Enfortumab Vedotin Antibody Drug Conjugate Targeting Nectin-4 … ·...

Therapeutics, Targets, and Chemical Biology

Enfortumab Vedotin Antibody–Drug ConjugateTargeting Nectin-4 Is a Highly Potent TherapeuticAgent in Multiple Preclinical Cancer ModelsPia M. Challita-Eid, Daulet Satpayev, Peng Yang, Zili An, Karen Morrison,Yuriy Shostak, Arthur Raitano, Rossana Nadell, Wendy Liu, Dawn Ratay Lortie,Linnette Capo, Alla Verlinsky, Monica Leavitt, Faisal Malik, Hector Avi~na,Claudia I. Guevara, Nick Dinh, Sher Karki, Banmeet S. Anand, Daniel S. Pereira,Ingrid B.J. Joseph, Fernando Do~nate, Kendall Morrison, and David R. Stover

Abstract

The identification of optimal target antigens on tumor cells iscentral to the advancement of new antibody-based cancertherapies. We performed suppression subtractive hybridizationand identified nectin-4 (PVRL4), a type I transmembrane pro-tein and member of a family of related immunoglobulin-likeadhesion molecules, as a potential target in epithelial cancers.We conducted immunohistochemical analysis of 2,394 patientspecimens from bladder, breast, lung, pancreatic, ovarian,head/neck, and esophageal tumors and found that 69% of allspecimens stained positive for nectin-4. Moderate to strongstaining was especially observed in 60% of bladder and 53% ofbreast tumor specimens, whereas the expression of nectin-4 innormal tissue was more limited. We generated a novel anti-body–drug conjugate (ADC) enfortumab vedotin comprising

the human anti-nectin-4 antibody conjugated to the highlypotent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin(also known as ASG-22ME) ADC were able to bind to cellsurface–expressed nectin-4 with high affinity and induced celldeath in vitro in a dose-dependent manner. Treatment of mousexenograft models of human breast, bladder, pancreatic, andlung cancers with enfortumab vedotin significantly inhibitedthe growth of all four tumor types and resulted in tumorregression of breast and bladder xenografts. Overall, thesefindings validate nectin-4 as an attractive therapeutic target inmultiple solid tumors and support further clinical develop-ment, investigation, and application of nectin-4–targetingADCs. Cancer Res; 76(10); 3003–13. �2016 AACR.

IntroductionCancers of epithelial origin represent a significant global med-

ical challenge that impacts patients, their families, and the healthcare system. Transitional cell carcinoma (TCC) of the bladder, acommonly diagnosed epithelial cancer, has an incidence rateestimated at >72,000 cases per year in the United States (1).Approximately 15,000 patients die annually from the metastaticform of the disease. Treatment options for metastatic TCC arequite limited andmost were developed decades ago (1, 2). Recentadvances in cancer genomics, such as identification of drivermutations and immune modulators that play a role in TCC, havemotivated the development of targeted therapies that inhibitgrowth factors, such as FGFR3 and immune checkpoint proteins,such as programmed cell death-1 ligand (PDL-1; ref. 3).

Antibody–drug conjugates (ADC) are an emerging class oftargeted therapeutic agents with the ability to deliver a highlycytotoxic payload to tumor sites by harnessing the exquisitespecificity of a mAb as a delivery vehicle (4, 5). The ADCsbrentuximab vedotin (Adcetris), which comprises an anti-CD30mAb conjugated to a microtubule inhibitor, and trastuzumabemtansine (Kadcyla), which comprises an anti-HER2 mAb con-jugated to amicrotubule-disrupting agent, received approval fromthe FDA for treatment of CD30-positive hematologic cancers (inpatients with classical Hodgkin lymphoma at high risk of relapseor progression as autologous hematopoietic stem cell transplan-tation consolidation and relapsed systemic anaplastic large celllymphoma) and HER2-positive breast cancer, respectively. Bothagents demonstrated durable and sustained responses in clinicaltrials (6–9).

Using suppression subtractive hybridization, we identified theprotein nectin-4, also known as poliovirus receptor–related pro-tein 4 (PVRL4), as a potential ADC target, as it showedhighmRNAexpression in bladder cancer. Nectin-4 is a member of the nectinfamily of immunoglobulin-like adhesionmolecules (10–12) thatare proposed tomediate Ca2þ-independent cell–cell adhesion viaboth homophilic and heterophilic trans-interactions at adherensjunctions, where they recruit cadherins and modulate cytoskele-ton rearrangements (12). On the basis of the differential expres-sion of nectin-4 in bladder cancers, we developed a novel ADC,enfortumab vedotin, comprising fusion between a fully human

Agensys Inc., Santa Monica, California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for D. Satpayev: ImaginAb, Inglewood, CA 90301.

Corresponding Author: Pia M. Challita-Eid, Agensys, Inc., 1800 Stewart Street,Santa Monica, CA 90404. Phone: 424-280-5000; Fax: 424-280-5044; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-1313

�2016 American Association for Cancer Research.

