A HER2-Targeting Antibody Drug Conjugate, Trastuzumab ...DS-8201a showed antitumor effects in...

Large Molecule Therapeutics

A HER2-Targeting Antibody–Drug Conjugate,Trastuzumab Deruxtecan (DS-8201a), EnhancesAntitumor Immunity in a Mouse ModelTomomi Nakayama Iwata, Chiaki Ishii, Saori Ishida, Yusuke Ogitani, Teiji Wada, andToshinori Agatsuma

Abstract

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting anti-body–drug conjugate with a topoisomerase I inhibitor exatecanderivative (DX-8951 derivative, DXd), has been reported toexert potent antitumor effects in xenograft mouse models andclinical trials. In this study, the immune system–activatingability of DS-8201a was assessed. DS-8201a significantlysuppressed tumor growth in an immunocompetent mousemodel with human HER2-expressing CT26.WT (CT26.WT-hHER2) cells. Cured immunocompetent mice rejected not onlyrechallenged CT26.WT-hHER2 cells, but also CT26.WT-mockcells. Splenocytes from the cured mice responded to both CT26.WT-hHER2 and CT26.WT-mock cells. Further analyses revealedthat DXd upregulated CD86 expression on bone marrow–

derived dendritic cells (DC) in vitro and that DS-8201a

increased tumor-infiltrating DCs and upregulated their CD86expression in vivo. DS-8201a also increased tumor-infiltratingCD8þ T cells and enhanced PD-L1 and MHC class I expressionon tumor cells. Furthermore, combination therapy withDS-8201a and anti–PD-1 antibody wasmore effective than eithermonotherapy. In conclusion, DS-8201a enhanced antitumorimmunity, as evidenced by the increased expression of DC mar-kers, augmented expression of MHC class I in tumor cells, andrejection of rechallenged tumor cells by adaptive immunecells, suggesting that DS-8201a enhanced tumor recognition byT cells. Furthermore, DS-8201a treatment benefited from combi-nation with anti–PD-1 antibody, possibly due to increased T-cellactivity and upregulated PD-L1 expression induced by DS-8201a.Mol Cancer Ther; 17(7); 1494–503. �2018 AACR.

IntroductionSeveral chemotherapeutic agents are known to induce acti-

vation of the immune system (1). Chemotherapeutic agentsinduce tumor cell death, and an "immunogenic cell death"activates the immune system (1–3). One of the mechanisms ofimmune activation by chemotherapeutic agents is activation ofdendritic cell (DC) function followed by activation of T cells(1, 2, 4). Interestingly, it has been reported that topoisomeraseI inhibitors are the agents that stimulate T-cell killing activity(5, 6) and topoisomerase I inhibitors are drawing attention asimmunomodulators.

Trastuzumab deruxtecan (DS-8201a) is a HER2-targeting anti-body–drug conjugate (ADC), structurally composed of a human-ized anti-humanHER2 (anti-hHER2) antibody, an enzymaticallycleavable peptide-linker, and a topoisomerase I inhibitor, exate-can derivative (DX-8951 derivative, DXd; SupplementaryFig. S1; refs. 7, 8). It has been shown to exhibit an antitumoreffect in mouse models (7). Moreover, in a phase I clinical trial,

DS-8201a showed antitumor effects in breast, gastric, gastro-esophageal, colorectal, salivary, and non–small cell lung cancerpatients (9–11), including ones who had previously been treatedwith trastuzumab emtansine (T-DM1), an ADC composed of atubulin polymerization inhibitor, and an anti-hHER2 antibody.Furthermore, DS-8201a was well-tolerated, and theMTDwas notreached during dose escalation (0.8–8.0 mg/kg; refs. 9–11).Accordingly, DS-8201a is expected to become a new therapy forHER2-positive tumors. Importantly, athymic nudemousemodelshadbeenused for those nonclinical evaluations, and involvementof the immune system in the efficacy ofDS-8201ahas not yet beenelucidated.

Recently, immune checkpoint inhibitors (ICI) have been dem-onstrated to show remarkable clinical benefits (12–14). Never-theless, there are many patients who are not sensitive or arerefractory to a single agent of ICI, and combination therapieswith other drugs are needed (15). Certain chemotherapeuticagents are suggested to show benefits in combination with ICIsin humans (16). This idea is also supported by preclinical mousestudies. Topoisomerase I inhibitors and other chemotherapeuticagents have successfully been combined with ICIs in syngeneicmouse models (4, 5, 17). However, there is a concern thatchemotherapeutic agents could induce lymphopenia and thusattenuate the effect of ICIs (2). ADCs could be an ideal alternativeoption for combination therapy because ADCs are intended totarget selectively cancer cells and spare normal cells. T-DM1 andalso several ADCs conjugated with tubulysin or pyrrolobenzo-diazepine dimer (PBD) showed immune-activating effects andbenefit in combination with ICIs in immunocompetent mousemodels (18, 19). Moreover, it was reported that a combination of

Biologics & Immuno-Oncology Laboratories, Oncology Function, R&D Division,Daiichi Sankyo Co., Ltd., Tokyo, Japan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Teiji Wada, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi,Shinagawa-ku, Tokyo 140-8710, Japan. Phone: 81-80-4357-4890; Fax:81-3-5740-3641; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0749

�2018 American Association for Cancer Research.

