a potent her3 monoclonal antibody that blocks …...large molecule therapeutics a potent her3...

Large Molecule Therapeutics

A Potent HER3 Monoclonal Antibody That BlocksBoth Ligand-Dependent and -IndependentActivities: Differential Impacts of PTEN Status onTumor ResponseZhan Xiao1, Rosa A. Carrasco1, Kevin Schifferli1, Krista Kinneer1, Ravinder Tammali1,Hong Chen1, Ray Rothstein1, Leslie Wetzel1, Chunning Yang2, Partha Chowdhury2,Ping Tsui2, Philipp Steiner1, Bahija Jallal1, Ronald Herbst1, Robert E. Hollingsworth1, andDavid A. Tice1

Abstract

HER3/ERBB3 is a kinase-deficient member of the EGFR familyreceptor tyrosine kinases (RTK) that is broadly expressed andactivated in human cancers. HER3 is a compelling cancer targetdue to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumorresistance to a variety of cancer therapies, especially targeteddrugs against EGFR and HER2. HER3 can be activated by itsligand (heregulin/HRG), which induces HER3 heterodimeriza-tion with EGFR, HER2, or other RTKs. Alternatively, HER3 canbe activated in a ligand-independent manner through hetero-dimerization with HER2 in HER2-amplified cells. We devel-oped a fully human mAb against HER3 (KTN3379) that effi-ciently suppressed HER3 activity in both ligand-dependent andindependent settings. Correspondingly, KTN3379 inhibited

tumor growth in divergent tumor models driven by eitherligand-dependent or independent mechanisms in vitro and invivo. Most intriguingly, while investigating the mechanisticunderpinnings of tumor response to KTN3379, we discoveredan interesting dichotomy in that PTEN loss, a frequently occur-ring oncogenic lesion in a broad range of cancer types, sub-stantially blunted the tumor response in HER2-amplified can-cer, but not in the ligand-driven cancer. To our knowledge, thisrepresents the first study ascertaining the impact of PTEN losson the antitumor efficacy of a HER3 mAb. KTN3379 is currentlyundergoing a phase Ib clinical trial in patients with advancedsolid tumors. Our current study may help us optimize patientselection schemes for KTN3379 to maximize its clinical ben-efits. Mol Cancer Ther; 15(4); 689–701. �2016 AACR.

IntroductionHER3; ERBB3 is a member of the EGFR/HER family of

receptor tyrosine kinases (RTK), which also includes EGFR(ERBB1/HER1), HER2 (ERBB2), and HER4 (ERBB4). EGFR andHER2 are well-established oncogenic RTKs driving the growthof multiple types of solid tumors, including breast, colorectal,lung, head and neck, glioblastoma, and gastric cancers (1, 2).Both EGFR and HER2 are clinically validated targets withapproved therapeutics in clinical practice (EGFR: cetuximab,panitumumab, erlotinib, gefitinib; HER2: trastuzumab, pertu-zumab, lapatinib). All four HER family RTKs are predicted to betype I single transmembrane domain proteins that have an N-terminal extracellular domain containing a ligand-binding site,

a transmembrane region, an intracellular cytoplasmic regionwith a tyrosine kinase domain, and a C-terminal signaling tail.Of the four EGFR/HER family members, HER3 is notable forhaving a functionally inactive tyrosine kinase domain (3) andHER2 is notable for not having a known ligand (4).

Tyrosine kinase activities of EGFR and HER2 have been showntobe essential for their oncogenic potentials. A consequence of thekinase-deficiency of HER3 is that it needs to form heterodimerswith kinase-proficient RTKs to become active (5). In the presenceof the HER3 ligand heregulin (HRG), HER3 preferentially hetero-dimerizes with EGFR or HER2, subsequently undergoing tyrosinephosphoylation by these partner molecules on the C-terminalregion of HER3, thereby enabling downstream signaling. HER3exhibits a similar capacity to heterodimerize with the activatedhepatocyte growth factor (HGF) receptor (MET; ref. 6) and fibro-blast growth factor receptor 2 (FGFR2; ref. 7), however, it iscurrently unclear to what degree heterodimerization with HER3is required for the full transforming capacity of MET or FGFR2.

