MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 2, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0032
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Establishment of a Novel Anti-Mouse CCR1
Monoclonal Antibody C1Mab-6

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Tsunenori Ouchida,* Yu Isoda,* Takuro Nakamura, Miyuki Yanaka, Tomohiro Tanaka, Saori Handa,
Mika K. Kaneko, Hiroyuki Suzuki, and Yukinari Kato

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors and is expressedon myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion
in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy.
Although CCR1 is an attractive target for solid tumors and hematological malignancies, therapeutic agents for
CCR1 have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody(mAb), C1Mab-6 (rat IgG2b, kappa), using the Cell-Based Immunization and Screening method. Flow cytometry and Western blot analyses showed that C1Mab-6 recognizes mCCR1 specifically. The dissociation
constant of C1Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9 · 10-9 M,
indicating that C1Mab-6 possesses a high affinity to mCCR1. These results suggest that C1Mab-6 could be auseful tool for targeting mCCR1 in preclinical mouse models.
Keywords: mouse CCR1, CBIS method, monoclonal antibody, flow cytometry, Western blot

and MMP9.7,8 Liver metastasis is observed in 25% of patientswith colorectal cancers and is the main cause of death.9,10
Analysis of disease-free survival (DFS) after curative liverresection showed that the DFS of patients with CCL15positive liver metastases is shorter than that of CCL15negative.8 More CCR1-positive myeloid cells accumulatedaround the CCL15-positive metastases than CCL15-negative
ones.8 CCR1 signaling also plays a critical role in the development of a carcinogen-induced hepatocellular carcinoma
model. The CCR1 deficiency in mice reduced the tumor
development through suppression of neovascularization andintratumoral Kupffer cell accumulation.11
Tumor microenvironment (TME) influences the tumor
progression and responsiveness to therapies.12 Modificationof TME is a strategy for improving the effect of antitumor
treatment.13 Although CCR1 is an attractive target for modifying TME and suppressing tumor progression, few preclinical studies using anti-mouse CCR1 (mCCR1)
monoclonal antibodies (mAbs) have been reported.14
We previously developed mAbs for mouse CCR3,15
CCR8,16 and CXCR417 using the Cell-Based Immunization

Introduction

C-C motif chemokine receptor 1 (CCR1), also known
as CD191, is a member of G-protein-coupled receptors
for many chemokines, such as CCL3, CCL5, CCL7, CCL8,
CCL13, CCL14, CCL15, CCL16, and CCL23 in humans.1
CCR1, which is expressed on neutrophils or macrophages, isimportant for the infiltration of these cells. The leukocyte
infiltration process consists of three steps: (i) tethering, rolling, and arrest on the vessel wall, (ii) crawling on the endothelium, and (iii) transendothelial migration.2 CCR1
contributes to stable adhesion of crawling neutrophils on theendothelium in the joints in the K/BxN serum transferred
mice (a murine model for rheumatoid arthritis [RA]).3,4
CCR1 signaling promotes tumor invasion and metastasis.
The cis-Apc(+/Delta716) Smad4(+/-) mutant mice develop
spontaneous invasive colorectal cancers.5 However, CCR1knockout in the background decreases the invasiveness ofthese tumors in vivo.6 CCR1-positive myeloid cells accumulate at the tumor invasive front and drive tumor invasion
by producing the matrix metalloproteinases, such as MMP2

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
*These authors contributed equally to this work.

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and Screening (CBIS) method.18–25 In this study, we established a novel anti-mCCR1 mAb using the CBIS method andevaluated its applications.
Materials and Methods
Antibodies

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The anti-mouse CCR1 mAb (clone S15040E)26 was purchased from BioLegend (San Diego, CA). We previously
developed RcMab-1 against isocitrate dehydrogenase 1.27 An
anti-DYKDDDDK mAb (clone 1E6) was purchased from
FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Alexa Fluor 488-conjugated anti-rat IgG was purchasedfrom Cell Signaling Technology, Inc. (Danvers, MA).
Horseradish peroxidase (HRP)-conjugated anti-rat IgG(A9542) and HRP-conjugated anti-mouse IgG (P0260) werepurchased from Sigma-Aldrich (St. Louis, MO) and Agilent
Technologies, Inc. (Santa Clara, CA), respectively.

