MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 2, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0031
Open camera or QR reader andscan code to access this article
and other resources online.

Cx1Mab-1:
A Novel Anti-mouse CXCR1 Monoclonal
Antibody for Flow Cytometry

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Guanjie Li,* Tomohiro Tanaka,* Tsunenori Ouchida, Mika K. Kaneko, Hiroyuki Suzuki, and Yukinari Kato

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on
the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6,CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several
diseases including tumors and diabetes mellitus, drugs targeting CXCR1 have been developed. Therefore, thedevelopment of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis andtreatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb, Cx1Mab-1 (rat IgG1, kappa),
using the Cell-Based Immunization and Screening method. Cx1Mab-1 reacted with mCXCR1-overexpressed
Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/
mCXCR1) in flow cytometry. Cx1Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/
mCXCR1 with a dissociation constant of 2.6 · 10-9 M and 2.1 · 10-8 M, respectively. Furthermore, Cx1Mab-1
could detect mCXCR1 by Western blot analysis. These results indicated that Cx1Mab-1 is useful for detecting
mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.
Keywords: mouse CXCR1, CBIS method, monoclonal antibody, flow cytometry

receptor for CXCL6, CXCL7, and IL-8, was also identified.6–8
CXCR2 is also a receptor for CXCL2 and CXCL3 and has
77% amino acids homology with CXCR1.9 Various cells
express CXCR1/CXCR2 on the cell surface, including
macrophages, neutrophils, basophils, natural killer cells,
mast cells, and other leukocytes.10,11 Furthermore, CXCR1/CXCR2 expression has also been confirmed in nonimmune
cells, such as endothelial cells, fibroblasts, and tumor
cells.5,12 Owing to their homology and ligand overlapping,
the functions of CXCR1/CXCR2 have been clarified in many
diseases.13
IL-8 plays a pivotal role in the tumor microenvironment(TME) as a factor of immunosuppressive cells’ recruitment,
angiogenesis, and induction of epithelial-to-mesenchymaltransition.4 Functional heterogeneity of cancer-associatedfibroblasts (CAFs) is regulated by IL-8 and other CXCR1/2
ligands.14 The normal ovarian fibroblast is converted to CAFs

Introduction

C

hemokines can be classified into four different subfamilies: C-C, C-X-C, C-X3-C, and X-C, depending on
the number and position of cysteine residues in theirN-terminus. C-X-C means the first two cysteine amino acids
are divided by other amino acids. Chemokines play pivotal
roles in immune responses, such as infiltration and migrationof immune cells by binding to its cognate receptors.1–3
The C-X-C motif chemokine receptor 1 (CXCR1), also
referred to as C-X-C motif chemokine ligand 8 (CXCL8)
receptor/interleukin-8 (IL-8) receptor alpha, is one of the
G-protein coupled receptors (GPCRs) composed of 350
amino acids with structural 7 transmembrane domains.4
CXCL6 and CXCL7 are also the chemokine ligands of
CXCR1.5 At the nearly same time that CXCR1 was discovered, the C-X-C motif chemokine receptor 2 (CXCR2), a

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
This article was previously published in preprint.org (doi: 10.20944/preprints202311.0501.v2).
*These authors contributed equally to this study.

