(LY6E) Provides Robust Tumor Killing in a Wi

Published OnlineFirst April 10, 2015; DOI: 10.1158/1078-0432.CCR-15-0156

Cancer Therapy: Preclinical Clinical Cancer Research An Antibody–Drug Conjugate Directed against Lymphocyte Antigen 6 Complex, Locus E (LY6E) Provides Robust Tumor Killing in a Wide Range of Solid Tumor Malignancies Jyoti Asundi1, Lisa Crocker2, Jarrod Tremayne2, Peter Chang3, Chie Sakanaka4, Josh Tanguay2, Susan Spencer2, Sreedevi Chalasani5, Elizabeth Luis6, Karen Gascoigne7, Rupal Desai8, Rajiv Raja8, Brad A. Friedman9, Peter M. Haverty9, Paul Polakis1, and Ron Firestein5

Abstract

Purpose: Chemotherapies are limited by a narrow therapeutic Results: Using in silico approaches, we found that LY6E was index resulting in suboptimal exposure of the tumor to the drug significantly overexpressed and amplified in a wide array of and acquired tumor resistance. One approach to overcome this is different human solid tumors. IHC analysis revealed high LY6E through antibody–drug conjugates (ADC) that facilitate greater protein expression in a number of tumor types, such as breast, potency via target-specific delivery of highly potent cytotoxic lung, gastric, ovarian, pancreatic, kidney and head/neck carcino- agents. mas. Characterization of the endocytic pathways for LY6E Experimental Design: In this study, we used a bioinformatics revealed that the LY6E-specific antibody is internalized into cells approach to identify the lymphocyte antigen 6 complex locus leading to lysosomal accumulation. Consistent with this, a LY6E- E (LY6E), an IFN-inducible glycosylphosphatidylinositol (GPI)- specific ADC inhibited in vitro cell proliferation and produced linked cell membrane protein as a promising ADC target. We durable tumor regression in vivo in clinically relevant LY6E- developed a monoclonal anti-LY6E antibody and characterized in expressing xenograft models. situ LY6E expression in over 750 cancer specimens and normal Conclusions: Our results identify LY6E as a highly promising tissues. Target-dependent anti-LY6E ADC killing was investigated molecularADCtargetfora varietyof solidtumortypeswithcurrent both in vitro and in vivo using patient-derived xenograft models. unmet medical need. Clin Cancer Res; 21(14); 3252–62. 2015 AACR.

Introduction modular elements: (i) a monoclonal antibody that targets antigens selectively expressed at the surface of tumor cells; (ii) a linker Traditional chemotherapeutic regimens are widely used in moiety; and (iii) a cytotoxic agent that mediates cell killing. In cancer therapy. Although efficacious, their use is limited by their principle, the ADC is systemically inert and functions as a pro- systemic toxic effects in patients leading to a narrow therapeutic drug, releasing its cytotoxic agent only after target engagement, window. One tactic to circumvent this limitation is the antibody– internalization, and subsequent proteolytic digestion in the can- drug conjugate (ADC) approach. ADCs are composed of three cer cell. The recent approval of two ADCs, trastuzumab emtansine (T-DM1; Kadcyla) targeting HER2-positive breast cancer and brentuximab vedotin for CD30-positive lymphomas (1, 2) has 1Department of Molecular Oncology, Genentech Research, South San led to a burgeoning interest in developing this technology for Francisco, California. 2Department of Translational Oncology, Genen- additional indications. 3 tech Research, South San Francisco, California. Touro University, The selection of an appropriate tumor antigen is of central California College of Pharmacy,California. 4Pharmaceuticals and Med- ical Devices Agency, Tokyo, Japan. 5Department of Pathology, Gen- importance to the activity of an ADC molecule. The ideal ADC entech Research, South San Francisco, California. 6Department of target should be highly and broadly expressed in cancer while Protein Chemistry, Genentech Research, South San Francisco, Cali- absent in normal tissues. Recent efforts by The Cancer Genome fornia. 7Department of Discovery Oncology, Genentech Research, South San Francisco, California. 8Department of Oncology Biomarker Atlas (TCGA) and other groups have empowered the systematic Development, Genentech Research, South San Francisco, California. characterization of molecular alterations in several key human 9 Department of Bioinformatics, Genentech Research, South San Fran- malignancies. These investigations have led to new opportunities cisco, California. to identify or reexamine cancer-specific antigens that may serve as Note: Supplementary data for this article are available at Clinical Cancer a springboard for a new generation of ADCs. Research Online (http://clincancerres.aacrjournals.org/). LY6E is a glycosylphosphatidylinositol (GPI)-linked cell-sur- Corresponding Authors: Ron Firestein, Genentech, 1 DNA Way, South San face protein that is induced by IFNa (3) and chemotherapy (4). Francisco, CA 94080. Phone: 650-225-8441; Fax: 650-225-8440; E-mail: LY6E mRNA was found to be overexpressed in colon and kidney ronf@gene.com; and Jyoti Asundi, E-mail: [email protected] cancer suggesting a role in cancer (5). In this report, we identify doi: 10.1158/1078-0432.CCR-15-0156 and characterize LY6E as an ADC target in a broad set of human 2015 American Association for Cancer Research. malignancies. We find that the LY6E locus is amplified and the

