J. Microbiol. Biotechnol.

J. Microbiol. Biotechnol. (2017), 27(7), 1336–1344https://doi.org/10.4014/jmb.1703.03066 Research Article jmbReview

Construction and Characterization of an Anti-Hepatitis B Virus preS1Humanized Antibody that Binds to the Essential Receptor BindingSiteJimin Wi1, Mun Sik Jeong1, and Hyo Jeong Hong1,2*

1Department of Systems Immunology, College of Biomedical Science, 2Institute of Bioscience and Biotechnology, Kangwon National

University, Chuncheon 24341, Republic of Korea

Introduction

Hepatitis B virus (HBV) is a small, enveloped, partially

double-stranded DNA virus with strict tropism to human

hepatocytes [1, 2]. Hepatitis B viral infection attacks the

liver and can cause both acute and chronic diseases [2]. An

estimated 240 million people are chronically infected with

hepatitis, and over one million people die every year owing

to complications of hepatitis B, including cirrhosis and

liver cancer [3, 4]. Hepatitis B prevalence is highest in sub-

Saharan Africa and East Asia, where between 5% and 10%

of the adult population is chronically infected [5, 6].

The genotypes of HBV are defined by an intergroup

divergence of more than 8% in the entire genome sequence

[7, 8]. To date, 10 distinct genotypes (A–J), which show a

distinct geographic distribution, have been described [9-

12]. Genotypes A and D are ubiquitous but most prevalent

in Europe and Africa, whereas genotypes B and C are

confined to Asia and Oceania. Genotypes E, F, G, and H are

also occasionally observed in Asia. Though rare, genotype I

is found in Laos, Vietnam, India, and China, and Genotype

J has been described in Japan. Genotypes A–D are common

and constitute 97% of the total patient population, whereas

genotypes E–G represent less than 2% [13, 14].

Received: March 30, 2017

Revised: April 19, 2017

Accepted: April 27, 2017

First published online

May 4, 2017

*Corresponding author

Phone: +82-33-250-8381;

Fax: +82-33-259-5643;

E-mail: hjhong@kangwon.ac.kr

pISSN 1017-7825, eISSN 1738-8872

Copyright© 2017 by

The Korean Society for Microbiology

and Biotechnology

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma. With

recent identification of HBV receptor, inhibition of virus entry has become a promising

concept in the development of new antiviral drugs. To date, 10 HBV genotypes (A–J) have

been defined. We previously generated two murine anti-preS1 monoclonal antibodies (mAbs),

KR359 and KR127, that recognize amino acids (aa) 19–26 and 37–45, respectively, in the

receptor binding site (aa 13–58, genotype C). Each mAb exhibited virus neutralizing activity in

vitro, and a humanized version of KR127 effectively neutralized HBV infection in

chimpanzees. In the present study, we constructed a humanized version (HzKR359-1) of

KR359 whose antigen binding activity is 4.4-fold higher than that of KR359, as assessed by

competitive ELISA, and produced recombinant preS1 antigens (aa 1–60) of different genotypes

to investigate the binding capacities of HzKR359-1 and a humanized version (HzKR127-3.2) of

KR127 to the 10 HBV genotypes. The results indicate that HzKR359-1 can bind to five

genotypes (A, B, C, H, and J), and HzKR127-3.2 can also bind to five genotypes (A, C, D, G,

and I). The combination of these two antibodies can bind to eight genotypes (A–D, G–J), and to

genotype C additively. Considering that genotypes A–D are common, whereas genotypes E

and F are occasionally represented in small patient population, the combination of these two

antibodies might block the entry of most virus genotypes and thus broadly neutralize HBV

infection.

Keywords: Hepatitis B virus, preS1, virus entry, humanized antibody, genotypes, binding

activity

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July 2017⎪Vol. 27⎪No. 7

The hepatotropism of HBV is partially attributed to the

entry step mediated by viral envelope proteins. The HBV

envelope contains three membrane glycoproteins, namely

the large, middle, and small surface proteins, which are

translated from a single open reading frame via different

in-frame start codons. The small surface protein is the

major component of the viral envelope. The middle protein

has an extra N-terminal preS2 region, whereas the large

protein, on the basis of the middle protein, harbors an

additional preS1 region of 108 aa (genotypes D and J) or

119 aa (the other subtypes) [15, 16]. Cell entry of HBV

presumably begins with reversible, low-affinity attachment

of HBV to the cell membrane via the interaction between

viral envelope proteins and heparin sulfate proteoglycan,

followed by high-affinity binding of viral envelope

proteins to a specific receptor, the bile-acid pump sodium-

taurocholate cotransporting polypeptide (NTCP) [17-20].

