RESUMO
Clinical studies of cell-based immunotherapies have included both patient-specific (autologous) and non-patient-specific (allogeneic) approaches. Major concerns in using allogeneic immunotherapies are that the induced immune responses may be predominantly directed against the allogeneic HLA molecules of the cellular immunotherapy and not against its potential tumor antigens and that only the allogeneic responses will be enhanced when the immunotherapies are combined with immune checkpoint regulators in an effort to enhance overall immunotherapy potency. To evaluate these possibilities, studies were performed using the GM-CSF-secreting B16F1 cell line as autologous immunotherapy (Auto) and the same cell line modified to over-express the MHC molecule K(d) to generate an immunotherapy that expresses an allogeneic component (Allo) when injected into C57/Bl6 mice. The goal was to compare the specific anti-tumor immune responses induced by these two immunotherapies, which share an identical antigen repertoire, with the exception of the allogeneic MHC class I molecule expressed by the Allo cells, and have identical GM-CSF-secretion levels. Both immunotherapies provided similar therapeutic benefit to tumor-bearing animals with a trend towards a more pronounced tumor growth delay in animals injected with the Allo immunotherapy. This correlated with a significant increase in the number of activated DCs and T-cells in the DLN of Allo-treated animals. In addition, persistent infiltration of effector CD8(+) T-cells was detected in the tumors of animals treated with the Allo immunotherapy, which correlated with a trend towards a greater antigen-specific T-cell response in these animals. When combined with the immune checkpoint regulator anti-PD-1, tumor-specific and allogeneic immune responses were equally enhanced. Thus, the ability of an allogeneic tumor cell immunotherapy to induce a therapeutic anti-tumor immune response is comparable, if not superior, to an autologous tumor cell immunotherapy and its anti-tumor potency can be enhanced when combined with immunomodulatory compounds.
Assuntos
Vacinas Anticâncer/imunologia , Transplante de Células/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Transplante Autólogo/imunologia , Transplante Homólogo/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Movimento Celular/imunologia , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoterapia/métodos , Linfonodos/citologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Baço/citologia , Baço/imunologia , Análise de Sobrevida , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , TransfecçãoRESUMO
Oncolytic adenoviral vectors that express immunostimulatory transgenes are currently being evaluated in clinic. Preclinical testing of these vectors has thus far been limited to immunodeficient xenograft tumor models since human adenoviruses do not replicate effectively in murine tumor cells. The effect of the immunostimulatory transgene on overall virus potency can therefore not be readily assessed in these models. Here, a model is described that allows the effective testing of mouse armed oncolytic adenovirus (MAV) vectors in immunocompetent syngeneic tumor models. These studies demonstrate that the MAV vectors have a high level of cytotoxicity in a wide range of murine tumor cells. The murine oncolytic viruses were successfully armed with murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) by a novel method which resulted in vectors with a high level of tumor-specific transgene expression. The mGM-CSF-armed MAV vectors showed an improved level of antitumor potency and induced a systemic antitumor immune response that was greater than that induced by unarmed parental vectors in immunocompetent syngeneic tumor models. Thus, the oncolytic MAV-1 system described here provides a murine homolog model for the testing of murine armed oncolytic adenovirus vectors in immunocompetent animals. The model allows evaluation of the impact of virus replication and the host immune response on overall virus potency and enables the generation of translational data that will be important for guiding the clinical development of these viruses.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Modelos Animais , Neoplasias/virologia , Vírus Oncolíticos/crescimento & desenvolvimento , Vírus Oncolíticos/imunologia , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Vírus Oncolíticos/genéticaRESUMO