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protocol for affinity assays is provided in the Supplementary Translational Relevance Methods section). Targeted cancer therapies exploit molecular alterations Human LY6E construct was subcloned into RCASBP retro- specific to the cancer cell, thus mitigating damage to normal viral expression vector and used to generate viral particles tissues. Antibody–drug conjugates (ADC) utilize a mono- expressing the gene of interest in DF-1 cells. PC-3-TVA-N- clonal antibody for efficient and specific delivery of a term-tagged gD human LY6E (PC3-LY6E) was generated as a cytotoxic agent. This new classoftherapeuticshasrecently pool of cells stably expressing LY6E, by infecting PC-3-TVA cell shown promising clinical activity in targeting tumors which line with viral particles expressing N-term-tagged human LY6E. harbor overexpressed or amplified (e.g., T-DM1) receptors Appropriate vector control cells (PC3-Vector) were also on the cancer cell surface. In this study, we identify and generated. characterize LY6E as a particularly attractive ADC target The Genentech in-house cell bank acquired cell lines over a based on its high expression in numerous human carcino- period of more than 10 years. Cell lines were maintained and mas, minimal expression in normal tissues, cell-surface propagated as described previously (6). localization, and swift endocytosis. In summary, our preclinical results suggest that the ADC targeting the LY6E may provide an effective new therapy for a wide range of solid Generation of anti-LY6E antibodies tumor malignancies. Anti-LY6E rabbit polyclonal antibody GEN-93-8-1 (8-1) was developed through a fee-for-service agreement with YenZym. The anti-LY6E rabbit monoclonal antibody, GEN-93-8-1 was developed by Epitomics (Abcam), from a rabbit originating from the YenZym collaboration. protein is broadly and highly expressed in diverse cancer types. Of Anti-LY6E antibody 9B12 was humanized from its parental note, LY6E is overexpressed in several cancers with limited ther- hybridoma clone; which was generated by immunizing Balb/c apeutic options, including ovarian, pancreatic, lung, gastric, and fi mice with puri ed LY6E protein (7). Immunization, hybridoma triple-negative [estrogen receptor (ER ), progesterone receptor fi growth and selection, and antibody puri cation methods (PR ), and (HER2 ) breast cancer (TNBC). To pursue LY6E as a used were as described previously (8). Anti-LY6E antibody cancer target, we developed an anti-LY6E monomethylauristatin E 9B12 was conjugated to auristatin MMAE by methods previously fi (MMAE) ADC that demonstrated potent ef cacy both in vitro and described (9). in vivo in different LY6E-expressing tumor models, including The drug to antibody ratio for anti-LY6E ADC (LY6E) was 3.6 to patient-derived xenografts (PDX) with heterogeneous expression. 3.8 and the drug to antibody ratio for control IgG-vc-MMAE (Ctrl) Our preclinical data suggest that anti-LY6E ADC has great poten- was 3.0 to 3.5. tial as a therapeutic agent for many cancers of unmet medical need in the clinical setting. Immunologic procedures FACS using 5 mg/mL anti-LY6E antibody 9B12 to detect LY6E Materials and Methods and cell viability assays were performed as described previously Tissue culture and cell lines (10). Internalization of anti-LY6E9B12antibodywasevaluated Human ovarian cancer cell lines A2780, COV318, COV362, using protocols as previously described (10). Ten thousand DOV13, EFO-27, KURAMOCHI, OVCAR433, OVISE, OAW42, KURAMOCHI cells were seeded onto each well of 4-well RMG-1, TOV-112D; human pancreatic cancer cell line chamber slides. Five days later, cells were washed and incubat- SU.86.86; human squamous non–smallcelllungcancer ed for 30 minutes, either in the presence or absence of endo- (NSCLC) cell line SW900; human malignant lymphoma cell cytic inhibitors/disruptors, 25 mmol/L chlorpromazine (CPZ) line RAMOS; human breast cancer cell lines HCC1569 and or 1 mmol/L methyl b cyclodextrin (MBC; C8138, C4555, MDA-MB-175 VII; human embryonic kidney cell line HEK-293; Sigma Life Sciences). Cells were further incubated for an addi- and human prostatic cell line PC-3 were obtained from cell tional 30 minutes with 5 mg/mL anti-LY6E antibody 9B12 or bank collections such as ECACC (European Collection of Cell with 25 mg/mL Transferrin directly conjugated to Alexa 555 cultures), JCRB (National Institute of Biomedical Innovation), (T-35352, Invitrogen). Incubation of cells with inhibitors, 9B12 DSMZ (German collection of micro-organisms and cell cul- antibody, transferring, and the appropriate negative controls tures), ATCC, or from the M.D. Anderson Cancer Center were conducted in growth media containing appropriate pro- (Houston, TX). Cell lines were tested and authenticated by tease inhibitors. Cells were permeabilized, washed, and incu- STR profiling at Genentech, Inc. bated for 60 minutes with 4 mg/mL rabbit antibody against The cell line PC-3-TVA is derived from the human prostatic cell lysosomal marker 1 (LAMP1; L1418, Sigma Life Sciences; line, PC-3 to ectopically express the tv-a gene, the receptor for ref. 10). LY6E antibodies 9B12 and LAMP1 were detected by avian leukosis virus subgroup A, which allows for viral entry and incubating cells with the appropriate secondary fluorescent chromosomal integration of the gene of interest. detection reagents for 60 minutes. Human LY6E (encoded by NM_002346) and cynomolgus Slides were mounted by applying ProLong gold antifade monkey LY6E (encoded by NM_002346.1) were cloned into a reagent with DAPI (P36935; Invitrogen) placed under a glass cytomegalovirus mammalian expression vector engineered to coverslip, and sealed with clear nail polish. Images were acquired encode an N-terminal gD (HSV-1 viral glycoprotein) tag. These on a Nikon TE-300 inverted microscope equipped with a 60 constructs were used in competition-binding assays to determine magnification 1.4 NA infinity-corrected Plan Apo oil objective affinity of anti-LY6E antibody to human and cynomolgus monkey (Technical Instruments) and a Retiga EX-cooled CCD camera (Q exogenous LY6E expressed transiently in HEK-293 cells (detailed Imaging), using the QCapture version 3.1.1 software application

