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1.
Cancer Immunol Immunother ; 68(5): 799-812, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30770959

RESUMO

CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400-1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD+CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8-6.3) and 10.8 months (8.1-16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.


Assuntos
Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia/métodos , Neoplasias Pulmonares/terapia , RNA Mensageiro/uso terapêutico , Linfócitos T/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Células Cultivadas , Feminino , Humanos , Imunoterapia/efeitos adversos , Reação no Local da Injeção/etiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Análise de Sobrevida
2.
J Immunol ; 198(4): 1595-1605, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28077601

RESUMO

Among innovative adjuvants conferring a Th1-shift, RNAdjuvant is a promising candidate. This adjuvant consists of a 547-nt uncapped noncoding ssRNA containing polyU repeats that is stabilized by a cationic carrier peptide. Whereas vaccination of mice with an influenza subunit vaccine induced moderate virus-specific IgG1, vaccination together with RNAdjuvant significantly enhanced this IgG1 and additionally promoted the formation of IgG2b/c, which is indicative of Th1 responses. Furthermore, such sera neutralized influenza virus, whereas this effect was not detected upon vaccination with the subunit vaccine alone. Similarly, upon vaccination with virus-like particles displaying vesicular stomatitis virus G protein, RNAdjuvant promoted the formation of virus-specific IgG2b/c and enhanced neutralizing IgG responses to an extent that mice were protected against lethal virus infection. RNAdjuvant induced dendritic cells to upregulate activation markers and produce IFN-I. Although these effects were strictly TLR7 dependent, RNAdjuvant-mediated augmentation of vaccine responses needed concurrent TLR and RIG-I-like helicase signaling. This was indicated by the absence of the adjuvant effect in vaccinated MyD88-/-Cardif-/- mice, which are devoid of TLR (with the exception of TLR3) and RIG-I-like helicase signaling, whereas in vaccinated MyD88-/- mice the adjuvant effect was reduced. Notably, i.m. RNAdjuvant injection induced local IFN-I responses and did not induce systemic effects, implying good tolerability and a favorable safety profile for RNAdjuvant.


Assuntos
Adjuvantes Imunológicos , Imunoglobulina G/sangue , Vacinas contra Influenza/imunologia , Glicoproteínas de Membrana/imunologia , RNA não Traduzido/imunologia , Receptor 7 Toll-Like/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Adjuvantes Imunológicos/efeitos adversos , Animais , Anticorpos Antivirais/sangue , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Imunoglobulina G/imunologia , Vacinas contra Influenza/administração & dosagem , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/metabolismo , Células Th1/imunologia , Receptor 7 Toll-Like/metabolismo , Vacinação , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/imunologia
3.
Int J Cancer ; 137(2): 372-84, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25530186

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 farmacológico
4.
BMC Cancer ; 14: 748, 2014 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-25288198

RESUMO

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) represents a significant unmet medical need. Despite advances with targeted therapies in a small subset of patients, fewer than 20% of patients survive for more than two years after diagnosis. Cancer vaccines are a promising therapeutic approach that offers the potential for durable responses through the engagement of the patient's own immune system. CV9202 is a self-adjuvanting mRNA vaccine that targets six antigens commonly expressed in NSCLC (NY-ESO-1, MAGEC1, MAGEC2, 5 T4, survivin, and MUC1). METHODS/DESIGN: The trial will assess the safety and tolerability of CV9202 vaccination combined with local radiation designed to enhance immune responses and will include patients with stage IV NSCLC and a response or stable disease after first-line chemotherapy or therapy with an EGFR tyrosine kinase inhibitor. Three histological and molecular subtypes of NSCLC will be investigated (squamous and non-squamous cell with/without EGFR mutations). All patients will receive two initial vaccinations with CV9202 prior to local radiotherapy (5 GY per day for four successive days) followed by further vaccinations until disease progression. The primary endpoint of the study is the number of patients experiencing Grade >3 treatment-related adverse events. Pharmacodynamic analyses include the assessment of immune responses to the antigens encoded by CV9202 and others not included in the panel (antigen spreading) and standard efficacy assessments. DISCUSSION: RNActive self-adjuvanted mRNA vaccines offer the potential for simultaneously inducing immune responses to a wide panel of antigens commonly expressed in tumors. This trial will assess the feasibility of this approach in combination with local radiotherapy in NSCLC patients. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01915524/EudraCT No.: 2012-004230-41.


