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Review article
Pharmacologically regulated cell therapy
Tobias Neff and C. Anthony Blau
Introduction
The development of cell and organ transplantation is one of the
great success stories of modern medicine. Transplantation of
various cells, tissues, and solid organs has entered the realm of the
clinically routine.1-4 Milestones in cell therapy include the development of transfusion medicine, hemopoietic stem cell transplantation, and adoptive immunotherapy. Progress in cell therapy outside
hematology has been slower to develop, but recent progress has
been made in Parkinson disease5,6 and diabetes mellitus.7 Most
recently, advances in stem cell biology may presage an impending
revolution in cell therapy. Several studies have demonstrated that
the stem cells of healthy adults retain enormous plasticity. For
example, hemopoietic stem cells can differentiate into muscle,8,9
liver cells,10 and neurons11,12; neural stem cells can differentiate
into blood,13 liver, kidney, or muscle cells14; and muscle stem cells
can differentiate into blood.15 Human embryonic stem cells, which
are capable of contributing to all human tissues, have been
generated for the first time.16 These discoveries make imaginable
the prospect of developing a new generation of cell therapeutics
applicable to a vast array of diseases. However, before this vision
can be realized many obstacles must be overcome, with one of the
most important obstacles resting in the present inability to control
what happens to a cell after it has been transplanted.
Currently available tools for controlling the survival, growth,
and differentiation of transplanted cell populations are blunt
instruments. In most cases, graft-versus-host disease (GVHD) is
only poorly controlled with immunosuppressive drugs, which in
turn cause a punishing array of side effects. Lineage-restricted
growth factors such as erythropoietin and granulocyte colonystimulating factor (G-CSF) have had a major effect on clinical
hematology, but growth factors are powerless in situations in which
it is desirable to preferentially expand a subpopulation of cells with
a particular lineage. These situations may arise in the setting of
mixed chimerism, in which donor and host hemopoietic elements
coexist; or in gene therapy, in which only a fraction of cells contain
the therapeutic gene. Under these circumstances, it would be useful
to have a means of specifically influencing the in vivo behavior of
genetically modified cells in terms of proliferation, death, or
differentiation. Toward this end, conditional systems have been
developed that rely on the principle that protein interactions control
virtually all cellular processes; in particular, protein dimerization
(or oligomerization) is at the heart of many signaling pathways
controlling cell death, proliferation, and differentiation.
These conditional systems share 2 main components: a fusion
protein and a drug. The fusion protein contains an intracellular
signaling molecule linked to a protein that provides a high-affinity
binding site for a drug called a chemical inducer of dimerization
(CID). Individually the signaling domains are functionally inert.
Activation occurs upon bringing 2 signaling domains into close
proximity with one another, a process termed dimerization. Proximity is controlled through addition of the CID, which in turn
activates signaling. The signaling fusion thus serves as a switch that
is turned on in the presence of the CID and off following
withdrawal of the CID. This method has the potential to control the
behavior of genetically modified cells both in vitro and in vivo. The
generation of pharmacologically regulated alleles has important
implications for analyzing the effect of signaling molecules such as
growth factor receptors, Janus kinases (JAK), signal transducers
and activators of transcription, and others. This review focuses
primarily on potential clinical applications of dimerizer systems
using intracellular growth factor–receptor sequences.
From the Division of Hematology, Department of Medicine, University of
Washington, Seattle, WA.
Hematology Junior Faculty Scholar Award, and an award from the Fanconi
Anemia Research Fund.
Submitted September 28, 2000; accepted January 18, 2001.
Reprints: C. Anthony Blau, Mailstop 357710, Health Sciences Building, University
of Washington, Seattle, WA 98195; e-mail: [email protected].
