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Lipid nanoparticles (LNPs) have been demonstrated to hold great promise for the clinical advancement of RNA therapeutics. Continued exploration of LNPs for application in new disease areas requires identification and optimization of leads in a high throughput way. Currently available high throughput in vivo screening platforms are well suited to screen for cellular uptake but less so for functional cargo delivery. We report on a platform which measures functional delivery of LNPs using unique peptide 'barcodes'. We describe the design and selection of the peptide barcodes and the evaluation of these for the screening of LNPs. We show that proteomic analysis of peptide barcodes correlates with quantification and efficacy of barcoded reporter proteins both in vitro and in vivo and, that the ranking of selected LNPs using peptide barcodes in a pool correlates with ranking using alternative methods in groups of animals treated with individual LNPs. We show that this system is sensitive, selective, and capable of reducing the size of an in vivo study by screening up to 10 unique formulations in a single pool, thus accelerating the discovery of new technologies for mRNA delivery.
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Several reports have highlighted a potential role of autoreactive B-cells and autoantibodies that correlates with increased disease severity in patients with idiopathic pulmonary fibrosis (IPF). Here we show that patients with IPF have an altered B-cell phenotype and that those subjects who have autoantibodies against the intermediate filament protein periplakin (PPL) have a significantly worse outcome in terms of progression-free survival. Using a mouse model of lung fibrosis, we demonstrate that introducing antibodies targeting the endogenous protein PPL (mimicking naturally occurring autoantibodies seen in patients) directly in the lung increases lung injury, inflammation, collagen and fibronectin expression through direct activation of follicular dendritic cells, which in turn activates and drives proliferation of fibroblasts. This fibrocyte population was also observed in fibrotic foci of patients with IPF and was increased in peripheral blood of IPF patients compared to aged-matched controls. This study reiterates the complex and heterogeneous nature of IPF, identifying new pathways that may prove suitable for therapeutic intervention.
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Autoanticuerpos , Fibrosis Pulmonar Idiopática , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Pulmón/metabolismo , Progresión de la Enfermedad , Fibroblastos/metabolismoRESUMEN
Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.
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Artritis Reumatoide/metabolismo , COVID-19/terapia , Proteinosis Alveolar Pulmonar/inmunología , Surfactantes Pulmonares/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Artritis Reumatoide/terapia , Autoanticuerpos/química , Líquido del Lavado Bronquioalveolar , COVID-19/inmunología , Colina/análogos & derivados , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Inflamación , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteinosis Alveolar Pulmonar/genética , SARS-CoV-2/inmunología , TensoactivosRESUMEN
Viral respiratory tract infections are known triggers of asthma exacerbations in both adults and children. The current standard of care, inhaled CS (corticosteroids) and LABAs (long-acting ß2-adrenoceptor agonists), fails to prevent the loss of control that manifests as an exacerbation. In order to better understand the mechanisms underlying viral asthma exacerbations we established an in vivo model using the clinically relevant aeroallergen HDM (house dust mite) and the viral mimetic/TLR3 (Toll-like receptor 3) agonist poly(I:C). Poly(I:C) alone induced a similar neutrophilic inflammatory profile in the BAL (bronchoalveolar lavage) to that of HRV1b (human rhinovirus 1b) alone, accompanied by both elevated BAL KC (keratinocyte-derived chemokine) and IL-1ß (interleukin-1ß). When mice allergic to HDM were also challenged with poly(I:C) the neutrophilic inflammatory profile was exacerbated. Increased CD8(+) T-cell numbers, increased CD4(+) and CD8(+) cell activation and elevated KC and IL-1ß were observed. No increases in Th2 cytokines or the eosinophil chemoattractant CCL11 [chemokine (C-C motif) ligand 11], above those induced by HDM alone, were observed. The poly(I:C)-exacerbated neutrophilia did not translate into changes in AHR (airways hyper-responsiveness), indicating that in this model inflammation and AHR are two mechanistically independent events. To test the clinical relevance of this model CS sensitivity was assessed using prednisone, a synthetic oral CS used to manage exacerbations in asthmatic patients already on maximal doses of inhaled CS. The increased neutrophils, and accompanying cytokines/chemokines KC and IL-1ß induced by poly(I:C) challenge of HDM-sensitized and challenged mice were insensitive to oral prednisone therapy. In summary we have described a CS-resistant mouse model mimicking the key aspects of viral asthma exacerbation using the clinically relevant aeroallergen HDM and the viral mimic poly(I:C). This model may provide better understanding of disease mechanisms underlying viral exacerbations and could be used to build early confidence in novel therapeutic axes targeting viral asthma exacerbations in Th2 asthmatics.
