vNARs as Neutralizing Intracellular Therapeutic Agents: Glioblastoma as a Target.

Glioblastoma is the most prevalent and fatal form of primary brain tumors. New targeted therapeutic strategies for this type of tumor are imperative given the dire prognosis for glioblastoma patients and the poor results of current multimodal therapy. Previously reported drawbacks of antibody-based therapeutics include the inability to translocate across the blood-brain barrier and reach intracellular targets due to their molecular weight. These disadvantages translate into poor target neutralization and cancer maintenance. Unlike conventional antibodies, vNARs can permeate tissues and recognize conformational or cryptic epitopes due to their stability, CDR3 amino acid sequence, and smaller molecular weight. Thus, vNARs represent a potential antibody format to use as intrabodies or soluble immunocarriers. This review comprehensively summarizes key intracellular pathways in glioblastoma cells that induce proliferation, progression, and cancer survival to determine a new potential targeted glioblastoma therapy based on previously reported vNARs. The results seek to support the next application of vNARs as single-domain antibody drug-conjugated therapies, which could overcome the disadvantages of conventional monoclonal antibodies and provide an innovative approach for glioblastoma treatment.

TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth.

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) remains a deadly cancer worldwide with a need for new therapeutic approaches. A dysregulation in the equilibrium between pro- and anti-inflammatory responses with a predominant immunosuppressive inflammatory reaction in advanced stage tumors seem to contribute to tumor growth and metastasis. The current therapies do not include strategies against pro-tumorigenic inflammation in cancer patients. We have shown that the upregulated cell surface expression of Toll-like Receptor (TLR) 2 and of TLR9 inside PDAC cells maintain chronic inflammatory responses, support chemotherapeutic resistance, and mediate tumor progression in human pancreatic cancer. We further demonstrated intracellular TLR2 and TLR9 targeting using specific intrabodies, which resulted in downregulated inflammatory signaling. In this study, we tested, for the first time, an intrabody-mediated TLR blockade in human TLR2- and TLR9-expressing pancreatic cancer cells for its effects on inflammatory signaling-mediated tumor growth. Newly designed anti-TLR2- and anti-TLR9-specific intrabodies inhibited PDAC growth. Co-expression analysis of the intrabodies and corresponding human TLRs showed efficient retention and accumulation of both intrabodies within the endoplasmic reticulum (ER), while co-immunoprecipitation studies indicated both intrabodies interacting with their cognate TLR antigen within the pancreatic cancer cells. Cancer cells with attenuated proliferation expressing accumulated TLR2 and TRL9 intrabodies demonstrated reduced STAT3 phosphorylation signaling, while apoptotic markers Caspases 3 and 8 were upregulated. To conclude, our results demonstrate the TLR2 and TLR9-specific intrabody-mediated signaling pathway inhibition of autoregulatory inflammation inside cancer cells and their proliferation, resulting in the suppression of pancreatic tumor cell growth. These findings underscore the potential of specific intrabody-mediated TLR inhibition in the ER relevant for tumor growth inhibition and open up a new therapeutic intervention strategy for the treatment of pancreatic cancer.

Emerging antibody-based therapies for Huntington's disease: current status and perspectives for future development.

INTRODUCTION: Being an inherited neurodegenerative disease with an identifiable genetic defect, Huntington's disease (HD) is a suitable candidate for early intervention, possibly even in the pre-symptomatic stage. Our recent advances in elucidating the pathogenesis of HD have revealed a series of novel potential therapeutic targets, among which immunotherapies are actively pursued in preclinical experiments. AREAS COVERED: This review focuses on the potential of antibody-based treatments targeting various epitopes (of mutant huntingtin as well as phosphorylated tau) that are currently evaluated in vitro and in animal experiments. The references used in this review were retrieved from the PubMed database, searching for immunotherapies in HD, and clinical trial registries were reviewed for molecules already evaluated in clinical trials. EXPERT OPINION: Antibody-based therapies have raised considerable interest in a series of neurodegenerative diseases characterized by deposition of aggregated of aberrantly folded proteins, HD included. Intrabodies and nanobodies can interact with mutant huntingtin inside the nervous cells. However, the conflicting results obtained with some of these intrabodies highlight the need for proper choice of epitopes and for developing animal models more closely mimicking human disease. Approval of these strategies will require a considerable financial and logistic effort on behalf of healthcare systems.

Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

Translation Dynamics of Single mRNAs in Live Cells.

The translation of messenger RNA (mRNA) into proteins represents the culmination of gene expression. Recent technological advances have revolutionized our ability to investigate this process with unprecedented precision, enabling the study of translation at the single-molecule level in real time within live cells. In this review, we provide an overview of single-mRNA translation reporters. We focus on the core technology, as well as the rapid development of complementary probes, tags, and accessories that enable the visualization and quantification of a wide array of translation dynamics. We then highlight notable studies that have utilized these reporters in model systems to address key biological questions. The high spatiotemporal resolution of these studies is shedding light on previously unseen phenomena, uncovering the full heterogeneity and complexity of translational regulation.

