RESUMEN
In humans, the natural killer (NK) cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αß T cell receptor-expressing subset with characteristic specificity for tissue-localized viruses. This subset also displays enhanced cytotoxic and memory phenotypes. Here, we characterized this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161-equivalent CD8+ T cell populations (CD8+NK1.1+) revealed substantial up-regulation of granzymes, perforin, killer lectin-like receptors, and innate signaling molecules in comparison to CD8+NK1.1- T cells. Adoptive transfer of CD8+NK1.1+ cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8+NK1.1- cells. Freshly isolated human CD8+CD61+ T cells exhibited heightened allogeneic killing activity in comparison to CD8+CD61- T cells or total peripheral blood mononuclear cells (PBMCs). To determine whether this subset might improve the antitumor efficacy of CAR T cell therapy against solid tumors, we compared bulk PBMCs, CD8+CD161-, and CD8+CD161+ T cells transduced with a human epidermal growth factor receptor-2 (HER2)-specific CAR construct. In vitro, CD8+CD161+ CAR-transduced T cells killed HER2+ targets faster and with greater efficiency. Similarly, these cells mediated enhanced in vivo antitumor efficacy in xenograft models of HER2+ pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.
Asunto(s)
Adenocarcinoma , Memoria Inmunológica , Animales , Linfocitos T CD8-positivos , Leucocitos Mononucleares , Ratones , Subgrupos de Linfocitos TRESUMEN
Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(-) disease. We report that CD19(-) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(-) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(-) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia Adoptiva , Leucemia de Células B/inmunología , Leucemia de Células B/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos CD19/química , Antígenos de Neoplasias , Biomarcadores , Línea Celular Tumoral , Citocinas/metabolismo , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunoterapia Adoptiva/métodos , Leucemia de Células B/genética , Leucemia de Células B/terapia , Ratones Transgénicos , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Relación Estructura-Actividad , Transducción Genética , Transgenes , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Refractory metastatic rhabdomyosarcoma is largely incurable. Here we analyze the response of a child with refractory bone marrow metastatic rhabdomyosarcoma to autologous HER2 CAR T cells. Three cycles of HER2 CAR T cells given after lymphodepleting chemotherapy induces remission which is consolidated with four more CAR T-cell infusions without lymphodepletion. Longitudinal immune-monitoring reveals remodeling of the T-cell receptor repertoire with immunodominant clones and serum autoantibodies reactive to oncogenic signaling pathway proteins. The disease relapses in the bone marrow at six months off-therapy. A second remission is achieved after one cycle of lymphodepletion and HER2 CAR T cells. Response consolidation with additional CAR T-cell infusions includes pembrolizumab to improve their efficacy. The patient described here is a participant in an ongoing phase I trial (NCT00902044; active, not recruiting), and is 20 months off T-cell infusions with no detectable disease at the time of this report.
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Inmunoterapia Adoptiva/métodos , Neoplasias de los Músculos/terapia , Recurrencia Local de Neoplasia/terapia , Receptor ErbB-2/inmunología , Rabdomiosarcoma/terapia , Linfocitos T/trasplante , Biopsia , Médula Ósea/patología , Niño , Ensayos Clínicos Fase I como Asunto , Humanos , Masculino , Neoplasias de los Músculos/inmunología , Neoplasias de los Músculos/patología , Recurrencia Local de Neoplasia/inmunología , Receptores Quiméricos de Antígenos/inmunología , Inducción de Remisión/métodos , Rabdomiosarcoma/inmunología , Rabdomiosarcoma/secundario , Linfocitos T/inmunología , Linfocitos T/metabolismo , Trasplante Autólogo/métodos , Resultado del TratamientoRESUMEN
This Article has been retracted; see accompanying Retraction.
RESUMEN
Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.