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(Q Imaging). Image analysis was performed using Photoshop CS Xenograft efficacy studies software (version 8.0; Adobe Systems). Efficacy of anti-LY6E ADC (9B12) or T-DM1 was evaluated in xenograft models established by implanting patient-derived RNAi tumor material or cancer cells subcutaneously in the dorsal right For gene depletion studies, PC3-LY6E cells were transiently flank of immunodeficient mice. When mean tumor volumes transfected for 48 hours with LY6E-specific siRNA (see Supple- reached approximately 90 to 200 mm3 (day 0), animals were mentary Table S1 for sequences) using lipofectamine RNAiMAX randomized into groups of 8 to 10 each and administered a single transfection reagent (13778150, Invitrogen) and methods sug- intravenous (i.v.) injection of either humanized anti-LY6E anti- gested by the manufacturer. body or ADC or T-DM1 or anti IgG (human isotype) ADC (Ctrl) conjugated to MMAE through the valine-citrulline linker (9). Gene expression and IHC Animal weights and tumor volumes were measured twice per For qRT-PCR, 200 ng RNA was amplified using the Invitrogen week until study end and all calculations were done as described Platinum Taq/Reverse Transcriptase enzyme mix as per the man- previously (11). ufacturer's protocol (Life Technologies). qPCR was conducted on For cell line xenograft studies, na€ve C.B-17 SCID or C.B-17 Fluidigm 96.96 Dynamic arrays using the Biomark HD system as SCID beige mice (Charles River Laboratories) were inoculated per the manufacturer's protocol. Samples were run in triplicate with either two million SU.86.86 cells suspended in HBSS and and cycle threshold (Ct) values were converted to relative expres- Matrigel (BD Biosciences) or 5 million SW900 cells suspended sion values (negative D Ct) by subtracting the mean of the two in HBSS, respectively. All studies were conducted in accordance reference genes (Transmembrane Protein 55B (TMEM55B) and with the Guide for the Care and Use of Laboratory Animals Vacuolar Protein Sorting Homolog B (VPS33B) from the mean of [Ref: Institute of Laboratory Animal Resources (NIH publica- target gene. tion no. 85-23), Washington, DC: National Academies Press; For normal tissue expression studies, Tissue Scan Normal 1996]. Tissue cDNA arrays for qPCR (HMRT103, OriGene Technologies) PAXF 1657 pancreatic cancer PDX model and MAXF 1162 and RNA extracted from various cancer tissues and xenografts (HER2-positive) primary breast cancer PDX model were estab- were probed for LY6E and GAPDH expression in triplicate on a lished at Oncotest GmbH. All experiments were approved by the 7500 Real Time PCR thermal cycler, (Life Technologies) using local authorities, and were conducted according to the guidelines manufacturer's reagents. Fluorophore-labeled probes used with of the German Animal Welfare Act (Tierschutzgesetz). Four- to 6- flanking primers for the detection of various genes are listed in week-old female immunodeficient NMRI nu/nu mice from Har- Supplementary Table S1. lan or Charles River were used. For immunoblotting, 1 mg/mL anti-LY6E rabbit monoclonal Triple-negative breast cancer ductal adenocarcinoma PDX antibody GEN-93-8-1 was used to detect LY6E and loading model HBCx-9 was established at XenTech. All experiments were control b-actin was detected using mouse monoclonal antibody performed in accordance with French legislation concerning the (Clone BH10D10, MA5-15452, Pierce, Thermo Scientific). Detec- protection of laboratory animals and in accordance with a cur- tion reagents Alexafluor 680 anti-rabbit IgG, (A21076, Invitro- rently valid license for experiments on vertebrate animals, issued gen) and IRDye800-conjugated anti-mouse IgG (610-132-121, by the French Ministry for Agriculture and Fisheries. Fourteen- Rockland Immunochemicals) were used. week-old female athymic nude mice (Hsd: Athymic Nude- IHC was performed on a Ventana Discovery XT autostainer Fox1nu) obtained from Harlan Laboratories were used. (Ventana Medical Systems). Formalin-fixed, paraffin-embedded whole tissue and tissue microarray sections were deparaffinized Cancer genomics analysis and pretreated with CC1 solution (Ventana Medical Systems) for For the analysis of LY6E mRNA expression, we used RNAseq 60 minutes followed by incubation with either 0.2 mg/mL anti- data from TCGA and Molecular Taxonomy of Breast Cancer LY6E rabbit monoclonal antibody or na€ve rabbit IgG (Clone International Consortium (METABRIC). TCGA RNA-seq data DA1E, Cell Signaling Technologies) for 60 minutes at 37C. were obtained from the Cancer Genomics Hub at UC Santa Cruz Detection was performed with 32-minute OmniMap anti-rabbit and processed and aligned with HTSeqGenie. Expression levels fi horseradish peroxidase (HRP) incubation and diaminobenzidine were quanti ed by RNASeq via Illumina sequencing (75 base fi (DAB; Ventana Medical Systems) followed by counter staining paired-end reads). Reads were ltered for quality and for rRNA with hematoxylin II (Ventana Medical Systems). contamination. These reads were aligned to the genome Staining intensity scores took into account both intensity of (GRCh37.1) using GSNAP (version 2013-03-31) with the follow- – staining and proportion of labeled cells. Positive expression was ing options: -M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1 pairmax-rna ¼ – defined as staining in greater than 50% of the tumor cells. 200000 clip-overlap. An average of 51.3 million uniquely Intensity scores are defined below: aligned reads with concordant read pairs was available per sample. Read counts for each transcript were processed with the DESeq variance stabilizing transform (VST) method to obtain Score Definition normalized expression levels. The resulting expression measure- Negative No detectable signal in >50% of tumor cells ments are referred to as "VST values." 1 þ Positive signal in >50% of tumor cells with majority of signal ¼ weak DNA copy number data 2 þ Positive signal in >50% of tumor cells with majority Illumina Human Omni 2.5-4 or Omni 2.5-8 arrays were of signal ¼ moderate processed with a modified version of the PICNIC (12) algorithm, 3 þ Positive signal in >50% of tumor cells with majority as published recently (13). In short, the modifications to PICNIC of signal ¼ strong increase the accuracy of global ploidy (and normal cell

