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ARTICLEOPEN

PD-L1 immunohistochemistry for canine cancers and clinicalbeneﬁt of anti-PD-L1 antibody in dogs with pulmonary
metastatic oral malignant melanoma

1234567890():,;

Naoya Maekawa1, Satoru Konnai 1,2 ✉, Maki Nishimura3, Yumiko Kagawa3, Satoshi Takagi4,5, Kenji Hosoya4, Hiroshi Ohta4,
Sangho Kim4, Tomohiro Okagawa1, Yusuke Izumi4, Tatsuya Deguchi4, Yukinari Kato6,7, Satoshi Yamamoto1,8, Keiichi Yamamoto1,8,
Mikihiro Toda1,8, Chie Nakajima1,9, Yasuhiko Suzuki1,9,10, Shiro Murata1,2 and Kazuhiko Ohashi1,2Immunotherapy targeting programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) represents promising treatments for humancancers. Our previous studies demonstrated PD-L1 overexpression in some canine cancers, and suggested the therapeutic potentialof a canine chimeric anti-PD-L1 monoclonal antibody (c4G12). However, such evidence is scarce, limiting the clinical application in
dogs. In the present report, canine PD-L1 expression was assessed in various cancer types, using a new anti-PD-L1 mAb, 6C11-3A11,
and the safety and efﬁcacy of c4G12 were explored in 29 dogs with pulmonary metastatic oral malignant melanoma (OMM). PD-L1
expression was detected in most canine malignant cancers including OMM, and survival was signiﬁcantly longer in the c4G12
treatment group (median 143 days) when compared to a historical control group (n = 15, median 54 days). In dogs withmeasurable disease (n = 13), one dog (7.7%) experienced a complete response. Treatment-related adverse events of any gradewere observed in 15 dogs (51.7%). Here we show that PD-L1 is a promising target for cancer immunotherapy in dogs, and dogs
could be a useful large animal model for human cancer research.
npj Precision Oncology (2021)5:10 ; https://doi.org/10.1038/s41698-021-00147-6

INTRODUCTION
Therapeutic antibodies targeting immune checkpoint molecules,
such as programmed cell death 1 (PD-1) and its ligand PD-ligand 1(PD-L1), have shown great promise in cancer treatment, by
reinvigorating immune responses against cancers1. PD-1 is an
immunoinhibitory receptor which belongs to the CD28 family, andexpressed predominantly in activated T cells2. PD-L1 is expressedin various cell types, including both hematopoietic and nonhematopoietic cells2. The interaction of PD-1 with PD-L1
attenuates T-cell effector functions, including cytokine secretionand cell proliferation, that are essential for robust immune
responses3. Of note, PD-L1 overexpression is often reported incancers, suggesting the PD-1/PD-L1 axis is a possible mechanism
adopted by cancers for immune evasion4,5. Blockade of thesemolecules using monoclonal antibodies (mAbs) enhances T-cell
responses against cancer antigens in both mouse models andin vitro human studies6–8.
Nivolumab, an anti-PD-1 mAb, demonstrated safety and efﬁcacy
in patients with advanced melanoma, non-small-cell lung cancer(NSCLC), and renal cell carcinoma. However, while the authors
reported treatment response in 36% of patients with PD-L1positive cancers, no objective response was found in PDL1–negative cancer patients9. In another study, NSCLC patientstreated with pembrolizumab showed objective response rate(ORR) of 45.2% if ≥50% of the tumor cells were PD-L1-positive on
immunohistochemistry (IHC), whereas only 16.5% of patients
responded to treatment if 1–49% of the tumor cells were PD-L1-

positive10. These studies suggest PD-L1 expression in cancers may
represent a useful biomarker for clinical response to PD-1/PD-L1inhibiting mAbs. To date, although PD-L1 IHC alone is notconsidered a precise biomarker11, the development of sensitive
PD-L1 IHC could provide a rationale for the use of PD-1/PD-L1inhibiting mAbs.
Malignant melanoma is a relatively common, but fatal disease indogs, especially with distant metastasis (stage IV: World HealthOrganization staging12). Among canine malignant melanoma, oralmalignant melanoma (OMM) is characterized by high invasivenessand metastatic propensity13,14. The median survival of dogs withstage IV OMM has been reported as 80 days, when treated withradiation15. The lungs are common sites for distant metastasis, anddogs with pulmonary metastatic OMM have the median survivalof less than 2 months, when treated with existing therapies
including surgery, radiation, and/or chemotherapy16. Metastatic
lesions appear resistant to chemotherapy17, and the lack ofeffective systemic therapy leads to very short expected survival.
Therefore, the development of new systemic therapy is crucial.
Recent studies have demonstrated that the PD-1/PD-L1 pathway is also involved in immune evasion of canine cancers. Some
canine cancers, including OMM, were reported to express PD-L1,
and speciﬁc anti-PD-1/PD-L1 mAbs induced immune-cell activation in vitro18–21. However, few studies have attempted to assess
PD-L1 expression in various canine cancers, and there is currently
no consensus on PD-L1 expression status in each cancer type. We
therefore aimed to assess PD-L1 expression in various cancer

