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Title: Discovery of anti-claudin-1 antibodies as candidate therapeutics against hepatitis C virus
Authors:
Mayo Yamashita, Manami Iida, Minoru Tada, Yohitaka Shirasago, Masayoshi
Fukasawa, Shotaro Nagase, Akihiro Watari, Akiko Ishii-Watabe, Kiyohito Yagi, Masuo Kondoh
Affiliations:
Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences,
Osaka University, Osaka 565-0871, Japan
(M.Y., M.I., S.N., A.W., K.K., M.K.)
158-0098, Japan (M.T., A.I.)
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo
162-8640, Japan (Y.S., M.F.)
Graduate School of Biological Science, Tokyo University of Science, Chiba 278-0022, Japan (Y.S.)
1
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Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo
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a) A running title: A claudin-1-targeted HTA
b) Corresponding author: Masuo Kondoh, PhD, Laboratory of Bio-Functional Molecular
Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871,
Japan; Tel: +81-6-6879-8196; Fax: +81-6-6879-8199; E-mail: masuo@phs.osaka-u.ac.jp
Masayoshi Fukasawa, PhD, Department of Biochemistry and Cell Biology, National Institute of
Infectious Diseases, Tokyo 162-8640, Japan; Tel: +81-3-4582-2733; Fax: +81-3-5285-1157; E-mail:
fuka@nih.go.jp
The number of tables: 0 tables
The number of figures: 8 figures
The number of references: 39 references
The number of words in the Abstract: 200 words
The number of words in the Introduction: 491 words
The number of words in the Discussion: 723 words
d) Abbreviations: CLDN, claudin; HCV, hepatitis C virus; mAb, monoclonal antibody; ADCC,
antibody-dependent cellular cytotoxicity; DAA, direct-acting antiviral agent; HTA, host-targeting
agent; SR-BI, scavenger receptor class B type I; PBS, phosphate-buffered saline; SDS-PAGE,
sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; ; HCVcc,
cell-culture-derived HCV; HCVpp, HCV pseudoparticle; NFAT, nuclear factor of activated T-cells;
PCR, polymerase chain reaction; CDC, complement-dependent cytotoxicity; TJ, tight junction.
e) A recommended section: Chemotherapy, Antibiotics, and Gene Therapy
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c) The number of text pages: 25 pages
JPET Fast Forward. Published on January 27, 2015 as DOI: 10.1124/jpet.114.217653
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Abstract
Claudin-1 (CLDN1), a known host factor for hepatitis C virus (HCV) entry and cell-to-cell
transmission, is a target molecule for inhibiting HCV infection. We previously developed 4 clones of
mouse anti-CLDN1 monoclonal antibody (mAb) that prevented HCV infection in vitro. Two of these
mAbs showed the highest anti-viral activity. Here, we optimized the anti-CLDN1 mAbs as
candidates for therapeutics by protein engineering. Although Fab fragments of the mAbs prevented
in vitro HCV infection, their inhibitory effects were much weaker than those of the whole mAbs. In
light and heavy chains inhibited in vitro HCV infection as efficiently as the parental mouse mAbs.
However, the chimeric IgG1 mAbs activated Fcγ receptor, suggesting that cytotoxicity against
mAb-bound CLDN1-expressing cells occurred through the induction of antibody-dependent cellular
cytotoxicity (ADCC). To avoid ADCC-induced side effects, we prepared human chimeric IgG4
mAbs. The chimeric IgG4 mAbs did not activate Fcγ receptor or induce ADCC, but they prevented
in vitro HCV infection as efficiently as did the parental mouse mAbs. These findings indicate that
IgG4 form of human chimeric anti-CLDN1 mAb may be a candidate molecule for clinically
applicable HCV therapy.