OUCHIDA ET AL.

performed without an adjuvant every week. We performed a
final booster immunization of 1 · 109 cells of CHO/mCCR1
intraperitoneally 2 days before harvesting splenocytes. We
fused the harvested splenocytes with P3U1 cells using
polyethylene glycol 1500 (PEG1500; Roche Diagnostics,
Indianapolis, IN).
Hybridoma cells were cultured in the RPMI-1640 medium,supplemented as shown above. We further added hypoxanthine, aminopterin, and thymidine (Thermo Fisher Scientific,Inc.), 5% Briclone (NICB, Dublin, Ireland), and 5 lg/mL ofplasmocin (InvivoGen) into the medium. The hybridoma
supernatants were screened by flow cytometry using LN229/
mCCR1 and parental LN229. The culture supernatants ofhybridomas were filtrated and purified using Ab-Capcher
Extra (ProteNova, Kagawa, Japan).
Flow cytometry

A 5-week-old Sprague–Dawley rat was purchased from
CLEA Japan (Tokyo, Japan). The animal was housed under
specific pathogen-free conditions. All animal experiments
were approved by the Animal Care and Use Committee of
Tohoku University (Permit No. 2022MdA-001).

CHO-K1, CHO/mCCR1, LN229, and LN229/mCCR1
cells were harvested by exposure to 1 mM EDTA (Nacalai
Tesque, Inc.). The cells were washed with 0.1% bovine serum
albumin in phosphate-buffered saline (PBS) and treated with
anti-mCCR1 mAbs for 30 minutes at 4C. After washing, thecells were treated with Alexa Fluor 488-conjugated anti-rat
IgG. Flow cytometric analysis was performed using theSA3800 Cell Analyzer (Sony Corp.).

Cell lines

Determination of the binding affinity by flow cytometry

LN229, Chinese hamster ovary (CHO)-K1, andP3X63Ag8U.1 (P3U1) cells were obtained from the American Type Culture Collection (Manassas, VA). pCMV6neomyc-DDK vector with mCCR1 (Accession No. NM_009912)
was purchased from OriGene Technologies, Inc. (Rockville,
MD). The plasmid was transfected into the cell lines using a
Neon transfection system (Thermo Fisher Scientific, Inc.,
Waltham, MA). Subsequently, LN229 and CHO-K1, whichstably overexpressed mCCR1 with C-terminal myc-DDK
tags (hereinafter described as LN229/mCCR1 and CHO/
mCCR1, respectively) were established using a cell sorter(SH800; Sony Corp., Tokyo, Japan), following cultivation ina medium containing 0.5 mg/mL G418 (Nacalai Tesque, Inc.,
Kyoto, Japan).
CHO-K1, P3U1, and CHO/mCCR1 were also cultured in a
Roswell Park Memorial Institute (RPMI)-1640 medium(Nacalai Tesque, Inc.) that was supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific,Inc.), 100 units/mL penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). LN229
and LN229/mCCR1 were cultured in a Dulbecco’s modified
Eagle medium (Nacalai Tesque, Inc.) that was supplemented
as shown above. Cells were grown in a humidified incubator,
which was supplied with 5% CO2 and 95% air at 37C.

CHO/mCCR1 and LN229/mCCR1 were suspended in
100 lL serially diluted anti-mCCR1 mAbs (100 lg/mL to
6 ng/mL), after which Alexa Fluor 488-conjugated anti-rat
IgG (1:200) were added. Fluorescence data were subsequently collected, using the SA3800 Cell Analyzer (SonyCorp.), following the calculation of the KD by fitting thebinding isotherms into the built-in; one-site binding model in
GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA).

