http://informahealthcare.com/drtISSN: 1061-186X (print), 1029-2330 (electronic)

J Drug Target, Early Online: 1–10! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2013.875030

REVIEW ARTICLE

Neonatal Fc receptor (FcRn): a novel target for therapeutic antibodiesand antibody engineering

Yuan Wang, Zehua Tian, Diraviyam Thirumalai, and Xiaoying Zhang

College of Veterinary Medicine, Northwest A&F University, Yangling, Shannxi Province, P.R. China

Abstract

The biomedical applications of antibodies as prophylactics, therapeutics and diagnostics aredeveloping rapidly. Neonatal Fc receptor (FcRn) is a major IgG Fc receptor capable of facilitatingthe translocation of IgG. FcRn can protect IgG from intracellular catabolism, thereby increasingits half-life. In recent decade, the interaction between FcRn and the Fc region has been reportedwith the focuses on either prolonging the plasma half-life of therapeutic IgG or shortening thehalf-life of pathogenic IgG. The FcRn–IgG interaction can be altered by modifying the Fc regionto change their affinity (increase or decrease), and/or by reducing the Fc fragments of IgG toenhance its penetration into tissues or cells. By over expression of FcRn, the exogenouscatabolism can be reduced, meanwhile the circulating IgG level could be enhanced. It has beenconfirmed in different FcRn over-expressed transgenic mice models, substantial humoralresponses against antigens with weak immunogenicity can be mounted. In addition, designinginhibitors for FcRn–IgG interaction is another application prospect for treating IgG-mediatedautoimmune diseases. Recent research advancements strengthen the understanding that FcRnis a key and promising drugable target in IgG intervention in the field of antibody engineering.

Keywords

Antibody drug, bioreactor, immunoglobulinG, neonatal Fc receptor, receptormodulation

History

Received 4 August 2013Revised 25 November 2013Accepted 10 December 2013Published online 9 January 2014

Introduction

Antibody molecules have been used for prophylactic, thera-

peutic and diagnostic purposes in medical field as promising

biopharmaceuticals and for many other experimental purposes

in biomedical researches because of their unique biological

properties. Remarkable advancements in hybridoma technol-

ogy and molecular biotechnology have facilitated the devel-

opment of various research domains in antibody engineering

such as murine, chimeric, humanized and human monoclonal

antibodies (mAbs) [1]. This development has reflected in the

availability of approved therapeutic MAbs in the pharma-

ceuticals markets. Thirty-five mAbs were approved as of

December 2013 [2], in which 29 mAbs are being currently

marketed in Europe and the USA as well as other countries

despite hundreds of other promising candidates are under

clinical trials [3]. The molecular properties and functions of

immunoglobulin (Ig) and their interaction with other biomol-

ecules should be thoroughly understood, in order to increase

the therapeutic antibodies production with enhanced efficacy

and plasma half-life. It is also very important to develop such

advanced techniques to manipulate these functional properties

desired for a particular application without exacerbating

undesirable ones [1], for instance, Neonatal Fc receptor

(FcRn) facilitates the translocation of IgG from mother to

fetus or offspring and protects IgG from intracellular catab-

olism thereby increasing the half-life of this class of antibody

molecules. In recent decades the interaction between FcRn

and the Fc region of IgG molecule has been reported with the

focus of either to increase the plasma half-life of therapeutic

antibodies or to decrease the half-life of pathogenic antibodies

[4]. In addition to this, a number of bio-engineering strategies

have been applied to modify the functionality of therapeutic

mAbs according to the therapeutic requirements of particular

biological mechanisms [5]. At this juncture, the present

review encompasses the current trends on the modification in

the therapeutic antibodies at Fc region, FcRn over-expression

in transgenic animals and production of inhibitor of

IgG–FcRn interaction.

IgG and its receptors

Igs play an important role in the animal innate and adaptive

immunity. Igs are classified into five isotypes in humans: IgM,

IgD, IgA, IgE and IgG based on the differences in the constant

region of heavy chain [6]. IgG is a major effector molecule of

the humoral immune response and it accounts nearly 75%

of the total Igs in plasma of healthy individuals [7]. IgGs have

an average half-life of 21 d in healthy humans, which is longer

than that of any other known serum proteins [8].

Human IgG can be further classified into IgG1, IgG2,

IgG3 and IgG4 mainly according to the differences in amino

acid composition and structure of the hinge region which

contains disulfide bonds between the g-heavy chains [9].

Address for correspondence: Prof. Dr. Xiao-Ying Zhang, College ofVeterinary Medicine, Northwest A&F University (North Campus), PostBox 19, Xinong Road 22, Yangling, Shaanxi Province 712100, P.R.China. Tel/Fax: +86 2987091239. E-mail: zhang.xy@nwsuaf.edu.cn

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The length and flexibility of the hinge region varies among

the IgG sub-classes [10,11]. The relative serum concentra-

tions of IgGs are as follows: IgG14IgG24IgG3& IgG4

[12]. The four sub-classes show more than 95% homology in

the amino acid sequences of constant domains of the g-heavy

chains [13].

IgG Fc receptors located on the cytomembrane of endo-

thelial cells, macrophages, lymphocytes, etc., play a central

role in both the afferent and efferent phase of an immune

response. The IgG Fc receptors are mainly composed of

FcRn, FcgIR, FcgRIIA, FcgRIIB, FcgRIIIA, FcgRIIIB,

moFcgRIV and boFcg2R [14,15] (Table 1).