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contrast, human chimeric IgG1 mAbs generated by grafting the variable domains of the mouse mAb
JPET Fast Forward. Published on January 27, 2015 as DOI: 10.1124/jpet.114.217653
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Introduction
One hundred and seventy million people worldwide are chronically infected with hepatitis C virus
(HCV). HCV is a leading cause of liver cirrhosis and hepatocellular carcinoma, and overcoming
HCV is an important healthcare issue (El-Serag, 2012; Murray and Rice, 2011). Anti-HCV agents are
classified into direct-acting antiviral agents (DAAs) and host-targeting agents (HTAs). Some DAAs,
such as protease inhibitors and RNA polymerase inhibitors, have been approved and are used
clinically (Scheel and Rice, 2013). However, the poor proofreading ability of HCV RNA polymerase
HTAs are expected to have high genetic barriers and the potential to overcome the development of
DAA-resistant viruses (Flisiak et al., 2009; Li et al., 2011; Scheel and Rice, 2013). Host factors
involved in HCV genome replication and the entry of HCV into hepatocytes are potent targets for
HTAs. For example, cyclophilin and miR-122 are host factors that are involved in the replication of
the HCV genome in hepatocytes. Cyclosporine A and miravirsen, inhibitors of cyclophilin and
miR-122, respectively, prevent in vitro and in vivo HCV replication (Hopkins et al., 2010; Lanford et
al., 2010; Paeshuyse et al., 2006). However, a clinical study of cyclosporine A revealed acute
pancreatitis in some participants, and the injection of miravirsen into chimpanzees led to reductions
in serum cholesterol (Flisiak et al., 2009; Lanford et al., 2010). Thus, HTAs with patient safety and
high genetic barriers to drug-resistance remain to be developed.
A host factor involved in HCV entry is an attractive target for a novel HTA. A series of HCV
studies revealed that heparan sulfate (Barth et al., 2006), CD81 (Pileri et al., 1998), scavenger
receptor class B type I (SR-BI) (Scarselli et al., 2002), claudin-1 (CLDN1) (Evans et al., 2007),
occludin (Ploss et al., 2009), and Niemann–Pick C1-like 1 cholesterol-absorption receptor (Sainz et
al., 2012) are responsible for HCV entry. Of these factors, CLDN1 is considered to be the most
potent target because CLDN1 is involved in both HCV entry into host cells via interaction with
CD81 and cell-to-cell HCV transmission (Harris et al., 2010; Timpe et al., 2008). Moreover,
CLDN1-targeted HTA showed pan-genotypic inhibition of HCV (genotypes 1 through 6) entry
(Fofana et al., 2010), indicating that CLDN1 may offer a high genetic barrier.
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leads to the appearance of viruses resistant to DAAs in patients (Sarrazin et al., 2012). In contrast,
JPET Fast Forward. Published on January 27, 2015 as DOI: 10.1124/jpet.114.217653
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We previously developed mouse anti-CLDN1 monoclonal antibodies (mAbs) by using a novel
method for immunization and screening of antibodies. Several of the resulting clones prevented in
vivo HCV infection without unwanted side effects, such as hepatotoxicity, loss of body weight, and
apparent abnormality (Iida et al., 2014; Nagase et al., 2013; the patent (WO2014132307A1)). We
thus provided proof-of-concept for the use of anti-CLDN1 mAbs as CLDN1-targeted HTAs. In the
current study, to improve the clinical applicability of anti-CLDN1 mAbs, we used them to create Fab
fragments, human chimeric IgG1 mAbs, and human chimeric IgG4 mAbs, and we investigated the
effects of these products on HCV infection.
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Materials and methods
Cells
Human CLDN-expressing HT1080 cells (Li et al., 2014) were cultured in Dulbecco’s modified
Eagle’s medium that contained 10% fetal bovine serum (referred to as “serum-contained medium”).
Human embryonic kidney 293T cells, human hepatic Huh7.5.1-8 cells, and S7-A cells
(Huh7.5.1-8–derived
CLDN1-deficient
cells) were
cultured
in serum-contained
medium
supplemented with 0.1 mM non-essential amino acids, 100 units/ml penicillin G, and 100 μg/ml
(Life Technologies, Carlsbad, CA) were cultured in FreeStyle CHO Expression Medium (Life
Technologies). Human peripheral blood mononuclear cells were obtained from Cellular Technology
(Cleveland, OH).
Mouse anti-CLDN1 mAbs and Fab fragments
Mouse anti-CLDN1 mAbs were purified from ascitic fluid produced in BALB/c mice that had
been inoculated intraperitoneally with hybridomas by using immobilized protein G columns. The
purified mAbs were dialyzed against phosphate-buffered saline (PBS), and the protein concentration
was determined by measuring absorbance at 280 nm. The purity of the mAbs was confirmed by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by staining with
Coomassie brilliant blue (CBB). Mouse IgG (Beckman Coulter, Brea, CA) was used as a control
IgG.