1
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan. 2Department of Disease Control, Faculty of Veterinary Medicine,
Hokkaido University, Sapporo, Japan. 3North Lab, Sapporo, Japan. 4Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
5
Department of Veterinary Surgery 1, School of Veterinary Medicine, Azabu University, Sagamihara, Japan. 6Department of Antibody Drug Development, Tohoku University
Graduate School of Medicine, Sendai, Japan. 7New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan. 8Fuso Pharmaceutical Industries, Ltd., Osaka, Japan.
9
Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan. 10Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education(GI-CoRE), Hokkaido University, Sapporo, Japan. ✉email: konnai@vetmed.hokudai.ac.jp

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N Maekawa et al.

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Table 1.

PD-L1 expression in various malignant cancers.

Pathology

Positive no./tested no.

Positive rate (%)

PD-L1 TPS
<1%

1–49%

≥50%

Squamous cell carcinoma (Skin)

18/20

90

2

2

16

Nasal adenocarcinoma

20/20

100

0

2

18

Transitional cell carcinoma
Anal sac gland carcinoma

20/20
19/20

100
95

0
1

0
4

20
15

Soft tissue sarcoma

14/20

70

6

2

12

Osteosarcoma

20/20

100

0

4

16

Malignant melanoma (Oral)

19/20

95

1

1

18

Mammary adenocarcinoma

20/20

100

0

0

20

Histiocytic sarcoma

18/20

90

2

12

6

Diffuse large B-cell lymphoma

17/20

85

3

5

12

Gastric adenocarcinoma
Transmissible venereal tumor

4/5
0/4

80
0

1
4

1
0

3
0

1234567890():,;TPS tumor proportion score

types, including OMM, using a novel anti-PD-L1 mAb (6C11-3A11)
and compared its sensitivity to 6G7-E1, a previously reported mAb
for canine IHC19.
In addition, a recent pilot study demonstrated antitumor
efﬁcacy of a canine chimeric anti-PD-L1 mAb (c4G12) against
canine OMM and undifferentiated sarcoma (n = 2), among asample of 9 dogs16. We therefore further aimed to assess the
efﬁcacy and safety of c4G12, in dogs with pulmonary metastatic
OMM, using a predetermined dosage regimen16. A secondary
objective included the exploration of factors predictive of survival,
including known correlates of improved survival in anti-PD-1/PDL1 therapy-treated patients; use of radiation therapy, C reactive
protein (CRP) level and lymphocyte-to-monocyte ratio (LMR)22–24.
RESULTS
PD-L1 IHC using 6C11-3A11 in various canine malignant
cancers
We previously established a canine PD-L1 IHC using 6G7-E1, an
IgM class mAb19. However, the sensitivity of our IHC seemed
insufﬁcient due to a lack of demonstrated PD-L1 expression inseveral cancer types, contrary to other reports19,20,25,26. To
resolve this inconsistency, we obtained a mAb from our
hybridoma pool, named 6C11-3A1127,28. In human, normal
tonsil is used as positive and negative control for PD-L1 IHC29.
In 6C11-3A11 IHC, canine tonsil epithelium was stained
positively (Supplementary Fig. 1), while squamous epitheliumin the same section showed no speciﬁc signals. The staining
pattern appeared to be consistent with that in human tonsils,
suggesting the speciﬁcity of 6C11-3A11 IHC. Next, we
performed IHC with both 6C11-3A11 and 6G7-E1 and directlycompared the staining intensity and patterns using the same
cancer samples. In squamous cell carcinoma, nasal adenocarcinoma, transitional cell carcinoma, anal sac gland carcinoma,
and soft tissue sarcoma, speciﬁc staining was not observed
using 6G7-E1. However, most of these samples showed clear
positive signals in 6C11-3A11 IHC. In osteosarcoma samples,
speciﬁc staining was conﬁrmed by both mAbs, and expectedly,
6C11-3A11 produced higher staining intensity (n = 5 for eachcancer type, Supplementary Fig. 2 and Supplementary Table 1).
Taken together, this suggests that 6C11-3A11 is more sensitive
than 6G7-E1 for the detection of PD-L1 in IHC.
Next, PD-L1 expression was further investigated in various
malignant cancers by 6C11-3A11 IHC. Among the 20 samples
npj Precision Oncology (2021) 10

