RESUMEN
There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT.
Asunto(s)
Antígenos de Plantas , Nanopartículas , Ratas , Humanos , Ratones , Animales , Polen , Inmunoglobulina E , Elastina , Micelas , Escherichia coli , Aluminio , Linfocitos T CD4-Positivos , Sales (Química) , Alérgenos , Inmunoglobulina G , Péptidos , CitocinasRESUMEN
Anionic liposomal formulations have previously shown to have intrinsic tolerogenic capacity and these properties have been related to the rigidity of the particles. The combination of highly rigid anionic liposomes to deliver tolerogenic adjuvants and antigen peptides has potential applications for the treatment of autoimmune and inflammatory diseases. However, the preparation of these highly rigid anionic liposomes using traditional methods such as lipid film hydration presents problems in terms of scalability and loading efficiency of some costly tolerogenic adjuvants like 1-α,25-dihydroxyvitaminD3. Here we propose the use of an off-the-shelf staggered herringbone micromixer for the preparation of these formulations and performed a systematic study on the effect of temperature and flow conditions on the size and polydispersity index of the formulations. Furthermore, we show that the system allows for the encapsulation of a wide variety of peptides and significantly higher loading efficiency of 1-α,25-dihydroxyvitaminD3 compared to the traditional lipid film hydration method, without compromising their non-inflammatory interaction with dendritic cells. Therefore, the microfluidics method presented here is a valuable tool for the preparation of highly rigid tolerogenic liposomes in a fast, size-tuneable and scalable manner.
Asunto(s)
Liposomas , Microfluídica , Adyuvantes Inmunológicos , Adyuvantes Farmacéuticos , Lípidos/química , Liposomas/química , Microfluídica/métodos , PéptidosRESUMEN
Subunit vaccines often contain colloidal aluminum salt-based adjuvants to activate the innate immune system. These aluminum salts consist of micrometer-sized aggregates. It is well-known that particle size affects the adjuvant effect of particulate adjuvants. In this study, the activation of human monocytes by hexagonal-shaped gibbsite (ø = 210 ± 40 nm) and rod-shaped boehmite (ø = 83 ± 827 nm) was compared with classical aluminum oxyhydroxide adjuvant (alum). To this end, human primary monocytes were cultured in the presence of alum, gibbsite, or boehmite. The transcriptome and proteome of the monocytes were investigated by using quantitative polymerase chain reaction and mass spectrometry. Human monocytic THP-1 cells were used to investigate the effect of the particles on cellular maturation, differentiation, activation, and cytokine secretion, as measured by flow cytometry and enzyme-linked immunosorbent assay. Each particle type resulted in a specific gene expression profile. IL-1ß and IL-6 secretion was significantly upregulated by boehmite and alum. Of the 7 surface markers investigated, only CD80 was significantly upregulated by alum and none by gibbsite or boehmite. Gibbsite hardly activated the monocytes. Boehmite activated human primary monocytes equally to alum, but induced a much milder stress-related response. Therefore, boehmite was identified as a promising adjuvant candidate.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Hidróxido de Aluminio/farmacología , Óxido de Aluminio/farmacología , Inmunidad Innata/efectos de los fármacos , Monocitos/efectos de los fármacos , Adyuvantes Inmunológicos/química , Hidróxido de Aluminio/química , Óxido de Aluminio/química , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Diferenciación Celular/efectos de los fármacos , Coloides , Composición de Medicamentos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Tamaño de la Partícula , Células THP-1 , TranscriptomaRESUMEN
Aluminium phosphate is a commonly used adjuvant consisting of heterogeneously sized aggregates up to several micrometers. However, aluminium phosphate nanoparticles may exhibit an improved adjuvant effect. In this study, nanoparticles were made by sonication of commercially available aluminium phosphate adjuvant, resulting in particles with a size (Z-average diameter) between 200-300â¯nm and a point of zero charge of 4.5. To prevent reaggregation, which occurred within 14 days, a screening of excipients was performed to identify stabilisers effective under physiological conditions (pH 7.4, 290 mOsm). The amino acids threonine, asparagine, and L-alanyl-L-1-aminoethylphosphonic acid (LAPA) stabilised sonicated aluminium phosphate. Particle sizes remained stable between 400-600â¯nm at 37⯰C during 106 days. Contrarily, arginine induced strong reaggregation to a particle size larger than 1000â¯nm. The stability of aluminium phosphate nanoparticles was strongly affected by the pH. Aggregation mainly occurred below pH 7. The adsorption capacity, a potentially relevant parameter for adjuvants, was slightly