PURPOSE: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy, which is known to stimulate potent and long-lasting antigen-specific immune responses, in combination with PD-1 blockade, which has been shown to augment cellular immune responses. EXPERIMENTAL DESIGN: Survival studies were done in the B16 melanoma and CT26 colon carcinoma tumor models. Immune monitoring studies were done in the B16 model. GM-CSF-secreting tumor cell immunotherapy was administered s.c. and the anti-PD-1 antibody was administered i.p. RESULTS: The studies reported here show that combining PD-1 blockade with GM-CSF-secreting tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with strong antigen-specific T-cell responses detected by tetramer staining and an in vivo CTL assay, higher secretion levels of proinflammatory cytokines by splenocytes, and the persistence of functional CD8+ T cells in the tumor microenvironment. Furthermore, in the biweekly multiple treatment setting, repeated antigen-specific T-cell expansion was only observed following administration of the cellular immunotherapy with the PD-1 blockade and not when the cellular immunotherapy or PD-1 blockade was used as monotherapy. CONCLUSION: The combination of PD-1 blockade with GM-CSF-secreting tumor cell immunotherapy leads to significantly improved antitumor responses by augmenting the tumor-reactive T-cell responses induced by the cellular immunotherapy. Readministration of the cellular immunotherapy with the anti-PD-1 antibody in subsequent immunotherapy cycles was required to reactivate these T-cell responses.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Neoplasias do Colo/terapia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Imunoterapia , Melanoma Experimental/terapia , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proliferação de Células , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Citocinas/metabolismo , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Receptor de Morte Celular Programada 1 , Ratos , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
PURPOSE: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy, which is known to stimulate a potent and long-lasting antigen-specific immune response in combination with lymphocyte activation gene-3 fusion protein (LAG-3Ig), which has been shown to act as an adjuvant for priming T helper type 1 and cytotoxic T-cell responses. EXPERIMENTAL DESIGN: Survival and immune monitoring studies were done in the B16 melanoma model. GM-CSF-secreting tumor cell immunotherapy was administered as a single s.c. injection and LAG-3Ig was administered s.c. at the immunotherapy site. RESULTS: The studies reported here show that combining LAG-3Ig with GM-CSF-secreting tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with increased numbers of systemic IFN gamma-secreting CD8+ T cells and a significantly increased infiltration of activated effector CD8+ T cells into the tumor. Moreover, an increase in antigen-specific IgG1 humoral responses was detected in serum of animals injected with the combination therapy compared with animals injected with either therapy alone. CONCLUSION: LAG-3Ig combined with a GM-CSF-secreting tumor cell immunotherapy stimulated both cellular and humoral antitumor immune responses that correlated with prolonged survival in tumor-bearing animals.
Assuntos
Antígenos CD/uso terapêutico , Imunoterapia/métodos , Fator Estimulador de Colônias de Macrófagos/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Animais , Anticorpos/sangue , Terapia Combinada , Citocinas/biossíntese , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Promising anti-tumor responses have been observed in the clinic using monoclonal antibodies (mAbs) that block immune checkpoints. One concern with these therapeutic agents remains the potential induction of immune breakthrough events (IBEs) resulting from the disruption of T cell homeostasis or the breaking of tolerance to self antigens. As an approach to maintaining anti-tumor responses but decreasing the likelihood of these events, the local expression of a mAb in combination with a GM-CSF-secreting cancer immunotherapy was evaluated. Using anti-cytotoxic T lymphocyte antigen (CTLA)-4 as a model antibody to test this hypothesis, tumor cell lines were generated that expressed the full-length mAb in addition to GM-CSF. Evaluation of these cell lines in two therapeutic tumor models revealed that local, cell-mediated delivery of anti-CTLA-4 from a GM-CSF-secreting tumor cell immunotherapy activated potent anti-tumor responses and prolonged overall survival at significantly lower serum mAb levels in the host. Furthermore, lowering the systemic exposure of the host to the immune modulatory mAb correlated with reduced evidence of systemic autoimmunity. This approach has broad utility for the delivery of mAbs or proteins locally from cellular immunotherapies to minimize IBEs while retaining the potent therapeutic effects of such combination treatments.
