MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 2, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0029
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Epitope Mapping of an Anti-Mouse CD39 Monoclonal
Antibody Using PA Scanning and RIEDL Scanning

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Yuki Okada, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K. Kaneko, and Yukinari Kato

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular adenosine triphosphate into
immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor
microenvironment attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied toantitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope ofmAbs is important. We previously established an anti-mouse CD39 (anti-mCD39) mAb, C39Mab-1 using the
Cell-Based Immunization and Screening method. In this study, we determined the critical epitope of C39Mab-1
using flow cytometry. We performed the PA tag (12 amino acids [aa])-substituted analysis (named PA scanning) and RIEDL tag (5 aa)-substituted analysis (named RIEDL scanning) to determine the critical epitope of
C39Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified theconformational epitope, spanning three segments of 275–279, 282–291, and 306–323 aa of mCD39. These
analyses would contribute to the identification of the conformational epitope of membrane proteins.
Keywords: mouse CD39, monoclonal antibody, epitope, PA scanning, RIEDL scanning

catalyze the hydrolysis of extracellular ATP and adenosine
diphosphate to adenosine monophosphate. Then, CD73 (5¢nucleotidase; encoded by NT5E) dephosphorylates AMP into
adenosine.9
Since CD39 mediates the dephosphorylation of extracellular ATP to immunosuppressive adenosine, anti-CD39
monoclonal antibodies (mAbs) have been generated tomodulate the adenosine metabolism.6 A preclinical study
showed that B66, an anti-mouse CD39 (mCD39) mAb, caninhibit mCD39 enzymatic activity in vitro and exerted theantitumor effect by the mono- or combination therapy with
the PD-1 blockade.10 The anti-human CD39 mAbs, such as
TTX-030, IPH5201, and SRF-617, were designed to inhibit
the CD39 enzymatic activity.7,10 These mAbs have been
evaluated in clinical trials for solid tumors in combination
with chemotherapeutic agents or immune checkpoint inhibitors.8 However, the relationship between the inhibitory
activity and the epitope has not been clarified.
We previously established a novel anti-mCD39 mAb(C39Mab-1) by the Cell-Based Immunization and Screening

Introduction

In the tumor microenvironment (TME), high concentrations of extracellular adenosine triphosphate (ATP)(100 to 500 mM) exist compared to nanomolar order in normal
tissues due to the passive release of cell death and active secretion by tumor cells and other subsets.1–3 The extracellularadenosine, produced by the hydrolysis of extracellular ATP, isinvolved in immunosuppressive TME4 and suppresses antitumor immune responses and enhances the immunologic escape of tumor cells.5 Therefore, the extent of ATP release andits degradation to adenosine should be controlled to restrict theimmunosuppressive TME and to facilitate the antitumor immunity during cancer immunotherapy.6,7CD39 (ectonucleoside triphosphate diphosphohydrolase 1;
encoded by ENTPD1) protein has 510 amino acids (aa) andharbors 7 potential N-linked glycosylation sites and 11 cysteine residues.8 Two transmembrane domains exist in the
CD39 protein. In the extracellular domain of CD39, fivehighly conserved segments mediate the enzymatic activity to

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.

44

EPITOPE MAPPING OF ANTI-MOUSE CD39 MAB(CBIS) method,11–36 and evaluated its applications, including
flow cytometry and western blot analyses.37 In this study, we
performed epitope mapping of C39Mab-1 using flow
cytometry-mediated novel strategies, named PA scanningand RIEDL scanning.
Materials and Methods
Antibodies

Plasmid construction and transfection

mCD39 cDNA was cloned into a pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan). For PA scanning, the substitution of PA tag(GVAMPGAEDDVV) in mCD39 was performed with oligonucleotides containing PA tag sequence at the desired
position. For example, for the substitution of the PA tag from
K288 to P299 of mCD39, we constructed E287GVAMPGAEDDVV-C300 (288-PA-299) in mCD39. For
RIEDL scanning, the substitution of the R* tag in mCD39
was performed with oligonucleotides containing the R* tagsequence at the desired position. For example, for the substitution of the R* tag from E287 to N291 of mCD39, weconstructed Y286-RIEDL-V292 (287-R*-291) in mCD39.
Alanine scanning in the mCD39 sequence was performed
with oligonucleotides containing the alanine sequence at the

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C39Mab-1 (an anti-mCD39 mAb),37 LpMab-7 (an antiRIEDL [R*] tag mAb),38–46 and NZ-1 (an anti-PA tagmAb)47–53 were described previously. An anti-mCD39 mAb(clone 5F2, mouse IgG1, kappa) was purchased from
BioLegend (San Diego, CA). Alexa Fluor 488-conjugatedanti-mouse IgG and anti-rat IgG were purchased from Cell
Signaling Technology, Inc. (Danvers, MA).

45

FIG. 1. The PA tag-substituted mutants of mCD39. The reactivities of C39Mab-1 are indicated: +, reactive; -, nonreactive.
mCD39, mouse CD39.

