Anaphylaxis caused by repetitive doses of a GITR

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Blood First Edition Paper, prepublished online February 20, 2014; DOI 10.1182/blood-2013-12-544742
Anaphylaxis caused by repetitive doses of a GITR agonist monoclonal antibody in mice
Short title: Repeated doses of anti-GITR cause anaphylaxis
Judith T. Murphy1,2, Andre P. Burey1, Amy M. Beebe3, Danling Gu3, Leonard G. Presta3, Taha
Merghoub1,5 and Jedd D. Wolchok1,2,4,5
1
Ludwig Collaborative Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY
10065;
2
Weill Cornell Graduate School of Medical Sciences, New York, NY 10065;
3
Merck Research Laboratories, Palo Alto, CA 94304;
4
Weill Cornell Medical College, New York, NY 10065
5
T.M. and J.D.W. contributed equally to this paper.
Address correspondence to Dr. Jedd D. Wolchok
1275 York Avenue, Room Z-1462, New York NY 10065
[email protected]
Phone number: 646-888-2395
Fax number: 646-422-0453
Copyright © 2014 American Society of Hematology
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Key Points
•
Repeated doses of agonist antibodies targeting the costimulatory receptors GITR and
OX40 results in anaphylaxis in mice.
•
Anaphylaxis caused by the GITR agonist antibody DTA-1 is dependent on GITR, IL-4,
basophils, and platelet activating factor.
Abstract
Immunotherapy for cancer using antibodies to enhance T cell function has been
successful in recent clinical trials. Many molecules which improve activation and effector
function of T cells have been investigated as potential new targets for immunomodulatory
antibodies, including the tumor necrosis factor receptor (TNFR) superfamily members GITR and
OX40. Antibodies engaging GITR or OX40 result in significant tumor protection in preclinical
models. In this study, we observed that the GITR agonist antibody DTA-1 causes anaphylaxis in
mice upon repeated intraperitoneal dosing. DTA-1-induced anaphylaxis requires GITR, CD4+ T
cells, B cells, and IL-4. Transfer of serum antibodies from DTA-1-treated mice, which contain
high levels of DTA-1-specific IgG1, can induce anaphylaxis in naïve mice upon administration
of an additional dose of DTA-1, suggesting that anaphylaxis results from anti-DTA-1 antibodies.
Depletion of basophils and blockade of platelet activating factor, the key components of the
IgG1 pathway of anaphylaxis, rescues the mice from DTA-1-induced anaphylaxis. These results
demonstrate a previously undescribed lethal side effect of repetitive doses of an agonist
immunomodulatory antibody as well as insight into the mechanism of toxicity, which may offer
a means of preventing adverse effects in future clinical trials using anti-GITR or other agonist
antibodies as immunotherapies.
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Introduction
Immune modulation using monoclonal antibodies has a significant impact on the overall
survival of cancer patients, based on the results of clinical trials using antibodies to block CTLA4 and PD-1(1-6). In an approach that differs from using antibodies to mitigate immune
checkpoint, agonist monoclonal antibodies can be used to directly stimulate T cell function.
Antibodies that engage members of the tumor necrosis factor receptor (TNFR) superfamily have
shown promising tumor protection in preclinical models (3, 7-13). Glucocorticoid-induced
TNFR related (GITR) is a costimulatory receptor in the TNFR superfamily with high homology
to the other TNFR superfamily members OX40, 4-1BB and CD27 (14). GITR and OX40 are
expressed primarily on activated CD4+ and CD8+ effector T cells as well as on CD4+Foxp3+
regulatory T cells (Tregs) (15, 16). Engagement of GITR and OX40 through agonist monoclonal
antibodies results in increased T cell activation, cytokine secretion, proliferation, and survival
(17-23). We and others have shown that the GITR agonist antibody DTA-1 and the OX40
agonist antibody OX86 are very effective anti-tumor therapies in murine tumor models, through
increasing anti-tumor CD4+ and CD8+ T cell effector function, as well as the destabilizing and
causing apoptosis of Tregs in the tumor microenvironment (7, 24-32). Additionally, B cells are
required for DTA-1-mediated protection from certain tumor models, indicating a humoral
component to the anti-tumor effects of DTA-1 (33).
