Biochemistry and Biophysics Reports 25 (2021) 100902

Contents lists available at ScienceDirect

Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

Establishment of a novel anti-TROP2 monoclonal antibody TrMab-29 for
immunohistochemical analysis
Yusuke Sayama a, Mika K. Kaneko a, Junko Takei a, Hideki Hosono a, Masato Sano a,
Teizo Asano a, Yukinari Kato a, b, *ab

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
TROP2
CBIS method
Monoclonal antibody
Breast cancer

TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overex­pressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop
a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, andimmunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immu­
nized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary(CHO)–K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2overexpressed CHO–K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO–K1 cells were selected using
flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse
IgG1 kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot
analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore,
TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may
be a valuable tool for the detection of TROP2 in cancer.

1. Introduction

immunization and screening [15]. CBIS has been employed to develop
sensitive and specific mAbs against many transmembrane proteins,
including CD19 [16], CD20 [17], CD44 [18], CD133 [15], PD-L1 [19],and podoplanin [20–26]. Importantly, those mAbs have proven to be
useful in flow cytometry, Western blot, and immunohistochemical an­
alyses. In this study, we developed a novel anti-TROP2 mAb (TrMab-29)
and evaluated its usefulness in flow cytometry, Western blot, andimmunohistochemical analyses.

The trophoblast cell-surface antigen (TROP2) is a type I trans­
membrane glycoprotein that was originally identified in humantrophoblast cells [1–3]. TROP2 is highly expressed in many cancers andmay play a critical role in tumor progression [4,5]. Because increased
TROP2 expression has been reported in more than 85% of all solidcancers, TROP2 may be a useful marker for cancer diagnosis andimmunotherapy [6–8]. It has also been identified in stem cells of various
tissues, including basal cells, all of which are capable of self-renewal,
regeneration, and differentiation [6,9,10]. Further, several monoclonal
antibodies (mAbs) against TROP2 are currently evaluated in clinicalcancer trials, including DS-1062a [4,11], PF-06664178 [4,12], and
IMMU-132 [4,13,14].
We previously established a Cell-Based Immunization and Screening(CBIS) method, in which cell lines are used exclusively for both

2. Materials and methods
2.1. Plasmid preparation
Human TROP2 DNA was synthesized commercially by Thermo
Fisher Scientific, Inc. (Waltham, MA, USA). TROP2 DNA with an Nterminal PA tag [27] and a C-terminal RAP tag [28]/MAP tag [29]Abbreviations: ADC, antibody-drug conjugates; ADCC, antibody-dependent cellular cytotoxicity; BSA, bovine serum albumin; CAR-T, chimeric antigen receptor Tcell; CBIS, Cell-Based Immunization and Screening; CDC, complement-dependent cytotoxicity; CHO, Chinese hamster ovary; DAB, 3,3′ -diaminobenzidine tetrahy­drochloride; mAb, monoclonal antibody; P3U1, P3X63Ag8U.1; PBS, phosphate-buffered saline; PIT, photoimmunotherapy; RIT, radioimmunotherapy; TROP2,
trophoblast cell-surface antigen; PVDF, polyvinylidene difluoride.
* Corresponding author. New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2020.100902
Received 25 July 2020; Received in revised form 8 December 2020; Accepted 27 December 2020
2405-5808/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Y. Sayama et al.

Biochemistry and Biophysics Reports 25 (2021) 100902(PA-TROP2-RAP-MAP) was subcloned into a pCAG-Ble expression vec­tor (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) using an
In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan) according to
the manufacturer’s instructions; the recombinant expression vector wasnamed pCAG/PA-TROP2-RAP-MAP. TROP2 DNA with a C-terminal PA
tag was also subcloned into a pCAG-Ble vector using an In-Fusion HD
Cloning Kit (Takara Bio, Inc.); this expression vector was named
pCAG/TROP2-PA. The amino acid sequences of each tag are as follows:
PA tag, consisting of 12 amino acids (GVAMPGAEDDVV); RAP tag, 12amino acids (DMVNPGLEDRIE); and MAP tag, 12 amino acids(GDGMVPPGIEDK).

Indianapolis, IN, USA), and the resulting hybridomas were selected in
RPMI 1640 medium containing hypoxanthine, aminopterin, andthymidine (Thermo Fisher Scientific, Inc.). Culture supernatants werescreened by flow cytometry. The isotype of mAbs were determined using
isotype-specific secondary antibodies (SouthernBiothech, Birmingham,AL).
2.4. Flow cytometry
Cells were collected following a brief exposure to 0.25% trypsin and
1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). The cells
were then washed with 0.1% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) and treated with primary mAbs or hybridoma
culture supernatants for 30 min at 4 ◦ C. After incubation, cells weretreated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell
Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data werecollected using SA3800 Cell Analyzer (Sony Corp.) and analyzed using
FlowJo (BD Biosciences, Franklin Lakes, NJ, USA).