In addition to the ligand-dependent HER3 heterodimerizationdescribed above, in cells with very high levels of HER2 expressionbut no HRG ligand, HER2:HER3 heterodimers form spontane-ously and enable downstream signaling in a ligand-independentmanner. The HER2:HER3 heterodimers, formed with or withoutHER3 ligand, are structurally and functionally distinct as eluci-dated by studies comparing the mechanism of actions of twodifferent anti-HER2 antibodies, trastuzumab and pertuzumab.

1Oncology Research, MedImmune, Inc., One MedImmune Way,Gaithersburg, MD. 2Antibody Development and Protein Engineering,MedImmune, Inc., Gaithersburg, Maryland.

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

Corresponding Author: Zhan Xiao, MedImmune, Inc., One Medimmune Way,Gaithersburg, MD 20878. Phone: 301-398-4344; Fax: 301-398-9344; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0555

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

www.aacrjournals.org 689

on June 25, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst February 15, 2016; DOI: 10.1158/1535-7163.MCT-15-0555

http://mct.aacrjournals.org/

Pertuzumab preferentially disrupts ligand-dependent heterodi-mer signaling, whereas trastuzumab disrupts only ligand-inde-pendent signaling (8).

The central role for HER3 in oncogenesis is to act as a scaffold-ing protein that couples to and enables the maximal induction ofPI3K, thereby driving the activation of the pro-oncogenic AKT/mTOR pathway. HER3 contains a cluster of six C-terminal tyr-osines that when phosphorylated, forms the consensus PI3K/p85binding site (9, 10). By forming heterodimers with HER3, theupstream tumor drivers (EGFR, HER2, cMET, and FGFR2) cancouple efficiently with and signal robustly through the PI3K/AKTpathway. Therefore, it is reasonable to expect that a loss of HER3activitymay block cancer progression in diverse systems driven bydivergent RTKs. Studies have shown that HER3 small-interferingRNA (siRNA) knockdown in HER2-amplified breast cancer cellsled to similar antiproliferative effects as HER2 siRNA knockdown,further demonstrating the importance ofHER3 and its interactionwith other RTKs (8).

Besides promoting tumor growth directly, HER3 has alsobeen implicated in conferring tumor resistance to several target-ed drugs, including EGFR inhibitors (6, 11), HER2 inhibitors(12–15) and inhibitors of downstream kinases (16–18). Thiscapacity to drive both tumor growth and therapeutic resistancemakes HER3 an attractive target for therapeutic intervention.

To this end, we developed a fully human IgG1 antibody againstHER3 that potently blocks ligand-dependent and -independentactivities of HER3 and the downstream PI3K/AKT pathway. Thistranslated into significant tumor growth suppression both in vitroand in vivo across multiple models with different mechanisms ofHER3 activation. Combination of our anti-HER3 antibody withapproved EGFRorHER2 inhibitors resulted in superior antitumoreffects. Interestingly, PTEN deficiency selectively interfered withthe efficacy of HER2 and HER3 antagonists in HER2-amplifiedcancers but not in ligand-driven cancers.

Materials and MethodsAntigens

Recombinant human HER1(ECD)/Fc chimera, human HER2(ECD)/Fc chimera, human HER3(ECD)/Fc chimera, and humanHER4(ECD)/Fc chimera were purchased from R&D Systems, andwere fused to the C-terminal 6�Histidine-tag via a linker peptide.Recombinant mouse HER3(ECD)/Fc chimera was generated atMedImmune.