While antibodies targeting costimulatory pathways have shown unquestionable potential
in preclinical models, clinical trials using a CD28 superagonist antibody and preclinical
experiments using a 4-1BB agonist antibody have had severe adverse immune-mediated side
effects (34, 35). This indicates that agonist monoclonal antibodies must be treated with great
caution, and potential side effects should be investigated comprehensively. In this study, we
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show that engagement of the TNFR superfamily members GITR and OX40 with repetitive
intraperitoneal (i.p.) doses of the agonist antibodies DTA-1 and OX86, respectively, causes
anaphylaxis in mice. Anaphylaxis induced by repetitive doses of DTA-1 is caused by serum
antibodies and is dependent on CD4+ T cells, B cells, basophils, platelet activating factor, and
GITR. We suggest a mechanism in which anaphylaxis results from generation of anaphylactic
anti-DTA-1 antibodies. Anaphylaxis caused by DTA-1 can be reduced or prevented by an
antibody that neutralizes IL-4, a platelet activating factor antagonist, or a basophil depleting
antibody. These results suggest that anaphylactic anti-drug antibody generation may be of
particular concern when using agonist antibodies targeting GITR and OX40.
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Methods
Mice and tumor cell lines. All mouse procedures were performed in accordance with IACUC
protocol guidelines at Memorial Sloan-Kettering Cancer Center (MSKCC) under an approved
protocol. Veterinary care was given to any animals requiring medical attention. C57BL/6J and
Kitw/Kitw-v mice were obtained from the Jackson Laboratory. MHC Class I deficient (strain
B2MN12) and MHC Class II deficient (strain ABBN12) were obtained from Taconic. GITR-/and littermate controls (Sv129 x C57BL/6 background)(36) were a gift from Dr. P. P. Pandolfi
(MSKCC, NY, NY) and were backcrossed >10 generations onto C57BL/6J background using a
speed congenic system(37). Mice with the µMT mutation were purchased from the Jackson
Laboratory and backcrossed > 10 generations onto C57BL/6J background and bred at MSKCC.
The B16-F10 mouse melanoma line was originally obtained from I. Fidler (M.D. Anderson
Cancer Center, Houston, TX). In therapeutic tumor protection experiments, mice were
challenged with 0.75-1.0 x 105 B16-F10 cells intradermally in the flank (50 µl/injection) and
monitored every 2 to 3 days for 80 days.
Monoclonal antibodies and drug treatments. DTA-1 (anti-GITR), OX86 (anti-OX40),
FGK45.5 (anti-CD40), GK1.5 (anti-CD4), 11B11 (anti-IL-4) and TA99 (anti-Tyrp-1) were
produced and purified by the Monoclonal Antibody Core Facility at MSKCC. LOB12.3 (anti-41BB), 1A8 (anti-Ly6G), LTF-2 (rat IgG2b isotype control), and HRPN (rat IgG1 isotype control)
were purchased from BioXcell. mDTA-1 was obtained from Merck Research Laboratories.
Ba103 (anti-CD200R3) was purchased from Hycult Biotech. Unless otherwise stated, all
antibodies were administered by i.p. injection in 200 µl of sterile PBS. For tumor protection
experiments, 0.2 mg of TA99 was injected on days 5 and 9 after tumor challenge, and 1 mg of
DTA-1 was injected on days 5, 9, and 16 after tumor challenge. For experiments investigating
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anaphylaxis, 1 mg of DTA-1, OX86, FGK45.5, LOB12.3, LTF-2, or OVA was injected on days
0, 4, and 11. For IL-4 neutralization experiments, 500 µg 11B11 was injected on day 0 of DTA1 treatment. For granulocyte depletion experiments, 500 µg 1A8 or 25 µg Ba103 (i.v. injection
into the tail vein) were administered on day 10 of DTA-1 treatment. For PAF inhibition
experiments, 100 µg CV6209 (Santa Cruz Biotech) was administered by i.p. injection 30 minutes
prior to the final dose of DTA-1. For histamine inhibition experiments, 125 µg triprolidine
(Sigma-Aldrich) was administered by i.p. injection 30 minutes prior to the final dose of DTA-1.
Temperature measurement. Temperature of each animal was measured at baseline and every
10-30 minutes following induction of anaphylaxis using a rectal probe (Physitemp) or infrared
thermometer (Fisher Scientific).
Cell transfer and passive transfer of immune sera. For cell transfer experiments, C57BL/6J
mice were sublethally irradiated (600 rad) 4 hours before transfer. Spleens and lymph nodes
from DTA-1-treated C57BL/6J mice were purified and 50 x 106 cells were administered by i.v.
tail vein injection in 200 µl of sterile PBS. For passive transfer of immune sera, blood was
collected from anesthetized mice by cardiac puncture as a terminal procedure. 200 µl of pooled
sera was then administered by i.v. injection into the tail vein of recipient mice.
