Inducible apoptosis as a safety switch for adoptive cell therapy

Abstract

Background: Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct.

Methods: We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch.

Results: Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence.

Conclusions: The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.).

Figure 1. Generation of Transgene and Function of Activated iCasp9

In Panel A, the suicide gene iCasp9 is shown to consist of the sequence of the human FK506-binding protein, FKBP12, with an F36V mutation, connected through a series of amino acids to the gene encoding human caspase 9. FKBP12-F36V binds with high affinity to a small-molecule dimerizing agent, AP1903. Panel B shows that physiological activation of the intrinsic apoptosis pathway requires a cytoplasmic protein, apoptotic peptidase activating factor 1 (APAF1), which binds with cytochrome c to form an oligomeric apoptosome, which in turn binds and cleaves caspase 9 preproprotein, releasing an activated form of the peptidase and resulting in a caspase cascade that ends in apoptosis. In transduced cells, the administration of AP1903 leads to dimerization of iCasp9, thereby bypassing activation of the initial mitochondrial apoptotic pathway. Panel C shows the structure of the iCaspase9.2A.ΔCD19 bicistronic transgene (i.e., one with two cistrons, the loci responsible for generating a protein), comprising the iCasp9 sequence, with truncated CD19 (ΔCD19) serving as the selectable marker. The sequence cassette is then incorporated into the SFG retroviral vector. Panel D shows the manufacturing process, along with the day of each manipulation. The arrows indicate the times of adding recombinant human interleukin-2 to the cultures. LTR denotes long terminal repeat, OKT3 mouse antihuman CD3 monoclonal antibody, and SGGGS Ser-Gly-Gly-Gly-Ser.

Figure 2. Detection of iCasp9-Transduced T Cells in Peripheral Blood

Panel A shows fluorescence-activated cell sorting (FACS) analysis for iCasp9-transduced T cells (CD3+, CD3+CD19+, CD4+CD19+, CD8+CD19+, or CD3−CD56+CD19+, with the cell count given per cubic millimeter of peripheral blood) from five patients who received cellular therapy after undergoing HLA-haploidentical stem-cell transplantation for relapsed leukemia. Skin or liver graft-versus-host disease (GVHD) developed in Patients 1, 2, 4, and 5, who had high levels of engraftment. Each of these patients received a single dose of AP1903. The tables beneath the graphs show the cell counts before and after treatment with AP1903. Panel B shows the number of copies of the iCasp9 transgene per microgram of DNA obtained from peripheral-blood mononuclear cells, evaluated by means of real-time quantitative polymerase-chain-reaction (PCR) amplification, for each patient at time points corresponding to those in Panel A before and after AP1903 infusion. The tables beneath the graphs show the actual quantitative PCR values before and after treatment, revealing a diminution by a factor of 100 (2 log) in the signal from the iCasp9 transgenic cells.

Figure 3. Rapid Reversal of GVHD after Treatment with AP1903

Panel A shows the normalization of bilirubin levels in Patient 1 within 24 hours after treatment with AP1903. To convert the values for bilirubin to micromoles per liter, multiply by 17.1. Panel B shows the disappearance of rash from the left arm of Patient 2 within 24 hours after treatment.

Figure 4. Persistence of Drug Sensitivity and Antiviral Function of CD3+CD19+ T Cells after Treatment with AP1903 in Vivo

Panel A shows the presence of CD3+CD19+ cells and the iCasp9 transgene in Patients 1 and 2. Cells were counted and incubated with antibodies labeled with fluorochrome (phycoerythrin [PE] or fluorescein isothiocyanate [FITC]) targeting CD19+ cells (PE) and CD3+ cells (FITC). CD3+CD19+ T cells remained within the CD3+ population in the peripheral blood after treatment with AP1903 for 5 months in Patient 1 and for 9 months in Patient 2. These CD3+CD19+ cells retained sensitivity to AP1903 in vitro, as assessed both by the reduction in the number of CD3+CD19+ cells on fluorescence-activated cell sorting (FACS) analysis (as measured per microliter of peripheral blood) (Panel A) and by quantitative polymerase-chain-reaction (PCR) analysis of the iCasp9 gene before and after exposure to the dimerizing drug (Panel B). As shown in Panel A, values for Patient 1 were 2290 lymphocytes per cubic millimeter with a CD3+ count of 41%, and values for Patient 2 were 1380 lymphocytes per cubic millimeter with a CD3+ count of 35%. As indicated on the y axis in Panel B, 3 is the minimum number of copies of iCasp9 that can be detected on quantitative PCR. CD3+CD19+ gene-modified T cells that were collected from Patient 2 (Panel C) and Patient 5 (Panel D) were responsive to viral peptide mixtures and survivin peptide mixture (in Patient 2) before the administration of AP1903, as shown by the presence of interferon-γ–positive CD3+CD19+ T cells in the peptide-stimulated cultures. The assessment of the recovering CD3+CD19+ population at 14 days in Patient 2 and at 14 and 30 days in Patient 5 after AP1903 infusion to treat GVHD showed the persistence of these antigen-specific cells in the absence of recurrent GVHD. ADV denotes adenovirus, BZLF1 BamHI Z leftward reading frame, EBNA Epstein–Barr nuclear antigen, IE1 immediate early protein, LMP latent membrane protein, perCP peridinin chlorophyll protein, and pp65 phosphoprotein 65.

Figure 5. Polyclonality of Recovering T Cells in Patient 1

The analysis of the repertoire of T-cell receptor variable beta chain (TCR Vβ) in the CD3+CD19+ selected population, which recovered 6 months after the administration of the dimerizing drug in Patient 1, shows a polyclonal pattern of expression, as measured by reactivity to monoclonal antibodies directed against TCR Vβ chain families and measured on FACS analysis (Panel A), as well as by multiplex polymerase-chain-reaction–based spectratyping to analyze the number of distinct TCR Vβ transcripts present (Panel B). PB denotes peripheral blood.