Production and First-in-Man Use of T Cells Engineered to Express a HSVTK-CD34 Sort-Suicide Gene

et al. (2013) Production and First-in-Man Use of T Cells Engineered to Express a HSVTK-CD34 Sort- Suicide Gene. PLoS ONE 8(10): e77106. doi:10.1371/journal.pone.0077106 Production and First-in-Man Use of T Cells Engineered to Express a HSVTK-CD34 Sort-Suicide Gene Hong Zhan 0 Kimberly Gilmour 0 Lucas Chan 0 Farzin Farzaneh 0 Anne Marie McNicol 0 Jin-Hua Xu 0 Stuart Adams 0 Boris Fehse 0 Paul Veys 0 Adrian Thrasher 0 Hubert Gaspar 0 Waseem Qasim 0 Evren Alici, Karolinska Institutet, Sweden 0 1 Molecular Immunology Unit, Institute of Child Health (ICH), University College London (UCL) , London , United Kingdom , 2 Department of Haematological Medicine, The Rayne institute, Kings College London (KCL) , London , United Kingdom , 3 Bone Marrow Transplant Unit, University of Hamburg , Hamburg , Germany Suicide gene modified donor T cells can improve immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), but can be eliminated in the event of graft versus host disease (GVHD) through the administration of prodrug. Here we report the production and first-in-man use of mismatched donor T cells modified with a gamma-retroviral vector expressing a herpes simplex thymidine kinase (HSVTK):truncated CD34 (tCD34) suicide gene/magnetic selection marker protein. A stable packaging cell line was established to produce clinical grade vector pseudotyped with the Gibbon Ape Leukaemia Virus (GALV). T cells were transduced in a closed bag system following activation with anti-CD3/CD28 beads, and enriched on the basis of CD34 expression. Engineered cells were administered in two escalating doses to three children receiving T-depleted, CD34 stem cell selected, mismatched allogeneic grafts. All patients had pre-existing viral infections and received chemotherapy conditioning without serotherapy. In all three subjects cell therapy was tolerated without acute toxicity or the development of acute GVHD. Circulating gene modified T cells were detectable by flow cytometry and by molecular tracking in all three subjects. There was resolution of virus infections, concordant with detectable antigen-specific T cell responses and gene modified cells persisted for over 12 months. These findings highlight the suitability of tCD34 as a GMP compliant selection marker and demonstrate the feasibility, safety and immunological potential of HSVTK-tCD34 suicide gene modified donor T cells. Trial Registration: ClinicalTrials.gov NCT01204502 ,http://clinicaltrials.gov/ct2/archive/NCT01204502. - Funding: Funders: 1. Moulton Trust; trial costs. 2. Department of Health,UK; vector costs. 3. Leukaemia and Lymphoma Research supported WQ through lecturership award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Allogeneic haematopoietic stem cell transplants (SCT) from mismatched unrelated donors or haploidentical family donors are high risk procedures, requiring rigorous T cell depletion to mitigate against graft versus host disease (GVHD) [1]. Strategies to remove donor T cells include in-vivo antibody based depletion through the inclusion of serotherapy (for example Alemtuzumab, Antithymocyte Globulin, or OKT3) in the conditioning regimen or by ex-vivo depletion of T cells by magnetic bead based graft manipulation (for example, through enrichment of stem cells expressing CD34, or by depletion of T cells expressing CD3 or abT cell receptors). Whilst stringently T-depleted grafts are less likely to cause GVHD, they also have reduced anti-viral properties and often lose graft versus leukaemia effects [2]. One approach designed to allow the infusion of mismatched donor T cells involves the stable introduction of a suicide gene to allow elimination of cells in the event of GVHD though the activation of specific prodrugs. The most extensively studied system uses gene modification with Herpes simplex thymdine kinase (HSVTK) which can activate Ganciclovir to induce cell death, and has now been tested in a number of clinical trials [310]. More recently a fusion gene encoding an inducible human caspase-9 apoptosis gene and modified human FK-binding protein has also been evaluated in pilot studies [11]. One prerequisite for this form of gene therapy, is the need to ensure that a very high proportion of infused cells encode the suicide gene, and thus all clinical trials to date have included linked selection marker genes. Bonini et al employed Neomycin based selection, subsequently switching to magnetic bead-antibody based selection of co-expressed truncated low affinity nerve growth factor receptor (DLNGFR) [3]. Alternatives include a truncated CD19 (DCD19) selection marker, used to enrich T cells expressing human caspase-9/FK-binding protein based suicide gene system [11]. Here we describe the first clinical data using a HSVTK suicide gene fused to a truncated splice variant of human CD34 (tCD34) [12]. Selection based on CD34 expression has an important advantage as it can be combined with Miltenyi CliniMacs reagents which are already widely used for CD34 stem cell selection. We, and others, have previously described pre-clinical variants of this system delivered by gamma-retroviral and HIV lentiviral vectors to human T cells [1215]. Here we describe gamma-retroviral gene modification, enrichment and clinical use of human T cells expressing a modified HSVTK-CD34 sort-suicide fusion gene in three subjects following T cell depleted allogeneic SCT. This small study 2. T cell transduction & selection Allogeneic donors had completed prior virological screening for peripheral blood SCT. Donor lymphocytes were subsequently obtained from ficolled whole blood (P1) or non-mobilised leukapheresis collection (P2, 3). Cells were re-suspended in gas Materials, Methods and Subject Details All subjects received treatment at Great Ormond Street Hospital, London under ethics approval from the UK Gene therapy advisory committee (GTAC) a national body overseeing ethical conduct of gene therapy studies. The study was regulated and monitored by the MHRA, UK. Parents provided written informed consent on behalf of all children. The protocol (see Protocol S1) for this study and supporting CONSORT checklist (see Checklist S1) are available as supporting information. 1. Plasmids and cell lines A gamma retroviral vector plasmid, encoding long terminal repeats from Myeloproliferative sarcoma virus (MPSV) and the leader 71 sequence from MESV and coding for a suicide/sort fusion gene comprising splice site corrected HSVTK fused to a truncated splice variant of human CD34 (Figure 1a) has been previously described [12] and was produced by Geneart (Germany) along with two independent accessory plasmids encoding ecotropic env and gag/pol, plasmids. Transiently produced ecotropic retroviral supernatant was produced in 293T cells (from a qualified master cell bank) (...truncated)