RESUMEN
PURPOSE: Pancreatic cancer remains one of the most lethal cancers, and late detection renders most tumors refractory to conventional therapies. Development of cancer prophylaxis may be the most realistic option for improving mortality associated with this disease. Here, we develop a novel individualized prophylactic and therapeutic vaccination regimen using induced pluripotent stem cells (iPSC), gene editing, and tumor-targeted replicating oncolytic viruses. EXPERIMENTAL DESIGN: We created a Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCs from healthy cells were induced to pancreatic tumor cells using in situ gene editing via stable provision of KRas G12D and p53 R172H tumor driver mutations. These cells were preinfected with oncolytic Adenovirus (AdV) as prime or Vaccinia virus (VV) as boost, to improve vaccine immunogenicity, prior to delivery of vaccines in a sequential regime to young KPC transgenic mice, genetically programmed to develop pancreatic cancer, to prevent and delay disease development. RESULTS: Tumor cells preinfected with oncolytic AdV as prime or VV as boost were the best regime to induce tumor-specific immunity. iPSC-derived tumor cells were highly related in antigen repertoire to pancreatic cancer cells of KPC transgenic mice, suggesting that an individual's stem cells can provide an antigenically matched whole tumor cell vaccine. The VIReST vaccination primed tumor-specific T-cell responses, resulting in delayed disease emergence and progression and significantly prolonged survival of KPC transgenic mice. Importantly, this regime was well-tolerated and nontoxic. CONCLUSIONS: These results provide both proof of concept and a robust technology platform for the development of personalized prophylactic cancer vaccines to prevent pancreatic malignancies in at-risk individuals.
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Vacunas contra el Cáncer/administración & dosificación , Células Madre Pluripotentes Inducidas/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Experimentales/prevención & control , Viroterapia Oncolítica , Virus Oncolíticos/inmunología , Neoplasias Pancreáticas/prevención & control , Animales , Vacunas contra el Cáncer/inmunología , Chlorocebus aethiops , Progresión de la Enfermedad , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Tasa de Supervivencia , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
The originally published version of this Article contained errors in Figure 4. In panel b, the square and diamond labels associated with the uppermost survival curve were incorrectly displayed as 'n' and 'u', respectively. These errors have now been corrected in both the PDF and HTML versions of the Article.
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Interleukin-12 (IL-12) has emerged as one of the most potent agents for anti-tumor immunotherapy. However, potentially lethal toxicity associated with systemic administration of IL-12 precludes its clinical application. Here we redesign the molecule in such a way that its anti-tumor efficacy is not compromised, but toxic effects are eliminated. Deletion of the N-terminal signal peptide of IL-12 can effect such a change by preventing IL-12 secretion from cells. We use a newly designed tumor-targeted oncolytic adenovirus (Ad-TD) to deliver non-secreting (ns) IL-12 to tumor cells and examine the therapeutic and toxic effects in Syrian hamster models of pancreatic cancer (PaCa). Strikingly, intraperitoneal delivery of Ad-TD-nsIL-12 significantly enhanced survival of animals with orthotopic PaCa and cured peritoneally disseminated PaCa with no toxic side effects, in contrast to the treatment with Ad-TD expressing unmodified IL-12. These findings offer renewed hope for development of IL-12-based treatments for cancer.
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Antineoplásicos/farmacología , Inmunoterapia/métodos , Interleucina-12/inmunología , Interleucina-12/farmacología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Adenoviridae/genética , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/química , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Cricetinae , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Humanos , Interleucina-12/efectos adversos , Interleucina-12/química , Neoplasias Pancreáticas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Oncolytic adenoviruses (Ad) represent an innovative approach to cancer therapy. Its efficacy depends on multiple actions, including direct tumor lysis and stimulation of antiviral and antitumor immune responses. In this study, we investigated the roles of T-cell responses in oncolytic adenoviral therapy. EXPERIMENTAL DESIGN: An immunocompetent and viral replication-permissive Syrian hamster tumor model was used. The therapeutic mechanisms of oncolytic Ad were investigated by T-cell deletion, immunohistochemical staining, and CTL assay. RESULTS: Deletion of T cells with an anti-CD3 antibody completely demolished the antitumor efficacy of oncolytic Ad. Intratumoral injection of Ad induced strong virus- and tumor-specific T-cell responses, as well as antiviral antibody response. Both antiviral and antitumor T-cell responses contributed to the efficacy of oncolytic Ad. Deletion of T cells increased viral replication and extended the persistence of infectious virus within tumors but almost abrogated the antitumor efficacy. Preexisting antiviral immunity promoted the clearance of injected oncolytic Ad from tumors but had no effect on antitumor efficacy. Strikingly, the repeated treatment with oncolytic Ad has strong therapeutic effect on relapsed tumors or tumors insensitive to the primary viral therapy. CONCLUSIONS: These results demonstrate that T cell-mediated immune responses outweigh the direct oncolysis in mediating antitumor efficacy of oncolytic Ad. Our data have a high impact on redesigning the regimen of oncolytic Ad for cancer treatment. Clin Cancer Res; 23(1); 239-49. ©2016 AACR.
