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From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
Perspectives
Preclinical models of acute and chronic graft-versus-host disease:
how predictive are they for a successful clinical translation?
Robert Zeiser1,* and Bruce R. Blazar2,*
1
Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Freiburg, Germany, and 2Department of
Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN
Despite major advances in recent years,
graft-versus-host disease (GVHD) remains
a major life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). To improve our therapeutic
armory against GVHD, preclinical evidence
is most frequently generated in mouse
and large animal models of GVHD. However, because every model has shortcomings, it is important to understand how
predictive the different models are and
why certain findings in these models could
not be translated into the clinic. Weaknesses of the animal GVHD models include the irradiation only-based conditioning
regimen, the homogenous donor/recipient
genetics in mice, canine or non-human
primates (NHP), anatomic site of T cells
used for transfer in mice, the homogenous
microbial environment in mice housed
under specific pathogen-free conditions,
and the lack of pharmacologic GVHD prevention in control groups. Despite these
major differences toward clinical allo-HCT,
findings generated in animal models of
GVHD have led to the current gold standards for GVHD prophylaxis and therapy.
The homogenous nature of the preclinical
models allows for reproducibility, which is
key for the characterization of the role of
a new cytokine, chemokine, transcription
factor, microRNA, kinase, or immune cell
population in the context of GVHD. Therefore, when carefully balancing reasons to
apply small and large animal models, it
becomes evident that they are valuable tools
to generate preclinical hypotheses, which
then have to be rigorously evaluated in the
clinical setting. In this study, we discuss
several clinical approaches that were motivated by preclinical evidence, novel NHP
models and their advantages, and highlight
the recent advances in understanding the
pathophysiology of GVHD. (Blood. 2016;
127(25):3117-3126)
Introduction
Our understanding of the roles of the innate immune system, the
adaptive immune system, and different epithelial and antigenpresenting cell types in graft-versus-host disease (GVHD) pathogenesis has made major advances over the last 2 decades. Despite
these advances and the prophylactic treatment with a wider array of
immunosuppressive medication, ;50% of the patients undergoing
allogeneic hematopoietic cell transplantation (allo-HCT) develop
grade 2-4 acute GVHD (aGVHD).1 aGVHD patients who are refractory to standard steroid treatment have a dismal long-term
prognosis with only 5% to 30% overall survival (OS).2-4 Chronic
GVHD (cGVHD) causes high morbidity, reduces the quality of life,
and is associated with a significantly higher risk of treatment-related
mortality and inferior OS.5 Clinical experience teaches that aGVHD
and cGVHD in humans are multilayer diseases, which are hard to
treat once they are fully established. The immunologic complexity
of the disease and the role of donor and recipient cell types has been
the focus of intensive research.6-10
In this review we discuss different prophylactic and therapeutic approaches against aGVHD and cGVHD that have been
developed in preclinical models and analyze how successful these
approaches were later in clinical trials. We divide the preclinical
approaches into pharmacologic and cellular therapy strategies
and connect them to the resulting clinical studies. Additionally,
promising novel approaches in mice and non-human primate
(NHP) models of GVHD that have not yet entered clinical studies
will be discussed.
Submitted February 9, 2016; accepted March 15, 2016. Prepublished online
as Blood First Edition paper, March 18, 2016; DOI 10.1182/blood-2016-02699082.
BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25
Pharmacologic prophylaxis and therapy
of aGVHD
Basics of aGVHD prophylaxis and therapy
The basic pharmacologic aGVHD prophylaxis with cyclosporine A
(CyA) and methotrexate (MTX) that is still used in a large proportion
of the currently applied immunosuppressive regiments following
allo-HCT was first studied in the dog model, where it showed potent
inhibitory effects on aGVHD.11 The studies in dogs were followed
by a clinical study that revealed that the combination of CyA and
MTX was superior to CyA alone with respect to protection from
GVHD and survival, and therefore the calcineurin inhibitor CyA
became the gold standard for GVHD prophylaxis.12 Later, the combination of tacrolimus and MTX after unrelated allo-HCT was
shown to significantly decrease the risk for aGVHD, but not OS and
relapse-free survival rates compared with CyA and MTX,13 and
therefore both CyA and tacrolimus are the backbone of most immunosuppressive regimens for patients currently undergoing alloHCT worldwide. Another established component for aGVHD
prophylaxis is anti-thymocyte globulin (ATG), which was initially
reported to be protective against GVHD in the canine model.14
Different types of ATG exist and for rabbit anti-thymocyte globulin
Fresenius (ATG-F), a randomized, open-label, multicenter phase 3
trial was performed that showed a decreased incidence of aGVHD
and cGVHD without an increase in relapse or nonrelapse mortality
*R.Z. and B.R.B. are equal contributors to this study.
