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GENE THERAPY
From ARIAD Gene Therapeutics, Cambridge, MA, and San
Raffaele Telethon Institute for Gene Therapy and Cancer Immunotherapy
and Gene Therapy Program, Istituto Scientifico H. S. Raffaele,
Milan, Italy.
Graft-versus-host disease (GVHD) is a major complication of
allogeneic bone marrow transplantation. One strategy to treat GVHD is
to equip donor T cells with a conditional suicide mechanism that can be
triggered when GVHD occurs. The herpes simplex virus thymidine kinase
(HSV-tk)/ganciclovir system used clinically has several
limitations, including immunogenicity and cell cycle dependence. An
alternative switch based on chemically inducible apoptosis was designed
and evaluated. A chimeric human protein was expressed comprising an
extracellular marker ( In allogeneic bone marrow transplantation (BMT),
the delayed infusion of donor lymphocytes plays a central therapeutic
role in the control of disease relapse (graft-versus-leukemia effect [GVL])1 and in the induction of immune
reconstitution,2,3 the latter especially in the subset of
T-depleted matched transplants and in the context of partially
mismatched transplants.4 However, graft-versus-host-disease (GVHD) represents a frequent and often lethal
complication of delayed lymphocyte infusions.1 Managing the threat of GVHD while maximizing the beneficial GVL effect would
broaden the scope and usefulness of allogeneic BMT procedures.
We and others have previously demonstrated that ex vivo genetic
manipulation of donor lymphocytes to insert a conditional, drug-inducible suicide gene provides a means for the specific elimination of donor T cells with the onset of GVHD while maximizing the therapeutic benefit of the GVL effect.2,5,6 Although a
number of suicide genes have been proposed,7,8 the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir
(GCV)-based suicide strategy appears to be the most effective and
specific and has been widely adopted.9,10 Cells are
engineered to express HSV-tk; the addition of ganciclovir
leads to cell death through tk-catalyzed metabolism of the
drug to a lethal product. In the current clinical trial,6
HSV-tk-engineered-donor T cells demonstrated an effective
antileukemic effect, and GVHD could be successfully treated through GCV
administration.2,11
Despite this demonstration of efficacy, the study revealed limitations
of the HSV-tk/GCV approach. First, in 8 of 24 treated patients, a specific cytotoxic CD8-mediated immune response developed against genetically engineered cells that led to the selective elimination of these cells (C.T., unpublished data). Although the cells
expressed both HSV-tk and the cell surface marker To circumvent these limitations, we assessed the suitability of a novel
suicide switch based on the human Fas receptor to trigger cell death in
primary human T lymphocytes. Fas (CD95, APO-1) is a member of the tumor
necrosis-nerve growth factor receptor superfamily.12
Cross-linking of Fas results in the recruitment of a death-inducing
signaling complex, activating a proteolytic cascade of caspases and
inducing cell death by apoptosis.12,13 We and others have
previously described a system for activating apoptosis at will and
demonstrated its function in engineered cell lines.14,15 A
chimeric protein containing the membrane-anchored intracellular domain
of Fas is fused to the FK506-binding protein, FKBP12. Cross-linking of
these proteins by the addition of a bivalent FKBP ligand (a
"dimerizer") triggers the apoptotic death signal. Recently, we
refined the system by designing a dimerizer drug, designated AP1903,
with specificity for the engineered FKBP over the endogenous
protein.16
In this report, we describe the experimental evaluation of the
Fas-based suicide switch for the elimination of primary human T
lymphocytes for the ultimate purpose of treating GVHD in BMT patients.
At the beginning of this work we identified several features that would
be required for successful use of the Fas switch Retroviral plasmid construction
F36V'-FKBP construction
Southern blot analysis Genomic DNA was isolated from T lymphocytes by standard phenol-chloroform extraction. DNA was digested with XbaI, which cuts between LNGFR and the first F36V-FKBP, and in the 3' LTR.
Blots (Hybond-N nylon membrane; Amersham, Buckinghamshire, United
Kingdom) were hybridized with the XbaI-NaeI
fragment of LV'VFas encoding for F36V'-F36V-Fas.
