An Antibody Drug Conjugate Directed against...

Cancer Therapy: Preclinical

An Antibody–Drug Conjugate Directed againstLymphocyte Antigen 6 Complex, Locus E (LY6E)Provides Robust Tumor Killing in aWide Range ofSolid Tumor MalignanciesJyoti 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, andRon Firestein5

Abstract

Purpose: Chemotherapies are limited by a narrow therapeuticindex resulting in suboptimal exposure of the tumor to the drugand acquired tumor resistance. One approach to overcome this isthrough antibody–drug conjugates (ADC) that facilitate greaterpotency via target-specific delivery of highly potent cytotoxicagents.

Experimental Design: In this study, we used a bioinformaticsapproach to identify the lymphocyte antigen 6 complex locusE (LY6E), an IFN-inducible glycosylphosphatidylinositol (GPI)-linked cell membrane protein as a promising ADC target. Wedeveloped amonoclonal anti-LY6E antibody and characterized insitu LY6E expression in over 750 cancer specimens and normaltissues. Target-dependent anti-LY6E ADC killing was investigatedboth in vitro and in vivo using patient-derived xenograft models.

Results: Using in silico approaches, we found that LY6E wassignificantly overexpressed and amplified in a wide array ofdifferent human solid tumors. IHC analysis revealed high LY6Eprotein expression in a number of tumor types, such as breast,lung, gastric, ovarian, pancreatic, kidney and head/neck carcino-mas. Characterization of the endocytic pathways for LY6Erevealed that the LY6E-specific antibody is internalized into cellsleading to lysosomal accumulation. Consistent with this, a LY6E-specific ADC inhibited in vitro cell proliferation and produceddurable tumor regression in vivo in clinically relevant LY6E-expressing xenograft models.

Conclusion: Our results identify LY6E as a highly promisingmolecular ADC target for a variety of solid tumor types withcurrent unmet medical need. Clin Cancer Res; 1–11. �2015 AACR.

IntroductionTraditional chemotherapeutic regimens are widely used in

cancer therapy. Although efficacious, their use is limited by theirsystemic toxic effects in patients leading to a narrow therapeuticwindow. One tactic to circumvent this limitation is the antibody–drug conjugate (ADC) approach. ADCs are composed of three

modular elements: (i) a monoclonal antibody that targets anti-gens selectively expressed at the surface of tumor cells; (ii) a linkermoiety; and (iii) a cytotoxic agent that mediates cell killing. Inprinciple, the ADC is systemically inert and functions as a pro-drug, releasing its cytotoxic agent only after target engagement,internalization, and subsequent proteolytic digestion in the can-cer cell. The recent approval of two ADCs, trastuzumab emtansine(T-DM1; Kadcyla) targeting HER2-positive breast cancer andbrentuximab vedotin for CD30-positive lymphomas (1, 2) hasled to a burgeoning interest in developing this technology foradditional indications.

The selection of an appropriate tumor antigen is of centralimportance to the activity of an ADC molecule. The ideal ADCtarget should be highly and broadly expressed in cancer whileabsent in normal tissues. Recent efforts by The Cancer GenomeAtlas (TCGA) and other groups have empowered the systematiccharacterization of molecular alterations in several key humanmalignancies. These investigations have led to new opportunitiesto identify or reexamine cancer-specific antigens that may serve asa springboard for a new generation of ADCs.

LY6E is a glycosylphosphatidylinositol (GPI)-linked cell-sur-face protein that is induced by IFNa (3) and chemotherapy (4).LY6E mRNA was found to be overexpressed in colon and kidneycancer suggesting a role in cancer (5). In this report, we identifyand characterize LY6E as an ADC target in a broad set of humanmalignancies. We find that the LY6E locus is amplified and the

1Department of Molecular Oncology, Genentech Research, South SanFrancisco, California. 2Department of Translational Oncology, Genen-tech Research, South San Francisco, California. 3Touro University,CaliforniaCollegeofPharmacy,California. 4Pharmaceuticals andMed-ical Devices Agency, Tokyo, Japan. 5Department of Pathology, Gen-entech Research, South San Francisco, California. 6Department ofProtein Chemistry, Genentech Research, South San Francisco, Cali-fornia. 7Department of Discovery Oncology, Genentech Research,South San Francisco, California. 8Department of Oncology BiomarkerDevelopment, Genentech Research, South San Francisco, California.9Department ofBioinformatics,GenentechResearch, SouthSanFran-cisco, California.

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

Corresponding Authors: Ron Firestein, Genentech, 1 DNA Way, South SanFrancisco, CA 94080. Phone: 650-225-8441; Fax: 650-225-8440; E-mail:[email protected]; and Jyoti Asundi, E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-0156

�2015 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org OF1

Research. on July 3, 2018. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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

http://clincancerres.aacrjournals.org/

protein is broadly and highly expressed in diverse cancer types. Ofnote, LY6E is overexpressed in several cancers with limited ther-apeutic options, including ovarian, pancreatic, lung, gastric, andtriple-negative [estrogen receptor (ER�), progesterone receptor(PR�), and (HER2�) breast cancer (TNBC). To pursue LY6E as acancer target, wedeveloped an anti-LY6Emonomethylauristatin E(MMAE) ADC that demonstrated potent efficacy both in vitro andin vivo in different LY6E-expressing tumor models, includingpatient-derived xenografts (PDX) with heterogeneous expression.Our preclinical data suggest that anti-LY6E ADC has great poten-tial as a therapeutic agent formany cancers of unmetmedical needin the clinical setting.

Materials and MethodsTissue culture and cell lines

Human ovarian cancer cell lines A2780, COV318, COV362,DOV13, EFO-27, KURAMOCHI, OVCAR433, OVISE, OAW42,RMG-1, TOV-112D; human pancreatic cancer cell lineSU.86.86; human squamous non–small cell lung cancer(NSCLC) cell line SW900; human malignant lymphoma cellline RAMOS; human breast cancer cell lines HCC1569 andMDA-MB-175 VII; human embryonic kidney cell line HEK-293;and human prostatic cell line PC-3 were obtained from cellbank collections such as ECACC (European Collection of Cellcultures), JCRB (National Institute of Biomedical Innovation),DSMZ (German collection of micro-organisms and cell cul-tures), ATCC, or from the M.D. Anderson Cancer Center(Houston, TX). Cell lines were tested and authenticated bySTR profiling at Genentech, Inc.

The cell line PC-3-TVA is derived from the human prostatic cellline, PC-3 to ectopically express the tv-a gene, the receptor foravian leukosis virus subgroup A, which allows for viral entry andchromosomal integration of the gene of interest.