59

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60

by IL-8 and triggers tumor growth in mice. Aberrant CXCR1/
2 ligands expression in CAFs is closely related to gastriccancer progression and poor prognosis in patients.15 In TMEof glioma, IL-8+CD4+ T cells function in a complex mannerwith other IL-8-producing cells to govern the immunosuppressive properties.16
IL-8 expression is positively correlated with CXCR1/2 andmight be a predictive marker of poor prognosis in glioma,
metastatic urothelial carcinoma, and metastatic renal cell
carcinoma patients.17,18 Furthermore, the blockade of the
IL-8-CXCR1/2 axis enhances the anti-programmed cell
death-1 (PD-1) therapeutic effects on glioma.19 Thus,CXCR1/2 molecules are promising novel cancer therapeutictargets for tumors.20 Reparixin, a noncompetitive allostericinhibitor of CXCR1/2, could prevent polymorphonuclear
cell recruitment.21
A phase 2 clinical trial using navarixin (SCH-527123), a
selective CXCR1/2 antagonist, has been conducted in combination with anti-PD-1 mAb, pembrolizumab, in advanced
solid tumors.22,23 Navarixin blocks neutrophil recruitment
and suppresses the tumor cell growth in a murine melanoma
model. CXCR1/2 are attractive targets for treatment against
multiple cancers.
CXCR1/2 and their chemokine ligands regulate the immune system, which mediates the progression of diseases
such as cancer. Therefore, CXCR1/2-targeting antibodies
will contribute to the diagnosis and therapy. Using the CellBased Immunization and Screening (CBIS) method, we havedeveloped many monoclonal antibodies (mAbs) against C-C
chemokine receptors, including mouse CCR3,24 mouse
CCR8,25–27 and human CCR928 mAbs. Next, we have been
trying to establish the anti-C-X-C motif chemokine receptor
mAbs.
We recently obtained an anti-mouse CXCR3 mAb,29 ananti-mouse CXCR4 mAb,30 and an anti-mouse CXCR6
mAb.31 In this study, we have successfully developed antimouse CXCR1 (mCXCR1) mAbs using the CBIS method thatis applicable to flow cytometry and Western blot analysis.

LI ET AL.

LN229 and LN229/mCXCR1 were cultured in a Dulbecco’s
modified Eagle’s medium (Nacalai Tesque, Inc.) that was
supplemented with 10% heat-inactivated FBS (Thermo
Fisher Scientific, Inc.), 100 U/mL penicillin, 100 lg/mL
streptomycin, and 0.25 lg/mL amphotericin B (Nacalai
Tesque, Inc.). Then, cells were grown in a humidified incubator, which was supplied with 5% CO2 and 95% air at 37C.
Antibodies

The anti-mCXCR1/IL-8 RA mAb (clone 1122A) was
purchased from R&D Systems, Inc., (Minneapolis, MN). An
anti-DYKDDDDK mAb (clone 1E6) and an anti-b-actinmAb (clone AC-15) were purchased from FUJIFILM Wako
Pure Chemical Corporation (Osaka, Japan) and SigmaAldrich Corp. (St. Louis, MO), respectively. Alexa Fluor
488-conjugated anti-rat IgG and Alexa Fluor 488-conjugatedanti-rabbit IgG were purchased from Cell Signaling Technology, Inc., (Danvers, MA).
Hybridoma production

The animal was housed under specific pathogen-free
conditions. All animal experiments were approved by theMaterials and Methods
Cell lines

LN229, Chinese hamster ovary (CHO)-K1, andP3X63Ag8U.1 (P3U1) cells were obtained from the American Type Culture Collection (Manassas, VA). pCMV6neomyc-DDK vector with mCXCR1 (Accession No.:
NM_178241) was purchased from OriGene Technologies,
Inc., (Rockville, MD). The plasmid was transfected into thecell lines using a Neon Transfection System (Thermo Fisher
Scientific, Inc., Waltham, MA). Subsequently, LN229 and
CHO-K1, which stably overexpressed mCXCR1 with
C-terminal myc-DDK tags (hereinafter described as LN229/
mCXCR1 and CHO/mCXCR1, respectively) were established using a cell sorter (SH800; Sony Corp., Tokyo, Japan),after cultivation in a medium containing 0.5 mg/mL G418
(Nacalai Tesque, Inc., Kyoto, Japan).
CHO-K1, P3U1, and CHO/mCXCR1 were also cultured ina Roswell Park Memorial Institute (RPMI)-1640 medium(Nacalai Tesque, Inc.) that was supplemented with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/mL penicillin, 100 lg/mL streptomycin,
and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.).