Supported by grants 5R01DK52997, 1R01DK57525, 2P01HL53750, and
2P01DK47754 from the National Institutes of Health, an American Society of
BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
Systems for achieving controlled proximity
Systems for achieving controlled proximity should meet several
criteria before being considered for clinical use. The first set of
criteria pertains to the CID. Obviously, the CID should not exist
naturally in humans, since constitutive activation would occur, and
it should be nontoxic. For testing in experimental systems, a
monomeric counterpart of the CID provides a valuable control for
competitive-inhibition assays. The second set of criteria pertains to
the fusion protein. The fusion should be entirely human in origin
because expression of nonhuman sequences can induce clinically
relevant immunologic responses.17 The fusion should be relatively
small because of the limited packaging capacity of many genedelivery systems and the anticipated need to incorporate a second
therapeutic gene into the vector in many cases. Finally, the fusion
should not undergo constitutive dimerization and must be strictly
dependent on the presence of the CID for spatial proximity and
therefore activation.
Steroid-based systems
The creation of steroid-regulated signaling molecules was first
described in studies using the N-terminal glucocorticoid-binding
domain (GBD) of the glucocorticoid receptor.18 Fusion proteins
containing the GBD are complexed with heat shock protein 90
(hsp90), which dissociates on hormone binding.19 This in turn has
various functional consequences, depending on the protein to
which the GBD is fused. When the GBD is fused to a transcription
factor, glucocorticoid binding relieves hsp90-mediated repression
of transactivation. Additionally, hsp90 dissociation allows nuclear
© 2001 by The American Society of Hematology
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BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
NEFF and BLAU
into clinical use. Additionally, coumermycin has not been developed for clinical use, mainly because of poor bioavailability,
adverse gastrointestinal effects of its oral form, and local irritation
on parenteral administration.28
FKBP12-based systems
Figure 1. Induction of dimerization by a CID. A chimeric growth switch consisting of
receptor sequences and a dimerization domain is activated on addition of a CID. The
CID enforces dimerization by binding 2 dimerization domains on 2 neighboring
molecules with a 1:2 stoichiometry. Dimerization causes signaling from the receptor
sequences. The principle is the same for the coumermycin-GyrB system and the
FK1012-FKBP system. The molecule shown also carries a myristylation domain
derived from c-src for targeting to the inner cell membrane.
translocation of the fusion protein and self-association of GBD
domains, leading to dimerization or multimerization.20 Similar
mechanisms appear to underlie other steroid-responsive systems,
the best characterized of which is the estrogen-binding domain
(EBD). Estrogen binding to a fusion protein incorporating the EBD
also allows dimerization.21 Of note, the self-association domains of
the GBD or EBD do not rely on the drug to enforce spatial
proximity directly. Rather, the steroid binding relieves hsp90mediated repression of dimerization. This contrasts with the
coumermycin-gyrase B (GyrB)–based system and the FK1012FK506 binding protein (FKBP)–based system described below. In
these systems, a symmetric small molecule forms a bridge between
2 dimerization domains (Figure 1). The steroid-based system was
described in detail by Picard.22
In response to the anticipated concern that binding by endogenous estrogen would produce constitutive activation in vivo, a
point mutation in the hormone-binding domain that decreases the
affinity for estrogen by 3 orders of magnitude was introduced.23
This modified domain has an unaltered affinity for tamoxifen, and a
system for generating conditionally active signaling proteins based
on the mutated EBD has been described.24,25 The clinical utility of
this system must be established in vivo, since the antagonism of
endogenous estrogen receptors by tamoxifen may be undesirable in
some settings. An advantage of the steroid-based system is that it
allows creation of conditional alleles by mechanisms other than
dimerization. For instance, Heyworth et al26 demonstrated that a
conditionally active GATA-2 allele induces cell-cycle exit and
differentiation in hemopoietic cell lines and primary murine bone
marrow cells.