Human LY6E (encoded by NM_002346) and cynomolgusmonkey LY6E (encoded by NM_002346.1) were cloned into acytomegalovirus mammalian expression vector engineered toencode an N-terminal gD (HSV-1 viral glycoprotein) tag. Theseconstructs were used in competition-binding assays to determineaffinity of anti-LY6E antibody tohumanand cynomolgusmonkeyexogenous LY6E expressed transiently in HEK-293 cells (detailed

protocol for affinity assays is provided in the SupplementaryMethods section).

Human LY6E construct was subcloned into RCASBP retro-viral expression vector and used to generate viral particlesexpressing the gene of interest in DF-1 cells. PC-3-TVA-N-term-tagged gD human LY6E (PC3-LY6E) was generated as apool of cells stably expressing LY6E, by infecting PC-3-TVA cellline with viral particles expressing N-term-tagged human LY6E.Appropriate vector control cells (PC3-Vector) were alsogenerated.

The Genentech in-house cell bank acquired cell lines over aperiod of more than 10 years. Cell lines were maintained andpropagated as described previously (6).

Generation of anti-LY6E antibodiesAnti-LY6E rabbit polyclonal antibody GEN-93-8-1 (8-1) was

developed through a fee-for-service agreement with YenZym. Theanti-LY6E rabbit monoclonal antibody, GEN-93-8-1 was devel-oped by Epitomics (Abcam), from a rabbit originating from theYenZym collaboration.

Anti-LY6E antibody 9B12 was humanized from its parentalhybridoma clone; which was generated by immunizing Balb/cmice with purified LY6E protein (7). Immunization, hybridomagrowth and selection, and antibody purification methodsused were as described previously (8). Anti-LY6E antibody9B12 was conjugated to auristatin MMAE by methods previouslydescribed (9).

The drug to antibody ratio for anti-LY6E ADC (LY6E)was 3.6 to3.8 and the drug to antibody ratio for control IgG-vc-MMAE (Ctrl)was 3.0 to 3.5.

Immunologic proceduresFACS using 5 mg/mL anti-LY6E antibody 9B12 to detect LY6E

and cell viability assays were performed as described previously(10). Internalization of anti-LY6E 9B12 antibody was evaluatedusing protocols as previously described (10). Ten thousandKURAMOCHI cells were seeded onto each well of 4-wellchamber slides. Five days later, cells were washed and incubat-ed for 30 minutes, either in the presence or absence of endo-cytic inhibitors/disruptors, 25 mmol/L chlorpromazine (CPZ)or 1 mmol/L methyl b cyclodextrin (MBC; C8138, C4555,Sigma Life Sciences). Cells were further incubated for an addi-tional 30 minutes with 5 mg/mL anti-LY6E antibody 9B12 orwith 25 mg/mL Transferrin directly conjugated to Alexa 555(T-35352, Invitrogen). Incubation of cells with inhibitors, 9B12antibody, transferring, and the appropriate negative controlswere conducted in growth media containing appropriate pro-tease inhibitors. Cells were permeabilized, washed, and incu-bated for 60 minutes with 4 mg/mL rabbit antibody againstlysosomal marker 1 (LAMP1; L1418, Sigma Life Sciences;ref. 10). LY6E antibodies 9B12 and LAMP1 were detected byincubating cells with the appropriate secondary fluorescentdetection reagents for 60 minutes.

Slides were mounted by applying ProLong gold antifadereagent with DAPI (P36935; Invitrogen) placed under a glasscoverslip, and sealed with clear nail polish. Images were acquiredon a Nikon TE-300 inverted microscope equipped with a �60magnification 1.4 NA infinity-corrected Plan Apo oil objective(Technical Instruments) and a Retiga EX-cooled CCD camera (QImaging), using the QCapture version 3.1.1 software application

Translational Relevance

Targeted cancer therapies exploit molecular alterationsspecific to the cancer cell, thus mitigating damage to normaltissues. Antibody–drug conjugates (ADC) utilize a mono-clonal antibody for efficient and specific delivery of acytotoxic agent. This new class of therapeutics has recentlyshown promising clinical activity in targeting tumors whichharbor overexpressed or amplified (e.g., T-DM1) receptorson the cancer cell surface. In this study, we identify andcharacterize LY6E as a particularly attractive ADC targetbased on its high expression in numerous human carcino-mas, minimal expression in normal tissues, cell-surfacelocalization, and swift endocytosis. In summary, our pre-clinical results suggest that the ADC targeting the LY6E mayprovide an effective new therapy for a wide range of solidtumor malignancies.

Asundi et al.

Clin Cancer Res; 2015 Clinical Cancer ResearchOF2




(Q Imaging). Image analysis was performed using Photoshop CSsoftware (version 8.0; Adobe Systems).

RNAiFor gene depletion studies, PC3-LY6E cells were transiently

transfected for 48 hours with LY6E-specific siRNA (see Supple-mentary Table S1 for sequences) using lipofectamine RNAiMAXtransfection reagent (13778150, Invitrogen) and methods sug-gested by the manufacturer.

Gene expression and IHCFor qRT-PCR, 200 ng RNA was amplified using the Invitrogen

Platinum Taq/Reverse Transcriptase enzyme mix as per the man-ufacturer's protocol (Life Technologies). qPCR was conducted onFluidigm 96.96 Dynamic arrays using the Biomark HD system asper the manufacturer's protocol. Samples were run in triplicateand cycle threshold (Ct) values were converted to relative expres-sion values (negative D Ct) by subtracting the mean of the tworeference genes (Transmembrane Protein 55B (TMEM55B) andVacuolar Protein Sorting Homolog B (VPS33B) from themean oftarget gene.

For normal tissue expression studies, Tissue Scan NormalTissue cDNA arrays for qPCR (HMRT103, OriGene Technologies)and RNA extracted from various cancer tissues and xenograftswere probed for LY6E and GAPDH expression in triplicate on a7500 Real Time PCR thermal cycler, (Life Technologies) usingmanufacturer's reagents. Fluorophore-labeled probes used withflanking primers for the detection of various genes are listed inSupplementary Table S1.

For immunoblotting, 1 mg/mL anti-LY6E rabbit monoclonalantibody GEN-93-8-1 was used to detect LY6E and loadingcontrol b-actin was detected using mouse monoclonal antibody(Clone BH10D10, MA5-15452, Pierce, Thermo Scientific). Detec-tion reagents Alexafluor 680 anti-rabbit IgG, (A21076, Invitro-gen) and IRDye800-conjugated anti-mouse IgG (610-132-121,Rockland Immunochemicals) were used.

IHC was performed on a Ventana Discovery XT autostainer(Ventana Medical Systems). Formalin-fixed, paraffin-embeddedwhole tissue and tissue microarray sections were deparaffinizedand pretreated with CC1 solution (Ventana Medical Systems) for60 minutes followed by incubation with either 0.2 mg/mL anti-LY6E rabbit monoclonal antibody or na€�ve rabbit IgG (CloneDA1E, Cell Signaling Technologies) for 60 minutes at 37�C.Detection was performed with 32-minute OmniMap anti-rabbithorseradish peroxidase (HRP) incubation and diaminobenzidine(DAB; Ventana Medical Systems) followed by counter stainingwith hematoxylin II (Ventana Medical Systems).

