RESUMEN
BACKGROUND: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). METHODS: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. RESULTS: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. CONCLUSIONS: The strong cytotoxic effect of a CD276-specific CAR in combination with an "off-the-shelf" NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.
Asunto(s)
Antígenos B7/genética , Regulación Neoplásica de la Expresión Génica , Células Asesinas Naturales/citología , Melanoma/inmunología , Receptores Quiméricos de Antígenos/genética , Neoplasias Cutáneas/inmunología , Microambiente Tumoral , Antígenos de Neoplasias/inmunología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Movimiento Celular , Citotoxicidad Inmunológica , Fibroblastos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hipoxia , Sistema Inmunológico , Terapia de Inmunosupresión , Inmunosupresores , Inmunoterapia Adoptiva/métodos , Técnicas In Vitro , Ácido Láctico/metabolismo , Lentivirus/genética , Melanoma/metabolismo , Invasividad Neoplásica , Neoplasias Cutáneas/metabolismoRESUMEN
Despite the recent success of CAR T cells targeting CD19 and CD22 in hematological malignancies, the production of CAR T cells still requires an extensive manufacturing process. The well-established NK-92 cell line provides a promising alternative to produce CAR-modified effector cells in a GMP-compliant, cost-effective way. NK-92 can be redirected against a variety of surface antigens by our adapter CAR (AdCAR) system utilizing biotinylated antibodies (bAb) as adapter molecules. Selected bAb were capable of inducing significant AdCAR NK-92-mediated lysis of non-Hodgkin lymphoma (NHL) and mantle-cell lymphoma (MCL) cell lines as well as primary MCL and chronic lymphocytic leukemia (CLL) cells. AdCAR specificity was proven using a JeKo-1 CD19/CD20 knockout antigen-loss model. Moreover, through combinations of bAb, AdCAR NK-92 cells are capable of combatting tumor antigen evasion mechanisms. In conclusion, we successfully generated the AdCAR NK-92 cell line which can be manufactured as an "off-the-shelf, on-demand" product allowing universal and tunable tumor targeting.