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A chimeric human-cat fusion protein blocks cat-induced
Daocheng Zhu1,3, Christopher L Kepley2,3, Ke Zhang1,3, Tetsuya Terada1,3, Takechiyo Yamada1 & Andrew Saxon1
Animal allergens are an important cause of asthma and allergic
rhinitis. We designed and tested a chimeric human-cat fusion
protein composed of a truncated human IgG Fcγ1 and the
major cat allergen Fel d1, as a proof of concept for a new
approach to allergy immunotherapy. This Fcγ-Fel d1 protein
induced dose-dependent inhibition of Fel d1-driven IgE-
mediated histamine release from cat-allergic donors’ basophils
and sensitized human cord blood-derived mast cells. Such
inhibition was associated with altered Syk and ERK signaling.
The Fcγ-Fel d1 protein also blocked in vivo reactivity in FcεRIα
transgenic mice passively sensitized with human IgE antibody
to cat and in Balb/c mice actively sensitized against Fel d1.
The Fcγ-Fel d1 protein alone did not induce mediator release.
Chimeric human Fcγ-allergen fusion proteins may provide a
new therapeutic platform for the immune-based therapy of
Traditionally, immune-based therapy for inhalant allergens relies upon
frequent injection of gradually increasing amounts of allergens. This
approach is time-consuming, protracted and marred by serious treatment
reactions. Immunotherapy for life-threatening food allergy (e.g., peanut
allergy) has proven unsuccessful1. Allergen-induced IgE-driven media-
tor release from mast cells and basophils is a key contributor in asthma,
allergic rhinitis and severe food reactions2. Cross-linking mast cell and
basophil FcεRI by multivalent antigen activates tyrosine phosphoryla-
tion of immunoreceptor tyrosine-based activation motifs (ITAMs) in the
β- and γ-FcεRI subunit cytoplasmic tails, thereby initiating downstream
signaling through Syk3. Mast cells and basophils also express FcγRIIb,
which contains a single conserved immunoreceptor tyrosine-based
inhibition motif (ITIM) within its cytoplasmic tail4,5. Studies indicate
that aggregating FcγRIIb to FcεRI leads to rapid tyrosine phosphoryla-
tion of the FcγRIIb ITIM tyrosine by FcεRI-associated Lyn and inhi-
bition of FcεRI signaling6–9. Experiments using a human Ig Fcγ-Fcε
fusion protein that directly cross-links FcεRI and FcγRIIb on human
basophils support this hypothesis10–13. We have developed and tested
a new form of immune therapy based on a chimeric fusion protein
(GFD) comprised of the human Fcγ plus the cat (Felis domesticus)
allergen14 Fel d1. This molecule is specifically designed to coaggregate
FcγRIIb with FcεRI-bound IgE, thereby inhibiting mediator release
while serving as allergen immunotherapy that could be given as a safe
series of high-dose injections.
We cloned human IgGγ1 constant region genomic DNA from the
hinge through the CH3 domain into a mammalian expression vec-
tor with a cytomegalovirus promoter and a mouse immunoglobulin
κ-chain leader sequence. We placed the cDNA encoding a Fel d1 con-
struct15 containing both chain 1 and chain 2 after the CHγ3 domain
with a 15 –amino acid (Gly4Ser)3 linker16 between Fcγ1 and Fel d1. The
fusion protein contained the binding site for FcγRII17. The Fcγ1-Fel d1
fusion protein was expressed as the predicted dimer of 140 kDa. Western
blot and ELISA testing showed that antibodies to human gamma chain
and Fel d1 recognize GFD. GFD binding to the human FcγRII was
shown using HMC-1, a human mast cell–like line that expresses FcγRII
but not FcεRI18. GFD bound to FcγRII in a fashion equivalent to human
IgG, as assessed by flow cytometry. ELISA results indicate that specific
IgE from cat-allergic patients’ sera recognized GFD (data not shown).
These results show that GFD is properly folded, has preserved FcR bind-
ing and is recognized by human IgE antibody to cat.
We purified basophils from cat-allergic subjects19 and cultured them
with 1 ng/ml to 1 µg/ml of GFD. Purified human IgG served as a control.
