MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 3, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0007

Original Articles

Development of Anti-Human CC Chemokine Receptor 9
Monoclonal Antibodies for Flow Cytometry

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

CC chemokine receptor 9 (CCR9) belongs to the beta chemokine receptor family and is mainly distributed on thesurface of immature T lymphocytes and enterocytes. This receptor is highly expressed in rheumatoid arthritis,
colitis, type 2 diabetes, and various tumors. Therefore, more sensitive monoclonal antibodies (mAbs) need to be
developed to predict the prognosis of many high CCR9 expression diseases. Because CCR9 is a structurally
unstable G protein-coupled receptor, it has been difficult to develop anti-CCR9 mAbs using the traditional method.
This study developed anti-human CCR9 (hCCR9) mAbs for flow cytometry using a Cell-Based Immunization andScreening (CBIS) method. Two mice were immunized with hCCR9-overexpressed Chinese hamster ovary (CHO)K1 cells (CHO/hCCR9), and hybridomas showing strong signals from CHO/hCCR9 and no signals from CHO-K1
cells were selected by flow cytometry. We established an anti-hCCR9 mAb, C9Mab-1 (IgG1, kappa), which
detected hCCR9 in MOLT-4 leukemia T lymphoblast cells and CHO/hCCR9 cells by flow cytometry. Our study
showed that an anti-hCCR9 mAb was developed more rapidly by the CBIS method than the previous method.
Keywords: CCR9, monoclonal antibody, CBIS, flow cytometry
Introduction

C

hemokines are classified into four groups, CXC, CC,
C, and CX3C chemokines, and are responsible forregulating cellular trafficking of various types of leukocytes.(1–4) Among them, CC chemokines are composed of 27
chemotactic cytokines(3,4) and have a C-C structure in which
the first two cysteines are adjacent to each other.(5) CC chemokine receptor 9 (CCR9) is a G protein-coupled receptor(GPCR) distributed mainly on the surface of immature
T lymphocytes and enterocytes. It belongs to the beta chemokine receptor family.(6) CC chemokine ligand 25 (CCL25), a
member of the CC chemokine subfamily, is also known as a
thymus-expressed chemokine and is the only ligand for
CCR9.(7) CCR9 and CCL25 play essential roles in inflammatory
cell proliferation and chemotaxis.(8)Overexpression of CCR9 and CCL25 is observed in many
cancers, including leukemia,(9) melanoma,(10) prostate cancer,(11) breast cancer,(12) lung cancer,(13) ovarian cancer,(14) andhepatocellular carcinoma.(12) CCR9 is also expressed in cells of
various other tissues, such as the trophoblast,(15) heart,(16) small
intestine,(17) and endometrial stroma,(18) and promotes cell proliferation,(19–22) tumorigenesis,(13,19,22) metastasis,(11,14,19,23,24)
and chemoresistance in tumors.(25–28) Furthermore, CCR9 isuseful for predicting lymph node metastasis in lung adenocarcinoma and may serve as a new prognostic biomarker.(13)12Also, CCR9 is highly expressed in rheumatoid arthritis,
colitis, and type 2 diabetes. In vivo studies have shown that it
may be an effective therapeutic target.(29–32) Therefore, CCR9
may be useful as a diagnostic agent to predict the prognosis ofmany diseases, and more sensitive monoclonal antibodies(mAbs) need to be developed. In this study, we established asensitive and specific mAb against human CCR9 (hCCR9).
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). MOLT-4 (a leukemia T
lymphoblast cell line) was obtained from the Japanese Collection of Research Bioresources ( JCRB) Cell Bank (Osaka,
Japan). The expression plasmid of hCCR9 (pCMV6neoCCR9Myc-DDK) was purchased from OriGene Technologies, Inc.,(Rockville, MD) and was transfected using Lipofectamine
LTX with Plus Reagent (Thermo Fisher Scientific, Inc.,
Waltham, MA). Stable transfectants were selected by limiting
dilution and cultivation in a medium containing 0.5 mg/mL
of G418 (Nacalai Tesque, Inc., Kyoto, Japan).
CHO-K1, P3U1, CHO/hCCR9, and MOLT-4 were
cultured in Roswell Park Memorial Institute (RPMI) 1640
medium (Nacalai Tesque, Inc.), supplemented with 10%