Fractionation of sera. All reagents were purchased from Thermo Scientific unless otherwise
stated. A disposable 5 mL polypropylene column was packed with 1 mL of Protein A/G agarose.
Sera were diluted 1:1 in (A/G) IgG Binding Buffer, added to the column, washed, and eluted in
IgG Elution Buffer according to the manufacturer’s instructions. Antibody and flowthrough
fractions were dialyzed using Slide-A-Lyzer MINI Dialysis Devices (3.5K MWCO) and
concentrated with Amicon Ultra Centrifugal Filters (50K) (Millipore).
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ELISA. Mice were treated with DTA-1 or LTF-2 on days -11 and -7 relative to sera collection.
Sera were analyzed by ELISA using plates coated with either DTA-1 or LTF-2 in PBS and
biotinylated rat anti-mouse IgG1 (BD Biosciences) as a secondary antibody. The SAv-HRP
enzyme reagent and the OptEIA detection reagent were purchased from BD Biosciences. Plates
were read with a SpectraMax 340PC (Molecular Devices) at 650 nm.
Statistical analysis. Statistical differences between experimental groups were determined by the
unpaired two-tailed Student’s t test using Graphpad Prism software.
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Results
Repetitive i.p. doses of DTA-1 cause anaphylaxis in a GITR-dependent manner
We and others have previously shown that DTA-1 is a very potent immunotherapy when
used as a single agent (24-27, 38, 39). A single dose of DTA-1 protects up to 60% of mice
bearing established tumors (25). To improve on the antitumor effects of DTA-1, we
administered multiple doses of DTA-1 in combination with a tumor antigen-specific antibody
(TA99) to mice bearing established tumors. While the combination therapy of TA99 and DTA-1
provided considerable protection from tumor challenge, we observed that about 20% of the mice
succumbed to a treatment-related toxicity (supplemental Figure 1). Upon monitoring mice
immediately following DTA-1 injections, we observed that most of the mice became illappearing and lethargic by 20 minutes after the final dose of DTA-1. Additionally, we observed
erythema of the footpads and piloerection in all affected animals, as well as very low blood
volume when the affected mice were euthanized. These effects were observed only following
the final dose and were not observed following the first or second doses of DTA-1. In addition,
the adverse effects of DTA-1 were independent of tumor challenge and TA99 treatment. The
symptoms and time course were suggestive of anaphylaxis (40-42), which we confirmed by
examining body temperature after each dose of DTA-1. Mice experienced a rapid and severe
drop in body temperature following the third i.p. dose of DTA-1 (Figure 1A, supplemental
Figure 2A). A murinized version of DTA-1 (mDTA-1) caused the same anaphylactic symptoms
as DTA-1, demonstrating that the foreign nature of DTA-1 is not the cause of anaphylaxis
(Figure 1B, supplemental Figure 2B). mDTA-1 caused more severe anaphylaxis symptoms
when administered at lower (75 µg) doses by intravenous (i.v.) injection into the tail vein
(supplemental Figure 3), but neither DTA-1 nor mDTA-1 caused anaphylaxis when administered
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subcutaneously. Three doses of isotype control antibody (rat IgG2b) did not cause symptoms of
anaphylaxis (Figure 1C).
We found that GITR-/- mice were protected from DTA-1-induced anaphylaxis (Figure
1D). GITR-/- mice showed symptoms of severe anaphylaxis in an anaphylaxis model caused by
ovalbumin, indicating that protection of GITR-/- mice from DTA-1-induced anaphylaxis is not a
result that can be generalized to all anaphylaxis models (Figure 1E). These results show that
GITR expression is critical for DTA-1-induced anaphylaxis.
Anaphylaxis is caused by repetitive dosing of antibodies targeting GITR and OX40
In order to determine whether anaphylaxis caused by DTA-1 was a general side effect of
repetitive dosing of antibodies targeting TNFR superfamily of costimulatory receptors, we
treated mice with agonist antibodies to OX40 (clone OX86), 4-1BB, and CD40. We observed
that mice treated with repetitive i.p. doses of OX86, but not anti-4-1BB or anti-CD40, underwent
anaphylaxis (Figure 2, supplemental Figure 4). Anaphylaxis caused by OX86 was comparable in
incidence and severity to anaphylaxis caused by DTA-1, suggesting the mechanism behind
anaphylaxis caused by both antibodies is similar. We used DTA-1 to study the mechanism of
anaphylaxis in the subsequent experiments.
