MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 2, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0005

Development of Anti-Mouse CC Chemokine Receptor 8
Monoclonal Antibodies for Flow Cytometry

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

Tomohiro Tanaka,1 Ren Nanamiya,1 Junko Takei,1 Takuro Nakamura,1 Miyuki Yanaka,1
Hideki Hosono,1 Masato Sano,1 Teizo Asano,1 Mika K. Kaneko,1 and Yukinari Kato1,2

CC chemokine receptor 8 (CCR8) belongs to the class A of G protein-coupled receptor. It is highly expressedon Treg and T helper 2 (TH2) cells recruited to the inflammation site and is implicated in allergy and asthma.
Recently, CCR8+Treg cells have been suggested to be a master regulator in the immunosuppressive tumor
microenvironment; therefore, developing sensitive monoclonal antibodies (mAbs) for CCR8 has been desired.
This study established a specific and sensitive mAb for mouse CCR8 (mCCR8), which is useful for flow
cytometry by using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mCCR8
mAb, C8Mab-2 (rat IgG2b, kappa), reacted with mCCR8-overexpressed Chinese hamster ovary-K1(CHO/mCCR8) cells and P388 (mouse lymphoid neoplasma) or J774-1 (mouse macrophage-like) cells, which
express endogenous mCCR8 by flow cytometry. C8Mab-2, which was established by the CBIS method, could
be useful for elucidating the mCCR8-related biological response by flow cytometry.
Keywords: CCR8, monoclonal antibody, CBIS, flow cytometry

CC chemokine receptor 8 (CCR8) is a seventransmembrane protein belonging to the CC chemokine receptor family. CCR8 is principally expressed on Treg and T
helper 2 (TH2) cells recruited to the inflammation site and is
implicated in allergy and asthma.(9–13) Recently, CCR8+Treg
cells have been suggested to be a master regulator in the
immunosuppressive TME.(3,9,14) Some reports insist that thehigher proportion of CCR8+Treg cells is often associatedwith poor prognosis in various cancer patients, including
breast cancer.(8,15,16) The frequency of peripheral blood Treg
cells is elevated in breast cancer patients and correlates with abetter anti-CTLA-4 antibody treatment.(17,18)
Furthermore, intratumoral Treg cells have high CCR8
expression, and this type of cells is endowed with higher
expressions of CD25, Foxp3, and CTLA-4 than CCR8-Treg
cells.(17) CCL1/I-309, the specific ligand for CCR8 in humans, is also involved in antitumor immunosuppression via
CCR8.(19–21) CCL1 is secreted by some cancer-related cells,such as TAMs, cancer-associated fibroblasts, and Treg cells,causing other Treg cells’ recruitment to the tumor site,leading to angiogenesis.(22,23) CCL1 accelerates the immunosuppressive capacity of CCR8+Treg cells. CCL1-CCR8
axis also functions in melanoma metastasis to the lymph
node.(24) Therefore, targeting CCR8 could show dramatic
antitumor effects.(2,3,6,25)

IntroductionT

he tumor microenvironment (TME) consists ofvarious elements, including immune and stromal cells,
which affect antitumor treatment effectiveness.(1) Immunosuppressive cells, such as regulatory T cells (Treg), myeloidderived suppressor cells, and tumor-associated macrophages(TAMs), play a pivotal role in tumor progression.(1–4) Recently, the development of cancer immunotherapy, including
immune checkpoint inhibitor and chimeric antigen receptor(CAR)-T therapy, has been accelerated, and much attention
has been focused on lymphocytes to develop an effective
treatment against tumors.(5)Treg cells were found as a CD4+ T cell member, which
maintains self-tolerance to suppress abnormal allergic reaction and autoimmune diseases.(2,6) Treg cells are known as
CD4+CD25+Foxp3+ T cells, which suppress immune response by inhibiting the proliferation of effector T cells, orsecreting immunosuppressive cytokines, such as transforming growth factor-b (TGF-b), IL-10, and granzyme/perforin,
which give cytotoxicity to cytotoxic T lymphocyte.(7) Treg
cells are also involved in developing an immunosuppressive
TME by inhibiting antitumor immunity.(8) Treg cells express
cytotoxic T lymphocyte-associated-4 (CTLA-4), whichblock dendritic cell maturation.(2)12Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

65

66

TANAKA ET AL.