Anti-HER3 antibody generationRecombinant human HER3 ECD-Fc fusion protein was bioti-

nylated and used for panning of Dyax 310 phagemid library (19).The phage library was deselected with streptavidin-coated mag-neticDynabeads,M-280 (Invitrogen), and 2mg/mLof polyclonalhuman IgG in PBS, pH 7.2, with 0.1% Tween 20 and 2%BSA. Thedeselected library was subjected to solution panning with bioti-nylated HER3-Fc fusion protein at 2 mg/mL in PBS, pH 7.2 with0.1% Tween 20 and 2%BSA. The biotinylated antigen and boundphages were captured by streptavidin-coated magnetic Dyna-beads, M-280, after each round of panning and washed 15 timeswith PBS, pH 7.2, with 0.1% Tween 20 and 2 times with PBS, pH7.2. Phages bound to the beads were eluted with 10 mmol/LTriethylamine (TEA) and amplified in E. coli for the next round ofpanning. Three rounds of panning were done to enrich HER3-binding phage. HER3-specific phage clones were identified by

ELISA using plates that were coated with one of the following:human HER3 ECD-Fc, murine HER3 ECD-Fc, human EGFRECD-Fc, human HER2 ECD-Fc, human HER4 ECD-Fc, or humanIgG at 5 mg/mL in PBS, pH 7.4, with the selection criterion beingpositive binding to human andmurineHer3without any bindingto other EGFR family members (EGFR, HER2, and HER4). Thelight chain and variable heavy chain from positive phage cloneswere generated by PCR and inserted into a pOE human IgG1expression vector. These IgG1-formated leads were furtherscreened in cell-based functional assays to select the most potentlead in suppressing ligand-induced HER3 activity (phospho-HER3) and conferring HER3 internalization. The selected anti-HER3 antibody was sequence engineered and affinity maturedusing methods described previously (20). The binding affinity ofthe anti-HER3 antibody to human andmurineHER3-ECD/Fcwasmeasured using surface plasmon resonance (SPR) and show Kd of0.5 and 1.7 nmol/L respectively. The anti-HER3 antibody under-went further engineering (YTE-mutation in the Fc domain) toextend half-life in human using methods described previously(21) and is named KTN3379. KTN3379 and the parental non-half-life extended clone behaved identically in in vitro and mousexenograft studies (KTN3379 only shows improved binding tohuman but not murine Fc-receptors), and therefore they wereused interchangeably in the studies herein and collectivelyreferred to as KTN3379.

Cell cultureAll cell lines used in this study have been sourced from ATCC.

Cell-lines were authenticated by the manufacturer based on thefollowing criteria: short tandemrepeat profiling, cellmorphology,and karyotyping. The human breast cancer cell lines BT474 andMCF7 were grown in RPMI1640 medium. The human breastcancer cell lines MDA-MB-175 VII and MDA-MB-361 were main-tained in DMEM/F12 and DMEM, respectively. The human mel-anoma cell lines HMCB and CHL-1 were maintained in EMEMand DMEM, respectively. The non–small cell lung carcinoma cellline A549 was maintained in Ham's F12 medium. The humanhead and neck squamous carcinoma cell line FADU was culturedin RPMI1640 medium. The human breast cancer cell line JIMT1was grown in DMEM. The human lung carcinoma cell line A549was cultured in Ham's F12 (Kaighn's modification). The coloncancer cell line HT29 was maintained in McCoy 5A medium. Thegastric cancer cell line Snu16 was maintained in RPMI1640medium. All media were supplemented with 10% heat-inacti-vated FBS (Invitrogen) except MDA-MB-361, medium for whichwas supplemented with 20% heat-inactivated FBS. All cells werecultured in the absence of antibiotics and in a humidified atmo-sphere of 5% CO2 and at a temperature of 37�C, and kept inculture for no more than 3 months prior to the studies detailedhere. The receipt times for each cell line from the cell bank were asfollows: BT474: February 14, 2012; MCF7: December 29, 2011;MDA-MB-361: November 30, 2011; HMCB:May 9, 2013; CHL-1:February 22, 2008; A549: September 20, 2011; FADU: March 29,2013; JIMT1: May 6, 2013; HT29: September 19, 2011; andSnu16: January 15, 2013.

Antibody treatments and immunoblot analysisHMCB and CHL-1 cells were plated in medium supplemented

with 10%heat-inactivated FBS. The next day, the platingmediumwas removed and cells were subjected to incubation with

Xiao et al.

Mol Cancer Ther; 15(4) April 2016 Molecular Cancer Therapeutics690




antibodies at the indicated concentrations for 72 hours, cells werewashed oncewith ice-cold PBS and then lysed by adding LaemmilReducing buffer (Boston BioProducts). After a brief incubation,cell lysates were collected, equal amounts were loaded onto BisNuPAGE Novex Bis-Tris gels (Invitrogen), and proteins trans-ferred onto polyvinylidene fluoride membranes (Invitrogen).Membranes were blocked with 5% nonfat dry milk and 0.1%Tween 20 (Sigma) in Tris-buffered saline pH 7.4 (TBS) andincubated overnight at 4�C with antibodies to pHER3-Tyr1289(4791, Cell Signaling Technology), pAKT-Ser473 (4060, CellSignaling Technology), and neuregulin-1/HRG (NRG1/HRG)antibody (sc-348, Santa Cruz Biotechnology). An antibody toGAPDH (G8795, Sigma) was used to ensure equal amount ofprotein was loaded across all wells. Membranes were washed inTBS containing 0.1% Tween 20 and then incubated for 1 hourwith horseradish peroxidase–conjugated streptavidin secondaryantibodies (GE Healthcare). After washing, protein bands weredetected on X-ray film by using SuperSignal West Femto Chemi-luminescent substrate or SuperSignal West Pico Chemilumines-cent substrate (Pierce/Thermo Scientific).

In Fig. 1B, MCF7 cells were pretreated with antibodies at 10 mg/mL for 1 hour and then treated with 20 ng/mL of HRG for 10minutes. Cells were then processed for immunoblotting similarlyas HMCB cells.

In Fig. 4A, the various tumor xenografts were grown to500–600 mm3 in mice and then harvested. The samples wereshredded and homogenized and then lysed by boiling in SDSsample buffer. Similar amounts were run on SDS-PAGE andimmunoblotted for HER3, HRG, and GAPDH as indicated above.

ELISAs for detection and quantification of phospho-HER3 andphospho-AKT

MCF7 cells were subjected to serum starvation for approxi-mately 16 hours and then treated with serial dilutions of anti-bodies prepared in serum-free medium. After 1-hour incubationwith the test antibodies, cells were stimulated with 25 ng/mLHRG for 20 minutes. The medium was then removed andcells were washed once with ice-cold PBS and then lysed byadding Triton X100 lysis buffer (Boston Bioproducts) containingprotease (Sigma) and phosphatase inhibitor cocktails (EMD

C

A B

No

treat

men

t

pHER3

GAPDH

Total HER3

pAKT

Con

trol I

gG

KTN

3379

D

Unt

reat

ed

Con

trol I

gG

10 1 0.1

0.01

0.00

1

µg/mL

pHER3

HER3

pAKT

AKT

GAPDH

KTN3379

- + +

−6 −5 −4 −3 −2 −1 10 2–20

0

20

40

60

80

100Control IgG

KTN3379

Control IgG

KTN3379

Antibody concentration log (µg/mL)

Antibody concentration log (µg/mL)

pHE

R3

bind

ing

(% in

hibi

tion

vs. c

ontro

l)

HRG

−5 −4 −3 −2 −1 10 2

−20

0

20

40

60

80

100

Cel

l sur

viva

l(%

inhi

bitio

n vs

. con

trol)

* P < 0.05

** P < 0.01

Figure 1.KTN3379 suppresses HER3 activity and tumor growth in ligand-driven cancers. A, MCF7 cells were starved overnight and pretreated with the indicated doses ofKTN3379 or control IgG for 1 hour. Cells were then stimulated with 20 ng/mL of HRG for 20 minutes followed by processing for ELISA measurement ofpHER3 signal. Control represents cells treated with HRG only. � , P < 0.05, t test. B, MCF7 cells were untreated or treated with 10 mg/mL of control IgG or KTN3379for 1 hour prior to stimulation with 20 ng/mL of HRG. Cells were harvested for immunoblot analysis for pHER3, total HER3, pAKT, and GAPDH. C, HMCB cellswere seeded at 2,000 cells/well and treated with the indicated dose range of KTN3379 or control IgG for 6 days. Cells were processed with CellTiter-Glo for totalviable cell counts (Promega). Control corresponds to untreated cells. �� , P < 0.01, t test. D, HMCB cells were untreated or treated with control IgG or the indicateddose of KTN3379 for 1 hour. Cells were harvested for immunoblot analysis of the HER3 pathway components: pHER3, total HER3, pAKT, total AKT, and GAPDH.