CD4+ T cells, B cells, and IL-4 are required for DTA-1-induced anaphylaxis
Because GITR is expressed most highly on T cells, we were particularly interested in
whether T cells are involved in anaphylaxis caused by repetitive dosing of DTA-1. We observed
that both RAG-/- and TCRβ-/- mice were protected from DTA-1-induced anaphylaxis (Figure 3A
and B). We found that MHC Class I deficient mice were not protected from DTA-1-induced
anaphylaxis, indicating CD8+ T cells do not play a major role in anaphylaxis caused by DTA-1
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(Figure 3C). Conversely, mice that were depleted of CD4+ T cells (supplemental Figure 5) as
well as MHC Class II deficient mice (Figure 3D) were protected from DTA-1-induced
anaphylaxis, suggesting a role for CD4+ T cells in anaphylaxis caused by DTA-1. Although
GITR is expressed at much lower levels on B cells than on CD4+ T cells (supplemental Figure
6), we also examined whether B cells were required for DTA-1-induced anaphylaxis because
GITR agonist antibodies have recently been shown to have potent humoral adjuvant effects (33,
43). We found that µMT mice, which lack mature B cells, were protected from anaphylaxis
caused by DTA-1 (Figure 3E). Because CD4+ T cells typically do not secrete vasoactive
mediators such as histamine that are important for the pathology of anaphylaxis, the role of CD4+
T cells in DTA-1-induced anaphylaxis is most likely to provide helper signals to B cells in order
to facilitate an anaphylactic antibody response. One such signal may be IL-4, a critical cytokine
for anaphylaxis as well as other Th2-type allergic responses (44, 45). We observed that
neutralizing IL-4 considerably reduced anaphylaxis severity (Figure 3F).
Anaphylactic activity can be transferred via serum antibodies
The findings that CD4+ T cells and B cells were required for DTA-1-induced anaphylaxis
suggested that an antibody response is the cause of the observed symptoms. Indeed, the
difference between anaphylaxis and other types of shock such as sepsis is the involvement of
antibodies, which trigger release of inflammatory mediators from innate immune cells through
Fc receptors (46). To further elucidate the mechanism underlying anaphylaxis induced by
repetitive dosing of DTA-1, we attempted to transfer the anaphylactic activity from one cohort of
mice to another through either cells or serum. Mice were treated with two doses of DTA-1
administered 4 days apart. Six days after the second dose of DTA-1, the mice were euthanized,
and cells from lymphoid organs or pooled sera were transferred into naïve animals following the
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schema in Figure 4A. No toxicity was observed immediately following transfer of cells or sera.
Three hours post-transfer, all mice were treated with a single dose of DTA-1. The mice that had
received the serum fraction displayed symptoms of anaphylaxis upon DTA-1 administration,
while the mice that had received cells did not (Figure 4B and C). Additionally, control mice that
had received sera from IgG-treated mice did not show signs of anaphylaxis upon an additional
dose of DTA-1 (Figure 4D). Although the sera transferred from DTA-1-treated mice most likely
contained DTA-1, we do not believe the transferred DTA-1 to be responsible for anaphylaxis,
because naïve mice that were treated with two doses of DTA-1 three hours apart did not undergo
anaphylaxis (supplemental Figure 7). Therefore, DTA-1 induces production of a factor present
in the serum that can elicit anaphylaxis in mice upon one additional dose of DTA-1. Serum from
DTA-1-treated wild type mice could also induce anaphylaxis upon an additional dose of DTA-1
in GITR-/- mice (Figure 4E), indicating that while GITR is required for anaphylaxis caused by
repeated doses of DTA-1 (Figure 1D), GITR triggering is not required for the actual symptoms
of anaphylaxis observed upon the final dose.
In order to determine which component in the serum of DTA-1-treated mice is
responsible for rendering naïve mice susceptible to DTA-1-induced anaphylaxis, we fractionated
sera from DTA-1-treated mice over a protein A/G column into a fraction containing only serum
antibodies and a fraction from which antibodies had been depleted (termed “flowthrough”
fraction). We observed that transfer of the antibody fraction, but not the flowthrough fraction,
from DTA-1-treated mice into naïve mice resulted in anaphylaxis when mice were treated with a
single dose of DTA-1 three hours later (Figure 4F and G). As mentioned above, while DTA-1 is
likely to be present in the antibody fraction we purified, we do not believe that the DTA-1
transferred in the antibody fraction is responsible for transferring the susceptibility to
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anaphylaxis. Rather, taking into account the humoral adjuvant properties of DTA-1, we believe
that the mice are making an anaphylactic antibody response to DTA-1 itself, and that the final
i.p. bolus injection of DTA-1 forms immune complexes with anti-DTA-1 antibodies and triggers
release of inflammatory mediators that cause vasodilation, leading to the observed drop in body
temperature and other anaphylactic symptoms in the affected mice.