Coumermycin-GyrB–based systems
A dimerizer system that uses the antibiotic coumermycin has also
been developed.27 This system relies on the binding of coumermycin to 2 copies of an N-terminal 24-kd fragment derived from
Escherichia coli DNA GyrB. Novobiocin, a competitive monomer
of coumermycin, enhances the usefulness of this system in
experimental models. However, the anticipated immunogenicity of
the bacterial sequences will likely hinder transition of this system
A dimerizer system that has advantages with respect to clinical
development is based on the immunophilin FKBP12, a rotamase
that binds the lipid-soluble immunosuppressive drug FK506 and its
dimeric counterpart, FK1012.29 Although the FK506-FKBP12
complex inhibits calcineurin and thus suppresses signaling by
means of the T-cell receptor, FKBP12 in complex with FK1012 (or
any of an increasing number of chemically designed FK1012
analogues) does not inhibit calcineurin and is therefore not
immunosuppressive.
Binding of FK1012 to endogenous FKBP12 sequences is
undesirable for 2 reasons. First, endogenous FKBP12 may act as a
sink, sequestering the drug so that it cannot perform its task of
binding FKBP12 motifs in the fusion protein. Second and more
important, binding to endogenous protein might interfere with the
physiologic involvement of FKBP12 in processes such as calciumchannel function30 and sperm motility.31 FKBP12 knockout mice
were found to have severe congenital cardiomyopathy, indicating
that FKBP12 has a role in the development and perhaps the
maintenance of the myocardium.32 To prevent binding to endogenous FKBP12, a new generation of dimerizers has been developed
in which a chemical modification or “bump” has been incorporated
into the CID, thereby precluding binding to FKBP12. To accommodate binding to bumped CIDs, a hole was engineered into FKBP12
by introducing a single phenylalanine-to-valine substitution at
amino acid position 36 (now termed F36V), thereby providing
lock-and-key specificity at the FKB12-CID interface.33,34 This
approach circumvents toxicity that might arise from binding to
endogenous FKBP12.
A bumped CID, AP1903, has been tested in a phase I trial in
healthy human subjects. A single intravenous dose achieved serum
levels predicted to be effective, without producing apparent toxic
effects.66 A possible disadvantage of the F36V substitution is
generation of a T-cell epitope that might lead to immune-mediated
rejection of the transduced cells. Although this has not been
observed in murine transplantation models, immune responses
must be carefully evaluated in studies using large animals and in
clinical trials.
Pharmacologic regulation of cell growth
A method for pharmacologically regulating the growth of genetically modified cells would have several possible applications. For
example, inefficient gene transfer into stem cells is a major obstacle
in gene therapy. To circumvent this obstacle, a gene encoding a
CID-responsive protein that regulates cell growth might be linked
to a second gene that encodes a therapeutic protein. After gene
transfer, the transduced population could be selectively expanded
by using CIDs either in vitro or in vivo (Figure 2). In vivo selection
using CIDs would have particular advantages in gene therapy. In
contrast to selection methods that rely on use of drug-resistance
genes, CIDs offer the possibility of achieving selection without the
toxic effects associated with administration of cytotoxic drugs.
Additionally, selection could bypass the need for myeloablative
conditioning, thereby further improving safety of the treatment.
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BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
PHARMACOLOGICALLY REGULATED CELL THERAPY
2537
that dimerization is necessary but not sufficient for activation of
receptor signaling48 and that preformed receptor complexes on the
cell surface are activated by a conformational change on ligand
binding.49,50 The extent to which these natural mechanisms are
mimicked by CIDs is unknown.
Applications in the hemopoietic system
Figure 2. In vivo selection of hemopoietic cells by using CID-selectable genes.
The vector encodes 2 genes, a therapeutic gene and a CID-selectable gene. After ex
vivo transduction and reinfusion of hemopoietic stem cells, administering a CID in
vivo signals a proliferative stimulus targeted exclusively to the transduced cell
population, which then selectively expands.
Finally, CID-mediated selection would probably circumvent the
variable transgene expression that is attributable to position
effects,35-37 since CID-responsiveness requires expression of the
transgene.
