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.
Asunto(s)
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
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
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.
Asunto(s)
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.
Asunto(s)
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
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
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
Despite advancements in antiretroviral therapy (ART) suppressing HIV-1 replication, existing antiviral drugs pose limitations, including lifelong medication, frequent administration, side effects and viral resistance, necessitating novel HIV-1 treatment approaches. CD4, pivotal for HIV-1 entry, poses challenges for drug development due to neutralization and cytotoxicity concerns. Nevertheless, Ibalizumab, the sole approved CD4-specific antibody for HIV-1 treatment, reignites interest in exploring alternative anti-HIV targets, emphasizing CD4's potential value for effective drug development. Here, we explore anti-CD4 nanobodies, particularly Nb457 from a CD4-immunized alpaca. Nb457 displays high potency and broad-spectrum activity against HIV-1, surpassing Ibalizumab's efficacy. Strikingly, engineered trimeric Nb457 nanobodies achieve complete inhibition against live HIV-1, outperforming Ibalizumab and parental Nb457. Structural analysis unveils Nb457-induced CD4 conformational changes impeding viral entry. Notably, Nb457 demonstrates therapeutic efficacy in humanized female mouse models. Our findings highlight anti-CD4 nanobodies as promising HIV-1 therapeutics, with potential implications for advancing clinical treatment against this global health challenge.
Asunto(s)
Antígenos CD4 , Camélidos del Nuevo Mundo , Anticuerpos Anti-VIH , Infecciones por VIH , VIH-1 , Anticuerpos de Dominio Único , VIH-1/inmunología , VIH-1/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología , Animales , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Humanos , Infecciones por VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Camélidos del Nuevo Mundo/inmunología , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/farmacología , Ratones , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Conformación Proteica , Femenino , Internalización del Virus/efectos de los fármacos , Células HEK293 , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Anticuerpos MonoclonalesRESUMEN
Nanobodies (Nbs), the smallest antigen-binding fragments with high stability and affinity derived from the variable domain of naturally occurring heavy-chain-only antibodies in camelids, have been shown as an efficient way to improve the specificity to tumors for photodynamic therapy (PDT). Nonetheless, the rapid clearance of Nbs in vivo restricts the accumulation and retention of the photosensitizer at the tumor site causing insufficient therapeutic outcome, especially in large-volume tumors. Herein, we develop photodynamic conjugates, MNB-Pyra Nbs, through site-specific conjugation between 7D12 Nbs and type I photosensitizer MNB-Pyra (morpholine-modified nile blue structure connected to pyrazolinone) in a 1:2 ratio. The photosensitizers with long-term retention can be released at the tumor site by reactive oxygen species cleavage after illumination, accompanied with fluorescence recovery for self-reporting the occurrence of PDT. Ultimately, a single dose of MNB-Pyra Nbs demonstrate highly effective tumor suppression with high biosafety in the large-volume tumor models after three rounds of PDT. This nanobody conjugate provides a paradigm for the design of precise long-time retention photosensitizers and is expected to promote the development of PDT.
Asunto(s)
Fotoquimioterapia , Fármacos Fotosensibilizantes , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Animales , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéutico , Fármacos Fotosensibilizantes/química , Ratones , Humanos , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Ratones Endogámicos BALB C , Femenino , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or ß-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.
Asunto(s)
Receptores CXCR4 , Anticuerpos de Dominio Único , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Receptores CXCR4/inmunología , Humanos , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Animales , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/antagonistas & inhibidores , Quimiocina CXCL12/inmunología , Células HEK293 , Afinidad de AnticuerposRESUMEN
The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe and fatal acute respiratory disease in humans. High fatality rates and continued infectiousness remain a pressing concern for global health preparedness. Antibodies targeted at the receptor-binding domain (RBD) are major countermeasures against human viral infection. Here, we report four potent nanobodies against MERS-CoV, which are isolated from alpaca, and especially the potency of Nb14 is highest in the pseudotyped virus assay. Structural studies show that Nb14 framework regions (FRs) are mainly involved in interactions targeting a novel epitope, which is entirely distinct from all previously reported antibodies, and disrupt the protein-carbohydrate interaction between residue W535 of RBD and hDPP4 N229-linked carbohydrate moiety (hDPP4-N229-glycan). Different from Nb14, Nb9 targets the cryptic face of RBD, which is distinctive from the hDPP4 binding site and the Nb14 epitope, and it induces the ß5-ß6 loop to inflect towards a shallow groove of the RBD and dampens the accommodation of a short helix of hDPP4. The particularly striking epitopes endow the two Nbs administrate synergistically in the pseudotyped MERS-CoV assays. These results not only character unprecedented epitopes for antibody recognition but also provide promising agents for prophylaxis and therapy of MERS-CoV infection.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones por Coronavirus , Epítopos , Coronavirus del Síndrome Respiratorio de Oriente Medio , Anticuerpos de Dominio Único , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Humanos , Epítopos/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Animales , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Camélidos del Nuevo Mundo/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Ratones , Receptores Virales/metabolismo , Receptores Virales/inmunologíaRESUMEN
Antibody pairs-based immunoassay platforms served as essential and effective tools in the field of pathogen detection. However, the cumbersome preparation and limited detection sensitivity of antibody pairs challenge in establishment of a highly sensitive detection platform. In this study, using COVID-19 testing as a case, we utilized readily accessible nanobodies as detection antibodies and further proposed an accurate design concept with a more scientific and efficient screening strategy to obtain ultrasensitive antibody pairs. We employed nanobodies capable of binding different antigenic epitopes of the nucleocapsid (NP) or receptor-binding domain (RBD) antigens sandwich as substitutes for monoclonal antibodies (mAbs) sandwich in fast detection formats and utilized time-resolved fluorescence (TRF) microspheres as the signal probe. Consequently, we developed a multi-epitope nanobody sandwich-based fluorescence lateral flow immunoassay (FLFA) strip. Our results suggest that the NP antigen had a detection limit of 12.01pg/mL, while the RBD antigen had a limit of 6.51 pg/mL using our FLFA strip. Based on double mAb sandwiches, the values presented herein demonstrated 4 to 32-fold enhancements in sensitivity, and 32 to 256-fold enhancements compared to commercially available antigen lateral flow assay kits. Furthermore, we demonstrated the excellent characteristics of the proposed test strip, including its specificity, stability, accuracy, and repeatability, which underscores its the prospective utility. Indeed, these findings indicate that our established screening strategy along with the multi-epitope nanobody sandwich mode provides an optimized strategy in the field of pathogen detection.
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Técnicas Biosensibles , COVID-19 , SARS-CoV-2 , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , COVID-19/diagnóstico , COVID-19/inmunología , COVID-19/virología , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Humanos , Técnicas Biosensibles/métodos , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Límite de Detección , Inmunoensayo/métodos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Prueba de COVID-19/métodos , Prueba Serológica para COVID-19/métodos , Antígenos Virales/inmunologíaRESUMEN
Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications.