Abstract

T cell therapy has proven effective to prevent and treat viral infections and immunogenic tumors. Application of this strategy to less immunogenic tumors that employ tumor evasion mechanisms may require genetic modification of the infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves have tempered enthusiasm. A safe and efficient means of destroying aberrant cells in vivo would ameliorate these concerns. The xenogeneic Herpes Simplex Virus I-derived thymidine kinase gene has been used as a suicide strategy for this purpose, but its immunogenicity results in immune elimination of the infused T cells and precludes administration of anti-viral drug ganciclovir for other indications. We adapted an alternative strategy using inducible human death molecules. Human caspase 9 was fused to a modified FK506 binding protein that allows for conditional dimerization using a chemical inducer of dimerization (CID). Administration of this small molecule pharmaceutical that has proven safe in healthy volunteers (Iuliucci et al, J Clin Pharmacol 2001, 41:870–879), activates the caspase molecule resulting in apoptosis of the cell. This system consists of human gene products with low potential immunogenicity. We chose caspase 9 as the death molecule since it is highly sensitive to dimerization and acts late in the apoptosis pathway, thereby reducing the risk of impairment of its function by cellular inhibitors of apoptosis. However, only low-level expression of the full-length inducible Caspase 9 (iCasp9) construct was compatible with survival of CTL. Deletion of the caspase recruitment domain (CARD) and removal of one of the two FKBPs allowed expression of iCasp9 in >70% of primary human T cells after a single transduction with a retrovirus vector. In vitro a single 10 nM dose of CID induced apoptosis in > 99% of T cells sorted for high transgene expression. A similarly high percentage of iCasp9-expressing T cells underwent apoptosis after CID administration in vivo in a mouse SCID human xenograft model. This functional activity is a log greater than previously available self-destruction systems. Importantly, even without sorting, activation of iCasp9 when co-expressed with transgenic IL-12 (a cytokine with potent anti-tumor activity) resulted in elimination of all IL-12 secreting cells. Unlike inducible Fas, iCasp9 maintained its function in MT-2 cells that over-express the anti-apoptotic molecule bclxl, demonstrating the importance of using a death molecule that acts downstream of the death inhibitors. Since iCasp9 does not impair T cell phenotype or function, these characteristics favor incorporation of inducible caspase 9 as safety feature in human T cell therapies.

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