Because growth factors provide a paradigm for controlling cell
proliferation, most efforts to regulate the growth of genetically
modified cells use the signaling domains of growth factor receptors. In theory, the simplest approach would involve use of genes
that encode naturally occurring full-length receptors, such as the
receptor for erythropoietin.38 However, this approach could not be
used clinically because the presence of endogenous ligand in vivo
induces sustained growth of cells bearing the transgene.39 Efforts to
bring cell proliferation under pharmacologic control have the
potential for clinical applicability in that growth factor receptors
can be stripped of their extracellular domains, thereby precluding
activation by endogenous ligands. Instead, the residual receptor
signaling domain is linked to motifs that render the fusion
dependent on CID. The feasibility of this approach was demonstrated by using the interleukin 3 (IL-3)–dependent murine pro-Bcell line Ba/F3. Ba/F3 cells have an especially useful property:
when forced to express a heterologous receptor, they can be
converted to dependency on the corresponding ligand. For example, expression of the erythropoietin receptor allows Ba/F3 cell
growth in erythropoietin despite the absence of IL-3.40 Similarly,
Ba/F3 cells expressing an FKBP12-erythropoietin receptor fusion
were shown to be dependent on FK1012, thereby demonstrating the
feasibility of pharmacologic control over cell proliferation.41
Similar results have been obtained with each of the dimerizer
systems described above, including signaling domains taken from
the G-CSF receptor,42,43 c-kit,44 mpl,45 flt-3, and gp130 (unpublished data). Dimerization of JAK was also shown to suffice for
Ba/F3 cell growth in the absence of IL-3.46,47 Recent data suggests
In applying pharmacologically regulated cell growth to the hemopoietic
system, stable expression of the transgene is essential. Stable expression
requires (1) vectors (such as retroviral vectors) that can stably integrate
into DNA, (2) gene transfer into a long-lived cell such as the
hemopoietic stem cell, and (3) incorporation of cis regulatory sequences
that guarantee expression in the desired cell type. In this regard,
sequences in the long-terminal repeats of murine stem cell virus–based
vectors were shown to direct expression in very early human hemopoietic cells (ie, severe combined immunodeficiency–nonobese diabetic
repopulating cells).51
In vitro studies. CID-mediated expansion of primary hemopoietic cells has been described in a few reports.34,42,45,52 After
retroviral transduction, cells are cultured in the absence of added
growth factors and in either the presence or absence of a CID. Only
a few receptors have been evaluated by using this approach, with
the most extensive studies involving the thrombopoietin receptor
mpl. Murine bone marrow cells transduced by using a vector
encoding a FKBP12-Mpl fusion failed to grow in the absence of
drug or added growth factors, with all cells dying in 7 to 14 days. In
contrast, FK1012 dramatically stimulated cell proliferation for up
to nearly a year of culture, with an initial brief phase of multilineage differentiation followed by a longer phase of preferential
megakaryocytic differentiation.45 Although mpl is capable of
inducing expansion of multipotential cells, including day-12 colonyforming units–spleen, mpl-expanded cells are incapable of longterm repopulation and show no potential for B or T lymphoid
differentiation (unpublished data). These findings are consistent
with an ability of mpl to induce self-renewal of multipotential
hemopoietic progenitor cells, which may be the functional equivalents of certain common myeloid progenitors.53 Studies using the
estrogen system found that CID-dependent dimerization of a
G-CSF receptor–EBD fusion supports the terminal differentiation
of committed murine progenitor cells.42
CID-dependent expansion of primary cells has also been
extended to CD34-selected human cord blood cells.52 After transduction, cells cultured in the presence of AP1903, a bumped CID,
expanded 13.8- to 186-fold relative to cells cultured in the absence
of AP1903, and the cell type that emerged in suspension culture
was erythroid. Contrary to results in the murine system, cell
expansion was transient. Possible reasons for the differences
between the murine and human systems are currently under
investigation.
In vivo studies. Use of the F36V-Mpl fusion for in vivo
selection of transduced murine hemopoietic cells has been reported.34 After transplantation of F36V-Mpl–transduced marrow
cells into lethally irradiated mice, administration of another
bumped CID, AP20187, produced a dramatic in vivo expansion of
transduced red cells, platelets, and to a lesser extent, granulocytes.