Repetitive dosing of DTA-1 induces anaphylaxis through the IgG1-basophil-PAF pathway
It has become apparent that two pathways of anaphylaxis exist in mice: the best
characterized is caused by antigen crosslinking of IgE on mast cells followed by release of
histamine (47-50), while the other pathway is mediated by IgG1 and basophil or neutrophil
release of platelet activating factor (PAF)(51-56). We observed that mast cell-deficient
Kitw/Kitw-v mice were not protected from anaphylaxis caused by DTA-1 (Figure 5A). This led us
to investigate the IgG1- and PAF-mediated pathway of anaphylaxis. Treatment with the PAF
inhibitor CV6209 thirty minutes before the final dose of DTA-1 resulted in decreased
anaphylaxis severity, while the histamine inhibitor triprolidine had no effect on anaphylaxis
caused by DTA-1 (Figure 5B), suggesting that PAF-secreting cells mediate anaphylaxis in our
model. Depletion of basophils, but not neutrophils, 24 hours before the final dose of DTA-1
resulted in protection from anaphylaxis (Figure 5C), supporting the finding of Tsujimura et al.
(56) that basophils, while present at very small numbers in peripheral blood, can be critical
effector cells of anaphylaxis.
To further assess the involvement of IgG1 antibodies in DTA-1-induced anaphylaxis, we
heated serum from DTA-1-treated mice at 56⁰ C to inactivate IgE antibodies. In further
agreement with the involvement of the IgG1 anaphylaxis pathway in DTA-1-induced
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anaphylaxis, we found that the heat-inactivated serum retained anaphylactic activity (Figure 5D).
Additionally, we found high levels of DTA-1-specific IgG1 antibodies in serum from individual
mice treated with DTA-1(Figure 5E), supporting the hypothesis stated above that DTA-1induced anaphylaxis results from an anti-DTA-1 anaphylactic antibody response.
Inhibiting anaphylaxis does not reduce the efficacy of DTA-1 as a tumor therapy
We next investigated whether anaphylaxis caused by repetitive dosing of DTA-1 could be
prevented while preserving the anti-tumor activity of DTA-1. Mice were challenged with B16
melanoma and treated with the combination therapy of DTA-1 and the tumor antigen-specific
antibody TA99 (supplemental Figure 1). As in Figure 5, PAF was inhibited thirty minutes prior
to the final dose of DTA-1. Blocking PAF protected tumor-bearing mice from anaphylaxis, as
observed in non-tumor-bearing mice (Figure 5B, Figure 6A). Additionally, protection from
anaphylaxis did not adversely affect the tumor protection resulting from TA99 + DTA-1 therapy
(Figure 6B). These results suggest that DTA-1-induced anaphylaxis can be realistically
mitigated in therapeutic settings.
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Discussion
Preclinical models have demonstrated that stimulation of the GITR and OX40 pathways
using DTA-1 or OX86 can result in striking immune modulation, leading to tumor protection and
induction of autoimmunity. In this report, we show that repetitive doses of the GITR agonist
antibody DTA-1 cause anaphylaxis in mice, which is mediated by IgG1, basophils, and PAF.
We observed similar effects using the OX40 agonist antibody OX86, suggesting that antibodies
engaging members of the TNFR superfamily may be particularly prone to anaphylaxis upon
repeated dosing. Interestingly, GITR was critically required for anaphylaxis caused by repetitive
dosing of DTA-1, but not for anaphylaxis caused by a single dose of DTA-1 after transfer of
serum antibodies from previously dosed mice. This strongly suggests that costimulation of T
cells through GITR drives anaphylactic sensitization. The critical mechanistic role for GITR in
DTA-1-mediated anaphylaxis is strongly supported by the observation that GITR-/- mice were
protected from DTA-1-induced, but not ovalbumin-induced, anaphylaxis. DTA-1-induced
anaphylaxis was mediated by serum antibodies generated after DTA-1 injection, and because no
additional foreign antigens were injected into the mice, the data suggest that anti-DTA-1
antibodies are responsible for the rapid symptoms observed after the final dose of DTA-1.
Therefore, it appears that engaging GITR using DTA-1 enhances the immunogenicity of DTA-1
itself, leading to anaphylaxis upon repetitive dosing.
