Enfortumab Vedotin Antibody–Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models Pia M

Published OnlineFirst March 24, 2016; DOI: 10.1158/0008-5472.CAN-15-1313 Cancer Therapeutics, Targets, and Chemical Biology Research

Enfortumab Vedotin Antibody–Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models Pia 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 Avina,~ Claudia I. Guevara, Nick Dinh, Sher Karki, Banmeet S. Anand, Daniel S. Pereira, Ingrid B.J. Joseph, Fernando Donate,~ Kendall Morrison, and David R. Stover

Abstract

The identification of optimal target antigens on tumor cells is the human anti-nectin-4 antibody conjugated to the highly central to the advancement of new antibody-based cancer potent microtubule-disrupting agent MMAE. Hybridoma (AGS- therapies. We performed suppression subtractive hybridization 22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin and identified nectin-4 (PVRL4), a type I transmembrane pro- (also known as ASG-22ME) ADC were able to bind to cell tein and member of a family of related immunoglobulin-like surface–expressed nectin-4 with high affinity and induced cell adhesion molecules, as a potential target in epithelial cancers. death in vitro in a dose-dependent manner. Treatment of mouse We conducted immunohistochemical analysis of 2,394 patient xenograft models of human breast, bladder, pancreatic, and specimens from bladder, breast, lung, pancreatic, ovarian, lung cancers with enfortumab vedotin significantly inhibited head/neck, and esophageal tumors and found that 69% of all the growth of all four tumor types and resulted in tumor specimens stained positive for nectin-4. Moderate to strong regression of breast and bladder xenografts. Overall, these staining was especially observed in 60% of bladder and 53% of findings validate nectin-4 as an attractive therapeutic target in breast tumor specimens, whereas the expression of nectin-4 in multiple solid tumors and support further clinical develop- normal tissue was more limited. We generated a novel anti- ment, investigation, and application of nectin-4–targeting body–drug conjugate (ADC) enfortumab vedotin comprising ADCs. Cancer Res; 76(10); 1–11. 2016 AACR.

Introduction Antibody–drug conjugates (ADC) are an emerging class of targeted therapeutic agents with the ability to deliver a highly Cancers of epithelial origin represent a significant global med- cytotoxic payload to tumor sites by harnessing the exquisite ical challenge that impacts patients, their families, and the health specificity of a mAb as a delivery vehicle (4, 5). The ADCs care system. Transitional cell carcinoma (TCC) of the bladder, a brentuximab vedotin (Adcetris), which comprises an anti-CD30 commonly diagnosed epithelial cancer, has an incidence rate mAb conjugated to a microtubule inhibitor, and trastuzumab estimated at >72,000 cases per year in the United States (1). emtansine (Kadcyla), which comprises an anti-HER2 mAb con- Approximately 15,000 patients die annually from the metastatic jugated to a microtubule-disrupting agent, received approval from form of the disease. Treatment options for metastatic TCC are the FDA for treatment of CD30-positive hematologic cancers (in quite limited and most were developed decades ago (1, 2). Recent patients with classical Hodgkin lymphoma at high risk of relapse advances in cancer genomics, such as identification of driver or progression as autologous hematopoietic stem cell transplan- mutations and immune modulators that play a role in TCC, have tation consolidation and relapsed systemic anaplastic large cell motivated the development of targeted therapies that inhibit lymphoma) and HER2-positive breast cancer, respectively. Both growth factors, such as FGFR3 and immune checkpoint proteins, agents demonstrated durable and sustained responses in clinical such as programmed cell death-1 ligand (PDL-1; ref. 3). trials (6–9). Using suppression subtractive hybridization, we identified the – Agensys Inc., Santa Monica, California. protein nectin-4, also known as poliovirus receptor related protein 4 (PVRL4), as a potential ADC target, as it showed high mRNA Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). expression in bladder cancer. Nectin-4 is a member of the nectin family of immunoglobulin-like adhesion molecules (10–12) that Current address for D. Satpayev: ImaginAb, Inglewood, CA 90301. þ are proposed to mediate Ca2 -independent cell–cell adhesion via Corresponding Author: Pia M. Challita-Eid, Agensys, Inc., 1800 Stewart Street, both homophilic and heterophilic trans-interactions at adherens Santa Monica, CA 90404. Phone: 424-280-5000; Fax: 424-280-5044; E-mail: junctions, where they recruit cadherins and modulate cytoskele- [email protected] ton rearrangements (12). On the basis of the differential expres- doi: 10.1158/0008-5472.CAN-15-1313 sion of nectin-4 in bladder cancers, we developed a novel ADC, 2016 American Association for Cancer Research. enfortumab vedotin, comprising fusion between a fully human

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antibody targeting nectin-4 and the potent microtubule-disrupt- and rehydration, tissue sections were treated for antigen retrieval ing agent monomethyl auristatin E (MMAE). Our findings dem- with EDTA for 30 minutes and incubated with the primary onstrate that the ADC specifically binds human, as well as rat and antibody M22-244b3 or isotype control antibody mouse IgG1k monkey nectin-4, inhibits in vitro growth of cell lines that express for 1 hour. Expression was then detected with the BioGenex Super cell surface nectin-4, and inhibits growth of several target-expres- Sensitive Polymer-HRP IHC Detection Kit (BioGenex Laborato- sing xenograft tumors, including patient-derived xenografts. Both ries Inc). The intensity and extent of nectin-4 expression was versions of enfortumab vedotin, hybridoma AGS-22M6E (also determined microscopically using the histochemical scoring known as ASG-22ME) and Chinese hamster ovary (CHO) ASG- system (H-score), defined as the sum of the products of the 22CE, show eradication of established tumor xenografts. Our staining intensity (score of 0–3) multiplied by the percentage of preclinical data suggest that anti-nectin-4 ADC has great potential cells (0–100) stained at a given intensity. Specimens were then as a therapeutic agent for multiple cancer indications in the classified as negative (H-score 0–14), weak (H-score 15–99), clinical setting. moderate (H-score 100–199), and strong (H-score 200–300).

