ORIGINAL PAPER
Nagoya J. Med. Sci. 83. 827–840, 2021
doi:10.18999/nagjms.83.4.827

Phase Ib study on the humanized anti-CCR4 antibody,
KW-0761, in advanced solid tumors
Takuro Saito1, Koji Kurose2, Takashi Kojima3, Takeru Funakoshi4,
Eiichi Sato5, Hiroyoshi Nishikawa6,7, Jun Nakajima8, Yasuyuki Seto9,
Kazuhiro Kakimi10, Shinsuke Iida11, Yuichiro Doki1, Mikio Oka12,
Ryuzo Ueda13 and Hisashi Wada14
Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Suita, Japan2
Department of Respiratory Medicine, Kawasaki Medical School, Kurashiki, Japan3
Department of Gastrointestinal Oncology, National Cancer Center Hospital East, Kashiwa, Japan
4
Department of Dermatology, Keio University School of Medicine, Tokyo, Japan
5
Department of Pathology, Institute of Medical Science (Medical Research Center), Tokyo Medical University,
Tokyo, Japan
6
Division of Cancer Immunology, Research Institute/Exploratory Oncology Research & Clinical Trial Center(EPOC), National Cancer Center, Tokyo/Kashiwa, Japan
7
Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Japan
8
Department of Thoracic Surgery, University of Tokyo Graduate School of Medicine, Tokyo, Japan
9
Department of Gastrointestinal Surgery, University of Tokyo Graduate School of Medicine, Tokyo, Japan10
Department of Immunotherapeutics, The University of Tokyo Hospital, Tokyo, Japan
11
Department of Hematology and Oncology, Nagoya City University Graduate School of Medical Sciences,
Nagoya, Japan
12
Department of Immuno-Oncology, Kawasaki Medical School, Kurashiki, Japan
13
Department of Tumor Immunology, Aichi Medical University School of Medicine, Nagakute, Japan
14
Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine,
Suita, Japan
1

ABSTRACT
Tregs infiltrate tumors and inhibit antitumor immunity. KW-0761 (Mogamulizumab) is a humanizedanti-CCR4 monoclonal antibody that could eliminate activated Tregs with high immunosuppressive activity
that express CCR4. In this phase Ib trial, KW-0761 was used as a cancer immunotherapeutic reagent todeplete Tregs in patients with advanced or recurrent solid CCR4-negative tumors. Thirty-nine patients withsolid cancer were treated with KW-0761 at a dose of 0.1 or 1.0 mg/kg. The safety, clinical responses,
and effects of Treg depletion were analyzed. Any grade and grade 3–4 treatment-related adverse events(AEs) were observed in 36 (92%) and 14 (36%) out of 39 patients, respectively. All treatment-related AEs
were manageable. One and 5 patients achieved a partial response and stable disease, respectively, during
treatment and were long survivors. The efficient depletion of Treg in peripheral blood was confirmed inboth cohorts. Therefore, the administration of KW-0761 was safe, resulting in the depletion of Tregs inperipheral blood and potential immune responses in patients with solid cancer. The combined use of KW-

Received: July 2, 2021; accepted: July 19, 2021
Corresponding Author: Ryuzo Ueda, MD, PhD
Department of Tumor Immunology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata,Nagakute 480-1195, Japan
Tel/Fax: +81-561-63-1514, E-mail: uedaryu@aichi-med-u.ac.jp
Hisashi Wada, MD, PhD
Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine,
2-2 Yamada-oka, Suita 565-0871, Japan
Tel/Fax: +81-6-6210-8413, E-mail: hwada@gesurg.med.osaka-u.ac.jp
827