The observation that the OX40 agonist antibody OX86 also caused anaphylaxis while the
4-1BB agonist antibody did not cause any anaphylactic symptoms supports further examination
of the similarities OX40 and GITR in signaling and expression. Interestingly, Tregs express
very high levels of GITR and OX40, but not 4-1BB; this expression pattern may explain the
more potent antibody responses caused by agonizing GITR and OX40, especially in light of
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recent findings that Tregs are involved in control of the germinal center response and that
depletion of Tregs can enhance anaphylaxis (57-63).
An important question is whether anaphylaxis caused by repetitive i.p. dosing of DTA-1
and OX86 in mice is translatable to humans treated with humanized versions of these antibodies.
Our preclinical observations will heighten awareness that anaphylaxis may be of particular
concern when using antibodies targeting GITR and OX40. Indeed, anaphylaxis caused by antidrug antibodies has been observed in patients retreated with OKT3 (mouse anti-CD3) and
basiliximab (chimeric anti-CD25) (64-66). Careful immune monitoring of sera from these
patients may have detected high levels of anti-drug IgE antibodies and could have precluded
these patients from receiving another dose of antibody. While the foreign and chimeric natures,
respectively, of OKT3 and basiliximab were considered the primary reasons for anaphylaxis
upon retreatment, it is interesting that both OKT3 and basiliximab target and modulate the
function of T lymphocytes. Together with our data showing anaphylaxis upon retreatment with
DTA-1 and OX86 in mice, this suggests that antibodies targeting T cells may be particularly
susceptible to anaphylactic sensitization. Our results indicate that caution should be exercised
when administering multiple doses of anti-GITR or anti-OX40 to patients, especially if high
levels of anti-drug IgE or IgG1 antibodies are observed after the first dose. Of additional
concern when translating anti-GITR to the clinic is our observation that anaphylaxis was also
observed in mice treated with mDTA-1. Also of note is that mDTA-1 and OX86 are different
isotypes from rat DTA-1; therefore, the observations that mDTA-1 and OX86 cause anaphylaxis
invalidates concerns that anaphylaxis caused by DTA-1 is an isotype-specific artifact. The
finding that repetitive dosing of mDTA-1 causes anaphylaxis when dosed by i.v. bolus, but not
when dosed subcutaneously, indicates that i.v. doses in the clinic should be delivered slowly.
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Regardless of the method of administration, however, administering a drug when high levels of
anti-drug antibodies are present will result in reduced bioavailability of the drug even if
anaphylaxis does not occur, because the drug will be cleared from the bloodstream by anti-drug
antibodies. Given the high costs of monoclonal antibodies as therapies, administering an
antibody to patients with high levels of anti-drug antibodies should be avoided from an economic
as well as a safety standpoint.
Additionally, several recent advances have been made in antibody engineering
technology that enables the creation of less immunogenic antibodies. Algorithms have been
developed to predict epitopes within a given protein sequence that will bind to MHC molecules,
as well has the structural consequences of mutations that will abrogate the immunogenic peptides
(67). Epitopes specific for Treg cells (Tregitopes) have also been determined in conserved
domains of Fc and F(ab) portions of antibodies in mice and humans (68). Activation of
regulatory T cells through engagement with Tregitope-MHC complexes results in expansion and
activation of regulatory T cells, along with enhancement of the immunosuppressive Treg
functions such as IL-10 and TGF-b secretion (69). Deimmunization strategies and engineering
to incorporate Treg epitopes may be of particular importance when designing antibodies
targeting GITR and OX40 for clinical use.
In conclusion, we found that repetitive i.p. bolus doses of DTA-1 cause anaphylaxis in a
manner that requires GITR, CD4+ T cells, B cells, basophils, PAF, IL-4, and serum antibodies.
These data are important because cancer patients are currently being treated with anti-GITR and
anti-OX40 in clinical trials, and anticipating immune-based side effects will result in safer trials
and will possibly expedite FDA approval for these and other immunotherapies. Understanding
anaphylaxis caused by anti-drug antibodies is particularly significant because it a phenomenon
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that has been observed in patients retreated with other antibodies. It is tempting to speculate that
antibodies targeting T cells, especially Tregs, are more susceptible to anaphylactic sensitization
than antibodies targeting tumor or stromal cells. It will be important to dose slowly and include
quantitative monitoring of anti-drug IgE and IgG1 antibodies in clinical trials using GITR
agonist antibodies. Moreover, the availability of anti-PAF drugs in the clinic for use in cardiac
rehabilitation (70) suggests that inclusion of these drugs in future clinical trials to mitigate
adverse immune-mediated events is a possibility.