Animals

Hybridoma production

For developing anti-mCCR1 mAbs, a rat was immunizedintraperitoneally with 1 · 109 cells of CHO/mCCR1. The
immunogen was harvested after brief exposure to 1 mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque,Inc.). We added Alhydrogel adjuvant 2% (InvivoGen, San
Diego, CA) as an adjuvant in the first immunization. Three
additional injections of 1 · 109 cells of CHO/mCCR1 wereWestern blot analysis

Cell lysates (10 lg) were boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.), after which
proteins were separated on 5%–20% polyacrylamide gels
(FUJIFILM Wako Pure Chemical Corporation) and transferred to polyvinylidene difluoride membranes (Merck
KGaA). After blocking with 4% skim milk (Nacalai Tesque,Inc.) in 0.05% Tween 20-containing PBS, they were incubated with 0.1 lg/mL of anti-DYKDDDDK or 1 lg/mL of theother primary mAbs. Then, they were incubated with HRPconjugated anti-rat immunoglobulins (for anti-mCCR1
mAbs and RcMab-1; diluted 1:10,000) or anti-mouse immunoglobulins (for an anti-DYKDDDDK mAb; diluted
1:1000). Finally, protein bands were detected using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation)with a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Results
Establishment of anti-mCCR1 antibodies

We used the CBIS method to establish novel anti-mCCR1
antibodies (Fig. 1). The CBIS method is a high-throughput
method for the establishment of antibodies against membrane proteins. CBIS method consists of two main steps:

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ANTI-MOUSE CCR1 MAB

69

FIG. 1. The scheme of establishment of C1Mab-6 by CBIS method. (A) CHO/mCCR1 cells were injected intraperitoneally into a Sprague–Dawley rat. The splenocytes of the rat were fused with P3U1 cells and seeded in 96 well plates.
(B) The culture supernatants of each well were screened through flow cytometry to distinguish anti-mCCR1 mAb-producing
hybridomas. C1Mab-6 was established by limiting dilution and some additional screenings. CBIS, Cell-Based Immunizationand Screening; CHO, Chinese hamster ovary; mAb, monoclonal antibody; mCCR1, mouse C-C motif chemokine receptor 1.
immunization of antigen-overexpressing cells (Fig. 1A) andscreening of hybridoma supernatants using flow cytometry(Fig. 1B). After immunization of CHO/mCCR1 and screening of hybridoma supernatants with LN229/mCCR1, C1Mab6 (rat IgG2b, kappa) was finally developed.

at a concentration of 0.01 lg/mL. C1Mab-6 and S15040E
did not react with LN229 and CHO-K1 even at 10 lg/mL(Figs. 2B and 3B), indicating that both mAbs are specific to
mCCR1.

Flow cytometry using anti-mCCR1 mAbs

Determination of dissociation constantsof anti-mCCR1 mAbs

To check the specificity and reactivity of C1Mab-6, we
performed flow cytometry against LN229/mCCR1, LN229,
CHO/mCCR1, and CHO-K1. S15040E is a commerciallyavailable anti-mCCR1 mAb.26 C1Mab-6 bound to LN229/
mCCR1 and CHO/mCCR1 cells in a dose-dependent manner (Figs. 2A and 3A). In contrast, S15040E exhibited
lower reactivity against both cells compared to C1Mab-6

Next, we determined the dissociation constants (KD) of
anti-mCCR1 mAbs, which is expressed on the cell surface
using flow cytometry. The geometric mean of the fluorescence intensity at each concentration of C1Mab-6 andS15040E was plotted. By fitting one-site binding models, theKD values of C1Mab-6 and S15040E for LN299/mCCR1
were determined as 1.0 · 10-8 and 1.7 · 10-7 M, respectively

OUCHIDA ET AL.