FIG. 1. A schematic procedure of anti-mCXCR1 mAbs
production. The procedure of CBIS. (A) LN229/mCXCR1
cells were immunized into a Sprague-Dawley rat using intraperitoneal injection. (B) The harvested splenocytes werefused with P3U1 cells using polyethylene glycol 1500.
(C) The culture supernatants of hybridoma were screened byflow cytometry. (D) Cx1Mab-1 was established by limiting
dilution. CBIS, Cell-Based Immunization and Screening;
mCXCR, mouse C-X-C motif chemokine receptor.

ANTI-MOUSE CXCR1 MAB

further added hypoxanthine, aminopterin, and thymidine(Thermo Fisher Scientific, Inc.), 5% Briclone (NICB, Dublin,
Ireland), and 5 lg/mL of plasmocin into the medium. The
hybridoma supernatants were screened by flow cytometry
using CHO/mCXCR1 and parental CHO-K1. The culture
supernatants of hybridomas were filtrated and purified using
Ab-Capcher Extra (ProteNova, Kagawa, Japan).
Flow cytometry

CHO-K1 and CHO/mCXCR1 cells were harvested after
brief exposure to 2.5 g/L-Trypsin/1 mmol/L-EDTA solution
with phenol red (Nacalai Tesque, Inc.). CHO-K1,
CHO/mCXCR1, LN229, and LN229/mCXCR1 cells were
washed with 0.1% bovine serum albumin in phosphate-bufferedsaline (PBS) and treated with primary mAbs for 30 min at 4C.
Afterward, cells were treated with Alexa Fluor 488-conjugatedDownloaded by Yukinari Kato from www.liebertpub.com at 04/09/24. For personal use only.

Animal Care and Use Committee of Tohoku University(Permit number: 2022MdA-001).
For developing anti-mCXCR1 mAbs, a 6-week-old female
Sprague-Dawley rat (CLEA Japan, Tokyo, Japan) was immunized intraperitoneally with 1 · 109 cells of LN229/mCXCR1.
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 LN229/mCXCR1 were performed
without an adjuvant every week. We performed a final booster
immunization of 1 · 109 cells of LN229/mCXCR1 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

61

FIG. 2. Flow cytometric analysis of anti-mCXCR1 mAbs against CHO/mCXCR1 and CHO-K1. CHO/mCXCR1 (A) andCHO-K1 cells (B) were treated with 0.01–10 mg/mL of Cx1Mab-1 and 1122A, followed by treatment with Alexa Fluor 488conjugated anti-rat IgG and Alexa Fluor 488-conjugated anti-rabbit IgG, respectively. CHO, Chinese hamster ovary.

62

anti-rat IgG (1:1000) or Alexa Fluor 488-conjugated anti-rabbit
IgG (1:1000). The fluorescence data was collected using an
SA3800 Cell Analyzer (Sony Corp.).
Determination of the binding affinity by flow cytometry

CHO/mCXCR1 and LN229/mCXCR1 were suspended in
100 lL serially diluted anti-mCXCR1 mAbs (25 to
0.006 lg/mL), after which Alexa Fluor 488-conjugated antirat IgG (1:200) or Alexa Fluor 488-conjugated anti-rabbit
IgG (1:200) was added. Fluorescence data were subsequently collected, using the SA3800 Cell Analyzer (SonyCorp.). The dissociation constant (KD) was calculated byfitting the binding isotherms into the built-in one-sitebinding model in GraphPad PRISM 10 (GraphPad Software,Inc., La Jolla, CA).

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Western blot analysis

Cell lysates were boiled in sodium dodecyl sulfate sample
buffer (Nacalai Tesque, Inc.), after which proteins were

LI ET AL.

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, membranes were incubated with
1 lg/mL of Cx1Mab-1, 1 lg/mL of an anti-mCXCR1/IL-8
RA mAb (clone 1122A), 1 lg/mL of an anti-DYKDDDDK
mAb (clone 1E6), or 1 lg/mL of an anti-b-actin mAb (clone
AC-15).
Then, they were incubated again with horseradish
peroxidase-conjugated anti-rat immunoglobulins (forCx1Mab-1; diluted 1:10,000; Sigma-Aldrich Corp.), antimouse immunoglobulins (for anti-DYKDDDDK and antib-actin; diluted 1:1000; Agilent Technologies, Inc., Santa
Clara, CA), or anti-rabbit immunoglobulins (for 1122A;
diluted 1:1000; Agilent Technologies, Inc.). Finally, protein
bands were detected using ECL Plus Western Blotting
Substrate (Thermo Fisher Scientific, Inc.) or ImmunoStar
LD (FUJIFILM Wako Pure Chemical Corporation) with a
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