828
Takuro Saito et al0761 to deplete Tregs and other immunotherapies is a promising approach to augment immune responses.
Keywords: solid cancer patients, mogamulizumab, regulatory T cells, clinical trial, CCR4
Abbreviations:Tregs: regulatory T cells
AEs: adverse events
TME: tumor microenvironment
mAbs: antibodies
PBMCs: blood mononuclear cells
CTLA-4: cytotoxic T-lymphocyte-associated protein 4
PD-1: programmed cell death protein 1
IDO: indoleamine 2,3-dioxygenase
VEGF: vascular endothelial growth factor
VEGFR: vascular endothelial growth factor receptor
ICOS: inducible T cell co-stimulator
GITR: glucocorticoid-induced TNFR-related protein
PI3K: phosphoinositide 3-kinase
TCR: T cell receptor
This is an Open Access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
License. To view the details of this license, please visit (http://creativecommons.org/licenses/by-nc-nd/4.0/).

INTRODUCTION
Immunotherapy, primarily with immune checkpoint inhibitors (ICIs) such as anti-cytotoxic
T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1)
monoclonal antibodies (mAbs), has markedly improved the survival rate of patients with cancer,
even in the advanced stage1,2,3; however, the efficacy of ICIs is unsatisfactory in the majority ofpatients and, thus, other therapies, including immunotherapy, are urgently needed. In the escapephase of the concept of cancer immunoediting, tumor cells have developed strategies to establish
an immunosuppressive state within the tumor microenvironment (TME) by producing immunosuppressive cytokines, such as vascular endothelial growth factor (VEGF), transforming growth
factor-β (TGF-β), and indoleamine 2,3-dioxygenase (IDO), and recruiting regulatory immune cells,
including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs).4 Therefore,
the manipulation of immunosuppressive cells or TME is an anticancer therapeutic strategy.
Tregs constitute 5–10% of CD4+ T cells in the periphery and play an important role inmaintaining immune tolerance.4,5 Tregs inhibit the development of antitumor immunity, thereby
hindering the immune surveillance of cancer and preventing effective antitumor immune responses
in tumor-bearing hosts. The presence of a large number of Tregs and a low ratio of CD8 + T cells
to Tregs in the TME correlate with a poor prognosis in various cancer types.6,7 Tregs promotetumor progression by suppressing antitumor immunity,8,9 manipulating Tregs, or targeting theimmunosuppressive factors produced by these cells, and, thus, have potential as an anticancer
treatment strategy. Despite promising pre-clinical studies, Treg cell-targeted therapy has not yet
been successfully applied to clinical settings.
KW-0761 (mogamulizumab) is a humanized anti-CCR4 immunoglobulin G1 (IgG1) mAb withenhanced antibody-dependent cellular cytotoxicity.10,11 It was developed as an orphan drug for
adult T-cell leukemia-lymphoma (ATLL), which expresses CCR4 on cell surfaces, and has been
approved for T-cell lymphomas, including ATLL and other peripheral T-cell lymphomas, by the
Pharmaceuticals and Medical Devices Agency, Food and Drug Administration, and European
Medicines Agency.12,13,14 Evidence is emerging to show that KW-0761 suppresses the function of

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Immunotherapy by CCR4 Treg depletion

effector Tregs (eTregs), which is a subpopulation of Tregs with high expression levels of FoxP3
and CCR4, and exhibits strong immunosuppressive activity against tumors, leading to robust
CD8+ T-cell proliferation in ATLL.15,16 Therefore, we conducted a clinical trial to examine thetreatment effects of KW-0761 on solid cancers based on a novel concept, namely, Treg depletion.
In a previous phase Ia clinical trial, KW-0761 was administered to 7 patients with lung cancerand 3 with esophageal cancer with a dose escalation design of 0.1, 0.5, and 1.0 mg/kg.17 All
doses were confirmed to be safe and well tolerated. Four out of the 10 patients achieved stabledisease (SD) during the treatment and were long survivors. The monitoring of FoxP3+ Tregs inperipheral blood mononuclear cells (PBMCs) during the treatment indicated efficient depletion
even at the lowest dose of 0.1 mg/kg.
To investigate the safety and efficacy of eTreg depletion and clinical responses in patients withsolid cancer in more detail, we conducted a phase Ib clinical trial on KW-0761 in 39 patientswith CCR4-negative cancers.

