MOLECULAR MEDICINE REPORTS 23: 92, 2021

Development and characterization of TrMab‑6,
a novel anti‑TROP2 monoclonal antibody for
antigen detection in breast cancer
YUSUKE SAYAMA1, MIKA K. KANEKO1 and YUKINARI KATO1,2
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine;
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980‑8575, Japan

2

Received June 26, 2020; Accepted September 15, 2020
DOI: 10.3892/mmr.2020.11731
Abstract. Trophoblast cell‑surface antigen 2 (TROP2) isa type I transmembrane glycoprotein that is overexpressedin a number of cancer types, including triple‑negative
breast cancer. The current study aimed to develop ahighly sensitive and specific monoclonal antibody (mAb)targeting TROP2, which could be used to evaluate TROP2
expression using flow cytometry, western blot analysis
and immunohistochemistry by employing the Cell‑Based
Immunization and Screening (CBIS) method. The estab‑lished anti‑TROP2 mAb, TrMab‑6 (mouse IgG 2b, κ),detected TROP2 on PA‑tagged TROP2‑overexpressing
Chinese hamster ovary‑K1 (CHO/TROP2‑PA) and breast
cancer cell lines, including MCF7 and BT‑474 using flow
cytometry. Western blot analysis indicated a 40 kDa bandin lysates prepared from CHO/TROP2‑PA, MCF7 andBT‑474 cells. Furthermore, TROP2 in 57/61 (93.4%) of thebreast cancer specimens was strongly detected using immu‑
nohistochemical analysis with TrMab‑6. In conclusion, thecurrent study demonstrated that TrMab‑6 may be a valuable
tool for the detection of TROP2 in a wide variety of breast
cancer types.

Correspondence to: Professor Yukinari Kato, New Industry

Creation Hatchery Center, Tohoku University, 2‑1 Seiryo‑machi,
Aoba‑ku, Sendai, Miyagi 980‑8575, JapanE‑mail: yukinarikato@med.tohoku.ac.jp

Abbreviations: ADCC, antibody‑dependent cellular cytotoxicity;

CBIS, Cell‑Based Immunization and Screening; CHO, Chinese
hamster ovary; DMEM, Dulbecco's modified Eagle's medium;
EDTA, ethylenediaminetetraacetic acid; mAb, monoclonal antibody;
H&E, hematoxylin and eosin; RPMI, Roswell Park Memorial
Institute; TACTD2, tumor‑associated calcium signal transducer 2;
TROP2, trophoblast cell‑surface antigen 2

Key words: TROP2, monoclonal antibody, CBIS, breast cancer

Introduction
Breast cancer is by far the most common malignant tumor inwomen. In 2018, there were 2,088,849 new breast cases (11.6%of the total cancer cases) and 626,679 deaths (6.6% of the totalcancer deaths) worldwide (1). Surgery, radiotherapy, chemo‑
therapy, and molecular targeted therapies are currently used
for breast cancer treatment; however, effective therapies forpatients diagnosed with triple‑negative breast cancer [TNBC;
i.e., those that are negative for estrogen receptor (ER), proges‑
terone receptor (PR), and human epidermal growth factor
receptor 2 (HER2)] remain limited (2‑4). TNBC accounts
for ~15% of invasive breast cancers; moreover, it tends to beaggressive and is associated with a poor prognosis (2,5,6).
TNBC is more common in young women than in older womenand is frequently associated with invasion and metastatic
disease (2,5‑7). As such, highly sensitive and specific mono‑clonal antibodies (mAbs) are required to facilitate the diagnosisof and treatment decisions for this breast cancer subtype.
The trophoblast cell‑surface antigen (TROP2), also
known as human tumor‑associated calcium signal transducer(TACSTD2), is a type I transmembrane glycoprotein origi‑
nally identified in human trophoblast cells (8‑10). Previously,
Schon and Orfanos reported that tunicamycin treatment ofliving cells and N‑glycanase digestion of immunopurified
TROP2 revealed that the molecular heterogeneity of TROP2 isdue to the different N‑glycosylation in normal and transformed
keratinocytes (11). In transformed keratinocytes, two distinct
precursor proteins at 38 and 42 kDa were detected, whereas innormal cells the 38‑kDa signal was dramatically decreased,
indicating that quantitative and qualitative changes of N‑glycanof TROP2 are associated with the transformation process ofhuman keratinocytes. TROP2 is highly expressed in several
cancers and may play a critical role in tumor progression inassociation with the pathways involving both the extracellularsignal‑related kinase (ERK) and c‑Jun N‑terminal kinase(JNK) (12,13). The expression of TROP2 has been reported in
more than 85% of all tumors; as such, TROP2 may be a usefulmarker for cancer diagnosis and immunotherapy (2,14,15). It
has also been identified in the stem cells of various tissues,
including basal cells, all of which are capable of self‑renewal,
regeneration, and differentiation (2,16,17). Several mAbs