NTCP is an integral membrane protein that is exclusively

expressed on the basolateral membrane of hepatocytes,

which explains the tropism of HBV for the liver. Multiple

lines of evidence have indicated that preS1, in particular,

the N-terminal half (aa 2–47, genotype D) and the

myristolyl moiety at its amino terminus, is critical for the

binding of viral particle to NTCP, whereas aa 9-18 of the

preS1 component (genotype D) are essential for HBV

attachment and subsequent infection [21, 22]. Therefore,

inhibition of virus entry that can neutralize the activities of

viral surface proteins or target the cellular receptors has

become a major concept in the development of new

antiviral drugs.

Monoclonal antibodies (mAbs) have great potential for

both diagnostic and therapeutic applications [23]. Murine

mAbs are easy to produce, but their therapeutic use in

humans is limited because of the human anti-mouse

antibody response during treatment [24]. To obviate this

problem, humanized antibodies have been constructed by

grafting the complementarity determining regions (CDRs)

of murine mAbs, which form an antigen-binding pocket,

onto homologous human antibody variable domains, while

retaining some murine residues in framework regions

(FRs) that are predicted to influence the conformation of

CDRs. This humanization technique is referred to as CDR

grafting [25, 26].

We previously generated two murine mAbs, KR359 and

KR127, which recognize aa 19–26 and 37–45 of preS1

(genotype C), respectively [27], and demonstrated that they

exhibit neutralizing activity in infection of human hepatocyte

primary culture by HBV in vitro [28]. Subsequently, we

constructed a humanized version (HzKR127) of KR127 by

CDR grafting and provided evidence that this humanized

antibody can neutralize HBV infection in chimpanzees [29].

In addition, we developed an improved version (HzKR127-

3.2) of HzKR127 through further humanization and affinity

maturation [30]. In the present study, we constructed a

humanized version (HzKR359-1) of KR359 via the CDR-

grafting method and characterized its binding capacities to

the preS1 region of HBV genotypes (A–J). In addition, we

analyzed the binding capacities of HzKR127-3.2 and

combined binding activities of HzKR359-1 and HzKR127-

3.2 to the preS1 region of HBV genotypes. The results

indicate that the combination of these two humanized

antibodies can bind to most HBV genotypes and thus may

broadly neutralize HBV infection.

Materials and Methods

Cell Culture

Murine hybridoma KR359 [27] cells were routinely cultured at

5% CO2, 37°C in IMDM (Gibco: Thermo Fisher Scientific, Inc.,

USA) supplemented with 10% (v/v) fetal bovine serum (Atlas,

USA). Suspension-adapted HEK293F cells (Invitrogen; Thermo

Fisher Scientific) were cultured in FreeStyle 293 Expression

medium (Gibco) in 125 ml Erlenmeyer flasks (Corning, USA) at

125 rpm on a shaker (N Biotek, Korea) in a humidified 37°C and

8% CO2 atmosphere.

Construction of Humanized Antibody HzKR359-1

For the cloning of the cDNAs encoding the mouse heavy chain

variable region (VH) or κ light chain variable region (Vκ) of KR359

antibody (IgG1, κ), total RNA was isolated from the KR359

hybridoma, and the first-strand cDNA was synthesized from the

total RNA using reverse transcriptase (Superscript II, Thermo

Fisher Scientific) and the 3’-primers specific to the γ1 heavy (Cγ1)

or κ light chain (Cκ) constant region of KR359. The cDNA

templates were subjected to PCR using the 5’-primer specific to

the N-terminal sequence of VH or Vκ of KR359 and the 3’-primer

specific to the mouse Cγ1 or Cκ. The resulting PCR products were

subcloned into the pBluescript vector to determine their nucleotide

sequences.

For the construction of a humanized version of KR359, the amino

acid sequences of the mouse VH and Vκ were compared with those

of human immunoglobulin germline segments in IMGT (http://

www.imgt.org), and the most homologous VH (VH4-59) and Vκ

(VK1-39) germline segments were selected. To construct a

humanized VH, the CDRs (HCDR1–HCDR3) of the mouse VH were

grafted onto the human VH4-59 germline segment and JH4, and

13 mouse FR residues that were predicted to influence the

conformation of HCDRs were also grafted. To construct a

humanized Vκ, the CDRs (LCDR1–LCDR3) of the mouse Vκ were

grafted onto the human VK1-39 germline gene segment and JK4.

The resulting humanized VH or Vκ sequence was synthesized and

1338 Wi et al.

J. Microbiol. Biotechnol.

subcloned into the EcoRI-ApaI or HindIII-BsiWI sites, respectively,

of pCMV-dhfrC [29] containing human Cγ1 or Cκ sequences,

respectively, to construct heavy or light chain expression plasmids,

respectively.