tested, 18 (90%), 20 (100%), 20 (100%), 19 (95%), 14 (70%), and20 (100%) were PD-L1-positive for squamous cell carcinoma,
nasal adenocarcinoma, transitional cell carcinoma, anal sac
gland carcinoma, soft tissue sarcoma, and osteosarcoma,
respectively. In 19 (95%) of OMM, 20 (100%) of mammary
adenocarcinoma, 18 (90%) of histiocytic sarcoma, 17 (85%) ofdiffuse large B-cell lymphoma, and 4 (80%) of gastric adenocarcinoma, PD-L1 was detected in the tumor cells (Supplementary Fig. 3a–e). In contrast, none of the transmissible venereal
tumor specimens expressed PD-L1 (Supplementary Fig. 3f). In
most cases, tumor cells were stained intracellularly, whereas cellsurface staining was obscure. Of note, inﬁltrating plasma cells
and lymphocytes were also PD-L1-positive in some specimens,
while stromal cells surrounding the tumor cells were predominantly PD-L1-negative.
To develop a scoring protocol for canine PD-L1 IHC, tumor
proportion score (TPS) for PD-L1 expression was calculated
according to the criteria described in the Methods section. The
majority of samples from each cancer type had TPS of ≥50%,
except for histiocytic sarcoma, in which most of the samples had
TPS of 1–49%. Table 1 summarizes the results of PD-L1 IHC with
6C11-3A11. Representative IHC results for malignant melanoma
and squamous cell carcinoma are shown in Fig. 1, with examples
for each TPS.

Characteristics of dogs enrolled in clinical study using c4G12
To evaluate the safety and clinical beneﬁts of anti-PD-L1 mAb in
canine pulmonary metastatic OMM, we conducted a veterinary
clinical study of c4G12 in our hospital involving 29 dogs (seeSupplementary Table 2 for details of each dog). At the time of
study enrollment, the median age was 13 years (range: 8–16
years). All dogs had primary OMM diagnosed by histopathological assessment and pulmonary metastases (PM) were conﬁrmed by chest X-ray or computed tomography (CT) scan. Prior
to the enrollment, most dogs underwent at least one priortreatment, including surgery, radiation, and/or chemotherapy.
The majority of dogs had PD-L1-positive cancers with TPS of≥50%, whereas only 2 dogs had PD-L1–negative cancers (TPS <1%). At baseline, 13 dogs (44.8%) had measurable disease asdeﬁned by cRECIST v1.030. The baseline characteristics of dogs
are summarized in Table 2.
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N Maekawa et al.

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( b)

(c)

(d)

(e)

(f)

Squamous cell
carcinoma

Malignantmelanoma(a)Fig. 1 Representative PD-L1 immunohistochemistry results with 6C11-3A11 for each tumor proportion score (TPS). The proportion of PDL1–expressing tumor cells was scored according to the percent of stained viable tumor cells (see “Methods”). Representative IHC results for
TPS < 1% (a, d), 1–49% (b, e), and ≥50% (c, f) are shown. Original magniﬁcation, ×200 (a–c) or ×100 (d–f).

Safety of c4G12 treatment
Dogs were treated with intravenous administration of c4G12 every
2 weeks. Median duration of c4G12 treatment was 98 days (range:
15–518 days). Concomitant therapy including radiation andsurgical excision was allowed in order to achieve local tumor
control (see Supplementary Table 3 for details of the treatment ofeach dog). Treatment-related adverse events (TRAEs) of any gradewere observed in 15–29 dogs (51.7%). TRAEs that occurred in atleast 10% of dogs included vomiting, diarrhea, and elevated ALT,
AST, and Lipase. Grade 3 TRAEs were observed in 4 dogs (13.8%),
including elevated ALT, AST, and Lipase without any clinicalsymptoms. One dog developed grade 3 pneumonitis after thesecond dose of c4G12, but recovered with treatment discontinuation and supportive care including glucocorticoid administration.
No grade 4 or 5 TRAEs were observed throughout the study. All
TRAEs are listed in Table 3.

days) in the control group (Fig. 3a; see Supplementary Table 4 fordetails of dogs in the historical control group). Subpopulation
analysis only involving dogs with measurable disease conﬁrmed asigniﬁcant difference in survival between the treatment (n = 13)
and historical control group (n = 8; P = 0.02). Similarly, in dogs with
non-measurable disease, survival was longer in the treatmentgroup (P = 0.01, Supplementary Fig. 4). In order to gain someinsights into the factors that could predict survival, the treatmentgroup was further dichotomized into subgroups according to theuse of radiation therapy and peripheral blood immunologicalmarkers. The concomitant or previous (within 8 weeks of treatment
initiation) use of radiation was signiﬁcantly associated withimproved overall survival (OS) in the c4G12 treatment group (P =0.02; Fig. 3b). Moreover, OS with c4G12 treatment was signiﬁcantly
longer in dogs that had low plasma CRP (cutoff 2.55, P = 0.01) or
high LMR (cutoff 1.41, P = 0.0002) at baseline (Fig. 3c, d).

