Biochemistry and Biophysics Reports 40 (2024) 101824

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Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

Development of specific anti-mouse atypical chemokine receptor 4
monoclonal antibodies
Miu Hirose , Hiroyuki Suzuki , Rena Ubukata , Tomohiro Tanaka , Mika K. Kaneko ,Yukinari Kato *Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Miyagi, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
Mouse ACKR4
Monoclonal antibody
Peptide immunization
Flow cytometry
Western blotting

Leukocyte migration is an essential function of innate and adaptive immune responses. Chemokines and their
receptors control the migration system. The abundance of chemokines is controlled by atypical chemokine re­
ceptors (ACKRs), chemokine receptor-like molecules that do not couple to the G protein signaling pathways.
Among them, ACKR4 regulates dendritic cell migration by controlling the ligands and is involved in tumor
development in mouse models. Because no anti-mouse ACKR4 (mACKR4) monoclonal antibody (mAb) for flow
cytometry has been reported, this study aimed to develop a novel mAb for mACKR4. Among the established antimACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab-2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa)
recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The disso­
ciation constant (KD) values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/mACKR4 were determined as 6.0 ×
10− 9 M, 1.3 × 10− 8 M, and 1.7 × 10− 9 M, respectively. Furthermore, A4Mab-1 and A4Mab-2 could detect
mACKR4 by western blotting. These results indicated that A4Mab-1, A4Mab-2, and A4Mab-3 help to detect
mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.

1. Introduction

internalization of receptor and ligand, which leads to degradation ofchemokine [2]. Therefore, ACKRs function as a chemokine scavenger
receptor that mediates chemokines’ rapid internalization anddegradation.
ACKR4 has been described as expressed in not only T lymphocytes[5], but also stromal cells [6,7]. ACKR4 binds to the chemokines,
including CCL19, CCL20, CCL21, CCL22, and CCL25. ACKR4 regulates
migratory responses driven through CCR7 (activated by CCL19 and
CCL21), CCR6 (activated by CCL20), CCR4 (activated by CCL22), andCCR9 (activated by CCL25) [2]. Therefore, ACKR4 controls thebioavailability of the abovementioned chemokines by creating a che­mokine gradient, which facilitates the directional migration of dendritic
cells (DCs) from the non-lymphatic tissue to the draining lymph node [6,8,9]. ACKR4 is also expressed in a flow-dependent manner in afferent
lymphatic collectors. The ACKR4 removes CCL21 from the collector
surface which prevents T cell migration to inflamed dermal collectors. In

The immune cell priming, memory responses, and effector functions
are controlled by chemokines and the restricted expression of the Gprotein-coupled receptors (GPCRs) [1]. The chemokine receptors are themost prominent family of receptors, which possess aseven-transmembrane domain. They can be categorized into a larger
subgroup of G protein-coupled receptors and a smaller subgroup of
atypical chemokine receptors (ACKR1 to ACKR4) [2,3]. Additional
candidates designated as CCRL2 (ACKR5), PITPNM3 (ACKR6), GPR182,
and CXCR3-B are identified, but they are required for further functional
characterization [2].
Upon a chemokine binding to its GPCR, the G protein subunits areusually activated [4]. The ACKRs are homologous to GPCRs. However,
ACKRs fail in inducing the classical signaling through G protein [2].
Instead, the signaling is transduced to β-arrestin-dependent

Abbreviations: ACKR, atypical chemokine receptor; mAb, monoclonal antibody; BSA, bovine serum albumin; CBIS, Cell-Based Immunization and Screening;
CHO–K1, Chinese hamster ovary-K1; CRC, colorectal cancer; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GPCR, G
protein-coupled receptor; KLH, keyhole limpet hemocyanin; PBS, phosphate-buffered saline.
* Corresponding author. Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi,
980-8575, Japan.
E-mail address: yukinari.kato.e6@tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2024.101824
Received 17 July 2024; Received in revised form 7 August 2024; Accepted 4 September 2024
2405-5808/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Biochemistry and Biophysics Reports 40 (2024) 101824