Purified mouse anti-CLDN1 mAbs were digested with papain by using the Fab Preparation kit
(Pierce Chemical, Rockford, IL) according to the manufacturer’s protocol. The purity of the Fab
fragments was checked by using SDS-PAGE with CBB staining. Normal mouse IgG Fab fragment
(Alpha Diagnostic Intl Inc., San Antonio, TX) was used as a control Fab.
Flow cytometric analysis
For analysis of mAbs and Fab fragments binding to CLDN-expressing HT1080 cells, Huh7.5.1-8
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streptomycin sulfate (referred to as “normal medium”). Suspension-adapted FreeStyle CHO-S cells
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cells and S7-A cells, cells were incubated in the presence or absence of mAbs or Fab fragments (5
μg/mL), followed by treatment with fluorescein-conjugated goat anti-mouse IgG (for mouse mAbs),
goat-anti mouse IgG (Fab) (for Fab fragments of mouse mAb), or goat anti-human IgG (for human
chimeric IgG1 and IgG4). The bound cells were analyzed by using a FACSCalibur flow cytometer
(BD Biosciences, San Jose, CA).
Inhibition assay for in vitro HCVcc infection
Cell culture-derived HCV-JFH1 (HCVcc) were prepared from the conditioned medium of
Huh7.5.1-8 cells (5 × 104 cells/well) were seeded in 48-well plates and cultured for 24 h at 37 °C.
The cells were treated with mAbs or Fab fragments at the various concentrations for 30 min at room
temperature, and then HCVcc were added to the wells. After an additional 2-h culture at room
temperature, the cells were washed with fresh normal medium and cultured in normal medium in the
presence or absence of each mAb or Fab fragment for 4 days at 37 °C. Total RNA was extracted
from the cells and culture supernatant. To measure HCV RNA in the total RNA fractions,
quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR) assays for HCV
was performed by using RNA-directTM Realtime PCR Master Mix (Toyobo Co. Ltd., Osaka, Japan)
as described previously (Murakami et al., 2009).
Inhibition assay for in vitro HCV pseudoparticle infection
HCV pseudoparticles (HCVpp) were generated as described previously (Murakami et al., 2013).
Briefly, a Gag–Pol packaging construct (Gag–Pol 5349), a transfer vector construct (Luc 126), and a
glycoprotein-expressing construct (HCV E1 and E2) [JFH1, genotype 2a; TH, genotype 1b
(Murakami et al., 2009)] were transduced into 293T cells (Logvinoff et al., 2004; Wakita et al., 2005).
The medium from the transfected cell cultures was collected and used as the HCVpp stock.
For the infection assay, Huh7.5.1-8 cells (5 × 104 cells/well) were seeded in 48-well plates and
cultured for 24 h at 37 °C. Cells were treated with mAbs or Fab fragments at the various
concentrations for 30 min at room temperature, and then HCVpp were added to the wells. The cells
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Huh7.5.1-8 cells as described previously (Murakami et al., 2009).
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were cultured for an additional 6 h at 37 °C. The cells were then washed with fresh normal medium
and cultured in the presence or absence of each mAb or Fab fragment for 2 days. The cells were
lysed, and the luciferase activity of the resulting cellular lysates was measured by using a
commercially available luciferase reporter assay kit (PicaGene, Toyo Ink Manufacturing, Tokyo,
Japan) according to the manufacturer’s instructions.
Activation of Fcγ receptor
To measure the activation of FcγRΙΙΙa by antigen-bound mAbs, Jurkat/FcγRΙΙΙa/ nuclear factor of
2014). CLDN1-expressing HT1080 cells (1 × 104 cells/well) were seeded in 96-well plates and
cultured for 24 h at 37 °C. Then, Jurkat/FcγRΙΙΙa/NFAT-Luc cells (1 × 105 cells/well) were added to
the wells in the presence or the absence of mAbs at the indicated concentrations. After 5 h of
incubation at 37 °C, luciferase activities were measured by using an EnSpire Multimode Plate
Reader (PerkinElmer, Downers Grove, IL) and the ONE-Glo Luciferase Assay System (Promega)
according to the manufacturer’s protocol.
