RESUMEN
Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.
Asunto(s)
Adenoviridae/genética , Antígenos de Superficie/genética , Antígenos CD40/metabolismo , Células Dendríticas/inmunología , Vectores Genéticos/genética , Glutamato Carboxipeptidasa II/genética , Neoplasias de la Próstata/prevención & control , Vacunación/métodos , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Adyuvantes Inmunológicos/metabolismo , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Antígenos de Superficie/metabolismo , Antígenos CD40/inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Glutamato Carboxipeptidasa II/metabolismo , Antígenos HLA-A/genética , Humanos , Interferón gamma/genética , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Terapia Molecular Dirigida , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virologíaRESUMEN
Adenovirus (Ad)-mediated transduction of dendritic cells (DC) is inefficient because of the lack of the primary Ad receptor, CAR. DC infection with Ad targeted to the CD40 results in increased gene transfer. The current report describes further development of the CD40-targeting approach using an adapter molecule that bridges the fiber of the Ad5 to CD40 on mouse DC. The adapter molecule, CFm40L, consists of CAR fused to mouse CD40 ligand via a trimerization motif. A stable cell line that secretes CFm40L at high levels was generated. Gene transfer to mouse bone marrow-derived DC (mBMDC) using CFm40L-targeted Ad was over 4 orders of magnitude more efficient than that for the untargeted Ad5. Gene transfer was achieved to over 70% of the mBMDC compared to undetectable transduction using untargeted Ad5. In addition to dramatically enhanced gene transfer, the CFm40L-targeted Ad5 induced phenotypical maturation and upregulated IL-12 expression. Most importantly, the CFm40L-targeted Ad5 elicited specific immune response against a model antigen in vivo. The results of this study demonstrate that Ad-mediated gene transfer to DC can be significantly enhanced using nonnative transduction pathways, such the CD40 pathway, which may have important applications in genetic vaccination for cancer and infectious diseases.
Asunto(s)
Adenoviridae/genética , Ligando de CD40/genética , Células Dendríticas/metabolismo , Vectores Genéticos , Receptores Virales/genética , Proteínas Recombinantes de Fusión/genética , Transducción Genética/métodos , Animales , Células de la Médula Ósea/citología , Linfocitos T CD4-Positivos/inmunología , Ligando de CD40/metabolismo , Linfocitos T CD8-positivos/inmunología , Línea Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Células Dendríticas/citología , Expresión Génica , Humanos , Interleucina-12/genética , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/análisis , Receptores Virales/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Most viruses exploit a variety of host cellular proteins as primary cellular attachment receptors in the context of successful execution of infection. Furthermore, many viral agents have evolved precise mechanisms to subvert host immune recognition to achieve persistence. Herein we present data indicating that adenovirus (Ad) serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. CD80 and CD86 are co-stimulatory molecules that are present on mature dendritic cells and B lymphocytes and are involved in stimulating T-lymphocyte activation. To our knowledge, this is one of the first demonstrations of a virus utilizing immunologic accessory molecules as a primary means of cellular entry. This finding suggests a mechanism whereby viral exploitation of these proteins as receptors may achieve both goals of cellular entry and evading the immune system.
Asunto(s)
Adenovirus Humanos/patogenicidad , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Adenovirus Humanos/clasificación , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/genética , Antígeno B7-1/química , Antígeno B7-1/genética , Antígeno B7-2 , Células CHO , Cricetinae , Células Dendríticas/virología , Vectores Genéticos , Células HeLa , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Péptidos/metabolismo , Proteínas Protozoarias , SerotipificaciónRESUMEN
PURPOSE: CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on various cell types in addition to hematopoietic cells. Recent studies show that CD40 expression is related to several carcinomas, although its role in cancer pathobiology is unknown. In this study, we demonstrate the expression of CD40 on several ovarian carcinoma cell lines and the ability of CD40 to mediate targeted adenoviral infection in vitro. EXPERIMENTAL DESIGN: CD40 expression on ovarian cancer cell lines and clinical patient samples was examined by reverse transcription-PCR and flow cytometry. To study the utilization of CD40 for gene delivery, we precomplexed a luciferase coding adenovirus (Ad), Ad5luc1, with a CD40-targeting molecule (CAR/G28). RESULTS: According to our studies, all of the examined ovarian cancer cell lines are expressing CD40. In addition, mRNA for CD40 was detected in every primary tumor sample, suggesting that CD40 is also expressed in vivo. Compared with nontargeted Ad, gene transfer was increased up to 40-fold in CD40+ cells when CD40-targeted Ad was used. Supporting the relation of targeted system to CD40, increasing the amount of targeting fusion protein results in dose response. Furthermore, blockade of CD40 receptors on cell surface decreases the infectability of CD40+ cells with CD40-targeted virus, indicating the specificity of the targeting system for CD40. CONCLUSIONS: These results suggest that CD40 is present in ovarian cancer cells and can be used for targeted gene delivery in a CAR-independent manner, circumventing the problem of the low expression levels of CAR in various cancer cells.