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Published OnlineFirst March 24, 2016; DOI: 10.1158/0008-5472.CAN-15-1313

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antibody targeting nectin-4 and the potent microtubule-disrupt-ing agent monomethyl auristatin E (MMAE). Our findings dem-onstrate that the ADC specifically binds human, as well as rat andmonkey nectin-4, inhibits in vitro growth of cell lines that expresscell surface nectin-4, and inhibits growth of several target-expres-sing xenograft tumors, including patient-derived xenografts. Bothversions of enfortumab vedotin, hybridoma AGS-22M6E (alsoknown as ASG-22ME) and Chinese hamster ovary (CHO) ASG-22CE, show eradication of established tumor xenografts. Ourpreclinical data suggest that anti-nectin-4 ADC has great potentialas a therapeutic agent for multiple cancer indications in theclinical setting.

Materials and MethodsCell lines and xenografts

Human cancer cell lines T-47D (breast cancer) and NCI-H322M (small-cell bronchoalveolar carcinoma) were obtainedfrom the NCI (2004; Frederick, MD). PC-3 (prostate cancer) waspurchased from the ATCC (2008) and cultured in RPMI1640media (Life Technologies) supplemented with 10% FBS (OmegaScientific) and 1% glutamine (Life Technologies). Rat1(E) fibro-blast cell line of rat origin, a gift fromDr. Robert Eisenman (2002;Fred Hutchinson Cancer Research Center, Seattle, WA), wascultured in DMEM (Life Technologies) supplemented with10% FBS and 1% glutamine (13). Cell lines were passaged inour laboratory for fewer than 6 months after their resuscitation.Human cell lines were confirmed utilizing short tandem repeatprofiling (Promega). AG-B1, AG-Br7, and AG-Panc4 are Agensys'proprietary, patient-derived tumor xenografts of bladder, breast,and pancreatic cancers, respectively. Tumors were propagated byserial passaging in SCID mice. Nectin-4 expression in the xeno-grafts was routinely monitored by qPCR and IHC.

Expression vectors, cloning, and purification of recombinantproteins

Human, cynomolgus monkey, and rat nectin-4 cDNAs werecloned into the retroviral vector pSRa, a gift from Dr. OwenWitte(University of California, Los Angeles, CA), or pBabe-puro (CellBiolabs, Inc.), using conventional recombinant DNA techniques.Viral particles for transduction were generated by cotransfectionof the recombinant retroviral vectors with helper plasmid encod-ing gag/pol/env proteins into 293FT cells (Life Technologies). AcDNA encoding amino acids 1–357 of the extracellular domain(ECD) of human nectin-4 was cloned into the pET vector forexpression in Escherichia coli. The complete ECDs of nectin-4 andnectin-1 were cloned into mammalian expression vectors witheither a myc-his tag or an IgG1 Fc fusion for expression in 293FTmammalian cells. Recombinant proteins were purified usingaffinity chromatography either onNi-NTAor proteinA/Gagarose.

IHCAmAb (M22-244b3) against the ECD of E. coli–derived recom-

binant human nectin-4 was generated using standard mousehybridoma technology. The antibody was validated for nectin-4 specificity by correlating staining patterns on formalin-fixed,paraffin-embedded (FFPE) sections of various cell lines, xeno-grafted tissues, and patient samples with available RNA data. Thespecificity of the antibody was also confirmed by Western blotanalysis. Tissue microarrays (TMA) were obtained from US Bio-max and TriStar Technology Group LLC. After deparaffinization

and rehydration, tissue sections were treated for antigen retrievalwith EDTA for 30 minutes and incubated with the primaryantibody M22-244b3 or isotype control antibody mouse IgG1kfor 1 hour. Expression was then detectedwith the BioGenex SuperSensitive Polymer-HRP IHC Detection Kit (BioGenex Laborato-ries Inc). The intensity and extent of nectin-4 expression wasdetermined microscopically using the histochemical scoringsystem (H-score), defined as the sum of the products of thestaining intensity (score of 0–3) multiplied by the percentage ofcells (0–100) stained at a given intensity. Specimens were thenclassified as negative (H-score 0–14), weak (H-score 15–99),moderate (H-score 100–199), and strong (H-score 200–300).

Flow cytometryAdherent cell monolayers were detached by treatment with 0.5

mmol/L EDTA in PBS, washed, and incubated with anti-nectin-4antibodies for 1 hour at 4�C. The cells were then washed andstained with phycoerythrin-conjugated goat anti-mouse IgG(Jackson ImmunoResearch Laboratories Inc.) and analyzed byflow cytometry.

Antibody and ADC generationThe fully humanmAb AGS-22M6 targeting the ECD of human

nectin-4 was generated using the Xenomouse platform developedby Amgen (formerly Abgenix; ref. 14). Briefly, human IgG1antibody–producing Xenomice were immunized with the puri-fied ECD of human nectin-4. B cells from lymph nodes andspleens were fused with the SP2/0 myeloma fusion partner.Hybridoma screening to identify antibodies specific for nectin-4 was performed using ELISA and FACS assays. For the recombi-nant expression of AGS-22M6 antibody in mammalian cells, theAGS-22M6 variable heavy chain and k light chains weresequenced from the corresponding hybridoma and cloned intothe pEE12.4 expression vector (Lonza), and the resulting plasmidwas transfected into CHO host cells (CHOK1SV, Lonza). Trans-fected cells were subjected to selection in the absence of glutamineto generate a stable antibody–producing cell line. The CHOK1SV-derived antibody is referred to as ASG-22C. ADCs referred to asAGS-22M6E (also known as ASG-22ME) and ASG-22CE weregenerated via conjugation of hybridoma-produced AGS-22M6andCHO-derived ASG-22C antibodies, respectively, to the small-molecule microtubule-disrupting agent MMAE via the cleavabledrug linker maleimidocaproylvaline-citrulline-p-aminobenzy-loxycarbonyl using technology licensed from Seattle Genetics,Inc. as previously published (15). The antibodies AGS-22M6 andASG-22C, as well as isotype control antibody, were conjugated toMMAE following partial reduction of interchain disulfide bondswith tris (2-carboxyethyl)-phosphine as described previously(16). The drug-to-antibody ratio was approximately 4:1.