Staining intensity scores took into account both intensity ofstaining and proportion of labeled cells. Positive expression wasdefined as staining in greater than 50% of the tumor cells.Intensity scores are defined below:

Score DefinitionNegative No detectable signal in >50% of tumor cells1 þ Positive signal in >50% of tumor cells with majority

of signal ¼ weak2 þ Positive signal in >50% of tumor cells with majority

of signal ¼ moderate3 þ Positive signal in >50% of tumor cells with majority

of signal ¼ strong

Xenograft efficacy studiesEfficacy of anti-LY6E ADC (9B12) or T-DM1 was evaluated in

xenograft models established by implanting patient-derivedtumor material or cancer cells subcutaneously in the dorsal rightflank of immunodeficient mice. When mean tumor volumesreached approximately 90 to 200 mm3 (day 0), animals wererandomized into groups of 8 to 10 each and administered a singleintravenous (i.v.) injection of either humanized anti-LY6E anti-body or ADC or T-DM1 or anti IgG (human isotype) ADC (Ctrl)conjugated to MMAE through the valine-citrulline linker (9).Animal weights and tumor volumes were measured twice perweek until study end and all calculations were done as describedpreviously (11).

For cell line xenograft studies, na€�ve C.B-17 SCID or C.B-17SCID beige mice (Charles River Laboratories) were inoculatedwith either two million SU.86.86 cells suspended in HBSS andMatrigel (BD Biosciences) or 5 million SW900 cells suspendedin HBSS, respectively. All studies were conducted in accordancewith the Guide for the Care and Use of Laboratory Animals[Ref: Institute of Laboratory Animal Resources (NIH publica-tion no. 85-23), Washington, DC: National Academies Press;1996].

PAXF 1657 pancreatic cancer PDX model and MAXF 1162(HER2-positive) primary breast cancer PDX model were estab-lished at Oncotest GmbH. All experiments were approved by thelocal authorities, and were conducted according to the guidelinesof the German Animal Welfare Act (Tierschutzgesetz). Four- to 6-week-old female immunodeficient NMRI nu/nu mice from Har-lan or Charles River were used.

Triple-negative breast cancer ductal adenocarcinoma PDXmodel HBCx-9 was established at XenTech. All experiments wereperformed in accordance with French legislation concerning theprotection of laboratory animals and in accordance with a cur-rently valid license for experiments on vertebrate animals, issuedby the French Ministry for Agriculture and Fisheries. Fourteen-week-old female athymic nude mice (Hsd: Athymic Nude-Fox1nu) obtained from Harlan Laboratories were used.

Cancer genomics analysisFor the analysis of LY6E mRNA expression, we used RNAseq

data from TCGA and Molecular Taxonomy of Breast CancerInternational Consortium (METABRIC). TCGA RNA-seq datawere obtained from the Cancer Genomics Hub at UC Santa Cruzand processed and aligned with HTSeqGenie. Expression levelswere quantified by RNASeq via Illumina sequencing (75 basepaired-end reads). Reads were filtered for quality and for rRNAcontamination. These reads were aligned to the genome(GRCh37.1) using GSNAP (version 2013-03-31) with the follow-ing options: -M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1 –pairmax-rna¼ 200000 –clip-overlap. An average of 51.3 million uniquelyaligned reads with concordant read pairs was available per sam-ple. Read counts for each transcript were processed with theDESeq variance stabilizing transform (VST) method to obtainnormalized expression levels. The resulting expression measure-ments are referred to as "VST values."

DNA copy number dataIllumina Human Omni 2.5-4 or Omni 2.5-8 arrays were

processed with a modified version of the PICNIC (12) algorithm,as published recently (13). In short, the modifications to PICNICincrease the accuracy of global ploidy (and normal cell

Preclinical Development of an anti-LY6E Antibody–Drug Conjugate

www.aacrjournals.org Clin Cancer Res; 2015 OF3




contamination) estimation and make the algorithm compatiblewith Illumina arrays. These modifications also allow for accuratetotal copy number estimates and dramatically reduce overseg-mentation. After PICNIC determines the sample ploidy and per-forms segmentation with its HMM to determine allele-specificabsolute copy number, we added an alternate segmentation withcghFLasso (14). This addition accounts for PICNIC's oversegmen-tation in regions where point copy number estimates indicatenoninteger total copy number. Segments with three or fewerprobes were merged with their neighboring segments.

For gene-specific copy number, this segmented total copynumber was averaged for each gene using all SNP intervals thatinclude a portion of that gene.We used the absolute copy numberscale (where 2 is normal for an autosome) in some applicationsrather than the traditional "relative" log2 (absolute/ploidy) copynumber. The significance of recurrent gains and losses wasassessed with Genomic Identification of Significant Targets inCancer (GISTIC; ref. 15). We used log2 (absolute copy number/ploidy) scale copy number data derived from the cghFLassosegmentation and analyzed only the autosomes. We used gainand loss cutoffs of 0.3 and �0.3, respectively, and the followingother options: "-cap 3 –rx 0 –smallmem 0 –savegene 0 –v 0."

ResultsTo identify new ADC targets that are broadly overexpressed

in cancer versus normal tissue, we analyzed a comprehensivegene expression dataset of 35 different human tumor types andnormal tissue controls. From this analysis, we identified lym-phocyte antigen 6 complex, locus E (LY6E) as an attractive ADCtarget. LY6E exhibited significant overexpression in breast,colon, ovarian, pancreatic, kidney, and gastric carcinomas(Fig. 1A). LY6E resides at the 8q24.3 locus and exhibits signif-icant copy number gain in a number of cancers where it isoverexpressed (Fig. 1B). Of these cancers, we further character-ized the LY6E locus in ovarian carcinomas as they had a highfrequency of LY6E amplification and gain (Fig. 1B). We usedGISTIC to analyze the LY6E amplicon from over 400 ovariantumors that were part of TCGA dataset (15). We found broadcopy number gain of the 8q arm, spanning both the MYC andLY6E locus (Fig. 1C). We found that LY6E amplification levelshighly correlated with its overexpression in both ovarian andbreast carcinomas (Fig. 1D). To examine LY6E in differentsubsets of breast cancer, we tested its expression in both markerdefined and molecularly subclassified breast cancers. LY6Eexpression was found to be significantly upregulated in tri-ple-negative/basal-like breast cancer by both quantitativereverse transcription PCR (qRT-PCR) and in silico analysis of1,730 breast samples from the METABRIC study (ref. 16; Sup-plementary Fig. S1A and S1B). These data illustrate LY6Eamplification and overexpression in a broad range of solidtumors.

