Cancer Therapy: Preclinical

Antitumor Effect of Programmed Death-1 (PD-1)Blockade in Humanized the NOG-MHC DoubleKnockout MouseTadashi Ashizawa1, Akira Iizuka1, Chizu Nonomura1, Ryota Kondou1, Chie Maeda1,Haruo Miyata1, Takashi Sugino2, Koichi Mitsuya3, Nakamasa Hayashi3,Yoko Nakasu3, Kouji Maruyama4, Ken Yamaguchi5, Ikumi Katano6,Mamoru Ito6, and Yasuto Akiyama1,3

Abstract

Purpose: Humanized mouse models using NOD/Shi-scid-IL2rgnull (NOG) and NOD/LtSz-scid IL2rgnull (NSG) mouse areassociated with several limitations, such as long incubation timefor stemcell engraftment and the development of xenograft versushost disease in mice injected with peripheral bloodmononuclearcells (PBMCs). To solve problems, we used humanized majorhistocompatibility class I- and class II-deficient NOG mice(referred to as NOG-dKO) to evaluate the antitumor effect ofanti-programmed death-1 (PD-1) antibody.

Experimental Design:Humanized NOG-dKO mice, in whichhuman PBMCs and human lymphoma cell line SCC-3, orglioblastoma cell line U87 were transplanted, were used as animmunotherapy model to investigate the effect of anti-PD-1antibody. A biosimilar anti-PD-1 mAb generated in our labo-ratory was administered to humanized NOG-dKO mice trans-planted with tumors.

Results: Within 4 weeks after transplantation, humanCD45þ cells in antibody-treated mice constituted approxi-mately 70% of spleen cells. The injection of anti-PD-1 anti-body reduced by more 50% the size of SCC-3 and U87 tumors.In addition, induction of CTLs against SCC-3 cells and upre-gulation of natural killer cell activity was observed in theantibody-treated group. Tumor-infiltrating lymphocyte profil-ing showed that more exhausted marker (PD1þTIM3þLAG3þ)positive T cells maintained in anti-PD-1 antibody–treatedtumor. A greater number of CD8þ and granzyme-producingT cells infiltrated the tumor in mice treated with the anti-PD-1antibody.

Conclusions: These results suggest that NOG-dKO micemight serve as a good humanized immunotherapy model toevaluate the efficacy of anti-PD-1 antibody prior to the clinicaltreatment. Clin Cancer Res; 1–10. �2016 AACR.

IntroductionWith the recent success of immune checkpoint antibodies,

such as ipilimumab and nivolumab, reported in patients withmetastatic melanoma, many ongoing clinical trials are under-way to evaluate their efficacy in various solid cancers otherthan melanomas (1–5). Specifically, a promising combinationtherapy of ipilimumab and nivolumab has demonstrated avery high response rate and long-term survival benefit in

patients with advanced cancers, including non–small cell lungcancer (6–8).

Despite these promising results, the response rate associat-ed with single-antibody treatment is approximately 20% to40%. Furthermore, it is difficult to accurately predict theresponders to antibody therapy based on the results of pre-clinical studies (9, 10).

Multiple types of humanized mice have been developed andused as a therapeutic model in preclinical studies of new cancertreatments. The severely immunodeficient mouse strains, such asNOD/Shi-scid-IL2rgnull (NOG; refs. 11, 12), NOD/LtSz-scidIL2rgnull (NSG; ref. 13), and BALB/c Rag2null IL2rgnull (14) areideal in vivo platforms for reconstituting the human hemato-lymphoid system due to their lack of an endogenous mouseimmune system. Several researchers demonstrated these human-ized mouse models are capable of temporary antigen-specificimmune responses, such as CTL activation (15) and humanantibody production (16, 17).

In addition, humanized mouse models transplanted with bothhuman peripheral blood mononuclear cells (PBMCs) and cancercells have been used in some preclinical studies to evaluateantibody-based therapies (18–20). These mouse models are ame-nable to hematopoietic stem cell transplantation; however, theycannot undergo human PBMC transplantation due to a severexenograft versus host disease (xeno-GVHD) response (21, 22).

1Immunotherapy Division, Shizuoka Cancer Center Research Institute, Nagai-zumi-cho, Sunto-gun, Shizuoka, Japan. 2Division of Pathology, Shizuoka CancerCenter Hospital, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan. 3Division of Neu-rosurgery, Shizuoka Cancer Center Hospital, Nagaizumi-cho, Sunto-gun, Shi-zuoka, Japan. 4Experimental Animal Facility, Shizuoka Cancer Center ResearchInstitute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan. 5Shizuoka Cancer CenterHospital, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan. 6Central Institute forExperimental Animals, Kawasaki-ku, Kawasaki, Kanagawa, Japan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Yasuto Akiyama, Shizuoka Cancer Center ResearchInstitute, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777,Japan. Phone: 815-5989-5222; Fax: 815-5989-6085; E-mail: y.akiyama@scchr.jp

doi: 10.1158/1078-0432.CCR-16-0122

�2016 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org OF1

In this study, we used MHC gene-double knockout (dKO)NOG mice, deficient in both the murine MHC class I and classII genes. MHC dKO NOGmice were generated by KO of the geneencoding b2-microglobulin, a component of the MHC class Imolecule, and the gene encoding IAb, the light chain of the MHCclass IImolecule (referred to asNOG-dKOorNOG-b2m, IAbdKOmice). Yaguchi and colleagues reported for the first time thatNOG-dKOmice that had undergone transplantationwith humanPBMCs exhibited a much milder xeno-GVHD response, with lessweight loss and a longer survival period, compared with controlNOG mice (23).

Consistent with these results, we were able to successfullytransplant PBMCs into NOG-dKO mice, whereas transplantedPBMCs were rejected by regular NOGmice. On the basis of theseobservations, we established humanized NOG-dKO mice trans-planted with both PBMCs and human cancer cells as a model toevaluate cancer immunotherapy. We used this therapeutic in vivomodel to evaluate antitumor activity of anti-PD-1 antibody withrespect to the immunologic response of human origin.

