RESUMEN
First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/inmunología , Vectores Genéticos/inmunología , Linfocitos T/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Adulto , Anciano , Anticuerpos Neutralizantes/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Antígeno Carcinoembrionario/genética , Estudios de Cohortes , Neoplasias Colorrectales/terapia , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos/genética , Humanos , Inmunización/métodos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Linfocitos T/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND: Targeted and effective treatment options are needed for solid tumors, including glioblastoma (GBM), where survival rates with standard treatments are typically less than 2 years from diagnosis. Solid tumors pose many barriers to immunotherapies, including therapy half-life and persistence, tumor penetrance, and targeting. Therapeutics delivered systemically may not traffic to the tumor site. If cellular therapies or drugs are able to access the tumor site, or can be delivered directly within the tumor, treatments may not persist for the duration necessary to reduce or eliminate tumor burden. An approach that allows durable and titratable local therapeutic protein delivery could improve antitumor efficacy while minimizing toxicities or unwanted on-target, off-tissue effects. METHODS: In this study, human monocyte-derived macrophages were genetically engineered to secrete a bispecific T cell engager (BiTE) specific to the mutated epidermal growth factor variant III (EGFRvIII) expressed by some GBM tumors. We investigated the ability of lentivirally modified macrophages to secrete a functional BiTE that can bind target tumor antigen and activate T cells. Secreted BiTE protein was assayed in a range of T cell functional assays in vitro and in subcutaneous and intracranial GBM xenograft models. Finally, we tested genetically engineered macrophages (GEMs) secreting BiTE and the proinflammatory cytokine interleukin (IL)-12 to amplify T cell responses in vitro and in vivo. RESULTS: Transduced human macrophages secreted a lentivirally encoded functional EGFRvIII-targeted BiTE protein capable of inducing T cell activation, proliferation, degranulation, and killing of antigen-specific tumor cells. Furthermore, BiTE secreting macrophages reduced early tumor burden in both subcutaneous and intracranial mouse models of GBM, a response which was enhanced using macrophages that were dual transduced to secrete both the BiTE protein and single chain IL-12, preventing tumor growth in an aggressive GBM model. CONCLUSIONS: The ability of macrophages to infiltrate and persist in solid tumor tissue could overcome many of the obstacles associated with systemic delivery of immunotherapies. We have found that human GEMs can locally and constitutively express one or more therapeutic proteins, which may help recruit T cells and transform the immunosuppressive tumor microenvironment to better support antitumor immunity.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Neoplasias Encefálicas/genética , Glioblastoma/genética , Inmunoterapia/métodos , Linfocitos T/inmunología , Animales , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Humanos , Ratones , Transfección , Microambiente TumoralRESUMEN
BACKGROUND: Though currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies. METHODS: Using lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies. RESULTS: Here, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins. CONCLUSIONS: Our data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.