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FIG. 2. Flow cytometry of anti-mCCR1 mAbs against LN229/mCCR1. LN229/mCCR1 (A) and LN229 (B) cells weretreated with 0.01–10 mg/mL of C1Mab-6 or S15040E, followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG
antibodies. The red lines show the cells treated with each mAb. The black lines show the cells treated with blocking bufferand Alexa Fluor 488-conjugated anti-rat IgG antibodies (negative control).

(Fig. 4A, B). Furthermore, the KD values of C1Mab-6 andS15040E for CHO/mCCR1 were calculated as 3.9 · 10-9
and 3.5 · 10-8 M, respectively (Fig. 4C, D). These results
indicate that C1Mab-6 possesses much higher affinity thanS15040E.

Western blot using anti-mCCR1 mAbs

Finally, we performed Western blot analysis using antimCCR1 mAbs. Lysates of CHO-K1 and CHO/mCCR1 wereprobed. The molecular weight of mCCR1-mycDDK is about

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ANTI-MOUSE CCR1 MAB

FIG. 3. Flow cytometry of anti-mCCR1 mAbs against CHO/mCCR1. CHO/mCCR1 (A) and CHO-K1 (B) cells weretreated with 0.01–10 mg/mL of C1Mab-6 or S15040E, followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG
antibodies. The red lines show the cells treated with each mAb. The black lines show the cells treated with blocking bufferand Alexa Fluor 488-conjugated anti-rat IgG antibodies (negative control).
44,000. C1Mab-6 and an anti-DYKDDDDK mAb (clone 1E6)
detected mCCR1 as about a 44-kDa band from lysates of
CHO/mCCR1 (Fig. 5), although S15040E could not detect
mCCR1 in the lysates of CHO/mCCR1. C1Mab-6 did not detect
any bands from the lysates of CHO-K1, indicating that C1Mab6 can recognize mCCR1 specifically in Western blot analysis.

Discussion

Anti-CCR1 therapies are potential treatments to improve
the quality of life of multiple myeloma (MM) patients. MM
is an incurable plasma B cell malignancy.28 Systemic osteopenia is observed in most MM patients.29 Osteopenia

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OUCHIDA ET AL.

FIG. 4. Determination of dissociation constants of anti-mCCR1 mAbs against mCCR1-overexpressed cells. The binding
affinity of C1Mab-6 and S15040E against LN229/mCCR1 cells (A, B) and CHO/mCCR1 cells (C, D) were determined byflow cytometry. The dots show the geometric mean of fluorescence intensity at each concentration. The solid lines are thefitting curve calculated by GraphPad PRISM 6.
causes bone fractures and impacts their quality of life.
Serum levels of CCL3 are elevated in MM patients andcorrelate with the extent of bone disease.30 MM cells produce CCL3, which activates osteoclast.31,32 Neutralizationof CCL3 with anti-CCL3 antibodies blocks osteolysis inmice.32 CCL3 is a ligand activating CCR1 and CCR5.1

CCX721, a small molecule antagonist against CCR1 not
CCR5, dramatically reduced the osteolysis induced by
5TGM1, a murine MM cell line.33 Additionally, CCX721
also reduces tumor burden of 5TGM1 cells inoculated intravenously.33 CCX721 does not block the proliferation of
5TGM1 cells in vitro or inoculated subcutaneously.33 These

FIG. 5. Western blot analysis using anti-mCCR1 mAbs. Cell lysates of CHO-K1 and CHO/mCCR1 cells were electrophoresed, and proteins were transferred onto PVDF membranes. After blocking, membranes were incubated with C1Mab-6,
S15040E, anti-DYKDDDDK mAb (1E6), or RcMab-1, and then incubated with HRP-conjugated anti-rat IgG or anti-mouse
IgG. RcMab-1 (an anti-IDH1 mAb) (an internal control). HRP, horseradish peroxidase; IDH1, isocitrate dehydrogenase 1;
PVDF, polyvinylidene difluoride.