Although the nature of the in vivo expanded cell (committed
progenitor or transplantable stem cell) was not conclusively
determined, stem cells containing the transgene were not exhausted
by CID treatment, as indicated by their persistence in recipients of
secondary transplants. Future work must determine the ideal stage
of hemopoiesis at which selection should be applied.
Clinical applicability of CID-mediated in vivo cell expansion
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NEFF and BLAU
will ultimately depend on achieving similar results in a large
animal. In addition to demonstrating efficacy, it will be particularly
important to show that CID-induced proliferation in this model
remains strictly drug dependent in vivo. It is noteworthy that a
single amino acid substitution in growth factor receptors,54 as well
as in FKBP-containing fusion proteins, can dramatically influence
the propensity for constitutive dimerization.55 Because a myeloproliferative syndrome or even frank leukemia might result from
constitutive activation of receptor sequences, the behavior of the
growth switch must be rigorously tested in mice and large animals
before a clinical trial can be contemplated. Progress has been made
in the development of an expression cassette for the ␤-globin
gene.56 Thalassemia may be an especially attractive target for this
approach because, as noted above, CID-mediated activation of the
mpl signaling domain induces a preferential in vivo expansion of
transduced erythroid cells. In view of the relatively few therapeutic
options available to many patients with end-stage thalassemia, a
clinical trial in this patient group may eventually be warranted.
Applications outside the hemopoietic system
The low efficiency of gene transfer is also an important obstacle to
successful gene therapy for diseases originating outside the hemopoietic
system. For example, CID-mediated expansion of transduced cells
might be applied to gene therapy for liver diseases. The feasibility of
using CIDs for liver cell expansion has been demonstrated both in vitro
and in vivo57 (A. Lieber, personal communication, January 2001). Other
investigations attempted to use CIDs to expand transduced muscle cells.
In one study, a murine myoblast cell line that is normally dependent on
fibroblast growth factor 1 (FGF1) was converted to AP20187 dependency on expression of an F36V-FGF1 receptor fusion (C. Murry and
M. Whitney, personal communication, February 2001).
Pharmacologic regulation of cell death
Complementing the use of CIDs to induce growth of genetically
modified cells are situations in which CIDs might be useful for
conditionally eliminating genetically modified cells. Transfer of a death
switch or suicide gene may have applications in the treatment of
malignant and other acquired diseases. The most notable application is
in adoptive immunotherapy for relapse after allogeneic marrow transplantation. For example, relapse after allogeneic transplantation for chronic
myelogenous leukemia (CML) can be treated by adoptive transfer of
donor lymphocytes, in which the graft-versus-leukemia effect produces
complete remissions in 60% to 70% of patients.58 However, the
development of life-threatening GVHD is a major impediment to this
approach. Introduction of a suicide gene into donor lymphocytes
provides a potential method for retaining specific control over survival
of those lymphocytes after adoptive transfer.59 This strategy was shown
to be feasible in a clinical trial using a gene encoding herpes simplex
virus thymidine kinase (HSV-TK).60 HSV-TK converts the antiviral
drug ganciclovir into a toxic metabolite, thereby eliminating the
transduced cell population while sparing nontransduced cells. A major
limitation to using HSV-TK clinically is that the viral protein induces
immunologic responses, as was observed in a clinical trial.17 An
additional limitation to use of HSV-TK in patients who have undergone
marrow transplantation is that a need for ganciclovir therapy may arise
because of cytomegalovirus infection and the HSV-TK–transduced T
cells would then become victims of collateral damage.
In addition to applications in adoptive immunotherapy, suicide-gene
therapy might be useful in treating disorders in which a locally confined
cell mass must be reduced—for example, benign prostate hyperplasia.61
Malignant cells are an obvious target for the suicide-gene approach, and
many investigators have experimentally assessed this strategy for cancer
gene therapy.62 This method faces serious difficulty, however, in that
complete and selective transduction of all tumor cells in vivo currently
appears to be impossible, notwithstanding the “bystander effect” that has
been claimed for some suicide systems.
