RESUMEN
The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1-5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.
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Proteínas Portadoras , Proteínas , Aminoácidos/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Unión Proteica , Proteínas/químicaRESUMEN
Hormone absence or inactivity is common in congenital disease, but hormone antagonism remains controversial. Here, we characterize two novel homozygous leptin variants that yielded antagonistic proteins in two unrelated children with intense hyperphagia, severe obesity, and high circulating levels of leptin. Both variants bind to the leptin receptor but trigger marginal, if any, signaling. In the presence of nonvariant leptin, the variants act as competitive antagonists. Thus, treatment with recombinant leptin was initiated at high doses, which were gradually lowered. Both patients eventually attained near-normal weight. Antidrug antibodies developed in the patients, although they had no apparent effect on efficacy. No severe adverse events were observed. (Funded by the German Research Foundation and others.).
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Leptina , Obesidad Mórbida , Niño , Humanos , Anticuerpos , Homocigoto , Leptina/genética , Obesidad Mórbida/genética , Transducción de SeñalRESUMEN
Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8-11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.
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ATP Citrato (pro-S)-Liasa/química , ATP Citrato (pro-S)-Liasa/metabolismo , Ciclo del Ácido Cítrico , Evolución Molecular , ATP Citrato (pro-S)-Liasa/genética , Biocatálisis , Chlorobium/enzimología , Chlorobium/genética , Cristalografía por Rayos X , Humanos , Methanosarcinales/enzimología , Methanosarcinales/genética , Modelos MolecularesRESUMEN
RATIONALE: Estimating the causal effect of an intervention at individual level, also called individual treatment effect (ITE), may help in identifying response prior to the intervention. OBJECTIVES: We aimed to develop machine learning (ML) models which estimate ITE of an intervention using data from randomised controlled trials and illustrate this approach with prediction of ITE on annual chronic obstructive pulmonary disease (COPD) exacerbation rates. METHODS: We used data from 8151 patients with COPD of the Study to Understand Mortality and MorbidITy in COPD (SUMMIT) trial (NCT01313676) to address the ITE of fluticasone furoate/vilanterol (FF/VI) versus control (placebo) on exacerbation rate and developed a novel metric, Q-score, for assessing the power of causal inference models. We then validated the methodology on 5990 subjects from the InforMing the PAthway of COPD Treatment (IMPACT) trial (NCT02164513) to estimate the ITE of FF/umeclidinium/VI (FF/UMEC/VI) versus UMEC/VI on exacerbation rate. We used Causal Forest as causal inference model. RESULTS: In SUMMIT, Causal Forest was optimised on the training set (n=5705) and tested on 2446 subjects (Q-score 0.61). In IMPACT, Causal Forest was optimised on 4193 subjects in the training set and tested on 1797 individuals (Q-score 0.21). In both trials, the quantiles of patients with the strongest ITE consistently demonstrated the largest reductions in observed exacerbations rates (0.54 and 0.53, p<0.001). Poor lung function and blood eosinophils, respectively, were the strongest predictors of ITE. CONCLUSIONS: This study shows that ML models for causal inference can be used to identify individual response to different COPD treatments and highlight treatment traits. Such models could become clinically useful tools for individual treatment decisions in COPD.
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Pulmón , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Administración por Inhalación , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Androstadienos/uso terapéutico , Androstadienos/farmacología , Alcoholes Bencílicos/uso terapéutico , Alcoholes Bencílicos/farmacología , Clorobencenos/uso terapéutico , Clorobencenos/farmacología , Broncodilatadores/uso terapéutico , Combinación de Medicamentos , Método Doble Ciego , Resultado del Tratamiento , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
BACKGROUND: Parameters from maximal expiratory flow-volume curves (MEFVC) have been linked to CT-based parameters of COPD. However, the association between MEFVC shape and phenotypes like emphysema, small airways disease (SAD) and bronchial wall thickening (BWT) has not been investigated. RESEARCH QUESTION: We analyzed if the shape of MEFVC can be linked to CT-determined emphysema, SAD and BWT in a large cohort of COPDGene participants. STUDY DESIGN AND METHODS: In the COPDGene cohort, we used principal component analysis (PCA) to extract patterns from MEFVC shape and performed multiple linear regression to assess the association of these patterns with CT parameters over the COPD spectrum, in mild and moderate-severe COPD. RESULTS: Over the entire spectrum, in mild and moderate-severe COPD, principal components of MEFVC were important predictors for the continuous CT parameters. Their contribution to the prediction of emphysema diminished when classical pulmonary function test parameters were added. For SAD, the components remained very strong predictors. The adjusted R2 was higher in moderate-severe COPD, while in mild COPD, the adjusted R2 for all CT outcomes was low; 0.28 for emphysema, 0.21 for SAD and 0.19 for BWT. INTERPRETATION: The shape of the maximal expiratory flow-volume curve as analyzed with PCA is not an appropriate screening tool for early disease phenotypes identified by CT scan. However, it contributes to assessing emphysema and SAD in moderate-severe COPD.
