RESUMEN
Neurodegenerative diseases (NDs) in mammals, such as Alzheimer's disease (AD), Parkinson's disease (PD), and transmissible spongiform encephalopathies (TSEs), are characterized by the accumulation of misfolded proteins in the central nervous system (CNS). Despite the presence of these pathogenic proteins, the immune response in affected individuals remains notably muted. Traditional immunological strategies, particularly those reliant on monoclonal antibodies (mAbs), face challenges related to tissue penetration, blood-brain barrier (BBB) crossing, and maintaining protein stability. This has led to a burgeoning interest in alternative immunotherapeutic avenues. Notably, single-domain antibodies (or nanobodies) and aptamers have emerged as promising candidates, as their reduced size facilitates high affinity antigen binding and they exhibit superior biophysical stability compared to mAbs. Aptamers, synthetic molecules generated from DNA or RNA ligands, present both rapid production times and cost-effective solutions. Both nanobodies and aptamers exhibit inherent qualities suitable for ND research and therapeutic development. Cross-seeding events must be considered in both traditional and small-molecule-based immunodiagnostic and therapeutic approaches, as well as subsequent neurotoxic impacts and complications beyond protein aggregates. This review delineates the challenges traditional immunological methods pose in ND research and underscores the potential of nanobodies and aptamers in advancing next-generation ND diagnostics and therapeutics.
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Aptámeros de Nucleótidos , Enfermedades Neurodegenerativas , Anticuerpos de Dominio Único , Humanos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/uso terapéutico , Aptámeros de Nucleótidos/uso terapéutico , Aptámeros de Nucleótidos/inmunología , Animales , Enfermedades Neurodegenerativas/inmunología , Enfermedades Neurodegenerativas/terapiaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, which caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lead to a crisis with devastating disasters to global public economy and health. Several studies suggest that the SARS-CoV-2 nucleocapsid protein (N protein) is one of uppermost structural constituents of SARS-CoV-2 and is relatively conserved which could become a specific diagnostic marker. In this study, eight single domain antibodies recognized the N protein specifically which were named pN01-pN08 were screened using human phage display library. According to multiple sequence alignment and molecular docking analyses, the interaction mechanism between antibody and N protein was predicted. ELISA results indicated pN01-pN08 with high affinity to protein N. To improve their efficacy, two fusion proteins were prepared and their affinity was tested. These finding showed that fusion proteins had higher affinity than single domain antibodies and will be used as diagnosis for the pandemic of SARS-CoV-2.
Asunto(s)
Anticuerpos Antivirales , COVID-19 , Proteínas de la Nucleocápside de Coronavirus , Simulación del Acoplamiento Molecular , SARS-CoV-2 , Anticuerpos de Dominio Único , Humanos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , SARS-CoV-2/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/química , COVID-19/inmunología , COVID-19/diagnóstico , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Fosfoproteínas/inmunología , Fosfoproteínas/química , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Biblioteca de PéptidosRESUMEN
Therapeutic antibody design has garnered widespread attention, highlighting its interdisciplinary importance. Advancements in technology emphasize the critical role of designing nanobodies and humanized antibodies in antibody engineering. However, current experimental methods are costly and time-consuming. Computational approaches, while progressing, faced limitations due to insufficient structural data and the absence of a standardized protocol. To tackle these challenges, our lab previously developed IsAb1.0, an in silico antibody design protocol. Yet, IsAb1.0 lacked accuracy, had a complex procedure, and required extensive antibody bioinformation. Moreover, it overlooked nanobody and humanized antibody design, hindering therapeutic antibody development. Building upon IsAb1.0, we enhanced our design protocol with artificial intelligence methods to create IsAb2.0. IsAb2.0 utilized AlphaFold-Multimer (2.3/3.0) for accurate modeling and complex construction without templates and employed the precise FlexddG method for in silico antibody optimization. Validated through optimization of a humanized nanobody J3 (HuJ3) targeting HIV-1 gp120, IsAb2.0 predicted five mutations that can improve HuJ3-gp120 binding affinity. These predictions were confirmed by commercial software and validated through binding and neutralization assays. IsAb2.0 streamlined antibody design, offering insights into future techniques to accelerate immunotherapy development.
