Abstract
Suicide gene therapy (SGT) is a powerful approach to exploit anti-host reactivity following allogeneic hemopoietic cell transplantation (allo-HCT) for a full graft-versus-leukemia (GvL) effect, while controlling graft-versus-host disease (GvHD). This is accomplished through genetic modification of donor lymphocytes with a suicide gene. The herpes simplex thymidine kinase (TK) suicide gene converts at a cellular level the pro-drug ganciclovir (GCV) into tri-phosphate toxic derivatives, thence conferring selective sensitivity. Clinical studies as well as animal models have substantiated the concept that a time-wise GCV administration is able to actually separate the therapeutic GvL effect from life-threatening GvHD. Genetic modification of lymphocytes with TK is currently pursued through retroviral vectors (RV). In vitro RV genetic modification requires proliferation, which is easily accomplished by polyclonal stimulation. Polyclonal stimulation with anti-CD3 antibodies (aCD3) has been show to reduce anti-host reactivity of gene-modified lymphocytes. This possibly limits the impact of the suicide gene strategy in allo-HCT. In this study we tackled the rules governing anti-host reactivity of TK+ human lymphocytes. We found that TK+ lymphocytes generated with aCD3 are mainly CD45RA−CCR7− effector memory (EM) cells (84,6±6,6%). Upon re-stimulation they produce interferon-γ, perforin B and granzyme A, but fail to up-regulate CD40L. EM TK+ lymphocytes have a mixed phenotype in regard to CD28/CD27 co-expression and displayed a limited ability to engraft and cause GvHD in a xenogeneic model using conditioned NOD/scid mice (take: 11%). In sharp contrast, TK+ lymphocytes generated with novel protocols taking advantage of anti-CD3 and anti-CD28 antibodies conjugated to para-magnetic cell-sized beads are enriched for CD45RA−CCR7+ central memory (CM) cells (65,3±6,2%) that are able to produce IL-2 and to strongly up-regulate CD40L. CM TK+ lymphocytes are homogenously CD28+CD27+ (91,1,3±2,5%). When infused in conditioned NOD/scid mice CM TK+ lymphocytes persistently engrafted and caused lethal GvHD in a significant fraction of mice (take: 55%, P=0,0017 vs EM TK+ cells). GCV administration to diseased animals resulted in the elimination of TK+ cells in blood and in target organs. Treated animals were rescued with survival up to 120 days (P=0,009 vs saline-treated mice). These results demonstrate that CM TK+ lymphocytes retain significant anti-host reactivity and provide a clue to their in vitro generation. CM TK+ lymphocytes are promising candidates for safe and effective allo-HCT for the cure of hematological malignancies.
Suicide gene therapy for human T cell mediated graft versus host disease in a murine xenograft model