Anti-tau intrabodies: From anti-tau immunoglobulins to the development of functional scFv intrabodies.

Over the last decade, there has been a growing interest in intrabodies and their therapeutic potential. Intrabodies are antibody fragments that are expressed inside a cell to target intracellular antigens. In the context of intracellular protein misfolding and aggregation, such as tau pathology in Alzheimer's disease, intrabodies have become an interesting approach as there is the possibility to target early stages of aggregation. As such, we engineered three anti-tau monoclonal antibodies into single-chain variable fragments for cytoplasmic expression and activity: PT51, PT77, and hTau21. Due to the reducing environment of the cytoplasm, single-chain variable fragment (scFv) aggregation is commonly observed. Therefore, we also performed complementarity-determining region (CDR) grafting into three different stable frameworks to rescue solubility and intracellular binding. All three scFvs retained binding to tau after cytoplasmic expression in HEK293 cells, in at least one of the frameworks. Subsequently, we show their capacity to interfere with either mouse or mutant human tau aggregation in two different primary mouse neuron models and organotypic hippocampal slice cultures. Collectively, our work extends the current knowledge on intracellular tau targeting with intrabodies, providing three scFv intrabodies that can be used as immunological tools to target tau inside cells.

Targeting intracellular galectins for cancer treatment.

Although considerable attention has been paid to the role of extracellular galectins in modulating, positively or negatively, tumor growth and metastasis, we have witnessed a growing interest in the role of intracellular galectins in response to their environment. This is not surprising as many galectins preferentially exist in cytosolic and nuclear compartments, which is consistent with the fact that they are exported outside the cells via a yet undefined non-classical mechanism. This review summarizes our most recent knowledge of their intracellular functions in cancer cells and provides some directions for future strategies to inhibit their role in cancer progression.

Quantitative Fluorescence Analysis Reveals Dendrite-Specific Thalamocortical Plasticity in L5 Pyramidal Neurons during Learning.

High-throughput anatomic data can stimulate and constrain new hypotheses about how neural circuits change in response to experience. Here, we use fluorescence-based reagents for presynaptic and postsynaptic labeling to monitor changes in thalamocortical synapses onto different compartments of layer 5 (L5) pyramidal (Pyr) neurons in somatosensory (barrel) cortex from mixed-sex mice during whisker-dependent learning (Audette et al., 2019). Using axonal fills and molecular-genetic tags for synapse identification in fixed tissue from Rbp4-Cre transgenic mice, we found that thalamocortical synapses from the higher-order posterior medial thalamic nucleus showed rapid morphologic changes in both presynaptic and postsynaptic structures at the earliest stages of sensory association training. Detected increases in thalamocortical synaptic size were compartment specific, occurring selectively in the proximal dendrites onto L5 Pyr and not at inputs onto their apical tufts in L1. Both axonal and dendritic changes were transient, normalizing back to baseline as animals became expert in the task. Anatomical measurements were corroborated by electrophysiological recordings at different stages of training. Thus, fluorescence-based analysis of input- and target-specific synapses can reveal compartment-specific changes in synapse properties during learning.SIGNIFICANCE STATEMENT Synaptic changes underlie the cellular basis of learning, experience, and neurologic diseases. Neuroanatomical methods to assess synaptic plasticity can provide critical spatial information necessary for building models of neuronal computations during learning and experience but are technically and fiscally intensive. Here, we describe a confocal fluorescence microscopy-based analytical method to assess input, cell type, and dendritic location-specific synaptic plasticity in a sensory learning assay. Our method not only confirms prior electrophysiological measurements but allows us to predict functional strength of synapses in a pathway-specific manner. Our findings also indicate that changes in primary sensory cortices are transient, occurring during early learning. Fluorescence-based synapse identification can be an efficient and easily adopted approach to study synaptic changes in a variety of experimental paradigms.

Recent advancements in targeted protein knockdown technologies-emerging paradigms for targeted therapy.