© 2016 by The American Society of Hematology
3117
From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
3118
ZEISER and BLAZAR
(NRM) when ATG-F was added to the standard GVHD prophylaxis.15
This was extended recently by a multicenter trial showing that
the rate of a composite end point of cGVHD-free survival and
relapse-free survival was higher with ATG.16 Besides ATG-F,
thymoglobulin was also shown to be protective against GVHD. 17
Steroids currently represent the gold-standard treatment of aGVHD
based on multiple prospective trials.18,19 Early evidence supporting the use of corticosteroids against aGVHD was provided in
the 1990s in a haploidentical parent into F1 mouse models of
GVHD.20,21
Cytokine and chemokine inhibition as acute
aGVHD prophylaxis and therapy
To avoid the broad spectrum of side effects caused by corticosteroids and to be able to offer a therapeutic option for patients
with GVHD that had failed corticosteroids, the role of multiple
cytokines in the pathophysiology of aGVHD was investigated
in the mouse model. Interleukin-11 (IL-11),22,23 IL-1b,24 tumor
necrosis factor-a (TNF-a),25,26 and IL-627,28 among others were
targeted in mouse models of GVHD leading to later clinical studies.
In the mouse model of GVHD, IL-11 promoted T-cell polarization
toward a T helper 2 (Th2) phenotype, was associated with a lower
level of IL-12, and reduced GVHD-related mortality.22,23 Consequently, recombinant human IL-11 was then investigated in a
phase 1/2 double-blind, placebo-controlled study for mucositis and
aGVHD prevention.29 Of 10 evaluable patients who received IL-11
in this trial, 4 died by day 40 and 1 died on day 85 due to transplantrelated toxicity. 29 The major adverse side effect in patients
receiving IL-11 was severe fluid retention that caused pulmonary
edema.29 This trial was not able to determine whether IL-11 given in
this schedule can reduce the rate of GVHD. The unexpected high
mortality showed that a cytokine that was well-tolerated by the mice
induced severe side effects in humans, sounding a note of caution
for investigators translating findings from preclinical models
into a trial on humans. IL-1b was shown to be a proinflammatory
cytokine in intestinal inflammation,30 to be released upon tissue
damage causing Nlrp3 inflammasome activation,31,32 which is connected to impaired suppressor function of myeloid-derived suppressor cells33 and to promote the severity of aGVHD in the mouse
model.24 Conversely, other studies in major histocompatibility
complex (MHC) disparate mouse models showed only a very
modest effect of IL-1 antagonists.34 Although initial studies using
IL-1 antagonism in the therapeutic setting suggested a benefit
for patients suffering from GVHD,24,35 the later prospective,
randomized, controlled trial failed to show a benefit from IL-1
blockade administered from day 24 to day 110 relative to alloHCT.36 In different mouse models of GVHD, TNF-a antagonism
reduced GVHD severity.37-39
The murine studies showed that TNF-a was derived from donor
T cells, regulated by microRNA-146a/TRAF638 and myeloid
cells.25 Mechanistically, TNF-a was shown to directly damage the
intestinal epithelium25,26 and to downmodulate the function of
regulatory T cells (Tregs),39 which protect against GVHD.40-42
Consequently, clinical studies using TNF-a antagonism with
etanercept43 or infliximab44 in the therapeutic setting against GVHD
were performed. Infliximab in addition to steroids reduced GVHD
severity; however, the reported NRM was unexpectedly high.44
Etanercept given as a combination therapy with inolimomab
(anti–IL-2Ra) for the treatment of steroid-refractory aGVHD yielded
BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25
a response rate of 48%.43 However, the estimated rates of 6-month and
2-year OS were 29% and 10%, respectively, leading the authors to
conclude that the combination failed to improve the dismal prognosis
of severe steroid-refractory aGVHD.43 These unfavorable results of
TNF-a blockade are connected to a high NRM and relapse, which is
consistent with reports showing a high incidence of fungal infections45
and reduced graft-versus-leukemia (GVL) effects46 when TNF-a
is antagonized. These findings could have been predicted by mouse
studies as TNF antagonism reduced GVL effects against P815 cells.25
IL-6 blockade was shown to potently reduce aGVHD in mice27,28 and
with the availability of the IL-6R antagonist tocilizumab, a prospective
single-institution phase 1/2 aGVHD prophylaxis trial was performed.47
This study showed an incidence of grade 2-4 aGVHD in patients treated
with tocilizumab at day 100 of 12%, which is lower than expected.47
These results are very promising, and several controlled trials assessing
tocilizumab in addition to standard GVHD prophylaxis or as GVHD
therapy are currently active (www.clinicaltrials.gov: #NCT01757197,
#NCT02447055, #NCT02206035, and #NCT02057770).
