MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 1, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0056
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TrMab-6 Exerts Antitumor Activity in Mouse Xenograft
Models of Breast Cancers

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Tomohiro Tanaka,1 Tomokazu Ohishi,2 Masaki Saito,3 Manabu Kawada,2 Mika K. Kaneko,1 and Yukinari Kato1,3

Trophoblast cell surface antigen 2 (TROP2) has been reported to be overexpressed in many cancers, andis involved in cancer cell proliferation, invasion, and metastasis. We previously developed a highly
sensitive anti-TROP2 monoclonal antibody (mAb) (clone TrMab-6; mouse IgG2b, kappa) using a CellBased Immunization and Screening method. TrMab-6 is useful for investigations using flow cytometry,
Western blotting, and immunohistochemistry and possesses antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) against TROP2-expressing triple-negative
breast cancer (TNBC) cell lines, such as MDA-MB-231 and MDA-MB-468. This study investigatedwhether TrMab-6 possesses in vivo antitumor activities via ADCC/CDC activities using mouse xenograft models of TNBC cell lines. In vivo experiments on MDA-MB-231 and MDA-MB-468 xenografts
revealed that TrMab-6 significantly reduced tumor growth compared with normal mouse IgG treatment.
The findings of this study suggest that TrMab-6 is a promising treatment option for TROP2-expressing
TNBC.
Keywords: TROP2, monoclonal antibody, antitumor activity, triple-negative breast cancer

inhibitors, phosphoinositide 3-kinase/Akt/mammalian targetof rapamycin inhibitors, and cyclin-dependent kinase inhibitors, are also used as the basic treatment strategy for
TNBC.(2,9,10) Immunotherapy is beginning to be introduced asa new treatment for breast cancers.(11,12)
The expression level of programmed cell death ligand 1 (PDL1) is higher in TNBC than in other breast cancers.(13–15) PDL1 expression is associated with malignancy and hormone
receptor-negative breast cancer.(16) Programmed cell death 1/
PD-L1-targeted antibodies, such as pembrolizumab and atezolizumab, have been actively tested on patients with breast
cancer.(12,17,18)
Trophoblast cell surface antigen 2 (TROP2), a type I
transmembrane glycoprotein, has been reported to be overexpressed in various solid tumors, including breast cancers.
A higher expression of TROP2 is involved in tumor growth,invasion, and proliferation.(19–21) Particularly, TROP2 expression has been described to be upregulated in 80%–95% ofIntroduction

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. It is defined by theabsence of estrogen receptor, progesterone receptor, andhuman epidermal growth factor receptor 2 (HER2) expressions.(1,2) TNBC formation is found in 10%–20% of breast
cancers and frequently occurs in young and African American women.(3–5) Patients with TNBC have a worse prognosis
than those with other breast cancers, with more than 30%
developing metastatic diseases or recurring cancer.(6,7)
Younger patients with TNBC have short disease-free survival
than older patients with TNBC.(3) Furthermore, *30% of
patients with metastatic TNBC have been recorded to have atleast one tumor metastasis to the brain.(8)Surgery, radiotherapy, and platinum-based chemotherapy
are primarily selected as the standard treatment for TNBC.
Molecular targeted drugs, such as poly ADP-ribose polymerase

123Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Microbial Chemistry Research Foundation, Institute of Microbial Chemistry (BIKAKEN), Numazu-shi, Japan.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.

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ANTITUMOR ACTIVITIES OF AN ANTI-TROP2 MAB

TNBC.(22,23) TROP2 has been identified as one of themarkers for epithelial cell adhesion molecule low/negative
circulating tumor cells, which are derived from patients withmetastatic TNBC.(24)
Sacituzumab govitecan (IMMU-132) is an antibody–drugconjugate (ADC) that combines an anti-TROP2 monoclonal
antibody (mAb) with the irinotecan metabolite SN-38 as a
payload.(25) The U.S. Food and Drug Administration (FDA)
approved sacituzumab govitecan for treatment against metastatic TNBC and designated it a breakthrough therapy.(26,27)
Another trial of this ADC was launched against some tumors,
including hormone receptor-positive and HER2-negative
breast cancers.(28)We have previously developed an anti-TROP2 mAb (clone
TrMab-6, mouse IgG2b, kappa), which has been applied tovarious experiments, including flow cytometry, Western
blotting, and immunohistochemistry.(29) TrMab-6 exerted
in vitro antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC) activities orin vivo antitumor activities using mouse xenograft models of
TROP2-overexpressed CHO-K1 (CHO/TROP2) or MCF7
breast cancer cells.(30) Furthermore, TrMab-6 exerted in vitro
ADCC and CDC activities against TNBC cell lines, such as
MDA-MB-231 and MDA-MB-468. However, whether
TrMab-6 exerted in vivo antitumor activities, using mouse
xenograft models of TNBC cell lines, has not been clarified.
In this study, we investigated whether TrMab-6 exerts
in vivo antitumor activities using mouse xenograft models of
TNBC cell lines, such as MDA-MB-231 and MDA-MB-468.
Materials and Methods
Cell lines