FcgR activates the phagocytes on interaction with IgG-

opsonized particles involving the ITMAs. This activation

signal may possibly be down-regulated by the immunorecep-

tor tyrosine-based inhibition motif (ITIM)-containing

FcgRIIb receptor on polymorphonuclear (PMN) neutrophils

and monocytes [14]. No other signaling motifs have been

implicated in FcgR-mediated phagocytosis. Cross linking of

ITAM-bearing receptors (e.g. T-cell receptor, FcgRI) does not

initiate phagocytosis, it generally triggers fusion and release

of granule contents into sealed immunologic synapses

between effector cells and targets [16].

The FcRn is a 52-kDa heterodimeric glycoprotein consist-

ing of two polypeptides: a 15-kDa soluble light chain

(b2-microglobulin) and a 50-kDa transmembrane anchored

heavy chain (a-FcRn) [17]. Both the FcRn a-chain and b2m

participate in IgG binding within the CH2–CH3 interface

[16]. The X-ray crystallography of soluble extracellular

domain of FcRn shows that its structure possesses a MHC-

class I-like fold, where the peptide groove is closed and

therefore unable to bind peptides at that site [18].

Distribution and function of FcRn

FcRn plays a central role in IgG biology at all stages of life

and has primary functions such as IgG transcytosis and

regulation of serum IgG homeostasis [19]. FcRn was origin-

ally isolated from the intestinal epithelium of neonatal rats

and shown to mediate the uptake of IgG derived from

mother’s milk [20]. FcRn is widely distributed in various

organs, tissues and cells of mammals and highly expressed in

epithelial cells, placental syncytiotrophoblasts and endothelial

cells (Figure 1). In these cells, FcRn has been shown to

involve in IgG transport across mucosal cells or from mother

to fetus, and regulation of IgG half-life, respectively [16].

In humans, FcRn is detected in both fetal and adult

intestines, and can mediate bidirectional transcytosis across

the intestinal epithelium in vitro as well as in vivo [21]. FcRn

is the vehicle that transports IgG across the intestinal

epithelial barrier into the lumen to combine with cognate

antigen (Figure 1B). FcRn then recycles the IgG/antigen

complex back into the lamina propria across the intestinal

barrier for the dendritic cells processing, and the complex is

presented to CD4+ T cells in regional organized lymphoid

structures [21].

FcRn plays major roles in the bidirectional transcytosis of

IgG at various sites including blood–retina barrier, blood–

brain barrier (Figure 1D), mammary gland and placenta. In

addition to translocation and protection from catabolism,

FcRn in the liver could prevent IgG and albumin loss through

the biliary system (Figure 1F). FcRn is also detected at high

levels in tracheal epithelial cells. This likely explains the

efficacy of Fc fusion protein delivery when inhalation is

directed to the upper air ways in non-human primate

experiments and human clinical trials [22–24].

FcRn as a biomarker for rational of modification intherapeutic antibody

The pharmacokinetic behaviors of antibodies could mainly be

influenced by regulating the metabolism and excretion in the

way of FcRn-mediated transportation [6]. Only in the acidic

environment of endocytic vacuoles (pH� 6.5), where histi-

dine residues in Fc fragment of IgG become protonated, can

FcRn bind to IgG with high affinity, while it does not bind to

IgG at physiologic pH (7.4) [25]. The FcRn-mediated IgG

recycling can be described as a three-step process: passive

pinocytosis of IgG into endothelial cells; acidification in

endosome by H+ adenosine triphosphatase (ATPase) at

approximately pH 6 that in-turn allows IgG binding to

FcRn; and unbound IgG destined to lysosomal degradation

while bound IgG is diverted back to circulation [4]. The

binding of Fc–FcRn in endosome may change FcRn distri-

bution and intracellular trafficking of FcRn-coated endosome

vesicles (Figure 1A). This directed endosome trafficking

mechanism may further explain the high efficiency of IgG

recycling [4].

Prolonging IgG half-life by mutating Fc fragment

Amino acid residues in CH2 and CH3 domains of IgG–Fc

fragment play important roles in the pH dependence of IgG–

FcRn binding. In order to characterize how by modulating

this interaction may influence antibody clearance and the

in vivo performance of therapeutic mAbs, a number of

investigations have focused on the mutation of amino acid

residues in CH2 and CH3 domains of IgG–Fc fragment.

Comparison of human IgG1 sequence with the crystal

structure of rat Fc fragment bound to murine FcRn shows

that in the human Fc, some of these residues, for example

Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435

could interact directly with human FcRn (Figure 2B), while

other nearby residues (e.g. Arg255, Thr256, Asp312, Glu380,

Glu382, His433 and Tyr436) interact indirectly [27].

The effect of altering the affinity of FcRn either at pH 6

and/or 7.4 values has been investigated. An interesting

phenomenon was found that in both cynomolgus and mice,

decreased serum clearance and increased half-life of muta-

tional antibody were showed by increasing the binding

affinity to FcRn at pH 6 with negligible change to binding

at pH 7.4 compared to that of wild-type (wt) antibody.

In contrast, mutational antibody with increased affinity to

FcRn at both pH 6 and pH 7.4 exhibited similar pharmaco-

kinetic behavior to that of wt antibody, showing that there is

no improvement in pharmacokinetics by increasing affinity at

pH 7.4 [28].

The study of alanine-scan of human IgG1 Fc against

human FcRn shows that the substitution of alanine at residue

N434 is favorable for improving the FcRn binding [15].