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL penicillin, 100 lg/mL streptomycin,
and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). Cells
were grown in a humidified incubator at 37C with an atmosphere of 5% CO2 and 95% air.
Antibodies

Anti-hCCR9 mAb (clone #112509) was purchased from
R&D systems (Minneapolis, MN). Secondary Alexa Fluor
488-conjugated anti-mouse IgG was purchased from Cell
Signaling Technology, Inc. (Danvers, MA).

NANAMIYA ET AL.
Results
Establishment of anti-hCCR9 mAbs

To produce anti-hCCR9 mAbs, we employed the CBIS
method using stable transfectants for both immunizationand flow cytometric screening (Fig. 1). Two mice were
immunized with CHO/hCCR9 cells, which overexpress
hCCR9. Hybridomas were seeded into 96-well plates, andCHO/hCCR9-positive and CHO-K1-negative wells wereselected. After limiting dilution, C9Mab-1 (IgG1, kappa)
was finally established.

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Hybridoma production

Female BALB/c mice (6 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). The animals were housed under
specific pathogen-free conditions. All animal experiments were
performed according to relevant guidelines and regulations tominimize animal suffering and distress in the laboratory. Animal experiments were approved by the Animal Care and Use
Committee of Tohoku University (Permit number: 2019NiA001). Mice were monitored daily for health during the full
4-week duration of the experiment. A reduction of more than
25% of total body weight was defined as a humane endpoint.
Mice were euthanized by cervical dislocation, and death was
verified by respiratory and cardiac arrest.
To develop mAbs against hCCR9, we employed theCell-Based Immunization and Screening (CBIS) method.(33)
Briefly, two BALB/c mice were immunized with CHO/hCCR9cells (1 · 108) by the intraperitoneal route together with the
Imject Alum (Thermo Fisher Scientific, Inc.). The procedureincluded three additional immunization 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 grownin an RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific, Inc.). Culture supernatants were screened by flow
cytometry.
Flow cytometry

Cells were harvested after brief exposure to 0.25% trypsin
and 1 mM ethylenediaminetetraacetic acid (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. The cells were then treated with Alexa Fluor
488-conjugated anti-mouse IgG (1:2000; Cell Signaling
Technology, Inc.). Fluorescence data were collected using theSA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of binding affinity by flow cytometry

CHO/hCCR9 was suspended in 100 lL of serially diluted
anti-hCCR9 mAbs (0.006–50 lg/mL), and Alexa Fluor 488conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.) was added. Fluorescence data were collected using
the BD FACSLyric (Becton, Dickinson and Company,
Franklin Lakes, NJ, USA). The dissociation constant (KD)was calculated by fitting binding isotherms to built-in, onesite binding models in GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA).

FIG. 1. Production of anti-hCCR9 mAbs. The procedureof the CBIS method. CHO/hCCR9 cells were immunized
into BALB/c mice using intraperitoneal injection. The
screening was performed by flow cytometry. CBIS, CellBased Immunization and Screening; CHO, Chinese hamster
ovary; hCCR9, human CC chemokine receptor 9; mAbs,
monoclonal antibodies.

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FIG. 2. Flow cytometry using anti-hCCR9. (A) CHO/hCCR9 cells were treated with 0.001–10 mg/mL of C9Mab-1 or#112509, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; filled, negative control. (B) CHO-K1
cells were treated with 0.001–10 mg/mL of C9Mab-1 or #112509, followed by treatment with Alexa Fluor 488-conjugatedanti-mouse IgG; filled, negative control. (C) MOLT-4 cells were treated with 0.001–10 mg/mL of C9Mab-1 or #112509,
followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; filled, negative control.
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Flow cytometry

