RESUMO
Natural tropism to the liver is a major obstacle in systemic delivery of adenoviruses in cancer gene therapy. Adenovirus binding to soluble coagulation factors and to cellular heparan sulphate proteoglycans via the fiber shaft KKTK domain are suggested to cause liver tropism. Serotype 5 adenovirus constructs with mutated KKTK regions exhibit liver detargeting, but they also transduce tumors less efficiently, possibly due to altered fiber conformation. We constructed Ad5/3lucS*, a 5/3 chimeric adenovirus with a mutated KKTK region. The fiber knob swap was hypothesized to facilitate tumor transduction. This construct was studied with or without additional coagulation factor ablation. Ad5/3lucS* exhibited significantly reduced transduction of human hepatic cells in vitro and mouse livers in vivo. Combination of coagulation factor ablation by warfarinization to Ad5/3lucS* seemed to further enhance liver detargeting. Cancer cell transduction by Ad5/3lucS* was retained in vitro. In vivo, viral particle accumulation in M4A4-LM3 xenograft tumors was comparable to controls, but Ad5/3lucS* transgene expression was nearly abolished. Coagulation factor ablation did not affect tumor transduction. These studies set the stage for further investigations into the effects of the KKTK mutation and coagulation factor ablation in the context of 5/3 serotype chimerism. Of note, the putative disconnect between tumor transduction and transgene expression could prove useful in further understanding of adenovirus biology.
Assuntos
Adenoviridae/genética , Heparitina Sulfato/metabolismo , Proteoglicanas/metabolismo , Vírus Reordenados/genética , Tropismo Viral/genética , Animais , Sítios de Ligação , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Fatores de Coagulação Sanguínea/metabolismo , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos , Heparitina Sulfato/química , Humanos , Injeções Intravenosas , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos Nus , Estrutura Terciária de Proteína , Proteoglicanas/genética , Transdução Genética , Transgenes , Varfarina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie-adenovirus receptor may be variable in advanced tumors, we developed Ad5-D24-RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD-4C modification of the fiber. This allows viral entry through alpha-v-beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5-RGD-D24-GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of human cancer was established. Treatments with Ad5-D24-RGD (N = 9) and Ad5-RGD-D24-GMCSF (N = 7) were well tolerated. Typical side effects were grade 1-2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5-RGD-D24-GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5-D24-RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5-RGD-D24-GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Neoplasias/terapia , Oligopeptídeos/metabolismo , Terapia Viral Oncolítica/métodos , Adenoviridae/genética , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , DNA Viral/genética , Resistencia a Medicamentos Antineoplásicos , Fadiga/etiologia , Feminino , Febre/etiologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Integrinas/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias/metabolismo , Neoplasias/virologia , Oligopeptídeos/genética , Terapia Viral Oncolítica/efeitos adversos , Vírus Oncolíticos/genética , Reação em Cadeia da Polimerase em Tempo Real , Resultado do Tratamento , Carga Viral , Replicação Viral/genéticaRESUMO
Augmenting antitumor immunity is a promising way to enhance the potency of oncolytic adenoviral therapy. Granulocyte-macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific CD8(+) cytotoxic T-lymphocytes. Serotype 5 adenoviruses (Ad5) are commonly used in cancer gene therapy. However, expression of the coxsackie-adenovirus receptor is variable in many advanced tumors and preclinical data have demonstrated an advantage for replacing the Ad5 knob with the Ad3 knob. Here, a 5/3 capsid chimeric and p16-Rb pathway selective oncolytic adenovirus coding for GMCSF was engineered and tested preclinically. A total of 21 patients with advanced solid tumors refractory to standard therapies were then treated intratumorally and intravenously with Ad5/3-D24-GMCSF, which was combined with low-dose metronomic cyclophosphamide to reduce regulatory T cells. No severe adverse events occurred. Analysis of pretreatment samples of malignant pleural effusion and ascites confirmed the efficacy of Ad5/3-D24-GMCSF in transduction and cell killing. Evidence of biological activity of the virus was seen in 13/21 patients and 8/12 showed objective clinical benefit as evaluated by radiology with Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Antiadenoviral and antitumoral immune responses were elicited after treatment. Thus, Ad5/3-D24-GMCSF seems safe in treating cancer patients and promising signs of efficacy were seen.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Adolescente , Adulto , Idoso , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Ciclofosfamida/uso terapêutico , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Imunossupressores/uso terapêutico , Masculino , Mesocricetus , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Granulocyte macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific cytotoxic T-cells through antigen-presenting cells. Oncolytic tumor cell-killing can produce a potent costimulatory danger signal and release of tumor epitopes for antigen-presenting cell sampling. Therefore, an oncolytic adenovirus coding for GMCSF was engineered and shown to induce tumor-specific immunity in an immunocompetent syngeneic hamster model. Subsequently, 20 patients with advanced solid tumors refractory to standard therapies were treated with Ad5-D24-GMCSF. Of the 16 radiologically evaluable patients, 2 had complete responses, 1 had a minor response, and 5 had disease stabilization. Responses were frequently seen in injected and noninjected tumors. Treatment was well tolerated and resulted in the induction of both tumor-specific and virus-specific immunity as measured by ELISPOT and pentamer analysis. This is the first time that oncolytic virus-mediated antitumor immunity has been shown in humans. Ad5-D24-GMCSF is promising for further clinical testing.