Later on an investigation [26] reported the saturation

2 Y. Wang et al. J Drug Target, Early Online: 1–10

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Tab

le1

.C

om

par

iso

no

fd

iffe

ren

tcl

asse

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Cla

ssA

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)

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Per

iph

eral

blo

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clea

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and

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and

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Figure 1. The important distributions and major transcytose mechanisms of FcRn. (A) FcRn is abundantly expressed by endothelial cells especiallyvascular endothelial cell. These cells internalize serum IgG, which binds to FcRn in an acidic endosomal compartment. FcRn then recycles IgG backinto circulation, thus extending its serum half-life. Serum proteins without a recycling receptor are destined for lysosomal degradation (Adopted fromRoopenian [4]). (B) In the adult human gut, enterocytes and lamina propria antigen-presenting cells (APCs) express FcRn. Enterocytes transcytose IgGinto the gut lumen where it binds to antigens. The IgG–antigen complex is then delivered to lamina propria dendritic cells (DCs) either directly or byreverse transcytosis across the epithelial-cell barrier. Antigen-loaded DCs then migrate to the draining lymph node to prime a T -cell response (Adoptedfrom Roopenian [4]). (C) FcRn is expressed in glomerular epithelial cells (podocytes), which form the main filtration barrier of the kidney. If IgGimmune complexes deposit at the kidney filter, podocyte FcRn may transcytose trapped immune complexes to prevent the filter from clogging. Furtherdownstream in the proximal convoluted tubule, FcRn may reclaim transcytosed IgG (not shown) (Adopted from Roopenian [4]). (D) FcRn is expressedin central nervous system (CNS) vascular endothelial cells. Therapeutic plaque-specific antibodies delivered systemically can enter the CNS throughtransient openings of the blood–brain barrier. Once in the CNS, these antibodies bind and dissolve plaque deposits. Subsequently FcRn mediatesefficient transport of the plaque-bound antibodies across the blood–brain barrier back into systemic circulation, thereby reducing CNS plaque burden(Adopted from Roopenian [4]). (E) The expression and distribution of FcRn in the upper airway epithelium cell of lung. High expression of FcRn inupper airway epithelium cell of lung indicates that pulmonary administration may be a potential path for antibody drugs (data not published). (F) Theexpression and distribution of FcRn in the hepatocyte.

4 Y. Wang et al. J Drug Target, Early Online: 1–10

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mutagenesis to evaluate the effects of all possible amino acid

substitutions at this position. By studying these variants along

with some previously described variants, the direct correl-

ation between pH 6 affinity improvement and neutral pH

improvement was revealed, thus it suggests that all tested

variants exhibit similar pH dependency in FcRn binding.

The pharmacokinetics of variants N434A and N434W was

evaluated and it resulted 4- and 80-fold improvements in pH 6

binding affinity to both human and nonhuman primate FcRn,

respectively (Figure 3). In cynomolgus monkey, the clearance

of N434A decreased by 2-fold, but intriguingly the clearance

of N434W was similar to that of wt. The result shows that

the half-life of IgG in vivo and its binding affinity for FcRn

is not directly proportional, and modest increase in pH 6

FcRn affinity can result in improved pharmacokinetics in

primates [26].

The antibody drugs are reformed by protein or genetic

engineering, and when designing these drugs the affinity

should be enhanced appropriately to extend the half-life of

antibody drugs in vivo. Further, exploring the dose–effect

relationship between the increase in affinity and improvement

of half-life can be a research direction of the transformation of

antibody drug. A series of published mutations in the Fc

domain, most with relative success in both increasing binding

to FcRn and extending serum half-life, are reviewed here

(Table 2).

Reducing the size of Fc fragment to enhance thefunction of IgG

Intact mAbs in certain applications is limited by their

pharmacological characteristics: slow blood clearance that

leads to significant exposure to normal tissues and relatively

poor tissue penetration that results in inadequate quantities

being delivery to the target tissue [30]. Antibody fragments

such as (fragment antigen-binding (Fab), single-chain variable

fragment (ScFv), variable domains of llama heavy chain

antibody (VHH), diabody, triabody and minibody could

penetrate with high frequency into tissues and bind to the

covert site of antigen compared to whole antibody molecule.

However, antibody fragments could not bind to FcRn since

they lack Fc fragment and have short plasma half-life [30].

The antibodies whose Fc fragments are engineered can bind

with antigens but preserve interactions with FcRn, and Fc

fragment fused with monomeric proteins like ScFv, VHH and

Fab are recently developed as candidates for therapeutic

applications with prolonged half-lives.

In order to reduce the size of Fc fragment without any

alteration in the FcRn’s binding site, three human soluble

monomeric IgG1 Fc fragments (mFc.1, mFc.23 and mFc.67)

were generated by using a combination of structure-based

rational protein design combined with multiple screening

strategies (Fc Monomer in Figure 4) [31]. Furthermore, the

Figure 2. FcRn and FcRn/Fc structures. (A) The structures of FcRn (http://www.its.caltech.edu/�bjorker/Structures/FCRN2.gif). (B) Affinityscreening of Fc fragment variants against human FcRn. Cited from Yeung et al. [26].

Figure 3. The mutations of amino acidsubstitutions at residue N434.

DOI: 10.3109/1061186X.2013.875030 Neonatal Fc receptor 5

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stability and binding ability to FcRn of these mFc was tested.

