RESUMO
Advanced therapy medicinal products (ATMP) in the European Union (EU) are regulated by Regulation 1394/2007 and comprise gene and cell therapy and tissue-engineered products. Under this framework, ATMP are authorised by the centralised procedure, coordinated by the European Medicines Agency (EMA), whereas clinical trial authorisations remain at the remit of each National Competent Authority. The Committee for Advanced Therapies is responsible for the scientific evaluation of the marketing authorisation applications and for generating a draft opinion that goes to the Committee for Human Medicinal Products for a final opinion. For every application, data and information relating to manufacturing processes and quality control of the active substance and final product have to be submitted for assessment together with data from non-clinical and clinical safety and efficacy studies. Technical requirements for ATMP are defined in the legislation, and guidance for different products is available through several EMA/CAT guidelines.Due to the diverse and complex nature of ATMP, a need for some regulatory flexibility was recognised. Thus, a risk-based approach was introduced in Regulation 1394/2007 allowing adapted regulatory requirements. This has led, for instance, to the development of good manufacturing practice (GMP) guidelines specific for ATMP. This, together with enhanced regulatory support, has allowed an increasing number of successful marketing authorisation applications resulting in 25 licensed ATMP in the EU, mainly gene therapy medicinal products. The promise of messenger RNA and genome editing technologies as therapeutic tools make the future for these innovative medicinal products look even brighter.This chapter reviews the regulatory landscape together with some of the support initiatives developed for ATMP in the EU.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Engenharia Tecidual , Humanos , Europa (Continente) , União Europeia , MarketingRESUMO
Chimeric antigen receptor (CAR)-engineered T-cell therapy is becoming one of the most promising approaches in the treatment of cancer. On June 28, 2018, the Committee for Advanced Therapies (CAT) and the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency adopted a positive opinion, recommending the granting of a marketing authorization for the medicinal product Kymriah for pediatric and young adult patients up to 25 years of age with B-cell acute lymphoblastic leukemia (ALL) that is refractory, in relapse after transplant, or in second or later relapse and for adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) after two or more lines of systemic therapy. Kymriah became one of the first European Union-approved CAR T therapies. The active substance of Kymriah is tisagenlecleucel, an autologous, immunocellular cancer therapy that involves reprogramming the patient's own T cells to identify and eliminate CD19-expressing cells. This is achieved by addition of a transgene encoding a CAR. The benefit of Kymriah was its ability to achieve remission with a significant duration in patients with ALL and an objective response with a significant duration in patients with DLBCL. The most common hematological toxicity was cytopenia in both patients with ALL and those with DLBCL. Nonhematological side effects in patients with ALL were cytokine release syndrome (CRS), infections, secondary hypogammaglobulinemia due to B-cell aplasia, pyrexia, and decreased appetite. The most common nonhematological side effects in patients with DLBCL were CRS, infections, pyrexia, diarrhea, nausea, hypotension, and fatigue. Kymriah also received an orphan designation on April 29, 2014, following a positive recommendation by the Committee for Orphan Medicinal Products (COMP). Maintenance of the orphan designation was recommended at the time of marketing authorization as the COMP considered the product was of significant benefit for patients with both conditions. IMPLICATIONS FOR PRACTICE: Chimeric antigen receptor (CAR)-engineered T-cell therapy is becoming the most promising approach in cancer treatment, involving reprogramming the patient's own T cells with a CAR-encoding transgene to identify and eliminate cancer-specific surface antigen-expressing cells. On June 28, 2018, Kymriah became one of the first EMA approved CAR T therapies. CAR T technology seems highly promising for diseases with single genetic/protein alterations; however, for more complex diseases there will be challenges to target clonal variability within the tumor type or clonal evolution during disease progression. Products with a lesser toxicity profile or more risk-minimization tools are also anticipated.
