title: Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application.
abstract: BACKGROUND
  Donor lymphocyte infusion is an effective form of adoptive immunotherapy for hematologic malignancies after allogeneic stem cell transplantation. Graft-versus-host disease, however, often develops due to recognition of ubiquitously-expressed minor histocompatibility antigens. Transfer of T-cell receptors recognizing hematopoiesis-restricted minor histocompatibility antigens to virus-specific T cells may be a powerful anti-tumor therapy with a low risk of graft-versus-host disease. The purpose of this study was to develop an optimal T-cell receptors-encoding multi-cistronic retroviral vector and an efficient method for generating T-cell receptors-engineered virus-specific T cells.
  
  
  METHODS
  Retroviral vectors encoding the T-cell receptors for the hematopoiesis-restricted minor histocompatibility antigen HA-2 with and without selection markers were compared for T-cell receptors surface expression and HA-2-specific lysis. In addition, two different methods, i.e. peptide stimulation of CD8(+) cells and Pro5 MHC pentamer-based isolation of antigen-specific T cells, were investigated for their efficiency to generate T-cell receptors-transduced virus-specific T cells.
  
  
  RESULTS
  Bi-cistronic vectors without selection markers most efficiently mediated T-cell receptors surface expression and HA-2-specific lysis. Furthermore, both methods were useful for generating gene-modified cells, but the purity of virus-specific T cells was higher after pentamer isolation. Finally, the capacity of gene-modified cells to express the transgenic T-cell receptors at the cell surface markedly differed between virus-specific T cells and was correlated with lysis of relevant target cells.
  
  
  CONCLUSIONS
  Our data support T-cell receptors gene transfer to pentamer-isolated virus-specific T cells using bi-cistronic retroviral vectors and illustrate the relevance of selection of gene-modified T cells with appropriate transgenic T-cell receptors surface expression for clinical gene therapy.
authors.affiliation: Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands. m.griffioen@lumc.nl
authors.forename: Marieke
authors.initials: M
authors.lastname: Griffioen
authors.forename: H M Esther
authors.initials: HM
authors.lastname: van Egmond
authors.forename: Helen
authors.initials: H
authors.lastname: Barnby-Porritt
authors.forename: Menno A W G
authors.initials: MA
authors.lastname: van der Hoorn
authors.forename: Renate S
authors.initials: RS
authors.lastname: Hagedoorn
authors.forename: Michel G D
authors.initials: MG
authors.lastname: Kester
authors.forename: Nikolai
authors.initials: N
authors.lastname: Schwabe
authors.forename: Roel
authors.initials: R
authors.lastname: Willemze
authors.forename: J H Frederik
authors.initials: JH
authors.lastname: Falkenburg
authors.forename: Mirjam H M
authors.initials: MH
authors.lastname: Heemskerk