The midpoint transition temperatures for Fc, mFc.1, mFc.23,

mFc.67 and CH2 were 75.1 ± 0.5, 45.0 ± 0.6, 45.2 ± 0.6,

51.0 ± 0.5 and 54.0 ± 0.8 �C, respectively. The 50% un-folding

of Fc occurred at higher urea concentrations (5.8 M) than that

of mFc.1 (4.1 M), mFc.23 (4.1 M), mFc.67 (4.3 M) and CH2

(4.2 M). The serum stability of the mFc proteins was also

evaluated by incubating the samples with human serum at

37 �C. The band for mFc.1 disappeared after three days

incubation, while the bands for Fc, mFc.23 and mFc.67 were

not evidently diminished even after 11-d incubations, sug-

gesting that Fc, mFc.23 and mFc.67 have high serum stability.

Surface plasmon resonance (SPR) experiments were used to

validate the pH-dependent FcRn binding and to obtain

reliable binding constants. At pH 6.0, the calculated binding

affinities of wt Fc, mFc.1, mFc.23 and mFc.67 to human

FcRn were 126, 204, 59 and 111 nM, respectively. At pH 7.4

neither wt Fc nor the mFcs showed detectable binding to

FcRn [31]. These results demonstrated that the highly soluble

mFcs maintain the characteristic pH dependent FcRn binding

and provide direct experimental evidence that efficient

binding to human FcRn does not require human Fc dimer-

ization. The newly identified mFcs are promising for the

development of mFc fusion proteins, and for novel types of

mFc-based therapeutic antibodies of small size with prolong

serum half-lives.

The Ig constant CH2 domain is critical for antibody

effector functions. Isolated CH2 domains are promising

scaffolds for the construction of libraries containing diverse

binders because it contains binding sites or portions for Fc

receptors and prolongs the half-life of antibody fragments

(Fc Dimer in Figure 4) [32]. The native CH2 domain has

significantly lower thermal stability when compared to other

small scaffolds, which could increase the probability of

instability when engineering binding to antigens and

enhanced effector functions. Previous studies showed that

the stability of an isolated human IgG1 CH2 can be

significantly increased by engineering an additional disulfide

bond between the A and G strands (m01) [33].

The stability of m01 was further increased by removing the

unstructured terminal residues (seven N-terminal residues that

are in a random coil) according to the isolated CH2 crystal

structure and NMR data [32]. After designing the plasmid

construction and yeast surface expression, CH2, m01, m01s,

Fc and CH3 were obtained for subsequent experiments.

Comparison of these antibody fragments showed that m01s is

remarkably stable with a melting temperature (Tm) of 82.6 �C,

which is about 10 and 30 �C higher than the original stabilized

CH2 (m01) and CH2, respectively. Due to the lack of CH3

domain, the binding of m01s to shFcRn is relatively weak

compared with the whole Fc, however, the affinity is higher

comparing with CH2 [32].

FcRn as a target for the improvement of therapeuticantibody production

Development of mutated IgG3 as an enhancedtherapeutic antibody

Human IgG3 can more effectively activate complement and

FcgR-mediated functions effectively when compared to anyTab

le2

.A

sum

mar

yo

fIg

Gm

uta

tio

ns

rep

ort

edto

enh

ance

the

FcR

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IgG

bin

din

gaf

fin

ity

and

exte

nd

IgG

seru

mh

alf-

life

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od

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uta

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teA

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gen

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ease

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ind

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affi

nit

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pH

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of

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(T1

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Ref

.

OS

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77

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other IgG subclass [13], which makes it an ideal candidate

for therapeutic applications. However, the shorter half-life of

IgG3 (one week) than other subclasses (three weeks) makes

IgG1 the therapeutic subclass of choice. The recycling of

IgG3 by FcRn may be less efficient due to the difference of

an amino acid at position 435, a key residue in IgG1, IgG2

and IgG4 for the pH-dependent formation of IgG–FcRn

complexes through histidine protonation around pH� 6.5

(IgG3 has an arginine at R435 position) [34].

In human in vitro and in vivo models, it was observed that

both IgG1 and IgG3 show pH-dependent binding to FcRn,

and that FcRn can transport IgG3 as efficiently as IgG1.

However, when IgG1 and IgG3 is co-present, IgG1 can inhibit

FcRn-mediated IgG3 transport leading to the degradation of

IgG3 because of intracellular competition for FcRn-mediated

transport. The data provide strong evidence that the presence

of an arginine at position 435 in IgG3 is sufficient to explain

its high rate of catabolism observed in vivo. Importantly,

it shows that the half-life of H435-containing IgG3 allotype

is comparable to IgG1 in humans. In a mouse model

for pneumococcal pneumonia, IgG3-R435H demonstrated a

significantly better protection against pneumonia than IgG1

and IgG3. It exhibited a proof of concept that IgG3-R435H

can be utilized for IgG-based immunotherapy aiming at

maximizing effector functions [35].

FcRn over-expressed transgenic animals as IgGbioreactors

In antibody therapy, by elevating serum concentration of

any IgG subclass can cause short serum half-life and high

catabolic rate, hence frequent immunization is required to

maintain high levels of Ag-specific Abs [36]. Several studies

have reported that the over expression of FcRn above normal

level could reduce the exogenous IgG catabolism in trans-

genic (Tg) animals, resulting in higher circulating levels of

IgG [37,38]. Furthermore, it was observed that the Tg mice

were able to mount robust humoral response against weakly

immunogenic antigens and also improved the hybridoma

production efficiently without any sign of autoimmunity [39].