PATIENTS AND METHODSPatients
Eligible patients had CCR4-negative treatment-refractory or advanced cancer with target
lesions. Archival or newly obtained tumor samples from patients were screened for the expression of CCR4 by immunohistochemistry (IHC) as previously described.18 CCR4 expression was
confirmed by the review committee with a central evaluation. Inclusion criteria were describedin the phase Ia study.17
Study Design
This study was designed as a multi-institutional, open-label, two-arm, investigator-initiatedphase Ib clinical trial on KW-0761. The investigational drug KW-0761was provided by Kyowa
Hakko Kirin. The study was registered with ClinicalTrials.gov as NCT01929486 and was approved
by local Institutional Review Boards. All participating patients provided written informed consent
before enrolment, in accordance with the Declaration of Helsinki. The primary objectives were tocharacterize the safety and efficacy of eTreg depletion in PBMCs by KW-0761 for patients withadvanced or recurrent solid cancer. The secondary objectives were to assess clinical responses,
including the overall response rate, progression-free survival (PFS), and overall survival (OS), andselect the recommended dose for a phase II trial. Twenty and nineteen patients were randomly
enrolled into cohorts with doses of 0.1 and 1.0 mg/kg, respectively, and received 8 intravenous
infusions of KW-0761 weekly followed by monthly infusions until disease progression. These
two doses of KW-0761 were established in the phase Ia study as the maximum-tolerated andminimal doses, respectively. Oral antihistamines and acetaminophen were administered prior toeach KW-0761 treatment, and hydrocortisone was simultaneously infused intravenously with thefirst KW-0761 treatment to prevent infusion reactions.
Toxicity Evaluation
Toxicity was evaluated according to the National Cancer Institute Common Terminology
Criteria for Adverse Events version 4.0. Patients were assessed for toxicity weekly during thefirst 8 treatments and every 4 weeks thereafter until 24 weeks after the last treatment. The
independent data monitoring committee evaluated safety data for each dose level.

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Takuro Saito et al

Clinical Response Evaluation
Responses were evaluated 12 weeks after the first KW-0761 treatment or at the study discontinuation based on computed tomography (CT) scans according to RECIST (ver. 1.1),19 and/or immune-related (ir) RECIST. OS was defined as the duration from the first day of KW-0761
treatment until the day of death from any cause. PFS was defined as the duration from thefirst day of KW-0761 treatment until either the day of progressive disease (PD) detection ordeath from any cause. Tumor responses, OS, and PFS were confirmed by a central evaluationfor each patient.
Evaluation of eTreg Depletion on PBMCs
Blood samples were obtained at baseline, 4 and 8 weeks after the first KW-0761 treatment,
and every 4 weeks during the continuous treatment until study-off. PBMCs were isolated fromheparinized blood by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare,
Fairfield, CT). Cells were stored in liquid N2 until used. Treg depletion was evaluated by flow
cytometry. After thawing, PBMCs were incubated with mAbs at 4°C for 20 minutes. Cells
were stained with anti-CD4-PerCP (clone SK3; BD Biosciences, San Jose, CA), anti-CD25-APC(clone 2A3; BD Biosciences), and anti-CD45RA-FITC (clone ALB11; Beckman Coulter, Brea,
CA) mAbs. The intracellular staining of FOXP3 was performed with anti-FoxP3-PE (clone
PCH101; eBioscience) mAb and a FoxP3/Transcription Factor Staining Buffer Set (eBioscience,
San Diego, CA) according to the manufacturer’s instructions. After the incubation, cells were
washed and analyzed by FACSCalibur (BD Biosciences). CD45RA+ FoxP3 lo resting/naïve Tregs,
CD45RA– FoxP3 hi activated/effector Tregs (eTregs), and CD45RA– FoxP3 lo, non-Tregs wereanalyzed as previously described.15

