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OPEN

Generation of a canine anti‑canine
CD20 antibody for canine
lymphoma treatment
Takuya Mizuno1*, Yukinari Kato 2,3, Mika K. Kaneko 2, Yusuke Sakai4, Toshinori Shiga5,
Masahiro Kato5, Toshihiro Tsukui5, Hirofumi Takemoto6, Akio Tokimasa6, Kenji Baba6,
Yuki Nemoto1, Osamu Sakai1 & Masaya Igase1
Lymphoma is the most common hematological cancer in dogs. Canine diffuse large B cell lymphoma
shows a relatively good response to treatment with multi-agent cyclophosphamide, doxorubicin,
vincristine, and prednisone (CHOP) chemotherapy; however, the 2-year survival rate is as low as
20%. For human B cell type lymphoma, the anti-CD20 chimeric antibody, rituximab, was developed
two decades ago. The combination of rituximab and CHOP chemotherapy was highly successful inimproving patient prognosis. However, no anti-canine CD20 antibody is available for the treatment ofcanine lymphoma. During this study, a rat anti-canine CD20 monoclonal antibody was established. We
also generated a rat-canine chimeric antibody against canine CD20 designed for clinical application.
This chimeric antibody (4E1-7-B) showed in vitro antibody-dependent cell-mediated cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) against the canine B cell lymphoma cell line
CLBL-1. Moreover, to obtain stronger ADCC activity, a defucosylated 4E1-7-B antibody (4E1-7-B_f)
was also generated, and it showed tenfold stronger ADCC activity compared with 4E1-7-B. 4E1-7-B_f
as well as 4E1-7-B suppressed the growth of CLBL-1 tumors in an immunodeficient xenotransplantmouse model. Finally, a single administration of 4E1-7-B_f induced considerable peripheral B cell
depletion in healthy beagles. Thus, 4E1-7-B_f is a good antibody drug candidate for canine B cell type
lymphoma.
Lymphoma is one of the most common hematological cancers in dogs, and is important as a point of comparison for human ­lymphoma1. Canine lymphomas are classified into T cell and B cell types according to their cellof origin. B cell lymphoma, the most common type which is similar to human diffused large B cell lymphoma(DLBCL), has a favorable prognosis compared to T cell lymphoma because the cyclophosphamide, doxorubicin,
vincristine, and prednisone (CHOP) regimen of chemotherapy leads to remission in most cases. However, the2-year survival rate is only 20%, and most cases recur after remission, finally proving refractory to ­chemotherapy2.
Therefore, a novel therapy is required. Recently, several novel treatment strategies for canine lymphoma, including low molecular weight inhibitors, have been ­established3, leading to the expectation of an improved prognosis
for dogs with lymphoma. Nevertheless, antibody treatment for canine lymphoma has not yet been established.
Among antibody therapies for cancer, rituximab, an anti-human CD20 antibody, is the oldest and mostwidely distributed. Rituximab is a chimeric antibody in which the constant region of the original anti-human
CD20 mouse antibody was replaced with a human constant r­ egion4. Rituximab binds to human CD20 moleculesand directly induces apoptotic cell death in addition to its function through antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)5. It has been used alongside multiple drug
CHOP chemotherapy (R-CHOP) in the treatment of B cell type lymphoid tumors, including non-Hodgkin B cell
lymphoma, with dramatic s­ uccess6,7. Following the success of R-CHOP therapy, several kinds of CD20 antibody

1

Laboratory of Molecular Diagnostics and Therapeutics, The United Graduate School of Veterinary Medicine,
Yamaguchi University, 1677‑1 Yoshida, Yamaguchi, Yamaguchi Prefecture 753‑8515, Japan. 2Department of
Antibody Drug Development, Tohoku University Graduate School of Medicine, Miyagi, Japan. 3New Industry
Creation Hatchery Center, Tohoku University, Miyagi, Japan. 4Laboratory of Veterinary Pathology, Joint Facultyof Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan. 5Nippon Zenyaku Kogyo Co., Ltd., Koriyama,
Fukushima, Japan. 6Laboratory of Veterinary Internal Medicine, Joint Faculty of Veterinary Medicine, Yamaguchi
University, Yamaguchi, Japan. *email: mizutaku@yamaguchi‑u.ac.jp
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drugs were developed to obtain a more potent cytopathic effect on B cell lymphoma cells, and among them,ofatumumab and obinutuzumab were recently ­launched8. Obinutuzumab, in particular, is a glycoengineered
CD20 antibody with reduced fucose modification of the heavy chain constant r­ egion9. Removal of fucose glycosylation from an antibody drug was expected to produce far greater ADCC a­ ctivity10; in fact, a defucosylated
anti-CCR4 humanized antibody drug was approved for the treatment of adult T cell l­eukemia11. Furthermore,
CD20 is also expressed on normal mature B cells, so anti-CD20 antibody drugs are able to eliminate normal
B cells. As a result, they have also been used for the treatment of autoimmune diseases, such as systemic lupus
erythematosus (SLE), nephrotic syndrome, and Wegener s­ yndrome12.
Human studies have suggested that anti-CD20 antibody therapy is an excellent candidate for the treatmentof canine B cell lymphoma because, as is the case in humans, CD20 is expressed in both canine B cell lymphoma
cells and canine normal mature B ­cells13. More than ten years ago, Impellizeri et al. reported that based on flow
cytometry analysis, rituximab did not bind to canine ­CD2014. However, there is an anti-human antibody thatcross-reacts with canine CD20 in immunohistochemistry but not flow cytometry, meaning that this antibody
is not capable of binding to the naïve canine CD20 ­molecule13. Since then, many laboratories have attempted todevelop monoclonal antibodies against canine CD20 in order to establish an antibody therapy for canine B cell
lymphoma. Aratana Therapeutics Inc. (Leawood, KS, USA) launched a therapeutic anti-canine CD20 antibody(Blontuvetmab) in 2015 in the United States and showed its clinical efficacy against B cell lymphoma in dogs in
a conference abstract; however, peer-reviewed data are not available. Ito et al. presented an anti-canine CD20
antibody (clone 6C8) and showed its induction of antibody-dependent cellular phagocytosis (ADCP) activity incanine B c­ ells15. Jain et al. developed an anti-canine CD20 antibody that cross reacted with human C­ D2016. Rue
et al. also developed an anti-canine CD20 antibody (clone 1E4) and generated a chimeric antibody for therapeutic ­use17. They observed the in vitro cytotoxicity of this antibody via CDC and a decrease in the number of
peripheral B cells in vivo in healthy beagles; however, the clinical efficacy in dogs with canine B cell lymphoma
remain unknown.
In this study, we took the novel approach of developing an anti-canine CD20 monoclonal antibody in rats asa host species. We show that this antibody induced cell death in canine B cell lymphoma cell lines. Moreover, we
generated a rat-canine chimeric version of this antibody and verified its function in vitro and in vivo.

