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Communication

C8Mab-21: A Novel Anti-Human CCR8 Monoclonal
Antibody for Flow Cytometry
Tomohiro Tanaka, Hiroyuki Suzuki, Guanjie Li, Mika K. Kaneko, Yukinari Kato *Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku,
Sendai, Miyagi 980-8575, Japan* Correspondence: yukinari.kato.e6@tohoku.ac.jp (Y.K.); Tel.: +81-22-717-8207

Abstract: C-C motif chemokine receptor-8 (CCR8) belongs to class A of G protein-coupled receptors (GPCRs).
CCR8 interacts with the specific chemokine ligand CCL1/I-309 in humans, which is produced by various cells,
including tumor-associated macrophages and regulatory T cells (Treg). CCR8 is highly expressed on Treg andT-helper 2 (TH2) cells recruited to the inflammation site and is implicated in allergy, asthma, and cancer
progression. The CCR8+Treg has been suggested to be an important regulator in the immunosuppressive tumor
microenvironment (TME); therefore, it has been desired to develop sensitive monoclonal antibodies (mAbs) for
CCR8. This study developed a specific mAb for human CCR8 (hCCR8), which is useful for flow cytometry by
employing the Cell-Based Immunization and Screening (CBIS) method. The established anti-hCCR8 mAb,
C8Mab-21, (mouse IgM, kappa) reacted with hCCR8-overexpressed Chinese hamster ovary-K1 (CHO/hCCR8)
cells, TALL-1 (acute T lymphoblastic leukemia), CCRF-HSB2 (human T-lymphoblastic leukemia), and naturalkiller cells, which express endogenous hCCR8 by flow cytometry. Furthermore, C8Mab-21 demonstrated amoderate binding affinity for CHO/hCCR8 and TALL-1 with a dissociation constant of 6.5×10-8 M and 2.0×10-8
M, respectively. C8Mab-21, which was established by the CBIS method, could be a useful tool for analyzing thehCCR8-related biological response using flow cytometry.
Keywords: CCR8; CBIS method; monoclonal antibody; flow cytometry

1. Introduction
Immune checkpoint inhibitors have become effective and powerful strategies for cancer
therapy.[1] In particular, the development of antibody drugs targeting immune checkpoint
molecules, such as programmed-cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), andPD-1 ligand 1 (PD-L1), has achieved remarkable therapeutic results.[2-4] PD-1 inhibits the excessive
activation of conventional T cells by suppressing costimulatory signaling and renders themdysfunctional or exhausted.[5] PD-1 and CTLA-4 are also expressed in regulatory T cells (Treg), oneof the immunosuppressors in the tumor microenvironment (TME). The inhibition could potentiate
the activation and immunosuppressive function of Treg cells.[6]Treg is defined as a CD4+T cell with CD25 and Foxp3 and maintains self-tolerance to prevent
excessive immune responses and autoimmune diseases.[7] Treg suppresses the effector functions ofT cells through the secretion of immunosuppressive cytokines, such as interleukin-10, transforming
growth factor-β, and cytotoxic granzyme/perforin.[7-11] Intratumoral Treg suppresses antitumor T
cell responses and thus resists the effects of immune checkpoint inhibitor therapy.[12,13] Antibodies
against T cell immunoreceptors with Ig and ITIM domains (TIGIT), one of the immune checkpoint
molecules, improve the effectiveness of PD-L1 antibodies by suppressing Treg.[14] Therefore, thedevelopment of immunotherapy targeting Treg is expected.[6,15]
Intratumoral Treg expresses a high level of C-C motif chemokine receptor-8 (CCR8).
Additionally, the CCR8-expressing Treg has also increased the expression of CD25 and Foxp3
compared with CCR8-negative Treg.[16] Therefore, CCR8-expressing Treg is considered to havepotent immunosuppressive functions. The CCR8-expressing Treg is known to be correlated withpoor prognosis in some cancer patients.[17,18] CCR8 is attractive as a target molecule for the next