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contamination) estimation and make the algorithm compatible endogenous LY6E-expressing luminal, ERþ breast cancer MDA- with Illumina arrays. These modifications also allow for accurate MB-175 VII) correlated well with flow-cytometric data demon- total copy number estimates and dramatically reduce overseg- strating high LY6E surface expression in these cells with an mentation. After PICNIC determines the sample ploidy and per- independent anti-LY6E antibody 9B12 (Fig 2A and B). Little to forms segmentation with its HMM to determine allele-specific no IHC signal was detected by 8-1 in LY6E-negative cell lines absolute copy number, we added an alternate segmentation with (PC3-Vector and Ramos; Fig. 2A and B). Specificity of 8-1 anti- cghFLasso (14). This addition accounts for PICNIC's oversegmen- body to LY6E was further confirmed by the reduced anti-LY6E 8-1 tation in regions where point copy number estimates indicate immunoblot signal (8 kDa) after siRNA-mediated LY6E deple- noninteger total copy number. Segments with three or fewer tion (Fig. 2C). Finally, we found a significant positive correlation probes were merged with their neighboring segments. between the 8-1 IHC signal and LY6E gene expression analysis For gene-specific copy number, this segmented total copy conducted on Fluidigm 96.96 dynamic arrays (Biomark HD number was averaged for each gene using all SNP intervals that system) on a panel of 106 serially cut human breast cancer tissues include a portion of that gene. We used the absolute copy number (Fig. 2D). Having established 8-1 as a specific and robust IHC scale (where 2 is normal for an autosome) in some applications reagent for LY6E, we evaluated in situ LY6E protein expression in rather than the traditional "relative" log2 (absolute/ploidy) copy 762 cancer samples, representing seven tumor types where high number. The significance of recurrent gains and losses was LY6E RNA levels were detected (Fig. 1A). LY6E expression was assessed with Genomic Identification of Significant Targets in scored using a 50% threshold cutoff (see Materials and Methods) Cancer (GISTIC; ref. 15). We used log2 (absolute copy number/ as follows: 0 (no staining/very weak staining); 1þ (weak staining); ploidy) scale copy number data derived from the cghFLasso 2þ (moderate staining); and 3þ (strong staining). Consistent segmentation and analyzed only the autosomes. We used gain with our RNA analysis, the highest level of LY6E expression was and loss cutoffs of 0.3 and 0.3, respectively, and the following found in triple-negative breast cancer samples (79% of samples other options: "-cap 3 –rx 0 –smallmem 0 –savegene 0 –v 0." showed 2þ or 3þ staining). In addition, breast, NSCLC, pancreatic, ovarian, head and neck, and gastric carcinomas all showed greater than 30% prevalence of moderate to high LY6E expression Results (Table 1). The LY6E expression pattern in cancer tissues was either To identify new ADC targets that are broadly overexpressed membranous or membranous/cytoplasmic in nature (Fig. 3). in cancer versus normal tissue, we analyzed a comprehensive An important hallmark of an ADC target is its overexpression in gene expression dataset of 35 different human tumor types and cancer tissues as compared with relatively low normal tissue normal tissue controls. From this analysis, we identified lym- expression. To this end, LY6E transcript and protein expression phocyte antigen 6 complex, locus E (LY6E) as an attractive ADC in normal tissues was assessed. Using qRT/PCR, low to no LY6E target. LY6E exhibited significant overexpression in breast, transcript expression was detected in a normal tissue panel when colon, ovarian, pancreatic, kidney, and gastric carcinomas compared with LY6E transcript detected on selected xenograft (Fig. 1A). LY6E resides at the 8q24.3 locus and exhibits signif- tumor models known to express LY6E by IHC analysis (Supple- icant copy number gain in a number of cancers where it is mentary Fig. S2). To further delineate LY6E expression in normal overexpressed (Fig. 1B). Of these cancers, we further character- tissues, a tissue microarray composed of 28 normal human tissues ized the LY6E locus in ovarian carcinomas as they had a high was stained with anti-LY6E 8-1 antibody. Consistent with our frequency of LY6E amplification and gain (Fig. 1B). We used RNA analysis of Ly6E expression, weak to no expression was GISTIC to analyze the LY6E amplicon from over 400 ovarian detected in normal tissues (Fig. 3 and Supplementary Table tumors that were part of TCGA dataset (15). We found broad S2). Thus, high LY6E expression across a broad range of solid copy number gain of the 8q arm, spanning both the MYC and tumor malignancies; coupled with relatively low to no LY6E LY6E locus (Fig. 1C). We found that LY6E amplification levels expression in most normal tissues tested underscores the potential highly correlated with its overexpression in both ovarian and utility of LY6E as an ADC target. breast carcinomas (Fig. 1D). To examine LY6E in different LY6E is a GPI-anchored protein and as such localizes to the subsets of breast cancer, we tested its expression in both marker plasma membrane. Given our data showing high LY6E expres- defined and molecularly subclassified breast cancers. LY6E sion in tumor compared with normal tissue, we surmised that expressionwasfoundtobesignificantly upregulated in tri- LY6E might serve as a tumor antigen target for an ADC. To ple-negative/basal-like breast cancer by both quantitative explore this, we first generated a panel of murine monoclonal reverse transcription PCR (qRT-PCR) and in silico analysis of antibodies against LY6E by using N-term His-tagged LY6E 1,730 breast samples from the METABRIC study (ref. 16; Sup- protein as immunogen. The anti-LY6E monoclonal antibodies plementary Fig. S1A and S1B). These data illustrate LY6E were characterized for their LY6E-binding affinity, cross species amplification and overexpression in a broad range of solid reactivity and nonreactivity against prostate stem cell antigen, tumors. the closest homolog (paralog) with 31% identity to LY6E. 9B12 Intrigued by the potential utility of using LY6E as an ADC target was identified as a LY6E-specific antibody with high affinity to in several types of cancers that have limited treatment options, we human LY6E (average dissociation constant K¸ d of 3.7 nmol/L; developed a monoclonal antibody to evaluate LY6E protein Scatchard analysis) and to cynomolgus monkey LY6E (average expression in various cancers by IHC. Anti-LY6E antibody dissociation constant K¸ d of 6.9 nmol/L; Scatchard analysis; GEN-93-8-1 (8-1) was derived from a rabbit immunized with Supplementary Table S3). 9B12 was therefore chosen for fur- N-term His-tagged LY6E protein. The sensitivity and specificity of ther development as a potential therapeutic molecule against the 8-1 antibody were validated using samples in which LY6E LY6E-positive cancers. expression was independently confirmed. Enriched IHC signal by Effective drug delivery of auristatins to cancer cells requires 8-1 in LY6E-expressing cells (exogenous LY6E expressing PC3 and internalization of the therapeutic molecule followed by efficient

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A Normal Cancer Not significant Significantly up Significantly down

2 1 0

Log −1 (fold change) 11 10 9 8 7

(Probe intensity) 6 2 5 Log

BCChromosome 8 100 24.12 12.3 24.23 23.3 24.21 11.1 12.1 23.1 13.2 21.13 21.3 22.2 50 11.22 0 3.0 2.5 2.0 1.5 1.0

