Because suicide systems can be designed not to evoke

Because suicide systems can be designed not to evoke cross-resistance to conventional agents, they can potentiate therapy—efficiently inducing apoptosis in transduced cells—without increasing toxicity. However, many suicide systems have drawbacks, proving less clinically effective than desired. HSV-TK, the gene encoding herpes simplex virus thymidine kinase, with ganciclovir (GCV), for example, is a well-known suicide gene system used as a safety switch for adoptive T cell therapy or cancer treatment (Ciceri et al., 2005, 2007). In fact, recent reports indicate that the HSV-TK/GCV approach effectively removes tumorigenic GSK503 among murine iPSCs (Chen et al., 2013; Lim et al., 2013). This system is nonetheless potentially limited as a safeguard system, because HSV-TK targets DNA synthesis in a cell-cycle-dependent manner and can kill only fast-dividing cells. This may leave populations of slowly dividing cells intact, with resistance to treatment. Cell killing may require many days and is usually incomplete, potentially delaying clinical benefit (Brenner et al., 2013; Hoyos et al., 2012; Leen et al., 2014). Its prodrug, GCV, may cause side effects such as renal dysfunction, liver dysfunction, and pancytopenia. Moreover, because HSV-TK is immunogenic, humoral immunity may reduce its efficacy (Berger et al., 2006; Traversari et al., 2007). On the other hand, inducible caspase-9 (iC9), encoded by a suicide gene engineered from human caspase-9 (CASP9), is not immunogenic, and can kill transduced cells in a cell-cycle-independent manner. iC9 is a fusion protein engineered by replacing the caspase recruitment domain (CARD) with a mutated FK506-binding protein (FKBP12) to allow conditional dimerization (Straathof et al., 2005b). In the presence of a chemical inducer of dimerization (CID; AP1903 or its functionally identical analog AP20187) (Clackson et al., 1998), dimerized iC9 directly activates intracytoplasmic caspase-3, bypassing activation of the mitochondrial apoptotic pathway, swiftly triggering apoptosis in transduced cells (Di Stasi et al., 2011). In a clinical study, infused iC9-transduced donor T cells underwent rapid apoptosis after CID treatment, and graft-versus-host disease (GVHD) symptoms receded dramatically without relapse of leukemia (Di Stasi et al., 2011; Zhou et al., 2014). iC9 is also a promising tool for cancer treatment, because its mesenchymal stromal cell-based delivery may effectively target non-small-cell lung cancer cells for elimination (Ando et al., 2014). Therefore, we decided to use iC9 to safeguard against problems during clinical and translational investigation of iPSC-based cell therapy. We succeeded in generating iPSCs from antigen-specific cytotoxic T lymphocytes (CTLs) in HIV1-infected patients (Nishimura et al., 2013). These T cell-derived iPSCs (T-iPSCs) were then redifferentiated into HIV1-specific CTLs. CTLs continuously exposed to viral or tumor antigens, with long-term expansion, may become exhausted (Klebanoff et al., 2006; Wherry, 2011). Whereas fully “rejuvenated” CTLs (rejCTLs) derived from T-iPSCs have higher proliferative capacity, younger memory phenotype, and longer telomeres than the original patient-derived CTLs (Nishimura et al., 2013), the actual in vivo efficacy of rejCTLs has not been tested formally. Moreover, some engineered T cell immunotherapies incurred GVHD after donor lymphocyte infusion, or “on-target, off-tumor toxicities” (Fedorov et al., 2013; Gargett and Brown, 2014) that might also be seen with iPSC-derived T cell therapy. With clinical trials in prospect, we explored the effectiveness and safety of an iC9 safeguard system using these CTLs. We here document that rejCTLs are effective against Epstein-Barr virus (EBV)-induced tumors in vivo and that these rejCTLs expressing iC9 can be eliminated by activation of this system in vivo, showing that the iC9 safeguard system both is practically useful and exhibits enhanced safety in the CTL therapy model. These results may facilitate broad application of this safeguard system in other branches of iPSC-derived regenerative medicine.