cancers
ArticleEnhanced Systemic Antitumour Immunity by HypofractionatedRadiotherapy and Anti-PD-L1 Therapy in Dogs with Pulmonary
Metastatic Oral Malignant Melanoma
Tatsuya Deguchi 1,† , Naoya Maekawa 2,† , Satoru Konnai 2,3, *, Ryo Owaki 1 , Kenji Hosoya 1 , Keitaro Morishita 1 ,Motoji Nakamura 1 , Tomohiro Okagawa 2 , Hiroto Takeuchi 3 , Sangho Kim 1 , Ryohei Kinoshita 1 ,Yurika Tachibana 1 , Madoka Yokokawa 1 , Satoshi Takagi 4 , Yukinari Kato 5,6 , Yasuhiko Suzuki 7,8 ,Shiro Murata 2,3 and Kazuhiko Ohashi 2,3
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2

3

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5

6

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8

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Citation: Deguchi, T.; Maekawa, N.;
Konnai, S.; Owaki, R.; Hosoya, K.;
Morishita, K.; Nakamura, M.;
Okagawa, T.; Takeuchi, H.; Kim, S.;et al. Enhanced Systemic Antitumour
Immunity by HypofractionatedRadiotherapy and Anti-PD-L1
Therapy in Dogs with Pulmonary

Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0819, Japan
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060-0818, Japan
Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060-0818, Japan
Department of Veterinary Surgery 1, School of Veterinary Medicine, Azabu University,
Sagamihara 252-5201, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan
International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan
Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE),
Hokkaido University, Sapporo 060-0808, Japan
Correspondence: konnai@vetmed.hokudai.ac.jp
These authors contributed to this work equally.

Simple Summary: Immune checkpoint inhibitors (ICIs) are promising treatment options for spontaneous cancers in dogs. To optimize the clinical benefit of ICIs, combination therapy with radiation has
been proposed for human cancers; however, the safety and efficacy of the combination approach have
not yet been reported in dogs. The retrospective analysis of dogs bearing pulmonary metastatic oralmalignant melanoma suggested that the use of previous (≤8 weeks) hypofractionated radiation therapy
is associated with a better clinical response to anti-PD-L1 immunotherapy. Radiotherapy before theinitiation of anti-PD-L1 therapy can be an effective approach to enhance antitumor immunity in dogs.

Metastatic Oral Malignant Melanoma.
Cancers 2023, 15, 3013. https://doi.org/10.3390/cancers15113013
Academic Editor: David Wong
Received: 7 April 2023
Revised: 9 May 2023
Accepted: 24 May 2023
Published: 1 June 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andAbstract: Although immune checkpoint inhibitors (ICIs), such as the anti-programmed death-ligand 1(PD-L1) antibody, have been developed for the treatment of canine malignant melanoma, desirable
clinical efficacies have not been achieved. Recent studies in humans have suggested that radiation therapy (RT) combined with ICIs induces robust systemic antitumour immunity in patientswith cancer. This study retrospectively examined the therapeutic efficacy of combination therapy(hypofractionated RT and anti-PD-L1 antibody [c4G12]) in dogs with pulmonary metastatic oralmalignant melanoma. The intrathoracic clinical benefit rate (CBR)/median overall survival (OS)in the no RT (n = 20, free from the effect of RT), previous RT (n = 9, received RT ≤8 weeks prior
to the first c4G12 dose), and concurrent RT (n = 10, c4G12 therapy within ±1 week of the first RT
fraction) groups were 10%/185 days, 55.6%/283.5 days (p < 0.05 vs. no RT group), and 20%/129 days
(p > 0.05 vs. no RT group), respectively. The adverse events were considered to be tolerable in the
combination therapy. Thus, hypofractionated RT before the initiation of c4G12 therapy can be aneffective approach for enhancing the therapeutic efficacy of immunotherapy, with acceptable safety
profiles. Further prospective clinical studies are required to confirm the findings of this study.

conditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Cancers 2023, 15, 3013. https://doi.org/10.3390/cancers15113013

https://www.mdpi.com/journal/cancers

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Keywords: hypofractionated radiation therapy; anti-PD-L1 antibody; immune checkpoint blockade;canine malignant melanoma; Immunotherapy