FIG. 3. Flow cytometric analysis of anti-mCXCR1 mAbs against LN229/mCXCR1 and LN229. LN229/mCXCR1 cells(A) and LN229 cells (B) were treated with 0.01–10 mg/mL of Cx1Mab-1 and 1122A, followed by treatment with Alexa
Fluor 488-conjugated anti-rat IgG and Alexa Fluor 488-conjugated anti-rabbit IgG, respectively.

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ANTI-MOUSE CXCR1 MAB

63

FIG. 4. The determination of the binding affinity of anti-mCXCR1 mAbs. CHO/mCXCR1 (A) or LN229/mCXCR1(B) cells were suspended in 100 mL serially diluted Cx1Mab-1 or 1122A (25 to 0.006 mg/mL). Then, cells were treatedwith Alexa Fluor 488-conjugated anti-rat IgG or Alexa Fluor 488-conjugated anti-rabbit IgG. Fluorescence data were
subsequently collected using the SA3800 Cell Analyzer. The dissociation constant (KD) was calculated by GraphPad
PRISM 10.

Results
Establishment of anti-mCXCR1 mAbs

To develop anti-mCXCR1 mAbs, we employed the CBIS
method using mCXCR1-overexpressed cells. Hybridoma
screening was conducted using flow cytometry (Fig. 1). A rat
was intraperitoneally immunized with LN229/mCXCR1.
Subsequently, hybridomas were seeded into 96-well plates,
after which flow cytometric analysis was used to extract
CHO/mCXCR1-reactive and CHO-K1-nonreactive supernatants. Afterward, we obtained CHO/mCXCR1-reactive
supernatants in 78 of 958 wells (8.1%). We finally establishedclone Cx1Mab-1 (rat IgG1, kappa) by limiting dilution andadditional screening.

acted with CHO/mCXCR1 and LN229/mCXCR1 even at
0.01 lg/mL concentration (Figs. 2, 3), indicating that bothmAbs are highly sensitive.
Determination of the binding affinity of Cx1Mab-1

The binding affinity of Cx1Mab-1 and 1122A was assessedwith CHO/mCXCR1 and LN229/mCXCR1 using flow cytometry. Results showed that the KD of Cx1Mab-1 for
CHO/mCXCR1 and LN229/mCXCR1 was 2.6 · 10-9 M and
2.1 · 10-8 M, respectively (Fig. 4). In contrast, the KD of 1122A
for CHO/mCXCR1 and LN229/mCXCR1 was 2.7 · 10-9 M
and 2.8 · 10-8 M, respectively (Fig. 4). These results indicatethat Cx1Mab-1 possesses a moderate affinity for exogenously
overexpressed mCXCR1 in CHO-K1 and LN229 cells.

Flow cytometric analysis

Flow cytometric analysis was conducted using Cx1Mab-1
and another anti-mCXCR1/IL-8 RA mAb (clone 1122A fromR&D Systems, Inc.) against CHO-K1, CHO/mCXCR1,
LN229, and LN229/mCXCR1. Results showed that Cx1Mab1 and 1122A recognized CHO/mCXCR1 dose dependently,
but not parental CHO-K1 cells (Fig. 2). Both mAbs also
recognized LN229/mCXCR1 dose dependently, but not parental LN229 cells (Fig. 3). Both Cx1Mab-1 and 1122A re-Western blot analysis

Western blot analysis was performed to further assess
the availability of Cx1Mab-1. Lysates of CHO-K1, CHO/
mCXCR1, LN229, and LN229/mCXCR1 cells were probed.
As demonstrated in Figure 5, Cx1Mab-1 detected the *48kDa band of mCXCR1 in lysates from CHO/mCXCR1 and
LN229/mCXCR1 cells, whereas this band was not present inlysates from CHO-K1 and LN229 cells, indicating that