Two hours later, we centrifuged the cells and assayed the histamine in
supernatants as ‘prerelease.’ We then washed and challenged the cells
with an optimal dose of Fel d1 (1.0 µg/ml) for 30 min and measured
the resulting histamine release. GFD inhibited release by more than 75%
(P < 0.002) at 10 ng/ml, whereas at 100 ng/ml inhibition was >90% (P < 0.001)
(Fig. 1a). Results without autologous serum during the first incubation were
similar, except overall histamine release was about 15% less. We observed
similar inhibition in cat allergen– sensitized cord blood–derived mast cells
wherein GFD (10 µg/ml) reduced degranulation by an average of 77%
(P < 0.05) (Fig. 1b). Thus, GFD inhibited allergen-driven histamine release in
a dose-dependent fashion. Notably, these results also show that GFD does not
function as an allergen because mediator prerelease was not observed with
GFD-incubated cat allergen–sensitized basophils.
Tyrosine phosphorylation is a key event connecting FcεRI cross-
linking to downstream signaling in human mast cells and basophils.
IgE stimulation in human FcεRI-positive cells quickly leads to
phosphorylation of ERK1/2 and Syk12,20. Cross-linking FcεRI on cord
1The Hart and Louise Lyon Laboratory, Division of Clinical Immunology/Allergy, Department of Medicine, UCLA School of Medicine, 10833 Le Conte Avenue, Los
Angeles, California 90095-1680, USA. 2Division of Rheumatology, Allergy and Immunology, Department of Internal Medicine, Room 4-115B, McGuire Hall, 1112
East Clay Street, Virginia Commonwealth University, Richmond, Virginia 23298-0263, USA. 3These authors contributed equally to this work. Correspondence should
be addressed to A.S. (firstname.lastname@example.org) or D.Z. (email@example.com).
Published online 27 March 2005; doi:10.1038/nm1219
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blood mast cells with IgE directed to Fel d1 induces substantial tyro-
sine phosphorylation of Syk and ERK, which was markedly reduced in
cells preincubated with GFD (Fig. 2). We observed inhibition 2 min
after antigen stimulation; inhibition persisted for 15 min. Thus, GFD
coaggregation of FcεRI and FcγRII through an Fcγ-Fel d1-IgE linkage
inhibits IgE-mediated Syk and ERK phosphorylation, which probably
contributes to inhibition of basophil and mast cell function.
Transgenic mice expressing the human FcεRIα chain (hFcεRIα+
mice) show allergic reactivity after administration of human IgE anti-
body and challenge with the appropriate antigen21, 22. Mast cells in these
transgenic mice also express the mouse FcγRIIb that can bind human
IgG. Using passive cutaneous anaphylaxis (PCA) in the hFcεRIα+ mice,
we tested the predicted inhibitory effects of GFD by co-cross-linking
the humanized FcεRI and the mouse FcγRIIb.
We primed transgenic mice (n = 12) intradermally with human
serum containing high-titer IgE antibody to Fel d1 (118 kU/L) and
measured PCA reactivity at sites injected with varying doses of GFD,
after intravenous challenge with purified Fel. We measured each PCA
reaction as the size of blue staining reaction of the skin at 30 min. GFD at
100 ng/spot consistently and completely blocked PCA reactivity
(Fig. 3a). The GE2 fusion protein, which directly crosslinks
FcεRI-FcγRII23 gave analogous inhibition (Fig. 3b). GFD blocked PCA
reactivity with 10-fold greater efficiency compared to GE2 (Fig. 3b).
