RESUMO
Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and T cell activation against a vesicular stomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) in vitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I T cells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 T cell response in vivo without altering the anti-viral CD8 T cell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.
Assuntos
Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/imunologia , Imunidade , Ativação Linfocitária , Ovalbumina/genética , Vesiculovirus/genética , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/química , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia Viral Oncolítica/métodos , Ovalbumina/química , Estabilidade ProteicaRESUMO
The ORF3 accessory protein has been shown to impede reverse genetics of cell-culture-adapted porcine epidemic diarrhea virus (PEDV). Its absence or truncated variants are also associated with viral attenuation in vivo. Here, three ORF3 variants (ORF3NP12, ORF3NP14 and ORF3RB14) and their truncated counterparts were investigated for their regulatory role in recovery of cell-adapted PEDV in vitro. We demonstrate that ORF3NP12, but not the truncated form, can inhibit recovery of reverse-genetics-derived PEDV when expressed in trans. When testing with other RNA viruses, ORF3 was found to inhibit rescue of porcine respiratory and reproductive syndrome virus (PRRSV), but not of influenza virus. Interestingly, results from mutagenesis of ORF3NP12 suggest that F81 and M167 are responsible for impairing PEDV rescue in vitro. By changing specific residues of ORF3, the recombinant PEDV bearing the modified ORF3NP12 can be productively propagated in VeroE6-APN cells. These results may provide mechanistic insights into ORF3-mediated inhibition of PEDV replication in new host cells.
Assuntos
Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Regulação Viral da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Mutação Puntual , Suínos , Doenças dos Suínos/virologia , Células Vero , Proteínas Virais/genéticaRESUMO
Porcine epidemic diarrhoea virus (PEDV) causes acute diarrhoea and dehydration in swine of all ages, with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America warrants an urgent search for effective vaccines. However, PEDV vaccine research has been hampered by difficulties in isolating and propagating the virus in mammalian cells, thereby complicating the recovery of infectious PEDV using a full-length infectious clone. Here, we engineered VeroE6 cells to stably express porcine aminopeptidase N (pAPN) and used them as a platform to obtain a high-growth variant of PEDV, termed PEDVAVCT12. Subsequently, the full-length cDNA clone was constructed by assembling contiguous cDNA fragments encompassing the complete genome of PEDVAVCT12 in a bacterial artificial chromosome. Infectious PEDV could be recovered, and the rescued virus displayed phenotypic properties identical to the parental virus. Interestingly, we found that PEDVAVCT12 contained a C-terminal deletion of the spike gene, resulting in disruption of the ORF3 start codon. When a functional ORF3 gene was restored, the recombinant virus could not be rescued, suggesting that ORF3 could suppress PEDV replication in vitro. In addition, a high-growth and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the ORF3 gene with the mCherry gene. Together, the results of this study provide a means to generate genetically defined PEDV as a promising vaccine candidate.
Assuntos
Infecções por Coronavirus/veterinária , DNA Complementar/genética , DNA Viral/genética , Diarreia/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/virologia , Animais , Sequência de Bases , Chlorocebus aethiops , Infecções por Coronavirus/virologia , DNA Complementar/metabolismo , DNA Viral/metabolismo , Diarreia/virologia , Genoma Viral , Dados de Sequência Molecular , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Células VeroRESUMO
Antibody-drug conjugates (ADC) achieve targeted drug delivery to a tumor and have demonstrated clinical success in many tumor types. The activity and safety profile of an ADC depends on its construction: antibody, payload, linker, and conjugation method, as well as the number of payload drugs per antibody [drug-to-antibody ratio (DAR)]. To allow for ADC optimization for a given target antigen, we developed Dolasynthen (DS), a novel ADC platform based on the payload auristatin hydroxypropylamide, that enables precise DAR-ranging and site-specific conjugation. We used the new platform to optimize an ADC that targets B7-H4 (VTCN1), an immune-suppressive protein that is overexpressed in breast, ovarian, and endometrial cancers. XMT-1660 is a site-specific DS DAR 6 ADC that induced complete tumor regressions in xenograft models of breast and ovarian cancer as well as in a syngeneic breast cancer model that is refractory to PD-1 immune checkpoint inhibition. In a panel of 28 breast cancer PDXs, XMT-1660 demonstrated activity that correlated with B7-H4 expression. XMT-1660 has recently entered clinical development in a phase I study (NCT05377996) in patients with cancer.