Polymerase chain reaction assay of FKBP region in Fas transgenes Genomic DNA from Fas-engineered cells was prepared by digestion of 0.5 × 106 cells with 50 µg/mL proteinase K. Using a standard PCR reaction, the FKBP region of the Fas suicide cassette was amplified using 2 FKBP flanking primers (D. J. Talbot, unpublished data).Retroviral transduction Retroviral plasmids were introduced into amphotropic Phoenix cells (G. P. Nolan, Stanford, CA) by lipofection. Stable transfectants were identified by resistance to 50 µg/mL puromycin. Retroviral supernatants were used to infect human peripheral blood lymphocytes as follows. Peripheral blood was collected from healthy donors, and T lymphocytes were purified by Ficoll-Histopaque (Sigma, St Louis, MO) density centrifugation followed by magnetic selection using anti-CD3 Dynabeads (Dynal, Oslo, Norway). Bead-adherent cells (2-4 × 106/mL) were cultured for 48 hours in RPMI 1640 supplemented with 10% fetal calf serum, 100 ng/mL anti-CD28 (Becton Dickinson, San Jose, CA), and 50 U/mL huIL-2. T lymphocytes (and NIH 3T3 cells) were transduced with retrovirus-containing supernatant either by plating on retronectin-coated wells (BioWhittaker, Walkersville, MD) or by centrifugation (90 minutes, 2000 rpm) in the presence of 4 µg/mL polybrene. Both methods gave similar transduction efficiencies; for T lymphocytes, this ranged from 10% to 50%.Immunomagnetic selection of Fas-transduced T lymphocytes Transduced T cells were immunoselected for expression of the cell surface markerLNGFR as previously described.2,6
Briefly, cells were incubated with mouse antihuman LNGFR monoclonal
antibody 20.4 (Chromaprobe, Mountain View, CA) and selected with goat
antimouse IgG-coated magnetic beads (Dynabeads M-450; Dynal). After
overnight incubation at 37°C, magnetic beads were separated from the
cells by pipetting and were removed magnetically from the culture. T lymphocytes purified in this manner were routinely more than 95% LNGFR-positive after a single round of selection. Cell recovery ranged
between 40% and 50%.
Apoptosis-cell death assays Elimination of T cells was measured by the following assay. Untransduced T cells were loaded with CellTracker Green CMFDA (Molecular Probes, Eugene, OR) as an internal control and were used to spike immunomagnetically sorted LV'VFas-transduced T cells. The cell mixture (2 × 106 cells/mL) was incubated with AP1903 for the indicated time, stained with 7-amino-actinomycin D (7-AAD, 2 µg/mL; Sigma) for 15 minutes on ice, and analyzed on a Becton Dickinson FACSort. The ratio (R) of live-gated (by forward/side scatter and 7-AAD), LV'VFas-transduced cells (nonfluorescent)-untransduced cells (green-fluorescent) was used to calculate specific cell survival using the following formula: %Survival = (R, drug-treated)/(R, untreated) × 100%. For annexin V assays, sorted LV'VFas-transduced T cells (2 × 106 cells/mL) were incubated with 10 nM AP1903. At the indicated time, an aliquot of 2 × 105 cells was taken, stained with annexin V-fluorescein isothiocyanate according to the manufacturer's instructions (Clontech, Palo Alto, CA), and analyzed by flow cytometry.To compare the AP1903/FAS suicide system with the standard
ganciclovir-HSV-tk system, human T lymphocytes transduced
with the LV'VFas retroviral vector or with the SFCMM-3 retroviral
vector11 (carrying the HSV-tk gene) were
immunoselected to more than 95% purity. Cells were cultivated for 5 days in the absence and in the presence of 10 nM AP1903 or 50 nM GCV in
triplicate. [3H]thymidine (1µ Ci/well; specific
activity 87 Ci/mmol; Dupont, Boston, MA) was added 16 hours before
harvesting the DNA and counting in a Analysis of antiallogeneic response Antiallogeneic cytotoxic T lymphocytes were induced in vitro in a mixed lymphocyte reaction (MLR) with 2 × 106 LV'VFas-transduced donor effector lymphocytes or untransduced cells as control and 1 × 106 irradiated (6 Gy) allogeneic peripheral blood mononuclear cells (PBMC) (fully mismatched). The MLR was performed in Iscoves modified Dulbecco medium supplemented with 10% human serum, glutamine, and antibiotics in the presence of 50 U/mL huIL-2. Equal numbers of lymphocytes were tested in a standard cytotoxicity assay 10 days later, using as target cells 51Cr-labeled phytohemagglutinin (PHA)-stimulated PBMCs from the same allogeneic donor and autologous PHA blasts as negative control cells. Natural killer cell-like activity was blocked by cold inhibition with a 30-fold excess of K562 cells over 51Cr-labeled specific target cells. A secondary MLR was performed by the addition of irradiated allogeneic PBMC (1 × 106) in the presence or absence of 10 nM AP1903. Ten days later cells were counted, and the cytotoxic activity of equivalent numbers of cells was measured as above.