2

SAYAMA et al: DEVELOPMENT OF ANTI-TROP2 MAB

targeting TROP2 are currently evaluated in clinical trials,
including PF‑06664178 (12,18), IMMU‑132 (12,19,20), andDS‑1062a (12,21).
In our previous studies, we developed the Cell‑Based
Immunization and Screening (CBIS) method; in this method,cell lines are used exclusively for both immunization andscreening (22). CBIS has been employed to develop sensitiveand specific mAbs against numerous transmembrane proteins,
including CD19 (23), CD20 (24), CD44 (25), CD133 (22), and
PD‑L1 (26). Of note, mAbs developed using this method have
proven to be extremely useful in flow cytometry, Western blot,
and immunohistochemical analyses.
In this study, we developed novel anti‑TROP2 mAbs andevaluated their capacity to target breast cancer cells using flow
cytometry, Western blot, and immunohistochemical analyses.
Materials and methods
Plasmid preparation. Human TROP2 DNA was synthesizedcommercially by Thermo Fisher Scientific (Waltham, MA, USA).
TROP2 DNA with an N‑terminal PA16 tag (27) and a C‑terminal
RAP tag (28)/MAP tag (29) (PA16‑TROP2‑RAP‑MAP) was
subcloned into the pCAG‑Ble expression vector (FUJIFILM
Wako Pure Chemical Corporation) using an In‑Fusion HD
Cloning Kit (Takara Bio, Inc.); the recombinant expression
vector was named pCAG/PA16‑TROP2‑RAP‑MAP. TROP2
DNA with a C‑terminal PA tag (27) alone was also subcloned
into the pCAG‑Ble vector using an In‑Fusion HD Cloning
Kit; this expression vector was named pCAG/TROP2‑PA.
The amino acid sequences of each tag are as follows: PA16
tag, 16 amino acids (GLEGGVAMPGAEDDVV); PA tag,
12 amino acids (GVAMPGAEDDVV); RAP tag, 12 aminoacids (DMVNPGLEDRIE); and MAP tag, 12 amino acids(GDGMVPPGIEDK).
Cell lines. Chinese hamster ovary (CHO)‑K1, P3X63Ag8U.1(P3U1), BT‑474, Lec1, Lec2, and Lec8 cell lines were obtained
from the American Type Culture Collection (ATCC; Manassas,
VA, USA). MCF7 was obtained from the Cell Resource Center
for Biomedical Research, Institute of Development, Aging andCancer, Tohoku University (Miyagi, Japan).
C H O ‑ K1 c el l s t h a t ove r ex p r e s s T RO P 2 ‑ PA
(C H O/ T RO P 2 ‑ PA) a n d PA16 ‑T RO P 2 ‑ R A P‑ M A P(CHO/PA16‑TROP2‑RAP‑MAP) were generated by transfec‑
tion of pCAG/TROP2‑PA and pCAG/PA16‑TROP2‑RAP‑MAP
to CHO‑K1 cells, respectively, using Lipofectamine
LTX Reagent (Thermo Fisher Scientific, Inc.). Cell lines
Lec1/TROP2, Lec2/TROP2, and Lec8/TROP2 were gener‑
ated by transfection of pCAG/TROP2‑PA to Lec1, Lec2, andLec8 cells, respectively, using the Neon Transfection System(Thermo Fisher Scientific, Inc.). Several days after the transfec‑
tion, the transfected cells were confirmed as TROP2‑positiveby flow cytometry (EC800, Sony Corp.) using a commercial
anti‑TROP2 antibody (Cat#LS‑C489657, LS Bio). The trans‑
fected cells were selected by limiting dilution culture andcultivation in the medium containing 0.5 mg/ml of zeocin(InvivoGen). We confirmed the transfection efficiency using
western blotting.
The TROP2 gene‑deleted cell line, MCF7/TROP2‑KO(BINDS‑29), was generated by transfection of CRISPR/Cas9