Tg mice were created using a bacterial artificial chromosome

that contains the bovine FcRn (bFcRn) a-chain gene [37]. The

clearance curves of the measured IgG were biphasic with

phase 1 (alpha phase) representing equilibration between the

intravascular and extravascular compartments, phase 2 (beta

phase) representing a slow elimination. Mathematical model-

ing of phases 1 and 2 up to hours 216 has shown good

correlation to the general scheme of FcRn-mediated IgG

pharmacokinetics [6]; on the basis of this finding Bender et al.

[37] estimated alpha phase half-lives in both wt mice and

Tg mice were similarly around 5 h. In contrast, there was a

significant difference (p50.05) in beta phase half-lives, as it

was 125.4 ± 3.2 h (mean ± SEM) in the wt and 165.1 ± 7.8 h

in the Tg animals (�30% longer half-life in Tg mice than in wt

mice). The results showed that FcRn over-expression can

prolong the half-life of Ag-specific Abs.

Rabbits have been used to produce polyclonal antibody and

mAbs [38]. By immunizing the FcRn over-expressed Tg

animals with ovalbumin, Tg rabbits exhibited the high serum

persistence of IgG in beta phase half-lives (7.1 ± 0.46 d), that

was longer than the control groups (5.3 ± 0.3 d). A difference

at the highest IgG levels was found i.e. 31.61 ± 2.7 mg/ml in

Tg rabbits versus 14.8 ± 2.6 mg/ml in wt (p50.01) [38]. FcRn

over-expression can also enhance effect on the humoral

immune response in Tg rabbits, indicating that the adaptation

of this technique to larger mammals will bring substantial

advantages for the production of both polyclonal and

monoclonal Abs [39].

Two of the Tg lines showed copy number-related bFcRn

expression and presented extended half-life of mouse IgG. It

shows that bFcRn a-chain forms a functional complex with

the mouse b2m, and thus binds and protects mouse IgG [37].

As there are plenty of b2m in vivo, FcRn a-chain analog may

be produced and taken before or with antibody to improve its

function.

FcRn as the target of medical interventions inIgG-mediated autoimmune diseases

Autoimmune diseases such as myasthenia gravis (MG), auto-

immune limbic encephalitis, epilepsy and spinal cord injury

are predominantly mediated by IgG auto-antibodies [40].

FcRn plays a critical role in the transportation and

protection of IgG due to the fact that it can regulate the

half-life of IgG. This led to the studies of FcRn as a potential

therapeutic target in autoimmune diseases. FcRn blockade

may have effects on the IgG-mediated diseases by saturating

and inhibiting FcRn. A series of FcRn blockades that can

inhibit interaction of FcRn–Ig, such as anti-FcRn antibody,

short peptide and organic compounds have been reported.

Figure 4. Digramatic illustration of monomeric and dimeric form of modified Fc domain.

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Anti-FcRn monoclonal antibodies

The 4C9, an anti-FcRn monoclonal antibody was produced to

investigate the effect on clearance of a model antibody anti-

methotrexate IgG (AMI) in rats [41]. Rats were instrumented

with jugular vein cannulas 2–3 d prior to investigation, and

4C9 was administered intravenously at the doses of 3, 15 and

60 mg/kg. AMI was then administered 4, 24 and 48 h after

administration of 4C9. AMI concentrations were determined

by assaying the blood samples. It was found that the 4C9

increased AMI systemic clearance in a dose-dependent

manner (from 0.99 ± 0.14 mg/h/kg in control animals to

1.27 ± 0.05, 1.73 ± 0.50 and 1.97 ± 0.49 mg/h/kg in animals

treated with 3, 15 and 60 mg/kg 4C9; p50.05). The effect of

4C9 was found to be transient; no significant effects on AMI

systemic clearance were observed when pre-treatment time

was increased to 24 or 48 h. As such, the data demonstrate that

4C9, a monoclonal anti-FcRn antibody, induces a transient,

dose-dependent increase in the elimination of IgG [41].

The therapeutic effects of the 1G3, an FcRn specific mAb,

were determined in rat MG models. Passive experimental

autoimmune MG was induced by administration of a mAb

specific for acetylcholine receptor (AChR), and it was shown

that treatment with 1G3 resulted in dose-dependent amelior-

ation of the disease symptoms [42]. In addition, the concen-

tration of pathogenic antibody in the serum was significantly

reduced. The effect of 1G3 was also studied in an active

model of experimental autoimmune MG, in which rats were

immunized with AChR. Treatment with 1G3 significantly

reduced the severity of the disease symptoms as well as the

levels of total IgG and anti-AChR IgG relative to untreated

animals [42]. These results suggested that FcRn blockade is

an effective way to treat IgG-mediated autoimmune diseases

and directs further research endeavors to develop improved

FcRn inhibitors.

Short peptides

To further reduce the molecular mass of FcRn–IgG inter-

action inhibitor, various short peptides were screened

by using phage display peptide library screening [43].

A consensus peptide sequence that binds to hFcRn was

discovered that contains no homology to the Fc domain of

IgG. The consensus motif consisted of GHFGGXY, where X

is preferably a hydrophobic amino acid and the motif is

enclosed by a disulfide loop. Chemical optimization of phage-

identified sequences yielded the 26-amino acid peptide dimer

SYN1436, which is capable of potent in vitro inhibition of the

hIgG–hFcRn interaction. SYN1436 binds to human FcRn

with subnanomolar affinity in SPR affinity binding experi-

ments. Activity experiments in vivo were performed in Tg

mice, where the mouse FcRn and b2m genes were replaced

with human homologs (TG32B mice) as the peptide binds to

human FcRn and not to rodent FcRn. SYN1436 was found to

accelerate the catabolism of exogenously administered human

IgG in doses as low as 1 mg/kg/d. In the treatment of

cynomolgus monkeys with repeated doses of 5 mg/kg

SYN1436 three times per week was found to reduce

endogenous IgG levels by approximately 80%, providing the

first evidence that FcRn inhibitors can affect IgG levels in

nonhuman primates [43].