Materials and Methods Flow cytometry Adherent cell monolayers were detached by treatment with 0.5 Cell lines and xenografts mmol/L EDTA in PBS, washed, and incubated with anti-nectin-4 Human cancer cell lines T-47D (breast cancer) and NCI- antibodies for 1 hour at 4 C. The cells were then washed and H322M (small-cell bronchoalveolar carcinoma) were obtained stained with phycoerythrin-conjugated goat anti-mouse IgG from the NCI (2004; Frederick, MD). PC-3 (prostate cancer) was (Jackson ImmunoResearch Laboratories Inc.) and analyzed by purchased from the ATCC (2008) and cultured in RPMI1640 flow cytometry. media (Life Technologies) supplemented with 10% FBS (Omega Scientific) and 1% glutamine (Life Technologies). Rat1(E) fibro- blast cell line of rat origin, a gift from Dr. Robert Eisenman (2002; Antibody and ADC generation Fred Hutchinson Cancer Research Center, Seattle, WA), was The fully human mAb AGS-22M6 targeting the ECD of human cultured in DMEM (Life Technologies) supplemented with nectin-4 was generated using the Xenomouse platform developed 10% FBS and 1% glutamine (13). Cell lines were passaged in by Amgen (formerly Abgenix; ref. 14). Briefly, human IgG1 our laboratory for fewer than 6 months after their resuscitation. antibody–producing Xenomice were immunized with the puri- Human cell lines were confirmed utilizing short tandem repeat fied ECD of human nectin-4. B cells from lymph nodes and profiling (Promega). AG-B1, AG-Br7, and AG-Panc4 are Agensys' spleens were fused with the SP2/0 myeloma fusion partner. proprietary, patient-derived tumor xenografts of bladder, breast, Hybridoma screening to identify antibodies specific for nectin- and pancreatic cancers, respectively. Tumors were propagated by 4 was performed using ELISA and FACS assays. For the recombi- serial passaging in SCID mice. Nectin-4 expression in the xeno- nant expression of AGS-22M6 antibody in mammalian cells, the grafts was routinely monitored by qPCR and IHC. AGS-22M6 variable heavy chain and k light chains were sequenced from the corresponding hybridoma and cloned into Expression vectors, cloning, and purification of recombinant the pEE12.4 expression vector (Lonza), and the resulting plasmid proteins was transfected into CHO host cells (CHOK1SV, Lonza). Trans- Human, cynomolgus monkey, and rat nectin-4 cDNAs were fected cells were subjected to selection in the absence of glutamine – cloned into the retroviral vector pSRa, a gift from Dr. Owen Witte to generate a stable antibody producing cell line. The CHOK1SV- (University of California, Los Angeles, CA), or pBabe-puro (Cell derived antibody is referred to as ASG-22C. ADCs referred to as Biolabs, Inc.), using conventional recombinant DNA techniques. AGS-22M6E (also known as ASG-22ME) and ASG-22CE were Viral particles for transduction were generated by cotransfection generated via conjugation of hybridoma-produced AGS-22M6 of the recombinant retroviral vectors with helper plasmid encod- and CHO-derived ASG-22C antibodies, respectively, to the small- ing gag/pol/env proteins into 293FT cells (Life Technologies). A molecule microtubule-disrupting agent MMAE via the cleavable cDNA encoding amino acids 1–357 of the extracellular domain drug linker maleimidocaproylvaline-citrulline-p-aminobenzy- (ECD) of human nectin-4 was cloned into the pET vector for loxycarbonyl using technology licensed from Seattle Genetics, expression in Escherichia coli. The complete ECDs of nectin-4 and Inc. as previously published (15). The antibodies AGS-22M6 and nectin-1 were cloned into mammalian expression vectors with ASG-22C, as well as isotype control antibody, were conjugated to fi either a myc-his tag or an IgG1 Fc fusion for expression in 293FT MMAE following partial reduction of interchain disul de bonds mammalian cells. Recombinant proteins were purified using with tris (2-carboxyethyl)-phosphine as described previously affinity chromatography either on Ni-NTA or protein A/G agarose. (16). The drug-to-antibody ratio was approximately 4:1.

IHC Cytotoxicity assay A mAb (M22-244b3) against the ECD of E. coli–derived recom- PC-3 cells engineered to express nectin-4 of human, monkey, or binant human nectin-4 was generated using standard mouse rat origin or T-47D breast carcinoma cells were incubated with hybridoma technology. The antibody was validated for nectin- serially diluted AGS-22M6E or isotype control ADC at 37Cin5% 4 specificity by correlating staining patterns on formalin-fixed, CO2. Cell viability was measured after 5 days using AlamarBlue paraffin-embedded (FFPE) sections of various cell lines, xeno- (Life Technologies). Percent survival was calculated as the number grafted tissues, and patient samples with available RNA data. The of live cells in treated wells divided by the number of live cells in specificity of the antibody was also confirmed by Western blot control wells. The IC50 was derived from the survival curve using analysis. Tissue microarrays (TMA) were obtained from US Bio- the sigmoid Emax nonlinear regression analysis function in max and TriStar Technology Group LLC. After deparaffinization GraphPad Prism (GraphPad).