reduced in the presence of asparagine, when using a model antigen (lysozyme). LAPA, arginine, threonine and aspartic acid reduced protein adsorption significantly. The adjuvant effect of aluminium phosphate nanoparticles was studied by immunisation of mice with diphtheria toxoid adjuvanted with the aluminium phosphate nanoparticles. The presence of LAPA, threonine, aspartic acid or asparagine did not alter diphtheria toxoid-specific antibody or toxin-neutralising antibody titres. Arginine increased diphtheria toxoid-specific antibody titres but not toxin-neutralising antibody titres. In conclusion, aluminium phosphate nanoparticles were stabilised by particular amino acids and induced an adjuvant effect comparable to that of aluminium phosphate microparticles.
Asunto(s)
Adyuvantes Inmunológicos , Compuestos de Aluminio/química , Toxoide Diftérico/química , Nanopartículas/química , Fosfatos/química , Compuestos de Aluminio/inmunología , Animales , Toxoide Diftérico/inmunología , Ratones , Tamaño de la Partícula , Fosfatos/inmunología , Propiedades de SuperficieRESUMEN
Worldwide, enteric infections rank third among all causes of disease burdens, and vaccines able to induce a strong and long-lasting intestinal immune responses are needed. Parenteral immunization generally do not generate intestinal IgA. Recently, however, injections of retinoic acid (RA) dissolved in oil, administered multiple times before vaccination to precondition the vaccine-draining lymph nodes, enabled a parenteral vaccine strategy to induce intestinal IgA. As multiple injections of RA before vaccination is not an attractive strategy for clinical practice, we aimed to develop a "one injection" vaccine formulation that upon parenteral administration induced intestinal IgA. Our vaccine formulation contained two liposomal delivery systems. One delivery system, based on 1,2-distearoyl- sn-glycero-3-phosphocholine stabilized with PEG, was designed to exhibit fast drainage of RA to local lymph nodes to precondition these for a mucosal immune response before being subjected to the vaccine antigen. The other delivery system, based on the cationic liposomal adjuvant CAF01 stabilized with cholesterol, was optimized for prolonged delivery of the antigen by migratory antigen-presenting cells to the preconditioned lymph node. Combined we call the adjuvant CAF23. We show that CAF23, administered by the subcutaneous route induces an antigen specific intestinal IgA response, making it a promising candidate adjuvant for vaccines against enteric diseases.
Asunto(s)
Adyuvantes Inmunológicos/química , Sistemas de Liberación de Medicamentos , Inmunidad Mucosa/inmunología , Inmunoglobulina A/inmunología , Liposomas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Rastreo Diferencial de Calorimetría , Femenino , Inmunización , Inyecciones Subcutáneas , Liposomas/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
PURPOSE: To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO). METHODS: Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins. RESULTS: The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified. CONCLUSION: The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos Inmunológicos/química , Cobre/química , Inmunoglobulina G/química , Agregado de Proteínas , Rituximab/química , Ácido Ascórbico/química , Ácido Ascórbico/farmacología , Catálisis , Humanos , Modelos Moleculares , Oxidación-Reducción/efectos de los fármacos , Agregado de Proteínas/efectos de los fármacos , SolucionesRESUMEN
Nanoparticle tracking analysis (NTA) has attracted great interest for application in the field of submicron particle characterization for biopharmaceuticals. It has the virtue of direct sample visualization and particle-by-particle tracking, but the complexity of method development has limited its routine applicability. We systematically evaluated data collection and processing parameters as well as sample handling methods using shake-stressed protein samples. The camera shutter and gain were identified as the key factors influencing NTA results. We also demonstrated that sample filtration was necessary for NTA analysis if there were high numbers of micron particles, whereas the choice of filter membrane was critical for data quality. Sample dilution into corresponding formulation buffer did not affect particle size distributions in our study. Finally, NTA analysis exhibited excellent repeatability in intraday comparison of multiple measurements on the same sample and interday comparison on different batches of samples. Shaking-induced protein aggregation could also be sensitively monitored by NTA. In conclusion, NTA analysis can be used as a robust stability-indicating method for the characterization of proteinaceous submicron particles and thereby complement other analytical methods, provided that consistent sample handling and parametric settings are established for the specific case study.