Results

Establishment of the anti‑canine CD20 antibody.

By immunization with NRK/cCD20 cells, an anticanine CD20 monoclonal antibody (clone 4E1-7) was obtained, and its subclass was determined to be rat ­IgG2a
by flow cytometry. 4E1-7 reacted with NRK/cCD20 cells, but not parental NRK cells (Fig. 1A). The antibody
also bound dose-dependently to the canine B cell lymphoma cell line CLBL-1 (Fig. 1B), but not to other canine
lymphoma cell lines: GL-1, CL-1, Ema, UL-1, CLC, CLK, CLGL90, and 17–71 cell lines (data not shown). The
antibody bound to the human lymphoma cell line Jurkat cells transduced to exogenously express canine CD20
(Jurkat/cCD20), but not to parental Jurkat cells (Fig. 1C). The anti-Flag antibody detected the bands of propermolecular weight (37 kDa) of canine CD20 in cell lysates from Jurkat/cCD20, but also in the 4E1-7 -immunoprecipitated cell lysates from Jurkat/cCD20 cells (Fig. 1D). However, the 4E1-7 antibody did not detect the bands
in the cell lysates from Jurkat/cCD20 (data not shown), indicating that 4E1-7 recognized the nonlinear epitope.
These results indicate that the 4E1-7 antibody is monoclonal and specific to the canine CD20 molecule.

Anti‑canine CD20 antibody binds to canine B cells and canine B cell lymphoma cells.

To testthe binding property of 4E1-7 to canine CD20 naturally expressed in canine B cells, canine PBMCs from 4
healthy beagle dogs were stained with 4E1-7 and anti-CD21 antibodies. Figure 1E showed that 4E1-7 boundspecifically to the ­CD21+ population in canine PBMCs. We also assessed the binding property of this antibody towhite blood cells, and neutrophils and monocytes were not stained by this antibody (data not shown). To provethat 4E1-7 also binds to B cell lymphoma cells from dogs, lymphoma cells were stained with 4E1-7 from 3 cases:
two B cell lymphoma cases and one T cell lymphoma case (Fig. 1F). The B cell lymphoma cells from both cases
were stained with 4E1-7, but those from the case with T cell lymphoma were not. This result shows that 4E1-7
also binds to canine CD20 naturally expressed in canine B cells.

Anti‑canine CD20 antibody has direct cell killing activity via apoptosis. To assess the functional

activity of 4E1-7, several in vitro studies were conducted using CLBL-1 cell lines. To evaluate ADCC activity,
CLBL-1 cells were treated with 4E1-7, followed by coculture with LAK cells for 4 h. Cell killing was not observedin this assay (Fig. 2A). Next, to examine CDC activity, CLBL-1 cells were treated with 4E1-7, followed by coculture with rabbit complement for 90 min. Again, cell killing was not observed in this assay (Fig. 2B). Finally,
CLBL-1 cells were treated with 4E1-7 and incubated for 48 h. Cell proliferation assay showed that cell proliferation of CLBL-1 cells were suppressed dose-dependently by 4E1-7 (Fig. 2C). Annexin V staining of CLBL-1 cells
treated with 4E1-7 showed that the annexin V­ + propidium iodide (PI)- population and annexin V­ + ­PI+ populations increased with 10 µg/ml 4E1-7 treatment (Fig. 2D), indicating that the anti-proliferative effect of 4E1-7 in
CLBL-1 cells was partly due to the induction of apoptotic cell death. These results suggested that 4E1-7 has ananti-proliferative function in CLBL-1 cells.

Dog chimeric anti‑canine CD20 antibody has strong ADCC activity.

To determine the potentialof the CD20 antibody as a therapeutic antibody for the treatment of canine B cell lymphoma, a rat-canine chimeric anti-canine CD20 antibody was prepared. As previously ­reported18, canine IgG has four classes, IgG-A,
IgG-B, IgG-C, and IgG-D. Since research shows that IgG-B and IgG-C antibodies function via ADCC and CDC­activity19, rat-dog chimeric anti-canine CD20 antibodies 4E1-7-B and 4E1-7-C were obtained. Previous research
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Figure 1.  4E1-7 binds to canine CD20. (A) NRK and NRK/cCD20 cells were stained with 10 µg/ml of isotype
control antibody (red) or anti-CD20 antibody, clone 4E1-7 (blue), followed by Dylight 649-labeled anti-rat
IgG secondary antibody. (B and C) CLBL-1 (B) as well as Jurkat and Jurkat/cCD20 cells (C) were stained with
the indicated amount of anti-CD20 antibody (4E1-7), followed by Dylight 649-labeled anti-rat IgG secondary
antibody. (D) Cell lysates were extracted from each Jurkat and Jurkat/cCD20 cells and immunoprecipitated with
1 µg of anti-canine CD20 antibody (4E1-7) or rat I­ gG2a antibody. Immunoprecipitated proteins (left) and wholecell lysate (right) were each separated by SDS-PAGE and transferred to the membrane, followed by western
blotting with an anti-FLAG antibody. (E) PBMCs were isolated from four healthy beagles and stained with ananti-CD21-PE antibody and an anti-canine CD20 antibody (4E1-7), followed by Dylight 649-labeled anti-rat
IgG secondary antibody. A representative image is shown. (F) Primary lymphoma cells were obtained fromlymph nodes of two dogs with B cell lymphoma or one dog with T cell lymphoma and stained with anti-canine
CD20 antibody (4E1-7) followed by an anti-rat IgG-PE secondary antibody. A representative image from the B
cell lymphoma dogs is shown.