Assuntos
Antineoplásicos , Neoplasias da Mama , Imunoconjugados , Humanos , Feminino , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Anticorpos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We show here, for the first time to our knowledge, that the antitumor therapy of oncolytic vesicular stomatitis virus (VSV) in the B16ova model depends upon signaling through myeloid differentiation primary response gene 88 (MyD88) in host cells. VSV-mediated therapy of B16ova tumors was abolished in MyD88(-/-) mice despite generation of antigen-specific T cell responses similar to those in immune-competent mice. Mice defective in only toll-like receptor 4 (TLR4), TLR7, or interleukin 1 (IL-1) signaling retained VSV-induced therapy, suggesting that multiple, redundant pathways of innate immune activation by the virus contribute to antitumor immune reactivity. Lack of MyD88 signaling was associated with decreased expression of proinflammatory cytokines and neutrophil infiltration in response to intratumoral virus, as well as decreased infiltration of draining lymph nodes (LN) with plasmacytoid dendritic cells (pDCs) (CD11b(-)GR1(+)B220(+)) and myeloid-derived suppressor cells (CD11b(+)GR1(+)F4/80(+)). MyD88 signaling in response to VSV was also closely associated with a type I interferon (IFN) response. This inhibited virus replication within the tumor but also protected the host from viral dissemination from the tumor. Therefore, the innate immune response to oncolytic viruses can be, simultaneously, protherapeutic, antioncolytic, and systemically protective. These paradoxically conflicting roles need to be carefully considered in future strategies designed to improve the efficacy of oncolytic virotherapy.
Assuntos
Melanoma Experimental/terapia , Fator 88 de Diferenciação Mieloide/metabolismo , Terapia Viral Oncolítica/métodos , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Interleucina-1/deficiência , Interleucina-1/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/virologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais , Linfócitos T/imunologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genética , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/metabolismo , Replicação Viral/genéticaRESUMO
Despite having potent oncolytic activity, in vitro, direct intratumoral injection of oncolytic vesicular stomatitis virus (VSV) into established AE17ova mesothelioma tumors in C57Bl/6 mice had no therapeutic effect. During studies to combine systemic cyclophosphamide (CPA) with VSV to suppress the innate immune reaction against VSV, we observed that CPA alone had highly significant antitumor effects in this model. However, against our expectations, the combination of CPA and VSV consistently reduced therapeutic efficacy compared to CPA alone, despite the fact that the combination increased intratumoral VSV titers. We show here that CPA-mediated therapy against AE17ova tumors was immune-mediated and dependent upon both CD4 T cells and natural killer (NK) cells. However, intratumoral VSV induced a transforming growth factor-ß (TGF-ß)-dependent suppressive activity, mediated by CD11b(+)GR-1(+) cells that significantly inhibited both antigen-specific T-cell activation, and CPA-activated, NK-dependent killing of AE17ova tumor cells. Overall, our results show that treatment with oncolytic viruses can induce a variety of immune-mediated consequences in vivo with both positive, or negative, effects on antitumor therapy. These underexplored immune consequences of treatment with oncolytic viruses may have significant, and possibly unexpected, impacts on how virotherapy interacts in combination with other agents which modulate antitumor immune effectors.
Assuntos
Ciclofosfamida/farmacologia , Terapia Genética/métodos , Mesotelioma/imunologia , Mesotelioma/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Terapia Combinada , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Melanoma Experimental/virologia , Mesotelioma/tratamento farmacológico , Mesotelioma/virologia , Camundongos , Camundongos Endogâmicos C57BL , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/metabolismo , Replicação ViralRESUMO
In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNß), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNß-escape tumors predictably express a point-mutated CSDE1P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNß evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.