Organic compounds

The inhibition of protein–protein interactions is a crucial

challenge for small molecular drug discovery, because such

interactions generally cover large protein surfaces. According

to present data, the rat Fc:FcRn protein–protein interface is

approximately 1870 A2 [27], while the short peptide inhibitor

of FcRn blocks IgG binding to FcRn only using 360 A2 of

buried surface area [18]. All these results showed that only

key ‘‘hot spots’’ on FcRn surface need to be targeted for

effective inhibition of the IgG–FcRn interaction. A novel

class of quinoxalines has been discovered as antagonists of

the IgG:FcRn protein–protein interaction through optimiza-

tion of a hit derived from the virtual ligand-based screen [44].

Using the coordinates from a crystal structure of shFcRn

and an antagonist peptide [18], a ligand-based virtual screen

of 2.5 million compounds generated a ranked list of virtual

hits. The top 500 compounds were tested in an IgG–FcRn

competition assay to identify compounds that could interfere

with this protein–protein interaction, and quinoxaline hit

compound 1 (Figure 5A) was identified with an IC50 of more

than 150 mM [44].

Various quinoxaline analogs were synthesized and

assessed using an FcRn–IgG competition ELISA assay.

First, the amide position was replaced as showed in

Figure 5(B) and the effects of various alkyl groups on

activity were explored. Un-substituted phenyl and heteroaryl

analogs showed no activity. However, introduction of either a

para or a meta-substituent on the phenyl ring had significant

effects on the activity. The 4-meoxyphenyl compound (R1

replaced by 4-MeO-Ph) increased the activity to 50 mM; the

3-methoxy phenyl compound (R1 replaced by 3-MeO-Ph) was

15-fold more active than the hit compound 1. Second, ring

size and modification at the azepane moiety was investigated

(Figure 5C). The results show that the un-substituted seven

(IC50¼ 10 mM) and eight (IC50¼ 6 mM) membered-alkyl

rings azepane and azocane provide optimal activity. Third,

the solubilizing morpholine group (Figure 5D) was

re-positioned to the benzyl carbon to generate a new

Figure 5. Some quinoxaline homologs of FcRn–IgG interactioninhibitor. (A) Chemical structure of quinoxaline hit compound-1. (B)structure–activity relationships (SAR) of the amide substituent ofcompound-1. (C) SAR of ring size of compound-1. (D) SAR of phenylring substitutions of compound-1.

8 Y. Wang et al. J Drug Target, Early Online: 1–10

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compound which improved the IC50 to 2 mM [44]. This family

of compounds may serve as useful tools in the study of FcRn

biology, as well as starting points for the further development

of orally available small molecule inhibitors of FcRn for

therapeutic use.

Conclusion and perspective

In the field of antibody engineering, extensive studies have

been conducted to improve the effector functions of Abs,

including the alteration of the key amino acid residue of Fc

region to improve the function and prolong the half-life;

reduction in the molecular weight of antibody for better

penetration and binding with antigens. It was reported that,

the prolonged half-life of therapeutic antibody ultimately

reduce the administration frequency or dosage requirements,

while maintaining or improving the efficacy of an Ab [5].

Some research attempts were made with the aim to increase

antibody half-life by mutating the Fc region at the important

amino acid essentially required for FcRn binding and

observed some relatively successful findings [45]. Though

the intact antibodies have long half-lives, they exhibit poor

penetration into tissues especially solid tumors, in some

occasion weak affinity or not binding with some regions of

antigens that are occluded and can only be accessed by

molecules of smaller sizes [46]. Antibody fragments that are

able to bind with the occluded site of antigen have short half-

lives, hence the alteration to increase the affinity of antibody

fragments towards FcRn has become requisite.

As discussed previously, the Tg animals which over-

express the FcRn can be used as antibody bioreactors. Further

expanding this technology to larger mammals will bring

substantial advantages for the production of polyclonal Ab

and mAbs [36,38,39,46,47].

Although many studies suggest that the half-life and

effector functions of antibodies could be enhanced by

modifying the FcRn–IgG interaction, there are many obs-

tacles that are likely to be overcome by research. The first and

foremost is enhancing the affinity between IgG and FcRn in

acidic condition (pH 6.0) without any change in their affinity

at pH 7.4 through genetic engineering.

The second important issue to be addressed is the

suspicious side effects in animals’ immune system due to

FcRn over expression. It has been documented that, FcRn

over-expression in mice can result in potent humoral response

against weakly immunogenic antigen [36], augment antigen-

presenting cell activity and robust immune response with

increased diversity of induced antibodies [47], enhancing the

expansion of Ag-specific B cells and plasma cells [36]. The

increased immune response by over-expressing FcRn may

disrupt the immune system of the animals. The product of the

inserted gene may interact with some metabolic pathway of

the animal, which induces the accumulation or disappearance

of certain metabolites in animals.

In conclusion, the FcRn could be a novel and important

target of therapeutic antibodies with appropriate modifica-

tion to alter the half-life (increase or decrease), bioavail-

ability, dosage form and accuracy and other limitations.

