In addition, they showed similar cytotoxic reactivity against disease peptide-pulsed T2 cells as non-transduced virus-specific T cells

In addition, they showed similar cytotoxic reactivity against disease peptide-pulsed T2 cells as non-transduced virus-specific T cells. Open in a separate window Figure 4. HA-1-TCR tranduced, virus-specific T cells demonstrate dose-dependent HA-1 specific reactivity and recognize HA-1-positive main leukemic cells. We have recently offered the results of our phase I clinical study in which the toxicity and the potential anti-leukemic effect of treatment with HA-1-specific cytotoxic T lymphocyte lines was examined in three individuals having a leukemic relapse following allogeneic SCT.14 The administration of HA-1-specific T-cell lines was demonstrated to be safe without induction of GvHD. However, HA-1-specific T-cell lines lacked persistence and anti-leukemic reactivity. This lack of persistence and anti-leukemic reactivity may be explained from the very long culture period of at least 4 weeks. TCR gene transfer is an attractive strategy to improve T cells with well-defined specificities in a short time period. Recently, the effectiveness of TCR transfer was shown in individuals with melanoma or synovial cell sarcoma who have been treated with TCR-modified autologous T cells.15C17 To engineer T cells that exert selective GvL without GvHD, we choose to transfer the HA-1-TCR into virus-specific T cells instead of polyclonal T cells. It has been explained that both cytomegalovirus (CMV)-specific18C23 and Epstein-Barr disease (EBV)-specific24C29 donor T cells can be securely reinfused into immunodeficient individuals at risk of developing CMV disease, EBV reactivation or EBV-positive B-cell lymphomas, respectively. This adoptive transfer was shown not only to be effective in avoiding or treating the viral diseases but also to be safe without inducing GvHD. In addition, long-term persistence of Rabbit Polyclonal to CKLF4 the virus-specific donor T cells was shown.26 We hypothesize Naspm trihydrochloride that activation of the endogenous TCR by viral antigens can result in both increased numbers of TCR-modified T cells, as well as with increased introduced TCR expression, as T-cell activation is followed by increased activation of the retroviral promotor.30C32 Previously, we demonstrated that we could reprogram virus-specific T cells into anti-leukemic effector T cells using TCR gene transfer without loss of their initial anti-virus specificity.33,34 Another possible advantage of the use of virus-specific T cells is the exclusion of regulatory T cells from your pool of TCR-modified lymphocytes that can possibly disturb the immune reaction. Since virus-specific T-cell populations consist of a restricted TCR repertoire,35,36 the number of different combined TCR dimers created will become limited and from data this appears a viable strategy to prevent neoreactivity37 caused by combined TCR dimers.37,38 Furthermore, we have modified the HA-1-TCR both to improve cell surface expression of the HA-1-TCR, and to diminish mixed TCR dimer expression with unknown and potentially unwanted reactivity.38,39 For the clinical study, we will selectively isolate permissive virus-specific T cells that highly communicate HA-1-TCR after gene transfer (Table 1).39,40 Table 1. List of different peptide-HLA complexes utilized for Naspm trihydrochloride FACS analysis and MACS-isolation. Open in a separate window Recently, Streptamers were used to selectively isolate CMV-specific T cells. 41 CMV-specific T cells were transferred directly after Streptamer-based isolation into individuals with CMV reactivation without toxicity, and individuals were able to manage CMV disease thereafter.41 Here, we describe a Good Manufacturing Practice (GMP) process to rapidly generate dual-specific, donor virus-specific T cells with high avidity anti-leukemic reactivity. The process of Streptamer-based isolation of genuine populations of virus-specific T cells and transduction with GMP-grade retroviral supernatant encoding the HA-1-TCR Naspm trihydrochloride has been validated with four large-scale test methods in the cleanroom. All HA-1-TCR-transduced, virus-specific T-cell products met the criteria for in process screening and quality control screening, and were highly reactive against HA-1-positive leukemic cells. Methods Selection and isolation of virus-specific T cells This study was authorized by the Leiden University or college Medical Center institutional review table and written educated consent was acquired according to the Declaration of Helsinki. From donor leukocytes from a leukapheresis product or total peripheral blood mononuclear cells either one or two virus-specific T-cell populations were isolated using Streptamers (Table 1) (Stage Therapeutics, G?tingen, Germany) according to the manufacturers instructions. Streptamer-incubated donor leukocytes were purified using autoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturers protocol, Naspm trihydrochloride or in the case of the test-runs using a CliniMACS instrument (Miltenyi Biotec) with the CD34 selection 1 system. Streptamers were dissociated from your eluted cells with 1 mM D-biotin. Naspm trihydrochloride Eluted cells purified by either auto-MACS or CliniMACS were cultured with irradiated, non-selected, autologous peripheral blood mononuclear cells (percentage 1:5) in T-cell medium consisting of IMDM supplemented with 10% ABOS, 100 IU/mL interleukin-2 (Chiron, Amsterdam, the Netherlands), and 10 ng/mL interleukin-15 (Peprotech, Rocky Hill, NJ, USA). Anti-CD3/CD28 beads (percentage 5:1, Dynabeads, Invitrogen) were added in some of the experiments. Transduction of the virus-specific T cells Some of the virus-specific T cells were transduced 2C3 days after MACS-isolation with vectors comprising only a NGF-R marker gene, or with GMP-grade retroviral supernatant encoding the HA-1-TCR (EUFETS GmbH, Idar Oberstein, Germany), as.