Expression and Purification of Humanized Antibody

The heavy and light chain expression plasmid DNAs were

mixed with polyethyleneimine (PEI, linear 25 kDa; Polysciences,

USA) at a ratio of 1:4 (60 μg:240 μg) and incubated at room

temperature for 25 min, as described previously [31]. The mixture

was added to 30 ml of HEK293F cells (1.0 × 106 cells/ml) and

cultured for 7 days. The culture supernatant was recovered by

centrifugation at 2,300 ×g for 30 min at 4°C and subjected to

indirect ELISA and quantitative ELISA to determine its antigen-

binding activity. For purification of the humanized antibody, the

culture supernatant was subjected to affinity chromatography on

Protein A-agarose beads (Amicogen, Inc., Korea). The antibody

was eluted from the column with 0.1 M sodium citrate buffer (pH

3.2) and neutralized with 1.0 M Tris-HCl buffer. Finally, the buffer

was changed to storage buffer (25 mM sodium citrate, 150 mM

NaCl, pH 6.4) by using Vivaspin (MWCO 30,000; Sartorius,

Germany), as described previously [32]. Antibody concentration

was determined with a NANO-DROP 2000 spectrophotometer

(Thermo Fisher Scientific) based on the molar extinction coefficient.

The integrity and purity of the purified antibody were analyzed

by SDS-PAGE and western blot analysis.

Construction, Expression, and Purification of Recombinant PreS1

Antigens

For the production of recombinant preS1 antigens of six HBV

genotypes (B–G), the preS1 sequences (aa 1–60) of the HBV

genotypes were individually synthesized and fused to the C-

terminus of the Ig1-Ig5 domains of human L1 cell adhesion

molecule (L1CAM), whereas a Strep-tag II sequence (Trp-Ser-His-

Pro-Gln-Phe-Glu-Lys) was fused to the C-terminus of the preS1

sequence for the detection and purification of the recombinant

preS1 antigen. The preS1 sequences were derived from the GenBank

database. The accession numbers of the genotype sequences are

genotype A (AB014370), genotype B (AB014366), genotype C

(AB014360), genotype D (AB090270), genotype E (AB091255),

genotype F (AB036950), genotype G (AB056513), genotype H

(AB298362.1), genotype I (AF241407), and genotype J (AB486012.1)

[12, 33, 34]. The preS1-Strep-tag II sequence was synthesized and

subcloned into the XhoI-ApaI sites of pIg5-hFc [32] to construct

pIg5-preS1-strep, and then the Ig1-Ig5 sequence was subcloned

into the EcoRI-XhoI sites of pIg5-preS1-strep to construct expression

plasmid pIg1-5-preS1-strep.

The expression plasmid was introduced into HEK293F cells

using PEI, and the recombinant preS1 antigen was transiently

expressed for 7 days, as described above. The culture supernatant

was subjected to indirect ELISA and quantitative ELISA to assess

the antibody-binding activity. For protein purification, the culture

supernatant was subjected to affinity chromatography on a

Streptactin Superflow high capacity column (Iba, Germany),

according to the protocol suggested by the supplier. Briefly, the

column was equilibrated with buffer W (100 mM Tris-HCl, pH

8.0, 150 mM NaCl, and 1 mM EDTA) and the bound protein was

eluted with 5 column volumes of buffer W supplemented with

2.5 mM desthiobiotin. Finally, the buffer was changed to PBS. The

protein concentration was determined using the NANO-DROP

2000 spectrophotometer, and the integrity of the purified protein

was assessed by western blot analysis.

Western Blot Analysis

The purified HzKR359-1 antibody was analyzed by 10% SDS-

PAGE under reducing conditions and 6% SDS-PAGE under non-

reducing conditions, followed by Coomassie Brilliant Blue G-250

staining. The protein bands were transferred to a nitrocellulose

membrane (GE Healthcare, France), and then the membrane was

incubated with anti-human IgG(H+L)-HRP conjugate (1:10,000

(v/v); Thermo Fisher Scientific). Finally, the bands were visualized

using a chemiluminescent substrate (WEST-ZOL plus; iNtRON

BioTechnology, Korea).

For the recombinant preS1 antigens, the purified proteins were

subjected to 10% SDS-PAGE and western blot analysis with a

murine mAb A10-A3 (0.5 μg/ml) that binds to the Ig1 domain of

human L1CAM [35], followed by anti-mouse IgG (Fc-specific)-

HRP conjugate (1:6,000 (v/v); Thermo Fisher Scientific).