Materials and MethodsAntibodies and flow cytometry

The following antibodies were used for a flow cytometricanalysis. The anti-mouse CD45 antibody used to label mousecells was purchased from BD Pharmingen. The anti-CD3-biotin(HIT3a), anti-CD4-PE (RPA-T4), anti-CD8-FITC (HIT8a), anti-CD11b-PE-Cy7 (ICRF44), anti-CD14-PerCP (MP9), anti-CD19-FITC (HIB19), anti-CD25-FITC (M-A251), anti-CD33-PE(WM53), anti-CD45-FITC (2D1), anti-CD45RA-FITC (HI100),anti-CD45RO-APC (UCHL1), anti-CD56-PE (B159), anti-CD127-PE-Cy7 (A019D5), anti-CD138-APC (MI15), anti-humanIgD-PE (IA6-2), anti-human IgM-PE-Cy5 (G20-127), and anti-human IgG-PE (G18-145) used to label human cells were alsopurchased from BD Pharmingen. Anti-FoxP3-PE (hFOXY) anti-body was purchased from eBioscience, Inc. Anti-TIM3-PE (F38-2E2) and anti-LAG3-FITC (17B4) antibodies were purchased fromMiltenyi Biotec and Adipogen. Propidium iodide (PI) was pur-

chased from Sigma-Aldrich Co. and used to distinguish living cellsfrom those that had undergone cell death. The anti-PD-1-APC(EH12.2H7), anti-PD-L1-APC (29E.2A3), and anti-Ki67-PE-Cy7(Ki-67) antibodies were purchased from BioLegend Inc.

Single-cell suspensions were obtained frommouse spleens andperipheral blood using ACK lysing buffer (Thermo Fisher Scien-tific). Tumor-infiltrating lymphocytes (TILs) were also separatedfrom the control or anti-PD-1 antibody–treated tumors by anti-human CD45-microbeads (Miltenyi Biotec) using autoMACSsystem (Miltenyi Biotec). Cells were stained with primary anti-bodies for 15minutes at 4�Candwashedwith cold PBSþ2%FBS.If applicable, cells were subsequently stained with the secondaryantibodies for 15 minutes at 4�C. Cells were then washed, fixedwith 0.5% paraform aldehyde–containing PBS (�), and analyzedon a FACSCanto II flow cytometer (BD Biosciences). Human cellswere identified by gating the mouse CD45�PI� and humanCD45þ fractions.

Production of the full-length anti-PD-1 mAbWe generated biosimilar of the anti-PD-1 mAb nivolumab

(manuscript in submission). Briefly, the amino acid sequence ofnivolumabwas downloaded from the J-PlatPat data base from theNational Center for Industrial Property Information and Training(INPIT; https://www.j-platpat.inpit.go.jp/web/tokujitsu/tkbs/TKBS_GM401_ToPDF.action). The anti-PD-1mAbwas producedusing the expi293 expression system, purified with a protein Acolumn, and used for the in vivo experiments.

Development of humanized NOG-dKO miceSix-week-old NOG-dKO mice were kindly supplied from Dr.

Mamoru Ito (The Central Institute for Experimental Animals,Kawasaki, Japan). All animals were cared for and treated humane-ly according to theGuidelines for thewelfare anduse of animals incancer research, and the experimental procedures approved by theAnimal Care and Use Committee of Shizuoka Cancer CenterResearch Institute.

The clinical experiments using PBMCs derived from patientswith glioma were approved by the Institutional Review Board ofShizuoka Cancer Center, Shizuoka, Japan. All patients providedwritten informed consent.

Eight-week-old NOG-dKO mice were irradiated with X-rays(2.5 Gy; HW-150; HITEX) on day 0, and 1 � 107 human PBMCscells with the HLA-A�0201 genotype were intravenously admin-istered to eachmouse via the tail vein. For human cellmonitoring,NOG-dKO mouse spleens and peripheral blood were obtainedfrom sacrificed mice at 4, 6, and 8 weeks after transplantation.Engraftment of human immune cells was investigated using FACSanalysis.

In the survival experiment, overall survival was compared inhumanized NOG-dKO mice and control NOG mice that hadundergone transplantation with human PBMCs. We set the nat-ural death as an endpoint in this study.

The studydesignof the experiment evaluatingmice treatedwiththe anti-PD-1 antibody is shown in Fig. 1. Human PBMCs (HLA-A�0201 positive) were injected on day 0, and 2 � 105 SCC-3human lymphoma cells (six mice) or U87 glioblastoma cell line(four mice) with HLA-A�0201 genotype were subcutaneouslyinjected into the flank region in each group on day 1. SCC-3 cellsexpressed high levels of phosphorylated STAT3 and PD-L1 (Sup-plementary Fig. S1). However, U87 cells showed low expressionlevels of both markers.

Translational Relevance

Despite the clinical success with immune checkpoint anti-body therapy against advanced cancers, there are still difficul-ties associated with predicting precisely the clinical responsesprior to the treatment. Biomarker studies are also performedintensively; however, these are not potent enough for theprecise prediction. In this study, we developed and used anovel NOG-MHC dKO mouse model. We found that thishumanized mouse model exhibited no obvious signs ofGVHD and that the injection of anti-PD-1 antibody inhibitedPD-L1–positive SCC-3 tumor growth and induced tumor-specific immune responses. Therefore, in vivo investigation ofanti-PD-1 antibody effect using humanizedNOG-dKOmousecan contribute to the profiling of patients to predict the efficacyof anti-PD-1 antibody prior to the clinical treatment. Theseobservations indicate that the NOG-dKO mouse might be agood tool giving a promising future to a translational researchof immune checkpoint antibody therapy.

Ashizawa et al.

