MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 1, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0016

Short Communication
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Development of a Sensitive Anti-Mouse CD39 Monoclonal
Antibody (C39Mab-1) for Flow Cytometry
and Western Blot Analyses

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

CD39 is involved in adenosine metabolism by converting extracellular ATP to adenosine. As extracellularadenosine plays a critical role in the immune suppression of the tumor microenvironment, the inhibition of
CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. This study
developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization andScreening method. The established anti-mCD39 mAb, C39Mab-1 (rat IgG2a, kappa), reacted with mCD39overexpressed Chinese hamster ovary-K1 (CHO/mCD39) by flow cytometry. The kinetic analysis using flow
cytometry indicated that the dissociation constant of C39Mab-1 for CHO/mCD39 was 7.3 · 10-9 M. Furthermore, C39Mab-1 detected the lysate of CHO/mCD39 by western blot analysis. These results indicated thatC39Mab-1 is useful for the detection of mCD39 in many functional studies.
Keywords: mouse CD39, monoclonal antibody, the Cell-Based Immunization and Screening, CBIS

nosuppression by activation of effectors, including protein
kinase A.6
Sitkovsky’s group first reported the immunosuppressive
effects of the A2A receptor in vivo.7 Inflammatory stimuli thatcaused minimal tissue damage in wild-type mice were sufficient to induce extensive tissue damage, more prolonged
and higher levels of proinflammatory cytokines, and individual death in mice lacking the A2A receptor.7 The groupalso showed genetic evidence of the importance of the A2A
receptor in tumor immunity.8 These findings impacted antitumor immunity by CD39–adenosine–A2 receptor axis andseveral landmark studies have developed multiple strategies
targeting adenosine metabolism.3,9The development of anti-CD39 monoclonal antibodies(mAbs) is a strategy to modulate the adenosine metabolism.
A preclinical study revealed that an anti-mouse CD39
(mCD39) mAb (clone B66), which can inhibit mCD39 activity in vitro, exhibited the antitumor effect in syngeneic
models by the monotherapy and combination therapy withthe programmed cell death-1 (PD-1) blockade.10 This
study also showed that B66 triggers an eATP–P2X7–
inflammasome–IL-18 pathway that promotes tumor immunity and overcomes anti-PD-1 resistance.10 The anti-humanIntroductionE

xtracellular adenosine, generated by the hydrolysis of extracellular ATP (eATP), mediates an immunosuppressive tumor microenvironment (TME).1 The highconcentration of eATP can be found in solid tumors due to thepassive release of cell death and active secretion by tumor
cells and other subsets in the TME.2 Following the release ofeATP, CD39 (ectonucleoside triphosphate diphosphohydrolase 1; encoded by ENTPD1) hydrolyzes eATP to
ADP and AMP. Then, another rate-limiting ectoenzyme,
CD73 (5¢-nucleotidase; encoded by NT5E), dephosphorylates AMP into adenosine.3
Growing body of evidence suggests that adenosinemediated immunosuppression is critical for tumor immune
evasion. Various tumors showed the elevated expression of
CD39, which promotes the local accumulation of adenosine
surrounding tumors.4 The adenosine-mediated immunosuppressive effect functions via four G protein-coupled type 1
purinergic (P1) receptors, A1, A2A, A2B, and A3 expressedon immune cells.5 Among the four P1 receptors, the A2A
and A2B are GS-coupled receptors and trigger intracellularcAMP accumulation. The cAMP signaling mediates immu-

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

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ANTI-MOUSE CD39 MAB

CD39 mAbs (clones TTX-030, IPH5201, and SRF-617) were
designed to inhibit CD39 enzymatic activity via allostericinhibition and minimize Fc receptor-mediated engagement toavoid the side effects.10,11 These mAbs have entered theclinical trials for solid tumors with a combination of chemotherapeutic agents or immune checkpoint inhibitors.3
Using the Cell-Based Immunization and Screening (CBIS)
method, we have developed many mAbs against membraneproteins, such as CD19,12 CD20,13,14 CD133,15 EpCAM,16,17
HER2,18 HER3,19 KLRG1,20 TIGIT,21 TROP2,22,23 programmed cell death ligand 1 (PD-L1),24 podoplanin,25–36 andCD44.37,38 The CBIS method includes the immunization of
antigen-overexpressed cells and high-throughput hybridoma
screening using flow cytometry. Anti-chemokine receptors
mAbs, including anti-mouse CCR3,39 anti-mouse CCR8,40
and anti-human CCR941 mAbs, were also successfully developed using the CBIS method.
In this study, novel anti-mCD39 mAbs were developed by
the CBIS method. We further evaluated its applications, including flow cytometry and western blot analyses.
Materials and Methods
Preparation of cell lines