Enzyme-Linked Immunosorbent Assays

All the incubations were performed at 37oC for 1 h. For the

quantitative ELISA of humanized antibody, antibody was serially

diluted in PBST (PBS with 0.05% Tween 20) and incubated with

goat anti-human IgG (κ-specific) antibody (200 ng in 50 mM

NaHCO3, pH 9.6; Thermo Fisher Scientific) coated on each well of

a 96-well Maxisorp plate (Nunc, Denmark). After washing, the

bound humanized antibody was incubated with anti-human IgG

(Fc-specific)-HRP (1:10,000; Thermo Fisher Scientific). For the

quantitative ELISA of the recombinant preS1 antigen, the preS1

antigen was serially diluted and incubated with anti-L1CAM

antibody A10-A3 (200 ng) coated on each well, and then bound

preS1 antigen was detected using anti-Strep-tag II antibody-HRP

(1:8,000; Iba). For indirect ELISA of humanized antibody, serially

diluted humanized antibody was incubated with the recombinant

preS1 antigen (200 ng) coated on each well, and then the bound

humanized antibody was incubated with anti-human IgG (Fc-

specific)-HRP (1:10,000; Thermo Fisher Scientific). Finally, TMB

substrate reagent (BD Biosciences, USA) was added and the

reaction was stopped with 2.5 M H2SO4. The optical density was

read at 450 nm in a microplate reader (Molecular Devices, USA).

For competitive ELISA, HzKR359-1 and increasing concentrations

of KR359 as a competing antibody were incubated with the preS1

antigen (genotype C) used as a coating antigen, and the bound

humanized antibody was detected using anti-human IgG (Fc-

specific)-HRP, while KR359-1 and increasing concentrations of

HzKR359-1 were incubated with the preS1 antigen, and the bound

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July 2017⎪Vol. 27⎪No. 7

mouse antibody was detected using anti-mouse IgG(Fc-specific)-

HRP (1:6,000; Thermo Fisher Scientific). Herceptin was used as an

irrelevant antibody control in this assay.

Analysis of Humanized Antibody Using Octet Red

The anti-human Fc-coated biosensor (Fortebio, Inc., USA) was

activated in 0.1% PBA (PBS containing 0.1% BSA) for 20 min by

agitating at 1,000 rpm. Antibody (1 μg/ml) was captured for

10 min and then washed with 0.1% PBA for 2 min. For affinity

determination, the sensor-loaded antibody was incubated with

the preS1 antigen (100, 50, 25, 12.5, or 6.25 nM in 0.1% PBA) for

10 min for the association step. Then, the dissociation step was

carried out in 0.1% PBA for 30 min. The sensor without loading

antibody was used as a reference well. The whole experiment

process was performed at 30oC and by agitating at 1,000 rpm.

Data were analyzed using a 1:1 interaction model with a ForteBio

data analysis software ver. 7.1.

For the analysis of additive binding of two mAbs to the preS1

antigen, the sensor-loaded humanized antibody was incubated

with the preS1 antigen (100, 50, or 25 nM in 0.1% PBA) for 10 min,

and then incubated with mouse antibody binding to a different

epitope. Baselines were established before and after the loading

step.

Results and Discussion

Construction, Expression, and Purification of Humanized

Antibody HzKR359-1

The cDNAs encoding the VH and Vκ of KR359 were

cloned from the hybridoma and their nucleotide sequences

were determined, as described in Materials and Methods.

Comparison of the amino acid sequences of the mouse VH

and Vκ with those of human immunoglobulin germline

gene segments in IMGT showed that the mouse VH and Vκ

were the most homologous to human VH4-59 and VK1-39

germline genes, respectively.

To construct a humanized VH, the CDRs (HCDR1–HCDR3)

of the mouse VH were grafted onto the human VH4-59 and

JH4, and 13 mouse FR residues that were predicted to

influence the conformation of HCDRs were also grafted.

Similarly, to construct a humanized Vκ, the CDRs (LCDR1–

LCDR3) of the mouse Vκ were grafted onto the human VK1-

39 and JK4. The resulting humanized VH and Vκ sequences

were codon-optimized and synthesized, and subsequently

separately subcloned into an antibody expression cassette

vector pCMV-dhfrC containing human Cγ1 or Cκ, respectively,

to construct humanized heavy or light chain expression

plasmids, respectively.

The expression plasmids were cotransfected into HEK293F

cells, and the resulting humanized antibody (HzKR359-1)

was transiently expressed and purified from the culture

supernatant by affinity chromatography on a Protein A

column. The purity and integrity of the purified antibody

were confirmed by SDS-PAGE (Fig. 1A) and western blot

analysis using anti-human IgG-HRP (Fig. 1B) under reducing

and non-reducing conditions.