plasmids targeting TROP2 using the Neon Transfection
System (Thermo Fisher Scientific, Inc.). Stable transfectants
were established by cell sorting using SH800 (Sony Corp.).
CHO‑K1,CHO/PA16‑TROP2‑RAP‑MAP, CHO/TROP2‑PA,
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); BT‑474 cells were cultured in Dulbecco's modified
Eagle's medium (DMEM; Nacalai Tesque, Inc.). All media
were supplemented with 10% heat‑inactivated fetal bovine
serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/ml peni‑
cillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B(Nacalai Tesque, Inc.). Cells were grown in an incubator
at 37˚C with humidity and 5% CO2 and 95% air atmosphere.
Animals. Female BALB/c mice (6 weeks old) were purchasedfrom CLEA Japan and kept under specific pathogen‑freeconditions. All animal experiments were conducted in accor‑
dance with the relevant guidelines and regulations in orderto minimize animal suffering and distress in the laboratory.
The Animal Care and Use Committee of Tohoku University
approved all the animal experiments (permit no. 2019NiA‑001).
Mice were euthanized by cervical dislocation under inhalation
anesthesia using 2% of isoflurane, and the death was verified
to be respiratory and cardiac arrest.
Hybridoma production. We employed CBIS to develop
new mAbs against TROP2. Two mice were immunizedwith CHO/PA16‑TROP2‑RAP‑MAP cells (1x10 8) via theintraperitoneal route (i.p.) together with the Imject Alum
(Thermo Fisher Scientific, Inc.). After several additional
immunizations, a booster immunization was administered
via the i.p. route 2 days before spleen cell collection. Mice
were euthanized by cervical dislocation under inhalation
anesthesia using isoflurane, and the death was verified tobe respiratory and cardiac arrest. We chopped spleens, andcollected spleen cells using serum‑free RPIM‑1640 medium.
We further broke the red blood cells with 3 ml of Red Blood
Cell Lysing Buffer Hybri‑Max (Sigma‑Aldrich Corp.) at 37˚C
for 1 min, and washed the spleen cells using serum‑free
RPIM‑1640 medium. The collected spleen cells were fusedwith P3U1 mouse myeloma cells using polyethylene glycol
1500 (Roche Diagnostics) (30,31); the resulting hybridomas
were selected in RPMI medium, including hypoxanthine,aminopterin, and thymidine (Thermo Fisher Scientific, Inc.).
The culture supernatants were screened via flow cytometry
using CHO/TROP2‑PA and CHO‑K1 cells.
Flow cytometry. Cells were collected following a brief expo‑
sure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic
acid (EDTA; Nacalai Tesque, Inc.). The cells were washedwith 0.1% bovine serum albumin in phosphate‑buffered saline(PBS) and treated with anti‑TROP2 mAbs, such as TrMab‑6(1 µg/ml) or EPR20043 (1/60 dilution; Abcam) for 30 min
at 4˚C. After incubation, the cells were treated with Alexa
Fluor 488‑conjugated anti‑mouse IgG (1:1,000; Cell Signaling
Technology, Inc.) or Alexa Fluor 488‑conjugated anti‑rabbit
IgG (1:1,000; Cell Signaling Technology, Inc.). Fluorescencedata were collected using SA3800 Spectral Cell Analyzer(Sony Corp.) and analyzed using FlowJo (BD Biosciences).