ANTI-MOUSE CCR1 MAB

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results indicated that CCR1 blockage inhibits tumor burden
and osteolysis by MM through modifying the bone marrow
microenvironment.
RA is a chronic inflammatory disease characterized bymassive infiltration of synovial tissue and synovial fluid withimmune cells in affected joints.34 CCR1 is abundantly expressed by monocytes/macrophages of RA, which mediate theinflammation at these sites.35 CCX354, an analog of CCX721,
was developed for the treatment of RA.36 CCX354 exhibited
good safety and clinical activities to RA patients in a phase II
study.37 However, once daily oral administration is needed
because the half-life of CCX354 in the plasma of humans
is *6 hours.36 Antibodies are therapeutic drugs stable in serum (the half-life of antibodies is around 3 weeks).38 C1Mab-6
can bind to mCCR1-expressed cells with a high affinity(Fig. 4). C1Mab-6 could be useful tools for developing antimCCR1 therapies in preclinical murine models.
Acknowledgment

This study was previously published in preprint.org (doi:
10.20944/preprints202312.2059.v1).
Authors’ Contributions

T.O., Y.I., T.N., M.Y., and T.T. performed the experiments. M.K.K. and Y.K. designed the experiments. T.O. and
T.T. analyzed the data. T.O., H.S., and Y.K. wrote themanuscript. All authors have read and agreed to the publishedversion of the manuscript.
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: JP23ama121008 (to Yukinari Kato), JP23am0401013(to Yukinari Kato), 23bm1123027h0001 (to Yukinari Kato),and JP23ck0106730 (to Yukinari Kato), and by the Japan Society for the Promotion of Science ( JSPS) Grants-in-Aid for
Scientific Research (KAKENHI) grant numbers: 23K19494(to Tsunenori Ouchida), 21K20789 (to Tomohiro Tanaka),
22K06995 (to Hiroyuki Suzuki), 21K07168 (to Mika K.
Kaneko), and 22K07224 (to Yukinari Kato).
References

1. Stone MJ, Hayward JA, Huang C, et al. Mechanisms ofregulation of the chemokine-receptor network. Int J Mol
Sci 2017;18:342; doi: 10.3390/ijms18020342
2. Ley K, Laudanna C, Cybulsky MI, et al. Getting to the siteof inflammation: The leukocyte adhesion cascade updated.
Nat Rev Immunol 2007;7:678–689; doi: 10.1038/nri2156
3. Miyabe Y, Miyabe C, Murooka TT, et al. Complement C5a
receptor is the key initiator of neutrophil adhesion igniting
immune complex-induced arthritis. Sci Immunol 2017;2:
eaaj2195; doi: 10.1126/sciimmunol.aaj2195
4. Christensen AD, Haase C, Cook AD, et al. K/BxN serumtransfer arthritis as a model for human inflammatory arthritis. Front Immunol 2016;7:213; doi: 10.3389/fimmu.2016.00213

