International Journal of Biological Macromolecules 199 (2022) 298–306
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
Targeting and neutralizing human epididymis protein 4 by novel
nanobodies to suppress ovarian cancer cells and attenuate
cisplatin resistance
Jianli Yu a, c, Yang Guo a, Yi Gu a, c, Fei Li b, Haipeng Song b, Rui Nian a, Xiying Fan a, *,
Wenshuai Liu a, *
a
CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao
266101, China
b
Shenzhen Innova Nanobodi Co., Ltd., No. 1301 Guanguang Road, Shenzhen 518110, China
c
University of Chinese Academy of Sciences, No. 19(A) Yuquan Road, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
HE4
Nanobody
Phage display
Apoptosis
Chemosensitivity
Human epididymis protein 4 (HE4) is a glycoprotein secreted by epithelial ovarian cancer (EOC) cells and is a
novel and specific biomarker for diagnosing and prognosing EOC. Previous studies have shown that over­
expression of HE4 is correlated with EOC tumorigenesis and chemoresistance. However, less has been reported
regarding the direct effect of the secreted HE4 protein as an autocrine factor in EOC cells. Here, we investigated
the molecular mechanism of the secretory form of HE4 on the growth of EOC cells by applying nanobodies with a
targeted interaction of free HE4. Three anti-HE4 nanobodies were selected from an immune library by phage
display. HE4 secreted by serum-free cultured OVCAR3 cells increased and was effectively neutralized by antiHE4 nanobodies, which inhibited cell viability. Treatment with the anti-HE4 nanobody 1G8 decreased Bcl-2
expression and increased BAX, cleaved PARP, and p53 levels, resulting in apoptosis of OVCAR3 cells. More­
over, 1G8 significantly improved the cisplatin response of OVCAR3 cells. Our data suggest that secretory HE4
played a novel pro-survival autocrine role and was a target of the anti-HE4 nanobody to improve the therapeutic
effects of cisplatin-based chemotherapy.
1. Introduction
Epithelial ovarian cancer (EOC) is a malignant tumor that causes a
high death rate in females [1]. An early EOC diagnosis and new treat­
ment schemes are needed to improve the EOC therapeutic effect. Human
epididymis protein 4 (HE4) is highly expressed in ovarian cancer tissues,
but not in normal ovarian tissues, and thus represents an important EOC
biomarker [2,3]. The diagnostic value of HE4 in EOC has been
confirmed, as it has become a gold standard for the early diagnosis of
EOC [4–6]. In addition, the roles of HE4 in ovarian cancer tumorigenesis
have drawn increasing attention. Several studies have suggested that
HE4 is positively correlated with EOC proliferation and growth [7–11].
Furthermore, HE4-induced chemoresistance has also been reported in
ovarian cancer cell lines [12–14]. However, these data were obtained
from HE4 gene overexpression or knockdown experiments. Less has
been studied on the effects of the natively secreted HE4 protein on EOC
cells.
Native HE4 is known to be a secretory glycoprotein. Thus, it is more
practical to explore the functions of the extracellular HE4 protein.
Zhuang et al. [15] reported that HE4 binds to Annexin II, a calciumdependent phospholipid-binding protein on the cell membrane. This
interaction has been attributed to Annexin II-assisted transfer of HE4
into the cell nucleus and the induction of invasion/metastasis-related
genes [14]. Furthermore, Wang et al. [16] reported that
HE4–ANXA2–MMP2 forms a triple protein complex that performs
together to promote the migration of various malignant cell lines.
Epidermal growth factor receptor is another transmembrane protein
that interacts with HE4 [11], resulting in the transduction of the growth
signal. These studies indicate that extracellular HE4 may directly bind to
a receptor or receptor-like protein on the cell membrane and act as an
autocrine factor to play important roles in the progression and chemo­
resistance of EOC. However, few studies have investigated the direct
* Corresponding authors.
E-mail addresses: fanxy@qibebt.ac.cn (X. Fan), liuws@qibebt.ac.cn (W. Liu).
https://doi.org/10.1016/j.ijbiomac.2022.01.015
Received 8 December 2021; Received in revised form 31 December 2021; Accepted 4 January 2022
Available online 8 January 2022
0141-8130/© 2022 Published by Elsevier B.V.
J. Yu et al.
International Journal of Biological Macromolecules 199 (2022) 298–306
effect of the native HE4 protein that is secreted outside of cells on the
development of EOC, and the exact molecular mechanisms are poorly
defined. Secretory HE4 could be targeted by anti-HE4 antibodies
resulting in loss of function, which is an effective strategy to investigate
the underlying mechanism of secretory HE4. In addition, the anti-HE4
antibody may be effective in treating ovarian cancer considering the
important roles of HE4 in ovarian carcinogenesis.
Nanobodies (Nbs) represent a new generation of antibodies usually
derived from camelids, which naturally possess a unique subset of im­
munoglobulins devoid of antibody light chains. The variable domain of
this heavy chain (approximately 15 kDa) is the smallest antigen-binding
repertoire found in any antibody [17,18]. This characteristic benefits
their easy production [19–21], rapid selection from immune or native
libraries [22], and straightforward construction into multivalent and
pluripotent antigen-binding formats using the same or different building
blocks [23,24]. Moreover, Nbs are extremely stable with antigenbinding affinity in the nanomolar range [22]. Studies on the structures
of Nbs have shown that complementarity determining region (CDR) 3 is
significantly longer than that of conventional antibodies [25]. They also
possess an additional disulfide bridge linking CDR1 and CDR3, enabling
the formation of a peptide loop with increased flexibility for enhanced
recognition of concave surfaces and a variety of more complex epitopes
[26–28] that cannot be identified and distinguished by conventional
antibodies. Thus, Nbs are more suitable for the development of functionblocking antibodies against HE4 because of the complex glycosylation
and multiple disulfide bonds of HE4.