Clinical efﬁcacy of c4G12 treatment
As more than half of the dogs did not have measurable lesions atbaseline, evaluation of tumor response was of secondary interestin this study. Tumor response as evidenced by diagnostic imaging
was observed in 5 of 29 dogs (17.2%). According to cRECIST v1.0,
one dog experienced a complete response (CR; dog #10) among
13 dogs that had measurable diseases at baseline, with ORR of
7.7% (95% conﬁdence interval (CI) = 0.2–36.0%) (Table 4, Fig. 2a,and Supplementary Table 3). Other 4 dogs that experienced tumor
response only had non-measurable lesions at baseline and thus
the response could not be evaluated by cRECIST. However, alldetectable tumors disappeared in 2 dogs (dog #12 and #19),
leading to numerically long survival time of more than 1 year
(417 days and >530 days, respectively; Fig. 2b and Supplementary
Table 3). In the other 2 dogs (dog #5 and #28), all lung metastatic
lesions disappeared in response to the treatment (Fig. 2c), but
residual tumors persisted in the lymph nodes and/or oral cavity.
Responses were durable, but all 5 dogs eventually had diseaseprogression at later time-point. All deaths were considered tumorrelated, except for dog #10 which died from chronic kidneydisease at day 168 of c4G12 treatment.
Survival from conﬁrmation of PM to death was compared by the
Kaplan–Meier method in the treatment group versus a historicalcontrol group (n = 15)16. Survival was signiﬁcantly longer in thetreatment group (P = 0.00006), with median survival of 143 days
(95% CI of 91–194 days), in contrast to 54 days (95% CI of 25–NA

DISCUSSION
Immunotherapy targeting the PD-1/PD-L1 axis is a promisingtreatment for several malignant cancers in humans. However,
although a subset of patients has a remarkable response totreatment, around 60–80% of patients do not respond1.
Exploration of resistance mechanisms and predictive biomarkers of immune checkpoint inhibitors (ICIs) is a growing ﬁeld ofstudy in human oncology. In dogs, only limited evidence for PDL1 expression in cancers is available and the safety and efﬁcacy
of anti-PD-L1 therapy have not yet been robustly established. In
the present report, we demonstrated PD-L1 expression in mosttypes of canine malignant cancers with IHC. In dogs withpulmonary metastatic OMM, c4G12 treatment was safe andinduced tumor response in some dogs. Importantly, the survivalof dogs with c4G12 treatment was signiﬁcantly longer than thatof the historical control group. Because no systemic therapy
that prolongs the survival of dogs with pulmonary metastatic
OMM is currently available, the results of this study strongly
encourage the further development of anti-PD-L1 therapy
in dogs.
Except for transmissible venereal tumor, PD-L1 expression was
detected in more than 70% of the samples, consistent withprevious reports using different mAbs19,20. Moreover, the majority
was classiﬁed as TPS ≥ 50%. These rates were high compared withhuman melanoma, squamous cell carcinoma, and urothelial
cancer, in which 51–71% was PD-L1-positive and only 14–31%

Published in partnership with The Hormel Institute, University of Minnesota

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Table 2.

Table 3.

Characteristics of the dogs at baseline.

Characteristic

Treatment-related adverse events (TRAEs).

TRAEs—no. (%)

Any grade Grade 3 Leading todiscontinuation

13

Any TRAEs

15 (51.7)

8–16

Anorexia

1 (3.4)

0 (0)

0 (0)

Vomiting

4 (13.8)

0 (0)

0 (0)

Age—yearMedianRange
Age category―no. (%)≥10 year<10 yearSex―no. (%)

4 (13.8) 1 (3.4)

27 (93.1)

Diarrhoea

3 (10.8)

0 (0)

0 (0)

2 (6.9)Thrombocytopenia

2 (6.9)

0 (0)

0 (0)

Albumin, low

1 (3.4)

0 (0)

0 (0)

8 (27.6)
3 (10.3)

1 (3.4)1 (3.4)

0 (0)
0 (0)
0 (0)

Intact8 (27.6)

ALT
AST

Castrated

11 (37.9)

Alkaline phosphatase

1 (3.4)0 (0)19 (65.5)

Lipase3 (10.3)

1 (3.4)

0 (0)

CPK

1 (3.4)

0 (0)

0 (0)

Conjunctivitis/ocularsurface disease

1 (3.4)

0 (0)

0 (0)

Pneumonitis/pulmonary
inﬁltrates

1 (3.4)1 (3.4)1 (3.4)

Male

All
FemaleIntact3 (10.3)

SpayedAll

7 (24.1)
10 (34.5)

PD-L1 status—no. (%)
Positive
TPS ≥ 50%

18 (62.1)

TPS 1–49%

1 (3.4)TPS ND

7 (24.1)All

26 (89.7)

Negative (TPS < 1%)ND

2 (6.9)1 (3.4)

Table 4.

Evaluation of response to c4G12 treatment.

Best overall response—no. (%)

Prior therapy―no. (%)
Surgery

Adverse events that were considered to be related to the treatment arelisted. Grading was performed according to VCOG-CTCAE v1.154.
ALT alanine aminotransferase, AST aspartate aminotransferase, CPK creatine
phosphokinase.