Clin Cancer Res; 2016 Clinical Cancer ResearchOF2

Starting on day 28, the anti-PD-1 antibody (2 mg/kg) wasadministered intraperitoneally biweekly six times. The antitu-mor activity of the anti-PD-1 antibody was evaluated by mea-suring tumor volume. Tumor volume was calculated on thebasis of the National Cancer Institute formula as follows: tumorvolume (mm3) ¼ length (mm) � [width (mm)]2 � 1/2. Mouseperipheral blood was collected from the retro-orbital venousplexus using heparinized pipettes on a weekly basis after anti-PD-1 antibody injection. One week after the last injection ofanti-PD-1 antibody, spleens and tumors were harvested fromthe control group and anti-PD-1 antibody–treated group.Blood and spleen cells from one set of three mice were usedfor in vitro assays, including the CTL induction assay, the naturalkiller (NK) cell assay, and PCR analysis of cytokine expression.Tumors from the other set of three mice were primarily used forTILs analysis and IHC analysis.

CTL induction assayCTL induction cultures were described previously (24). Brief-

ly, spleen cells harvested from control and anti-PD-1 antibody-treated mice were restimulated with irradiated (180 Gy) SCC-3cells at a ratio of 10:1 in the presence of IL2 for 7 days.Stimulated CTLs (1 � 105) and living SCC-3 cells (1 � 105)were coincubated in a round-bottom 96-well microcultureplate for 24 hours. The supernatants were subsequently col-lected and IFNg levels were measured using an ELISA Kitspecific for human IFNg (Biosource).

NK-cell assayHarvested splenocytes were used as effector cells, and K562

cells, purchased fromATCC, were used as target cells. The effector:target (E:T) ratio ranged from 100:1 to 11:1. The cytotoxic activityof NK cells was measured using the DELFIA nonradioactivecytotoxicity assay (PerkinElmer Inc.) as reported previously(24). The percentage of specific lysis was determined by thefollowing formula: percentage of specific lysis ¼ (experimentalrelease – spontaneous release)/(maximal release – spontaneousrelease) � 100.

RT-PCR analysis of cytokine expressionThe RT-PCR analysis of cytokines, stem cell, and epithelial–

mesenchymal transition (EMT)marker genes using the 7500 RealTime PCR System (Thermo Fisher Scientific) was performed asdescribed previously. Briefly, all PCR primers [IL2, IL4, IL10,TNFa, IFNg , IL12A, TGFb1, GAPDH for cytokines, c-Myc,NANOG, NES, Oct3/4, SOX2 for stem cell markers; BIRC5(Survivin), CCND1 (CyclinD1), FOXC2, MMP2, SMAD2,SNAIL1, SNAIL2, TCF4 and TWIST1 for EMT-associated genes]and TaqMan probes were purchased from Thermo Fisher Scien-tific. Total RNA was isolated from peripheral blood cells,spleen cells, and SCC-3 tumors using the NucleoSpin RNA Kit(Macherey-Nagel GmBH & Co.). Complementary DNA wassynthesized using SuperScript III RTase and Oligo (dT)20 primer.

ImmunohistochemistryAnti-CD4 (4B12) and anti-CD8 (C8/144B) antibodies

(Thermo Fisher Scientific), anti-granzyme B antibody (GrB-7;DAKO), anti-IL17 antibody (H-132; Santa Cruz BiotechnologyInc.), anti-FoxP3 antibody (236A/E7; Abcam), anti-CD204antibody (SRA-C6; TransGenic Inc.), and anti-phospho-STAT3antibody (D3A7; Cell Signaling Technology, Inc.) were pur-chased and used for immunohistochemical analysis. More than10 areas of tumor at a high magnification (�200) in eachsection stained with various antibodies was calculated usingimage analysis software, Winroof (Mitani Corporation). TILswere analyzed using various antibodies on sections from con-trol and anti-PD-1 antibody–treated groups.

Statistical analysisSignificant difference was analyzed using Student t test and

Mann–Whitney U test. Values of P < 0.05 were consideredstatistically significant. Survival curves of NOGmice transplantedwith human PBMCs were estimated using Kaplan–Meier methodand log-rank test was used to compare the survival curves.

ResultsPBMC transplantation in NOG-dKO mice

NOG-dKO mice received 2.5 Gy radiation and an intravenousinjection of 1 � 107 PBMCs on day 0, as shown in Fig. 1. The

6w4w2w1w0

X-rayHumanPBMC iv

5w3w

Tumor, spleenharvest

8w

orSCC-3U87 cells

sc

Anti-PD-1Ab i.p. (2 mg/kg)

MHC Double KO mice

3. PBMCFACSCytokine PCR (IL2, IL4, IL10, TNFα, IFNγ,

TGFβ1)

1. Tumor IHC (CD4, CD8, Granzyme, Treg etc.)Real-time PCR (antigens, cytokines)TIL FACS (hCD45)

2. Spleen FACS (hCD45)Cytotoxicity assay (CTL, NK assay)Real-time PCR (cytokines)

Figure 1.

Experimental design and treatmentschedule of anti-PD-1 therapy againstSCC-3 and U87 tumors. Human PBMCs(HLA-A�0201 positive)were injected onday 0, and SCC-3 or U87 cells with theHLA-A�0201 genotype weresubcutaneously injected into six or fourmice from each group on day 1.Beginning on day 28, anti-PD-1 wasadministered intraperitoneallybiweekly until six injections had beenadministered. Mouse peripheral bloodwas obtained weekly following theinitiation of anti-PD-1 injections. Oneweek after the last injection of anti-PD-1antibody, spleens and tumors wereharvested from the control group andthe anti-PD-1–treated group.

Antitumor Effect of Anti-PD-1 Antibody in NOG-MHC dKO Mice

www.aacrjournals.org Clin Cancer Res; 2016 OF3

engraftment of PBMCs was measured every 2 weeks for up to 8weeks. The percentage of human CD45þ cells in the treated micewas approximately 30% in the blood and 60% in the spleens upto 6 to 8 weeks (Supplementary Table S1). Importantly, the totalnumber of spleen cells increased severalfold compared with pre-transplantation levels, which indicated injected human PBMCexpanded similarly in mice. As to subpopulation of spleen cells,the ratio of human CD19þ cells in CD45þ human leukocytesreached over 30% at 4 weeks in spleens, but those cells rapidlydiminished at 6weeks. Interestingly, approximatelymore thanhalfof CD19þ B cells were CD138þ during the entire period ofmonitoring. In contrast, CD3þ T cells reached more than 50% ofhumanCD45þ cells at 4weeks and increasedup to90%at 6weeks.With regard to the subpopulations of CD3þ cells, CD8þ cells weredominant compared with CD4þ cells (78.7% vs. 19.8%).