Intrigued by the potential utility of using LY6E as an ADC targetin several types of cancers that have limited treatment options, wedeveloped a monoclonal antibody to evaluate LY6E proteinexpression in various cancers by IHC. Anti-LY6E antibodyGEN-93-8-1 (8-1) was derived from a rabbit immunized withN-term His-tagged LY6E protein. The sensitivity and specificity ofthe 8-1 antibody were validated using samples in which LY6Eexpression was independently confirmed. Enriched IHC signal by8-1 in LY6E-expressing cells (exogenous LY6E expressing PC3 and

endogenous LY6E-expressing luminal, ERþ breast cancer MDA-MB-175 VII) correlated well with flow-cytometric data demon-strating high LY6E surface expression in these cells with anindependent anti-LY6E antibody 9B12 (Fig 2A and B). Little tono IHC signal was detected by 8-1 in LY6E-negative cell lines(PC3-Vector and Ramos; Fig. 2A and B). Specificity of 8-1 anti-body to LY6E was further confirmed by the reduced anti-LY6E 8-1immunoblot signal (�8 kDa) after siRNA-mediated LY6E deple-tion (Fig. 2C). Finally, we found a significant positive correlationbetween the 8-1 IHC signal and LY6E gene expression analysisconducted on Fluidigm 96.96 dynamic arrays (Biomark HDsystem) on a panel of 106 serially cut human breast cancer tissues(Fig. 2D). Having established 8-1 as a specific and robust IHCreagent for LY6E, we evaluated in situ LY6E protein expression in762 cancer samples, representing seven tumor types where highLY6E RNA levels were detected (Fig. 1A). LY6E expression wasscored using a 50% threshold cutoff (see Materials and Methods)as follows: 0 (no staining/veryweak staining); 1þ (weak staining);2þ (moderate staining); and 3þ (strong staining). Consistentwith our RNA analysis, the highest level of LY6E expression wasfound in triple-negative breast cancer samples (79% of samplesshowed 2þ or 3þ staining). In addition, breast, NSCLC, pancre-atic, ovarian, head and neck, and gastric carcinomas all showedgreater than 30%prevalence ofmoderate to high LY6E expression(Table 1). The LY6E expression pattern in cancer tissues was eithermembranous or membranous/cytoplasmic in nature (Fig. 3).

An important hallmark of anADC target is its overexpression incancer tissues as compared with relatively low normal tissueexpression. To this end, LY6E transcript and protein expressionin normal tissues was assessed. Using qRT/PCR, low to no LY6Etranscript expression was detected in a normal tissue panel whencompared with LY6E transcript detected on selected xenografttumor models known to express LY6E by IHC analysis (Supple-mentary Fig. S2). To further delineate LY6E expression in normaltissues, a tissuemicroarray composed of 28normal human tissueswas stained with anti-LY6E 8-1 antibody. Consistent with ourRNA analysis of Ly6E expression, weak to no expression wasdetected in normal tissues (Fig. 3 and Supplementary TableS2). Thus, high LY6E expression across a broad range of solidtumor malignancies; coupled with relatively low to no LY6Eexpression inmost normal tissues tested underscores thepotentialutility of LY6E as an ADC target.

LY6E is a GPI-anchored protein and as such localizes to theplasma membrane. Given our data showing high LY6E expres-sion in tumor compared with normal tissue, we surmised thatLY6E might serve as a tumor antigen target for an ADC. Toexplore this, we first generated a panel of murine monoclonalantibodies against LY6E by using N-term His-tagged LY6Eprotein as immunogen. The anti-LY6E monoclonal antibodieswere characterized for their LY6E-binding affinity, cross speciesreactivity and nonreactivity against prostate stem cell antigen,the closest homolog (paralog) with 31% identity to LY6E. 9B12was identified as a LY6E-specific antibody with high affinity tohuman LY6E (average dissociation constant Kd of 3.7 nmol/L;Scatchard analysis) and to cynomolgus monkey LY6E (averagedissociation constant Kd of 6.9 nmol/L; Scatchard analysis;Supplementary Table S3). 9B12 was therefore chosen for fur-ther development as a potential therapeutic molecule againstLY6E-positive cancers.

Effective drug delivery of auristatins to cancer cells requiresinternalization of the therapeutic molecule followed by efficient

Asundi et al.





A

D

Log 2

(f

old

chan

ge)

Log 2

(P

robe

inte

nsity

)

5

6

7

8

910

11

−101

Significantly downSignificantly upNot significantCancerNormal

B C

Rel

ativ

e co

py n

umbe

r sa

mpl

e (%

)

−log

10 Q

val

ue

Chromosome 8

LY6EMYC

Copy number

1413121110987654321

Ly6E

nR

PK

M

5,00

02,

000

1,00

050

020

010

0

Ovarian cancer ρ = 0.41, P = 6.42x10 −15

Copy number

121110987654321

Ly6E

nR

PK

M

1,00

0 2

,000

500

200

100

5,00

050

2010

Breast cancer ρ = 0.327, P = 9.96x10−22

100

50

03.02.52.01.51.00.50.0

−0.5−1.0−1.5−2.0−2.5

05

1015

2025

3011

.1

11.2

2

12.1

12.3

13.2

21.1

3

21.3

22.2

23.1

23.3

24.1

2

24.2

1

24.2

3

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 averagedifference. Horizontal black bars represent median expression across each set of tissues. (Continued on the following page.)






cleavage of the linker molecule in the lysosomes to mediateintracellular release of free drug (17).

GPI-anchored proteins are organized into lipid rafts and areendocytosed via the CLIC/GEEC (clathrin-independent carrier/GPI anchored protein enriched early endosomal compartment)internalization route (18, 19). To test whether 9B12 would beinternalized effectively into cancer cells, we used immunofluo-rescence detection to evaluate uptake of the antibody into ovariancancer cells KURAMOCHI. We observed uptake of 9B12 into livecells within a 30-minute incubation period. Postincubation with9B12 antibody, cells were fixed and permeabilized. 9B12 colo-calized to the intracellular vesiclesmarked by the late endosomal/

lysosomal marker LAMP1 (Supplementary Fig. S3A). Weobserved that uptake of antibody was inhibited by preincubatingcells with lipid raft disruptor, 1 mmol/L MBC (20), whereasuptake of 9B12 into cells remained unaffected by inhibitor ofclathrin-mediated endocytosis, 25 mmol/L CPZ (21). Uptake oftransferrin, a ligand endocytosed via clathrin, was indeed inhib-ited by preincubating cells with CPZ (Supplementary Fig. S3B).9B12was also endocytosed to the lysosomes of breast cancer cells(HCC1569) within a 2-hour period (unpublished data). Thesedata demonstrate that 9B12 would effectively deliver the drug tolysosomes; where the ADC could be degraded to release freeMMAE toxin to mediate cell killing. These data strongly support

D

LY6E IHCIHC 0 IHC 1 IHC 2 IHC 3

Rel

ativ

e LY

6E R

NA

exp

ress

ion

(Δ

Ct)