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

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cancer immunotherapy.[19] Anti-CCR8 drugs, including S-531011,[20] IPG7236,[21] and SRF114[22]are undergoing clinical trials.
CCR8 is one of the seven transmembrane-spanning G protein-coupled receptors (GPCRs).
Human CCR8 is known to bind to five C-C chemokine ligands (CCLs): CCL1/I-309, CCL4, CCL16,
CCL17, and CCL18.[23] CCR8 is upregulated in not only Treg, but also various cancers, including
breast, non-small cell lung (NSCLC), bladder, and colorectal cancer.[17,18] CCR8 mediates bladdercancer cell migration, invasion, and epithelial-mesenchymal transition by interacting with
CCL18.[24] CCR8 and its specific ligand CCL1/I-309 regulate the immune system, which mediates
the progression of diseases such as cancers. CCR8-CCL1/I-309 axis promotes migration and inhibits
apoptosis in Treg and lymphomas.[25,26] Therefore, CCR8-targeting antibodies will contribute to theelucidation of pathological mechanisms, diagnosis, and therapy.
Using the Cell-Based Immunization and Screening (CBIS) method, we previously developed
numerous monoclonal antibodies (mAbs) against chemokine receptors, including mouse
CCR3,[27,28] mouse CCR8,[29-31] human CCR9,[32] and mouse CXCR4.[33] In this study, we havesuccessfully developed an anti-human CCR8 (hCCR8) mAb using the CBIS method, which isapplicable to flow cytometry.
2. Materials and Methods
2.1. Preparation of Cell Lines
LN229, Chinese hamster ovary (CHO)-K1, and P3X63Ag8U.1 (P3U1) cells were obtained fromthe American Type Culture Collection (Manassas, VA). TALL-1 and CCRF-HSB2 cells were obtained
from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). The humannatural killer (NK) cells (donor lot. 4022602, purity > 70%) were purchased from Takara Bio (Shiga,
Japan). pCMV6neo-myc-DDK vector with hCCR8 (Accession No.: NM_005201) 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, which stably overexpressed hCCR8 with C-terminal myc-DDK tags (hereinafter describedas LN229/hCCR8 and CHO/hCCR8, respectively) were established using a cell sorter (SH800; Sony
Corp., Tokyo, Japan), following cultivation in a medium containing 0.5 mg/mL G418 (Nacalai Tesque,Inc., Kyoto, Japan).
2.2. Production of Hybridomas
For developing anti-hCCR8 mAbs, two female 6-week-old BALB/c mice were immunizedintraperitoneally with 1 × 108 cells of LN229/hCCR8. The immunogen was harvested after brief
exposure to 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). We added Imject
Alum Adjuvant (Thermo Fisher Scientific Inc.) as an adjuvant in the first immunization. Three
additional injections of 1 × 108 cells of LN229/hCCR8 were performed without an adjuvant every
week. We performed a final booster immunization of 1 × 108 cells of LN229/hCCR8 intraperitoneally
two days before harvesting splenocytes from mice. We conducted cell-fusion of the harvested
splenocytes with P3U1 cells using polyethylene glycol 1500 (PEG1500; Roche Diagnostics,
Indianapolis, IN).
2.3. Flow Cytometric Analyses
CHO-K1 and CHO/hCCR8 cells were harvested after brief exposure to 1 mM EDTA. CHO-K1,
CHO/hCCR8, TALL-1, and CCRF-HSB2 cells were washed with 0.1% bovine serum albumin in
phosphate-buffered saline and treated with primary mAbs for 30 min at 4°C. Afterward, cells weretreated with Alexa Fluor 488-conjugated anti-mouse IgG (1:1000) following the collection offluorescence data, using the SA3800 Cell Analyzer (Sony Corp.). The anti-human CD198 (CCR8)
mAbs (clones S19017D and L263G8) were purchased from BioLegend (San Diego, CA). The Alexa
Fluor 488-conjugated anti-mouse IgG was purchased from Cell Signaling Technology, Inc. (Danvers,
MA).

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2.4. Determination of the Binding Affinity by Flow Cytometry
CHO/hCCR8 and TALL-1 were suspended in 100 μL serially diluted of C8Mab-21 (100 μg/mL to
0.006 μg/mL), S19017D (10 μg/mL to 0.0006 μg/mL for CHO/hCCR8, 10 μg/mL, 2.5 μg/mL to 0.0006
μg/mL for TALL-1), or L263G8 (10 μg/mL to 0.0006 μg/mL for CHO/hCCR8, 0.625 μg/mL to 0.0006
μg/mL for TALL-1), after which Alexa Fluor 488-conjugated anti-mouse IgG (1:200) was added.
Fluorescence data were subsequently collected, using the BD FACSLyric (BD Biosciences, Franklin
Lakes, NJ), following the calculation of the dissociation constant (KD) by fitting the binding isotherms
into the built-in; one-site binding model in GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla,CA).
3. Results
3.1. Establishment of Anti-hCCR8 mAbs by the CBIS Method
To develop anti-hCCR8 mAbs, we employed the CBIS method using hCCR8-overexpressed
cells. Anti-hCCR8 mAbs-producing hybridoma screening was conducted using flow cytometry(Figure 1). Two mice were intraperitoneally immunized with LN229/hCCR8 weekly for a total of 5
times. Subsequently, hybridomas were seeded into 96-well plates, after which flow cytometricanalysis was used to select CHO/hCCR8-reactive and CHO-K1-nonreactive supernatants ofhybridomas. Afterward, we obtained CHO/hCCR8-reactive supernatant in only 1 of 956 wells(0.10%). We finally established clone C8Mab-21 (mouse IgM, kappa) by limiting dilution andadditional screening.

