mAbs

ISSN: 1942-0862 (Print) 1942-0870 (Online) Journal homepage: http://www.tandfonline.com/loi/kmab20

Monoclonal antibodies directed against human

FcRn and their applications

Gregory J. Christianson, Victor Z. Sun, Shreeram Akilesh, Emanuele

Pesavento, Gabriele Proetzel & Derry C. Roopenian

To cite this article: Gregory J. Christianson, Victor Z. Sun, Shreeram Akilesh, Emanuele
Pesavento, Gabriele Proetzel & Derry C. Roopenian (2012) Monoclonal antibodies directed
against human FcRn and their applications, mAbs, 4:2, 208-216, DOI: 10.4161/mabs.4.2.19397

To link to this article: http://dx.doi.org/10.4161/mabs.4.2.19397

Published online: 01 Mar 2012.

Submit your article to this journal

Article views: 271

View related articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=kmab20

Download by: [121.207.210.105] Date: 04 October 2015, At: 20:58

 Report

mAbs 4:2, 208-216; March/April 2012; © 2012 Landes Bioscience

Monoclonal antibodies directed

against human FcRn and their applications
Gregory J. Christianson, Victor Z. Sun, Shreeram Akilesh,† Emanuele Pesavento,‡ Gabriele Proetzel and Derry C. Roopenian*
The Jackson Laboratory; Bar Harbor, ME USA; †Current Address: Barnes-Jewish Hospital; St. Louis, MO USA; ‡Current Address: VIB Department of Molecular and Cellular
Interactions; Laboratory for Cellular and Molecular Immunology; Vrije Universiteit; Brussels, Belgium

Key words: FcRn, IgG, monoclonal antibody, albumin, therapy

Abbreviations: MHC, major histocompatibility complex; IgG, immunoglobulin G (h for human, m for mouse); FcRn, Fc receptor

©2013 Landes Bioscience. Do not distribute.

neonatal (h for human, m for mouse); mAb, monoclonal antibody; β2M, beta-2 microglobulin; HSA, human serum albumin;
GFP, enhanced green fluorescent protein; IF, immunofluorescence

The MHC class I-like Fc receptor (FcRn) is an intracellular trafficking Fc receptor that is uniquely responsible for the extended
serum half-life of antibodies of the IgG subclass and their ability to transport across cellular barriers. By performing
Downloaded by [121.207.210.105] at 20:58 04 October 2015

these functions, FcRn affects numerous facets of antibody biology and pathobiology. Its critical role in controlling IgG
pharmacokinetics has been leveraged for the design of therapeutic antibodies and related biologics. FcRn also traffics
serum albumin and is responsible for the enhanced pharmacokinetic properties of albumin-conjugated therapeutics.
The understanding of FcRn and its therapeutic applications has been limited by a paucity of reliable serological reagents
against human FcRn. Here, we describe the properties of a new panel of highly specific monoclonal antibodies (mAbs)
directed against human FcRn with diverse epitope specificities. We show that this antibody panel can be used to study
the tissue expression pattern of human FcRn, to selectively block IgG and serum albumin binding to human FcRn in vitro
and to inhibit FcRn function in vivo. This mAb panel provides a powerful resource for probing the biology of human FcRn
and for the evaluation of therapeutic FcRn blockade strategies.

Introduction and bioavailability of IgG monoclonal antibodies (mAbs) and

Although originally referred to as the neonatal Fc receptor, FcRn
influences IgG serum levels and tissue distribution at all stages
of life.1-5 This divergent member of the MHC class I family mol-
ecule is a heterodimer composed of an evolutionally distinct
α-chain in complex with the β2-microglobulin (β2M) light chain
that is common to most other class I molecules. FcRn is an intra-
cellular trafficking, integral membrane Fc receptor for IgG. It
resides primarily in the early acidic endosomes where it captures
endocytosed IgG by binding to the Fc region only at a low pH.6-8
FcRn rescues bound IgG from degradation in the lysosomal com-
partment and transports its ligand to the cell surface for release
at neutral extracellular pH. Through this mechanism, FcRn is
responsible for the long serum half-life of IgG.9 FcRn transports
IgG across the tight epithelial barriers of the endothelium and
mucosa, thus influencing its bioavailability.7,10 In antigen pre-
senting cells, FcRn controls the presentation of antigens in IgG
immune complexes to T cells11,12 and IgG-Fc fusion protein vac-
cines.13,14 FcRn also controls serum albumin homeostasis by an
analogous trafficking mechanism as for IgG.5,15,16 These properties
have proven to be critical for maximizing the pharmacokinetics
IgG-Fc fusion proteins and albumin conjugated protein thera-
peutics.5,17,18 While the biological and therapeutic importance of
FcRn’s varied and expanding functional attributes are increas-
ingly documented, major gaps remain in the understanding of
the cell biology of FcRn, its tissue sites of action, and methods to
exploit this biology for therapeutic purposes.
Of particular relevance is the need for reliable serological
reagents for detection of human FcRn (hFcRn) and for prob-
ing its biology in normal and pathological states. This report
describes the generation, characterization and applications of a
part of mAbs with varied epitope specificities directed against
human (h)FcRn. Our data demonstrate that the mAbs can read-
ily detect hFcRn by flow cytometry and on tissue sections, which
makes them valuable tools for defining the sites of hFcRn expres-
sion. Furthermore, the ability of certain mAbs to differentially
block hIgG or human serum albumin binding supports a model
of distinct binding sites for these ligands to hFcRn.5,16 Finally,
treatment of hFcRn transgenic mice with a hIgG blocking mAb
provides a proof-of-principle that anti-hFcRn mAbs can be
applied therapeutically to specifically control the serum persis-
tence of human IgG in vivo.

*Correspondence to: Derry C. Roopenian; Email: derry.roopenian@jax.org

Submitted: 12/26/11; Revised: 01/17/12; Accepted: 01/18/12
http://dx.doi.org/10.4161/mabs.4.2.19397

208 mAbs Volume 4 Issue 2

 Report Report

©2013 Landes Bioscience. Do not distribute.

Downloaded by [121.207.210.105] at 20:58 04 October 2015

Figure 1. hFcRn binding and blocking activity of anti-hFcRn antisera. (A) Representation of the construct design lacking the cytoplasmic targeting
domain (left). Confocal images of HeLa cells transiently transfected with the hFcRn-GFP after incubation with hIgG at the indicated pH, fixed and
stained with goat anti-hIgGAF568 (right). (B) Flow cytometric data of 293hFcRn-GFP cells incubated with hIgGAF647 at pH 6.0 (left) or 7.2 (right). (C) Representa-
tive results from evaluation of antisera from mice primed with hFcRn. Binding of the antisera to hFcRn-GFP was detected with goat anti-mouse IgG-PE
at pH 7.2 (left). Functional blockade of hIgG binding to hFcRn was determined by the ability to inhibit the binding of hIgGAF647 at pH 6.0 (right). Values
within scatter plots represent the AF647 mean fluorescence intensity (MFI) of each hFcRn-GFP+-gated cell population. Data are representative of at
least two experiments.

