Work

CMV-Specific HER2-Redirected T Cells For The Adoptive Immunotherapy Of Glioblastoma

Year: 2010

Type: article

Abstract: We demonstrated that T cells from GBM patients engineered to express a HER2-specific chimeric antigen receptor (CAR) kill autologous HER2+GBM cells ex vivo, including the CD133+GBM stem cell population. In vivo, the adoptive transfer of HER2-specific T cells resulted in regression of established GBM in the brain of mice, yet tumors recurred in a number of treated animals. This limitation in T-cell efficacy is most likely due to a) limited T-cell persistence in vivo and/or b) tumor antigen loss variants in treated tumors. Since the majority of GBMs are positive for CMVpp65 we expressed HER2.CARs in CMV-specific cytotoxic T cells (CTLs) to a) enhance their persistence in vivo following native T-cell receptor (αβTCR) engagement with CMV-antigens and b) improve their antitumor activity by simultaneously targeting CMV and HER2. To study the feasibility of generating CMV CTLs from GBM patients, we determined the precursor frequency of CMV-specific T cells in 12 patients using IFN-g ELIspot assays. To generate CMV CTL ex vivo, peripheral blood mononuclear cells were transduced with an adenovirus vector; encoding the immunodominant CMV-pp65 antigen (Ad5f35pp65) then stimulated with autologous EBV transformed Ad5f35pp65-transduced irradiated lymphoblastic cell lines (EBV-LCL). CTLs were then transduced with a retroviral vector encoding a HER2.CAR (FRP5.CD28.ζ). We detected the expression of the CAR transgene using flowcytometry. To concomitantly identify the CMV-specific T cell population we used pentamer analysis for CMVpp65. We performed 51Cr-release assays to characterize the functionality of HER2.CAR CMV.CTLs ex vivo. CMV-specific T cells were readily detectable in the blood of GBM patients as judged by IFN-g secretion after stimulation with CMVpp65 PepMixes™. We were able to generate CTLs that are specific for CMV through their endogenous ab TCR and specific for HER2 through the CAR. Approximately 50% of CMVpp65-specific CTLs expressed HER2.CARs on the cell surface. CMV-specific CTLs expressing the HER2-specific CAR recognized and killed the HER2-positive GBM cell line (U373) as well as autologous OKT3-blasted T cells pulsed with CMVpp65 PepMixes™ in a standard 4 hour 51Cr-release assays. Neither the HER2 –ve cell lines nor autologous OKT3-blasted T cells were killed. Our data demonstrates the feasibility of generating CAR expressing CMVpp65-specific CTLs that are functional against HER2 and CMV expressed in GBM.

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.bbmt.2009.12.107