Development of a conditional Fas suicide switch for use in primary human T cells The original format of the inducible Fas suicide system was described by Spencer et al15 and comprised the cytoplasmic domain of the human Fas death receptor fused to 2 copies of human wild-type FKBP12 and an epitope tag. This chimeric protein was fused to an N-terminal myristoylation motif to localize it to the plasma membrane. For this study, we made several modifications to optimize the construct for clinical use in T cells. We replaced the 2 FKBP12 domains with the FKBP point mutant, F36V-FKBP, to allow use of the more potent and specific dimerizer drug, AP1903.16 We removed the C-terminal epitope tag to minimize the potential immunogenicity of the protein. In addition, we replaced the myristoylation motif with the extracellular and transmembrane domains of LNGFR to localize the chimeric protein to the plasma membrane and simultaneously provide a cell surface marker.6,17 The approach physically links the conditional apoptosis cassette with an affinity handle that can be used to purify transduced cells, providing a means to ensure that all resultant cells can be eliminated by adding AP1903. The resultant gene cassette is herein called LVVFas (Figure 1A).
One of the major safety considerations of using any suicide gene approach for the treatment of GVHD is that all the ex vivo-engineered T lymphocytes to be infused into the patient must express the functional suicide gene. This is of particular concern with the AP1903/Fas system because of the presence of repetitive sequences (2 copies of F36V-FKBP).16 Retrovirally introduced transgenes containing repetitive sequences tend to have a high frequency of rearrangement, which very likely would compromise the function of the chimeric Fas protein. Selection of transduced cells using the LNGFR surface marker does not necessarily prevent exclusion of cells expressing a rearranged transgene because the rearrangement may not affect the LNGFR portion of the protein. Previous studies that used the conditional Fas suicide approach did not report problems with rearrangement, but in these cases nonretroviral approaches or clones of transduced cells were used.14-17,19 To reduce the risk for rearrangement associated with the 2 identical copies of F36V-FKBP, we designed a "codon-wobbled" variant of F36V-FKBP, termed F36V'-FKBP. These 2 constructs encode identical polypeptide chains but share only 62% homology at the nucleotide level by virtue of silent changes of the wobble bases in most codons. The construct LV'VFas incorporates one copy of F36V'-FKBP and one copy of F36V-FKBP and is anticipated to be less recombinogenic than LVVFas (Figure 1A). A control construct, LV'V, lacks the cytoplasmic domain of Fas and thus is incapable of signaling. These transgenes were cloned into the retroviral vector pMX,18 which uses the MoMLV LTR to drive expression and which was found to provide a higher-level expression of the chimeric protein than LXSN-based vectors (data not shown). F36V'-FKBP prevents rearrangement of the Fas transgene during retroviral transduction To test the performance and genetic stability of these constructs, they were transiently transfected into amphotropic Phoenix packaging cells, and supernatant collected 48 hours later was used to transduce NIH 3T3 cells. To examine the integrity of the FKBP portion of the Fas transgene, we used a PCR-based assay in which the FKBP portion is amplified from the genomic DNA of transduced cells. Cells transduced with LV'VFas yielded a single PCR product of the expected size, 724 bp (Figure 1B, lane 