Besides cytokines that promote the activation of T cells,
chemokines that guide the migration of T cells toward GVHD target
organs were identified as a pharmacologic target in mouse models of
aGVHD.48,49 However, this strategy is seen controversial as high
radiation can break the principles of chemokine-mediated selected
tissue migration and trapping. For example, CCR5 inhibition was
protective against GVHD in a non-irradiated GVHD mouse model,48
whereas in the presence of total body irradiation (TBI) an earlier time
to onset and a worsening of GVHD was observed when CCR52/2
T cells were used.50 This knowledge was later applied in a GVHD
prophylaxis setting where a single institution phase 1 trial reported
that CCR5 inhibition prevents aGVHD of the liver and gut before
day 100,51 which has to be confirmed in a randomized prospective
multicenter study. However, recent data using CCR5 inhibition in
the setting of reduced-intensity conditioning revealed no protection
from GVHD.52 Therefore, the potential efficacy of CCR5 inhibition
may be context dependent and has yet to be fully tested. Inhibition
of T-cell egress from the lymph node53,54 and dendritic cell (DC)
migration55 was potently inhibited by the sphingosine 1-phosphate
receptor agonist FTY720 in the mouse model of GVHD, a therapeutic
concept that is currently investigated by using KRP-203, the sphingosine 1-phosphate receptor type 1 agonist,56 in a clinical study on patients
undergoing allo-HCT (www.clinicaltrials.gov: #NCT01830010) with
the advantage that upon discontinuation of the drug, T-cell effector
function can be unleashed from suppression,57 as has been demonstrated for rodent T-effector responses in allo-bone marrow (BM)
transplantation.18
Besides blocking T-cell migration, the co-stimulation of T cells was
recognized as a potential powerful target during aGVHD. In the 1990s,
it was shown that CTLA4-immunoglobulin (Ig) reduces lethal murine
GVHD,58 which later motivated a trial showing that CD28:CD80/86
co-stimulation blockade with abatacept leads to low GVHD rates.59
The opponent of CTLA4-Ig, the CTLA-4 blocking antibody ipilimumab
was recently applied in the posttransplant setting for patients with
refractory malignancies and did not lead to an unacceptable high
GVHD rate when given at a median of 1 year (range, 125-2368 days)
after the last allogeneic cell infusion.60 Recent studies on another
negative regulator of T-cell activation, namely programmed death-1
using checkpoint inhibition showed promising results in the mouse
model61,62 that should be further investigated in the clinic.
The multiple approaches developed from the mouse model into a
clinical application for aGVHD are summarized in Figure 1 and the
summary of translation of each application is provided in Tables 1
and 2.
From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25
CLINICAL TRANSLATION OF GVHD MODEL-DERIVED DATA
Chemokine-R inhibitors
(marivaroc)
CTLA-4
agonists Cytokines
TCR
Chemokine
receptors
e.g. CCR5
Sphingosin-1P-R
agonists
3119
Cytokine antagonists
(infliximab, eternacept)
Cytokine receptor
antagonists (daclizumab,
basiliximab, tocilizumab,
anakinra, inolimomab)
CTLA-4
Cytokine-R
Calcineurin
inhibitors
(Tac, CyA)
T cell depletion
(ATG-F, thymoglobulin,
ECP, cyclophosphamid)
S-1P-R
Calcineurin
NFAT
(inactive) P
T cell polarization
(IL-11, statins)
P
mTOR
G2
Immune modulatory
cells (MSCs, Treg,
rapamycin induced Th2)
-GalCer => NKT, Treg
NFAT
(activ)
G1
Cell
cycle
M
T cell
proliferation
mTOR inhibitors
(sirolimus,
everolimus)
Inhibitors of cell
proliferation
(MTX, MMF)
Corticosteroids
S
Transcription
of cytokine
Histone
genes
deacetylase
Tissue protection
& regenration
(KGF/palifermin)
Janus kinase 1, 2
inhibitors (ruxolitinib)
ITAMs Inhibitory
signal
Proinflammatory
cytokines
HDAC inhibitors
(vorinostat)
Figure 1. aGVHD. Simplified sketch showing the mode of action of multiple immunosuppressive strategies that were all developed from animal models into a clinical
application for aGVHD. Ab, antibody; ECP, extracorporal photophoresis; ITAM, immunoreceptor tyrosine-based activation motif; MMF, mycophenolate mofetil; MSC,
mesenchymal stroma cells; mTOR, mammalian target of rapamycin complex; NKT, natural killer T cells.
Targeting multiple layers of aGVHD
In contrast to the approaches against GVHD that aim at targeting T cells
or their cytokines, other studies were performed in the 1990s that aimed
at improving the regeneration of the epithelial barrier by using a growth
factor called keratinocyte growth factor (KGF).63,64 KGF reduced
aGVHD in mouse models as shown by different groups.63,64 However,
the survival benefit did vary between the different reports, raging from a
modest improvement of the survival63 to highly protective effects.64
The drug palifermin did not reduce aGVHD in patients, but it did reduce
the need for parenteral nutrition after TBI.65,66 The concept of enhancing epithelial regeneration via stimulation of intestinal stem cells, eg, via
R-spondin-167 is still actively investigated and may have the advantage
of sparing GVL effects as donor T cells are not blocked.