GFD blocked PCA reactivity equally well when injected 4 h after or
simultaneously with cat-allergic donors’ serum (Fig. 3b). PCA reactivity
of purified cat-allergic donors’ serum was destroyed by heat inactiva-
tion at 56 οC for 30 min23 (data not shown). To test the specificity of
GFD, we sensitized hFcεRIα+ mice with chimeric human IgE antibody
to NP (4-hydroxy-3-nitrophenylacetyl) and induced PCA reactivity by
intravenous challenge with NP–bovine serum albumin. GFD did not
block IgE-induced reactivity to NP (data not shown). These data show
that the GFD specifically inhibits cat allergen–induced IgE-mediated
mediator release in vivo. To prove that GFD was not functioning as an
allergen, we gave mice Evans blue dye 15 min after local administration
of GFD at sites initially injected with cat-allergic donors’ serum or puri-
fied IgE from cat-allergic subjects’ serum (data not shown). We observed
no reaction as evidenced by lack of dye extravasation, which shows that
GFD itself did not induce mast cell release and such an effect does not
account for the failure of GFD-treated sites to react upon later systemic
We developed a model of systemic reactivity to Fel d1 in actively sen-
sitized Balb/c mice to test the immunotherapeutic potential of GFD. The
value of this model is based on the binding of human Fcγ by the mouse
FcγRs11 (data not shown). Thus, the Fcγ portion of GFD binds to mouse
FcγRs, including FcγRIIb, and drives inhibitory signaling through its
ITIM motif. Simultaneously, the Fel d1 portion of GFD will bind to
mouse Fel d1-specific IgE and/or IgG1 on the surface of sensitized
mast cells or basophils with the expectation that GFD- mediated cross-
linking of these FcεRIs and FcγRs will generate a negative signal for
mast cells or basophils.
We sensitized BALB/c mice with Fel d1 and treated them up to day
21 (Fig. 4a). GFD treatment completely blocked Fel d1-induced air-
way hyper-responsiveness at days 30 (Fig. 4b) and 44 (data not shown),
assessed by increased pulmonary resistance after methacholine challenge.
Similarly, we observed blunted eosinophilic airway inflammation in
Fel d1-sensitized and intratracheally challenged mice, evident as decreased
eosinophils in bronchoalveolar lavage fluid after GFD treatment (Fig. 4c).
Using a sensitization and GFD immunotherapy model that used intense
Figure 1 GFD inhibits human basophil and mast cell degranulation.
Basophils from an atopic donor (a) or cat serum–sensitized, cord blood–
derived mast cells (b) were incubated for 2 h with GFD and the supernatant
assayed for prerelease of histamine (a) or β-hexosaminidase (b). Washed
cells were challenged with Fel d1 and histamine or β-hexosaminidase
measured in the supernatant (Fel d1 release). Nonspecific human IgG (hIgG)
and IgE (hIgE) were used as controls. The results from one experiment
are representative of three separate experiments. The asterisk indicates a
statistically significant difference when comparing the two conditions.
*P < 0.05.
Figure 2 GFD inhibits FcεRI-mediated Syk and ERK phosphorylation. Cord
blood–derived mast cells were sensitized with cat-allergic donors’ serum,
washed, activated as described, and western blotted with the indicated
antibodies. The top panel (Syk) represents an independent experiment
from the bottom panel (Erk). Results are representative of at least three
NATURE MEDICINE ADVANCE ONLINE PUBLICATION
Fel d1 sensitization, we tested GFD for its ability
to block systemic allergic reactivity as indicated by
a decrease in challenged animals’ core body tem-
perature24. Core temperatures dropped an aver-
age of 1.7 ± 0.2 οC over the hour after intratracheal
Fel d1 challenge. This was completely blocked following GFD treatment
(P < 0.001) (Fig. 4d). These results indicate that GFD administrated in a
regime similar to allergen immunotherapy ameliorates allergic responses
to Fel d1 in previously sensitized animals.
Recognition of the Fel d1 portion of GFD by cat allergen-specific
IgE is predicted to lead to the formation of FcγR-(GFD)-IgE-FcεRI
complexes on basophils and mast cells. This probably accounts for
the ability of GFD to inhibit mediator release from basophils of cat
allergen–sensitive humans and to inhibit PCA reactivity in transgenic
mice, and is analogous to what we found with a human bifunctional
Fcγ-Fcε protein (GE2), which directly cross-links FcγRIIb and FcεRI
to induce antigen nonspecific inhibitory signaling10,23. In contrast
to GE2, GFD indirectly cross-links FcγRIIb and FcεRI through anti-
gen-specific IgE and inhibits basophil and mast cell reactivity in an
allergen-specific fashion. GFD also contains the allergen (Fel d1) that
should induce a protective immune response, as seen with standard
immunotherapy25,26 and as observed in mice with Fel d1-induced sys-
temic and airway reactivity (Fig. 4). The advantage of an Fcγ-allergen
construct (e.g., GFD) versus allergen is that the chimeric protein does
not drive mediator release.