Survival benefit from humanized NOG-dKO mice comparedwith humanized NOG mice

In another transplantation experiment using human PBMCs,the survival time between NOG-dKO and control NOG mice

under the human PBMCs transplantation was compared. All fivehumanized NOG-dKO mice were alive 10 weeks after the trans-plantation. In contrast, all the control humanized NOGmice haddied within 10 weeks after transplantation (SupplementaryFig. S2).

Antitumor effect of the anti-PD-1 antibody against SCC-3 andU87 tumors

After the initiation of treatment with the anti-PD-1 antibody,SCC-3 and U87 tumors decreased in size by more than 50%during six injection treatments on day 18 in SCC-3 tumorand on day 18 and 20 in U87 tumor between control and anti-PD-1 antibody–treated mice (Fig. 2). In addition, anti-PD-1antibody treatment showed a tendency of preventing weightloss in the treated mice through the treatment period. How-ever, antitumor effects of anti-PD-1 antibody againstboth tumors were not statistically significant compared withthe control group. Of particular significance, anti-PD-1 anti-body treatment did not significantly affect the engraftment of

A

C

B

DDays a�er ini�al administra�onDays a�er ini�al administra�on

Days a�er ini�al administra�on

V/V0

Body

wei

ght c

hang

e (g

)

Body

wei

ght c

hang

e (g

)

–8

–6

–4

–2

0

2

181411740–8

–6

–4

–2

0

2

20181411740

0

2

4

6

8

10

12

181411740

V/V0

0

2

4

6

8

10

12

20181411740

Days a�er ini�al administra�on

Figure 2.

Inhibitory effect of anti-PD-1 on the growth of SCC-3 and U87 tumors in vivo. A, V/V0 values of anti-PD-1 antibody-treated SCC3 tumors (n ¼ 6) are shown.The efficacy of the antibody treatment was expressed as the mean V/V0 value, where V is the tumor volume on the day of evaluation and V0 is thetumor volume on the day of treatment. B, The mean tumor volume of anti-PD-1 antibody–treated U87 tumors (n ¼ 4) is shown. Body weight change inanti-PD-1–treated mice bearing SCC-3 tumors (C), and U87 tumors (D). (*) Control group, (*) Anti-PD-1–treated group. Anti-PD-1 was administeredintraperitoneally six times. Each point represents the mean value derived from six mice in SCC-3 tumors or four mice in U87 tumors.

Ashizawa et al.

Clin Cancer Res; 2016 Clinical Cancer ResearchOF4

human PBMCs compared with the control group (Supplemen-tary Table S2).

Flow cytometry analysis of TILs from SCC-3 tumorsThe total number TILs from each tumor significantly

increased in anti-PD-1 antibody–treated tumor compared withthe control (Supplementary Table S3). Among CD45þ TILs inanti-PD-1 Ab-treated tumors, the frequency of CD3þCD8þ cellsand PD-1þTIM3þLAG3þ cells increased in anti-PD-1 Ab–trea-ted tumors. These results might suggest that anti-PD-1 Abpromoted the infiltration of CTLs and maintained exhaustedT-cell populations. Meanwhile, activation marker positive cells

(CD4þCD45ROþCD127þ) showed a tendency of increase inanti-PD-1 Ab-treated group, but it was not significant (Fig. 3and Supplementary Table S3). In addition, the frequency ofCD4þKi67þ and CD8þKi67þ T cells was not different betweenthe control and anti-PD-1 Ab–treated group.

Induction of CTL and NK activity in anti-PD-1–treatedhumanized mice

CTL activity against SCC-3 cells was identified using an IFNgproduction assay in spleen cells derived from the anti-PD-1-treated humanized NOG-dKO (Fig. 4A and Supplementary Fig.S3). NK-cell cytotoxic activity targeting K562 cells was observed in

Control

α-PD-1 Ab

A

PE

-Cy7

-CD

8

PE-CD4

PE

-Cy7

-CD

8

PE-CD4

PE

-Cy7

-CD

127

APC-CD45RO

PE

-Cy7

-CD

127

APC-CD45RO

AP

C-P

D-1

PerCP-CD3

FITC

-LA

G-3

PE-TIM-3

AP

C-P

D-1

PerCP-CD3

FITC

-LA

G-3

PE-TIM-3

CD3+ Gated CD4+ Gated PD-1+ Gated

Control

α-PD-1 Ab

B

AP

C-K

i-67

PE-CD4

AP

C-K

i-67

FITC-CD8

AP

C-K

i-67

PE-CD4

AP

C-K

i-67

FITC-CD8

CD3+ Gated CD3+ Gated

Figure 3.

The profiling of TILs using flow cytometry in the control and anti-PD-1 antibody–treated tumors. A, The frequency of CD4þ and CD8þ cells in CD3þ-gatedpopulation, the frequency of activation T-cell marker (CD45ROþCD127þ)-positive cells in CD4þ-gated population, and the frequency of exhaustedT-cell marker (TIM3þLAG3þ)-positive cells in PD-1þ-gated population were shown. B, The frequency of CD4þKi67þ and CD8þKi67þ cells in CD3þ-gatedpopulation was shown. Representative data were shown in each cell population.

Antitumor Effect of Anti-PD-1 Antibody in NOG-MHC dKO Mice

www.aacrjournals.org Clin Cancer Res; 2016 OF5

all the anti-PD-1–treatedmice with an E:T ratio of greater than 33(Fig. 4B and Supplementary Fig. S3).