In the present study, a series of anti-HE4 Nbs were generated by
phage display technology and characterized by surface plasmon reso­
nance (SPR) technology. Then, the anti-HE4 Nbs were used to neutralize
secreted HE4 in EOC cells. After blocking the binding activity of auto­
crine HE4, the viability of OVCAR3 cells was determined with the Cell
Counting Kit-8 (CCK-8) assay, and the molecular mechanism of secre­
tory HE4 was investigated by flow cytometry and western blot assay.
Moreover, we evaluated the therapeutic effects of a combination of the
Nb and cisplatin in OVCAR3 cells. We expect to explain cisplatin resis­
tance in EOC and, more importantly, provide insight into a treatment
strategy for EOC while exploring the biological mechanism of secretory
HE4.
2.2. Construction of the nanobody library
The HE4 DNA sequence was synthesized and fused with an Fc tag at
the C-terminus followed by transient transfection into HEK293 cells.
After a 3-day suspension culture, the cell culture supernatant was har­
vested and loaded onto the HiTrap rProtein A prepacked column for
purification. The purified protein (400 μg) mixed with an equal volume
of Gerbu adjuvant was injected into a healthy alpaca five times at 2week intervals. Four days after the final injection, peripheral blood
lymphocytes (PBLs) were collected from 50 mL of blood by Ficoll density
gradient centrifugation. The anti-HE4 Nb library was constructed ac­
cording to a previous study [29]. Briefly, total RNA was extracted from
PBLs with the Tipure Isolation Reagent and transcribed into cDNA.
Then, the Nb gene was amplified using nested PCR, digested with the Pst
I and BstE II restriction enzymes, and ligated into the pMES4 phagemid.
Subsequently, the recombinant phagemids were transformed into E. coli
TG1 competent cells. Library capacity was calculated by counting the
number of colonies after gradient dilution. 18 colonies were randomly
chosen to test the correct insertion rate by PCR amplification.
2.3. Biopanning of the nanobody library
The anti-HE4 Nbs were selected by phage display. The Nb library was
infected with 109 colony forming units (CFU) of VCSM13 helper phages
to express Nbs at the tip of phage particles. Biopanning was performed
according to a previous study [30]. HE4 (10 μg/mL) was coated onto 96well plates at 4 ◦ C overnight. After blocking with 5% bovine serum al­
bumin (BSA) in phosphate-buffered saline (PBS) for 2 h, the 96-well
plates were incubated with phage particles for 1 h at 37 ◦ C. The
bound phages were eluted with 100 mM glycine solution for 10 min at
room temperature (RT), and then immediately neutralized with 1.0 M
Tris-HCl (pH 9.1). The eluted phages were titrated by limited gradient
dilution and subsequently rescued with VCSM13 for the next round of
panning. After four rounds of panning, 90 individual colonies were
randomly selected to grow in LB medium and infected with VCSM13.
The cell supernatants were harvested and added to a 96-well plate
coated with HE4 (1 μg/mL). After a 1-h incubation at 37 ◦ C, the 96-well
plate was washed with PBS containing 0.05% (v/v) Tween 20 (PBST)
and incubated with anti-M13-HRP antibody (1:10,000) at RT for 1 h.
TMB was added, and absorbance was measured at 450 nm. Then, the
identified clones were sent for sequencing.
2. Materials and methods
2.1. Reagents
2.4. Expression and characterization of the selected nanobodies
The VCSM13 helper phage, horseradish peroxidase (HRP)-conju­
gated mouse anti-M13 monoclonal antibody (#27-9421-01), and the
rProtein A prepacked column (#17508002) were purchased from GE
(Milwaukee, WI, USA). The Tipure Isolation Reagent (#11667165001)
and Transcriptor First Strand cDNA Synthesis Kit (#04896866001) were
obtained from Roche (Indianapolis, IN, USA). The protein markers
(#26616) were purchased from Thermo Fisher Scientific Inc. (Waltham,
MA, USA). All polymerase chain reaction (PCR) primers and DNA mol­
ecules were synthesized by Genwiz (Beijing, China). The Ni-NTA resin
(L00250) was obtained from GeneScript (Nanjing, China). The OVCAR3
human ovarian cancer cell line and the HEK293 human embryonic
kidney cell line were purchased from ATCC (Manassas, VA, USA). The
96-well cell culture plates (#3988) were obtained from Corning
(Corning, NY, USA). The cell culture reagents were purchased from
Gibco (Grand Island, NY, USA). The DNA Content Quantitation Assay Kit
(CA1510), Annexin II-FITC Apoptosis Detection Kit (CA1020), and Goat
Anti-rabbit IgG/HRP antibody (SE134) were purchased from Solarbio
(Beijing, China). The anti-p53 antibody (#10442-1-AP), anti-BAX anti­
body (#50599-2-lg), anti-Bcl-2 antibody (#12789-1-AP), and anti-PARP
antibody (#13371-1-AP) were purchased from Proteintech (Rocky Hill,
NJ, USA).
The plasmids of the identified clones were extracted from TG1 cells
and transformed into E. coli BL21 cells. The cells were grown in LB
medium with 100 μg/mL ampicillin until the OD600 value reached 0.8.
Expression of the Nbs was subsequently induced with 1 mM IPTG for 20
h at 30 ◦ C.The culture was centrifuged at 8000 ×g for 10 min, and the
cell pellets were resuspended in TES buffer (20 mM Tris-HCl, 1 mM
EDTA, 20% sucrose, pH 8.0). Periplasmic proteins were released by
osmotic shock and purified on a Ni-NTA affinity column. After eluting
with 500 mM imidazole, the Nbs were dialyzed against PBS and
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Nb affinity was estimated according to the BIACORE T100
(GE) instrument manual. Briefly, HE4 was immobilized on the CM5 chip
surface. Then, five series of Nb dilutions (0.16, 0.32, 0.64, 1.28, and
2.56 nM) were sequentially injected at a flow rate of 30 μL/min for 600
s. Data were processed and fit to a 1:1 binding model using BIACORE
T100 Evaluation Software to determine the binding kinetics rate
constants.
2.5. Cell line and cell culture
OVCAR3 cells were cultured in RPMI-1640 medium with 10% fetal
bovine serum (FBS) and 1% penicillin/streptomycin at 37 ◦ C with 5%
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International Journal of Biological Macromolecules 199 (2022) 298–306
CO2 in an incubator (ICO, Memert, Germany).