Assuntos
Anticorpos Monoclonais/biossíntese , Antígenos CD/imunologia , Autoimunidade/imunologia , Imunoterapia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Animais , Anticorpos Monoclonais/genética , Antígeno CTLA-4 , Linhagem Celular Tumoral , Clonagem Molecular , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVE: Acute myeloid leukemia (AML) is a highly malignant neoplasm responsible for nearly 10,000 cancer-related deaths annually in the United States. Treatment options for elderly patients with AML remain limited. Standard regimens using cytarabine (cytosine arabinoside [AraC]), a nucleotide analogue, result in significant toxicity with poor overall response. Combination of a cytotoxic chemotherapy and tumor-specific immunotherapy has the potential to improve overall efficacy by inducing an anti-tumor immune response against minimal residual disease. The studies reported here were performed to evaluate the therapeutic benefit of combining a granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy with AraC treatment. MATERIALS AND METHODS: C57Bl/6 mice were challenged with C1498-luc cells intravenously and evaluated by in vivo imaging throughout the study to monitor the systemic progression of the tumor. Individual animals were euthanized when in vivo total photon counts exceeded 5 x 10(8) and/or when they were in poor clinical condition. Cytotoxicity assay was performed to evaluate effector function and flow cytometry was used for phenotyping of splenocytes from experimental animals. RESULTS: Administration of GM-CSF-secreting tumor cell immunotherapy during AraC -induced cytopenia enhanced the anti-tumor efficacy of the immunotherapy, resulting in prolonged survival. AraC treatment did not negatively impact antigen-specific T-cell activation elicited by the immunotherapy and surviving animals treated with the combination demonstrated strong tumor-specific memory responses. CONCLUSION: GM-CSF-secreting tumor cell immunotherapy in combination with AraC prolongs survival of tumor-bearing mice, with a median survival time of 61 days observed in mice treated with AraC alone and 90% of mice treated with the combination therapy still alive by day 150.
Assuntos
Antineoplásicos/administração & dosagem , Citarabina/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Animais , Antineoplásicos/efeitos adversos , Linhagem Celular Tumoral , Terapia Combinada , Citarabina/efeitos adversos , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Técnicas de Transferência de Genes , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Memória Imunológica , Injeções Intraperitoneais , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Luciferases/biossíntese , Luciferases/genética , Linfopenia/induzido quimicamente , Linfopenia/imunologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , Taxa de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
Immunization with irradiated tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates potent, specific and long lasting anti-tumor immunity in clinical and preclinical settings. Efforts to further increase immunotherapy efficacy with immune-modulatory agents are under evaluation. Based on the immune-modulatory properties of 4-1BB (CD137), it has been postulated that agonistic 4-1BB antibodies may add additional anti-tumor efficacy to GM-CSF-secreting tumor cell immunotherapy. The combination of GM-CSF-secreting tumor cell immunotherapy and anti-4-1BB monoclonal antibody (mAb) treatment resulted in rejection of established tumors in the B16 melanoma model. These anti-tumor effects correlated with persistent tumor-specific CD8(+) T cell responses. In addition, early tumor infiltration of functional CD8(+) T cells and a greater expansion of antigen-specific memory T cells were found in mice treated with the combination therapy. In summary, an agonistic anti-4-1BB mAb combined with GM-CSF-secreting tumor cell immunotherapy may provide a novel and potent treatment strategy for patients with cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Fatores Imunológicos/uso terapêutico , Imunoterapia Adotiva/métodos , Melanoma Experimental/terapia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fatores Imunológicos/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Selective replication of oncolytic viruses in tumor cells provides a promising approach for the treatment of human cancers. One of the limitations observed with oncolytic viruses currently used in the treatment of solid tumors is the inefficient spread of virus throughout the tumor mass following intratumoral injection. Data are presented showing that oncolytic adenoviruses expressing the relaxin gene and containing an Ad5/Ad35 chimeric fiber showed significantly enhanced transduction and increased virus spread throughout the tumor when compared with non-relaxin-expressing, Ad5-based viruses. The increased spread of such viruses throughout tumors correlated well with improved antitumor efficacy and overall survival in two highly metastatic tumor models. Furthermore, nonreplicating viruses expressing relaxin did not increase metastases, suggesting that high level expression of relaxin will not enhance metastatic spread of tumors. In summary, the data show that relaxin may play a role in rearranging matrix components within tumors, which helps recombinant oncolytic adenoviruses to spread effectively throughout the tumor mass and thereby increase the extent of viral replication within the tumor. Expressing relaxin from Ad5/Ad35 fiber chimeric adenoviruses may prove a potent and novel approach to treating patients with cancer.
