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Blood, 15 February 2004, Vol. 103, No. 4, pp. 1261-1269. Prepublished online as a Blood First Edition Paper on October 16, 2003; DOI 10.1182/blood-2003-08-2908.
GENE THERAPY Pharmacologically regulated Fas-mediated death of adoptively transferred T cells in a nonhuman primate modelFrom the Clinical Cooperative Group Hemopoietic Cell Transplantation, and the Institute of Molecular Immunology, GSF-National Research Center for Environment and Health, Department of Medicine, Klinikum Grosshadern, University of Munich, Munich, Germany; Division of Hematology and Department of Medicine, University of Washington, Seattle; Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA; and ARIAD Pharmaceuticals, Cambridge, MA.
Conditional suicide genes derived from pathogens have been developed to confer drug sensitivity and enhance safety of cell therapy, but this approach is limited by immune responses to the transgene product. We examined a strategy to regulate survival of transferred cells based on induction of apoptosis through oligomerization of a modified human Fas receptor by a bivalent drug (AP1903). Three macaques (Macaca nemestrina) received autologous T cells retrovirally engineered to express a Fas suicide-construct (LV'VFas). High levels of transduced cells were present in blood following cell transfer, but LV'VFas+ cells declined rapidly after AP1903 administration. A small fraction of LV'VFas+ cells resisted elimination by AP1903, in part due to insufficient levels of transgene expression in resting T cells, because reactivation of these cells in vitro enhanced sensitivity to AP1903. An immune response to the transgene product was observed, but epitope mapping indicated the response was directed to discrete components of human LV'VFas that were variant with the corresponding macaque sequences. These data demonstrate that chemically induced dimerization can be used to regulate survival of adoptively transferred T cells in vivo.
Novel therapeutic approaches based on the adoptive transfer of T cells have been developed for the treatment of infections or malignancies.1-6 Cellular immunotherapy is not without risk because transferred T cells may recognize both normal and diseased tissues.7,8 For example, the infusion of donor lymphocytes to treat recurrent malignancy in allogeneic hematopoietic stem cell (HSC) transplant recipients may have antitumor activity but can cause fatal graft-versus-host disease (GVHD) or bone marrow aplasia.9,10 In the nontransplantation setting, many candidate tumor-associated antigens for T-cell therapy are self-proteins,11,12 and serious toxicity to normal tissues may occur.8,13 Thus, it would be advantageous to have a means for controlling the fate of adoptively transferred cells in vivo. One approach to enhance the safety of cell therapy is the introduction of conditional suicide genes, such as those encoding the herpes simplex virus thymidine kinase (HSV-TK) or bacterial cytidine deaminase enzymes.14-16 Expression of the HSV-TK has been the most extensively studied approach and renders proliferating cells sensitive to the nucleoside analog ganciclovir, which is efficiently phosphorylated by the viral TK and incorporated into elongating DNA, resulting in chain termination.14,16 HSV-TK has been introduced into allogeneic donor lymphocytes that were adoptively transferred to patients after T-cell depleted HSC transplantation, and the administration of ganciclovir successfully eliminated transferred T cells and resolved the GVHD that developed in a subset of the recipients.17 However, HSV-TK is an immunogenic viral protein, and, like other foreign proteins encoded by transgene products, can result in immune-mediated rejection of transduced cells.18-22 The issue of immunogenicity, and the need to use ganciclovir to control cytomegalovirus infection after HSC transplantation may confound the broad clinical use of this approach. Genetic modification of human T cells with a human CD20 cDNA renders them susceptible to lysis by anti-CD20 immunoglobulin (Ig) G1 monoclonal antibody (mAb) and has been proposed as a nonimmunogenic strategy for regulating cell survival.23 However, ligation of CD20 initiates signaling events that modulate cell growth and proliferation,24,25 and a CD20+ T-cell leukemia has been described.26 Moreover, systemic anti-CD20 mAb treatment would not be selective for transduced cells but would also ablate normal CD20+ B cells.27 The ideal system for conditional elimination of T cells in a clinical setting would use a nontoxic inducer and a human-derived target protein to minimize immunogenicity. One promising approach uses a derivative of the human Fas receptor to deliver a conditional death signal in response to a nontoxic bivalent drug.28,29 In this system, a Fas chimeric molecule termed LV'VFas, in which the intracellular death signaling domain of Fas is linked to 2 copies of a modified 12-kDa human FK506-binding protein (FKBP12), is introduced into cells.29 The LV'VFas chimeric molecule remains inert until addition of AP1903, a bivalent "dimerizer" drug that cross-links the FKBP12 domains and activates Fas-mediated apoptosis.29,30 To date, inducible elimination of T cells with the LV'VFas system has been demonstrated only in vitro.30,31 In this study, we examined in a nonhuman primate model whether artificially activating Fas-receptor functions through LV'VFas may provide a means for controlling survival of adoptively transferred T cells in vivo. Autologous macaque T cells were isolated, modified to express LV'VFas, and transferred back into the animals. The administration of AP1903 induced rapid, selective, and nontoxic elimination of transferred T cells expressing LV'VFas. These results establish the efficacy of the inducible Fas system in vivo and suggest this approach may be useful for controlling the fate of cells in clinical cell therapy.