Cytotoxicity assayPC-3 cells engineered to express nectin-4 of human,monkey, or

rat origin or T-47D breast carcinoma cells were incubated withserially diluted AGS-22M6Eor isotype control ADC at 37�C in 5%CO2. Cell viability was measured after 5 days using AlamarBlue(Life Technologies). Percent survivalwas calculated as the numberof live cells in treated wells divided by the number of live cells incontrol wells. The IC50 was derived from the survival curve usingthe sigmoid Emax nonlinear regression analysis function inGraphPad Prism (GraphPad).

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In vivo studiesAll experimental protocols were approved by Agensys' Insti-

tutional Animal Care and Use Committee. The bladder cancerAG-B1 xenograft model was established at Agensys by seriallypassaging a patient specimen of bladder cancer in SCID mice.The AG-Br7 xenograft model was derived from a specimen of apatient with triple-negative breast cancer. AG-Panc4 was estab-lished from a patient with moderately differentiated adenocar-cinoma of the pancreas (17). NCI-H322M lung adenocarcino-ma cell line was used as a lung cancer model. Five- to 6-week-old ICR-SCID mice were obtained from Taconic BioSciences.For experiments with subcutaneous models of AG-B1, AG-Br7,and AG-Panc4, xenograft tumor fragments (5–6 pieces, 1 mm3

each) were implanted subcutaneously into the flank of eachmouse. For experiments using the orthotopic breast tumormodel AG-Br7, tumor pieces were enzymatically digested tosingle-cell suspension using Liberase Blendzyme (RocheApplied Science), and 3 � 106 cells were injected into themammary fat pad of individual female SCID mice. For experi-ments with NCI-H322M lung adenocarcinoma–derived xeno-grafts, mice were injected with 2.5 � 106 tumor cells subcuta-neously into the flank of the mice. Number of mice used foreach experiment is indicated in Fig. 6. Treatment by intrave-nous administration was initiated when tumors reachedapproximately 200 mm3 in size. Tumor length (L) and width(W) were measured with a caliper, and tumor volume wascalculated using the formula W2 � L/2. Percent tumor regres-sion was calculated as the tumor volume in a mouse treatedwith AGS-22M6E relative to the tumor volume in mice treatedwith control ADC or vehicle control. Percent tumor regressionat the end of the study was calculated as the tumor volume ineach mouse one day before sacrifice relative to the tumor

volume in the same mouse on day 1 of the study. A statisticalanalysis of the tumor volumes at the start of treatment and oneday before animal sacrifice was performed using the nonpara-metric Kruskal–Wallis test. Pairwise comparisons were madeusing the Tukey–Kramer method (two sided) on the ranks ofthe data to protect experiment-wise error rate.

ResultsNectin-4 expression in normal tissues and tumor specimens

In a series of discovery experiments using suppression sub-tractive hybridization as described previously (18), we identi-fied nectin-4 (also known as PVRL4) as a gene markedlyupregulated in bladder cancer. Nectin-4 is a type I transmem-brane polypeptide member of the nectin family of adhesionmolecules and was first identified through a bioinformaticssearch by Reymond and colleagues (19). We generated a mouseantibody, referred to as M22-244b3, directed against the ECDof human nectin-4 expressed in E. coli. M22-244b3 is specificfor nectin-4 and does not crossreact with other nectin familymembers (data not shown). Immunohistochemical analysisusing M22-244b3 of a panel of 294 normal tissue specimensrepresenting 36 human organs showed homogenous weak tomoderate staining mainly in human skin keratinocytes, skinappendages (sweat glands and hair follicles), transitional epi-thelium of bladder, salivary gland (ducts), esophagus, breast,and stomach. Representative specimens are shown in Fig. 1.Weak staining was detected in samples of larynx, pituitary,placenta, testis, ureter, and uterus.

Next, we conducted an extensive immunohistochemical ana-lysis of nectin-4 expression in a total of 2,394 cases of hu-man cancers from 34 tumor TMAs representing 7 different

Figure 1.Nectin-4 expression in human normaltissues. Positive immunohistochemicalstaining for nectin-4 in FFPE humantissue sections is indicated by brown-color precipitate in skin (A); salivaryglands (B); bladder (C); and esophagus(D). Lack of nectin-4 staining is shownin ovary (E) and spleen (F).