Figure 1. A schematic procedure of anti-hCCR8 mAbs production. The procedure of the Cell-Based
Immunization and Screening (CBIS) method for antibody development. (A) LN229/hCCR8 cells were
immunized into two mice using intraperitoneal injection. (B) The spleen cells from mice were fusedwith P3U1 myeloma cells. (C) The culture supernatants of hybridoma were screened by flow
cytometry using CHO-K1 and CHO/hCCR8. (D) After limiting dilution of hybridomas and additional
analysis, C8Mab-21 was finally established.

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3.2. Flow Cytometric Analysis
Flow cytometric analysis was conducted using C8Mab-21 and commercially available antihuman CD198 (CCR8) mAbs (clone S19017D and L263G8) against CHO-K1, CHO/hCCR8, TALL-1,
CCRF-HSB2, and NK cells. Results showed that C8Mab-21, S19017D, and L263G8 recognize
CHO/hCCR8 dose-dependently (Figure 2A). C8Mab-21, and L263G8 did not react with parental CHOK1 cells even at 20 μg/mL of mAbs. S19017D slightly reacted to CHO-K1 cells at 20 μg/mL and also 2
μg/mL (Figure 2B). Regarding endogenously hCCR8-expressing cells in Figure 3, C8Mab-21
recognized TALL-1, CCRF-HSB2, and NK cells at a concentration of 2 μg/mL or 20 μg/mL (Figure 3).
S19017D reacted to TALL-1 and CCRF-HSB2 dose-dependently even at a concentration of 0.02 μg/mL.
However, NK cells were not recognized by S19017D even at a concentration of 20 μg/mL (Figure 3).
L263G8 reacted to TALL-1 and CCRF-HSB2 even at 0.02 μg/mL, and also NK cells at a concentrationof 2 μg/mL or higher of mAb (Figure 3). C8Mab-21 could detect exogenously and endogenously
expressed naïve conformational hCCR8 in flow cytometry.

Figure 2. Flow cytometric analysis of anti-hCCR8 mAbs against CHO/hCCR8 and CHO-K1.
CHO/hCCR8 (A) and CHO-K1 cells (B) were treated with 0.02–20 μg/mL of C8Mab-21, S19017D, andL263G8 (red line), followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG.
Fluorescence data were collected using the SA3800 Cell Analyzer. Black line, control (no primary
antibody treatment).

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Figure 3. Flow cytometric analysis of anti-hCCR8 mAbs against endogenously hCCR8-expressing
cells. TALL-1 (A), CCRF-HSB2 (B), and NK cells (C) were treated with 0.02–20 μg/mL of C8Mab-21,
S19017D, and L263G8 (red line), followed by treatment with Alexa Fluor 488-conjugated anti-mouse
IgG. Fluorescence data were collected using the SA3800 Cell Analyzer. Black line, control (no primary
antibody treatment).

3.3. Determination of the Binding Affinity of Anti-hCCR8 mAbs to CHO/hCCR8
The binding affinity of C8Mab-21, S19017D, and L263G8 was assessed with exogenously hCCR8expressed CHO/hCCR8 using flow cytometry. Results showed that the KD values of C8Mab-21,
S19017D, and L263G8 for CHO/hCCR8 are 6.5×10-8 M, 2.6×10-9 M, and 1.2×10-9 M, respectively (Figure4). These results indicate that C8Mab-21 possesses a moderate affinity for exogenously overexpressed
hCCR8 in CHO-K1 cells.

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Figure 4. The analysis of the binding affinity of anti-hCCR8 mAbs for CHO/hCCR8. CHO/hCCR8cells were suspended in 100 μL of serially diluted C8Mab-21 (100 μg/mL to 0.006 μg/mL) (A), S19017D(10 μg/mL to 0.0006 μg/mL) (B), or L263G8 (10 μg/mL to 0.0006 μg/mL) (C). Then, cells were treatedwith Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were subsequently collectedusing the BD FACSLyric, following the calculation of the dissociation constant (KD) by GraphPad
PRISM 6.

3.4. Determination of the Binding Affinity of Anti-hCCR8 mAbs to TALL-1
The binding affinity of C8Mab-21, S19017D, and L263G8 was analyzed with endogenously
hCCR8-expressing TALL-1 using flow cytometry. Results indicated that the KD values of C8Mab-21,
S19017D, and L263G8 for TALL-1 are 2.0×10-8 M, 4.6×10-10 M, and 7.8×10-11 M, respectively (Figure 5).
These results showed that C8Mab-21 possesses a moderate affinity for endogenously expressing
hCCR8 in TALL-1 leukemia cells.