MolecularCancerTherapeutics

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brentuximab vedotin, an ADC composed of a tubulin polymer-ization inhibitor and an anti-human CD30 antibody, and nivo-lumab has synergistic activity in humans (20). To our knowledge,the payloads of ADCs that are currently suggested to be involvedin activation of immune system are tubulin inhibitors and aDNA-crosslinking agent, and an immunomodulatory effect ofthe ADC composed of a topoisomerase I inhibitor has not beenreported yet.

In this study, we examined the immunologic effect of DS-8201a and the combination benefit of DS-8201a with anti–PD-1-blocking antibody in a mouse model. Here, we identified anew role of DS-8201a, an immunostimulatory activity that isdistinct from the DXd's cytotoxic activity against tumor cells.

Materials and MethodsAntibodies and compounds

DS-8201a, DXd, and its parental anti-human HER2 (anti-hHER2) antibody were prepared as previously described (7, 8,21). Its drug-to-antibody ratio was 7.6, as determined by reversephase chromatography. Anti–PD-1 antibody (clone; RMP1-14)was purchased from Bio X Cell. For flow cytometric analyses,FITC-labeled anti-hHER2 antibody (340553; Becton Dickinson),Pacific Blue–labeled anti-mouse CD45 antibody (103126; Bio-Legend), PE-labeled anti-mouse CD3e antibody (553064; BectonDickinson), PerCP/Cy5.5-labeled anti-mouse CD4 antibody(100434; BioLegend), PE-Cy 7–labeled anti-mouse CD8a anti-body (552877; Becton Dickinson), Alexa FluorR 647–labeledanti-human/mouse Granzyme B antibody (515405; BioLegend),PE-labeled anti-mouse CD86 (B7-2) antibody (553692; BectonDickinson), APC-labeled anti-mouse CD11c antibody (550261;Becton Dickinson), FITC-labeled anti-mouse MHC Class II (I-A/I-E) antibody (11-5321-85; Thermo Fisher Scientific), PE-labeledanti-human Her2/neu antibody (340552; Becton Dickinson),APC-labeled anti-mouse CD274 (B7-H1, PD-L1) antibody(124312; BioLegend), FITC-labeled anti-mouse H-2Dd antibody(110606; BioLegend), FITC-labeledmouse IgG1k isotype control(555909; Becton Dickinson), Pacific Blue-labeled rat IgG2bkisotype control (400627; BioLegend), PE-labeled Hamster IgG1lisotype control (553954; Becton Dickinson), PerCP/Cy5.5 RatIgG2bk isotype control (400632; BioLegend), PE/Cy7 Rat IgG2akisotype control (400522; BioLegend), Alexa-647 mouse IgG1kisotype control (400136; BioLegend), PE-labeled rat IgG2ak iso-type control (12-4321-81; Thermo Fisher Scientific), APC-labeledhamster IgG1l1 isotype control (553956; Becton Dickinson),FITC-labeled rat IgG2b isotype control (11-4031-82; ThermoFisher Scientific), PE-labeled mouse IgG1k isotype control(551436; Becton Dickinson), APC-labeled rat IgG2bk isotypecontrol (553991; Becton Dickinson), and FITC-labeled mouseIgG2ak isotype control (400207; BioLegend) were used.

Flow cytometryThe LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo

Fisher Scientific) was used to exclude dead cells. Flow cytometricdata were acquired with the FACSCanto II (Becton Dickinson),and data were analyzed with FlowJo 7.6.5 software (TOMYDIGITAL BIOLOGY CO., LTD.).

Cell linesHuman breast cancer cell line MDA-MB-453 (HTB-131) was

purchased from the American Type Culture Collection and was

cultured in Leibovitz's L-15 Medium (Thermo Fisher Scientific)supplementedwith 10%FBS (ThermoFisher Scientific) at 37�C ina free gas exchangewith atmospheric air.Humanbreast cancer cellline KPL-4 was provided from Dr. Kurebayashi at the KawasakiMedical University (Japan) and was cultured with RPMI1640medium (Thermo Fisher Scientific) supplemented with 10% FBSat 37�C and 5% CO2 atmosphere.

Mouse cancer cell line CT26.WT (CRL 2638) was purchasedfrom the American Type Culture Collection. An empty vector(pQCXIN; Clontech) or human HER2 gene (NM_004448.3) wasretrovirally introduced into CT26.WT cells (CT26.WT-mock andCT26.WT-hHER2, respectively). The cells were cultured with inRPMI 1640medium supplementedwith 10%FBS and 250 mg/mLGeneticin (Thermo Fisher Scientific) at 37�C and 5% CO2

atmosphere. Human HER2 expression was confirmed by flowcytometry.

