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ArticleEpitope Mapping of an Anti-Mouse CCR8
Monoclonal Antibody C8Mab-2 Using Flow
Cytometry
Hiyori Kobayashi, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K. Kaneko and Yukinari Kato *Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan; hiyori.kobayashi.r2@dc.tohoku.ac.jp (H.K.);
hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); tomohiro.tanaka.b5@tohoku.ac.jp (T.T.);mika.kaneko.d4@tohoku.ac.jp (M.K.K.)* Correspondence: yukinari.kato.e6@tohoku.ac.jp; Tel.: +81-22-717-8207

Abstract: The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed inregulatory T (Treg) cells and is associated with tumor progression. The massive accumulation ofTreg cells into tumors suppresses the effector function of CD8+ cells against tumor cells. Therefore,
selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates
antitumor immune responses and improves responses to cancer immunotherapy. Previously, wedeveloped an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the Cell-Based Immunization andScreening (CBIS) method. In this study, the binding epitope of C8Mab-2 was investigated using flow
cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C8Mab-2
recognizes the N-terminal region (1–33 amino acids) of mCCR8. Next, 1×alanine (or glycine)scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results
revealed that the 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is acentral amino acid of the epitope. These results revealed the involvement of the N-terminus of
mCCR8 in the recognition by C8Mab-2.
Keywords: mouse CCR8; monoclonal antibody; epitope mapping; alanine scanning; flow cytometry

1. Introduction
The C-C motif chemokine receptor 8 (CCR8) is a member of G protein-coupled receptors(GPCRs) family. The C-C motif chemokine ligands (CCLs), including CCL1, CCL8, CCL16, andCCL18 are known as ligands for human CCR8. CCL1 is the only ligand for CCR8,[1] which is
produced by CD11b+CD14+ myeloid cells during the infiltration of regulatory T (Treg) cells into
tumor.[2] Upon binding of CCL1 to CCR8, the FOXP3 is upregulated by the STAT3 pathway, and theactivated CCR8+ Treg cells potently suppress antitumor immunity through secretion of granzyme B
and IL-10.[3] Increased expression of CCR8 is observed in Treg cells, especially in cancer patients.[4]Patients with high levels of Treg cells exhibit poor prognoses and clinical outcomes in several
cancers.[5] Therefore, it has been proposed that depletion of tumor-infiltrated Treg cells could restore
antitumor immunity and improve responses to tumor immunotherapy.[6] Recent preclinical mousemodels have revealed that depletion of Treg cells using an anti-mouse CCR8 (mCCR8) monoclonal
antibody (mAb) exhibited strong antitumor responses through dramatic changes of the intratumor
CD8+ T cell profile[7] or enhanced the antitumor effects of anti-programmed cell death 1 (PD-1)
therapy.[8]The understanding of the structural-based CCR activation is important for the development oftherapeutic agents. Among the CCR family members, CCR2 and CCR5 have been structurally solvedin both inactive and active states,[9–12] while inactive-state of CCR7 and CCR9, and active-state of
CCR1 and CCR6 structures are also characterized.[13–16] Furthermore, the structures of CCR8 in© 2024 by the author(s). Distributed under a Creative Commons CC BY license.