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Figure 2.  The 4E1-7 antibody induces cell death in CLBL-1 cells. (A) ADCC activity of 4E1-7 CLBL-1/luc cells
were treated with the indicated amount of anti-CD20 antibody (4E1-7) or rat I­ gG2a isotype control antibody
for 20 min, followed by the addition of LAK cells. After 4 h of incubation, luciferase activity in living cells wasmeasured using a luminometer after cell lysis. Mean ± SD of three independent experiments are shown. (B)CDC activity of 4E1-7 CLBL-1 cells were treated with the indicated amount of anti-CD20 antibody (4E1-7)
or rat ­IgG2a isotype control antibody for 15 min, followed by the addition of rabbit complement. After 90 min,viability was counted by trypan blue dye exclusion assay. Mean ± SD of three independent experiments areshown. (C) Direct cell killing activity of 4E1-7 CLBL-1 cells were incubated with the indicated amount ofanti-CD20 antibody (4E1-7) or rat ­IgG2a isotype control antibody for 72 h, followed by addition of the CCK-8
reagent. Cell proliferation rate was calculated. Mean ± SD of three independent experiments are shown. *Indicates P < 0.05. (D) Annexin V assay of 4E1-7 CLBL-1 cells were incubated with the indicated amount ofanti-CD20 antibody (4E1-7) or rat ­IgG2a isotype control antibody for 72 h, collected, and finally stained withannexin V and PI. The percentages of each annexin V positive and PI negative cells (A + /P-), annexin V negativeand PI positive cells (A-/P +), annexin V and PI double positive cells (A + /P +), and annexin V and PI double
negative cells (A-/P-) are shown. Mean ± SD of three independent experiments are shown. * Indicates P < 0.05.
revealed that the anti-canine CD20 antibody (clone 1E4) had killing activity in CLBL-1 cells and the decreased
number of peripheral B cells in healthy ­beagles17. Thus, we also prepared the chimeric antibody constructed fromthe variable region of 1E4 and from canine IgG-B for comparison. Flow cytometric analysis showed that both
antibodies bound to CLBL-1 cell lines in a dose-dependent manner (Fig. 3A). These antibodies also bound to
the EL-4/cCD20 cells, but Kd value of 1E4-B, 4E1-7-B and 4E1-7-C showed no difference (7.31 × 109, 2.56 × 108,
and 3.83 × 108, respectively) (Fig. 3B). We also confirmed the binding ability of 4E1-7-B to B cell lymphoma cells
from 5 high grade B cell lymphoma cases (Supplementary Fig. 1).
Next, to establish the functional activity of the chimeric antibody, its ADCC and CDC activity as well as
direct killing activity were examined. First, assessment of the ADCC mechanism showed, unexpectedly, that the
4E1-7-B and 4E1-7-C chimeric antibodies induced cell lysis of CLBL-1/luc cells in an antibody dose-dependent
manner, and 4E1-7-B activity was significantly stronger than that of 4E1-7-C (Fig. 3C). The extent of cell lysis
between the LAK cells varied in each dog, but the trends between the different dogs were similar. However, the

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Figure 3.  Chimeric antibody, 4E1-7-B, induces ADCC and CDC in CLBL-1 cells. (A) CLBL-1 cells were
stained with the indicated amount of one of the anti-CD20 chimeric antibodies (4E1-7-B, 4E1-7-C, or 1E4-B),
followed by Alexa 647-labeled anti-dog IgG secondary antibody. (B) EL-4/cCD20 cells were stained with 10 µg/
ml of one of the anti-CD20 chimeric antibodies (4E1-7-B, 4E1-7-C, or 1E4-B), followed by Alexa 647-labeledanti-dog IgG secondary antibody. (C) ADCC activity of chimeric antibodies CLBL-1/luc cells were treatedwith the indicated amount of one of the anti-CD20 chimeric antibodies (4E1-7-B, 4E1-7-C, or 1E4-B) or the
rat-dog IgG-B chimeric isotype control antibody for 20 min, followed by the addition of LAK cells. After 4 hof incubation, luciferase activity in living cells was measured using a luminometer after the cells were lysed.
Three independent experiments were performed and means ± SD of cell cytotoxicity in triplicate from one
representative result are shown. (D) CDC activity of chimeric antibodies CLBL-1/luc cells were treated with oneof the anti-CD20 antibodies (4E1-7-B, 4E1-7-C, or 1E4-B) or the rat I­ gG2a isotype control antibody for 15 min,followed by the addition of rabbit complement. After 90 min, luciferase activity in living cells was measured
using a luminometer after the cells were lysed. Three independent experiments were performed and means ± SD
are shown. * and ** indicate P < 0.0001 and P < 0.05, respectively. (E) Suppressive activity of cell proliferation ofchimeric antibodies CLBL-1 cells were incubated with the indicated amount of one of the anti-CD20 antibodies(4E1-7-B, 4E1-7-C, or 1E4-B) or the rat I­ gG2a isotype control antibody, for 15 min followed by treatment with
10 µg/ml of anti-dog IgG secondary antibody (black bars) for crosslinking, or mock control (empty bars). After
72 h of incubation, a CCK-8 reagent was added, and the cell proliferation rate was calculated. Three independent
experiments were performed and means ± SD are shown. * indicates P < 0.05.
1E4-B antibody did not show any ADCC activity. Second, assessment of CDC by chimeric antibodies showed thatin addition to 1E4-B and isotype control antibodies, 4E1-7-C induced no cell death in CLBL-1/luc cells. However,
4E1-7-B induced some cell death in a dose-dependent manner (Fig. 3D). Finally, the antibodies’ suppression ofcell proliferation was assessed. Unexpectedly, treatment of CLBL-1 cells with 4E1-7-B and 4E1-7-C showed very
weak suppression of cell proliferation compared with the parental 4E1-7 antibody (Fig. 3E). However, this effectwas enhanced by crosslinking 4E1-7-B and 4E1-7-C antibodies with an anti-dog antibody. Conversely, 1E4-B
showed no effects on cell proliferation, even in the presence of anti-dog IgG crosslinking.

Defucosylated dog chimeric anti‑canine CD20 antibody showed stronger ADCC activity. To
obtain stronger ADCC activity, the defucosylated chimeric antibody was generated using the cell lines deficient in ­FUT820,21. The defucosylated antibody (4E1-7-B_f) bound to CLBL-1 cells in a similar manner to the
4E1-7-B antibody by showing the similar Kd value (2.41 × 108) (Fig. 4A). Assessment of ADCC activity (Fig. 4B)
showed that both 4E1-7-B and 4E1-7-B_f antibodies induced cytopathic effects in CLBL-1/luc cells, but 4E1-7B_f showed almost 10 times greater cytotoxicity than 4E1-7-B. Assessment of CDC activity showed that theantibodies had similar effects (Fig. 4C). 4E1-7-B_f also suppressed cell proliferation of CLBL-1 cells, and thisScientific Reports |(2020) 10:11476 |https://doi.org/10.1038/s41598-020-68470-9