Mouse models, treatments, and analysis of intratumoral cellsAll mouse studies were carried out in an Association for

Assessment and Accreditation of Laboratory Animal Care Inter-national–approved veterinary research facility and in accordancewith the local guidelines of the Institutional Animal Care andUseCommittee. Three to 6 mice were housed together in sterilizedcages and maintained under specific pathogen-free conditions.The mice were euthanized with CO2 gas when they reachedendpoints (tumor volume exceeding 3,000 mm3, 10% reductionof body weight, or clinical signs indicating that mice should beeuthanized for ethical reasons). Female BALB/cAnNCrlCrlj(BALB/c) mice, aged 4 weeks to 5 weeks, were purchased fromCharles River Laboratories Japan Inc. Mice (aged 5 to 6 weeks)were inoculated with 5 million CT26.WT-hHER2 cells sus-pended in saline into the right flank by s.c. injection. Tumorvolume was defined as 1/2 � length � width2. When theaverage volume of tumors reached approximately 100 to 200mm3, the mice were divided into control and treatment groupsbased on tumor volumes by using the randomized blockmethod, and treatment was initiated (day 0). DS-8201a(10 mg/kg), anti-hHER2 antibody (10 mg/kg), and anti–PD-1 antibody (5 mg/kg) were administered i.v. at a volume of10 mL/kg to mice. As a control, ABS buffer (10 mmol/L AcetateBuffer, 5% sorbitol, and pH5.5) was administered at the samevolume as the DS-8201a. DS-8201a and anti-hHER2 antibodywere administered on days 0 and 7. Anti–PD-1 antibody wasadministered on days 0, 3, 7, and 10.

For a rechallenge study, mice whose CT26.WT-hHER2 tumorshad been cured by DS-8201a treatment (10 mg/kg, once a week,twice, i.v.) were divided into two groups. The mice were thensubcutaneously inoculated with 5 million CT26.WT-mock orCT26.WT-hHER2 cells into the left flank. Na€�ve (never tumor-inoculated) mice were also inoculated with each cell line forcomparison.

For flow cytometric analysis of T cells, DCs, and tumor cells,when the average volume of tumors reached approximately 250–400 mm3 (8 days after tumor inoculation), the mice were treatedwith vehicle or DS-8201a (10 mg/kg, once, i.v.; day 0). The micewere euthanized with CO2 asphyxiation on day 8, and tumorswere cut into small pieces and dissociated with the TumorDissociation Kit (Miltenyi Biotec) by the gentleMACS OctoDissociator with Heaters (Miltenyi Biotec). The resultant singlecells were blocked with Mouse BD Fc Block reagent (BectonDickinson) and stained with antibodies against mouse CD3,

Immunostimulatory Activity of DS-8201a, a HER2-Targeting ADC

www.aacrjournals.org Mol Cancer Ther; 17(7) July 2018 1495




CD4, CD8, CD11c, CD45, CD86,GranzymeB,MHC class I,MHCclass II, and PD-L1 and human HER2.

The Enzyme-Linked Immunospot (ELISPOT) assaySplenocytes from CT26.WT-mock or CT26.WT-hHER2 tumor–

bearing na€�vemicewithout drug treatment andmicewhoseCT26.hHER2 tumors had been cured by DS-8201a treatment weresubsequently cocultured with CT26.WT-mock or CT26.WT-hHER2 cells at 37�C, 5% CO2, for 24 hours in high-protein-binding PVDF filter plates precoated with anti-mouse IFNg mAb(mIFNgp-1M/10; Cellular Technology Limited). IFNg secretedfrom cocultured cells was determined by a Murine IFNg Single-Color Enzymatic ELISPOT Assay followed by ImmunoSpot S6ENTRY Analyzer (Cellular Technology Limited) and Immuno-Spot 5.0 software (version 5.1.36; Cellular Technology Limited).

In vitro DC analysisTo obtain bonemarrow–derived dendritic cells (BMDC), bone

marrow cells from femurs of BALB/c mice were cultured withRPMI 1640 medium supplemented with 10% FBS, 55 mmol/L of2-melcaptoethanol, 100 U/mL of penicillin, 100 mg/mL of strep-tomycin, 1 mmol/L sodium pyruvate, 1� nonessential aminoacid, 2 mmol/L of L-glutamine, and 10 ng/mL of recombinantmurine GM-CSF (DC culture medium) for 11 days. The resultantBMDCs were cultured in DC culture medium supplemented withDXd (0, 0.0625, 0.125, 0.25, 0.5, and 1.0 mmol/L), or dimethylsulfoxide as a vehicle control. After 24 hours of cell culture, thecellswereharvested, blockedwithMouseBDFcBlock reagent, andstained with antibodies against mouse CD11c, CD45, CD86, andMHC class II.