Another approach aimed at leaving effector T cells that mediate
GVL effects intact was based on studies in the mouse model of aGVHD
showing that memory CD41 T cells cause less or almost no aGVHD
but mediate GVL effects.68,69,113 The clinical study showed that the
use of naive T-cell–depleted stem cell grafts with tacrolimus only
suppression was connected to the same incidence of but more steroid
responsive aGVHD, along with a markedly reduced cGVHD incidence, the latter consistent with rodent studies of Tn depletion
in a cGVHD model.70,113 An early study performed in 1991 in mice
showed that photoinactivation of T-cell function with psoralen
and UV radiation suppresses aGVHD.71 Meanwhile, extracorporal
photophoresis has become an important treatment option for patients
with aGVHD.72,73 Statins that inhibit the rate-limiting enzyme of
the L-mevalonate pathway were shown to reduce farnesyl- and
geranylgeranyl-residues that are required for the correct attachment
of different small GTPases to the cell membrane and thereby modulate
the allogeneic immune response.74 Consistently different independent
groups could show that statins reduce aGVHD in mouse models.75,76
In patients undergoing allo-HCT, some studies showed that statin
intake by the donor77 or host78 was connected to a reduced GVHD
incidence,77-79 whereas another trial showed that the addition of
atorvastatin to standard aGVHD prophylaxis did not provide a benefit
with respect to GVHD rates.80 Comparable to statins that have a broad
range of inhibitory effects, histone deacetylase (HDAC) inhibitors were
shown to modify multiple layers of the allogeneic immune response.
Mechanistically, it was shown that not only the phenotype of T cells
was polarized toward Tregs but also that DCs treated ex vivo with
HDAC inhibitors displayed increased expression of indoleamine 2,3dioxygenase, which reduces both DC and T-cell function.81 Consistently, administration of the HDAC inhibitor vorinostat in combination
with standard aGVHD prophylaxis in a phase 1/2 study after relateddonor reduced-intensity conditioning allo-HCT was associated with a
relatively low incidence of grade 2-4 aGVHD by day 100 of 22%.82
Signaling of multiple cytokine receptors requires intact Janus
kinase 1/2 (JAK1/2) activity (Figure 1) and different groups could
show that pharmacologic inhibition of JAK1/2 reduced aGVHD in
the mouse.83,84 In a retrospective survey, 19 stem cell transplant
48,49
53,54
Anti-CCR5 antibody treatment protects against aGVHD-related mortality (1999, 2003)
The sphingosine 1-phosphate receptor agonist FTY720 reduces GVHD (2003, 2007)
92
CP, cyclophosphamide; DLA, dog leukocyte antigen.
GVHD severity in mice (2014)95
93,95
a-GalCer reduces GVHD (2005)
CP can induce tolerance toward skin allografts (1989)93 and posttransplant CP reduced
87
83,84
81
75,76
Proteasome inhibition with bortezomib reduces GVHD (2004)
JAK1/2 inhibition reduces aGVHD (2014, 2015)
HDAC inhibition reduced GVHD severity in mice (2008)
Statins reduce GVHD severity (2007, 2009)
71
68,69,113
63,64
multi-institutional trial
Posttransplantation CP is effective as single-agent aGVHD prophylaxis (2014). Open label
RGI-2001 is tested for GVHD prevention (2016). Phase 1/2 trial
Phase 1 trial (2009); prospective phase 1/2 trial (2012)
Short-course, bortezomib-based GVHD prophylaxis yields low aGVHD rates (2009, 2012).
Retrospective analysis
JAK1/2 inhibition reduces aGVHD in patients refractory to multiple therapies (2015).
Phase 1/2 trial
of severe aGVHD (2014).
Vorinostat in combination with standard GVHD prophylaxis is associated with a low incidence
Retrospective analysis (2010); prospective phase 2 trial, donor and recipient treatment (2013)
Statin intake is connected to reduced GVHD incidence in patients (2010, 2010, 2013).
prospective studies for ECP (2015)
ECP is an effective therapy for aGVHD (2000, 2015). Pilot study (2000); meta-analysis of
Single-arm, 2 site clinical trial
incidence though more steroid responsiveness (2015).