73

5. Takaku K, Oshima M, Miyoshi H, et al. Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4)
and Apc genes. Cell 1998;92:645–656; doi: 10.1016/s00928674(00)81132-0
6. Kitamura T, Kometani K, Hashida H, et al. SMAD4-deficient
intestinal tumors recruit CCR1+ myeloid cells that promoteinvasion. Nat Genet 2007;39:467–475; doi: 10.1038/ng1997
7. Hirai H, Fujishita T, Kurimoto K, et al. CCR1-mediatedaccumulation of myeloid cells in the liver microenvironment
promoting mouse colon cancer metastasis. Clin Exp Metastasis 2014;31:977–989; doi: 10.1007/s10585-014-9684-z
8. Itatani Y, Kawada K, Fujishita T, et al. Loss of SMAD4
from colorectal cancer cells promotes CCL15 expression to
recruit CCR1+ myeloid cells and facilitate liver metastasis.
Gastroenterology 2013;145:1064–1075.e1011; doi: 10.1053/j.gastro.2013.07.033
9. Engstrand J, Nilsson H, Stromberg C, et al. Colorectal
cancer liver metastases—A population-based study on incidence, management and survival. BMC Cancer 2018;18:
78; doi: 10.1186/s12885-017-3925-x
10. Zhou H, Liu Z, Wang Y, et al. Colorectal liver metastasis:
Molecular mechanism and interventional therapy. Signal
Transduct Target Ther 2022;7:70; doi: 10.1038/s41392022-00922-2
11. Yang X, Lu P, Fujii C, et al. Essential contribution of achemokine, CCL3, and its receptor, CCR1, to hepatocellular carcinoma progression. Int J Cancer 2006;118:1869–
1876; doi: 10.1002/ijc.21596
12. de Visser KE, Joyce JA. The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. Cancer
Cell 2023;41:374–403; doi: 10.1016/j.ccell.2023.02.016
13. Cheng YQ, Wang SB, Liu JH, et al. Modifying the tumour
microenvironment and reverting tumour cells: New strategies for treating malignant tumours. Cell Prolif 2020;53:
e12865; doi: 10.1111/cpr.12865
14. Kiyasu Y, Kawada K, Hirai H, et al. Disruption of CCR1mediated myeloid cell accumulation suppresses colorectal
cancer progression in mice. Cancer Lett 2020;487:53–62;
doi: 10.1016/j.canlet.2020.05.028
15. Asano T, Suzuki H, Tanaka T, et al. C(3)Mab-3: A
monoclonal antibody for mouse CC chemokine receptor 3
for flow cytometry. Monoclon Antib Immunodiagn Immunother 2022;41:74–79; doi: 10.1089/mab.2021.0062
16. Tanaka T, Nanamiya R, Takei J, et al. Development of antimouse CC chemokine receptor 8 monoclonal antibodies forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:65–70; doi: 10.1089/mab.2021.0005
17. Ouchida T, Suzuki H, Tanaka T, et al. Cx(4)Mab-1: A
novel anti-mouse CXCR4 monoclonal antibody for flow
cytometry. Monoclon Antib Immunodiagn Immunother2024;43(1):10–16; doi: 10.1089/mab.2023.0023
18. Hosono H, Asano T, Takei J, et al. Development of an antielephant Podoplanin monoclonal antibody PMab-265 forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:141–145; doi: 10.1089/mab.2021.0015
19. Takei J, Asano T, Nanamiya R, et al. Development of antihuman T cell immunoreceptor with Ig and ITIM domains(TIGIT) monoclonal antibodies for flow cytometry.
Monoclon Antib Immunodiagn Immunother 2021;40:71–
75; doi: 10.1089/mab.2021.0006
20. Tanaka T, Asano T, Sano M, et al. Development of
monoclonal antibody PMab-269 against California Sea
Lion Podoplanin. Monoclon Antib Immunodiagn Immunother 2021;40:124–133; doi: 10.1089/mab.2021.0011

Downloaded by Yukinari Kato from www.liebertpub.com at 04/09/24. For personal use only.