Statistical analysisAll statistical analyses were performed using SAS System

Release 9.02.300 (SAS Institute Inc.). Antitumor effects, rechal-lenge study, and ELISPOT assays were analyzed with a Wilcoxonrank-sum test, and flow cytometric data were analyzed with theStudent t test. To analyze efficacy of combination therapy, theKaplan–Meier analysis followed by the log-rank test was per-formed for comparisons of the survival curve. Bonferroni correc-tion was applied for multiple comparisons. A P value less than0.05 was considered to be statistically significant.

ResultsAntitumor effect of DS-8201a in an immunocompetent mousemodel

Because the parental anti-hHER2 antibody of DS-8201a doesnot cross-react with mouse HER2, we prepared a mouse cell linethat stably expresses the human target gene (22–24) and usedthose cells for an immunocompetent mouse tumor model. Thehuman HER2 gene was introduced into CT26.WT cells, andthe expression level of human HER2 on the resultant CT26.WT-hHER2 cells was determined by flow cytometric analysis.Human HER2 was expressed on CT26.WT-hHER2 cells at acomparable level to other representative human HER2-positive(around 2–3þ determined by Herceptest) cancer cell lines, MDA-MB-453 (25, 26) and KPL-4 (refs. 7, 27; Fig. 1A). The CT26.WT-hHER2 cells were inoculated into BALB/c mice subcutaneously,and tumor growth was confirmed (Fig. 1B). When mice withCT26.WT-hHER2 tumors were i.v. treated with vehicle, 10 mg/kgof DS-8201a, or 10mg/kg of its parental anti-hHER2 antibody on

days 0 and 7, themean tumor volumes at day 9 of vehicle-treated,DS-8201a-treated, and anti-hHER2 antibody–treated groupswere936, 402, and 969 mm3, respectively (Fig. 1B). Compared withanti-hHER2 antibody, DS-8201a showed a significant antitumoreffect (P¼ 0.0091 at day 9). Importantly, DS-8201a also exerted asignificant antitumor effect compared with the isotype controlnontargeted ADC (Supplementary Fig. S2). These results indicatethat the CT26.WT-hHER2 model can be used for further studiesand that the antitumor effect of DS-8201a in this model dependson mainly the payload delivered into tumor by anti-hHER2antibody and not on the anti-hHER2 antibody itself.

Contribution of DS-8201a to immune memory formationIn a different experiment, a complete response (CR)was seen in

23%(18/80) ofCT26.WT-hHER2 (s.c. inoculation)–bearingmicetreated with DS-8201a (10 mg/kg, once a week, 2 times, i.v.).These cured mice were rechallenged with CT26.WT-hHER2 orCT26.WT-mock cells (s.c. inoculation). Na€�ve mice (previously

Figure 1.

Antitumor effect of DS-8201a in a syngeneic mouse model. A, Expression ofexogenously introduced human HER2 gene was confirmed in mouse CT26.WT-hHER2 cells (gray-filled peak). B, Antitumor effect was examined in asyngeneic mouse model with CT26.WT-hHER2 cells (s.c. inoculation) upontreatmentwith anti-hHER2 antibody orDS-8201a (10mg/kg, once aweek, twice,i.v.) at the time points indicated by arrows. When tumors were established, thetreatments were initiated (day 0). The graph shows mean tumor volumes andstandard errors (n ¼ 10). A Wilcoxon rank-sum test was conducted for thestatistical analysis of tumor volumes to compare anti-HER2 antibody–treatedand DS-8201a–treated groups on day 9.

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not inoculated with tumor cells) were used as a control cohort.CT26.WT-hHER2 cells were completely rejected in the rechal-lenged mice (Fig. 2A and B). Furthermore, CT26.WT-mock cellswere also rejected in the cured mice from the CT26.WT-hHER2

tumor, although to a lesser extent (Fig. 2A and C). We confirmedthat the CT26.WT-mock and CT26.WT-hHER2 cells can grownormally in na€�ve mice (Fig. 2A, D, and E). These results suggestthat multiple antigens other than human HER2 were recognized

Figure 2.

Antitumor immunity caused by DS-8201a. Mice cured of CT26.WT-hHER2 tumors with DS-8201a (10 mg/kg, once a week, twice, i.v.) were rechallengedwith CT26.WT-mock or CT26.WT-hHER2 cells (s.c. inoculation), and tumor growth was determined. Na€�ve (never tumor-inoculated) mice were used as controls.A, Mean tumor volumes and standard errors (n ¼ 9). B–E, Spider plots of individual data. A Wilcoxon rank-sum test was conducted for the comparisonbetween na€�ve mice and cured mice at 17 days after inoculation.






by the immune system inmice cured of CT26.WT-hHER2 tumorsbyDS-8201a. Thiswas also supported by an increase of IFNg fromsplenocytes. IFNg is a cytokine that is produced mainly byactivated T cells and natural killer (NK) cells and which hasantitumor effects (28, 29). At the endpoint of the rechallengestudy, splenocytes were isolated from each mouse. The spleno-cytes from the na€�ve mice challenged with CT26.WT-HER2 with-out DS-8201a treatment rarely reacted to CT26.WT-hHER2 and

CT26.WT-mock cells in terms of IFNg secretion, whereas thesplenocytes from the mice that had been cured from CT26.WT-hHER2 tumor by DS-8201a treatment and rechallenged withCT26.WT-hHER2 cells were significantly activated by not onlyCT26.WT-hHER2 cells but alsoCT26.WT-mock cells (Fig. 3A). Theresults suggest that DS-8201a treatment induced T cells thatrecognize not only humanHER2 but also other antigens of tumorcells. Similarly, the splenocytes from the na€�ve mice challenged

Figure 3.