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64

FIG. 5. Western blot analysis using anti-mCXCR1 mAbs.
Cell lysates (10 mg) of CHO-K1, CHO/mCXCR1, LN229,
and LN229/mCXCR1 cells were electrophoresed and proteins were transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were incubated with
1 mg/mL of Cx1Mab-1, 1122A, an anti-DYKDDDDK mAb(1E6), or an anti-b-actin mAb (AC-15), and then incubatedwith peroxidase-conjugated anti-rat IgG or anti-mouse IgG.
Cx1Mab-1 specifically detects mCXCR1. Clone 1122A
showed a weak nonspecific band in CHO-K1 (Fig. 5);
therefore, Cx1Mab-1 is more specific than clone 1122A in
Western blot analysis.

LI ET AL.

their receptors expressed in these cells modulate a complex
TME.23,32 Neutrophil is one of the major mediators of inflammation. IL-8 secreted from CD4+ T cells recruited
CXCR1/2-expressed neutrophils to the inflammation site.33
The neutrophils promote inflammation by generating reactive
oxygen species.19,34 In this study, we established Cx1Mab-1
that recognizes mCXCR1 with high sensitivity (Figs. 2, 3).
Therefore, Cx1Mab-1 would contribute to detecting andisolating the mCXCR1-positive lymphocytes.
Tumor-associated macrophage (TAM) plays a vital role as
a tumor immune regulator in TME.35 TAM produces IL-8
that attenuates cancer immunity by suppressing the infiltration of CD8+ T cells and enhances programmed cell death-1
ligand-1 (PD-L1) expression in TAM itself.36 Blocking IL-8
in TAM by neutralizing antibody suppresses epithelial
ovarian cancer migration.37 Furthermore, CXCR1/2-positive
neutrophils and myeloid-derived suppressor cells in cancer
patients also play an important role in immune suppression
by obstructing the contact between CD8+ T cells and thetarget tumor cells.19 Therefore, it is interesting to investigate
whether Cx1Mab-1 interferes with the IL-8-CXCR1 interaction. Furthermore, the identification of epitope is essential
to know the property of Cx1Mab-1.
Therapeutic drugs that target CXCR1/2 have been developed for cancers.12 SCH527123 is an orally available allosteric inhibitor of CXCR1/2 that can inhibit the neutrophil
activity.22,38 Reparixin selectively blocks CXCR1 that isexpressed in human breast cancer stem cells (CSCs) in preclinical models.39 Furthermore, reparixin significantly decreases tumor growth in combination with paclitaxel ordocetaxel compared with chemotherapy alone.39,40 AntiCXCR1 mAbs also have the potential to affect CSCs
in vitro.39 Furthermore, the blockers for CXCR1/2 potentiate
the efficacy of immune checkpoint inhibitors (ICIs) throughsuppression of neutrophils and myeloid-derived suppressor cells.19,41,42
Several clinical trials demonstrate the promise of combination therapies treating CXCR1/2-targeted drugs and
ICIs.4,43 We have previously enhanced antibody-dependent
cellular cytotoxicity (ADCC) activity complement-dependent
cytotoxicity (CDC) by modifying mAb isotypes and defucosylation in mAbs.44–46 Since Cx1Mab-1 is rat IgG1 that shows
no ADCC and CDC activities, it will be converted into a mouse
IgG2a version to verify the effect of depletion of mCXCR1positive immunosuppressive cells in preclinical models.
Authors’ Contributions

G.L., T.T., and T.O. performed the experiments. M.K.K.
and Y.K. designed the experiments. G.L., T.T., and M.K.K.
analyzed the data. T.T., H.S., and Y.K. wrote the article. All
authors have read and agreed to the published version of thearticle.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

Discussion

The TME is composed of various cells including cancer
cells, immune cells, and stromal cells.32 Chemokines andThis research was supported in part by Japan Agency
for Medical Research and Development (AMED) underGrant Numbers JP23ama121008 (to Y.K.), JP23am0401013

ANTI-MOUSE CXCR1 MAB(to Y.K.), 23bm1123027h0001 (to Y.K.), andJP23ck0106730 (to Y.K.), and by the Japan Society for the
Promotion of Science ( JSPS) Grants-in-Aid for Scientific
Research (KAKENHI) Grant Numbers 21K20789 (to T.T.),
23K19494 (to T.O.), 22K06995 (to H.S.), 21K07168(to M.K.K.), and 22K07224 (to Y.K.).