Results rather than through the Fc region. Next, to determine whether

Screening assay validation and antisera generation. We first
established a flow cytometry-based assay suitable for detecting
antibody activity specific for human and mouse FcRn and for the
assessment of the binding and functional blockade of hIgG bind-
ing. An enhanced green fluorescent protein (GFP) was spliced
between the signal sequences and the N-terminus of FcRn
constructs with truncated cytoplasmic, endosomal targeting
domains (Fig. 1A). This truncation resulted in robust expression
of the hFcRn-GFP chimeric proteins on the plasma membrane
as first validated by confocal immunofluorescence (IF) imaging
of transiently transfected HeLa cells (Fig. 1A) and then by flow
cytometric analysis of HEK293 cells stably transfected with the
same construct (Fig. 1B). Epitope tagging with GFP did not
alter the normal pH-dependent binding of hFcRn to hIgG. The
hFcRn-GFP fusion protein bound hIgG at acidic pH 6 but not
neutral pH (Fig. 1A and B). Similar acidic pH-dependent bind-
ing of hIgG was observed for HeLa and 293 cells transfected with
the mFcRn-GFP construct (data not shown).
To generate anti-hFcRn antisera, we immunized B6 wild-type
(WT) and FcRn-/- mice with splenocytes from hFcRn transgenic
mice. Plasma samples from immunized mice were first screened
for their ability to bind hFcRn at a neutral pH to ensure that
the antibodies bound hFcRn by their antigen binding domains
the antisera could inhibit the binding of hIgG, we assessed their
ability to compete with Alexa Fluor® 647-labeled hIgG (hIg-
GAF647) for binding to GFP-tagged human FcRn expressed on
the surface of 293 cells (293hFcRn-GFP) cells at pH 6.0. The antisera
showed different binding and blocking properties. For example,
antiserum 26,162 bound hFcRn at pH 7.2 and blocked hIgG AF647
binding at pH 6.0 (Fig. 1C, top). In contrast, antiserum 26898
bound, but did not block, hIgGAF647 binding to hFcRn (Fig. 1C,
middle). Lastly, some antisera, such as antiserum 26902, showed
neither hFcRn binding nor blocking activity (Fig. 1C, bottom).
Generation of anti-FcRn antibody-producing hybridomas.
Mice with antisera showing hFcRn binding or blocking activity
were used for hybridoma generation. Supernatants from hybrid-
omas were assayed for their binding to hFcRn using 293hFcRn-GFP
cells in a cellular enzyme-linked immunosorbent assay (ELISA),
and those showing activity against hFcRn in this assay were sub-
cloned to ensure stable antibody production. mAbs from these
hybridomas were purified, conjugated to Alexa Fluor 647, and
tested in the previously described flow cytometry assay for their
ability to bind at pH 7.2 to human or mouse FcRn. In total, nine
unique hybridoma clones were established. Only one, DVN24,
showed appreciable cross-reactivity to mFcRn (Fig. 2A). The
relative binding affinity of each mAb for hFcRn was estimated
by its ability to bind 293hFcRn-GFP cells by quantifying the mean

www.landesbioscience.com mAbs 209

 ©2013 Landes Bioscience. Do not distribute.
Downloaded by [121.207.210.105] at 20:58 04 October 2015

Figure 2. Anti-hFcRn mAb specificities and relative affinities. 293hFcRn-GFP, 293mFcRn-GFP, or control 293 cells were incubated with AF647-labeled anti-hFcRn
mAbs (A) at a fixed concentration of 200 ng/106 cells to determine specificity or (B) with a range of concentrations to assess relative mAb affinities for
hFcRn. Values within scatter plots represent the AF647 MFIs of each hFcRn-EGFP+-gated cell population. Data are representative of at least two experi-
ments.

Table 1. Competitive binding of anti-hFcRn mAbsa

BLOCK DVN1 ADM11 DVN21 DVN22 DVN23 DVN24 ADM31 ADM32
DVN1 b
100 33 32 17 55 0 85 67
ADM11 47 100 21 8 13 8 58 48
DVN21 0 32 100 10 4 55 57 42
DVN22c 0 34 66 100 103 1 37 7
DVN23 c
0 14 11 96 100 31 38 29
DVN24 0 13 0 0 0 100 54 13
ADM31b 106 7 0 12 0 10 100 100
ADM32b 94 13 1 4 4 4 101 100
293hFcRn-GFP cells were exposed to unlabeled anti-hFcRn mAbs (blocking) followed by a 1,000-fold lower concentration of AF647-labeled anti-hFcRn
a

mAbs in a matrix at pH 7.2. AF647 MFIs of the hFcRn-GFP+-gated cell populations were then determined by flow cytometric analyses. Values represent
% inhibition of labeled mAb binding as described in the Materials and Methods; bADM31, ADM32, DVN1 reciprocal blockade; cDVN22, DVN23 reciprocal
blockade.

fluorescent intensity (MFI) of titered mAb concentrations. Most
of the mAbs demonstrated comparably high affinity for hFcRn,
with the exception of ADM12, which demonstrated a somewhat
lower affinity (Fig. 2B).
To evaluate the epitopes of hFcRn targeted by the mAbs, the
ability of the mAbs to cross-compete for binding to hFcRn on
293hFcRn-GFP cells was determined (Table 1). The binding patterns
demonstrated a spectrum of epitopes that were consistent with
both unique and shared hFcRn binding sites. ADM11, DVN21
and DVN24 appear to have unique epitopes on hFcRn since they
did not compete with the other mAbs in the competitive binding
assay. DVN1, ADM31 and ADM32 were able to compete with
each other, which suggests that they bind to overlapping epitopes.
DVN22 and DVN23 were each capable of blocking the other’s
binding to hFcRn. Taken together, this mAb panel identified at
least five distinct epitopes on hFcRn.
Confocal IF imaging of hFcRn using the mAbs. We then
assessed the ability of the mAbs to stain hFcRn in fresh frozen
tissue sections from hFcRn transgenic mice (on a mFcRn-/- back-
ground) or from patient samples (Fig. 3). ADM31AF647 readily
detected hFcRn in the hFcRn transgenic mouse and human
kidney sections (Fig. 3A and D). Using ADM31AF647, the FcRn
signal was strongest in the proximal tubules and the the glom-
erulus (Fig. 3B and E), consistent with the known expression
pattern of FcRn in the kidney.19,20 As a negative control, ADM31
failed to stain kidney sections from B6 mice, confirming its bind-
ing specificity for hFcRn (Fig. 3C). Other tissue types, includ-
ing heart, brain, liver, muscle and spleen, similarly demonstrated
patterns of hFcRn staining consistent with previous reports (data