17 (58.6)

RadiationCR

1 (7.7)PR

0 (0)≤8 weeksa>8 weeks

8 (27.6)

SD

0 (0)

4 (13.8)PD

10 (76.9)

Unknown

2 (6.9)All
Chemotherapy

14 (48.3)
1 (3.4)None

2 (6.9)Measurable lesion(s)―no. (%)
Present

13 (44.8)

Absent16 (55.2)

TPS tumor proportion score, ND not determined.
aDogs that received previous radiation ≤8 weeks before the ﬁrst doseof c4G12.

was TPS ≥ 50%31, suggesting the differences in etiology or
immune microenvironment of cancers between dogs andhumans. The reason PD-L1 expression was not detected in our
samples of transmissible venereal tumor remains unclear. In
another contagious cancer known as the Tasmanian devil facial
tumor disease, tumor cells can express PD-L1 in vitro, but its
expression is minimal in naturally infected animals32. As theseunique cancers possess alternative mechanisms to evade the hostimmune system (e.g., downregulation of MHC molecules)33–35,
immunosuppression via the PD-1/PD-L1 pathway might not be
important, a topic to be further clariﬁed. To date, PD-L1 expression
in tumor biopsies is utilized to identify the eligible patient for antiPD-1/PD-L1 therapy. For example, TPS ≥ 1% is required for NSCLC
patients to receive pembrolizumab as ﬁrst-line treatment. However, its reported predictive utility varies among studies9–11,36,37. In
our study, the association between tumor PD-L1 expression andclinical outcome remains unclear, because most dogs had PD-L1npj Precision Oncology (2021) 10

NE
ORR―% (95% CI)

2 (15.4)
7.7 (0.2–36.0)

Tumor response to c4G12 treatment was deﬁned and recorded according
to cRECIST v1.030.
CR complete response, PR partial response, SD stable disease, PD
progressive disease, NE not evaluable, ORR objective response rate (CR +PR), CI conﬁdence interval.

positive cancers and TPS ≥ 50%. Notably, among 5 dogs that
responded to treatment, 1 had PD-L1 TPS of <1%, suggesting that
PD-L1 IHC alone might not be an adequate biomarker. Larger
studies are needed to conﬁrm the predictive utility of PD-L1 IHC in
canine anti-PD-L1 therapy.
The c4G12 treatment was well-tolerated, and the frequency andseverity of TRAEs were consistent with previous reports of humanclinical trials using anti-PD-1/PD-L1 mAbs9,38. TRAEs with potential
immune-related causes included pneumonitis and thrombocytopenia. Hepatitis, pancreatitis, and colitis were suspected fromother TRAEs in several dogs, although none of those was clinicallyconﬁrmed. Since pneumonitis, colitis, hepatitis, endocrinopathies,
and infusion-related reaction are reported in human anti-PD-1/PDL1 therapies9,38, these events should be assessed in future canine
studies. The ORR of 7.7% in c4G12 treatment was low comparedwith previous human studies of advanced melanoma using antiPD-L1 mAbs, with 17.3%–30.2% patients having objected
responses38,39. Tumor mutational burden (TMB) was reported tocorrelate with improved response to ICIs including anti-PD-L1
mAbs40,41. In human cutaneous melanoma, mean TMB was 49.17
mutations/Mb, while acral or mucosal melanoma had mean TMB
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N Maekawa et al.

5(b) Pekingese, castrated male, 8-year-old (#12)(a) Pomeranian, male, 12-year-old (#10)
Before treatment

7 weeks

4 weeks

12 weeks(c) Beagle, spayed female, 11-year-old (#5)
Before treatment

6 weeks

18 weeks

Fig. 2 Antitumor efﬁcacies of c4G12 in dogs with oral malignant melanoma. a Representative tumor response in dogs with measurable
disease. The dog (Pomeranian, male, 12-year-old, #10 in Supplementary Tables 2, 3) received c4G12 at a dose of 5 mg/kg every 2 weeks. Oral
recurrent tumor (upper panel) and pulmonary metastatic lesions (lower panel) responded to treatment at week 7. b, c Representative tumor
response in dogs with non-measurable disease. b The dog (Pekingese, castrated male, 8-year-old, #12) received c4G12 at a dose of 5 mg/kg
every 2 weeks. Pulmonary metastatic lesions were conﬁrmed at week 4. Subsequently, the lesions responded to treatment at week 12. c The
dog (Beagle, spayed female, 11-year-old, #5) received c4G12 at a dose of 5 mg/kg every 2 weeks until week 12 and thereafter the dose waschanged to 2 mg/kg. A metastatic lesion in the lung (upper panel) disappeared at week 6. In contrast, the other lesion (lower panel) showedslight increase in tumor size at week 6, followed by complete regression at week 18 (an immune-related pattern of response). Contrastenhanced and matched transverse CT images are shown. Arrow heads indicate tumor lesions.