We performed flow cytometry using C9Mab-1 against
CHO/hCCR9, CHO-K1, and MOLT-4. C9Mab-1 recognized
CHO/hCCR9 dose-dependently (Fig. 2A), but not CHO-K1(Fig. 2B). Another anti-hCCR9 mAb (clone #112509 from R&D
systems: positive control) also recognized CHO/hCCR9 dosedependently (Fig. 2A), but not CHO-K1 (Fig. 2B). Although
C9Mab-1 reacted with CHO/hCCR9 even at 0.001 lg/mL,
clone #112509 reacted with it in more than 0.1 lg/mL(Fig. 2A), indicating that the sensitivity of C9Mab-1 is higher
than that of clone #112509. C9Mab-1 also reacted with
MOLT-4 cells, which express endogenous hCCR9 dosedependently even in 0.001 lg/mL (Fig. 2C). In contrast, clone#112509 reacted with it in more than 0.1 lg/mL (Fig. 2C).

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Determination of the binding affinity of C9Mab-1

We then assessed the apparent binding affinity of C9Mab-1
with CHO/hCCR9 using flow cytometry. The KD of C9Mab-1
for CHO/hCCR9 was 1.2 · 10-9 M (Fig. 3), indicating that
C9Mab-1 possesses a high affinity for CHO/hCCR9 cells.
Discussion

Because the CBIS method does not require purified
proteins (Fig. 1), it has been possible to develop antibodies
against type I transmembrane and multitransmembraneproteins, such as GPCRs, which are difficult to be purified.
We have developed anti-CD20 mAbs(34,35) and anti-CD133
mAbs,(33) and many anti-podoplanin (PDPN) mAbs against
pig,(36) Tasmanian devil,(37) alpaca,(38) tiger,(39) whale,(40)goat,(41) horse,(42) and bear(43) PDPNs, anti-PD-L1 mAbs,(44)anti-CD19 mAbs,(45) and anti-CD44 mAbs(46) using the CBIS
method. This study established a highly sensitive and specificanti-hCCR9 mAb (clone C9Mab-1) that can be used in flow
cytometry using the CBIS method (Fig. 2). Importantly,
C9Mab-1 recognized endogenous hCCR9, which is expressedin MOLT-4 cells. Furthermore, the KD of C 9Mab-1 for
CHO/hCCR9 was 1.2 · 10-9 M (Fig. 3), indicating that
C9Mab-1 possesses a high binding affinity for hCCR9.
Chamorro et al.(47) and Somovilla-Crespo et al.(48) previously established an anti-hCCR9 mAb (clone 91R, mouse
IgG2b) useful for flow cytometry and Western blotting, and an

NANAMIYA ET AL.

anti-hCCR9 mAb (clone 92R, mouse IgG2a) useful only forflow cytometry, respectively, by immunizing mice with the
eukaryotic expression vector, including full-length hCCR9using a gene gun. The development of antibodies using thisgene gun was first reported by Tang et al.(49) for human
growth hormone. This immunization method does not require the adjustment of immunogens and can utilize thepost-translational modification system of immunized animals. Therefore, it is possible to develop antibodies thatrecognize the three-dimensional structure of the target molecule and can be applied to multiple transmembraneproteins.(50–55) However, the immune system might not respond in several conditions: (1) the expression level of thetarget molecule is low; (2) the target molecule is localized
intracellularly; (3) the extracellular region is too small. Also,
the gene gun requires expensive equipment.(56)Several commercially available anti-hCCR9 mAbs weredeveloped using hCCR9-transfected cells as an immunogen
without the need for protein purification (Supplementary
Table S1). An anti-hCCR9 mAb (clone #112509, mouse
IgG2a), used as a positive control in this study, was also immunized using hCCR9-transfected BaF3 mouse proB cellline. Those mAbs are often not applicable to Western blotting
because they might recognize the conformation of proteins at
the cell membrane, although they are useful for immunohistochemistry or flow cytometry.
In conclusion, we established the anti-hCCR9 mAb,
C 9Mab-1, applicable to flow cytometry using the CBIS
method. The results of our study could also be applied to themore efficient development of other anti-GPCR mAbs.
Authors’ Contributions