MOLECULAR MEDICINE REPORTS 23: 92, 2021

3

Determination of the binding affinity. MCF7 or BT‑474 cells(2x105) were suspended in 100 µg of serially diluted TrMab‑6(6 ng/ml‑100 µg/ml) for 30 min at 4˚C, followed by the addi‑
tion of Alexa Flour 488‑conjugated anti‑mouse IgG (1:200;
Cell Signaling Technologies, Inc.). Fluorescence data werecollected using a cell analyzer (EC800). The dissociation
constant (KD) was calculated by fitting the binding isotherms
to built‑in, one‑site binding models in GraphPad Prism 8(GraphPad Software, Inc.).
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 trans‑ferred onto polyvinylidene difluoride (PVDF) membranes(Merck KGaA). After blocking with 4% skim milk (Nacalai
Tesque, Inc.) in PBS with 0.05% Tween‑20, the membranes
were incubated with 1 or 5 µg/ml of TrMab‑6, 1/2000 dilu‑
tion of EPR20043 (Abcam), 1 µg/ml of NZ‑1 (anti‑PA tag), or
1 µg/ml of anti‑β‑actin (clone AC‑15; Sigma‑Aldrich Corp.).
This was followed by incubation with peroxidase‑conjugatedanti‑mouse immunoglobulins (Agilent Technologies
Inc.; diluted 1:1,000) to detect TrMab‑6 and anti‑ β ‑actin,
peroxidase‑conjugated anti‑rabbit immunoglobulins (Agilent
Technologies Inc.; diluted 1:1,000) to detect EPR20043, oranti‑rat IgG (Sigma‑Aldrich Corp; diluted 1:10,000) to detect
NZ‑1, respectively. Finally, protein bands were detectedwith ImmunoStar LD (FUJIFILM Wako Pure Chemical
Corporation) using a Sayaca‑Imager (DRC Co. Ltd.).
Immunohistochemical analysis. Paraffin‑embedded tissue
sections of the breast cancer tissue array (Cat#T8235721‑5,
Lot#B104066; BioChain, San Francisco, CA, USA) wereautoclaved in EnVision FLEX Target Retrieval Solution HighpH (Agilent Technologies, Inc.) for 20 min. After blocking
with SuperBlock T20 (Thermo Fisher Scientific, Inc.), tissue
sections were incubated with TrMab‑6 (5 µg/ml) or EPR20043(1/500 dilution; Abcam) for 1 h at room temperature andthen treated with the EnVision+ Kit for mouse (Agilent
Technologies Inc.) and EnVision+ Kit for rabbit (Agilent
Technologies Inc.) for 30 min, respectively. Color wasdeveloped using 3,3'‑diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies Inc.) for 2 min. Counterstaining
was performed with hematoxylin (FUJIFILM Wako Pure
Chemical Corporation).
Results
Development of novel anti‑TROP2 mAbs using the CBIS
method. We immunized two mice with CHO/PA16‑TROP2‑​
RAP‑MAP cells and anti‑TROP2 mAbs were screened via
flow cytometry (Fig. 1). The first screening approach identi‑
fied strong signals from CHO/TROP2‑PA cells and weak tono signals from CHO‑K1 cells using hybridoma supernatantsfrom 90 of the 956 wells (9.4%). The second screening
approach identified strong signals from MCF7 cells from 84 of
the 90 hybridoma supernatants identified in the earlier step
(93.3%). After limiting dilution, we established 30 positive
clones. Further screening via Western blot and immunohisto‑
chemistry led to the establishment of TrMab‑6. The subclass

Figure 1. Illustration of Cell‑Based Immunization and Screening. A total of
two mice were immunized with CHO/PA16‑TROP2‑RAP‑MAP cells, andanti‑TROP2 monoclonal antibodies were screened using flow cytometry.
TROP2, TROP2, trophoblast cell‑surface antigen 2.

of TrMab‑6 was determined to be mouse IgG2b as shown in
Fig. S1A.
Flow cytometry analysis. We developed several transfectants,
such as CHO/TROP2‑PA, Lec1/TROP2, Lec2/TROP2, andLec8/TROP2, and the transfection efficiency was confirmed
using an anti‑PA tag mAb (NZ‑1) by Western blot analysis(Fig. S1B). Then, we performed flow cytometry targeting several
relevant cell lines in order to characterize antigen detection
using TrMab‑6 (Fig. 2). TrMab‑6 detected CHO/TROP2‑PA
cells, but not parental CHO‑K1 cells. TrMab‑6 also detectedendogenous TROP2 on human breast cancer cell lines,
including MCF7 and BT‑474. Contrarily, TrMab‑6 did not reactwith BINDS‑29 (TROP2‑gene‑deleted MCF7 cells). Taken
together, these results suggested that TrMab‑6 is specific for
TROP2. As shown in Fig. S2, another anti‑TROP2 mAb (clone
EPR20043) weakly reacted with MCF7, but did not react with
BT‑474 although TrMab‑6 strongly reacted with both MCF7
and BT‑474, indicating that TrMab‑6 is more useful for flow
cytometry than EPR20043 although EPR20043 was shown to
be useful in all applications, such as flow cytometry, Western
blot, and immunohistochemical analyses (Table SI).
Next, we investigated whether the epitope of TrMab‑6 isassociated with glycans. Thus, we performed flow cytom‑
etry using TROP2‑transfected glycan‑deficient CHO cells,
including those deficient in Lec1 (N‑glycan‑deficient),
Lec2 (sialic acid‑deficient), and Lec8 (galactose‑deficient)cells. As presented in Fig. 2, TrMab‑6 reacted with
Lec1/TROP2, Lec2/TROP2, and Lec8/TROP2 cells to an extent

4

SAYAMA et al: DEVELOPMENT OF ANTI-TROP2 MAB

Figure 3. Flow cytometric determination of the binding affinity of TrMab‑6for breast cancer cells. (A) MCF7 or (B) BT‑474 cells were suspended in
100 ul of serially diluted antibodies (6 ng/ml to 100 µg/ml), followed by incu‑
bation with Alexa Fluor 488‑conjugated secondary antibodies. Fluorescencedata were collected using a cell analyzer; GeoMean, geometric mean offluorescence intensity.