IFNg secretion by splenocytes fromthe rechallenged mice. Splenocyteswere obtained from rechallengedmicepreviously cured of CT26.WT-hHER2tumors by DS-8201a and from na€�vemice challenged with CT26.WT-mockor CT26.WT-hHER2 cells. IFNgsecretion was examined by ELISPOTassays. The number of spots wasregarded as number of IFNg-secretingsplenocytes. A, Splenocytes fromthe rechallenged mice withCT26.WT-hHER2 cells were culturedwith CT26.WT-HER2 or CT26.WT-mock cells, and immune cell reactivityto tumor cells was determined by IFNgsecretion. B, Splenocytes from therechallenged mice with CT26.WT-mock cells were cultured withCT26.WT-HER2 or CT26.WT-mockcells, and immune cell reactivity totumor cells was determined by IFNgsecretion. Graphs show the meannumber of IFNg-secreting splenocytesand standard errors (n ¼ 9).A Wilcoxon rank-sum test wasconducted for the comparisonbetween na€�ve mice and cured mice.

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with CT26.WT-mock without DS-8201a treatment rarely reactedto CT26.WT-mock and CT26.WT-hHER2 cells in terms of IFNgsecretion, whereas the splenocytes from the mice that had beencured of CT26.WT-hHER2 tumors by DS-8201a treatment andrechallenged with CT26.WT-mock cells were significantly activat-ed by not only CT26.WT-mock cells but also CT26.WT-hHER2cells compared with cells from the na€�ve mice (Fig. 3B).

Contribution of adaptive immunity for effect of DS-8201aTo examinewhether adaptive immune system is involved in the

antitumor effect of DS-8201a, athymic nudemicewith defective Tand B cells and robust NK cells (30–32) were s.c. inoculated withCT26.WT-hHER2 cells and treated with DS-8201a (10 mg/kg,once a week, twice, i.v.). In contrast with the immunocompetentmousemodel (Fig. 1B), only a slight antitumor effect ofDS-8201awas observed in the immunocompromised athymic nude mousemodel (Supplementary Fig. S3). Actually, a cell proliferation assayof CT26.WT-hHER2 showed it to be less sensitive to DS-8201aand its payloadDXd, comparedwith human cancer cell line KPL-4and human HER2 stably expressing mouse cancer cell line EMT6-hHER2 (Supplementary Fig. S4). These data suggested that even inthe tumors less sensitive to cytotoxic activity of DS-8201a, adap-tive immunity was activated and played important roles in anti-tumor effect observed in the immunocompetent mouse model.

Next, intratumoral T cells were examined in the immunocom-petent mouse model with CT26.WT-hHER2 (s.c. inoculation).Eight days after DS-8201a (10 mg/kg, once, i.v.) treatment, theratio of CD8þ T cells (CD45þCD3þCD8þ cells) andGranzyme Bþ

CD8þ T cells significantly increased compared with vehicle con-trols (Fig. 4). The increased CD8þ cells were also observed by

immunohistochemical staining (Supplementary Fig. S5). Nota-bly, when CD8þ cells were depleted by an anti-CD8 depletionantibody, the antitumor effect of DS-8201a was negated (Sup-plementary Fig. S6). These data suggested that adaptive immu-nity, especially CD8þ T cells, was involved in the antitumor effect.

Increased expression of DC activation markers bytopoisomerase I inhibitor, DXd

It has been reported that various chemotherapeutic agents,including topoisomerase I inhibitors, activate DCs (1, 2, 4,6, 33). Therefore, DC maturation and activation markers, CD86andMHC class II, onmouse BMDCswere evaluated to investigatethe direct effect of DXd, the payload of DS-8201a, on BMDCs. Inthe presence of DXd, CD86 andMHC class II expression increasedin a concentration-dependent manner 24 hours after culture(Fig. 5A). These results showed that the payload of DS-8201adirectly increased DC activation markers and suggested that thismechanism was associated with increased antitumor immunity.

Upregulation of activation marker levels on intratumoralDCs by DS-8201a

Because DXd upregulated activation markers of BMDCsin vitro, the effect ofDS-8201a on intratumoralDCswas examinedin the immunocompetent mouse model with CT26.WT-hHER2(s.c. inoculation). Eight days after DS-8201a (10 mg/kg, once,i.v.) treatment, the ratio of DCs (CD45þCD11cþMHC classIIþ cells) to lymphocytes (CD45þ cells) significantly increasedin DS-8201a–treated tumors compared with vehicle controls(Fig. 5B). Moreover, the percentage of CD86þ cells in DCs aswell as mean fluorescence intensity (MFI) of CD86 on DCs

Figure 4.