210 mAbs Volume 4 Issue 2

 ©2013 Landes Bioscience. Do not distribute.
Downloaded by [121.207.210.105] at 20:58 04 October 2015

Figure 3. Immunofluorescent detection of hFcRn (red channel) in kidney tissue sections from (A) hFcRn transgenic mice, (B) hFcRn transgenic mice
costained for proximal convoluted tubule (blue channel) and podocytes (green channel), (C) wild-type B6 mice costained for proximal convoluted
tubule and podocytes, (D) human tissue, (E) human tissue costained for proximal convoluted tubule and podocytes, and (F) human tissue showing
only proximal convoluted tubule and podocytes (GM, Glomerulus, PCT, proximal convoluted tubules).

Figure 4. hFcRn blocking activity. 293hFcRn-GFP cells were incubated with a range of concentrations of DVN1 (○), ADM11 (●), DVN21 (▲), DVN22 (□),
DVN23 (■), DVN24 (◇), ADM31 (◆) or ADM32 (━) at pH 6, then stained for functional binding with (A) 20 μg/mL hIgGAF647, or (B) 200 μg/mL HSAbiotin
followed by streptavidin-PE. Nonfunctional binding was indicated by the AF647 or PE MFIs of cells stained with labeled ligands at pH 7.2 (X). (C) Scatter
plots of 293hFcRn-GFP cells that were untreated at pH 6.0 (upper left) or at pH 7.2 (upper right), exposed to 200 ng/mL of DVN24 (lower left) or ADM11 (low-
er right) at pH 6.0, and then stained for functional binding of hIgGAF647 at the corresponding pH. (D) Scatter plots of 293hFcRn-GFP cells that were untreated
at pH 6.0 (upper left) or at pH 7.5 (upper right), exposed to 200 ng/mL of ADM31 (lower left) or ADM11 (lower right), and then stained for functional
binding with HSA-biotin and counterstained with streptavidin-PE at the corresponding pH. AF647 or PE MFI values of the hFcRn-GFP+-gated cell popu-
lations are indicated. Data are representative of three independent experiments.

not shown). The other mAbs in the part displayed similar tis-
sue staining profiles for hFcRn, with the exception of DNV24,
which cross-reacted with mFcRn (data not shown). These results
demonstrate the utility of the mAbs for defining the cellular and
tissue distribution of human FcRn.
Inhibition of hIgG and HSA binding in vitro. The potential
of each mAb clone to inhibit the pH-dependent binding of hIgG
or human serum albumin (HSA) to hFcRn was analyzed using an
in vitro binding competition assay. In this assay, 293hFcRn-GFP cells
were treated with increasing concentrations of each mAb clone

www.landesbioscience.com mAbs 211

 before labeled hIgG or HSA was added. Under these
competitive conditions, the bound hIgG or HSA was
quantified by flow cytometry (Fig. 4). Both DVN24
and DVN21 potently blocked hIgG binding to hFcRn,
with the maximum effect at 3 μg/mL and 10 μg/mL,
respectively (Fig. 4A; representative scatter plots results
for DVN24, B). In contrast, DVN23, ADM31 and
ADM32 most efficiently blocked HSA binding, leav-
ing IgG binding intact (Fig. 4C; representative scat-
ter plots results for ADM31, D). These results showed
that certain mAbs block the pH-dependent binding
of hIgG and albumin selectively. Thus, the different

©2013 Landes Bioscience. Do not distribute.

epitopes on hFcRn recognized by our mAb part are
consistent with published results that support spatially
distinct IgG-Fc and HSA binding sites on FcRn.5,15,16,21
Treatment with anti-hFcRn mAbs in vivo. We
next investigated whether the newly generated mAbs
that selectively blocked hIgG binding to hFcRn
Downloaded by [121.207.210.105] at 20:58 04 October 2015

could accelerate the clearance of IgG antibodies

in vivo. For these studies, we focused primarily on
DVN24 because it was the most effective inhibitor
of hIgG binding to hFcRn in vitro (Fig. 4A). Tracer
amounts of hIgG, mIgG1 and mIgA were injected
into hFcRn transgenic mice to establish β-phase Figure 5. Blockade of IgG by anti-hFcRn mAbs in vivo. (A) B6 Tg276 mice were co-
injected ip with 100 μg each of hIgG, mIgG1 (1B7.11) and mIgA (2F11-10) as tracers.
clearance baselines before the mice were treated with The mice were then injected with 1,000 μg (◇), 300 μg (◆), 100 μg (△), 30 μg (▲)
varying doses of DVN24 or an isotype-matched mAb or 0 μg (□) of DVN24 or with 1,000 μg (X) of a control IgG2a mAb into groups of 3
(Fig. 5A and B). Mice treated with DVN24 showed mice on days 2, 3 and 4 d after tracer injection. (B) B6 Tg276 mice (n = 3) were co-
2.6- to 8.2-fold reduced concentrations of hIgG in injected ip with 100 μg hIgG and 1 mg of HSA tracers followed by injections with
serum compared with mice treated with a similar 1 mg doses of ADM32 (-), DVN24 (◇) or isotype matched mIgG2a (X). (C) B6 Tg276
mice (n = 4–5) were injected with 100 μg hIgG tracer and then treated on days 5–7
amount of isotype control mAb, with the maximal or 10–12 with 1 mg of DVN24 (◇) or mIgG2a (X). Significant differences (A–C) are
effect observed at 1 mg dosages. We also determined indicated as *p < 0.05 and **p < 0.01 comparing 1,000 μg injections of DVN24 with
whether the therapeutic administration of DVN24 the control isotype matched mIgG2a. (D) B6 WT mice (n = 8) were injected ip with
affected the serum persistence of HSA. DVN24 failed 200 μg of mIgG1 (1B7.11) as a tracer, and then injected ip on days 4–6 with 1 mg
to promote the clearance of tracer HSA while promot- doses of DVN24 (◇), ADM31 (◆) or vehicle only (X). Tracer plasma concentrations
were determined by ELISA and plotted either as percent remaining as compared
ing the clearance of hIgG in the same mice (Fig. 5B). with the first time point plasma concentrations or as plasma concentrations ±SD.
These results are consistent with the in vitro block- Significant differences (D) are indicated as **p < 0.01 comparing 1,000 μg injections
ade studies described above (Fig. 4), and support the of DVN24 with the vehicle only control.
binding specificity of DVN24 to a site on hFcRn that
specifically interacts with hIgG and results in the accel-
eration of hIgG clearance in vivo without affecting the half-life primarily by the need for reliable serological reagents to probe
of HSA. the biology of hFcRn. Immunization of mice with cells from
Since DVN24 cross-reacts with mFcRn (Fig. 2A), we hFcRn-transgenic mice resulted in a panel of nine independently
addressed whether administration of this mAb could influence derived, stable hybridoma clones that secrete mAbs specific for
the serum persistence of mIgG in mice. Treatment of B6 WT native heavy chain determinants of hFcRn. The applications of
mice with DVN24 resulted in an approximate 2.8-fold reduction the antibody part are summarized in Table 2. We show here that
of tracer mIgG1 compared with treatment with the HSA-blocking these mAbs have promise as incisive reagents capable of unlock-
mAb ADM31, which had a negligible effect (Fig. 5D). While the ing a deeper understanding of the tissue and cellular expression
treatment was not as efficient as that found in the hFcRn studies, patterns of hFcRn, which may facilitate a better understanding of
the results indicate that the mAbs show both species and epitope FcRn biology and lead to development of improved therapeutics.
specificity in this in vivo system. Whether the mAb panel may be applied to the study of FcRn
in other mammalian species, including primates, remains to be
Discussion determined.
The major function of FcRn is to bind IgG and serum albu-
The overall goal of this study was to develop and characterize a min in endosomal compartments and rescue these ligands by
diverse panel of mAbs with specificity for hFcRn and to explore returning them intact to the cell surface. This trafficking is
their applications in vitro and in vivo. This effort was motivated highly dependent on pH because FcRn requires an acidic pH