1. Introduction
Melanoma is the most common oral malignancy observed in dogs. The biological behaviour of canine oral melanoma has been considered locally invasive and highly
metastatic [1,2] and is comparable to that of human oral melanoma [3]. Radiation therapy (RT) is an effective treatment for primary oral melanoma and involved lymph nodes.
Hypofractionated protocols (e.g., 3–6 once-weekly fractions of 6–9 Gy) have often beenused to treat canine oral malignant melanoma (OMM) [4–6]; however, the optimal dose per
fraction, fraction numbers, and intervals have yet to be determined. The reported rangeof overall response rate of RT was 73–94%, whereas the progression-free interval ranged
from 3.6–8.6 months [7–11]. The high metastatic potential, especially to the lung, makes it
difficult to achieve long-term control of the tumour and ultimately leads to the death of
the affected dogs [4,12]. The establishment of systemic therapy is necessary to improve
the prognosis of canine OMM because conventional chemotherapy does not improve thesurvival time [13,14]. Although several immunotherapies have been proposed for thetreatment of canine OMM, desirable clinical efficacies have not been achieved [15].
RT has been associated with enhanced antitumour immunity not only locally but alsosystemically. Damage-associated molecular pattern molecules (DAMPs) are released whenirradiation causes immunogenic cell death (ICD) in tumour tissues. Dendritic cells arestimulated by DAMPs through pattern recognition receptors, such as toll-like receptors,
and activate cytotoxic T lymphocytes. Subsequently, activated T lymphocytes systemically attack tumour cells [16,17]. The enhancement of systemic antitumour immunity byirradiation has been recognised as an abscopal effect where metastatic lesions outside theirradiation field shrink after local RT. Apart from mouse cancer models, only 46 casesof confirmed abscopal effects have been reported in the literature from 1969 to 2014 [18].
Moreover, the induction of the abscopal effect in naturally occurring canine cancers has
not been reported [19]. Thus, the enhancement of antitumour immunity by irradiation
rarely occurs in spontaneous cancers in both humans and animals. The immune evasion
mechanisms of tumours can counteract irradiation-induced antitumour immunity in thevast majority of cancer patients [20–24].
Programmed death 1 (PD-1) and its ligands, programmed death-ligand 1 (PD-L1) and
PD-L2, are prominent molecules involved in tumour immune evasion and are called immune checkpoint molecules. These molecules play important roles in immune suppression
to maintain immune homeostasis and protect the host from excessive immune responses or
autoimmune diseases. PD-L1 and PD-L2 are type I transmembrane proteins that belong to
the B7 family. Although the expression of PD-L2 is limited to dendritic cells, macrophages,
and bone-marrow-derived mast cells, PD-L1 is expressed in various cell types, including
haematopoietic and nonhematopoietic cells [25]. Overexpression of PD-L1 is a common feature of various human cancers, and it causes immune evasion of tumour cells by inhibitingcell proliferation and cytokine secretion of PD-1–expressing tumour-specific T cells [26,27].
Several types of canine malignant tumours also express PD-L1, including squamous cell
carcinoma, nasal adenocarcinoma, transitional cell carcinoma, osteosarcoma, and malignant
melanoma, suggesting that canine tumours have an immune evasion mechanism throughPD-L1 overexpression [28–36]. In our previous reports, almost all OMM cases tested expressed PD-L1 (19/20 cases, 95%) [35]. Recent studies have demonstrated that treatment
with canine chimeric anti-PD-L1 antibody (c4G12) is effective against canine OMM [33,35].
c4G12 binds to canine PD-L1 with high avidity, efficiently inhibits the interaction between
canine PD-1 and PD-L1, and enhances cytokine production and T-cell proliferation in canine peripheral blood mononuclear cell cultures [33]. The response rate to c4G12 treatment
in canine OMM was found to be 14.3% (1/7 dogs) [33], which is comparable to that ofCancers 2023, 15, 3013