RESULTS
Patient Characteristics
In this phase Ib study, 39 patients with advanced CCR4-negative solid cancer were randomly
assigned to receive a treatment with KW-0761 at 0.1 mg/kg (n=20) or 1.0 mg/kg (n=19) between
October 2013 and April 2016 (Table 1). The median age of patients was 65 years. The cohort
included 11 patients with esophageal cancer, 9 with lung cancer, 6 with malignant melanoma,
5 with gastric cancer, 5 with ovarian cancer, and 3 with mesothelioma with 2 to 23 infusionsof KW-0761. Eleven patients dropped out before the completion of the first cycle with 8 infusions of KW-0761 due to the withdrawal of consent in 1 patient, disease progression in 4, anda decision by the principal investigators in 6. The median number of infusions was 8 (2–14)
in the 0.1 mg/kg cohort and 8 (2–23) in the 1.0 mg/kg cohort. The median follow-up was 94(21–498) days in the 0.1 mg/kg cohort and 99 (25–511) in the 1.0 mg/kg cohort. Seven (35%)
patients in the 0.1 mg/kg cohort and 7 (36%) in the 1.0 mg/kg cohort died, and the cause of
all deaths was disease progression.

B-24

B-26

B-28

B-32

B-33

B-35

B-37

B-39

0.1

0.1

0.1

0.1

0.1

0.1

0.1

B-23

0.1

0.1

B-21

B-22

0.1

0.1

B-19

0.1

B-11

0.1

B-12

B-10

0.1

B-15

B-07

0.1

0.1

B-05

0.1

0.1

B-01

B-03

0.1

0.1

ID

Dose
(mg/kg)

59

65

47

63

65

71

85

70

61

53

67

76

57

66

70

64

68

67

70

66

Age

F

M

F

F

F

M

M

M

M

F

M

M

M

M

M

F

F

M

F

M

Sex

Esophageal cancer (SCC)

Esophageal cancer (SCC)

Esophageal cancer (Other)

Ovarian cancer

NSCLC

Mesothelioma

Gastric cancer

Gastric cancer

NSCLC

Ovarian cancer

Melanoma

NSCLC

NSCLC

Melanoma

Esophageal cancer (Other)

Esophageal cancer (SCC)

Melanoma

Melanoma

NSCLC

Esophageal cancer (SCC)

Tumor Type

14

9

4

8

8

4

2

8

8

8

8

8

8

8

4

8

8

5

8

6

No. ofInfusions

PD

SD

PD

PD

PD

SD

PD

PD

PD

PD

PD

SD

PD

PD

PD

PD

PD

PD

PD

PD

Best
overall
response

70

96

29

69

63

40

21

70

63

67

77

63

67

76

25

63

77

28

63

42

Time toprogression(days)

Table 1 Patient characteristics

251

284

31

77

395

40

21

271

498

224

91

92

77

150

391

249

77

73

422

96

OS
(days)

+

+

+

+

+

–

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

–

–

–

–

–

–

–

+

+

+

+

–

–

+

+

–

–

–

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

NA

+

+

+

+

+

Treatment- TreatmentTreg
related AEs related AEs
depletion(Grade 3–4)
(All)

831

Immunotherapy by CCR4 Treg depletion

B-14

B-16

1.0

1.0

B-34

B-36

B-38

1.0

1.0

1.0

73

80

65

68

77

45

64

61

54

68

67

57

68

54

64

51

49

68

65

F

M

M

M

M

M

M

M

M

M

M

M

F

F

M

F

M

F

F

NSCLC: non-small cell lung cancer
SCLC: small cell lung cancer
SCC: squamous cell carcinoma
PD: progressive disease
SD: stable disease
PR: partial response
AEs: adverse events
NA: not assessed