10

8

6

4

− 1 2 3 4

LY6E

LY6E siRNA

Actin

C

A B

% o

f Max

% o

f Max

MDA-MB-175

RAMOS

FL1-H: FL1-height

FL1-H: FL1-height

% o

f Max

% o

f Max

PC3-Ly6E

PC3-Vector

FL1-H: FL1-height

FL1-H: FL1-height

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

100 100 101 102 103 104101 102 103 104

100 101 102 103 104101 102 103 104

Figure 2.Development and characterization ofanti-LY6E monoclonal antibody forIHC. A and B, correlating the detectionof LY6E protein by 8-1 IHC to surfaceLY6E expression detected by 9B12flow cytometry. Flow-cytometricdetection of surface LY6E expression(red line) on live cells by 9B12 antibodyis depicted on the left panel.Secondary antibody alone is used ascontrol (gray line). IHC detection ofLY6E total protein by 8-1 antibody isdepicted on the corresponding rightpanel for each cell line. Cell linesdepicted are PC3-LY6E (A, top); PC3-Vector (A, bottom); MDA-MB-175-VII,high endogenous LY6E expressing cellline (B, top); and RAMOS, non-LY6E–expressing cell line (B, bottom). C,total LY6E protein detection byimmunoblotting with 8-1 antibody onPC3-LY6E cells. Independent LY6EsiRNAs labeled 1, 2, 3, and 4 [or nosiRNA (–) as control], were used toknock down LY6E expression. Lysateswere analyzed for LY6E protein 48hours post-siRNA transfection. b-actinserves as loading control (D). LY6Eprotein detection by 8-1 IHC iscompared with LY6E gene expressionin the same 106 serially cut breastcancer samples. The graph correlatesIHC detection of LY6E protein by 8-1antibody (IHC scoring on the x-axis)with LY6E gene expression analysisagainst two custom-designed assaystargeting the reference genes,TMEM55B and VPS33B on FluidigmDynamic arrays. LY6E RNA expressionon the y-axis is shown as DCt

(calculated using geomean of tworeference genes: TMEM55B andVPS33B). �� , P < 0.001 for IHC 3 groupas compared with IHC-0 or IHC-1þ.

(Continued.) Top panel represents overexpression in cancer tissues as fold change over normal samples from the same tissueswhere green or blue shading indicatesstatistical 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 colored bydiscretized 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 eachcopy number category. C, GISTIC was used to assess the statistical significance of recurrent gain. A large portion of the q-arm of chromosome 8 showshighly significant recurrent gain. This region encompasses both MYC and LY6E. D, absolute total copy number (Materials and Methods) at the LY6E locus shows ahighly significant positive correlation with LY6E transcript levels. The correlation coefficient (Spearman) and its significance are shown above for cancer panels ofbreast and ovarian carcinoma.

Asundi et al.





the use of 9B12 as a drug delivery agent targeting LY6E-positivecancers.

An important aspect of an ADC is its ability to mediatetarget-dependent cell killing. Because LY6E amplification levelshighly correlated with its overexpression in ovarian carcino-mas, we chose to evaluate the ability of anti-LY6E ADC toprovide targeted chemotherapy against a selected panel ofovarian cancer cell lines. We identified cell lines that weresensitive to the free MMAE drug and displayed a range of LY6Esurface expression that correlated positively to LY6E copynumber gain (Supplementary Table S4). Figure 4A and B

highlights anti-LY6E ADC activity in three ovarian cancer celllines with varying LY6E surface expression and copy number(CN) gain, (low expression, DOV 13; moderate expression,COV362; and high expression, KURAMOCHI); but with equiv-alent sensitivity to free MMAE (EC50 values of 0.38, 0.33, and0.3 nmol/L; Fig. 4C). The data demonstrated an overall cor-relation between LY6E surface expression and anti-LY6E ADCcell killing activity (Fig. 4A and B and Supplementary TableS4). Characterization of LY6E in 17 breast cancer cell linesshowed a similar correlation where LY6E expression predictedsensitivity to anti-LY6E ADC (Fig. 4D). In summary, these data

Table 1. Prevalence of LY6E in various cancers

Prevalence IHC (%) % IHCTumor types N 0 1þ 2þ 3þ 2þ/3þHer2� breast cancers 90 17 19 40 24 64SubtypesTNBC 39 5 158 33 46 79HRþ 51 20 20 41 20 61

NSCLC 276 13 30 46 10 56SubtypesAdenocarcinoma 205 11 25 51 13 64Squamous 71 17 37 39 7 46Pancreatic adenocarcinoma 78 12 26 50 13 63Ovarian carcinoma 57 23 24 44 9 53Head/neck squamous carcinoma 61 15 43 39 3 42Gastric adenocarcinoma 94 30 39 27 4 31Colorectal adenocarcinoma 106 36 53 10 1 11

Normal 0 1+ 2+ 3+

Bre

ast

Lung

Pan

crea

s

Tumor tissue

Ova

rian

HN

SC

CG

astr

ic

Figure 3.Expression of LY6E in human tumorsand matched normal tissues. IHCdetection of LY6E protein using anti-LY6E GEN-93-8-1 antibody is shownfrom a series of normal and cancertissues in indications where LY6Etranscript was found to beupregulated in a tumor-specificmanner. Photomicrograph imagesdepict tumor tissues representative of0, 1þ, 2þ, and 3þ staining intensity.Black scale line, 100 mm.






demonstrate the ability of anti-LY6E ADC to deliver a targetedcytotoxic agent.

To determine the therapeutic potential of anti-LY6E ADC, weevaluated its preclinical activity against murine xenograft modelsderived from various cancer cell lines and patient-derived tumormodels (PDX). We chose models that allowed us to generaterobust uniform sized tumors; and most closely represented themoderate (IHC 2þ) LY6E protein expression levels (Fig. 5A–D;inset panels) that could be expected in cancers in clinicalsettings. A single dose of anti-LY6E ADC demonstrated excel-lent efficacy against both a pancreatic (SU.86.86) and a NSCLCxenograft model (SW900) that homogenously express Ly6E atweak to moderate levels. Anti-LY6E ADC provided clear tumorinhibitory activity in both models; a 2-mg/kg dose yielded 8 of10 partial responses (PR) and 1/10 complete response (CR) inthe SU.86.86 xenograft (Fig. 5A); whereas a 1-mg/kg dose of theADC yielded 8 PRs with tumor regression lasting for about amonth postdose in the SW900 xenograft model (Fig. 5B). Anti-LY6E ADC activity was 1. Dependent on LY6E expression in allmodels; as a control non-specific antibody, conjugated to

MMAE had little activity at the highest dose; and 2. RequiredMMAE, since 8 to 12 mg/kg unconjugated anti-LY6E antibodyhad no activity in the three models tested (SupplementaryTable S5).