3 of 12human anti-PD-L1 antibody therapy in patients with advanced melanoma [37]. Althoughanti-PD-L1 therapy has achieved a breakthrough for this extremely progressive tumour,
further analysis is needed to improve the response rate and survival benefit.
In irradiation-induced antitumour immunity, the PD-1/PD-L1 axis may play a crucialrole in the suppression of cytotoxic T cell responses within the tumour microenvironment
because PD-L1 expression is upregulated after irradiation [38]. Therefore, the combinationof RT and anti-PD-1/PD-L1 therapy has gained attention for achieving synergistic antitumour efficacy. Indeed, the combination therapy of RT and immune checkpoint inhibitors(ICIs) induced an abscopal effect in mouse models of cancer [24,39], and early clinical studies have demonstrated that patients with non-small-cell lung cancer who received previous
RT had an improvement in overall survival (OS) after anti-PD-1 antibody treatment ascompared to those without previous RT [40].
In this study, we evaluated the synergistic antitumour efficacy of hypofractionatedRT and c4G12 treatment in dogs with pulmonary metastatic OMM. To investigate whether
the combination therapy induced better systemic antitumour immunity, dogs treatedwith c4G12 at the Hokkaido University Veterinary Teaching Hospital (HUVTH) wereretrospectively evaluated for the response rate of intrathoracic (i.e., outside the irradiation
field) metastatic lesions and OS. Dogs were divided into three groups based on the presenceor absence of RT, as well as the timing of RT, to examine whether the temporal order of the
combinational irradiation influences the outcome of c4G12 immunotherapy.
2. Materials and Methods
2.1. Entry Requirement
Dogs with pulmonary metastatic OMM (stage IV in the TNM-based staging scheme)that underwent c4G12 treatment at HUVTH between March 2016 and September 2021
were retrospectively examined in this study. All dogs were diagnosed with OMM basedon histopathological examination and PD-L1 expression was assessed by immunohistochemical staining as described previously [35]. Pulmonary metastatic sites were detectedusing chest radiography or computed tomography (CT) in all dogs. Dogs with concurrent
cancer of different origins or severe systemic illness unrelated to the cancer were excluded
from the analysis. The clinical study using c4G12 was conducted with the approval of theInstitutional Animal Care Committee of Hokkaido University (Approval number: 15-0149
and 20-0041). The use of animals throughout the clinical study was approved by the Ethics
Committee of the Faculty of Veterinary Medicine, Hokkaido University. Prior to enrolment
in the clinical study, written informed consent was obtained from the dog owners.
2.2. c4G12 Treatment
c4G12 treatment was performed as described previously [35]. Dogs received intravenous administration of c4G12 every 2 weeks at 5 mg/kg (in some cases, 2 mg/kg waschosen by veterinarians) over 1 h. Results of physical examination, complete blood count,and blood chemistry were used to evaluate adverse events. Adverse events were graded
and recorded according to the Veterinary Cooperative Oncology Group–Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 [41].
2.3. Radiation Therapy
Most RT treatments were conducted using the Elekta Synergy Integrity R LINAC (Elekta
AB, Stockholm, Sweden), whereas some dogs were treated using an orthovoltage machine,
TITAN-320S (Shimadzu Industrial System, Kyoto, Japan). In all dogs, general anaesthesia
was induced with propofol and maintained with sevoflurane delivered in oxygen. The oral
tumour and involved lymph nodes, if present, received four fractions of RT (approximately
26–32 Gy in total) at one-week intervals. For the linac radiation treatment, the gross tumour
volume (GTV), clinical target volume (CTV), planning target volume (PTV), and organ at risk
(OAR), including the eyes, brain, and larynx, were delineated using the CT dataset. Inverse
planning was designed with isocentrically placed 4 or 6 MV radiation beams using volumetricCancers 2023, 15, 3013