Assuntos
Proteínas de Ligação a DNA/imunologia , Interferon beta/imunologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/imunologia , Proteínas de Ligação a RNA/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação Viral/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Imunoterapia/métodos , Interferon beta/metabolismo , Camundongos Endogâmicos C57BL , Mutação , Vírus Oncolíticos/metabolismo , Vírus Oncolíticos/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
BACKGROUND: Diffuse midline glioma, formerly DIPG (diffuse intrinsic pontine glioma), is the deadliest pediatric brainstem tumor with median survival of less than one year. Here, we investigated (i) whether direct delivery of adenovirus-expressing cluster of differentiation (CD)40 ligand (Ad-CD40L) to brainstem tumors would induce immune-mediated tumor clearance and (ii) if so, whether therapy would be associated with a manageable toxicity due to immune-mediated inflammation in the brainstem. METHODS: Syngeneic gliomas in the brainstems of immunocompetent mice were treated with Ad-CD40L and survival, toxicity, and immune profiles determined. A clinically translatable vector, whose replication would be tightly restricted to tumor cells, rAd-Δ24-CD40L, was tested in human patient-derived diffuse midline gliomas and immunocompetent models. RESULTS: Expression of Ad-CD40L restricted to brainstem gliomas by pre-infection induced complete rejection, associated with immune cell infiltration, of which CD4+ T cells were critical for therapy. Direct intratumoral injection of Ad-CD40L into established brainstem tumors improved survival and induced some complete cures but with some acute toxicity. RNA-sequencing analysis showed that Ad-CD40L therapy induced neuroinflammatory immune responses associated with interleukin (IL)-6, IL-1ß, and tumor necrosis factor α. Therefore, to generate a vector whose replication, and transgene expression, would be tightly restricted to tumor cells, we constructed rAd-Δ24-CD40L, the backbone of which has already entered clinical trials for diffuse midline gliomas. Direct intratumoral injection of rAd-Δ24-CD40L, with systemic blockade of IL-6 and IL-1ß, generated significant numbers of cures with readily manageable toxicity. CONCLUSIONS: Virus-mediated delivery of CD40L has the potential to be effective in treating diffuse midline gliomas without obligatory neuroinflammation-associated toxicity.
Assuntos
Neoplasias do Tronco Encefálico , Glioma , Adenoviridae , Animais , Neoplasias do Tronco Encefálico/terapia , Linfócitos T CD4-Positivos , Ligante de CD40 , Glioma/terapia , Humanos , CamundongosRESUMO
The application of adoptive T cell therapies, including those using chimeric antigen receptor (CAR)-modified T cells, to solid tumors requires combinatorial strategies to overcome immune suppression associated with the tumor microenvironment. Here we test whether the inflammatory nature of oncolytic viruses and their ability to remodel the tumor microenvironment may help to recruit and potentiate the functionality of CAR T cells. Contrary to our hypothesis, VSVmIFNß infection is associated with attrition of murine EGFRvIII CAR T cells in a B16EGFRvIII model, despite inducing a robust proinflammatory shift in the chemokine profile. Mechanistically, type I interferon (IFN) expressed following infection promotes apoptosis, activation, and inhibitory receptor expression, and interferon-insensitive CAR T cells enable combinatorial therapy with VSVmIFNß. Our study uncovers an unexpected mechanism of therapeutic interference, and prompts further investigation into the interaction between CAR T cells and oncolytic viruses to optimize combination therapy.
Assuntos
Imunoterapia Adotiva , Interferon beta/metabolismo , Vírus Oncolíticos/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Terapia Combinada , Feminino , Interferon beta/genética , Ativação Linfocitária , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Baço/imunologiaRESUMO
APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade.
