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Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.
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Tecnología Química Verde , Cuerpos de Inclusión/metabolismo , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Dominio Catalítico , Caveolina 1/química , Caveolina 1/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diseño de Equipo , Tecnología Química Verde/instrumentación , Muramidasa/química , Muramidasa/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, escape coronavirus disease 2019 therapeutics and vaccines, and jeopardize public health. To combat SARS-CoV-2 antigenic escape, we developed a rapid, high-throughput pipeline to discover monospecific VHH antibodies and iteratively develop VHH-Fc-VHH bispecifics capable of neutralizing emerging SARS-CoV-2 variants. By panning VHH single-domain phage libraries against ancestral or beta spike proteins, we discovered high-affinity VHH antibodies with unique target epitopes. Combining two VHHs into a tetravalent bispecific construct conferred broad neutralization activity against multiple variants and was more resistant to antigenic escape than the monospecific antibody alone. Following the rise of the Omicron variant, a VHH in the original bispecific construct was replaced with another VHH discovered against the Omicron BA.1 receptor binding domain; the resulting bispecific exhibited neutralization against both BA.1 and BA.5 sublineage variants. A heavy chain-only tetravalent VHH-Fc-VHH bispecific platform derived from humanized synthetic libraries held a myriad of unique advantages: (i) synthetic preconstructed libraries minimized risk of liabilities and maximized discovery speed, (ii) VHH scaffolds allowed for a modular "plug-and-play" format that could be rapidly iterated upon as variants of concern arose, (iii) natural dimerization of single VHH-Fc-VHH polypeptides allowed for straightforward bispecific production and purification methods, and (iv) multivalent approaches enhanced avidity boosting effects and neutralization potency, and conferred more robust resistance to antigenic escape than monovalent approaches against specific variants. This iterative platform of rapid VHH discovery combined with modular bispecific design holds promise for long-term viral control efforts.
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New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries-two synthetic and two immunized-against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.
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Antagonistas del Receptor de Adenosina A2 , Receptor de Adenosina A2A , Humanos , Animales , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2A/inmunología , Células HEK293 , Ratones , Antagonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/farmacología , Macaca fascicularis , Biblioteca de PéptidosRESUMEN
Congenital hyperinsulinism (HI) is a genetic disorder in which pancreatic ß-cell insulin secretion is excessive and results in hypoglycemia that, without treatment, can cause brain damage or death. Most patients with loss-of-function mutations in ABCC8 and KCNJ11, the genes encoding the ß-cell ATP-sensitive potassium channel (KATP), are unresponsive to diazoxide, the only U.S. Food and Drug Administration-approved medical therapy and require pancreatectomy. The glucagon-like peptide 1 receptor (GLP-1R) antagonist exendin-(9-39) is an effective therapeutic agent that inhibits insulin secretion in both HI and acquired hyperinsulinism. Previously, we identified a highly potent antagonist antibody, TB-001-003, which was derived from our synthetic antibody libraries that were designed to target G protein-coupled receptors. Here, we designed a combinatorial variant antibody library to optimize the activity of TB-001-003 against GLP-1R and performed phage display on cells overexpressing GLP-1R. One antagonist, TB-222-023, is more potent than exendin-(9-39), also known as avexitide. TB-222-023 effectively decreased insulin secretion in primary isolated pancreatic islets from a mouse model of hyperinsulinism, Sur1-/- mice, and in islets from an infant with HI, and increased plasma glucose levels and decreased the insulin to glucose ratio in Sur1-/- mice. These findings demonstrate that targeting GLP-1R with an antibody antagonist is an effective and innovative strategy for treatment of hyperinsulinism. ARTICLE HIGHLIGHTS: Patients with the most common and severe form of diazoxide-unresponsive congenital hyperinsulinism (HI) require a pancreatectomy. Other second-line therapies are limited in their use because of severe side effects and short half-lives. Therefore, there is a critical need for better therapies. Studies with the glucagon-like peptide 1 receptor (GLP-1R) antagonist, avexitide (exendin-(9-39)), have demonstrated that GLP-1R antagonism is effective at lowering insulin secretion and increasing plasma glucose levels. We have optimized a GLP-1R antagonist antibody with more potent blocking of GLP-1R than avexitide. This antibody therapy is a potential novel and effective treatment for HI.