Figure 2. Flow cytometric detection of TROP2 using TrMab‑6. (A) CHO‑K1,(B) CHO/TROP2‑PA, (C) MCF7, (D) BINDS‑29 (MCF7/TROP2‑KO),(E) BT‑474, (F) Lec1/TROP2, (G) Lec2/TROP2 and (H) Lec8/TROP2 cells
were incubated with TrMab‑6 (1 µg/ml; red line) or 0.1% BSA in PBS (grey)
for 30 min, followed by Alexa Fluor 488‑conjugated secondary antibodies.
Fluorescence data were collected using a cell analyzer. TROP2, trophoblast
cell‑surface antigen 2.

indistinguishable from that observed with CHO/TROP2‑PA.
These results indicated that the binding epitope recognized by
TrMab‑6 was unlikely to be associated with glycans.
Determination of the binding affinity using TrMab‑6 against
breast cancers by flow cytometry. To determine the binding
affinity of TrMab‑6, we conducted kinetic analysis of theinteraction of TrMab‑6 with MCF7 and BT‑474 cells via flow
cytometry. The KD of TrMab‑6 was determined to be 6.5x10‑9 M
when targeting MCF7 cells and 1.1x10‑10 M for BT‑474 cells(Fig. 3). These results indicated that TrMab‑6 binds with highaffinity to TROP2‑expressing breast cancer cells.
Western blot analyses. TrMab‑6 binding identified TROP2 as
an immunoreactive band with an estimated 40 kDa band inlysates prepared from CHO/TROP2‑PA, MCF7, and BT‑474
cells; no immunoreactive bands were found in CHO‑K1 andTROP2‑gene‑deleted MCF7 (BINDS‑29) cells (Fig. 4), again
confirming its specificity for TROP2. TrMab‑6 also detected

TROP2 of Lec1/TROP2, Lec2/TROP2, and Lec8/TROP2.
Although TROP2 proteins, which were expressed in
Lec1/TROP2 and Lec8/TROP2, were detected in lower
molecular weight compared with CHO/TROP2‑PA andLec2/TROP2, the intensity by TrMab‑6 was similar among
those cell lines, indicating that the binding epitope of TrMab‑6
is independent of glycans. An anti‑PA tag mAb (NZ‑1) also
detected TROP2 bands in lysates of CHO/TROP2‑PA,
Lec1/TROP2, Lec2/TROP2, and Lec8/TROP2 cells. These
results indicated that TrMab‑6 could be used to detect TROP2
expressed by breast cancer cells via Western blot.
We compared the reactivity of TrMab‑6 and anotheranti‑TROP2 mAb (clone EPR20043) in Western blot analysis.
As shown in Fig. S3, both TrMab‑6 and EPR20043 strongly
detected TROP2 from both MCF7 and BT‑474, indicatingthat both TrMab‑6 and EPR20043 are useful for Western blot
analysis.
Immunohistochemical analyses against breast cancer. We thenused TrMab‑6 to target clinical specimens of human breast
cancer tissue via immunohistochemical analysis (Table I).
TrMab‑6 detected TROP2 in 57/61 of the breast cancer speci‑
mens (93.4%; Table II). Among these specimens were 50/54
cases (93.1%) of invasive ductal carcinoma (Table II). Typical
TrMab‑6‑associated staining patterns in the specimens ofinvasive ductal carcinomas are presented in Fig. 5A and B.
Hematoxylin and eosin (H&E) staining of invasive ductal
carcinoma tissue is presented in Fig. 5C and D. Furthermore,MOLECULAR MEDICINE REPORTS 23: 92, 2021

5

Figure 5. Detection of TROP2 in breast cancer specimens using immuno‑
histochemical analysis with TrMab‑6. (A and B) Tissue sections of patientswith human breast cancer (invasive ductal carcinoma, Case No. 5) wereincubated with 5 µg/ml of TrMab‑6 and then treated with the EnVision+
kit. Tissues were counterstained with hematoxylin. (C and D) H&E staining
was performed using consecutive breast cancer tissues (invasive ductal
carcinoma, Case No. 14). (E and F) Tissue sections of patients with humanbreast cancer (invasive lobular carcinoma) were incubated with 5 µg/ml of
TrMab‑6 and treated with a EnVision+ kit. Counterstaining was performed
with hematoxylin. (G and H) H&E staining was performed using consecu‑
tive breast cancer tissues (invasive lobular carcinoma). Scale bar, 100 µm.
TROP2, trophoblast cell‑surface antigen 2; H&E, hematoxylin and eosin.
Figure 4. Detection of TROP2 with TrMab‑6 using western blot analysis.
The cell lysates (10 µg) of CHO‑K1, CHO/TROP2‑PA, MCF7, BINDS‑29(MCF7/TROP2‑KO), BT‑474, Lec1/TROP2, Lec2/TROP2 and Lec8/TROP2
were subjected to SDS‑PAGE and transferred onto the PVDF membranes.
The membranes were incubated with l µg/ml of (A) TrMab‑6, an (B) anti‑PA
mAb (NZ‑1) and an (C) anti‑β ‑actin mAb (AC‑15), followed by secondary
antibodies. TROP2, trophoblast cell‑surface antigen 2.