LN229, Chinese hamster ovary (CHO)-K1, andP3X63Ag8U.1 (P3U1) were obtained from the American
Type Culture Collection (Manassas, VA).
The synthesized DNA (Eurofins Genomics KK) encoding
mCD39 (Accession No.: NM_009848) was subsequently
subcloned into a pCAGzeo_nPA-cRAPMAP vector, which isderived from a pCAGzeo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), N-terminal PA tag,42–44 andC-terminal RAP tag45,46 + MAP tag.47,48 The amino acid sequences of the tag system were as follows: PA tag, 12 aminoacids (GVAMPGAEDDVV); RAP tag, 12 amino acids(DMVNPGLEDRIE); and MAP tag, 12 amino acids(GDGMVPPGIEDK). The PA tag can be detected by an antihuman podoplanin mAb (clone NZ-1).42–44,49–61 The mCD39
plasmid was transfected into CHO-K1 and LN229 cells, using a
Neon transfection system (Thermo Fisher Scientific Inc., Waltham, MA). Stable transfectants were established through cellsorting using a cell sorter (SH800; Sony Corp., Tokyo, Japan),after which cultivation in a medium, containing 0.5 mg/mL of
Zeocin (InvivoGen, San Diego, CA) was conducted.
CHO-K1, mCD39-overexpressed CHO-K1 (CHO/
mCD39), and P3U1 were cultured in a Roswell Park Memorial
Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan), with 10% heat-inactivated fetal bovine serum (FBS;
Thermo Fisher Scientific Inc.), 100 units/mL of penicillin,
100 mg/mL of streptomycin, and 0.25 mg/mL of amphotericin
B (Nacalai Tesque, Inc.). LN229 and mCD39-overexpressed
LN229 (LN229/mCD39) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Nacalai Tesque, Inc.), supplemented with 10% FBS, 100 U/mL of penicillin, 100 mg/mL
streptomycin, and 0.25 mg/mL amphotericin B.
All cells were grown in a humidified incubator at 37C, at
an atmosphere of 5% carbon dioxide and 95% air.
Antibodies

An anti-mCD39 mAb (clone 5F2, mouse IgG1, kappa) was
purchased from BioLegend (San Diego, CA). Alexa Fluor

25488-conjugated anti-rat IgG and Alexa Fluor 488-conjugatedanti-mouse IgG secondary Abs were purchased from Cell
Signaling Technology, Inc. (Danvers, MA).
Production of hybridomas

A 5-week-old Sprague–Dawley rat was purchased from
CLEA Japan (Tokyo, Japan). The animal was housed under
specific pathogen-free conditions. All animal experiments
were performed according to the relevant guidelines andregulations to minimize animal suffering and distress in thelaboratory. The Animal Care and Use Committee of Tohoku
University (Permit No.: 2019NiA-001) approved animal
experiments. The rat was monitored daily for health during
the complete 4-week duration of the experiment. A reductionof more than 25% of the total body weight was defined as ahumane endpoint. During the sacrifice, the rat was euthanizedthrough cervical dislocation, after which death was verified
through respiratory and cardiac arrest.
To develop mAbs against mCD39, we intraperitoneallyimmunized one rat with LN229/mCD39 (1 · 109 cells) plus
Imject Alum (Thermo Fisher Scientific Inc.). The procedureincluded three additional injections every week (1 · 109
cells/rat), which were followed by a final booster intraperitoneal injection (1 · 109 cells/rat), 2 days before harvesting
spleen cells. The harvested spleen cells were subsequently
fused with P3U1 cells, using PEG1500 (Roche Diagnostics,
Indianapolis, IN), after which hybridomas were grown in the
RPMI-1640 medium with 10% FBS, 100 units/mL of penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin B. For the hybridoma selection, hypoxanthine,aminopterin, and thymidine (Thermo Fisher Scientific Inc.)
were added into the medium. The supernatants were subsequently screened using flow cytometry using CHO/mCD39
and CHO-K1.
Purification of mAbs

The cultured supernatants of C39Mab-1-producing hybridomas were collected through centrifugation at 2330 g for
5 minutes, followed by filtration using Steritop (0.22 mm,
Merck KGaA, Darmstadt, Germany). The filtered supernatants were subsequently applied to 1 mL of Protein G Sepharose 4 Fast Flow (GE Healthcare, Chicago, IL). Afterwashing with phosphate-buffered saline (PBS), bound antibodies were eluted with an IgG elution buffer (Thermo Fisher
Scientific Inc.), followed by immediate neutralization of eluates, using 1 M Tris-HCl (pH 8.0). Finally, the eluates wereconcentrated, after which the elution buffer was replacedwith PBS using Amicon Ultra (Merck KGaA).
Flow cytometric analysis

