1. ¹Division of Oncology, Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO
2. ²Xcyte Therapies, Inc., Seattle, WA

^|[ast]|MLB is an employee of Xcyte Therapies, Inc., and has equity (stock) interests in Xcyte Therapies, Inc.

Abstract

One approach to prevent graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) is to genetically modify the donor T cells with a HSV thymidine kinase (TK) suicide gene. We and others have previously demonstrated that early administration of the prodrug ganciclovir (GCV) protects mice from lethal GVHD after BMT with allogeneic na|[iuml]|ve (uncultured) TK-expressing transgenic (Tg) T cells. However, the efficacy of GCV control of GVHD following BMT with ex-vivo activated, TK transduced and selected (Td) donor T cells has not been thoroughly characterized because of the significantly reduced GVHD-inducing potential of cultured cells compared to na|[iuml]|ve T cells in both preclinical models and initial clinical trials. In this study, we evaluated the GVHD-inducing potential and GCV sensitivity of Td and na|[iuml]|ve TK-expressing T cells after allogeneic BMT. Murine T cells were Td with a chimeric fusion suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full length TK (CD34-TK). High efficiency (>50%) transduction of T cells with CD34-TK retrovirus was accomplished 24 h after activation with anti-CD3/anti-CD28 coated beads and gene-modified cells were purified to >98% by CD34 immunoselection 2 days post-infection. Na|[iuml]|ve B6 CD34-TK⁺ Tg T cells were isolated the day of BMT. Purified T cells were then injected (4e6 total T cells), along with T cell depleted B6 (CD45.1⁺) BM, into lethally irradiated allogeneic (BALB/c, CD45.2⁺) recipients. Untreated animals (No GCV) that received either na|[iuml]|ve or Td T cells died of GVHD, with a median survival of 25 days and 26 days, respectively. Consistent with GVHD, these untreated mice lost >20% of their pretransplant body weight and exhibited impaired lymphoid reconstitution. To evaluate the ability of GCV to prevent this GVHD, we administered a 7 day course of GCV beginning 1, 4, or 10 days after BMT. Treatment with GCV from days 1-7 after BMT protected the allogeneic recipients from lethal GVHD, irrespective of the donor T cell source. However, animals that received Td T cells and GCV from days 1-7 had significantly decreased weight gain compared to both the BM only controls and mice that received na|[iuml]|ve T cells and GCV from days 1-7 (p < 0.01 from day 60 onwards). This sustained weight loss suggests that moderate, non-lethal, GVHD may be ongoing in these mice. We observed similar protection from lethal GVHD with significantly reduced weight gain in animals that received Td T cells and treatment with GCV from days 4-10. In contrast, lethal GVHD was evident in day 4-10 GCV treated mice that received na|[iuml]|ve T cells and in all recipients (irrespective of donor T cell source) that were treated with GCV from days 10-16 after BMT. In summary, these studies demonstrated important differences in the efficacy of GCV control of GVHD after allogeneic BMT with na|[iuml]|ve or Td CD34-TK-expressing T cells. These observations may have important implications in future clinical trials using TK-expressing T cells to control GVHD after BMT.