Naive T-cell–depleted stem cell graft transfer is connected to less cGVHD and no change aGVHD
analysis (2013)
after TBI (2013). Radomized, double-blind, placebo-controlled trial (2012); retrospective
Palifermin does not reduce aGVHD severity (2012) but there is a need for parenteral nutrition
Single-arm feasibility study
CD28:CD80/86 co-stimulation blockade with abatacept leads to low GVHD rates (2013).
open-label phase 1/2 study
Active clinical study on KRP203 in patients undergoing allo-HCT (2016). Randomized,
institution trial
CCR5 inhibition prevents aGVHD of liver and gut before day 100 (2012). Phase 1/2 single
institution trial
Early IL-6 inhibition with tocilizumab leads to a low risk of aGVHD (2014). Phase 1/2 single
study (2009); retrospective analysis (2011)
Infliximab and corticosteroids are effective as initial treatment of GVHD. Prospective phase 3
placebo-controlled study
IL-1 antagonist is not effective in the GVHD prophylaxis setting (2002). Phase 3 prospective
controlled study
IL-11 leads to increased mortality in patients (2002). Phase 1/2 double-blind, placebo-
multicenter randomized trial (1998); retrospective analysis (2009)
Corticosteroids are effective as first-line therapy for aGVHD in patients (1998, 2009). Phase 3
Phase 3 prospective, randomized trials (2009, 2016)
Different types of ATG reduce the risk of acute and cGVHD in patients (1979, 2009, 2016).
randomized trial
MTX and CyA is superior to CyA alone for GVHD prophylaxis (1986). Phase 3 prospective,
Main conclusion from the clinical trials (y)
97,98
51
88,89
85
82
77-79
72,73
70
65,66
59
57
129
47
44,128
36
29
18,19
15,16,127
12
Reference
ZEISER and BLAZAR
(1991)
Photoinactivation of T-cell function with psoralen and UV radiation suppresses GVHD in mice
Memory CD41 T cells cause less aGVHD and cGVHD (2003, 2007, 2009)
KGF reduces but does not uniformly eliminate GVHD lethality in mice (1998, 1999)
58
27,28
IL-6 blockade reduces aGVHD in mice (2009, 2011)
CTLA4-Ig reduces lethal murine GVHD (1994)
25,26
TNF-a antagonism reduces GVHD (1999, 2003)
24
22,23
IL-11 downregulated IL-12, and reduced aGVHD-related mortality (1998, 1999)
IL-1 blockade reduces GVHD in mice in some but not all models (1991)
20,21
14
ATG prevents GVHD in DLA haplotype mismatched littermate dogs (1979)
Corticosteroids reduce GVHD severity in mice (1990, 1995)
11
Reference
CyA and MTX reduce GVHD in the canine GVHD model (1982)
Main conclusion from the preclinical model of GVHD (y)
3120
Table 1. Translation of immunosuppressive strategies from animal models of GVHD into clinical trials
From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25
From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25
CLINICAL TRANSLATION OF GVHD MODEL-DERIVED DATA
3121
Table 2. Translation of cellular therapies from aGVHD mouse models into clinical trials
Main conclusion from the mouse model of GVHD
Reference
Clinical trial
Reference
Treg transfer reduces GVHD (2002, 2003)
40,42,100
Treg transfer is associated with low GVHD rates (2011, 2011, 2016). Open
101-103
label and phase 1 trials
Th2 cells generated by rapamycin exposure cause
107
GVHD protection (2005)
Rapamycin-resistant donor CD41 Th2/Th1 transfer after allo-HCT is
108
well-tolerated and connected to low aGVHD day 100 (2013). Phase 2
clinical trial
MSCs reduce GVHD (2012)
130
MSC reduces GVHD in open-label studies and phase 2 trials (2004, 2014)
centers in Europe and the United States reported their data on the
use of the JAK1/2 inhibitor ruxolitinib for steroid refractory GVHD.85
The overall response rate was 81.5% (44/54) in steroid refractory
aGVHD including 25 complete responses (46.3%), whereas for steroid
refractory cGVHD the overall response rate was 85.4% (35/41), consistent with data in a cGVHD mouse model.85 Ruxolitinib will be investigated in a prospective trial in Germany (#NCT02396628) and a
clinical trial using the JAK1 selective inhibitor INCB39110 has
begun for the treatment of GVHD (#NCT02614612). Potential
differences between JAK1 and JAK2 inhibition include a potentially
lower risk of cytopenia when only JAK1 is inhibited, although this
may come with a reduced efficacy as JAK2 inhibition alone was
shown to reduce GVHD. Also, a recent study suggests that topical
ruxolitinib suppresses GVHD and protects skin follicular stem cells,
whereas topical corticosteroids inhibit skin stem cells and niche
pre-adipocytes.86 A promising approach to reduce aGVHD is the
proteasome inhibitor bortezomib that was shown to reduce aGVHD
in the mouse model.87 Mechanistically bortezomib inhibits nuclear
factor-kB, thereby reducing inflammatory protein production. Clinical studies using a short-course, bortezomib-based GVHD prophylaxis yielded low aGVHD rates.88,89
a-GalCer is a glycolipid that functions as a CD1d ligand. Because
a-GalCer was found to expand and activate natural killer T cells, and
subsequently Tregs,90 it was developed as an immune modulator.