0.5 Q value 0.0 10 −

0.5 − log −1.0 −1.5 Relative copy number sample (%) −2.0 − 0 5 10 15 20 25 30 2.5 MYC LY6E

− D Ovarian cancer ρ = 0.41, P = 6.42x10 −15 Breast cancer ρ = 0.327, P = 9.96x10 22 5,000 5,000 1,000 2,000 2,000 500 1,000 200100 Ly6E nRPKM 500 Ly6E nRPKM 502010 200 100

1413121110987654321 987654321 121110 Copy number Copy number

Figure 1. LY6E gene expression and ampliﬁcation in tumor and normal tissues. A, LY6E gene expression proﬁle: Each dot represents LY6E gene expression in normal (black), cancer (red), and diseased (blue) human tissues. Measurements were carried out on the Affymetrix U133P chip and are expressed as scaled average difference. Horizontal black bars represent median expression across each set of tissues. (Continued on the following page.)

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Figure 2. AB Development and characterization of anti-LY6E monoclonal antibody for 100 100 IHC. A and B, correlating the detection 80 of LY6E protein by 8-1 IHC to surface 80 LY6E expression detected by 9B12 60 60 ﬂow cytometry. Flow-cytometric detection of surface LY6E expression 40 40 % of Max % of Max (red line) on live cells by 9B12 antibody 20 20 is depicted on the left panel. Secondary antibody alone is used as 0 PC3-Ly6E 0 0 1 2 3 4 MDA-MB-175 control (gray line). IHC detection of 100 101 102 103 104 10 10 10 10 10 LY6E total protein by 8-1 antibody is FL1-H: FL1-height FL1-H: FL1-height depicted on the corresponding right panel for each cell line. Cell lines 100 100 depicted are PC3-LY6E (A, top); PC3- 80 80 Vector (A, bottom); MDA-MB-175-VII, high endogenous LY6E expressing cell 60 60 line (B, top); and RAMOS, non-LY6E– 40 expressing cell line (B, bottom). C, 40 % of Max % of Max total LY6E protein detection by 20 20 immunoblotting with 8-1 antibody on 0 PC3-LY6E cells. Independent LY6E 0 PC3-Vector RAMOS 0 1 2 3 4 100 101 102 103 104 siRNAs labeled 1, 2, 3, and 4 [or no 10 10 10 10 10 siRNA (–) as control], were used to FL1-H: FL1-height FL1-H: FL1-height knock down LY6E expression. Lysates were analyzed for LY6E protein 48 hours post-siRNA transfection. b-actin serves as loading control (D). LY6E C D protein detection by 8-1 IHC is LY6E siRNA 10 compared with LY6E gene expression in the same 106 serially cut breast 8 cancer samples. The graph correlates − 1 2 3 4 ) IHC detection of LY6E protein by 8-1 t LY6E 6 antibody (IHC scoring on the x-axis) ( Δ C with LY6E gene expression analysis against two custom-designed assays Actin 4 targeting the reference genes, TMEM55B and VPS33B on Fluidigm IHC 0 IHC 1 IHC 2 IHC 3

Dynamic arrays. LY6E RNA expression Relative LY6E RNA expression LY6E IHC on the y-axis is shown as DCt (calculated using geomean of two reference genes: TMEM55B and VPS33B). , P < 0.001 for IHC 3 group as compared with IHC-0 or IHC-1þ. cleavage of the linker molecule in the lysosomes to mediate lysosomal marker LAMP1 (Supplementary Fig. S3A). We intracellular release of free drug (17). observed that uptake of antibody was inhibited by preincubating GPI-anchored proteins are organized into lipid rafts and are cells with lipid raft disruptor, 1 mmol/L MBC (20), whereas endocytosed via the CLIC/GEEC (clathrin-independent carrier/ uptake of 9B12 into cells remained unaffected by inhibitor of GPI anchored protein enriched early endosomal compartment) clathrin-mediated endocytosis, 25 mmol/L CPZ (21). Uptake of internalization route (18, 19). To test whether 9B12 would be transferrin, a ligand endocytosed via clathrin, was indeed inhib- internalized effectively into cancer cells, we used immunoﬂuo- ited by preincubating cells with CPZ (Supplementary Fig. S3B). rescence detection to evaluate uptake of the antibody into ovarian 9B12 was also endocytosed to the lysosomes of breast cancer cells cancer cells KURAMOCHI. We observed uptake of 9B12 into live (HCC1569) within a 2-hour period (unpublished data). These cells within a 30-minute incubation period. Postincubation with data demonstrate that 9B12 would effectively deliver the drug to 9B12 antibody, cells were ﬁxed and permeabilized. 9B12 colo- lysosomes; where the ADC could be degraded to release free calized to the intracellular vesicles marked by the late endosomal/ MMAE toxin to mediate cell killing. These data strongly support

(Continued.) Top panel represents overexpression in cancer tissues as fold change over normal samples from the same tissues where green or blue shading indicates statistical significant expression change. Red bold typeface on the x-axis denotes tumors that were further characterized for LY6E expression by IHC analysis (see Fig. 3). B, relative copy number (Materials and Methods) derived from TCGA tumors is plotted by tissue. Each point represents a tumor and is coloredby discretized absolute total copy number (black: 2 copies, blue: <2 copies, orange: 3 or 4 copies, red: >4 copies). A bar plot indicates the proportions of tumors in each copy number category. C, GISTIC was used to assess the statistical significance of recurrent gain. A large portion of the q-arm of chromosome 8 shows highly significant recurrent gain. This region encompasses both MYC and LY6E. D, absolute total copy number (Materials and Methods) at the LY6E locus shows a highly significant positive correlation with LY6E transcript levels. The correlation coefficient (Spearman) and its significance are shown above for cancer panels of breast and ovarian carcinoma.