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Figure 5. The analysis of the binding affinity of anti-hCCR8 mAbs for TALL-1. TALL-1 cells weresuspended in 100 μL of serially diluted C8Mab-21 (100 μg/mL to 0.006 μg/mL) (A), S19017D (10 μg/mL,
2.5 μg/mL to 0.0006 μg/mL) (B), or L263G8 (0.625 μg/mL to 0.0006 μg/mL) (C). Then, cells were treatedwith Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were subsequently collectedusing the BD FACSLyric, following the calculation of the dissociation constant (KD) by GraphPad
PRISM 6.

4. Discussion
GPCRs, including chemokine receptors, are focused as targets for many diseases, including
inflammatory disorders and cancers.[34,35] GPCRs transmit signals to intracellular molecules about
extracellular conditions and govern broad cell dynamics, such as proliferation, homeostasis,
migration, and motility of the cells.[34,35] Therapeutic drugs, including mAbs targeting GPCRs, havebeen developed to date; however, the complexity of the structure, the small area of epitope regions,and the difficulty of purifying the protein as an immunogen pose high hurdles.[36,37]

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Unlike protein purifications, the preparation of antigens is not complicated in the CBIS method.
Furthermore, it is possible to retain the antigen structure and modification such as glycosylation andfolding using the CBIS method. We have successfully developed multiple mAbs by the CBIS method
against human epidermal growth factor receptor 1 (HER1; EGFR),[38] HER3,[39] trophoblast cellsurface antigen 2,[40] epithelial cell adhesion molecule,[41] CD44,[42-44] and podoplanin.[45]Furthermore, some of the developed mAbs by the CBIS method exhibited cancer specificity byrecognizing unique cancer-specific epitopes.[46-51] Therefore, the CBIS method is an efficient anduseful tactic for generating diverse antibodies targeting membrane proteins.
In the immunosuppressive TME, CD8+T cells are exhausted along with the induction of
CCR8+Treg. CCR8+Treg infiltration is confirmed to be associated with high TOX, an exhaustion
marker of T cells, expression in CD8+T cells in some types of cancer patients.[52,53] Elimination of
CCR8+Treg using antibodies is expected to advance the treatment of these cancer patients. Targeting
CCR8 might be more specific in antitumor activity than other approaches aimed at Treg
removal.[53,54] In mice, CCR8+T cell depletion therapy using anti-CCR8 mAbs induces tumorspecific immune responses without triggering autoimmune responses or immune reactions in
TME.[55] Since C8Mab-21 could recognize cell surface hCCR8, we plan to investigate the function of
C8Mab-21 against Treg, such as detection and interfering effects in future studies. Furthermore, wehave previously enhanced antibody-dependent cellular cytotoxicity (ADCC) activity andcomplement-dependent cytotoxicity (CDC) by modifying isotypes and defucosylation in
mAbs.[56,57] Since C8Mab-21 is mouse IgM, which has no ADCC activity, it will be converted into amouse IgG2a version to evaluate the effect of antitumor activities in xenograft models.
Interestingly, a correlation between cancer-associated fibroblasts (CAFs) and CCR8 has beenfound from the results of omics analysis.[22] CCR8 is suggested to be involved in the pathogenesisof various cancer types. CAFs are one of the tumor suppressor factors, the same as Treg, that are
known to interfere with the function of tumor immune cells by promoting fibrosis and constructing
an extracellular matrix in the TME.[58,59] CAFs with a myofibroblastic-like phenotype transfer large
amounts of proteins to the surrounding endothelial cells through matrix-bound vesicles, which might
contribute to cancer progression and treatment resistance.[60,61] Recently, the phenotypes of CAF
have been reported to be associated with better or worse outcomes in NSCLC patients.[62] Althoughfurther functional analysis of CCR8-expressing CAFs is required, targeting CCR8 might suppress
Treg and CAFs, and lead to synergistic antitumor immunotherapy results. In that case, it is well worth
evaluating the impact of C8Mab-21 on CAFs as well in future studies.
Authorship confirmation/contribution statement: T.T. and G.L. performed the experiments. M.K.K. and Y.K.
designed the experiments. T.T. analyzed the data. T.T., H.S., and Y.K. wrote the manuscript. All authors haveread and agreed to the published version of the manuscript.
Author Disclosure Statement: The authors have no conflict of interest.
Funding Information: This research was supported, in part, by Japan Agency for Medical Research andDevelopment (AMED) under Grant Nos.: JP23ama121008 (to Y.K.), JP23am0401013 (to Y.K.), JP23bm1123027 (toY.K.), and JP23ck0106730 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid
for Scientific Research (KAKENHI) Grant Nos.: 21K20789 (to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and22K07224 (to Y.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.

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