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

No competing financial interests exist.
Funding Information

This research was supported in part by AMED under Grant
Nos. JP20am0401013 (Y.K.) and JP20am0101078 (Y.K.),
and by JSPS KAKENHI Grant Nos. 20K16322 (M.S.) and
19K07705 (Y.K.).
Supplementary Material

Supplementary Table S1References

FIG. 3. Determination of the binding affinity of C9Mab-1.
CHO/hCCR9 was suspended in 100 mL of serially diluted
C9Mab-1 (0.006–50 mg/mL). Alexa Fluor 488-conjugatedanti-mouse IgG was added. Fluorescence data were collected using the BD FACSLyric.

1. Strydom N, and Rankin SM: Regulation of circulating
neutrophil numbers under homeostasis and in disease.
J Innate Immun 2013;5:304–314.
2. Zlotnik A, and Yoshie O: Chemokines: A new classification system and their role in immunity. Immunity 2000;12:
121–127.
3. Roy I, Evans DB, and Dwinell MB: Chemokines andchemokine receptors: Update on utility and challenges forthe clinician. Surgery 2014;155:961–973.

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

ANTI-HUMAN CCR9

4. Griffith JW, Sokol CL, and Luster AD: Chemokines andchemokine receptors: Positioning cells for host defense andimmunity. Annu Rev Immunol 2014;32:659–702.
5. Miller MC, and Mayo KH: Chemokines from a structural
perspective. Int J Mol Sci 2017;18:2088.
6. Tu Z, Xiao R, Xiong J, Tembo KM, Deng X, Xiong M, Liu
P, Wang M, and Zhang Q: CCR9 in cancer: Oncogenic roleand therapeutic targeting. J Hematol Oncol 2016;9:10.
7. Zaballos A, Gutiérrez J, Varona R, Ardavı́n C, and Márquez G: Cutting edge: Identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for thechemokine TECK. J Immunol 1999;162:5671–5675.
8. Qiuping Z, Jei X, Youxin J, Wei J, Chun L, Jin W, Qun W,
Yan L, Chunsong H, Mingzhen Y, Qingping G, Kejian Z,
Zhimin S, Qun L, Junyan L, and Jinquan T: CC chemokine
ligand 25 enhances resistance to apoptosis in CD4+ T cells
from patients with T-cell lineage acute and chronic lymphocytic leukemia by means of livin activation. Cancer Res
2004;64:7579–7587.
9. Mirandola L, Chiriva-Internati M, Montagna D, Locatelli
F, Zecca M, Ranzani M, Basile A, Locati M, Cobos E, Kast
WM, Asselta R, Paraboschi EM, Comi P, and Chiaramonte
R: Notch1 regulates chemotaxis and proliferation by controlling the CC-chemokine receptors 5 and 9 in T cell acutelymphoblastic leukaemia. J Pathol 2012;226:713–722.
10. Kühnelt-Leddihn L, Müller H, Eisendle K, Zelger B, andWeinlich G: Overexpression of the chemokine receptors
CXCR4, CCR7, CCR9, and CCR10 in human primary
cutaneous melanoma: A potential prognostic value for
CCR7 and CCR10? Arch Dermatol Res 2012;304:185–193.
11. Singh S, Singh UP, Stiles JK, Grizzle WE, and Lillard JW:
Expression and functional role of CCR9 in prostate cancer
cell migration and invasion. Clin Cancer Res 2004;10:8743–8750.
12. Zhang Z, Sun T, Chen Y, Gong S, Sun X, Zou F, and Peng
R: CCL25/CCR9 Signal promotes migration and invasionin hepatocellular and breast cancer cell lines. DNA Cell
Biol 2016;35:348–357.
13. Zhong Y, Jiang L, Lin H, Li B, Lan J, Liang S, Shen B, Lei
Z, and Zheng W: Expression of CC chemokine receptor 9
predicts poor prognosis in patients with lung adenocarcinoma. Diagn Pathol 2015;10:101.
14. Johnson EL, Singh R, Singh S, Johnson-Holiday CM,
Grizzle WE, Partridge EE, and Lillard JW: CCL25-CCR9interaction modulates ovarian cancer cell migration, metalloproteinase expression, and invasion. World J Surg
Oncol 2010;8:62.
15. Weingrill RB, Hoshida MS, Martinhago CD, Correa-Silva
S, Cardoso E, Palmeira P, Marinho CRF, and Bevilacqua E:
Chemokine (C-C motif) ligand 25 expressed by trophoblast
cells and leukocytes bearing its receptor Ccr9: An allianceduring embryo implantation? Am J Reprod Immunol 2018;
79:e12783.
16. Xu Z, Mei F, Liu H, Sun C, and Zheng Z: C-C motif chemokine receptor 9 exacerbates pressure overload-induced
cardiac hypertrophy and dysfunction. J Am Heart Assoc
2016;5:e003342.
17. Amiya T, Nakamoto N, Irie J, Taniki N, Chu PS, Koda Y,
Miyamoto K, Yamaguchi A, Shiba S, Morikawa R, Itoh H,
and Kanai T: C-C motif chemokine receptor 9 regulates
obesity-induced insulin resistance via inflammation of thesmall intestine in mice. Diabetologia 2021;64:603–617.
18. Wang Y, Yu J, Luo X, Wang X, Li M, Wang L, and Li D:
Abnormal regulation of chemokine TECK and its receptor