Recent studies have suggested that PDX models would betterrepresent the clinical behavior of human tumors as comparedwith cell line-derived xenografts (22, 23). We, therefore, testedanti-LY6E ADC activity in a pancreatic PDX model, PAXF 1657,which expresses LY6E (IHC 2þ) at moderate levels (Fig. 5C).Anti-LY6E ADC showed remarkable efficacy with 8 of 10 PRsand 2 of 10 CRs following a single anti-LY6EADCdose of 4mg/kg(Fig. 5C)

Many ADC targets are heterogeneously expressed in humantumors but are only validated in preclinical models that showrobust homogenous target expression (24, 10). Therefore, tobetter represent tumors exhibiting nonuniform target expression,we chose two PDX models HBCx-9 (TNBC) and MAXF 1162(HER2þ breast cancer) where LY6E was heterogeneouslyexpressed at weak–moderate levels (Fig. 5D and SupplementaryFig. S4). Nevertheless, robust tumor killing was observed in both

A

C

DOV 13 (CN = 2)

% o

f Max

COV362 (CN = 3)

% o

f Max

KURAMOCHI (CN = 4)

% o

f Max

1.0

2.0

2.5

0

LY6E-vc-MMAE

IgG-vc-MMAE

LY6E-vc-MMAE

IgG-vc-MMAE

1.0

2.0

2.5

0

B

Per

cent

sur

viva

l

Free MMAE (nmol/L)

0100101.00.10.01

25

50

75

100

DOV 13

COV362

KURAMOCHI

*

*

* ** *

**

101.00.10.010.001 101.00.10.010.001

1.5

0.5

1.5

0.5

D MMAE-sensitive breast cancer cell lines

ρ= 0.61; P = 0.009

LY6E

exp

ress

ion

(RP

KM

)

Viability

400

200

600

40

60

100

1,000

00.2 0.4 0.6 0.8 1.0

ADC (μg/mL) ADC (μg/mL) ADC (μg/mL)

Rel

ativ

e lig

ht u

nits

(×10

–5)

Rel

ativ

e lig

ht u

nits

(×10

–5)

Rel

ativ

e lig

ht u

nits

(×10

–5)

LY6E-vc-MMAE

IgG-vc-MMAE

3.0

4.0

5.0

1.0

2.0

101.00.10.01

*

0.0010

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

FL1-H FL1-H FL1-H

Figure 4.Correlation of LY6E surface expression tocell killing in vitro LY6E surface expressioncorrelates to anti-LY6E ADC cell killing invitro. A, shows the relative amounts of cellsurface LY6E detected by flow cytometrywith 9B12 antibody (red line) on ovariancell lines DOV 13 (left), COV362 (middle),and KURAMOCHI (right). Secondaryantibody alonewas used as a control (grayline). LY6E copy number (CN) for each cellis shown at the top of the flow-cytometricplot. B, cell killing by anti-LY6E ADC(9B12) titration is presented below eachflow-cytometric plot for thecorresponding cell line. The indicatedconcentrations of anti-LY6E-vc-MMAE(red line) or control IgG-vc-MMAE (grayline) were incubated with cells for 5 daysand relative cell viability (y-axis) assessedusingCellTiter-Glo. P values are calculatedbased on two tailed, Student t test.Asterisks depict P value <0.01. C, the celllines DOV 13 (dotted line, round markers),COV362 (solid line, round markers), andKURAMOCHI (dotted line, squaremarkers) were treated with increasingconcentrations of free MMAE for 5 daysand cell viability was determined by theCellTiter-Glo assay. D, correlationbetween LY6E expression and anti-LY6EADC killing (at 5 mg/mL anti-LY6E ADC) ina panel of 17 MMAE-sensitive breastcancer cell lines. Each dot represents onecell line. Viability effects were determined3 days after single-dose treatment withthe anti-LY6E-ADC. Correlation wasdetermined by Pearson analysis and the Pvalue of the correlation is indicated. RPKMis defined as Reads Per Kilobase perMillion mapped reads.

Asundi et al.





models. In HBCx-9, all animals achieved a complete response at adose of 12 mg/kg. Tumor regression was sustained for approxi-mately 25 days after treatment, even in the lower dose (8 mg/kg).To further test the limits of anti-LY6E ADC, we used the MAXF1162 HER2þ breast cancer PDX model. Interestingly, the MAXF1162 model, despite displaying high (IHC 3þ, data not shown)HER2expression, demonstratedno response toHER2 targeting byT-DM1 (Supplementary Fig. S4B). Remarkably, we observedstrong efficacy of anti-LY6E ADC in MAXF 1162 with a singledose at 4mg/kg yielding 4 of 10 PRs. Higher doses of the ADC ledto complete responses. (Supplementary Fig. S4C and Supplemen-tary Table S5 and S6). In summary, potent dose-dependent anti-LY6E ADC efficacy was achieved in vivo against a range of tumorindications with different LY6E expression patterns and responseto other ADC agents.

DiscussionIn this study, we used an in silico approach to identify LY6E

transcript overexpression in a wide array of solid tumor malig-nancies. LY6E protein overexpression in these tumors was con-firmed by developing a robust, LY6E-specific IHC assay. Further-more, overexpression of LY6E was noted in several cancer typesthat have limited treatment options with the highest level of LY6Eexpression being found in triple-negative breast cancer samples,(79% of samples showed 2þ or 3þ staining). It is worth notingthat while a number of ADC targets have been offered in specificcancer target indications, LY6E is unique in its broad expressionpattern across a wide range of different human cancers. Thesefindings enhanced our interest in exploring the therapeutic poten-tial of using LY6E as an ADC target.

Vehicle

LY6E(6)

LY6E(1)LY6E(3)

Ctrl(6)

Tum

or v

olum

e (1

00 ×

mm

3)

4

6

10

2

8

10 30 400

Day20

BA

Tum

or v

olum

e (1

00 ×

mm

3)

Vehicle

Ctrl(8)

LY6E(8)

LY6E(2)LY6E(4)

LY6E(1)

LY6E(0.5)Ctrl(4)

Naked Ab

10 20 30 400

5

10

15

20

25

Day

SU.86.86 SW900

D

Vehicle

LY6E(12)

LY6E(4)

LY6E(2)

Ctrl(12)

Ctrl(8)

LY6E(8)

Ctrl(2)

Tum

or v

olum

e (1

00 ×

mm

3 )

10 20 30 400

4

8

12

16

Day

HBCx-9C

Vehicle

LY6E(12)

LY6E(4)

LY6E(2)

Ctrl(12)

Ctrl(8)

LY6E(8)

Ctrl(2)

10 20 30 400

Tum

or v

olum

e (1

00 ×

mm

3 )

5

10

15

20

25

Day

PAXF 1657

1+/2+

2+

2+

1+/2+ (heterogeneous)

