‘2a’ and ‘Motifs’ constructs interacted with 96 and 25 library constructs, respectively, while the ‘GDD’ construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.

The spatiotemporal organization of chromatin is regulated at different levels in the nucleus. Epigenetic modifications such as DNA methylation and histone modifications are involved in chromatin regulation and play fundamental roles in genome function. While the one-dimensional epigenomic landscape in many cell types has been revealed by chromatin immunoprecipitation and sequencing, the dynamic changes of chromatin modifications and their relevance to chromatin organization and genome function remain elusive. Live-cell probes to visualize chromatin and its modifications have become powerful tools to monitor dynamic chromatin regulation. Bulk chromatin can be visualized by both small fluorescent dyes and fluorescent proteins, and specific endogenous genomic loci have been detected by adapting genome-editing tools. To track chromatin modifications in living cells, various types of probes have been developed. Protein domains that bind weakly to specific modifications, such as chromodomains for histone methylation, can be repeated to create a tighter binding probe that can then be tagged with a fluorescent protein. It has also been demonstrated that antigen-binding fragments and single-chain variable fragments from modification-specific antibodies can serve as binding probes without disturbing cell division, development and differentiation. These modification-binding modules are used in modification sensors based on fluorescence/Förster resonance energy transfer to measure the intramolecular conformational changes triggered by modifications. Other probes can be created using a bivalent binding system, such as fluorescence complementation or luciferase chemiluminescence. Live-cell chromatin modification imaging using these probes will address dynamic chromatin regulation and will be useful for assaying and screening effective epigenome drugs in cells and organisms.

Nanobodies (nAbs) are recombinant antigen-binding variable domain fragments obtained from heavy-chain-only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well-established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Total RNA isolation from camelid leukocytes Basic Protocol 2: First-strand cDNA synthesis; VH H and VH repertoire PCR Basic Protocol 3: Preparation of the phage display library Basic Protocol 4: Panning of the phage display library Basic Protocol 5: Small-scale nAb expression Basic Protocol 6: Sequence analysis of selected nAb clones Basic Protocol 7: Nanobody validation as immunolabels Basic Protocol 8: Generation of nAb-pEGFP mammalian expression constructs Basic Protocol 9: Nanobody validation as intrabodies Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones Support Protocol 2: Amplification of helper phage stock Support Protocol 3: nAb expression in amber suppressor E. coli bacterial strains.

Neurodegenerative diseases are commonly generated by intracellular accumulation of misfolded/aggregated mutated proteins. These abnormal protein aggregates impair the functions of mitochondria and induce oxidative stress, thereby resulting in neuronal cell death. In turn, neuronal damage induces chronic inflammation and neurodegeneration. Thus, reducing/eliminating these abnormal protein aggregates is a priority for any anti-neurodegenerative therapeutic approach. Although several antibodies against mutated neuronal proteins have been already developed, how to efficiently deliver them inside the target cells remains an unmet issue. Extracellular vesicles/exosomes incorporating intrabodies against the pathogenic products would be a tool for innovative therapeutic approaches. In this review/perspective article, we identify and describe the major molecular targets associated with neurodegenerative diseases, as well as the antibodies already developed against them. Finally, we propose a novel targeting strategy based on the endogenous engineering of extracellular vesicles/exosomes constitutively released by cells of the central nervous system.

Beta-arrestins (βarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr–clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications. A synthetic antibody fragment can disrupt the interaction of a GPCR regulatory protein β-arrestin with clathrin, and thereby inhibit induced internalization of GPCRs.

Conformational or misfolding diseases are a large class of devastating human disorders associated with protein misfolding and aggregation. Most conformational diseases are caused by a combination of genetic and environmental factors, suggesting that spontaneous events can destabilize the protein involved in the pathology or impair the clearance mechanisms of misfolded aggregates. Aging is one of the risk factors associa-ted to these events, and the clinical relevance of conformational disorders is growing dramatically, as they begin to reach epidemic proportions due to increases in mean lifespan. Currently, there are no effective strategies to slow or prevent these diseases. Intrabodies are promising therapeutic agents for the treatment of misfolding diseases, because of their virtually infinite ability to specifically recognize the different conformations of a protein, including pathological isoforms, and because they can be targeted to the potential sites of aggregation (both intra- and extracellular sites). These molecules can work as neutralizing agents against amylo-idogenic proteins by preventing their aggregation, and/or as molecular shunters of intracellular traffic by re-routing the protein from its potential aggregation site. The fast-developing field of recombinant antibody technology provides intrabodies with enhanced binding specificity and stability, together with lower immunogenicity, for use in a clinical setting. This review provides an update on the applications of intrabodies in misfolding diseases, with particular emphasis on an evaluation of their multiple and feasible modes of action.

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPC) into the infectious scrapie isoform (PrPSc). We have recently demonstrated that anti-prion intrabodies targeted to the lumen of the endoplasmic reticulum provide a simple and effective means to inhibit the transport of PrPC to the cell surface. Here, we report that they completely block the traffic of mature full-length PrPC molecules, impair prion lysosomal degradation, and interfere with the early phase of scrapie formation. Since anti-prion intrabodies efficiently block PrPSc accumulation in vitro, we investigated whether they could also antagonize scrapie infectivity in vivo. We found that mice intracerebrally injected with KDEL-8H4-NGF-differentiated PC12 cells infected with scrapie neither develop scrapie clinical signs nor brain damage. Furthermore, no protease-resistant PrPSc is detectable in brains of inoculated animals. These results indicate that anti-prion intrabody strategy may be effective against prion infection.

Many attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the periplasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K(d)=15nM) to that of the cognate scFv(D1.3) fragment (K(d)=16nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm.

IMPACT: 1.868; International audience; Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single-chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti-PCNA scFvs were screened in vitro after being tagged with dimeric glutathione-S-transferase. Anti-PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error-prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C-terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide-conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody-based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright (c) 2014 John Wiley & Sons, Ltd.

International audience; Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.