The breast cancer cell lines MDA-MB-231 and MDA-MB468 were obtained from the American Type Culture Collection(Manassas, VA). MDA-MB-231 and MDA-MB-468 were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai
Tesque, Inc., Kyoto, Japan), supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific, Inc.,
Waltham, MA), 100 U/mL of penicillin (Nacalai Tesque, Inc.),
100 lg/mL of streptomycin (Nacalai Tesque, Inc.), and0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.), and incubated at 37C in a humidified atmosphere containing 5% CO2.
Primary antibodies

Purified mouse IgG (Cat. No. I8765) was purchased from
Sigma-Aldrich (St. Louis, MO). TrMab-6 was purified using
Protein G-Sepharose (GE Healthcare Biosciences, Pittsburgh, PA).
Antitumor activity of an anti-TROP2 mAb in a mouse
xenograft model

A total of 32 five-week-old female BALB/c nude mice(mean weight, 15 – 3 g) were purchased from Charles River
Laboratories, Inc. (Kanagawa, Japan). The mice were divided
into (i) MDA-MB-231-bearing mice and (ii) MDA-MB-468bearing mice (n = 16 in each group). On day 7, each groupwas subdivided into two groups (n = 8 in each group) withequal mean tumor volume: a control mouse IgG-treated or
TrMab-6-treated group. All animal experiments were performed following institutional guidelines and regulations to33

minimize animal suffering and distress in the laboratory. The
Institutional Committee for experiments of the Institute ofMicrobial Chemistry (Permit No. 2021-015) approved theanimal studies for antitumor activity.
The mice were maintained in a pathogen-free environment, on an 11-hour light/13-hour dark cycle at a temperatureof 23C – 2C and humidity of 55% – 5%, with food andwater supplied ad libitum throughout the experiments. The
mice were monitored for health and weight every 3 or 4 days.
The experiments on mice were conducted after 4 weeks.
Weight loss of >25% or tumor volume of >3000 mm3 was
identified as humane endpoints for euthanasia. At humane
and experimental endpoints, the mice were euthanized bycervical dislocation, and death was verified by validating
respiratory and cardiac arrest.
After an acclimation period of 1 week, these mice wereused in experiments at 7 weeks of age (mean weight, 19 – 2g).
The cells (0.3 mL of 1.33 · 108 cells/mL in DMEM) weremixed with 0.5 mL of BD Matrigel Matrix Growth Factor
Reduced (BD Biosciences, San Jose, CA). A total of 100 mLof this suspension (5 · 106 cells) was subcutaneously injected
into the left flank of each animal. On day 7 postinoculation,
100 mg of TrMab-6 or control mouse IgG in 100 mL ofphosphate-buffered saline (PBS) was intraperitoneally (i.p.)
injected. Additional antibody inoculations were performedon days 14 and 21. All mice were euthanized by cervicaldislocation 23 days after cell implantation, and tumor diameters and volumes were measured and recorded.
Statistical analysis

Data are expressed as mean – standard error of the mean.
Statistical analysis was conducted with ANOVA and Sidak’s
multiple comparison tests for tumor volume and mouseweight, and Welch’s t-test for tumor weight. All calculations
were performed using GraphPad Prism 7 (GraphPad Software, Inc.). p < 0.05 was considered statistically significant.
Results
Antitumor effect of TrMab-6 in mouse xenograftsof a TROP2-expressing TNBC cell line (MDA-MB-231)