Changes in cytokine and other genes expression in anti-PD-1–treated mice

A RT-PCR analysis revealed that TGFb1 gene expression wasdownregulated inPBMCs and SCC-3 tumor tissues in the anti-PD-1–treated group (Fig. 5A). In contrast, IFNg gene expression onday 7 was upregulated in PBMCs in the anti-PD-1–treated group(Fig. 5B). Meanwhile, the expression of stem cell marker genes,such as nestin, Oct3/4, and SOX2 and EMT-associated genes, suchas survivin, FOXC2, and MMP2 were suppressed by more than90% in anti-PD-1–treated SCC3 tumor (Fig. 5C).

Immunohistochemical analysis of SCC-3 tumor tissuesHematoxylin–eosin (H&E) stained tumor specimens did not

exhibit obvious histologic differences in the anti-PD-1–treatedgroup compared with the control group, with the exception ofpatchy lymphoid cell infiltrations observed in antibody-treatedgroup (Fig. 6A). CD8þ and granzyme Bþ lymphocytes were morefrequently observed in the anti-PD-1–treated group; however,CD4þ, FoxP3þ lymphocyte, and CD204þ immune cell levels didnot significantly differ (Fig. 6B).

DiscussionCancer immunotherapy studies using immunocompetent

mice serve as an excellent platform for the development ofnew therapeutic strategies and for providing insight into thefunctions of the immune system. However, these studies can beassociated with significant limitations, as a mouse model is notcapable of precisely predicting the clinical outcomes of pati-ents with cancer. Inevitably, the mouse immune system elicitsdifferent responses to allogenic human tumor cells, some ofwhich do not resemble the antitumor reaction in the humanimmune system at all.

In the past decade, severely immunodeficient mouse strainssuch as NOG, NSG, and BALB/c Rag2null IL2rgnull have beendeveloped and used to establish humanized immunodeficientmouse models. However, allogenic PBMC transplantation inthese mice is known to induce a strong xeno-GVHD responsethat results in lethality. Therefore, allogeneic transplantationstudies in these models must be completed before the onset ofxeno-GVHD, hereby limiting the time period available toconduct these experiments to 3 to 4 weeks after transplantation(21, 22). Sanmamed and colleagues (20) reported that acombination of the anti-human CD137 antibody (urelumab)and the anti-PD-1 antibody (nivolumab) inhibited tumorgrowth and increased IFNg production in conjunction withallogeneic (HT29) or autologous (from a gastric cancer patient)stem cell transplantation in the Rag2�/�IL2Rgnull humanizedmouse model of gastric cancer. Interestingly, they also reportedthat tumor growth was slowed in a strictly autologous trans-plantation model using PBMCs and surgically resected tumortissue from the same patient. They concluded that these resultsvalidated the significance of this humanized mouse model forevaluating the human immune response; however, the experi-ments were still limited to a time period of 3 to 4 weeksposttransplantation due to the issue of xeno-GVHD onset.

In this study, we developed and used a novel NOG-dKOmouse model. We found that this humanized mousemodel exhibited no obvious signs of xeno-GVHD and sur-vived up to 12 weeks longer than control NOG mice. Thisobservation suggests that the NOG-dKO mouse meritsfurther investigations to evaluate the response of the humanimmune system to antibodies targeting immune checkpointmolecules.

In our humanized NOG-dKOmice transplanted with SCC-3 orU87 tumor cells, and treated with the anti-PD-1 antibody, tumorgrowth was inhibited. Furthermore, this antitumor response wasnot observed in humanized tumor-bearing mice that had notreceived human PBMC transplantation (data not shown),

Cyt

otox

icity

(%)

GroupsGroups

IFN

γ Le

vels

(pg/

mL)

C2C1T3T2T1 C30.0

50.0

100.0

150.0BA

0

20

40

60

80

100K562 (1:11)K562 (1:33)K562 (1:100)

T3T2T1C2C1 C3

Figure 4.

Induction of cytotoxic activity in humanized SCC3 tumor-bearingmice treatedwith anti-PD-1 antibody.A,CTL induction against SCC3 cells from three spleens of anti-PD-1–treatedmice. Levels of IFNg producedbyCTL cells stimulatedwith irradiated SCC-3cellsweremeasuredusing anELISAKit. Open column, control group; closedcolumn, anti-PD-1–treated group. B, NK-cell cytotoxic activity of three spleens from anti-PD-1–treated mice. Cytotoxic activity was measured using theNonradioactive Cytotoxicity Assay Kit. C, control group; T, anti-PD-1–treated group. Each column represents the mean value of the triplicate experiments.

Ashizawa et al.

Clin Cancer Res; 2016 Clinical Cancer ResearchOF6

indicating that anti-PD-1 antibody-induced tumor growth wasmediated by the engrafted PBMCs.

With respect to immunologic responses, the following fourresponses were observed in this study: (i) an SCC-3–specific CTLactivity induction, (ii) upregulation ofNK-cell activity, (iii) down-regulation of TGFb1 expression in PBMCs and SCC-3 tumors, and(iv) an increase in CD8þ and granzyme Bþ T cells and mainte-nance of exhausted marker (PD-1þTIM3þLAG3þ)-positive T cellsin the tumor.

The induction of CTL activity targeting tumor cells and intra-tumoral infiltration of immune effector cells are responses fre-quently observed in patients with cancer treated with antibodiestargeting immune checkpoint molecules (25–29). Although anupregulation of NK-cell activity is not a common observationof clinical trials with anti-PD-1 therapy, this response mightdepend on the time point at which tumors are harvest and thenumber of antibody injection. In general, activated NK cellsexpress PD-1 on the cell surface, and IFNg secretion from effectorT cells strongly upregulates the expression of PD-L1 on tumorcells. These events might contribute to the increased resistance oftumor cells to NK-cell lysis (30). Anti-PD-1 treatment has beenreported by Benson and colleagues to enhance NK-cell killingactivity against multiple myeloma cells by blocking PD-1/PD-L1signaling (31). Moreover, Das and colleagues demonstrated thatan upregulation of genes involved in cytokine and NK-cell func-tions was observed in patients with cancer given a combination ofanti-PD-1 and anti-CTLA-4 treatment (32). This observation

suggests that NK-cell activation occurs early in the course ofantibody treatment.