2.10. Chemosensitivity analysis
OVCAR3 cells were seeded in 96-well plates (2500 cells/well) in
1640 medium containing 10% FBS. After an overnight attachment
period, the cells incubated with or without 5 nM 1G8 were treated with
different doses of cisplatin (0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 μg/mL) in
1640 medium containing 2% FBS. After 48-h of incubation, the CCK-8
reagent (10 μL/well) was added and incubated for 1 h at 37 ◦ C. Absor­
bance was measured at 450 nm. The half-maximal inhibitory concen­
tration (IC50) was calculated from the dose-response curve. Three
independent experiments were performed, each with three technical
replicates.
2.6. Enzyme-linked immunosorbent assay (ELISA)
The OVCAR3 cells were seeded in 96-well plates at a density of 2500
cells/well in RPMI-1640 medium containing 10% FBS and allowed to
adhere overnight. Then, the medium was replaced with serum-free
RPMI-1640 medium or fresh RPMI-1640 medium containing 10% FBS
for further culture. The culture supernatants were harvested after 24, 48,
and 72 h respectively, and the concentrations of secretory HE4 were
measured using an ELISA kit (Beijing Savant Biotechnology Co., Ltd.,
Beijing, China) approved by National Medical Products Administration
of China. Briefly, 25 μL of the supernatant or calibrator was added to a
96-well plate coated with the capture antibody. After adding 25 μL of the
conjugate (HRP-labeled antibody), the plate was incubated at 37 ◦ C for
45 min. The plate was washed five times with PBST and the luminescent
substrate was added. Luminescence was measured in a microplate
reader (Spark, Tecan, Basel, Switzerland).
2.11. Statistical analysis
Data were analyzed using Prism 5.0 software (GraphPad Software,
Inc., La Jolla, CA, USA) and presented as mean ± standard deviation.
Differences between groups were evaluated using one-way analysis of
variance. Two-sided P-values <0.05 were considered significant.
2.7. Cell viability assay
3. Results
The OVCAR3 cells were seeded in 96-well plates as described above.
The culture medium was replaced with RPMI-1640 medium containing
different concentrations of Nbs (0.5, 5, 50, 500, 5000, and 50,000 pM).
After 48 h, the culture medium was replaced with 100 μL of fresh 1640
medium containing the same concentration of Nbs. Control experiments
were carried out using PBS as the untreated group and 5 × 104 pM of
anti-procalcitonin (anti-PCT) Nb screened in our laboratory as the
negative group to match the 50,000 pM Nb concentration mentioned
above. The CCK-8 reagent (10 μL/well) was added after 24, 48, and 72 h,
respectively, and incubated for 1 h at 37 ◦ C. Absorbance was measured
at 450 nm using the microplate reader. Cell viability was expressed as a
percentage of the untreated group.
3.1. Selection of anti-HE4 Nbs by phage display
A 2-year-old healthy alpaca was immunized with HE4 over 8 weeks
to isolate the Nbs against HE4. After the final immunization, the HE4specific antibody titer in the sera had reached 1:800,000. This indi­
cated that HE4-specific B lymphocytes were stimulated to express an­
tibodies, including heavy-chain-only antibodies. Total mRNA was
extracted from PBLs and used to synthesize cDNA followed by the first
PCR with the CALL001 and CALL002 primers [29]. PCR products
including a ~1000 bp fragment for the VH-CH1-CH2 exons and a ~700
bp fragment for the VHH-CH2 exons were obtained (Fig. 1A). The ~700
bp fragments were purified by gel extraction and used as the template
for the second PCR. The purified second PCR products were ligated into
the pMES4 phagemid after digestion with the Pst I and BstE II restriction
enzymes and then electroporated into competent E. coli TG1 cells. The
library capacity was 1.4 × 109 CFU according to the plating and gradient
dilution methods (Fig. 1B). The colony PCR analysis of 18 randomly
picked colonies revealed that the correct insertion rate was nearly 100%
(Fig. 1C).
According to a procedure described by our previous study [30], antiHE4 Nbs were selected by phage display. Approximately 1 × 1012
phages were used in each round of panning. After the fourth round of
panning, 90 individual colonies were randomly picked to identify HE4specific Nbs by phage ELISA. After sequencing, the positive colonies
were classified into three families based on the diversity of the amino
acid sequences (Fig. 2A), which were called 1G8, 3F10, and 4G8.
2.8. Flow cytometry analysis
OVCAR3 cells were plated in a T25 flask at a density of 80,000 cells/
flask and grown in RPMI-1640 medium containing 10% FBS at 37 ◦ C for
18 h. As described above, the cells were treated with 5 nM 1G8, the antiPCT Nb or PBS. The cells were harvested by trypsinization 72 h posttreatment, washed in PBS, and fixed in cold 70% ethanol for 2 h at
4 ◦ C. After a 30-min RNase A treatment at 37 ◦ C, the cells were stained
with propidium iodide (PI) at 4 ◦ C for 30 min and then analyzed for DNA
content by FACSAria flow cytometry (BD Biosciences, Brea, CA, USA).
The percentages of cells containing different DNA contents were quan­
tified using Modfit software (version 3.2.0, Verity Software House,
Topsham, ME, USA). The samples for the apoptosis analysis were
washed in the binding buffer from the Annexin V-FITC/ PI Apoptosis
Detection Kit. The cells in the binding buffer were stained with Annexin
V-FITC and PI for 10 and 5 min at RT in the dark, respectively. The
apoptotic cells were counted by FACSAria flow cytometry.