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FIG. 2. Epitope determination of C39Mab-1 using PA tag-substituted mutants of mCD39. The PA tag-substituted mutantsof mCD39 were transiently expressed in CHO-K1 cells. The mutants-expressed CHO-K1 cells were incubated with 1 mg/mL
of C39Mab-1 (A, red line), 1 mg/mL of NZ-1 (B, red line), or control blocking buffer (black line), followed by secondary
antibodies treatment. The data were analyzed using the SA3800 Cell Analyzer. CHO, Chinese hamster ovary; P. C., positive
control (CHO/PA-CD133); WT, wild type mCD39.
46

EPITOPE MAPPING OF ANTI-MOUSE CD39 MAB

47

desired position. The PCR fragments bearing the desired
mutations were inserted into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation) using an In-Fusion
HD Cloning Kit (Takara Bio, Inc., Shiga, Japan).
The mCD39 mutant plasmids were transiently transfected
into Chinese hamster ovary (CHO)-K1 cells (the American
Type Culture Collection, Manassas, VA) using the Neon
Transfection System (Thermo Fisher Scientific, Inc., Waltham, MA). CHO-K1 cells and transfectants were cultured in
Roswell Park Memorial Institute-1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan) supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific,Inc.), 100 units/mL of penicillin, 100 lg/mL of streptomycin,
and 0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.).
The cells were cultured at 37C in a humidified atmosphere
containing 5% carbon dioxide and 95% air.

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Flow cytometry

CHO-K1 cells and transfectants were harvested after abrief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.) and washed with 0.1%
bovine serum albumin in phosphate-buffered saline.
C39Mab-1 (1 lg/mL), LpMab-7 (10 lg/mL), or NZ-1
(1 lg/mL) were incubated for 30 minutes at 4C. The cells
were further treated with Alexa Fluor 488-conjugated antimouse IgG (1:2000 for LpMab-7 and 5F2) or anti-rat IgG(1:2000 for C39Mab-1 and NZ-1). Fluorescence data werecollected using the SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Results

FIG. 3. The RIEDL tag-substituted mutants of mCD39.
The reactivities of C39Mab-1 are indicated: +, reactive; -,nonreactive.

Epitope mapping of C39Mab-1 using flow cytometry
with PA tag-substituted mCD39

We previously established an anti-mCD39 mAb (C39Mab1) by the CBIS method.37 To determine the C39Mab-1 epitope, we first examined the reactivity to the peptides thatcover the extracellular domain of mCD39. However,C39Mab-1 did not react with the peptides (data not shown),suggesting that C39Mab-1 recognizes a conformational and/or modified epitope.
To identify the binding epitope of C39Mab-1, we generated
PA tag (GVAMPGAEDDVV)-substituted mCD39 mutants
as shown in Figure 1. We analyzed the reactivity of C39Mab1 against the PA tag-substituted mCD39 (named PA scanning). As shown in Figure 2A, the reactivity of C39Mab-1
almost completely disappeared in 255-PA-266, 267-PA-278, 269PA-280, 282-PA-293, 288-PA-299, 301-PA-312, 312-PA-323, 325PA-336, and 415-PA-426 mutants of mCD39. In contrast, thereactivity of C39Mab-1 was observed in the PA tagsubstituted mutants of 38–253, 343–413, and 427–462 aa.
The cell surface expression of each mutant was confirmed byan anti-PA tag mAb, NZ-1 (Fig. 2B). The reactivity wassummarized in Figure 1. These results indicated that the
epitope of C39Mab-1 contains 255–336 and 415–426 aa of
mCD39.
Epitope mapping of C39Mab-1 using flow cytometry
with RIEDL tag-substituted mCD39

As shown in Figure 2A, C39Mab-1 did not react with thecontinuous PA tag-substituted region of 255–336 aa in

mCD39. We generated the R* tag-substituted mCD39 asshown in Figure 3 to narrow down the C39Mab-1 epitope. We
analyzed the reactivity of C39Mab-1 against the R* tagsubstituted mCD39 (named RIEDL scanning). As shown in
Figure 4A, the reactivity of C39Mab-1 almost completely
disappeared in 275-R*-279, 282-R*-286, 287-R*-291, 306-R*-310,
311-R*-315, 316-R*-320, and 319-R*-323 mutants of mCD39. In
contrast, the reactivity of C39Mab-1 was observed in R* tagsubstituted mutants of 255–274, 276–280, 292–305, and
325–336 aa. The cell surface expression of each mutant was
confirmed by an anti-R* tag mAb, LpMab-7 (Fig. 4B). The
reactivity was summarized in Figure 3. These results narrowed down the epitope of C39Mab-1 in 275–279, 282–291,
and 306–323 aa of mCD39.
Epitope mapping of C39Mab-1 using flow cytometry
with 1 · alanine- or 2 · alanine-substituted mCD39