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indications—bladder, lung, breast, pancreatic, ovarian, head/neck, and esophageal cancers. As shown in Table 1, across allevaluated cancer indications, 69% of TMA cancer specimens werepositive for nectin-4. Highest frequencies for overall expression ofnectin-4were observed for bladder, breast, andpancreatic tumors.Moderate or strong staining (indicated by H-score) was mostfrequently observed in bladder and breast specimens: 60% ofbladder and 53%of breast cancer specimens showed either strongor moderate staining, with approximately equal percentageswithin each tumor type. In ovarian, head/neck, and esophagealtumor specimens, the prevalence of nectin-4–positive specimenswith moderate or strong staining was generally lower and signif-icantlymore pronounced in themucinous type. Strong expressionwas detected in both lung adenocarcinoma and squamous cellcarcinoma (SCC; 5% and 10%, respectively). Representativeimages of moderate and strong immunohistochemical stainingacross tumor specimens are shown in Fig. 2. Notably, specificimmunoreactivity for nectin-4 was localized predominantly oncell membrane or cell membrane and cytoplasm of tumor cells.Metastatic tissue specimens (typically lymph nodes of bladder,breast, lung, and ovarian cancers) included in some TMAs showedsimilar H-score of nectin-4 expression as observed for the primarytumors. Nectin-4 was detected in 60% of lung cancer metastasis(22/31 adenocarcinoma, 24/34 SCC, and 0/12 small-cell carci-noma). In ovarian cancer, nectin-4 was detected in 77%of patientmetastasis specimens (30/38 serous adenocarcinoma, 1/1mucin-ous adenocarcinoma, and 0/1 transitional carcinoma).

Antibody and ADC characterizationA fully humanmAb, AGS-22M6,was generated by immunizing

human IgG1–producing strain of XenoMouse with the humannectin-4 ECD. AGS-22M6 was selected among a panel of >50distinct anti-nectin-4 mAb produced in the XenoMouse. Thisantibody binds to transfected human, monkey, or rat nectin-4

expressed on the cell surface of PC-3 cells (Fig. 3A) and endog-enous nectin-4 expressed on the surface of NCI-H322M and T-47D cells (Fig. 3B). The PC-3 cell line was selected for transfectionas it does not express endogenous nectin-4. The apparent affinity-binding constant (KD) value for nectin-4 expressed on T-47Dbreast cancer cells was 0.01 nmol/L (Fig. 3C). We also found asimilar KD for AGS-22M6E, the ADC produced by conjugation tovcMMAE (Fig. 3C). Thus, the conjugation process did not alterbinding characteristics of parental antibody AGS-22M6 to nectin-4 on the surface of cancer cell lines.

To further characterize AGS-22M6, we constructed deletionmutants consisting of the membrane-distant (V) or membrane-proximal (C0C00) domains of nectin-4 fused to its transmembraneand intracellular domains. We expressed the domain-deletedproteins on the surface of Rat1(E) fibroblasts and conductedbinding experiments with AGS-22M6 or another anti-nectin-4antibody, AGS-22M104, which had been found to bind nectin-4within its C0C00 and not the V region. Notably, AGS-22M6 recog-nized an epitope located in the V-domain with the same apparentaffinity as to the full-length nectin-4 expressed in the same hostcells (Fig. 4A). AGS-22M6 did not bind to the C0C00 domain. Proofthat lack of binding of AGS-22M6 was not due to lack of expres-sion of the C0C00 domain mutant was demonstrated by the abilityof AGS-22M104 to exhibit binding in the assay.

The V-domains of nectin molecules were previously demon-strated tomediate homo- andheterodimerizationof nectins in cis-and trans- configurations, and nectin-4was shown to interact withnectin-1 with nanomolar affinity (20). Therefore, we evaluatedthe ability of AGS-22M6 to block the interaction of nectin-1 withnectin-4 expressed on the surface of Rat1(E) fibroblasts. As shownin Fig. 4B, a biotinylated ECDof nectin-1 expressed as an Fc fusionprotein was able to bind to cell surface nectin-4, and this inter-action was effectively disrupted by the addition of either AGS-22M6 or the AGS-22M6E ADC.

Table 1. Expression of nectin-4 in human tumor specimens

Intensity of staininga

Strong Moderate Low Negative Overall positiveCancer type N (%) N (%) N (%) N (%) (%)

Bladder (N ¼ 524) 162 (31) 154 (29) 118 (23) 90 (17)Transitional (N ¼ 467) (34) (30) (21) (16) 83Metastasis (N ¼ 25) (12) (44) (36) (8)Others (N ¼ 32) (6) (16) (31) (47)

Breast (N ¼ 654) 174 (27) 168 (26) 170 (26) 142 (22)Ductal carcinoma (N ¼ 386) (31) (25) (23) (21)Lobular carcinoma (N ¼ 30) (20) (27) (33) (20) 78Metastasis (N ¼ 203) (18) (27) (31) (24)Others (N ¼ 35) (31) (26) (23) (20)

Pancreatic (N ¼ 164) 21 (13) 39 (24) 56 (34) 48 (29) 71Lung (N ¼ 618) 46 (7) 121 (20) 173 (28) 278 (45)Squamous (N ¼ 235) (10) (22) (30) (38)Adenocarcinoma (N ¼ 212) (5) (25) (34) (36)Small cell carcinoma (N ¼ 50) (0) (0) (2) (98) 55Metastasis (N ¼ 77) (12) (19) (29) (40)Others (N ¼ 44) (7) (5) (18) (70)

Ovarian (N ¼ 118) 0 21 (18) 46 (39) 51 (43)Serous (N ¼ 60) 0 (2) (45) (53)Mucinous (N ¼ 14) 0 (50) (7) (43) 57Metastasis (N ¼ 40) 0 (32) (45) (23)Others (N ¼ 4) 0 (0) (0) (100)

Head & Neck (N ¼ 135) 3 (2) 22 (16) 54 (40) 56 (41) 59Esophageal (N ¼ 181) 7 (4) 37 (20) 55 (30) 82 (45) 55Total (N ¼ 2,394) 413 (17) 562 (24) 672 (28) 747 (31) 69aIntensity of staining: strong, H-score ¼ 200–300; moderate, H-score ¼ 100–199; low, H-score ¼ 15–99; negative, H-score ¼ 0–14.