3.2. Characterization of the anti-HE4 Nbs
The pMES4 phagemid containing the Nb gene was transformed into
the E. coli BL21 strain to suppress the amber stop codon between genes
III and Nb of the phagemid. Only the Nbs containing the C-terminus
His6-tag were expressed in the periplasm of BL21 cells. After extraction
using the osmotic shock method, soluble Nbs were purified using a NiNTA Superflow Sepharose column. The SDS-PAGE analysis showed
that the molecular weight of the Nbs was about 14 kDa, and purity was
90% (Fig. 2B). The affinities of the three Nbs to the HE4 antigen were
measured by SPR on the BIACORE T100 system. As shown in Fig. 2C, the
equilibrium dissociation constants (KD) of 1G8, 3F10, and 4G8 were 1.6
× 10− 9, 2.9 × 10− 8, and 9.9 × 10− 8 mol/L respectively. These results
indicate that the three Nbs showed high affinity and strong binding to
HE4.
2.9. Western blot assay
After the incubation with 1G8, anti-PCT Nb or PBS for 72 h, the cells
were digested with trypsin and lysed in RIPA buffer. Total proteins were
separated by SDS-PAGE and transferred to a polyvinylidene difluoride
membrane (Millipore, Billerica, MA, USA). After blocking in 5% BSA at
RT for 1 h, the membranes were incubated with primary antibodies at
4 ◦ C overnight. The membranes were washed and incubated with HRPconjugated secondary antibody at RT for 1 h. The protein bands were
visualized with the DAB detection system, and the expression levels of
these proteins were evaluated using ImageJ software (version 1.4.3;
National Institutes of Health, Bethesda, MD, USA).
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Fig. 1. Construction of the nanobody library. (A) The Nb gene was obtained by two-step PCR. (B) The size of the library was determined by counting the number of
clones after serial dilution. (C) Colonies were randomly picked to estimate the correct insertion rate of the VHH genes by PCR amplification.
3.3. OVCAR3 cells secreting HE4 increase in the absence of serum
3.4. Neutralization of secretory HE4 by anti-HE4 Nbs suppresses cell
viability
Uncontrolled proliferation is a feature of cancer that potentially
contributes to cancer progression. As shown in Fig. 3A, the OVCAR3
cells proliferated in the serum-free and serum-containing medium for
72 h. However, the serum-free culture condition did not suppress the
proliferation of OVCAR3 cells, indicating that certain OVCAR3 cell
factors may play important roles in maintaining active cells. OVCAR3
cells displayed increased HE4 secretion during serum-free culture,
compared to those in medium containing 10% FBS, and secreted up to
56.2 pM HE4 into the extracellular medium after 72 h (Fig. 3B). These
results indicate that increased secretion of HE4 was a cell-protective
response and may contribute to maintaining cell survival in the
absence of serum. Furthermore, the absence of serum eliminated inter­
ference by external factors, such as hormones and growth factors, and
gave prominence to the roles of secretory HE4 as an autocrine factor in
the growth of OVCAR3 cells.
To explore the function of secreted HE4, the OVCAR3 cells in serumfree medium were treated with 0, 5, 50, 500, 5000, or 50,000 pM antiHE4 Nbs for 72 h. Then, the CCK-8 assay was performed to measure cell
viability. The Nb-treated cells exhibited decreased viability compared
with untreated cells, and no change was observed in the cell viability
between anti-PCT Nb-treated cells and untreated cells (Fig. 3C). When
OVCAR3 cells were treated with 5 nM of anti-HE4 Nbs, cell viability
decreased significantly with treatment time (Fig. 3D). Notably, 1G8
exhibited the strongest inhibitory effect on proliferation among the
three tested Nbs. The highest inhibitory rate was approximately 30%
when OVCAR3 cells were treated with 5 nM 1G8. Thus, we focused
primarily on 1G8 for the remaining studies.
3.5. Treatment with anti-HE4 Nbs induces apoptosis and cell cycle arrest
The effects of 1G8 on the induction of cell cycle arrest and apoptotic
cell death in OVCAR3 cells were analyzed by flow cytometry. Control
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Fig. 2. Characterization of the Nbs selected from the library. (A) Three anti-HE4 Nbs were identified (called 1G8, 3F10, and 4G8), with different amino acid se­
quences in the CDRs. Amino acids positions of the three CDRs were indicated according to the IMGT Scientific chart for the V-Domain. (B) SDS-PAGE analysis of the
purified Nbs. (C) The equilibrium dissociation constant (KD) between the HE4 antigen and Nbs was determined by the SPR technique. Nbs of different dilutions
(0.16–2.56 nM) were interacted with immobilized HE4 for each sensorgram.
Fig. 3. Cell viability assay and ELISA for HE4
secreted by OVCAR3 cells. The OVCAR3 cells were
cultured in medium with or without FBS for 24, 48,
and 72 h. (A) Cell viability was measured using the
CCK-8 assay and (B) the culture supernatant was
harvested to determine the concentration of HE4
using the HE4 ELISA kit. In the absence of FBS, the
viability of OVCAR3 cells (C) treated with various
concentrations (0.5, 5, 50, 500, 5000, and 50,000
pM) of Nbs or (D) with the same concentration of Nbs
for different times was determined. Data are
expressed as mean ± SD of three independent
experiments.
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experiments were carried out using PBS-treated cells as the untreated
group and anti-PCT Nb-treated cells as the negative group. After 72 h of
treatment with 5 nM 1G8, the population of OVCAR3 cells in the G2/M
phase was more than double compared to the control group (Fig. 4A).
Furthermore, the cell apoptosis assay was performed by double staining
with Annexin V-FITC and PI. Early apoptotic cells stained positively with
Annexin V-FITC only, and late apoptotic cells stained positively with
Annexin-V-FITC and PI. As shown in Fig. 4B, 1G8 significantly increased
the percentage of the apoptotic population at the early apoptotic
(Annexin V+/PI− ) and late apoptotic phases (Annexin V+/PI+). These
results suggest that the inhibitory effect of 1G8 on OVCAR3 cell prolif­
eration was due to cell cycle arrest and the induction of apoptosis.