Assuntos
Adenoviridae/fisiologia , Terapia Viral Oncolítica/métodos , Neoplasias da Próstata/terapia , Relaxina/biossíntese , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Linhagem Celular Tumoral , Colágeno/metabolismo , Feminino , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Metástase Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/virologia , Relaxina/genética , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Animais , Apoptose , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Proteínas Recombinantes , Linfócitos T/imunologiaRESUMO
Monoclonal antibody (mAb) delivery by gene transfer in vivo may be an attractive alternative to current mAb therapies for applications that require long-term therapy. This article describes a transfer system that allows inducible high-level expression of unmodified mAbs in vivo. A recombinant adeno-associated viral (rAAV) vector is used that comprises an expression cassette consisting of a dimerizer-regulated promoter that drives expression of the antibody heavy and light chains linked by a 2A self-processing peptide and a furin cleavage site. Following intravenous injection of the rAAV vector, serum mAb levels >1 mg/ml were attained by administration of the inducer, rapamycin. Antibody expression could be rapidly shut off by discontinuing treatment with rapamycin. By optimizing the furin cleavage sequence, this system generated native antibody in vivo, decreasing the likelihood of a host immune response to foreign sequences. In summary, this optimized mAb expression system allows regulated high-level expression of native full-length mAbs in vivo and may offer a new opportunity for delivery of therapeutic mAbs in the clinic.
Assuntos
Anticorpos Monoclonais/genética , Dependovirus/genética , Vetores Genéticos/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Western Blotting , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática , Feminino , Furina/metabolismo , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/administração & dosagem , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Dados de Sequência Molecular , Sirolimo/farmacologiaRESUMO
IL-7 is known for its role in lymphopoiesis and T-cell homeostasis. In addition, its capacity to augment the immune response to weak or low affinity antigens makes it an ideal candidate to evaluate in combination with a GM-CSF-secreting tumor cell immunotherapy, which has been shown to elicit broad humoral and cellular immune responses. The studies reported here show that IL-7, when combined with a GM-CSF-secreting tumor cell immunotherapy, significantly prolonged the survival of tumor-bearing mice. The enhanced anti-tumor protection correlated with an increased number of activated dendritic cells (DC) and T cells in lymphoid tissues, such as the draining lymph nodes (DLN) and spleen. Moreover, an increased number of activated effector T cells were found in the tumor microenvironment, correlating with a more potent systemic tumor-specific T-cell response than each monotherapy alone. Taken together, these studies demonstrate that IL-7 augments the anti-tumor response of a GM-CSF-secreting tumor cell immunotherapy in preclinical models.
Assuntos
Vacinas Anticâncer/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-7/uso terapêutico , Melanoma Experimental/terapia , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Animais , Antígeno CD11c/análise , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Contagem de Células , Linhagem Celular Tumoral , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interferon gama/análise , Interleucina-7/genética , Interleucina-7/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/análise , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Análise de Sobrevida , Antígeno gp100 de MelanomaRESUMO
PURPOSE: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. EXPERIMENTAL DESIGN: Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a soluble VEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory T cells were quantified by their CD4+CD25hi and CD4+FoxP3+ phenotype. RESULTS: The present study established that GM-CSF-secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4+ and CD8+ tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory T cells in the tumor, suggesting a novel mechanism of VEGF-mediated immune suppression at the tumor site. CONCLUSION: GM-CSF-secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunoterapia/métodos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Linfócitos T Reguladores/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose , Carcinoma/terapia , Contagem de Células , Neoplasias do Colo/terapia , Terapia Combinada , Células Dendríticas/citologia , Proteína Ligante Fas/fisiologia , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Linfócitos do Interstício Tumoral/citologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/mortalidade , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Análise de Sobrevida , Linfócitos T Reguladores/citologia , Resultado do Tratamento , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor fas/análiseRESUMO
The presence of the blood-brain barrier complicates drug delivery in the development of therapeutic agents for the treatment of glioblastoma multiforme (GBM). The use of local gene transfer in the brain has the potential to overcome this delivery barrier by allowing the expression of therapeutic agents directly at the tumor site. In this study, we describe the development of a recombinant adeno-associated (rAAV) serotype 8 vector that encodes an optimized soluble inhibitor, termed sVEGFR1/R2, of vascular endothelial growth factor (VEGF). VEGF is an angiogenic factor highly up-regulated in GBM tumor tissue and correlates with disease progression. In subcutaneous models of GBM, VEGF inhibition following rAAV-mediated gene transfer significantly reduces overall tumor volume and increases median survival time following a single administration of vector. Using orthotopic brain tumor models of GBM, we find that direct intracranial administration of the rAAV-sVEGFR1/R2 vector to the tumor site demonstrates anti-tumor efficacy at doses that are not efficacious following systemic delivery of the vector. We propose that rAAV-mediated gene transfer of a potent soluble VEGF inhibitor in the CNS represents an effective antiangiogenic treatment strategy for GBM.