Retroviral vectors and generation of transduced macaque T cells
The LV'VFas vector has been described previously.29,30 This construct encodes a fusion protein consisting of the extracellular and transmembrane domains of the human low-affinity nerve growth factor receptor (
Animals and experimental design Adult macaques (Macaca nemestrina) were housed at the University of Washington Regional Primate Research Center, under conditions approved by the American Association for Accreditation of Laboratory Animal Care. Protocols were approved by the Institutional Review Board and Animal Care and Use Committee. Autologous LV'V+ or LV'VFas+ T cells were given to each macaque at a dose of 5 x 109/m2 by intravenous infusion. AP1903 (0.1 or 0.2 mg/kg, as indicated in the text) was administered by intravenous infusion for 2 hours beginning 1 day after the T-cell transfer, and then every other day for 5 or 10 doses. AP1903 plasma levels were determined using a liquid chromatography/tandem mass spectrometry method.33 Complete blood counts and serum chemistries were performed in accredited clinical laboratories. Flow cytometry
PBMCs and T cells were stained with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or PE-Cy5–conjugated mAbs to CD3, CD4, CD8, CD14, CD16, CD20, CD25 (PharMingen), and LNGFR (Chromaprobe, Mountain View, CA). Transferred Cell death assays
Cells were exposed for 2 hours to AP1903 (0.01-100 nM) and viability was examined after 24 hours by trypan blue exclusion. Alternatively, cells were stained with 7-amino-actinomycin D (7-AAD; 2 µg/mL, 15 minutes on ice) and viability was assessed by flow cytometry.30 The ratio of live-gated (by forward/side scatter and 7-AAD) unmodified or LV'VFas+ T cells was used to calculate the specific cell survival: percent survival = (R, drug-treated)/(R, untreated) x 100%.30 In some experiments, recombinant nerve growth factor Cytotoxicity assays
Transgene-specific cytotoxic responses were examined and T-cell clones were generated as described.18,19 Briefly, PBMCs obtained before and after infusion were stimulated twice 1 week apart with aliquots of Fluorescent probe PCR assay (TaqMan) Polymerase chain reaction (PCR) amplifications and analyses were performed using a quantitative real-time PCR assay (Perkin-Elmer Applied Biosystems, Foster City, CA).19 DNA (0.3-1 µg) was amplified in duplicate using PCR primers and TaqMan probes (Synthegen, Houston, TX) designed to detect unique LV'V or LV'VFas sequences. For LV'VFas, PCR primers 5'-ATCCCACCACATGCCACTCT-3' and 5'-TTTCTGCATGTTTTCTGTACTTCCTTT-3' were used with a fluorescent-tagged probe 5'-FAMTCTAGTTTCCAGTTTTAGAAGCTCCACATCGAAGA-TAMRA-3' encompassing the junction of the F36V and Fas genes. For LV'V, the primers were 5'-TCATCCCACCACATGCCAC-3' and 5'-TCTGGTACGTCGTACGGATAACTAGT-3', and the probe 5'-FAM-TCGTCTTCGATGTGGAGCTTCTAAAACTGGA-TAMRA-3' encompassed the junction of F36V and the retroviral vector pMX. Standards consisted of DNA derived from the infused LV'V+ or LV'VFas+ T cells.