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In vitro cytotoxicityThe ability of the AGS-22M6E ADC to inhibit cell survival was

evaluated in the PC-3 cell lines engineered to express human,monkey, or rat nectin-4, as well as in the T-47D human breastcarcinoma cells that endogenously express nectin-4. Serial dilu-tions of AGS-22M6, AGS-22M6E ADC, and an isotype controlADC were added to cells, and cell viability was measured after 5days. As shown in Fig. 5, AGS-22M6E ADC, but not unconjugatedAGS-22M6 or control ADC, induced dose-dependent inhibitionof cell viability both in T-47D cells and in PC-3 cells transfectedwith human, monkey, or rat nectin-4. AGS-22M6E ADC had noeffect onmock-transfected PC-3 cell controls. We did not observeany significant cytotoxic activity using an isotype control ADC onthe various PC-3 cell lines.

In vivo efficacyTo evaluate the therapeutic potential of AGS-22M6E ADC,

we performed a set of in vivo efficacy studies in murine modelsof xenografted human cancers. Both cell line–derived andproprietary patient tumor–derived xenografts were selected onthe basis of the expression of human nectin-4. A total of 13xenograft models were evaluated across 4 cancer indications(bladder, breast, pancreas, and lung). Tumor size ranged from100 to 200 mm3 at the start of the treatment in all models, andADC was administered in all studies by intravenous injection.Overall, AGS-22M6E ADC induced tumor regression in 5 of the13 models and resulted in significant inhibition of establishedtumor growth in 12 of 13 models examined. Representativestudies using an AG-B1 bladder cancer model, two AG-Br7

Bladdercancer

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Figure 2.Nectin-4 expression in human cancerpatient specimens. RepresentativeFFPE tissue sections analyzed by IHCfor nectin-4 are shown. A, transitionalbladder carcinoma with strongexpression. B, transitional bladdercancer with moderate expression.C, breast carcinoma with strongexpression. D, breast carcinoma withmoderate expression. E, lungsquamous carcinoma. F, lungadenocarcinoma. G, pancreaticadenocarcinoma with strongexpression. H, pancreaticadenocarcinoma with moderateexpression. I, ovarian serous carcinoma.J, ovarian mucinous carcinoma.






breast cancer models, and an AG-Panc4 pancreatic cancermodel are described below. Nectin-4 expression in these xeno-graft tumors is shown in Fig. 6A. In all these in vivo antitumorefficacy studies, the ADCs were well tolerated. No reduction inbody weight or signs of distress was observed in any of thetreatment groups (data not shown).

Treatment of AG-B1 xenograft tumors with single 4 mg/kgdoses of AGS-22M6E or two other anti-nectin-4 ADCs causedregression of established tumors (Fig. 6B). Immunohistochemicalevaluation of the tumor site at the end of the study showedcomplete tumor eradication in 5 of the 6 mice treated withAGS-22M6E. Depending on the treatment regimen, AGS-22M6Ewas able to eradicate or significantly inhibit tumor growthin this bladder cancer model. As shown in Fig. 6C, AGS-22M6E at0.8 mg/kg dosed every 4 days for a total of 5 doses significantlyinhibited growth of established AG-B1 tumor xenografts whencompared with control ADC at 0.8 mg/kg (P < 0.0001), whereasAGS-22M6E given at the lower dose of 0.4 mg/kg did not showsignificant antitumor activity when compared with any of thecontrols (P > 0.05). IHC analysis of xenografts from treatedanimals showed strong AGS-22M6E ADC localization to thexenograft tumorswithin 6hours of treatment, whereas the control

ADC is trapped in the stroma (Supplementary Fig. S1). Localiza-tion of ADC peaks at 24 hours and starts diffusing at 72 hours(Supplementary Fig. S1).