PTEN as a Context-Dependent Predictive Marker for HER3 Therapy

www.aacrjournals.org Mol Cancer Ther; 15(4) April 2016 691




Millipore). A commercially available sandwich ELISA kit (R&DSystems)was used todetermine the level of phosphorylatedHER3(pHER3) in the MCF7 cell lysates based on manufacturer'srecommendations.

BT474 cells were plated in 96-well plates at a density of30,000 cells per well in RPMI medium (Invitrogen) containing10% heat-inactivated FBS (Invitrogen). The next day, the plat-ing medium was removed and cells were treated with theindicated doses of antibody prepared in culture medium. After4-hour incubation at 37�C, media were removed and cells werewashed once with ice-cold PBS (Invitrogen) and then lysed bythe addition of Triton X lysis buffer (Boston BioProducts)containing protease inhibitor (Sigma) and phosphatase inhib-itor cocktails (EMD Millipore). A commercially available sand-wich ELISA kit (R&D Systems) was used to determine thelevel of pHER3 based on the manufacturer's recommendations.A commercially available electrochemiluminescence (ECL)immunoassay was used to quantify the level of phosphorylatedAKT (pAKT) and total AKT (AKT; Meso Scale Discovery). Briefly,cell lysates were added to multiplex Meso Scale Discovery(MSD) plates precoated with capture antibodies for pAKT(Ser473) and AKT. After incubation for 1 hour at room tem-perature, unbound material was removed by washing, and ananti-total AKT antibody conjugated with an electrochemilumi-nescent compound, SulfoTAG (included with MSD kit), wasadded. SulfoTAG results in light emission when electrochem-ically stimulated. Following a 1-hour incubation period atroom temperature, the unbound material was removed bywashing. The ECL signal was captured and recorded by MSDSector Imager 6000.

Cell proliferation assaysHMCB and CHL1 cells with or without PTEN knockdown were

seeded at a density of 2,000 cells/well in complete mediumcontaining 10% heat-inactivated FBS and in white polystyrenetissue culture–treated 96-well plates with flat bottoms (Corning).The next day, plating medium was removed and antibody treat-ments in complete medium were added. Plates were then incu-bated in 5% CO2 at 37�C for 6 days. Equal volumes of CellTiter-Glo reagent (Promega) were added to each well at the end ofeach time point. Plates were rocked on a plate shaker for 10minutes at room temperature to ensure complete cell lysis.Luminescence was measured using an EnVision 2104 MultilabelReader (PerkinElmer).

BT474 cells were seeded at a density of 2,000 cells in 100 mLof RPMI cell culture medium (Invitrogen) containing 10%heat-inactivated FBS (Invitrogen) in white polystyrene tissueculture–treated 96-well plates. The next day, the plating medi-um was removed and the indicated antibodies were added inRPMI containing 10% heat-inactivated FBS to a final volume of100 mL per well. The plates were incubated in 5% CO2 at 37�Cfor 6 days. Equal volumes of CellTiter Glo reagent (Promega)were added to each well. Plates were incubated for 10 minutesat room temperature to ensure complete cell lysis. Lumines-cence was measured using an EnVision 2104 Multilabel Reader(PerkinElmer).

Colony formation assayBT474 cells were seeded at a density of 1,500 cells per well in

complete containing 10% heat-inactivated FBS (Invitrogen) intissue culture–treated 6-well plates. The next day, 200 mL of

media was removed from each well and replaced with 200 mL ofantibodies prepared at 10� concentration. Plates were incu-bated in 5% CO2 at 37�C for 3 weeks, and antibody treatmentwas changed once a week. At the end of the treatment period,medium was removed from each well and replaced with 1-mLmedium. An equal volume (1 mL) of CellTiter Glo reagent(Promega) was then added to each well. Plates were incubatedfor 10 minutes at room temperature to ensure complete celllysis. Luminescence was measured using an EnVision 2104Multilabel Reader (PerkinElmer).