Fig. 1. Flow cytometry of mACKR4-overexpressed CHO–K1 cells using anti-mACKR4 mAbs. CHO/mACKR4 (A) and CHO–K1 (B) cells were treated with 0.01–1
μg/mL of A4Mab-1, A4Mab-2, or A4Mab-3 (red line). The mAbs-treated cells were further incubated with anti-rat IgG conjugated with Alexa Fluor 488. The black line
represents the negative control (blocking buffer). The dose-dependent reactivities of A4Mabs to CHO/mACKR4 were investigated at least three times.

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Biochemistry and Biophysics Reports 40 (2024) 101824

Fig. 2. A peptide-blocking assay using A4Mab-1, A4Mab-2, and A4Mab-3 with mACKR4 peptide. A4Mab-1, A4Mab-2, or A4Mab-3 (0.1 μg/mL) plus mACKR4
peptide (10 μg/mL, blue line) or control (1 % DMSO in blocking buffer, red line) were reacted with CHO/mACKR4 for 30 min at 4 ◦ C, followed by treatment with
Alexa Fluor 488-conjugated anti-rat IgG. The black line represents the negative control (blocking buffer). DMSO, dimethyl sulfoxide.

the absence of ACKR4, the T cell migration to draining lymph nodes isreduced [10].
ACKR4 expression is reduced in human colorectal cancer (CRC)
compared with normal colon epithelial cells [11]. The downregulationof ACKR4 in CRC is associated with a weak antitumor immune response[11]. Loss of ACKR4 in mouse colorectal cancer cells impairs the DC
migration to the tumor-draining lymph nodes, which leads to the
reduced number of tumor-specific T-cells and resistance to immune
checkpoint blockades [11]. In the MMTV-PyMT transgenic mousemodel, which recapitulates the step-wise progression of human breast
cancer [12], the loss of mACKR4 led to an increase in intratumor CD8+ T
cells and CD103+ DCs and inhibited tumor development [12]. Thesefindings promise further study of the targeting of ACKR4 to improve theefficacy of immunotherapy. Therefore, a specific monoclonal antibody(mAb) against mouse ACKR4 (mACKR4) is essential to identify andtarget the ACKR4-expressing cells in the preclinical tumor models.
We have employed the Cell-Based Immunization and Screening(CBIS) method and developed anti-mouse chemokine receptor mAbs
against CCR1 (clone C1Mab-6) [13], CCR3 (clones C3Mab-2, C3Mab-3,
and C3Mab-4) [14–16], CCR5 (clone C5Mab-2) [17], CCR8 (clones
C8Mab-1, C8Mab-2, and C8Mab-3) [18–20], CXCR1 (clone Cx1Mab-1)[21], CXCR3 (clone Cx3Mab-4) [22], and CXCR4 (clone Cx4Mab-1) [23].

Furthermore, we established anti-mouse chemokine receptor mAbs
against CCR2 (clone C2Mab-6) [24], CCR3 (clones C3Mab-6 andC3Mab-7) [25], CCR4 (clone C4Mab-1) [26], CCR5 (clones C5Mab-4 andC5Mab-8) [27], CCR9 (clone C9Mab-24) [28], and CXCR6 (clone
Cx6Mab-1) [29] using the N-terminal peptide immunization. Because noanti-mACKR4 mAb was commercially available, we aimed to develop
novel anti-mACKR4 mAbs.
2. Materials and methods
2.1. Cell lines and plasmids
P3X63Ag8U.1 (P3U1), LN229, and Chinese hamster ovary (CHO)–K1
cell lines were obtained from the American Type Culture Collection(Manassas, VA).
The synthesized mACKR4 (Accession No.: NM_145700.2) cDNA
(Eurofins Genomics KK, Tokyo, Japan) was subsequently subcloned intopCAGzeo-nMAP vectors (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan) and was deposited to RIKEN Bioresource Center (Tsu­kuba, Japan). The MAP tag can be detected by an anti-mouse podoplaninmAb (clone PMab-1) [30]. The mACKR4 plasmids were transfected into
CHO–K1 and LN229. Stable clones were sorted using a cell sorter3