Assuntos
Linfoma Difuso de Grandes Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Criança , Humanos , Imunoterapia Adotiva , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genéticaRESUMO
We have engineered the polymeric vaccine BLSOmp31 by decorating the highly immunogenic and decameric Brucella lumazine synthase with an exposed loop of the Brucella outer membrane protein Omp31. In the present study, we have immunized different groups of rams with the recombinant chimera rBLSOmp31 in two different adjuvants (Incomplete Freund Adjuvant-IFA and QUIL A) and with the plasmid pCIBLSOmp31 administered either by i.m. injection alone or by using electroporation. In addition, we have used a heterologous prime-boost strategy consisting of repeated pCIBLSOmp31 electroporation priming followed by a single protein boost. Both, chimera rBLSOmp31 in IFA and the prime-boost strategy induced the highest IgG specific antibodies with bacteriolytic activity. While electroporation-enhanced humoral immune responses as compared to pCIBLSOmp31 injection alone, the highest levels of specific IFN-gamma and protection against bacterial challenge were achieved with prime-boost (76%) and chimera rBLSOmp31 in IFA (63%). Taken together these results strongly support the usefulness of the chimera BLSOmp31 as a vaccine against Brucella ovis in ovine brucellosis.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacina contra Brucelose/imunologia , Brucelose/veterinária , Doenças dos Ovinos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Testes de Aglutinação , Animais , Anticorpos Antibacterianos/sangue , Brucella ovis/imunologia , Brucelose/imunologia , Brucelose/prevenção & controle , Proteínas do Sistema Complemento/imunologia , Eletroporação , Adjuvante de Freund/imunologia , Imunidade Humoral , Imunoglobulina G/sangue , Interferon gama/imunologia , Lipídeos/imunologia , Masculino , Complexos Multienzimáticos/imunologia , Plasmídeos , Saponinas de Quilaia , Proteínas Recombinantes/imunologia , Saponinas/imunologia , Ovinos , Doenças dos Ovinos/prevenção & controleRESUMO
We are presently aware of two early-phase DNA vaccine clinical trials in humans using electroporation-enhanced vaccine delivery. Moreover, two phase I immunogenetherapy studies are in progress and several tolerability studies have been performed on healthy volunteers. We have used knowledge from these studies to compose a template for clinical protocols involving electroporation-mediated gene delivery. In this template the emphasis will be on aspects related to electroporation. In addition, we will discuss general topics concerning electroporation-augmented DNA vaccination in human subjects.
Assuntos
Eletroquimioterapia/métodos , Vacinas de DNA/administração & dosagem , Protocolos Clínicos , Ensaios Clínicos como Assunto , Eletroquimioterapia/instrumentação , Humanos , Injeções Intramusculares , Educação de Pacientes como Assunto , Seleção de PacientesRESUMO
In vivo electroporation (EP) has been shown to augment the immunogenicity of plasmid DNA vaccines, but its mechanism of action has not been fully characterized. In this study, we show that in vivo EP augmented cellular and humoral immune responses to a human immunodeficiency virus type 1 Env DNA vaccine in mice and allowed a 10-fold reduction in vaccine dose. This enhancement was durable for over 6 months, and re-exposure to antigen resulted in anamnestic effector and central memory CD8(+) T-lymphocyte responses. Interestingly, in vivo EP also recruited large mixed cellular inflammatory infiltrates to the site of inoculation. These infiltrates contained 45-fold-increased numbers of macrophages and 77-fold-increased numbers of dendritic cells as well as 2- to 6-fold-increased numbers of B and T lymphocytes compared to infiltrates following DNA vaccination alone. These data suggest that recruiting inflammatory cells, including antigen-presenting cells (APCs), to the site of antigen production substantially improves the immunogenicity of DNA vaccines. Combining in vivo EP with plasmid chemokine adjuvants that similarly recruited APCs to the injection site, however, did not result in synergy.
Assuntos
Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Movimento Celular/imunologia , HIV-1/imunologia , Imunogenética , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Adjuvantes Imunológicos , Animais , Proliferação de Células , Quimiocinas/genética , Quimiocinas/imunologia , Eletroporação , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Plasmídeos/genéticaRESUMO
The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.