of only 2.64 mutations/Mb42. Because canine oral melanoma arises
independent of ultra-violet light exposure with TMBs comparable
to human mucosal melanoma43,44, low response rate to c4G12
treatment might reﬂect low neoantigen expression in canine
OMM. Further research is required to elucidate resistant mechanisms to anti-PD-L1 therapy in dogs.
The OS of dogs with c4G12 treatment was enhanced with theuse of radiation therapy, as previously seen in mouse preclinicalPublished in partnership with The Hormel Institute, University of Minnesota

models45 and a human clinical study, where the previous
radiotherapy was associated with the improved OS in NSCLC
patients treated with pembrolizumab22. Because radiation induces
immunogenic cell death in the tumor microenvironment (TME)and subsequently activates antitumor immune responses46,
combined radiation and ICIs represent a promising strategy to
improve treatment outcome, the topic of our future study on
canine OMM using c4G12. Peripheral blood CRP level was also
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N Maekawa et al.

6

(b)

(a)

1

Treatment group
Control group

Proportion survival

Proportion survival

1
0.8

P = 0.00006
0.6
0.4
0.2

Radiation (+)
Radiation (-)

0.8
P = 0.02
0.6
0.4
0.2
0

0
0

100

200

300

400

100

200

500

400

300

Time (days)

Time (days)

Number at risk

0500

Number at risk

Treatment

29

15

5

3

2

1

Radiation (+) 13

9

4

2

1

1

Control

15

1

0

0

0

0

Radiation (-) 14

5

1

1

0

0

(d)

(c)

1

CRP > 2.55
CRP ≤ 2.55

Proportion survival

Proportion survival

1
0.8

P = 0.01
0.6
0.4
0.2

LMR > 1.41
LMR ≤ 1.41

0.8
P = 0.0002
0.6
0.4
0.2
0

0
0

100

200

300

400

0

500

100

200

300

400

500

Time (days)

Time (days)
Number at risk

Number at risk
CRP > 2.55

5

1

0

0

0

0

LMR > 1.41 24

15

5

3

1

1

CRP ≤ 2.55

23

14

5

3

1

1

LMR ≤ 1.41

0

0

0

0

0

5

Fig. 3 Survival of dogs treated with c4G12. a Comparison of survival between c4G12 treatment group (n = 29) and historical control group(n = 15). Survival (days) was deﬁned as time from conﬁrmation of pulmonary metastasis to death. For subgroup analysis, the treatment group
was dichotomized according to (b) the use of previous/concomitant radiation, (c) plasma C reactive protein (CRP) levels (cutoff 2.55 mg/dL), or(d) the lymphocyte-to-monocyte ratio (LMR, cutoff 1.41), and overall survival was compared between the subgroups. Dogs that received
radiation therapy concomitantly or ≤8 weeks before the initiation of antibody treatment were classiﬁed into radiation (+) subgroup. Marks on
the line indicate censored data. Statistical analysis was performed using the log-rank test.

associated with improved OS in the c4G12 treatment group,
consistent with a previous report of cancer patients who weretreated with anti-PD-1/PD-L1 therapy23. In cancer patients, CRP
levels can be increased47, possibly reﬂecting the inﬂammatory
TME. Although inﬂammation can be tumor-promoting48, thereason animals with high CRP levels have poor survival remains
unclear. Similarly, OS with c4G12 treatment was signiﬁcantly
longer in dogs with high LMR, as with a previous report on
pembrolizumab treatment in metastatic melanoma patients24.
LMR is a systemic immune status marker that, at least in part,reﬂects the TME49,50. Because tumor-inﬁltrating lymphocytes playa pivotal role in antitumor immunity and circulating monocytes
can differentiate into tumor-associated macrophages that haveprotumor propensity51, increased LMR could be an indicator for
preferable antitumor immune responses. Because these analyses
were conducted in a univariate manner and were exploratory innature with a limited sample size, further studies are needed tovalidate these ﬁndings.
npj Precision Oncology (2021) 10

The use of a small-sized historical control group in survival
comparison is a limitation in this clinical study. Only dogs withpulmonary metastatic OMM which were recently treated in thesame hospital were included in the control group; however, theresults should be interpreted carefully because the control group
was retrospective and non-randomized population that may
contain multiple biases. Because prolongation of survival by
c4G12 treatment was strongly suggested, further studies involving
more appropriate control (e.g. double-blinded, randomized, and/or placebo-treated) are warranted to conﬁrm the beneﬁt insurvival.
Although tumor-bearing mice are often utilized as preclinicalmodels, results are not always reproduced in humans52. Becausesome canine naturally occurring cancers, including malignant
melanoma, resemble human cancers53 and dogs are geneticallyoutbred and immunocompetent, dogs might be useful large
animal models, especially for immunological studies including ICIs.
In conclusion, PD-L1 appears to be a promising target for canine
cancer immunotherapy, and c4G12, an immune checkpointPublished in partnership with The Hormel Institute, University of Minnesota

N Maekawa et al.