The tumor formation of 16 MDA-MB-231-bearing mice wasobserved on day 7 before the mice were divided into TrMab-6treated and control groups. On days 7, 14, and 21, after MDAMB-231 cell injections into the mice, either TrMab-6 (100 lg)
or control mouse IgG (100 lg) was i.p. injected into the mice.
Tumor volume was measured on days 7, 10, 14, 16, 21, and 23
after MDA-MB-231 cell injection. The TrMab-6-treated mice
exhibited significantly less tumor growth on days 14 ( p < 0.01),
16 ( p < 0.01), 21 ( p < 0.01), and 23 ( p < 0.01) compared with
the IgG-treated control mice (Fig. 1A).
On day 23, a 35% reduction in tumor volume was seen in
the TrMab-6-treated mice (Fig. 1A). Tumors from the
TrMab-6-treated mice weighed significantly less than tumors
from the IgG-treated control mice on day 23 (50% reduction,p < 0.01; Fig. 1B). Figure 1C demonstrates each tumor. These
results indicated that TrMab-6 reduced the growth of MDAMB-231 xenografts, but did not contribute toward their totalelimination. Total body weights did not significantly differ
between the treatment and control groups (Fig. 1D).
Figure 1E demonstrates the body appearance of the mice.

TANAKA ET AL.

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34

FIG. 1. Evaluation of the antitumor activity of TrMab-6 in MDA-MB-231 xenografts. (A) MDA-MB-231 cells (5 · 106 cells)
were subcutaneously injected into the left flank. After day 7, 100 mg of TrMab-6 and control mouse IgG in 100 mL of PBS was i.p.
injected into the treated and control mice, respectively. Additional antibodies were then injected on days 14 and 21. Tumor
volume was measured on days 7, 10, 14, 16, 21, and 23. Values are mean – SEM. Asterisk indicates statistical significance(**p < 0.01, ANOVA and Sidak’s multiple comparisons test). (B) MDA-MB-231 xenograft tumors were resected from the
TrMab-6 and control mouse IgG groups. Tumor weight on day 23 was measured from excised xenografts. Values are mean –SEM. Asterisk indicates statistical significance (**p < 0.01, Welch’s t-test). (C) Resected tumors of MDA-MB-231 xenografts
from the control mouse IgG and TrMab-6 groups on day 23. Scale bar, 1 cm. (D) Body weights of the mice implanted with MDAMB-231 xenografts were recorded on days 7, 10, 14, 16, 21, and 23. (E) Body appearance of mice on day 23. Scale bar, 1 cm. i.p.,
intraperitoneally; n.s., not significant; PBS, phosphate-buffered saline; SEM, standard error of the mean.

Antitumor effect of TrMab-6 in mouse xenograftsof a TROP2-expressing TNBC cell line (MDA-MB-468)

The tumor formation of 16 MDA-MB-468-bearing mice
was observed on day 7 before the mice were divided into
TrMab-6-treated and control groups. On days 7, 14, and 21,
after MDA-MB-468 cell injections into the mice, either

TrMab-6 (100 lg) or control mouse IgG (100 lg) was i.p.
injected into the mice. Tumor volume was measured on days
7, 10, 14, 16, 21, and 23 after MDA-MB-468 cell injection.
The TrMab-6-treated mice exhibited significantly less tumor
growth on days 10 ( p < 0.05), 14 ( p < 0.01), 16 ( p < 0.01), 21( p < 0.01), and 23 ( p < 0.01) compared with the IgG-treatedcontrol mice (Fig. 2A).

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ANTITUMOR ACTIVITIES OF AN ANTI-TROP2 MAB

FIG. 2. Evaluation of the antitumor activity of TrMab-6 in MDA-MB-468 xenografts. (A) MDA-MB-468 cells (5 · 106
cells) were subcutaneously injected into the left flank. After day 7, 100 mg of the TrMab-6 and control mouse IgG in 100 mL
of PBS was i.p. injected into the treated and control mice, respectively. Additional antibodies were then injected on days 14
and 21. Tumor volume was measured on days 7, 10, 14, 16, 21, and 23. Values are mean – SEM. Asterisk indicatesstatistical significance (**p < 0.01, *p < 0.05, ANOVA and Sidak’s multiple comparisons test). (B) MDA-MB-468 xenograft
tumors were resected from the TrMab-6 and control mouse IgG groups. Tumor weight on day 23 was measured fromexcised xenografts. Values are mean – SEM. Asterisk indicates statistical significance (**p < 0.01, Welch’s t-test).
(C) Resected tumors of MDA-MB-468 xenografts from control mouse IgG and TrMab-6 groups on day 23. Scale bar, 1 cm.
(D) Body weights of the mice implanted with MDA-MB-468 xenografts were recorded on days 7, 10, 14, 16, 21, and 23.
(E) Body appearance of mice on day 23. Scale bar, 1 cm. n.s., not significant.