Assuntos
Vacinas Anticâncer/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , RNA Mensageiro/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Quimioterapia Adjuvante , Terapia Combinada , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , RNA Mensageiro/efeitos adversos , RNA Mensageiro/uso terapêutico , Doses de Radiação , Radioterapia , Resultado do Tratamento
5.
J Gene Med ; 14(6): 428-39, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22262664

RESUMO

Direct vaccination with mRNA encoding tumor antigens is a novel and promising approach in cancer immunotherapy. CureVac's mRNA vaccines contain free and protamine-complexed mRNA. Such two-component mRNA vaccines support both antigen expression and immune stimulation. These self-adjuvanting RNA vaccines, administered intradermally without any additional adjuvant, induce a comprehensive balanced immune response, comprising antigen specific CD4+ T cells, CD8+ T cells and B cells. The balanced immune response results in a strong anti-tumor effect and complete protection against antigen positive tumor cells. This tumor inhibition elicited by mRNA vaccines is a result of the concerted action of different players. After just two intradermal vaccinations, we observe multiple changes at the tumor site, including the up-regulation of many genes connected to T and natural killer cell activation, as well as genes responsible for improved infiltration of immune cells into the tumor via chemotaxis. The two-component mRNA vaccines induce a very fast and boostable immune response. Therefore, the vaccination schedules can be adjusted to suit the clinical situation. Moreover, by combining the mRNA vaccines with therapies in clinical use (chemotherapy or anti-CTLA-4 antibody therapy), an even more effective anti-tumor response can be elicited. The first clinical data obtained from two separate Phase I/IIa trials conducted in PCA (prostate cancer) and NSCLC (non-small cell lung carcinoma) patients have shown that the two-component mRNA vaccines are safe, well tolerated and highly immunogenic in humans.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias da Próstata/terapia , Animais , Antígenos de Neoplasias/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular , Terapia Combinada , Células HeLa , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , RNA Mensageiro/genética , Vacinas de DNA
6.
RNA Biol ; 9(11): 1319-30, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23064118

RESUMO

mRNA vaccines combine desirable immunological properties with an outstanding safety profile and the unmet flexibility of genetic vaccines. Based on in situ protein expression, mRNA vaccines are capable of inducing a balanced immune response comprising both cellular and humoral immunity while not subject to MHC haplotype restriction. In addition, mRNA is an intrinsically safe vector as it is a minimal and only transient carrier of information that does not interact with the genome. Because any protein can be expressed from mRNA without the need to adjust the production process, mRNA vaccines also offer maximum flexibility with respect to development. Taken together, mRNA presents a promising vector that may well become the basis of a game-changing vaccine technology platform. Here, we outline the current knowledge regarding different aspects that should be considered when developing an mRNA-based vaccine technology.


Assuntos
Adjuvantes Imunológicos , RNA Mensageiro/imunologia , Vacinas Sintéticas/imunologia , Animais , Transporte Biológico , Expressão Gênica , Humanos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/metabolismo
7.
J Surg Res ; 157(2): 235-42, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19589542

RESUMO

BACKGROUND: Immune function after hemorrhagic shock and subsequent sepsis is characterized by an early proinflammatory burst of IL-6, and high IL-6 levels have been linked to high mortality after trauma and in sepsis. Trans-signaling is defined as the activation of cells that do not express the membrane bound IL-6 receptor by the complex of IL-6 and the soluble IL-6 receptor (sIL-6R). Gp130-Fc is able to bind the IL-6/sIL-6R complex, and beneficial effects of IL-6 blockade in chronic inflammatory diseases have been shown. The first aim of this study was to investigate the potential effect of a gp130 blockade via the gp130-Fc antibody causing impairment of IL-6 signaling. The second aim was to find out what role the IL-6/sIL-6R complex can play in the context of hemorrhagic shock and subsequent sepsis as an acute inflammatory disease. MATERIAL AND METHODS: Male CBA/J mice were subjected to hemorrhagic shock (35+/-5 mmHg for 90min and fluid resuscitation) or sham operation. At resuscitation each animal received either 0.5mg gp130-Fc or placebo (PL) i.p. At 48 h after resuscitation, both splenocytes and peritoneal macrophages (pMphi) were harvested or polymicrobial sepsis was induced by cecal ligation and puncture. Survival over 10 d was determined. Release of IL-6, TNF-alpha, and IL-10 of pMphi and release of IL-2, IL-10, and IFN-gamma of splenocytes was assessed by ELISA. Proliferation of splenocytes and their morphologic damage were determined. RESULTS: Binding of the IL-6/sIL-6R complex by gp130-Fc led to significant lower IL-6 levels compared with placebo treated animals. Placebo treated males showed depressed proinflammatory immune response (IL-2, IL-6) after hemorrhagic shock. While splenocyte proliferation was significantly reduced directly after hemorrhagic shock and restored after 48 h by gp130-Fc, pMphi cytokine release was not influenced. Finally, survival appeared to be unaffected. CONCLUSION: Transsignaling does not seem to play a pivotal role in the development of the immune dysfunction and mortality in our model of hemorrhage and subsequent sepsis.


