RESUMO
First attempts to use exogenous mRNA for protein expression in vivo were made more than 25 years ago. However, widespread appreciation of in vitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such in vivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient in vivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.
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Anticorpos Monoclonais/genética , Sistemas de Liberação de Medicamentos/métodos , Imunização Passiva/métodos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/uso terapêutico , Animais , Doenças Transmissíveis/terapia , Composição de Medicamentos/métodos , Humanos , Lipídeos/química , Nanopartículas/química , Neoplasias/terapia , Toxinas Biológicas/imunologiaRESUMO
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 SobrevidaRESUMO
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/genéticaRESUMO
BACKGROUND: Tumor metastasis and immune evasion present major challenges of cancer treatment. Radiotherapy can overcome immunosuppressive tumor microenvironments. Anecdotal reports suggest abscopal anti-tumor immune responses. This study assesses abscopal effects of radiotherapy in combination with mRNA-based cancer vaccination (RNActive®). METHODS: C57BL/6 mice were injected with ovalbumin-expressing thymoma cells into the right hind leg (primary tumor) and left flank (secondary tumor) with a delay of 4 days. Primary tumors were irradiated with 3 × 2 Gy, while secondary tumors were shielded. RNA and combined treatment groups received mRNA-based RNActive® vaccination. RESULTS: Radiotherapy and combined radioimmunotherapy significantly delayed primary tumor growth with a tumor control in 15 and 53% of mice, respectively. In small secondary tumors, radioimmunotherapy significantly slowed growth rate compared to vaccination (p = 0.002) and control groups (p = 0.01). Cytokine microarray analysis of secondary tumors showed changes in the cytokine microenvironment, even in the non-irradiated contralateral tumors after combination treatment. CONCLUSION: Combined irradiation and immunotherapy is able to induce abscopal responses, even with low, normofractionated radiation doses. Thus, the combination of mRNA-based vaccination with irradiation might be an effective regimen to induce systemic anti-tumor immunity.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Modelos Animais de Doenças , Ovalbumina/imunologia , RNA Mensageiro/imunologia , Radioimunoterapia , Timoma/terapia , Neoplasias do Timo/terapia , Animais , Terapia Combinada , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/genética , RNA Mensageiro/genética , Timoma/genética , Timoma/imunologia , Neoplasias do Timo/genética , Neoplasias do Timo/imunologiaRESUMO
The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA-lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA-mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.
Assuntos
Antitoxina Botulínica/imunologia , Botulismo/prevenção & controle , Imunização Passiva/métodos , Profilaxia Pós-Exposição , RNA Mensageiro/administração & dosagem , Vacina Antirrábica/imunologia , Raiva/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antitoxina Botulínica/administração & dosagem , Antitoxina Botulínica/sangue , Botulismo/terapia , Relação Dose-Resposta Imunológica , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Raiva/terapia , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/sangue , Vírus da Raiva/imunologiaRESUMO
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/imunologiaRESUMO
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.
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.
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
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 TratamentoRESUMO
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 PolimeraseRESUMO
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/métodosRESUMO
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 DNARESUMO
Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.
Assuntos
Endocitose , Lisossomos/metabolismo , RNA Mensageiro/metabolismo , Transporte Biológico , Carbocianinas , Cavéolas/metabolismo , Células HEK293 , Células HeLa , Humanos , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Receptores Depuradores/metabolismoRESUMO
Direct vaccination with messenger RNA (mRNA) molecules encoding tumor-associated antigens is a novel and promising approach in cancer immunotherapy. The main advantage of using mRNA for vaccination is that the same molecule not only provides an antigen source for adaptive immunity, but can simultaneously bind to pattern recognition receptors, thus stimulating innate immunity. However, achieving both features remains challenging, as the complexation of mRNA required for immune-stimulating activity may inhibit its translatability. In this study, we present a new and more effective vaccine design: a two-component mRNA-based tumor vaccine that supports both: antigen expression and immune stimulation, mediated by Toll like receptor 7 (TLR7). The two-component mRNA vaccines, containing free and protamine-complexed mRNA, induce balanced adaptive immune responses providing humoral as well as T cell mediated immunity. This balanced immune response is based on the induction of antigen-specific CD4(+) T helper cells and cytotoxic CD8(+) T cells. Once activated, these CD4(+) and CD8(+) T cells secrete a wide set of cytokines, which drive a TH1 response. Immunization with the two-component vaccines induces sustained memory responses, mediated by antigen-specific memory T cells. Moreover, treatment of mice with the two-component mRNA vaccine mediates a strong antitumor response against OVA-expressing tumor cells, not only in a prophylactic but also in a therapeutic setting. In conclusion, two-component mRNA vaccines with self-adjuvanting activity induce balanced adaptive immune responses and mediate sustained antitumor activity.
Assuntos
Imunidade Adaptativa , Vacinas Anticâncer/imunologia , Neoplasias Experimentais/terapia , RNA Mensageiro , Animais , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/genética , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Imunidade Celular , Imunidade Humoral , Memória Imunológica , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/prevenção & controle , Protaminas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so-called cell-penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis-independent cellular entry. In this study, we provide evidence that the Antennapedia-homeodomain-derived antennapedia (Antp) peptide, nona-arginine and the HIV-1 Tat-protein-derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis-independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant-negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.