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Figure 4.  Defucosylated chimeric antibody, 4E1-7-B_f, induces more potent ADCC in CLBL-1 cells.
(A) CLBL-1 cells were stained with the indicated amount of either of two anti-CD20 chimeric antibodies,
4E1-7-B or 4E1-7-B_f, followed by Alexa 647-labeled anti-dog IgG secondary antibody. (B) ADCC activityof defucosylated chimeric antibodies CLBL-1/luc cells were treated with the indicated amount of either of thetwo anti-CD20 chimeric antibodies (4E1-7-B or 4E1-7-B_f) or the rat-dog IgG-B chimeric isotype control
antibody for 20 min, followed by the addition of LAK cells. After 4 h of incubation, luciferase activity inliving cells was measured using a luminometer after the cells were lysed. Three independent experiments
were performed, and the means ± SD of cell cytotoxicity in triplicate from a representative result are shown. *and ** indicate P < 0.0001 and P < 0.0004, respectively, compared to the isotype control. (C) CDC activity ofdefucosylated chimeric antibodies CLBL-1/luc cells were treated with either of the two anti-CD20 antibodies(4E1-7-B or 4E1-7-B_f) or the rat I­ gG2a isotype control antibody for 15 min, followed by the addition of rabbit
complement. After 90 min, luciferase activity in living cells was measured using a luminometer after the cells
were lysed. Three independent experiments were performed and means ± SD are shown. * Indicates P < 0.0001.
(D) Suppressive activity of cell proliferation of defucosylated chimeric antibodies CLBL-1 cells were incubatedwith the indicated amount of either of two anti-CD20 antibodies (4E1-7-B or 4E1-7-B_f) or the rat I­ gG2a isotype
control antibody for 15 min, followed by treatment with 10 µg/ml of anti-dog IgG antibody (black bars) forcrosslinking, or a mock control (empty bars). After 72 h of incubation, a CCK-8 reagent was added, and cellproliferation rate was calculated. Three independent experiments were performed and means ± SD are shown.
was enhanced by the addition of anti-dog IgG (Fig. 4D). However, the suppression rate was slightly higher in
4E1-7-B_f-treated CLBL-1 cells.

Chimeric anti‑canine CD20 antibody showed an anti‑tumor effect in xenotranplanted
mice. To assess the anti-tumor effect of these chimeric antibodies in vivo, CLBL-1 cells were xenotransplanted

into NOD/SCID mice, which were then treated with the chimeric antibody plus PBMCs (Fig. 5). Sole treatment
with PBMCs (Fig. 5B) showed a slight delay in tumor growth compared with PBS treatment (Fig. 5A). However,administration of 4E1-7-B plus PBMCs (Fig. 5C) delayed tumor growth in two of four mice, and administration

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Figure 5.  The defucosylated chimeric antibody, 4E1-7-B_f, suppresses tumor growth in NOD/SCID mice.
(A–D) NOD/SCID mice were subcutaneously injected with CLBL-1 cells. After tumor size reached 150–250­mm3, mice were randomized into four groups to receive intraperitoneal treatment with either dog IgG (A), dog
IgG (B), 4E1-7-B (C), or 4E1-7-B_f (D), as well as PBMCs around the tumor (B–D) every other day. Mice wereeuthanized as humane endpoints were reached or after 13 days of treatment.
of 4E1-7-B_f suppressed tumor growth in all four mice (Fig. 5D), however tumor sizes at endpoint did not show
a statistical significance between 4E1-7-B and 4E1-7-B_f groups (Supplementary Fig. 2).

Chimeric anti‑canine CD20 antibody decreases peripheral B cells in healthy beagles. To provethat chimeric antibodies have the ability to lyse C­ D20+ B cells in dogs, two groups of four healthy beagles were
injected intravenously once with chimeric antibodies (either 0.5 or 5.0 mg/kg), and then the percentages of
peripheral ­CD21+ B cells were determined using flow cytometry (Fig. 6A and Supplementary Fig. 3). On the
day after injection, the percentages and numbers of ­CD21+ B cells decreased to almost zero, and all dogs had
no ­CD21+ B cells on day 3, which remained the case until day 7 in the 0.5 mg/kg group. On Day 14, one of thedogs of 0.5 mg/kg group still showed no ­CD21+ B cells, but the other three dogs showed a slight increase in
the percentage of B cells. Compared with the 0.5 mg/kg dosage group, all four dogs in the 5.0 mg/kg dosage
group showed no ­CD21+ B cells initially, but these cells gradually increased in two of the four dogs from day 21
onwards, one dog still had no C
­ D21+ B cells on day 28 (when it was euthanized), and the remaining dog showed+no ­CD21 B cells until day 56. In both groups, the percentages of C
­ D21+ B cells had not returned to the original
percentages by day 119.
We also surgically removed the lymph nodes from one dog from each group on days 14 and 28. Immunohistochemical staining of B cells in the lymph nodes with the anti-CD79a antibody showed shrinkage of the positive
area in the samples collected on day 14 and day 28 from the dog in the 5.0 mg/kg dose group and day 14 fromthe dog in the 0.5 mg/kg dose group, but the dogs in the 0.5 mg/kg dose group on day 28 showed no obvious
effects on the CD79-positive area (Fig. 6B).

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Figure. 6.  The defucosylated chimeric antibody, 4E1-7-B_f, depletes B cells in healthy beagle dogs. (A, B) Eight
healthy beagles were intravenously inoculated with either 0.5 mg/kg or 5.0 mg/kg of the 4E1-7-B_f antibody(four dogs per dose) once on day 0. ­CD21+ B lymphocytes in peripheral blood were counted by flow cytometry
every other day throughout day 1–7 then weekly after day 7. (A) In both groups, each line indicates a dog. Oneof the four dogs (orange line) from each group was euthanized on day 28 for analysis. (B) Lymph nodes were
surgically obtained at days 14 and 28 from one dog from each group, and immunohistologically stained with ananti-CD79a antibody. Immunohistological staining of lymph node sample from a healthy beagle is shown as acontrol sample (Cont.) for comparison. Scale bar = 200 µm.