Assuntos
Anticorpos/farmacologia , Receptor gp130 de Citocina/imunologia , Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/antagonistas & inibidores , Sepse/metabolismo , Choque Hemorrágico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Receptor gp130 de Citocina/antagonistas & inibidores , Receptor gp130 de Citocina/metabolismo , Modelos Animais de Doenças , Íleo/patologia , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/fisiologia , Interleucina-6/metabolismo , Rim/patologia , Fígado/patologia , Pulmão/patologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos CBA , Monocinas/metabolismo , Receptores de Interleucina-6/metabolismo , Sepse/imunologia , Sepse/patologia , Choque Hemorrágico/imunologia , Choque Hemorrágico/patologia , Transdução de Sinais/fisiologia , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia
8.
J Immunother Cancer ; 7(1): 38, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30736848

RESUMO

BACKGROUND: Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses. METHODS: We describe a phase Ib clinical trial evaluating treatment with BI1361849 combined with local radiation in 26 stage IV NSCLC patients with partial response (PR)/stable disease (SD) after standard first-line therapy. Patients were stratified into three strata (1: non-squamous NSCLC, no epidermal growth factor receptor (EGFR) mutation, PR/SD after ≥4 cycles of platinum- and pemetrexed-based treatment [n = 16]; 2: squamous NSCLC, PR/SD after ≥4 cycles of platinum-based and non-platinum compound treatment [n = 8]; 3: non-squamous NSCLC, EGFR mutation, PR/SD after ≥3 and ≤ 6 months EGFR-tyrosine kinase inhibitor (TKI) treatment [n = 2]). Patients received intradermal BI1361849, local radiation (4 × 5 Gy), then BI1361849 until disease progression. Strata 1 and 3 also had maintenance pemetrexed or continued EGFR-TKI therapy, respectively. The primary endpoint was evaluation of safety; secondary objectives included assessment of clinical efficacy (every 6 weeks during treatment) and of immune response (on Days 1 [baseline], 19 and 61). RESULTS: Study treatment was well tolerated; injection site reactions and flu-like symptoms were the most common BI1361849-related adverse events. Three patients had grade 3 BI1361849-related adverse events (fatigue, pyrexia); there was one grade 3 radiation-related event (dysphagia). In comparison to baseline, immunomonitoring revealed increased BI1361849 antigen-specific immune responses in the majority of patients (84%), whereby antigen-specific antibody levels were increased in 80% and functional T cells in 40% of patients, and involvement of multiple antigen specificities was evident in 52% of patients. One patient had a partial response in combination with pemetrexed maintenance, and 46.2% achieved stable disease as best overall response. Best overall response was SD in 57.7% for target lesions. CONCLUSION: The results support further investigation of mRNA-based immunotherapy in NSCLC including combinations with immune checkpoint inhibitors. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01915524 .


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/terapia , Pemetrexede/uso terapêutico , Protaminas/uso terapêutico , RNA Mensageiro/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Terapia Combinada , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mucina-1/genética , Proteínas de Neoplasias/genética , Survivina/genética
9.
J Neuroimmunol ; 205(1-2): 64-72, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18950871