RESUMEN
Triple-negative breast cancer (TNBC) is an aggressive disease lacking targeted therapy. In this study, we developed a CAR T cell-based immunotherapeutic strategy to target TEM8, a marker initially defined on endothelial cells in colon tumors that was discovered recently to be upregulated in TNBC. CAR T cells were developed that upon specific recognition of TEM8 secreted immunostimulatory cytokines and killed tumor endothelial cells as well as TEM8-positive TNBC cells. Notably, the TEM8 CAR T cells targeted breast cancer stem-like cells, offsetting the formation of mammospheres relative to nontransduced T cells. Adoptive transfer of TEM8 CAR T cells induced regression of established, localized patient-derived xenograft tumors, as well as lung metastatic TNBC cell line-derived xenograft tumors, by both killing TEM8+ TNBC tumor cells and targeting the tumor endothelium to block tumor neovascularization. Our findings offer a preclinical proof of concept for immunotherapeutic targeting of TEM8 as a strategy to treat TNBC.Significance: These findings offer a preclinical proof of concept for immunotherapeutic targeting of an endothelial antigen that is overexpressed in triple-negative breast cancer and the associated tumor vasculature. Cancer Res; 78(2); 489-500. ©2017 AACR.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Inmunoterapia , Neoplasias Pulmonares/terapia , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Superficie Celular/metabolismo , Linfocitos T/trasplante , Neoplasias de la Mama Triple Negativas/terapia , Animales , Apoptosis , Biomarcadores de Tumor , Estudios de Casos y Controles , Proliferación Celular , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Ratones , Proteínas de Microfilamentos , Pronóstico , Tasa de Supervivencia , Linfocitos T/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Background: Glioblastoma (GBM) is the most common primary malignant brain cancer, and is currently incurable. Chimeric antigen receptor (CAR) T cells have shown promise in GBM treatment. While we have shown that combinatorial targeting of 2 glioma antigens offsets antigen escape and enhances T-cell effector functions, the interpatient variability in surface antigen expression between patients hinders the clinical impact of targeting 2 antigen pairs. This study addresses targeting 3 antigens using a single CAR T-cell product for broader application. Methods: We analyzed the surface expression of 3 targetable glioma antigens (human epidermal growth factor receptor 2 [HER2], interleukin-13 receptor subunit alpha-2 [IL13Rα2], and ephrin-A2 [EphA2]) in 15 primary GBM samples. Accordingly, we created a trivalent T-cell product armed with 3 CAR molecules specific for these validated targets encoded by a single universal (U) tricistronic transgene (UCAR T cells). Results: Our data showed that co-targeting HER2, IL13Rα2, and EphA2 could overcome interpatient variability by a tendency to capture nearly 100% of tumor cells in most tumors tested in this cohort. UCAR T cells made from GBM patients' blood uniformly expressed all 3 CAR molecules with distinct antigen specificity. UCAR T cells mediated robust immune synapses with tumor targets forming more polarized microtubule organizing centers and exhibited improved cytotoxicity and cytokine release over best monospecific and bispecific CAR T cells per patient tumor profile. Lastly, low doses of UCAR T cells controlled established autologous GBM patient derived xenografts (PDXs) and improved survival of treated animals. Conclusion: UCAR T cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.
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Variación Antigénica/inmunología , Glioblastoma/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Receptor EphA2/inmunología , Receptor ErbB-2/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Apoptosis , Proliferación Celular , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Receptor ErbB-2/metabolismo , Linfocitos T/metabolismo , Escape del Tumor , Animales , Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva , Interleucina-13/metabolismo , Activación de Linfocitos , Ratones , Ratones SCID , Recurrencia Local de Neoplasia , Trasplante de Neoplasias , Unión Proteica , Multimerización de Proteína , Receptores de Antígenos de Linfocitos T/metabolismo , TransgenesRESUMEN
Compensation is a critical process for the unbiased analysis of flow cytometry data. Numerous compensation strategies exist, including the use of bead-based products. The purpose of this study was to determine whether beads, specifically polystyrene microspheres (PSMS) compare to the use of primary leukocytes for single color based compensation when conducting polychromatic flow cytometry. To do so, we stained individual tubes of both PSMS and leukocytes with panel specific antibodies conjugated to fluorochromes corresponding to fluorescent channels FL1-FL10. We compared the matrix generated by PSMS to that generated using peripheral blood mononuclear cells (PBMC). Ideal for compensation is a sample with both a discrete negative population and a bright positive population. We demonstrate that PSMS display autofluorescence properties similar to PBMC. When comparing PSMS to PBMC for compensation PSMS yielded more evenly distributed and discrete negative and positive populations to use for compensation. We analyzed three donors' PBMC stained with our 10-color T cell subpopulation panel using compensation generated by PSMS vs.PBMC and detected no significant differences in the population distribution. Panel specific antibodies bound to PSMS represent an invaluable valid tool to generate suitable compensation matrices especially when sample material is limited and/or the sample requires analysis of dynamically modulated or rare events.
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Citometría de Flujo , Inmunofenotipificación , Leucocitos Mononucleares/citología , Microesferas , Anticuerpos/metabolismo , Color , Citometría de Flujo/métodos , Colorantes Fluorescentes/metabolismo , Humanos , Inmunofenotipificación/métodos , Leucocitos/citología , Leucocitos/inmunología , Poliestirenos/inmunologíaRESUMEN
Substantial progress has been made in the treatment of pediatric solid tumors over the past 4 decades. However, children with metastatic and or recurrent disease continue to do poorly despite the aggressive multi-modality conventional therapies. The increasing understanding of the tumor biology and the interaction between the tumor and the immune system over the recent years have led to the development of novel immune-based therapies as alternative options for some of these high-risk malignancies. The safety and anti-tumor efficacy of various tumor vaccines and tumor-antigen specific immune cells are currently being investigated for various solid tumors. In early clinical trials, most of these cellular therapies have been well tolerated and have shown promising clinical responses. Although substantial work is being done in this field, the available knowledge for pediatric tumors remains limited. We review the contemporary early phase cell-based immunotherapy efforts for pediatric solid tumors and discuss the rationale and the challenges thereof.