Preclinical models have demonstrated efficacy of a-GalCer in many
autoimmune disorders and GVHD.91,92 Although inhibition was found
when the N-acyl variant C20:2 was used, another form of a-GalCer
exacerbated GVHD.91 Based on these preclinical studies, the liposomal
formulation of a-GalCer named RGI-2001 is currently investigated in a
phase 1/2a, open-label, multicenter, dose-escalation study for patients
undergoing allo-HCT (www.clinicaltrials.gov: #NCT01379209).51
An important observation made in murine BM chimera was that
CP given on day 2 after transplantation induced tolerance toward
skin allografts.93 The authors concluded that the form of CP posttransplantation conditioning most likely decreased the number of
MHC-alloreactive T cells.93 Consistent with that concept, analyses
of T-cell receptor Vb subunits that recognize endogenous superantigens in disparate murine allo-combinations showed that CP
deletes alloreactive T cells.94 Posttransplant CP reduced aGVHD
severity in mice in a Treg-dependent manner, which was shown by
using transgenic mice in whom Foxp31 Tregs can be selectively
depleted.95 In patients, it was shown that posttransplant CP is highly
effective in preserving human Tregs, and in aGVHD prophylaxis96
when used in combination with sirolimus or as a single agent.97-99 The
multiple approaches developed from the mouse model into a clinical
application for aGVHD are summarized in Figure 1.
A goal of preclinical models is to develop new approaches to
prevent and treat GVHD. Although numerous reagents and cell
therapies have progressed from preclinical studies into clinical
trials, these were predominantly phase 1 and 2 studies. Alternatively,
some approaches have been moved into the clinic based upon
biological underpinnings and targets without in vivo preclinical
110,111
testing. Although both approaches have merit and the true predictive
value of either for successfully completing phase 3 studies or changing
practice has yet to be determined, there are as yet a paucity of examples
in which uniformly negative data in preclinical models have proven to
be robustly positive in clinical studies. However, it is also true that welldesigned phase 1 and 2 clinical trials are fundamentally important in
deciding whether and how to best move forward new advances in
GVHD prevention and therapy.
Cellular approaches to prevent or treat aGVHD
translated from the mouse into the clinic
In contrast to pharmacologic approaches against aGVHD, that are by
their nature short lived unless a state of deep tolerance is acquired
during drug therapy, the transfer of a tolerogenic cell population that
persists in the body could ideally lead to long-term tolerance. To exploit
this concept, the transfer of tolerogenic Foxp31 Tregs in mice was
performed and led to an impressive reduction of aGVHD.40,42,100
Clinical studies using Treg transfer in the prophylactic setting was
found to be connected to low aGVHD rates and adequate immune
reconstitution.101-103
The administration of mogamulizumab besides reducing adult
T-cell leukemia cells also induced prolonged suppression of normal
Tregs.104 Consistent with a suppressive role of Tregs against GVHD in
patients undergoing allo-HCT, pretransplant use of mogamulizumab
induces severe aGVHD.105 This observation supports the clinical
relevance of the finding in the mouse model that Tregs are potent
suppressors of GVHD.
Rapamycin (sirolimus) was shown to be more potent in suppressing
conventional T-cell expansion compared with Treg expansion due to
differential dependence on mammalian target of rapamycin complex/
Akt expanded Treg cells106 and to polarize T cells toward a Th2
cytokine profile that was protective against aGVHD in mice.107
Motivated by these and other preclinical murine studies, a phase 2
multicenter clinical trial of ex vivo expanded rapamycin-resistant
donor CD41 Th2/Th1 cells after allogeneic-matched sibling donor
HCT was performed.108 The cumulative incidence probability of
aGVHD was 20% and 40% at days 100 and 180 post–allo-HCT
indicating a potential benefit of this strategy that will have to be
compared with other immunosuppressive interventions in future
studies. Another cell population that holds promise to protect from
aGVHD are MSCs, based on findings in a humanized mouse model
of T-cell activation.109 However, the mouse data are controversial
and differ between groups, which was also the case for the later
studies in humans, as MSCs reduced aGVHD in some trials and a
randomized trial failed to show a benefit against aGVHD for patients
undergoing allo-HCT.110,111 The reported controversies could be
due to the differences in the preparation process of the MSCs, as well
as the time point of transfer.
From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
3122
BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25
ZEISER and BLAZAR
BCR
SYK inhibitors
(fostamatinib
entospletinib)
BTK inhibitors
(ibrutinib)
B cell depletion
(rituximab)
BTK
CD20
Cytokine-R
SYK
ITK
Janus kinase 1, 2
inhibitors (ruxolitinib)
Immune modulatory
cells (MSCs, Treg)
transfer.
Expand Treg by
retinoids (AM80G)
or IL-2.
mTOR
Blockade of T cell help:
Cacineurin inhibitors,
MMF, mTOR inhibitors
corticosteroids,
cyclophosphamide
T cell
G2
Ab
production
G1
B cell
proliferation
Cell
cycle
M
mTOR inhibitors
(sirolimus,
everolimus)
Inhibitors of cell
proliferation
(MTX, MMF)
S
Supportive
cytokines
(e.g. IL-4)
Figure 2. cGVHD. Simplified sketch showing the mode of action of multiple immunosuppressive strategies that were all developed from animal models into a clinical
application for cGVHD. BCR, B-cell receptor; M, M phase; S, S phase; SYK, spleen tyrosine kinase.