Construction, Expression, and Purification of Recombinant

PreS1 Antigens of Six Different HBV Genotypes

To produce the recombinant preS1 antigens, the Ig1–Ig5

domains of human L1CAM were used as a carrier protein

because these domains were found to be stably expressed

in mammalian cells and secreted well in our previous

study [35], and a Strep tag was fused to the C-terminus of

Fig. 1. SDS-PAGE and western blot analysis of purified HzKR359-1 antibody.

(A) 10% SDS-PAGE under non-reducing (NR) or reducing (R) conditions of purified antibody. Bands that correspond to a heavy chain (~50 kDa),

light chain (~25 kDa), and whole antibody (150 kDa) are shown. (B) Western blot analysis of purified antibody under non-reducing (NR, 6%) or

reducing (R, 10%) conditions with anti-human IgG (H+L)-HRP.

1340 Wi et al.

J. Microbiol. Biotechnol.

preS1 for the detection and purification of the recombinant

preS1 antigen (Fig. 2A). The amino acid sequences of the

preS1 region (aa 1–60) of 10 HBV genotypes (A–J), including

the essential NTCP-binding site (aa 20–29) and the epitopes

of HzKR359-1 (aa 19–26) and HzKR127-3.2 (aa 37–45), are

shown in Fig. 2B. Comparison of the epitope sequences

among the genotypes indicated that five genotypes (A, B,

C, H, and J) have the same epitope sequence of HzKR359-1,

whereas three genotypes (A, C, and I) have the same

epitope sequence of HzKR127-3.2. Therefore, the preS1

antigens of six HBV genotypes (B–G) were produced to

examine the antigen-binding activity of HzKR359-1 or

HzKR127-3.2 to all of the HBV genotypes (A–J).

For the production of the preS1 antigens, the preS1

sequences of the six genotypes (B–G) with Strep tags were

synthesized and individually fused to the C-terminus of

the Ig1-Ig5 domains. The recombinant preS1 fusion proteins

were transiently expressed in HEK293F cells, and the

culture supernatants were subjected to quantitative ELISA

using a murine anti-L1CAM mAb (A10-A3) as a coating

antigen and anti-Strep Tag-HRP to measure their expression

levels. As a result, the preS1 antigens of the genotypes B–D

(2.4–4.2 µg/ml) were expressed at higher levels than those

of genotypes E–G (0.7–0.9 µg/ml; data not shown). The

preS1 antigens were purified from the culture supernatants

using a Strep tag and subjected to western blot analysis

using A10-A3 antibody. The result showed that the preS1

antigens were stably expressed and purified (Fig. 2C).

Analyses of Antigen-Binding Activity and Affinity of

HzKR359-1

The antigen-binding activity of the purified HzKR359-1

antibody was analyzed by indirect ELISA using the purified

preS1 antigen (genotype C) as a coating antigen. HzKR359-

1 bound to the preS1 antigen in a dose-dependent manner,

and its antigen-binding activity was slightly lower than

that of HzKR127-3.2, which was developed through affinity

maturation of a humanized KR127 antibody constructed by

CDR grafting (Fig. 3A). The data indicate that the HzKR359-1

antibody was successfully constructed.

To compare the antigen-binding activity of HzKR359-1

with that of KR359, competitive ELISAs were performed.

HzKR359-1 and increasing concentrations of KR359 as a

competing antibody were incubated with the preS1 antigen

(genotype C) coated on the well, and the bound humanized

antibody was detected using anti-human IgG (Fc-specific)-

HRP, whereas anti-HER2 humanized antibody (Herceptin)

was used as an irrelevant antibody control (Fig. 3B). Similarly,

KR359-1 and increasing concentrations of HzKR359-1 were

incubated with the preS1 antigen, and the bound mouse

antibody was detected using anti-mouse IgG-HRP (Fig. 3C).

The results indicated that HzKR359-1 and KR359 competed

Fig. 2. Construction, expression, and purification of recombinant preS1 antigens of different HBV genotypes.

(A) Schemes of L1CAM (upper) and recombinant preS1 antigen (lower). L1CAM has six Ig domains (circles), five Fn domains (boxes), a

transmembrane domain (TM), and a short cytoplasmic tail. N-linked glycosylation sites (Y) are represented. (B) The preS1 sequences (aa 1-60) of

10 HBV genotypes (A–J). The dotted line above the sequences demarcates an essential sodium taurocholate cotransporting polypeptide-binding

site (amino acid 20–29). The black box indicates the epitope of HzKR359-1. The gray box indicates the epitope of HzKR127-3.2. (C) Western blot

analysis of purified recombinant preS1 antigens with anti-L1CAM A10-A3 antibody as a primary antibody. Lane 1, standard protein marker; lane

2, genotype C; lane 3, genotype B; lane 4, genotype D; lane 5, genotype E; lane 6, genotype F; lane 7, genotype G.