Figure 5.Anti-LY6E ADC inhibits tumorgrowth in vivo. A–D, graphs depictefficacy of anti-LY6E ADC in variouspancreatic, NSCLC, andbreast cancerxenografts. Mice bearing tumorsderived from SU.86.86 pancreaticcancer cell line (A) or SW900 NSCLCsquamous cell carcinoma (B),patient-derived primary tumors,PAXF 1657 (C), and HBCx-9 (D) wereadministered a single i.v. injection ofeither vehicle, anti-LY6E antibody9B12, either as unconjugated [NakedAb, depicted in (A) only, seesupplementary Table S5 for NakedAb in other xenografts], or as ADC(LY6E), control ADC (Ctrl) at mg/kgdoses indicated in parentheses.Photomicrographic insets show theintensity and heterogeneity of LY6Eexpression as detected by anti-LY6EIHC. Scale bar, 100 mm. Averagetumor volumes with SDs weredetermined from 9 to 10 animals pergroup and depicted on the y-axiswhereas day of study was depictedon the x-axis; using GraphPad Prismv6.0e software. Detailed tumorgrowth and statistical analyses forthe efficacy xenograft modelsare provided in SupplementaryTables S5 and S6.






We chose to use LY6E as a target antigen for the synthetic,potent antimitotic analogue of dolstatin 10, MMAE, which bindsand disrupts the microtubule network in proliferating cells(9, 17). The use of MMAE offered distinct advantages over usingDNA-binding cytotoxic agents such as duocarmycin and calichea-micin analogues, as these potent molecules lack selectivity totarget cells (25). MMAE was further chosen over other clinicallyapproved drugs such as doxorubicin, vinca alkaloids, etc., as thesedrugs were marred by relatively low potencies, linker instability,and compositional heterogeneity (9, 25, 26). MMAE offered usthe further advantage of using conjugation and cleavable linkerchemistries that allowed the ADC to remain relatively stable inplasma but released the drug efficiently in the acidic lysosomalcompartment of target cells, thereby minimizing systemic drugrelease and damage to nontarget cells (17, 25).

Internalization of the antibody for effective drug delivery to theacidic lysosomal compartment of a cancer cell, where intracellularcleavage releases free drug, remains a key feature of MMAE-conjugated ADC-targeted chemotherapy. In principle, such ADCsshould have minimal toxicities toward normal cells due to lowtarget expression on these cells resulting in lowuptake of the drug.In addition, MMAEs interfere with microtubule dynamics individing cells; the rate of cell division of normal cells is generallylower than that for cancer cells; which furthermoderates potentialtoxicities of the ADCs to normal cells (8).

We elucidated LY6E trafficking in an effort to maximize anti-tumor efficacy by selecting the appropriate antibody to bestmediate ADC drug delivery. We observed uptake of anti-LY6Eantibody 9B12 into lysosomes within a 30-minute incubationperiod, via mechanisms distinct from the classical traffickingpathways involving clathrin-coated pits. Our data demonstrateeffective endocytosis of a GPI-anchored protein and illustrate thatthe 9B12 anti-LY6E ADC would serve as an effective mediator ofdrug delivery to lysosomes.

We found an overall trend toward increased LY6E gene copynumber correlating to increased LY6E cell-surface expression andenhanced in vitro anti-LY6E ADC activity in breast and ovariancancer cell lines. However, in vitro killing by ADC was also clearlyinfluenced by the relative sensitivity of the various cell lines to thefree drug. For instance, OAW42, an ovarian cell line with highLY6E gene copynumber of 5,was resistant to killing by freeMMAEand therefore resistant to killing by anti-LY6E ADC.

Having observed excellent efficacy of anti-LY6E ADC againstcancer cell line derived xenografts, we further evaluated its efficacyin physiologically relevant PDX models with moderate homog-enous and heterogeneous LY6E expression. Efficacy of anti-LY6EADC at 4 mg/kg in the pancreatic PDX model PAXF 1657 wasparticularly encouraging in view of the fact that an ADC targetinganother GPI-anchored protein mesothelin (MSLN) yielded com-parable efficacy in the samemodel at a substantially higher dose of20 mg/kg (27). Both LY6E and MSLN demonstrated moderate(IHC 2þ) scores within the dynamic range of individual targetexpression in the PAXF 1657 PDX model. On the basis of ourinternalization data, we may speculate that the higher efficacy ofanti-LY6E ADC as compared with anti-MSLN ADC in this model

could be due to a more efficient endocytosis process for LY6E (30minutes for LY6E as compared with 20 hours for MSLN), (27)resulting in robust drug delivery and therefore, efficacy of theADC.

MAXF1162 (40% IHCþ) andHBCx-9 (�15%IHCþ) representhighbarmodelswith less than50%of tumor cells exhibiting LY6Eexpression by IHC. The remarkable efficacy of anti-LY6E ADC inthese heterogeneous disease settings could likely be due to abystander effect, whereby the efficient release of the drug in targetcells is followed by diffusion of the membrane permeable MMAEinto neighboring cells lacking target (28). The primary mecha-nism of action of MMAE is by the disruption of the microtubulenetwork in proliferating cells (9, 17). This, combined with lowLY6E expression in normal tissues may reduce potential systemictoxicities due to anti-LY6E ADC.

Moreover, the expression of LY6E in HER2þ breast cancerssuggests that anti-LY6E ADC could represent a treatment optionforHER2þ patients, unresponsive to existing treatments such as T-DM1. Indeed, the anti-LY6E ADCwas effective in the MAXF 1162PDX model, which responds only weakly to T-DM1 despiteIHC3þ expression of HER2 (Supplementary Fig. S4B). Furtherinvestigation will be required to elucidate the mechanism thataccounts for LY6E activity in T-DM1–resistant breast cancers.

In summary, we find that LY6E protein is overexpressed in awide range of solid tumor malignancies and anti-LY6E ADC haspotential utility as a treatment option for patients with unmetmedical need in clinical settings.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Asundi, J. Tremayne, P. Chang, R. Raja, R. FiresteinDevelopment of methodology: J. Asundi, P. Chang, C. Sakanaka, R. Desai,R. Raja, R. FiresteinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Asundi, L. Crocker, J. Tremayne, P. Chang,C. Sakanaka, J. Tanguay, S. Chalasani, E. Luis, R. Raja, R. FiresteinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Asundi, L. Crocker, P. Chang, C. Sakanaka,J. Tanguay, E. Luis, K. Gascoigne, B.A. Friedman, P.M. Haverty, P. Polakis,R. FiresteinWriting, review, and/or revision of the manuscript: J. Asundi, L. Crocker,R. Desai, R. Raja, P. Polakis, R. FiresteinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Asundi, L. Crocker, P. Chang, C. Sakanaka,R. FiresteinStudy supervision: P. Chang, S. Spencer, R. Raja, R. FiresteinOther (generated qPCR data for the manuscript): R. Desai

AcknowledgmentsThe authors thank Zeran Wang for help with Scatchard analysis, Anneleen

Damaen and Zhaoshi Jiang for Bioinformatics support, and Linda Rangell,Debra Dunlap, and Thinh Pham for tissue staining and analysis.