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In vivo studies volume in the same mouse on day 1 of the study. A statistical All experimental protocols were approved by Agensys' Insti- analysis of the tumor volumes at the start of treatment and one tutional Animal Care and Use Committee. The bladder cancer day before animal sacrifice was performed using the nonpara- AG-B1 xenograft model was established at Agensys by serially metric Kruskal–Wallis test. Pairwise comparisons were made passaging a patient specimen of bladder cancer in SCID mice. using the Tukey–Kramer method (two sided) on the ranks of The AG-Br7 xenograft model was derived from a specimen of a the data to protect experiment-wise error rate. patient with triple-negative breast cancer. AG-Panc4 was established from a patient with moderately differentiated adenocar- Results cinoma of the pancreas (17). NCI-H322M lung adenocarcinoma cell line was used as a lung cancer model. Five- to 6-week- Nectin-4 expression in normal tissues and tumor specimens old ICR-SCID mice were obtained from Taconic BioSciences. In a series of discovery experiments using suppression sub- For experiments with subcutaneous models of AG-B1, AG-Br7, tractive hybridization as described previously (18), we identi- and AG-Panc4, xenograft tumor fragments (5–6pieces,1mm3 fied nectin-4 (also known as PVRL4) as a gene markedly each) were implanted subcutaneously into the flank of each upregulated in bladder cancer. Nectin-4 is a type I transmem- mouse. For experiments using the orthotopic breast tumor brane polypeptide member of the nectin family of adhesion model AG-Br7, tumor pieces were enzymatically digested to molecules and was first identified through a bioinformatics single-cell suspension using Liberase Blendzyme (Roche search by Reymond and colleagues (19). We generated a mouse Applied Science), and 3 106 cells were injected into the antibody, referred to as M22-244b3, directed against the ECD mammary fat pad of individual female SCID mice. For experi- of human nectin-4 expressed in E. coli. M22-244b3 is specific ments with NCI-H322M lung adenocarcinoma–derived xeno- for nectin-4 and does not crossreact with other nectin family grafts,micewereinjectedwith2.5 106 tumor cells subcuta- members (data not shown). Immunohistochemical analysis neously into the flank of the mice. Number of mice used for using M22-244b3 of a panel of 294 normal tissue specimens each experiment is indicated in Fig. 6. Treatment by intrave- representing 36 human organs showed homogenous weak to nous administration was initiated when tumors reached moderate staining mainly in humanskinkeratinocytes,skin approximately 200 mm3 in size. Tumor length (L)andwidth appendages (sweat glands and hair follicles), transitional epi- (W) were measured with a caliper, and tumor volume was thelium of bladder, salivary gland (ducts), esophagus, breast, calculated using the formula W2 L/2. Percent tumor regres- and stomach. Representative specimens are shown in Fig. 1. sion was calculated as the tumor volume in a mouse treated Weak staining was detected in samples of larynx, pituitary, with AGS-22M6E relative to the tumor volume in mice treated placenta, testis, ureter, and uterus. with control ADC or vehicle control. Percent tumor regression Next, we conducted an extensive immunohistochemical ana- at the end of the study was calculated as the tumor volume in lysis of nectin-4 expression in a total of 2,394 cases of hu- each mouse one day before sacrifice relative to the tumor man cancers from 34 tumor TMAs representing 7 different

Figure 1. Nectin-4 expression in human normal tissues. Positive immunohistochemical staining for nectin-4 in FFPE human tissue sections is indicated by brown- color precipitate in skin (A); salivary glands (B); bladder (C); and esophagus (D). Lack of nectin-4 staining is shown in ovary (E) and spleen (F).

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Table 1. Expression of nectin-4 in human tumor specimens Intensity of staininga Strong Moderate Low Negative Overall positive Cancer 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) 83 Metastasis (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) 78 Metastasis (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) 71 Lung (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) 55 Metastasis (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) 57 Metastasis (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) 59 Esophageal (N ¼ 181) 7 (4) 37 (20) 55 (30) 82 (45) 55 Total (N ¼ 2,394) 413 (17) 562 (24) 672 (28) 747 (31) 69 aIntensity 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.