3.7. Anti-HE4 Nb enhances the cisplatin sensitivity of OVCAR3 cells
Based on our finding of the tumor-suppressing effect of 1G8, we
further examined whether 1G8 mediated the chemosensitivity of
OVCAR3 cells. In the presence or absence of 1G8 (5 nM), OVCAR3 cells
were treated with up to 8 μg/mL cisplatin to determine the 48-h IC50 by
the CCK-8 assay. Cell viability decreased in the co-treatment group
compared to the group treated with cisplatin alone (Fig. 6A). The IC50 of
cisplatin in the co-treatment group was 1.23 μg/mL, which was nearly
20% less than the control group (Fig. 6B), demonstrating that 1G8
decreased cisplatin resistance in OVCAR3 cells.
4. Discussion
3.6. Treatment with anti-HE4 Nbs initiates the caspase-dependent
apoptosis pathway
HE4 is secreted by cancer cells in EOC patients and plays a critical
role in the progression of EOC. Previous studies have suggested that
overexpression and knockout of the HE4 gene affect cell proliferation
and tumor growth [7–14], but less is known about the function of the
secretory HE4 protein. Ovarian cancer cells are generally cultured in
serum-containing medium and proliferate dependent on a large number
of factors, such as hormones, lipids, and growth factors, in the serum.
Thus, the HE4 concentration is maintained at a low level, which ham­
pers research on HE4. Many researchers have used genetic engineering
techniques to increase HE4 expression to a pathological concentration to
study HE4. A recombinant HE4 protein was used to treat ovarian cancer
cells and investigate the effect of extracellular HE4 protein, but
controversial results were obtained due to the difference in cell status.
The recombinant HE4 protein does not affect cell viability or chemo­
resistance of ovarian cancer cells continuously cultured in serumcontaining medium [12,32]. Similarly, we found no change in the
viability of OVCAR3 cells treated with the anti-HE4 antibody in serumcontaining medium (data not shown). However, ovarian cancer cells
treated with recombinant HE4 exhibited higher viability after 12–24 h
To investigate the mechanism of 1G8-induced apoptosis in OVCAR3
cells, the apoptosis-related biomarkers were determined by western blot
analysis. Poly ADP ribose polymerase (PARP) is the substrate of cysteine
protease 3 (caspase 3), which is the core apoptotic executioner. Cleaved
PARP increased significantly in OVCAR3 cells treated with 5 nM 1G8 for
72 h (Fig. 5), indicating activation of caspase 3. Although cleaved PARP
also appeared in the control groups, this was largely due to the pro­
longed serum starvation leading to cell apoptosis. 1G8 accelerated cell
apoptosis and no full PARP appeared in the treatment group. In addition,
1G8 caused a significant increase in pro-apoptotic BAX and a decrease in
anti-apoptotic Bcl-2. The BAX/Bcl-2 ratio increased in 1G8-treated cells
compared to the control group. P53 is a tumor suppressor protein that
mediates a variety of anti-proliferative processes through cell cycle
checkpoints and apoptosis [31]. In this study, p53 expression was
upregulated following the 1G8 treatment (Fig. 5).
Fig. 4. Cell cycle and apoptosis assay. OVCAR3 cells were treated with 5 nM 1G8 for 72 h. Control experiments were carried out using 5 nM of the anti-PCT
nanobody as the control group and using PBS as the untreated group. (A) The cells were stained with PI, and DNA content for the cell cycle distribution analysis
was measured by flow cytometry. (B) Apoptotic cell death was measured by flow cytometry after staining with Annexin V-FITC and PI. Data are mean ± SD of three
experiments. *, p < 0.05.
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International Journal of Biological Macromolecules 199 (2022) 298–306
Fig. 5. Western blot analysis of the cell proteins in the different groups. (A) Representative western blot analysis of whole-cell lysates. (B) Quantification of the
corresponding blots by densitometry. Data are mean ± SD of three replicate experiments and are expressed as relative intensity units. *, p < 0.05.
Fig. 6. IC50 of cisplatin in OVCAR3 cells. OVCAR3 cells were treated with 0–8 μg/mL of cisplatin in the presence or absence of 1G8 (5 nM) for 48 h. (A) Cell viability
was measured by the CCK-8 assay, and the values are expressed relative to the cell viability values in untreated cells normalized to 100%. (B) IC50 values were
calculated using Prism 5.0 software. Data are mean ± SD of three independent experiments. *, p < 0.05.
of serum starvation [8,33]. Serum starvation could induce a cellprotective stress response, which has been ignored by previous
studies. In the current study, serum starvation stimulated OVCAR3 cells
to secrete more native HE4, which may have produced a superposition
or competitive effect on the action of recombinant HE4. In addition,
there is no unified standard for the biological activities of recombinant
HE4 in different experiments, which may have caused discrepancies in
the results. Thus, native HE4 secreted by ovarian cancer cells is the best
choice to investigate the functions of the HE4 protein and explore its
potential as a therapeutic target. In the present study, OVCAR3 cells
displayed increased secretion of HE4 while maintaining activity in the
absence of serum for 72 h, which provided an ideal cell culture model for
research on native HE4 secreted by cancer cells. Serum is vitally
important as a source of growth factors when culturing cells in basal
media. Thus, the cell culture model highlighted the roles of secretory
HE4 in cancer cell biology by eliminating interference from serum in the
growth medium.
The serum concentration is low around the tumor at the early clinical
stage, and a variety of proteins secreted by cancer cells are involved in
establishing the tumor microenvironment for tumor cell proliferation,
adhesion, motility, and intercellular communication [34]. HE4 is
secreted by ovarian cancer cells of patients and has been accepted as a
biomarker for the early stage of ovarian cancer. Thus, knowledge of the
secretory HE4 protein in the tumor microenvironment is important for
studies on ovarian cancer cell biology. In this study, we used anti-HE4
Nbs to neutralize secretory HE4, which resulted in the loss of auto­
crine function of the HE4 protein. After blocking the binding activity of
autocrine HE4, the viability of OVCAR3 cells decreased and cell
apoptosis occurred in the conditioned medium. The western blot assay
showed that the anti-HE4 Nb 1G8 increased BAX expression but
decreased Bcl-2 expression. These factors initiated the mitochondrial
apoptosis pathway and evoked upregulation of activated caspase-3,
increasing cleaved PARP (Fig. 6). These data suggest that secretory
HE4 inhibited apoptosis to protect ovarian cancer cells from death.