Asunto(s)
Química Farmacéutica/métodos , Inmunoglobulina G/química , Nanopartículas/química , Tamaño de la Partícula , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Evaluación Preclínica de Medicamentos/métodos , Inmunoglobulina G/análisis , Nanopartículas/análisis , Proteínas/análisis , Proteínas/químicaRESUMEN
All therapeutic proteins are potentially immunogenic. Antibodies formed against these drugs can decrease efficacy, leading to drastically increased therapeutic costs and in rare cases to serious and sometimes life threatening side-effects. Many efforts are therefore undertaken to develop therapeutic proteins with minimal immunogenicity. For this, immunogenicity prediction of candidate drugs during early drug development is essential. Several in silico, in vitro and in vivo models are used to predict immunogenicity of drug leads, to modify potentially immunogenic properties and to continue development of drug candidates with expected low immunogenicity. Despite the extensive use of these predictive models, their actual predictive value varies. Important reasons for this uncertainty are the limited/insufficient knowledge on the immune mechanisms underlying immunogenicity of therapeutic proteins, the fact that different predictive models explore different components of the immune system and the lack of an integrated clinical validation. In this review, we discuss the predictive models in use, summarize aspects of immunogenicity that these models predict and explore the merits and the limitations of each of the models.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Proteínas/inmunología , Proteínas/uso terapéutico , Animales , Simulación por Computador , Humanos , Sistema Inmunológico/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Modelos BiológicosRESUMEN
Our study aimed to comparatively evaluate Micro-Flow Imaging (MFI) and the recently introduced technique of resonant mass measurement (Archimedes, RMM) as orthogonal methods for the quantitative differentiation of silicone oil droplets and protein particles. This distinction in the submicron and micron size range is highly relevant for the development of biopharmaceuticals, in particular for products in prefilled syringes. Samples of artificially generated silicone oil droplets and protein particles were quantified individually and in defined mixtures to assess the performance of the two techniques. The built-in MFI software solution proved to be suitable to discriminate between droplets and particles for sizes above 2 µm at moderate droplet/particle ratios (70:30-30:70). A customized filter developed specifically for this study greatly improved the results and enabled reliable discrimination also for more extreme mixing ratios (95:5-15:85). RMM showed highly accurate discrimination in the size range of about 0.5-2 µm independent of the ratio, provided that a sufficient number of particles (>50 counted particles) were counted. We recommend applying both techniques for a comprehensive analysis of biotherapeutics potentially containing silicone oil droplets and protein particles in the submicron and micron size range.
Asunto(s)
Proteínas/aislamiento & purificación , Aceites de Silicona/aislamiento & purificación , Procesamiento de Imagen Asistido por Computador , Tamaño de la Partícula , Programas InformáticosRESUMEN
An immune response to a therapeutic protein that compromises the biopharmaceutical activity or cross-reacts with an endogenous protein is a serious clinical event. The role of protein aggregates and particles in biopharmaceutical formulations in mediating this immune response has gained considerable attention over the recent past. Model systems that could consistently and reliably predict the relative immunogenicity of biopharmaceutical protein formulations would be extremely valuable. Several approaches have been developed in an attempt to provide this insight, including in silico algorithms, in vitro tests utilizing human leukocytes and in vivo animal models. This commentary provides an update of these various approaches as well as the author's perspectives on the pros and cons of these different methods.