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Asma/inmunología , Modelos Animales de Enfermedad , Infecciones por Picornaviridae/inmunología , Rhinovirus/inmunología , Animales , Asma/tratamiento farmacológico , Asma/virología , Hiperreactividad Bronquial/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Quimiocinas/inmunología , Femenino , Glucocorticoides/uso terapéutico , Células HeLa , Interacciones Huésped-Patógeno/inmunología , Humanos , Interleucina-1beta/inmunología , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Infecciones por Picornaviridae/virología , Neumonía/inmunología , Poli I-C/inmunología , Prednisona/uso terapéutico , Pyroglyphidae/inmunología , Rhinovirus/fisiología , Receptor Toll-Like 3/inmunologíaRESUMEN
Introduction: Oxyntomodulin (Oxm) hormone peptide has a number of beneficial effects on nutrition and metabolism including increased energy expenditure and reduced body weight gain. Despite its many advantages as a potential therapeutic agent, Oxm is subjected to rapid renal clearance and protease degradation limiting its clinical application. Previously, we have shown that subcutaneous administration of a fibrillar Oxm formulation can significantly prolong its bioactivity in vivo from a few hours to a few days. Methods: We used a protease resistant analogue of Oxm, Aib2-Oxm, to form nanfibrils depot and improve serum stability of released peptide. The nanofibrils and monomeric peptide in solution were characterized by spectroscopic, microscopic techniques, potency assay, QCM-D and in vivo studies. Results: We show that in comparison to Oxm, Aib2-Oxm fibrils display a slower elongation rate requiring higher ionic strength solutions, and a higher propensity to dissociate. Upon subcutaneous administration of fibrillar Aib2-Oxm in rodents, a 5-fold increase in bioactivity relative to fibrillar Oxm and a significantly longer bioactivity than free Aib2-Oxm were characterized. Importantly, a decrease in food intake was observed up to 72-hour post-administration, which was not seen for free Aib2-Oxm. Conclusion: Our findings provides compelling evidence for the development of long-lasting peptide fibrillar formulations that yield extended plasma exposure and enhanced in vivo pharmacological response.
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Péptido 1 Similar al Glucagón , Glucagón , Ingestión de Alimentos/fisiología , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Oxintomodulina/química , Oxintomodulina/farmacología , Péptido Hidrolasas , Péptidos/farmacología , Receptores de Glucagón/metabolismo , AnimalesRESUMEN
Interleukin (IL)-33 is a broad-acting alarmin cytokine that can drive inflammatory responses following tissue damage or infection and is a promising target for treatment of inflammatory disease. Here, we describe the identification of tozorakimab (MEDI3506), a potent, human anti-IL-33 monoclonal antibody, which can inhibit reduced IL-33 (IL-33red) and oxidized IL-33 (IL-33ox) activities through distinct serum-stimulated 2 (ST2) and receptor for advanced glycation end products/epidermal growth factor receptor (RAGE/EGFR complex) signalling pathways. We hypothesized that a therapeutic antibody would require an affinity higher than that of ST2 for IL-33, with an association rate greater than 107 M-1 s-1, to effectively neutralize IL-33 following rapid release from damaged tissue. An innovative antibody generation campaign identified tozorakimab, an antibody with a femtomolar affinity for IL-33red and a fast association rate (8.5 × 107 M-1 s-1), which was comparable to soluble ST2. Tozorakimab potently inhibited ST2-dependent inflammatory responses driven by IL-33 in primary human cells and in a murine model of lung epithelial injury. Additionally, tozorakimab prevented the oxidation of IL-33 and its activity via the RAGE/EGFR signalling pathway, thus increasing in vitro epithelial cell migration and repair. Tozorakimab is a novel therapeutic agent with a dual mechanism of action that blocks IL-33red and IL-33ox signalling, offering potential to reduce inflammation and epithelial dysfunction in human disease.
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Inflamación , Proteína 1 Similar al Receptor de Interleucina-1 , Ratones , Humanos , Animales , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Inflamación/metabolismo , Interleucina-33/metabolismo , Citocinas/metabolismo , Receptores ErbB/metabolismo , Transducción de SeñalRESUMEN
Diabetes mellitus is a major healthcare challenge. Pramlintide, a peptide analogue of the hormone amylin, is currently used as an adjunct with insulin for patients who fail to achieve glycemic control with only insulin therapy. However, hypoglycemia is the dominant risk factor associated with such approaches and careful dosing of both drugs is needed. To mitigate this risk factor and compliance issues related to multiple dosing of different drugs, sustained delivery of Pramlintide from silica depot administered subcutaneously (SC) was investigated in a rat model. The pramlintide-silica microparticle hydrogel depot was formulated by spray drying of silica sol-gels. In vitro dissolution tests revealed an initial burst of pramlintide followed by controlled release due to the dissolution of the silica matrix. At higher dosing, pramlintide released from subcutaneously administered silica depot in rats showed a steady concentration of 500 pM in serum for 60 days. Released pramlintide retained its pharmacological activity in vivo, as evidenced by loss of weight. The biodegradable silica matrix offers a sustained release of pramlintide for at least two months in the rat model and shows potential for clinical applications.