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Acknowledgements
We thank Xia Yang and Hong Zhong for technical help, and Dr. Daniel Hirschhorn-Cymerman
for critically reading the manuscript. This study was supported in part by research funding from
Merck to A.M.B, D.G., and L.G.P. This work was supported by grants from the National Cancer
Institute (R01 CA56821, P01 CA33049, and P01 CA59350), Swim Across America, the Lita
Annenberg Hazen Foundation, the T.J. Martell Foundation, the Mr. William H. Goodwin and
Mrs. Alice Goodwin and the Commonwealth Cancer Foundation for Research, the Joanna M.
Nicolay Melanoma Foundation, and the Experimental Therapeutics Center of MSKCC.
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Authorship contributions
J.T.M, A.M.B., T.M., and J.D.W designed research; J.T.M., A.P.B., D.G., and L.G.P. performed
research; A.M.B, D.G, and L.G.P. contributed a new reagent; J.T.M, A.M.B, D.G., L.G.P, T.M.,
and J.D.W. analyzed data; and J.T.M., T.M., and J.D.W. wrote the paper.
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Disclosure of Conflicts of Interest
Amy M. Beebe, Danling Gu, and Leonard G. Presta are employees of Merck Research
Laboratories. This does not alter the authors’ adherence to the Blood policies on sharing data
and materials.
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Figure legends
FIGURE 1. DTA-1 causes anaphylaxis in a GITR-dependent manner. C57BL/6J mice were
treated with 1 mg DTA-1 (A), mDTA-1 (B), or isotype control (rat IgG2b, clone LTF-2) (C) on
Days 0, 4, and 11. GITR-/- mice were treated with 1 mg DTA-1 (D) or OVA (E). Rectal
temperatures of individual mice were monitored for 1 hour after each dose, represented by
individual lines in the graphs. Data are representative of three independent experiments (n=5
mice/group).
FIGURE 2. Repeated i.p. doses of the TNFR superfamily agonist antibodies DTA-1 and
OX86 cause anaphylaxis. C57BL/6J mice were treated with 1 mg of DTA-1 (A), OX86 (B),
anti-4-1BB (clone LOB.12) (C), or anti-CD40 (clone FGK45.5) (D) on days 0, 4, and 11. Rectal
temperatures of individual mice were monitored for 1 hour following the final injection,
represented by individual lines in the graphs. Data are representative of three independent
experiments (n=5 mice/group).
FIGURE 3. CD4+ T cells, B cells, and IL-4 are required for DTA-1-induced anaphylaxis.
(A) RAG-/- mice, (B) TCRβ deficient mice, (C) MHC Class I deficient mice, (D) MHC Class II
deficient mice, or (E) µMT mice were injected with 1 mg DTA-1 on days 0, 4, and 11. Rectal
temperatures of mice were monitored for 1 hour following the final injection. (F) C57BL/6J
mice were treated with 1 mg DTA-1 on days 0, 4, and 11. Concurrently with the day 0 injection,
mice also received 0.5 mg anti-IL-4 (clone 11B11) or isotype control (rat IgG1, clone HRPN).
Rectal temperatures were monitored following the day 11 dose of DTA-1. Lines in the graphs
represent the mean of each group of mice. The data are representative of two independent
experiments (n=5 mice/group). *p < 0.05, ** p < 0.0001 by unpaired two-tailed Student t test.
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
FIGURE 4. Serum antibodies from DTA-1-treated animals transfer anaphylactic activity to
naïve mice. (A-E) C57BL/6J mice were treated with 1 mg DTA-1 or isotype control (rat
IgG2b, clone LTF-2) on day -10 and day -6. On day 0, mice were euthanized and sera, spleens,
and lymph nodes were removed. Either 50 x 106 cells from pooled spleens and lymph nodes (B)
or 200 µl pooled sera (C-E) were transferred by i.v. tail vein injection into naïve C57BL/6J (C,
D) or GITR-/- (E) mice. Three hours later, a single dose of DTA-1 was administered by i.p.
injection and rectal temperatures were monitored for one hour, represented by individual lines in
the graphs. Data are representative of three independent experiments (n = 5 mice/group). (F-G)
Donor mice were treated as in A. After sera were harvested, antibodies were fractionated from
the pooled sera using a protein A/G column. Either 1 mg of the antibody fraction (F) or 11 mg
of the antibody-depleted fraction (termed “flowthrough” fraction) (G) was transferred by i.v. tail
vein injection into naïve mice. Three hours later, 1 mg DTA-1 was administered by i.p. injection
and rectal temperatures of individual mice were monitored, represented by individual lines in the
graphs. Data are representative of two independent experiments (n = 3-5 mice/group).