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Inteligencia Artificial , Ingeniería de Proteínas , Humanos , Ingeniería de Proteínas/métodos , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Diseño de Fármacos , Simulación por ComputadorRESUMEN
BACKGROUND: T cell-based immunotherapies are facing great challenges in the recruitment and activation of tumor-specific T cells against solid tumors. Among which, utilizing nanobody (Nb) or nanobodies (Nbs) to construct T cell engager has emerged as a more practical potential for enhancing the anti-tumor effectiveness of T cells. Here, we designed a new Nb-guided multifunctional T cell engager (Nb-MuTE) that not only recruited effector T cells into the tumor tissues, but also efficiently activated T cells anti-tumor immunity when synergies with photothermal effect. RESULTS: The Nb-MuTE, which was constructed based on an indocyanine green (ICG)-containing liposome with surface conjugation of CD105 and CD3 Nbs, and showed excellent targetability to both tumor and T cells, following enhancement of activation, proliferation and cytokine secretion of tumor-specific T cells. Notably, the immunological anti-tumor functions of Nb-MuTE-mediated T cells were further enhanced by the ICG-induced photothermal effect in vitro and in vivo. CONCLUSIONS: Such a new platform Nb-MuTE provides a practical and "all-in-one" strategy to potentiate T cell responses for the treatment of solid tumor in clinic.
Asunto(s)
Inmunoterapia , Verde de Indocianina , Anticuerpos de Dominio Único , Linfocitos T , Animales , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Ratones , Linfocitos T/inmunología , Verde de Indocianina/química , Inmunoterapia/métodos , Línea Celular Tumoral , Humanos , Neoplasias/terapia , Neoplasias/inmunología , Femenino , Ratones Endogámicos BALB C , Terapia Fototérmica/métodos , Liposomas/química , Activación de Linfocitos , Ratones Endogámicos C57BL , Complejo CD3/inmunologíaRESUMEN
Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders.
Asunto(s)
Factor XII , Inflamación , Ratones Noqueados , Neutrófilos , Anticuerpos de Dominio Único , Trombosis , Animales , Humanos , Trombosis/inmunología , Trombosis/metabolismo , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología , Masculino , Factor XII/metabolismo , Factor XII/antagonistas & inhibidores , Inflamación/metabolismo , Ratones , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/efectos de los fármacos , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Agregación Plaquetaria/efectos de los fármacos , Factor XIIa/metabolismo , Factor XIIa/antagonistas & inhibidores , Fibrina/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno/metabolismoRESUMEN
Oral delivery of proteins faces challenges due to the harsh conditions of the gastrointestinal (GI) tract, including gastric acid and intestinal enzyme degradation. Permeation enhancers are limited in their ability to deliver proteins with high molecular weight and can potentially cause toxicity by opening tight junctions. To overcome these challenges, we propose the use of montmorillonite (MMT) as an adjuvant that possesses both inflammation-oriented abilities and the ability to regulate gut microbiota. This adjuvant can be used as a universal protein oral delivery technology by fusing with advantageous binding amino acid sequences. We demonstrated that anti-TNF-α nanobody (VII) can be intercalated into the MMT interlayer space. The carboxylate groups (-COOH) of aspartic acid (D) and glutamic acid (E) interact with the MMT surface through electrostatic interactions with sodium ions (Na+). The amino groups (NH2) of asparagine (N) and glutamine (Q) are primarily attracted to the MMT layers through hydrogen bonding with oxygen atoms on the surface. This binding mechanism protects VII from degradation and ensures its release in the intestinal tract, as well as retaining biological activity, leading to significantly enhanced therapeutic effects on colitis. Furthermore, VII@MMT increases the abundance of short-chain fatty acids (SCFAs)-producing strains, including Clostridia, Prevotellaceae, Alloprevotella, Oscillospiraceae, Clostridia_vadinBB60_group, and Ruminococcaceae, therefore enhance the production of SCFAs and butyrate, inducing regulatory T cells (Tregs) production to modulate local and systemic immune homeostasis. Overall, the MMT adjuvant provides a promising universal strategy for protein oral delivery by rational designed protein.