7). However, cells transduced with LVVFas produced an additional band reduced in size by approximately 300 bp, a size difference consistent with the deletion of one FKBP (Figure 1B, lane 6). This result indicates that there is a significant frequency of rearrangement with the Fas construct containing 2 identical copies of F36V-FKBP but that the use of the modified F36V'-FKBP/ F36V-FKBP combination eliminates this problem.Engineering of primary human T lymphocytes with Fas suicide genes and immunomagnetic selection of transduced cells We next evaluated the expression and stability of the conditional Fas constructs in T cells. Primary human T lymphocytes were isolated from peripheral blood obtained from volunteer donors using anti-CD3 magnetic beads. Purified T lymphocytes were stimulated for 48 hours before infection with Fas recombinant retroviruses. We observed high-level expression of the transgene as monitored by flow cytometry to detect LNGFR-positive cells (Figure 2A), but there was a complete absence of "toxicity" of the construct (data not shown). To test whether theLNGFR cell surface marker could be used to isolate transduced T
cells, LNGFR-positive cells were immunoselected 48 hours after
infection with anti-LNGFR magnetic beads as previously
described.2 We found that cells sorted in this manner were
routinely more than 95% to 99% LNGFR-positive in a single round of
purification (Figure 2A). Of note, cell viability was not compromised
during the selection process (data not shown). This is an important
observation because cross-linking of LNGFR by the antibody-coated beads
might be expected to cluster the Fas domains and activate signaling, an
outcome that would complicate the use of this procedure to isolate
transduced cells. Similarly, ligation of LNGFR on the surface of
LV'VFas-transduced T cells with human  nerve growth factor (-NGF)
had no effect on cell viability (data not shown). The fact that these
procedures do not trigger apoptosis suggests that the
cross-linking of LNGFR brings together the Fas death domains in a
structural context that is unproductive for signaling.
To assess the integrity of the Fas transgene in transduced human T
lymphocytes, Southern blot analysis was performed. Similar to our
findings in 3T3 cells, primary human T lymphocytes transduced with
LVVFas yielded LV'VFas transgene confers susceptibility to AP1903-induced cell death to primary human T lymphocytes We next assessed the susceptibility of LV'VFas-transduced T cells to AP1903-induced cell death. The Fas system has previously been tested in vitro in several cell lines, including HT1080, 293, Jurkat, and HeLa cells,14,16,17,19 and in vivo in a transgenic mouse model.15 Interestingly, the killing efficiency varied widely in these diverse cell types, perhaps reflecting an intrinsic, cell type-specific susceptibility to Fas-induced apoptosis. This emphasizes that the performance of the Fas system must be optimized for each target cell type in the case of this study, primary human
T cells.
LV'VFas-transduced T lymphocytes were exposed to different
concentrations of the dimerizer AP1903, and survival was analyzed 24 and 48 hours later. As shown in Figure
3A, the induction of cell death was
highly dose dependent. Maximal killing occurred in the presence of 3 to
10 nM AP1903, and the IC50 was approximately 0.2 nM. In a
large number of experiments, the maximal killing efficiency was
consistently in the range of 60% to 80%, and the IC50 was
reproducibly approximately 0.2 nM. We observed no significant donor-to-donor variability of the killing efficiency (data not shown).