We next evaluated potency of AGS-22M6E against breast cancerusing the AG-Br7 subcutaneous xenograftmodel. As shown in Fig.6D, AGS-22M6E at 3 mg/kg had a potent tumor inhibitory effectwhen compared with the control ADC, the unconjugated anti-body AGS-22M6 or the vehicle control: 91.3% inhibition versuscontrol ADC (P < 0.0001); 94.2% inhibition versus AGS22M6(P< 0.0001); and 93.9% inhibition versus vehicle (P < 0.0001). Inaddition, treatment with 3 mg/kg AGS-22M6E resulted in regres-sion of the tumor by 62.9% at the end of the study relative to thestarting tumor size (P < 0.0001). AGS-22M6E given at the lowerdose of 1 mg/kg also showed significant effect when comparedwith either control ADC at the equivalent dose (P ¼ 0.0371) orvehicle control (P¼0.0003), resulting in 44.5%and34.6% tumorinhibition, respectively. A statistically significant difference (P ¼0.0147) was detected when the two doses of AGS-22M6E werecompared, indicating a dose-dependent effect of AGS-22M6E. Theefficacy of AGS-22M6E was also examined against an orthotopicxenograft, in which AG-Br7 tumors were established inmammaryfat pads of SCID mice. As shown in Fig. 6E, AGS-22M6E

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Figure 3.AGS-22M6 antibody specificallyrecognizes cell-surface human nectin-4and its orthologs. PC-3 cells engineered toexpress nectin-4 on the cell surface, andcell lines NCI-H322M and T-47D, bothof which express endogenous nectin-4on the cell surface, were subjected toimmunocytological staining with AGS-22M6 and analyzed by flow cytometry. A,cell counts of PC-3 cells expressing human,monkey, and rat nectin-4 (openhistograms) were overlaid against mock-transfected PC-3 cells (solid histogram).B, counts of NCI-H322M and T-47D (openhistogram) were overlaid against isotypecontrol–stained cells (solid histogram).C, saturation curve of AGS-22M6 and AGS-22M6E binding to nectin-4 expressed on T-47D by flow cytometry shows KD of 0.01nmol/L. MFI, mean fluorescence intensity.

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Figure 4.Identification of the AGS-22M6–binding domain. A, Rat1(E) cells recombinantly expressing either full-length nectin-4, or domain-deleted mutants containing onlythe V domain or C0-C00 domains were stained with AGS-22M6 or AGS-22M104 at 10 mg/mL for 1 hour at 4�C, followed by binding of phycoerythrin (PE)-conjugated anti-human IgG antibody. The cells were then analyzed by flow cytometry and histogram results (open histogram) were overlaid against Rat1(E)mock-transfected cells (solid histogram). B, a fusion of the Fc of human IgG1 with the ECD of recombinant nectin-1 (nectin-1-Fc) or a control-Fc protein werebiotinylated and incubated with nectin-4–expressing Rat1(E) cells at 300 ng/mL in the absence or presence of increasing concentrations of AGS-22M6 orAGS-22M6E for 1 hour. Cells were washed, stained with streptavidin-PE (Jackson ImmunoResearch Laboratories), and analyzed by flow cytometry. Histogramof nectin-1-Fc compared with control-Fc binding in the absence of antibody is shown in the insert.






administered either as a single 10 mg/kg dose or as two 5 mg/kgdoses resulted in complete eradication of established tumors.Lower doses were not explored in this model.

In the AG-Panc4 model, AGS-22M6E at 3 mg/kg led to tumorstasis, with significant tumor growth inhibition compared withisotype control ADC at equivalent dosage (Fig. 6F). In the NCI-H322M lung adenocarcinoma xenograft model, AGS-22M6E at 3mg/kg resulted in 83.6% tumor regression compared with isotypecontrol ADC at the same dose (P < 0.0001, Fig. 6G). In addition,treatment with AGS-22M6E at 3 mg/kg resulted in 20.5% tumorregression (P¼ 0.03461). AGS-22M6E given at a lower dose of 1.0mg/kg did not show a significant effect when compared withcontrol ADC at the equivalent dose (P ¼ 0.1455).

In support of the clinical development of an ADC targetingnectin-4, the AGS-22M6 antibody produced in the hybridomasystem was converted into a human recombinant productexpressed in CHO cells as detailed in Materials and Methods. Acomparability analysis between the hybridoma-derived AGS-22M6E ADC and the CHO-derived ASG-22CE ADC was per-formed in vitro and in vivo. Both ADCs showed identical in vitrocharacteristics: binding constants for nectin-4 were 0.057 and0.060 nmol/L for AGS-22M6E and ASG-22CE, respectively, andIC50 of 1.523 and 1.674 nmol/L, respectively. When injected inmice, both AGS-22M6E and ASG-22CE showed comparablepharmacokinetic profile up to 10 days (Supplementary Fig.S2; Table 1). ADC and total IgG exhibited similar exposure andserum elimination half-life ranging from 1.5 to 2 days. They alsoexhibited equivalent dose-dependent tumor growth inhibition inthe breast cancer AG-Br7 xenograft model (Fig. 6H).