Downregulation of TGFb1 mediated by PD-1/PD-L1 block-ade has not been commonly observed in clinical trials andother studies of the immune system. TGFb1 is a blood markerassociated with a poor prognosis and this molecule mediatesimmunosuppressive activity in the tumor microenvironmentvia the mechanistic PD-L1 upregulation and activation ofSTAT3 signaling (33–35). In addition, TGFb1 is an importantfactor for inducing regulatory T cells or IL17-producing helperT cells (Th17) in association with IL23 (36). Therefore, it isreasonable to hypothesize that TGFb1 downregulation con-tributes to the accumulation of CD8þ and granzymeþ CTLs inthe tumor of humanized mice treated with anti-PD-1.

Furthermore, TGFb1 is also known to be a promoting factorto mediate EMT and maintain stemness, which prefer to cancerprogression and metastasis. A RT-PCR study revealed thedownregulation of the expression level of several stem cellmarker and EMT-associated genes including FOXC2, nestin,Oct4, and SOX2. Therefore, the association of TGFb1 withthese genes in cancer-favoring signal cascade can be indicated(37, 38), which might be blocked by anti-PD-1 antibodytreatment. This observation should be the novel result in thestudy investigating the therapeutic mechanism of anti-PD-1antibody. Importantly, to verify TGFb1 downregulation,TGFb1 protein levels in the tumor should be assessed usingIHC.

Tumor

Controlday19

α-PD-1 Abday19

% o

f Con

trol

0

50

100

150

*

IFNgBlood (PBMC)

α-PD-1 Ab

Day14Day0 Day14Day0Day7 Day7

Control

% o

f Con

trol

0

50

100

150

200

250*

% o

f Con

trol

Blood (PBMC)TGFb1A

C

B

α-PD-1 AbDay14Day0 Day14Day0Day7 Day7

Control

0

50

100

150

200

250

300

* *

0.001

0.01

0.1

1

10

**

**

**

**

** **

Rat

io o

f con

trol

GenesSurvivin

CyclinD1

FOXC2MMP2

NANOG

NESOct3/4

SMAD2

SNAI1SNAI2

SOX2

TCF4

TWIST1

Stem cell and EMT markers

Figure 5.

Analysis of various gene expression in peripheral blood cells and SCC-3 tumors using RT-PCR. A, Downregulation of TGFb1 gene expression in PBMC andtumor tissue. Open column, control group; closed column, anti-PD-1–treated group. B, Upregulation IFNg gene expression in PBMCs on day7. Open column,control group; shaded column, anti-PD-1–treated group. C, Stem cell marker and EMT-associated genes expression. Open column, control group; closedcolumn, anti-PD-1–treated group. Each column represents the mean value of triplicate experiments. �� , P < 0.01; � , P < 0.05, statistically significant.

Antitumor Effect of Anti-PD-1 Antibody in NOG-MHC dKO Mice

www.aacrjournals.org Clin Cancer Res; 2016 OF7

Immunohistochemical analysis in this study revealed anincrease in CD8þ and granzymeþ T-cell infiltration of tumors.In clinical trials evaluating anti-PD-1, CD8þ TILs appear to beprimarily observed in antibody-treated patients. In contrast, inpatients treated with anti-CTLA-4 antibody alone or a combina-tion of anti-CTLA-4 and anti-PD-1, both an increase in CD8þ Tcells and a decrease in regulatory T cells are frequently observed(25, 29). In addition, TIL profiling showed an increase ofexhaustedmarker (PD-1þTIM3þLAG3þ)-positive T cell frequencyin anti-PD-1 antibody-treatedmice,whichwas consistentwith theobservation that anti-PD-1 antibody promoted exhausted mark-er-positive T-cell expansion and survival (39).

Pham and colleagues reported that TAM, referred to asCD11bþF4/80þ cells, significantly increased in murine spon-taneously arising medulloblastoma tumors as demonstrated byIHC; however, the effect of anti-PD-1 on TAM infiltration wasnot well characterized (40). There have been very few clinicalstudies specifically evaluating the role of TAM in immunecheckpoint antibody therapy. However, in this study,CD204þ cell level did not significantly differ in anti-PD-1antibody–treated tumors. The TIL profiling might depend onnot only the strength of immune response induced by single orcombination antibody therapy, but the immunologic status ofthe tumor in individual patient.

The significant advantage that our humanized NOG-dKOmice can provide to the development of novel human immu-notherapy because they evade the limitations imposed bylethal xeno-GVHD should be stressed. The utilization of the

humanized NOG-dKO mouse model for in vivo experimentswill enhance the value of immunotherapeutic studies, such asthose evaluating novel immune checkpoint antibody treat-ment and combinations of tumor-based vaccines or othersmall molecules. In the near future, we plan to use thehumanized NOG-dKO mouse system to identify novelbiomarkers that can serve as prognostic indicators of theantitumor response to antibodies targeting immune check-point molecules, such as PD-1 and CTLA-4, prior to theinitiation of treatment.

Disclosure of Potential Conflicts of InterestY. Nakasu has provided expert testimony. No potential conflicts of

interest were disclosed by the other authors.

Authors' ContributionsConception and design: Y. AkiyamaDevelopment of methodology: T. AshizawaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Ashizawa, A. Iizuka, C. Nonomura, R. Kondou,C. Maeda, H. Miyata, T. Sugino, K. Mitsuya, Y. NakasuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Iizuka, R. Kondou, N. HayashiWriting, review, and/or revision of the manuscript: Y. AkiyamaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Maruyama, I. Katano, M. Ito, Y. AkiyamaStudy supervision: K. Yamaguchi, Y. AkiyamaOther (provided recombinant antibodies): A. Iizuka

CD8 Granzyme

Control

Anti-PD-1 Ab

H-E Stain CD204A

B

CD204

0.0

4.0

8.0

12.0

16.0IL17

0

20

40

60

80

100FoxP3

Control An�-PD-1 Ab

0.0

20.0

40.0

60.0CD4

Control An�-PD-1 Ab

Cel

l num

ber/f

ield

Cel

l num

ber/f

ield

0.0

10.0

20.0

30.0

40.0

CD8

Control An�-PD-1 Ab

Granzyme B

Control An�-PD-1 Ab

**

Pos

itive

are

a (m

m2 ) /

field

0

0.1

0.2

0.3

0.4

**

0

20

40

60

Control An�-PD-1 Ab Control An�-PD-1 Ab

Figure 6.