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for the same epitope, indicating that HzKR359-1 retained

the same epitope specificity as that of KR359. The IC50 of

HzKR359-1 was calculated to be 0.37 µg/ml (Fig. 3C),

whereas that of KR359 was 1.62 µg/ml (Fig. 3B), indicating

that the antigen-binding activity of HzKR359-1 is higher

than that of KR359.

To precisely compare the affinity of HzKR359-1 with that

of HzKR127-3.2, the affinities of these antibodies for the

preS1 antigen (genotype C) were measured using Octet Red.

As shown in Fig. 4, the affinity (3.28 × 10-9 M) of HzKR359-1

was 5.8-fold lower than that (5.69 × 10-10 M) of HzKR127-3.2.

Analyses of the Antigen-Binding Activities of HzKR359-1

or HzKR127-3.2 to Different HBV Genotypes

Having analyzed the antigen-binding activities of

HzKR359-1 and HzKR127-3.2 to the preS1 of genotype C, we

further extended the analysis to other genotypes. Because

the epitope sequences of HzKR359-1 and HzKR127-3.2 in

genotype A are identical to those in genotype C, we

Fig. 3. Indirect ELISA of HzKR359-1 and HzKR127-3.2 (A) and

competitive ELISA of HzKR359-1 and KR359 using KR359 (B)

or HzKR359-1 (C) as a competitor.

The data are presented in Sigma plots. (B and C) The optical density

obtained from the control (no competitor) was considered as 100%.

Herceptin was used as an irrelevant antibody (negative control).

Fig. 4. Affinity determination of HzKR359-1 (A) and

HzKR127-3.2 (B) for preS1 (genotype C) using Octet Red.

PreS1 concentrations used are 100, 50, 25, 12.5, and 6.25 nM. (C)

Calculated association and dissociation rates and the KD value.

1342 Wi et al.

J. Microbiol. Biotechnol.

exempted the expression of the preS1 antigen of genotype

A. HzKR359-1 or HzKR127-3.2 was incubated with each of

the purified recombinant preS1 antigens of genotypes (B–

G), bound by anti-L1CAM antibody (A10-A3) coated on the

well, to perform indirect ELISA. The results showed that

HzKR359-1 bound to the preS1 of genotypes B and C

(Fig. 5A), whereas HzKR127-3.2 bound to genotypes C, D,

and G (Fig. 5B). Accordingly, HzKR359-1 plus HzKR127-3.2

bound to genotypes B, C, D, and G (Fig. 5C). Considering

that the epitope sequences of HzKR359-1 and HzKR127-3.2 in

genotype A are identical to those in genotype C, HzKR359-

1 can bind to the preS1 of three genotypes (A–C), whereas

HzKR127-3.2 can bind to four genotypes (A, C, D, and G).

Given that the epitope sequences of HzKR359-1 in

genotypes H and J are identical to those in genotypes A–C,

HzKR359-1 is expected to bind to five genotypes (A–C, H, and

J). Likewise, because the epitope sequence of HzKR127-3.2

in genotype I is identical to that in genotypes A and C,

HzKR127-3.2 is predicted to bind to five genotypes (A, C,

D, G, and I). Collectively, the combination of HzKR359-1

and HzKR127-3.2 is expected to bind to eight genotypes

(A-D and G-J), except genotypes E and F. Considering

that genotypes E and F are occasionally represented in

small patient populations, the combination of HzKR359-1

and HzKR127-3.2 may bind to HBV particles of most

genotypes.

Additive Binding of HzKR359-1 and HzKR127-3.2 to

Genotype C

Because the epitope of HzKR359-1 is only 11 aa distant

from that of HzKR127-3.2, we examined whether these two

antibodies compete in binding to the preS1 antigen of

genotype C, using competitive binding assay. As shown in

Fig. 6A, HzKR359-1 or Herceptin as a negative control did

not inhibit the binding between KR127 and the preS1

antigen, whereas HzKR127-3.2 efficiently inhibited binding,

suggesting that HzKR359-1 and HzKR127-3.2 can bind to

genotype C simultaneously. To demonstrate this, HzKR359-1

or HzKR127-3.2 was incubated with different concentrations

of the preS1 antigen followed by KR127 or KR359, respectively,

using Octet Red (Figs. 6B and 6C). Indeed, HzKR359-1 and

KR127 (or HzKR127-3.2 and KR359) bound to the preS1

antigen additively and, thus, showed enhanced binding

activity to genotype C compared with either antibody alone.

In conclusion, we successfully constructed a humanized

antibody (HzKR359-1) that binds to the essential receptor

binding site and demonstrated that the affinity of

HzKR359-1 is higher than that of the original murine mAb.