In addition, when CD4 and CD8 T-cell populations were examined
separately, no difference was found in the killing efficiency or in the
IC50 value (data not shown). This information is relevant because CD4 and CD8 donor lymphocytes contribute to GVHD, as shown by
donor lymphocyte infusion protocols involving the infusion of either
CD4-depleted20 or CD8-depleted donor
lymphocytes.21 LV'VFas-transduced T lymphocytes
demonstrated normal viability in the absence of drug, indicating the
absence of autotoxicity
As shown in Figure 3B, the extent of cell death of drug-treated Fas-engineered T cells is comparable to that of HSV-tk-engineered cells. In this experiment a second-generation vector expressing wild-type HSV-tk (SFCMM-3) was used.11 Because a previous version of this vector (SFCMM-2, which expresses HSV-tk-neo fusion) confers significantly lower sensitivity to GCV11 but was used successfully to ablate T cells and treat GVHD in clinical trials,2 these data suggest that the AP1903-Fas system may also be suitable for clinical use to treat GVHD. Induction of apoptosis by the AP1903/Fas system occurs within 1 hour of drug addition To provide rapid mitigation of GVHD, it is important that the donor T cells be neutralized as quickly as possible. In contrast to HSV-tk-induced cell death, Fas-induced apoptosis is expected to be a rapid event that occurs within hours of the appropriate signal. Some of the earliest detectable events in the apoptotic pathway are the loss of the cytoplasmic polarization of plasma membrane phosphatidylserine and the fragmentation of nuclear DNA.13,22,23 We used the former readout to determine the kinetics of AP1903-induced cell death. LV'VFas-transduced T lymphocytes were treated with 10 nM AP1903 and stained with annexin V24 at different times (Figure 4). In as little as 1 hour, apoptotic cells were clearly detectable. The percentage of apoptotic cells continued to increase over time; after 24 hours of drug treatment, 68% of the cells were apoptotic (Figure 4). In contrast, LV'VFas-transduced lymphocytes cultured in the absence of AP1903 showed only a small increase in the proportion of apoptotic cells that could be attributed to spontaneous cell death during culture (Figure 4). Similarly, no significant change in the annexin V staining profile was observed when untransduced T cells were incubated with AP1903 (data not shown). These findings show that AP1903-induced apoptosis is extremely fast. Furthermore, the kinetics observed with AP1903-induced apoptosis are similar to those of primary human lymphocytes (our unpublished observations) or the human Jurkat T-cell line treated with anti-Fas antibodies,24 suggesting that the same signaling cascade is triggered by both agents.
Maximal killing is achieved even by short exposures to AP1903, and additional doses increase the efficiency To explore the AP1903 dosing regimen and administration schedule likely to be required in a clinical setting, we determined the length of time Fas-engineered T cells must be exposed to the drug to trigger apoptosis. LV'VFas-transduced T lymphocytes were treated with 10 nM AP1903 for 1, 2, or 4 hours, the drug was washed away, and the cells were further incubated until analysis at 24 or 48 hours. Surprisingly, even a 1-hour exposure resulted in near-maximal cell death (Figure 5A). Prolonging the duration to 2 or 4 hours did not significantly increase the killing efficiency, and the continuous presence of the drug had only a small additional effect at the 48-hour time point (Figure 5A).
The fact that a single AP1903 dose eliminates 60% to 80% of engineered cells raises the question of whether the remaining cells are intrinsically resistant to AP1903-induced apoptosis. This is a significant question because clinical use to mitigate GVHD would be optimal if all engineered cells could be eliminated. We investigated whether cells surviving the first drug challenge could be eliminated by a second drug administration. As shown in Figure 5B, a first dose of AP1903 (2-hour pulse with 10 nM) resulted in the elimination of approximately 72% of LV'VFas-transduced T lymphocytes. Forty-eight hours after the first dose, the surviving cells were treated with a second 2-hour pulse of AP1903. This second dose resulted in the killing of more than 60% of the remaining cells. Together, the 2 pulses (doses) resulted in an aggregate killing efficiency of approximately 90% (Figure 5B). In contrast, control LV'VFas-engineered cells subjected to the same procedure but without the addition of AP1903 showed only a small decrease in viability (Figure 5B). These findings indicate that cells not killed by the first AP1903 treatment have not acquired resistance and are only temporarily refractory to