Discussion

In this study, the hybridoma producing anti-canine CD20 antibody, clone 4E1-7, was established. This antibody
suppressed cell growth of CD20-positive cell lines, partly due to apoptosis, but showed no ADCC and CDC
activity. A few anti-canine CD20 antibodies, clone ­6C815, clone ­1E417,22, NCD1.216 and Blontuvetmab (meeting abstract) have previously been described. Besides clone 1E4, none of these were shown to have an in vitro
cytotoxic effect against canine CD20 positive cells. Clone 1E4, however, showed in vitro antibody-dependent
cell phagocytosis (ADCP) activity and CDC a­ ctivity22, and administration of a chimeric version of this antibody
also induced peripheral B cell depletion in healthy b­ eagles17. Unfortunately, the antibody cloned in our study,
4E1-7, did not exhibit ADCC and CDC activity, but did show direct cell killing activity. 4E1-7 turned out to bea rat ­IgG2a subclass, and, generally, rat I­ gG2a has been shown to exhibit ADCC and CDC a­ ctivity23. The lack of
ADCC activity of 4E1-7 may be attributable to the use of canine LAK cells as effector cells for the ADCC assay.
Canine CD16 on LAK cells may not interact effectively with rat ­IgG2a. However, the reason for defective CDC
activity remains unknown.
Interestingly, chimerization of the 4E1-7 antibody with canine IgG-B or IgG-C changed the characteristics ofthis antibody such that it gained potent ADCC and CDC activity but had reduced the suppressive effect on cellproliferation compared with the 4E1-7 antibody. We also compared these effects with the previously described
1E4 chimeric antibody (1E4-B)17,22, the only antibody shown to have in vivo B cell-reducing ability. 4E1-7-B had
the highest ADCC activity followed by 4E1-7-C; meanwhile, 1E4-B showed no ADCC activity, even though itreportedly has ADCP a­ ctivity17,22. This discrepancy might be because ADCC activity depends on CD16 expression on NK cells. On the other hand, ADCP activity depends on CD32 expression on macrophages. We observed
CDC activity in 4E1-7-B-treated CLBL-1 cells, but not in 4E1-7-C or 1E4-B-treated cells. This was unexpected
because previous research has shown that canine IgG-C has similar binding ability to human C1q as IgG-B19. The
reason for this discrepancy is unknown; however, previous study only showed the binding ability to human C1q,
but not CDC activity, and then canine IgG-B and IgG-C may have differential CDC activity, and the 4E1-7-C
antibody was not strong enough to induce CDC in our system. Although 1E4-B displayed CDC activity during
previous ­research17,22, no activity was detected in this study. We inferred that this results from differences inScientific Reports |Vol:.(1234567890)(2020) 10:11476 |https://doi.org/10.1038/s41598-020-68470-98

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experimental conditions, such as incubation time; however, it was conclusively shown that 4E1-7-B displayed
greater CDC activity than 1E4-B.
For therapeutic use of an antibody drug in the veterinary field, cost of the antibody is a pertinent issue, andso an antibody with high efficiency is desirable. For this reason, the defucosylated anti-canine CD20 antibody(4E1-7-B_f) was also generated and its activity compared with the 4E1-7-B antibody. As expected, CDC activity
was not altered by defucosylation, but the 4E1-7-B_f antibody exhibited almost ten times greater ADCC activity
than the 4E1-7-B antibody. We also observed slightly stronger suppressive effect on cell proliferation; however,
the reason behind this discrepancy is not clear, although defucosylation may change the antibody structure andthereby influence the CDC activity.
To test the antibody in vivo, NOD/SCID mice were used for xenotransplantation of CLBL-1 cells. PBMCs
were inoculated with an antibody to complement NK cell activity in the NOD/SCID m­ ice24, in which NK activity is very l­ ow25 and rat NK cells also might not work efficiently with canine IgG. Treatment with PBMCs alone
slowed tumor growth, which may be due to the small amount of NK activity in PBMCs. However, treatment
with 4E1-7-B or 4E1-7-B_f and PBMC suppressed tumor growth. We did not show the statistical significanceof the reduced tumor sizes at endpoints between two antibodies, which may be due to the small sample sizes,
although 4E1-7-B_f tended to suppressthe tumor growth in four out of four mice.
Effects of the 4E1-7-B_f antibody on B cell depletion were impressive because even a single injection of
0.5 mg/kg induced transient disappearance of peripheral C­ D21+ B cells. Previously, the 1E4-B antibody was+
found to reduce peripheral C­ D21 B cells after four sequential injections at a dosage of 10–30 mg/kg17, and thisreduction was not to the levels that we observed in this study. Moreover, a single injection of 5.0 mg/kg 4E1-7-B_f
induced a more sustainable decrease in peripheral C­ D21+ B cells with lymphoid depletion in the lymph nodes
according to analysis of biopsies. We did not compare the efficacy of 4E1-7-B with 4E1-7-B_f in this study;
however, the defucosylated antibody gave a remarkably strong effect of B cell depletion. In addition, despite thepreliminary safety study using healthy beagle dogs, throughout this study, we had not seen any adverse events,
such as general conditions, physical examination, complete blood counts except the slight and transient declineof platelet counts on the next day of treatment (supplementary Fig. 4), and serum biochemistry (data not shown).
In conclusion, we generated an anti-canine CD20 chimeric antibody, which had a high cytopathic effect on a
­CD20+ canine cell line. We also showed that defucosylation provided additive ADCC activity. This antibody also
decreased the B cells efficiently in healthy dogs. Clinical trials for canine patients with B cell lymphoma using
this antibody are now being prepared. We anticipate that this will become a treatment option and a candidate
for a new therapeutic approach in the near future.

