Retroviral Gene Transfer of T Cell Receptors (TCR) Specific for Minor Histocompatibility Antigens to Virus-Specific T Cells as Cellular Immunotherapy of Patients with Relapsed Hematological Malignancies after Allogeneic Stem Cell Transplantation.
Authors: Griffioen Marieke , van Egmond H.M. Esther , van der Hoorn Menno A.W.G. , Hagedoorn Renate S. , Kester Michel , Falkenburg J.H. Frederik , Heemskerk Mirjam H.M. 
Published: October 2019

Abstract
Patients with hematological malignancies can be successfully treated by T cell-depleted allogeneic stem cell transplantation (alloSCT) followed by donor lymphocyte infusion (DLI). Failure of some relapsed hematological malignancies to respond to DLI is probably due to immune tolerance induced by regulatory and/or anergic T cells. We previously showed that functional T cells with redirected anti-leukemic reactivity can be generated by transfer of TCRs specific for minor histocompatibility antigens (mHags) to total peripheral blood mononuclear cells (PBMC) as well as CMV-specific T cells. By introducing TCRs into CMV or EBV specific T cells, T cells with proper memory/effector phenotypes are targeted, and due to virus persistence these T cells may show prolonged survival in vivo. The purpose of this study is to develop an efficient method for the isolation, retroviral transduction and expansion of TCR-transduced CMV- and EBV-specific T cells for cellular immunotherapy of patients with relapsed hematological malignancies after alloSCT.
For clinical application, construction of single retroviral vectors coding for the α as well as β chains of mHag-specific TCRs is required. We used the MP71 retroviral vector for TCR gene transfer, since this vector contains Myeloproliferative Sarcoma Virus LTR sequences and a Mouse Embryonic Stem Cell Virus leader, which has been optimized for use in the clinic. The MP71 vector also contained a Woodchuck Hepatitis Response Element (WPRE). The WPRE, which is used as an element enhancing transgene expression at the post-transcriptional level, has recently been described to encode 60 amino acids of a protein with potential oncogenic activity. Therefore, we reconstructed the MP71 vector by introduction of a multiple cloning site (MCS) and, for safety issues, deleted the WPRE. The TCR α and β genes specific for the hematopoietic-restricted mHag HA-2 were linked by a 50-bp sequence encoding a “self-cleaving” 2A-like peptide and introduced into the MCS of the MP71 vector. Linkage of the TCR α and β genes by the 2A-like sequence allowed additional linkage of the low affinity nerve growth factor receptor (LNGFR) or human CD20 selection marker genes by an IRES sequence. The advantage of the human CD20 gene is that it can also function as suicide gene, allowing elimination of transduced cells in vivo if undesired side effects occur. Introduction of the single MP71 retroviral vector coding for the HA-2 TCR α and β chains as well as LNGFR into a TCR α- and β-deficient Jurkat T cell line led to high levels of TCR surface expression correlating with LNGFR marker gene expression. These data indicate proper cleavage and assembly of the transduced TCR α and β chains. Moreover, removal of the WPRE did not affect the surface expression level of the transduced TCR. Furthermore, CMV- and EBV-specific T cells were isolated from human individuals by the IFN-γ capture assay and subsequently transduced with a single retroviral vector coding for the HA-2-TCR α and β chains as well as LNGFR. CMV- and EBV-specific T cells from different human individuals could be successfully isolated to 60–90% purity and the TCR-transduced CMV- and EBV-specific T cells were shown to be fully functional, recognizing the viral peptides as well as the endogenously-processed HA-2 mHag.