In our studies, we have developed many useful monoclonal
antibodies (mAbs) against membrane proteins using the CellBased Immunization and Screening (CBIS) method.(26–28)
Here, we developed a novel anti-mouse CCR8 (mCCR8)
mAb, which can be used for flow cytometry.
Materials and Methods

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). P388 and J774-1 wereobtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku
University (Miyagi, Japan). Synthesized DNA (Eurofins
Genomics KK, Tokyo, Japan) encoding mCCR8 (Accession
No.: NM_007720.2) was subcloned into a pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The mCCR8 plasmid was transfected using the Neontransfection system (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were established by cellsorting using a cell sorter (SH800; Sony Corp., Tokyo, Japan)
and cultivation in a medium containing 0.5 mg/mL of Zeocin(InvivoGen, San Diego, CA).
CHO-K1, P3U1, CHO/mCCR8, P388, and J774-1 were
cultured in Roswell Park Memorial Institute (RPMI) 1640
medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc.), 100 U/mL of penicillin, 100 lg/mL of
streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai
Tesque, Inc.). Cells were grown in a humidified incubator at
37C with 5% CO2 and 95% air.
Antibodies

An anti-mCCR8 mAb (clone SA214G2) was purchasedfrom BioLegend (San Diego, CA). Secondary Alexa Fluor
488-conjugated anti-rat IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA).
Hybridoma production

A female Sprague-Dawley (SD) rat (6-week-old) was purchased from CLEA Japan (Tokyo, Japan). The animal washoused under specific pathogen-free conditions. All animal
experiments were performed according to relevant guidelines
and regulations to minimize animal suffering and distress in
the laboratory. Animal experiments were approved by the
Animal Care and Use Committee of Tohoku University(Permit number: 2019NiA-001). The rat was monitored daily
for health during the full 4-week duration of the experiment.

FIG. 1. Production of anti-mCCR8 mAbs. The procedureof CBIS. CHO/mCCR8 cells were immunized into an SD rat
using intraperitoneal injection. Screening was performed byflow cytometry. CBIS, Cell-Based Immunization andScreening; CHO, Chinese hamster ovary; mAbs, monoclonal antibodies; mCCR8, mouse CC chemokine receptor 8;
SD, Sprague-Dawley.

‰
FIG. 2. Flow cytometry using anti-mCCR8. (A) CHO/mCCR8 cells were treated with 0.01–10 mg/mL of C8Mab-2 orSA214G2, followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG; black line, negative control. (B) CHO-K1
cells were treated with 0.01–10 mg/mL of C8Mab-2 or SA214G2, followed by treatment with Alexa Fluor 488-conjugatedanti-rat IgG; black line, negative control. (C) P388 cells were treated with 0.01–10 mg/mL of C8Mab-2 or SA214G2,
followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG; black line, negative control. (D) J774-1 cells weretreated with 0.01–10 mg/mL of C8Mab-2 or SA214G2, followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG;
black line, negative control.

67

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

68

A reduction of >25% of total body weight was defined as ahumane endpoint. The rat was euthanized by cervical dislocation, and death was verified by respiratory and cardiac arrest.
To develop mAbs against mCCR8, the CBIS method(26)was used. Briefly, one SD rat was immunized with
CHO/mCCR8 cells (1 · 109) by the intraperitoneal route together with the Imject Alum (Thermo Fisher Scientific, Inc.).
The procedure included three additional immunizations followed by a final booster intraperitoneal injection administered 2 days before spleen cells were harvested. Harvested
spleen cells were subsequently fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in an RPMI medium supplemented
with hypoxanthine, aminopterin, and thymidine for selection(Thermo Fisher Scientific, Inc.). Culture supernatants werescreened by flow cytometry.

TANAKA ET AL.

FIG. 3. Determination of the binding affinity of C8Mab-2.
CHO/mCCR8 was suspended in 100 mL of serially diluted
C8Mab-2 (0.006–100 mg/mL). Then, Alexa Fluor 488conjugated anti-rat IgG was added. Fluorescence data werecollected using the BD FACSLyric.