Methods

Cell culture. Canine B cell lymphoma cell line, CLBL-126 (kindly provided by Dr. Barbara Rütgen from
University of Veterinary Medicine Vienna) 17–7127 (kindly provided by Dr. Ilene D. Kurzman from Universityof Wisconsin) and GL-128 (kindly provided by Dr. Munekazu Nakaichi from Yamaguchi University), canine T
cell lymphoma cell lines: CL-129 (kindly provided by Dr. Yasuyuki Momoi from The University of Tokyo), UL-1,
Nody-1, Ema, CLK, and C­ LC30, CLGL-9031 (kindly provided by Dr. Maxey Wellman from The Ohio State University), , human lymphoblastoid cell line (Jurkat), mouse lymphoblastoid cell line (EL-4), and mouse myeloma
cell line (P3U1) were all maintained in R10 medium, consisting of RPMI 1,640 supplemented with 10% FBS,
100 U/ml penicillin, 100 µg/ml streptomycin, and 55 µM β-mercaptoethanol. A rat kidney cell line (NRK), a
human kidney cell line (HEK293T), and packaging cell lines (PLAT-E, PLAT-gp, and PG13) were cultured in
D10 medium, consisting of DMEM with the same supplements as the R10 medium. These cell lines were cultured at 37 °C in a humidified incubator with 5% ­CO2.
Canine peripheral blood mononuclear cells (PBMCs) were isolated from healthy beagles that were kept as
blood donors at the Yamaguchi University Animal Medical Center using lymphoprep (Axis-Shield Ltd., Dundee,
Scotland) according to conventional methods. PBMCs were directly used for flow cytometry staining, as effector cells for inoculation of mice for tumor xenotransplant study, or were cultured in R10 medium containing
1,000 IU/ml of human recombinant IL-2 (Proleukin®, Chiron Therapeutics, Emeryville, CA) for 7 days prior touse in an ADCC assay as the lymphokine-activated killer (LAK) effector cells.
Tumor cells from lymphoma cases. Primary lymphoma cells were obtained from enlarged lymph nodes
from dogs that visited Yamaguchi University Animal Medical Center for diagnosis and treatment. Cytology wasused to identify samples with greater than 90% of lymphoblasts. After diagnosis, the remaining sample cells were
centrifuged and used for flow cytometry staining with owners’ consent.
Molecular cloning of canine CD20 and canine IgG heavy chain and light chain constantregions. Canine CD20 and canine IgG heavy chain and light chain constant regions were molecularlycloned using stored cDNA from the cervical lymph node (for CD20) and spleen (for IgG) of a healthy beagle, as
described in Doc S1.

Establishment of stable cell lines. To obtain stable cell lines, the retroviral and lentiviral expression sys-

tems were used. The construction of expression vectors is described in Doc S2. Retroviral canine CD20 expressing vector (pMx-IP-cCD20-flag#4) with pCAGGS-VSVG, or lentiviral canine CD20 expressing vector (CSIICMV-cCD20-Flag-IP#4) with pCVSVG and p8.9QV were transfected into the packaging cell line, PLAT-gp or
HEK293T. NRK, Jurkat, and EL-4 cells were infected with the produced virus and cultured in the presence of
puromycin (10 µg/ml, 4 µg/ml, and 4 µg/ml, respectively) to obtain the stable transfectants, NRK/cCD20, Jurkat/
cCD20 and EL-4/cCD20, respectively.

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To obtain a luciferase gene-expressing CLBL-1 cell line, Retroviral luciferase gene expressing vector (pMxluc-IP#9) was transfected into PLAT-E cells. The produced retrovirus was added to a PG13 packaging cell line
(PG13/luc). Retrovirus produced by PG13 was further added to CLBL-1 cells, followed by selection in the presence of 0.5 µg/ml of puromycin to obtain CLBL-1/luc cells.

Generation of a monoclonal antibody. Hybridoma producing the monoclonal antibody against canine
CD20 was obtained by immunizing the rat with NRK/cCD20 cells, according to our published ­protocol32, in
accordance with the Yamaguchi University Animal Care and Use guidelines. Supernatant from the resultant
hybridoma was collected and screened for reactivity to NRK/cCD20 and Jurkat/cCD20 using flow cytometry.
Finally, a single hybridoma clone (4E1-7) was isolated using the limiting dilution method, followed by the adaptation of culture media to serum free media, Hybridoma SFM (Thermo Fisher Scientific Inc.). The supernatant
was pooled, and the monoclonal antibody was purified using a HiTrap Protein A HP column (GE Healthcare
UK Ltd, Buckinghamshire, England). Finally, the subclass of the antibody was determined using flow cytometry.
Production and purification of recombinant antibody.

To obtain the recombinant antibodies(4E1-7-B, 4E1-7-C, and 1E4-B), expression plasmids for the heavy chain and light chain of each chimeric antibody (Doc S3) were transfected into 293 cells using the Expi293 expression system (Thermo Fisher ScientificInc.). The collected supernatant was purified using a HiTrap Protein A HP column (GE Healthcare) and desaltedusing a PD10 column (GE Healthcare). For production of the defucosylated antibody, BINDS-09 (core-fucose
KO ExpiCHO-S) cells (https​://www.med-tohok​u-antib​ody.com/topic​s/001_paper​_cell.htm) were used insteadof HEK293T cells (29).

Flow cytometry. Each cell was collected and washed with flow cytometry buffer (PBS with 2% FBS and 0.1%
NaN3). 2 × 105 cells were stained with the primary antibody: an anti-canine CD20 antibody (4E1-7, 4E1-7-B,
4E1-7-C, or 1E-4-B), a PE-labeled anti-CD21 antibody (Bio-Rad Laboratories, Inc., Berkeley, CA), or an isotype
control (rat I­ gG2a, BioLegend, San Diego, CA, USA), followed by a secondary antibody: PE-labeled anti-rat IgG(SouthernBiotech, Birmingham, AL), Dylight 649-labeled anti-rat IgG (BioLegend), or Alexa 647-labeled antidog IgG (Jackson ImmunoResearch, West Grove, PA, USA).
To determine the subclass of 4E1-7, NRK/cCD20 cells were first stained with the anti-canine CD20 antibody(4E1-7), and then stained with either biotin-labeled anti-rat IgG-κ, anti-rat IgG-λ, anti-rat IgG1, anti-rat I­ gG2a,or anti-rat ­IgG2b antibody, followed by incubation with streptavidin-PE (Thermo Fisher Scientific Inc.). Results
obtained using a BD Accuri C5 flow cytometer (BD Biosciences) were analyzed using FlowJo v.10 software (TreeStar Inc., Ashland, OR, USA).
Binding affinity of each antibody was determined by flow cytometry. Each cell was stained by the seriallydiluted antibody, as described above. Kd value was determined using the Nonlinear Regression Michaelis–Menten
curve fit by JMP14.0 software (JMP Japan, Tokyo, Japan).
Western blotting and Immunoprecipitation. Cell lines were collected and stored at − 80 °C until use.

SDS-PAGE and western blotting were done as described ­previously33. The anti-Flag antibody (clone M2, SigmaAldrich Crop., St. Louis, MO) and the anti-actin mouse monoclonal antibody (clone AC-15, Sigma-AldrichCorp.) were used as primary antibodies. The horseradish peroxidase (HRP)-conjugated secondary anti-rat(Zymed, San Francisco, CA) and anti-mouse (Bio-rad Laboratories, Inc.) antibodies were used as secondary
antibodies. The membrane was visualized by immersion in Western Lightning chemiluminescence reagent (Perkin Elmer, Foster City, CA, USA). Immunoreactive bands were visualized using the Luminescent Image Analyzer LAS 3,000 Mini instrument (FIJIFILM, Tokyo, Japan).
For immunoprecipitation, clarified cell lysates were pre-cleared with 10 µl of protein A/G agarose (Santa
Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at 4 °C with rotation. The resultant supernatant was mixed at
4 °C overnight with 1 µg of each antibody, which was premixed with 10 µl of protein A/G agarose for 1 h at 4 °C.
The immunoprecipitates were washed three times with PBS and used for Western blotting as described above.