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modulated arc therapy (VMAT) techniques with one arc on the CMS Monaco 3.0 treatment
planning system (CMS-Elekta Ltd., Crawley, UK). It utilises a Monte Carlo algorithm with
continuous dose rate variation to determine the dose with heterogeneity correction. Forward
planning was performed with 4, 6, and 10 MV beams using 3-dimentional conventional
RT on a Pinnacle 3 treatment planning system (Philips Healthcare, Best, The Netherlands).
Orthovoltage treatment planning was designed based on hand calculation for a 200 kV photon
beam. A few dogs (n = 3) received RT in other facilities before their first visit to HUVTH, withunspecified protocols and instruments.
2.4. Evaluation of Intrathoracic Response
Tumour response in the thoracic cavity (i.e., pulmonary metastatic lesions) to c4G12
treatment was recorded according to the response evaluation criteria for solid tumours indogs (cRECIST) v1.0 [42]. Baseline assessments were performed within three weeks prior
to the first c4G12 dose. During treatment, tumours were evaluated at least every 6 weeks
using the same modality (chest X-ray or CT). For dogs with non-measurable disease (i.e.,
all metastatic lesions in the lung <10 mm on CT or <20 mm on X-ray), tumour response
was classified as either CR (disappearance of all detectable tumours), PD (unequivocal
progression of the tumour or appearance of new lesions), or SD/PR (reduction or stable
persistence of the tumour: not classified as CR or PD). The CBR was defined as thepercentage of dogs with the best intrathoracic response of CR, PR, SD, or SD/PR.
2.5. Evaluation of Survival
OS (days) of dogs was defined as the time from the first c4G12 dose to death. In
comparison with historical data of dogs treated with RT only, OS was defined as the timefrom the first dose of RT to death. Dogs with pulmonary metastatic OMM treated with
RT only at HUVTH between 2014 and 2019 were included in the historical control group(n = 10, RT-only group). Kaplan–Meier curves were generated to estimate the mediansurvival time with a 95% CI. Dogs that were still alive at the end of the study period were
included in the survival analysis as censored data.
2.6. Statistical Analysis
Statistical analyses of binary variables, continuous variables, and survival times were
performed using Fisher’s exact test, the Kruskal–Wallis test followed by the Steel-Dwass
test, and the log-rank test, respectively. The statistical software EZR (version 1.35) [43] wasused for all the analyses. p < 0.05 was considered statistically significant.
3. Results
3.1. Baseline Characteristics of Dogs
In total, 39 dogs with pulmonary metastatic OMM were eligible, including the 26 dogs
described in a previous report [35], and included 12 miniature dachshunds, five mixedbreed dogs, three pugs, three golden retrievers, three toy poodles, two American cocker
spaniels, two Chihuahuas, two Labrador retrievers, and one each of the following: beagle,
Yorkshire terrier, kaninchen dachshund, papillon, flat-coated retriever, Pomeranian, andPekingese. The median age was 13 years (range, 8–15 years), with 21 males (15 castrated)and 18 females (13 spayed). The median body weight was 6.0 kg (range, 2.0–29.0 kg). The
primary tumour locations were mandible (n = 20), maxilla (n = 17), and unspecified (n = 2)
(Table S1). Dogs treated with c4G12 were separated into the following three groups: (1) no
RT group, dogs that were considered free from the effect of RT, with no history of RT or
the previous RT terminated >8 weeks before the initiation of c4G12 therapy, (2) previous
RT group, dogs that were treated with RT ≤8 weeks prior to the first c4G12 dose, and(3) concurrent RT group, which received RT along with the c4G12 therapy, with the firstc4G12 dose administered within ±1 week of the first fraction of RT (Figure 1a). Among the
39 dogs, 20, nine, and ten belonged to the no RT, previous RT, and concurrent RT groups,

Cancers 2023, 15, 3013

specified (n = 2) (Table S1). Dogs treated with c4G12 were separated into the followi
three groups: (1) no RT group, dogs that were considered free from the effect of RT, w
no history of RT or the previous RT terminated >8 weeks before the initiation of c4G
therapy, (2) previous RT group, dogs that were treated with RT ≤8 weeks prior to the fi
c4G12 dose, and (3) concurrent RT group, which received RT along with the c4G12 th
5 of 12
apy, with the first c4G12 dose administered within ±1 week of the first fraction of RT (Fure 1a). Among the 39 dogs, 20, nine, and ten belonged to the no RT, previous RT, aconcurrent RT groups, respectively. No statistically significant differences were observ
sex, age, bodyweight,differences
or primary were
tumourlocationinamong
thebodytreatmentgroups (Tarespectively. Noinstatisticallysignificant
observed
sex, age,weight,1). location among the treatment groups (Table 1).
or primary tumour