A growing body of evidence has demonstrated that HE4 is closely
related to the poor prognosis and recurrence of EOC caused by cisplatin
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International Journal of Biological Macromolecules 199 (2022) 298–306
J. Yu et al.
resistance [35–37]. Cisplatin primarily induces cell death by apoptosis,
and a defect in apoptotic signaling also confers cisplatin resistance [38].
Our data show that cell apoptosis was inhibited by secretory HE4, which
may have promoted collateral resistance to cisplatin in OVCAR3 cells.
Furthermore, p53 expression was markedly upregulated after the antiHE4 Nb treatment in the present study. The increased p53 expression
optimizes cisplatin treatment efficacy by inducing apoptosis in various
cancer cell types [39,40]. Thus, we validated the synergistic effect be­
tween the anti-HE4 Nb 1G8 and cisplatin in serum-containing medium
and found that 1G8 improved the cisplatin response of OVCAR3 cells.
Improving cisplatin sensitivity is necessary for ovarian cancer treatment,
as it could reduce the cisplatin dosage and minimize serious adverse
drug reactions, such as nephrotoxicity, emesis, and renal impairment
[39]. Cisplatin combination therapy is more effective than single-agent
therapies for treating EOC. Nbs have more advantages as biological
drugs in combination therapy compared to chemical drugs because they
have less possibility of causing drug resistance and fewer side effects due
to specific binding to the target. Considering their good tumor pene­
tration and lower immunogenicity, Nbs are expected to outperform
monoclonal antibodies. Previous studies proposed that HE4 could be a
potential gene therapy target, but there are still many challenges when
applying gene silencing technology in clinical practice, such as targeted
transport and security [41,42]. Our findings suggest that secretory HE4
is a druggable protein and that anti-HE4 Nbs were more suitable for
developing a simple and practical strategy for treating EOC. Further
studies are required to investigate the inhibitory effect of 1G8 on other
ovarian cancer cells or the synergistic inhibitory effect of 1G8 with other
commonly used drugs in vivo.
Taken together, we report for the first time that OVCAR3 cells
secreted a large amount of HE4 into the extracellular microenvironment
in conditioned medium without serum. The secretory HE4 protein was
effectively neutralized by anti-HE4 Nbs generated from a phage display
library, resulting in OVCAR3 cell apoptosis and an enhanced chemo­
therapy response to cisplatin. Our data show that the anti-HE4 Nb 1G8
was a potential chemosensitizer and could provide a potential new
strategy for treating EOC.
[2] C. Zhang, H. Hu, X. Wang, Y. Zhu, M. Jiang, W.F.D.C. Protein, A promising
diagnosis biomarker of ovarian cancer, J. Cancer 12 (18) (2021) 5404–5412,
https://doi.org/10.7150/jca.57880.
[3] G. Scaletta, F. Plotti, D. Luvero, S. Capriglione, R. Montera, A. Miranda, S. Lopez,
C. Terranova, C.D.C. Nardone, R. Angioli, The role of novel biomarker HE4 in the
diagnosis, prognosis and follow-up of ovarian cancer: a systematic review, Expert.
Rev. Anticancer. Ther. 17 (9) (2017) 827–839, https://doi.org/10.1080/
14737140.2017.1360138.
[4] C. Zhu, N. Zhang, A. Zhong, K. Xiao, R. Lu, L. Guo, A combined strategy of TK1,
HE4 and CA125 shows better diagnostic performance than risk of ovarian
malignancy algorithm (ROMA) in ovarian carcinoma, Clin. Chim. Acta 524 (2022)
43–50, https://doi.org/10.1016/j.cca.2021.11.018.
[5] E.C. Kight, I. Hussain, A.K. Bowden, F.R. Haselton, Recurrence monitoring for
ovarian cancer using a cell phone-integrated paper device to measure the ovarian
cancer biomarker HE4/CRE ratio in urine, Sci. Rep. 11 (1) (2021) 21945, https://
doi.org/10.1038/s41598-021-01544-4.
[6] V. Dochez, H. Caillon, E. Vaucel, J. Dimet, N. Winer, G. Ducarme, Biomarkers and
algorithms for diagnosis of ovarian cancer: CA125, HE4, RMI and ROMA, a review,
J. Ovarian Res. 12 (1) (2019) 28, https://doi.org/10.1186/s13048-019-0503-7.
[7] A. Wang, C. Jin, X. Tian, Y. Wang, H. Li, Knockdown of HE4 suppresses aggressive
cell growth and malignant progression of ovarian cancer by inhibiting the JAK/
STAT3 pathway, Biol. Open 8 (9) (2019) bio043570, https://doi.org/10.1242/
bio.043570.
[8] H. Wang, L. Zhu, J. Gao, Z. Hu, B. Lin, Promotive role of recombinant HE4 protein
in proliferation and carboplatin resistance in ovarian cancer cells, Oncol. Rep. 33
(1) (2015) 403–412, https://doi.org/10.3892/or.2014.3549.
[9] L. Zhu, H. Zhuang, H. Wang, M. Tan, C.L. Schwab, L. Deng, J. Gao, Y. Hao, X. Li,
S. Gao, J. Liu, B. Lin, Overexpression of HE4 (human epididymis protein 4)
enhances proliferation, invasion and metastasis of ovarian cancer, Oncotarget 7 (1)
(2016) 729–744, https://doi.org/10.18632/oncotarget.6327.
[10] Y.F. Zhu, G.L. Gao, S.B. Tang, Z.D. Zhang, Q.S. Huang, Effect of WFDC 2 silencing
on the proliferation, motility and invasion of human serous ovarian cancer cells in
vitro, Asian Pac. J. Trop. Med. 6 (4) (2013) 265–272, https://doi.org/10.1016/
S1995-7645(13)60055-3.
[11] R.G. Moore, E.K. Hill, T. Horan, N. Yano, K. Kim, S. MacLaughlan, G. LambertMesserlian, Y.D. Tseng, J.F. Padbury, M.C. Miller, HE4 (WFDC2) gene
overexpression promotes ovarian tumor growth, Sci. Rep. 4 (2014) 3574, https://
doi.org/10.1038/srep03574.