Pharmacologic prophylaxis and therapy of
cGVHD translated from the mouse into
the clinic
In a mouse model of cGVHD, it was shown that animals lacking Bruton
tyrosine kinase (BTK) in B cells or IL-2 inducible kinase in T cells did
not develop cGVHD, indicating that these molecules play a central role
in the pathophysiology of cGVHD.134 In addition to the findings in the
cGVHD mouse model, activation of T and B cells from patients with
active cGVHD was inhibited by BTK and IL-2 inducible kinase
blockade by ibrutinib.134 Based on these data, a multicenter open-label
phase 1b/2 study of ibrutinib in GVHD is being performed.112 Based on
a potent inhibitor effect of ruxolitinib in an aGVHD mouse model,83
patients with cGVHD having failed multiple previous therapies were
treated with the JAK1/2 inhibitor and yielded a response rate of
more than 80%85; however, these results need to be confirmed in a
prospective trial. Studies in mice showed that IL-2 is critical for Treg
expansion, activity, and survival during GVHD,41 which was later
followed by a phase 1/2 study showing that exogenous IL-2 increases
Treg numbers and improves disease in patients with cGVHD. 114
A central role for B cells in the pathogenesis of cGVHD was shown
by studies in a mouse model of non-sclerodermatous cGVHD,115
which motivated the successful use of the B-cell–depleting antibody
rituximab in patients with cGVHD.116,117 Posttransplant CP applied as
described above was shown to reduce rates of cGVHD. Additionally,
the synthetic retinoid tamibarotene (AM80G) was found to reduce skin
scores and pathology of cGVHD in a mouse model,118 and has been
Table 3. Translation of immunosuppressive strategies from animal models of cGVHD into clinical trials
Main conclusion from the mouse model of cGVHD
BTK inhibition reduces cGVHD in mice (2014)
IL-2 is critical for Treg expansion, activity, and survival during
Reference
Clinical trial
134
BTK inhibition reduces cGVHD in patients (2016). Phase 1b/2 study
112
41,131
Exogenous IL-2 increases Treg numbers and improves disease in
114
115
The B-cell–depleting antibody rituximab is effective in patients with
GVHD (2004, 2006)
B cells play a central role in the pathogenesis of cGVHD (2006)
Reference
patients with cGVHD (2011). Phase 1/2 study
116,117
cGVHD. Retrospective analysis (2003); phase 1/2 study (2006)
JAK1/2 inhibition reduces cGVHD in the mouse (2015,
85
Supplemental Figures)
Syk inhibition reduces cGVHD in the mouse (2014, 2015)
JAK1/2 inhibition reduces cGVHD in patients refractory to multiple
85
therapies (2015). Retrospective analysis
124,125,132
Open clinical trials for Syk inhibitors (fostamatinib, entospletinib) (2016)
135
From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25
CLINICAL TRANSLATION OF GVHD MODEL-DERIVED DATA
3123
Table 4. Translation of cellular therapies from cGVHD mouse models into clinical trials
Main conclusion from the mouse model of GVHD
Treg transfer reduces cGVHD in mice (2007)
Reference
Clinical trial
Reference
133
Treg transfer in human cGVHD (2015). Open label trial
119
tested consecutively in a phase 2 trial for cGVHD (#UMIN 000020363)
in Japan. The multiple approaches developed from the mouse model into
a clinical application for cGVHD are summarized in Figure 2 and the
summary of translation of each approach is provided in Tables 3 and 4.
Cellular therapy approaches against cGVHD
Because Tregs were shown to reduce aGVHD in the mouse
model,40,42,100 investigators used human donor Tregs that were cultivated for 7 to 12 days and then given to patients with cGVHD.118
Two of 5 patients showed a clinical response with improvement of
cGVHD symptoms and 3 patients showed stable cGVHD symptoms
for up to 21 months.119
Trials are in progress and planned to extend these studies, and to
incorporate low-dose IL-2 to treat cGVHD resistant to conventional
therapies.