7
inhibiting antibody for dogs, deserves further investigation. We
believe that this study is a substantial step toward putting theimmune checkpoint blockade into veterinary clinical practice andmaking dogs as a translational model for human cancer research.
METHODS
Specimens and mAbs used for IHC
Formalin-ﬁxed and parafﬁn-embedded (FFPE) tissue samples were used for
PD-L1 IHC. The tissue samples had been submitted for histologicaldiagnosis and stored in a commercial pathology laboratory (North Lab,
Hokkaido, Japan). The study included samples of normal tonsil (n = 4), skinsquamous cell carcinoma (n = 20), nasal adenocarcinoma (n = 20),transitional cell carcinoma (n = 20), anal sac gland carcinoma (n = 20),
soft tissue sarcoma (n = 20), osteosarcoma (n = 20), OMM (n = 20),
mammary adenocarcinoma (n = 20), histiocytic sarcoma (n = 20), diffuse
large B-cell lymphoma (n = 20), gastric adenocarcinoma (n = 5) andtransmissible venereal tumor (n = 4). Inﬂammatory mammary carcinoma
samples were excluded.
A rat anti-PD-L1 mAb, 6G7-E1, has been described previously19. A new
anti-PD-L1 mAb 6C11-3A11 was selected from previously establishedhybridoma clones27. Rat immunization, hybridoma establishment, isotyping, and mAb puriﬁcation were performed at Cell Engineering Corporation(Osaka, Japan). The binding speciﬁcity of 6C11-3A11 was conﬁrmed using
recombinant canine PD-L1–expressing cells by ﬂow cytometry in ourprevious paper28.

were allowed (e.g., NSAIDs, antibiotics, anodynes, and antitussives). In
all dogs treated by concomitant or previous radiation therapy, the oral
tumor and involved lymph nodes, if present, received approximately
26–32 Gy in total, which were delivered in 4 fractions at one-weekintervals using Elekta Synergy integrity R LINAC (Elekta AB, Stockholm,
Sweden) or TITAN-320S (Shimadzu Industrial System, Kyoto, Japan). The
study period was from 11th March 2016 to 11th March 2020. Dogs still
alive at the end of the study period were included in the survival
analysis as censored data.

Evaluation of adverse events
Adverse events, that emerged after initiation of therapy with a reasonable
possibility that the event was caused by c4G12 treatment, were deﬁned astreatment-related events. These events were graded and recorded
following the veterinary cooperative oncology group–common terminology criteria for adverse events (VCOG-CTCAE) v1.154.

Evaluation of tumor response
Tumor response to the treatment was deﬁned and recorded according to the
response evaluation criteria in solid tumors in dogs (cRECIST) v1.030.
Assessment of the tumor burden was performed by X-ray or CT within
3 weeks prior to the ﬁrst c4G12 dose (baseline) and was scheduled every
6 weeks during the treatment. The dogs that have no measurable (target)
lesion at baseline were considered “with non-measurable disease” andexcluded from the response evaluation. The ORR was calculated as thepercentage of dogs that had the best overall response of CR and partial
response (PR).

PD-L1 IHCFFPE sections underwent immunohistochemical staining with 6G7-E1 aspreviously described19, except for the use of non-decalciﬁed specimens
for osteosarcoma. Similarly, 6C11-3A11 staining was performed at a ﬁnal
concentration of 5 μg/mL. Histoﬁne simple stain MAX PO (rat) (Nichirei,
Tokyo, Japan) was used as a secondary antibody, and Mayer’s
hematoxylin stain was used as a counterstain. When histologicalevidence of cell staining was observed by an optical microscope, theassessed sections were considered positive for PD-L1 expression.
Stained sections without the primary antibody served as negative
control for 6C11-3A11 IHC (Supplementary Fig. 5). Comparison of theanti-PD-L1 mAbs was performed by staining sections obtained from thesame cancer samples. For 6C11-3A11 IHC, the PD-L1 TPS was deﬁned
and calculated as follows: TPS = the number of viable tumor cells with
PD-L1 expression/the number of total viable tumor cells × 100 (%). The
evaluation included a minimum of 100 viable tumor cells.