B-30

B-31

1.0

B-29

1.0

1.0

B-25

B-27

1.0

1.0

B-20

B-13

1.0

1.0

B-09

1.0

B-17

B-08

1.0

B-18

B-06

1.0

1.0

B-04

1.0

1.0

B-02

1.0

(Continued)

Ovarian cancer

Mesothelioma

Esophageal cancer (Other)

Mesothelioma

Esophageal cancer (SCC)

Gastric cancer

Esophageal cancer (SCC)

NSCLC

SCLC

NSCLC

Gastric cancer

NSCLC

Gastric cancer

Ovarian cancer

Esophageal cancer (SCC)

Esophageal cancer (SCC)

Melanoma

Melanoma

Ovarian cancer

8

10

8

10

8

8

5

8

8

8

5

3

4

8

23

2

8

8

8

PD

SD

PD

SD

PD

PD

PD

PD

PD

PD

PD

PD

PD

PD

PR

PD

PD

PD

PD

76

160

81

124

63

70

36

63

56

62

47

25

35

65

491

27

56

79

66

84

168

88

124

68

272

99

497

58

65

47

25

35

318

511

93

233

142

413

+

+

+

+

+

+

+

+

+

+

+

+

–

+

+

+

+

–

+

–

–

+

+

–

–

+

–

–

+

–

+

–

–

+

–

–

–

–

+

+

+

+

+

+

+

+

+

+

+

NA

+

+

+

+

+

+

+

832
Takuro Saito et al833
Immunotherapy by CCR4 Treg depletion

Adverse events (AEs)
Any grade treatment-related AEs occurred in 19 (95%) out of 20 patients in the 0.1 mg/kg
cohort and in 17 (89%) out of 19 in the 1.0 mg/kg cohort; grade 3–4 treatment-related AEs
occurred in 7 (35%) patients in the 0.1 mg/kg cohort and in 6 (32%) in the 1.0 mg/kg cohort(Table 1). Table 2 lists all treatment-related AEs, with 65 and 49 AEs being observed in the 0.1
and 1.0 mg/kg cohorts, respectively. The most frequently observed categories of treatment-related
AEs were skin disorders and lymphopenia. Grade 3–4 treatment-related AEs were lymphopenia(4 [20%] in the 0.1 mg/kg cohort and 4 [21%] in the 1.0 mg/kg cohort), rash, increased alanine
aminotransferase, increased aspartate aminotransferase, hypophosphatemia, increased gammaglutamyltransferase, and appetite loss. All treatment-related AEs were manageable or recovered
without any treatment, and no drug-related deaths were observed.

Table 2 Treatment-related adverse eventsGrade

0.1 mg/kg (N=20)

1.0 mg/kg (N=19)

1

2

3

4

1

2

3

4

Cases

16

12

6

1

10

14

4

2

Total events

34

23

7

1

23

20

4

2

2

Non-hematological
General
Fever

1

1

Fatigue

1

2

Appetite loss

1

Edema

1

Weight loss

1
1

Skin and subcutaneous tissue
Rash

8

Pruritus

1

5

1

5

Drug eruption

5
1

Gastrointestinal
Diarrhea

1

Nausea1

Vomiting

1

1

Ear and labyrinth
Vertigo positional1

Ear discomfort

1

Cardiac disorders
Arrhythmia
Electrocardiogram T wave inversion
Hypertension

1

2
1
1

834
Takuro Saito et al(Continued)
Thoracic disorders
Pleural effusion

1

Nervous system disorders
Dizziness

1

Dysgeusia

1

Headache3

Neuropathy peripheral

1

Infections
Oral candidiasis

1

Upper respiratory tract Inflammation

1

Cheilitis

1

Endocrine
Hypothyroidism

1

Gynecomastia

11

Hematological
Leukopenia

2

Lymphopenia

1

1
10

3

Thrombocytopenia
1

Hypokalemia

1

Hyponatremia

1

Hypophosphatemia

1

Hypoalbuminemia

1

2

2

1

GGT increased1

ALT increased

3

1

AST increased

21Amylase increased

1

LDH increased1

ALP increased1

TSH increased

ALT: alanine aminotransferase
AST: aspartate aminotransferase
LDH: lactate dehydrogenase
TSH: thyroid-stimulating hormone
GGT: gamma-glutamyltransferase
ALP: alkaline phosphatase