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Enfisema , Enfermedad Pulmonar Obstructiva Crónica , Enfisema Pulmonar , Humanos , Análisis de Componente Principal , Fumar , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/genética , Espirometría , Fenotipo , Volumen Espiratorio ForzadoRESUMEN
Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.
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Glicoproteínas , Schistosoma mansoni , Animales , Proteínas de Unión al ADN , Epítopos/química , Fucosa/metabolismo , Glicoproteínas/metabolismo , Humanos , Inmunoglobulina M , Mamíferos/metabolismo , Proteínas de la Membrana , Ratones , Polisacáridos/química , Schistosoma mansoni/química , Schistosoma mansoni/metabolismoRESUMEN
RATIONALE: While American Thoracic Society (ATS)/European Respiratory Society (ERS) quality control criteria for spirometry include several quantitative limits, it also requires manual visual inspection. The current approach is time consuming and leads to high intertechnician variability. We propose a deep-learning approach called convolutional neural network (CNN), to standardise spirometric manoeuvre acceptability and usability. METHODS AND METHODS: In 36â873 curves from the National Health and Nutritional Examination Survey USA 2011-2012, technicians labelled 54% of curves as meeting ATS/ERS 2005 acceptability criteria with satisfactory start and end of test, but identified 93% of curves with a usable forced expiratory volume in 1â s. We processed raw data into images of maximal expiratory flow-volume curve (MEFVC), calculated ATS/ERS quantifiable criteria and developed CNNs to determine manoeuvre acceptability and usability on 90% of the curves. The models were tested on the remaining 10% of curves. We calculated Shapley values to interpret the models. RESULTS: In the test set (n=3738), CNN showed an accuracy of 87% for acceptability and 92% for usability, with the latter demonstrating a high sensitivity (92%) and specificity (96%). They were significantly superior (p<0.0001) to ATS/ERS quantifiable rule-based models. Shapley interpretation revealed MEFVC<1â s (MEFVC pattern within first second of exhalation) and plateau in volume-time were most important in determining acceptability, while MEFVC<1â s entirely determined usability. CONCLUSION: The CNNs identified relevant attributes in spirometric curves to standardise ATS/ERS manoeuvre acceptability and usability recommendations, and further provides individual manoeuvre feedback. Our algorithm combines the visual experience of skilled technicians and ATS/ERS quantitative rules in automating the critical phase of spirometry quality control.
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Aprendizaje Profundo , Algoritmos , Espiración , Volumen Espiratorio Forzado , Humanos , Espirometría , Estados Unidos , Capacidad VitalAsunto(s)
Pólipos Nasales , Neutrófilos , Trampas Extracelulares/inmunología , Femenino , Galectinas/inmunología , Humanos , Inflamasomas/inmunología , Inflamación , Interleucina-1beta/inmunología , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Pólipos Nasales/inmunología , Pólipos Nasales/patología , Neutrófilos/inmunología , Neutrófilos/patologíaRESUMEN
Glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram-negative bacteria, the molecular and structural basis of GSH import into Gram-positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS-I-leg of GSSG is accommodated tightly via extensive interactions contributed by the N- and C-terminal lobes of GshT, while the GS-II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine-sulfur or the glycine-carboxylate. Finally, we show that GshT primes glutathione import via the L-cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of L-cystine.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Glutatión/metabolismo , Bacterias Grampositivas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Streptococcus mutans/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Sitios de Unión , Transporte Biológico , Cristalografía , Cistina/metabolismo , Glutatión/análogos & derivados , Glutatión/química , Disulfuro de Glutatión/metabolismo , Bacterias Grampositivas/química , Bacterias Grampositivas/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Streptococcus mutans/química , Streptococcus mutans/genética , Streptococcus mutans/crecimiento & desarrolloRESUMEN
Spontaneous protein crystallization is a rare event, yet protein crystals are frequently found in eosinophil-rich inflammation. In humans, Charcot-Leyden crystals (CLCs) are made from galectin-10 (Gal10) protein, an abundant protein in eosinophils. Although mice do not encode Gal10 in their genome, they do form pseudo-CLCs, made from the chitinase-like proteins Ym1 and/or Ym2, encoded by Chil3 and Chil4 and made by myeloid and epithelial cells respectively. Here, we investigated the biological effects of pseudo-CLCs since their function is currently unknown. We produced recombinant Ym1 crystals which were shown to have identical crystal packing and structure by X-ray crystallography as in vivo native crystals derived from murine lung. When administered to the airways of mice, crystalline but not soluble Ym1 stimulated innate and adaptive immunity and acted as a type 2 immune adjuvant for eosinophilic inflammation via triggering of dendritic cells (DCs). Murine Ym1 protein crystals found at sites of eosinophilic inflammation reinforce type 2 immunity and could serve as a surrogate model for studying the biology of human CLCs.