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2.3. Production of hybridomas
The approval of animal experiments was obtained from the Animal
Care and Use Committee of Tohoku University (Permit number:
2022MdA-001). Two five-week-old Sprague-Dawley rats (CLEA Japan,
Tokyo, Japan) were intraperitoneally immunized with 100 μg of the KLHconjugated mACKR4 peptide (mACKR4-KLH) with Alhydrogel adjuvant 2% (InvivoGen). After three additional weekly immunizations (100 μg/rat)
and final booster immunizations (100 μg/rat), hybridomas were produced
and cultured in the medium containing hypoxanthine, aminopterin, andthymidine (HAT; Thermo Fisher Scientific Inc., Waltham, MA), 10 % FBS,
and 5 % BriClone (NICB, Dublin, Ireland). The supernatants were screenedusing enzyme-linked immunosorbent assay (ELISA) using the mACKR4
peptide. The supernatants were further screened by flow cytometry using
CHO–K1 and CHO/mACKR4. Established anti-mACKR4 mAbs can be ob­tained from the Antibody Bank of Tohoku University (http://www.
med-tohoku-antibody.com/topics/001_paper_antibody_PDIS.
htm#ACKR4).
2.4. Purification of antibodies
The cultured supernatants of A4Mab-1, A4Mab-2, and A4Mab-3 hy­
bridomas were purified using 1 mL of Ab-Capcher (ProteNova, Kagawa,Japan). The mAbs were eluted with an IgG elution buffer (Thermo Fisher
Scientific Inc.), and replaced with phosphate-buffered saline (PBS) using
Amicon Ultra (Merck KGaA, Darmstadt, Germany).
2.5. ELISA
The synthesized peptide (MALELNQSAEYYYEENEMNC) was immo­
bilized on 96 well immunoplates (Thermo Fisher Scientific Inc.).
Blocking was performed with 1 % bovine serum albumin (BSA)-PBS
containing 0.05 % Tween20 (PBST; Nacalai Tesque, Inc.). The plates
were incubated with supernatants, followed by peroxidase-conjugatedanti-rat IgG (Sigma-Aldrich Corp., St. Louis, MO). The peroxidase re­actions were performed using ELISA POD Substrate TMB Kit (Nacalai
Tesque, Inc.).
2.6. Flow cytometric analysis
Cells were harvested after brief exposure to 1 mM EDTA. The cells
were washed with blocking buffer (0.1 % BSA in PBS) and treated with
1, 0.1, and 0.01 μg/mL of A4Mab-1, A4Mab-2, and A4Mab-3 for 30 min at
4 ◦ C. For peptide inhibition assay, A4Mab-1, A4Mab-2, and A4Mab-3(0.1 μg/mL) were pre-incubated with 10 μg/mL of mACKR4 peptide ordimethyl sulfoxide (DMSO) for 25 min at 4 ◦ C, and incubated with thecells for 30 min at 4 ◦ C. Alexa Fluor 488-conjugated anti-rat IgG wasused as a secondary antibody. The SA3800 Cell Analyzer (Sony Corp.)
was used for fluorescence data collection and analysis. The dissociation
constant (KD) was determined as described previously [27].

Fig. 3. The binding affinity of anti-mACKR4 mAbs. CHO/mACKR4 cells
were suspended in serially diluted A4Mab-1, A4Mab-2, or A4Mab-3. The cells
were treated with anti-rat IgG conjugated with Alexa Fluor 488. The fluores­
cence data were subsequently collected using the SA3800 Cell Analyzer, fol­
lowed by the calculation of the KD using GraphPad PRISM 6. The experiments
were performed three times independently and the representative results wereshown. The KD values (mean ± SD [M]) were determined from the three in­dependent experiments.