Asunto(s)
Productos Biológicos/efectos adversos , Productos Biológicos/inmunología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Proteínas/efectos adversos , Proteínas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Animales , Formación de Anticuerpos/inmunología , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Evaluación Preclínica de Medicamentos , Humanos , Proteínas/farmacología , Proteínas/uso terapéutico , Vacunas Sintéticas/farmacología , Vacunas Sintéticas/uso terapéuticoRESUMEN
In the search for an optimal approach for the transcutaneous immunization (TCI) of hepatitis B surface antigen (HBsAg), two vesicle formulations, L595 vesicles (composed of sucrose-laurate ester and octaoxyethylene-laurate ester) and sPC vesicles (composed of soybean-phosphatidylcholine and Span-80) were prepared and characterized in vitro and in vivo. HBsAg was associated to the vesicles, resulting in sPC-HBsAg vesicles (±170nm) with 79% HBsAg association and L595-HBsAg vesicles (±75nm) with only 29% HBsAg association. The vesicles induced in mice via TCI an antibody response only when the skin was pretreated with microneedles. This response was improved by the adjuvant cholera toxin. The sPC-HBsAg vesicle formulations showed to be the most immunogenic for TCI, which was related to the higher degree of HBsAg association.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B/prevención & control , Vacunación/métodos , Adyuvantes Inmunológicos/administración & dosificación , Administración Cutánea , Animales , Toxina del Cólera/administración & dosificación , Cromatografía en Gel , Femenino , Hepatitis B/sangre , Hepatitis B/inmunología , Hexosas/química , Inmunoglobulina G/sangre , Lauratos/química , Liposomas/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Tamaño de la Partícula , Fosfatidilcolinas/química , Polietilenglicoles/química , Piel/metabolismo , Sacarosa/químicaRESUMEN
Immunogenicity of therapeutic proteins lowers patient well-being and drastically increases therapeutic costs. Preventing immunogenicity is an important issue to consider when developing novel therapeutic proteins and applying them in the clinic. Animal models are increasingly used to study immunogenicity of therapeutic proteins. They are employed as predictive tools to assess different aspects of immunogenicity during drug development and have become vital in studying the mechanisms underlying immunogenicity of therapeutic proteins. However, the use of animal models needs critical evaluation. Because of species differences, predictive value of such models is limited, and mechanistic studies can be restricted. This review addresses the suitability of animal models for immunogenicity prediction and summarizes the insights in immunogenicity that they have given so far.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Modelos Animales , Proteínas/inmunología , Proteínas/farmacología , Animales , Productos Biológicos/inmunología , Productos Biológicos/farmacología , Humanos , Valor Predictivo de las PruebasRESUMEN
PEGylation has been suggested to improve the stability of insulin, but evidence for that is scarce. Here, we compared the forced aggregation behavior of insulin and mono-PEGylated insulin. Therefore, recombinant human insulin was conjugated on lysine B29 with 5-kDa PEG. PEG-insulin was purified by size-exclusion chromatography (SEC) and characterized by mass spectrometry (MS). Next, insulin and PEG-insulin were subjected to heating at 75 °C, metal-catalyzed oxidation, and glutaraldehyde cross-linking. The products were characterized physicochemically by complementary analytical methods. Mono-PEGylation of insulin was confirmed by SEC and MS. Under each of the applied stress conditions, insulin and PEG-insulin showed comparable degradation profiles. All the stressed samples showed submicron aggregates in the size range between 50 and 500 nm. Covalent aggregates and conformational changes were found for both oxidized products. Insulin and its PEGylated counterpart also exhibited similar characteristics when exposed to heat stress, that is, slightly changed secondary and tertiary structures, covalent aggregates with partially intact epitopes, and separation of chain A from chain B. Both glutaraldehyde-treated insulin and PEG-insulin contained covalent and noncovalent aggregates with intact epitopes, showed partially perturbed secondary structure, and substantial loss of tertiary structure. From these results, we conclude that PEGylation does not protect insulin against forced aggregation.