Asunto(s)
Ingeniería Genética/métodos , Inmunoterapia/métodos , Macrófagos/metabolismo , Neoplasias/inmunología , Microambiente Tumoral/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Adult brain tumors establish an immunosuppressive tumor microenvironment as a modality of immune escape, with several immunotherapies designed to overcome this barrier. However, the relationship between tumor cells and immune cells in pediatric brain tumor patients is not as well-defined. In this study, we sought to determine whether the model of immune escape observed in adult brain tumors is reflected in patients with pediatric brain tumors by evaluating NKG2D ligand expression on tissue microarrays created from patients with a variety of childhood brain tumor diagnoses, and infiltration of Natural Killer and myeloid cells. We noted a disparity between mRNA and protein expression for the 8 known NKG2D ligands. Surprisingly, high-grade gliomas did not have increased NKG2D ligand expression compared to normal adjacent brain tissue, nor did they have significant myeloid or NK cell infiltration. These data suggest that pediatric brain tumors have reduced NK cell-mediated immune surveillance, and a less immunosuppressive tumor microenvironment as compared to their adult counterparts. These data indicate that therapies aimed to improve NK cell trafficking and functions in pediatric brain tumors may have a greater impact on anti-tumor immune responses and patient survival, with fewer obstacles to overcome.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Células Mieloides/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/patología , Niño , Citotoxicidad Inmunológica/inmunología , Humanos , Inmunohistoquímica , Ligandos , ARN Mensajero/metabolismo , Análisis de Matrices TisularesRESUMEN
HIV infection of the central nervous system leads to HIV-associated dementia (HAD) in a substantial subset of infected individuals. The pathogenesis of neuronal dysfunction in HAD is not well understood, but previous studies have demonstrated evidence for activation of apoptotic pathways. The tumor suppressor transcription factor p53 is an apical mediator of neuronal apoptosis following a variety of injurious stimuli. To determine whether p53 participates in HAD, we exposed cerebrocortical cultures from wild-type and p53 deficient mice to the neurotoxic HIV envelope protein gp120. Using neuron/microglia co-culture of mixed p53 genotype, we observed that both neurons and microglia require p53 for gp120 induced neuronal apoptosis. Additionally, accumulation of p53 protein in neurons was recently reported in post-mortem cortical tissue from a small group of HAD patients. Using a much larger cohort of HAD cases, we extend this finding and report that p53 protein also increases in non-neuronal cells, including microglia. Taken together these findings demonstrate a novel role for p53 in the microglial response to gp120. Additionally, these findings, in conjunction with a recent report that monocytes expressing HIV-Tat also secrete neurotoxins that promote p53 activation, suggest that distinct HIV proteins may converge on the p53 pathway to promote neurotoxicity.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Lóbulo Frontal/patología , Proteína gp120 de Envoltorio del VIH/fisiología , Microglía/patología , Neuronas/metabolismo , Complejo SIDA Demencia/patología , Animales , Apoptosis , Biomarcadores , Calcio/análisis , Células Cultivadas , Técnicas de Cocultivo , Lóbulo Frontal/metabolismo , Genes p53 , Humanos , Ratones , Ratones Noqueados , Microglía/metabolismo , Degeneración Nerviosa , Neuronas/patología , Receptores CCR5/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal , Método Simple CiegoRESUMEN
Microglia are the resident immune cells of the CNS: they are activated rapidly by CNS damage and perform the function of tissue macrophages. The first steps during microglial activation are currently under intense study, and it is widely believed that substances released from damaged brain tissue can trigger this process. We recently reported that the blood coagulation factor thrombin, which enters the CNS during breakdown of the blood-brain barrier, activates microglial cells. The cellular effects of thrombin and trypsin-like proteases are mediated by proteinase-activated receptors (PARs). Based on our prior data we hypothesized that microglial cells express these receptors. Using RT-PCR and flow cytometry, we report that primary mouse microglial cells, as well as the murine microglial cell lines BV-2 and N9, indeed express PARs, albeit at different levels. Demonstrating multiple PARs on microglia may enhance the attractiveness of PARs as therapeutic targets in neuroinflammatory disorders.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Microglía/metabolismo , Receptores Proteinasa-Activados/biosíntesis , Animales , Corteza Cerebral/metabolismo , Ratones , Unión Proteica/fisiología , Receptores Proteinasa-Activados/genéticaRESUMEN
Anti-vector immunity mitigates immune responses induced by recombinant adenovirus vector vaccines, limiting their prime-boost capabilities. We have developed a novel gene delivery and expression platform (Ad5 [E1-, E2b-]) that induces immune responses despite pre-existing and/or developed concomitant Ad5 immunity. In the present study, we evaluated if this new Ad5 platform could overcome the adverse condition of pre-existing Ad5 immunity to induce effective immune responses in prime-boost immunization regimens against two different infectious diseases in the same animal. Ad5 immune rhesus macaques (RM) were immunized multiple times with the Ad5 [E1-, E2b-] platform expressing antigens from simian immunodeficiency virus (SIV). Immunized RM developed cell-mediated immunity against SIV antigens Gag, Pol, Nef and Env as well as antibody against Env. Vaccinated and vector control RMs were challenged intra-rectally with homologous SIVmac239. During a 7-week follow-up, there was perturbation of SIV load in some immunized RM. At 7 weeks post-challenge, eight immunized animals (53%) did not have detectable SIV, compared to two RM controls (13%) (P<0.02; log-rank Mantel-Cox test). There was no correlation of protective MHC contributing to infection control. The RM without detectable circulating SIV, now hyper immune to Ad5, were then vaccinated with the same Ad5 [E1-, E2b-] platform expressing H1N1 influenza hemagglutinin (HA). Thirty days post Ad5 [E1-, E2b-]-HA vaccination, significant levels of influenza neutralizing antibody were induced in all animals that increased after an Ad5 [E1-, E2b-]-HA homologous boost. These data demonstrate the versatility of this new vector platform to immunize against two separate disease targets in the same animal despite the presence of immunity against the delivery platform, permitting homologous repeat immunizations with an Ad5 gene delivery platform.
