RESUMO
BACKGROUND: Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic messenger RNA (mRNA) encoding a CD40 ligand, a constitutively active Toll-like receptor 4 and CD70, together with mRNA encoding fusion proteins of a human leukocyte antigen (HLA)-class II targeting signal (DC-LAMP) and a melanoma-associated antigen (MAA); either MAGE-A3, MAGE-C2, tyrosinase or gp100) (TriMixDC-MEL) are superiorly immunogenic. PATIENTS AND METHODS: In this phase IB clinical trial, 24 million viable DCs were administered by four biweekly combined intradermal (id) and intravenous (iv) administrations, and a fifth administration on week 16. The number of iv-administered DCs was escalated in four sequentially treated cohorts. Immune responses were assessed by analysis of antigen specificity of blood-derived T-cells and skin infiltrating lymphocytes (SKILs). RESULTS: Fifteen patients with pretreated advanced melanoma tolerated administration of TriMixDC-MEL well. Two patients achieved a complete response and two patients a partial response. All objective responders are progression-free after a follow-up of, respectively, 24+, 28+, 33+, and 34+ months. Post-therapy antigen-specific SKILs were documented in 6 of 12 patients, and antigen-specific CD8(+) T-cells were detected in the blood of 4 of 5 patients. CONCLUSIONS: Cellular immunotherapy with TriMixDC-MEL is safe and immunogenic. Antitumor activity with durable disease control is observed across the investigated iv-dose levels. CLINICALTRIALSGOV IDENTIFIER: NCT01066390.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Dendríticas/imunologia , Imunoterapia/métodos , Melanoma/terapia , Neoplasias Cutâneas/terapia , Adulto , Idoso , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Ligante CD27/genética , Ligante CD27/metabolismo , Ligante de CD40/genética , Ligante de CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/citologia , Intervalo Livre de Doença , Eletroporação , Feminino , Humanos , Proteína 3 de Membrana Associada ao Lisossomo/genética , Proteína 3 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.
Assuntos
Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/imunologia , Terapia Genética/métodos , Imunoterapia/métodos , Melanoma Experimental/terapia , Neoplasias Cutâneas/terapia , Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Testes Imunológicos de Citotoxicidade , Células Dendríticas/imunologia , Eletroporação/métodos , Citometria de Fluxo , Engenharia Genética , Humanos , Interferon gama/imunologia , Ativação Linfocitária , Antígeno MART-1 , Melanoma Experimental/imunologia , Proteínas de Neoplasias/genética , RNA/administração & dosagem , RNA de Cadeia Dupla/administração & dosagem , Neoplasias Cutâneas/imunologia , Células Tumorais CultivadasRESUMO
Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.
Assuntos
Células Dendríticas/imunologia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Imunoterapia/métodos , Lentivirus/genética , Neoplasias/terapia , Animais , Antígenos Glicosídicos Associados a Tumores/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Células Dendríticas/virologia , Feminino , Memória Imunológica , Interferon gama/imunologia , Linfonodos/imunologia , Linfonodos/virologia , Ativação Linfocitária , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Ovalbumina/genética , Ovalbumina/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/imunologia , Transdução Genética/métodosRESUMO
Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.