2.7. Western blotting
Sodium dodecyl sulfate-treated cell lysates from LN229 andmACKR4-overexpressed LN229 (LN229/mACKR4) were separated onpolyacrylamide gels. The proteins were transferred onto polyvinylidene
difluoride membranes (Merck KGaA). The membranes were incubatedwith 1 μg/mL of A4Mab-1, A4Mab-2, A4Mab-3, or AC-15 (an anti-β-actinmAb; Sigma-Aldrich Corp.). For peptide blocking assay, 1 μg/mL of
A4Mab-1 and A4Mab-2 were pre-incubated with 1 μg/mL of mACKR4
peptide or DMSO for 15 min and incubated with the membranes.
Peroxidase-conjugated anti-rat IgG or anti-mouse IgG (Agilent Tech­
nologies Inc., Santa Clara, CA) were used as secondary antibodies. The
signals were detected using ImmunoStar LD (FUJIFILM Wako Pure
Chemical Corporation) and a Sayaca-Imager (DRC Co. Ltd., Tokyo,
Japan).

(SH800; Sony Corp., Tokyo, Japan). These cells were cultured as
described previously [29].
2.2. Peptides
The N-terminal extracellular region of mACKR4 (1-MAL­
ELNQSAEYYYEENEMN-19) plus C-terminal cysteine was synthesized by
Eurofins Genomics KK. The keyhole limpet hemocyanin (KLH) was
subsequently conjugated at the C-terminus.

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Biochemistry and Biophysics Reports 40 (2024) 101824

Fig. 4. Western blotting using anti-mACKR4 mAbs. The lysates of LN229 and LN229/mACKR4 cells were electrophoresed and transferred onto polyvinylidene
difluoride membranes. The membranes were incubated with 1 μg/mL of A4Mab-1 (A), 1 μg/mL of A4Mab-2 (B), 1 μg/mL of A4Mab-3 (C), or 1 μg/mL of AC-15 (ananti-β-actin mAb) (D). The membranes were subsequently incubated with peroxidase-conjugated anti-rat IgG (for A4Mab-1, A4Mab-2, and A4Mab-3) or anti-mouse
IgG (for AC-15). Note that the exposure time of A4Mab-1, A4Mab-2, and A4Mab-3 blotting was the same. (E) The membranes were incubated with 1 μg/mL of A4Mab1 plus the mACKR4 peptide (1 μg/mL) or DMSO. (F) The membranes were incubated with 1 μg/mL of A4Mab-2 plus the mACKR4 peptide (1 μg/mL) or DMSO. They
were subsequently incubated with peroxidase-conjugated anti-rat IgG. These experiments were performed at least twice.

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Biochemistry and Biophysics Reports 40 (2024) 101824