In vivo persistence of autologous LV'VFas+ T cells
Autologous macaque T cells were transduced using a retrovirus encoding LV'VFas, at efficiencies ranging from 22% to 50%, and highly pure preparations of
LV'VFas-transduced T cells were cultured in vitro for 14 days after selection and then adoptively transferred in a dose of 5 x 109/m2 to a macaque to define the duration that transduced T cells would persist in the absence of AP1903. This dose of T cells is approximately equal to the number of lymphocytes in the blood pool and represents 1% to 2% of the total body lymphocyte pool.35 Real-time PCR for the LV'VFas sequence and flow cytometry using an anti-LNGFR mAb were used to detect transduced T cells and track persistence in vivo. The frequency of transduced cells determined by PCR in PBMCs 1 day after infusion was 4.9%, and this increased to 8.7% by day 3 after infusion (Figure 2A). This level would correspond to about 5% to 10% of the infused cells being present in the circulation 1 day after transfer. The transferred cells were maintained at a frequency of more than 2.4% of PBMCs for 9 days and then gradually declined (Figure 2A). Cytofluorometric quantification of
To assess the effects of AP1903 on cell survival, an identical dose of LV'VFas+ T cells was given to the same animal 42 days after the first infusion. AP1903 (0.1 mg/kg) was administered by intravenous 2-hour infusion every other day for 10 doses beginning 1 day after the T-cell transfer. Consistent with previous findings in human volunteers,33 the plasma level of AP1903 immediately after infusion was 86 nM and greatly exceeded the concentration required for maximum killing of LV'VFas+ T cells in vitro (3-10 nM).30 The frequency of LV'VFas+ T cells in PBMCs 1 day after the T-cell infusion and prior to administration of AP1903 was 2.5%, but declined precipitously to less than 0.05% after only 3 doses of AP1903 (Figure 2A). The human LV'VFas construct can be a target for immune recognition in macaques
The results in this first macaque were consistent with effective ablation of transferred T cells by AP1903. However, clearance of LV'VFas-modified T cells might also be explained by an immune response to epitopes derived from the fusion sites between the protein domains or from the human LV'VFas fusion protein because the cytoplasmic domains of human and M nemestrina Fas (GenBank accession no. M67454
[GenBank]
and AF344850
[GenBank]
, residues 192-319) are only 84% identical at the amino acid level, and additional polymorphisms may exist between macaque and human FKBP12 and Survival of LV'VFas+ T cells can be regulated by AP1903 in vivo
The observation that human LV'VFas is immunogenic in macaques required a modified experimental approach that would permit evaluation of the effect of AP1903 early after T-cell infusion prior to the induction of an immune response. To provide an internal control against which elimination of LV'VFas+ cells could be quantitatively assessed, we also transduced an aliquot of T cells from a second macaque with the LV'V vector, which lacks the Fas domain.30 The transduction efficiency using LV'V (Phoenix A) was 60% as compared to 30% with the LV'VFas (PG13) virus, but the transduced cells contained a very similar phenotypic distribution. The LV'V-transduced population contained 38.4% CD4+ and 60.1% CD8+ T cells, and the LV'VFas-transduced population contained 45.4% CD4+ and 53.4% CD8+ T cells. The T cells transduced with each vector also proliferated equivalently well in response to stimulation with anti-CD3 and anti-CD28 mAbs. The LV'V-transduced T cells were insensitive to AP1903 in vitro (data not shown) and could be distinguished from LV'VFas+ cells by PCR. Equal numbers (5 x 109/m2) of LV'V+ and LV'VFas+ T cells were simultaneously administered to the second macaque, and treatment with AP1903 (0.2 mg/kg) was begun 1 day after the T-cell transfer and then every other day for 5 doses. High levels of both LV'V+ and LV'VFas+ T cells (5.4% versus 8.4%) were detected 1 day after the cell infusions (Figure 3A). The frequency of control LV'V+ T cells peaked at day 3 at 7.5% and persisted for 14 days at equivalent or slightly higher levels than observed on day 1 (Figure 3A-B). In contrast, the levels of LV'VFas+ cells dropped substantially (