indications—bladder, lung, breast, pancreatic, ovarian, head/ expressed on the cell surface of PC-3 cells (Fig. 3A) and endog- neck, and esophageal cancers. As shown in Table 1, across all enous nectin-4 expressed on the surface of NCI-H322M and T- evaluated cancer indications, 69% of TMA cancer specimens were 47D cells (Fig. 3B). The PC-3 cell line was selected for transfection positive for nectin-4. Highest frequencies for overall expression of as it does not express endogenous nectin-4. The apparent affinity- nectin-4 were observed for bladder, breast, and pancreatic tumors. binding constant (KD) value for nectin-4 expressed on T-47D Moderate or strong staining (indicated by H-score) was most breast cancer cells was 0.01 nmol/L (Fig. 3C). We also found a frequently observed in bladder and breast specimens: 60% of similar KD for AGS-22M6E, the ADC produced by conjugation to bladder and 53% of breast cancer specimens showed either strong vcMMAE (Fig. 3C). Thus, the conjugation process did not alter or moderate staining, with approximately equal percentages binding characteristics of parental antibody AGS-22M6 to nectin- within each tumor type. In ovarian, head/neck, and esophageal 4 on the surface of cancer cell lines. tumor specimens, the prevalence of nectin-4–positive specimens To further characterize AGS-22M6, we constructed deletion with moderate or strong staining was generally lower and signif- mutants consisting of the membrane-distant (V) or membrane- icantly more pronounced in the mucinous type. Strong expression proximal (C0C00) domains of nectin-4 fused to its transmembrane was detected in both lung adenocarcinoma and squamous cell and intracellular domains. We expressed the domain-deleted carcinoma (SCC; 5% and 10%, respectively). Representative proteins on the surface of Rat1(E) fibroblasts and conducted images of moderate and strong immunohistochemical staining binding experiments with AGS-22M6 or another anti-nectin-4 across tumor specimens are shown in Fig. 2. Notably, specific antibody, AGS-22M104, which had been found to bind nectin-4 immunoreactivity for nectin-4 was localized predominantly on within its C0C00 and not the V region. Notably, AGS-22M6 recog- cell membrane or cell membrane and cytoplasm of tumor cells. nized an epitope located in the V-domain with the same apparent Metastatic tissue specimens (typically lymph nodes of bladder, affinity as to the full-length nectin-4 expressed in the same host breast, lung, and ovarian cancers) included in some TMAs showed cells (Fig. 4A). AGS-22M6 did not bind to the C0C00 domain. Proof similar H-score of nectin-4 expression as observed for the primary that lack of binding of AGS-22M6 was not due to lack of expres- tumors. Nectin-4 was detected in 60% of lung cancer metastasis sion of the C0C00 domain mutant was demonstrated by the ability (22/31 adenocarcinoma, 24/34 SCC, and 0/12 small-cell carci- of AGS-22M104 to exhibit binding in the assay. noma). In ovarian cancer, nectin-4 was detected in 77% of patient The V-domains of nectin molecules were previously demon- metastasis specimens (30/38 serous adenocarcinoma, 1/1 mucin- strated to mediate homo- and heterodimerization of nectins in cis- ous adenocarcinoma, and 0/1 transitional carcinoma). and trans- configurations, and nectin-4 was shown to interact with nectin-1 with nanomolar affinity (20). Therefore, we evaluated Antibody and ADC characterization the ability of AGS-22M6 to block the interaction of nectin-1 with A fully human mAb, AGS-22M6, was generated by immunizing nectin-4 expressed on the surface of Rat1(E) fibroblasts. As shown human IgG1–producing strain of XenoMouse with the human in Fig. 4B, a biotinylated ECD of nectin-1 expressed as an Fc fusion nectin-4 ECD. AGS-22M6 was selected among a panel of >50 protein was able to bind to cell surface nectin-4, and this inter- distinct anti-nectin-4 mAb produced in the XenoMouse. This action was effectively disrupted by the addition of either AGS- antibody binds to transfected human, monkey, or rat nectin-4 22M6 or the AGS-22M6E ADC.

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A B

Bladder cancer

C D

Breast cancer

Figure 2. Nectin-4 expression in human cancer patient specimens. Representative FFPE tissue sections analyzed by IHC for nectin-4 are shown. A, transitional E F bladder carcinoma with strong expression. B, transitional bladder cancer with moderate expression. Lung C, breast carcinoma with strong cancer expression. D, breast carcinoma with moderate expression. E, lung squamous carcinoma. F, lung adenocarcinoma. G, pancreatic adenocarcinoma with strong expression. H, pancreatic adenocarcinoma with moderate G H expression. I, ovarian serous carcinoma. J, ovarian mucinous carcinoma. Pancreac cancer

I J Ovarian cancer

In vitro cytotoxicity In vivo efficacy The ability of the AGS-22M6E ADC to inhibit cell survival was To evaluate the therapeutic potential of AGS-22M6E ADC, evaluated in the PC-3 cell lines engineered to express human, we performed a set of in vivo efficacy studies in murine models monkey, or rat nectin-4, as well as in the T-47D human breast of xenografted human cancers. Both cell line–derived and carcinoma cells that endogenously express nectin-4. Serial dilu- proprietary patient tumor–derived xenografts were selected on tions of AGS-22M6, AGS-22M6E ADC, and an isotype control the basis of the expression of human nectin-4. A total of 13 ADC were added to cells, and cell viability was measured after 5 xenograft models were evaluated across 4 cancer indications days. As shown in Fig. 5, AGS-22M6E ADC, but not unconjugated (bladder, breast, pancreas, and lung). Tumor size ranged from AGS-22M6 or control ADC, induced dose-dependent inhibition 100 to 200 mm3 at the start of the treatment in all models, and of cell viability both in T-47D cells and in PC-3 cells transfected ADC was administered in all studies by intravenous injection. with human, monkey, or rat nectin-4. AGS-22M6E ADC had no Overall, AGS-22M6E ADC induced tumor regression in 5 of the effect on mock-transfected PC-3 cell controls. We did not observe 13 models and resulted in significant inhibition of established any significant cytotoxic activity using an isotype control ADC on tumorgrowthin12of13modelsexamined.Representative the various PC-3 cell lines. studies using an AG-B1 bladder cancer model, two AG-Br7