Asunto(s)
Adenoviridae/inmunología , Portadores de Fármacos , Vectores Genéticos/inmunología , Inmunización/métodos , Vacunas contra la Influenza/inmunología , Vacunas contra el SIDAS/inmunología , Adenoviridae/genética , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/inmunología , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Macaca mulatta , Masculino , Vacunas contra el SIDAS/administración & dosificación , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Carga ViralRESUMEN
The effectiveness of recombinant Adenovirus serotype 5 (Ad5) vectors to induce immune responses against targeted antigens has been limited by the presence of pre-existing or Ad5 vaccine induced anti-vector immunity. The Ad5 [E1-, E2b-] platform, a recombinant Ad5 with additional deletions, has been previously reported by us to induce immune responses in the presence of Ad5 immunity. In an Ad5 immune non-human primate (NHP) model, an Ad5 [E1-, E2b-] construct expressing HIV-1 Gag induced immune responses in the presence of pre-existing Ad5 immunity. In the present study we expand on these prior observations by comparing the cell mediated immune (CMI) responses induced by Ad5 [E1-, E2b-]-SIV-gag/nef in Ad5 naïve and Ad5 immune NHP. Additionally, NHP were immunized with an Ad5 [E1-, E2b-]-HIV-pol construct following two homologous administrations of Ad5 [E1-, E2b-]-SIV-gag/nef to determine if an immune response could be induced against a third antigen in the presence of vaccine induced Ad5 immunity. Positive CMI responses, as assessed by interferon-gamma (IFN-γ) secreting lymphocytes, were induced against all three antigens. These CMI responses increased over a course of multiple immunizations and the response profiles observed in Ad5 naïve and Ad5 immune NHP were similar. No influence of the major histocompatibility complex on CMI responses was observed. These data indicate that the new Ad5 [E1-, E2b-] platform based vaccine could be used for homologous vaccination regimes to induce robust CMI responses in the presence of Ad5 vector immunity.
Asunto(s)
Adenoviridae/genética , Adenoviridae/inmunología , Portadores de Fármacos , Vectores Genéticos , Vacunas contra el SIDAS/inmunología , Animales , Femenino , Eliminación de Gen , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Productos del Gen nef/genética , Productos del Gen nef/inmunología , Productos del Gen pol/genética , Productos del Gen pol/inmunología , Interferón gamma/metabolismo , Linfocitos/inmunología , Macaca mulatta , Masculino , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The variant surface antigen VAR2CSA is a pregnancy malaria vaccine candidate, but its size and polymorphism are obstacles to development. We expressed 3D7-type VAR2CSA domains in Escherichia coli as insoluble His-tagged proteins (Duffy binding-like [DBL] domains DBL1, DBL3, DBL4, and DBL5) that were denatured and refolded or as soluble glutathione S-transferase-tagged protein (DBL6). Anti-DBL5 antiserum cross-reacted with surface proteins of chondroitin sulfate A (CSA)-binding laboratory strains (3D7-CSA and FCR3-CSA) and a clinical pregnancy malaria isolate, whereas anti-DBL6 antiserum reacted only to 3D7 surface protein. This is the first report that E. coli-expressed VAR2CSA domains induce antibody to native VAR2CSA.