Assuntos
Vacinas Anticâncer/farmacologia , Citidina Desaminase/imunologia , Imunoterapia/métodos , Antígenos de Histocompatibilidade Menor/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Resistencia a Medicamentos Antineoplásicos , Epitopos/imunologia , Feminino , Humanos , Células Matadoras Naturais/imunologia , Melanoma/imunologia , Melanoma/terapia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação , Evasão Tumoral/efeitos dos fármacosRESUMO
There are several roadblocks that hinder systemic delivery of oncolytic viruses to the sites of metastatic disease. These include the tumor vasculature, which provides a physical barrier to tumor-specific virus extravasation. Although interleukin-2 (IL-2) has been used in antitumor therapy, it is associated with endothelial cell injury, leading to vascular leak syndrome (VLS). Here, we demonstrate that IL-2-mediated VLS, accentuated by depletion of regulatory T cells (Treg), facilitates localization of intravenously (i.v.) delivered oncolytic virus into established tumors in immune-competent mice. IL-2, in association with Treg depletion, generates "hyperactivated" natural killer (NK) cells, possessing antitumor activity and secreting factors that facilitate virus spread/replication throughout the tumor by disrupting the tumor architecture. As a result, the combination of Treg depletion/IL-2 and systemic oncolytic virotherapy was found to be significantly more therapeutic against established disease than either treatment alone. These data demonstrate that it is possible to combine biological therapy with oncolytic virotherapy to generate systemic therapy against established tumors.
Assuntos
Interleucina-2/uso terapêutico , Depleção Linfocítica , Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Terapia Combinada , Endotélio Vascular/efeitos dos fármacos , Interleucina-2/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Vírus Oncolíticos/efeitos dos fármacos , Vírus Oncolíticos/fisiologia , Linfócitos T Reguladores/imunologia , Internalização do Vírus , Replicação Viral/efeitos dos fármacosRESUMO
There are several roadblocks that hinder systemic delivery of oncolytic viruses to the sites of metastatic disease. These include the tumor vasculature, which provides a physical barrier to tumor-specific virus extravasation. Although interleukin-2 (IL-2) has been used in antitumor therapy, it is associated with endothelial cell injury, leading to vascular leak syndrome (VLS). Here, we demonstrate that IL-2-mediated VLS, accentuated by depletion of regulatory T cells (Treg), facilitates localization of intravenously (IV) delivered oncolytic virus into established tumors in immune-competent mice. IL-2, in association with Treg depletion, generates "hyperactivated" natural killer (NK) cells, possessing antitumor activity and secreting factors that facilitate virus spread/replication throughout the tumor by disrupting the tumor architecture. As a result, the combination of Treg depletion/IL-2 and systemic oncolytic virotherapy was found to be significantly more therapeutic against established disease than either treatment alone. These data demonstrate that it is possible to combine biological therapy with oncolytic virotherapy to generate systemic therapy against established tumors.
RESUMO
To protect viral particles from neutralization, sequestration, nonspecific adhesion, and mislocalization following systemic delivery, we have previously exploited the natural tumor-homing properties of antigen-specific CD8+ T cells. Thus, OT-I T cells, preloaded in vitro with the oncolytic vesicular stomatitis virus (VSV), can deliver virus to established B16ova tumors to generate significantly better therapy than that achievable with OT-I T cells, or systemically delivered VSV, alone. Here, we demonstrate that preconditioning immune-competent mice with Treg depletion and interleukin-2 (IL-2), before adoptive T-cell therapy with OT-I T cells loaded with VSV, leads to further highly significant increases in antitumor therapy. Therapy was associated with antitumor immune memory, but with no detectable toxicities associated with IL-2, Treg depletion, or systemic dissemination of the oncolytic virus. Efficacy was contributed by multiple factors, including improved persistence of T cells; enhanced delivery of VSV to tumors; increased persistence of OT-I cells in vivo resulting from tumor oncolysis; and activation of NK cells, which acquire potent antitumor and proviral activities. By controlling the levels of virus loaded onto the OT-I cells, adoptive therapy was still effective in mice preimmune to the virus, indicating that therapy with virus-loaded T cells may be useful even in virus-immune patients. Taken together, our data show that it is possible to combine adoptive T-cell therapy, with biological therapy (Treg depletion+IL-2), and VSV virotherapy, to treat established tumors under conditions where none of the individual modalities alone is successful.