Figure1. OS of dogs
receiving
c4G12 immunotherapy
with previous/concurrent
or without previous/concurrent
RT.
Figure 1. OS of dogs
receiving
c4G12
immunotherapy
with or without
RT.
TimingofRT
andanti-PD-L1
antibody(c4G12)
therapyforeachtreatmentgroup.
(b)
Kaplan–Me(a) Timing of RT and anti-PD-L1 antibody (c4G12) therapy for each treatment group. (b) Kaplan–Meiercurves for OS from the initiation of c4G12 immunotherapy. Marks on the line indicate censo
curves for OS from the initiation of c4G12 immunotherapy. Marks on the line indicate censored data.
data. Statistical analysis was performed using the log-rank test.
Statistical analysis was performed using the log-rank test.

Table 1. Characteristics of dogs included in this study.
No RT (n = 20)

Previous RT (n = 9)

Concurrent RT (n = 10)

American cocker spaniel

1 (5)1 (11)

0

Beagle

1 (5)

0

0

Chihuahua

1 (5)

0

1 (10)

Flat-coated retriever

1 (5)

0

0

Golden retriever

1 (5)2 (22)

0

Kaninchen dachshund

1 (5)

0

0

Labrador retriever

1 (5)1 (11)0BreedCancers 2023, 15, 3013

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Table 1. Cont.
No RT (n = 20)

Previous RT (n = 9)

Concurrent RT (n = 10)

Miniature dachshund

6 (30)

2 (22)

4 (40)

Papillon

1 (5)

0

0

Pekingese0

1 (11)

0

Pomeranian0

1 (11)0Pug

2 (10)0

1 (10)

Toy poodle

1 (5)

0

2 (20)

Yorkshire terrier

1 (5)

0

0

Mix

2 (10)

1 (11)

2 (20)

10 (50)7 (78)

4 (40)

Castrated

6 (30)

5 (56)

4 (40)

Intact4 (20)

2 (22)

0

Female10 (50)2 (22)

6 (60)

Spayed8 (40)

1 (11)

4 (40)

Intact2 (10)

1 (11)

2 (20)≤10 years

2 (10)

2 (22)

1 (10)>10 years

18 (90)7 (78)

9 (90)

Sex
Male

Age

Body Weight≤5 kg

6 (30)

2 (22)

1 (10)

5–10 kg

10 (50)3 (33)

9 (90)>10 kg4 (20)

4 (44)

0

Mandible

10 (50)2 (22)

5 (50)

Maxilla8 (40)

7 (78)

5 (50)

Unspecified

2 (10)

00Primary tumour site

No. of dogs (%) are shown.

3.2. Treatment and Adverse Events
All dogs received at least one dose of c4G12, administered every 2 weeks at 2 or
5 mg/kg. The median duration of c4G12 treatment was 91 days (range, 2–750 days) witha median number of c4G12 dosage of six times (range, 1–54 times) (Table S2). In the no
RT group, adverse events of any grade related to c4G12 treatment occurred in 12 dogs(60%). Most events were classified as grade 1–2; however, grade 3 events (pneumonitis andanaphylaxis) occurred in two dogs (10%). Both grade 3 events were resolved after shortterm supportive care. In the previous RT group, adverse events of any grade were observedin four dogs (44%), including grade 3 events (elevation in alanine aminotransferase [ALT],
aspartate aminotransferase [AST], or lipase) in three dogs (33%). In the concurrent RT
group, adverse events of any grade occurred in six dogs (60%), including grade 3 events(elevation in alkaline phosphatase/AST or blood urea nitrogen [BUN]) in two dogs (20%).
None of the grade 3 events in the previous and concurrent RT groups were associatedwith clinical symptoms; thus, additional care was not indicated. The frequency of grade 3
adverse events did not differ significantly among the groups (Table 2).

