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Development of Highly Sensitive Anti-Mouse CD39 Monoclonal Antibodies C39Mab-1 and C39Mab-2 for flow cytometry

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03 April 2023

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04 April 2023

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Abstract
CD39 is involved in adenosine metabolism through conversion of extracellular ATP to adenosine. Because extracellular adenosine plays a critical role in the immune suppression of tumor microenvironment, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. In this study, we developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mCD39 mAbs, which were established by the CBIS method including C39Mab-1 (rat IgG2a, kappa) and C39Mab-2 (rat IgG2a, lambda), reacted with not only mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) but also endogenously mCD39-expressed cell lines, such as L1210 (mouse lymphocytic leukemia) and J774-1 (mouse macrophage-like) cell lines through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constant (KD) of C39Mab-1 and C39Mab-2 for CHO/mCD39 was 7.3 × 10−9 M and 5.5 × 10−9 M, respectively. KD of C39Mab-1 and C39Mab-2 for L1210 was 3.3 × 10−9 M and 3.6 × 10−10 M, respectively. Furthermore, C39Mab-1 could detect the lysate of CHO/mCD39 by western blot analysis. These results indicate that C39Mab-1 and C39Mab-2 are useful for the detection of mCD39 in many functional studies.
Keywords: 
Subject: Medicine and Pharmacology  -   Oncology and Oncogenics

1. Introduction

Extracellular adenosine (eADO), which is generated by the hydrolysis of extracellular ATP (eATP), mediates immunosuppressive tumor microenvironment (TME) [1]. High concentration of eATP can be found in solid tumors due to not only passive release of cell death, but also active secretion by tumor cells and other subsets in the TME [2]. Following the release of eATP, 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].
A growing body of evidence suggests that adenosine-mediated immunosuppression is a critical mechanism of 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 are expressed on immune cells [5]. Among four P1 receptors, the A2A and A2B are GS-coupled receptors and trigger intracellular cAMP accumulation. The cAMP signaling mediates immunosuppression through the activation of effectors including protein kinase A [6].
Sitkovsky’s group first reported the immunosuppressive effect of A2A receptor in vivo [7]. Inflammatory stimuli that caused minimal tissue damage in wild-type mice were sufficient to induce extensive tissue damage, more prolonged and higher levels of pro-inflammatory cytokines, and individual death in mice lacking the A2A receptor [7]. The group also showed genetic evidence of the importance of the A2A receptor in tumor immunity [8]. These findings impact on antitumor immunity by CD39–adenosine–A2 receptor axis and several landmark studies have developed the multiple strategies targeting adenosine metabolism [3,9].
The development of anti-CD39 monoclonal antibodies (mAbs) is one of the strategies 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 with the 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-human CD39 mAbs (clones TTX-030, IPH5201, and SRF-617) were designed to inhibit CD39 enzymatic activity via allosteric inhibition and minimize Fc receptor-mediated engagement to avoid the side effect [10,11]. These mAbs have entered into clinical trials for solid tumors with combination of chemotherapeutic agents or immune checkpoint inhibitors [3].
The Cell-Based Immunization and Screening (CBIS) method includes the immunization of antigen-overexpressing cells and the high-throughput hybridoma screening using flow cytometry. Using the CBIS method, we have developed mAbs against membrane proteins, including 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 [25,26,27,28,29,30,31,32,33,34,35,36], CD44 [37,38], mouse CCR3 [39], mouse CCR8 [40], and human CCR9 [41] mAbs.
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.