Human chimeric IgG mAbs
cDNA encoding the heavy-chain and light-chain variable domains of CLDN1 mAb clones 2C1
and 3A2 (Iida et al., 2014; Nagase et al., 2013; the patent (WO2014132307A1)) were amplified by
polymerase chain reaction (PCR), and the PCR products were subcloned into pFUSE-CHIg-hG1 (for
IgG1), pFUSE-CHIg-hG4 (for IgG4), and pFUSE2-CLIg-hk vectors (InVivoGen, San Diego, CA).
To prepare a human chimeric IgG4 mutant, we substituted the codon of serine at position 228 (EU
numbering scheme) in pFUSE-CHIg-hG4 with proline, resulting in pFUSE-CHIg-hG4 mutant.
cDNA encoding the heavy chain region of clones 2C1 and 3A2 was subcloned into the
pFUSE-CHIg-hG4 mutant.
Human chimeric mAbs were prepared by using the FreeStyle MAX CHO Expression System (Life
Technologies). Briefly, CHO-S cells were co-transfected with pFUSE-CHIgs and pFUSE-CLIg of
2C1 or 3A2 by using the FreeStyle MAX Reagent, and then the transfected cells were cultured for 6
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activated T-cells (NFAT)-Luc cells were used as effector cell as described previously (Tada et al.,
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days in FreeStyle CHO Expression Medium. The conditioned medium was recovered and was
applied to a protein G column. The column was washed with 20 mM sodium phosphate buffer (pH
6.8), and the mAbs were eluted with 0.1 M glycine-HCl (pH 3.0). The eluted fraction containing
mAbs was neutralized with 1 M Tris-HCl (pH 8.0), followed by desalting using a PD-10 column (GE
Healthcare, Cleveland, OH) and PBS as the exchange solvent. The concentration of the purified
antibody was determined by measuring the absorbance at 280 nm.
Statistical analysis
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Data were analyzed by the Student t-test. The significant difference was set at p < 0.05.
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Results
Effects of Fab fragments on HCV entry
We previously created 4 clones of mouse anti-CLDN1 mAbs, among which 2 clones (2C1 and
3A2) showed the highest preventive activity for HCV infection (Iida et al., 2014; Nagase et al., 2013;
the patent (WO2014132307A1)). In this study we therefore optimized mAbs 2C1 and 3A2 toward
clinical applications. We first digested these mAbs with papain because the resulting Fab fragments
have improved tissue penetration and no activation of effector systems (Stockwin and Holmes,
Like the parental mAbs, the corresponding Fab fragments bound to CLDN1-expressing cells but
not to those that expressed CLDN2, 3, 4, 6, 7, or 9 (Suppl. Fig. 1). To confirm the specificity of the
Fab fragments, we investigated their interaction with Huh7.5.1-8 cells and the CLDN1-deficient
S7-A cell line derived from those cells. The Fab fragments bound to Huh7.5.1-8 cells but not to S7-A
cells (Fig. 1). Therefore, Fab fragmentation of the mAbs did not affect their CLDN1-specific
recognition.
Although treatment of Huh7.5.1-8 cells with the parental mAb (25 μg/mL (0.17 μM)) completely
prevented infection of HCVcc, Fab fragments 2C1 and 3A2 at 25 μg/mL (0.5 μM) attenuated HCVcc
infection by only 51% and 75% of control antibody-treated infection levels, respectively (Fig. 2A).
To clarify the mode of action of Fabs, we investigated their effect on HCV infection by surrogate
HCV entry assay using HCVpp (genotypes 1b and 2a). Treatment of cells with the intact parental
mAb (25 μg/mL (0.17 μM)) prevented infection of HCVpp (1b and 2a) to less than 5% of the control
level (Fig. 2B and 2C). However, Fab fragments of 2C1 (0.5 μM) reduced HCVpp entry to 46%
(genotype 1b) and 49% (genotype 2a) of the level observed after control Fab treatments (Figs. 2B).
3A2 Fab fragments (0.5 μM) did not attenuate HCVpp (1b) entry appreciably and only limited
HCVpp (2a) infection to 73% of the control level (Fig. 2C). Taking these results together, Fab
fragmentation may not be a suitable method for engineering the CLDN1 mAbs for clinical use
because the inhibitory activity of the Fab fragments on HCV infection was much weaker than that of
the intact parental mAbs.
