iCaspase 9 Suicide Gene System

Abstract

Although cellular therapies may be effective in cancer treatment, their potential for expansion, damage of normal organs, and malignant transformation is a source of concern. The ability to conditionally eliminate aberrant cells in vivo would ameliorate these concerns and broaden the application of cellular therapy. We devised an inducible T-cell safety switch that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This system is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization using a small-molecule drug. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iC9) becomes activated and leads to the rapid apoptosis of cells expressing this construct. We have demonstrated the clinical feasibility and efficacy of this approach after haploidentical hematopoietic stem cell transplant (haplo-HSCT). A single dose of a small-molecule drug (AP1903) eliminated more than 90 % of the modified T cells within 30 min after administration and symptoms resolved without recurrence. This system has the potential to broaden the clinical applications of cellular therapy.

Trial registration: ClinicalTrials.gov NCT00710892.

Fig. 1

The structure of the iCaspase9.2A.ΔCD19 transgene. The transgene consists of a suicide gene, inducible caspase 9 (iCasp9), and a selectable marker, truncated CD19 (.ΔCD19), linked by a 2A-like sequence, which encodes a cleavable peptide. iCasp9 consists of a drug-binding domain (FKBP12-F36V) connected via a short linker (SGGGS) to human caspase 9. The sequence cassette is then incorporated into the SFG retroviral vector

Fig. 2

Overview of the manufacturing process. Selective allodepletion was performed by co-culturing donor PBMC with recipient EBV-LCL to activate alloreactive cells: activated cells expressed CD25 and were subsequently eliminated by anti-CD25 immunotoxin. The allodepleted cells were activated by OKT3 and transduced with the retroviral vector 48 h later. Immunomagnetic selection was performed on day 4 of transduction; the positive fraction could be expanded for a further 4 days and cryopreserved. The arrows indicate the times of adding recombinant human interleukin-2 to the cultures

Fig. 3

Detection of allodepleted T cells expressing iC9 by CD19 after transduction and selection. Suicide gene-modified cells could be enriched to high purity by CD19 immunomagnetic selection. CD19 immuno-magnetic selection was performed on day 4 post-transduction using CliniMACS Plus automated selection device. Shown here are FACS analyses performed 2 days after immunomagnetic selection for non-transduced, transduced, and CD19-selected T cells. Region analyzed on viable cells

Fig. 4

Gene-modified allodepleted T cells were rapidly and efficiently eliminated by CID. The day after immu-nomagnetic selection, cells were treated with 10 nM dimerizer (AP20187). FACS analysis for annexin V and 7-AAD was performed at 22–30 h. The percentages of viable cells are indicated in the plots. AP20187 resulted in ≥90 % killing of CD19-selected cells but had no effect on non-transduced controls. (a) Non-transduced cells without CID; (b ) non-transduced cells treated with CID; (c ) CD19-selected cells without CID; and (d) CD19-selected cells treated with CID