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A Recombinant cells Human necn-4 Monkey necn-4 Rat necn-4 Counts Counts Counts 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Figure 3. FL2-H FL2-H FL2-H AGS-22M6 antibody speciﬁcally recognizes cell-surface human nectin-4 Endogenously expressing cells and its orthologs. PC-3 cells engineered to B express nectin-4 on the cell surface, and NCI-H322M T-47D cell lines NCI-H322M and T-47D, both of which express endogenous nectin-4 on the cell surface, were subjected to immunocytological staining with AGS- 22M6 and analyzed by ﬂow cytometry. A, cell counts of PC-3 cells expressing human, monkey, and rat nectin-4 (open Counts Counts

5101520 histograms) were overlaid against mock- transfected PC-3 cells (solid histogram). B, counts of NCI-H322M and T-47D (open 0 0 5 10 15 20 100 101 102 103 104 100 101 102 103 104 histogram) were overlaid against isotype – FL2-H FL2-H control stained cells (solid histogram). C, saturation curve of AGS-22M6 and AGS- C Affinity binding 22M6E binding to nectin-4 expressed on T- 47D by ﬂow cytometry shows KD of 0.01 300 ﬂ K nmol/L. MFI, mean uorescence intensity. D = 0.01 nmol/L

200

MFI AGS-22M6E 100 AGS-22M6

0 0.00001 0.0001 0.001 0.01 0.1 1 10 100 nmol/L

breast cancer models, and an AG-Panc4 pancreatic cancer ADC is trapped in the stroma (Supplementary Fig. S1). Localiza- model are described below. Nectin-4 expression in these xeno- tion of ADC peaks at 24 hours and starts diffusing at 72 hours grafttumorsisshowninFig.6A.Inallthesein vivo antitumor (Supplementary Fig. S1). efficacy studies, the ADCs were well tolerated. No reduction in We next evaluated potency of AGS-22M6E against breast cancer body weight or signs of distress was observed in any of the using the AG-Br7 subcutaneous xenograft model. As shown in Fig. treatment groups (data not shown). 6D, AGS-22M6E at 3 mg/kg had a potent tumor inhibitory effect Treatment of AG-B1 xenograft tumors with single 4 mg/kg when compared with the control ADC, the unconjugated anti- doses of AGS-22M6E or two other anti-nectin-4 ADCs caused body AGS-22M6 or the vehicle control: 91.3% inhibition versus regression of established tumors (Fig. 6B). Immunohistochemical control ADC (P < 0.0001); 94.2% inhibition versus AGS22M6 evaluation of the tumor site at the end of the study showed (P < 0.0001); and 93.9% inhibition versus vehicle (P < 0.0001). In complete tumor eradication in 5 of the 6 mice treated with addition, treatment with 3 mg/kg AGS-22M6E resulted in regres- AGS-22M6E. Depending on the treatment regimen, AGS- sion of the tumor by 62.9% at the end of the study relative to the 22M6E was able to eradicate or significantly inhibit tumor growth starting tumor size (P < 0.0001). AGS-22M6E given at the lower in this bladder cancer model. As shown in Fig. 6C, AGS-22M6E at dose of 1 mg/kg also showed significant effect when compared 0.8 mg/kg dosed every 4 days for a total of 5 doses significantly with either control ADC at the equivalent dose (P ¼ 0.0371) or inhibited growth of established AG-B1 tumor xenografts when vehicle control (P ¼ 0.0003), resulting in 44.5% and 34.6% tumor compared with control ADC at 0.8 mg/kg (P < 0.0001), whereas inhibition, respectively. A statistically significant difference (P ¼ AGS-22M6E given at the lower dose of 0.4 mg/kg did not show 0.0147) was detected when the two doses of AGS-22M6E were significant antitumor activity when compared with any of the compared, indicating a dose-dependent effect of AGS-22M6E. The controls (P > 0.05). IHC analysis of xenografts from treated efficacy of AGS-22M6E was also examined against an orthotopic animals showed strong AGS-22M6E ADC localization to the xenograft, in which AG-Br7 tumors were established in mammary xenograft tumors within 6 hours of treatment, whereas the control fat pads of SCID mice. As shown in Fig. 6E, AGS-22M6E

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Necn-4 Necn-4-V Necn-4-C′C′′ A 300 300

300

200 200 AGS-22M6 200 Count Count Count 100 100 100

0 0 0 0 1 2 3 4 1 3 1 3 10 10 10 10 10 100 10 102 10 104 100 10 102 10 104 FL2-H FL2-H FL2-H 300 300 300

200 200 200 AGS-22M104 Count Count Count 100 100 100

0 0 0 0 1 2 3 4 1 3 1 3 10 10 10 10 10 100 10 102 10 104 100 10 102 10 104 FL2-H FL2-H FL2-H

V V

C′ C′ C′ C′

C′′ C′′ VV C′′ C′′

200 B Rat1-necn-4

80 Control-Fc 150 60 Necn-1-Fc 40 Count

100 0 0 1 2 3 4 10 10 10 10 10 FL2-H AGS-22M6 50 AGS-22M6E Mean fluorescence intensity

0 0.01 0.1 1 10 100 1,000 Antibody concentration (ng/mL)

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 only the V domain or C0-C00 domains were stained with AGS-22M6 or AGS-22M104 at 10 mg/mL for 1 hour at 4C, 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 were biotinylated and incubated with nectin-4–expressing Rat1(E) cells at 300 ng/mL in the absence or presence of increasing concentrations of AGS-22M6 or AGS-22M6E for 1 hour. Cells were washed, stained with streptavidin-PE (Jackson ImmunoResearch Laboratories), and analyzed by flow cytometry. Histogram of nectin-1-Fc compared with control-Fc binding in the absence of antibody is shown in the insert. www.aacrjournals.org Cancer Res; 76(10) May 15, 2016 OF7