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2003).
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Effects of human-mouse chimeric IgG1 on HCV entry
To
our
knowledge,
36
antibodies
have
been
approved
as
therapeutic
agents
(http://www.immunologylink.com/FDA-APP-Abs.html), of which 27 (75%) are human chimeric or
human IgG1 immunoglobulins. We therefore next developed human-mouse chimeric IgG1 constructs
of mAbs 2C1 and 3A2 by genetically grafting the variable regions of the heavy and light chains of
the mouse mAbs into human IgG1. The human chimeric mAbs (2C1 and 3A2) bound to CLDN1 but
not to cells expressing CLDN2, 3, 4, 6, 7, or 9 (Suppl. Fig. 2). In addition, the chimeric mAbs also
retained the CLDN-specificity of the parental mouse mAbs.
We then investigated the effects of the chimeric mAbs on in vitro HCVcc infection. Each chimeric
mAb dose-dependently reduced intracellular HCVcc (Fig. 4A). Treatment of cells with the chimeric
mAbs also decreased the amount of HCVcc in the conditioned medium of Huh7.5.1-8 cells in a
dose-dependent fashion (Suppl. Fig. 3). We obtained similar results regarding HCVpp infection. The
chimeric mAbs dose-dependently prevented infection of cells with HCVpp (genotype 1b and 2a)
(Fig. 4B and Suppl. Fig. 4). The inhibitory activity of the chimeric mAbs was slightly weaker than
those of the mouse mAbs.
HCV is not toxic itself, but the inflammation caused by immune response against HCV-infected
hepatocytes leads to hepatitis (El-Serag, 2012). Because anti-HCV agents must not induce
inflammatory responses if they are to be clinically useful, we next investigated whether the human
chimeric IgG1 mAbs activate effector systems, including complement-dependent cytotoxicity (CDC)
and antibody-dependent cellular cytotoxicity (ADCC). The chimeric mAbs (2C1 and 3A2) did not
activate CDC (Suppl. Fig. 5). Activation of Fcγ receptor IIIa is associated with the activation of
ADCC (Stockwin and Holmes, 2003). Hence, we investigated whether Fcγ receptor IIIa was
activated by the chimeric mAbs using Jurkat/FcγRIIIa/NFAT-Luc cells, in which luciferase
expression was associated with activation of Fcγ receptor IIIa (Houot et al., 2011; Tada et al., 2014).
The chimeric mAbs activated Fcγ receptor IIIa through the interaction of the chimeric IgG1 mAbs
with CLDN1-expressing cells, suggesting induction of ADCC by natural killer cells against
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bound to Huh7.5.1-8 cells but not to S7-A cells (Fig. 3). Therefore, the human chimeric IgG1 mAbs
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CLDN1-expressing cells (Fig. 5).
Effects of human-mouse chimeric IgG4 on HCV entry
To avoid the adverse effects due to inflammation via ADCC, we selected IgG4 among IgG
subclasses because IgG4 is reportedly a poor trigger of CDC and ADCC (Stockwin and Holmes,
2003). One problem associated with IgG4 is that bispecific antibodies arise frequently in vivo, due to
the ease with which monovalent portions containing one heavy chain and one light chain can be
exchanged among IgG4 molecules (Aalberse and Schuurman, 2002). Substitution of the serine at
molecules (Aalberse and Schuurman, 2002). We prepared both human chimeric IgG4 and IgG4
mutants (Ser228Pro) of 2C1 and 3A2 by grafting the heavy and light chains of the variable regions.
Denaturing SDS-PAGE analysis showed that IgG half-molecules occurred with the IgG4
constructs but not the IgG4 mutants (Fig. 6A), indicating that the IgG4 mutant is more stable than
IgG4. Both the IgG4 and IgG4 mutant versions of 2C1 and 3A2 retained specific binding to CLDN1
(Suppl. Fig. 6A and 6B). The IgG4 and IgG4 mutant constructs also bound to Huh7.5.1-8 cells but
not to S7-A cells (data not shown, Fig. 6B ). Together these results indicate that the IgG4 and IgG4
mutant versions of 2C1 and 3A2 retained CLDN-binding specificity.