Asunto(s)
Bentonita , Microbioma Gastrointestinal , Factor de Necrosis Tumoral alfa , Bentonita/química , Animales , Administración Oral , Factor de Necrosis Tumoral alfa/metabolismo , Ratones , Microbioma Gastrointestinal/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/inmunología , Anticuerpos de Dominio Único/administración & dosificación , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Humanos , Inflamación/tratamiento farmacológico , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacologíaRESUMEN
Near-infrared (NIR) probes are highly sought after as fluorescent tags for multicolor cellular and in vivo imaging. Here we develop small NIR fluorescent nanobodies, termed NIR-FbLAG16 and NIR-FbLAG30, enabling background-free visualization of various GFP-derived probes and biosensors. We also design a red-shifted variant, NIR-Fb(718), to simultaneously target several antigens within the NIR spectral range. Leveraging the antigen-stabilizing property of the developed NIR-Fbs, we then create two modular systems for precise control of gene expression in GFP-labeled cells. Applying the NIR-Fbs in vivo, we target cells expressing GFP and the calcium biosensor GCaMP6 in the somatosensory cortex of transgenic mice. Simultaneously tracking calcium activity and the reference signal from NIR-FbLAGs bound to GCaMP6 enables ratiometric deep-brain in vivo imaging. Altogether, NIR-FbLAGs present a promising approach for imaging and manipulating various processes in live cells and behaving animals expressing GFP-based probes.
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Técnicas Biosensibles , Proteínas Fluorescentes Verdes , Ratones Transgénicos , Anticuerpos de Dominio Único , Animales , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Técnicas Biosensibles/métodos , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/química , Ratones , Humanos , Colorantes Fluorescentes/química , Calcio/metabolismo , Espectroscopía Infrarroja Corta/métodos , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/diagnóstico por imagen , Células HEK293RESUMEN
COVID-19 has caused millions of deaths and many times more infections worldwide, emphasizing the unpreparedness of the global health system in the face of new infections and the key role for vaccines and therapeutics, including virus-neutralizing antibodies, in prevention and containment of the disease. Continuous evolution of the SARS-CoV-2 coronavirus has been causing its new variants to evade the action of the immune system, which highlighted the importance of detailed knowledge of the epitopes of already selected potent virus-neutralizing antibodies. A single-chain antibody ("nanobody") targeting the SARS-CoV-2 receptor-binding domain (RBD), clone P2C5, had exhibited robust virus-neutralizing activity against all SARS-CoV-2 variants and, being a major component of the anti-COVID-19 formulation "GamCoviMab", had successfully passed Phase I of clinical trials. However, after the emergence of the Delta and XBB variants, a decrease in the neutralizing activity of this nanobody was observed. Here we report on the successful crystal structure determination of the RBD:P2C5 complex at 3.1 Å, which revealed the intricate protein-protein interface, sterically occluding full ACE2 receptor binding by the P2C5-neutralized RBD. Moreover, the structure revealed the developed RBD:P2C5 interface centered around residues Leu452 and Phe490, thereby explaining the evasion of the Delta or Omicron XBB, but not Omicron B.1.1.529 variant, as a result of the single L452R or F490S mutations, respectively, from the action of P2C5. The structure obtained is expected to foster nanobody engineering in order to rescue neutralization activity and will facilitate epitope mapping for other neutralizing nanobodies by competition assays.
Asunto(s)
Anticuerpos Neutralizantes , SARS-CoV-2 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/química , Humanos , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , COVID-19/virología , Dominios Proteicos , Unión Proteica , Epítopos/inmunología , Epítopos/química , Modelos Moleculares , Evasión Inmune , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Sitios de UniónRESUMEN
The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells. We have previously shown that selective inhibition of plasma membrane V-ATPases in breast tumor cells inhibits the invasion of these cells in vitro. We have now developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. We show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro. We further find that injection of this nanobody into mice implanted with 4T1-12B cells orthotopically in the mammary fat pad inhibits metastasis of tumor cells to lung. These results suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis.
