RESUMEN
SARS-CoV-2 infection is initiated by Spike glycoprotein binding to the human angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain. Blocking this interaction has been proven to be an effective approach to inhibit virus infection. Here we report the discovery of a neutralizing nanobody named VHH60, which was directly produced from an engineering nanobody library based on a commercialized nanobody within a very short period. VHH60 competes with human ACE2 to bind the receptor binding domain of the Spike protein at S351, S470-471and S493-494 as determined by structural analysis, with an affinity of 2.56 nM. It inhibits infections of both ancestral SARS-CoV-2 strain and pseudotyped viruses harboring SARS-CoV-2 wildtype, key mutations or variants at the nanomolar level. Furthermore, VHH60 suppressed SARS-CoV-2 infection and propagation 50-fold better and protected mice from death for twice as long as the control group after SARS-CoV-2 nasal infections in vivo. Therefore, VHH60 is not only a powerful nanobody with a promising profile for disease control but also provides evidence for a highly effective and rapid approach to generating therapeutic nanobodies.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , COVID-19 , 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/farmacología , Anticuerpos de Dominio Único/inmunología , Humanos , Animales , COVID-19/inmunología , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Neutralizantes/farmacología , Tratamiento Farmacológico de COVID-19 , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Células HEK293 , Ratones Endogámicos BALB C , Unión Proteica , FemeninoRESUMEN
Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.
Asunto(s)
Anticuerpos Bloqueadores/química , Anticuerpos Monoclonales/química , Proteína Accesoria del Receptor de Interleucina-1/química , Interleucina-1beta/química , Modelos Moleculares , Conformación Proteica , Receptores Tipo I de Interleucina-1/química , Animales , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Mapeo Epitopo/métodos , Epítopos/inmunología , Humanos , Inmunoglobulina G/química , Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Biblioteca de Péptidos , Unión Proteica/efectos de los fármacos , Receptores Tipo I de Interleucina-1/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
TDP-43 is a highly conserved and ubiquitously expressed nuclear protein. It has been implicated in the regulation of transcription, alternative splicing, translation, and neuronal plasticity. TDP-43 has also been shown to be a disease signature protein associated with several neurodegenerative diseases including amyotrophic lateral sclerosis. However, the correlation of the physiological functions of TDP-43 with these diseases remains unknown. We have used the gene targeting approach to disrupt the expression of TDP-43 in mouse. Loss of the TDP-43 expression results in peri-implantation lethality of mice between embryonic days (E) 3.5 and 6.5. Blastocysts of the homozygous Tardbp null mutants are morphologically normal, but exhibit defective outgrowth of the inner cell mass in vitro. Our data demonstrate the essential function of TDP-43 in peri-implantation stage during the embryo development, likely because of its involvement in multiple biological processes in a variety of cell types.
Asunto(s)
Proteínas de Unión al ADN/genética , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Western Blotting , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/embriología , Femenino , Genes Esenciales , Genotipo , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The therapeutic efficacy of an antibody drug depends on the variable domains and on the constant crystallizable fragment (Fc). IgG variable domains have been the targets of extensive molecular engineering in search of more specific binders with higher affinities for their targets. Similarly, Fc engineering approaches have led to modulating both the immune effector responses and serum half-lives of therapeutic antibodies. A high-affinity interaction between the IgG Fc and neonatal Fc receptor (FcRn) at a slightly acidic pH can protect IgG molecules from undergoing lysosomal or serum proteinase-induced degradation. Here we describe an optimized protocol for the development of a tailored, synthetic human Fc repertoire to select Fc mutants which show highly pH-restricted FcRn binding with high affinity.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/metabolismo , Ingeniería de Proteínas/métodos , Receptores Fc/metabolismo , Técnicas de Visualización de Superficie Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Mutación/genética , Biblioteca de PéptidosRESUMEN
Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.
Asunto(s)
Bacteriófagos/genética , Fragmentos Fab de Inmunoglobulinas/genética , Biblioteca de Péptidos , Animales , HumanosRESUMEN
Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Inmunoconjugados/inmunología , Inmunotoxinas/inmunología , Biblioteca de Péptidos , Anticuerpos de Cadena Única/inmunología , Secuencia de Aminoácidos , Afinidad de Anticuerpos/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Epítopos/química , Epítopos/inmunología , Células HEK293 , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Inmunotoxinas/química , Células MCF-7 , Receptor ErbB-2/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
The insulin-like growth factor-1 receptor (IGF-1R) plays a pivotal role in transformation, growth, and survival of glioblastoma multiforme (GBM) cells, and has emerged as a general and promising target for cancer treatment. In this study, we examined the anti-tumor effects of CHM-1, a synthetic 6,7-methylenedioxy substituted 2-phenyl-4-quinolone derivative, on GBM cells in vitro and in vivo. CHM-1 selectively blocked IGF-1R auto-phosphorylation, with an ability to distinguish between IGF-1R and related tyrosine kinase receptors, such as insulin receptor (IR), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR). Further investigation revealed that, the phosphorylation of ERK1/2 as well as Akt was inhibited in CHM-1 treated GBM8401 cells possibly through the selective blockage of IGF-1R auto-phosphorylation. Our study also showed that p.o. treatment with the hydrophilic dihydrogen phosphate CHM-1P reduced the tumor volumes of the GBM8401 derived tumors in mouse brain and also prolonged the survival rate. The results provided potential opportunities for effective chemotherapy for GBM.
