In Vivo comparison of 3 Suicide Gene-Prodrug Combinations in a Mouse Graft-Versus-Host-Disease Model

Abstract 3121

Cellular therapies through allogeneic hematopoietic stem cell transplants (allo-HSCT) and infusion of genetically modified T cells targeted against tumor associated antigens hold great promise for cancer therapy. However, significant risk of graft-versus-host-disease (GvHD) with allo-HSCT and potential toxicities from the transgene products in the case of genetically modified T cells have generated considerable interest in developing safeguards in the form of suicide genes to allow for the efficient in vivo abrogation of infused T cells in case of unanticipated adverse events. We have previously reported the comparable in vitro function of 3 different suicide gene-prodrug combinations (Park et al. ASH 2010 abstract #3771). The three suicide genes examined included herpes simplex virus thymidine kinase (HSV-TK SR39) with the prodrug ganciclovir, inducible caspase 9 (iCasp9) with the chemical inducer of dimerization (CID), and a novel suicide gene nitroreductase (NTR) derived from E.coli combined with the prodrug metronidazole. Here, we report the comparison of in vivo efficacy of these 3 suicide gene-prodrug combinations using our previously published mouse GvHD model (Santos et al. Nat Med 2009). SCID-Beige mice were injected i.v. with 1×10⁶ FACS-sorted major histocompatibility complex mismatched C57/BL6 T cells transduced to express each suicide gene (HSV-TK SR39, iCasp9 or NTR) cloned in frame with external Gaussia Luciferase (extGLuc), separated by the 2A peptide in an SFG-based vector. Seven days after intravenous injection of modified T cells, SCID-Beige mice developed GvHD as evidenced by in vivo expansion of the transferred T cells detected by bioluminescent imaging (BLI) after a bolus injection of coelenterazine. Subsequently, the mice injected with the HSV-TK SR39-transduced T cells were treated intraperitoneally (i.p.) twice daily for 5 days with ganciclovir at 25mg/kg; the mice with the NTR-transduced T cells were treated i.p. three times daily for 5 days with metronidazole at 200μg; the mice with the iCasp9-transduced T cells were treated i.p. daily for 3 days with CID at 50μg; and the mice with the HSV-TK SR39-transduced T cells treated i.p. twice daily for 5 days with normal saline served as a control group. All treated mice were imaged by BLI on days 3, 7, 11, 18, 25, 32, and weekly thereafter for a total of 3 months following the first dose of each prodrug. All of the control mice treated with normal saline developed rapidly progressing GvHD both clinically and by BLI, meeting the criteria for euthanasia by day 18. In contrast, the administration of prodrugs successfully eradicated the modified T cells expressing respective suicide genes by day 3 and no evidence of GvHD was detected. However, a long-term follow-up of these mice revealed the re-expansion of the transferred T cells or relapse of GvHD in all mice in the NTR group (by day 39) and in 2 out of 3 mice in the HSV-TK SR39 group (by day 53), but none in the mice in the iCasp9 group. The relapsed mice in the NTR and HSV-TK SR groups were treated again with the prodrugs, but none responded to the re-treatment. At the end of the 3 month follow-up, all mice in the NTR group and 2 out of 3 mice from the HSV-TK SR39 group died from progressive GvHD, while all of the mice in the iCasp9 group survived with no evidence of GvHD. In conclusion, our results suggest that the long-term safety and efficacy data of iCasp9, together with its lack of immunogenicity and the absence of reported side effects of CID (Di Stasi et al. ASH 2010 abstract #559), might be advantageous as a promising suicide gene-based strategy in ensuring the rapid and sustained eradication of the adoptively transferred T cells in case of unanticipated severe toxicities of cellular therapies. Based on this in vivo data, we are planning to incorporate the iCasp9 suicide gene in our future clinical trials with T cells genetically modified to express a chimeric antigen receptor targeted against a tumor specific antigen.