RESUMO

Interleukin-6 (IL-6) plays a crucial role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). It exerts its cellular effects by a membrane-bound IL-6 receptor (IL-6R), or, alternatively, by forming a complex with the soluble IL-6R (sIL-6R), a process named IL-6 transsignalling. Here we investigate the role of IL-6 transsignalling in myelin basic protein (MBP)-induced EAE in the Lewis rat. In vivo blockade of IL-6 transsignalling by the injection of a specifically designed gp130-Fc fusion protein significantly delayed the onset of adoptively transferred EAE in comparison to control rats injected with PBS or isotype IgG. Histological evaluation on day 3 after immunization revealed reduced numbers of T cells and macrophages in the lumbar spinal cord of gp130-Fc treated rats. At the same time, blockade of IL-6 transsignalling resulted in a reduced expression of vascular cell adhesion molecule-1 on spinal cord microvessels while experiments in cell culture failed to show a direct effect on the regulation of endothelial adhesion molecules. In experiments including active EAE and T cell culture, inhibition of IL-6 transsignalling mildly increased T cell proliferation, but did not change severity of active MBP-EAE or regulate Th1/Th17 responses. We conclude that IL-6 transsignalling may play a role in autoimmune inflammation of the CNS mainly by regulating early expression of adhesion molecules, possibly via cellular networks at the blood-brain barrier.


Assuntos
Barreira Hematoencefálica/fisiopatologia , Encefalomielite Autoimune Experimental/patologia , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais/fisiologia , Animais , Células CHO , Proliferação de Células , Células Cultivadas , Cricetinae , Cricetulus , Receptor gp130 de Citocina/imunologia , Receptor gp130 de Citocina/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/fisiopatologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Cobaias , Proteína Básica da Mielina/imunologia , Ratos , Ratos Endogâmicos Lew , Medula Espinal/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo
10.
Oncoimmunology ; 5(12): e1249560, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28123889

RESUMO

We recently completed a phase I/IIa trial of RNActive® CV9201, a novel mRNA-based therapeutic vaccine targeting five tumor-associated antigens in non-small cell lung cancer (NSCLC) patients. The aim of the study presented here was to comprehensively analyze changes in peripheral blood during the vaccination period and to generate hypotheses facilitating the identification of potential biomarkers correlating with differential clinical outcomes post RNActive® immunotherapy. We performed whole-genome expression profiling in a subgroup of 22 stage IV NSCLC patients before and after initiation of treatment with CV9201. Utilizing an analytic approach based on blood transcriptional modules (BTMs), a previously described, sensitive tool for blood transcriptome data analysis, patients segregated into two major clusters based on transcriptional changes post RNActive® treatment. The first group of patients was characterized by the upregulation of an expression signature associated with myeloid cells and inflammation, whereas the other group exhibited an expression signature associated with T and NK cells. Patients with an enrichment of T and NK cell modules after treatment compared to baseline exhibited significantly longer progression-free and overall survival compared to patients with an upregulation of myeloid cell and inflammatory modules. Notably, these gene expression signatures were mutually exclusive and inversely correlated. Furthermore, our findings correlated with phenotypic data derived by flow cytometry as well as the neutrophil-to-lymphocyte ratio. Our study thus demonstrates non-overlapping, distinct transcriptional profiles correlating with survival warranting further validation for the development of biomarker candidates for mRNA-based immunotherapy.

11.
Protein Sci ; 14(3): 783-90, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15722452

RESUMO

Cytokines of the interleukin-6 (IL-6)-type family all bind to the glycoprotein gp130 on the cell surface and require interaction with two gp130 or one gp130 and another related signal transducing receptor subunit. In addition, some cytokines of this family, such as IL-6, interleukin-11, ciliary neurotrophic factor, neuropoietin, cardiotrophin-1, and cardiotrophin-1-like-cytokine, interact with specific ligand binding receptor proteins. High- and low-affinity binding sites have been determined for these cytokines. So far, however, the stoichiometry of the signaling receptor complexes has remained unclear, because the formation of the cytokine/cytokine-receptor complexes has been analyzed with soluble receptor components in solution, which do not necessarily reflect the situation on the cellular membrane. Consequently, the binding affinities measured in solution have been orders of magnitude below the values obtained with whole cells. We have expressed two gp130 extracellular domains in the context of a Fc-fusion protein, which fixes the receptors within one dimension and thereby restricts the flexibility of the proteins in a fashion similar to that within the plasma membrane. We measured binding of IL-6 and interleukin-b receptor (IL-6R) by means of fluorescence-correlation spectroscopy. For the first time we have succeeded in recapitulating in a cell-free condition the binding affinities and dynamics of IL-6 and IL-6R to the gp130 receptor proteins, which have been determined on whole cells. Our results demonstrate that a dimer of gp130 first binds one IL-6/IL-6R complex and only at higher ligand concentrations does it bind a second IL-6/IL-6R complex. This view contrasts with the current perception of IL-6 receptor activation and reveals an alternative receptor activation mechanism.