3. Results

showed the usefulness for flow cytometry (Fig. 1− 3) and western blotting(Fig. 4) to detect mACKR4. Because no anti-mACKR4 mAb for flow
cytometry has been reported, these mAbs could be the first ones for
detecting mouse ACKR4 in flow cytometry. The information on antiACKR4 mAbs is available from the Antibody Bank of Tohoku University(http://www.med-tohoku-antibody.
com/topics/001_paper_antibody_PDIS.htm#ACKR4).
ACKR4 has emerged as an essential regulator of DC migration via
CCR7, as ACKR4 binds to the CCR7 ligands, CCL19 and CCL21 [31].
ACKR4 does not induce classical GPCR signaling and instead leads to thedegradation of chemokine [32,33]. ACKRs are linked to endocytic ma­chinery via β-arrestin. Upon chemokine ligation to ACKRs, the complex
is generally internalized to endosomal machinery and subsequent che­mokine degradation by lysosome [34]. A4Mab-1, A4Mab-2, andA4Mab-3 recognize the N-terminal region of mACKR4 with different KD
values from 1.3 × 10− 8 M to 1.7 × 10− 9 M (Fig. 3). Although the ACKR4
N-terminus has not been determined as a ligand-binding region, it would
be interesting to investigate whether these mAbs possess the neutral­izing activity to the ligands or promote the internalization of mACKR4.
These functions are expected to inhibit the mACKR4 activity, which may
increase the number of mACKR4 ligands.
A4Mab-1, A4Mab-2, and A4Mab-3 are applicable for flow cytometry(Fig. 1− 3), and A4Mab-1, A4Mab-2, but not A4Mab-3 are useful forwestern blotting (Fig. 4). These results suggest that A4Mab-3 possesses
the different epitope from A4Mab-1 and A4Mab-2. Previously, we
determined the epitope of Cx6Mab-1 (an anti-mouse CXCR6 mAb) using
1 × and 2 × alanine scanning methods [35]. In future studies, we should
evaluate the epitopes of A4Mab-1, A4Mab-2, and A4Mab-3. The identi­
fication of epitopes would contribute to understanding the properties ofmAbs.
Immune checkpoint blockades have developed for cancer treatments[36]. The extent of intratumor CD8+ cell infiltration is correlated withimproved responsiveness of anti-PD-1/PD-L1 therapy in melanoma [37]and improved outcomes in colorectal cancer [38]. A study investigated
the roles of host mACKR4 in the development of mammary tumor bycrossing MMTV-PyMT transgenic mice with the mACKR4-knockout
strain [12]. The mammary tumor development was delayed in themACKR4-knockout compared to control mACKR4-sufficient transgenic
mice [12]. Mechanistically, the loss of mACKR4 leads to increased
intra-tumor CCL21 levels and elevated numbers of CD103+ DCs andCD8+ cells within tumors. However, the contribution ofmACKR4-expressing host cells to tumor microenvironment (TME) has
not been investigated. Therefore, A4Mab-1, A4Mab-2, and A4Mab-3
would help identify the host cells that contribute to the formation of
immunosuppressive TME. Furthermore, these mAbs could contribute to
the preclinical studies for the depletion of mACKR4-expressing cells toenhance responsiveness to immune checkpoint blockade or T-cellco-stimulation.