Asunto(s)
Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Dioxoles/uso terapéutico , Glioblastoma/tratamiento farmacológico , Quinolonas/uso terapéutico , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Dioxoles/farmacología , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos BALB C , Ratones SCID , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinolonas/farmacología , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Humoral immunity against diverse pathogens is rapidly elicited from natural antibody repertoires of limited complexity. But the organizing principles underlying the antibody repertoires that facilitate this immunity are not well-understood. We used HER2 as a model immunogen and reverse-engineered murine antibody response through constructing an artificial antibody library encoded with rudimentary sequence and structural characteristics learned from high throughput sequencing of antibody variable domains. Antibodies selected in vitro from the phage-displayed synthetic antibody library bound to the model immunogen with high affinity and specificities, which reproduced the specificities of natural antibody responses. We conclude that natural antibody structural repertoires are shaped to allow functional antibodies to be encoded efficiently, within the complexity limit of an individual antibody repertoire, to bind to diverse protein antigens with high specificity and affinity. Phage-displayed synthetic antibody libraries, in conjunction with high-throughput sequencing, can thus be designed to replicate natural antibody responses and to generate novel antibodies against diverse antigens.
Asunto(s)
Reacciones Antígeno-Anticuerpo/inmunología , Inmunidad Innata/inmunología , Receptor ErbB-2/química , Receptor ErbB-2/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Protein loops are frequently considered as critical determinants in protein structure and function. Recent advances in high-throughput methods for DNA sequencing and thermal stability measurement have enabled effective exploration of sequence-structure-function relationships in local protein regions. Using these data-intensive technologies, we investigated the sequence-structure-function relationships of six complementarity-determining regions (CDRs) and ten non-CDR loops in the variable domains of a model vascular endothelial growth factor (VEGF)-binding single-chain antibody variable fragment (scFv) whose sequence had been optimized via a consensus-sequence approach. The results show that only a handful of residues involving long-range tertiary interactions distant from the antigen-binding site are strongly coupled with antigen binding. This implies that the loops are passive regions in protein folding; the essential sequences of these regions are dictated by conserved tertiary interactions and the consensus local loop-sequence features contribute little to protein stability and function.
Asunto(s)
Regiones Determinantes de Complementariedad/química , Anticuerpos de Cadena Única/química , Factor A de Crecimiento Endotelial Vascular/química , Secuencia de Aminoácidos , Regiones Determinantes de Complementariedad/inmunología , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión Proteica , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Anticuerpos de Cadena Única/inmunología , Proteína Estafilocócica A/química , Proteína Estafilocócica A/inmunología , Relación Estructura-Actividad , Termodinámica , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Conformational disorders are involved in various neurodegenerative diseases. Reactive oxygen species (ROS) are the major contributors to neurodegenerative disease; however, ROS that affect the structural changes in misfolded disease proteins have yet to be well characterized. Here we demonstrate that the intrinsic propensity of TDP-43 to aggregate drives the assembly of TDP-43-positive stress granules and soluble toxic TDP-43 oligomers in response to a ROS insult via a disulfide crosslinking-independent mechanism. Notably, ROS-induced TDP-43 protein assembly correlates with the dynamics of certain TDP-43-associated chaperones. The heat-shock protein (HSP)-90 inhibitor 17-AAG prevents ROS-induced TDP-43 aggregation, alters the type of TDP-43 multimers and reduces the severity of pathological TDP-43 inclusions. In summary, our study suggests that a common mechanism could be involved in the pathogenesis of conformational diseases that result from HSP dysregulation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Muerte Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Células HEK293 , Proteínas de Choque Térmico/genética , Humanos , Procesamiento de Imagen Asistido por Computador , Estrés Oxidativo , Especies Reactivas de OxígenoRESUMEN
The degraded, misfolded C terminus of TAR DNA-binding protein-43 is associated with a wide spectrum of neurodegenerative diseases, particularly frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. However, the precise mechanism of pathological cleavage of the TAR DNA-binding protein-43 remains unknown. Here we show that the TAR DNA-binding protein-43 C-terminal protein physically interacts with itself or with the cellular-folded yeast prion domain of Sup35 forming dynamic aggregates. This prion-like nature governs known cellular functions of the TAR DNA-binding protein-43, including subcellular localisation and exon skipping of the cystic fibrosis transmembrane conductance regulator. Significantly, mutants with a failure to engage in prion-like interactions are processed into an ~24-kDa C-terminal fragment of the TAR DNA-binding protein-43. The estimated cleavage site of degraded TAR DNA-binding protein-43 fragments corresponds to the pathological cleavage site identified in patients with the TAR DNA-binding protein-43 proteinopathies. Consistently, epigallocatechin gallate constrains prion-like interactions, attenuating pathological-like degradation. Thus, the native folding of TAR DNA-binding protein-43 C terminus acts as a guardian of pathogenesis, which is directly associated with loss-of-function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Secuencias de Aminoácidos , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Exones , Humanos , Enfermedades Neurodegenerativas/genética , ProteolisisRESUMEN