FIGURE 5. Anaphylaxis caused by DTA-1 is mediated by platelet activating factor,
basophils, and IgG1 antibodies. (A) C57BL/6J or Kitw/KitW-v mice were treated with 1 mg
DTA-1 on days 0, 4, and 11. Rectal temperatures of individual mice were monitored for 1 hour
following the final injection, represented by individual lines in the graphs. Data are
representative of two independent experiments (n=5 mice/group). (B) C57BL/6J mice were
treated with 1 mg DTA-1 on days 0, 4, and 11. Thirty minutes prior to the final DTA-1
injection, mice were injected intraperitoneally with either 125 µg of the histamine inhibitor
triprolidine or 125 µg of the PAF inhibitor CV6209. Rectal temperatures of individual mice
were monitored for 1 hour following the final dose of DTA-1, represented by individual lines in
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
the graphs. Data are representative of three independent experiments (n=5 mice/group). (C)
C57BL/6J mice were treated with 1 mg DTA-1 on days 0, 4, and 11. One day before the final
dose of DTA-1, mice were either injected intraperitoneally with 0.5 mg of anti-Ly6G (clone
1A8) or intravenously with 25 µg of anti-CD200R3 (clone Ba103). Rectal temperatures of
individual mice were monitored for 1 hour following the final dose of DTA-1, represented by
individual lines in the graphs. Data are representative of three independent experiments (n=5
mice/group). (D) Sera from DTA-1 treated mice were collected as in Fig. 4A. The pooled sera
were heat inactivated at 56⁰ C for three hours. Heat inactivated sera was then transferred by i.v.
injection into the tail vein of recipient mice. Three hours later, a single dose of DTA-1 was
administered and rectal temperatures were monitored, represented by individual lines in the
graphs. Data are representative of two independent experiments (n=5 mice/group). (E)
C57BL/6J mice were treated with DTA-1 or isotype control (rat IgG2b, clone LTF-2) on days 0
and 4. On day 11, sera were collected from individual mice and assayed by ELISA for IgG1
antibodies recognizing DTA-1 or LTF-2. Data are representative of three independent
experiments.
FIGURE 6. Inhibition of platelet activating factor protects mice from anaphylaxis while
maintaining tumor protection provided by DTA-1 as part of a combination therapy.
C57BL/6J mice were challenged intradermally with 1 x 105 B16 melanoma cells. Groups of
mice were either left untreated (Naïve), or treated with 200 µg TA99 on days 5 and 9 after tumor
challenge, 1 mg DTA-1 on days 5, 9, and 16 after tumor challenge, or a combination of both
TA99 and DTA-1 at the aforementioned doses and schedules. One group treated with TA99 +
DTA-1 received 125 µg CV6209 by i.p. injection in PBS thirty minutes before the day 16 dose
of DTA-1. (A) Rectal temperatures were monitored following the day 16 dose of DTA-1. (B)
From www.bloodjournal.org by guest on February 4, 2015. For personal use only.
Tumor growth was measured every 2-3 days, represented by individual lines in the graphs. Mice
were euthanized when tumor diameter reached 1 cm. Data are representative of three
independent experiments (n = 15 mice/group). * p < 0.05, unpaired two-tailed Student t test.
Figure 1.
A.
B.
C.
D.
E.
DTA-1-induced
anaphylaxis
OVA-induced
anaphylaxis
Figure 2.
A.
B.
C.
D.
Figure 3.
A.
B.
C.
D.
E.
F.
Figure 4.
A.
Donor mice
Day -10
DTA-1
Day -6
DTA-1
B.
C.
Day 0
Cells
Sera
3 hrs
Recipient
mice
(group 1)
D.
E.
Recipient
mice
(group 2)
DTA-1
administered
to groups 1 & 2
F.
G.
Figure 5.
A.
D.
B.
E.
C.
Figure 6.
A.
B.
(14/15)
(12/15)
(14/15)
(8/15)
(10/15)
Day 16
PAF inhibitor
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Prepublished online February 20, 2014;
doi:10.1182/blood-2013-12-544742
Anaphylaxis caused by repetitive doses of a GITR agonist monoclonal
antibody in mice
Judith T. Murphy, Andre P. Burey, Amy M. Beebe, Danling Gu, Leonard G. Presta, Taha Merghoub and
Jedd D. Wolchok
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