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Hiperinsulinismo Congénito , Receptor del Péptido 1 Similar al Glucagón , Hiperinsulinismo , Animales , Ratones , Anticuerpos/uso terapéutico , Glucemia , Hiperinsulinismo Congénito/tratamiento farmacológico , Hiperinsulinismo Congénito/genética , Diazóxido/farmacología , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón/antagonistas & inhibidores , Hiperinsulinismo/inmunología , Hiperinsulinismo/terapia , Mutación , Receptores de Sulfonilureas/genéticaRESUMEN
Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Técnicas de Visualización de Superficie Celular , Inmunoglobulina G/inmunología , Biblioteca de Péptidos , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , COVID-19/metabolismo , COVID-19/prevención & control , COVID-19/virología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Epítopos , Femenino , Interacciones Huésped-Patógeno , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Mesocricetus , SARS-CoV-2/patogenicidad , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Células VeroRESUMEN
G protein-coupled receptors (GPCRs) are a group of seven-transmembrane receptor proteins that have proven to be successful drug targets. Antibodies are becoming an increasingly promising modality to target these receptors due to their unique properties, such as exquisite specificity, long half-life, and fewer side effects, and their improved pharmacokinetic and pharmacodynamic profiles compared to peptides and small molecules, which results from their more favorable biodistribution. To date, there are only two US Food and Drug Administration-approved GPCR antibody drugs, namely erenumab and mogamulizumab, and this highlights the challenges encountered in identifying functional antibodies against GPCRs. Utilizing Twist's precision DNA writing technologies, we have created a GPCR-focused phage display library with 1 × 1010 diversity. Specifically, we mined endogenous GPCR binding ligand and peptide sequences and incorporated these binding motifs into the heavy chain complementarity-determining region 3 in a synthetic antibody library. Glucagon-like peptide-1 receptor (GLP-1 R) is a class B GPCR that acts as the receptor for the incretin GLP-1, which is released to regulate insulin levels in response to food intake. GLP-1 R agonists have been widely used to increase insulin secretion to lower blood glucose levels for the treatment of type 1 and type 2 diabetes, whereas GLP-1 R antagonists have applications in the treatment of severe hypoglycemia associated with bariatric surgery and hyperinsulinomic hypoglycemia. Here we present the discovery and creation of both antagonistic and agonistic GLP-1 R antibodies by panning this GPCR-focused phage display library on a GLP-1 R-overexpressing Chinese hamster ovary cell line and demonstrate their in vitro and in vivo functional activity.
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Anticuerpos Monoclonales/farmacología , Glucemia/efectos de los fármacos , Técnicas de Visualización de Superficie Celular , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/antagonistas & inhibidores , Control Glucémico , Hipoglucemiantes/farmacología , Incretinas/farmacología , Biblioteca de Péptidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacocinética , Sitios de Unión de Anticuerpos , Biomarcadores/sangre , Glucemia/metabolismo , Células CHO , Cricetulus , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Ensayos Analíticos de Alto Rendimiento , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacocinética , Incretinas/genética , Incretinas/metabolismo , Incretinas/farmacocinética , Ligandos , Masculino , Ratones Endogámicos C57BL , Dominios y Motivos de Interacción de Proteínas , Ratas Sprague-DawleyRESUMEN
The Wnt signaling pathway is intricately connected with bone mass regulation in humans and rodent models. We designed an antibody-based platform that generates potent and selective Wnt mimetics. Using this platform, we engineer bi-specific Wnt mimetics that target Frizzled and low-density lipoprotein receptor-related proteins and evaluate their effects on bone accrual in murine models. These synthetic Wnt agonists induce rapid and robust bone building effects, and correct bone mass deficiency and bone defects in various disease models, including osteoporosis, aging, and long bone fracture. Furthermore, when these Wnt agonists are combined with antiresorptive bisphosphonates or anti-sclerostin antibody therapies, additional bone accrual/maintenance effects are observed compared to monotherapy, which could benefit individuals with severe and/or acute bone-building deficiencies. Our data support the continued development of Wnt mimetics for the treatment of diseases of low bone mineral density, including osteoporosis.
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Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/tratamiento farmacológico , Fracturas del Fémur/tratamiento farmacológico , Osteoporosis Posmenopáusica/tratamiento farmacológico , Proteínas Wnt/agonistas , Anciano , Envejecimiento/fisiología , Animales , Densidad Ósea/efectos de los fármacos , Densidad Ósea/fisiología , Conservadores de la Densidad Ósea/uso terapéutico , Resorción Ósea/fisiopatología , Difosfonatos/farmacología , Difosfonatos/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Femenino , Fracturas del Fémur/patología , Fémur/efectos de los fármacos , Fémur/lesiones , Fémur/patología , Humanos , Ratones , Osteoporosis Posmenopáusica/fisiopatología , Vía de Señalización Wnt/efectos de los fármacos , Adulto JovenRESUMEN
BACKGROUND: Development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences. METHODS AND RESULTS: A rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature. CONCLUSIONS: Critically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library's full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment.
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Phage display offers a powerful approach to engineer protein affinity. A naturally occurring analog to phage display, the Bordetella bronchiseptica bacteriophage (BP) employs a highly variable protein termed the major tropism determinant (Mtd) to recognize its dynamic host. Propagation of BP provides a self-made phage library (SMPL) with vast numbers of phage particles, each displaying a single Mtd variant. We report applying the diversity of the BP-SMPL to access a tyrosine-rich library of Mtd variants. Expression of the SMPL-engineered Mtd variant as a GST-bound fusion protein demonstrated specific binding to the target T4 lysozyme with dissociation constants in the sub-micromolar range. The results guide future experiments with SMPLs applied to protein engineering.
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Bacteriófagos/metabolismo , Bordetella bronchiseptica/virología , Ingeniería de Proteínas , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Phage display libraries are widely used as tools for identifying, dissecting and optimizing ligands. Development of a simple method to access greater library diversities could expedite and expand the technique. This paper reports progress toward harnessing the naturally occurring diversity generating retroelement used by Bordetella bronchiseptica bacteriophage to alter its tail-fiber protein. Mutagenesis and testing identified four sites amenable to the insertion of <19-residue heterologous peptides within the variable region. Such sites allow auto-generation of peptide libraries surrounded by a scaffold with additional variations. The resultant self-made phage libraries were used successfully for selections targeting anti-FLAG antibody, immobilized metal affinity chromatography microtiter plates and HIV-1 gp41. The reported experiments demonstrate the utility of the major tropism determinant protein of B.bronchiseptica as a natural scaffold for diverse, phage-constructed libraries with heterologous self-made phage libraries.