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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
An Anti-Hepatitis B Virus PreS1 Humanized Antibody 1337
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
An Anti-Hepatitis B Virus PreS1 Humanized Antibody 1339
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.
An Anti-Hepatitis B Virus PreS1 Humanized Antibody 1341
July 2017⎪Vol. 27⎪No. 7
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.
An Anti-Hepatitis B Virus PreS1 Humanized Antibody 1343
July 2017⎪Vol. 27⎪No. 7
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.
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