RESUMO
Protein- and peptide-based tumor vaccines depend on strong adjuvants to induce potent immune responses. Here, we demonstrated that a recently developed novel adjuvant based on a non-coding, long-chain RNA molecule, termed RNAdjuvant(®) , profoundly increased immunogenicity of both antigen formats. RNAdjuvant(®) induced balanced, long-lasting immune responses that resulted in a strong anti-tumor activity. A direct comparison to Poly(I:C) showed superior efficacy of our adjuvant to enhance antigen-specific multifunctional CD8(+) T-cell responses and mediate anti-tumor responses induced by peptide derived from HPV-16 E7 protein in the syngeneic TC-1 tumor, a murine model of human HPV-induced cervical cancer. Moreover, the adjuvant was able to induce functional memory responses that mediated complete tumor remission. Despite its remarkable immunostimulatory activity, our RNA-based adjuvant exhibited an excellent pre-clinical safety profile. It acted only locally at the injection site where it elicited a transient but strong up-regulation of pro-inflammatory and anti-viral cytokines as well as cytoplasmic RNA sensors without systemic cytokine release. This was followed by the activation of immune cells in the draining lymph nodes. Our data indicate that our RNA-based adjuvant is a safe and potent immunostimulator that may profoundly improve the efficacy of a variety of cancer vaccines.
Assuntos
Adjuvantes Imunológicos , Vacinas Anticâncer/imunologia , RNA Longo não Codificante/imunologia , Neoplasias do Colo do Útero/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/farmacologia , Linhagem Celular Transformada , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/imunologia , Peptídeos/imunologia , Peptídeos/farmacologia , Poli I-C/imunologia , Poli I-C/farmacologia , RNA Longo não Codificante/genética , Resultado do Tratamento , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
Biologicals including monoclonal antibodies are the current flagships in pharmaceutical industry. However, they are exposed to a multitude of destabilization conditions like for instance hydrophobic interfaces, leading to reduced biological activity. Polysorbates are commonly applied to effectively stabilize these active pharmaceutical ingredients against colloidal stress. Nevertheless, chemical instability of polysorbate via hydrolysis or oxidation results in degradation products that might form particles via phase separation. Polysorbates are mixtures of hundreds of individual components, and recently purer quality grades with reduced variations in the fatty acid composition are available. As the protective function of polysorbate itself is not completely understood, even less is known about its individual components, raising the question of the existence of a superior polysorbate species in respect to protein stabilization or degradation susceptibility. Here, we evaluated the protective function of four main fractions of polysorbate 20 (PS20) in agitation studies with monoclonal antibodies, followed by particle analysis as well as protein and polysorbate content determination. The commercially-available inherent mixtures PS20 high purity and PS20 all-laurate, as well as the fraction isosorbide-POE-monolaurate showed superior protection against mechanical-induced stress (visual inspection and turbidity) at the air-water interface in comparison to sole sorbitan-POE-monolaurate, -dilaurate, and -trilaurate. Fractions composed mainly of higher-order esters like sorbitan-POE-dilaurate and sorbitan-POE-trilaurate indicated high turbidities as indication for subvisible and small particles accompanied by a reduced protein monomer content after agitation. For the isosorbide-POE-monolaurates as well as for the inherent polysorbate mixtures no obvious differences in protein content and protein aggregation (SEC) were observed, reflecting the observations from visual appearance. However, absolute polysorbate concentrations vary drastically between different species in the actual formulations. As there are still open questions in respect to protein specificity or regarding mixtures versus individual components of PS20, further studies must be performed, to gain a better understanding of a "generalized" stabilizing effect of polysorbates on monoclonal antibodies. The knowledge of the characteristics of individual polysorbate species can have the potential to pave the way to superior detergents in respect to protein stabilization and/or degradation susceptibility.
Assuntos
Ácidos Graxos , Polissorbatos , Polissorbatos/química , Composição de Medicamentos , Oxirredução , Ácidos Graxos/química , Anticorpos Monoclonais/química , Tensoativos/químicaRESUMO
Lipoproteins from gram-positive and -negative bacteria, mycoplasma, and shorter synthetic lipopeptide analogues activate cells of the innate immune system via the Toll-like receptor TLR2/TLR1 or TLR2/TLR6 heterodimers. For this reason, these compounds constitute highly active adjuvants for vaccines either admixed or covalently linked. The lanthionine scaffold has structural similarity with the S-(2,3-dihydroxypropyl)cysteine core structure of the lipopeptides. Therefore, lanthionine-based lipopeptide amides were synthesized and probed for activity as potential TLR2 agonists or antagonists. A collection of analytically defined lipolanthionine peptide amides exhibited an inhibitory effect of the TLR2-mediated IL-8 secretion when applied in high molar excess to the agonistic synthetic lipopeptide Pam3Cys-Ser-(Lys)4-OH. Structure-activity relationships revealed the influence of the chirality of the two alpha-carbon atoms, the chain lengths of the attached fatty acids and fatty amines, and the oxidation level of the sulfur atom on the inhibitory activity of the lipolanthionine peptide amides.