AP1903/Fas-induced cell death. This is supported by the absence of rearranged transgene-bearing cells before or after AP1903 treatment (Figure 2B, lanes 4 to 7). Rather, the presence of cells surviving AP1903 treatment is probably attributable to properties intrinsic to T-cell biology25 and is consistent with previous observations that cross-linking of endogenous Fas on the surface of stimulated primary human T lymphocytes26,27 or human Jurkat T cells28,29 kills only 40% to 80% of the cells. Although the phenomenon is not yet fully understood, temporary resistance of T cells to Fas-induced cell death may reflect stochastic variations in intracellular levels of antiapoptotic molecules such as bcl-2 and bcl-xL,30,31 XIAP,32 or c-FLIP,33 leading to transient blockage of the Fas signal. Interestingly, AP1903 treatment of Fas-engineered lymphocytes caused the rapid down-regulation of the LV'VFas protein in cells that were not killed by the drug (data not shown). This disappearance from the surface is dependent on the Fas signaling domain; the expression level of the LV'V control transgene is not affected by the drug (data not shown). However, the down-regulation of LV'VFas expression is only temporary. Expression levels recover within 24 to 48 hours after withdrawal of the drug (data not shown). AP1903/Fas-induced cell death of primary human T lymphocytes is not dependent on the proliferation state of the cell T lymphocytes are a highly heterogeneous population of cells with different antigen specificities, and they exist in various states of activation and differentiation. One of the inherent limitations of the HSV-tk suicide system is that only cells that progress through the S-phase of the cell cycle are killed by GCV administration. In essence, this means that only activated, rapidly proliferating T cells are eliminated efficiently, whereas cells that are in a resting, nonproliferation state are relatively resistant. In the clinical trial involving the infusion of HSV-tk-engineered lymphocytes, this resulted in the partial resistance of chronic GVHD to GCV treatment.2,11 By contrast, Fas-induced apoptosis does not require S-phase entry.27 The AP1903/Fas suicide system is, therefore, expected to function independently of the proliferative state of the cell. In addition, the transgene-derived Fas-fusion protein is expressed constitutively, and there is less than a 2-fold difference in expression levels between activated and resting T cells (data not shown). Therefore, AP1903-induced apoptosis should operate independently of activation-induced Fas-receptor up-regulation,34 and it should be dependent only on the sequential activation of caspases, which are known to be constitutively expressed.35-37The cell cycle dependence of the AP1903/Fas system was tested by taking
advantage of the transient nature of the activation of in
vitro-stimulated T lymphocytes. After the initial stimulation, cells
proliferated rapidly and remained in a highly activated state for
approximately 2 weeks, after which proliferation slowed down and they
gradually returned to a less activated ("resting") state. To
measure the proliferation/activation state of the Fas-engineered T
cells, we examined 2 parameters: the expression of the IL-2 receptor
As shown in Figure 6A, LV'VFas-transduced
T lymphocytes expressing high levels of CD25 (top panel) were
eliminated by AP1903 with 66% ± 7.5% (n = 10) efficiency. When
cells were examined after CD25 expression returned to basal levels
(Figure 6A, bottom panel), 63% ± 4.7% (n = 9) killing was observed
after AP1903 treatment. Taken together, these results show that there
is no statistically significant difference (P = .17)
between the killing efficiencies of highly activated
(CD25hi) T cells and cells that have returned to a less
activated state (CD25
Fas-transduced T lymphocytes are able to mount a specific antiallogeneic response To determine whether Fas-transduced T lymphocytes are immune functional, and in particular whether they retain the ability to generate a specific antiallogeneic response, an MLR was performed. LV'VFas-transduced lymphocytes were stimulated with irradiated, allogeneic PBMCs, and specific cytotoxic activity was measured in a chromium release assay 10 days later. As shown in Figure 7A, LV'VFas-transduced T cells exhibited the same specific lytic activity against allogeneic target cells as untransduced T cells, suggesting that transduced cells are immune functional and that activation on encounter with a specific antigen does not trigger spontaneous apoptosis. Because donor T cells play a central therapeutic role during allo-BMT in providing a GVL effect, these properties are important for the eventual use of the AP1903/Fas system to combat GVHD.