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Table 1. Prevalence of LY6E in various cancers Prevalence IHC (%) % IHC Tumor types N 01þ 2þ 3þ 2þ/3þ Her2 breast cancers 90 17 19 40 24 64 Subtypes TNBC 39 5 158 33 46 79 HRþ 51 20 20 41 20 61 NSCLC 276 13 30 46 10 56 Subtypes Adenocarcinoma 205 11 25 51 13 64 Squamous 71 17 37 39 7 46 Pancreatic adenocarcinoma 78 12 26 50 13 63 Ovarian carcinoma 57 23 24 44 9 53 Head/neck squamous carcinoma 61 15 43 39 3 42 Gastric adenocarcinoma 94 30 39 27 4 31 Colorectal adenocarcinoma 106 36 53 10 1 11

the use of 9B12 as a drug delivery agent targeting LY6E-positive highlights anti-LY6E ADC activity in three ovarian cancer cell cancers. lines with varying LY6E surface expression and copy number An important aspect of an ADC is its ability to mediate (CN) gain, (low expression, DOV 13; moderate expression, target-dependent cell killing. Because LY6E ampliﬁcation levels COV362; and high expression, KURAMOCHI); but with equiv- highly correlated with its overexpression in ovarian carcino- alent sensitivity to free MMAE (EC50 values of 0.38, 0.33, and mas, we chose to evaluate the ability of anti-LY6E ADC to 0.3 nmol/L; Fig. 4C). The data demonstrated an overall cor- provide targeted chemotherapy against a selected panel of relation between LY6E surface expression and anti-LY6E ADC ovarian cancer cell lines. We identiﬁed cell lines that were cell killing activity (Fig. 4A and B and Supplementary Table sensitive to the free MMAE drug and displayed a range of LY6E S4). Characterization of LY6E in 17 breast cancer cell lines surface expression that correlated positively to LY6E copy showed a similar correlation where LY6E expression predicted number gain (Supplementary Table S4). Figure 4A and B sensitivity to anti-LY6E ADC (Fig. 4D). In summary, these data

Tumor tissue

Normal 0 1+ 2+ 3+ Breast Lung Figure 3. Expression of LY6E in human tumors and matched normal tissues. IHC detection of LY6E protein using anti- LY6E GEN-93-8-1 antibody is shown from a series of normal and cancer Pancreas tissues in indications where LY6E transcript was found to be upregulated in a tumor-speciﬁc manner. Photomicrograph images depict tumor tissues representative of 0, 1þ,2þ, and 3þ staining intensity. Ovarian Black scale line, 100 mm. HNSCC Gastric

3258 Clin Cancer Res; 21(14) July 15, 2015 Clinical Cancer Research

Preclinical Development of an anti-LY6E Antibody–Drug Conjugate

A DOV 13 (CN = 2) COV362 (CN = 3) KURAMOCHI (CN = 4) 100 100 100

Figure 4. 80 80 80 Correlation of LY6E surface expression to 60 cell killing in vitro LY6E surface expression 60 60 correlates to anti-LY6E ADC cell killing 40 40 40 % of Max % of Max in vitro. A, shows the relative amounts of % of Max cell surface LY6E detected by ﬂow 20 20 20 cytometry with 9B12 antibody (red line) 0 0 0 on ovarian cell lines DOV 13 (left), COV362 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 (middle), and KURAMOCHI (right). FL1-H FL1-H FL1-H Secondary antibody alone was used as a control (gray line). LY6E copy number (CN) for each cell is shown at the top of the B

2.5 ﬂ ) 2.5 ) ow-cytometric plot. B, cell killing by anti- 5.0 ) –5 IgG-vc-MMAE –5 IgG-vc-MMAE LY6E ADC (9B12) titration is presented –5 2.0 ﬂ 4.0 IgG-vc-MMAE (×10 2.0 (×10 below each ow-cytometric plot for the (×10 * corresponding cell line. The indicated * 3.0 1.5 * 1.5 * concentrations of anti-LY6E-vc-MMAE LY6E-vc-MMAE * (red line) or control IgG-vc-MMAE (gray * LY6E-vc-MMAE 1.0 2.0 1.0 * * line) were incubated with cells for 5 days * and relative cell viability (y-axis) assessed 1.0 0.5 0.5 LY6E-vc-MMAE Relative light units using CellTiter-Glo. P values are calculated Relative light units

Relative light units 0 based on two tailed, Student t test. 0 0 0.001 1.00.10.01 10 0.001 101.00.10.01 Asterisks depict P value <0.01. C, the cell 0.001 0.01 101.00.1 μ μ ADC (μg/mL) lines DOV 13 (dotted line, round markers), ADC ( g/mL) ADC ( g/mL) COV362 (solid line, round markers), and KURAMOCHI (dotted line, square markers) were treated with increasing C D concentrations of free MMAE for 5 days MMAE-sensitive breast cancer cell lines ρ and cell viability was determined by the 1,000 = 0.61; P = 0.009 CellTiter-Glo assay. D, correlation 100 between LY6E expression and anti-LY6E 600 ADC killing (at 5 mg/mL anti-LY6E ADC) in DOV 13 400 a panel of 17 MMAE-sensitive breast 75 COV362 cancer cell lines. Each dot represents one 200 cell line. Viability effects were determined KURAMOCHI 50 3 days after single-dose treatment with 100 the anti-LY6E-ADC. Correlation was 60 determined by Pearson analysis and the Percent survival 25