Moreover, IgG4 and mutant IgG4 constructs of 2C1 and 3A2 mAbs dose-dependently decreased
HCVcc levels in the cells (Suppl. Fig. 7 and Fig. 7A,). We also confirmed the effects of the IgG4 and
IgG4 mutant mAbs on infection of HCVpp (1b and 2a). As expected in light of the data from the
HCVcc analysis, the IgG4 and IgG4 mutant versions of 2C1 and 3A2 effectively prevented HCVpp
infection in a dose-dependent manner (data not shown and Fig. 7B). We further investigated whether
the IgG4 and IgG4 mutant mAbs activated Fcγ receptor IIIa, potentially leading to undesirable
inflammation. The human chimeric IgG4 and IgG4 mutant versions of 2C1 and 3A2 activated Fcγ
receptor IIIa 100-fold less than did their human chimeric IgG1 counterparts (Fig. 8). These findings
indicate that the conversion of anti-CLDN1 mAbs 2C1 and 3A2 to the IgG4 subclass enables the
resulting reagents to retain the inhibitory activity of the parental mouse mAbs on HCV infection and
potentially reduces the side effects due to activation of Fcγ receptor IIIa, thereby avoiding ADCC
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position 228 (EU numbering scheme) with proline can avoid the formation of half-monoclonal IgG4
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against mAb-bound cells.
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Discussion
Previously, we developed mouse anti-CLDN1 mAbs that prevented in vitro and in vivo HCV
infection (Iida et al., 2014; Nagase et al., 2013; the patent (WO2014132307A1)). We used parental
mouse mAbs, their Fab fragments, and human chimeric IgG1, human chimeric IgG4, and human
chimeric IgG4 mutant constructs as lead compounds to optimize for the development of
CLDN1-targeted HTAs. In general, Fab fragments have better tissue penetration than do full-length
mAbs (Stockwin and Holmes, 2003), but Fab fragments (molecular weight, ~50 kDa) offer less steric
weight, ~150 kDa). We found that the Fab fragments of our mouse mAbs had much weaker
inhibitory activity against HCV infection than did the parental mAbs. In addition, the affinity to
CLDN1 also may be weaker than that of the intact mAbs because Fab fragments are monovalent.
Furthermore, in other virus models, Fab fragments neutralized viral infection by a different
mechanism from that used by the full-length IgG (Edwards and Dimmock, 2000; Edwards and
Dimmock, 2001; McInerney et al., 1997). A detailed analysis addressing this difference between Fab
fragments and the intact mAbs is needed. Taken together, our findings indicate that Fab
fragmentation of anti-CLDN1 mAbs is unsuitable for future clinical application.
In contrast to the Fab fragments, the human chimeric IgG1 and IgG4 forms of the anti-CLDN1
mAbs were as effective against HCV infection as were the parental mouse mAbs. Hepatotoxicity is
an exacerbating factor for hepatitis C, and therefore the induction of ADCC by therapeutic Abs has to
be avoided. The human chimeric IgG1 form of our mAbs activated Fcγ receptor IIIa, indicating the
activation of ADCC to the mAb-bound cells. However, the human chimeric IgG1 form activated
ADCC, the human chimeric IgG4 forms did not (Suppl. Fig. 8). One potential drawback of the IgG4
form is that it frequently exchanges IgG half-molecules, resulting in the generation of bispecific
IgG4 molecules in plasma (Neergaard et al., 2014). Therefore, we created a mutant human chimeric
IgG4 construct by substituting serine at position 228 with proline; this point mutation generates
inter-H-chain binding in the hinge region, resulting in the formation of stable divalent IgG4
(Aalberse and Schuurman, 2002). The mutant form of human chimeric IgG4 mAbs retained the full
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interference of the binding of virus to cellular receptors than do intact IgG molecules (molecular
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anti-HCV activity of the parental mouse mAbs without activation of Fcγ receptor IIIa and subsequent
ADCC against hepatocytes.