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Mock Human nectin-4 Monkey nectin-4 Rat nectin-4 125 125 125 125

100 100 100 100

75 75 75 75

50 50 50 50 % Survival % Survival % Survival % Survival 25 25 25 25 IC50 = 1.2 ng/mL IC50 = 3.4 ng/mL IC50 = 4.7 ng/mL (95% CI, 0.8–1.9) (95% CI, 2.5–5.3) (95% CI, 3.2–6.9) 0 0 0 0 1 1 1 1 10 0.1 10 0.1 10 0.1 10 0.1 100 0.01 100 0.01 100 0.01 100 0.01 Antibody (ng/mL) Antibody (ng/mL) Antibody (ng/mL) Antibody (ng/mL)

T-47D 125

100 AGS22M6 75 AGS22M6E ADC Figure 5. Isotype control ADC AGS-22M6E ADC shows potent and speciﬁc in vitro cytotoxicity. 50 PC-3 cells engineered to express human, monkey, or rat nectin- 4, as well as T-47D breast cancer cells, were incubated with increasing concentrations of unconjugated AGS-22M6,

% Survival (± SEM) 25 IC50 = 37.8 ng/mL conjugated AGS-22M6E ADC, or isotype control ADC for (95% CI, 25.8–55.4) 5 days. CI, conﬁdence interval. 0 1 10 100 1,000 10,000 Antibody (ng/mL)

administered either as a single 10 mg/kg dose or as two 5 mg/kg in normal tissues (19). More recently, nectin-4 was identified as doses resulted in complete eradication of established tumors. an entry point for measles and other viruses and was proposed Lower doses were not explored in this model. as a target for oncolytic viral therapy (21, 22). Although several In the AG-Panc4 model, AGS-22M6E at 3 mg/kg led to tumor groups reported the detection of nectin-4 expression in breast, stasis, with significant tumor growth inhibition compared with ovarian, and lung cancers (23–26), no comprehensive analysis isotype control ADC at equivalent dosage (Fig. 6F). In the NCI- has been reported to date. Using suppression subtractive hybrid- H322M lung adenocarcinoma xenograft model, AGS-22M6E at 3 ization, we identified nectin-4 as one of the genes dramatically mg/kg resulted in 83.6% tumor regression compared with isotype upregulated in bladder cancer specimens. Our RNA expression control ADC at the same dose (P < 0.0001, Fig. 6G). In addition, analysis of a larger cancer sample panel and of normal tissues treatment with AGS-22M6E at 3 mg/kg resulted in 20.5% tumor also indicated that nectin-4 transcript is present at low levels in regression (P ¼ 0.03461). AGS-22M6E given at a lower dose of 1.0 various normal tissues and is upregulated in a subset of breast, mg/kg did not show a significant effect when compared with pancreatic, and lung cancer specimens (Agensys, unpublished control ADC at the equivalent dose (P ¼ 0.1455). data). In this report, we used IHC to demonstrate nectin-4 In support of the clinical development of an ADC targeting protein expression in normal tissue specimens and in TMAs nectin-4, the AGS-22M6 antibody produced in the hybridoma from various types of cancer. Our results showed broader system was converted into a human recombinant product expression in normal tissues than was previously reported. expressed in CHO cells as detailed in Materials and Methods. A Nectin-4 expression was detected in the epithelia of bladder, comparability analysis between the hybridoma-derived AGS- breast, stomach, esophagus, salivary gland (ducts), and skin 22M6E ADC and the CHO-derived ASG-22CE ADC was per- (epidermis and sweat glands). Epithelial components of other formed in vitro and in vivo. Both ADCs showed identical in vitro organs showed weaker and less consistent staining for nectin-4. characteristics: binding constants for nectin-4 were 0.057 and Investigation of normal tissue antigen expression is essential for 0.060 nmol/L for AGS-22M6E and ASG-22CE, respectively, and the clinical progression of an ADC. Prohibitive on-target toxi- IC50 of 1.523 and 1.674 nmol/L, respectively. When injected in cities have been observed for several ADCs, such as skin toxi- mice, both AGS-22M6E and ASG-22CE showed comparable cities (27). The extensive expression profiling of nectin-4 pharmacokinetic profile up to 10 days (Supplementary Fig. described herein allowed us to identify normal tissues that may S2; Table 1). ADC and total IgG exhibited similar exposure and raise the potential risk of eliciting nectin-4 on-target toxicities. serum elimination half-life ranging from 1.5 to 2 days. They also These have been investigated in two cross-reactive species (rat exhibited equivalent dose-dependent tumor growth inhibition in and cynomolgus monkey), as well as in the clinical setting. the breast cancer AG-Br7 xenograft model (Fig. 6H). Preliminarily, the findings suggest a favorable therapeutic win- dow, but details will be reported as part of a complete analysis of phase I results. Discussion In cancer patient specimens, >50% samples tested positive Nectin-4 was originally cloned from human trachea and for nectin-4 protein expression, and the level of expression, described as an antigen with a restricted pattern of expression based on IHC staining intensity, was highest in bladder cancer

OF8 Cancer Res; 76(10) May 15, 2016 Cancer Research

ADC Cancer Therapeutic Targeting Nectin-4

Control AG-B1AG-Br7 AG-Panc4 NCI-H322M AG-B1 stain

1,400 BCDAG-B1 Bladder cancer 1,400 AG-B1 Bladder cancer 1,600 AG-Br7 Breast cancer Vehicle control ) ) Vehicle control ) 1,400 Vehicle control 3 1,200

