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Communication

Development of specific Anti-Mouse Atypical
Chemokine Receptor 4 Monoclonal Antibodies
Miu Hirose, Hiroyuki Suzuki, Rena Ubukata, Tomohiro Tanaka, Mika K. Kaneko
and Yukinari Kato *Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai, Miyagi 980-8575, Japan* Correspondence: yukinari.kato.e6@tohoku.ac.jp (Y.K.); Tel.: +81-22-717-8207

Abstract: Leukocyte migration is an essential function of innate and adaptive immune responses.
Chemokines and their receptors control the migration system. The abundance of chemokines iscontrolled by atypical chemokine receptors (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-mACKR4 mAb for flow cytometry has been reported, this study aimed to develop
a novel monoclonal antibody (mAb) for mouse ACKR4 (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 dissociation constant (KD) values of A4Mab-1, A4Mab-2, and A4Mab-3 for
CHO/mACKR4 were determined as 3.5 × 10−9 M, 6.0 × 10−9 M, and 1.6 × 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 andwestern blotting and obtain the proof of concept in preclinical models.
Keywords: mouse ACKR4; monoclonal antibody; peptide immunization; flow cytometry; western
blotting

1. Introduction
The immune cell priming, memory responses, and effector functions are controlled bychemokines and the restricted expression of the G protein-coupled receptors (GPCRs) [1]. The
chemokine receptors are the most prominent family of receptors, which possess a seventransmembrane 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 are usually activated [4]. The
ACKRs are homologous to GPCRs. However, ACKRs fail in inducing the classical signaling throughG protein [2]. Instead, the signaling is transduced to β-arrestin-dependent internalization of receptor
and ligand, which leads to degradation of chemokine [2]. Therefore, ACKRs function as a chemokine
scavenger receptor that mediates chemokines’ rapid internalization and degradation.
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), and CCR9 (activated by CCL25) [2]. Therefore,
ACKR4 controls the bioavailability 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 [6–9].

© 2024 by the author(s). Distributed under a Creative Commons CC BY license.

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ACKR4 expression is reduced in human colorectal cancer (CRC) compared with normal colon
epithelial cells [10]. The downregulation of ACKR4 in CRC is associated with a weak antitumor
immune response [10]. 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 andresistance to immune checkpoint blockades [10]. In the MMTV-PyMT transgenic mouse model,
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 [11].
These findings promise further study of the targeting of ACKR4 to improve the efficacy of
immunotherapy. Therefore, a specific monoclonal antibody (mAb) against mouse ACKR4 (mACKR4)
is essential to identify and target 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) [12], CCR3 (clones C3Mab-2,
C3Mab-3, and C3Mab-4) [13–15], CCR5 (clone C5Mab-2) [16], CCR8 (clones C8Mab-1, C8Mab-2, andC8Mab-3) [17–19], CXCR1 (clone Cx1Mab-1) [20], CXCR3 (clone Cx3Mab-4) [21], and CXCR4 (clone
Cx4Mab-1) [22]. Furthermore, we established anti-mouse chemokine receptor mAbs against CCR2
(clone C2Mab-6) [23], CCR3 (clones C3Mab-6 and C3Mab-7) [24], CCR4 (clone C4Mab-1) [25], CCR5
(clones C5Mab-4 and C5Mab-8) [26], CCR9 (clone C9Mab-24) [27], and CXCR6 (clone Cx6Mab-1) [28]using the N-terminal peptide immunization. Because no anti-mACKR4 mAb was commerciallyavailable, 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 into pCAGzeo-nMAP vectors (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan). The MAP tag can be detected by an anti-mouse podoplaninmAb (clone PMab-1) [29]. The mACKR4 plasmids were transfected into CHO-K1 and LN229. Stableclones were sorted using a cell sorter (SH800; Sony Corp., Tokyo, Japan). These cells were cultured
as described previously [28].
2.2. Peptides
The N-terminal extracellular region of mACKR4 (1-MALELNQSAEYYYEENEMN-19) plus Cterminal cysteine was synthesized by Eurofins Genomics KK. The keyhole limpet hemocyanin (KLH)
was subsequently conjugated at the C-terminus.
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). A five-week-old Sprague-Dawley rat (CLEA
Japan, Tokyo, Japan) was intraperitoneally immunized with 100 µg of the KLH-conjugated 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, and thymidine (HAT;
Thermo Fisher Scientific Inc., Waltham, MA), 10% FBS, and 5% BriClone (NICB, Dublin, Ireland). The
supernatants were screened using enzyme-linked immunosorbent assay (ELISA) using the mACKR4
peptide. The supernatants were further screened by flow cytometry using CHO-K1 andCHO/mACKR4.
2.4. Purification of Antibodies
The cultured supernatants of A4Mab-1, A4Mab-2, and A4Mab-3 hybridomas were purified using
1 mL of Ab-Capcher (ProteNova, Kagawa, Japan). The mAbs were eluted with an IgG elution bufferPreprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 19 July 2024