Asunto(s)
Neoplasias Pulmonares , Anticuerpos de Dominio Único , ATPasas de Translocación de Protón Vacuolares , Animales , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología , Femenino , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Línea Celular Tumoral , Neoplasias de la Mama/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Ratones Endogámicos BALB C , Humanos , Invasividad NeoplásicaRESUMEN
Macroporous three-dimensional (3D) framework structured melamine foam-based Enzyme-Linked Immunosorbent Assay (f-ELISA) biosensors were developed for rapid, reliable, sensitive, and on-site detection of trace amount of biomolecules and chemicals. Various ligands can be chemically immobilized onto the melamine foam, which brings in the possibility of working with antibodies, nanobodies, and peptides, respectively, as affinity probes for f-ELISA biosensors with improved stability. Different chemical reagents can be used to modify the foam materials, resulting in varied reactivities with antibodies, nanobodies, and peptides. As a result, the f-ELISA sensors produced from these modified foams exhibit varying levels of sensitivity and performance. This study demonstrated that the chemical reagents used for immobilizing antibodies, nanobodies, and peptides could affect the sensitivities of the f-ELISA sensors, and their storage stabilities under different temperatures varied depending on the sensing probes used, with f-ELISA sensors employing nanobodies as probes exhibiting the highest stability. This study not only showcases the versatility of the f-ELISA system but also opens new avenues for developing cost-effective, portable, and user-friendly diagnostic tools with optimized sensitivity and stability.
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Técnicas Biosensibles , Ensayo de Inmunoadsorción Enzimática , Anticuerpos de Dominio Único , Triazinas , Triazinas/análisis , Triazinas/química , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Técnicas Biosensibles/métodos , Péptidos/química , Anticuerpos/inmunología , Anticuerpos/química , Anticuerpos Inmovilizados/inmunología , Anticuerpos Inmovilizados/química , Límite de DetecciónRESUMEN
BACKGROUND: Due to the serious issue of ofloxacin (OFL) abuse, there is an increasingly urgent need for accurate and rapid detection of OFL. Immunoassay has become the "golden method" for detecting OFL in complex matrix beneficial to its applicability for a large-scale screening, rapidity, and simplicity. However, traditional antibodies used in immunoassay present challenges such as time-consuming preparation, unstable sensitivity and specificity, and difficulty in directional evolution. In this paper, we successfully developed an OFL detection method based on a shark-derived single-domain antibody (ssdAb) to address these issues. RESULTS: Using phage display technology and a heterologous expression system, OFL-specific clones 1O11, 1O13, 1O17, 1O19, 1O21, and 2O26 were successfully isolated and expressed in soluble form. Among all OFL-specific ssdAbs, the 1O17 ssdAb exhibited the highest binding affinity to OFL in a concentration-dependence manner. The limit of detection (IC10) of 1O17 ssdAb was calculated as 0.34 ng/mL with a detection range of 3.40-1315.00 ng/mL, and its cross reactivity with other analogs was calculated to be less than 5.98 %, indicating high specificity and sensitivity. Molecular docking results revealed that 100Trp and 101Arg located in the CDR3 region of 1O17 ssdAb were crucial for OFL binding. In fish matrix performance tests, the 1O17 ssdAb did not demonstrate severe matrix interference in OFL-negative fish matrix, achieving satisfactory recovery rates ranging from 83.04 % to 108.82 % with high reproducibility. SIGNIFICANCE: This research provides a new and efficient OFL detection recognition element with significant potential in immunoassay applications, broadening the application scenarios of ssdAbs. It offers valuable insights into the structure-activity relationship between ssdAbs and small molecules, laying a theoretical foundation for the further directional modification and maturation of ssdAbs in subsequent applications.
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Ofloxacino , Tiburones , Anticuerpos de Dominio Único , Animales , Ofloxacino/análisis , Ofloxacino/inmunología , Ofloxacino/química , Tiburones/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Límite de Detección , Inmunoensayo/métodosRESUMEN
Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development.