Ligand-dependent and -independent dimerization studiesT47D and BT474 cells were plated at a density of 1� 106 cells

per well in a 6-well plate in complete medium with 10% heat-inactivated FBS. The next day, plating medium was removedand replaced with medium containing a saturating dose oftesting antibodies. The indicated antibodies were tested at aconcentration of 5 mg/mL for 2 hours at 37�C. T47D cells areused as a ligand-dependent model. T47D cells require stimu-lation by heregulin (HRG) to induce HER2:HER3 heterodimerformation. At the end of the 2-hour incubation with antibodies,media were removed and replaced with medium alone or withmedium containing 50 ng/mL HRG and incubated for 10minutes at 37�C. After HRG stimulation, cells were washedtwice with cold PBS, and lysed with cell lysis buffer (CellSignaling Technologies) containing protease inhibitors on icefor 30 minutes. BT474 cells represent a ligand-independentmodel, and therefore do not require HRG stimulation forHER2:HER3 heterodimer formation. At the end of the 1.5-hourincubation with antibodies, media were removed, and cellswere washed once with cold PBS. The crosslinker 3, 30-dithiobis[sulfosuccinimidylpropionate] (DTSSP) was added ata concentration of 2 mmol/L in 1-mL cold PBS. Cells wereincubated for at least 1 hour on ice. Cells were then washed3 times with cold PBS. Cell lysis buffer (500 mL) containingprotease and phosphatase inhibitors was added and the cellswere placed on ice for at least 30 minutes to allow forlysis before harvesting with a cell scraper. HER2 and HER3were immunoprecipitated from cell lysates. Cell lysates(500 mL) were combined with 50 mL protein A sepharose beads(50% slurry; Invitrogen) preconjugated to 1 mg of HER3 mAb(MAB3481, R&D Systems) in a SigmaPrep spin column (Sig-ma). The mixture was incubated with rotation at 4�C overnight.The next day, beads were separated from the cell lysateby centrifugation. Beads in the columns were washed fourtimes with cold cell lysis buffer (Cell Signaling Technology)containing protease (Sigma) and phosphatase inhibitors(EMD Millipore). After the wash procedure, 50 mL 2� SDSsample buffer containing 50 mmol/L dithiothreitol (DTT; EMDChemicals) was added (for BT474 cells only) into the spincolumns. The columns were then boiled for 4 minutes. ForT47D cells, no DTT was added in the 2� SDS sample buffer.Proteins were eluted by centrifugation and used immediatelyfor immunoblotting.

PTEN RNA interference studiesHMCB, CHL-1, and BT474 cells were stably transduced with

PTEN-targeted shRNA vectors (Open Biosystems). Stable cloneswere selected and successful target knockdown was confirmedby PTEN immunoblot. Cells were then used for either cell

Xiao et al.





proliferation study or immunoblotting for HER3 signalingpathway activity with the indicated antibody treatment.

Spheroid assayBxPC3 cells were transfected with ON-TARGETplus Non-

targeting (NT) or PTEN siRNA SMARTpools (GE Life Sciences)using RNAiMAX transfection reagent (Thermo Fisher), follow-ing the manufacturer's protocol. Cells were placed into spher-oid culture 48 hours after transfection with siRNA. Cell lysateswere also collected 48 hours after transfection with siRNA toassess knockdown efficiency by immunoblotting as describedin the "Antibody Treatments and Western Blotting" sectionherein.