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Anticuerpos Monoclonales/uso terapéutico , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos , Neoplasias del Sistema Nervioso Central/terapia , Inmunoterapia , Humanos , PediatríaRESUMEN
Separase, an enzyme that cleaves the chromosomal cohesin during mitosis, is overexpressed in a wide range of human epithelial cancers of breast, bone and prostate (Meyer et al., Clin Cancer Res 15(8):2703-2710, 2009). Overexpression of Separase in animal models results in aneuploidy and tumorigenesis. We have examined the expression and localization of Separase protein in adult and pediatric glioblastoma and normal brain specimens. Immunofluorescence microscopy and Western blot analysis showed significant overexpression of Separase in all adult and a subset of pediatric glioblastoma cells. Tumor status and patient survival strongly correlate with the mislocalization of Separase into the nucleus throughout all stages of the cell cycle. Unlike exclusively nuclear localization in mitotic control cells, glioblastoma samples have a significantly higher number of resting (interphase) cells with strong nuclear Separase staining. Additionally, patient survival analysis demonstrated a strong correlation between overexpression of Separase protein in adult glioblastoma and a high incidence of relapse and reduced overall survival. These results further strengthen our hypothesis that Separase is an oncogene whose overexpression induces tumorigenesis, and indicate that Separase overexpression and aberrant nuclear localization are common in many tumor types and may predict outcome in some human malignancies.
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Neoplasias Encefálicas/metabolismo , Núcleo Celular/metabolismo , Glioblastoma/metabolismo , Separasa/metabolismo , Regulación hacia Arriba , Neoplasias Encefálicas/mortalidad , Ciclo Celular , Glioblastoma/mortalidad , Humanos , Pronóstico , Recurrencia , Tasa de SupervivenciaRESUMEN
Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.
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Traslado Adoptivo , Antígenos de Neoplasias/metabolismo , Glioblastoma/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Terapia Combinada , Glioblastoma/inmunología , Glioblastoma/patología , Células HEK293 , Humanos , Subunidad alfa2 del Receptor de Interleucina-13/genética , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Ratones , Ratones SCID , Modelos Biológicos , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales Cultivadas , Escape del Tumor , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor-the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment.Molecular Therapy-Nucleic Acids (2013) 2, e105; doi:10.1038/mtna.2013.32; published online 9 July 2013.
RESUMEN
Atopic asthma is a chronic inflammatory disease of the lungs generally marked by excessive Th2 inflammation. The role of allergen-specific IgG in asthma is still controversial; however, a receptor of IgG-immune complexes (IgG-ICs), FcγRIII, has been shown to promote Th2 responses through an unknown mechanism. Herein, we demonstrate that allergen-specific IgG-ICs, formed upon reexposure to allergen, promoted Th2 responses in two different models of IC-mediated inflammation that were independent of a preformed T cell memory response. Development of Th2-type airway inflammation was shown to be both FcγRIII and TLR4 dependent, and T cells were necessary and sufficient for this process to occur, even in the absence of type 2 innate lymphoid cells. We sought to identify downstream targets of FcγRIII signaling that could contribute to this process and demonstrated that bone marrow-derived DCs, alveolar macrophages, and respiratory DCs significantly upregulated IL-33 when activated through FcγRIII and TLR4. Importantly, IC-induced Th2 inflammation was dependent on the ST2/IL-33 pathway. Our results suggest that allergen-specific IgG can enhance secondary responses by ligating FcγRIII on antigen-presenting cells to augment development of Th2-mediated responses in the lungs via an IL-33-dependent mechanism.
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Inflamación/metabolismo , Interleucinas/metabolismo , Pulmón/patología , Receptores de IgG/metabolismo , Animales , Asma/metabolismo , Células de la Médula Ósea/citología , Células Dendríticas/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Hipersensibilidad , Hipersensibilidad Inmediata/metabolismo , Inmunoglobulina G/metabolismo , Interleucina-33 , Leucocitos Mononucleares/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Células Th2RESUMEN
Medulloblastoma (MB) is a cancer of the cerebellum and the most common primary pediatric malignancy of the central nervous system. Classified as a primitive neural ectoderm tumor; it is thought to arise from granule cell precursors in the cerebellum. The standard of care consists of surgery, chemotherapy and age-dependent radiation therapy. Despite aggressive multimodality therapy; approximately 30% of MB patients remain incurable. Moreover, for long-term survivors, the treatment related sequelae are often debilitating. Side effects include cerebellar mutism, sterility, neurocognitive deficits, and a substantial risk of developing secondary cancers. In a quest for more effective and targeted therapies, scientists have begun to investigate the biological events that not only initiate but also sustain the malignant phenotype in MB. Of particular interest is, the role of the tumor microenvironment in tumor pathogenesis. This review seeks to highlight several key processes observed in cancer biology, particularly the involvement of the tumor microenvironment, with relevant examples from MB.