TrMab‑6 detected TROP2 in 4/4 cases (100%) of invasive
lobular carcinoma, 2/2 cases (100%) of adenocarcinoma, and
1/1 case (100%) of medullary carcinoma (Table II); the typicalstaining patterns of invasive lobular carcinoma are presented inFig. 5E and F, and H&E staining was performed as presented inFig. 5G and H. Among the 61 breast cancer cases, 30/61 cases(49.2%) were stained strongly positive, 18/61 cases (29.5%)
were stained moderately positive, and 9/61 cases (14.8%) were
stained weakly positive by TrMab‑6 (Table II). We obtained
the information about ER, PR, and HER2 (Table I). To deter‑mine HER2 expression, we used an anti‑HER2 mAb (clone
H2Mab‑77) (32). Among 61 breast cancers, 31 cases (50.8%)
were determined to be triple‑negative (Table III). Interestingly,
28/31 (90.3%) were stained by TrMab‑6; especially, 17/31(54.8%) were stained strongly positive by TrMab‑6 (Table III),
indicating that triple‑negative breast cancers should be an ideal
target of anti‑TROP2 mAbs, including TrMab‑6.

We compared the reactivity of TrMab‑6 and anotheranti‑TROP2 mAb (clone EPR20043) in immunohistochemical
analysis. As shown in Table I, EPR20043 stained 60/61 (98.4%)
breast cancer tissues, although TrMab‑6 stained 57/61 (93.4%)
breast cancer tissues, indicating that EPR20043 is more usefulfor immunohistochemical analysis than TrMab‑6.
Discussion
Generating a mAb that can be utilized for multiple applications,
including flow cytometry, Western blot, and immunohis‑
tochemistry, is usually difficult. Using the CBIS method, inwhich antigen‑expressing cell lines are used for both immuni‑
zation and screening (22), we have developed numerous usefulmAbs that target membrane proteins, including CD19 (23),CD20 (24), CD44 (25), CD133 (22), PD‑L1 (26), and podoplanin(PDPN) (33‑36). Among these unique targets, CD20 has four
membrane‑spanning domains and only two small extracellulardomains that include amino acids 72‑80 and 142‑182 (37,38).
Although there are several commercially available mAbs thatinteract with amino acids 142‑182 of CD20 and are specificallyuseful in flow cytometry, there are no available anti‑CD20
mAbs that are effective not only in flow cytometry but also inWestern blot and immunohistochemical analyses. We recently

SAYAMA et al: DEVELOPMENT OF ANTI-TROP2 MAB

6Table I. Results of TrMab‑6 and EPR20043 immunostaining in 61 breast cancers.
								HER2/
Case Age Sex
Pathological diagnosis
Differentiation
TNM
ER PR H2Mab‑77 TrMab‑6 EPR20043
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51

44
58
40
52
60
57
48
66
58
63
32
59
44
60
44
82
58
57
41
44
78
60
N/A
46
41
59
45
43
40
51
45
45
47
55
58
47
38
40
57
42
60
58
41
50
60
53
65
43
57
37
50

F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F

Invasive ductal carcinoma
Medullary carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive lobular carcinoma
Adenocarcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive lobular carcinoma
Invasive lobular carcinoma
Invasive lobular carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Adenocarcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma

Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Well
Well
Moderately
Moderately
Moderately
Moderately
Well
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
PoorlyPoorlyN/A
N/A
Moderately
PoorlyPoorlyModerately‑Poorly
Moderately
Moderately
Moderately
PoorlyPoorlyPoorlyModerately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately

T2 N2 M1T2 N2 M1
T2 N1 M0
T2 N2 M1
T2 N1 M1T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N2 M1T2 N0 M0
T2 N0 M0
T2 N2 M0
T2 N2 M0
T2 N1 M0
T2 N2 M0
T2 N1 M1
T2 N1 M1T3 N3 M0
T2 N1 M0
T2 N2 M0
T2 N1 M0
T2 N0 M0
T2 N1 M1T2 N3 M1T2 N2 M0
T2 N0 M0
T2 N0 M0
T2 N1 M1
T1 N0 M0
T2 N2 M0
T2 N0 M0
T2 N1 M0
T2 N1 M0
T2 N3 M1T3 N3 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N2 M1T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N0 M0
T2 N3 M0