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Adenoviridae , Vectores Genéticos , Neoplasias/inmunología , Neoplasias/patología , Viroterapia Oncolítica , Virus Oncolíticos , Linfocitos T/inmunología , Replicación Viral/inmunología , Virus/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Anticuerpos Antivirales/inmunología , Línea Celular Tumoral , Cricetinae , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Depleción Linfocítica , Neoplasias/terapia , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Carga Tumoral/genética , Carga Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor resistance is a major hurdle to anti-Her2/neu Ab-based cancer therapy. Current strategies to overcome tumor resistance focus on tumor cell-intrinsic resistance. However, the extrinsic mechanisms, especially the tumor microenvironment, also play important roles in modulating the therapeutic response and resistance of the Ab. In this study, we demonstrate that tumor progression is highly associated with TAMs with immune-suppressive M2 phenotypes, and deletion of TAMs markedly enhanced the therapeutic effects of anti-Her2/neu Ab in a HER2/neu-dependent breast cancer cell TUBO model. Tumor local delivery of IL-21 can skew TAM polarization away from the M2 phenotype to a tumor-inhibiting M1 phenotype, which rapidly stimulates T cell responses against tumor and dramatically promotes the therapeutic effect of anti-Her2 Ab. Skewing of TAM polarization by IL-21 relies substantially on direct action of IL-21 on TAMs rather than stimulation of T and NK cells. Thus, our findings identify the abundant TAMs as a major extrinsic barrier for anti-Her2/neu Ab therapy and present a novel approach to combat this extrinsic resistance by tumor local delivery of IL-21 to skew TAM polarization. This study offers a therapeutic strategy to modulate the tumor microenvironment to overcome tumor-extrinsic resistance.
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Resistencia a Antineoplásicos/inmunología , Interleucinas/inmunología , Macrófagos/inmunología , Neoplasias Mamarias Experimentales/inmunología , Microambiente Tumoral/inmunología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Femenino , Citometría de Flujo , Inmunohistoquímica , Interleucinas/administración & dosificación , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor ErbB-2/antagonistas & inhibidores , TrastuzumabRESUMEN
PURPOSE: Vaccinia virus has strong potential as a novel therapeutic agent for treatment of pancreatic cancer. We investigated whether arming vaccinia virus with interleukin-10 (IL10) could enhance the antitumor efficacy with the view that IL10 might dampen the host immunity to the virus, increasing viral persistence, thus maximizing the oncolytic effect and antitumor immunity associated with vaccinia virus. EXPERIMENTAL DESIGN: The antitumor efficacy of IL10-armed vaccinia virus (VVLΔTK-IL10) and control VVΔTK was assessed in pancreatic cancer cell lines, mice bearing subcutaneous pancreatic cancer tumors and a pancreatic cancer transgenic mouse model. Viral persistence within the tumors was examined and immune depletion experiments as well as immunophenotyping of splenocytes were carried out to dissect the functional mechanisms associated with the viral efficacy. RESULTS: Compared with unarmed VVLΔTK, VVLΔTK-IL10 had a similar level of cytotoxicity and replication in vitro in murine pancreatic cancer cell lines, but rendered a superior antitumor efficacy in the subcutaneous pancreatic cancer model and a K-ras-p53 mutant-transgenic pancreatic cancer model after systemic delivery, with induction of long-term antitumor immunity. The antitumor efficacy of VVLΔTK-IL10 was dependent on CD4(+) and CD8(+), but not NK cells. Clearance of VVLΔTK-IL10 was reduced at early time points compared with the control virus. Treatment with VVLΔTK-IL10 resulted in a reduction in virus-specific, but not tumor-specific CD8(+) cells compared with VVLΔTK. CONCLUSIONS: These results suggest that VVLΔTK-IL10 has strong potential as an antitumor therapeutic for pancreatic cancer.