Overall, GFD inhibited allergen-driven IgE-mediated mediator
release in vitro from human basophils and cord blood–derived mast
cells, and in vivo from both passively cat allergen–sensitized FcεRIα
transgenic mice and in actively cat allergen–sensitized mice. Chimeric
human Fcγ-allergen proteins such as GFD provide a new platform for
antigen-specific immunotherapy in a host of human allergic diseases
and may be a particularly powerful approach
for treatment of severe food-induced allergy.
GFD construction and expression. To construct
the human Fcγ-Fel d1 chimeric gene, we amplified
cDNA encoding Fel d1 from a recombinant Fel d1
cDNA clone (chain 1 + chain 2)15. The 5´-end primer
contained a flexible linker sequence. We cloned
amplified products into pCR2.1 vector (Invitrogen),
sequenced and inserted the Sal I-Not I fragment
into pAN expression vector (Invitrogen pDisplay).
We transfected the expression vector containing the
new Ig Fcγ-Fel d1 chimeric gene into SP2/0 cells and
purified the Fcγ-Fel d1 fusion protein in the culture
supernatants by protein A affinity chromatography.
Figure 3 GFD inhibits IgE-mediated degranulation
in FcεRIα transgenic mice. (a) Dose-dependent
inhibition of PCA by GFD. The skin sites were
sensitized with cat-allergic donors’ serum,
followed by the administration of: (I) saline;
(II–IV), GFD 1, 10 and 100 ng, respectively.
(b) Comparison of GFD and GE2 for inhibiting
PCA. The skin sites were sensitized and treated
as follows: (I) saline 4 h later; (II) 100 ng GFD
4 h later; (III) 100 ng GFD simultaneously with
serum; (IV) 1 µg GE2 simultaneously with serum;
(V) 100 ng GE2 4 h later; (VI) 100 ng GE2
simultaneously with serum.
Figure 4 GFD blocks Fel d1-induced allergic
response in mice. (a) Schematic diagram of the
experimental protocol. (b) Inhibitory effect of GFD
on Fel d1-induced AHR. The numbers represent
the average values from three measurements of
airway resistance. (c) Inhibitory effect of GFD Fel
d1-induced pulmonary eosinophilic inflammation.
Total and differential numbers of bronchoalveolar
lavage fluid cells were counted. (d) Inhibitory
effect of GFD on Fel d1-induced systemic allergic
reactivity evidenced as core body temperature.
Core body temperature change was measured at
5-min intervals immediately after Fel d1 challenge.
The asterisk indicates a statistically significant
difference between the two conditions. *P < 0.05.
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Western blots. We ran purified GFD on SDS PAGE and transferred it to membranes
where we probed it with either mouse antibody to human IgG (γ chain–specific) or
antibody to Fel d1 and goat antibody to mouse IgG conjugated to horseradish per-
oxidase. For Syk and Erk measurements, we treated IgE-sensitized cord-blood mast
cells with GFD or control materials, and followed with activation, lysis and blotting
as described5. We probed blots with rabbit antibodies that recognize phosphorylated
Syk (Cell Signaling) or mouse antibodies to phosphorylated ERK 1/2 (Upstate).
We developed blots using enhanced chemiluminescence reagents (ECL, Amersham
Biosciences) and exposed them to BioMax film (Eastman Kodak). We quantified
band intensities using a molecular imaging package (Bio-Rad, Discovery Series;
Quantity One, Quantitative Software) and presented them as a ratio of Syk or Erk to
actin to compensate for gel loading and blot stripping-reprobing variations.
