RESUMEN
The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.
Asunto(s)
Células Madre Hematopoyéticas/citología , Inmunoterapia/métodos , Leucemia Mieloide Aguda/terapia , ARN Guía de Kinetoplastida/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Linfocitos T/inmunología , Animales , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Electroporación , Femenino , Hematopoyesis , Humanos , Leucemia Mieloide Aguda/inmunología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante de Neoplasias , Especies Reactivas de Oxígeno , Linfocitos T/citologíaRESUMEN
Epstein-Barr Virus (EBV) can establish latent infections with distinct gene expression patterns referred to as latency types. These different latency types are epigenetically stable and correspond to different promoter utilization. Here we explore the three-dimensional conformations of the EBV genome in different latency types. We employed Chromosome Conformation Capture (3C) assay to investigate chromatin loop formation between the OriP enhancer and the promoters that determine type I (Qp) or type III (Cp) gene expression. We show that OriP is in close physical proximity to Qp in type I latency, and to Cp in type III latency. The cellular chromatin insulator and boundary factor CTCF was implicated in EBV chromatin loop formation. Combining 3C and ChIP assays we found that CTCF is physically associated with OriP-Qp loop formation in type I and OriP-Cp loop formation in type III latency. Mutations in the CTCF binding site located at Qp disrupt loop formation between Qp and OriP, and lead to the activation of Cp transcription. Mutation of the CTCF binding site at Cp, as well as siRNA depletion of CTCF eliminates both OriP-associated loops, indicating that CTCF plays an integral role in loop formation. These data indicate that epigenetically stable EBV latency types adopt distinct chromatin architectures that depend on CTCF and mediate alternative promoter targeting by the OriP enhancer.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Herpesvirus Humano 4/fisiología , Proteínas Represoras/metabolismo , Transcripción Genética , Latencia del Virus/fisiología , Factor de Unión a CCCTC , Línea Celular , Cromatina/genética , Cromatina/virología , Elementos de Facilitación Genéticos/genética , Humanos , Regiones Promotoras Genéticas/genética , Proteínas Represoras/genéticaRESUMEN
Despite the success of CAR-T cell cancer immunotherapy, challenges in efficacy and safety remain. Investigators have begun to enhance CAR-T cells with the expression of accessory molecules to address these challenges. Current systems rely on constitutive transgene expression or multiple viral vectors, resulting in unregulated response and product heterogeneity. Here, we develop a genetic platform that combines autonomous antigen-induced production of an accessory molecule with constitutive CAR expression in a single lentiviral vector called Uni-Vect. The broad therapeutic application of Uni-Vect is demonstrated in vivo by activation-dependent expression of (1) an immunostimulatory cytokine that improves efficacy, (2) an antibody that ameliorates cytokine-release syndrome, and (3) transcription factors that modulate T cell biology. Uni-Vect is also implemented as a platform to characterize immune receptors. Overall, we demonstrate that Uni-Vect provides a foundation for a more clinically actionable next-generation cellular immunotherapy.
Asunto(s)
Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Humanos , Inmunoterapia Adoptiva/métodos , Linfocitos T , Vectores Genéticos/genética , Citocinas/metabolismoRESUMEN
Cellular immunotherapies represent a promising approach for the treatment of cancer. Engineered adoptive cell therapies redirect and augment a leukocyte's inherent ability to mount an immune response by introducing novel anti-tumor capabilities and targeting moieties. A prominent example of this approach is the use of T cells engineered to express chimeric antigen receptors (CARs), which have demonstrated significant efficacy against some hematologic malignancies. Despite increasingly sophisticated strategies to harness immune cell function, efficacy against solid tumors has remained elusive for adoptive cell therapies. Amongst cell types used in immunotherapies, however, macrophages have recently emerged as prominent candidates for the treatment of solid tumors. In this review, we discuss the use of monocytes and macrophages as adoptive cell therapies. Macrophages are innate immune cells that are intrinsically equipped with broad therapeutic effector functions, including active trafficking to tumor sites, direct tumor phagocytosis, activation of the tumor microenvironment and professional antigen presentation. We focus on engineering strategies for manipulating macrophages, with a specific focus on CAR macrophages (CAR-M). We highlight CAR design for macrophages, the production of CAR-M for adoptive cell transfer, and clinical considerations for their use in treating solid malignancies. We then outline recent progress and results in applying CAR-M as immunotherapies. The recent development of engineered macrophage-based therapies holds promise as a key weapon in the immune cell therapy armamentarium.