The suicide genes described above make S-phase–dependent drugs
toxic and thus target only cycling cells. FKBP12-based death switches
that theoretically should be independent of the S phase have been
developed.61,63-65 Unlike suicide systems that rely on expression of viral
proteins, CID-based death switches are entirely human in origin and
therefore have a lower probability of provoking immunogenicity. The
fas receptor belongs to the tumor necrosis factor receptor family and is
believed to signal on trimerization. Likewise, intracellular fas-receptor
sequences fused to 2 tandem-oriented dimerizer domains63,65 can
mediate controlled apoptosis in cell lines and transduced human T cells
on CID-mediated multimerization. Of note, a single FKBP domain
allows only dimerization and therefore cannot induce efficient cell
killing. One disadvantage of a fas-based system may be the highly
variable susceptibility of different cell types to fas-mediated apoptosis.61
AP1903-induced multimerization of caspases 1, 3, 8, and 9, as well as
Fadd sequences, also yields apoptosis switches, and the efficacy of these
apoptotic proteins appears to be less dependent on cell type.61,65
Phase I and phase II clinical trials are currently in preparation at
the Fred Hutchinson Cancer Research Center in Seattle and in
Milan, Italy, to evaluate a FKBP-fas fusion suicide gene in the
setting of adoptive immunotherapy. The fusion is labeled with an
extracellular low-affinity nerve growth factor receptor (NGFR) tag
to allow transduced cells to be selected by using an anti-NGFR
antibody. In vitro, this death switch can induce apoptosis of a
considerable proportion of transduced lymphocytes after only an
hour of exposure to AP1903.67 Patients who have relapse after
allogeneic bone marrow transplantation for CML will receive
donor leukocyte infusions with genetically altered lymphocytes
containing the novel death switch. Administration of AP1903 in
this situation is expected to eliminate the modified lymphocytes in
vivo and thus serve as a specific treatment for severe GVHD.
Future prospects
Studies using murine bone marrow have suggested that different
receptor sequences produce different outcomes (unpublished data).
A question that must be resolved is whether desired cell types can
be specifically elicited by introducing the appropriate signal. For
instance, mpl sequences allow preferential expansion of erythroid
cells and, to a lesser extent, megakaryocytes. Which signaling
domains would allow controlled expansion of myeloid cells? Is it
possible to equip hemopoietic or even more primitive stem cells
with a receptor or other signaling molecule that preferentially
induces their in vivo differentiation toward a particular cell type or
hemopoietic lineage?
Because of the plasticity of stem cells derived from bone marrow,
delivering the right signal at the right time to these cells may enhance
their contribution to tissues such as liver10 and muscle.8,9 Delivery of the
signal in vivo might allow subsequent local expansion of these cells to a
therapeutically relevant proportion. In addition to liver and muscle cells,
the system could be used for other nonhemopoietic cell types, such as
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BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
PHARMACOLOGICALLY REGULATED CELL THERAPY
dopaminergic cells, for treatment of Parkinson disease; or ␤ islet cells,
for the treatment of diabetes mellitus—diseases for which a shortage of
available donors currently creates major limitations to cell therapy.5,6
Finally, with the development of human embryonic stem cells, it may
eventually be possible to engineer virtually every tissue to replace failing
organs. Many of these strategies face important technical obstacles and
raise ethical and regulatory questions beyond the scope of this review.
However, the feasibility of pharmacologic regulation of genetically
2539
modified cell populations in vivo has been demonstrated.34 The stage is
now set for a wide range of potential applications.
Acknowledgments
We thank T. Papayannopoulou and J. Abkowitz for helpful
comments and suggestions on the manuscript.
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2001 97: 2535-2540
doi:10.1182/blood.V97.9.2535
Pharmacologically regulated cell therapy
Tobias Neff and C. Anthony Blau
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