doi:10.20944/preprints202407.1576.v1

3(Thermo Fisher Scientific Inc.), and replaced with phosphate-buffered saline (PBS) using Amicon
Ultra (Merck KGaA, Darmstadt, Germany).
2.5. ELISA
The synthesized mACKR4 peptide (MALELNQSAEYYYEENEMNC) was immobilized 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 wereincubated with supernatants, followed by peroxidase-conjugated anti-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 A4Mab3 for 30 min at 4°C. For peptide inhibition assay, A4Mab-1, A4Mab-2, and A4Mab-3 (0.1 µg/mL) werepre-incubated with 10 µg/mL of mACKR4 peptide or dimethyl sulfoxide (DMSO) for 25 min at 4°C,
and incubated with the cells for 30 min at 4°C. Alexa Fluor 488-conjugated anti-rat IgG was used asa secondary antibody. The SA3800 Cell Analyzer (Sony Corp.) was used for fluorescence datacollection and analysis. The dissociation constant (KD) was determined as described previously [26].
2.7. Western Blotting
Sodium dodecyl sulfate-treated cell lysates from LN229 and LN229/mACKR4 were separated
on polyacrylamide gels. The proteins were transferred onto polyvinylidene difluoride membranes(Merck KGaA). The membranes were incubated with 1 µg/mL of A4Mab-1, A4Mab-2, PMab-1, or AC15 (an anti-β-actin mAb; Sigma-Aldrich Corp.). For peptide blocking assay, 1 µg/mL of A4Mab-1 andA4Mab-2 were pre-incubated with 1 µg/mL of mACKR4 peptide or dimethyl sulfoxide (DMSO) for
15 min and incubated with the membranes. Peroxidase-conjugated anti-rat IgG or anti-mouse IgG(Agilent Technologies Inc., Santa Clara, CA) were used as secondary antibodies. The signals weredetected using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) and a Sayaca-Imager(DRC Co. Ltd., Tokyo, Japan).
3. Results
3.1. Development of Anti-mACKR4 mAbs Using N-Terminal Peptide Immunization
To develop anti-mACKR4 mAbs, one rat was immunized with mACKR4-KLH (Figure 1A). The
splenocytes were fused with 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 CHOK1-non-reactive supernatants were selected using flow cytometry (Figure 1B). After the cloning bylimiting dilution and additional screenings, anti-mACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) were finally established (Figure 1C).

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Figure 1. The production of anti-mACKR4 mAbs. (A) mACKR4 N-terminal peptide conjugated with
KLH (mACKR4-KLH) was immunized into a Sprague–Dawley rat. The spleen cells were fused with
P3U1 cells. (B) To select anti-mACKR4 mAb-producing hybridomas, the supernatants were screenedby ELISA and flow cytometry using CHO-K1 and CHO/mACKR4 cells. (C) After limiting dilution,
anti-mACKR4 mAbs, A4Mab-1, A4Mab-2, and A4Mab-3 were finally established. ELISA, enzymelinked immunosorbent assay.

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 A4Mab3 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 (Figure 2A). A4Mab-3 exhibited asuperior reactivity against CHO/mACKR4 cells compared to A4Mab-1 and A4Mab-2 (Figure 2A).
Parental CHO-K1 cells were not recognized by any mAbs even at 1 µg/mL (Figure 2B). The superior
reactivity of A4Mab-3 was also observed in LN229/mACKR4 cells (supplementary Figure 1).
We next performed a peptide-blocking assay. As shown in Figure 3, A4Mab-1, A4Mab-2, andA4Mab-3 reacted with the CHO/mACKR4. The mACKR4 peptide wholly neutralized these reactions(Figure 3).

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Figure 2. 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, orA4Mab-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).

Figure 3. 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.

3.3. Determination of the Binding Affinity of A4Mab-1, A4Mab-2, and A4Mab-3 Using Flow Cytometry

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To determine the KD values of A4Mab-1, A4Mab-2, and A4Mab-3 against CHO/mACKR4, we
conducted flow cytometry, and the geometric 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 3.5 × 10−9 M, 6.0 × 10−9 M, and 1.6 × 10−9 M, respectively. (Figure 4). These results indicatethat A4Mab-3 possesses the most superior affinity to CHO/mACKR4.

Figure 4. The binding affinity of anti-mACKR4 mAbs. CHO/mACKR4 cells were suspended inserially diluted A4Mab-1, A4Mab-2, or A4Mab-3. The cells were treated with anti-rat IgG conjugatedwith Alexa Fluor 488. The fluorescence data were subsequently collected using the SA3800 Cell
Analyzer, followed by the calculation of the KD using GraphPad PRISM 6.