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

Flow cytometry analysis

Cells were harvested after brief exposure to 0.25% trypsin
and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai
Tesque, Inc.). They were washed with 0.1% bovine serum
albumin in phosphate-buffered saline and treated with primary mAbs for 30 minutes at 4C. Then, the cells weretreated with Alexa Fluor 488-conjugated anti-rat IgG(1:2000; Cell Signaling Technology, Inc.). Fluorescence data
were collected using the SA3800 Cell Analyzer (Sony Corp.,
Tokyo, Japan).
Determination of binding affinity by flow cytometry

CHO/mCCR8 was suspended in 100 lL of serially diluted
anti-mCCR8 mAbs, and Alexa Fluor 488-conjugated anti-rat
IgG (1:200; Cell Signaling Technology, Inc.) was added.
Fluorescence data were collected using the BD FACSLyric(BD Biosciences, Franklin Lakes, NJ). The dissociation
constant (KD) was calculated by fitting binding isotherms tobuilt-in, one-site binding models in GraphPad PRISM 8(GraphPad Software, Inc., La Jolla, CA).
Results
Establishment of anti-mCCR8 mAbs

To develop anti-mCCR8 mAbs, we employed the CBIS
method, using stable transfectants for immunization and flow
cytometry (Fig. 1). A rat was immunized with CHO/mCCR8cells, which overexpress mCCR8. Hybridomas were seeded
into 96-well plates, and CHO/mCCR8-positive and CHOK1-negative wells were selected. The first screening approach identified strong signals from CHO/mCCR8 cells andweak or no signals from CHO-K1 cells in 73 of 1916 wells(3.8%). After limiting dilution, C8Mab-2 (IgG2b, kappa) was
finally established.
Flow cytometry

We performed flow cytometry using C8Mab-2 against
CHO/mCCR8 and CHO-K1. C8Mab-2 recognized CHO/
mCCR8 dose dependently (Fig. 2A), but not CHO-K1(Fig. 2B). Another anti-mCCR8 mAb (clone SA214G2 from
BioLegend: positive control) also recognized CHO/mCCR8
dose dependently (Fig. 2A), but not CHO-K1 (Fig. 2B). Both
C8Mab-2 and SA214G2 reacted with CHO/mCCR8 even in

0.01 lg/mL (Fig. 2A). Both C8Mab-2 and SA214G2 also
reacted with P388 (Fig. 2C) and J774-1 (Fig. 2D), expressing
endogenous mCCR8 dose dependently.
Determination of the binding affinity of C8Mab-2

We then assessed the apparent binding affinity of C8Mab-2
with CHO/mCCR8 by flow cytometry. The KD of C8Mab-2
for CHO/mCCR8 was 1.4 · 10-8 M (Fig. 3), indicating that
C8Mab-2 possesses moderate affinity for CHO/mCCR8 cells.
Discussion

G protein-coupled receptors (GPCRs), including CCR8,
are expressed on the cell surface, transmit signals to intracellular molecules about various extracellular conditions, andgovern broad cell dynamics, such as homeostasis, proliferation, and migration. GPCRs are also deeply involved incancer and inflammatory diseases.(29) Therefore, GPCRs arefocused as targets for many diseases, and the development oftherapeutic agents, such as anti-GPCR antibodies, has been
investigated. The development of anti-GPCR antibodies has
been reported to be difficult due to the complexity of its
folded three-dimensional structure, the small exposed area of
extracellular epitopes, and the difficulty of purifying afunctional protein as an antigen.(30) The preparation of immunogens that maintain a biological and functional structureis vital for obtaining competent antibodies.
CC chemokine receptor belongs to the GPCR family andregulates various cellular dynamics. CCR8 is a 41-kDa seventransmembrane CC chemokine receptor in humans, which
possesses 355 amino acids. mCCR8 shares over 70% aminoacid sequence identity with the human protein. It has beenreported that CCR8+Treg cells function to suppress the immune response in the TME.(2) The presence of CCR8+Treg
cells in a tumor is often associated with poor prognosis invarious cancer patients, including breast cancer.(15) Several
mAbs for CCR8 were initially developed in the research forthe detection or neutralization of CCR8(31–33) and are alsocommercially available (Supplementary Table S1). Half ofthem were developed by immunizing rabbits with synthetic
peptides, and applications are limited to Western blotting.
Maintaining the structure of the GPCR, such as CC chemokine