Clinical study using c4G12
The present clinical study was conducted in the Veterinary Teaching
Hospital, Hokkaido University as research aiming to demonstrate that
PD-L1 is a promising target for cancer immunotherapy in dogs. The
study was approved by the Institutional Animal Care Committee,
Hokkaido University (Approval number: 15–0149) and the Faculty of
Veterinary Medicine, Hokkaido University (Approval number: 15028).
The use of animals throughout the clinical study was approved by the
ethics committee, Faculty of Veterinary Medicine, Hokkaido University.
Prior to study enrollment, written informed consent was obtained fromboth the dogs’ owner and veterinarian. Dogs were required to meet thefollowing criteria for inclusion; (a) histologically conﬁrmed malignant
melanoma that originated in the oral cavity (b) with pulmonary
metastatic lesion(s) as evidenced by chest X-ray or CT. Dogs withirrelevant severe systemic illness or autoimmune disease were excluded
from the study. In total, 29 dogs were enrolled, including 4 dogs
described in the previous report16. PD-L1 expression in the primarytumors, obtained by surgical excision at prior surgeries or biopsies, wasassessed by 6C11-3A11 IHC. PD-L1 TPS was calculated as describedabove, as long as an adequate tumor section was available for
evaluation. Dogs were treated with c4G12 every 2 weeks at 2 mg or
5 mg/kg by intravenous administration using a syringe pump over 1 h.
The optimization of the dose and delivery regimen was not a scope of
this study; the treatment dose was basically set as 5 mg/kg, but
determined by veterinarians on a case-by-case basis. During thetreatment, physical examination, complete blood count, and blood
chemistry were performed at intervals of 2–6 weeks. When clinicallynecessary, concomitant drugs not considered to be immunosuppressive
Published in partnership with The Hormel Institute, University of Minnesota

Evaluation of survival
Survival duration (days) of dogs in the c4G12 treatment group was deﬁned
as time from conﬁrmation of PM to death. Dogs with pulmonary metastatic
OMM treated by standard therapies at the Veterinary Teaching Hospital of
Hokkaido University during 2013 to 2016 were used as a historical control
group16. All deaths in the control group were considered tumor-related.
There was no statistically signiﬁcant difference in sex, age, and bodyweight between the two groups (Fisher’s exact test for sex and
Mann–Whitney U test for age/body weight). For subgroup analysis, OS(days) of the treated dogs was deﬁned as time from the ﬁrst dose of c4G12
to death. The dogs were divided into subgroups according to the use ofprevious (≤8 weeks from the ﬁrst dose of c4G12) or concomitant radiation,and baseline plasma CRP and LMR. LMR was deﬁned as the ratio of
absolute lymphocyte count to absolute monocyte count. The baseline
blood test was performed on the day of the ﬁrst dose of c4G12. The
optimal cutoff values for the CRP level and LMR was determined using
receiver operating characteristic (ROC) curves (Supplementary Fig. 6). The
survival curves were generated using the Kaplan–Meier method andstatistical analysis was performed using the log-rank test. A statisticalsoftware EZR (version 1.35)55 was used in all analyses and P < 0.05 wasconsidered statistically signiﬁcant.

Reporting summary
Further information on research design is available in the Nature Research
Reporting Summary linked to this article.

DATA AVAILABILITY
High-resolution immunohistochemistry (IHC) and computerized tomography (CT)images supporting Figs. 1 and 2 of the article, and baseline blood test analysis data
supporting Fig. 3, are available in the ﬁgshare repository: https://doi.org/10.6084/m9.
ﬁgshare.1338544156. The datasets supporting the survival analysis plots in Fig. 3, andall other data supporting the ﬁndings of the current study, are included in the
supplementary ﬁles that accompany the article.

Received: 13 July 2020; Accepted: 13 January 2021;

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ACKNOWLEDGEMENTS
This work was supported by the Grant-in-Aid for Scientiﬁc Research (Grant Numbers
16K15042, 19H03114, and 19K15969) and Grant-in-aid for JSPS Fellows (Grant
Number 15J01989) from the Japan Society for the Promotion of Science (JSPS) and by
Japan Agency for Medical Research and Development (AMED) under Grant Number:

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JP20am0101078. Funders had no role in the study design, data collection andanalysis, decision to publish, and manuscript preparation.

Correspondence and requests for materials should be addressed to S.K.
Reprints and permission information is available at http://www.nature.com/
reprints

AUTHOR CONTRIBUTIONS
S. Konnai, Y.S., S.M., and K.O. designed and supervised the project. N.M., S. Konnai,
MN, Y. Kagawa, S.T., K.H., H.O., S. Kim, T.O., Y.I., and T.D. performed the experiments.
N.M., S.K., M.N., Y. Kagawa, S.T., C.N., Y.S., S.M., and K.O. analyzed the data. Y. Kagawa,Y. Kato, S.Y., K.Y., M.T., C.N., and Y.S. provided materials, reagents and/or analysis tools.
N.M. prepared the manuscript. All authors reviewed and approved the manuscript.

COMPETING INTERESTS
Authors S.Y., K.Y., and M.T. were employed by Fuso Pharmaceutical Industries, Ltd. S.
Konnai, K.O., S.M., Y.S., C.N., T.O., N.M., S.T., and Y. Kagawa are the authors of patentapplications covering materials and techniques described in this paper (PCT/JP2017/029055, PCT/JP2018/11895). All other authors declare no competing interests.

ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41698-021-00147-6.

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