To establish spheroid cultures, cells were removed from culturevessels with 0.05% trypsin and collected by centrifugation. CellswerewashedoncewithPBS and resuspended in0.25%MethoCult(Stem Cell Technologies) at a density of 0.25 � 106 cells/mL.Cells were then added to the inner wells of a non-tissue culture–treated U-bottomed 96-well plate (Costar), 20 mL per well. Thecells were pelleted via centrifugation at 1,200 rpm for 5 minutes,followed by an overnight incubation at 37�C. Treatments ofindicated antibodies were added in 100 mL of RPMI þ 2% FBSwith or without 100 ng/mL HRG and 100 mg/mL antibodies asdescribed in the figure legends, and plates were incubated for 7days. To evaluate cell survival, 100 mL of CellTiter-Glo reagent(Promega) was added to each well. Plates were rocked on a plateshaker for 20 minutes at room temperature to ensure completecell lysis. Luminescence was measured using an EnVision 2104Multilabel Reader (PerkinElmer).

In vivo studiesAll cell lines used for in vivo studies were grown in monolayer

culture as described above and maintained at 37�C in a 5%CO2

incubator and harvested by trypsinization. Xenografts wereestablished by subcutaneously injecting 5 � 106 (FADU,H460, or A549) cells per mouse, using a 27-gauge needle.Breast carcinoma xenografts were established orthotopicallyby subcutaneously injecting 1 � 107 (BT474) or 5 � 106

(MDA-MB-361, JIMT1) cells per mouse into the right fat pad.For BT474 and MDA-MB-361, estrogen pellets (0.36 mg) wereplaced under the skin of the left flank 1–2 days prior to cellimplantation. All xenografts were established in 4- to 6-week-old athymic nude mice (Harlan). All cells were suspended in50% Matrigel/PBS except for H460, which were suspended inPBS alone. Mice were staged and randomized into 10 mice pergroup when tumors reached 200–300 mm3. KTN3379, cetux-imab, and a nonspecific IgG1 control (MedImmune) wereprepared by diluting with PBS, trastuzumab and pertuzumabwere prepared by diluting with 0.9% saline. All mice treatedwith antibody therapeutics were dosed intraperitoneally twice aweek according to body weight (10 mL/kg). Both tumor andbody weight measurements were collected twice weekly andtumor volume calculated using the equation (L �W2)/2, whereL and W refer to the length and width dimensions, respectively.The general health of mice was monitored daily and all experi-ments were conducted in accordance to the Associationfor Assessment and Accreditation of Laboratory Animal Careand MedImmune Institutional Animal Care and Use Commit-tee guidelines for humane treatment and care of laboratoryanimals.

Pharmacodynamic analysis of tumorsTumor-bearing mice were dosed (i.p.) with KTN3379

(30 mg/kg), trastuzumab (10 mg/kg), pertuzumab (10 mg/kg),isotype control (30 mg/kg), or in combination on day 0 andday 3, and tumors were collected 24 hours after the seconddose. Harvested tumor samples were homogenized using aFastPrep-24 instrument (MP Bio). Briefly, tumors were placedin lysing matrix A vials (MP Bio) containing RIPA lysis buffer(500 mL) with protease and phosphatase inhibitors and pulsedfor 30 to 45 seconds, then immediately placed on ice. Lysateswere then centrifuged, clarified, and supernatants quantitatedby bicinchoninic acid (BCA) protein assay (Pierce).

ResultsDiscovery of fully human antagonistic mAbs against HER3

We set out to identify potent HER3 antagonistic mAbs. Theextracellular domain of human HER3 was used to pan a Dyaxhuman Fab phage library. Antibodies were then selected on thebasis of (i) their ability to bind to human HER3 withoutbinding to other EGFR/HER family members including EGFR,HER2, and HER4 (Supplementary Fig. S1); and (ii) similarbinding to murine HER3 (Supplementary Table S1). Uniqueclones were reformatted into human immunoglobulin G1(IgG1) and screened in a variety of cell-based assays. Theanti-HER3 antibody with highest potency suppressing HER3activity (phosphor-HER3/pHER3) and internalized HER3 invarious cell lines was further optimized for affinity. The finalanti-HER3 antibody, KTN3379, bound to human HER3 andmurine Her3 proteins with a Kd of 0.5 nmol/L and 1.7 nmol/L,respectively (Supplementary Table S1).