Received January 21, 2015; revisedMarch 17, 2015; acceptedMarch 19, 2015;published OnlineFirst April 10, 2015.

References1. Burris HA. Trastuzumab emtansine: a novel antibody-drug conjugate for

HER2-positive breast cancer. Expert Opin Biol Ther 2011;11:807–19.2. Foyil KV, Bartlett NL. Brentuximab vedotin for the treatment of CD30

þlymphomas. Immunotherapy 2011;3:475–85.


Asundi et al.




3. Schmeisser H, Mejido J, Balinsky CA, Morrow AN, Clark CR, Zhao T, et al.Identification of alpha interferon-induced genes associated with antiviralactivity in Daudi cells and characterization of IFIT3 as a novel antiviralgene. J Virol 2010;84:10671–80.

4. Rubinfeld B, Upadhyay A, Clark SL, Fong SE, Smith V, Koeppen H, et al.Identification and immunotherapeutic targeting of antigens induced bychemotherapy. Nature Biotech 2006;24:205–9.

5. Bresson-MazetC,GandrillonO,Gonin-Giraud S. Stem cell antigen2: a newgene involved in the self-renewal of erythroid progenitors. Cell Prolif2008;41:726–38.

6. Asundi J, Lacap JA, Clark S, Nannini M, Roth L, Polakis P. MAPK pathwayinhibition enhances the efficacy of an anti-endothelin B receptor drugconjugate by inducing target expression in melanoma. Mol Cancer Ther2014;13:1599–610.

7. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, et al.Humanization of an anti-p185HER2 antibody for human cancer therapy.Proc Natl Acad 1992;89:4285–9.

8. Chen Y, Chalouni C, Tan C, Clark R, Venook R, Ohri R, et al. Themelanosomal protein PMEL17 as a target for antibody drug conjugatetherapy in melanoma. J Biol Chem 2012;287:24082–91.

9. Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG, ChaceDF, et al. Development of potent monoclonal antibody auristatin con-jugates for cancer therapy. Nature Biotech 2003;21:778–84.

10. Asundi J, Reed C, Arca J, McCutcheon K, Ferrando R, Clark S, et al. Anantibody-drug conjugate targeting the endothelin B receptor for the treat-ment of melanoma. Clin Cancer Res 2011;17:965–75.

11. Adler AS, McCleland ML, Truong T, Lau S, Modrusan Z, Soukup TM, et al.CDK8 maintains tumor dedifferentiation and embryonic stem cell plur-ipotency. Cancer Res 2012;72:2129–39.

12. Greenman CD, Bignell G, Butler A, Edkins S, Hinton J, Beare D, et al.PICNIC: an algorithm to predict absolute allelic copy number variationwith microarray cancer data. Biostatistics 2010;11:164–75.

13. Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS,et al. Comprehensive genomic analysis identifies SOX2 as a frequentlyamplified gene in small-cell lung cancer. Nature Genetics 2012;44:1111–6.

14. Tibshirani R, Wang P. Spatial smoothing and hot spot detection for CGHdata using the fused lasso. Biostatistics 2008;9:18–29.

15. Mermel CH, Schumacher SE, Hill B, Meyerson ML, Beroukhim R, Getz G.GISTIC2.0 facilitates sensitive and confident localization of the targets of

focal somatic copy-number alteration in human cancers. Genome Biol2011;12:R41.

16. Curtis C, Shah SP, Chin SF, Turashvili G, RuedaOM,DunningMJ, et al. Thegenomic and transcriptomic architecture of 2,000 breast tumours revealsnovel subgroups. Nature 2012;486:346–52.

17. Francisco JA, Cerveny CG, Meyer DL, Mixan BJ, Klussman K, Chace DF,et al. cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conju-gate with potent and selective antitumor activity. Blood 2003;102:1458–65.

18. Kumari S, Mg S, Mayor S. Endocytosis unplugged: multiple ways to enterthe cell. Cell Res 2010;20:256–75.

19. Sangiorgio V, PittoM, Palestini P,MasseriniM. GPI-anchored proteins andlipid rafts. Ital J Biochem 2004;53:98–111.

20. Smart EJ, Anderson RG. Alterations in membrane cholesterol thataffect structure and function of caveolae. Methods Enzymol 2002;353:131–9.

21. Subtil A, Hemar A, Dautry-Varsat A. Rapid endocytosis of interleukin 2receptors when clathrin-coated pit endocytosis is inhibited. J Cell Sci1994;107:3461–8.

22. Kopetz S, Lemos R, PowisG. The promise of patient-derived xenografts: thebest laid plans of mice and men. Clin Cancer Res 2012;18:5160–2.

23. Tentler JJ, Tan AC, Weekes CD, Jimeno A, Leong S, Pitts TM, et al. Patient-derived tumour xenografts as models for oncology drug development. NatRev Clin Onc 2012;9:338–50.

24. Chen Y, Clark S, Wong T, Chen Y, Chen Y, Dennis MS, et al. Armedantibodies targeting the mucin repeats of the ovarian cancer antigen,MUC16, are highly efficacious in animal tumor models. Cancer Res2007;67:4924–32.

25. Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy. AnnuRev Med 2010;64:15–29.

26. Senter PD. Potent antibody drug conjugates for cancer therapy. Curr OpinChem Biol 2009;14:235–44.

27. Scales SJ, GuptaN, PachecoG, Firestein R, FrenchDM, KoeppenH, et al. Ananti-mesothelin-monomethyl auristatin E conjugate with potent anti-tumor activity in ovarian, pancreatic and mesothelioma models. MolCancer Ther 2014;13:2630–40.

28. Okeley NM, Miyamoto JB, Zhang X, Sanderson RJ, Benjamin DR, SieversEL, et al. Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clin Cancer Res 2010;16:888–97.






Published OnlineFirst April 10, 2015.Clin Cancer Res Jyoti Asundi, Lisa Crocker, Jarrod Tremayne, et al. Killing in a Wide Range of Solid Tumor MalignanciesAntigen 6 Complex, Locus E (LY6E) Provides Robust Tumor

Drug Conjugate Directed against Lymphocyte−An Antibody

Updated version

10.1158/1078-0432.CCR-15-0156doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2015/04/14/1078-0432.CCR-15-0156.DC1Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/early/2015/05/30/1078-0432.CCR-15-0156To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-15-0156

http://clincancerres.aacrjournals.org/content/suppl/2015/04/14/1078-0432.CCR-15-0156.DC1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2015/05/30/1078-0432.CCR-15-0156


An Antibody Drug Conjugate Directed against...

Documents

Transcript of An Antibody Drug Conjugate Directed against...