Asunto(s)
Anticuerpos Antivirales , Microscopía por Crioelectrón , Epítopos , Infecciones por Henipavirus , Virus Nipah , Anticuerpos de Dominio Único , Virus Nipah/inmunología , Humanos , Animales , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/prevención & control , Infecciones por Henipavirus/virología , Epítopos/inmunología , Ratones , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos Antivirales/inmunología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/química , Femenino , Células HEK293RESUMEN
Single-domain antibodies (sdAbs) or nanobodies have received widespread attention due to their small size (~ 15 kDa) and diverse applications in bio-derived therapeutics. As many modern biotechnology breakthroughs are applied to antibody engineering and design, nanobody thermostability or melting temperature (Tm) is crucial for their successful utilization. In this study, we present TEMPRO which is a predictive modeling approach for estimating the Tm of nanobodies using computational methods. Our methodology integrates various nanobody biophysical features to include Evolutionary Scale Modeling (ESM) embeddings, NetSurfP3 structural predictions, pLDDT scores per sdAb region from AlphaFold2, and each sequence's physicochemical characteristics. This approach is validated with our combined dataset containing 567 unique sequences with corresponding experimental Tm values from a manually curated internal data and a recently published nanobody database, NbThermo. Our results indicate the efficacy of protein embeddings in reliably predicting the Tm of sdAbs with mean absolute error (MAE) of 4.03 °C and root mean squared error (RMSE) of 5.66 °C, thus offering a valuable tool for the optimization of nanobodies for various biomedical and therapeutic applications. Moreover, we have validated the models' performance using experimentally determined Tms from nanobodies not found in NbThermo. This predictive model not only enhances nanobody thermostability prediction, but also provides a useful perspective of using embeddings as a tool for facilitating a broader applicability of downstream protein analyses.
Asunto(s)
Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Temperatura de Transición , Modelos Moleculares , Estabilidad Proteica , Biología Computacional/métodosRESUMEN
Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation with no cure and limited treatment options that often have systemic side effects. In this study, we developed a target-specific system to potentially treat IBD by engineering the probiotic bacterium Escherichia coli Nissle 1917 (EcN). Our modular system comprises three components: a transcription factor-based sensor (NorR) capable of detecting the inflammation biomarker nitric oxide (NO), a type 1 hemolysin secretion system, and a therapeutic cargo consisting of a library of humanized anti-TNFα nanobodies. Despite a reduction in sensitivity, our system demonstrated a concentration-dependent response to NO, successfully secreting functional nanobodies with binding affinities comparable to the commonly used drug Adalimumab, as confirmed by enzyme-linked immunosorbent assay and in vitro assays. This newly validated nanobody library expands EcN therapeutic capabilities. The adopted secretion system, also characterized for the first time in EcN, can be further adapted as a platform for screening and purifying proteins of interest. Additionally, we provided a mathematical framework to assess critical parameters in engineering probiotic systems, including the production and diffusion of relevant molecules, bacterial colonization rates, and particle interactions. This integrated approach expands the synthetic biology toolbox for EcN-based therapies, providing novel parts, circuits, and a model for tunable responses at inflammatory hotspots.
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Escherichia coli , Enfermedades Inflamatorias del Intestino , Probióticos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/terapia , Óxido Nítrico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos de Dominio Único/genética , Adalimumab/genética , Inflamación/metabolismoRESUMEN
The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating ß-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.
Asunto(s)
Receptores Frizzled , Anticuerpos de Dominio Único , Receptores Frizzled/metabolismo , Receptores Frizzled/química , Humanos , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Unión Proteica , Cristalografía por Rayos X , Células HEK293 , Sitios de Unión , Microscopía por Crioelectrón , Animales , Modelos Moleculares , Dominios Proteicos , Proteínas Dishevelled/metabolismo , Proteínas Dishevelled/química , Proteínas Dishevelled/genética , Vía de Señalización Wnt , beta Catenina/metabolismo , beta Catenina/químicaRESUMEN
While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor-binding domain to direct lentivirus infection of Spike-expressing cells. Using four different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. Targeting is dependent on the fusion function of the Spike protein, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery by using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections.IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.
Asunto(s)
Vectores Genéticos , Lentivirus , SARS-CoV-2 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos de Dominio Único/inmunología , Lentivirus/genética , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , Vectores Genéticos/genética , Células HEK293 , COVID-19/virología , COVID-19/inmunología , Unión Proteica , Internalización del Virus , Fusión Celular , Técnicas de Transferencia de GenRESUMEN
Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 µg of recombinant nanobody could neutralize the toxic effects of 20 µg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.