71

Eosinophilia

Glucose urine present

1

1
1
1

1

2

835
Immunotherapy by CCR4 Treg depletion

Clinical Responses
Confirmed objective responses with RECIST were SD in 3 patients with NSCLC, esophageal
cancer, or mesothelioma in the 0.1 mg/kg cohort, and a partial response in 1 patient withesophageal cancer and SD in 2 patients with mesothelioma in the 1.0 mg/kg cohort (Table 1,
Fig. 1). Median PFS was 67 and 65 days and OS was 271 and 272 days in the 0.1 and 1.0
mg/dl cohorts, respectively (Table 1). A durable response was observed in 2 patients (B-09,
B-39) (Fig. 2).

Fig. 1Best percentage change in the target lesion tumor burden from baseline in patients witha CT assessment
Waterfall plot for the maximum percentage reduction in the target lesion tumor burden until disease progression(study-off) according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 progression. A positive change in the tumor burden indicates tumor growth; a negative change in the tumor burden indicates a tumor
reduction. The horizontal dotted lines denote a 30% decrease and 20% increase, indicating an objective response
and progressive disease, respectively, as per RECIST version 1.1. The tumor type and presence or absence ofadverse events (AEs) are annotated for each patient. 0.1 mg/ml (n=18), blue bar; 1.0 mg/ml (n=17), orange bar.

836
Takuro Saito et al

Fig. 2 Percentage change in the target lesion tumor burden from baseline over time
A spaghetti plot for changes from the baseline in the tumor burden, measured as the sum of the longest diametersof the target lesions by patients over time. The black triangle indicates the presence of a new lesion. Horizontal
dotted lines denote a 30% decrease and 20% increase. 0.1 mg/ml (n=20), blue line; 1.0 mg/ml (n=19), orange line.

Efficacy of eTreg Depletion on PBMCs
eTreg depletion by KW-0761 was examined with PBMCs at baseline and during the KW-0761
treatment using flow cytometry. The percentage of eTregs in CD4+ T cells markedly decreased
in all patients, except for 2 for whom blood samples were not available after the start of theKW-0761 treatment (Table 1). The median percentages of eTregs in CD4+ T cells in the 0.1
and 1.0 mg/kg cohorts were 2.1 and 2.1% at baseline and 0.22 and 0.21% after 4 infusions,
respectively. The percentage of eTregs in blood remained low during the treatment with KW-0761.

DISCUSSION
The results of this phase Ib study confirmed the safety of KW-0761. Some treatment-related
AEs were observed, including lymphopenia, which was the most frequent grade 3–4 AE; however,these AEs were all manageable and no drug-related deaths occurred. All AEs related to skinreactions in the present study were grades 1–2, except for one patient with a rash, which wereless severe than those previously reported in ATLL patients.11,13 No significant difference wasnoted in the incidence of AEs between the 0.1 and 1.0 mg/kg cohorts.
The manipulation of Tregs has attracted increasing attention as a novel therapeutic strategy forcancer. In this phase Ib study, eTregs were efficiently depleted on PBMCs after the KW-0761
treatment. Several potential strategies to target Tregs are currently being clinically and preclinically investigated alone or in combination, mostly with ICIs. One approach is the depletion ofTregs by targeting molecules specifically expressed on Tregs, such as CD25, CTLA-4, CCR4,
OX40 (CD134), inducible T cell co-stimulator (ICOS), and glucocorticoid-induced TNFR-related