Assuntos
Adjuvantes Imunológicos/síntese química , Alanina/análogos & derivados , Ácidos Graxos/química , Interleucina-8/antagonistas & inibidores , Oligopeptídeos/síntese química , Sulfetos/síntese química , Receptor 2 Toll-Like/antagonistas & inibidores , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Alanina/síntese química , Alanina/química , Alanina/farmacologia , Linhagem Celular , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Interleucina-8/metabolismo , Espectroscopia de Ressonância Magnética , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/farmacologiaRESUMO
Lipoproteins activate cells of the innate immune system via heteromers of Toll-like receptor (TLR) 2 with either TLR1 or TLR6. In spite of progress in understanding TLR-dependent signal transduction and the pathophysiological relevance of TLR2, the molecular basis of ligand recognition by this receptor is poorly defined. Here, we show that the bioactivity of lipopeptides (LP) critically depends on the dilution protocol and especially the presence of proteins or detergents acting as solubilizers. Fluorescence correlation spectroscopy of fluorescently labeled analogs of synthetic LP revealed that the LP form aggregates in solution. Dilution into protein- and serum-free buffers led to a complete loss of activity due to formation of large and highly heterogeneous aggregates. When dimethylsulfoxide stock solutions were diluted into BSA or serum-containing buffers particles of strongly reduced size were obtained. For some LP, an intermediary dilution step either with tert.-butyl alcohol/H2O (4:1) or with octyl-beta-D-glucopyranoside further increased activity. For a panel of LP exhibiting very different activities when diluted directly into protein-containing solutions, introduction of this dilution step resulted in comparable bioactivities. These results demonstrate the significance of solubilizing agents for the bioactivity of LP and are highly relevant for analyzing structure-activity relationships of LP-dependent TLR2 activation.
Assuntos
Glucosídeos/farmacologia , Lipoproteínas/farmacologia , Solventes/farmacologia , Receptor 2 Toll-Like/agonistas , terc-Butil Álcool/farmacologia , Soluções Tampão , Linhagem Celular Tumoral , Corantes Fluorescentes/análise , Humanos , Lipoproteínas/química , Monócitos , Concentração Osmolar , Rodaminas/análise , Soroalbumina Bovina , Solubilidade , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
For the detection of bioanalytes, there is an ongoing search for synthetic sensors to replace enzyme-based assays which are sensitive to contaminants or suboptimal storage conditions. Lipopolysaccharide (LPS), a bacteria-borne endotoxin that may lead to life-threatening conditions such as septic shock, is one such case. Fluorescently labeled analogues of two peptide variants derived from the putative ligand-binding domain of the LPS-binding protein CD14 were developed that detect and discriminate LPS and lipids down to the submicromolar concentration range. Peptides are terminally labeled with carboxyfluorescein and tetramethylrhodamine. For one given peptide, sensitivity and specificity for the detection of LPS and discrimination from other lipids are achieved by spectral signatures that combine changes in the fluorescence resonance energy transfer (FRET) between both dyes and the total emission of tetramethylrhodamine. Alternatively, specificity is obtained by combining the FRET efficiencies of both peptide variants. In comparison to published synthetic LPS sensors, the CD14-derived sensors yield an increase in sensitivity by about 3 orders of magnitude and exhibit specificity for analytes for which the design of synthetic recognition elements is a challenging task. Moreover, one of the sensors enabled the detection of LPS in the presence of up to 50% fetal calf serum, thereby demonstrating the feasibility of this peptide-based approach for clinically relevant samples.
Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Lipopolissacarídeos/química , Peptídeos/química , Sítios de Ligação , Fluoresceínas/química , Ligantes , Lipídeos/química , Rodaminas/químicaRESUMO
The interaction of lipopolysaccharide with CD14 plays a key role in signaling that activates an early defense against pathogens but also contributes to the development of sepsis and septic shock. Here we have mapped the entire 356-amino-acid protein with synthetic 20-amino-acid peptides and have identified a new lipopolysaccharide-binding domain with a strong LPS-neutralizing activity. Moreover, analysis of the structure-activity relationship of this peptide, which corresponds to amino acids 81-100 of human CD14, revealed that leucines 87, 91, and 94 are essential for these activities. The functional relevance of these residues was confirmed by cellular expression of mutant CD14 proteins that are no longer able to bind LPS. Furthermore, the peptide provided a basis for the generation of highly soluble analogues with stronger lipopolysaccharide-neutralizing activity.
Assuntos
Receptores de Lipopolissacarídeos/química , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Estrutura Terciária de Proteína , Sítios de Ligação , Linhagem Celular , Dicroísmo Circular , Humanos , Receptores de Lipopolissacarídeos/genética , Modelos Moleculares , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Reprodutibilidade dos Testes , Relação Estrutura-AtividadeRESUMO
Microarrays that mediate the uptake of small molecules into living cells are described. Tissue culture cells were seeded onto glass substrates functionalized locally with fluorescently labelled test substances. In order to enable a localized transfer of substances after contact of cells with the substrate, substances were immobilized on the surface either by non-covalent interactions or chemolabile linker groups. These chemolabile linker groups were incorporated into covalently immobilized compounds. Different ester linkages were evaluated as chemolabile linker groups. As model compounds, esters of the carboxy group of a cysteine with the hydroxy groups of carboxyfluorescein-labelled serine amide and tyrosine amide residues or the thiol group of another fluorescein-labelled cysteine amide were generated. Covalent immobilization occurred on maleimide-functionalized glass cover slips. The surface functionalization and release kinetics were assessed by confocal laser scanning microscopy. The fastest release was obtained for the phenolic tyrosine ester. Alternatively, fluorescently labelled peptides were immobilized by non-covalent interactions on glass and on a hydrogel matrix. In order to increase the efficiency of cellular uptake, peptides were N-terminally extended with a cell-penetrating peptide. Uptake of these peptides into cells was confined to the functionalized spots, and was specific for peptides extended with the cell-penetrating peptide.