The concepts of HTAs have been proved in human-liver-chimeric mice by using antibodies against
for CD81, SR-BI, and CLDN1 (Iida et al., 2014; the patent (WO2014132307A1); Meuleman et al.,
2008; Timpe et al., 2008). CD81 is a widely expressed cell-surface protein that is involved in signal
transduction and cell adhesion in the immune system (Levy et al., 1998). SR-BI is a receptor highly
expressed in live hepatocytes and steroidogenic tissues and is associated with the metabolism of
high-density lipoproteins (Krieger, 2001). Therefore, HCV binds to CD81 or SR-BI, which are
the tight junctions (TJs) between adjacent cells and therefore plays a role in the TJ seal, preventing
the free movement of solutes between the apical and basolateral sides of epithelial cell sheets (Furuse
and Tsukita, 2006). In the liver, CLDN1 is predominantly expressed at the apical bile canalicular
membrane in normal liver tissue, consistent with its function in the TJ seal. However, HCV infection
perturbs the TJ seal, leading to basolateral translocation of CLDN1 (Reynolds et al., 2008).
Therefore, antibodies against CLDN1 may interact with the cell-surface CLDN1 that is involved in
HCV entry, without modulation of the CLDN1 embedded in TJ seals. Therefore, CLDN1 is a safe
and effective target for the development of HTAs. Indeed, treatment of cells with 2C1 or 3A2 did not
perturb TJ-integrity. 2C1 and 3A2 also inhibited HCV infection of human liver chimeric mice
without adverse effects (Iida et al., 2014; the patent (WO2014132307A1)).
In summary, we optimized mouse anti-CLDN1 mAbs toward the clinical anti-HCV application by
using antibody engineering to successfully create human chimeric IgG4 mAb constructs. The
chimeric IgG4 mutant forms prevented HCV infection as well as did the parental mAbs. Moreover,
the chimeric IgG4 mAbs did not induce ADCC against mAb-bound cells. Therefore, the chimeric
IgG4 forms may be potential therapeutic Abs for the development of HTAs for HCV therapy.
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localized in the cellular surface but also play biologic functions in the liver. CLDN1 is embedded in
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Acknowledgements
We thank all the members of our laboratory for their technical support and useful comments.
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Authorship contribution
M.Y., M.I. and M.T. equally contributed to this study.
Participated in research design: Yamashita, Iida, Tada, Fukasawa, Kondoh
Conducted experiments: Yamashita, Iida, Shirasago
Contributed new reagents or analytic tools: Tada, Shirasago, Nagase, Watari, Ishii-Watabe,
Fukasawa
Wrote or contributed to the writing of the manuscript: Yamashita, Tada, Ishii-Watabe, Fukasawa,
Kondoh
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Performed data analysis: Yamashita, Iida, Tada, Fukasawa, Ishii-Watabe, Yagi, Kondoh
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Footnotes
Financial supports: This work was supported by a Health and Labor Sciences Research Grant from
the Ministry of Health, Labor, and Welfare of Japan; a Grant-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science, and Technology of Japan (24390042 (MK) and
23590104 (MF)); and funds from the Adaptable and Seamless Technology Transfer Program through
Target-driven
R&D,
Japan
Science
and
Technology
Agency
(AS242Z01694Q
and
AS251Z00905Q); the Takeda Science Foundation; the Nakatomi Foundation; and the Platform for
Sports, Science, and Technology, Japan; and the Advanced Research for Medical Products Mining
Programme of the National Institute of Biomedical Innovation (NIBIO).
23
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Drug Discovery, Informatics, and Structural Life Science of the Ministry of Education, Culture,
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Figure legends
Figure 1. Claudin specificity of Fab fragment
Huh7.5.1-8 cells (CLDN1-expressing) or S7-A cells (CLDN1-deficient cells derived from
Huh7.5.1-8 cells) were incubated with Fab fragments (5 μg/mL), and then treated with
fluorescein-isothiocyanate-conjugated goat anti-mouse IgG (VH + VL). Solid and open histograms
represent vehicle- and Fab-treated cells, respectively.
A) HCVcc inhibition assay. Huh7.5.1-8 cells were pretreated with whole Abs or Fab fragments (25
μg/mL) of control or anti-CLDN1 (2C1 or 3A2) for 30 min at room temperature, and infected with
HCVcc (2.52 × 107 copies/mL) in the presence or absence of the whole Abs or Fab fragments (25
μg/mL) for 2 h at room temperature. The treated cells then were cultured for an additional 4 days in
normal medium that contained the whole Abs or Fab fragments (25 μg/mL). HCV infection was
evaluated by qRT-PCR assays to measure the HCV genomic RNA copies in the cells. Values are
expressed as percentages. Data are presented as means ± SD (n = 4). *Significant difference between
whole Ab and Fab fragment (p < 0.05). B and C) HCVpp inhibition assay. Huh7.5.1-8 cells were
pretreated with whole Abs or Fab fragments (25 μg/mL) of control or anti-CLDN1 (2C1 or 3A2) for
30 min at room temperature, and incubated for 6 h with HCVpp (panel B, genotype 1b; panel C,
genotype 2a) in the presence or absence of the whole Abs or Fab fragments at 25 μg/mL at 37 °C.