Isotype control ADC 3 1,200 3 Isotype control ADC 0.4 mg/kg AGS-22M6E Control ADC 1 mg/kg 1,000 AGS-22M22E Isotype control ADC 0.8 mg/kg 1,200 Control ADC 3 mg/kg AGS-22M104E 1,000 AGS-22M6E 0.4 mg/kg AGS-22M6 3 mg/kg 1,000 N = 6 per group AGS-22M6E 0.8 mg/kg AGS-22M6E 1 mg/kg 800 4 mg/kg ×1 800 N = 10 per group AGS-22M6E 3 mg/kg 800 q4d × 5 600 600 N = 10 per group 600 q4d × 6 400 400 P < 0.0001 400 Mean tumor volume (mm Mean tumor volume 200 (mm Mean tumor volume 200 (mm Mean tumor volume 200 P < 0.0001 0 0 0 0 5 10 15 20 25 30 0 5 10 15 20 0 5 10 15 20 25 Time (days) Time (days) Time (days) E FG 1,200 NCI-H322M lung cancer 1,600 AG-Br7 Breast cancer OT 1,400 AG-Panc4 Pancreatic cancer Vehicle control Vehicle control ) 1,400 ) Vehicle control ) 1,000

3 3 1,200 3 Control ADC 1 mg/kg Control ADC 5 mg/kg q4d × 2 Control ADC 1 mg/kg 1,200 AGS-22M6E 5 mg/kg q4d × 2 Control ADC 3 mg/kg Control ADC 3 mg/kg AGS-22M6E 10 mg/kg ×1 1,000 AGS-22M6 3 mg/kg 800 AGS-22M6 3 mg/kg AGS-22M6E 1 mg/kg AGS-22M6E 1 mg/kg 1,000 N = 8 per group 800 AGS-22M6E 3 mg/kg AGS-22M6E 3 mg/kg 800 600 N = 10 per group N = 10 per group 600 q4d × 6 q4d × 5 600 400 400 400 P = 0.0298 Mean tumor volume (mm Mean tumor volume Mean tumor volume (mm Mean tumor volume Mean tumor volume (mm Mean tumor volume P < 0.0001 200 200 200 P < 0.0001 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 Time (days) Time (days) 0 5 10 15 20 Time (days) H 2,500 AG-Br7 Breast cancer

Vehicle control ) 3 2,000 Control ADC 1 mg/kg Control ADC 3 mg/kg AGS-22M6E 1 mg/kg 1,500 AGS-22M6E 3 mg/kg ASG-22CE 1 mg/kg ASG-22CE 3 mg/kg 1,000 N = 10 per group

500 Mean tumor volume (mm Mean tumor volume

0 0 5 10 15 20 25 Time (days)

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 xenograft models 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 growth over time for AG-B1 subcutaneous xenografts treated with three different ADC at 4 mg/kg (B); AG-B1 subcutaneous xenografts treated with AGS-22M6E (or controls) 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 orthotopic xenografts treated with AGS-22M6E (or controls) at 5 and 10 mg/kg (E); AG-Panc4 subcutaneous xenografts treated with AGS-22M6E (or controls) at 1 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 by AGS-22M6E ADC and ASG-22CE ADC in the breast cancer AG-Br7 xenograft model. Treatment was initiated on day 0 as indicated by arrows. Mean tumor volume data for each group were plotted over time with SE bars.

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Challita-Eid et al.