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References

1. Hughes CE, Nibbs RJB. A guide to chemokines and their
receptors. FEBS J 2018;285:2944–2971; 10.1111/febs.14466
2. Nagarsheth N, Wicha MS, Zou W. Chemokines in thecancer microenvironment and their relevance in cancer
immunotherapy. Nat Rev Immunol 2017;17:559–572; 10.1038/nri.2017.49
3. White GE, Iqbal AJ, Greaves DR. CC chemokine receptors
and chronic inflammation—Therapeutic opportunities andpharmacological challenges. Pharmacol Rev 2013;65:47–
89; 10.1124/pr.111.005074
4. Han ZJ, Li YB, Yang LX, et al. Roles of the CXCL8CXCR1/2 axis in the tumor microenvironment and immunotherapy. Molecules 2021;27:10137; 10.3390/molecules
27010137
5. Russo RC, Garcia CC, Teixeira MM, et al. The CXCL8/
IL-8 chemokine family and its receptors in inflammatory
diseases. Expert Rev Clin Immunol 2014;10:593–619; 10.1586/1744666x.2014.894886
6. Murphy PM, Tiffany HL. Cloning of complementary DNA
encoding a functional human interleukin-8 receptor. Science 1991;253:1280–1283; 10.1126/science.1891716
7. Holmes WE, Lee J, Kuang WJ, et al. Structure and functional expression of a human interleukin-8 receptor. Science 1991;253:1278–1280; 10.1126/science.1840701
8. Wang W, Deng Z, Liu G, et al. Platelet-derived extracellular vesicles promote the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes via CXCR2
signaling. Exp Ther Med 2021;22:1120; 10.3892/etm.2021.10554
9. Bi H, Zhang Y, Wang S, et al. Interleukin-8 promotes cell
migration via CXCR1 and CXCR2 in liver cancer. Oncol
Lett 2019;18:4176–4184; 10.3892/ol.2019.10735
10. Chuntharapai A, Lee J, Hébert CA, et al. Monoclonal antibodies detect different distribution patterns of IL-8 receptor A and IL-8 receptor B on human peripheral blood
leukocytes. J Immunol 1994;153:5682–5688.
11. Tavares LP, Garcia CC, Machado MG, et al. CXCR1/2
antagonism is protective during influenza and postinfluenza pneumococcal infection. Front Immunol 2017;8:
1799; 10.3389/fimmu.2017.01799
12. Sitaru S, Budke A, Bertini R, et al. Therapeutic inhibition
of CXCR1/2: Where do we stand? Intern Emerg Med 2023;
18:1647–1664; 10.1007/s11739-023-03309-5
13. Fu X, Wang Q, Du H, et al. CXCL8 and the peritoneal
metastasis of ovarian and gastric cancer. Front Immunol
2023;14:1159061; 10.3389/fimmu.2023.1159061
14. Chen Y, McAndrews KM, Kalluri R. Clinical and therapeutic relevance of cancer-associated fibroblasts. Nat Rev
Clin Oncol 2021;18:792–804; 10.1038/s41571-02100546-5
15. Naito Y, Yamamoto Y, Sakamoto N, et al. Cancer extracellular vesicles contribute to stromal heterogeneity by
inducing chemokines in cancer-associated fibroblasts. Oncogene 2019;38:5566–5579; 10.1038/s41388-019-0832-4