Assuntos
Sistema Nervoso Central/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Glioblastoma/terapia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular , Linhagem Celular Tumoral , Dependovirus/classificação , Feminino , Terapia Genética/métodos , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Transplante de Neoplasias , Ratos , Ratos Nus , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Sorotipagem , Solubilidade , Transplante HeterólogoRESUMO
Docetaxel has demonstrated therapeutic efficacy against breast, prostate, and ovarian cancer and other solid tumors. The tumoricidal activity of docetaxel is mainly attributed to its ability to block microtubule depolymerization, thus inducing G(2)-M arrest and apoptosis. Mounting evidence indicates that docetaxel also possesses immunomodulatory activity such as augmenting macrophage and lymphokine activated killer activity and inducing pro-inflammatory cytokines, suggesting that docetaxel may be a good chemotherapeutic agent to combine with cancer immunotherapies, assuming that it does not inhibit the vaccine-induced immune response. The anti-tumor activity of the combination of docetaxel and a GM-CSF-secreting B16F10 tumor cell vaccine (B16.GM) was evaluated in the murine B16 melanoma model. Dose levels of docetaxel and the B16.GM vaccine known to be ineffective when used as single agents were selected. Three iv treatments of 6 mg/kg docetaxel per injection given on days 5, 9, and 13 after tumor challenge or a single vaccination with 2-3 x 10(6) B16.GM cells on day 3 were ineffective at inhibiting tumor growth when used as single agents [median survival time (MST)=24 days in both treatment groups and in control animals]. However, combination of docetaxel and B16.GM vaccine significantly delayed tumor growth, increasing MST to 45 days. A similar improvement in anti-tumor efficacy was observed using multiple treatment cycles of the B16.GM vaccine/docetaxel combination. Administration of docetaxel every 4 days between bi-weekly B16.GM vaccinations increased the median survival of tumor-bearing mice from 31 to 52 days compared to multiple B16.GM vaccinations alone. In summary, these data demonstrate that rather than inhibiting the anti-tumor effects of a GM-CSF-secreting tumor cell vaccine, docetaxel combined with a whole cell vaccine significantly inhibits tumor growth, increases survival time and does not impede T-cell activation in the murine B16F10 melanoma tumor model. GM-secreting tumor cell vaccines in combination with docetaxel may represent a new strategy for combining chemo and immunotherapy for cancer.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Melanoma Experimental/terapia , Taxoides/uso terapêutico , Animais , Terapia Combinada , Docetaxel , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Melanoma Experimental/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sobrevida , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
PURPOSE: Transcriptionally controlled oncolytic adenovirus CG5757 is engineered with two tumor-specific promoters from E2F-1 and human telomerase reverse transcriptase genes. This virus has broad anticancer spectrum and higher specificity. The objective of the current study is to show its antitumor selectivity and therapeutic potential. EXPERIMENTAL DESIGN: The antitumor specificity of E2F-1 and human telomerase reverse transcriptase promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and cytotoxicity assays followed by a newly developed ex vivo tumor culture assay. Subsequently, in vivo antitumor efficacy and toxicology studies were carried out to assess the therapeutic potential of this oncolytic agent. RESULTS: In a broad panel of cells, E2F-1 and human telomerase reverse transcriptase promoters were activated in a tumor-selective manner. Under the control of these promoters, expression of E1a and E1b genes appears only in tumor cells. This specificity is extended to viral replication and hence the cytotoxicity in a broad range of cancer cells. Furthermore, CG5757 only replicates in cancer tissues but not in normal tissues that are derived from clinical biopsies. The safety profile was further confirmed in in vivo toxicology studies, and strong efficacy was documented in several tumor xenograft models after CG5757 was given via different routes and regimens. CONCLUSIONS: CG5757 has strong antitumor selectivity and potency. It has low toxicity and has great potential as a therapeutic agent for different types of cancers.