2. Materials and methods

2.1. Preparation of cell lines

P3X63Ag8U.1 (P3U1), LN229, and Chinese hamster ovary (CHO)-K1 cells were obtained from the American Type Culture Collection (Manassas, VA). J774-1 (mouse macrophage-like) and L1210 (mouse lymphocytic leukemia) cells were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University (Miyagi, Japan).
The synthesized DNA (Eurofins Genomics KK) encoding mCD39 (Accession No.: NM_ 009848) was subcloned into a pCAGzeo_nPA-cRAPMAP vector. The amino acid sequences of the tag system were as follows: PA tag [42,43,44], 12 amino acids (GVAMPGAEDDVV); RAP tag [45,46], 12 amino acids (DMVNPGLEDRIE); and MAP tag [47,48], 12 amino acids (GDGMVPPGIEDK). The PA tag can be detected by an anti-human podoplanin mAb (clone NZ-1) [42,43,44,49,50,51,52,53,54,55,56,57,58,59,60,61]. Then, the mCD39 plasmid was transfected into CHO-K1 and LN229 cells, as described previously [39].
Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) were used to culture CHO-K1, mCD39-overexpressed CHO-K1 (CHO/mCD39), P3U1, L1210, and J774-1 cells. Dulbecco’s Modified Eagle Medium (DMEM) (Nacalai Tesque, Inc.) were used to culture LN229 and mCD39-overexpressed LN229 (LN229/mCD39). These media were supplemented with 10% FBS, 100 U/mL of penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B.
All cells were grown in a humidified incubator at 37°C, at an atmosphere of 5% CO2 and 95% air.

2.2. Antibodies

An anti-mCD39 mAb (clone 5F2, mouse IgG1, kappa) was purchased from BioLegend (San Diego, CA). Alexa Fluor 488-conjugated anti-rat IgG and Alexa Fluor 488-conjugated anti-mouse IgG secondary Abs were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

2.3. Production of hybridomas

A five-weeks old Sprague–Dawley rat was purchased from CLEA Japan (Tokyo, Japan). All animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001).
To develop mAbs against mCD39, we intraperitoneally immunized one rat with LN229/mCD39 (1 × 109 cells) plus Imject Alum (Thermo Fisher Scientific Inc.). The procedure included three additional injections every week (1 × 109 cells/rat), which were followed by a final booster intraperitoneal injection (1 × 109 cells/rat), two days before the harvest of spleen cells. Hybridomas were produced using PEG1500 (Roche Diagnostics, Indianapolis, IN) as described previously [39]. Supernatants were subsequently screened using flow cytometry, using CHO/mCD39, CHO-K1, L1210, and J774-1 cells.

2.4. Purification of mAbs

The cultured supernatants of C39Mab-1 and C39Mab-2-producing hybridomas were filtered using Steritop (0.22 μm, Merck KGaA, Darmstadt, Germany). Filtered supernatants were subsequently applied to 1 mL of Protein G Sepharose 4 Fast Flow (GE Healthcare, Chicago, IL). The bound antibodies were eluted with an IgG elution buffer (Thermo Fisher Scientific Inc.). Finally, eluates were replacd with the elution buffer concentrated, after which PBS was used to replace with phosphate-buffer saline (PBS), using Amicon Ultra (Merck KGaA).

2.5. Flow cytometric analyses

CHO/mCD39 and CHO-K1 were harvested after a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA, Nacalai Tesque, Inc.). Cells were subsequently washed with 0.1% bovine serum albumin (BSA) in PBS and treated with 0.001, 0.01, 0.1, and 1 μg/mL primary mAbs for 30 min at 4°C. Then, cells were treated with anti-rat IgG conjugated with Alexa Fluor 488 or anti-mouse IgG conjugated with Alexa Fluor 488 (1:2000). The fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp.).

2.6. Determination of dissociation constant (KD) through flow cytometry

CHO/mCD39 and L1210 were suspended in 100 μL serially-diluted C39Mab-1 and C39Mab-2 for 30 min at 4°C. Then, cells were treated with 50 μL Alexa Fluor 488-conjugated anti-rat IgG (1:200). Then, fluorescence data were collected, using the SA3800 Cell Analyzer. The 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).