Assuntos
Antígenos CD/metabolismo , Membrana Celular/metabolismo , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/isolamento & purificação , Células CHO , Cromatografia em Gel , Cricetinae , Cricetulus , Receptor gp130 de Citocina , Interpretação Estatística de Dados , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Modelos Biológicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência
12.
Biochim Biophys Acta ; 1592(3): 323-43, 2002 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-12421676

RESUMO

The activation of cells that do not express the membrane bound interleukin-6 6 receptor (IL-6R) by IL-6 and the soluble IL-6 receptor (sIL-6R) is termed transsignalling. Transsignalling may be an pathogenetic factor in human diseases as diverse as multiple myeloma (MM), Castleman's disease, prostate carcinoma, Crohn's disease, systemic sclerosis, Still's disease, osteoporosis and cardiovascular diseases. IL-6 and sIL-6R may directly or indirectly enhance their own production on endothelial or bone marrow stromal cells. Positive feedback autocrine loops thus created in affected organs may either cause or maintain disease progression. In autoimmune or vasculitic disease, the ability of the IL-6/sIL-6R complex to inhibit apoptosis of autoreactive T-cells may be central to the development of tissue specific autoimmunity. The anti-apoptotic effect of the IL-6/sIL-6R complex may be involved in tumour genesis and resistance to chemotherapy. Only in rare cases, where counterregulation has failed, there is a notable systemic effect of IL-6/sIL-6R. Appropriate animal models are necessary to establish the pathogenetic role of the IL-6/sIL-6R complex. A specific treatment option for diseases influenced by the sIL-6R could be based on gp130-Fc, a soluble gp130 (sgp130) linked to the Fc-fragment of IgG1. gp130-Fc has shown efficacy in vivo in animal models of Crohn's disease.


Assuntos
Interleucina-6/fisiologia , Receptores de Interleucina-6/fisiologia , Transdução de Sinais/fisiologia , Animais , Anticorpos/uso terapêutico , Antígenos CD/genética , Antígenos CD/fisiologia , Doenças Autoimunes/fisiopatologia , Doenças Ósseas/fisiopatologia , Doenças do Sistema Nervoso Central/fisiopatologia , Receptor gp130 de Citocina , Modelos Animais de Doenças , Endometriose/fisiopatologia , Feminino , Cardiopatias/fisiopatologia , Humanos , Hepatopatias/fisiopatologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias/fisiopatologia , Receptores de Interleucina-6/análise , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/química , Solubilidade , Doenças Vasculares/fisiopatologia
13.
J Mol Biol ; 315(4): 637-46, 2002 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-11812136

RESUMO

To elucidate the function of the two cytokine-binding modules (CBM) of the leukemia inhibitory factor receptor (LIFR), receptor chimeras of LIFR and the interleukin-6 receptor (IL-6R) were constructed. Either the NH(2)-terminal (chimera RILLIFdeltaI) or the COOH-terminal LIFR CBM (chimera RILLIFdeltaII) were replaced by the structurally related CBM of the IL-6R which does not bind LIF. Chimera RILLIFdeltaI is functionally inactive, whereas RILLIFdeltaII binds LIF and mediates signalling as efficiently as the wild-type LIFR. Deletion mutants of the LIFR revealed that both the NH(2)-terminal CBM and the Ig-like domain of the LIFR are involved in LIF binding, presumably via the LIF site III epitope. The main function of the COOH-terminal CBM of the LIFR is to position the NH(2)-terminal CBM and the Ig-like domain, so that these can bind to LIF. In analogy to a recently published model of the IL-6R complex, a model of the active LIFR complex is suggested which positions the COOH-terminal CBM at LIF site I and the NH(2)-terminal CBM and the Ig-like domain at site III. An additional contact is postulated between the Ig-like domain of gp130 and the NH(2)-terminal CBM of the LIFR.