‑
‑
1+
1+
‑
‑
1+
2+
1+
3+
‑
‑
1+
3+
‑
‑
‑
2+
‑
‑
2+
‑
3+
‑
‑
2+
1+
‑
1+
1+
1+
2+
‑
‑
1+
‑
‑
‑
‑
‑
‑
‑
‑
‑
1+
‑
‑
‑
‑
‑
‑

‑
‑
‑
1+
‑
‑
2+
1+
‑
3+
‑
‑
1+
2+
3+
‑
‑
1+
‑
‑
1+
‑
‑
‑
‑
1+
1+
‑
1+
1+
2+
3+
‑
‑
1+
‑
‑
‑
‑
‑
‑
1+
3+
‑
1+
‑
‑
‑
‑
1+
‑

‑
‑
1+
‑
‑
‑
2+
‑
‑
‑
‑
‑
1+
‑
3+
‑
1+
‑
‑
‑
‑
1+
2+
‑
‑
‑
‑
‑
‑
‑
1+
3+
‑
1+
‑
‑
‑
‑
‑
2+
‑
‑
‑
‑
‑
‑
‑
‑
3+
‑
‑

3+
3+
2+
1+
3+
2+
2+
2+
3+
3+
2+
1+
2+
3+
3+
3+
3+
3+
3+
3+
3+
1+
3+
3+
3+
2+
‑
1+
2+
2+
2+
3+
3+
3+
2+
1+
3+
3+
1+
3+
3+
1+
2+
3+
3+
3+
1+
3+
3+
2+
‑

3+
2+
3+
2+
3+
3+
3+
2+
3+
3+
3+
1+
3+
3+
3+
3+
3+
3+
3+
1+
3+
1+
2+
3+
3+
2+
1+
1+
1+
3+
3+
3+
3+
3+
3+
2+
3+
3+
1+
3+
3+
1+
2+
3+
2+
3+
2+
3+
3+
3+
2+

MOLECULAR MEDICINE REPORTS 23: 92, 2021

7

Table I. Continued.
								HER2/Case Age Sex
Pathological diagnosis
Differentiation
TNM
ER PR H2Mab‑77 TrMab‑6 EPR20043
52
53
54
55
56
57
58
59
60
61

48
50
53
49
65
43
58
48
N/A
N/A

F
F
F
F
F
F
F
F
F
F

Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma

PoorlyModerately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately‑Poorly

T2 N1 M0 ‑T2 N0 M0 ‑T2 N0 M0 ‑T2 N0 M0 ‑T2 N1 M0 1+
T2 N0 M0 ‑T2 N0 M0 ‑T2 N0 M0 ‑
N/A
‑
N/A
1+

‑
‑
‑
‑
1+
‑
‑
‑
‑
1+

‑
‑
‑
‑
‑
‑
‑
‑
‑
‑

1+
3+
3+
2+
2+
‑
‑
2+
2+
2+

1+
3+
3+
2+
3+
‑
1+
3+
2+
3+

N/A, not available; TNM, tumor node metastasis; F, female; ER, estrogen receptor; PR, progesterone receptor.

Table II. Results of TrMab‑6 immunostaining in 61 breast cancers.

Pathological diagnosis
Invasive ductal carcinoma
Invasive lobular carcinoma
Adenocarcinoma
Medullary carcinoma
Total

TrMab‑6‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑
No. of cases
3+
2+
1+
‑
No. of positive cases (%)
54
4
2
1
61

26
16
8
4
1
2
1
0
2
0
0
0
1
0
0
0
30/61 (49.2%) 18/61 (29.5%) 9/61 (14.8%) 4/61 (6.6%)

50/54 (92.6)
4/4 (100)
2/2 (100)
1/1 (100)
57/61 (93.4%)

Table III. Results of TrMab‑6 immunostaining in 31 triple negative breast cancers.