Effect of anti-PD-1 on immune cellsinfiltrating the SCC-3 tumors.A, Imagesof control and anti-PD-1–treatedtumors stained with H&E, the anti-CD8,anti-granzyme B, and anti-CD204antibodies. Magnification: �400 forH&E, granzyme B, �200 for CD8 andCD204 staining. B, Effect of anti-PD-1on the number of infiltrating immunecells in SCC-3 tumors. More than 10fields of view in sections of each tumorat a high magnification (�200). Thesections were stained with variousantibodies and the images wereevaluated using image analysissoftware (Winroof). Positive cell countper field was compared betweencontrol and anti-PD-1–treated groups.�� , P < 0.01, statistically significant.

Ashizawa et al.

Clin Cancer Res; 2016 Clinical Cancer ResearchOF8

AcknowledgmentsWe thank Mr. Koji Takahashi for his excellent assistance in maintaining

NOG-dKO mice in the animal facility.

Grant SupportThis work was supported by a grant from JSPS KAKENHI, Japan (grant no.

26430178; to A. Iizuka).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 14, 2016; revised June 20, 2016; accepted June 29, 2016;published OnlineFirst June 29, 2016.

References1. Weber JS, O'Day S, UrbaW, Powderly J, Nichol G, Yellin M, et al. Phase I/II

study of ipilimumab for patients with metastatic melanoma. J Clin Oncol2008;26:5950–56.

2. Hodi FS,O'Day SJ,McDermott DF,Weber RW, Sosman JA,Haanen JB, et al.Improved survivalwith ipilimumab in patients withmetastaticmelanoma.N Engl J Med 2005;363:711–23.

3. Topalian SL,Hodi FS, Brahmer JR,Gettinger SN, SmithDC,McDermottDF,et al. Safety, activity, and immune correlates of anti-PD-1 antibody incancer. N Engl J Med 2012;366:2443–54.

4. Brahmer JR, Tykodi SS, ChowLQ,HwuWJ, Topalian SL,HwuP, et al. Safetyand activity of anti-PD-L1 antibody in patients with advanced cancer.N Engl J Med 2012;366:2455–65.

5. Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, SharfmanWH, et al. Survival, durable tumor remission, and long-term safety inpatients with advanced melanoma receiving nivolumab. J Clin Oncol2014;32:1020–30.

6. Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM,et al. Nivolomab plus ipilimumab in advanced melanoma. N Engl J Med2013;369:122–33.

7. Larkin J, Chiarion-Sileni V, Gonzaliz R, Grob JJ, Cowey CL, Lao CD, et al.Combined nivolumab and ipilimumab or monotherapy in untreatedmelanoma. N Engl J Med 2015;373:23–34.

8. Antonia SJ, Gettinger SN, Chow LQM, Juergens RA, Borghaei H, Shen Y,et al. Nivolumab (anti-PD-1; BMS-936558, ONO-4538) and ipilimumabinfirst-lineNSCLC: interimphase I results. J ClinOncol 32:5s, 2014 (suppl;abstr 8023).

9. Wang C, Thudium KB, Han M, Wang XT, Huang H, Feingersh D, et al. Invitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558,and in vivo toxicology in nonhuman primates. Cancer Immunol Res2014;2:846–56.

10. Nomi T, Sho M, Akahori T, Hamada K, Kubo A, Kanehiro H, et al. Clinicalsignificance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer. Clin CancerRes 2007;13:2151–57.

11. Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K, et al.NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse modelfor engraftment of human cells. Blood 2002;100:3175–82.

12. Hiramatsu H, Nishikomori R, Heike T, Ito M, Kobayashi K, Katamura K,et al. Complete reconstitution of human lymphocytes from cord bloodCD34þ cells using the NOD/SCID/gammacnull mice model. Blood 2003;102:873–80.

13. Shultz LD, Lyons BL, Burzenski LM,Gott B, Chen X, Chaleff S, et al. Humanlymphoid and myeloin cell development in NOD/LtSz-scid IL2R gammanull mice engrafted with mobilized human hematopoietic stem cells.J Immunol 2005;174:6477–89.

14. Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffarentti JC, LanzavecchiaA, et al. Development of a human adaptive immune system in cordblood cell-transplanted mice. Science 2004;304:104–7.

15. InoueM, Senju S,Hirata S, Irie A, BabaH,Nishimura Y. An in vivomodel ofpriming of antigen-specific human CTL by Mo-DC in NOD/Shi-scidIL2gamma(null) (NOG) mice. Immunol Lett 2009;126:67–72.

16. Ito R, Shiina M, Saito Y, Tokuda Y, Kametani Y, Habu S. Antigen-specificantibody production of human B cells in NOGmice reconstituted with thehuman immune system. Curr Top Microbiol Immunol 2008;324:95–107.

17. Tonomura N, Habiro K, Shimizu A, Sykes M, Yang YG. Antigen-specifichuman T-cell responses and T cell-dependent production of human anti-bodies in a humanized mouse model. Blood 2008;111:4293–6.

18. Wege AK, Schmidt M, Ueberham E, Ponnath M, Ortmann O, BrockhoffG, et al. Co-transplantation of human hematopietic stem cells and

human breast cancer cells in NSG mice: a novel approach to generatetumor cell specific human antibodies. MAbs 2014;6:968–77.

19. Ito A, Ishida T, Yano H, Inagaki A, Suzuki S, Sato F, et al. Defucosylatedanti-CCR4 monoclonal antibody exercises potent ADCC-mediated anti-tumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rgamma(null) mouse model. Cancer Immunol Immunother 2009;58:1195–206.