Assuntos
Complexo CD3/metabolismo , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Hepacivirus/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas não Estruturais Virais/biossíntese , Animais , DNA Viral/genética , Eletroporação , Feminino , Hepacivirus/genética , Hepacivirus/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interferon gama/biossíntese , Fígado/imunologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Coelhos , Testes de Toxicidade , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismoRESUMO
UNLABELLED: Liver sinusoidal endothelial cells (LSECs) display a number of receptors for efficient uptake of potentially injurious molecules. The receptors for the Fc portion of immunoglobulin G (IgG) antibodies (FcgammaRs) regulate a number of physiological and pathophysiological events. We used reverse transcription polymerase chain reaction (RT-PCR) and Western blotting to determine the expression of different types of FcgammaRs in LSECs. Biochemical approaches and immunofluorescence microscopy were used to characterize the FcgammaR-mediated endocytosis of immune complexes (ICs). FcgammaRIIb2 was identified as the main receptor for the efficient uptake of ICs in LSECs. The receptor was shown to use the clathrin pathway for IC uptake; however, the association with lipid rafts may slow the rate of its internalization. Moreover, despite trafficking through lysosomal integral membrane protein-II (LIMP-II)-containing compartments, the receptor was not degraded. Finally, it was shown that the receptor recycles to the cell surface both with and without IC. CONCLUSION: FcgammaRIIb2 is the main receptor for endocytosis of ICs in rat LSECs. Internalized ICs are degraded with slow kinetics, and IC internalization is not linked to receptor downregulation. After internalization, the receptor recycles to the cell surface both with and without ICs. Thus, FcgammaRIIb2 in rat LSECs is used as both a recycling receptor and a receptor for efficient IC clearance.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Antígenos CD/metabolismo , Endocitose/imunologia , Fígado/imunologia , Receptores de IgG/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/análise , Antígenos CD/genética , Western Blotting , Membrana Celular/química , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/genética , Células Endoteliais/imunologia , Cinética , Fosforilação , Transporte Proteico , Ratos , Ratos Wistar , Receptores de IgG/análise , Receptores de IgG/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/metabolismoRESUMO
Since human immunodeficiency virus (HIV)-specific cell-mediated immune (CMI) responses are critical in the early control and resolution of HIV infection and correlate with postchallenge outcomes in rhesus macaque challenge experiments, we sought to identify a plasmid DNA (pDNA) vaccine design capable of eliciting robust and balanced CMI responses to multiple HIV type 1 (HIV-1)-derived antigens for further development. Previously, a number of two-, three-, and four-vector pDNA vaccine designs were identified as capable of eliciting HIV-1 antigen-specific CMI responses in mice (M. A. Egan et al., Vaccine 24:4510-4523, 2006). We then sought to further characterize the relative immunogenicities of these two-, three-, and four-vector pDNA vaccine designs in nonhuman primates and to determine the extent to which in vivo electroporation (EP) could improve the resulting immune responses. The results indicated that a two-vector pDNA vaccine design elicited the most robust and balanced CMI response. In addition, vaccination in combination with in vivo EP led to a more rapid onset and enhanced vaccine-specific immune responses. In macaques immunized in combination with in vivo EP, we observed a 10- to 40-fold increase in HIV-specific enzyme-linked immunospot assay responses compared to those for macaques receiving a 5-fold higher dose of vaccine without in vivo EP. This increase in CMI responses translates to an apparent 50- to 200-fold increase in pDNA vaccine potency. Importantly, in vivo EP enhanced the immune response against the less immunogenic antigens, resulting in a more balanced immune response. In addition, in vivo EP resulted in an approximate 2.5-log(10) increase in antibody responses. The results further indicated that in vivo EP was associated with a significant reduction in pDNA persistence and did not result in an increase in pDNA associated with high-molecular-weight DNA relative to macaques receiving the pDNA without EP. Collectively, these results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/sangue , HIV-1/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas contra a AIDS/genética , Animais , Eletroporação , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/genética , Humanos , Interferon gama/biossíntese , Leucócitos Mononucleares/imunologia , Macaca mulatta , Masculino , Modelos Animais , Testes de Neutralização , Plasmídeos , Vacinas de DNA/genéticaRESUMO
DNA vaccines represent a new and promising technology that uses DNA to encode the antigen(s) of interest, instead of inoculating with attenuated or inactivated microbes or isolated antigens. Antigen is produced within the transfected cells minaicking a real life viral infection. This vaccine modality has been shown to elicit strong cellular immune responses and is promising for treating diseases where traditional vaccine approaches have failed. In spite of promising results in small animal models, DNA vaccines have so far proven less potent in human clinical trials. In this review we provide a general overview on the mechanisms of action for DNA vaccines, discuss potential benefits of traditional vaccine approaches and review current strategies for improving the immunogenicity of DNA vaccines to enable the successful transfer of the technology can be successfully transferred from mice to men.