Increase of intratumoral CD8þ T cells. Effect of DS-8201a(10 mg/kg, once, i.v.) was examined in theimmunocompetent mouse model subcutaneouslyinoculated with the CT26.WT-hHER2 cells. Theexpression levels of CD45, CD3, CD8, CD4, andGranzyme B in cells from tumors were determined atday 8 by flow cytometry after the treatment. A,Percentages of CD4þ T cells (CD45þ CD3þ CD4þ cells)among live cells. B, Percentages of CD8þ T cells(CD45þ CD3þ CD8þ cells) among live cells. C,Percentages of Granzyme Bþ cells among CD8þ T cells.D, Percentages of Granzyme Bþ CD8þ T cells among livecells. The open circles show individual data, and theblack squares show mean with standard errors (n ¼ 8).The Student t test was conducted for the comparisonbetween the DS-8201a–treated and vehicle-treatedgroups.






significantly increased in DS-8201a–treated tumors comparedwith those of vehicle control (Fig. 5C and D). Based on theseresults, we confirmed that DS-8201a activates DCs in vivo. This is anew role of DS-8201a that is distinct from its previously observedcytotoxic activity (7).

Increased expression of immune-associated molecules ontumor cells by DS-8201a

To examine whether DS-8201a modulates immune-associatedmarkers on tumor cells, PD-L1 and MHC class I expression levelswere determined in the immunocompetent mouse model with

CT26.WT-hHER2 (s.c. inoculation). Eight days after DS-8201atreatment (10mg/kg, once, i.v.),MFI of PD-L1 andMHCclass I onhHER2þ cells was higher in DS-8201a–treated tumors than invehicle controls (Fig. 5E and F). These results are consistent withprevious reports of certain chemotherapeutic agents upregulatingPD-L1 andMHC class I expression on tumor cells (8, 14). Furtherexperiments in vitro suggested that DXd did not obviously changePD-L1 expression on CT26.WT-hHER2 cells (unpublished obser-vation). In contrast, DS-8201a directly increased MHC class Iexpression on CT26.WT-hHER2 cells in vitro (SupplementaryFig. S7A); the effect was comparable at lower dose and evenmore

Figure 5.

Upregulation of activationmarkers onDCs and increase of MHC class I andPD-L1 on tumor cells treated withDS-8201a. A, Mouse BMDCs werecultured in the presence of DXd (openpeaks) or dimethyl sulfoxide (DMSO,dashed lines) for 24 hours, and CD86,CD11c, CD45, and MHC class IIexpression was determined by flowcytometry. DCs were identified asCD45þ CD11cþ cells. Representativeresults are shown. Grey filled peaksindicate non-treated cell population.B–F, Effect of DS-8201a (10 mg/kg,once, i.v.) was examined in theimmunocompetent mouse model s.c.inoculated with the CT26.WT-hHER2cells. Expression of CD86, CD11c,CD45, MHC class II, hHER2, PD-L1, andMHC class I on cells from tumors wasdetermined at 8 days after treatmentby flow cytometry. B, Percentages ofDCs (CD45þ CD11cþ MHC class IIþ

cells) among CD45þ cells. C,Percentages of CD86þ cells amongDCs. D, MFI values of CD86 on DCs.E, MFI values of PD-L1 on hHER2þ

cells. F, MFI values of MHC class I onhHER2þ cells. Open circles showindividual data, and black squaresshow means with standard errors (n¼ 7). The Student t test wasconducted for the comparisonbetween DS-8201a–treated andvehicle-treated groups.

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pronounced at higher concentrations, compared with other ADCpayloads, including DM1, DM4, and MMAE (SupplementaryFig. S7B). The increased MHC class I expression would promotetumor recognition by T cells, as antigen presentation by MHCclass I activates T-cell immunity (34). On the other hand,increased PD-L1 expression is likely a consequence of T-cellactivation and may decrease antitumor immunity in vivo becauseof its inhibitory signals. These results suggest the rationale ofcombining DS-8201a, an immunomodulatory compound, withICIs, especially anti–PD-1/PD-L1–blocking agents, to block theinhibitory signals.