212 mAbs Volume 4 Issue 2

 Table 2. Summary of anti-hFcRn mAb characteristics
mAb Specificity Shared Blockade in vitro
Name Isotype hFcRn mFcRn pH a Bindingb hIgG HSA mIgG FACSc IFc WBc Straind
DVN1 IgG1 + - 7–8 31, 32 - - - + + - WT
ADM11 IgG2b + - 6–8 - - - - + + - WT
ADM12 IgG2b + - 6–8 NDe - - - + + - WT
DVN21 IgG1 + - 6–8 - + - - + + - FcRn-/-
DVN22 IgG1 + - 7–8 23 - - - + + - FcRn-/-
DVN23 IgG2b + - 6–8 22 - - - + + - FcRn-/-
DVN24 IgG2a + + 6–8 - + - + + + - FcRn-/-

©2013 Landes Bioscience. Do not distribute.

ADM31 IgG2b + - 6–8 1, 32 - + - + + - FcRn-/-
ADM32 IgG2b + - 6–8 1, 31 - + - + + - FcRn-/-
a
mAbs binding hFcRn on 293 hFcRn-GFP
cells; Reciprocally compete for binding hFcRn; Detection by cytometric analysis and immunofluorescence (IF), but
b c

fail by western blot (WB); dMouse strains used as responders to generate mAbs; eNot determined.

(~6) for either ligand to bind with high affinity. While bind- FcRn can be used to treat disease. The palliative effects of high
Downloaded by [121.207.210.105] at 20:58 04 October 2015

ing of both IgG and HSA are pH-dependent, their binding to
FcRn appear to occur at geographically distinct sites.5,15,16,21 FcRn
binding to the Fc region of IgG is mediated by protonation of
histidine residues on the Fc fragment that then bind to acidic
residues on the a1 and a2 domains of FcRn.22,23 Less is known
about the FcRn-albumin interaction surface, but recent stud-
ies support a similar histidine-dependent mechanism in which
protonated histidine residue (H166) on FcRn engages acidic Fc
residues.15,21 As summarized in Table 2, the binding patterns of
the mAb part observed in competition experiments in vitro iden-
tified both unique and overlapping epitopes on hFcRn. These
studies, in combination with the ability of certain anti-hFcRn
mAbs to inhibit the pH-dependent binding of hFcRn to hIgG
(DVN21 and DVN24) with others inhibiting the binding of
HSA (DVN23, ADM31 and ADM32), suggest that the mAb
epitope footprints are discrete and spatially distinct. Direct bio-
chemical measurements using methods including surface plas-
mon resonance could be used more to exactly define the mAb
affinities and epitope specificities. The anti-FcRn mAb panel
can therefore serve as a novel and useful tool for probing interac-
tions of hFcRn with its ligands and their trafficking patterns. For
example, two mAbs, DVN1 and DVN22, require a neutral pH
to bind hFcRn. At this neutral pH, one (DVN1) cross-competes
with ADM31 and ADM32. The fact that ADM31 and ADM32
are able to bind hFcRn and block HSA binding at an acidic pH
while DVN1 binds this site only at a neutral pH may be of use for
probing pH-sensitive HSA/FcRn interactions along the recycling
pathway using ultramicroscopic/ultrastructural techniques.
Autoantibodies with IgG isotypes contribute to a wide range
of autoimmune disorders, including Goodpasture’s syndome,
myasthenia gravis, idiopathic thrombocytopenic purpura (ITP),
lupus and rheumatoid arthritis. Through its ability to rescue IgG
from destruction in the lysosomal compartment, FcRn is respon-
sible for the maintenance of high concentrations of IgG in cir-
culation. FcRn-deficient mice are more resistant to autoimmune
diseases caused by pathogenic IgG autoantibodies because they
are unable to maintain the high concentrations of serum IgG.24-26
These studies lead to the concept that the therapeutic blockade of
dose administration of IgG in a number of autoimmune disorders
can be explained at least partially by the ability of this treatment
to saturate FcRn-mediated protection of IgG, resulting in rapid
clearance of pathogenic IgG autoantibodies.27-29 Blockade strate-
gies specifically directed at FcRn include the use of IgG mAbs
genetically engineered in the Fc region for high affinity binding
to FcRn (referred to as AbDegs),30-32 and synthetic peptides that
bind the Fc engagement site of FcRn.33
Blockade using mAbs specifically directed against FcRn is an
obvious alternative therapeutic approach to promote the clear-
ance of potentially pathogenic IgG. Proof of principle for mAb
blockade of FcRn has been demonstrated previously in a rat
model of experimentally-induced autoimmune myasthenia gra-
vis in which treatment of the FcRn heavy chain-specific mAb
1G3 lead to a transient reduction of serum IgG concentrations
as well as the severity of disease.34 However, similar studies were
not conducted for hFcRn. This approach is therapeutically rele-
vant because hFcRn has more stringent binding requirements for
IgG than rodent FcRn. Therefore, a ‘humanized’ FcRn system
is needed to test antibodies and therapies that may be applied to
patients. To this end, we utilized hFcRn-transgenic mice to test
the feasibility of hFcRn blockade by epitope-specific antibodies.
We first screened each anti-hFcRn mAb that we had generated
for its ability to block IgG binding to hFcRn in vitro. IgG bind-
ing blockade was not a consistent feature in that of the seven anti-
hFcRn mAbs tested that bound hFcRn at an acidic (endosomal)
pH, only DVN21 and DVN24 effectively blocked hIgG binding
in vitro (Table 2), and only DVN24 demonstrated therapeutic
efficacy by accelerating the clearance of hIgG in vivo (Fig. 5;
negative data for DVN21 are not shown). The lack of therapeu-
tic effect with the other anti-hFcRn mAbs, which bind hFcRn
avidly and, therefore, presumably remain bound to hFcRn but
do not impede the endosomal trafficking of its normal molecu-
lar cargo, can be interpreted as support for the durability of the
FcRn recycling pathway.
DVN24 is the only mAb identified to have a therapeutic affect
on hIgG clearance in the hFcRn Tg model. Since the Fc frag-
ment of mIgG minimally binds hFcRn,35 this therapeutic effect