20. Sanmamed MF, Rodriguez I, Schalper KA, Onate C, Azpilikueta A, Rodri-guez-Ruiz ME, et al. Nivolumab and urelumab enhance antitumor activityof human T lymphocytes engrafted in Rag2-/-IL2Rgnull immunodeficientmice. Cancer Res 2015;75:3466–78.

21. Ito R, Katano I, Kawai K, Hirata H, Ogura T, Kamisako T, et al. Highlysensitive model for xenogenic GVHD using severe immunodeficient NOGmice. Transplantation 2009;87:1654–8.

22. Alcantar-Orozco EM, Gornall H, Baldan V, Hawkins RE, Gilham DE.Potential limitations of the NSG humanized mouse as a model systemto optimize engineered human T cell therapy for cancer. Hum Gene TherMethods 2013;24:310–20.

23. Yaguchi T, Kobayashi A, Katano I, Ka Y, Ito M, Kawakami Y. MHC class I/IIdeficient NOGmice are useful for analysis of human T/B cell responses forhuman tumor immunology research. J Immunother Cancer 2013;1:P39

24. Inoue K, Saegusa N, Omiya M, Ashizawa T, Miyata H, Komiyama M, et al.Immunologically augmented skin flap as a novel dendritic cell vaccineagainst head and neck cancer in a rat model. Cancer Sci 2015;106:143–50.

25. Ascierto PA, KalosM, Schaer DA, CallahanMK,Wolchok JD. Biomarker forimmunostimulatory monoclonal antibodies in combination strategies formelanoma and other tumor types. Clin Cancer Res 2013;19:1009–20.

26. Lipson EJ, SharfmanWH, Drake CG,Wollner I, Taube JM, Anders RA, et al.Durable cancer regression off-treatment and effective reinduction therapywith an anti-PD-1 antibody. Clin Cancer Res 2013;19:462–8.

27. Taube JM, Klein A, Brahmer JR, Xu H, Pan X, Kim JH, et al. Association ofPD-1, PD-1 ligand, and other features of the tumor immune microenvi-ronment with response to anti-PD-1 therapy. Clin Cancer Res 2014;20:5064–74.

28. Duraiswamy J, Freeman GJ, Coukos G. Therapeutic PD-1 pathway block-ade augments with other modalities of immunotherapy T-cell function toprevent immune decline in ovarian cancer. Cancer Res 2013;73:6800–912.

29. Duraiswamy J, Kaluza KM, Freeman GJ, Coukos G. Dual blockade of PD-1and CTLA-4 combined with tumor vaccine effectively restores T-cell rejec-tion function in tumors. Cancer Res 2013;73:3591–603.

30. Bellucci R,Martin A, BommaritoD,Wang K, Hansen SH, FreemanGJ, et al.Interferon-g-induced activation of JAK1 and JAK2 suppresses tumor cellsusceptibility to NK cells through upregulation of PD-L1 expression.Oncoimmunol 2015;4:e1008824.

31. Benson DM, Bakan CE, Mishra A, Hofmeister CC, Efebera Y, Becknell B,et al. The PD-1/PD-L1 axis modulates the natural killer cell versus multiplemyeloma effect: a therapeutic target for CT-011, a novel monoclonal anti-PD-1 antibody. Blood 2010;116:2286–94.

32. Das R, Verma R, Sznol M, Boddupalli CS, Gettinger SN, Kluger H, et al.Combination therapy with anti-CTLA-4 and anti-PD-1 leads to distinctimmunologic change in vivo. J immunol 2015;194:950–9.

33. Song S, Yuan P, Wu H, Chen J, Fu J, Li P, et al. Dendritic cells with anincreased PD-L1 by TGF-b induce T-cell energy for the cytotoxicity ofhepatocellular carcinoma cells. Int Immunopharmacol 2014;20:117–23.

34. Fu BH, Fu ZZ, Meng W, Gu T, Sun XD, Zhang Z. Platelet VEGF and serumTGF-b1 levels predict chemotherapy response in non-small cell lung cancerpatients. Tumor Biol 2015;36:6477–83.

35. Cheng JC, Graber MS, Hsu FM, Tsai CL, Castaneda L, Lee JM, et al. Highserum levels of vascular endothelial growth factor-A and transforming

Antitumor Effect of Anti-PD-1 Antibody in NOG-MHC dKO Mice

www.aacrjournals.org Clin Cancer Res; 2016 OF9

growth factor-b1 before neoadjuvant chemoradiotherapy predict pooroutcomes in patients with esophageal squamous cell carcinoma receivingcombined modality therapy. Ann Surg Oncol 2014;21:2361–8.

36. Li S, Li Y, Qu X, Liu X, Liang J. Detection and significance of Treg FoxP3(þ)and Th17 cells in peripheral blood of nonsmall cell lung cancer patients.Arch Med Sci 2014;10:232–9.

37. Mani SA, Yang J, Brooks M, Schwaninger G, Zhou A, Miura N, et al.Mesenchymal Forkhead1 (FOXC2) plays a key role in metastasis and isassociated with aggressive basal-like breast cancers. Proc Natl Acad Sci USA2007;104:10069–74.

38. Su HT, Weng CC, Hsiao PJ, Chen LH, Kuo TL, Chen YW, et al. Stem cellmarker nestin is critical for TGF-b1-mediated tumor progression in pan-creatic cancer. Mol Cancer Res 2013;11:768–79.

39. Fourcade J, Sun Z, Pagliano O, Chauvin JM, Sander C, Janjic B, et al. PD-1and Tim-3 regulate the expansion of tumor antigen-specific CD8þ T cellsinduced by melanoma vaccines. Cancer Res 2014;74:1045–55.

40. Pham CD, Flores C, Yang C, Pinheiro EM, Yearley JH, Sayour EJ, et al.Differential immune microenvironments and response to immune check-point blockade among molecular subtypes of murine medulloblastoma.Clin Cancer Res 2015;22:582–95.

Clin Cancer Res; 2016 Clinical Cancer ResearchOF10

Ashizawa et al.