Basophil and cord blood–derived mast cell purification. We collected blood from
donors who were skin test–positive to Fel d1 and had a self-reported history of cat
allergy. The basophils were purified by Percoll gradient centrifugation, followed
by negative selection using magnetic beads. Basophil purities were ≥95% as deter-
mined with Wrights-Giemsa stain. Cord blood–derived mast cells were derived as
described12. We obtained informed consent for all human subjects as approved by
the Institutional Review Board at Virginia Commonwealth University.
Measurement of degranulation. We sensitized cord blood–derived mast cells
with human IgE antibody to Fel d1 in serum from cat-allergic donors (Plasma
Labs) for 24 h. After washing the mast cells or basophils, we added GFD (0–10
µg/ml) for 2 h at 37 °C and centrifuged the cells and used the supernatants to
determine any mediator prerelease. We washed and activated cells in Tyrodes
(mast cells) or DMEM (basophils), with or without optimal concentrations of
Fel d1 (Indoor Biotechnologies) (10–200 ng/ml). As a control, we substituted
nonspecific human IgG for GFD. After 30 min, we centrifuged cells and removed
the supernatant for β-hexosaminidase or histamine analysis, as described27.
Passive cutaneous anaphylaxis. We injected transgenic mice intradermally
with 50 µl of 1:5 diluted cat allergic serum or purified IgE from that serum.
We injected different doses of purified GFD at the same sites 4 or 24 h later. We
injected GFD simultaneously with the allergic serum (or purified IgE) in selected
experiments. We injected mice intravenously with 10 µg of purified Fel d1, plus
Evans blue dye, 4 or 24 h later. The mice were generally killed 30 min after the
intravenous challenge although in some experiments, this was done at 60 and
120 min to assess and confirm the blocking activity of GFD.
Mouse models. To measure airway changes shown in Fig. 4b and 4c, we sensitized
and treated 6–8-week-old BALB/c mice using the protocol diagrammed in Fig. 4a. To
assess systemic allergic reactivity, we sensitized mice by intraperitoneal injection with
5 µg of Fel d1 on d 1 and 14, then boosted them intratracheally with 1 µg of Fel d1 on
days 28, 29, 30 and 33. We treated mice subcutaneously with 5 µg of GFD or saline on
days 37, 38 and 39. We subjected the mice then to intratracheal challenge with 1 µg
of Fel d1 on day 40 (Fig. 4d) and monitored the animals’ core temperature rectally
using a rectal probe digital thermometer (YSI Inc.). We obtained approval from the
Animal Research Committee at UCLA for all the animal experiments performed.
Airway response to methacholine. We measured airway responsiveness 48 h
after intratracheal challenge using a modified forced oscillation method28. We
connected anesthetized mice to a computer-controlled small-animal ventilator
(FlexiVent, SCIREQ) and calibrated it to remove resistance of the tracheal, cannula
and tubing. We obtained measurements of pulmonary resistance at 10-s intervals,
both before and after intravenous administration of acetyl-β-methylcholine-chlo-
ride (1.67 µg/g body weight) by using the forced oscillatory technique with the
conventional primewave 8 with a peak to peak amplitude of 0.17442 ml.
Bronchoalveolar lavage. Two days after intratracheal challenge, the mice were
killed, their lungs lavaged and total numbers of bronchoalveolar lavage cells
counted after cells were stained with trypan blue. Differential cell counts mea-
sured on at least 300 cells stained with Wright-Giemsa.
Supported by an USPHS-NIH grant, AI-15251 to A.S. We thank S.L. Morrison, R. Trinh
and L. A. Chan for technical advice and for providing some experimental materials.
C.L.K. was supported by a grant from the American Lung Association and the AD
Williams Foundation at VCU, and the Food Allergy and Anaphylaxis Network.
We also thank J.P. Kinet for providing the human FcεRIα transgenic mice. We are
grateful to T.H. Sulahian and P.M. Guyre for the Fel d1 cDNA construct. We thank
M. Jyrala, L. Zhang, and M. Rainof for technical assistance.
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests (see the Nature Medicine website
Received 5 January 2005; accepted 3 March 2005
Published online at http://www.nature.com/naturemedicine/
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