The treated cells were cultured for an additional 2 days. Cellular luciferase activities were measured
and expressed as percentages of the control value. Data in each graph are presented as means ± SD
(n = 4). *Significant difference between whole Ab and Fab fragment (p < 0.05).
Figure 3. Claudin specificity of human-mouse chimeric IgG1
Huh7.5.1-8 or S7-A cells were incubated with the chimeric IgG1 mAb (5 μg/mL) and then treated
with fluorescein-isothiocyanate-conjugated goat anti-human IgG. Solid and open histograms
represent vehicle- and chimeric mAb-treated cells, respectively.
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Figure 2. In vitro HCV infection analysis of Fab fragments
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Figure 4. In vitro HCV infection analysis of human-mouse chimerig IgG1
A) HCVcc assay. Huh7.5.1-8 cells were treated with or without human chimeric or mouse
anti-CLDN1 mAbs at the indicated concentrations as described in the legend of Fig. 2A. HCV RNA
contents in the cells were quantified by qRT-PCR and expressed as percentages of the control value.
Data are presented as means ± SD (n = 4). *Significant difference between mouse mAb and chimeric
IgG1 (p < 0.05). B) HCVpp assay. Huh7.5.1-8 cells were treated with or without human chimeric or
mouse anti-CLDN1 mAbs (2C1 or 3A2) at the indicated concentrations as described in the legend of
value. Data in each graph are presented as means ± SD (n = 4). *Significant difference between
mouse mAb and chimeric IgG1 (p < 0.05).
Figure 5. Activation of Fcγ receptor IIIa
CLDN1-expressing HT1080 cells were co-incubated for 5 h with Jurkat/FcγRIIIa/NFAT-Luc cells in
the presence or absence of human chimeric mAbs at the indicated concentrations, after which the
luciferase activity was determined as described in Materials and Methods. Data are shown as means
± SD (n = 3).
Figure 6. Characterization of human chimeric IgG4 mutant
A) SDS-PAGE analysis. Chimeric IgG4 and IgG4 mutant were subjected to 10% SDS-PAGE in
non-reducing condition, followed by staining with CBB. The upper and lower arrows indicate
divalent and monovalent form of IgG, respectively.
B) CLDN-specificity. Huh7.5.1-8 or S7-A cells
were incubated with the chimeric IgG4 mutant mAbs (5 μg/mL) and then treated with
fluorescein-isothiocyanate-conjugated goat anti-human IgG. Solid and open histograms represent the
vehicle- and the chimeric mAb-treated cells, respectively.
Figure 7. Effects of chimeric IgG4 mutant mAbs on HCV infection
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Fig. 2B. Cellular luciferase activities were measured and expressed as percentages of the control
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A) HCVcc assay. Huh7.5.1-8 cells were treated with or without chimeric IgG4 mutant at the
indicated concentrations as described in the legend of Fig. 2A. The cellular HCV RNA contents were
quantified by qRT-PCR and expressed as percentages of the control value. Data are presented as
means ± SD (n = 4). *Significant difference between mouse mAb and chimeric IgG4 mutant (p <
0.05). B) HCVpp assay. Huh7.5.1-8 cells were treated with or without chimeric mAb at the indicated
concentrations as described in the legends of Fig. 2B. Cellular luciferase activities were measured
and expressed as percentages of the control values. Data are presented as means ± SD (n = 4).
*Significant difference between mouse mAb and chimeric IgG4 mutant (p < 0.05).
CLDN1-expressing HT1080 cells were co-incubated with Jurkat/FcγRIIIa/NFAT-Luc cells in the
presence or the absence of mAbs at the indicated concentrations for 5 h, and then the luciferase
activity was determined as described in Materials and Methods. Data are shown as means ± SD (n =
3).
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Figure 8. Effects of chimeric Abs on the activation of Fcγ receptor IIIa
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