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

ANTI-MOUSE CCR8

receptors, is essential, primarily when it is used for flow cytometry, in which mAbs recognize the extracellular epitopes of
the GPCR protein in immunology.(30)
Using the CBIS method, unlike immunizing synthetic
peptides, the biological structure and modification of proteins, such as glycosylation and folding, could be retained. In
commercial products, most of the mAbs applicable for flow
cytometry have also been developed by cell-based immunization (Supplementary Table S1). The cell-based method
might be presenting a more natural and functional antigen
than synthetic peptides as an immunogen. Furthermore, it iseasier to acquire mAbs against multipass-transmembraneproteins because no purified proteins are required for immunogens and screening.
Previously, we developed mAbs, such as programmed celldeath ligand 1 (PD-L1),(27) EpCAM,(34) TROP2,(35) CD20,(36)CD44,(28) and CD133,(26) using the CBIS method. In thisstudy, we successfully developed a sensitive anti-mCCR8
mAb, C8Mab-2, that can be used for flow cytometry using the
CBIS method. C8Mab-2 recognized overexpressed and endogenously expressed mCCR8 in each cell line (Fig. 2). Because CCR8 is considered the next pivotal immune checkpoint
molecule,(3) C8Mab-2 might be useful for investigating themolecular mechanism in various CCR8-related diseases using
mouse in vivo models.
Authors’ Contributions

T.T., R.N., J.T., T.N., M.Y., H.H., M.S., and T.A. performed experiments; M.K.K. designed the experiments; andT.T. and Y.K. wrote the article.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under Grant
Numbers: JP20am0401013 (Y.K.) and JP20am0101078(Y.K.), and by the Japan Society for the Promotion of
Science (JSPS) KAKENHI Grant Numbers: 20K16322(M.S.) and 19K07705 (Y.K.).
Supplementary Material

Supplementary Table S1References

1. Demaria O, Cornen S, Daeron M, Morel Y, Medzhitov R,
and Vivier E: Harnessing innate immunity in cancer therapy. Nature 2019;574:45–56.
2. Ohue Y, and Nishikawa H: Regulatory T (Treg) cells incancer: Can Treg cells be a new therapeutic target? Cancer
Sci 2019;110:2080–2089.
3. Karin N: Chemokines and cancer: New immune checkpoints for cancer therapy. Curr Opin Immunol 2018;51:
140–145.
4. Martens A, Wistuba-Hamprecht K, Geukes Foppen M,
Yuan J, Postow MA, Wong P, Romano E, Khammari A,
Dreno B, Capone M, Ascierto PA, Di Giacomo AM, Maio
M, Schilling B, Sucker A, Schadendorf D, Hassel JC, Ei-