HzKR359-1 can bind to five genotypes (A–C, H, and J), and

HzKR127-3.2 binds to six genotypes. The combination of

HzKR359-1 and HzKR127-3.2 can bind to most HBV

genotypes, with the exception of genotypes E and F, and

showed enhanced binding to genotype C compared with

that of either antibody alone. Given that HzKR127 exhibited

Fig. 5. Analyses of the antigen-binding activities of HzKR359-

1 (A), HzKR127-3.2 (B), and HzKR359-1 plus HzKR127-3.2 (C)

to the preS1 of different genotypes.

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neutralizing activity to genotype C in chimpanzees and

that the epitopes of HzKR359-1 and HzKR127-3.2 are

located in the receptor-binding site, these two humanized

antibodies might effectively block the entry of most virus

genotypes and thus broadly neutralizing HBV infection.

Acknowledgments

This work was supported by a grant from the Leaders

INdustry-university Cooperation Plus Project supported

by the Ministry of Education and a grant from the Ministry

of Science, ICT and Future Planning.

References

1. Ganem D, Varmus HE. 1987. The molecular biology of the

hepatitis B viruses. Annu. Rev. Biochem. 56: 651-693.

2. Rehermann B, Nascimbeni M. 2005. Immunology of

hepatitis B virus and hepatitis C virus infection. Nat. Rev.

Immunol. 5: 215-229.

3. Ott JJ, Stevens GA, Groeger J, Wiersma ST. 2012. Global

epidemiology of hepatitis B virus infection: new estimates

of age-specific HBsAg seroprevalence and endemicity.

Vaccine 30: 2212-2219.

4. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K,

Aboyans V, et al. 2012. Global and regional mortality from

235 causes of death for 20 age groups in 1990 and 2010: a

systematic analysis for the Global Burden of Disease Study

2010. Lancet 380: 2095-2128.

5. McGlynn KA, London WT. 2011. The global epidemiology

of hepatocellular carcinoma: present and future. Clin. Liver

Dis. 15: 223-243, vii-x.

6. GBD 2013 Mortality and Causes of Death Collaborators

(Naghavi M, Wang H, Lozano R, Davis A, Liang X, Zhou M,

et al.). 2015. Global, regional, and national age-sex specific

all-cause and cause-specific mortality for 240 causes of

death, 1990-2013: a systematic analysis for the Global

Burden of Disease Study 2013. Lancet 385: 117-171.

7. Kramvis A, Kew M, Francois G. 2005. Hepatitis B virus

genotypes. Vaccine 23: 2409-2423.

8. Schaefer S. 2007. Hepatitis B virus taxonomy and hepatitis B

virus genotypes. World J. Gastroenterol. 13: 14-21.

9. Kramvis A, Kew MC. 2007. Epidemiology of hepatitis B

virus in Africa, its genotypes and clinical associations of

genotypes. Hepatol. Res. 37: S9-S19.

10. Norder H, Courouce AM, Coursaget P, Echevarria JM, Lee

SD, Mushahwar IK, et al. 2004. Genetic diversity of hepatitis

B virus strains derived worldwide: genotypes, subgenotypes,

and HBsAg subtypes. Intervirology 47: 289-309.

11. Kramvis A. 2014. Genotypes and genetic variability of

hepatitis B virus. Intervirology 57: 141-150.

12. Tatematsu K, Tanaka Y, Kurbanov F, Sugauchi F, Mano S,

Fig. 6. Binding characteristics of HzKR359-1 and HzKR127-3.2

for genotype C.

(A) Competition ELISA of KR127 using HzKR359-1 or HzKR127-3.2

as competing antibody. The data are presented in Sigma plots. The

optical density obtained from the control (no competitor) was

considered as 100%. Herceptin was used as an irrelevant antibody

(negative control). (B and C) Additive binding analysis of HzKR127-

3.2 and KR359 (B) or HzKR359-1 and KR127 (C) using Octet Red.

PreS1 concentrations used are 100, 50, and 25 nM. As a control, PBS

containing 0.1% BSA was used in place of the antigen or antibodies.

1344 Wi et al.

J. Microbiol. Biotechnol.

Maeshiro T, et al. 2009. A genetic variant of hepatitis B virus

divergent from known human and ape genotypes isolated

from a Japanese patient and provisionally assigned to new

genotype J. J. Virol. 83: 10538-10547.

13. Sunbul M. 2014. Hepatitis B virus genotypes: global

distribution and clinical importance. World J. Gastroenterol.

20: 5427-5434.

14. Westland C, Delaney W, Yang H, Chen SS, Marcellin P,

Hadziyannis S, et al. 2003. Hepatitis B virus genotypes and

virologic response in 694 patients in phase III studies of

adefovir dipivoxil. Gastroenterology 125: 107-116.