3.1. Development of anti-mACKR4 mAbs using N-terminal peptide
immunization
To develop anti-mACKR4 mAbs, two rats were immunized with
mACKR4-KLH (Supplementary Fig. 1A). The splenocytes were fusedwith P3U1 myeloma cells and seeded into 96-well plates. Then, positive
wells for the naked mACKR4 peptide were selected using ELISA. Then,
CHO/mACKR4-reactive and CHO–K1-non-reactive supernatants wereselected using flow cytometry (Supplementary Fig. 1B). After the clon­ing by limiting dilution from the independent wells and additional
screenings, anti-mACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab-2(rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) were finally estab­lished (Supplementary Fig. 1C). A4Mab-1 was first established from aseries of immunization and screening. A4Mab-2 and A4Mab-3 were ob­tained from another one.
3.2. Flow cytometric analysis using A4Mab-1, A4Mab-2, and A4Mab-3
We conducted flow cytometry using three anti-mACKR4 mAbs:
A4Mab-1, A4Mab-2, and A4Mab-3 against CHO/mACKR4 and CHO–K1
cells. A4Mab-1, A4Mab-2, and A4Mab-3 recognized CHO/mACKR4 cells
dose-dependently at 1, 0.1, and 0.01 μg/mL (Fig. 1A). A4Mab-3
exhibited a superior reactivity against CHO/mACKR4 cells compared to
A4Mab-1 and A4Mab-2 (Fig. 1A). Parental CHO–K1 cells were notrecognized by any mAbs even at 1 μg/mL (Fig. 1B). The superior reac­tivity of A4Mab-3 was also observed in LN229/mACKR4 cells(Supplementary Fig. 2).
We next performed a peptide-blocking assay. As shown in Fig. 2,
A4Mab-1, A4Mab-2, and A4Mab-3 reacted with the CHO/mACKR4. The
mACKR4 peptide wholly neutralized these reactions (Fig. 2).
3.3. Determination of the binding affinity of A4Mab-1, A4Mab-2, andA4Mab-3 using flow cytometry
To determine the KD values of A4Mab-1, A4Mab-2, and A4Mab-3
against CHO/mACKR4, we conducted flow cytometry, and the geo­
metric mean of the fluorescence intensity was plotted versus the con­centration. The KD values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/
mACKR4 were determined as 6.0 × 10− 9 M, 1.3 × 10− 8 M, and 1.7 ×
10− 9 M, respectively. (Fig. 3). These results indicate that A4Mab-3
possesses the most superior affinity to CHO/mACKR4.
3.4. Detection of mACKR4 using A4Mab-1, A4Mab-2, and A4Mab-3 bywestern blotting
Western blotting was performed to assess the reactivity of A4Mab-1,
A4Mab-2, and A4Mab-3. Lysates of LN229 and LN229/mACKR4 cells
were probed. A4Mab-1 (Fig. 4A) and A4Mab-2 (Fig. 4B) detected
mACKR4 as a ~50-kDa band. In contrast, A4Mab-1 and A4Mab-2 did notshow any bands from the lysates of LN229 cells. A4Mab-2 exhibited asuperior reactivity against LN229/mACKR4 cell lysate compared to
A4Mab-1 (Fig. 4A and B, the same exposure time). In contrast, A4Mab-3
could not detect any bands in the LN229/mACKR4 cell lysate (Fig. 4C).
An anti-β-actin was used as an internal control (Fig. 4D). We also per­formed the peptide blocking experiment. We found that the 50-kDa banddetected by A4Mab-1 and A4Mab-2 was blocked in the presence ofmACKR4 peptide (Fig. 4E and F, respectively). These results suggest that
A4Mab-1 and A4Mab-2 are useful for detecting mACKR4 by western
blotting.

Author disclosure statement
The authors have no conflict of interest.
Funding information
This research was supported in part by Japan Agency for Medical
Research and Development (AMED) under Grant Numbers:
JP24am0521010 (to Y.K.), JP24ama121008 (to Y.K.), JP23am0401013(to Y.K.), JP24bm1123027 (to Y.K.), and JP24ck0106730 (to Y.K.), andby the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid
for Scientific Research (KAKENHI) grant nos. 22K06995 (to H.S.),
21K20789 (to T.T.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).

4. Discussion

CRediT authorship contribution statement

In this study, we developed novel anti-mACKR4 mAbs (A4Mab-1,
A4Mab-2, and A4Mab-3) using the N-terminal peptide immunization and

Miu Hirose: Investigation. Hiroyuki Suzuki: Writing – original6

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Biochemistry and Biophysics Reports 40 (2024) 101824

draft, Investigation, Funding acquisition. Rena Ubukata: Investigation.
Tomohiro Tanaka: Investigation, Funding acquisition. Mika K.
Kaneko: Funding acquisition, Conceptualization. Yukinari Kato:
Writing – review & editing, Project administration, Funding acquisition,
Conceptualization.

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Declaration of competing interest
The authors declare the following financial interests/personal re­lationships which may be considered as potential competing interests:
Yukinari Kato reports financial support was provided by Japan Agency
for Medical Research and Development. Hiroyuki Suzuki reports finan­cial support was provided by Japan Society for the Promotion of Science.
Mika K. Kaneko reports financial support was provided by Japan Society
for the Promotion of Science. Tomohiro Tanaka reports financial support
was provided by Japan Society for the Promotion of Science. If there areother authors, they declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbrep.2024.101824.
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