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Inmunidad Adaptativa , Quitinasas , Animales , Humanos , Ratones , Adyuvantes Inmunológicos , Cristalización , InflamaciónRESUMEN
The class III receptor tyrosine kinase (RTKIII) Fms-like tyrosine kinase receptor 3 (Flt3) and its cytokine ligand (FL) play central roles in hematopoiesis and the immune system, by establishing signaling cascades crucial for the development and homeostasis of hematopoietic progenitors and antigen-presenting dendritic cells. However, Flt3 is also one of the most frequently mutated receptors in hematologic malignancies and is currently a major prognostic factor and clinical target for acute myeloid leukemia. Here, we report the structural basis for the Flt3 ligand-receptor complex and unveil an unanticipated extracellular assembly unlike any other RTKIII/V complex characterized to date. FL induces dimerization of Flt3 via a remarkably compact binding epitope localized at the tip of extracellular domain 3 of Flt3, and it invokes a ternary complex devoid of homotypic receptor interactions. Comparisons of Flt3 with homologous receptors and available mutagenesis data for FL have allowed us to rationalize the unique features of the Flt3 extracellular assembly. Furthermore, thermodynamic dissection of complex formation points to a pronounced enthalpically driven binding event coupled to an entropic penalty. Together, our data suggest that the high-affinity Flt3:FL complex is driven in part by a single preformed binding epitope on FL reminiscent of a "lock-and-key" binding mode, thereby setting the stage for antagonist design.
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Citocinas/química , Citocinas/metabolismo , Hematopoyesis/fisiología , Transducción de Señal/fisiología , Tirosina Quinasa 3 Similar a fms , Secuencia de Aminoácidos , Cristalografía por Rayos X , Espacio Extracelular/química , Espacio Extracelular/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos , Ligandos , Datos de Secuencia Molecular , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Relación Estructura-Actividad , Termodinámica , Tirosina Quinasa 3 Similar a fms/química , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Intriguingly, the signaling-competent isoform of LEP-R is only lowly abundant amid several inactive short LEP-R isoforms contributing to a mechanistic conundrum. Here we show by X-ray crystallography and cryo-EM that, in contrast to long-standing paradigms, Leptin induces type I cytokine receptor assemblies featuring 3:3 stoichiometry and demonstrate such Leptin-induced trimerization of LEP-R on living cells via single-molecule microscopy. In mediating these assemblies, Leptin undergoes drastic restructuring that activates its site III for binding to the Ig domain of an adjacent LEP-R. These interactions are abolished by mutations linked to obesity. Collectively, our study provides the structural and mechanistic framework for how evolutionarily conserved Leptin:LEP-R assemblies with 3:3 stoichiometry can engage distinct LEP-R isoforms to achieve signaling.