69

gentler TK, Martus P, Wolchok JD, Blank C, Pawelec G,
Garbe C, and Weide B: Baseline peripheral blood biomarkers associated with clinical outcome of advanced
melanoma patients treated with ipilimumab. Clin Cancer
Res 2016;22:2908–2918.
5. Yano H, Andrews LP, Workman CJ, Vignali DAA: Intratumoral regulatory T cells: Markers, subsets and theirimpact on anti-tumor immunity. Immunology 2019;157:
232–247.
6. Bianchini R, Karagiannis SN, Jordakieva G, and JensenJarolim E: The role of IgG4 in the fine tuning of tolerancein IgE-mediated allergy and cancer. Int J Mol Sci 2020;21:5017.
7. Tanaka A, and Sakaguchi S: Regulatory T cells in cancer
immunotherapy. Cell Res 2017;27:109–118.
8. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram
P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow
M, Zhu Y, Wei S, Kryczek I, Daniel B, Gordon A, Myers
L, Lackner A, Disis ML, Knutson KL, Chen L, and Zou W:
Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced
survival. Nat Med 2004;10:942–949.
9. Barsheshet Y, Wildbaum G, Levy E, Vitenshtein A,
Akinseye C, Griggs J, Lira SA, and Karin N: CCR8(+)FOXp3(+) Treg cells as master drivers of immune
regulation. Proc Natl Acad Sci U S A 2017;114:6086–
6091.
10. Chensue SW, Lukacs NW, Yang TY, Shang X, Frait KA,
Kunkel SL, Kung T, Wiekowski MT, Hedrick JA, Cook
DN, Zingoni A, Narula SK, Zlotnik A, Barrat FJ, O’Garra
A, Napolitano M, and Lira SA: Aberrant in vivo T helper
type 2 cell response and impaired eosinophil recruitment in
CC chemokine receptor 8 knockout mice. J Exp Med 2001;
193:573–584.
11. Mikhak Z, Fukui M, Farsidjani A, Medoff BD, Tager AM,
and Luster AD: Contribution of CCR4 and CCR8 to
antigen-specific T(H)2 cell trafficking in allergic pulmonary inflammation. J Allergy Clin Immunol 2009;123:67–
73.e63.
12. Soler D, Chapman TR, Poisson LR, Wang L, Cote-Sierra J,
Ryan M, McDonald A, Badola S, Fedyk E, Coyle AJ,
Hodge MR, and Kolbeck R: CCR8 expression identifies
CD4 memory T cells enriched for FOXP3+ regulatory andTh2 effector lymphocytes. J Immunol 2006;177:6940–
6951.
13. Coghill JM, Fowler KA, West ML, Fulton LM, van Deventer H, McKinnon KP, Vincent BG, Lin K, PanoskaltsisMortari A, Cook DN, Blazar BR, and Serody JS: CC
chemokine receptor 8 potentiates donor Treg survival andis critical for the prevention of murine graft-versus-hostdisease. Blood 2013;122:825–836.
14. Iellem A, Mariani M, Lang R, Recalde H, PaninaBordignon P, Sinigaglia F, and D’Ambrosio D: Uniquechemotactic response profile and specific expression ofchemokine receptors CCR4 and CCR8 by CD4(+)CD25(+)
regulatory T cells. J Exp Med 2001;194:847–853.
15. Plitas G, Konopacki C, Wu K, Bos PD, Morrow M, Putintseva EV, Chudakov DM, and Rudensky AY: Regulatory
T cells exhibit distinct features in human breast cancer.
Immunity 2016;45:1122–1134.
16. De Simone M, Arrigoni A, Rossetti G, Gruarin P, Ranzani
V, Politano C, Bonnal RJP, Provasi E, Sarnicola ML,
Panzeri I, Moro M, Crosti M, Mazzara S, Vaira V, Bosari S,
Palleschi A, Santambrogio L, Bovo G, Zucchini N, Totis

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/21. For personal use only.

70

M, Gianotti L, Cesana G, Perego RA, Maroni N, Pisani
Ceretti A, Opocher E, De Francesco R, Geginat J, Stunnenberg HG, Abrignani S, and Pagani M: Transcriptionallandscape of human tissue lymphocytes unveils uniquenessof tumor-infiltrating T regulatory cells. Immunity 2016;45:
1135–1147.
17. Wang L, Simons DL, Lu X, Tu TY, Solomon S, Wang R,
Rosario A, Avalos C, Schmolze D, Yim J, Waisman J, and
Lee PP: Connecting blood and intratumoral Treg cell activity in predicting future relapse in breast cancer. Nat
Immunol 2019;20:1220–1230.
18. Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V,
Doherty G, Drebin JA, Strasberg SM, Eberlein TJ, Goedegebuure PS, and Linehan DC: Prevalence of regulatory T
cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 2002;169:2756–2761.
19. Goya I, Gutiérrez J, Varona R, Kremer L, Zaballos A, andMárquez G: Identification of CCR8 as the specific receptor
for the human beta-chemokine I-309: Cloning and molecular characterization of murine CCR8 as the receptor for
TCA-3. J Immunol 1998;160:1975–1981.
20. Hoelzinger DB, Smith SE, Mirza N, Dominguez AL, Manrique SZ, and Lustgarten J: Blockade of CCL1 inhibits T
regulatory cell suppressive function enhancing tumor immunity without affecting T effector responses. J Immunol
2010;184:6833–6842.
21. Li Z, Chan K, Qi Y, Lu L, Ning F, Wu M, Wang H, Wang
Y, Cai S, and Du J: Participation of CCL1 in snail-positive
fibroblasts in colorectal cancer contribute to 5fluorouracil/paclitaxel chemoresistance. Cancer Res Treat
2018;50:894–907.
22. Korbecki J, Grochans S, Gutowska I, Barczak K, andBaranowska-Bosiacka I: CC chemokines in a tumor: A
review of pro-cancer and anti-cancer properties of receptors
CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 Ligands.
Int J Mol Sci 2020;21:7619.
23. Bernardini G, Spinetti G, Ribatti D, Camarda G, Morbidelli
L, Ziche M, Santoni A, Capogrossi MC, and Napolitano M:
I-309 binds to and activates endothelial cell functions andacts as an angiogenic molecule in vivo. Blood 2000;96:4039–4045.
24. Das S, Sarrou E, Podgrabinska S, Cassella M, Mungamuri
SK, Feirt N, Gordon R, Nagi CS, Wang Y, Entenberg D,
Condeelis J, and Skobe M: Tumor cell entry into the lymph
node is controlled by CCL1 chemokine expressed by lymph
node lymphatic sinuses. J Exp Med 2013;210:1509–1528.
25. Villarreal DO, L’Huillier A, Armington S, Mottershead C,
Filippova EV, Coder BD, Petit RG, and Princiotta MF:
Targeting CCR8 induces protective antitumor immunityand enhances vaccine-induced responses in colon cancer.
Cancer Res 2018;78:5340–5348.
26. Itai S, Fujii Y, Nakamura T, Chang YW, Yanaka M, Saidoh
N, Handa S, Suzuki H, Harada H, Yamada S, Kaneko MK,
and Kato Y: Establishment of CMab-43, a sensitive andspecific Anti-CD133 monoclonal antibody, for immunohistochemistry. Monoclon Antib Immunodiagn Immunother 2017;36:231–235.