65

16. Teijeira A, Garasa S, Ochoa MC, et al. IL8, neutrophils,
and NETs in a collusion against cancer immunity and immunotherapy. Clin Cancer Res 2021;27:2383–2393; 10.1158/1078-0432.Ccr-20-1319
17. Schalper KA, Carleton M, Zhou M, et al. Elevated serum
interleukin-8 is associated with enhanced intratumor neutrophils and reduced clinical benefit of immune-checkpoint
inhibitors. Nat Med 2020;26:688–692; 10.1038/s41591020-0856-x
18. Yuen KC, Liu LF, Gupta V, et al. High systemic andtumor-associated IL-8 correlates with reduced clinicalbenefit of PD-L1 blockade. Nat Med 2020;26:693–698; 10.1038/s41591-020-0860-1
19. Liu H, Zhao Q, Tan L, et al. Neutralizing IL-8 potentiates immune checkpoint blockade efficacy for glioma. Cancer
Cell 2023;41:693–710.e698; 10.1016/j.ccell.2023.03.004
20. Ruffini PA. The CXCL8-CXCR1/2 axis as a therapeutictarget in breast cancer stem-like cells. Front Oncol 2019;9:40; 10.3389/fonc.2019.00040
21. Bertini R, Allegretti M, Bizzarri C, et al. Noncompetitive
allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: Prevention of reperfusion injury.
Proc Natl Acad Sci U S A 2004;101:11791–11796; 10.1073/pnas.0402090101
22. Chapman RW, Minnicozzi M, Celly CS, et al. A novel,
orally active CXCR1/2 receptor antagonist, Sch527123,
inhibits neutrophil recruitment, mucus production, andgoblet cell hyperplasia in animal models of pulmonary inflammation. J Pharmacol Exp Ther 2007;322:486–493; 10.1124/jpet.106.119040
23. Singh S, Sadanandam A, Nannuru KC, et al. Smallmolecule antagonists for CXCR2 and CXCR1 inhibit
human melanoma growth by decreasing tumor cell proliferation, survival, and angiogenesis. Clin Cancer Res 2009;
15:2380–2386; 10.1158/1078-0432.Ccr-08-2387
24. Asano T, Nanamiya R, Takei J, et al. Development of antimouse CC chemokine receptor 3 monoclonal antibodies forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:107–112; 10.1089/mab.2021.0009
25. 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; 10.1089/mab.2021.0005
26. Suzuki H, Saito M, Asano T, et al. C(8)Mab-3: An antimouse CCR8 monoclonal antibody for immunocytochemistry. Monoclon Antib Immunodiagn Immunother 2022;41:
110–114; 10.1089/mab.2022.0002
27. Saito M, Suzuki H, Tanaka T, et al. Development of ananti-mouse CCR8 monoclonal antibody (C(8)Mab-1) forflow cytometry and immunocytochemistry. Monoclon Antib Immunodiagn Immunother 2022;41:333–338; 10.1089/
mab.2021.0069
28. Nanamiya R, Takei J, Asano T, et al. Development of antihuman CC chemokine receptor 9 monoclonal antibodies forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:101–106; 10.1089/mab.2021.0007
29. Ouchida T, Isoda Y, Tanaka T, et al. Cx3Mab-4: A Novel
Anti-mouse CXCR3 Monoclonal Antibody for Flow Cytometry. Monoclon Antib Immunodiagn Immunother 2024;[Epub ahead of print] 10.1089/mab.2023.0024.
30. Ouchida T, Suzuki H, Tanaka T, et al. Cx4Mab-1: A Novel
Anti-Mouse CXCR4 Monoclonal Antibody for Flow Cytometry. Monoclon Antib Immunodiagn Immunother 2023;
43:10–16; 10.1089/mab.2023.0023