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Interleucina-10/genética , Virus Oncolíticos/genética , Neoplasias Pancreáticas/terapia , Virus Vaccinia/genética , Animales , Línea Celular Tumoral , Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trasplante de Neoplasias , Viroterapia Oncolítica , Neoplasias Pancreáticas/inmunología , Replicación ViralRESUMEN
PURPOSE: The efficacy of oncolytic viruses depends on multiple actions including direct tumor lysis, modulation of tumor perfusion, and stimulation of tumor-directed immune responses. In this study, we investigated whether a sequential combination of immunologically distinct viruses might enhance antitumor efficacy through the induction of tumor-specific immunity and circumvention or mitigation of antiviral immune responses. EXPERIMENTAL DESIGN: The Syrian hamster as an immune-competent model that supports replication of both adenovirus and vaccinia virus was evaluated in vitro and in vivo. The antitumor efficacy of either virus alone or sequential combination of the two viruses was examined in pancreatic and kidney cancer models. The functional mechanism of the regimen developed here was investigated by histopathology, immunohistochemistry staining, CTL assay, and T-cell depletion. RESULTS: The Syrian hamster is a suitable model for assessment of oncolytic adenovirus and vaccinia virus. Three low doses of adenovirus followed by three low doses of vaccinia virus resulted in a superior antitumor efficacy to the reverse combination, or six doses of either virus alone, against pancreatic and kidney tumors in Syrian hamsters. A total of 62.5% of animals bearing either tumor type treated with the sequential combination became tumor-free, accompanied by the induction of effective tumor-specific immunity. This enhanced efficacy was ablated by CD3+ T-cell depletion but was not associated with humoral immunity against the viruses. CONCLUSION: These findings show that sequential treatment of tumors with oncolytic adenovirus and vaccinia virus is a promising approach for cancer therapy and that T-cell responses play a critical role.
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Neoplasias Renales/terapia , Viroterapia Oncolítica , Neoplasias Pancreáticas/terapia , Adenoviridae/genética , Adenoviridae/inmunología , Adenoviridae/fisiología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Apoptosis , Células Cultivadas , Cricetinae , Femenino , Humanos , Inmunocompetencia , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Mesocricetus , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , Virus Oncolíticos/fisiología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , Carga Tumoral , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus Vaccinia/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It has been well established that immune surveillance plays critical roles in preventing the occurrence and progression of tumor. More and more evidence in recent years showed the host anti-tumor immune responses also play important roles in the chemotherapy and radiotherapy of cancers. Our previous study found that tumor- targeting therapy of anti-HER2/neu mAb is mediated by CD8(+) T cell responses. However, we found here that enhancement of CD8(+) T cell responses by combination therapy with IL-15R/IL-15 fusion protein or anti-CD40, which are strong stimultors for T cell responses, failed to promote the tumor therapeutic effects of anti-HER2/neu mAb. Analysis of tumor microenviornment showed that tumor tissues were heavily infiltrated with the immunosuppressive macrophages and most tumor infiltrating T cells, especially CD8(+) T cells, expressed high level of inhibitory co-signaling receptor PD-1. These data suggest that tumor microenvironment is dominated by the immunosuppressive strategies, which thwart anti-tumor immune responses. Therefore, the successful tumor therapy should be the removal of inhibitory signals in the tumor microenvironment in combination with other therapeutic strategies.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Tolerancia Inmunológica/inmunología , Receptor ErbB-2/inmunología , Microambiente Tumoral/inmunología , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Trasplante de NeoplasiasRESUMEN