Asunto(s)
Antígenos de Protozoos/inmunología , Plasmodium falciparum/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/genética , ADN Protozoario/química , ADN Protozoario/genética , Eritrocitos/parasitología , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Humanos , Malaria Falciparum/parasitología , Datos de Secuencia Molecular , Filogenia , Plasmodium falciparum/genética , Reacción en Cadena de la Polimerasa , Embarazo , Complicaciones Parasitarias del Embarazo/parasitología , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de SecuenciaRESUMEN
Microglial cells are the resident immune cells of the central nervous system. Their function resembles that of tissue macrophages and, as such, they share many properties with both peripheral macrophages and monocytes. One striking similarity is the difficulty with which these cells can be genetically manipulated via transfection or transduction. We have sought to overcome this challenge and generate stably transduced microglial cell lines. Based on encouraging results from macrophages, we hypothesized that lentiviral vectors might provide a valuable tool in the transduction of microglial cells. Using a lentiviral-based vector system expressing enhanced green fluorescent protein (eGFP) under the control of the murine stem cell virus promoter (MSCV), we found that multiplicities of infection (MOIs) of 1, 10, and 100 transduce >70%, >88%, and >95% of the cells, respectively. From the pool of transduced cells, we established lines of N9 and BV-2 microglial cells with distinct fluorescence intensities. Using real time-polymerase chain reaction (PCR), we correlated the integrated eGFP copy numbers to eGFP fluorescence measured by flow cytometry. When mixed, up to three lines with different eGFP intensities could be separated by flow cytometry and fluorescence microscopy. Neither infection nor transgene expression influenced microglial activation as assessed by nitric oxide (NO) production, cytokine release, and surface antigen expression. Our findings that microglial cells are easily transduced by lentiviral based vectors will facilitate research depending on genetic manipulation and help generate transgenic cell lines. In addition, the availability of microglial cell lines with defined fluorescence properties could replace elaborate staining procedures for microglial identification in co-culture experiments.
Asunto(s)
Vectores Genéticos/genética , Lentivirus/genética , Microglía/virología , Transducción Genética/métodos , Transgenes/genética , Animales , Células Cultivadas , Citometría de Flujo , Dosificación de Gen , Regulación Viral de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Interleucina-6/metabolismo , Ratones , Microglía/metabolismo , Óxido Nítrico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In Vbeta5 transgenic mice, mature Vbeta5(+)CD4(+) T cells are tolerized upon recognition of a self Ag, encoded by a defective endogenous retrovirus, whose expression is confined to the lymphoid periphery. Cells are driven by the tolerogen to enter one of two tolerance pathways, deletion or TCR revision. CD4(+) T cells entering the former pathway are rendered anergic and then eliminated. In contrast, TCR revision drives gene rearrangement at the endogenous TCR beta locus and results in the appearance of Vbeta5(-), endogenous Vbeta(+), CD4(+) T cells that are both self-tolerant and functional. An analysis of the molecules that influence each of these pathways was conducted to understand better the nature of the interactions that control tolerance induction in the lymphoid periphery. These studies reveal that deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule, Fas, CD4, and CD8 independent. In contrast, TCR revision is radiosensitive, B cell, CD28, and inducible costimulatory molecule dependent, Fas and CD4 influenced, and CD8 independent. Our data demonstrate the differential regulation of these two divergent tolerance pathways, despite the fact that they are both driven by the same tolerogen and restricted to mature CD4(+) T cells.