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complex with either the antagonistic mAb or the endogenous ligand CCL1 were determined, which
provides the specific activation mechanism by CCL1 and inhibition by mAb.[17]We have developed anti-mouse GPCR mAbs against CCR1 (clone C1Mab-6),[18] CCR3 (clones
C3Mab-2, C3Mab-3, and C3Mab-4),[19–21] CCR8 (clones C8Mab-1, C8Mab-2, and C8Mab-3),[22–24]
CXCR1 (clone Cx1Mab-1),[25] CXCR3 (clone Cx3Mab-4),[26] and CXCR4 (clone Cx4Mab-1)[27] using
the Cell-Based Immunization and Screening (CBIS) method. For the determination of the epitopes,
we have faced difficulty using conventional methods such as enzyme-linked immunosorbent assay.
In this study, epitope mapping of the an anti-mCCR8 mAb was conducted by flow cytometry-based
approaches.
2. Materials and Methods
2.1. Plasmid construction and cell lines
Chimeric mutants including mCCR3 (mCCR8p1–33), mCCR3 (mCCR8p92–105), mCCR3(mCCR8p170–200), and mCCR3 (mCCR8p262–278) and alanine (or glycine) substituted mutants inthe mCCR8 N-terminal region were produced with a PA16 tag at their N-terminus. Stabletransfectants of Chinese hamster ovary (CHO)-K1 and CHO/mCCR8 cells were established in ourprevious study.[24] The chimeric and the point mutant plasmids were transfected into CHO-K1 cells.
Stable transfectants were selected using a cell sorter (SH800; Sony Corp., Tokyo, Japan).
2.2. Antibodies
C8Mab-2 was established by the CBIS method.[23] NZ-1 (an anti-PA16 tag mAb) was described
previously.[28] A secondary Alexa Fluor 488-conjugated anti-rat IgG was purchased from Cell
Signaling Technology, Inc. (Danvers, MA, USA).
2.3. Flow cytometry
Cells were treated with C8Mab-2 (10 μg/mL) or NZ-1 (1 μg/mL) for 30 min at 4℃ andsubsequently with Alexa Fluor 488-conjugated anti-rat IgG (1:2000; Cell Signaling Technology, Inc.).
Fluorescence data were obtained using the SA3800 Cell Analyzer (Sony Corp.).
3. Results
3.1. Determination of the epitope of an anti-mCCR8 mAb by flow cytometry using chimeric proteins
C8Mab-2 is applicable for flow cytometry and immunocytochemistry.[23] To investigate thebinding epitope of C8Mab-2, we focused on four extracellular regions of mCCR8, including the Nterminal region [1–33 amino acids (aa)], extracellular loop 1 (ECL1; 92–105 aa), ECL2 (170–200 aa),and ECL3 (262–278 aa). The four extracellular regions of mCCR8 were substituted into thecorresponding regions of mCCR3, which possesses a similar structure to mCCR8. As shown in Figure
1, mCCR3 (mCCR8p1–33), mCCR3 (mCCR8p92–105), mCCR3 (mCCR8p170–200), and mCCR3(mCCR8p262–278) were generated. The chimeric proteins were transiently expressed on CHO-K1
cells, and the reactivities to C8Mab-2 were analyzed using flow cytometry (Figure 2A). C8Mab-2
reacted with mCCR3 (mCCR8p1–33) and CHO/mCCR8 cells, but not with other chimeric proteins(Figure 2A). The cell surface expression of each mutant was confirmed by an anti-PA16 tag mAb, NZ1 (Figure 2B). These results indicated that the N-terminal region of mCCR8 is recognized by C8Mab2.

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Figure 1. Schematic illustration of chimeric proteins. The four extracellular regions of mCCR8,
including the N-terminal region (1–33 aa), ECL1 (92–105 aa), ECL2 (170–200 aa), and ECL3 (262–278
aa) were substituted into the corresponding regions of mCCR3. ECL, extracellular loop.

Figure 2. Determination of the epitope of an anti-mCCR8 mAb by flow cytometry using chimeric
proteins. C8Mab-2 (10 μg/mL) (A) and an anti-PA16 tag mAb, NZ-1 (1 μg/mL) (B) were treated with
CHO-K1 cells which were transiently expressed the chimeric proteins, CHO/mCCR8, or CHO-K1 cells
for 30 min at 4°C, followed by the addition of Alexa Fluor 488-conjugated anti-rat IgG. Red lines show
the cells with C8Mab-2 or NZ-1 treatment, and black lines show cells treated with a blocking buffer asa negative control.

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3.2. Determination of the C8Mab-2 epitope by flow cytometry using 1×alanine scanning
Thirty-two 1×alanine (or glycine) substitution mutants of mCCR8 were constructed, and themutant proteins were stably expressed on CHO-K1 cells. The reactivity against C8Mab-2 was assessedusing flow cytometry. As shown in Figure 3A, C8Mab-2 did not react with a mutant (T20A). In
contrast, C8Mab-2 reacted with the other 31 mutants. The cell surface expression of each mutant was
confirmed by NZ-1 (Figure 3B). These results showed that Thr20 of mCCR8 is important for C8Mab2 binding.

Figure 3. Determination of the C8Mab-2 epitope by flow cytometry using 1×alanine scanning.
C8Mab-2 (10 μg/mL) (A) and NZ-1 (1 μg/mL) (B) were treated with CHO-K1 cells which were stably
expressed mutant proteins or CHO-K1 cells for 30 min at 4°C, followed by the addition of Alexa Fluor
488-conjugated anti-rat IgG. Red lines show the cells with C8Mab-2 or NZ-1 treatment, and black lines
show cells treated with a blocking buffer as a negative control.