www.landesbioscience.com mAbs 213

 is facilitated solely by its antigen specificity for FcRn. However,
the effect proved to be transient and required multiple dosages
of >50/mg/kg to achieve an appreciable therapeutic effect. This
transience is very likely the result of the short serum persistence
of native DVN24, which virtually disappears from the circu-
lation within hours after administration (results not shown).
Whether the therapeutic efficiency of DVN24 will be enhanced
by the replacement of the mouse Fc with the human IgG-Fc, thus
enabling it to capitalize on hFcRn’s normal recycling function,
remains to be determined. Finally, it is of interest that DVN24
was also the only mAb identified that cross-reacts with mFcRn.
Administration of this mAb accelerated the clearance of mIgG1
in B6 WT mice, albeit less efficiently than its effect on hIgG
clearance in hFcRn Tg mice. DVN24, therefore, has the dual
potential for investigating the effects of the therapeutic blockade
of FcRn in humanized and standard mouse models.
Materials and Methods
Downloaded by [121.207.210.105] at 20:58 04 October 2015
Mice. C57BL6/J (B6) mice. FcRn knockout mice on the
C57BL6/J background (designated B6.129X1-Fcgrttm1Dcr /DcrJ)
carrying a null allele of the α-chain of FcRn have been previously
described (FcRn-/-).9,36 Mice carrying hFcRn transgene (desig-
nated B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ) carry
the null allele of mFcRn and the hFcRn cDNA transgene under
the control of the CAG promoter and are referred to as Tg276 or
hFcRn transgenic mice.31,36 All mice were maintained under spe-
cific pathogen-free conditions and the procedures were approved
by the Jackson Laboratory Animal Care and Use Committee.
Generation of hFcRn and mFcRn constructs for trans-
fection. To produce the hFcRn construct, the 5' non-coding
sequence (118 bp) including the sequence encoding the 23
AA hFcRn signal sequence was PCR-amplified from hFcRn
cDNA (kindly provided by Clark Anderson, Ohio State
University) using forward CCC CCC CCG CTA GCG AAG
CCC CTC CTC GGC GTC CTG GT (NheI site underlined)
and reverse CCC CCC CCA CCG GTC CGC CCA GGC
TCC CAG GAA GGA GAA A (AgeI site underlined) prim-
ers. Extra cytosine nucleotides were included at the 5' ends of
primers to increase the efficiency of restriction endonuclease
activity. This PCR product was digested with NheI and AgeI,
and inserted downstream of the CMV-IE promoter, between
the NheI and AgeI restriction sites upstream and in-frame with
the EGFP coding sequence of the pEGFP-Cl vector (Clontech,
6084-1) to produce an N-terminal EGFP-tagged hFcRn con-
struct lacking the C-terminal endosomal targeting domain.
The mFcRn construct was generated from RNA isolated from
ten day old C57BL/6J neonatal proximal small intestine and
PCR amplified using forward (CCC CCC CCC TCG AGG
GTC AGA GAC CCG CCC CCA, XhoI site underlined) and
reverse (CCC CCC CCG AAT TCG CGC ATC CTG CCC
CAC AA, EcoRI site underlined) primers. As described above,
cytosine nucleotides were added to the 5' ends of the primers
to improve restriction endonuclease cleavage of the PCR prod-
uct. A 944 bp product was digested with XhoI and EcoRI, and
cloned into the corresponding sites of the pEGFP-C1 vector
214 mAbs
into which we had already inserted 118 bp of sequence includ-
ing the sequence encoding the 23 amino acid hFcRn signal
sequence downstream of the CMV-IE promoter, and upstream
and in-frame with GFP to produce an N-terminal GFP-tagged
tailless mFcRn construct. All PCR-amplified inserts were bi-
directionally sequence-verified across the cloning sites.
Cell lines. Purified hFcRn-GFP and mFcRn-GFP plasmids
were transfected into HeLa or HEK293 cells using Lipofectamine
Plus (Invitrogen, 15338-100) following the manufacturer’s proto-
col. Stably transfected HEK293 cells were selected using 400 μg/
mL G418 (Sigma-Aldrich, G5013) in 10% FBS-supplemented
DMEM. Since the constructs lacked the endosomal targeting
©2013 Landes Bioscience. Do not distribute.
domain, the resulting 293hFcRn-GFP and 293mFcRn-GFP cells expressed
FcRn at high levels on their plasma membrane, as verified by
immunofluorescence imaging and FACS analysis after staining
with anti-FcRn mAbs.
Antisera, hybridoma generation and screening. To generate
anti-hFcRn antibody responses, B6 WT or FcRn null mice were
immunized multiple times via intraperitoneal (i.p.) injection
with spleen cells from Tg276 mice in and complete or incomplete
Freund’s adjuvant. Plasma samples collected from boosted mice
were assayed by flow cytometry for anti-FcRn binding and the
functional blockade of IgG binding. To assess hFcRn binding,
293hFcRn-GFP cells were incubated with antisera, stained with goat
anti-mouse IgG-R-phycoeythrin (Southern Biotech, 1031-09),
and then analyzed using a FACSCalibur (BD Biosciences, San
Jose, CA USA). The ability to block hFcRn function was assessed
by adding antisera to 293hFcRn-EGFP cells in FACS buffer (PBS with
1% BSA and 0.05% NaN3), pH 6 or pH 7.2, followed by the
addition of hIgGAF647, and data were similarly acquired using a
FACSCalibur.
Spleen cells from immunized mice with high binding or block-
ing activity were fused with SP2/0-Ag14 myeloma cells accord-
ing to standard protocols. Selection for hybridomas was provided
by refeeding cells on days 2, 3, 4, 5, 7, 9 and 11 with DMEM-20
supplemented with HAT and 100 U/mL IL-6. A cellular ELISA
was used to screen the hybridomas for hFcRn binding activity
by incubating culture supernatant with 293hFcRn-GFP cells, stain-
ing with goat anti-mouse IgG-alkaline phosphatase (Southern
Biotech, 1031-04), and measured by adding p-nitrophenyl phos-
phate (AMRESCO, 0617) at 1 mg/mL in substrate buffer (50
mM sodium bicarbonate, 5 mM MgCl2, pH 8.6) followed by the
addition of 1% sodium dodecyl sulfate (Sigma-Aldrich, L4390)
to solubilize cells prior to OD405 determination on a SpectraMAX
190 plate reader (Molecular Devices, Sunnyvale, CA). Productive
hybridomas were expanded from anti-hFcRn positive wells, and