Assuntos
Inibidores do Crescimento/metabolismo , Imunoglobulinas/química , Interleucina-6 , Linfocinas/metabolismo , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação , Células COS , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Inibidores do Crescimento/farmacologia , Humanos , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Ligantes , Linfocinas/farmacologia , Oncostatina M , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Estrutura Terciária de Proteína , Receptores de Citocinas/genética , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Receptores de OSM-LIF , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Transfecção
14.
J Immunother Cancer ; 3: 26, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26082837

RESUMO

BACKGROUND: CV9103 is a prostate-cancer vaccine containing self-adjuvanted mRNA (RNActive®) encoding the antigens PSA, PSCA, PSMA, and STEAP1. This phase I/IIa study evaluated safety and immunogenicity of CV9103 in patients with advanced castration-resistant prostate-cancer. METHODS: 44 Patients received up to 5 intra-dermal vaccinations. Three dose levels of total mRNA were tested in Phase I in cohorts of 3-6 patients to determine a recommended dose. In phase II, 32 additional patients were treated at the recommended dose. The primary endpoint was safety and tolerability, the secondary endpoint was induction of antigen specific immune responses monitored at baseline and at weeks 5, 9 and 17. RESULTS: The most frequent adverse events were grade 1/2 injection site erythema, injection site reactions, fatigue, pyrexia, chills and influenza-like illness. Possibly treatment related urinary retention occurred in 3 patients. The recommended dose was 1280 µg. A total of 26/33 evaluable patients treated at 1280 µg developed an immune response, directed against multiple antigens in 15 out of 33 patients. One patient showed a confirmed PSA response. In the subgroup of 36 metastatic patients, the Kaplan-Meier estimate of median overall survival was 31.4 months [95 % CI: 21.2; n.a]. CONCLUSIONS: The self-adjuvanted RNActive® vaccine CV9103 was well tolerated and immunogenic. The technology is a versatile, fast and cost-effective platform allowing for creation of vaccines. The follow-up vaccine CV9104 including the additional antigens prostatic acid phosphatase (PAP) and Muc1 is currently being tested in a randomized phase IIb trial to assess the clinical benefit induced by this new vaccination approach. TRIAL REGISTRATION: EU Clinical Trials Register: EudraCT number 2008-003967-37, registered 27 Jan 2009.

15.
J Invest Dermatol ; 123(1): 124-31, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15191552

RESUMO

Interleukin-6 (IL-6) is involved in the growth and differentiation of numerous cell types. In the skin it is produced primarily by keratinocytes. The transcription factor STAT3 is activated by cytokines of the IL-6 family. In this study, we examined the involvement of IL-6, soluble IL-6-receptor, and STAT3 in epidermal barrier repair after injury to the stratum corneum by tape-stripping. After barrier disruption in wild-type mice we found an increased immunostaining of IL-6 and IL-6R on epidermal keratinocytes at 15 min to 5 h after treatment. The increase in IL-6 and IL-6R was confirmed by western blotting using epidermal homogenates and was partially prevented by occlusion immediately after barrier disruption. In IL-6-deficient mice, epidermal barrier repair was reduced at 3-24 h after treatment. Topical application of IL-6 or Hyper-IL-6, a complex of IL-6 linked to the soluble IL-6 receptor, enhanced epidermal barrier repair in wild-type mice. Application of the fusion protein gp130-FC, a specific inhibitor of the agonist IL-6/sIL-6 receptor complex, delayed barrier repair in wild, but not in IL-6-deficient mice. STAT3 tyrosine phosphorylation was induced after barrier disruption in wild-type, but markedly reduced in IL-6-deficient mice. Our results indicate that the IL-6 cytokine system, particularly transsignalling via the soluble IL-6R, is critically involved in barrier repair after skin injury.


Assuntos
Epiderme/metabolismo , Homeostase/fisiologia , Interleucina-6/metabolismo , Queratinócitos/metabolismo , Administração Tópica , Animais , Antígenos CD/genética , Antígenos CD/farmacologia , Receptor gp130 de Citocina , Proteínas de Ligação a DNA/metabolismo , Células Epidérmicas , Epiderme/lesões , Interleucina-6/genética , Interleucina-6/farmacologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Pelados , Camundongos Mutantes , Permeabilidade , Fosforilação , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3 , Transativadores/metabolismo , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
16.
FEBS Lett ; 538(1-3): 113-6, 2003 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-12633863

RESUMO

The recently solved X-ray structure of the extracellular portion of the interleukin-6 (IL-6) receptor (IL-6R) revealed an IL-6R dimer in the crystal lattice which probably represents a physiological dimer. Performing coprecipitation experiments with two differently tagged IL-6R variants expressed in COS-7 cells, we show that an IL-6R dimer exists in the plasma membrane in the absence of IL-6. Ligand binding does not seem to affect the dimerization status. When lysates of COS-7 cells expressing only one of the IL-6R variants are mixed, spontaneous dimerization occurs. Thus, the IL-6R dimer observed in the crystal structure represents a physiologically occurring phenomenon.