and breast cancer. In general, good correlation between protein nism of action of such antibody will need to be further expression based on IHC and previously observed transcript investigated. levels was observed. Overall, IHC analyses demonstrated mod- On the basis of its ability to induce tumor regression in erate to strong expression (H-score > 100) of nectin-4 in 60% of human breast and bladder cancerspecimens,aswellitsfavor- bladder cancer and 53% of breast cancer specimens. In addi- able toxicology profile in various species (Agensys, unpub- tion, 37% and 27% of pancreatic and lung cancer specimens, lished data), AGS-22M6E, the hybridoma-derived ADC, was respectively, demonstrated moderate to strong expression. Gen- investigated in early-phase clinical studies for safety and phar- erally, we observed similar overall frequency of nectin-4 expres- macokinetics in patients with solid tumors that express nectin-4 sion in ovarian cancer as previously published by Derycke and (ClinicalTrials.gov identifier NCT01409135). Because pivotal colleagues (26). registration trials will require an antibody produced at high Soluble form of Nectin-4 in patient serum and in ascites in titers using mammalian cells, we have subsequently developed case of ovarian cancer has been reported (24–26). Although a comparable CHO-derived anti-nectin-4 antibody, ASG-22CE. the levels varied between reports, all investigators show an Our preclinical studies with ASG-22CE ADC showed that it has association between higher detected levels of soluble nectin-4 equivalent binding and potency as the hybridoma product. and disease progression. Many other membrane-anchored ASG-22CE entered the clinic in 2014 as monotherapy in proteins release their ECD through proteolytic cleavage, such patients with metastatic urothelial cancer, as well as other solid as HER2 and EGFR (28, 29). However, clinical significance of tumors that express nectin-4 (ClinicalTrials.gov identifier such form has been difficult to establish, and effect on NCT02091999), and is currently under clinical development response to targeted therapy has not been fully elucidated as enfortumab vedotin. (30–32). Overall, the data presented herein indicate that nectin-4 is a AGS-22M6, a fully human antibody targeting nectin-4, was viable target for ADC therapy in carcinomas expressing nectin-4 selected from a panel of >50 antibodies for further evaluation and support the evaluation of such ADCs in the clinical setting. as a drug conjugate based on its high affinity, broad species The preclinical data demonstrate growth inhibition across cross-reactivity, and ability to mediate very potent cell death multiple human xenograft tumor models and tumor regression both in vitro and in vivo. The ADC AGS-22M6E, composed of in bladder and breast cancer models. Our finding of nectin-4 AGS-22M6E conjugated to MMAE, exhibited dose-dependent overexpression in human tumor specimens, particularly those antitumor activity in vivo.Atlowerdoses(1mg/kg),theADC from patients with breast, bladder, pancreatic, or lung cancer, inhibited growth of bladder and breast cancer xenografts. At suggests that a significant proportion of patients may benefit higher doses (3mg/kg),AGS-22M6Einducedtumorregres- from nectin-4 targeted delivery of a potent cytotoxic agent to sion of the subcutaneous bladder cancer xenograft, the sub- the tumor site. cutaneous breast cancer xenograft, and the orthotopic breast cancer xenograft. In addition, AGS-22M6E inhibited the Disclosure of Potential Conflicts of Interest growth of both the pancreatic cancer xenograft and the lung Enfortumab Vedotin is being codeveloped in conjunction with Agensys' adenocarcinoma xenograft, with associated modest tumor partner, Seattle Genetics, Inc. No potential conflicts of interest were regression in the lung tumor model. The in vivo efficacy of disclosed. AGS-22M6E generally correlated well with the expression level of nectin-4 in xenografts and cell lines. In most of the studies Authors' Contributions described herein, the parental unconjugated antibody AGS- Conception and design: P.M. Challita-Eid, D. Satpayev, Z. An, Y. Shostak, I.B.J. 22M6 was included as a control for efficacy. We concluded that Joseph, F. Donate,~ K. Morrison, D.R. Stover binding of unconjugated antibody to nectin-4 does not result Development of methodology: P.M. Challita-Eid, D. Satpayev, Z. An, in antitumor activity in either in vitro or in vivo preclinical K. Morrison, A. Raitano, H. Avina,~ C.I. Guevara, S. Karki, K. Morrison models. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Satpayev, P. Yang, K. Morrison, Y. Shostak, Our data also demonstrated that under the conditions of an R. Nadell, W. Liu, D.R. Lortie, L. Capo, A. Verlinsky, M. Leavitt, F. Malik, in vitro binding assay, AGS-22M6 and AGS-22M6E blocked the H. Avina,~ C.I. Guevara, N. Dinh, I.B.J. Joseph, F. Donate,~ K. Morrison interaction of nectin-4 with nectin-1, its putative binding Analysis and interpretation of data (e.g., statistical analysis, biostatistics, partner. The functional significance of this observation is pres- computational analysis): P.M. Challita-Eid, D. Satpayev, K. Morrison, ently unknown, as the role of nectin-4 in normal epithelial Y. Shostak, A. Raitano, D.R. Lortie, M. Leavitt, H. Avina,~ C.I. Guevara, S. Karki, ~ tissue structure and morphogenesis is poorly understood. We B.S. Anand, I.B.J. Joseph, F. Donate, K. Morrison, D.R. Stover Writing, review, and/or revision of the manuscript: P.M. Challita-Eid, and others hypothesize that nectin-4 plays a role in the estab- D. Satpayev, Z. An, L. Capo, B.S. Anand, D.S. Pereira, I.B.J. Joseph, F. Donate,~ lishment and maintenance of adherens junctions, which in K. Morrison, D.R. Stover normal epithelium define cell polarity, a characteristic often Administrative, technical, or material support (i.e., reporting or organizing lost during cancer progression (12). We have attempted, but data, constructing databases): P.M. Challita-Eid, D. Satpayev, S. Karki, not yet succeeded, in identifying an anti-nectin-4 antibody that D.R. Stover intrinsically modulates tumor growth. In a recent publication, Study supervision: P.M. Challita-Eid, D. Satpayev, Z. An, A. Raitano, I.B.J. Joseph, F. Doñate, D.R. Stover Pavlova and colleagues (33) demonstrated that nectin-4 pro- Other (developed the antibody): R. Nadell motes anchorage-independent growth in breast cancer cells by activation of the matrix-independent integrin b4/SHP-2/c-Src Acknowledgments and that the transformation of breast cancer cells to anchorage The authors thank the hardworking and thoughtful members of the Agensys independence is dependent on nectin-4. The authors also research departments that did not appear in this article but that contributed to reported modest inhibition of growth of a breast cancer xeno- previous conference presentations or contributed to historical data that ulti- graft by a mAb targeted against human nectin-4. The mecha- mately led to this final article.

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ADC Cancer Therapeutic Targeting Nectin-4

Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate This study was funded by Agensys, Inc., an afﬁliate of Astellas Pharma, this fact. Inc. The costs of publication of this article were defrayed in part by the Received May 14, 2015; revised January 7, 2016; accepted February 26, 2016; payment of page charges. This article must therefore be hereby marked published OnlineFirst March 24, 2016.

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www.aacrjournals.org Cancer Res; 76(10) May 15, 2016 OF11

Enfortumab Vedotin Antibody−Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models

Pia M. Challita-Eid, Daulet Satpayev, Peng Yang, et al.

Cancer Res Published OnlineFirst March 24, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-1313

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