105

CCR9 in the endometriotic milieu is involved in pathogenesis of endometriosis by way of enhancing invasivenessof endometrial stromal cells. Cell Mol Immunol 2010;7:
51–60.
19. Li B, Wang Z, Zhong Y, Lan J, Li X, and Lin H: CCR9CCL25 interaction suppresses apoptosis of lung cancer
cells by activating the PI3K/Akt pathway. Med Oncol
2015;32:66.
20. Shen X, Mailey B, Ellenhorn JD, Chu PG, Lowy AM, andKim J: CC chemokine receptor 9 enhances proliferation inpancreatic intraepithelial neoplasia and pancreatic cancer
cells. J Gastrointest Surg 2009;13:1955–1962; discussion
1962.
21. Youn BS, Kim YJ, Mantel C, Yu KY, and Broxmeyer HE:
Blocking of c-FLIP(L)—independent cycloheximide-induced
apoptosis or Fas-mediated apoptosis by the CC chemokine
receptor 9/TECK interaction. Blood 2001;98:925–933.
22. Zhang Z, Qin C, Wu Y, Su Z, Xian G, and Hu B: CCR9 as
a prognostic marker and therapeutic target in hepatocellular
carcinoma. Oncol Rep 2014;31:1629–1636.
23. Zhou B, Leng J, Hu M, Zhang L, Wang Z, Liu D, Tong X,
Yu B, Hu Y, Deng C, Liu Y, and Zhang Q: Ezrin is a key
molecule in the metastasis of MOLT4 cells induced by
CCL25/CCR9. Leuk Res 2010;34:769–776.
24. Heinrich EL, Arrington AK, Ko ME, Luu C, Lee W, Lu J,
and Kim J: Paracrine activation of chemokine receptor
CCR9 enhances the invasiveness of pancreatic cancer cells.
Cancer Microenviron 2013;6:241–245.
25. Johnson EL, Singh R, Johnson-Holiday CM, Grizzle WE,
Partridge EE, Lillard JW, and Singh S: CCR9 interactions
support ovarian cancer cell survival and resistance tocisplatin-induced apoptosis in a PI3K-dependent and FAKindependent fashion. J Ovarian Res 2010;3:15.
26. Sharma PK, Singh R, Novakovic KR, Eaton JW, Grizzle
WE, and Singh S: CCR9 mediates PI3K/AKT-dependent
antiapoptotic signals in prostate cancer cells and inhibitionof CCR9-CCL25 interaction enhances the cytotoxic effectsof etoposide. Int J Cancer 2010;127:2020–2030.
27. Johnson-Holiday C, Singh R, Johnson EL, Grizzle WE,
Lillard JW, and Singh S: CCR9-CCL25 interactions promote cisplatin resistance in breast cancer cell through Akt
activation in a PI3K-dependent and FAK-independent
fashion. World J Surg Oncol 2011;9:46.
28. Lee S, Heinrich EL, Li L, Lu J, Choi AH, Levy RA,
Wagner JE, Yip ML, Vaidehi N, and Kim J: CCR9mediated signaling through b-catenin and identification of anovel CCR9 antagonist. Mol Oncol 2015;9:1599–1611.
29. Yokoyama W, Kohsaka H, Kaneko K, Walters M, Takayasu
A, Fukuda S, Miyabe C, Miyabe Y, Love PE, Nakamoto N,
Kanai T, Watanabe-Imai K, Charvat TT, Penfold ME, Jaen
J, Schall TJ, Harigai M, Miyasaka N, and Nanki T: Abrogation of CC chemokine receptor 9 ameliorates collageninduced arthritis of mice. Arthritis Res Ther 2014;16:445.
30. Wurbel MA, McIntire MG, Dwyer P, and Fiebiger E:
CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis. PLoS One 2011;
6:e16442.
31. Schmutz C, Cartwright A, Williams H, Haworth O, Williams JH, Filer A, Salmon M, Buckley CD, and Middleton
J: Monocytes/macrophages express chemokine receptor
CCR9 in rheumatoid arthritis and CCL25 stimulates theirdifferentiation. Arthritis Res Ther 2010;12:R161.
32. Atanes P, Lee V, Huang GC, and Persaud SJ: The role of
the CCL25-CCR9 axis in beta-cell function: Potential forDownloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