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Table 2. Adverse events related to c4G12 treatment.
No RT (n = 20)
AnyPrevious RT (n = 9)

Concurrent RT (n = 10)

Any Grade

Grade 3

Any Grade

Grade 3

Any Grade

Grade 3

12 (60)2 (10)

4 (44)

3 (33)

6 (60)

2 (20)

Anorexia

00

1 (11)

0

0

0

Diarrhoea

2 (10)0

1 (11)0

2 (20)

0

Vomiting

3 (15)

0

2 (22)0

2 (20)0
1 (10)

Alkaline phosphatase

1 (5)

0

0

0

2 (20)ALT

5 (25)

0

3 (33)

1 (11)

1 (10)

0

AST

0

0

3 (33)

1 (11)

1 (10)

1 (10)

BUN

0

0

0

0

2 (20)

1 (10)

Albumin, low00

1 (11)000Glucose, low00

1 (11)

000Lipase2 (10)0

1 (11)

1 (11)

1 (10)

0

CPK

0

0

1 (11)

0

0

0Conjunctivitis/ocularsurface disease

1 (5)

0

0

0

0

0

Thrombocytopenia

2 (10)

0

0

0

0

0

0

0

0

0

1 (10)

0

1 (5)

1 (5)

0

0

0

0

0

0

0

0

1 (10)

0

Pancreatitis
Pneumonitis/pulmonary
infiltrates
Allergic reaction0000Anaphylaxis
1 (5)
1 (5)
No. of dogs with event (%) are shown. Adverse events considered to be related to c4G12 immunotherapy by
the investigators are listed. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea
nitrogen; CPK, creatine phosphokinase.

All dogs except for one in the concurrent RT group completed the RT protocol asplanned (four fractions of 6.5–8 Gy at one-week intervals). Adverse events related to RT
were typically grades 1–2 and were characterised by alopecia, pruritus, skin ulceration,and mucositis, which were resolved without treatment. To examine whether combination
therapy increases the risk of side effects associated with RT, the frequency of adverse
events related to RT was compared with the RT-only group (n = 10, historical control which
consisted of dogs with pulmonary metastatic OMM treated with RT only, Table S3). No
significant differences were observed in the frequency of adverse events related to RT
among the RT-only, previous RT, and concurrent RT groups (Table S4).
Surgical resection of the metastatic lesion was performed during c4G12 treatment in
one dog in the no RT group (lymph node), two in the previous RT group (lung and eye),and one in the concurrent RT group (spleen). No additional treatment was performed in
the remaining 35 dogs (Table S1).
3.3. Intrathoracic Response and Overall Survival
Intrathoracic metastatic lesions in two dogs responded to c4G12 treatment (one complete response [CR] and one stable disease [SD]) and the clinical benefit rate (CBR) was
10.0% (2/20) in the no RT group. The dog that achieved CR survived for 362 days, while
another dog with SD survived for 152 days. In the previous RT group, four, one, and fourdogs exhibited CR, SD/partial response (PR), and progressive disease (PD), respectively.
The intrathoracic CBR was 55.6% (5/9), which was significantly higher than that of the no
RT group (p = 0.016, Table 3). Among the dogs with a clinical benefit (n = 5), the median
OS was 399 days (95% confidence interval [CI]: 98–NA days). In the concurrent RT group,
two dogs experienced SD/PR and the CBR was 20.0% (2/10), which was not significantly
different from that of the other groups (p = 0.584 vs. no RT group and p = 0.170 vs. previous
RT group). The OS of the two SD/PR dogs was 131 and 194 days, respectively.

Cancers 2023, 15, 3013

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Table 3. Intrathoracic response to c4G12 treatment.
No RT (n = 20)

Previous RT (n = 9)

Concurrent RT (n = 10)

CR

1 (5)

4 (44)

0

SD or SD/PR *1 (5)

1 (11)

2 (20)

PD

15 (75)4 (44)

6 (60)

NE

3 (15)0

2 (20)

Clinical benefit **2 (10)

5 (56) †2 (20)

No. of dogs (%) are shown. * Non-CR/non-PD in dogs with non-measurable pulmonary lesions at baseline.
** Clinical benefit = CR + PR + SD (SD/PR). † p < 0.05 by Fisher’s exact test (vs. No RT group). CR, complete
response; PR, partial response; SD, stable disease; PD, progressive disease; NE, not evaluable.