DiscussionNectin-4 was originally cloned from human trachea and

described as an antigen with a restricted pattern of expression

in normal tissues (19). More recently, nectin-4 was identified asan entry point for measles and other viruses and was proposedas a target for oncolytic viral therapy (21, 22). Although severalgroups reported the detection of nectin-4 expression in breast,ovarian, and lung cancers (23–26), no comprehensive analysishas been reported to date. Using suppression subtractive hybrid-ization, we identified nectin-4 as one of the genes dramaticallyupregulated in bladder cancer specimens. Our RNA expressionanalysis of a larger cancer sample panel and of normal tissuesalso indicated that nectin-4 transcript is present at low levels invarious normal tissues and is upregulated in a subset of breast,pancreatic, and lung cancer specimens (Agensys, unpublisheddata). In this report, we used IHC to demonstrate nectin-4protein expression in normal tissue specimens and in TMAsfrom various types of cancer. Our results showed broaderexpression in normal tissues than was previously reported.Nectin-4 expression was detected in the epithelia of bladder,breast, stomach, esophagus, salivary gland (ducts), and skin(epidermis and sweat glands). Epithelial components of otherorgans showed weaker and less consistent staining for nectin-4.Investigation of normal tissue antigen expression is essential forthe clinical progression of an ADC. Prohibitive on-target toxi-cities have been observed for several ADCs, such as skin toxi-cities (27). The extensive expression profiling of nectin-4described herein allowed us to identify normal tissues that mayraise the potential risk of eliciting nectin-4 on-target toxicities.These have been investigated in two cross-reactive species (ratand cynomolgus monkey), as well as in the clinical setting.Preliminarily, the findings suggest a favorable therapeutic win-dow, but details will be reported as part of a complete analysisof phase I results.

In cancer patient specimens, >50% samples tested positivefor nectin-4 protein expression, and the level of expression,based on IHC staining intensity, was highest in bladder cancer

T-47D

AGS22M6AGS22M6E ADCIsotype control ADC

IC50 = 37.8 ng/mL(95% CI, 25.8–55.4)

IC50 = 1.2 ng/mL(95% CI, 0.8–1.9)

IC50 = 3.4 ng/mL(95% CI, 2.5–5.3)

IC50 = 4.7 ng/mL(95% CI, 3.2–6.9)

Rat nectin-4Monkey nectin-4Human nectin-4Mock

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Figure 5.AGS-22M6E ADC shows potent and specific in vitro cytotoxicity.PC-3 cells engineered to express human, monkey, or rat nectin-4, as well as T-47D breast cancer cells, were incubated withincreasing concentrations of unconjugated AGS-22M6,conjugated AGS-22M6E ADC, or isotype control ADC for5 days. CI, confidence interval.


Challita-Eid et al.




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Figure 6.Nectin-4–targeted ADC shows significant tumor growth inhibition in multiple xenograft models. A, examples of nectin-4 staining in the in vivo xenograftmodels of AG-B1, AG-Br7, AG-Panc4, and NCI-H322M, as well as mouse IgG1 isotype staining of AG-B1 as background staining control. Tumor growthover time for AG-B1 subcutaneous xenografts treated with three different ADC at 4 mg/kg (B); AG-B1 subcutaneous xenografts treated with AGS-22M6E (orcontrols) at 0.8 and 0.4 mg/kg (C); AG-Br7 subcutaneous xenografts treated with AGS-22M6E (or controls) at 1 and 3 mg/kg (D); AG-Br7 orthotopicxenografts treated with AGS-22M6E (or controls) at 5 and 10 mg/kg (E); AG-Panc4 subcutaneous xenografts treated with AGS-22M6E (or controls) at1 and 3 mg/kg (F); and NCI-H322M subcutaneous xenografts treated with AGS-22M6E (or controls) at 1 and 3 mg/kg (G). H, tumor growth inhibition byAGS-22M6E ADC and ASG-22CE ADC in the breast cancer AG-Br7 xenograft model. Treatment was initiated on day 0 as indicated by arrows. Meantumor volume data for each group were plotted over time with SE bars.






and breast cancer. In general, good correlation between proteinexpression based on IHC and previously observed transcriptlevels was observed. Overall, IHC analyses demonstrated mod-erate to strong expression (H-score > 100) of nectin-4 in 60% ofbladder cancer and 53% of breast cancer specimens. In addi-tion, 37% and 27% of pancreatic and lung cancer specimens,respectively, demonstrated moderate to strong expression. Gen-erally, we observed similar overall frequency of nectin-4 expres-sion in ovarian cancer as previously published by Derycke andcolleagues (26).

Soluble form of Nectin-4 in patient serum and in ascites incase of ovarian cancer has been reported (24–26). Althoughthe levels varied between reports, all investigators show anassociation between higher detected levels of soluble nectin-4and disease progression. Many other membrane-anchoredproteins release their ECD through proteolytic cleavage, suchas HER2 and EGFR (28, 29). However, clinical significance ofsuch form has been difficult to establish, and effect onresponse to targeted therapy has not been fully elucidated(30–32).

AGS-22M6, a fully human antibody targeting nectin-4, wasselected from a panel of >50 antibodies for further evaluationas a drug conjugate based on its high affinity, broad speciescross-reactivity, and ability to mediate very potent cell deathboth in vitro and in vivo. The ADC AGS-22M6E, composed ofAGS-22M6E conjugated to MMAE, exhibited dose-dependentantitumor activity in vivo. At lower doses (�1 mg/kg), the ADCinhibited growth of bladder and breast cancer xenografts. Athigher doses (�3 mg/kg), AGS-22M6E induced tumor regres-sion of the subcutaneous bladder cancer xenograft, the sub-cutaneous breast cancer xenograft, and the orthotopic breastcancer xenograft. In addition, AGS-22M6E inhibited thegrowth of both the pancreatic cancer xenograft and the lungadenocarcinoma xenograft, with associated modest tumorregression in the lung tumor model. The in vivo efficacy ofAGS-22M6E generally correlated well with the expression levelof nectin-4 in xenografts and cell lines. In most of the studiesdescribed herein, the parental unconjugated antibody AGS-22M6 was included as a control for efficacy. We concluded thatbinding of unconjugated antibody to nectin-4 does not resultin antitumor activity in either in vitro or in vivo preclinicalmodels.