A generalized therapeutic strategy for various disease conditions, including cancer, is to deplete or inactivate harmful protein targets. Various forms of protein or gene silencing molecules, e.g., small molecule inhibitors, RNA interference (RNAi), and microRNAs (miRNAs) have been used against druggable targets. Over the past few years, targeted protein degradation (TPD) approaches have been developed for direct degradation of candidate proteins. Among the TPD approaches, proteolysis targeting chimeras (PROTACs) have emerged as one of the most promising approaches for the selective elimination of proteins via the ubiquitin-proteasome system. Other than PROTACs, TPD methods with potential therapeutic use include intrabody-mediated protein knockdown and tripartite motif-21 (TRIM-21) mediated TRIM-Away. In this review, protein knockdown approaches, their modes of action, and their advantages over conventional gene knockdown approaches are summarized. In cancers, disease-associated protein functions are often executed by specific post-translational modifications (PTMs). The role of TRIM-Away is highlighted in the direct knockdown of PTM forms of target proteins. Moreover, the application challenges and the prospective clinical use of TPD approaches in various diseases are also discussed.

Study of spatiotemporal regulation of kinase signaling using genetically encodable molecular tools.

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.

Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions.

Tumor cells are characterized by overexpressed tumor-associated antigens or mutated neoantigens, which are expressed on the cell surface or intracellularly. One strategy of cancer immunotherapy is to target cell-surface-expressed tumor-associated antigens (TAAs) with therapeutic antibodies. For targeting TAAs or neoantigens, adoptive T-cell therapies with activated autologous T cells from cancer patients transduced with novel recombinant TCRs or chimeric antigen receptors have been successfully applied. Many TAAs and most neoantigens are expressed in the cytoplasm or nucleus of tumor cells. As alternative to adoptive T-cell therapy, the mRNA of intracellular tumor antigens can be depleted by RNAi, the corresponding genes or proteins deleted by CRISPR-Cas or inactivated by kinase inhibitors or by intrabodies, respectively. Intrabodies are suitable to knockdown TAAs and neoantigens without off-target effects. RNA sequencing and proteome analysis of single tumor cells combined with computational methods is bringing forward the identification of new neoantigens for the selection of anti-cancer intrabodies, which can be easily performed using phage display antibody repertoires. For specifically delivering intrabodies into tumor cells, the usage of new capsid-modified adeno-associated viruses and lipid nanoparticles coupled with specific ligands to cell surface receptors can be used and might bring cancer intrabodies into the clinic.

Targeting the "undruggable" RAS with biologics.

RAS proteins represent critical drivers of tumor development and thus are the focus of intense efforts to pharmacologically inhibit these proteins in human cancer. Although recent success has been attained in developing clinically efficacious inhibitors to KRASG12C, there remains a critical need for developing approaches to inhibit additional mutant RAS proteins. A number of anti-RAS biologics have been developed which reveal novel and potentially therapeutically targetable vulnerabilities in oncogenic RAS. This review will discuss the growing field of anti-RAS biologics and potential development of these reagents into new anti-RAS therapies.

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies.

Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.

Interference of p53:Twist1 interaction through competing nanobodies.

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.

Live-cell imaging probes to track chromatin modification dynamics.

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.

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment.

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.

Selection and Modelling of a New Single-Domain Intrabody Against TDP-43.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated to deteriorating motor and cognitive functions, and short survival. The disease is caused by neuronal death which results in progressive muscle wasting and weakness, ultimately leading to lethal respiratory failure. The misbehaviour of a specific protein, TDP-43, which aggregates and becomes toxic in ALS patient's neurons, is supposed to be one of the causes. TDP-43 is a DNA/RNA-binding protein involved in several functions related to nucleic acid metabolism. Sequestration of TDP-43 aggregates is a possible therapeutic strategy that could alleviate or block pathology. Here, we describe the selection and characterization of a new intracellular antibody (intrabody) against TDP-43 from a llama nanobody library. The structure of the selected intrabody was predicted in silico and the model was used to suggest mutations that enabled to improve its expression yield, facilitating its experimental validation. We showed how coupling experimental methodologies with in silico design may allow us to obtain an antibody able to recognize the RNA binding regions of TDP-43. Our findings illustrate a strategy for the mitigation of TDP-43 proteinopathy in ALS and provide a potential new tool for diagnostics.

Treating Parkinson's Disease with Antibodies: Previous Studies and Future Directions.

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 Proteins Co-Expressed and Co-Purified in the Presence of Antibody Fragments.

Recombinant antibodies in single-domain format (VHHs) have been recently used for stabilizing antigens during their purification and crystallization. VHHs are also known for their structural stability and a significant part of them share the characteristic of remaining functionally folded also in the absence of the internal disulfide bond. Therefore, they can be expressed as intrabodies in the cell cytoplasm as well as in the bacterial periplasm. This evidence means that, in theory, VHHs can be co-expressed with their antigens independently on the redox constrains. It has also suggested the idea of using co-expression and co-purification of antigen-antibody complexes for maximizing the stabilizing effect of the antibody on its antigen during all the production steps for both cytoplasmic and periplasmic expression strategies.

Development, Screening, and Validation of Camelid-Derived Nanobodies for Neuroscience Research.

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.