2.7. Western blot analysis

Cell lysates were prepared as described previously, and were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). Protein lysates (10 µg) were separated on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% Tween 20, membranes were incubated with 10 μg/mL of C39Mab-1 and C39Mab-2, 1 μg/mL of an anti-isocitrate dehydrogenase 1 (IDH1) (clone RcMab-1) [62,63] or 1 μg/mL of NZ-1 (anti-PA tag mAb). Membranes were then incubated with peroxidase-conjugated anti-rat immunoglobulins (diluted 1:10000; Sigma-Aldrich Corp., St. Louis, MO, USA). Finally, protein bands were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

3. Results

3.1. Development of anti-mCD39 mAbs by the CBIS method

To develop anti-mCD39 mAbs, one rat was immunized with LN229/mCD39 cells. (Figure 1A). The spleen was excised, and the splenocytes were fused with myeloma P3U1 cells (Figure 1B). Then, positive wells were screened by the selection of mCD39-expressing cell-reactive and CHO-K1-non-reactive supernatants, using flow cytometry (Figure 1C). After the limiting dilution and several additional screenings, anti-mCD39 mAbs, such as C39Mab-1 (rat IgG2a, kappa) and C39Mab-2 (rat IgG2a, lambda) were finally established (Figure 1D).

3.2. Flow cytometric analyses

We performed flow cytometry using three anti-mCD39 mAbs: C39Mab-1, C39Mab-2, and 5F2 against CHO/mCD39, L1210, and J774-1 cell lines. Both C39Mab-1 and C39Mab-2 recognized CHO/mCD39 cells dose-dependently at 1, 0.1, 0.01, and 0.001 μg/ml. In contrast, 5F2 needed more than 0.01 μg/ml for the detection of CHO/mCD39 (Figure 2A). Parental CHO-K1 cells were not recognized even at 1 μg/ml of all mAbs (Figure 2B).
Furthermore, we investigated the reactivity of C39Mab-1 and C39Mab-2 against endogenously mCD39-expressed cell lines, such as L1210 (mouse lymphocytic leukemia) and J774-1 (mouse macrophage-like). C39Mab-1 and C39Mab-2 reacted with L1210 and J774-1 cells at more than 0.1 μg/ml (Figure 3A and B). In contrast, 5F2 could not clearly reacted with L1210 and J774-1 cells at 1 μg/ml. 5F2 could detect them at 10 μg/ml (data not shown). These results suggested that C39Mab-1 and C39Mab-2 specifically recognizes mCD39, and is also useful for detecting endogenous mCD39 by flow cytometry.

3.3. Kinetic analyses of C39Mab-1 and C39Mab-2 against mCD39-expressing cells using flow cytometry

To determine the KD of C39Mab-1 and C39Mab-2 with mCD39-expressing cells, we conducted kinetic analysis by flow cytometry using CHO/mCD39 and L1210 cells. The KD values of C39Mab-1 and C39Mab-2 for CHO/mCD39 were determined as 7.3 × 10−9 M and 5.5 × 10−9 M, respectively (Figure 4A and B). Furthermore, The KD values of C39Mab-1 and C39Mab-2 for L1210 were determined as 3.3 × 10−9 M and 3.6 × 10−10 M, respectively (Figure 4C and D). These results indicate that C39Mab-1 and C39Mab-2 possess the high affinity for both CHO/mCD39 and L1210 cells.

3.4. Western blot analyses

Western blotting was performed to further assess the specificity of C39Mab-1 and C39Mab-2. Cell lysates of CHO-K1 and CHO/mCD39 were probed. As shown in Figure 5A, C39Mab-1 detected mCD39 as a ~100-kDa band. However, C39Mab-2 did not detect any bands from lysates of CHO-K1 and CHO/mCD39 cells (Figure 5B). An anti-PA tag mAb (clone NZ-1) recognized the lysates from CHO/ mCD39 (~100 kDa, mainly) (Figure 5C). These results indicated that C39Mab-1 can detect mCD39 specifically by western blot analysis.