CHO-K1 and CHO/mCD39 were harvested after a brief
exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). The cells were subsequently washed with 0.1% bovine serum albumin in PBS andtreated with 0.001, 0.01, 0.1, and 1 lg/mL of primary mAbs
for 30 minutes at 4C. The cells were treated with Alexa
Fluor 488-conjugated anti-rat IgG or Alexa Fluor 488conjugated anti-mouse IgG (1:2,000). The fluorescence data
were collected using the SA3800 Cell Analyzer (Sony Corp.).

26

OKADA ET AL.

Determination of dissociation constantthrough flow cytometry

CHO/mCD39 were suspended in 100 lL serially diluted
C39Mab-1 for 30 min at 4C. The cells were treated with
50 lL of Alexa Fluor 488-conjugated anti-rat IgG (1:200).
The fluorescence data were collected, using the SA3800 Cell
Analyzer. The dissociation constant (KD) was subsequently
calculated by fitting saturation binding curves to the built-in;
one-site binding models in GraphPad PRISM 8 (GraphPad
Software, Inc., La Jolla, CA).

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Western blot analysis

Cell lysates were boiled in sodium dodecyl sulfate sample
buffer (Nacalai Tesque, Inc.). The protein lysates (10 lg)
were separated on 5%–20% polyacrylamide gels (FUJIFILM
Wako Pure Chemical Corporation) and transferred onto
polyvinylidene difluoride membranes (Merck KGaA). After
blocking with 4% skim milk (Nacalai Tesque, Inc.) in PBS
with 0.05% Tween 20, the membranes were incubated with
10 lg/mL of C39Mab-1, 1 lg/mL of an anti-isocitrate dehydrogenase 1 (IDH1) mAb (clone RcMab-1),62,63 or 1 lg/mL
of NZ-1 (an anti-PA tag mAb). The membranes were thenincubated with peroxidase-conjugated anti-rat immunoglobulins (diluted 1:10,000; Sigma-Aldrich Corp., St. Louis,
MO). Finally, the protein bands were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation)using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Results
Development of anti-mCD39 mAbs
by the CBIS method

To develop anti-mCD39 mAbs, one rat was immunizedwith LN229/mCD39 cells (Fig. 1A). The spleen was thenexcised from the rat, and splenocytes were fused with P3U1
cells (Fig. 1B). The developed hybridomas were subsequently seeded into ten 96-well plates and cultivated for 6
days. The positive wells were screened by the selection of
mCD39-expressing cell-reactive and CHO-K1-nonreactive
supernatants, using flow cytometry (Fig. 1C). After the limiting dilution and several additional screenings, an antimCD39 mAb, C39Mab-1 (rat IgG2a, kappa), was finallyestablished (Fig. 1D).

FIG. 1. A schematic illustration demonstrating the production of anti-mCD39 mAbs. (A) CD39 is anchored to themembrane by two transmembrane domains at the two endsof the molecule. LN229/mCD39 cells were immunized intoa Sprague–Dawley rat, using an intraperitoneal injection.
(B) The spleen cells were then fused with P3U1 cells.
(C) Subsequently, the culture supernatants were screened
through flow cytometry to select anti-mCD39 mAbproducing hybridomas. (D) After limiting dilution and some
additional screenings, anti-mCD39 mAbs were finally established. mAbs, monoclonal antibodies.
concentration of C39Mab-1. The KD value of C39Mab-1 for
CHO/mCD39 was determined as 7.3 · 10-9 M.

Flow cytometric analyses

We conducted flow cytometry using anti-mCD39 mAbs
(C39Mab-1 and 5F2) against CHO/mCD39 and CHO-K1 celllines. C39Mab-1 recognized CHO/mCD39 cells dosedependently at 1, 0.1, 0.01, and 0.001 lg/mL. In contrast, 5F2
needed more than 0.01 lg/mL for the detection of
CHO/mCD39 (Fig. 2A). Parental CHO-K1 cells were notrecognized even at 1 lg/mL of all mAbs (Fig. 2B).
Kinetic analyses of C39Mab-1 against
mCD39-overexpressed cells using flow cytometry

To determine the KD of C39Mab-1 with mCD39overexpressed cells, we conducted kinetic analysis by flow
cytometry using CHO/mCD39 (Fig. 3). The geometricmean of the fluorescence intensity was plotted versus theWestern blot analysis