837
Immunotherapy by CCR4 Treg depletion

protein (GITR), or signals that are crucial for Treg cell survival and function, including T cell
receptor (TCR) and IL-2 receptor signaling. Another approach for targeting Tregs is to control
or modulate Treg cell function and infiltration, such as TGF-β, tyrosine kinase, phosphoinositide
3-kinase (PI3K), IDO, the CD39-CD73 axis, and vascular endothelial growth factor receptor(VEGFR) signaling. In these strategies, anti-CTLA-4 mAbs has attracted special attention
because it yielded durable responses in a subset of cancer patients.3,20 Recent findings fromseveral preclinical studies indicated that Treg depletion is one of the mechanisms underlying theantitumor effects of anti-CTLA-4 mAbs used as a checkpoint inhibitor.21,22 Anti-CTLA-4 mAbreportedly deplete Tregs from the TME, which may contribute to the clinical benefits of thisagent.20 However, direct Treg cell-targeted therapy has not yet been applied to clinical settings.
Treg recruitment at local sites is driven by combinations of chemokines and their receptors, such
as CCL22 to CCR4 and CCL1 to CCR8.23,24 CCR4 is differentially expressed on the surface ofvarious types of lymphocyte subpopulations. In a previous phase Ia study, we confirmed the highexpression of CCR4 on eTregs and CCR4+ eTreg enrichment in tumor specimens.17,25 Moreover,an in vitro experiment on an anti-CCR4 mAb treatment with PBMCs showed the efficientinduction of NY-ESO-specific CD4+ and CD8+ T cells due to the depletion of CCR4+ eTregs.
ATLL patients treated with KW-0761 showed the induction of NY-ESO-1-specific CD8+ T cells,
potentially contributing to the prolongation of survival. These findings suggest the contributionof KW-0761 to antitumor immune enhancements through Treg depletion.
In this clinical trial, while KW-0761 effectively depleted eTregs in PBMCs, most of the treatedpatients did not exhibit tumor regression, which is consistent with the findings of our previous
phase Ia study; however, a durable clinical response was observed in 2 patients with esophageal
cancer. A possible explanation for the low clinical efficacy of KW-0761 is that KW-0761 may
deplete another type of CCR4+ T-cell subset. The comprehensive assessment conducted in theprevious phase Ia study showed the high expression of CCR4 on eTregs, Th2 CD4+ T cells, andTh17 CD4+ T cells and the low expression of CCR4 on Th1 CD4+ T cells and CD8+ T cells.17
The efficacy of KW-0761 depends on the balance between the depletion of eTregs, which may
improve antitumor immunity, and the depletion of other types of cells, including CD8+ T cells
or Th1 CD4+ T cells, which may attenuate antitumor immunity. Another plausible explanation
for impaired clinical responses by KW-0761 is that eTregs were not sufficiently depleted in the
TME. It currently remains unclear whether these drugs selectively deplete Tregs in the TME. The
density of Tregs in the TME is not always reflected in peripheral blood. In this clinical trial, weobserved Treg reductions in the tumor of a patient with biopsy specimens that were collected atbaseline and post-treatment (data not shown). Therefore, further basic and translational research iswarranted to obtain a more detailed understanding of Treg cell functions, particularly in the TME.
In summary, KW-0761 was safely administered and efficiently depleted eTreg in PBMCs,
with potential immune responses in solid CCR4-negative cancers. We are currently conducting
a phase I clinical trial on the preoperative administration of KW-0761 combined with anti-PD-1
mAb to patients with advanced or recurrent solid cancer, with the expectation of the synergistic
effects of these immunologically different functional treatments. Although further refinement of theregimen is required, the combined use of KW-0761 to deplete Tregs and other immunotherapies
is a promising strategy to augment immune responses.

IN MEMORIAM
This study is dedicated to the memory of the late Dr. E. Nakayama.

