Abstract
Abstract 559
Adverse effects from cellular therapies may be prolonged and progressively worsen as cells expand and persist long-term, unlike most small molecule toxicities, which diminish following drug withdrawal. “Suicide genes” can be introduced into administered cells and activated – generally by a small molecule pro-drug - in the event of adverse effects. Although one such approach using a transferred Herpes Simplex Virus thymidine kinase (HSV-tk) gene may provide effective control, its mechanism of action requires interference with DNA synthesis. As a consequence, cell killing may be protracted over several days, with an even longer delay in clinical benefit. Moreover, an anti-viral therapeutic pro-drug (such as ganciclovir) is required for cell elimination, removing this class of agents from the therapeutic repertoire. Finally, HSV-tk is virus-derived and hence potentially immunogenic. We now report the clinical evaluation of a human suicide gene, inducible Caspase-9 (iCasp9), which is designed to interface with the physiological apoptotic pathway. To generate the iCasp9 molecule we modified human caspase 9 to dimerize and activate upon exposure to a synthetic, otherwise bioinert, small molecule dimerizing agent, AP1903.
We infused iCasp9-expressing T lymphocytes, in an effort to enhance immune recovery and reduce infection/relapse following transplantation of HLA-haploidentical hemopoietic (CD34+) stem cells as treatment for high-risk, relapsed leukemia. The infused T cells were first depleted of alloreactive progenitor cells after stimulation with recipient irradiated, EBV-transformed lymphoblastoid cells (40:1) for 72 hrs, and subsequent exposure to a CD25-directed immunotoxin (RFT5-dgA). The allodepleted cells were then transduced with a retroviral vector encoding the iCasp9 suicide gene and a selection marker (DCD19), which allowed enrichment to >95% purity.
Four subjects received 1–3 x106 gene-modified T cells/kg. Forced expression of a transgenic caspase-derived molecule did not preclude in vivo survival or expansion of infused T cells, which became detectable by flow cytometry (CD3+DCD19+cells) and by Q-PCR (for iCasp9) within 7 days of infusion. By day 14 these cells expanded to a median cell number/μ l of 175 (range 2–348) (dose level 1) and 49 (range 4–93) (dose level 2). The iCasp9+ T cells contained both CD4+ and CD8+ subsets, were viral-reactive (CMV, EBV, and ADV) and polyclonal, and have now persisted beyond 240 days. Three of the 4 subjects subsequently developed grade I/II acute GvHD of skin, one of whom also had a rising bilirubin, attributed to liver GvHD. As per protocol, these subjects received a single dose of dimerizer agent. Within 30 minutes of completing AP1903 administration, we observed a circa 90% reduction of transgenic T cells, as assessed by flow cytometry for CD3+CD19+ T cells and by Q-PCR amplification for iCasp9. This effect was followed within 36 hrs by resolution of all aGvHD, showing that the iCasp9 transgene can be functional in vivo and can rapidly deplete sufficient T cells to control GvHD. Of note, the residual allodepleted T cells were no longer associated with GvHD but were still able to re-expand within 21 days (median CD3+CD19+ cells/μ l: 77 (range 38–87)), and contained subpopulations that preserved reactivity to viruses (CMV) and fungi (Aspergillus fumigatus), as assessed by IFN-γ production.
In conclusion, administration of small numbers of iCasp9+ allodepleted T cells may produce CD4+ and CD8+ T-cell reconstitution after haplo-identical, CD34+ SCT, while administration of a small molecule dimerizer agent has rapidly ablated residual allo-reactive T cells and abrogated early GvHD, whilst preserving anti-viral specificity.
Supported by NIH-NHLBI U54HL081007
Disclosures:
No relevant conflicts of interest to declare.
Flow Cytometry and FTIR Spectroscopy for Detection of Early Apoptosis in Human T Cells