Novel promising targets and approaches
In order to generate hypotheses that match the human situation as
closely as possible, recent studies on novel targets for GVHD, the RNA
expression profiles of CD31 T cells from NHP with aGVHD were
analyzed.20 The study included cohorts of allo-HCT as an untreated
control and recipients were given autologous HCT or allo-HCT with no
immunoprophylaxis, sirolimus monotherapy, or tacrolimus-MTX.20
The authors found that aurora kinase A was more abundant in the
GVHD group, and then directly applied this knowledge in the mouse
model of GVHD where they could show that pharmacologic inhibition
of aurora kinase A reduced GVHD severity. One strength of this study
lies in the fact that the NHP used were evolutionary closer to resembling
humans and therefore, the identified targets will most likely match the
human situation much better than targets found in mice developing
GVHD. Another potent strategy of GVHD prevention is the common
g-chain blockade,120 as this chain is a subunit of the receptors for IL-2,
IL-4, IL-7, IL-9, IL-15, and IL-21. However, although GVHD is
reduced, the effect on T cells that reject allogeneic leukemia cells needs
to be considered, making this approach most interesting for patients
undergoing allo-HCT for nonmalignant diseases. Other lines of
promising future translation for aGVHD are: besides others, natural
killer T-cell therapy,121 IL-22 proteins that help to protect the intestinal
stem cell mice,122 and modifications of the microbiome.123 cGVHD
promising approaches include among others: Syk inhibitors that
were shown to reduce disease severity in the mouse model124,125 and
are currently developed into a clinical trial, BTK inhibition,
targeting of IL-21, and B-cell activating factor that may help to
control this major complication after allo-HCT.
Conclusion
It is unlikely that the mouse model will ever fully reflect the human
situation even if chemotherapy based conditioning, minor antigen
mismatch models, and granulocyte colony-stimulating factor mobilized
peripheral blood stem cells will be used in humanized mouse
models, because the situation in humans is much more complex, in
particular with respect to genetic differences that lie outside the
MHC loci and environmental conditions. Additionally, a major
reason why certain novel pharmacologic approaches against GVHD
that were shown to be successful in the mouse model then failed in
the clinical setting is that they were applied to patients as treatment
of steroid-refractory GVHD, which shares some similarities with
severe murine GVHD but is per se a different disease. Moreover,
generally, mouse models use BM that contains few T cells and are
supplemented typically by splenocytes or purified T cells from
secondary lymphoid organs. In contrast, patients will receive BM
grafts that are “contaminated” with peripheral blood T cells,
mobilized peripheral blood grafts, or cord blood grafts. T cells in
each instance may have distinct functional characteristics compared
with mouse T cells situated in secondary lymphoid organs.
Therefore, it may be advisable to directly search for correlates in
patient samples when a finding in the mouse model of GVHD has
been made. This can be done by a prospective quantification of
the potential target (eg, a cytokine or a kinase such as BTK) in
human material, and a correlation with the incidence and severity
of GVHD. Conversely, the novel “omics”-driven approaches (eg,
proteomics, genomics) applied to human samples that are now
increasingly used in many areas of medicine will still need a counterpart to functionally address the role of the identified candidate
molecules before a translation into the clinic is possible. Additionally, there is now increasing use of “omics”-based discovery approaches in animal models that may provide new mechanisms
and insights for interrogation and potential validation in GVHD
patients. There are multiple examples for proteomics- or genomicsbased approaches in mice driving human studies.20,21,126 Finally,
another important aspect to potentially improve the predictive value
of preclinical GVHD models may be to incorporate GVHD treatment
models into testing rather than GVHD prevention models alone,
because GVHD treatment has been and continues to be an important
research field in the future, in particular with the high medical need
for GVHD patients who have failed conventional therapies such as
steroids. By combining insights from small and large animal models,
as well as human clinical laboratory and interventional studies, we
believe that this collective approach will have the highest likelihood
for success in improving the outcome for patients who are in need of
new therapies.
Acknowledgments
This study was supported by a grant from the Deutsche Forschungsgemeinschaft Germany (DFG); Heisenberg professorship to R.Z. (DFG ZE
872/3-1); a DFG individual grant to R.Z. (DFG ZE 872/1-2); a European
Research Council consolidator grant to R.Z. (681012 GVHDCure);
grants from the National Institutes of Health, National Heart, Lung and
Blood Institute (R01 HL56067, HL11879), National Institute of Allergy
and Infectious Diseases (AI 34495 and AI 056299), and National Cancer
Institute (P01 CA142106); and a Leukemia and Lymphoma Translational
From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
3124
BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25
ZEISER and BLAZAR
Research grant (6458). The authors apologize to those investigators whose
work could not be cited due to space restrictions.
Authorship
Contribution: Both R.Z. and B.R.B. collected literature, discussed
the studies, and contributed equally to the writing of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Robert Zeiser, Department of Hematology and
Oncology, Freiburg University Medical Center, Albert-Ludwigs
University, Hugstetter Strasse 55, 79106 Freiburg, Germany; e-mail:
[email protected]; and Bruce R. Blazar, Department of Pediatrics, Division of Blood and Marrow Transplantation,
MMC 366, 420 Delaware St SE, Minneapolis, MN 55455; e-mail:
[email protected].
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From www.bloodjournal.org by Fredrik Schjesvold on August 18, 2016. For personal use only.
2016 127: 3117-3126
doi:10.1182/blood-2016-02-699082 originally published
online March 18, 2016
Preclinical models of acute and chronic graft-versus-host disease: how
predictive are they for a successful clinical translation?
Robert Zeiser and Bruce R. Blazar
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