TANAKA ET AL.

27. Yamada S, Itai S, Nakamura T, Yanaka M, Chang YW,
Suzuki H, Kaneko MK, and Kato Y: Monoclonal antibody
L(1)Mab-13 detected human PD-L1 in lung cancers. Monoclon Antib Immunodiagn Immunother 2018;37:110–115.
28. Yamada S, Itai S, Nakamura T, Yanaka M, Kaneko MK,
and Kato Y: Detection of high CD44 expression in oral
cancers using the novel monoclonal antibody, C(44)Mab-5.
Biochem Biophys Rep 2018;14:64–68.
29. Schöneberg T, and Liebscher I: Mutations in G proteincoupled receptors: Mechanisms, pathophysiology and potential
therapeutic approaches. Pharmacol Rev 2021;73:89–119.
30. Jo M, and Jung ST: Engineering therapeutic antibodies
targeting G-protein-coupled receptors. Exp Mol Med 2016;
48:e207.
31. Vila-Caballer M, González-Granado JM, Zorita V, Abu
Nabah YN, Silvestre-Roig C, Del Monte-Monge A,
Molina-Sánchez P, Ait-Oufella H, Andrés-Manzano MJ,Sanz MJ, Weber C, Kremer L, Gutiérrez J, Mallat Z, andAndrés V: Disruption of the CCL1-CCR8 axis inhibits
vascular Treg recruitment and function and promotes atherosclerosis in mice. J Mol Cell Cardiol 2019;132:154–163.
32. Haque NS, Fallon JT, Pan JJ, Taubman MB, and Harpel PC:
Chemokine receptor-8 (CCR8) mediates human vascularsmooth muscle cell chemotaxis and metalloproteinase-2 secretion. Blood 2004;103:1296–1304.
33. Haque NS, Fallon JT, Taubman MB, and Harpel PC: The
chemokine receptor CCR8 mediates human endothelial cellchemotaxis induced by I-309 and Kaposi sarcoma
herpesvirus-encoded vMIP-I and by lipoprotein(a)stimulated endothelial cell conditioned medium. Blood
2001;97:39–45.
34. Kaneko MK, Ohishi T, Takei J, Sano M, Nakamura T,
Hosono H, Yanaka M, Asano T, Sayama Y, Harada H,
Kawada M, and Kato Y: Anti-EpCAM monoclonal antibody exerts antitumor activity against oral squamous cell
carcinomas. Oncol Rep 2020;44:2517–2526.
35. Sayama Y, Kaneko MK, Takei J, Hosono H, Sano M,
Asano T, and Kato Y: Establishment of a novel antiTROP2 monoclonal antibody TrMab-29 for immunohistochemical analysis. Biochem Biophys Rep 2021;25:100902.
36. Furusawa Y, Kaneko MK, and Kato Y: Establishment ofan Anti-CD20 Monoclonal Antibody (C(20)Mab-60) for
Immunohistochemical Analyses. Monoclon Antib Immunodiagn Immunother 2020;39:112–116.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: February 6, 2021
Accepted: February 28, 2021