Allogeneic Suicide Gene-Modified T Cells (aSGMTC) Are Highly Cytotoxic to Non-Hematopoietic Tumor Cell Lines: Rationale for the Production of a Bank of aSGMTC for the Treatment of Solid Tumors.

Abstract 2459

Poster Board II-436

Suicide gene therapy using alloreactive T cells is an efficient immunotherapeutic strategy for hematologic malignancies (Tiberghien et al, Blood 2001). We hypothesized that solid tumors, such as hepatocellular carcinoma (HCC) could be targeted by intratumoral infusion of allogeneic T cells produced from normal donors: the tumor cells would be recognized, not as being tumoral, but as being allogeneic. The prior introduction into such T cells of a suicide gene encoding the herpes simplex virus thymidine kinase (HSV-tk) that confers sensitivity to a pro-drug, ganciclovir, would allow controlling their potentially deleterious alloreactivity toward normal patient's tissues, as we demonstrated in bone marrow-transplanted patients with >10 years follow-up (Deschamps et al, Blood 2007). Our aim is to demonstrate the feasibility of this approach and to create a bank of “ready-to-use” allogeneic gene-modified T cells (GMC) for potential future in vivo evaluation and clinical use. Normal donors' peripheral blood mononuclear cells (PBMC) activated by CD3 monoclonal antibodies and Interleukin (IL)-2 were retrovirally transduced at d3 with a vector encoding a CD34/HSV-tk fusion protein. CD34+ sorted GMC were expanded with IL-2 until d14 and assessed for cytotoxicity against HeLa cells (human epithelial adenocarcinoma) and Huh7 cells (human hepatoma) by coincubation for 1-6 days at different effector:target (E:T) ratios, then staining of residual viable adherent targets by crystal violet. Allogeneic GMC (>90% T cells with a reversed CD4:CD8 ratio (0.58+/-0.06, n=9) and a predominant CD45RA- CD27- effector/memory phenotype) exhibited a strong and ganciclovir-sensitive cytotoxicity against Huh7 cells : >80% cell lysis of Huh7 cells were usually observed at an E:T ratio = 4:1 after 24h of co-incubation. Ex vivo-expanded, but non transduced, control cells were similarly cytotoxic, indicating that the retroviral transduction did not affect the cytotoxic activity of GMC. By contrast, PBMC were weakly cytotoxic, since no lysis of Huh7 cells was detectable after 24h co-incubation and <30% lysis were observed at d6 at an E:T ratio = 40:1. Similar results were observed with HeLa cells, that were even more sensitive to GMC-mediated lysis than Huh7. Acquisition of a cytotoxic activity was dependent upon the cell culture conditions: replacing the CD3 activation by CD3/CD28 or IL-2 by IL-7 was associated with increased proliferative potential and frequencies of cells with a so-called “naïve” (CD45RA+ CD27+) and central memory (CD45RA- CD27+) phenotype, but with a decreased cytotoxic activity. In vivo studies demonstrated that GMC induce tumor cell regression when coinfused with luciderase-expressing HeLa cells. Our results suggest that alloreactive GMC may represent a novel strategy for treatment of solid tumors which deserves further evaluation in vivo.