cloned 3 times by limiting dilution to verify stable productive
secretion of the resulting subclones. All mAb supernatants were
protein G (GE Healthcare, 17-0404-01) purified and labeled
with Alexa Fluor 674 (Invitrogen, A-20173). Specificity and rela-
tive affinity were measured by staining 293hFcRn-GFP, 293mFcRn-GFP
and 293 cells with AF647-labeled anti-hFcRn mAbs at a fixed
concentration of 200 ng/106 cells to determine specificity, or
with a range of indicated concentrations to determine affinity.
Data were acquired using a FACSCalibur and relative affinities
for hFcRn were represented as ratios of the Alexa Fluor 647 MFI
Volume 4 Issue 2
 from each mAb concentration to the MFI yielded from its high-
est concentration.
Competitive binding assay. Anti-hFcRn mAbs were screened
for their ability to compete for binding to hFcRn on ice. In matrix
fashion, unlabeled mAbs (blocking) were individually added
to 106 293hFcRn-EGFP cells at a final concentration of 500 μg/mL
for 30 min, and cells were washed with 4 mL FACS buffer, pH
7.2. The same set of mAbs labeled with AF647 were then added
individually to antibody treated 293hFcRn-GFP cells at a final con-
centration of 500 ng/mL, incubated for 30 min, and cells were
washed with 4 mL FACS buffer, pH 7.2. Events were acquired
on a FACSCalibur and the percent inhibition of labeled antibody
binding was calculated from the Alexa Fluor 647 MFI of each
hFcRn-GFP+ -gated cell population as follows: % Inhibition =
(1 - ((MFIEXP - MFISELF)/MFINONE)) x 100, where MFIEXP is the
MFI of a given labeled mAb in the presence of a competing unla-
beled blocking mAb, MFISELF is the MFI of a given labeled mAb
in the presence of the same unlabeled blocking mAb, MFINONE is
Downloaded by [121.207.210.105] at 20:58 04 October 2015
the MFI of a given labeled mAb in the absence of an unlabeled
blocking mAb.
Antibody blockade of hIgG and HSA binding in vitro.
Antisera and mAbs were screened for the ability to block hFcRn
function on ice by adding either 2 μL of heteroantisera, or
defined amounts of purified mAbs, to 106 293hFcRn-GFP cells in
25 μL FACS buffer, pH 6 or 7.2 for 30 min, then washed with
corresponding pH FACS buffer. The cells were incubated for 60
min with hIgG-Alexa Fluor 647 (1 μg) or 10 μg human serum
albumin-biotin (HSA-biotin), and washed with corresponding
pH FACS buffer. For assessment of HSA-biotin binding, cells
were incubated for 30 min with streptavidin-phycoerythrin
(Southern Biotech, 7100-09) followed by washing with cor-
responding pH FACS buffer. HSA binding FACS buffer was
albumin-free. MFI was determined by acquiring events on a
FACSCalibur.
Immunofluorescence and confocal microscopy. The expres-
sion pattern of hFcRn in various tissues was determined using
immunofluorescence (IF) techniques. Organs from age and
sex-matched mice were collected and immediately embedded
with OCT and frozen using Precision Cryo-embedding System
(IHC world, Woodstock, MD). Sectioning of these tissues was
performed with a Leica CM 1950 Cryostat (Buffalo Grove, IL).
Seven to 10 μm sections were applied to Fisherbrand Superfrost®
Plus slides and stored at -20°C. Quality control of the prepa-
rations was evaluated by H&E staining of consecutive sections.
Normal human kidney tissue was harvested from nephrectomy
specimens and snap frozen in OCT medium and sectioned
as described previously. Use of human tissue specimens was
approved by the Washington University Institutional Review
Board (IRB #201107288).
www.landesbioscience.com mAbs
For the IF studies shown, hFcRn was detected using Alexa
Fluor 647 labeled mAbs. Mouse kidney tissues were counter-
stained with rat anti-mouse podocalyxin (clone 192703) (R&D
Systems Inc., MAB1556) coupled with a goat anti-rat FITC
(Invitrogen, 629511) and biotin Lotus Tetragonolobus Lectin
(LTL) (Vector Laboratories, B-1325) coupled with streptavidin
DyLite 488 (Thermo Scientific, 21832) to highlight podocytes
and proximal convoluted tubules. IF labeling was performed
using standard protocols and imaged with a Leica SP5 confo-
cal microscope within 24 h of mounting. Human tissues were
stained in a similar fashion.
mAb blockade of FcRn in vivo. hFcRn Tg276 mice carry-
©2013 Landes Bioscience. Do not distribute.
ing one copy of the hFcRn or B6 WT mice were first injected
i.p. with 100 μg of tracer mIgG1 (1B7-11, anti-TNP, American
Type Culture Collection, TIB-191), mIgA (2F11-15, anti-TNP,
American Type Culture Collection, TIB-194), human IgG
(Gammagard, Baxter, 1501375) or 1 mg HSA (Sigma-Aldrich,
A-7284) as tracers. ELISA of serial blood samples was used to
assess the plasma concentrations of each tracer. The tracer anti-
bodies were captured with TNP-bovine IgG (EMD Chemicals,
324101), mouse anti-human IgG Fc (Southern Biotech, 9040-
01), or rabbit anti-HSA (United States Biological, A1327-46),
respectively. Goat anti-mouse IgG1-AP (Southern Biotech, 1070-
04), rat anti-mouse IgA-AP (Southern Biotech, 1165-04), mouse
anti-human kappa-AP (Southern Biotech, 9230-04) or goat
anti-HSA-AP (Bethy Laboratories, A80-229AP), respectively,
were used for detection. The ELISA plates were washed 3 times
with 250 μL ELISA wash (PBS, 0.05% Tween 20, 0.05% NaN3)
between each step, and both the block and all dilutions, were
made in ELISA wash with 1% BSA. The ELISA was developed
using p-nitrophenyl phosphate substrate added at 1 mg/mL in
substrate buffer prior to OD405 measurement on a SpectraMAX
190 plate reader.
Disclosure of Potential Conflicts of Interest
D.C.R and S.A. have applied for US and European patents that
include some of the mAbs described.
Acknowledgments
This work was supported by the grants from the Alliance for
Lupus Research and the National Institutes of Health (NIH RO1
DK56597).
Authors’ Contributions
G.J.C., V.Z.S., S.A., E.P. and S.P. designed and performed exper-
iments and analyzed data. D.C.R. designed experiments and
analyzed data. G.J.C., V.Z.S., S.A., G.P. and D.C.R. contributed
to the writing of the manuscript.
215