Western blotting was performed to further assess the
specificity of C39Mab-1. The cell lysates of CHO-K1 andCHO/mCD39 were probed. As shown in Figure 4A, C39Mab1 detected mCD39 as a *100-kDa band. An anti-PA tagmAb (clone NZ-1) recognized the lysates from CHO/mCD39(*100 kDa, mainly) (Fig. 4B). These results indicated thatC39Mab-1 can detect mCD39 specifically by western blot
analysis.
Discussion

In the TME, extracellular levels of ATP can reach 100–
500 mM compared to the nanomolar order in normal tissues.9
CD39 can rapidly hydrolyze and convert to adenosine in27

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ANTI-MOUSE CD39 MAB

FIG. 2. Flow cytometry to mCD39-overexpressing cells using anti-mCD39 mAbs. CHO/mCD39 (A) and CHO-K1(B) cells were treated with 0.001–1 mg/mL of C39Mab-1 and 5F2, followed by treatment with Alexa Fluor 488-conjugatedanti-rat IgG (for C39Mab-1) or Alexa Fluor 488-conjugated anti-mouse IgG (for 5F2). The black line represents the negative
control.
cooperation with CD73. In this TME, an enzymatic inhibitorof CD39 is the rational mechanism to inhibit the productionof immunosuppressive adenosine. The clinically tested ananti-CD39 mAb, TTX-030 (human IgG4), had a subnanomolar EC50 for human CD39-overexpressed CHO cells
in the flow cytometry-based assay like in Figure 3. Furthermore, TTX-030 allosterically inhibited the enzymatic activity of CD39 in the recombinant human CD39 extracellulardomain and membrane-bound cellular CD39.64 We will investigate the effect of C39Mab-1 on the enzymatic activity of
mCD39 in future studies.
Recently, Zhang et al.65 demonstrated the application of ananti-mCD39 mAb for tumor therapy by the depletion of
immunosuppressive cells through enhanced Fcg receptor–

mediated antibody-dependent cellular cytotoxicity (ADCC).
They found that mCD39 expression on tumor-infiltrating
immune and vascular endothelial cells was markedly higher
than that in normal tissues. They used a nonneutralizing antimCD39 mAb (clone 5F2, mouse IgG1) and screened anisotype-switched hybridoma subline of the IgG2c isotype
which has more potent ADCC activities. To enhance the effector functions, the fucosyltransferase 8 gene was deleted in
the 5F2 hybridomas using clustered regularly interspaced
short palindromic repeats technology to produce the afucosylated antibody. They showed that the afucosylated antimCD39 IgG2c exerted the potent antitumor effect againstmouse melanoma and colorectal tumor models through thedepletion of regulatory/exhausted T cells, tumor-associated28

OKADA ET AL.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

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This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Nos.: 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 ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Grant Nos. 22K06995 (to H.S.), 21K07168(to M.K.K.), and 22K07224 (to Y.K.).

FIG. 3. The determination of the binding affinity of
C39Mab-1. CHO/mCD39 cells were suspended in 100 mL
serially diluted C39Mab-1 at the indicated concentrations.
The cells were treated with Alexa Fluor 488-conjugatedanti-rat IgG. The fluorescence data were subsequently collected using the SA3800 Cell Analyzer, following the calculation of the KD by GraphPad PRISM 8. KD, dissociation
constant.

macrophages, and tumor vasculature with high mCD39
expression.
We previously produced recombinant antibodies, whichwere converted into mouse IgG2a subclass from mouse IgG1.
Furthermore, we produced afucosylated IgG2a mAbs using
Fut8-deficient CHO-K1 cells to potentiate the ADCC activity. The afucosylated mAbs showed potent antitumor activity
in mouse xenograft models.66–73 Therefore, a class-switched
and afucosylated version of C39Mab-1 could be used toevaluate the antitumor activity in vivo.

FIG. 4. Western blotting using C39Mab-1. The cell lysatesof CHO-K1 and CHO/mCD39 were electrophoresed andtransferred onto PVDF membranes. The membranes wereincubated with 10 mg/mL of C39Mab-1 (A), 1 mg/mL of NZ1 (an anti-PA tag mAb) (B), or 1 mg/mL of RcMab-1(an anti-IDH1 mAb) (C). The membranes were subsequently incubated with peroxidase-conjugated anti-rat immunoglobulins. The arrows indicate the predicted size of
mCD39 (*100 kDa). PVDF, polyvinylidene fluoride.

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or.2020.7735

31

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: September 1, 2023
Accepted: September 15, 2023