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Adipoquinas , Leptina , Leptina/genética , Leptina/metabolismo , Leptina/farmacología , Isoformas de Proteínas/genética , Transducción de SeñalRESUMEN
Background and aims: Pulmonary hypertension due to left heart disease (PH-LHD) is the most frequent form of PH. As differential diagnosis with pulmonary arterial hypertension (PAH) has therapeutic implications, it is important to accurately and noninvasively differentiate PH-LHD from PAH before referral to PH centres. The aim was to develop and validate a machine learning (ML) model to improve prediction of PH-LHD in a population of PAH and PH-LHD patients. Methods: Noninvasive PH-LHD predictors from 172 PAH and 172 PH-LHD patients from the PH centre database at the University Hospitals of Leuven (Leuven, Belgium) were used to develop an ML model. The Jacobs score was used as performance benchmark. The dataset was split into a training and test set (70:30) and the best model was selected after 10-fold cross-validation on the training dataset (n=240). The final model was externally validated using 165 patients (91 PAH, 74 PH-LHD) from Erasme Hospital (Brussels, Belgium). Results: In the internal test dataset (n=104), a random forest-based model correctly diagnosed 70% of PH-LHD patients (sensitivity: n=35/50), with 100% positive predicted value, 78% negative predicted value and 100% specificity. The model outperformed the Jacobs score, which identified 18% (n=9/50) of the patients with PH-LHD without false positives. In external validation, the model had 64% sensitivity at 100% specificity, while the Jacobs score had a sensitivity of 3% for no false positives. Conclusions: ML significantly improves the sensitivity of PH-LHD prediction at 100% specificity. Such a model may substantially reduce the number of patients referred for invasive diagnostics without missing PAH diagnoses.
RESUMEN
Short-chain dehydrogenases/reductases (SDRs) are a rapidly expanding superfamily of enzymes that are found in all kingdoms of life. Hallmarked by a highly conserved Asn-Ser-Tyr-Lys catalytic tetrad, SDRs have a broad substrate spectrum and play diverse roles in key metabolic processes. Locus tag VVA1599 in Vibrio vulnificus encodes a short-chain dehydrogenase (hereafter referred to as SDRvv) which lacks the signature catalytic tetrad of SDR members. Structure-based protein sequence alignments have suggested that SDRvv may harbour a unique binding site for its nicotinamide cofactor. To date, structural studies of SDRs with altered catalytic centres are underrepresented in the scientific literature, thus limiting understanding of their spectrum of substrate and cofactor preferences. Here, the expression, purification and crystallization of recombinant SDRvv are presented. Two well diffracting crystal forms could be obtained by cocrystallization in the presence of the reduced form of the phosphorylated nicotinamide cofactor NADPH. The collected data were of sufficient quality for successful structure determination by molecular replacement and subsequent refinement. This work sets the stage for deriving the identity of the natural substrate of SDRvv and the structure-function landscape of typical and atypical SDRs.
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Biocatálisis , Oxidorreductasas/química , Vibrio vulnificus/enzimología , Cristalización , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
The extracellular complex between the haematopoietic receptor Flt3 and its cytokine ligand (FL) is the cornerstone of signalling cascades that are central to early haematopoiesis and the immune system. Here, efficient protocols for the production of two ectodomain variants of human Flt3 receptor, Flt3D1-D5 and Flt3D1-D4, for structural studies are reported based on tetracycline-inducible stable cell lines in HEK293S cells deficient in N-acetylglycosaminyltransferase I (GnTI-/-) that can secrete the target proteins with limited and homogeneous N-linked glycosylation to milligram amounts. The ensuing preparative purification of Flt3 receptor-ligand complexes yielded monodisperse complex preparations that were amenable to crystallization. Crystals of the Flt3D1-D4-FL and Flt3D1-D5-FL complexes diffracted to 4.3 and 7.8â Å resolution, respectively, and exhibited variable diffraction quality even within the same crystal. The resulting data led to the successful structure determination of Flt3D1-D4-FL via a combination of molecular-replacement and density-modification protocols exploiting the noncrystallographic symmetry and high solvent content of the crystals.
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Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tirosina Quinasa 3 Similar a fms/química , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Cristalografía por Rayos X , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Conformación Proteica , Proteínas Recombinantes/genética , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Protein crystals derived from innate immune cells have been synonymous with a Type-2 immune response in both mouse and man for over 150 years. Eosinophilic Galectin-10 (Charcot-Leyden) crystals in humans, and Ym1/Ym2 crystals in mice are frequently found in the context of parasitic infections, but also in diseases such as asthma and chronic rhinosinusitis. Despite their notable presence, these crystals are often overlooked as trivial markers of Type-2 inflammation. Here, we discuss the source, context, and role of protein crystallization. We focus on similarities observed between Galectin-10 and Ym1/2 crystals in driving immune responses; the subsequent benefit to the host during worm infection, and conversely the detrimental exacerbation of inflammation and mucus production during asthma.