2.2. Cell lines
MCF7 was obtained from the Cell Resource Center for Biomedical
Research, Institute of Development, Aging and Cancer, Tohoku Uni­
versity (Miyagi, Japan). Chinese hamster ovary (CHO)–K1,
P3X63Ag8U.1 (P3U1), Lec1, Lec2, and Lec8 cell lines were obtained
from the American Type Culture Collection (Manassas, VA, USA).
CHO–K1 cells overexpressing TROP2-PA (CHO/TROP2-PA) and PATROP2-RAP-MAP (CHO/PA-TROP2-RAP-MAP) were established bytransfecting pCAG/TROP2-PA and pCAG/PA-TROP2-RAP-MAP in
CHO–K1 cells, respectively, using Lipofectamine LTX Reagent (Thermo
Fisher Scientific, Inc.) according to the manufacturer’s protocol. The
transfected cells were selected by limiting dilution in the medium con­taining 0.5 mg/mL of zeocin (InvivoGen, San Diego, CA, USA). The
transfected cells were confirmed as TROP2-positive by flow cytometry(EC800, Sony Corp., Tokyo, Japan) using an anti-TROP2 antibody(Cat#LS-C489657, LS Bio, Seattle, WA, USA).
Lec1/TROP2, Lec2/TROP2, and Lec8/TROP2 were established bytransfecting pCAG/TROP2-PA to Lec1, Lec2, and Lec8 cells, respec­tively, using the Neon Transfection System (Thermo Fisher Scientific,Inc.). Stable transfectants were established by cell sorting using SH800(Sony Corp.), and were cultivated in the medium containing 0.5 mg/mLof zeocin (InvivoGen).
The TROP2-knockout cell line, MCF7/TROP2-KO (BINDS-29), wasestablished by transfecting CRISPR/Cas9 plasmids for TROP2 into MCF7
cells using the Neon Transfection System (Thermo Fisher Scientific,Inc.). Stable transfectants were established by cell sorting using SH800(Sony Corp.).
CHO–K1, CHO/TROP2-PA, CHO/PA-TROP2-RAP-MAP, P3U1,
MCF7, Lec1/TROP2, Lec2/TROP2, Lec8/TROP2, and BINDS-29 cells
were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium(Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/
mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B(Nacalai Tesque, Inc.). Cells were grown in a humidified incubator at
37 ◦ C with 5% CO2.

2.5. Western blot analysis
Cell lysates (10 μg) were boiled in sodium dodecyl sulfate (SDS)
sample buffer (Nacalai Tesque, Inc.). Proteins were separated on 5%–
20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation)and transferred onto polyvinylidene difluoride (PVDF) membranes(Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.) in PBS with 0.05% Tween 20, membranes wereincubated with 5 μg/mL TrMab-29, 1 μg/mL NZ-1 (an anti-PA tag mAb;
rat IgG2a, lambda) [27,30], or 1 μg/mL anti-β-actin (clone AC-15; Sig­
ma-Aldrich Corp., St. Louis, MO). Membranes were then incubated witheither peroxidase-conjugated anti-mouse immunoglobulins (diluted
1:1000; Agilent Technologies, Inc., Santa Clara, CA, USA) to detect
TrMab-29 and anti-β-actin, or with an anti-rat IgG (diluted 1:10,000;
Sigma-Aldrich Corp.) to detect NZ-1. Finally, protein bands weredetected with a chemiluminescence reagent, ImmunoStar LD (FUJIFILM
Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd.,Tokyo, Japan).
2.6. Immunohistochemical analysis
Paraffin-embedded tissue sections of a breast cancer tissue array(Cat#T8235721-5, Lot#B104066; BioChain, San Francisco, CA, USA)
were autoclaved in EnVision FLEX Target Retrieval Solution High pH(Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock
T20 (Thermo Fisher Scientific, Inc.), tissue sections were incubated with
TrMab-29 (5 μg/mL) for 1 h at room temperature and then treated with
the EnVision + Kit for mouse (Agilent Technologies Inc.) for 30 min.
Color was developed using 3,3′ -diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies Inc.) for 2 min. Counterstaining was per­formed with hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
Hematoxylin & eosin (HE) staining (FUJIFILM Wako Pure Chemical
Corporation) was performed using consecutive tissue sections. Leica
DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to
examine the sections and obtain images.