Asunto(s)
Terapia Genética , Inmunoterapia Adoptiva , Macrófagos/trasplante , Monocitos/trasplante , Neoplasias/terapia , Receptores Quiméricos de Antígenos/genética , Animales , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Fenotipo , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
Adoptive cell therapy with genetically modified T cells has generated exciting outcomes in hematologic malignancies, but its application to solid tumors has proven challenging. This gap has spurred the investigation of alternative immune cells as therapeutics. Macrophages are potent immune effector cells whose functional plasticity leads to antitumor as well as protumor function in different settings, and this plasticity has led to notable efforts to deplete or repolarize tumor-associated macrophages. Alternatively, macrophages could be adoptively transferred after ex vivo genetic modification. In this review, we highlight the role of macrophages in solid tumors, the progress made with macrophage-focused immunotherapeutic modalities, and the emergence of chimeric antigen receptor macrophage cell therapy.
Asunto(s)
Inmunoterapia/métodos , Macrófagos , Neoplasias/terapia , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Humanos , Inmunoterapia Adoptiva/métodos , Macrófagos/patología , Macrófagos/trasplante , Neoplasias/patología , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/patologíaRESUMEN
Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1-4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity.
Asunto(s)
Inmunoterapia Adoptiva , Macrófagos/fisiología , Neoplasias/terapia , Animales , Línea Celular Tumoral , Supervivencia Celular , Humanos , Inmunoterapia , Neoplasias Pulmonares/terapia , Ratones , Microscopía por Video , Neoplasias ExperimentalesRESUMEN
We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19- leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.
Asunto(s)
Antígenos CD19/inmunología , Resistencia a Antineoplásicos/inmunología , Epítopos/inmunología , Leucemia/tratamiento farmacológico , Adulto , Antígenos CD19/uso terapéutico , Linfocitos B/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Leucemia/inmunología , Leucemia/patología , Masculino , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/uso terapéutico , Receptores Quiméricos de Antígenos , Linfocitos T/inmunología , Adulto JovenRESUMEN
Patients with otherwise treatment-resistant Hodgkin lymphoma could benefit from chimeric antigen receptor T-cell (CART) therapy. However, Hodgkin lymphoma lacks CD19 and contains a highly immunosuppressive tumor microenvironment (TME). We hypothesized that in Hodgkin lymphoma, CART should target both malignant cells and the TME. We demonstrated CD123 on both Hodgkin lymphoma cells and TME, including tumor-associated macrophages (TAM). In vitro, Hodgkin lymphoma cells convert macrophages toward immunosuppressive TAMs that inhibit T-cell proliferation. In contrast, anti-CD123 CART recognized and killed TAMs, thus overcoming immunosuppression. Finally, we showed in immunodeficient mouse models that CART123 eradicated Hodgkin lymphoma and established long-term immune memory. A novel platform that targets malignant cells and the microenvironment may be needed to successfully treat malignancies with an immunosuppressive milieu.Significance: Anti-CD123 chimeric antigen receptor T cells target both the malignant cells and TAMs in Hodgkin lymphoma, thereby eliminating an important immunosuppressive component of the tumor microenvironment. Cancer Discov; 7(10); 1154-67. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.
Asunto(s)
Enfermedad de Hodgkin/terapia , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Macrófagos/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/trasplante , Animales , Diferenciación Celular , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Enfermedad de Hodgkin/inmunología , Humanos , Células K562 , Macrófagos/citología , Macrófagos/patología , Ratones , Linfocitos T/inmunología , Microambiente TumoralRESUMEN
PURPOSE: Responses to therapy with chimeric antigen receptor T cells recognizing CD19 (CART19, CTL019) may vary by histology. Mantle cell lymphoma (MCL) represents a B-cell malignancy that remains incurable despite novel therapies such as the BTK inhibitor ibrutinib, and where data from CTL019 therapy are scant. Using MCL as a model, we sought to build upon the outcomes from CTL019 and from ibrutinib therapy by combining these in a rational manner. EXPERIMENTAL DESIGN: MCL cell lines and primary MCL samples were combined with autologous or normal donor-derived anti-CD19 CAR T cells along with ibrutinib. The effect of the combination was studied in vitro and in mouse xenograft models. RESULTS: MCL cells strongly activated multiple CTL019 effector functions, and MCL killing by CTL019 was further enhanced in the presence of ibrutinib. In a xenograft MCL model, we showed superior disease control in the CTL019- as compared with ibrutinib-treated mice (median survival not reached vs. 95 days, P < 0.005) but most mice receiving CTL019 monotherapy eventually relapsed. Therefore, we added ibrutinib to CTL019 and showed that 80% to 100% of mice in the CTL019 + ibrutinib arm and 0% to 20% of mice in the CTL019 arm, respectively, remained in long-term remission (P < 0.05). CONCLUSIONS: Combining CTL019 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Clin Cancer Res; 22(11); 2684-96. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Linfoma de Células del Manto/tratamiento farmacológico , Pirazoles/farmacología , Pirimidinas/farmacología , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa , Animales , Línea Celular Tumoral , Terapia Combinada , Resistencia a Antineoplásicos , Humanos , Inmunoterapia Adoptiva , Ratones Endogámicos NOD , Ratones SCID , Piperidinas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.
