RESUMEN
OBJECTIVE: To test the killing effect of type â receptor tyrosine kinase-like orphan receptor (ROR1) chimeric antigen receptor T cell (CAR-T) on several ROR1-expressing tumor cells in vitro. METHODS: The CAR gene was designed and synthesized by constructing the lentiviral vector plasmid, and BamHâ /EcoRâ was used to identify the plasmid. The expression levels of ROR1 among a variety of tumor cell lines were compared using flow cytometry (FCM). The killing effect of CAR-T on positive cells was detected by FCM, the LDH assay and ELISA. RESULTS: The double enzyme digestion identified CAR gene was successfully constructed to the lentivirus vector plasmid. FCM detection showed that the efficiency of CAR-T infection was about 47.23%. Multiple tumor cells expressed ROR1 in varying degrees. The FCM and the LDH assay indicated that CAR-T specifically killed ROR1-positive tumor cells. On positive target cells, more interferonI-γ (FN-γ) could be released during the CAR-T killing process than control T (P<0.05). CONCLUSION: We successfully constructed ROR1 CAR-T. CAR-T can specifically kill ROR1-positive tumor cells and cause the release of large amounts of IFN-γ, providing an experimental basis for clinical application.
Asunto(s)
Inmunoterapia Adoptiva , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/inmunología , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Linfocitos T/citología , Línea Celular Tumoral , Humanos , LentivirusRESUMEN
Chimeric antigen receptor-modified T cells (CAR-T cells) have emerged as a promising cancer immunotherapy for solid tumors. Epithelial cell adhesion molecule (EpCAM) is overexpressed in a variety of tumors and is recognized as a biomarker for circulating tumor cells and cancer stem cells, representing an attractive target for adoptive T-cell immunotherapy. This study generated third-generation CAR-T cells with redirected specificity to EpCAM (EpCAM CAR-T) by lentiviral vector. The study demonstrated that EpCAM CAR-T cells can elicit lytic cytotoxicity to target cells in an EpCAM-dependent manner and secrete cytotoxic cytokines, including interferon gamma and tumor necrosis factor alpha. Furthermore, adoptive transfer of EpCAM CAR-T cells significantly delayed tumor growth and formation in xenograft models. In addition, the safety evaluation showed that CAR-T cells have no systemic toxicity in mice. The data confirmed the antitumor ability and safety of CAR-T cells targeting EpCAM and may provide a new target for CAR-T cell therapies in treating solid tumors.
Asunto(s)
Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Molécula de Adhesión Celular Epitelial/inmunología , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Animales , Biomarcadores , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/terapia , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Molécula de Adhesión Celular Epitelial/antagonistas & inhibidores , Humanos , Inmunofenotipificación , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
T-lymphocytes genetically engineered with the chimeric antigen receptor (CAR-T) have shown great therapeutic potential in cancer treatment. A variety of preclinical researches and clinical trials of CAR-T therapy have been carried out to lay the foundation for future clinical application. In these researches, several gene-transfer methods were used to deliver CARs or other genes into T-lymphocytes, equipping CAR-modified T cells with a property of recognizing and attacking antigen-expressing tumor cells in a major histocompatibility complex-independent manner. Here, we summarize the gene-transfer vectors commonly used in the generation of CAR-T cell, including retrovirus vectors, lentivirus vectors, the transposon/transposase system, the plasmid-based system, and the messenger RNA electroporation system. The following aspects were compared in parallel: efficiency of gene transfer, the integration methods in the modified T cells, foreground of scale-up production, and application and development in clinical trials. These aspects should be taken into account to generate the optimal CAR-gene vector that may be suitable for future clinical application.
Asunto(s)
Vectores Genéticos , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/fisiología , Transducción Genética/métodos , Transfección/métodos , Animales , Ensayos Clínicos como Asunto , Elementos Transponibles de ADN , Electroporación/métodos , Humanos , Inmunoterapia/métodos , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
T cells, genetically modified by chimeric antigen receptors (CAR-T), are endowed with specificity to a desired antigen and are cytotoxic to cells expressing the targeted antigen. CAR-T-based cancer immunotherapy is a promising therapy for curing hematological malignancy, such as acute lymphoid leukemia, and is promising for extending their efficacy to defeat solid tumors. To date, dozens of different CAR-T cells have been evaluated in clinical trials to treat tumors; this necessitates the establishment of guidelines for the production and application of CAR-T cells. However, it is challenging to standardize CAR-T cancer therapy because it involves a combination of gene therapy and cell therapy. In this review, we compare the existing guidelines for CAR-T cells and discuss the challenges and considerations for establishing guidance for CAR-T-based cancer immunotherapy.
Asunto(s)
Guías como Asunto , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/trasplante , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/normas , Citometría de Flujo , Humanos , Inmunoterapia Adoptiva/normas , Neoplasias/genética , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transfección/métodos , Transfección/normasRESUMEN
Recent reports on the impressive efficacy of chimeric antigen receptor (CAR)-modified T cells against hematologic malignancies have inspired oncologists to extend these efforts for the treatment of solid tumors. Clinical trials of CAR-T-based cancer immunotherapy for solid tumors showed that the efficacies are not as remarkable as in the case of hematologic malignancies. There are several challenges that researchers must face when treating solid cancers with CAR-T cells, these include choosing an ideal target, promoting efficient trafficking and infiltration, overcoming the immunosuppressive microenvironment, and avoiding associated toxicity. In this review, we discuss the obstacles imposed by solid tumors on CAR-T cell-based immunotherapy and strategies adopted to improve the therapeutic potential of this approach. Continued investigations are necessary to improve therapeutic outcomes and decrease the adverse effects of CAR-T cell therapy in patients with solid malignancies in the future.