References
1. Ghetie V, Ward ES. Multiple roles for the major
histocompatibility complex class I-related recep-
tor FcRn. Annu Rev Immunol 2000; 18:739-66;
PMID:10837074; http://dx.doi.org/10.1146/annurev.
immunol.18.1.739.
2. Roopenian DC, Akilesh S. FcRn: the neonatal Fc
receptor comes of age. Nat Rev Immunol 2007; 7:715-
25; PMID:17703228; http://dx.doi.org/10.1038/
nri2155.
3. Ward ES, Ober RJ. Chapter 4: Multitasking by
exploitation of intracellular transport functions the
many faces of FcRn. Adv Immunol 2009; 103:77-115;
PMID:19755184; http://dx.doi.org/10.1016/S0065-
2776(09)03004-1.
4. Kuo TT, Baker K, Yoshida M, Qiao SW, Aveson VG,
Lencer WI, et al. Neonatal Fc receptor: from immu-
nity to therapeutics. J Clin Immunol 2010; 30:777-89;
PMID:20886282; http://dx.doi.org/10.1007/s10875-
010-9468-4.
5. Andersen JT, Sandlie I. The versatile MHC class
I-related FcRn protects IgG and albumin from deg-
radation: implications for development of new diag-
nostics and therapeutics. Drug Metab Pharmacokinet
2009; 24:318-32; PMID:19745559; http://dx.doi.
Downloaded by [121.207.210.105] at 20:58 04 October 2015
org/10.2133/dmpk.24.318.
6. Tesar DB, Björkman PJ. An intracellular traffic
jam: Fc receptor-mediated transport of immuno-
globulin G. Curr Opin Struct Biol 2010; 20:226-
33; PMID:20171874; http://dx.doi.org/10.1016/j.
sbi.2010.01.010.
7. He W, Ladinsky MS, Huey-Tubman KE, Jensen
GJ, McIntosh JR, Björkman PJ. FcRn-mediated
antibody transport across epithelial cells revealed
by electron tomography. Nature 2008; 455:542-
6; PMID:18818657; http://dx.doi.org/10.1038/
nature07255.
8. Gan Z, Ram S, Vaccaro C, Ober RJ, Ward ES.
Analyses of the recycling receptor, FcRn, in live cells
reveal novel pathways for lysosomal delivery. Traffic
2009; 10:600-14; PMID:19192244; http://dx.doi.
org/10.1111/j.1600-0854.2009.00887.x.
9. Roopenian DC, Christianson GJ, Sproule TJ, Brown
AC, Akilesh S, Jung N, et al. The MHC class I-like IgG
receptor controls perinatal IgG transport, IgG homeo-
stasis and fate of IgG-Fc-coupled drugs. J Immunol
2003; 170:3528-33; PMID:12646614.
10. Baker K, Qiao SW, Kuo T, Kobayashi K, Yoshida M,
Lencer WI, et al. Immune and non-immune functions
of the (not so) neonatal Fc receptor, FcRn. Semin
Immunopathol 2009; 31:223-36; PMID:19495758;
http://dx.doi.org/10.1007/s00281-009-0160-9.
11. Baker K, Qiao SW, Kuo TT, Aveson VG, Platzer B,
Andersen JT, et al. Neonatal Fc receptor for IgG (FcRn)
regulates cross-presentation of IgG immune complexes
by CD8-CD11b+ dendritic cells. Proc Natl Acad Sci
USA 2011; 108:9927-32; PMID:21628593; http://
dx.doi.org/10.1073/pnas.1019037108.
12. Liu X, Lu L, Yang Z, Palaniyandi S, Zeng R, Gao
LY, et al. The neonatal FcR-mediated presentation
of immune-complexed antigen is associated with
endosomal and phagosomal pH and antigen stabil-
ity in macrophages and dendritic cells. J Immunol
2011; 186:4674-86; PMID:21402891; http://dx.doi.
org/10.4049/jimmunol.1003584.
216
13. Ye L, Zeng R, Bai Y, Roopenian DC, Zhu X.
Efficient mucosal vaccination mediated by the neo-
natal Fc receptor. Nat Biotechnol 2011; 29:158-
63; PMID:21240266; http://dx.doi.org/10.1038/
nbt.1742.
14. Lu L, Palaniyandi S, Zeng R, Bai Y, Liu X, Wang Y,
et al. A neonatal Fc receptor-targeted mucosal vac-
cine strategy effectively induces HIV-1 antigen-specific
immunity to genital infection. J Virol 2011; 85:10542-
53; PMID:21849464; http://dx.doi.org/10.1128/
JVI.05441-11.
15. Andersen JT, Dee Qian J, Sandlie I. The conserved
histidine 166 residue of the human neonatal Fc
receptor heavy chain is critical for the pH-dependent
binding to albumin. Eur J Immunol 2006; 36:3044-
51; PMID:17048273; http://dx.doi.org/10.1002/
eji.200636556.
16. Anderson CL, Chaudhury C, Kim J, Bronson CL,
Wani MA, Mohanty S. Perspective—FcRn transports
albumin: relevance to immunology and medicine.
Trends Immunol 2006; 27:343-8; PMID:16731041;
http://dx.doi.org/10.1016/j.it.2006.05.004.
17. Neumann E, Frei E, Funk D, Becker MD, Schrenk
HH, Müller-Ladner U, et al. Native albumin for
targeted drug delivery. Expert Opin Drug Deliv 2010;
7:915-25; PMID:20586704; http://dx.doi.org/10.151
7/17425247.2010.498474.
18. Andersen JT, Pehrson R, Tolmachev V, Daba MB,
Abrahmsén L, Ekblad C. Extending half-life by indi-
rect targeting of the neonatal Fc receptor (FcRn) using
a minimal albumin binding domain. J Biol Chem