106

therapeutic intervention in type 2 diabetes. Metabolism
2020;113:154394.
33. 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 Immunother2017;36:231–235.
34. Furusawa Y, Kaneko MK, and Kato Y: Establishment of
C20Mab-11, a novel anti-CD20 monoclonal antibody, forthe detection of B cells. Oncol Lett 2020;20:1961–1967.
35. Furusawa Y, Kaneko MK, and Kato Y: Establishment of ananti-CD20 monoclonal antibody (C20Mab-60) for immunohistochemical analyses. Monoclon Antib Immunodiagn
Immunother 2020;39:112–116.
36. Kato Y, Yamada S, Furusawa Y, Itai S, Nakamura T, Yanaka M, Sano M, Harada H, Fukui M, and Kaneko MK:
PMab-213: A monoclonal antibody for immunohistochemical analysis against pig podoplanin. Monoclon AntibImmunodiagn Immunother 2019;38:18–24.
37. Furusawa Y, Yamada S, Itai S, Nakamura T, Takei J, Sano
M, Harada H, Fukui M, Kaneko MK, and Kato Y: Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin.
Biochem Biophys Rep 2019;18:100631.
38. Kato Y, Furusawa Y, Yamada S, Itai S, Takei J, Sano M,
and Kaneko MK: Establishment of a monoclonal antibody
PMab-225 against alpaca podoplanin for immunohistochemical analyses. Biochem Biophys Rep 2019;18:100633.
39. Furusawa Y, Kaneko MK, Nakamura T, Itai S, Fukui M,
Harada H, Yamada S, and Kato Y: Establishment of a
monoclonal antibody PMab-231 for tiger podoplanin. Monoclon Antib Immunodiagn Immunother 2019;38:89–95.
40. Kato Y, Furusawa Y, Itai S, Takei J, Nakamura T, Sano M,
Harada H, Yamada S, and Kaneko MK: Establishment ofan anticetacean podoplanin monoclonal antibody PMab237 for immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2019;38:108–113.
41. Furusawa Y, Yamada S, Nakamura T, Sano M, Sayama Y,
Itai S, Takei J, Harada H, Fukui M, Kaneko MK, and Kato
Y: PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin. Heliyon 2019;5:e02063.
42. Furusawa Y, Yamada S, Itai S, Nakamura T, Yanaka M,
Sano M, Harada H, Fukui M, Kaneko MK, and Kato Y:
PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin. Biochem Biophys
Rep 2019;18:100616.
43. Furusawa Y, Takei J, Sayama Y, Yamada S, Kaneko MK,
and Kato Y: Development of an anti-bear podoplanin
monoclonal antibody PMab-247 for immunohistochemical
analysis. Biochem Biophys Rep 2019;18:100644.
44. Yamada S, Itai S, Nakamura T, Yanaka M, Chang YW,
Suzuki H, Kaneko MK, and Kato Y: Monoclonal antibody
L1Mab-13 detected human PD-L1 in lung cancers. Monoclon Antib Immunodiagn Immunother 2018;37:110–115.
45. Yamada S, Kaneko MK, Sayama Y, Asano T, Sano M,
Yanaka M, Nakamura T, Okamoto S, Handa S, Komatsu Y,
Nakamura Y, Furusawa Y, Takei J, and Kato Y: Development of novel mouse monoclonal antibodies against
human CD19. Monoclon Antib Immunodiagn Immunother2020;39:45–50.