Assuntos
Receptores de Interleucina-6/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Membrana Celular/metabolismo , Dimerização , Humanos , Testes de Precipitina , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética
17.
J Immunol Methods ; 291(1-2): 93-100, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15345308

RESUMO

The proinflammatory cytokine IL-6 binds to the membrane bound or soluble IL-6 receptor (IL-6R) and activates an intracellular signaling cascade after complex formation with two gp130 molecules. These mediate general homeostasis and orchestrates the immune response during disease. Trans-signalling via the soluble IL-6R has importance for the development and maintenance of human diseases like Crohn's disease, peritonitis and rheumatoid arthritis. We have developed an enzyme-linked immunoabsorbent assay (ELISA) that detects the binding of gp130 to the IL-6/sIL-6R complex. Competitive binding of sgp130-Fc, viral IL-6, and the inhibitory drug Suramin to gp130 has been demonstrated. The assay can be used for high-throughput screening of peptide and chemical compound libraries for the identification of new agonists and antagonists of the binding between gp130 and IL-6/sIL-6R.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/análise , Antígenos CD/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Interleucina-6/imunologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/imunologia , Receptores de Interleucina-6/imunologia , Ligação Competitiva , Linhagem Celular , Receptor gp130 de Citocina , Humanos , Interleucina-6/antagonistas & inibidores , Substâncias Macromoleculares , Glicoproteínas de Membrana/antagonistas & inibidores , Ligação Proteica , Receptores de Interleucina-6/antagonistas & inibidores
18.
Ther Adv Vaccines ; 2(1): 10-31, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24757523

RESUMO

Recent advances strongly suggest that mRNA rather than DNA will be the nucleotide basis for a new class of vaccines and drugs. Therapeutic cancer vaccines against a variety of targets have been developed on this basis and initial clinical experience suggests that preclinical activity can be successfully translated to human application. Likewise, prophylactic vaccines against viral pathogens and allergens have demonstrated their activity in animal models. These successes could be extended preclinically to mRNA protein and gene replacement therapy as well as the induction of pluripotent stem cells by mRNA encoded transcription factors. The production of mRNA-based vaccines and drugs is highly flexible, scalable and cost competitive, and eliminates the requirement of a cold chain. mRNA-based drugs and vaccines offer all the advantages of a nucleotide-based approach at reduced costs and represent a truly disruptive technology that may start a revolution in medicine.

19.
Radiat Oncol ; 9: 180, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25127546

RESUMO

BACKGROUND: The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of messenger RNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics. METHODS: The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The molecular mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells. RESULTS: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression analysis of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor associated factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors. CONCLUSION: Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.


Assuntos
Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Imunoterapia/métodos , RNA Mensageiro/imunologia , Radioterapia/métodos , Animais , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
20.
Hum Vaccin Immunother ; 9(10): 2263-76, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23921513

RESUMO

Nucleotide based vaccines represent an enticing, novel approach to vaccination. We have developed a novel immunization technology, RNActive(®) vaccines, that have two important characteristics: mRNA molecules are used whose protein expression capacity has been enhanced by 4 to 5 orders of magnitude by modifications of the nucleotide sequence with the naturally occurring nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) that do not affect the primary amino acid sequence. Second, they are complexed with protamine and thus activate the immune system by involvement of toll-like receptor (TLR) 7. Essentially, this bestows self-adjuvant activity on RNActive(®) vaccines. RNActive(®) vaccines induce strong, balanced immune responses comprising humoral and cellular responses, effector and memory responses as well as activation of important subpopulations of immune cells, such as Th1 and Th2 cells. Pre-germinal center and germinal center B cells were detected in human patients upon vaccination. RNActive(®) vaccines successfully protect against lethal challenges with a variety of different influenza strains in preclinical models. Anti-tumor activity was observed preclinically under therapeutic as well as prophylactic conditions. Initial clinical experiences suggest that the preclinical immunogenicity of RNActive(®) could be successfully translated to humans.


Assuntos
Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , RNA/administração & dosagem , RNA/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Humanos , Receptor 7 Toll-Like/imunologia , Vacinação/métodos
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