Assuntos
Adenoviridae/genética , Proteínas de Ligação a DNA/genética , Neoplasias/terapia , Regiões Promotoras Genéticas/genética , Telomerase/genética , Animais , Linhagem Celular Tumoral , Expressão Gênica , Genes Virais/genética , Terapia Genética , Humanos , Masculino , Camundongos , Camundongos Nus , Replicação Viral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival in many types of cancer. It has recently been shown that the lymphangiogenic growth factor, vascular endothelial growth factor-C (VEGF-C), and its receptor, VEGF receptor-3 (VEGFR3), may play a pivotal role in the promotion of metastasis to regional lymph nodes. In this study, human prostate and melanoma tumor models that preferentially metastasize to the lymph nodes following s.c. tumor cell implantation were established from lymph node metastases via in vivo selection. Melanoma tumor cell sublines established from lymph node metastasis express higher amounts of VEGF-C than the parental tumor cells. The inhibition of tumor-derived VEGF-C with a soluble VEGFR3 decoy receptor, sVEGFR3-Fc, expressed via a recombinant adeno-associated viral vector, potently blocks tumor-associated lymphangiogenesis and tumor metastasis to the lymph nodes, when the treatment was initiated before the tumor implantation. In addition, sVEGFR3-Fc serum levels required for efficient blockade of lymph node metastases are strictly dependent on the VEGF-C levels generated by the primary tumor. Recombinant adeno-associated virus-mediated gene transfer of sVEGFR3-Fc may represent a feasible therapeutic strategy for blockade of lymphogenous metastasis.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Neoplasias/terapia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Linhagem Celular Tumoral , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Neoplasias Pulmonares/secundário , Metástase Linfática , Masculino , Melanoma/genética , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/sangueRESUMO
Therapeutic monoclonal antibodies (mAbs) are currently being developed for the treatment of cancer and other diseases. Despite clinical success, widespread application of mAb therapies may be limited by manufacturing capabilities. In this paper, we describe a mAb delivery system that allows continuous production of a full-length antibody at high-concentrations in vivo after gene transfer. The mAb is expressed from a single open reading frame by linking the heavy and light chains with a 2A self-processing peptide derived from the foot-and-mouth disease virus. Using this expression system, we generated a recombinant adeno-associated virus vector encoding the VEGFR2-neutralizing mAb DC101 (rAAV8-DC101). A single dose of rAAV8-DC101 resulted in long-term expression of >1,000 microg/ml of DC101 in mice, demonstrating significant anti-tumor efficacy. This report describes the first feasible gene therapy approach for stable delivery of mAbs at therapeutic levels, which may serve as an attractive alternative to direct injection of mAbs.
Assuntos
Adenoviridae/genética , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Formação de Anticorpos/genética , Terapia Genética/métodos , Rim/metabolismo , Neoplasias/terapia , Proteínas Virais/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Linhagem Celular , Técnicas de Transferência de Genes , Humanos , Hibridomas , Rim/imunologia , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Proteínas Virais/genéticaRESUMO
IFN-alpha is approved for the treatment of multiple cancers. Its pleiotropic properties include inhibition of proliferation and angiogenesis and induction of apoptosis. Type I IFNs also exert immunomodulatory effects, which make it an appropriate candidate to combine with cancer vaccines. The studies reported herein show that 50% of mice reject established B16 tumors following treatment with the combination of a granulocyte macrophage colony-stimulating factor-secreting tumor cell vaccine (B16.GM) and subclinical doses of recombinant murine IFN-alpha delivered at the vaccine site. Similarly, 80% of mice treated with the combination reject established B16 tumors when recombinant murine IFN-alpha is given at the challenge site, suggesting that in the latter case its antiproliferative, proapoptotic, and antiangiogenic properties may be involved in controlling tumor growth. In contrast, fewer than 10% of mice reject the tumors when either one is used as a monotherapy. Furthermore, a 30-fold increase in the frequency of melanoma-associated antigen (Trp-2 and gp100) specific T cells was observed in mice treated with the combination when compared with unvaccinated controls. These data show that IFN-alpha combined with a granulocyte macrophage colony-stimulating factor-secreting tumor cell vaccine significantly enhances vaccine potency and may represent a potential new approach for tumor immunotherapy.