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Combination therapy in Type 2 Diabetes Mellitus is necessary to achieve tight glycaemic control and reduce complication risk. Current treatment plans require patients to take several drugs concomitantly leading to low therapy adherence. This study describes the development and characterisation of a stable parenteral co-formulation of a sodium glucose co-transporter 2 inhibitor (dapagliflozin) and a therapeutic lipidated peptide, using hydroxypropyl-ß-cyclodextrin as an enabling excipient. Using NMR, calorimetry, computational modelling and spectroscopic methods, we show that besides increasing the solubility of dapagliflozin, cyclodextrin prevents self-association of the peptide through interaction with the lipid chain and amino acids prone to aggregation including aromatic groups and ionisable residues. While those interactions cause a dramatic secondary structure change, no impact on potency was seen in vitro. A subcutaneous administration of the co-formulation in rat showed that both drugs reach exposure levels previously shown to be efficacious in clinical mono-therapy studies. Interestingly, a faster absorption rate was observed for the peptide formulated within the cyclodextrin vehicle with respect to the buffer vehicle, which could trigger an earlier onset of action. The cyclodextrin based co-formulation is therefore a promising approach to develop a fixed dose combination of a therapeutic peptide and a small molecule drug for increased patient adherence and better blood glucose control.
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2-Hidroxipropil-beta-Ciclodextrina/química , Compuestos de Bencidrilo/farmacocinética , Glucemia/efectos de los fármacos , Excipientes/química , Glucósidos/farmacocinética , Hipoglucemiantes/farmacocinética , Péptidos/farmacocinética , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacocinética , Animales , Compuestos de Bencidrilo/química , Glucemia/metabolismo , Células CHO , Cricetulus , Combinación de Medicamentos , Composición de Medicamentos , Absorción Gastrointestinal , Glucósidos/química , Hipoglucemiantes/química , Inyecciones Subcutáneas , Masculino , Péptidos/administración & dosificación , Péptidos/química , Agregado de Proteínas , Estructura Secundaria de Proteína , Ratas , Inhibidores del Cotransportador de Sodio-Glucosa 2/química , SolubilidadRESUMEN
GM-CSF is important in regulating acute, persistent neutrophilic inflammation in certain settings, including lung injury. Ligand binding induces rapid internalization of the GM-CSF receptor (GM-CSFRα) complex, a process essential for signaling. Whereas GM-CSF controls many aspects of neutrophil biology, regulation of GM-CSFRα expression is poorly understood, particularly the role of GM-CSFRα in ligand clearance and whether signaling is sustained despite major down-regulation of GM-CSFRα surface expression. We established a quantitative assay of GM-CSFRα surface expression and used this, together with selective anti-GM-CSFR antibodies, to define GM-CSFRα kinetics in human neutrophils, and in murine blood and alveolar neutrophils in a lung injury model. Despite rapid sustained ligand-induced GM-CSFRα loss from the neutrophil surface, which persisted even following ligand removal, pro-survival effects of GM-CSF required ongoing ligand-receptor interaction. Neutrophils recruited to the lungs following LPS challenge showed initially high mGM-CSFRα expression, which along with mGM-CSFRß declined over 24 hr; this was associated with a transient increase in bronchoalveolar lavage fluid (BALF) mGM-CSF concentration. Treating mice in an LPS challenge model with CAM-3003, an anti-mGM-CSFRα mAb, inhibited inflammatory cell influx into the lung and maintained the level of BALF mGM-CSF. Consistent with neutrophil consumption of GM-CSF, human neutrophils depleted exogenous GM-CSF, independent of protease activity. These data show that loss of membrane GM-CSFRα following GM-CSF exposure does not preclude sustained GM-CSF/GM-CSFRα signaling and that this receptor plays a key role in ligand clearance. Hence neutrophilic activation via GM-CSFR may play an important role in neutrophilic lung inflammation even in the absence of high GM-CSF levels or GM-CSFRα expression.
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Lesión Pulmonar Aguda/inmunología , Regulación de la Expresión Génica/inmunología , Neutrófilos/inmunología , Alveolos Pulmonares/inmunología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Adulto , Animales , Línea Celular Tumoral , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neutrófilos/patología , Alveolos Pulmonares/patología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factores de TiempoRESUMEN
Interleukin (IL)-33 is an IL-1 family alarmin released from damaged epithelial and endothelial barriers to elicit immune responses and allergic inflammation via its receptor ST2. Serine proteases released from neutrophils, mast cells and cytotoxic lymphocytes have been proposed to process the N-terminus of IL-33 to enhance its activity. Here we report that processing of full length IL-33 can occur in mice deficient in these immune cell protease activities. We sought alternative mechanisms for the proteolytic activation of IL-33 and discovered that exogenous allergen proteases and endogenous calpains, from damaged airway epithelial cells, can process full length IL-33 and increase its alarmin activity up to ~60-fold. Processed forms of IL-33 of apparent molecular weights ~18, 20, 22 and 23 kDa, were detected in human lungs consistent with some, but not all, proposed processing sites. Furthermore, allergen proteases degraded processed forms of IL-33 after cysteine residue oxidation. We suggest that IL-33 can sense the proteolytic and oxidative microenvironment during tissue injury that facilitate its rapid activation and inactivation to regulate the duration of its alarmin function.