2.3. Hybridoma production
Female BALB/c mice (6 weeks old) were purchased from CLEA Japan(Tokyo, Japan) and kept under specific pathogen-free conditions. All
animal experiments were conducted in accordance with relevant
guidelines and regulations to minimize animal suffering and distress in
the laboratory. The Animal Care and Use Committee of Tohoku Uni­
versity approved all animal experiments performed in this study (Permit
number: 2019NiA-001). Mice were euthanized by cervical dislocation
under anesthesia, and death was verified by respiratory and cardiac
arrest. We employed CBIS to develop new mAbs against TROP2. Twomice were immunized with CHO/PA-TROP2-RAP-MAP cells (1 × 108
cells/mouse) via intraperitoneal (i.p.) injection with Imject Alum
(Thermo Fisher Scientific, Inc.). After several additional immunizations,a booster immunization was administered by i. p. injection 2 days before
spleen cells were collected. The collected spleen cells were fused with
P3U1 cells using polyethylene glycol 1500 (Roche Diagnostics,

3. Results
3.1. Establishment of anti-TROP2 mAbs
Two mice were immunized with CHO/PA-TROP2-RAP-MAP cells todevelop anti-TROP2 mAbs. Hybridomas showing strong signals from
CHO/TROP2-PA cells and weak-to-no signals from CHO–K1 cells wereselected using flow cytometry. After additional Western blot andimmunohistochemical analyses, we established a novel anti-TROP2
mAb, TrMab-29 (mouse IgG1 kappa). Hybridoma cells of TrMab-29
could stably grow and secret mAbs using serum-free medium.
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Biochemistry and Biophysics Reports 25 (2021) 100902

Fig. 1. TROP2 detection by TrMab-29 using flow
cytometry and Western blot. (A) CHO–K1, CHO/
TROP2-PA, MCF7, and BINDS-29 (MCF7/TROP2KO) cells were incubated with TrMab-29 (1 μg/mL;
red line) or 0.1% BSA in PBS (gray line) for 30 min,followed by incubation with Alexa Fluor 488-conju­
gated secondary antibodies. Fluorescence data werecollected using a cell analyzer. (B) Detection of
TROP2 with TrMab-29 by Western blot analysis. Cell
lysates (10 μg) of CHO–K1, CHO/TROP2-PA, MCF7,
and BINDS-29 were subjected to SDS-PAGE andtransferred to PVDF membranes. Membranes werethen incubated with 5 μg/mL of TrMab-29, 1 μg/mLof anti-PA (NZ-1), and 1 μg/mL of anti-β-actin (AC15), followed by incubation with secondary anti­
bodies and detection.

3.2. Characterization of TrMab-29

deficient CHO cells, namely N-glycan-deficient Lec1, sialic aciddeficient Lec2, and galactose-deficient Lec8 cells. As shown in Supple­
mentary Fig. 1, we found that TrMab-29 reacted with Lec1/TROP2,
Lec2/TROP2, and Lec8/TROP2 cells similar to that found with CHO/
TROP2-PA cells, indicating that the TrMab-29 epitope is not associ­ated with glycans.
Further, we evaluated the usefulness of TrMab-29 for Western blot
analysis. We found a TrMab-29-positive 40 kDa band in lysates fromboth CHO/TROP2-PA and MCF7 cells (Fig. 1B). In contrast, no band wasobserved in lysates from CHO–K1 and TROP2-knockout MCF7 (BINDS-

We first investigated whether TrMab-29 can specifically detect
TROP2 by flow cytometry (Fig. 1A). We detected TROP2 in CHO/
TROP2-PA cells, but not parental CHO–K1 cells. TrMab-29 also detec­
ted endogenous TROP2 on MCF7, a human breast cancer cell line. In
contrast, we found that TrMab-29 did not react with TROP2-knockout
MCF7 cells (BINDS-29), indicating that TrMab-29 is specific for TROP2.
Next, we determined if the TrMab-29 epitope is associated withglycans. We performed flow cytometry using TROP2-transfected glycan3

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Biochemistry and Biophysics Reports 25 (2021) 100902

Fig. 2. Detection of TROP2 in breast cancer
specimens by immunohistochemical analysis with
TrMab-29. (A, B) Invasive ductal carcinoma tissue
sections from patients with breast cancer were incu­
bated with 5 μg/mL TrMab-29 and then treated with
the EnVision + Kit. Tissues were counterstained withhematoxylin. (C, D) Hematoxylin and eosin (H&E)
staining was performed using consecutive invasive
ductal carcinoma tissue sections. (E, F) Adenocarci­
noma tissue sections from patients with breast cancer
were incubated with 5 μg/mL TrMab-29, followed bytreatment with EnVision + Kit. Counterstaining was
performed with hematoxylin. (G, H) H&E staining
was performed using consecutive adenocarcinoma
tissue sections. Scale bar = 100 μm.