15. Heermann KH, Goldmann U, Schwartz W, Seyffarth T,

Baumgarten H, Gerlich WH. 1984. Large surface proteins of

hepatitis B virus containing the pre-S sequence. J. Virol.

52: 396-402.

16. Stibbe W, Gerlich WH. 1983. Structural relationships

between minor and major proteins of hepatitis B surface

antigen. J. Virol. 46: 626-628.

17. Glebe D, Urban S. 2007. Viral and cellular determinants

involved in hepadnaviral entry. World J. Gastroenterol. 13: 22-38.

18. Engelke M, Mills K, Seitz S, Simon P, Gripon P, Schnolzer

M, et al. 2006. Characterization of a hepatitis B and hepatitis

delta virus receptor binding site. Hepatology 43: 750-760.

19. Xie Y, Zhai J, Deng Q, Tiollais P, Wang Y, Zhao M. 2010.

Entry of hepatitis B virus: mechanism and new therapeutic

target. Pathol. Biol. (Paris) 58: 301-307.

20. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, et al. 2012.

Sodium taurocholate cotransporting polypeptide is a functional

receptor for human hepatitis B and D virus. eLife 1: e00049.

21. Glebe D, Urban S, Knoop EV, Cag N, Krass P, Grun S, et al.

2005. Mapping of the hepatitis B virus attachment site by

use of infection-inhibiting preS1 lipopeptides and tupaia

hepatocytes. Gastroenterology 129: 234-245.

22. Schulze A, Schieck A, Ni Y, Mier W, Urban S. 2010. Fine

mapping of pre-S sequence requirements for hepatitis B

virus large envelope protein-mediated receptor interaction.

J. Virol. 84: 1989-2000.

23. Ecker DM, Jones SD, Levine HL. 2015. The therapeutic

monoclonal antibody market. mAbs 7: 9-14.

24. Shawler DL, Bartholomew RM, Smith LM, Dillman RO.

1985. Human immune response to multiple injections of

murine monoclonal IgG. J. Immunol. 135: 1530-1535.

25. Queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF,

Duncan JF, et al. 1989. A humanized antibody that binds to

the interleukin 2 receptor. Proc. Natl. Acad. Sci. USA 86:

10029-10033.

26. Riechmann L, Clark M, Waldmann H, Winter G. 1988.

Reshaping human antibodies for therapy. Nature 332: 323-327.

27. Ryu CJ, Kim YK, Hur H, Kim HS, Oh JM, Kang YJ, et al.

2000. Mouse monoclonal antibodies to hepatitis B virus

preS1 produced after immunization with recombinant preS1

peptide. Hybridoma 19: 185-189.

28. Maeng CY, Ryu CJ, Gripon P, Guguen-Guillouzo C, Hong

HJ. 2000. Fine mapping of virus-neutralizing epitopes on

hepatitis B virus PreS1. Virology 270: 9-16.

29. Hong HJ, Ryu CJ, Hur H, Kim S, Oh HK, Oh MS, et al.

2004. In vivo neutralization of hepatitis B virus infection by

an anti-preS1 humanized antibody in chimpanzees. Virology

318: 134-141.

30. Kim JH, Gripon P, Bouezzedine F, Jeong MS, Chi SW, Ryu SE,

et al. 2015. Enhanced humanization and affinity maturation

of neutralizing anti-hepatitis B virus preS1 antibody based on

antigen-antibody complex structure. FEBS Lett. 589: 193-200.

31. Backliwal G, Hildinger M, Hasija V, Wurm FM. 2008. High-

density transfection with HEK-293 cells allows doubling of

transient titers and removes need for a priori DNA complex

formation with PEI. Biotechnol. Bioeng. 99: 721-727.

32. Cho S, Park I, Kim H, Jeong MS, Lim M, Lee ES, et al. 2016.

Generation, characterization and preclinical studies of a

human anti-L1CAM monoclonal antibody that cross-reacts

with rodent L1CAM. mAbs 8: 414-425.

33. Faleye TO, Adewumi OM, Ifeorah IM, Akere A, Bakarey

AS, Omoruyi EC, et al. 2015. Detection and circulation of

hepatitis B virus immune escape mutants among asymptomatic

community dwellers in Ibadan, southwestern Nigeria. Int. J.

Infect. Dis. 39: 102-109.

34. Tran TT, Trinh TN, Abe K. 2008. New complex recombinant

genotype of hepatitis B virus identified in Vietnam. J. Virol.

82: 5657-5663.

35. Wei CH, Lee ES, Jeon JY, Heo YS, Kim SJ, Jeon YH, et al.

2011. Structural mechanism of the antigen recognition by

the L1 cell adhesion molecule antibody A10-A3. FEBS Lett.

585: 153-158.