[12] J.R. Ribeiro, C. Schorl, N. Yano, N. Romano, K.K. Kim, R.K. Singh, R.G. Moore, HE4
promotes collateral resistance to cisplatin and paclitaxel in ovarian cancer cells,
J. Ovarian Res. 9 (1) (2016) 28, https://doi.org/10.1186/s13048-016-0240-0.
[13] S. Lee, S. Choi, Y. Lee, D. Chung, S. Hong, N. Park, Role of human epididymis
protein 4 in chemoresistance and prognosis of epithelial ovarian cancer, J. Obstet.
Gynaecol. Res. 43 (1) (2017) 220–227, https://doi.org/10.1111/jog.13181.
[14] Q. Liu, D.W. Liu, M.J. Zheng, L. Deng, H.M. Wang, S. Jin, J.J. Liu, Y.Y. Hao, L.
C. Zhu, B. Lin, Human epididymis protein 4 promotes P-glycoprotein-mediated
chemoresistance in ovarian cancer cells through interactions with annexin II, Mol.
Med. Rep. 24 (1) (2021) 496, https://doi.org/10.3892/mmr.2021.12135.
[15] H. Zhuang, M. Tan, J. Liu, Z. Hu, D. Liu, J. Gao, L. Zhu, B. Lin, Human epididymis
protein 4 in association with annexin II promotes invasion and metastasis of
ovarian cancer cells, Mol. Cancer 13 (2014) 243, https://doi.org/10.1186/14764598-13-243.
[16] J. Wang, L. Deng, H. Zhuang, J. Liu, D. Liu, X. Li, S. Jin, L. Zhu, H. Wang, B. Lin,
Interaction of HE4 and ANXA2 exists in various malignant
cells—HE4–ANXA2–MMP2 protein complex promotes cell migration, Cancer Cell
Int. 19 (2019) 161, https://doi.org/10.1186/s12935-019-0864-4.
[17] S. Muyldermans, Nanobodies: natural single-domain antibodies, Annu. Rev.
Biochem. 82 (2013) 775–797, https://doi.org/10.1146/annurev-biochem-063011092449.
[18] S. Muyldermans, Applications of nanobodies, Annu. Rev. Anim. Biosci. 9 (2021)
401–421, https://doi.org/10.1146/annurev-animal-021419-083831.
[19] A. Noor, G. Walser, M. Wesseling, P. Giron, A.M. Laffra, F. Haddouchi, J.D. Grève,
P. Kronenberger, Production of a mono-biotinylated EGFR nanobody in the E. Coli
periplasm using the pET22b vector, BMC res, Notes 11 (1) (2018) 751, https://doi.
org/10.1186/s13104-018-3852-1.
[20] Q. Chen, Y. Zhou, J. Yu, W. Liu, F. Li, M. Xian, R. Nian, H. Song, D. Feng, An
efficient constitutive expression system for anti-CEACAM5 nanobody production in
the yeast Pichia pastoris, Protein Expr. Purif. 155 (2019) 43–47, https://doi.org/
10.1016/j.pep.2018.11.001.
[21] T. Iwaki, K. Hara, K. Umemura, Nanobody production can be simplified by direct
secretion from Escherichia coli, Protein Expr. Purif. 170 (2020), 105607, https://
doi.org/10.1016/j.pep.2020.105607.
[22] W. Liu, H. Song, Q. Chen, J. Yu, M. Xian, R. Nian, D. Feng, Recent advances in the
selection and identification of antigen-specific nanobodies, Mol. Immunol. 96
(2018) 37–47, https://doi.org/10.1016/j.molimm.2018.02.012.
[23] P.S.L. Santos, P. Quintero-Campos, S. Morais, C. Echaides, Á. Maquieira,
G. Lassabe, G. Gonzalez-Sapienza, Bispecific single-domain antibodies as highly
standardized synthetic calibrators for immunodiagnosis, Anal. Chem. (2021),
https://doi.org/10.1021/acs.analchem.1c04603.
[24] P. Koenig, H. Das, H. Liu, B.M. Kümmerer, F.N. Gohr, L. Jenster, L.D.J. Schiffelers,
Y.M. Tesfamariam, M. Uchima, J.D. Wuerth, K. Gatterdam, N. Ruetalo, M.
H. Christensen, C.I. Fandrey, S. Normann, J.M.P. Tödtmann, S. Pritzl, L. Hanke,
J. Boos, M. Yuan, X. Zhu, J.L. Schmid-Burgk, H. Kato, M. Schindler, I.A. Wilson,
M. Geyer, K.U. Ludwig, B.M. Hällberg, N.C. Wu, F.I. Schmidt, Structure-guided
multivalent nanobodies block SARS-CoV-2 infection and suppress mutational
CRediT authorship contribution statement
Jianli Yu: Conceptualization, data curation, methodology, and
writing of the original draft. Yang Guo: Methodology and review and
editing of the manuscript. Yi Gu: Data curation and visualization. Fei Li:
Data curation and visualization. Haipeng Song: Investigation and
formal analysis. Rui Nian: Investigation, resources, and review and
editing of the manuscript. Xiying Fan: Methodology and supervision.
Wenshuai Liu: Funding acquisition, project administration, and review
and editing of the manuscript.
Declaration of competing interest
The authors declare no conflict of interest.
Data availability
Data will be made available on request.
Acknowledgements
This work was supported by Shandong Energy Institute (SEI) [SEI
I202128] and QIBEBT and Dalian National Laboratory for Clean Energy
(DNL), CAS [QIBEBT ZZBS 201807].
References
[1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2020, CA Cancer J. Clin. 70 (1)
(2020) 7–30, https://doi.org/10.3322/caac.21590.
305
J. Yu et al.
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
International Journal of Biological Macromolecules 199 (2022) 298–306
[33] Y. Shen, Y. Wang, X. Jiang, L. Lu, C. Wang, W. Luo, Y. Zhang, P. Li, Z. Du, T. Dai,
Preparation and characterization of a high-affinity monoclonal antibody against
human epididymis protein-4, Protein Expr. Purif. 141 (2018) 44–51, https://doi.
org/10.1016/j.pep.2017.09.005.