Breast cancer subtype
Triple negative breast cancer

No. of cases
31

TrMab‑6‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑
3+ (%)
2+ (%)
1+ (%)‑ (%)
17 (54.8)

developed clone C20Mab‑11, which can detect CD20 associ‑ated with B‑cell lymphoma by flow cytometry, Western blot,
and immunohistochemical analyses (24).
Likewise, we herein aimed to establish one or more
multipurpose anti‑TROP2 mAbs because the applications ofcommercially available anti‑TROP2 mAbs were somewhat
limited (Table SI). Using the CBIS method, we success‑fully developed a sensitive and specific novel anti‑TROP2
mAb (clone TrMab‑6) that can be used in every application,
including flow cytometry (Fig. 3), Western blot (Fig. 4), andimmunohistochemical analyses (Fig. 5). Another anti‑TROP2
mAb (clone EPR20043 from Abcam) is more sensitive than
TrMab‑6 in immunohistochemical analysis (Table I), but not
useful in flow cytometry (Fig. S2). EPR20043 might reactwith intracellular region of TROP2 although the immunogen

5 (16.1)

6 (19.4)

3 (9.7)

No. of positive cases
28/31 (90.3%)

was not clearly shown in its application sheet. Clone SP293(Abcam) was also shown to be useful for flow cytometry,
Western blot, and immunohistochemical analyses in its appli‑
cation sheet; however, the intracellular region of TROP2 wasalso used as immunogen (Table SI).
We conclude that TrMab‑6 is more advantageous thanthe other anti‑TROP2 mAbs, such as EPR20043 and SP293
because TrMab‑6 is useful to detect TROP2 in all applications,such as flow cytometry in non‑fixed condition, Western blot,
and immunohistochemical analysis. However, in this study,
TrMab‑6 was shown to be useful for only in vitro experiments.
In the future, we will determine whether TrMab‑6 would besuitable for use as targeted molecular therapy against breast
cancers. The subclasses of mouse IgG, IgG2a and IgG2b, both
induce antibody‑dependent cellular cytotoxicity (ADCC) or8

SAYAMA et al: DEVELOPMENT OF ANTI-TROP2 MAB

complement‑dependent cytotoxicity (CDC) (39,40). TrMab‑6
was determined to be of the mouse IgG2b subclass (Fig. S1).
Although we performed ADCC reporter assay using TrMab‑6,
ADCC activity was not observed unexpectedly (data notshown). To promote antibody therapy using TrMab‑6, we
will utilize antibody‑drug conjugates (ADCs), radioimmu‑
notherapy (RIT), photoimmunotherapy (PIT), or chimericantigen receptor T‑cell (CAR‑T) therapy. About ADCs
targeting TROP2, conjugation of the irinotecan metabolite,
SN‑38, to a humanized anti‑TROP2 antibody (sacituzumab
govitecan) promotes broad and potent antitumor effects inhuman cancer xenografts and in patients with advanced
triple‑negative breast, non‑small‑cell and small‑cell lung, andurothelial cancers (14). About RIT, van Rij et al (41) previ‑
ously reported that TROP2‑expressing prostate cancer can betargeted efficiently with TF12 [anti‑TROP2 x anti‑HSG (hista‑mine‑succinyl‑glycine)] and 177 Lu‑labeled diHSG‑peptide(IMP288). Furthermore, Nishimura et al (42) selected TROP2
as a molecular target for PIT, and utilized a newly developed
humanized anti‑TROP2 mAb conjugated to the photosensitizer
IR700 (TROP2‑IR700) for the treatment of pancreatic carci‑
noma and cholangiocarcinoma. Growth of tumor xenografts
was significantly inhibited in response to TROP2‑targeted
PIT relative to controls, suggesting that TROP2‑targeted
PIT is also an important means for improving treatment for
TROP2‑expressing cancers. About CAR‑T, Zhao et al (43)reported that novel bi‑specific TROP2/PD‑L1 CAR‑T cells
could target TROP2/PD‑L1 and checkpoint blockade, resulting
in cytotoxicity for gastric cancer cells. These results alsosuggested that CAR‑T cell therapy featuring TROP2 can be
developed to target TROP2‑expressing cancers. These various
modalities will allow us to explore TrMab‑6‑mediated anti‑
tumor activities in the mouse xenograft model of breast cancer.
Acknowledgements
The authors would like to thank Mr. Takuro Nakamura,
Ms. Miyuki Yanaka, Ms. Saori Handa, Ms. Saki Okamoto, andMr. Yu Komatsu (Department of Antibody Drug Development,
Tohoku University Graduate School of Medicine) for technicalassistance in the in vitro experiments.
Funding
The present study was supported in part by Japan Agency
for Medical Research and Development (AMED; grantnos. JP20am0401013, JP20am0101078 and JP20ae0101028).
Availability of data and materials
The datasets used and/or analyzed during the current study
are available from the corresponding author on reasonable
request.
Authors' contributions
YS performed experiments, and analyzed experimental data.
YS and YK wrote the manuscript. MKK and YK designed
the current study. All authors read and approved the final
manuscript.

Ethics approval and consent to participate
Animal studies were approved by The Animal Care and Use
Committee of Tohoku University (Permit no. 2019NiA‑001).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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