3.4. Detection of mACKR4 Using A4Mab-1, A4Mab-2, and A4Mab-3 by Western 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 (Figure 5A) and A4Mab-2 (Figure5B) detected mACKR4 as a ~50-kDa band. In contrast, A4Mab-1 and A4Mab-2 did not show any bands
from the lysates of LN229 cells. A4Mab-2 exhibited a superior reactivity against LN229/mACKR4 celllysate compared to A4Mab-1 (Figure 5A and B, the same exposure time). In contrast, A4Mab-3 could
not detect any bands in the LN229/mACKR4 cell lysate (data not shown). A similar molecular weightband was also detected by PMab-1 (Figure 5C). An anti-β-actin was used as an internal control (Figure
5D). We also performed the peptide blocking experiment. We found that the 50-kDa band detectedby A4Mab-1 and A4Mab-2 was blocked in the presence of mACKR4 peptide (Figure 5E and 5F,
respectively). These results suggest that A4Mab-1 and A4Mab-2 are useful for detecting mACKR4 bywestern blotting.

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Figure 5. Western blotting using anti-mACKR4 mAbs. The lysates of LN229 and LN229/mACKR4
cells were electrophoresed and transferred onto polyvinylidene fluoride membranes. The membranes
were incubated with 1 µg/mL of A4Mab-1 (A), 1 µg/mL of A4Mab-2 (B), 1 µg/mL of PMab-1 (an antiMAP tag mAb) (C), or 1 µg/mL of AC-15 (an anti-β-actin mAb) (D). The membranes were
subsequently incubated with peroxidase-conjugated anti-rat IgG (for A4Mab-1, A4Mab-2, and PMab1) or anti-mouse IgG (for AC-15). Note that the exposure time of A4Mab-1 and A4Mab-2 blotting was
the same. (E) The membranes were incubated with 1 µg/mL of A4Mab-1 plus the mACKR4 peptide (1µg/mL) or DMSO. (F) The membranes were incubated with 1 µg/mL of A4Mab-2 plus themACKR4peptide (1 µg/mL) or DMSO. They were subsequently incubated with peroxidaseconjugated anti-rat IgG.

4. Discussion
In this study, we developed novel anti-mACKR4 mAbs (A4Mab-1, A4Mab-2, and A4Mab-3) using
the N-terminal peptide immunization and showed the usefulness for flow cytometry (Figures 2−4)
and western blotting (Figure 5) 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.
ACKR4 has emerged as an essential regulator of DC migration via CCR7, as ACKR4 binds to the
CCR7 ligands, CCL19 and CCL21 [30]. ACKR4 dose not induce classical GPCR signaling and instead
leads to the degradation of chemokine [31,32]. ACKRs are linked to endocytic machinery via βarrestin. Upon chemokine ligation to ACKRs, the complex is generally internalized to endosomal
machinery and subsequent chemokine degradation by lysosome [33]. A4Mab-1, A4Mab-2, andA4Mab-3 recognize the N-terminal region of mACKR4 with different KD values from 6.0 × 10−9 M to
1.6 × 10−9 M (Figure 4). 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 themACKR4 activity, which may increase the number of mACKR4 ligands.
A4Mab-1, A4Mab-2, and A4Mab-3 are applicable for flow cytometry (Figures 2−4), and A4Mab-1,
A4Mab-2, but not A4Mab-3 are useful for western blotting (Figure 5). These results suggest that

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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 [34]. In
future studies, we should evaluate the epitopes of A4Mab-1, A4Mab-2, and A4Mab-3. The
identification of epitopes would contribute to understanding the properties of mAbs.
Immune checkpoint blockades have developed for cancer treatments [35]. The extent of
intratumor CD8+ cell infiltration is correlated with improved responsiveness of anti-PD-1/PD-L1
therapy in melanoma [36] and improved outcomes in colorectal cancer [37]. A study investigated theroles of host mACKR4 in the development of mammary tumor by crossing MMTV-PyMT transgenic
mice with the mACKR4-knockout strain [11]. The mammary tumor development was delayed in themACKR4-knockout compared to control mACKR4-sufficient transgenic mice [11]. Mechanistically,
the loss of mACKR4 leads to increased intra-tumor CCL21 levels and elevated numbers of CD103+
DCs and CD8+ cells within tumors. However, the contribution of mACKR4-expressing host cells totumor microenvironment (TME) has not been investigated. Therefore, A4Mab-1, A4Mab-2, andA4Mab-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 ofmACKR4-expressing cells to enhance responsiveness to immune checkpoint blockade or T-cell costimulation.
Author Contributions: Miu Hirose: Investigation. Hiroyuki Suzuki: Investigation, Funding acquisition,
Writing – original draft. Rena Ubukata: Investigation. Tomohiro Tanaka: Investigation, Funding acquisition.
Mika K. Kaneko: Conceptualization, Funding acquisition
Yukinari Kato: Conceptualization, Funding acquisition, Project administration, Writing – review and editing.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by Japan Agency for Medical Research and Development (AMED)
under Grant Numbers: JP24ama121008 (to Y.K.), JP23am0401013 (to Y.K.), JP24bm1123027 (to Y.K.), andJP24ck0106730 (to Y.K.), and by 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 (toY.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal Care and Use
Committee of Tohoku University (Permit number: 2022MdA-001) for studies involving animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: All related data and methods are presented in this paper. Additional inquiries
should be addressed to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest involving this article.

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