At the end of the study period, zero, one, and one dogs were still alive in the no RT,
previous RT, and concurrent RT groups, respectively. Twelve dogs were censored because
they were lost to follow-up. One dog that experienced CR in the previous RT group died ofan unrelated cause (chronic kidney disease). Twenty dogs died of metastasis progression,
while the remaining four died of local progression of OMM (Table S2). The median OS fromthe first c4G12 treatment to death in all 39 dogs was 129 days (95% CI: 93–185 days). The
median OS in the no RT group was 185 days (95% CI: 53–152 days). OS was significantly
longer in the previous RT group than that in the no RT group (p = 0.036), with a median
OS of 283.5 days (95% CI: 61–NA days). The median OS of the concurrent RT group was
129 days (95% CI: 2–NA days), with no significant difference in OS as compared to the no
RT group (p = 0.294, Figure 1b). However, OS from the first fraction of RT was significantly
longer in the concurrent RT group than that of the RT-only group (median OS: 48 days,
95% CI: 14–NA days, p = 0.036, Figure S1).
4. Discussion
This study showed that in dogs with pulmonary metastatic OMM, the intrathoracic
tumour response rate and OS were significantly improved in the previous RT group comparedwith that of the no RT group. These results strongly suggest that systemic antitumour immunity reinvigorated by anti-PD-L1 therapy was enhanced by ICD induced by RT. Furthermore,since concurrent RT did not improve the response rate and OS, the timing of RT is found to beof great importance for achieving synergistic efficacy. As the types, frequency, and severity ofadverse events related to c4G12 or RT in combination therapy were similar to those of eachtreatment alone, hypofractionated RT before c4G12 therapy can be an effective combination
therapy with acceptable toxicity to treat canine OMM.
The abscopal effect has been shown in many types of human malignant neoplasms,
such as hepatocellular carcinoma, lymphoma, renal cell carcinoma, and melanoma. Although the reported cases were substantially limited, seven of 46 cases (15.2%) wererecorded in patients with melanoma. Additionally, the lungs are the most common
metastatic sites, exhibiting an abscopal effect [18]. Thus, dogs with pulmonary metastatic
OMM, which we focused on in this study, are considered a suitable model to investigate
the abscopal effects or enhancement of antitumour systemic immunity after irradiation. Although the abscopal effect itself rarely occurs and has been poorly studied, immunotherapy
combined with RT may increase the frequency of cases with an abscopal effect. As ICIs suchas the anti-PD-1/PD-L1/cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody enhancesystemic antitumour immunity and local immunosuppression caused by RT is considered
to be partly due to the upregulation of immune checkpoints [38], the combination of ICIs
and RT has been gaining attention for their expected synergistic efficacies.
One retrospective study in humans showed the outcomes of 21 patients with advanced
melanoma treated with ipilimumab, a CTLA-4 inhibitor, followed by RT. An abscopal effectwas observed in 11 patients (52%), nine of whom had PR (43%) and two had SD (10%) [44].
Additionally, RT during anti-PD-1 therapy achieved a 48% response rate (including CR, PR,
and SD) for non-radiated lesions in advanced melanoma patients [45]. Although the timing