P value of the correlation is indicated. LY6E expression (RPKM) 40 RPKM is defined as Reads Per Kilobase per Million mapped reads. 0 0 0.01 0.1 1.0 10 100 0.2 0.4 0.6 0.8 1.0 Free MMAE (nmol/L) Viability demonstrate the ability of anti-LY6E ADC to deliver a targeted MMAE had little activity at the highest dose; and 2. Required cytotoxic agent. MMAE, since 8 to 12 mg/kg unconjugated anti-LY6E antibody To determine the therapeutic potential of anti-LY6E ADC, we had no activity in the three models tested (Supplementary evaluated its preclinical activity against murine xenograft models Table S5). derived from various cancer cell lines and patient-derived tumor Recent studies have suggested that PDX models would better models (PDX). We chose models that allowed us to generate represent the clinical behavior of human tumors as compared robust uniform sized tumors; and most closely represented the with cell line-derived xenografts (22, 23). We, therefore, tested moderate (IHC 2þ) LY6E protein expression levels (Fig. 5A–D; anti-LY6E ADC activity in a pancreatic PDX model, PAXF 1657, inset panels) that could be expected in cancers in clinical which expresses LY6E (IHC 2þ) at moderate levels (Fig. 5C). settings. A single dose of anti-LY6E ADC demonstrated excel- Anti-LY6E ADC showed remarkable efficacy with 8 of 10 PRs lent efficacy against both a pancreatic (SU.86.86) and a NSCLC and 2 of 10 CRs following a single anti-LY6E ADC dose of 4 mg/kg xenograft model (SW900) that homogenously express Ly6E at (Fig. 5C) weak to moderate levels. Anti-LY6E ADC provided clear tumor Many ADC targets are heterogeneously expressed in human inhibitory activity in both models; a 2-mg/kg dose yielded 8 of tumors but are only validated in preclinical models that show 10 partial responses (PR) and 1/10 complete response (CR) in robust homogenous target expression (24, 10). Therefore, to theSU.86.86xenograft(Fig.5A);whereasa1-mg/kgdoseofthe better represent tumors exhibiting nonuniform target expression, ADC yielded 8 PRs with tumor regression lasting for about a we chose two PDX models HBCx-9 (TNBC) and MAXF 1162 þ month postdose in the SW900 xenograft model (Fig. 5B). Anti- (HER2 breast cancer) where LY6E was heterogeneously LY6E ADC activity was 1. Dependent on LY6E expression in all expressed at weak–moderate levels (Fig. 5D and Supplementary models; as a control non-specific antibody, conjugated to Fig. S4). Nevertheless, robust tumor killing was observed in both

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Asundi et al.

A SU.86.86 B SW900

1+/2+ 2+

25 Vehicle 10 Figure 5. ) ) Ctrl(4) 3

3 Vehicle LY6E(0.5) Anti-LY6E ADC inhibits tumor 20 8 growth in vivo.A–D, graphs depict Ctrl(6) Naked Ab efﬁcacy of anti-LY6E ADC in various Ctrl(8) pancreatic, NSCLC, and breast cancer 15 6 xenografts. Mice bearing tumors derived from SU.86.86 pancreatic 10 LY6E(1) 4 cancer cell line (A) or SW900 NSCLC squamous cell carcinoma (B), patient-derived primary tumors, 5 2 LY6E(1) LY6E(2) PAXF 1657 (C), and HBCx-9 (D) were

Tumor volume (100 × mm LY6E(3) Tumor volume (100 × (100 × volume mm Tumor administered a single i.v. injection of LY6E(4) LY6E(6) LY6E(8) either vehicle, anti-LY6E antibody 0 10 20 30 40 0 10 20 30 40 9B12, either as unconjugated [Naked Day Day Ab, depicted in (A) only, see supplementary Table S5 for Naked Ab in other xenografts], or as ADC C PAXF 1657 D HBCx-9 (LY6E), control ADC (Ctrl) at mg/kg doses indicated in parentheses. Photomicrographic insets show the intensity and heterogeneity of LY6E expression as detected by anti-LY6E IHC. Scale bar, 100 mm. Average 2+ 1+/2+ (heterogeneous) tumor volumes with SDs were determined from 9 to 10 animals per group and depicted on the y-axis Ctrl(8) 16 25 Ctrl(2) whereas day of study was depicted ) ) on the x-axis; using GraphPad Prism

3 Vehicle

Vehicle 3 20 v6.0e software. Detailed tumor 12 Ctrl(12) growth and statistical analyses for Ctrl(2) ﬁ Ctrl(12) LY6E(2) the ef cacy xenograft models 15 Ctrl(8) are provided in Supplementary 8 Tables S5 and S6. 10 LY6E(4) LY6E(2) 5 4 LY6E(4)

Tumor volume (100 × mm × Tumor volume (100 LY6E(8) LY6E(8) Tumor volume (100 × mm LY6E(12) LY6E(12) 0 10 20 30 40 0 10 20 30 40 Day Day

models. In HBCx-9, all animals achieved a complete response at a Discussion dose of 12 mg/kg. Tumor regression was sustained for approxi- In this study, we used an in silico approach to identify LY6E mately 25 days after treatment, even in the lower dose (8 mg/kg). transcript overexpression in a wide array of solid tumor malig- To further test the limits of anti-LY6E ADC, we used the MAXF þ nancies. LY6E protein overexpression in these tumors was con- 1162 HER2 breast cancer PDX model. Interestingly, the MAXF firmed by developing a robust, LY6E-specific IHC assay. Further- 1162 model, despite displaying high (IHC 3þ, data not shown) more, overexpression of LY6E was noted in several cancer types HER2 expression, demonstrated no response to HER2 targeting by that have limited treatment options with the highest level of LY6E T-DM1 (Supplementary Fig. S4B). Remarkably, we observed expression being found in triple-negative breast cancer samples, strong efficacy of anti-LY6E ADC in MAXF 1162 with a single (79% of samples showed 2þ or 3þ staining). It is worth noting dose at 4 mg/kg yielding 4 of 10 PRs. Higher doses of the ADC led that while a number of ADC targets have been offered in specific to complete responses. (Supplementary Fig. S4C and Supplemen- cancer target indications, LY6E is unique in its broad expression tary Table S5 and S6). In summary, potent dose-dependent anti- pattern across a wide range of different human cancers. These LY6E ADC efficacy was achieved in vivo against a range of tumor findings enhanced our interest in exploring the therapeutic poten- indications with different LY6E expression patterns and response tial of using LY6E as an ADC target. to other ADC agents.