Assuntos
Membrana Celular/metabolismo , Peptídeos/química , Apoptose , Arginina/química , Cátions , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Endocitose , Produtos do Gene tat/metabolismo , HIV-1/metabolismo , Células HeLa , Humanos , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Transporte Proteico , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Inside the cell, proteases act in concert in the degradation of proteins and peptides. In order to understand the significance of an individual proteolytic activity within an ensemble of proteases, protocols and probes are required that enable a quantitative determination of the contribution of a protease to the break-down of a given substrate. Here we present a fluorescence resonance energy transfer-based probe and protocols for a quantitative determination of proteolytic activities inside the endolysosomal compartment. A peptide substrate that is readily cleaved by different cathepsins is flanked by fluorescein and tetramethylrhodamine-labeled lysine residues. Efficient endolysosomal targeting of the substrate is achieved by N-terminal elongation with the cell-penetrating peptide nona-arginine. The proteasome inhibitor lactacystin has a small, but significant effect on the break-down of the substrate, thus demonstrating that only a minor fraction of the peptide reaches the cytoplasm in its intact form. Nona-arginine therefore constitutes a highly efficient low-molecular-weight moiety for targeting the endolysosomal compartment.
Assuntos
Endossomos/enzimologia , Lisossomos/enzimologia , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Catálise/efeitos dos fármacos , Catepsina B/metabolismo , Catepsina D/metabolismo , Catepsina L , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fluoresceínas/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Células HeLa , Humanos , Oligopeptídeos/química , Pepstatinas/farmacologia , Peptídeos/síntese química , Inibidores de Proteases/farmacologia , Rodaminas/química , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Especificidade por SubstratoRESUMO
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
Cell-penetrating peptides (CPPs) have become widely used vectors for the cellular import of molecules in basic and applied biomedical research. Despite the broad acceptance of these molecules as molecular carriers, the details of the mode of cellular internalization and membrane permeation remain elusive. Within the last two years endocytosis has been demonstrated to be a route of uptake shared by several CPPs. These findings had a significant impact on CPP research. State-of-the-art cell biology is now required to advance the understanding of the intracellular fate of the CPP and cargo molecules. Owing to their presumed ability to cross lipid bilayers, CPPs also represent highly interesting objects of biophysical research. Numerous studies have investigated structure-activity relationships of CPPs with respect to their ability to bind to a lipid bilayer or to cross this barrier. Endocytosis route only relocates the membrane permeation from the cell surface to endocytic compartments. Therefore, biophysical experiments are key to a mechanistic molecular understanding of the cellular uptake of CPPs. However, biophysical investigations have to consider the molecular environment encountered by a peptide inside and outside a cell. In this contribution we will review biophysical and cell-biology data obtained for several prominent CPPs. Furthermore, we will summarize recent findings on the cell-penetrating characteristics of antimicrobial peptides and the antimicrobial properties of CPPs. Peptides of both groups have overlapping characteristics. Therefore, both fields may greatly benefit from each other. The review will conclude with a perspective of how biophysics and cell biology may synergize even more efficiently in the future.
Assuntos
Permeabilidade da Membrana Celular , Peptídeos/farmacocinética , Sequência de Aminoácidos , Animais , Transporte Biológico , Sistemas de Liberação de Medicamentos , Endocitose , HumanosRESUMO
Cationic cell-penetrating peptides (CPPs) have been used widely as delivery vectors for the import of molecules that otherwise do not cross the plasma membrane of eukaryotic cells. In this work, we demonstrate that the three cationic CPPs, Antennapedia homeodomain-derived peptide (Antp), nona-arginine and Tat-derived peptide, inhibit tumour necrosis factor (TNF)-mediated signal transduction. This inhibition is based on the downregulation of TNF receptors at the cell surface by induction of internalization. In contrast to TNF-dependent receptor internalization, no receptor activation occurs. The receptor downregulation is not restricted to the CPPs. Remarkably, the HIV-1 Tat protein itself also induces the internalization of TNF receptors. The dynamin dependence of the internalization, as well as the fact that epidermal growth factor receptors are also internalized, suggest a general induction of clathrin-dependent endocytosis as the mechanism of action. The significance of these findings for the use of cationic CPPs in the import of bioactive peptides is demonstrated here using a conjugate consisting of Antp and a Smac protein-derived cargo peptide. The cargo alone, when introduced into cells by electroporation, enhanced TNF-induced apoptosis by inhibiting the anti-apoptotic action of IAPs (inhibitor of apoptosis proteins). For the Antp-Smac conjugate at concentrations below 40 muM the inhibitory effect of the Antp peptide compensated for the pro-apoptotic activity of the cargo, and led to the protection of cells against TNF-mediated apoptosis. These data provide important new information for the use of cationic CPPs for the cellular delivery of bioactive molecules.