BACKGROUND: Agonist antibodies against CD137 (4-1BB) on T lymphocytes are used to increase host anti-tumor immunity, but often leading to severe liver injury in treated mice or in patients during clinical trials. Interleukin-6 (IL-6) has been reported to protect hepatocyte death, but the role of IL-6 in protecting chronic T cell-induced liver diseases is not clearly defined due to lack of relevant animal models. We aimed to define the role of IL-6 in CD8+ T cell-mediated liver injury induced by a CD137 agonistic mAb (clone 2A) in mice. METHODS/PRINCIPAL FINDINGS: We expressed IL-6 in the liver by hydrodynamic gene delivery in mice treated with 2A or control mAb and studied how IL-6 treatment affected host immunity and T cell-mediated liver injury. We found that ectopic IL-6 expression in the liver elevated intrahepatic leukocyte infiltration but prevented CD8+ T cell-mediated liver injury. In IL-6 treated mice, CD8+ T cells proliferation and IFN-γ expression were inhibited in the liver. We discovered that IL-6 increased accumulation of Gr-1+CD11b+ myeloid derived suppressor cells (MDSCs) in the liver and spleen. These MDSCs had the ability to inhibit T cells proliferation and activation. Finally, we showed that the MDSCs were sufficient and essential for IL-6-mediated protection of anti-CD137 mAb-induced liver injury. CONCLUSIONS/SIGNIFICANCE: We concluded that IL-6 induced Gr-1+CD11b+ MDSCs in the liver to inhibit T cell-mediated liver injury. The findings have defined a novel mechanism of IL-6 in protecting liver from CD8+ T cell-mediated injury.
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Antígeno CD11b/metabolismo , Linfocitos T CD8-positivos/inmunología , Interleucina-6/farmacología , Hepatopatías/prevención & control , Hígado/patología , Células Mieloides/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Interleucina-6/administración & dosificación , Hígado/efectos de los fármacos , Hígado/inmunología , Hepatopatías/inmunología , Hepatopatías/patología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Sustancias Protectoras/farmacología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , EsplenomegaliaRESUMEN
Chronic hepatitis B virus (HBV) infection is characterized by sustained liver inflammation with an influx of lymphocytes, which contributes to the development of cirrhosis and hepatocellular carcinoma. The mechanisms underlying this immune-mediated hepatic pathogenesis remain ill defined. We report in this article that repetitive infusion of anti-CD137 agonist mAb in HBV-transgenic mice closely mimics this process by sequentially inducing hepatitis, fibrosis, cirrhosis, and, ultimately, liver cancer. CD137 mAb initially triggers hepatic inflammatory infiltration due to activation of nonspecific CD8(+) T cells with memory phenotype. CD8(+) T cell-derived IFN-γ plays a central role in the progression of chronic liver diseases by actively recruiting hepatic macrophages to produce fibrosis-promoting cytokines and chemokines, including TNF-α, IL-6, and MCP-1. Importantly, the natural ligand of CD137 was upregulated significantly in circulating CD14(+) monocytes in patients with chronic hepatitis B infection and closely correlated with development of liver cirrhosis. Thus, sustained CD137 stimulation may be a contributing factor for liver immunopathology in chronic HBV infection. Our studies reveal a common molecular pathway that is used to defend against viral infection but also causes chronic hepatic diseases.
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Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/inmunología , Virus de la Hepatitis B/inmunología , Neoplasias Hepáticas Experimentales/inmunología , Monocitos/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/metabolismo , Humanos , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/inmunología , Receptores de Lipopolisacáridos/metabolismo , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/virología , Ratones , Ratones Transgénicos , Monocitos/metabolismo , Monocitos/patología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/biosíntesis , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
BACKGROUND: PD-1, encoded by PDCD1, is highly expressed on virus-specific T cells and plays critical roles in modulating anti-virus immune responses in chronic viral infection. It is unknown, however, whether polymorphisms of the PDCD1 are associated with viral clearance during chronic viral infections. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we used the polymerase chain reaction-restriction fragment length polymorphism method to genotype two single nucleotide polymorphisms (SNPs) of PDCD1 in 502 patients with chronic hepatitis B virus (HBV) infection and 359 healthy controls to determine the association between PDCD1 genotypes and serum viral load as well as the risk of chronic infection. Our results showed that although neither the P7209(C/T) SNP site nor the P8737(A/G) site was associated with the risk of chronic HBV infection, the P7209 (T) allele in intron 4 is significantly associated with lower viral burden in the blood. Using a luciferase reporter assay, we demonstrated that the P7209 (T) allele creates a negative cis-element for gene transcription. CONCLUSIONS AND SIGNIFICANCE: Our data provide the first evidence that PDCD1 polymorphisms is a genetic factor in pathogenesis of chronic viral infection and reveal the functional significance of the P7209 SNP of the PDCD1.