To test whether the cytotoxic activity of Fas-engineered T cells can be inhibited by AP1903 treatment, cells from the primary MLR were restimulated with allogeneic PBMCs in the absence or presence of 10 nM AP1903. As expected, 60% to 80% of the Fas-engineered cells were killed by the drug in the secondary MLR (data not shown). Interestingly, cells surviving the drug treatment exhibited significant reductions in lytic activity relative to Fas-engineered cells restimulated in the absence of AP1903 (Figure 7B). This result suggests that in addition to the 60% to 80% reduction of cytotoxic activity associated with AP1903-induced cell death, the lytic activity against an allogeneic target cell of Fas-engineered T cells is significantly reduced after exposure to AP1903, an issue particularly relevant in the treatment of GVHD. This result is in good agreement with the previous observation that antiallogeneic lymphocytes challenged with the relevant target cell showed a reduction in proliferation of approximately 35% when they were preincubated with anti-Fas antibody.38
In this report, we examined the feasibility of using the AP1903/Fas suicide strategy for the treatment of GVHD after allogeneic BMT by examining its performance in vitro in genetically engineered primary human T lymphocytes. We showed that a modified Fas cassette that incorporates a cell surface marker allows transduced cells to be purified to homogeneity without activating apoptosis. We demonstrated that a "codon-wobbled" version of F36V-FKBP is a crucial element to prevent the rearrangement of the Fas transgene, an important safety consideration in gene therapy applications using retroviral vectors. We showed that primary human T lymphocytes can be engineered to express high levels of the Fas transgene and can then be eliminated by the AP1903/Fas system with high efficiency, potency, and specificity. Importantly, Fas-transduced lymphocytes retain their immune potential, a relevant issue for the GVL effect. The redesigned Fas cassette, therefore, fulfills the criteria we identified at the beginning of this study, required for clinical usefulness for combating GVHD. Spencer et al15,19 have found that expression of Fas in transiently transfected cells induces apoptosis in the absence of drug addition. Most likely this basal toxicity ("autotoxicity") is caused by the self-association of the death domain.39 Consistent with these findings, we observed high autotoxicity with the LV'VFas construct in transiently transfected cell lines. However, when the LV'VFas construct was introduced in cells by retroviral infection, there was a complete lack of autotoxicity, both in cell lines and in primary human T lymphocytes. These findings suggest that autotoxicity is only observed when Fas is expressed at extremely high levels, as is the case in transiently transfected cells. This is in good agreement with the finding that Fas toxicity is significantly decreased when the amount of DNA transfected is reduced.19 Although this study was undertaken to investigate potential clinical use of the Fas T-cell suicide system, the conditional elimination of T is also useful as a research tool. Conditional ablation of subpopulations of T cells, for example in transgenic mouse models,15,40 provides a means to probe their importance in specific aspects of the immune response. Our data suggest that the modified Fas construct with an integral cell surface marker could also be useful in these research applications. Recent clinical experience with the HSV-tk suicide system has identified several significant limitations to its use in treating GVHD associated with allo-BMT.11,41 Immune responses against infused HSV-tk-engineered T lymphocytes developed in several patients, resulting in the elimination of the cells and precluding the possibility of future infusions. In addition, chronic GVHD could only be partially controlled by GCV administration, a finding that was attributed to the cell cycle dependence of HSV-tk killing.11 Finally, GCV administration for concurrent clinical conditions other than GVHD resulted in the undesired clearance of HSV-tk donor lymphocytes and, hence, in the loss of the GVL effect. The AP1903/Fas system offers several potential advantages over the
HSV-tk/GCV strategy. Unlike HSV-tk, all the
functional protein components of AP1903/Fas are of human origin and are
less likely to elicit immune responses. The only potential for
immunogenicity in the construct are the point mutation in F36V-FKBP and
3 junction peptides. We demonstrate that with a single treatment of
AP1903, 60% to 80% of Fas-engineered cells are eliminated. This is in comparison with 30% to 50% in vitro killing of
HSV-tk-neo-engineered T cells11 using the same
construct (SFCMM-2) that was used successfully in clinical
trials.2 The most recent retroviral vectors carrying the
wild-type HSV-tk suicide gene are more efficient in killing highly proliferating cells Our experiments provide information that will be useful in defining the dosing regimen for use in GVHD applications. Specifically, delivery of short (1- to 2-hour) pulses of drug that are repeated at approximately 24- to 48-hour intervals should mimic the conditions found to induce efficient elimination of engineered T cells in vitro. The pharmacologic properties of AP1903 in healthy human volunteers were recently determined in a phase 1 trial that demonstrated the safety of the drug and established a direct relation between the plasma concentrations and the dose administered (J. D. Iuliucci, unpublished data). No clinically meaningful adverse effects were observed at plasma AP1903 concentrations up to 100-fold above the maximally effective in vitro concentration. Based on our experiments showing safety, specificity, killing efficiency, and immune competence of LV'VFas-engineered lymphocytes and on the results of the phase 1 clinical trial of AP1903, we propose the AP1903/Fas system as a novel, safe, and efficacious suicide strategy to induce a controlled GVL effect in allogeneic marrow transplantation. Our data indicate that Fas-engineered donor lymphocytes should be as efficacious as HSV-tk-engineered donor lymphocyte infusions in controlling disease relapse but able to bypass the intrinsic limitations of the HSV-tk/GCV suicide system.