3260 Clin Cancer Res; 21(14) July 15, 2015 Clinical Cancer Research

Preclinical Development of an anti-LY6E Antibody–Drug Conjugate

We chose to use LY6E as a target antigen for the synthetic, could be due to a more efficient endocytosis process for LY6E (30 potent antimitotic analogue of dolstatin 10, MMAE, which binds minutes for LY6E as compared with 20 hours for MSLN), (27) and disrupts the microtubule network in proliferating cells resulting in robust drug delivery and therefore, efficacy of the (9, 17). The use of MMAE offered distinct advantages over using ADC. DNA-binding cytotoxic agents such as duocarmycin and calichea- MAXF 1162 (40% IHCþ) and HBCx-9 (15% IHCþ) represent micin analogues, as these potent molecules lack selectivity to high bar models with less than 50% of tumor cells exhibiting LY6E target cells (25). MMAE was further chosen over other clinically expression by IHC. The remarkable efficacy of anti-LY6E ADC in approved drugs such as doxorubicin, vinca alkaloids, etc., as these these heterogeneous disease settings could likely be due to a drugs were marred by relatively low potencies, linker instability, bystander effect, whereby the efficient release of the drug in target and compositional heterogeneity (9, 25, 26). MMAE offered us cells is followed by diffusion of the membrane permeable MMAE the further advantage of using conjugation and cleavable linker into neighboring cells lacking target (28). The primary mecha- chemistries that allowed the ADC to remain relatively stable in nism of action of MMAE is by the disruption of the microtubule plasma but released the drug efficiently in the acidic lysosomal network in proliferating cells (9, 17). This, combined with low compartment of target cells, thereby minimizing systemic drug LY6E expression in normal tissues may reduce potential systemic release and damage to nontarget cells (17, 25). toxicities due to anti-LY6E ADC. þ Internalization of the antibody for effective drug delivery to the Moreover, the expression of LY6E in HER2 breast cancers acidic lysosomal compartment of a cancer cell, where intracellular suggests that anti-LY6E ADC could represent a treatment option þ cleavage releases free drug, remains a key feature of MMAE- for HER2 patients, unresponsive to existing treatments such as T- conjugated ADC-targeted chemotherapy. In principle, such ADCs DM1. Indeed, the anti-LY6E ADC was effective in the MAXF 1162 should have minimal toxicities toward normal cells due to low PDX model, which responds only weakly to T-DM1 despite þ target expression on these cells resulting in low uptake of the drug. IHC3 expression of HER2 (Supplementary Fig. S4B). Further In addition, MMAEs interfere with microtubule dynamics in investigation will be required to elucidate the mechanism that dividing cells; the rate of cell division of normal cells is generally accounts for LY6E activity in T-DM1–resistant breast cancers. lower than that for cancer cells; which further moderates potential In summary, we find that LY6E protein is overexpressed in a toxicities of the ADCs to normal cells (8). wide range of solid tumor malignancies and anti-LY6E ADC has We elucidated LY6E trafficking in an effort to maximize anti- potential utility as a treatment option for patients with unmet tumor efficacy by selecting the appropriate antibody to best medical need in clinical settings. mediate ADC drug delivery. We observed uptake of anti-LY6E antibody 9B12 into lysosomes within a 30-minute incubation Disclosure of Potential Conflicts of Interest fi period, via mechanisms distinct from the classical traf cking No potential conflicts of interest were disclosed. pathways involving clathrin-coated pits. Our data demonstrate effective endocytosis of a GPI-anchored protein and illustrate that the 9B12 anti-LY6E ADC would serve as an effective mediator of Authors' Contributions drug delivery to lysosomes. Conception and design: J. Asundi, J. Tremayne, P. Chang, R. Raja, R. Firestein Development of methodology: J. Asundi, P. Chang, C. Sakanaka, R. Desai, We found an overall trend toward increased LY6E gene copy R. Raja, R. Firestein number correlating to increased LY6E cell-surface expression and Acquisition of data (provided animals, acquired and managed patients, enhanced in vitro anti-LY6E ADC activity in breast and ovarian provided facilities, etc.): J. Asundi, L. Crocker, J. Tremayne, P. Chang, cancer cell lines. However, in vitro killing by ADC was also clearly C. Sakanaka, J. Tanguay, S. Chalasani, E. Luis, R. Raja, R. Firestein influenced by the relative sensitivity of the various cell lines to the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, free drug. For instance, OAW42, an ovarian cell line with high computational analysis): J. Asundi, L. Crocker, P. Chang, C. Sakanaka, J. Tanguay, E. Luis, K. Gascoigne, B.A. Friedman, P.M. Haverty, P. Polakis, LY6E gene copy number of 5, was resistant to killing by free MMAE R. Firestein and therefore resistant to killing by anti-LY6E ADC. Writing, review, and/or revision of the manuscript: J. Asundi, L. Crocker, Having observed excellent efficacy of anti-LY6E ADC against R. Desai, R. Raja, P. Polakis, R. Firestein cancer cell line derived xenografts, we further evaluated its efficacy Administrative, technical, or material support (i.e., reporting or organizing in physiologically relevant PDX models with moderate homog- data, constructing databases): J. Asundi, L. Crocker, P. Chang, C. Sakanaka, enous and heterogeneous LY6E expression. Efficacy of anti-LY6E R. Firestein Study supervision: P. Chang, S. Spencer, R. Raja, R. Firestein ADC at 4 mg/kg in the pancreatic PDX model PAXF 1657 was Other (generated qPCR data for the manuscript): R. Desai particularly encouraging in view of the fact that an ADC targeting another GPI-anchored protein mesothelin (MSLN) yielded com- Acknowledgments parable efficacy in the same model at a substantially higher dose of The authors thank Zeran Wang for help with Scatchard analysis, Anneleen 20 mg/kg (27). Both LY6E and MSLN demonstrated moderate þ Damaen and Zhaoshi Jiang for Bioinformatics support, and Linda Rangell, (IHC 2 ) scores within the dynamic range of individual target Debra Dunlap, and Thinh Pham for tissue staining and analysis. expression in the PAXF 1657 PDX model. On the basis of our internalization data, we may speculate that the higher efficacy of Received January 21, 2015; revised March 17, 2015; accepted March 19, 2015; anti-LY6E ADC as compared with anti-MSLN ADC in this model published OnlineFirst April 10, 2015.

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3262 Clin Cancer Res; 21(14) July 15, 2015 Clinical Cancer Research

An Antibody−Drug Conjugate Directed against Lymphocyte Antigen 6 Complex, Locus E (LY6E) Provides Robust Tumor Killing in a Wide Range of Solid Tumor Malignancies

Jyoti Asundi, Lisa Crocker, Jarrod Tremayne, et al.

Clin Cancer Res 2015;21:3252-3262. Published OnlineFirst April 10, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-15-0156

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