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Antígenos CD/genética , Proteínas Reguladoras de la Apoptosis/genética , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/genética , Inteínas/genética , Carga Viral/genética , Adulto , Antígenos CD/inmunología , Antígenos CD/metabolismo , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Genotipo , Virus de la Hepatitis B/patogenicidad , Hepatitis B Crónica/inmunología , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Receptor de Muerte Celular Programada 1 , Virulencia/genéticaRESUMEN
In this study, we demonstrate that an effective immune response against prostate tumors in mouse tumor model can be elicited using a strategy that combines CTLA-4 blockade and pSLC-3P-Fc-modified tumor cell vaccine (named B16F10-SLC-3P-Fc). Treatment of B16F10-3P-bearing mice resulted in a significant reduction in tumor incidence as assessed 2 months after treatment. In vivo Ab depletion confirmed that the antitumor effect was primarily CD8+ T cells and CD4+ T lymphocytes were required for the induction of CD8+ CTL response in B16F10-SLC-3P-Fc+anti-CTLA-4 mAb-immunized mice. Moreover, mice that were cured of an established tumor were protected against a rechallenge with the same tumor for at least 4 months, suggesting the generation of memory responses. Adoptive transfer experiments further indicate that antitumor reactivity can be transferred to naïve mice by splenocytes. These findings demonstrate that this combinatorial treatment can elicit a potent anti-tumor immune response and suggest potential of this approach for treatment of prostate cancer.
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Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Antígenos de Diferenciación/inmunología , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Melanoma Experimental/inmunología , Neoplasias de la Próstata/terapia , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígeno CTLA-4 , Vacunas contra el Cáncer/inmunología , Inmunoterapia , Inmunoterapia Adoptiva , Masculino , Ratones , Ratones Endogámicos C57BL , Próstata/inmunología , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
We previously reported that several DNA fragments from human prostate-specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate-specific antigen (hPSA) genes were selected and fused to create a novel hPSM-mPAP-hPSA fusion gene (named 3P gene), and human secondary lymphoid tissue chemokine (SLC), 3P, and human IgG Fc genes were inserted into pcDNA3.1 to construct a DNA vaccine, designated pSLC-3P-Fc. In this report, to establish a more efficient treatment for immunotherapy against prostate cancer, the construct was transfected into B16F10 to generate gene-modified tumor cell vaccine (named B16F10-SLC-3P-Fc). In poorly immunogenic B16F10 mouse melanoma model, the immunization with B16F10-SLC-3P-Fc resulted in a strong antitumor response and 50% of tumor-bearing mice achieved long-term survival (>120 days). In vivo depletion of lymphocytes indicated that CD8(+) T cells were involved in the direct tumor killing, whereas CD4(+) T lymphocytes were required for the induction of CD8(+) CTL response in B16F10-SLC-3P-Fc-immunized mice. Splenocytes from B16F10-SLC-3P-Fc-immunized mice specifically recognized and lysed PSM, PAP, PSA, and 3P expressing tumor cells. The combined therapy of B16F10-SLC-3P-Fc plus anti-B7-H1 MAbs further enhanced the immune response. Rechallenge experiment showed that a persistent memory response was successfully induced by the combined therapy. These observations suggest pSLC-3P-Fc-modified tumor cells could serve as a vaccine against prostate cancer, and the therapy combined with anti-B7-H1 MAbs further enhanced the antitumor immune response.
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Antígenos CD/inmunología , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Quimiocinas/inmunología , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/inmunología , Vacunas de ADN/inmunología , Fosfatasa Ácida , Animales , Presentación de Antígeno/inmunología , Antígenos de Superficie/inmunología , Antígeno B7-H1 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Quimiotaxis de Leucocito/inmunología , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Humanos , Inmunoterapia/métodos , Inyecciones Subcutáneas , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Proteínas Tirosina Fosfatasas/inmunología , Tasa de Supervivencia , Células Tumorales Cultivadas , Vacunas de ADN/administración & dosificaciónRESUMEN
The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.