We thank Zulma Magnani, Franklin Cerasoli Jr, and Patrizia Rovere for helpful discussion; Carl Rollins, Daniela Maggioni, and Scott Wardwell for technical assistance; and Dale Talbot for developing the FKBP PCR assay. In addition, we thank the various blood donors for enabling us to conduct these experiments.
Submitted May 30, 2000; accepted October 16, 2000.
Supported in part by a grant from the Italian Association for Cancer Research, Milano, Italy.
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: Daniel C. Thomis or Tim Clackson, ARIAD Gene Therapeutics, 26 Landsdowne St, Cambridge, MA 02139; e-mail: clackson{at}ariad.com.
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-LNGFR-2x(F36V-FKBP)-Fas-E
Val36 point mutant of human FKBP12;
F36V', codon-wobbled F36V; FAS, cytoplasmic domain
(residues 175-319) of human Fas. Sizes (in amino acids) of the
individual components are indicated. Expression of the transgenes was
driven by the MoMLV LTR using the retroviral vector pMX. (B) PCR
analysis of FKBP region in Fas-transduced NIH 3T3 cells. Fas-transduced
3T3 cells were lysed, and the integrity of the FKBP region of the Fas
transgene was analyzed by PCR. Lane 1, molecular weight standard; lane
2, control LV'VFas plasmid; lane 3, no DNA control; lane 4, untransduced 3T3 cells; lane 5, LV'V-transduced 3T3 cells; lane 6, LVVFas-transduced 3T3 cells; lane 7, LV'VFas-transduced 3T3 cells.
Arrows on the right indicate the sizes of the expected band (724 bp)
and a band truncated by approximately 300 bp.
). DNA was
digested with XbaI, separated by electrophoresis and probed
with an FKBP-Fas-specific probe. Lane 1, untransduced control
lymphocytes; lanes 2 and 3, LVVFas-transduced lymphocytes; lanes 4 and
5, donor A LV'VFas-transduced lymphocytes; lanes 6 and 7, donor B
LV'VFas-transduced lymphocytes; lane 8, control plasmid. Arrows
indicate the size of the expected band (1627 bp) and a band truncated
by approximately 300 bp.
),
LV'VFas-transduced cells analyzed after 24 hours (
), and
LV'VFas-transduced cells analyzed after 48 hours (
). Values are the
mean ± SD of duplicate points (except for LV'V). The result shown
is representative of at least 5 independent experiments. (B) Comparison
of killing of human T cells engineered with LV'VFas or
HSV-tk. Primary human T lymphocytes were transduced with
LV'VFas or SFCMM-3 (HSV-tk), immunomagnetically purified and
treated with either 10 nM AP1903 or 50 nM GCV for 5 days. Survival was
measured as described in "Materials and methods." Open bars
represent untreated cells; filled bars represent cells treated with
drug. Values are the mean ± SD of 3 independent
points.
). We then analyzed cycling and
noncycling LV'VFas-transduced T cells based on their incorporation of
[3H]thymidine. Again, there was no significant difference
between rapidly cycling and noncycling cells with respect to their
killing efficiencies (Figure 
) and antiautologous (
) and antiautologous (