3.3. Determination of the C8Mab-2 epitope by flow cytometry using 2×alanine scanning
We also examined the reactivity of C8Mab-2 against 2×alanine (or glycine)-substituted mCCR8.
We constructed thirty 2×alanine (or glycine)-substituted mutants in the N-terminal region of mCCR8
except for Cys23. The reactivity against C8Mab-2 was assessed using flow cytometry. As shown in
Figure 4A, C8Mab-2 did not react with the four mutants (D17A-F18A, F19A-T20A, T20A-A21G, andPreprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 January 2024

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A21G-P22A). In contrast, C8Mab-2 reacted with the other 26 mutants. The cell surface expression ofeach mutant was confirmed by NZ-1 (Figure 4B). These results showed that a motif from Asp17 to
Pro22 in mCCR8 is important for C8Mab-2 recognition.

Figure 4. Determination of the C8Mab-2 epitope by flow cytometry using 2×alanine scanning.
C8Mab-2 (10 μg/mL) (A) and NZ-1 (1 μg/mL) (B) were treated with CHO-K1 cells which weretransiently expressed mutant proteins or CHO-K1 cells for 30 min at 4°C, followed by the addition of
Alexa Fluor 488-conjugated anti-rat IgG. Red lines show the cells with C8Mab-2 or NZ-1 treatment,
and black lines show cells treated with a blocking buffer as a negative control.

4. Discussion
In this study, we performed the flow cytometry-based epitope mapping of an anti-mouse CCR8mAb (C8Mab-2) using the chimeric proteins (Figures 1 and 2). Furthermore, we determined that the
17-DFFTAP-22 is important for the recognition by C8Mab-2 in the 2×alanine scanning (Figure 4), andThr20 is a central amino acid of the epitope by the 1×alanine scanning (Figure 3). Figure 5 summarizes
the epitope of C8Mab-2. In the epitope mapping of an anti-human CCR8 mAb (clone mAb1), CCR8
chimeras, in which the N-terminus, ECL1, ECL 2, or ECL 3 were replaced with the corresponding
sequences from human CCR5, were used.[17] The mAb1 did not recognize ECL1-replaced CCR8 andPreprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 January 2024

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ECL2-replaced CCR8, suggesting that both ECL1 and ECL2 are required for mAb1 binding.[17]Because we could not determine the binding epitope of our other anti-mCCR8 mAbs (C8Mab-1 andC8Mab-3) in this study (data not shown), the substitution of two ECLs may be required for theidentification of the epitopes of those anti-mCCR8 mAbs.

Figure 5. The schematic illustration of the C8Mab-2 epitope, which was identified by 1×alanine
scanning and 2×alanine scanning. The 17-DFFTAP-22 is important for the recognition by C8Mab-2 in the
2×alanine scanning (underlined). The Thr20 (red) is determined to be a central amino acid of the
epitope by the 1×alanine scanning.

The “hot” tumor is characterized by the massive infiltration of CD8+ effector T cells, which isimportant for the antitumor immune responses. Immune checkpoint inhibitors such as anti-PD-1
mAbs are effective in hot tumors. However, the response rate is still low due to the lack of CD8+
effector T cell infiltration or accumulation of Treg cells suppressing the effector activities, which ischaracterized as “cold tumors”.[29,30] Since CCR8 expression is increased in tumor-infiltrated Treg
cells, CCR8 is one of the promising target for depleting of Treg cells selectively in tumors.[4] AntimCCR8 mAbs have been used to suppress cancer growth in several cancer models.[7,8,31]
Furthermore, an anti-human CCR8 mAb (S-531011) was developed.[32] S-531011 has antibodydependent cell-mediated cytotoxicity activity against tumor-infiltrating CCR8+ Treg cells andneutralization activity against the CCR8 signaling.[32] Meanwhile, there is no information about therelationship between the Treg cells-depleting activity and epitope of the mAbs. Our strategy for
epitope identification would contribute not only to the understanding of mAb-epitope interaction
but also to the improvement of those therapeutic mAbs.
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Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely thoseof the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s)disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or
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