Assuntos
Vacinas Anticâncer/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interferon-alfa/farmacologia , Melanoma Experimental/terapia , Células 3T3 , Animais , Vacinas Anticâncer/imunologia , Processos de Crescimento Celular/efeitos dos fármacos , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interferon-alfa/imunologia , Linfonodos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/imunologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/terapia , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Antígeno gp100 de MelanomaRESUMO
Adenovirus has been used widely as a gene transfer vector in the laboratory and clinic for the purpose of gene therapy. Conditionally replication-competent oncolytic adenoviruses are capable of multiplying up to a thousand old in target cells, a property that might prove to be of tremendous potential in the area of cancer therapy. Intravesicular therapy of refractory superficial bladder cancer employing an oncolytic adenovirus would allow for local administration and efficient delivery of virus to bladder tumor. The glycosaminoglycan layer on the surface of the bladder urothelium acts as a nonspecific antiadherence barrier and may be a significant roadblock to efficient infection of the urothelium by adenoviruses. Several laboratories have investigated the potential utility of bladder pretreatment with chemical agents to enhance the adenovirus infection of bladder urothelium but with limited success. A class of compounds has been identified that is effective for pretreatment of urothelium, permitting efficient adenoviral infection. In a murine model, pretreatment of the bladder with 0.1% dodecyl-beta-D-maltoside (DDM) or sodium dodecyl sulfate (SDS) for 5 min resulted in >90% transduction of the urothelial layer within 15 min after exposure to a replication-defective adenovirus compared to =5% transduction in untreated bladders. DDM could be coformulated with adenovirus, and complete transduction of the urothelium was achieved following retention of the admixture in the bladder for 45 min. A similar enhancement of adenoviral infection following pretreatment of bladder with DDM and SDS was observed in a rat model. The use of these compounds may facilitate the development of adenovirus-based therapy for bladder cancer.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Glucosídeos/farmacologia , Dodecilsulfato de Sódio/farmacologia , Bexiga Urinária/citologia , Urotélio/efeitos dos fármacos , Animais , Benzenossulfonatos/farmacologia , Feminino , Vetores Genéticos/genética , Glicosaminoglicanos/metabolismo , Camundongos , Ratos , Bexiga Urinária/virologia , Urotélio/metabolismo , Urotélio/virologiaRESUMO
Angiostatin is a potent endogenous inhibitor of angiogenesis and tumor growth in vivo. The therapeutic potential of adeno-associated viral (AAV) gene delivery of angiostatin in modulating tumor growth in vivo was evaluated. Sustained levels of angiostatin were detected in the sera of mice for up to 6 months after they received a single injection of AAV-angiostatin. AAV-mediated stable expression of angiostatin inhibited tumor burden in the highly aggressive B16F10 melanoma and Lewis lung carcinoma (LLC) models of experimental metastasis. Moreover, AAV-angiostatin prolonged survival in B16F10 and LLC tumor-bearing mice compared to control groups. Anti-tumor efficacy was consistently observed when angiostatin serum levels of 15-50 ng/ml were detected following gene transfer, but the effect was minimal when the levels were lower or higher than this range. The combination of AAV-angiostatin gene therapy with chemotherapy was also shown to extend marginally the survival of mice bearing preestablished human tumors; however, the effect was evident only within a narrow dose of circulating angiostatin. These studies demonstrate the feasibility of using AAV anti-angiogenic gene therapy as a cancer treatment modality and suggest that the optimal anti-tumor efficacy of angiostatin following gene transfer may be limited to a narrow dose range.