2011; 286:5234-41; PMID:21138843; http://dx.doi.
org/10.1074/jbc.M110.164848.
19. Akilesh S, Huber TB, Wu H, Wang G, Hartleben B,
Kopp JB, et al. Podocytes use FcRn to clear IgG from
the glomerular basement membrane. Proc Natl Acad
Sci USA 2008; 105:967-72; PMID:18198272; http://
dx.doi.org/10.1073/pnas.0711515105.
20. Haymann JP, Levraud JP, Bouet S, Kappes V, Hagège
J, Nguyen G, et al. Characterization and localization of
the neonatal Fc receptor in adult human kidney. J Am
Soc Nephrol 2000; 11:632-9; PMID:10752522.
21. Andersen JT, Dalhus B, Cameron J, Daba MB,
Plumridge A, Evans L, et al. Structure-based mutagen-
esis reveals the albumin-binding site of the neonatal Fc
receptor. Nat Commun 2012; 3:610; PMID:22215085;
http://dx.doi.org/10.1038/ncomms1607.
22. Burmeister WP, Huber AH, Bjorkman PJ. Crystal
structure of the complex of rat neonatal Fc receptor
with Fc. Nature 1994; 372:379-83; PMID:7969498;
http://dx.doi.org/10.1038/372379a0.
23. Martin WL, West AP Jr, Gan L, Bjorkman PJ.
Crystal structure at 2.8 A of an FcRn/heterodimeric Fc
complex: mechanism of pH-dependent binding. Mol
Cell 2001; 7:867-77; PMID:11336709; http://dx.doi.
org/10.1016/S1097-2765(01)00230-1.
24. Christianson GJ, Blankenburg RL, Duffy TM, Panka
D, Roths JB, Marshak-Rothstein A, et al. beta2-
microglobulin dependence of the lupus-like autoim-
mune syndrome of MRL-lpr mice. J Immunol 1996;
156:4932-9; PMID:8648144.
mAbs
25. Ghetie V, Hubbard JG, Kim JK, Tsen MF, Lee Y, Ward
ES. Abnormally short serum half-lives of IgG in beta
2-microglobulin-deficient mice. Eur J Immunol 1996;
26:690-6; PMID:8605939; http://dx.doi.org/10.1002/
eji.1830260327.
26. Israel EJ, Wilsker DF, Hayes KC, Schoenfeld D,
Simister NE. Increased clearance of IgG in mice that
lack beta 2-microglobulin: possible protective role of
FcRn. Immunology 1996; 89:573-8; PMID:9014824;
http://dx.doi.org/10.1046/j.1365-2567.1996.d01-
775.x.
27. Jin F, Balthasar JP. Mechanisms of intravenous immuno-
globulin action in immune thrombocytopenic purpura.
Hum Immunol 2005; 66:403-10; PMID:15866704;
http://dx.doi.org/10.1016/j.humimm.2005.01.029.
28. Akilesh S, Petkova S, Sproule TJ, Shaffer DJ,
©2013 Landes Bioscience. Do not distribute.
Christianson GJ, Roopenian D. The MHC class
I-like Fc receptor promotes humorally mediated auto-
immune disease. J Clin Invest 2004; 113:1328-33;
PMID:15124024.
29. Li N, Zhao M, Hilario-Vargas J, Prisayanh P, Warren S,
Diaz LA, et al. Complete FcRn dependence for intrave-
nous Ig therapy in autoimmune skin blistering diseases.
J Clin Invest 2005; 115:3440-50; PMID:16284651;
http://dx.doi.org/10.1172/JCI24394.
30. Vaccaro C, Zhou J, Ober RJ, Ward ES. Engineering
the Fc region of immunoglobulin G to modulate in
vivo antibody levels. Nat Biotechnol 2005; 23:1283-8;
PMID:16186811; http://dx.doi.org/10.1038/nbt1143.
31. Petkova SB, Akilesh S, Sproule TJ, Christianson GJ,
Al Khabbaz H, Brown AC, et al. Enhanced half-life
of genetically engineered human IgG1 antibodies in
a humanized FcRn mouse model: potential applica-
tion in humorally mediated autoimmune disease. Int
Immunol 2006; 18:1759-69; PMID:17077181; http://
dx.doi.org/10.1093/intimm/dxl110.
32. Patel DA, Puig-Canto A, Challa DK, Perez Montoyo H,
Ober RJ, Ward ES. Neonatal Fc receptor blockade by
Fc engineering ameliorates arthritis in a murine model.
J Immunol 2011; 187:1015-22; PMID:21690327;
http://dx.doi.org/10.4049/jimmunol.1003780.
33. Mezo AR, Sridhar V, Badger J, Sakorafas P, Nienaber
V. X-ray crystal structures of monomeric and dimeric
peptide inhibitors in complex with the human neonatal
Fc receptor, FcRn. J Biol Chem 2010; 285:27694-
701; PMID:20592032; http://dx.doi.org/10.1074/jbc.
M110.120667.
34. Liu L, Garcia AM, Santoro H, Zhang Y, McDonnell K,
Dumont J, et al. Amelioration of experimental autoim-
mune myasthenia gravis in rats by neonatal FcR block-
ade. J Immunol 2007; 178:5390-8; PMID:17404325.
35. Ober RJ, Radu CG, Ghetie V, Ward ES. Differences
in promiscuity for antibody-FcRn interactions across
species: implications for therapeutic antibodies. Int
Immunol 2001; 13:1551-9; PMID:11717196; http://
dx.doi.org/10.1093/intimm/13.12.1551.
36. Roopenian DC, Christianson GJ, Sproule TJ. Human
FcRn transgenic mice for pharmacokinetic evalua-
tion of therapeutic antibodies. Methods Mol Biol
2010; 602:93-104; PMID:20012394; http://dx.doi.
org/10.1007/978-1-60761-058-8_6.
Volume 4 Issue 2