Cell proliferation assay.

CLBL-1 cells (2 × 104 in 100 µl per well) were seeded into 96-well flat bottom
microtiter plates containing each antibody in triplicate. In crosslinking experiments, CLBL-1 cells and antibodies were incubated for 15 min on ice, followed by the addition of 10 µg/ml of anti-dog IgG Fc-specific antibody(Jackson ImmunoResearch). After incubating for 72 h, 10 µl of CCK-8 solution (Dojindo, Kumamoto, Japan)
was added to each well followed by incubation for a further 2 h. Absorbance at 450 nm was measured using an
ARVO X4 microplate reader (Perkin Elmer).

ADCC assay.

ADCC assay was performed as previously described ­method21, using CLBL-1/luc cells (5 × 103
in 100 µl per well), either rat I­ gG2a (Thermo Fisher Scientific Inc.), 4E1-7, or chimeric antibodies, and IL-2-stimulated PBLs (an effector to target ratio of 20:1). Cell-mediated cytotoxicity was calculated as follows; triplicate
wells were averaged, and percent lysis was calculated from the relative luminometer unit (RLU) data using thefollowing equation: percent specific lysis = 100 × (spontaneous death RLU – test RLU)/(spontaneous death RLU– maximal killing RLU).

CDC assay.

CDC assay was performed as previously described m­ ethod21, using CLBL-1/luc cells (5 × 105
in 100 µl per well), either rat ­IgG2a (Thermo Fisher Scientific Inc.), 4E1-7, or chimeric antibodies, and LOWTOX®-H rabbit complement (Cedarlane, Burlington, Canada; final concentration 1:40). Live and dead cells were
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in 100 µl per well) were used instead of CLBL-1 cells, and live and dead cells were counted as described above.

Apoptosis assay. CLBL-1 cells (1 × 105 in 500 µl per well) were seeded into 24-well plates containing either
rat ­IgG2a or 4E1-7 antibodies. After 72 h of incubation, cells were stained using the FITC Annexin V Apoptosis
Detection kit I (BD Biosciences) according to manufacturer instructions and were analyzed by flow cytometry.
Mouse study.

6–8 week-old NOD.CB17-Prkdcscid/J (NOD/SCID) mice were obtained from Charles River
Laboratories Japan, Inc. (Kanagawa, Japan). Studies were conducted in a specific pathogen-free area in accordance with the Yamaguchi University Animal Care and Use guidelines. The CLBL-1 cells (1 × 106 cells in 30 µl ofPBS) were subcutaneously transplanted into the flank of NOD/SCID mice. After inoculation, the two perpendicular dimensions of the tumors were measured daily with calipers. Tumor volume was calculated from themeasurements using the formula for the volume of an ellipsoid sphere [Tumor volume (­mm3) = 1/2 × Length(mm) × Width2 ­(mm2)]. Once the tumors reached 150–250 ­mm3, the chimeric antibody (150 µg in 500 µl ofPBS) was intraperitoneally injected every four days. Each time antibodies were injected, freshly isolated canine
PBMCs (5 × 106 cells each) from healthy beagles were suspended in 60 µl of PBS and injected into the flanksurroundings of the tumors to complement the NK cell activity in t­ umors24 because NOD/SCID mice have lowlevels of NK ­activity25. After tumors reached 2,000 ­mm3, or 13 days has passed since the start of treatment, mice
were sacrificed, and this is set as endpoints.

Dog study. Eight healthy beagles (of four dogs each) were bred in house at the Zenoaq animal facility, andstudies were conducted in a specific pathogen-free area and approved by the Animal Care and Use Committeeof Zenoaq Nippon Zenyaku Kogyo Co., Ltd (Fukushima, Japan). One of two different dosages (either 0.5 mg/kg
or 5.0 mg/kg) of defucosylated canine-rat chimeric anti-canine CD20 antibody (4E1-7-B_f) was intravenously
injected once on day 0, forming the low and high dose groups, respectively. The percentages of CD21 + B cells
in the peripheral blood were monitored at the indicated times using flow cytometry analysis (Animal Allergy
Clinical LABORATORIES, Kanagawa, Japan). On day 14 and day 28, lymph nodes were obtained for immunohistopathological analysis from one dog from each the low and high dose groups under general anesthesia.
Lymph nodes were sliced at 3 µm thickness and stained with an anti-CD79a antibody (clone HM57, dilution
1:50, Agilent Technologies Inc., Santa Clara, CA, USA) as previously ­described34.
Statistical analysis. All statistical analyses were performed using JMP14.0 software (JMP Japan, Tokyo,

Japan). Data were analyzed using one-way ANOVA followed by Tukey–Kramer multiple comparison tests.
P < 0.05 was considered as significantly different.