NANAMIYA ET AL.

46. 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, C44Mab-5.
Biochem Biophys Rep 2018;14:64–68.
47. Chamorro S, Vela M, Franco-Villanueva A, Carramolino L,
Gutierrez J, Gomez L, Lozano M, Salvador B, Garcia-Gallo
M, Martinez AC, and Kremer L: Antitumor effects of a
monoclonal antibody to human CCR9 in leukemia cell
xenografts. MAbs 2014;6:1000–1012.
48. Somovilla-Crespo B, Martin Monzon MT, Vela M, CorralizaGorjon I, Santamaria S, Garcia-Sanz JA, and Kremer L:
92R monoclonal antibody inhibits human CCR9(+) leukemia cells growth in NSG mice xenografts. Front Immunol 2018;9:77.
49. Tang DC, DeVit M, and Johnston SA: Genetic immunization is a simple method for eliciting an immune response.
Nature 1992;356:152–154.
50. Hazen M, Bhakta S, Vij R, Randle S, Kallop D, Chiang V,
Hotzel I, Jaiswal BS, Ervin KE, Li B, Weimer RM, Polakis
P, Scheller RH, Junutula JR, and Hongo JA: An improved
and robust DNA immunization method to develop antibodies against extracellular loops of multi-transmembraneproteins. MAbs 2014;6:95–107.
51. Liu S, Wang S, and Lu S: DNA immunization as a technology platform for monoclonal antibody induction. Emerg
Microbes Infect 2016;5:e33.
52. Vij R, Lin Z, Schneider K, Seshasayee D, and Koerber JT:
Analysis of the effect of promoter type and skin pretreatment on antigen expression and antibody response
after gene gun-based immunization. PLoS One 2018;13:e0197962.
53. Palomba ML, Roberts WK, Dao T, Manukian G, GuevaraPatino JA, Wolchok JD, Scheinberg DA, and Houghton
AN: CD8+ T-cell-dependent immunity following xenogeneic DNA immunization against CD20 in a tumor challenge model of B-cell lymphoma. Clin Cancer Res 2005;
11:370–379.
54. Eden T, Menzel S, Wesolowski J, Bergmann P, Nissen M,
Dubberke G, Seyfried F, Albrecht B, Haag F, and KochNolte F: A cDNA immunization strategy to generate nanobodies against membrane proteins in native conformation.
Front Immunol 2017;8:1989.
55. Wang S, and Lu S: DNA immunization. Curr Protoc Microbiol 2013;31:18.3.1–18.3.24.
56. O’Brien JA, and Lummis SC: Biolistic transfection ofneuronal cultures using a hand-held gene gun. Nat Protoc
2006;1:977–981.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: February 9, 2021
Accepted: February 25, 2021