Assuntos
Vacinas de DNA , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Biotecnologia/tendências , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Celular/genética , Imunidade Celular/imunologia , Imunogenética , Camundongos , Vacinação/tendências , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologiaRESUMO
DNA vaccines have shown to induce strong immune response in small animals; however, its capacity of inducing robust antigen-specific immune responses in large animals is limited. In the present study, in vivo electroporation (EP) was applied and the effect of EP on humoral immune response against tuberculosis (TB) induced by DNA vaccination was tested in mice and rhesus macaques. Mice injected with 10 microg DNA encoding Ag85A and ESAT-6 followed by EP showed a reproducible humoral immunity which was equal to that obtained by using 100 microg DNA without EP. Boosting the DNA/EP treated animals with corresponding recombinant protein (50 microg of either Ag85A or ESAT-6) without adding adjuvant gave more than a 7-8-fold increase in the antibody titre but only 3-4-fold increase was found in the mice receiving 100 microg DNA without EP followed by protein boost. In concordance with the results obtained in mice, the monkeys received less DNA achieved equal high antibody responses to those induced by high dosage of DNA. Boosting the the DNA/EP treated monkeys with TB protein (500 microg of either Ag85A or ESAT-6) improved the humoral response by 7-8-fold increase in antibody titre, indicating electroporation's ability to compensate lower DNA concentration and enhance humoral immunity of TB DNA vaccines in mice and non-human primates.
Assuntos
Anticorpos Antivirais/biossíntese , Eletroporação , Vacinas de DNA/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Camundongos , PrimatasRESUMO
Intramuscular injection of plasmid DNA followed by electrical stimulation (electroporation) is an efficient method for achieving therapeutic levels of encoded proteins or eliciting efficient immune responses in smaller animals such as mice and rats. Electroporation in larger animals and humans poses new technical challenges, the main difficulty being to maintain efficacy while limiting invasiveness and pain. Here we present data using a new device for combined injection and electroporation in large animals and humans. The device injects DNA through two needles during insertion into the muscle and thus distributes the injection volume along the needles which also serve as electrodes. Since the electrical field is strongest close to the needle-electrode, a near perfect match between the DNA and the electric field is achieved. We show that using moderate amounts of DNA: (1) muscle tissue is transfected along the entire length of the needle path, (2) the efficacy is higher compared to when the DNA is injected between the electrodes, (3) level of protein expression can be tightly controlled by the number of treatments, and (4) efficient immunization is achieved.
Assuntos
Eletroporação/instrumentação , Terapia Genética , Animais , Formação de Anticorpos , Eletrodos , Desenho de Equipamento , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Agulhas , Ratos , beta-Galactosidase/genéticaRESUMO
The process by which clathrin-coated vesicles are produced involves interactions of multifunctional adaptor proteins with the plasma membrane, as well as with clathrin and several accessory proteins and phosphoinositides. Here we review recent findings highlighting new insights into mechanisms underlying clathrin-dependent endocytosis.
Assuntos
Proteínas de Ligação ao Cálcio , Clatrina/fisiologia , Endocitose , Complexo 2 de Proteínas Adaptadoras/fisiologia , Animais , Arrestinas/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Clatrina/análise , Invaginações Revestidas da Membrana Celular/química , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Vesículas Revestidas/química , Glicoproteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Sinaptotagminas , beta-ArrestinasRESUMO
Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37 degrees C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.
Assuntos
Actinas/metabolismo , Endossomos/metabolismo , Macrófagos/metabolismo , Fusão de Membrana/fisiologia , Fagossomos/metabolismo , Trifosfato de Adenosina , Animais , Células Cultivadas , Microscopia Crioeletrônica , Citoesqueleto/metabolismo , Citosol/metabolismo , Endocitose/fisiologia , Camundongos , Microscopia Confocal , Modelos Moleculares , Organelas/metabolismo , Timosina/metabolismoRESUMO
To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [(125)I]tyramine cellobiose ([(125)I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [(125)I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [(125)I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [(125)I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [(125)I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce 'hybrid organelles' (active lysosomes) that are unable to mature into dense lysosomes.