On the whole, manipulation and the effective interventions

of FcRn with respect to drugable antibodies may become

a revolutionary change in the field of antibody

engineering.

Acknowledgements

The authors thank Minjie Hefang and Xuemei Jiang for their

skillful technical assistance in preparing the figures.

Declaration of interest

This work was supported by the Ph.D. Programs Foundation

of Ministry of Education of China (20100204120019) and the

Ministry of Education and State Administration of Foreign

Experts Affairs ‘‘overseas teacher’’ project

(MS2011XBNL057), China. The authors report no conflicts

of interest. The authors alone are responsible for the content

and writing of this article.

References

1. Boswell CA, Tesar DB, Mukhyala K, et al. Effects of charge onantibody tissue distribution and pharmacokinetics. BioconjugateChem 2010;21:2153–63.

2. Background: monoclonal antibody therapeutics. Availablefrom: http://www.landesbioscience.com/journals/mabs/about/#background [modified in text and last accessed 18 Dec 2013].

3. Reichert JM. Marketed therapeutic antibodies compendium. MAbs2012;4:413–15.

4. Roopenian DC, Akilesh S. FcRn: the neonatal Fc receptor comes ofage. Nat Rev Immunol 2007;7:715–25.

5. Vincent KJ, Zurini M. Current strategies in antibody engineering:Fc engineering and pH-dependent antigen binding, bispecificantibodies and antibody drug conjugates. Biotechnol J 2012;7:1444–50.

6. Lobo ED, Hansen RJ, Balthasar JP. Antibody pharmacokinetics andpharmacodynamics. J Pharm Sci. 2004;93:2645–68.

7. Spiegelberg HL. Biological activities of Igs of different classes andsubclasses. Adv Immunol 1974;19:259–94.

8. Wang W, Lu P, Fang Y, et al. Monoclonal antibodies with identicalFc sequences can bind to FcRndifferentially with pharmacokineticconsequences. Drug Metab Dispos 2011;39:1469–77.

9. Grey HM, Kunkel HG. H Chain subgroups of myeloma proteinsand normal 7S g-globulin. J Exp Med 1964;120:253–66.

10. Burton DR, Gregory L, Jefferis R. Aspects of the molecularstructure of the IgG subclasses. Monogr Allergy 1986;19:7–35.

11. Pumphrey R. Computer models of the human immunoglobulinsshape and segmental flexibility. Immunol Today 1986;7:174–8.

12. Shakib F, Stanworth DR. Human IgG subclasses in health anddisease (a review). Part I. Ric Clin Lab 1980;10:463–79.

13. Meulenbroek AJ. Human IgG subclasses: useful diagnostic markersfor immunocompetence. Amsterdam, Netherlands: Sanquin; 2008.

14. Nimmerjahn F, Ravetch JV. Fcg receptors: old friends and newfamily members. Immunity 2006;24:19–28.

15. Shields RL, Namenuk AK, Hong K, et al. High resolution mappingof the binding site on human IgG1 for FcgRI, FcgRII, FcgRIII, andFcRn and design of IgG1 variants with improved binding to theFcgR. J Biol Chem 2001;276:6591–604.

16. Vidarsson G, Stemerding AM, Stapleton NM, et al. FcRn: an IgGreceptor on phagocytes with a novel role in phagocytosis. Blood2006;108:3573–9.

17. Simister NE, Mostov KE. An Fc receptor structurally related toMHC class I antigens. Nature 1989;337:184–7.

18. Mezo AR, Sridhar V, Badger J, et al. X-ray crystal structures ofmonomeric and dimeric peptide inhibitors in complex with thehuman neonatal Fc receptor, FcRn. J Biol Chem 2010;258:27694–701.

19. Bai Y, Ye L, Tesar DB, et al. Intracellular neutralization of viralinfection in polarized epithelial cells by neonatal Fc receptor(FcRn)-mediated IgG transport. Proc Natl Acad Sci USA 2011;108:18406–11.

20. Rodewald R, Kraehenbuhl J-P. Receptor-mediated transport of IgG.J Cell Biol 1984;99:159–64.

DOI: 10.3109/1061186X.2013.875030 Neonatal Fc receptor 9

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y 11

3.14

0.84

.105

on

01/0

9/14

For

pers

onal

use

onl

y.

21. Yoshida M, Claypool SM, Wagner JS, et al. Human neonatal Fcreceptor mediates transport of IgG into luminal secretions fordelivery of antigens to mucosal dendritic cells. Immunity 2004;20:769–83.

22. Bitonti AJ, Dumont JA. Pulmonary administration of therapeuticproteins using an immunoglobulin transport pathway. Adv DrugDeliv Rev 2006;58:1106–18.

23. Kuo TT, Baker K, Yoshida M, et al. Neonatal Fc receptor: fromimmunity to therapeutics. J Clin Immunol 2010;30:777–89.

24. Vallee S, Rakhe S, Reidy T, et al. Pulmonary administration ofinterferon Beta-1a-fc fusion protein in non-human primates usingan immunoglobulin transport pathway. J Interferon Cytokine Res2012;32:178–84.

25. Raghavan M, Gastinel LN, Bjorkman PJ. The class I major histo-compatibility complex related Fc receptor shows pH-dependentstability differences correlating with immunoglobulin binding andrelease. Biochemistry 1993;32:8654–60.

26. Yeung YA, Leabman MK, Marvin JS, et al. Engineering humanIgG1 affinity to human neonatal Fc receptor: impact of affinityimprovement on pharmacokinetics in primates. J Immunol 2009;182:7663–71.