4. Discussion

In this study, we developed novel anti-mCD39 mAbs (C39Mab-1 and C39Mab-2) using the CBIS method, and investigated the usefulness for flow cytometry and western blotting for detecting mCD39. C39Mab-1 is available for both flow cytometry (Figure 2 and 3) and western blot analysis (Figure 5). C39Mab-2 is available only for flow cytometry (Figure 2 and 3), but exhibited the higher affinity to mCD39 expressing cells than C39Mab-1 (Figure 4). To assess the difference of the properties, the identification of the epitope is essential. We have developed the REMAP method [64,65,66,67] to determine the epitope of mAbs. The conformational epitopes of anti-EGFR mAbs (EMab-51 and EMab-134) [65,67] and anti-CD44 mAbs (C44Mab-5 and C44Mab-46) [64,66] could be determined using the REMAP method. Therefore, further studies are required to determine the epitope of C39Mab-1 and C39Mab-2.
In the TME, extracellular levels of ATP can reach to 100 to 500 μM compared to nanomolar order in normal tissues [9]. CD39 can rapidly hydrolyzed and convert to adenosine in cooperation with CD73. In this TME, an enzymatic inhibitor of CD39 is the rational mechanism to inhibit the production of immunosuppressive adenosine. The clinically tested anti-CD39 mAb, TTX-030 (human IgG4), had a subnanomolar EC50 for human CD39-overexpressed CHO cells in flow cytometry-based assay like Figure 3. Furthermore, TTX-030 allosterically inhibited the enzymatic activity of CD39 in recombinant human CD39 extracellular domain and membrane-bound cellular CD39 [68]. We will investigate the effect of C39Mab-1 and C39Mab-2 on the enzymatic activity of mCD39 in the future study.
Recently, Zhang et al. [69] demonstrated the application of an anti-mCD39 mAb for tumor therapy by the depletion of immunosuppressive cells through enhanced Fcγ receptor–mediated antibody-dependent cellular cytotoxicity (ADCC). They found that mCD39 expression on tumor-infiltrating immune and vascular endothelial cells is markedly higher than in normal tissues. They used a non-neutralizing anti-mCD39 mAb (clone 5F2, mouse IgG1), and screened an isotype-switched hybridoma subline of the IgG2c isotype which has more potent ADCC activities. To enhance the effector functions, the fucosyltransferase 8 (Fut8) gene was deleted in the 5F2 hybridomas using CRISPR to produce the afucosylated antibody. They showed that the afucosylated anti-mCD39 IgG2c exerted the potent antitumor effect against mouse melanoma and colorectal tumor models through the depletion of regulatory/exhausted T cells, tumor-associated macrophages and tumor vasculature with high mCD39 expression.
We previously produced recombinant antibodies, which are 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 [70,71,72,73,74,75,76,77]. Therefore, a class-switched and afucosylated version of C39Mab-1 or C39Mab-2 could be used to evaluate the antitumor activity in vivo.

Author Contributions

HS, Y.Kudo, MT, NG, KI, TO, TT, TA, TN, and MY performed the experiments. MKK and Y.Kato designed the experiments. HS, and Y.Kato analyzed the data. HS, and Y.Kato wrote the manuscript. All authors read and approved the final manuscript and agreed to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is appropriately investigated and resolved.

Funding

This research was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Numbers: JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (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.), and 22K07224 (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: 2019NiA-001) for studies involving animals.