Our data also demonstrated that under the conditions of anin vitro binding assay, AGS-22M6 and AGS-22M6E blocked theinteraction of nectin-4 with nectin-1, its putative bindingpartner. The functional significance of this observation is pres-ently unknown, as the role of nectin-4 in normal epithelialtissue structure and morphogenesis is poorly understood. Weand others hypothesize that nectin-4 plays a role in the estab-lishment and maintenance of adherens junctions, which innormal epithelium define cell polarity, a characteristic oftenlost during cancer progression (12). We have attempted, butnot yet succeeded, in identifying an anti-nectin-4 antibody thatintrinsically modulates tumor growth. In a recent publication,Pavlova and colleagues (33) demonstrated that nectin-4 pro-motes anchorage-independent growth in breast cancer cells byactivation of the matrix-independent integrin b4/SHP-2/c-Srcand that the transformation of breast cancer cells to anchorageindependence is dependent on nectin-4. The authors alsoreported modest inhibition of growth of a breast cancer xeno-graft by a mAb targeted against human nectin-4. The mecha-

nism of action of such antibody will need to be furtherinvestigated.

On the basis of its ability to induce tumor regression inhuman breast and bladder cancer specimens, as well its favor-able toxicology profile in various species (Agensys, unpub-lished data), AGS-22M6E, the hybridoma-derived ADC, wasinvestigated in early-phase clinical studies for safety and phar-macokinetics in patients with solid tumors that express nectin-4(ClinicalTrials.gov identifier NCT01409135). Because pivotalregistration trials will require an antibody produced at hightiters using mammalian cells, we have subsequently developeda comparable CHO-derived anti-nectin-4 antibody, ASG-22CE.Our preclinical studies with ASG-22CE ADC showed that it hasequivalent binding and potency as the hybridoma product.ASG-22CE entered the clinic in 2014 as monotherapy inpatients with metastatic urothelial cancer, as well as other solidtumors that express nectin-4 (ClinicalTrials.gov identifierNCT02091999), and is currently under clinical developmentas enfortumab vedotin.

Overall, the data presented herein indicate that nectin-4 is aviable target for ADC therapy in carcinomas expressing nectin-4and support the evaluation of such ADCs in the clinical setting.The preclinical data demonstrate growth inhibition acrossmultiple human xenograft tumor models and tumor regressionin bladder and breast cancer models. Our finding of nectin-4overexpression in human tumor specimens, particularly thosefrom patients with breast, bladder, pancreatic, or lung cancer,suggests that a significant proportion of patients may benefitfrom nectin-4 targeted delivery of a potent cytotoxic agent tothe tumor site.

Disclosure of Potential Conflicts of InterestEnfortumab Vedotin is being codeveloped in conjunction with Agensys'

partner, Seattle Genetics, Inc. No potential conflicts of interest weredisclosed.

Authors' ContributionsConception and design: P.M. Challita-Eid, D. Satpayev, Z. An, Y. Shostak, I.B.J.Joseph, F. Do~nate, K. Morrison, D.R. StoverDevelopment of methodology: P.M. Challita-Eid, D. Satpayev, Z. An,K. Morrison, A. Raitano, H. Avi~na, C.I. Guevara, S. Karki, K. MorrisonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Satpayev, P. Yang, K. Morrison, Y. Shostak,R. Nadell, W. Liu, D.R. Lortie, L. Capo, A. Verlinsky, M. Leavitt, F. Malik,H. Avi~na, C.I. Guevara, N. Dinh, I.B.J. Joseph, F. Do~nate, K. MorrisonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.M. Challita-Eid, D. Satpayev, K. Morrison,Y. Shostak, A. Raitano, D.R. Lortie, M. Leavitt, H. Avi~na, C.I. Guevara, S. Karki,B.S. Anand, I.B.J. Joseph, F. Do~nate, K. Morrison, D.R. StoverWriting, review, and/or revision of the manuscript: P.M. Challita-Eid,D. Satpayev, Z. An, L. Capo, B.S. Anand, D.S. Pereira, I.B.J. Joseph, F. Do~nate,K. Morrison, D.R. StoverAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P.M. Challita-Eid, D. Satpayev, S. Karki,D.R. StoverStudy supervision: P.M. Challita-Eid, D. Satpayev, Z. An, A. Raitano, I.B.J.Joseph, F. Doñate, D.R. StoverOther (developed the antibody): R. Nadell

AcknowledgmentsThe authors thank the hardworking and thoughtful members of the Agensys

research departments that did not appear in this article but that contributed toprevious conference presentations or contributed to historical data that ulti-mately led to this final article.


Challita-Eid et al.




Grant SupportThis study was funded by Agensys, Inc., an affiliate of Astellas Pharma,

Inc.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 14, 2015; revised January 7, 2016; accepted February 26, 2016;published OnlineFirst March 24, 2016.

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2016;76:3003-3013. Published OnlineFirst March 24, 2016.Cancer Res Pia M. Challita-Eid, Daulet Satpayev, Peng Yang, et al. ModelsIs a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer

Drug Conjugate Targeting Nectin-4−Enfortumab Vedotin Antibody

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