Received: 9 September 2019; Accepted: 25 June 2020

References

1. Ito, D., Frantz, A. M. & Modiano, J. F. Canine lymphoma as a comparative model for human non-Hodgkin lymphoma: recent
progress and applications. Vet. Immunol. Immunopathol. 159, 192–201 (2014).
2. Aresu, L. Canine lymphoma, more than a morphological diagnosis: what we have learned about diffuse large B-cell lymphoma.
Front Vet Sci. 3, 77. https​://doi.org/10.3389/fvets​.2016.00077​ (2016).
3. Thamm, D. H. Novel treatments for lymphoma. Vet. Clin. North Am. Small Anim. Pract. 49, 903–915 (2019).
4. Reff, M. E. et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 83, 435–445 (1994).
5. Glennie, M. J., French, R. R., Cragg, M. S. & Taylor, R. P. Mechanisms of killing by anti-CD20 monoclonal antibodies. Mol. Immunol.
44, 3823–3837 (2007).
6. Salles, G. et al. Rituximab in B-cell hematologic malignancies: a review of 20 years of clinical experience. Adv. Ther. 34, 2232–2273(2017).
7. Bello, C. & Sotomayor, E. M. Monoclonal antibodies for B-cell lymphomas: rituximab and beyond. Hematol. Am. Soc. Hematol.
Educ. Program. 2007, 233–242 (2007).
8. Freeman, C. L. & Sehn, L. Anti-CD20 directed therapy of b cell lymphomas: are new agents really better?. Curr. Oncol. Rep. 20,
103 (2018).
9. Golay, J. et al. Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B
more efficiently than rituximab. Blood 122, 3482–3491 (2013).
10. Liu, S. D. et al. Afucosylated antibodies increase activation of FcγRIIIa-dependent signaling components to intensify processes
promoting ADCC. Cancer Immunol. Res. 3, 173–183 (2015).
11. Ishii, T. et al. Defucosylated humanized anti-CCR4 monoclonal antibody KW-0761 as a novel immunotherapeutic agent for adult
T-cell leukemia/lymphoma. Clin. Cancer Res. 16, 1520–1531 (2010).
12. Kao, D., Lux, A., Schwab, I. & Nimmerjahn, F. Targeting B cells and autoantibodies in the therapy of autoimmune diseases. Semin.
Immunopathol. 36, 289–299 (2014).
13. Jubala, C. M. et al. CD20 expression in normal canine B cells and in canine non-Hodgkin lymphoma. Vet. Pathol. 42, 468–476(2005).
14. Impellizeri, J. A., Howell, K., McKeever, K. P. & Crow, S. E. The role of rituximab in the treatment of canine lymphoma: an ex vivo
evaluation. Vet. J. 171, 556–558 (2006).
15. Ito, D., Brewer, S., Modiano, J. F. & Beall, M. J. Development of a novel anti-canine CD20 monoclonal antibody with diagnostic
and therapeutic potential. Leuk Lymphoma 56, 219–225 (2015).
16. Jain, S. et al. The development of a recombinant scFv monoclonal antibody targeting canine CD20 for use in comparative medicine.
PLoS ONE 11, e0148366. https​://doi.org/10.1371/journ​al.pone.01483​66 (2016).
17. Rue, S. M. et al. Identification of a candidate therapeutic antibody for treatment of canine B-cell lymphoma. Vet. Immunol. Immunopathol. 164, 148–159 (2015).
18. Tang, L., Sampson, C., Dreitz, M. J. & McCall, C. Cloning and characterization of cDNAs encoding four different canine immunoglobulin gamma chains. Vet. Immunol. Immunopathol. 80, 259–270 (2001).

Scientific Reports |(2020) 10:11476 |https://doi.org/10.1038/s41598-020-68470-9

11Vol.:(0123456789)

www.nature.com/scientificreports/
19. Bergeron, L. M. et al. Comparative functional characterization of canine IgG subclasses. Vet. Immunol. Immunopathol. 157, 31–41(2014).
20. Yamane-Ohnuki, N. et al. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing
completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng. 87, 614–622(2004).
21. Kato, Y. et al. Establishment of P38Bf, a core-fucose-deficient mouse-canine chimeric antibody against dog podoplanin. Monoclon.
Antib Immunodiagn. Immunother. 37, 218–223 (2018).
22. Weiskopf, K. et al. Eradication of canine diffuse large B-cell lymphoma in a murine xenograft model with CD47 blockade andanti-CD20. Cancer Immunol. Res. 4, 1072–1087 (2016).
23. Miklós, K., Tolnay, M., Bazin, H. & Medgyesi, G. A. Antibody mediated lysis of hapten-conjugated target cells by macrophages
and by complement: the influence of IgG subclass, antibody and hapten density. Mol. Immunol. 29, 379–384 (1992).
24. Kaneko, M. K. et al. Antipodocalyxin antibody chPcMab-47 exerts antitumor activity in mouse xenograft models of colorectal
adenocarcinomas. Monoclon. Antib. Immunodiagn. Immunother. 36, 157–162 (2017).
25. Shultz, L. D. et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J. Immunol. 154, 180–191(1995).
26. Rütgen, B. C. et al. Establishment and characterization of a novel canine B-cell line derived from a spontaneously occurring diffuse
large cell lymphoma. Leuk Res. 34, 932–938 (2010).
27. Steplewski, Z., Jeglum, K. A., Rosales, C. & Weintraub, N. Canine lymphoma-associated antigens defined by murine monoclonal
antibodies. Cancer Immunol. Immunother. 24, 197–201 (1987).
28. Nakaichi, M. et al. Establishment and characterization of a new canine B-cell leukemia cell line. J. Vet. Med. Sci. 58, 469–471 (1996).
29. Momoi, Y. et al. Establishment and characterization of a canine T-lymphoblastoid cell line derived from malignant lymphoma.
Vet. Immunol. Immunopathol. 59, 11–20 (1997).
30. Umeki, S. et al. Establishment of five canine lymphoma cell lines and tumor formation in a xenotransplantation model. J. Vet. Med.
Sci. 75, 467–474 (2013).
31. Suter, S. E. et al. In vitro canine distemper virus infection of canine lymphoid cells: a prelude to oncolytic therapy for lymphoma.
Clin. Cancer Res. 11, 1579–1587 (2005).
32. Umeki, S. et al. Characterization of monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (PSGL-1). Vet. Immunol.
Immunopathol. 142, 119–125 (2011).
33. Okawa, T. et al. Calreticulin expression in neoplastic versus normal dog mammary glands: a cDNA subtraction-based study. Res.
Vet. Sci. 92, 80–91 (2012).
34. Thalheim, L., Williams, L. E., Borst, L. B., Fogle, J. E. & Suter, S. E. Lymphoma immunophenotype of dogs determined by immunohistochemistry, flow cytometry, and polymerase chain reaction for antigen receptor rearrangements. J. Vet. Intern. Med. 27,
1509–1516 (2013).

Acknowledgements

The authors would like to acknowledge the technical expertise of the DNA Core facility of the Center for Gene
Research, Yamaguchi University, supported by a grant-in-aid from the Ministry of Education, Culture, Sports,
Science and Technology of Japan. We thank to Drs. Barbara Rütgen, Maxey Wellman, and Ilene D. Kurzman
for providing cell lines.

Author contributions

T.M. designed this study, performed experiments and wrote the manuscript. Y.K., M.K.K., M.K, and T.T produced and purified antibody. Y.S., T.S., H.T., A.T. and K.B. performed experiments. Y.N., O.S., and M.I. edited
the manuscript.

Competing interests

Y.K. and T.M. have received research funding from Nippon Zenyaku Kogyo Co., Ltd. The rest of authors declare
no other potential conflicts of interest.

Additional information

Supplementary information is available for this paper at https​://doi.org/10.1038/s4159​8-020-68470​-9.
Correspondence and requests for materials should be addressed to T.M.
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