[34] F. Mbeunkui, D.J.J. Jr, Cancer and the tumor microenvironment: a review of an
essential relationship, Cancer Chemother. Pharmacol. 63 (4) (2009) 571–582,
https://doi.org/10.1007/s00280-008-0881-9.
[35] V.D. Gonzalez, N. Samusik, T.J. Chen, E.S. Savig, N. Aghaeepour, D.A. Quigley, Y.
W. Huang, V. Giangarra, A.D. Borowsky, N.E. Hubbard, Commonly occurring cell
subsets in high-grade serous ovarian tumors identified by single-cell mass
cytometry, Cell Rep. 22 (2018) 1875–1888, https://doi.org/10.1016/j.
celrep.2018.01.053.
[36] R. Angioli, F. Plotti, S. Capriglione, A. Aloisi, R. Montera, D. Luvero, A. Miranda, E.
V. Cafa, P. Damiani, P. Benedetti-Panici, Can the preoperative HE4 level predict
optimal cytoreduction in patients with advanced ovarian carcinoma? Gynecol.
Oncol. 128 (2013) 579–583, https://doi.org/10.1016/j.ygyno.2012.11.040.
[37] C. Yuan, R. Li, S. Yan, B. Kong, Prognostic value of HE4 in patients with ovarian
cancer, Clin. Chem. Lab. Med. 56 (7) (2018) 1026–1034, https://doi.org/10.1515/
cclm-2017-1176.
[38] D. Shaloam, B.T. Paul, Cisplatin in cancer therapy: molecular mechanisms of
action, Eur. J. Pharmacol. 740 (2014) 364–378, https://doi.org/10.1016/j.
ejphar.2014.07.025.
[39] A. Leekha, B.S. Gurjar, A. Tyagi, M.A. Rizvi, A.K. Verma, Vitamin C in synergism
with cisplatin induces cell death in cervical cancer cells through altered redox
cycling and p53 upregulation, J. Cancer Res. Clin. Oncol. 142 (12) (2016)
2503–2514, https://doi.org/10.1007/s00432-016-2235-z.
[40] X. Wang, Y. Bao, Z. Dong, Q. Chen, H. Guo, C. Xiang, J. Shao, WP1130 attenuates
cisplatin resistance by decreasing P53 expression in non-small cell lung
carcinomas, Oncotarget 8 (30) (2017) 49033–49043, https://doi.org/10.18632/
oncotarget.16931.
[41] X.M. Angeula, K.A. High, Entering the modern era of gene therapy, Annu. Rev.
Med. 70 (2019) 273–288, https://doi.org/10.1146/annurev-med-012017-043332.
[42] H. Brody, Gene therapy, Nature 564 (7735) (2018) S5, https://doi.org/10.1038/
d41586-018-07639-9.
escape, Science 371 (6530) (2021) eabe6230, https://doi.org/10.1126/science.
abe6230.
S. Muyldermans, V.V. Smider, Distinct antibody species: structural differences
creating therapeutic opportunities, Curr. Opin. Immunol. 40 (2016) 7–13, https://
doi.org/10.1016/j.coi.2016.02.003.
K.E. Conrath, M. Lauwereys, M. Galleni, A. Matagne, J.M. Frère, J. Kinne, L. Wyns,
S. Muyldermans, Beta-lactamase inhibitors derived from single-domain antibody
fragments elicited in the camelidae, Antimicrob. Agents Chemother. 45 (10) (2001)
2807–2812, https://doi.org/10.1128/AAC.45.10.2807-2812.2001.
M.L.V. Hendrickx, A. DE Winter, K. Buelens, G. Compernolle, G. HassanzadehGhassabeh, S. Muyldermans, A. Gils, P.J. Declerck, TAFIa inhibiting nanobodies as
profibrinolytic tools and discovery of a new TAFIa conformation, J. Thromb.
Haemost. 9 (11) (2011) 2268–2277, https://doi.org/10.1111/j.15387836.2011.04495.x.
G. Hassaine, C. Deluz, L. Grasso, R. Wyss, M.B. Tol, R. Hovius, A. Graff,
H. Stahlberg, T. Tomizaki, A. Desmyter, C. Moreau, X.D. Li, F. Poitevin, H. Vogel,
H. Nury, X-ray structure of the mouse serotonin 5-HT3 receptor, Nature 512 (7514)
(2014) 276–281, https://doi.org/10.1038/nature13552.
E. Pardon, T. Laeremans, S. Triest, S.G.F. Rasmussen, A. Wohlkoenig, A. Ruf,
S. Muyldermans, W.G.J. Hol, B.K. Kobilka, J. Steyaert, A general protocol for the
generation of nanobodies for structural biology, Nat. Protoc. 9 (3) (2014) 674–693,
https://doi.org/10.1038/nprot.2014.039.
J. Lin, J. Yu, H. Wang, Y. Xu, F. Li, X. Chen, Y. Liang, J. Tang, L. Wu, Z. Zhou,
Development of a highly thermostable immunoassay based on a nanobody-alkaline
phosphatase fusion protein for carcinoembryonic antigen detection, Anal. Bioanal.
Chem. 412 (8) (2019) 1723–1728, https://doi.org/10.1007/s00216-020-02456-4.
X. Wang, E.R. Simpson, K.A. Brown, p53: protection against tumor growth beyond
effects on cell cycle and apoptosis, Cancer Res. 75 (23) (2015) 5001–5007, https://
doi.org/10.1158/0008-5472.CAN-15-0563.
X. Kong, X. Chang, H. Cheng, R. Ma, X. Ye, H. Cui, Human epididymis protein 4
inhibits proliferation of human ovarian cancer cells via the mitogen-activated
protein kinase and phosphoinositide 3-kinase/AKT pathways, Int. J. Gynecol.
Cancer 24 (3) (2014) 427–436, https://doi.org/10.1097/IGC.0000000000000078.
306