Residual LV'VFas+ T cells display enhanced sensitivity to AP1903 after activation in vitro
The results in macaque no. 2 demonstrated the persistence of a small subset of LV'VFas+ T cells after treatment with AP1903. The resistance of a subset of transduced T cells to AP1903 in vivo might potentially be due to antiapoptotic effects of
We next evaluated whether expression of the LV'VFas transgene was dependent on the activation state of T cells. We found that LV'VFas+ T cells rested for more than 7 days in vitro in the absence of T-cell receptor or cytokine stimulation exhibited a reversible reduction in
Isolation and selective analysis of residual LV'VFas+ T cells in vivo from macaque no. 2 using anti-LNGFR mAb was impossible because both LV'V+ and LV'VFas+ T cells had been infused. Thus, a third macaque was given an infusion with LV'VFas+ T cells (5 x 109/m2) alone and treated with AP1903 every other day for 10 doses beginning on day 1. As before, LV'VFas+ T cells were present in PBMCs at high levels on day 1 prior to AP1903 (7.6% of PBMCs), and the majority (81%) were eliminated over 10 days of treatment with AP1903 (Figure 6A). The few
Identification of the antigenic epitope within the human LV'VFas protein
The in vivo experiments demonstrate that LV'VFas-transduced T cells can be effectively ablated in vivo with AP1903, but the observation that the human LV'VFas protein was immunogenic in macaques raised the possibility that this construct might also be immunogenic in humans, similar to suicide genes derived from pathogens. In principle, the immune response to human LV'VFas could be due to T-cell recognition of peptides derived from the fusion sites of the LV'VFas chimeric protein or from sequences that differ between the human protein components of the LV'VFas construct and the homologous macaque proteins. Therefore, peptides of 18 amino acids in length that overlap the sequences at the fusion sites between
We next assessed the specificity of the cytolytic T-cell response to components of the transgene product. LV'VFas-reactive T-cell clones were isolated from macaque no. 1 by limiting dilution and examined for recognition of target cells transduced with retroviral vectors encoding either
The introduction and expression of genes that confer an inducible death phenotype to somatic cells is an attractive strategy for controlling cell survival.16,17,44 Chemically induced dimerization of receptors is a general approach for controlling signal transduction and has been used to drive cell proliferation45,46 and to induce apoptotic cell death.28-30 A construct termed LV'VFas, which consists exclusively of human proteins to reduce the possibility that transgene products will be immunogenic, has been generated for inducing apoptosis of human cells. In this study, the efficiency of LV'VFas for regulating survival of transferred T cells was explored in a nonhuman primate model. The transfer of large numbers of both LV'V+ and LV'VFas+ T cells resulted in high levels of modified cells in peripheral blood, allowing us to examine the efficacy of AP1903 for signaling cell death. Our studies show that administration of AP1903 results in a rapid elimination of transduced T cells in vivo without causing toxicity. These data in a large animal model provide the first demonstration that a chemical dimerizing agent can regulate viability of transferred gene-modified cells.