29) cells, indicating that TrMab-29 specifically detected both exogenousand endogenous TROP2 in Western blot. An anti-PA tag mAb (NZ-1) also
detected a 40-kDa band in the lysates of CHO/TROP2-PA cells.
Finally, we investigated whether TrMab-29 is suitable for immuno­
histochemical analysis. As shown in Fig. 2, we found a TrMab-29positive membrane-staining pattern in invasive ductal carcinoma(Fig. 2A and B) and adenocarcinoma (Fig. 2E and F) tissue sections frompatients with breast cancer. Hematoxylin and eosin (H&E) staining ofinvasive ductal carcinoma sections (Fig. 2C and D) and adenocarcinoma
sections (Fig. 2G and H) are also presented for reference. These findings
demonstrate that TrMab-29 is well suited for immunohistochemical
analysis using paraffin-embedded tissue sections.

4. Discussion
Using the CBIS method, in which antigen-expressing cell lines areused for both immunization and screening [15], we have developed
many useful mAbs against membrane proteins, including CD19 [16],CD20 [17], CD44 [18], CD133 [15], and PD-L1 [19], for research ap­
plications. Of these mAbs, we recently developed clone C20Mab-11 toreliably detect CD20 by flow cytometry, Western blot, and immuno­
histochemical analysis [17]. CD20 has four membrane-spanning do­mains and only two small extracellular domains that include aminoacids 72–80 and 142–182 [31,32]. Although there are several
commercially available mAbs that interact with amino acids 142–182 ofCD20 and are specifically useful in flow cytometry, there are no avail­able anti-CD20 mAbs that are effective not only in flow cytometry but
also in Western blot and immunohistochemical analyses [17], indicating
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Biochemistry and Biophysics Reports 25 (2021) 100902

that establishing multipurpose mAbs remains difficult. In this study, we
aimed to establish a multipurpose anti-TROP2 mAb using the CBIS
method. To that end, we successfully developed a sensitive and specificnovel anti-TROP2 mAb (TrMab-29), which can be used in different
research applications, such as flow cytometry (Fig. 1A), Western blot
(Fig. 1B), and immunohistochemical analyses (Fig. 2).
As a next step, it would be of interest to determine whether TrMab-29
is suitable for use in targeted molecular therapy against TROP2expressing cancers. Among mouse immunoglobulins, IgG2a and IgG2b
can induce antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) [33,34]. As TrMab-29 was
determined to belong to the mouse IgG1 subclass, which does not exert
ADCC/CDC, it should be changed to the IgG2a or IgG2b subclass.
Recently, we have focused on various modalities that can be used topromote antibody therapy, including antibody-drug conjugates (ADCs),
radioimmunotherapy (RIT), photoimmunotherapy (PIT), and chimericantigen receptor T-cell (CAR-T) therapy. ADCs include a mAb, a linker,
and a “payload,” providing a means in which a cytotoxic drug can bedelivered directly to cancer tissues via antibody targeting [35]. RIT is amodality permitting selective internal radiation therapy using radio­
labeled mAbs against cancer-associated antigens [36], whereas
near-infrared PIT (NIR-PIT) is a novel cancer therapy that combines the
specificity of mAbs with directed toxicity induced by a photoabsorber
following exposure to NIR light [37]. Alternatively, CAR-T therapy en­
ables T-cells to detect predefined surface antigens on cancer cells inde­
pendent of major histocompatibility restriction [38]. Following theestablishment and characterization of our novel anti-TROP2 mAb
described in the current study, we will investigate different treatment
modalities, including ADC, RIT, PIT, and CAR-T-cell therapy, to explore
TrMab-29-mediated antitumor activities in TROP2-expressing cancers.

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Funding
This research was supported in part by AMED [grant numbers:
JP20am0401013, JP20am0101078, JP20ae0101028] (Y⋅K.).
Author contributions
Y.S: performed the experiments. M.K.K: designed the experiments. Y.
K: designed the experiments. J.T: Formal analysis, analyzed the data. H.
H: Formal analysis, analyzed the data. M.S: Formal analysis, analyzed
the data. T.A: Formal analysis, analyzed the data. Y.S: Writing – originaldraft, wrote the manuscript. Y.K: Writing – original draft, wrote themanuscript.
Declaration of competing interest
The authors declare no conflicts of interest involving this article.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbrep.2020.100902.
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