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Alérgenos/inmunología , Hipersensibilidad/etiología , Inmunidad , Proteínas/inmunología , Animales , Biomarcadores , Cristalinas/inmunología , Susceptibilidad a Enfermedades , Humanos , Hipersensibilidad/metabolismo , Mediadores de Inflamación/metabolismo , Ratones , Especificidad de Órganos/inmunología , Proteínas/químicaRESUMEN
Antibody fragments are promising building blocks for developing targeted therapeutics, thus improving treatment efficacy while minimising off-target toxicity. Despite recent advances in targeted therapeutics, patients with Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), a high-risk malignancy, lack specific and effective targeted treatments. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in 50% of Ph-like ALL cases, conferring the survival of leukemia blasts through activation of the JAK/STAT signalling pathway. Targeting such a vital cell-surface protein could result in potent anti-leukaemic efficacy and reduce the likelihood of relapse associated with antigen loss. Herein, we developed a novel single-chain variable fragment (scFv) against CRLF2 based on a monoclonal antibody raised against the recombinant extracellular domain of human TSLPRα chain. The scFv fragment demonstrated excellent binding affinity with CRLF2 protein in the nanomolar range. Cellular association studies in vitro using an inducible CRLF2 knockdown cell line and ex vivo using patient-derived xenografts revealed the selective association of the scFv with CRLF2. The fragment exhibited significant receptor antagonistic effects on STAT5 signalling, suggesting possible therapeutic implications in vivo. This study is the first to describe the potential use of a novel scFv for targeting Ph-like ALL.
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Fragmentos de Inmunoglobulinas/metabolismo , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Línea Celular Tumoral , Niño , Endocitosis , Células HEK293 , Humanos , Ratones , Fosforilación , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Anticuerpos de Cadena Única/aislamiento & purificaciónRESUMEN
Cytokines are small secreted proteins that among many functions also play key roles in the orchestration of inflammation in host defense and disease. Over the past years, a large number of biologics have been developed to target cytokines in disease, amongst which soluble receptor fusion proteins have shown some promise in pre-clinical studies. We have previously shown proof-of-concept for the therapeutic targeting of interleukin (IL)-33 in airway inflammation using a newly developed biologic, termed IL-33trap, comprising the ectodomains of the cognate receptor ST2 and the co-receptor IL-1RAcP fused into a single-chain recombinant fusion protein. Here we extend the biophysical and biological characterization of IL-33trap variants, and show that IL-33trap is a stable protein with a monomeric profile both at physiological temperatures and during liquid storage at 4°C. Reducing the N-glycan heterogeneity and complexity of IL-33trap via GlycoDelete engineering neither affects its stability nor its inhibitory activity against IL-33. We also report that IL-33trap specifically targets biologically active IL-33 splice variants. Finally, we document the generation and antagonistic activity of a single-chain IL-4/13trap, which inhibits both IL-4 and IL-13 signaling. Collectively, these results illustrate that single-chain soluble receptor fusion proteins against IL-4, IL-13, and IL-33 are novel biologics that might not only be of interest for research purposes and further interrogation of the role of their target cytokines in physiology and disease, but may also complement monoclonal antibodies for the treatment of allergic and other inflammatory diseases.
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Antiinflamatorios , Interleucina-33/antagonistas & inhibidores , Proteínas Recombinantes de Fusión , Células HEK293 , Humanos , Interleucina-13/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidoresRESUMEN
Flt3 ligand (FL) is an early-acting hematopoietic cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells by activating its cognate receptor, Flt3. Recently, FL was shown to potently contribute to the development and expansion of antigen-presenting dendritic cells and CD34(+) natural killer cell progenitors in vivo. Here, we report a comprehensive method for the production of bioactive recombinant human FL (rhFL) in E. coli, suitable for structural, biophysical and physiological studies. A soluble form of human FL capable of binding to the Ftl3 receptor could be overexpressed in the E. coli strain Rosetta-gami(DE3) as inclusion bodies. We have established protocols for the efficient in vitro refolding and ensuing purification of rhFL to homogeneity (>95%), with yields approaching 5 mg of pure rhFL per liter of culture. The ability of rhFL to adopt a bioactive conformation was confirmed via a cell-proliferation assay and the activation of the Flt3 receptor in the human leukemic cell line, OCI-AML3.