On day 23, a 48% reduction in tumor volume was seen in
the TrMab-6-treated mice (Fig. 2A). Tumors from the
TrMab-6-treated mice weighed significantly less than thosefrom the IgG-treated control mice on day 23 (49% reduction,p < 0.01; Fig. 2B). Figure 2C demonstrates each tumor. These

results indicated that TrMab-6 reduced the growth of MDAMB-468 xenografts, but did not contribute toward their totalelimination. Total body weights did not significantly differ
between the treatment and control groups (Fig. 2D).
Figure 2E demonstrates the body appearance of the mice.

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Discussion

Precision medicine has recently received considerable attention in personalized cancer treatment.(31,32) The conceptof precision medicine targets proteins that drive carcinogenesis based on the abnormal molecular profiles of individual patients with cancer rather than the types of cancers,
leading to the improved prognosis of patients.(33) Increasing
evidence has indicated that TROP2 influences tumor development and progression. TROP2 overexpression enhances
cancer cell proliferation and invasion, and predicts poor
prognosis in solid tumors.(22,34,35) Alternatively, the inhibition of TROP2 expression impaired cell proliferation, migration, and invasion.(34,36) Therefore, TROP2 has been
regarded as a potential target for cancer immunotherapy andprecision medicine.
The therapeutic potential of mAbs and their derivatives has
been well recognized for decades in treating various diseases,
including cancer.(37) Ikeda et al. have established two
recombinant fucosylated and defucosylated chimeric
TROP2-targeting antibodies (cAR47A6.4.2 and cPr1E11,
respectively) with a human IgG1 constant region. Althoughboth antibodies showed significant anticancer activities
against human cancer cell lines in vitro and in vivo, only
ADCC, but not CDC, was a substantive contributor to theefficacy of antibodies.(38)
As far as we know, TrMab-6 is the first anti-TROP2 mAb
that shows anticancer activity in vivo (Figs. 1 and 2) andpossesses both ADCC and CDC activities.(30) Stein et al.
established RS7, a mouse IgG1 mAb raised against humanlung carcinoma, which could recognize human TROP2.(39–41)
Since RS7 is rapidly internalized from the surface of cells
after binding to TROP2, this antibody provided an opportunity to deliver cytotoxins into TROP2-expressing cells. The
radiolabeled RS7 significantly showed antitumor activity
in vivo,(42–45) and mutant ranpirnase, an amphibian RNase
toxin-conjugated humanized RS7 (hRS7), inhibited cancer
cell growth in vivo.(46,47) Sacituzumab govitecan is an FDAapproved first-in-class ADC consisting of hRS7, conjugatedto a topoisomerase I inhibitor (SN-38) for treating advanced
breast cancer.(48) These results indicate that antibody-based
immunotherapy is a promising therapeutic strategy for
TROP2-expressing cancers.
Intriguingly, TROP2 expression was detected in cancer
cells and normal human tissues, including the prostate, cervix, lung, breast, uterus, kidney, skin, pancreas, liver, salivary
grand, and esophagus.(49,50) These results suggest that
TROP2 has important functions in normal tissues, thereby
raising concerns that TROP2-targeting therapies may have
unexpected effects on normal tissues. We recently developed
a novel technology to produce a cancer-specific mAb (CasMab), which could react only to the antigen on cancer
cells.(51) We have also successfully produced several
CasMabs.(52–55) In the future, TROP2 CasMab production
can be applicable as a basis for designing and optimizing
potent immunotherapy modalities, such as ADCs and chimeric antigen receptor T cell therapy.
Authors’ Contributions

T.T., T.O., M.S., and M.K. performed the experiments;
M.K.K. designed the experiments; and T.T. and Y.K. wrote
the article.

TANAKA ET AL.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by the Japan Agency
for Medical Research and Development (AMED) under grant
numbers JP21am0401013 (to Yukinari Kato) andJP21am0101078 (to Yukinari Kato).
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: November 17, 2021
Accepted: January 11, 2022