Assuntos
Adaptação Biológica/imunologia , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Terapia Viral Oncolítica , Linfócitos T/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Antígenos/imunologia , Linhagem Celular Tumoral , Memória Imunológica/imunologia , Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Neoplasias/genética , Linfócitos T/efeitos dos fármacos , Vesiculovirus/genéticaRESUMO
Relatively little attention has been paid to the role of virotherapy in promoting antitumor immune responses. Here, we show that CD8+ T cells are critical for the efficacy of intratumoral vesicular stomatitis virus virotherapy and are induced against both virally encoded and tumor-associated immunodominant epitopes. We tested three separate immune interventions to increase the frequency/activity of activated antitumoral T cells. Depletion of Treg had a negative therapeutic effect because it relieved suppression of the antiviral immune response, leading to early viral clearance. In contrast, increasing the circulating levels of tumor antigen-specific T cells using adoptive T cell transfer therapy, in combination with intratumoral virotherapy, generated significantly improved therapy over either adoptive therapy or virotherapy alone. Moreover, the incorporation of a tumor-associated antigen within the oncolytic vesicular stomatitis virus increased the levels of activation of naïve T cells against the antigen, which translated into increased antitumor therapy. Therefore, our results show that strategies which enhance immune activation against tumor-associated antigens can also be used to enhance the efficacy of virotherapy.
Assuntos
Imunoterapia Adotiva/métodos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Terapia Viral Oncolítica/métodos , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Melanoma Experimental/virologia , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Immunotherapy has shown remarkable clinical promise in the treatment of various types of cancers. However, clinical benefits derive from a highly inflammatory mechanism of action. This presents unique challenges for use in pediatric brainstem tumors including diffuse intrinsic pontine glioma (DIPG), since treatment-related inflammation could cause catastrophic toxicity. Therefore, the goal of this study was to investigate whether inflammatory, immune-based therapies are likely to be too dangerous to pursue for the treatment of pediatric brainstem tumors. METHODS: To complement previous immunotherapy studies using patient-derived xenografts in immunodeficient mice, we developed fully immunocompetent models of immunotherapy using transplantable, syngeneic tumors. These four models - HSVtk/GCV suicide gene immunotherapy, oncolytic viroimmunotherapy, adoptive T cell transfer, and CAR T cell therapy - have been optimized to treat tumors outside of the CNS and induce a broad spectrum of inflammatory profiles, maximizing the chances of observing brainstem toxicity. RESULTS: All four models achieved anti-tumor efficacy in the absence of toxicity, with the exception of recombinant vaccinia virus expressing GMCSF, which demonstrated inflammatory toxicity. Histology, imaging, and flow cytometry confirmed the presence of brainstem inflammation in all models. Where used, the addition of immune checkpoint blockade did not introduce toxicity. CONCLUSIONS: It remains imperative to regard the brainstem with caution for immunotherapeutic intervention. Nonetheless, we show that further careful development of immunotherapies for pediatric brainstem tumors is warranted to harness the potential potency of anti-tumor immune responses, despite their possible toxicity within this anatomically sensitive location.
Assuntos
Neoplasias do Tronco Encefálico/terapia , Glioma Pontino Intrínseco Difuso/terapia , Terapia Genética/métodos , Imunoterapia Adotiva/métodos , Terapia Viral Oncolítica/métodos , Linfócitos T/transplante , Animais , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/imunologia , Linhagem Celular Tumoral , Glioma Pontino Intrínseco Difuso/imunologia , Feminino , Genes Transgênicos Suicidas , Terapia Genética/efeitos adversos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Imunoterapia Adotiva/efeitos adversos , Camundongos , Terapia Viral Oncolítica/efeitos adversos , Resultado do Tratamento , Vaccinia virus/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antitumor T-cell responses raised by first-line therapies such as chemotherapy, radiation, tumor cell vaccines, and viroimmunotherapy tend to be weak, both quantitatively (low frequency) and qualitatively (low affinity). We show here that T cells that recognize tumor-associated antigens can directly kill tumor cells if used at high effector-to-target ratios. However, when these tumor-reactive T cells were present at suboptimal ratios, direct T-cell-mediated tumor cell killing was reduced and the ability of tumor cells to evolve away from a coapplied therapy (oncolytic or suicide gene therapy) was promoted. This T-cell-mediated increase in therapeutic resistance was associated with C to T transition mutations that are characteristic of APOBEC3 cytosine deaminase activity and was induced through a TNFα and protein kinase C-dependent pathway. Short hairpin RNA inhibition of endogenous APOBEC3 reduced rates of tumor escape from oncolytic virus or suicide gene therapy to those seen in the absence of antitumor T-cell coculture. Conversely, overexpression of human APOBEC3B in tumor cells enhanced escape from suicide gene therapy and oncolytic virus therapy both in vitro and in vivo Our data suggest that weak affinity or low frequency T-cell responses against tumor antigens may contribute to the ability of tumor cells to evolve away from first-line therapies. We conclude that immunotherapies need to be optimized as early as possible so that, if they do not kill the tumor completely, they do not promote treatment resistance.