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

66

31. Kitamura K, Suzuki H, Kaneko MK, et al. Cx(6)Mab-1: A
novel anti-mouse CXCR6 monoclonal antibody establishedby N-terminal peptide immunization. Monoclon AntibImmunodiagn Immunother 2022;41:133–141; 10.1089/mab.2022.0010
32. Borlongan MC, Saha D, Wang H. Tumor microenvironment: A niche for cancer stem cell immunotherapy. Stem
Cell Rev Rep2024;20(1):3–24; 10.1007/s12015-02310639-6
33. Hammond ME, Lapointe GR, Feucht PH, et al. IL-8 induces neutrophil chemotaxis predominantly via type I IL-8
receptors. J Immunol 1995;155:1428–1433.
34. Thai TD, Chuenchom C, Donsa W, et al. Helicobacter
pylori extract induces purified neutrophils to produce reactive oxygen species only in the presence of plasma.
Biomed Rep 2023;19:89; 10.3892/br.2023.1671
35. Qin R, Ren W, Ya G, et al. Role of chemokines in the
crosstalk between tumor and tumor-associated macrophages. Clin Exp Med 2023;23:1359–1373; 10.1007/
s10238-022-00888-z
36. Lin C, He H, Liu H, et al. Tumour-associated macrophagesderived CXCL8 determines immune evasion through autonomous PD-L1 expression in gastric cancer. Gut 2019;68:1764–1773; 10.1136/gutjnl-2018-316324
37. Zhang T, Ma Z, Wang R, et al. Thrombin facilitates invasionof ovarian cancer along peritoneum by inducing monocyte
differentiation toward tumor-associated macrophage-likecells. Cancer Immunol Immunother 2010;59:1097–1108; 10.1007/s00262-010-0836-y
38. Gonsiorek W, Fan X, Hesk D, et al. Pharmacological characterization of Sch527123, a potent allosteric CXCR1/CXCR2
antagonist. J Pharmacol Exp Ther 2007;322:477–485; 10.1124/jpet.106.118927
39. Ginestier C, Liu S, Diebel ME, et al. CXCR1 blockade
selectively targets human breast cancer stem cells in vitro
and in xenografts. J Clin Invest 2010;120:485–497; 10.1172/jci39397
40. Brandolini L, Cristiano L, Fidoamore A, et al. Targeting
CXCR1 on breast cancer stem cells: Signaling pathways
and clinical application modelling. Oncotarget 2015;6:43375–43394; 10.18632/oncotarget.6234

LI ET AL.

41. Gulhati P, Schalck A, Jiang S, et al. Targeting T cell
checkpoints 41BB and LAG3 and myeloid cell
CXCR1/CXCR2 results in antitumor immunity and durable
response in pancreatic cancer. Nat Cancer 2023;4:62–80;
10.1038/s43018-022-00500-z
42. Teijeira Á, Garasa S, Gato M, et al. CXCR1 and CXCR2
chemokine receptor agonists produced by tumors induce
neutrophil extracellular traps that interfere with immune
cytotoxicity. Immunity 2020;52:856–871.e858; 10.1016/j.immuni.2020.03.001
43. Kargl J, Zhu X, Zhang H, et al. Neutrophil content predicts
lymphocyte depletion and anti-PD1 treatment failure in
NSCLC. JCI Insight 2019;4(24):e130850; 10.1172/jci.insight.130850
44. Li G, Suzuki H, Ohishi T, et al. Antitumor activities of adefucosylated anti-EpCAM monoclonal antibody in colorectal carcinoma xenograft models. Int J Mol Med 2023;
51(2):18; 10.3892/ijmm.2023.5221
45. Tateyama N, Nanamiya R, Ohishi T, et al. Defucosylated
anti-epidermal growth factor receptor monoclonal antibody
134-mG(2a)-f exerts antitumor activities in mouse xenograft models of dog epidermal growth factor receptoroverexpressed cells. Monoclon Antib Immunodiagn
Immunother 2021;40:177–183; 10.1089/mab.2021.0022
46. Tanaka T, Suzuki H, Ohishi T, et al. Antitumor activities
against breast cancers by an afucosylated anti-HER2
monoclonal antibody H(2) Mab-77-mG(2a)-f. Cancer Sci
2024;115(1):298–309; 10.1111/cas.16008

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
MiyagiJapan
E-mail: yukinari.kato.e6@tohoku.ac.jp
Received: December 26, 2023
Accepted: February 24, 2024