Asunto(s)
Antivenenos , Venenos Elapídicos , Naja naja , Anticuerpos de Dominio Único , Mordeduras de Serpientes , Animales , Venenos Elapídicos/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Ratones , Antivenenos/farmacología , Antivenenos/inmunología , Mordeduras de Serpientes/tratamiento farmacológico , Camelus , Cromatografía Líquida de Alta Presión , Pruebas de NeutralizaciónRESUMEN
This study introduces an efficient on-column refolding and purification method for preparing nanobodies (Nbs) expressed as inclusion bodies and fusion proteins. The HisTrapTM FF system was successfully employed for the purification of the fusion protein FN1-ΔI-CM-2D5. The intein ΔI-CM cleavage activity was activated at 42 °C, followed by incubation for 4 h. Leveraging the remarkable thermal stability of Nbs, 2D5 was further purified through heat treatment at 80 °C for 1h. This method yielded up to 107.2 mg of pure 2D5 with a purity of 99.2 % from just 1L of bacterial culture grown in a shaker flask. Furthermore, this approach successfully restored native secondary structure and affinity of 2D5. Additionally, the platform was effectively applied to the refolding and purification of a polystyrene-binding nanobody (B2), which exhibited limited expression in the periplasmic and cytoplasmic spaces of E. coli. This endeavor resulted in the isolation of 53.2 mg of pure B2 Nb with a purity exceeding 99.5 % from the same volume of bacterial culture. Significantly, this approach restored the native secondary structure of the Nbs, highlighting its potential for addressing challenges associated with expressing complex Nbs in E. coli. Overall, this innovative platform provides a scientifically rigorous and reproducible method for the efficient preparation of Nbs, offering a valuable tool for antibody research and development.
Asunto(s)
Escherichia coli , Cuerpos de Inclusión , Replegamiento Proteico , Proteínas Recombinantes de Fusión , Anticuerpos de Dominio Único , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificación , Anticuerpos de Dominio Único/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Within the past decade, single domain antibodies (sdAbs) have been recognized as unique affinity binding reagents that can be tailored for performance in a variety of immunoassay formats. Luminex MagPlex color-coded magnetic microspheres provide a high-throughput platform that enables multiplexed immunoassays. We developed a MagPlex bead-based assay for the detection of SARS-CoV-2, using sdAbs against SARS-CoV-2 nucleocapsid (N) protein in which we engineered the sdAb capture reagents to orient them on the beads. The oriented sdAbs provided an increase in sensitivity over randomly oriented sdAbs for samples of N diluted in buffer, which also translated into better detection of SARS-CoV-2 in clinical samples. We assessed the specificity of the assay by examining seasonal coronavirus clinical samples. In summary, we provide a proof-of-concept that a bead-based assay using sdAbs to detect SARS-CoV-2 is feasible and future research combining it with other sdAb-coated beads that can detect other viruses may provide a useful diagnostic tool.
Asunto(s)
Anticuerpos Antivirales , COVID-19 , Proteínas de la Nucleocápside de Coronavirus , SARS-CoV-2 , Anticuerpos de Dominio Único , Humanos , SARS-CoV-2/inmunología , COVID-19/diagnóstico , COVID-19/inmunología , COVID-19/virología , Anticuerpos de Dominio Único/inmunología , Anticuerpos Antivirales/inmunología , Inmunoensayo/métodos , Proteínas de la Nucleocápside de Coronavirus/inmunología , Prueba Serológica para COVID-19/métodos , Fosfoproteínas/inmunología , Sensibilidad y Especificidad , MicroesferasRESUMEN
Selective degradation of pathological protein aggregates while sparing monomeric forms is of major therapeutic interest. The E3 ligase tripartite motif-containing protein 21 (TRIM21) degrades antibody-bound proteins in an assembly state-specific manner due to the requirement of TRIM21 RING domain clustering for activation, yet effective targeting of intracellular assemblies remains challenging. Here, we fused the RING domain of TRIM21 to a target-specific nanobody to create intracellularly expressed constructs capable of selectively degrading assembled proteins. We evaluated this approach against green fluorescent protein-tagged histone 2B (H2B-GFP) and tau, a protein that undergoes pathological aggregation in Alzheimer's and other neurodegenerative diseases. RING-nanobody degraders prevented or reversed tau aggregation in culture and in vivo, with minimal impact on monomeric tau. This approach may have therapeutic potential for the many disorders driven by intracellular protein aggregation.