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia , Melanoma Experimental/terapia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Feminino , Ganciclovir/uso terapêutico , Orthoreovirus Mamífero 3 , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Mutação , Terapia Viral Oncolítica , Evasão TumoralRESUMO
Tumor cells frequently evade applied therapies through the accumulation of genomic mutations and rapid evolution. In the case of oncolytic virotherapy, understanding the mechanisms by which cancer cells develop resistance to infection and lysis is critical to the development of more effective viral-based platforms. Here, we identify APOBEC3 as an important factor that restricts the potency of oncolytic vesicular stomatitis virus (VSV). We show that VSV infection of B16 murine melanoma cells upregulated APOBEC3 in an IFN-ß-dependent manner, which was responsible for the evolution of virus-resistant cell populations and suggested that APOBEC3 expression promoted the acquisition of a virus-resistant phenotype. Knockdown of APOBEC3 in B16 cells diminished their capacity to develop resistance to VSV infection in vitro and enhanced the therapeutic effect of VSV in vivo. Similarly, overexpression of human APOBEC3B promoted the acquisition of resistance to oncolytic VSV both in vitro and in vivo. Finally, we demonstrate that APOBEC3B expression had a direct effect on the fitness of VSV, an RNA virus that has not previously been identified as restricted by APOBEC3B. This research identifies APOBEC3 enzymes as key players to target in order to improve the efficacy of viral or broader nucleic acid-based therapeutic platforms.
RESUMO
Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvß6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues.Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvß6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed.Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvß3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvß6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non-tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20-treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5.Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvß6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215-24. ©2018 AACR.
Assuntos
Antígenos de Neoplasias/genética , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/terapia , Integrinas/genética , Terapia Viral Oncolítica , Adenoviridae/genética , Animais , Carboidratos Epimerases/genética , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/virologia , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Feminino , Genes cdc/genética , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Cetona Oxirredutases/genética , Camundongos , Vírus Oncolíticos/genética , Distribuição Tecidual , Transdução Genética , Tropismo/genéticaRESUMO
The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein.
Assuntos
Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Proteínas da Matriz Viral/metabolismo , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas , Genes Reporter , Teste de Complementação Genética , Humanos , Fenótipo , Recombinação Genética , Proteínas da Matriz Viral/genéticaRESUMO
Emergence of the porcine epidemic diarrhea virus (PEDV) as a global threat to the swine industry underlies the urgent need for deeper understanding of this virus. To date, we have yet to identify functions for all the major gene products, much less grasp their implications for the viral life cycle and pathogenic mechanisms. A major reason is the lack of genetic tools for studying PEDV. In this review, we discuss the reverse genetics approaches that have been successfully used to engineer infectious clones of PEDV as well as other potential and complementary methods that have yet to be applied to PEDV. The importance of proper cell culture for successful PEDV propagation and maintenance of disease phenotype are addressed in our survey of permissive cell lines. We also highlight areas of particular relevance to PEDV pathogenesis and disease that have benefited from reverse genetics studies and pressing questions that await resolution by such studies. In particular, we examine the spike protein as a determinant of viral tropism, entry and virulence, ORF3 and its association with cell culture adaptation, and the nucleocapsid protein and its potential role in modulating PEDV pathogenicity. Finally, we conclude with an exploration of how reverse genetics can help mitigate the global impact of PEDV by addressing the challenges of vaccine development.