In vivo combination effect of DS-8201a with an anti–PD-1antibody

Whereas DS-8201a demonstrated immune-modulating activ-ity, the expression of PD-L1, an immune-inhibiting molecule,increased during DS-8201a treatment. Therefore, the combina-tion of DS-8201a with an anti–PD-1 antibody was examined inthe immunocompetentmousemodel with CT26.WT-hHER2 (s.c.inoculation). When mice were treated with vehicle, DS-8201a(10mg/kg, once a week, 2 times, i.v.), anti–PD-1 antibody (5mg/kg, twice a week, 2 cycles, i.v.), or a combination of the two, thesurvival rates at day 38were 0%, 20%, 20%, and 80%, respectively(Fig. 6). DS-8201a or anti–PD-1 antibodymonotherapy extendedthe overall survival time (vs. vehicle, P¼ 0.0001 and P¼ 0.0010,respectively), and the combination of DS-8201a and anti–PD-1antibody further increased the overall survival time comparedwith eachmonotherapy (P¼ 0.0006 vs. DS-8201a, P < 0.0001 vs.anti–PD-1 antibody). The number of mice that achieved a CRwith vehicle, DS-8201a, anti–PD-1 antibody, and combination ofDS-8201a and anti–PD-1 antibody were 0, 4, 2, and 13 out of 20,respectively. These results suggest that the PD-1/PD-L1 pathway

inhibits activation of the immune system by DS-8201a. Thus, theanti–PD-1 antibody can block the PD-1/PD-L1 inhibitory signaland increase the efficacy of DS-8201a. Therefore, the benefit ofcombination therapy with DS-8201a and anti–PD-1 antibody isstrongly supported. This combination therapy is expected to be anew effective treatment for HER2-positive tumors.

DiscussionA variety of preclinical studies have been conducted using

immunocompromisedmousemodels (xenograftmousemodels)to see the direct effect of agents on human-derived tumor cells. Insuch experiments, the importance of the immune system, espe-cially T and B cells, tended to be overlooked, as their functionsand/or numbers are impaired in the models. In our studies, weexamined the immunologic effect of DS-8201a by using animmunocompetent mouse model and identified a novel role ofDS-8201a. DS-8201a activated the mouse immune system andattenuated tumor growth. The importance of the immune systemfor the antitumor effect of DS-8201a was supported by theincreased expression of MHC class I in tumor cells, the increasedexpression of DC activation markers, the increase of tumor-infiltrating CD8þ T cells, the negation of DS-8201a antitumoreffect by CD8þ cell depletion or in athymic nude mice, and therejection of rechallenged tumor cells in the immunocompetentmouse model.

The parental anti-hHER2 antibody and isotype control non-targeted ADC did not show an obvious antitumor effect in themodel. Therefore, the antitumor effect of DS-8201a is primarilydependent on the payload delivered into HER2-expressingtumors by the antibody. Since it has been reported that anti-ErbB2 antibody itself can promote antitumor immunity andshows combination benefit with anti–PD-1 antibody in a mousemodel, a larger DS-8201a effect would be expected if tumors areHER2 signal dependent (35). Remarkably, the mice whose CT26.WT-hHER2 tumors had been cured by DS-8201a treatmentrejected not only rechallenged CT26.WT-hHER2 but also CT-26.WT-mock cells, suggesting that the antigens were not limitedto human HER2 but also to other CT26.WT cell–derived mole-cules. Multiple recognition of tumor antigens by the immunesystem was also suggested by the experiments where splenocytesfrom the CT26.WT-hHER2–rejected mice were reactive evenagainst CT26.WT-mock cells. Previous evidence also suggests thespreading of antigens by chemotherapeutic agents or antibodies(15), and the mode of action of DS-8201a is possibly associatedwith antigen spreading where tumor cells are killed by the pay-load, and tumor antigens are recognizedby theDS-8201a–elicitedimmune response. Notably, although direct antitumor effect ofDS-8201a on CT26.WT-hHER2 cells was not substantial in thecultured cells (Supplementary Fig. S4) and thenudemousemodel(Supplementary Fig. S3), immune cells recognized the tumorantigens (Fig. 3) and mediated antitumor effect in the immuno-competent mouse model (Fig. 1B; Supplementary Fig. S6). Theseresults support the idea that small fraction of tumor cell deathcould be enough to induce immune reaction and subsequentimmune-mediated antitumor activity in case of DS-8201a. Thisidea is also supported by another type of payload (19).

The payload of DS-8201a has 10 times more potent topoisom-erase I inhibitory activity than SN-38 (7), and this could lead tomore immunologic cell death than treatment with SN-38 andother existing topoisomerase I inhibitors. In addition, DS-8201a

Figure 6.

Combination effect of DS-8201a and anti–PD-1 blocking antibody. In vivo effectof DS-8201a (10 mg/kg, once a week, twice, i.v.) combined with anti–PD-1antibody (5 mg/kg, twice a week, 2 cycles, i.v.) was compared with each agentindividually in the immunocompetent mouse model s.c. inoculated with theCT26.WT-hHER2 cells. Compounds were administered at the time pointsindicated by arrows. Survival was analyzed by the Kaplan–Meier method, andgroups were compared with the log-rank test (n ¼ 20). Vehicle-treated groupwas compared with DS-8201a–treated group and anti–PD-1 antibody–treatedgroup (P ¼ 0.0001 and P ¼ 0.0010, respectively). DS-8201a and anti–PD-1antibody–treated group was also compared with the DS-8201a–treated groupand the anti–PD-1 antibody–treated group (P ¼ 0.0006 and P < 0.0001,respectively).






works selectively at the tumor site; therefore, it kills tumor cellswith minimal damage to immune cells compared with tradi-tional systemic treatment with chemotherapeutic agents, pos-sibly leading to efficient activation of antitumor immunity viarelease of tumor cell antigens (2, 36). Therefore, DS-8201a canbe considered a potent immune stimulator, and further anal-yses to show whether DS-8201a induces immunologic celldeath are needed.