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Takuro Saito et al

ACKNOWLEDGMENTS
We thank all the patients and their families who participated in this clinical trial. We also
thank all the nurses, clinical research coordinators in Kawasaki Medical School Hospital, Nagoya
City University Hospital, Osaka University Hospital, and National Cancer Center Hospital East,
Tokyo University Hospital, Keio University Hospital, review committees, and medical experts who
were involved in this study. We are grateful to Drs. Y. Ueda, H. Nagase, Y. Ohue, T. Oguri, A.
Arakawa, M. Nakamura, Y. Mori, T. Ishida, H. Matsushita, M. Anraku, and M. Sugaya for theirclinical support, Dr. H. Ito for statistical support, and Drs. K. Iwata, S. Kihara, and T. Hida fordata and safety monitoring support. We are also grateful to Kyowa Hakko Kirin for providing
us with the investigational drug KW-0761.

FUNDING/SUPPORTThis study was supported by Grants-in-Aid for Scientific Research (S) grant no. 17H06162(H.N.), Challenging Exploratory Research grant no. 16K15551 (H.N.), Research Activity Start-up
grant no. 15H06878 (Y.M.), Young Scientists (B) grant no. 17K15738 (Y.M.), and Scientific
Research (B) grant no. 19H03729 (H.W.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by the Projects for Cancer Research by Therapeutic Evolution[P-CREATE, no. 16cm0106301h0001 (H.N.) and no. 17cm0106322h0002 (Y.M.)], by the Development of Technology for Patient Stratification Biomarker Discovery grant [no.19ae0101074s0401(R.U. and H.N.)] from the Japan Agency for Medical Research and Development (AMED), andby the National Cancer Center Research and Development Fund [no. 28-A-7 and 31-A-7 (H.N.)].

CONFLICTS OF INTEREST
T.K. received honoraria and research funding from Ono Pharmaceutical, MSD, Shionogi,
Bristol-Myers Squibb, Chugai Pharmaceutical, Amgen, Astellas Pharmaceutical, Oncolys BioPharma, Parexel, and Merck Serono outside of this study. T.F. received research funding fromOno Pharmaceutical outside of this study. H.N. received honoraria and research funding from Ono
Pharmaceutical, Chugai Pharmaceutical, MSD, and Bristol-Myers Squibb, and research funding
from Taiho Pharmaceutical, Daiichi-Sankyo, Kyowa Kirin, Zenyaku Kogyo, Oncolys BioPharma,
Debiopharma, Asahi-Kasei, Sysmex, Fujifilm, SRL, Astellas Pharmaceutical, Sumitomo Dainippon
Pharma, and BD Japan outside of this study. K.K. received research funding from TAKARA BIO
and MSD outside of this study. The Department of Immunotherapeutics, The University of Tokyo
Hospital is endowed by TAKARA BIO. S.I. received honoraria and research funding from Ono
Pharmaceutical, Takeda, Sanofi, Bristol-Myers Squibb, Janssen, Celgene and Daichi-Sankyo, andresearch funding from Kyowa Kirin, Abbvie, Chugai Pharmaceutical, MSD, and Gilead outsideof this study. Y.D. received honoraria and research funding from Ono Pharmaceutical, Taiho
Pharmaceutical, and research funding from Chugai Pharmaceutical, Covidien Japan, Johnson &Johnson, and honoraria from Otsuka Pharmaceutical outside of this study. M.O. received researchfunding from Thyas, Sysmex, and Pole Star outside of this study. R.U. received research funding
from Ono Pharmaceutical, Chugai Pharmaceutical, and Kyowa Kirin outside of this study. H.W.
received research funding from Ono Pharmaceutical and Kyowa Kirin, and honoraria from Ono
Pharmaceutical, Chugai Pharmaceutical, MSD, and Bristol-Myers Squibb outside of this study.
The Department of Clinical Research in Tumor Immunology, Osaka University Graduate Schoolof Medicine is a joint research laboratory with Shionogi & Co., Ltd.

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Immunotherapy by CCR4 Treg depletion

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