74

21. Goto N, Suzuki H, Tanaka T, et al. Development of a
monoclonal antibody PMab-292 against ferret Podoplanin.
Monoclon Antib Immunodiagn Immunother 2022;41:101–
109; doi: 10.1089/mab.2021.0067
22. Li G, Suzuki H, Asano T, et al. Development of a novelanti-EpCAM monoclonal antibody for various applications. Antibodies (Basel) 2022;11:20041; doi: 10.3390/
antib11020041
23. Nanamiya R, Suzuki H, Takei J, et al. Development of
monoclonal antibody 281-mG(2a)-f against Golden Hamster Podoplanin. Monoclon Antib Immunodiagn Immunother 2022;41:311–319; doi: 10.1089/mab.2021.0058
24. Goto N, Suzuki H, Tanaka T, et al. EMab-300 detects
mouse epidermal growth factor receptor-expressing cancer
cell lines in flow cytometry. Antibodies (Basel) 2023;12:30042; doi: 10.3390/antib12030042
25. Suzuki H, Tanaka T, Kudo Y, et al. A rat anti-mouse CD39
monoclonal antibody for flow cytometry. Monoclon AntibImmunodiagn Immunother 2023;42(6):203–208; doi: 10.1089/mab.2023.0018
26. Du X, Li F, Zhang C, et al. Eosinophil-derived chemokine(hCCL15/23, mCCL6) interacts with CCR1 to promoteeosinophilic airway inflammation. Signal Transduct Target
Ther 2021;6:91; doi: 10.1038/s41392-021-00482-x
27. Kato Kaneko M, Ogasawara S, Kato Y. Establishment of amulti-specific monoclonal antibody MsMab-1 recognizing
both IDH1 and IDH2 mutations. Tohoku J Exp Med 2013;
230:103–109; doi: 10.1620/tjem.230.103
28. Puertas B, Gonzalez-Calle V, Sobejano-Fuertes E, et al.
Novel agents as main drivers for continued improvement insurvival in multiple myeloma. Cancers (Basel) 2023;15:
1558; doi: 10.3390/cancers15051558
29. Hiasa M, Harada T, Tanaka E, et al. Pathogenesis andtreatment of multiple myeloma bone disease. Jpn Dent Sci
Rev 2021;57:164–173; doi: 10.1016/j.jdsr.2021.08.006
30. Terpos E, Politou M, Szydlo R, et al. Serum levels ofmacrophage inflammatory protein-1 alpha (MIP-1alpha)correlate with the extent of bone disease and survival inpatients with multiple myeloma. Br J Haematol 2003;123:
106–109; doi: 10.1046/j.1365-2141.2003.04561.x
31. Vallet S, Pozzi S, Patel K, et al. A novel role for CCL3
(MIP-1alpha) in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast
function. Leukemia 2011;25:1174–1181; doi: 10.1038/leu.2011.43

OUCHIDA ET AL.

32. Oyajobi BO, Franchin G, Williams PJ, et al. Dual effects ofmacrophage inflammatory protein-1alpha on osteolysis andtumor burden in the murine 5TGM1 model of myelomabone disease. Blood 2003;102(1):311–319; doi: 10.1182/
blood-2002-12-3905
33. Dairaghi DJ, Oyajobi BO, Gupta A, et al. CCR1 blockade
reduces tumor burden and osteolysis in vivo in a mousemodel of myeloma bone disease. Blood 2012;120:1449–
1457; doi: 10.1182/blood-2011-10-384784
34. Haringman JJ, Ludikhuize J, Tak PP. Chemokines in joint
disease: The key to inflammation? Ann Rheum Dis 2004;
63:1186–1194; doi: 10.1136/ard.2004.020529
35. Katschke KJ, Jr., Rottman JB, Ruth JH, et al. Differential
expression of chemokine receptors on peripheral blood,
synovial fluid, and synovial tissue monocytes/macrophages
in rheumatoid arthritis. Arthritis Rheum 2001;44:1022–
1032; doi: 10.1002/1529-0131(200105)44:5<1022::Aidanr181>3.0.Co;2-n
36. Dairaghi DJ, Zhang P, Wang Y, et al. Pharmacokinetic andpharmacodynamic evaluation of the novel CCR1 antagonist
CCX354 in healthy human subjects: Implications for selection of clinical dose. Clin Pharmacol Ther 2011;89:726–
734; doi: 10.1038/clpt.2011.33
37. Tak PP, Balanescu A, Tseluyko V, et al. Chemokine receptor CCR1 antagonist CCX354-C treatment for rheumatoid arthritis: CARAT-2, a randomised, placebo controlled
clinical trial. Ann Rheum Dis 2013;72:337–344; doi: 10.1136/annrheumdis-2011-201605
38. Morell A, Terry WD, Waldmann TA. Metabolic propertiesof IgG subclasses in man. J Clin Invest 1970;49:673–680;
doi: 10.1172/JCI106279

Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: December 26, 2023
Accepted: February 5, 2024