The 1 · alanine- or 2 · alanine-substituted mutant analyses
are important strategies to determine the center of the epitope.54–56 We next generated 33 alanine-substituted mCD39
in 275th to 323rd aa of mCD39 and investigated the reactivityof C39Mab-1 against CHO-K1 cells, which overexpressed themCD39 mutants transiently. As a result, C39Mab-1 reactedwith all alanine-substituted mutants and wild type (Supplementary Fig. S1).
We also examined the reactivity of C39Mab-1 against
2 · alanine-substituted mCD39; however, C39Mab-1 reacted

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FIG. 4. Epitope determination of C39Mab-1 using RIEDL tag-substituted mutants of mCD39. The RIEDL (R*) tagsubstituted mutants of mCD39 were transiently expressed in CHO-K1 cells. The mutants-expressed CHO-K1 cells wereincubated with 1 mg/mL of C39Mab-1 (A, red line), 10 mg/mL of LpMab-7 (B, red line), or control blocking buffer (blackline), followed by secondary antibodies treatment. The data were analyzed using the SA3800 Cell Analyzer. P. C., positive
control (CHO/2·RIEDL-seaPDPN).24
48

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EPITOPE MAPPING OF ANTI-MOUSE CD39 MAB

49

FIG. 5. Structure of CD39 and putative epitope of C39Mab-1. (A) Rat CD39 structure (PDB ID: 3ZX3). The corresponding sequence to the three segments of C39Mab-1 epitope segments is highlighted. Apyrase-conserved regions (ACR1–
5) are also indicated. (B) The alignment of rat and mouse CD39 sequence around C39Mab-1 epitope segments. The
underlined sequences form a b-sheet as shown in (A). (C) Putative b-sheet structure of C39Mab-1 epitope segments.
with all mutants (Supplementary Fig. S2). Therefore, we
could not determine the epitope of C39Mab-1 using 1 · alanine or 2 · alanine scanning methods.
Discussion

In this study, we performed the flow cytometry-based
epitope mapping of C39Mab-1 using the PA scanning(Figs. 1, 2) and the RIEDL scanning (Figs. 3, 4). We foundthat the three segments, 275–279, 282–291, and 306–323 aa
of mCD39 are important for the recognition by C39Mab-1.
Zebisch et al. demonstrated the crystal structure of rat
CD39 between the two lobes of the catalytic domain.57
Figure 5A shows the structure of rat CD39 (PDB ID: 3ZX3).
Apyrase-conserved regions (ACR1–5) form the active-sitecleft,58 which is distant from the three segments identified as
C39Mab-1 epitope. As shown in Figure 5B, the corresponding rat CD39 sequence to the three segments forms ab-sheet structure. In mouse sequence motifs, 276-VLKD-279,
287-EKVVN-291, and 312-QFRIQG-317 could contribute to

the formation of the b-sheet (Fig. 5C). The substitution of
the R* tag on not only the above three motifs but also surrounding sequence may disrupt the b-sheet structure, which
results in the impaired recognition by C39Mab-1. Although
we could not determine the critical aa of the epitope by
1 · alanine or 2 · alanine scanning methods (SupplementaryFigs. S1, S2), it would be interesting to introduce mutations
between some b-sheet segments and examine the reactivityof C39Mab-1.
Therapeutic anti-CD39 mAbs, including TTX-030, were
designed to inhibit CD39 enzymatic activity through theuncompetitive allosteric mechanism of action.7,10 The epitope of TTX-030 was determined as E142 to Y159 using thehuman-mouse CD39 chimeric protein by flow cytometry.59
The region is distal to the ATP-binding residues (E174 andS218), supporting the allosteric mechanism of action by
TTX-030. However, the optimal mAb-binding sites to inhibit
the CD39 enzymatic activity have not been identified.
Therefore, the detailed relationship between each mAb epitope and the inhibitory activity should be determined. The PA

50

OKADA ET AL.

scanning and RIEDL scanning would contribute to the determination of the conformational epitope of not only CD39
but also other membranous antigens.
Acknowledgment

This article was previously published in preprint.org (doi:
10.20944/preprints202312.0369.v1).
Authors’ Contributions

Y.O. and T.T. performed the experiments. M.K.K. and
Y.K. designed the experiments. H.S. and M.K.K. analyzed
the data. H.S. and Y.K. wrote the article. All authors haveread and agreed to the published version of the article.
Author Disclosure Statement

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No competing financial interests exist.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development under Grant numbers
JP23ama121008 (to Y.K.), JP23am0401013 (to Y.K.),
23bm1123027h0001 (to Y.K.), and JP23ck0106730(to Y.K.), and by the Japan Society for the Promotion of
Science Grants-in-Aid for Scientific Research (KAKENHI)
Grant numbers 21K20789 (to T.T.), 22K06995 (to H.S.),
21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Supplementary Material

Supplementary Figure S1
Supplementary Figure S2
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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: December 5, 2023
Accepted: February 5, 2024