RESUMEN
PURPOSE: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. EXPERIMENTAL DESIGN: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR(+) T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR(+) T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. RESULTS: We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K(+) AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env(+) tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env-specific CAR(+) T cells, we observed a significant antitumor effect. CONCLUSIONS: Adoptive cellular immunotherapy with HERV-K env-specific CAR(+) T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors.
Asunto(s)
Terapia Genética/métodos , Inmunoterapia Adoptiva/métodos , Melanoma/virología , Linfocitos T/trasplante , Proteínas Virales/inmunología , Animales , Ingeniería Genética/métodos , Humanos , Inmunohistoquímica , Melanoma/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recent advances in Genomics and Proteomics have necessitated a constant supply of DNA derived from specific genotypes. Lymphoblastoid cell lines (LCLs) are of great practical value as an unlimited source of stable genomic DNA and viable cells, which can be used to perform a variety of biochemical and molecular studies. LCLs are typically generated by infection of primary lymphocytes with Epstein-Barr virus (EBV). In this unit, we describe simple procedures for production of EBV, isolation of lymphocytes, EBV infection of lymphocytes, and isolation of the resulting LCLs.