Cancers 2023, 15, 3013

9 of 12of RT and ICI therapy was different, a CBR of 55.6% for non-irradiated lesions of canine
OMM in the previous RT group in the present study was comparable or preferable to thosein the combination therapy in human advanced melanoma. Additionally, the combinationof ICIs and RT was associated with prolonged OS as compared to monotherapy in humancancer patients [40,46,47]. Consistent with these observations, in this study, the OS wassignificantly longer in the previous RT group than that in the no RT group. Interestingly,
the OS in the concurrent RT group was similar to that of the no RT group; however, it wassignificantly longer than that of the historical RT-only group. These results suggest that
c4G12 therapy is somewhat effective in all situations, but concurrent RT does not result in asynergistic effect with c4G12 treatment.
The optimal RT protocol (timing, dose, and fractionation) for combined therapy
remains a topic of debate [48]. An early study using mouse models of cancer revealed that
concurrent but not sequential (starting 7 days after the last dose of RT) anti-PD-L1 therapy
was associated with improved survival, indicating that concurrent RT may induce bettersynergistic effects [49]. However, in human clinical studies, controversial results have beenreported, suggesting that the optimal timing of RT depends on various clinical conditions,
including cancer types, patient characteristics, and RT protocols. For example, in patientswith metastatic melanoma, concurrent RT combined with anti-PD-1 antibody resulted ina similar OS as compared to sequential therapy (RT followed by anti-PD-1 therapy) [50].
Another study in patients with stage III non-small-cell lung cancer showed that sequential
anti-PD-L1 therapy after chemoradiotherapy was associated with better OS than that ofchemoradiotherapy alone [47,51]. More recently, using mouse models, administration ofanti-PD-1 antibody before irradiation has been shown to increase irradiation-induced deathof polyfunctional effector CD8+ T cells within the tumour microenvironment, leading to
the loss of systemic antitumour immunity [52]. As the RT protocol used in this study wasfour fractions at one-week intervals and c4G12 was administered every 2 weeks, immune
activation by the first two administrations of anti-PD-L1 antibody in the concurrent RT
group may have been abrogated by the following fractions of RT, diminishing the synergistic
antitumour efficacy. Therefore, we propose that the optimal timing of RT for canine OMM
would be immediately before the initiation of anti-PD-L1 therapy, with the last dose of RT
completed before the first dose of anti-PD-L1 antibody, although this hypothesis should befurther examined in prospective clinical studies.
5. Conclusions
Combination therapy using hypofractionated RT and an anti-PD-L1 antibody may be a
promising strategy to induce robust systemic antitumour immunity. This study provides thefirst evidence which suggests that a sequential combination (hypofractionated RT followedby c4G12 therapy) is better than a concurrent combination to elicit synergistic therapeutic
efficacy against canine OMM. The findings may be extrapolated not only to other canine
cancers but also human cancers where hypofractionated protocols are used for RT, as dogs
are considered clinically relevant models of human cancer with various genetic backgrounds
and spontaneous development of cancer in immunocompetent settings. Further studies arenecessary to determine the optimal protocol for such combination therapy.
Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/cancers15113013/s1, Table S1. Baseline characteristics of c4G12-treateddogs; Table S2. Summary of c4G12 treatment; Table S3. Summary of dogs in RT-only group; Table S4.
Adverse events related to RT; Figure S1. OS of dogs treated with RT only or c4G12 immunotherapy
plus concurrent RT.
Author Contributions: T.D., N.M., S.K. (Satoru Konnai), T.O., S.M. and K.O. designed and supervised
this project. T.D., N.M., R.O., M.N., H.T., Y.T. and M.Y. collected clinical data. T.D., N.M., S.K.
(Satoru Konnai) and R.O. analysed the data. K.H., K.M., S.T., S.K. (Sangho Kim), R.K., Y.K. and Y.S.
provided materials, reagents and/or analysis tools. T.D., N.M. and S.K. (Satoru Konnai) prepared themanuscript. All authors have read and agreed to the published version of the manuscript.

Cancers 2023, 15, 3013

10 of 12Funding: This work was supported by Grant-in-Aid for Scientific Research (Grant Numbers 16K15042,
19H03114, 19K15969, 19K23702, and 21K1498301) and Grant-in-Aid for JSPS Fellows (Grant Number
15J01989) from the Japan Society for the Promotion of Science (JSPS) and by the Japan Agency for Medical Research and Development (AMED) under Grant Numbers: JP22ama121008 and JP21am0101078.
The funders had no role in the study design, data collection and analysis, decision to publish, ormanuscript preparation.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board of Hokkaido University (Approval number: 15-0149 and 20-0041).
Data Availability Statement: All data generated or analysed during this study are included in thispublished article (and its Supplementary Information files).
Acknowledgments: We would like to thank all the staff at the Veterinary Teaching Hospital, Hokkaido
University for their excellent assistance throughout the clinical study.
Conflicts of Interest: N.M., S.K., T.O., Y.S., S.M. and K.O. are authors of a patent covering materials
and techniques described in this paper (PCT/JP2017/029055 and JP2022-99097). All other authors
declare no competing interests.

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