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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Figure 1. A schematic illustration showing the production of anti-mCD39 mAbs. (A) CD39 is anchored to the membrane by two transmembrane domains at the two ends of the molecule. LN229/mCD39 cells was immunized into an SD rat, using an intraperitoneal injection. (B) Spleen cells were then fused with P3U1 cells. (C) The culture supernatants were screened through flow cytometry to select anti-mCD39 mAb-producing hybridomas. (D) After limiting dilution, anti-mCD39 mAbs were finally established.
Figure 1. A schematic illustration showing the production of anti-mCD39 mAbs. (A) CD39 is anchored to the membrane by two transmembrane domains at the two ends of the molecule. LN229/mCD39 cells was immunized into an SD rat, using an intraperitoneal injection. (B) Spleen cells were then fused with P3U1 cells. (C) The culture supernatants were screened through flow cytometry to select anti-mCD39 mAb-producing hybridomas. (D) After limiting dilution, anti-mCD39 mAbs were finally established.
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Figure 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 µg/mL of C39Mab-1, C39Mab-2 and 5F2, followed by treatment with anti-rat IgG conjugated with Alexa Fluor 488 (for C39Mab-1 and C39Mab-2) or anti-mouse IgG conjugated with Alexa Fluor 488 (for 5F2). Black line represents the negative control.
Figure 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 µg/mL of C39Mab-1, C39Mab-2 and 5F2, followed by treatment with anti-rat IgG conjugated with Alexa Fluor 488 (for C39Mab-1 and C39Mab-2) or anti-mouse IgG conjugated with Alexa Fluor 488 (for 5F2). Black line represents the negative control.
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Figure 3. Flow cytometry to endogenously mCD39-expressing cells using anti- mCD39 mAbs. J-774-1 (A) and L1210 (B) cells were treated with 0.001–1 µg/mL of C39Mab-1, C39Mab-2, and 5F2, followed by treatment with anti-rat IgG conjugated with Alexa Fluor 488 (for C39Mab-1 and C39Mab-2) or anti-mouse IgG conjugated with Alexa Fluor 488 (for 5F2). Black line represents the negative control.
Figure 3. Flow cytometry to endogenously mCD39-expressing cells using anti- mCD39 mAbs. J-774-1 (A) and L1210 (B) cells were treated with 0.001–1 µg/mL of C39Mab-1, C39Mab-2, and 5F2, followed by treatment with anti-rat IgG conjugated with Alexa Fluor 488 (for C39Mab-1 and C39Mab-2) or anti-mouse IgG conjugated with Alexa Fluor 488 (for 5F2). Black line represents the negative control.
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Figure 4. The determination of the binding affinity of Cx6Mab-1. CHO/mCD39 (A and B) and L1210 (C and D) cells were suspended in 100 µL serially diluted C39Mab-1 and C39Mab-2 at indicated concentrations. Then, cells were treated with Alexa Fluor 488-conjugated anti-rat IgG. Fluorescence data were subsequently collected using the SA3800 Cell Analyzer, following the calculation of the dissociation constant (KD) by GraphPad PRISM 8.
Figure 4. The determination of the binding affinity of Cx6Mab-1. CHO/mCD39 (A and B) and L1210 (C and D) cells were suspended in 100 µL serially diluted C39Mab-1 and C39Mab-2 at indicated concentrations. Then, cells were treated with Alexa Fluor 488-conjugated anti-rat IgG. Fluorescence data were subsequently collected using the SA3800 Cell Analyzer, following the calculation of the dissociation constant (KD) by GraphPad PRISM 8.
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Figure 5. Western blotting using C39Mab-1 and C39Mab-2. Cell lysates of CHO-K1 and CHO/mCD39 were electrophoresed and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 10 µg/mL of C39Mab-1 (A), 10 µg/mL of C39Mab-2 (B), 1 μg/mL of NZ-1 (an anti-PA tag mAb) (C), or 1 µg/mL of RcMab-1 (an anti-IDH mAb) (D). Membranes were subsequently incubated with peroxidase-conjugated anti-rat immunoglobulins. Arrows indicate the predicted size of mCD39 (~100 kDa).
Figure 5. Western blotting using C39Mab-1 and C39Mab-2. Cell lysates of CHO-K1 and CHO/mCD39 were electrophoresed and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 10 µg/mL of C39Mab-1 (A), 10 µg/mL of C39Mab-2 (B), 1 μg/mL of NZ-1 (an anti-PA tag mAb) (C), or 1 µg/mL of RcMab-1 (an anti-IDH mAb) (D). Membranes were subsequently incubated with peroxidase-conjugated anti-rat immunoglobulins. Arrows indicate the predicted size of mCD39 (~100 kDa).
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