In this study, we examined the effects of several plant-derived natural compounds on head and neck squamous cell carcinoma (HNSCC) cells. The results revealed that (-)-epigallocatechin gallate (EGCG) demonstrated the most efficient cytotoxic effects on HNSCC cells. We then investigated the underlying molecular mechanism for the potent proapoptotic effect of EGCG on HNSCC. Cell apoptosis was observed in the EGCG-treated SAS and Cal-27 cells in a time- and dose-dependent manner. In concert with the caspase-8 activation by EGCG, an enhanced expression in functional Fas/CD95 was identified. Consistent with the increased Fas/CD95 expression, a drastic decrease in the Tyr705 phosphorylation of STAT3, a known negative regulator of Fas/CD95 transcription, was shown within 15 min in the EGCG-treated cells, leading to downregulation of the target gene products of STAT3, such as bcl-2, vascular endothelial growth factor (VEGF), mcl-1, and cyclin D1. An overexpression in STAT3 led to resistance to EGCG, suggesting that STAT3 was a critical target of EGCG. Besides inhibiting constitutive expression, EGCG also abrogated the interleukin-6 (IL-6)-induced JAK/STAT3 signaling and further inhibited IL-6-induced proliferation on HNSCC cells. In comparison with apigenin, curcumin, and AG490, EGCG was a more effective inhibitor of IL-6-induced proliferation on HNSCC cells. Overall, our results strongly suggest that EGCG induces Fas/CD95-mediated apoptosis through inhibiting constitutive and IL-6-induced JAK/STAT3 signaling. This mechanism may be partially responsible for EGCG's ability to suppress proliferation of HNSCC cells. These findings provide that EGCG may be useful in the chemoprevention and/or treatment of HNSCC.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/metabolismo , Catequina/análogos & derivados , Proteína Ligando Fas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/fisiopatología , Catequina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Proteína Ligando Fas/genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/fisiopatología , Humanos , Interleucina-6/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Ankyrin repeat domain 17 (Ankrd17) encodes an ubiquitously expressed protein with two clusters of ankyrin repeats. We have used gene targeting strategy to ablate the Ankrd17 gene in mouse. The Ankrd17-deficient mice died between embryonic day (E) 10.5 and E11.5 due to cardiovascular defects. Serious hemorrhages were detected and the vascular smooth muscle cells (vSMCs) surrounding the vessels were drastically reduced in the Ankrd17-deficient embryos, suggesting that the vascular maturation was not completed. Interestingly, vSMC differentiation marker genes were up-regulated in the mutant embryos. Our data have demonstrated the indispensability of Ankrd17 functioning for vascular maturation during early development. The Ankrd17-deficient mice also provide a new animal model for the analysis of the regulatory pathways of the differentiation of vSMC precursor cells.
Asunto(s)
Ancirinas/metabolismo , Vasos Sanguíneos/embriología , Desarrollo Embrionario/fisiología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/fisiología , Proteínas/metabolismo , Animales , Ancirinas/genética , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Diferenciación Celular/fisiología , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Hemorragia , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/citología , Proteínas/genética , Proteínas de Unión al ARNRESUMEN
NF-kappa B/Rel transcription factors play essential roles to mediate the immune response and apoptosis, and they have also been implicated in cellular differentiation such as erythropoiesis. To elucidate the possible role(s) of NF-kappa B in erythroid gene regulation and erythropoiesis, we have carried out transient transfection studies of the human embryonic/fetal erythroid cell line K562 and mouse adult erythroid MEL cells. It is shown that tumor necrosis factor-alpha represses the transcription activity directed by either alpha or zeta globin promoter in a dose-dependent manner. Furthermore, different NF-kappa B family members could effectively repress the transfected alpha-like globin promoters in K562 as well as in MEL cells. The involvement of NF-kappa B pathway is supported by the ability of a NF-kappa B-specific, dominant negative mutant to block the tumor necrosis factor-alpha or p65-mediated suppression of the alpha-like globin promoter activities. The suppression appears to be mediated through cis-linked HS-40 enhancer. Finally, stably transfected K562 cells overexpressing p65 contain reduced amounts of the p45/NF-E2 RNA and functional NF-E2 proteins. Our studies have identified a new set of targets of NF-kappa B. We suggest that the relatively high activity of the NF-kappa B pathway in early erythroid progenitors is involved in the suppression of erythroid-specific genes. Later in differentiation, together with other changes, the decline of the amounts of the NF-kappa B family of factors leads to derepression and consequent increase of NF-E2, which in turn would activate a subset of erythroid-specific genes.