A small fraction of LV'VFas-modified cells resisted the effects of AP1903 and persisted in vivo. Several factors may explain the observed resistance of a subset of LV'VFas+ cells to AP1903 in vivo. First, the activity of the MoMLV-LTR promoter is reduced in resting T cells,40,47 and the residual LV'VFas-modified T cells that survived exposure to AP1903 in vivo exhibited reduced levels of transgene expression. The activation of antigen-specific T cells after target recognition has been demonstrated to enhance transgene expression in animal studies of adoptive therapy48-50 and in vitro activation of T cells that resisted AP1903 enhanced both transgene expression and sensitivity to AP1903. Thus, AP1903 may have enhanced efficacy for eliminating adoptively transferred T cells that cause toxicity following encounter with cognate antigen in vivo, such as those T cells participating in GVHD. An alternative to ensure the maintenance of AP1903 sensitivity would be to use the activation-independent CD2 promotor51 or to incorporate human interferon-
An alternative explanation for the partial elimination of LV'VFas+ T cells is that expression of Finally, evidence indicates that Fas signaling does not always lead to apoptosis59,60 in part due to expression of antiapoptotic proteins such as BCL-XL,61 c-Flips,62 or members of the inhibitor-of-apoptosis family.63 Up-regulation of such proteins could also contribute to the transient insensitivity of a subset of T cells to AP1903. This could potentially be overcome by modifying T cells to express inducible versions of downstream effectors of apoptosis, such as caspase-1 or caspase-3, which can bypass most of these regulatory checkpoints.64,65 Induction of apoptosis through chemical-induced dimerization of caspase-1 and caspase-3 has been demonstrated in transiently transfected cell lines,65 and these inducible constructs could potentially be used to control the fate of primary T cells. The persistence of LV'VFas-modified T cells in macaques was sufficient to evaluate AP1903-mediated T-cell elimination, but long-term persistence of the gene-modified T cells in the absence of AP1903 was not achieved due to the development of host CD8+ CTL responses specific to epitopes derived from the human LV'VFas protein. Cytolytic responses to epitopes derived from the fusion sites of the chimeric LV'VFas protein were not observed, supporting the potential clinical utility of this approach. However, we identified a T-cell response that targeted an epitope within human Fas that differed by 2 amino acids from its macaque homolog. Because this is a macaque antihuman response, it is reasonable to conclude that immune responses to the human protein will be less of an impediment to persistence of LV'VFas+ T cells in humans. In conclusion, our studies demonstrate the utility of small molecules for initiating intracellular signaling events from transgene products and regulating survival of adoptively transferred T cells in vivo. AP1903 was found to be safe in a phase 1 trial in human volunteers.33 Therefore, this nontoxic and potentially nonimmunogenic suicide system holds significant clinical promise for controlling cellular therapies.
We thank Carole Elliott and the staff of the University of Washington Regional Primate Research Center for excellent technical assistance. We thank Selvi Pradeepan and Carly Graytock for technical assistance with bioanalytical work. We also thank Jin Zhang, Lilith A. Reeves, and Kenneth G. Cornetta for producer cell line and vector production.
Submitted August 25, 2003; accepted October 6, 2003.
Prepublished online as Blood First Edition Paper, October 16, 2003; DOI 10.1182/blood-2003-08-2908.
Supported by National Institutes of Health grants HL66947 (S.H., S.R.R.) and CA18029 (S.H., S.R.R.). J.D.I., D.C.D., J.G., and T.C. have declared commercial interest in a company (ARIAD Pharmaceuticals Inc) whose potential product was studied.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stanley R. Riddell, Program in Immunology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA 98109; e-mail: sriddell{at}fhcrc.org.
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Abstract
Introduction
LNGFR) as a cell surface marker, 2 copies of a human FKBP12 containing a single point mutation at position 36 (F36V) to provide a high-affinity receptor for AP1903,
) or LV'VFas+ T cells (
) were exposed 4 times (48 hours apart) for 2 hours to medium alone or to medium containing 10 nM AP1903. Cells were washed, and the survival of LV'VFas+ T cells as compared to the untreated cells was evaluated 24 hours later by trypan blue exclusion.
(NGF-
-irradiated autologous T cells transduced with 
) or LV'VFas+ (
) target cells. Data are shown for an effector-to-target (E/T) ratio of 20:1.
75%) after the first dose of AP1903, and 82% of infused cells were eliminated over the ensuing 10 days (
) and LV'VFas+ (
) T cells was examined for the frequency of vector-containing cells by TaqMan PCR. The 100% value was assigned to the frequency of LV'V+ or LV'VFas+ T cells among PBMCs at day 1, prior to the initial dose of AP1903. Values for subsequent dates are calculated as the percent change in transduced cells among PBMCs compared with the day 1 value. The arrow indicates the day of the T-cell infusion. (C) Transgene product-specific cytolytic T-cell responses after the transfer of LV'V+ and LV'VFas+ T cells. PBMCs were collected from macaque no. 2 on the indicated days and stimulated with autologous 
chain (CD25) as a marker of T-cell activation, and for surface expression of 