Asunto(s)
Antígenos CD19/metabolismo , Antineoplásicos/administración & dosificación , Subunidad alfa del Receptor de Interleucina-3/administración & dosificación , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T/administración & dosificación , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Inmunoterapia/métodos , Ratones Endogámicos NOD , Ratones SCID , Recurrencia Local de Neoplasia/prevención & control , Células Madre Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Linfocitos T/inmunología , Linfocitos T/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Asthma, a common disorder that affects >250 million people worldwide, is defined by exaggerated bronchoconstriction to inflammatory mediators including acetylcholine (ACh), bradykinin and histamine-also termed airway hyper-responsiveness. Nearly 10% of people with asthma have severe, treatment-resistant disease, which is frequently associated with immunoglobulin-E sensitization to ubiquitous fungi, typically Aspergillus fumigatus (Af). Here we show that a major Af allergen, Asp f13, which is a serine protease, alkaline protease 1 (Alp 1), promotes airway hyper-responsiveness by infiltrating the bronchial submucosa and disrupting airway smooth muscle (ASM) cell-extracellular matrix (ECM) interactions. Alp 1-mediated ECM degradation evokes pathophysiological RhoA-dependent Ca(2+) sensitivity and bronchoconstriction. These findings support a pathogenic mechanism in asthma and other lung diseases associated with epithelial barrier impairment, whereby ASM cells respond directly to inhaled environmental allergens to generate airway hyper-responsiveness.
Asunto(s)
Alérgenos/inmunología , Aspergillus fumigatus/química , Asma/inmunología , Proteínas Bacterianas/inmunología , Broncoconstricción/efectos de los fármacos , Endopeptidasas/inmunología , Proteínas Fúngicas/inmunología , Inmunoglobulina E/biosíntesis , Administración por Inhalación , Alérgenos/administración & dosificación , Animales , Aspergillus fumigatus/inmunología , Asma/patología , Proteínas Bacterianas/administración & dosificación , Bronquios/efectos de los fármacos , Bronquios/inmunología , Bronquios/patología , Broncoconstricción/inmunología , Calcio/inmunología , Calcio/metabolismo , Modelos Animales de Enfermedad , Endopeptidasas/administración & dosificación , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/inmunología , Femenino , Proteínas Fúngicas/administración & dosificación , Expresión Génica , Histamina/biosíntesis , Histamina/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/patología , Cultivo Primario de Células , Tráquea/efectos de los fármacos , Tráquea/inmunología , Tráquea/patología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/inmunología , Proteína de Unión al GTP rhoARESUMEN
Epstein-Barr virus (EBV), which is associated with multiple human tumors, persists as a minichromosome in the nucleus of B lymphocytes and induces malignancies through incompletely understood mechanisms. Here, we present a large-scale functional genomic analysis of EBV. Our experimentally generated nucleosome positioning maps and viral protein binding data were integrated with over 700 publicly available high-throughput sequencing data sets for human lymphoblastoid cell lines mapped to the EBV genome. We found that viral lytic genes are coexpressed with cellular cancer-associated pathways, suggesting that the lytic cycle may play an unexpected role in virus-mediated oncogenesis. Host regulators of viral oncogene expression and chromosome structure were identified and validated, revealing a role for the B cell-specific protein Pax5 in viral gene regulation and the cohesin complex in regulating higher order chromatin structure. Our findings provide a deeper understanding of latent viral persistence in oncogenesis and establish a valuable viral genomics resource for future exploration.