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Alarminas/metabolismo , Alérgenos/metabolismo , Inmunidad Innata , Interleucina-33/metabolismo , Necrosis/patología , Proteolisis , Mucosa Respiratoria/patología , Animales , Calpaína/metabolismo , Línea Celular , Humanos , Interleucina-33/química , Pulmón/patología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Biológicos , Peso MolecularRESUMEN
The use of peptides as therapeutic agents is undergoing a renaissance with the expectation of new drugs with enhanced levels of efficacy and safety. Their clinical potential will be only fully realised once their physicochemical and pharmacokinetic properties have been precisely controlled. Here we demonstrate a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo. We show that oxyntomodulin, a peptide with potential to treat obesity and diabetes, self-assembles into a stable nanofibril formulation which subsequently dissociates to release active peptide and produces a pharmacological effect in vivo. The subcutaneous administration of the nanofibrils in rats results in greatly prolonged exposure, with a constant oxyntomodulin bioactivity detectable in serum for at least 5 days as compared to free oxyntomodulin which is undetectable after only 4 h. Such an approach is simple, cost-efficient and generic in addressing the limitations of peptide therapeutics.
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Obesidad/tratamiento farmacológico , Oxintomodulina/farmacocinética , Hormonas Peptídicas/farmacocinética , Animales , Glucosa/metabolismo , Inyecciones Subcutáneas , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Oxintomodulina/administración & dosificación , Oxintomodulina/sangre , Oxintomodulina/química , Hormonas Peptídicas/administración & dosificación , Hormonas Peptídicas/sangre , Hormonas Peptídicas/química , Ratas , Ratas Sprague-DawleyRESUMEN
Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.
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Anticuerpos Monoclonales de Origen Murino/inmunología , Especificidad de Anticuerpos , Epítopos de Linfocito T/inmunología , Receptores de Interleucina/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Asma/tratamiento farmacológico , Asma/inmunología , Asma/patología , Línea Celular Transformada , Toxina Diftérica/química , Toxina Diftérica/inmunología , Epítopos de Linfocito T/química , Femenino , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Ratones , Ratones Endogámicos BALB C , Ratas , Receptores de Interleucina/química , Toxina Tetánica/química , Toxina Tetánica/inmunologíaRESUMEN
In response to infections and irritants, the respiratory epithelium releases the alarmin interleukin (IL)-33 to elicit a rapid immune response. However, little is known about the regulation of IL-33 following its release. Here we report that the biological activity of IL-33 at its receptor ST2 is rapidly terminated in the extracellular environment by the formation of two disulphide bridges, resulting in an extensive conformational change that disrupts the ST2 binding site. Both reduced (active) and disulphide bonded (inactive) forms of IL-33 can be detected in lung lavage samples from mice challenged with Alternaria extract and in sputum from patients with moderate-severe asthma. We propose that this mechanism for the rapid inactivation of secreted IL-33 constitutes a 'molecular clock' that limits the range and duration of ST2-dependent immunological responses to airway stimuli. Other IL-1 family members are also susceptible to cysteine oxidation changes that could regulate their activity and systemic exposure through a similar mechanism.
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Asma/inmunología , Interleucina-33/metabolismo , Receptores de Superficie Celular/inmunología , Receptores de Interleucina/inmunología , Animales , Asma/genética , Asma/metabolismo , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33/genética , Interleucina-33/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Oxidación-Reducción , Receptores de Superficie Celular/genética , Receptores de Interleucina/genéticaRESUMEN
Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling. Using rational engineering of the IgG4 Fc domain to disrupt key interactions at the CH3-CH3 interface, we identified a number of point mutations that abolish Fc dimerization and created half-antibodies, a novel monovalent antibody format that retains a monomeric Fc domain. Introduction of these mutations into an IgG1 framework also led to the creation of half-antibodies. These half-antibodies were shown to be soluble, thermodynamically stable and monomeric, characteristics that are favorable for use as therapeutic proteins. Despite significantly reduced FcRn binding in vitro, which suggests that avidity gains in a dimeric Fc are critical to optimal FcRn binding, this format demonstrated an increased terminal serum half-life compared with that expected for most alternative antibody fragments.