Mechanisms underlying the increased immunity and theimmunologic memory effect would rely on a series of processesincluding cell death, activation of DCs, T-cell activation, andpossibly others. DS-8201a upregulated the expression of DCmaturation and activation markers both in vitro and in vivo andincreased the intratumoral DC population in vivo. A recent reportshows that a topoisomerase I inhibitor activates DC via cancercells (6). These results indicated that topoisomerase I inhibitorsincluding DXd were potentially associated with DC activation.In addition, DXd directly upregulated MHC class I expression ontumor cells in vitro, and DS-8201a enhanced MHC class I expres-sion on tumor cells in vivo. The increased MHC class I expressionpossibly contributed to the increased immune activity and immu-nologic memory formation. Recently, three different topoisom-erase I inhibitors (camptothecin, topotecan, irinotecan) havebeen shown to enhance tumor-killing activity of T cells (5). Ourresults in this study also support this idea. Although the antitumoreffect of DS-8201a was significant, the effect was not sufficientto show CR, as only approximately 20 % of tumor-bearing micewith DS-8201a achieved CR in the CT26.WT-hHER2 model. Thereason could be that DS-8201a also upregulated tumor PD-L1expression as the consequence of T-cell activation. Therefore, wecombined DS-8201a and anti–PD-1 antibody, and the combina-tion increased the antitumor effect.

ICIs show remarkable antitumor effects in patientswith variouscancers (12–14). However, only a limited number of patientsrespond to ICIs (15). Therefore, new treatments for poor respon-ders are needed. One solution to this problem is combinationtherapy. Combination benefits of HER2-targeting therapy withICIs have been shown inmousemodels (18, 35) and are currentlybeing evaluated in clinical trials (37). For combination with ICIs,ADCs are considered to be better than chemotherapeutic agentsbecause ADCs work selectively at the tumor site with reduced sideeffect to other organs. T-DM1 has shown immune activation andbenefits in combination with ICIs in vivo, and DM1, a componentof T-DM1, activates DCs in vitro (18).

In this study, DS-8201a, a HER2-targeting ADC with atopoisomerase I inhibitor, showed an antitumor effect with

immunostimulatory activity in a mouse model. DS-8201a alsobenefited from combination with anti–PD-1 antibody, as evi-denced by the following results: (1) DS-8201a increased MHCclass I expression on tumor cells, in addition to their cytotoxicactivity, (2) DS-8201a upregulated DC activation markers, (3)DS-8201a activated an adaptive immune response via (1) and(2), followed by PD-L1 expression, and (4) the combination ofDS-8201a and anti–PD-1 antibody showed higher antitumoreffect. Further, DXd-based ADC technology is widely capable ofbeing applied to various antibodies. Theoretically, other DXd-based ADCs with the same payload, but different antibodies,would show similar immunostimulatory activity. The results ofthis study provide preliminary evidence for the potential role ofDXd-based ADCs and ICIs. Further studies including clinicalstudies are needed to better understand the role of this com-bination in cancers.

Disclosure of Potential Conflicts of InterestT.N. Iwata, C. Ishii, S. Ishida, and T. Wada have ownership interest

(including patents) in a patent application. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: T.N. Iwata, T. Wada, T. AgatsumaDevelopment of methodology: T.N. IwataAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.N. Iwata, C. Ishii, S. IshidaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.N. IwataWriting, review, and/or revision of the manuscript: T.N. Iwata, Y. Ogitani,T. Wada, T. AgatsumaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. OgitaniStudy supervision: Y. Ogitani, T. Wada, T. Agatsuma

AcknowledgmentsThis study was funded by Daiichi Sankyo Co., Ltd.The authors would like to thank Yuki Abe, Kiyoshi Sugihara, Masaya

Ishizaki, Kyoko Harikae, Masato Hata, Takafumi Otsuka, Jun Harada,Hirokazu Ishikawa, Megumi Minami, Yoshinori Kashimoto, and othercolleagues in the facility for critical review, comments, and materials.

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 August 9, 2017; revised December 7, 2017; accepted April 17, 2018;published first April 27, 2018.

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2018;17:1494-1503. Published OnlineFirst April 27, 2018.Mol Cancer Ther Tomomi Nakayama Iwata, Chiaki Ishii, Saori Ishida, et al. ModelDeruxtecan (DS-8201a), Enhances Antitumor Immunity in a Mouse

Drug Conjugate, Trastuzumab−A HER2-Targeting Antibody

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