27. Martin WL, West Jr AP, Gan L, Bjorkman PJ. Crystal structureat 2.8 A of an FcRn/heterodimeric Fc complex: mechanism ofpH-dependent binding. Mol Cell 2001;7:867–77.

28. Bumbaca D, Boswell CA, Fielder PJ, Khawli LA. Physiochemicaland biochemical factors influencing the pharmacokinetics ofantibody therapeutics. AAPS J 2012;14:554–8.

29. Zalevsky J, Chamberlain AK, Horton HM, et al. Enhanced antibodyhalf-life improves in vivo activity. Nat Biotechnol 2010;28:157–9.

30. Xie XM, Richard G, Hall JC. Antibody fragment engineering andapplications in diagnosis and therapeutics. In: Meulenberg EP, ed.Antibodies applications and new developments. Illinois: BenthamScience Publishers; 2012:225–79.

31. Ying T, Chen W, Gong R, et al. Soluble monomeric IgG1 Fc. J BiolChem 2012;287:19399–408.

32. Gong R, Wang Y, Feng Y, et al. Shortened engineered humanantibody CH2 domains increased stability and binding to the humanneonatal Fc receptor. J Biol Chem 2011;286:27288–93.

33. Gong R, Vu BK, Feng Y, et al. Engineered human antibodyconstant domains with increased stability. J Biol Chem 2009;284:14203–10.

34. Shimizu A, Honzawa M, Ito S, et al. H NMR studies of the Fcregion of human IgG1 and IgG3 immunoglobulins: assignment ofhistidine resonances in the CH3 domain and identification of IgG3protein carrying G3m (st) allotypes. Mol Immunol 1983;20:141–8.

35. Stapleton NM, Andersen JT, Stemerding AM, et al. Competitionfor FcRn-mediated transport gives rise to short half-life of humanIgG3 and offers therapeutic potential. Nat Commun 2011;2:599.doi:10.1038/ncomms1608.

36. Cervenak J, Bender B, Schneider Z, et al. Neonatal FcRoverexpression boosts humoral immune response in transgenicmice. J Immunol 2011;186:959–68.

37. Bender B, Bodrogi L, Mayer B, et al. Position independent andcopy-number-related expression of the bovine neonatal Fc receptora-chain in transgenic mice carrying a 102 kb BAC genomicfragment. Transgenic Res 2007;16:613–27.

38. Catunda Lemos AP, Cervenak J, Bender B, et al. Characterizationof the rabbit neonatal Fc receptor (FcRn) and analyzing theimmunophenotype of the transgenic rabbits that overexpressesFcRn. PLoS One 2012;7:1–14.

39. Kacskovics I, Cervenak J, Erdei A, et al. Recent advances usingFcRn over-expression in transgenic animals to overcome impedi-ments of standard antibody technologies to improve the generationof specific antibodies. MAbs 2012;3:431–9.

40. Sesarman A, Vidarsson G, Sitaru C. The neonatal Fc receptor astherapeutic target in IgG-mediated autoimmune diseases. Cell MolLife Sci 2010;67:2533–50.

41. Getman KE, Balthasar JP. Pharmacokinetic effects of 4C9, an anti-FcRn antibody, in rats: implications for the use of FcRn inhibitorsfor the treatment of humoral autoimmune and alloimmune condi-tions. J Pharm Sci 2005;94:718–29.

42. Liu L, Garcia AM, Santoro H, et al. Amelioration of experimentalautoimmune myasthenia gravis in rats by neonatal FcR blockade.J Immunol 2007;178:5390–8.

43. Mezo AR, McDonnell KA, Hehir CA, et al. Reduction of IgG innonhuman primates by a peptide antagonist of the neonatal Fcreceptor FcRn. Proc Natl Acad Sci USA 2008;105:2337–42.

44. Wang Z, Fraley C, Mezo AR. Discovery and structure–activityrelationships of small molecules that block the human immuno-globulin G–human neonatal Fc receptor (hIgG–hFcRn) protein–protein interaction. Bioorg Med Chem Lett 2013;23:1253–6.

45. Kuo TT, Aveson VG. Neonatal Fc receptor and IgG-basedtherapeutics. MAbs 2011;3:422–30.

46. Vegh A, Cervenak J, Jankovics I, Kacskovics I. FcRn over-expression in mice results in potent humoral response againstweakly immunogenic antigen. MAbs 2011;3:173–80.

47. Vegh A, Farkas A, Kovesdi D, et al. FcRn over-expression intransgenic mice results in augmented APC activity and robustimmune response with increased diversity of induced antibodies.PLoS One 2012;7:1–11.

48. Dall’Acqua WF, Kiener PA, Wu H. Properties of human IgG1sengineered for enhanced binding to the neonatal Fc receptor(FcRn). J Biol Chem 2006;281:23514–24.

49. Hinton PR, Johlfs MG, Xiong JM, et al. Engineered human IgGantibodies with longer serum half-lives in primates. J Biol Chem2004;279:6213–6.

50. Hinton PR, Xiong JM, Johlfs MG, et al. An engineered humanIgG1 antibody with longer serum half-Life. J Immunol 2006;176:346–56.

51. Petkova SB, Akilesh S, Sproule TJ, et al. Enhanced half-life ofgenetically engineered human IgG1 antibodies in a humanizedFcRn mouse model: potential application in humorally mediatedautoimmune disease. Int Immunol 2006;18:1759–69.

10 Y. Wang et al. J Drug Target, Early Online: 1–10

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