KTN3379 blocks ligand-dependent and ligand-independentHER3 signaling and tumor cell growth

To ascertainwhetherKTN3379 is effective in suppressing ligand(heregulin/HRG) driven HER3 signaling, we treated the breastcancer cell line MCF7 with HRG and measured the resultingphospho-HER3 signal using an ELISA assay (pHER3). MCF7 cellsdo not express detectable endogenousHRG, so exogenousHRG isneeded to induce HER3 phosphorylation. Pretreatment withKTN3379, but not the control IgG, for 1 hour prior to the ligandstimulation efficiently blocked pHER3 in a dose-dependent man-ner (Fig. 1A), demonstrating suppression of ligand-inducedHER3activation. We additionally confirmed this ELISA data with animmunoblot study, showing that KTN3379 not only efficientlyblocked ligand-induced pHER3, but also pAKT, the downstreameffector of HER3 (Fig. 1B).

To determine the functional impact of KTN3379 on cancer cellsdriven by endogenousHRG-dependent HER3 activity, we used anestablished HRG-HER3 autocrine model, melanoma cell lineHMCB (22), to characterize the antiproliferative effect ofKTN3379 in a 6-day cell growth assay. KTN3379, but not controlIgG, inhibited cell growth in a dose-dependent manner (Fig. 1C).The reason for the large difference in time of treatment betweenthe pHER3 inhibition assay (1 hour, Fig. 1A) versus this cellgrowth assay (6 days) is that the effect of an antibody on itsproximal target receptor's phosphorylation status is much moredirect and rapid than its distal impact on overall cell proliferation,which usually takes much longer to be demonstrable.

To explain this antiproliferative effect, we additionally charac-terized the impact of the KTN3379 on the HER3 signaling






pathway in HMCB cells via immunoblot (Fig. 1D). KTN3379abrogated pHER3 and the pAKT signals in a dose-dependentmanner, suggesting the antiproliferative effect was due to inhi-bition of HER3-AKT pathway. Interesting, the total HER3 proteinblot showed partial degradation of HER3 by theHER3mAb at thehigher dose levels, suggesting target proteolysis constitutes one ofits mechanisms of action in this model.

We also observed a similar growth inhibition effect in anotherknown HRG-autocrine model, the breast cancer cell line MDA-MB-175 (Supplementary Fig. S2A). Besides directly sustainingcancer cell growth, we also found that HRG/HER3 pathway couldinduce cancer cell secretion of VEGF, the master driver of tumorangiogenesis, thereby contributing to the multifaceted tumor-promoting capacity of HER3 (Supplementary Fig. S2B). Asexpected, KTN3379 was able to substantially block the VEGFinduction effect.

Next we studied the ability of our anti-HER3 mAb to sup-press HER3 signaling in the ligand-independent setting usingthe HER2-amplified breast cancer model BT474. This modelhas high HER3 expression and activity (as demonstrated bypHER3) but minimal HRG expression (23). As mentionedearlier, HER2-HER3 heterodimerization in models such as thisone is not induced by ligand and occurs in a constitutivemanner, likely driven by the extremely high levels of HER2

protein. Using pHER3 ELISA assay, we were able to show thatthe KTN3379 treatment attenuated the high basal pHER3 signalin a dose-dependent manner (Fig. 2A), which translated intoinhibition of the downstream effector pAKT (Fig. 2B). To studythe consequence of this inhibition of HER3 activity, we assessedthe impact of KTN3379 on BT474 cell proliferation in both ashort-term cell growth assay (6 days, Fig. 2C) and a long-termcolony formation study (3 weeks: Fig. 2D). Moderate suppres-sion of cell growth was observed in the 6 day proliferation assay(�40% inhibition, Fig. 2C), consistent with the attenuationof basal pHER3. In contrast, we saw more drastic suppressionof colony formation (�80%, Fig. 2D), perhaps due to theprolonged incubation time (3 weeks vs. 6 days). Cell-cycleanalysis indicated that the antiproliferative effect of KTN3379in HER2-amplified cancers is likely conferred through induc-tion of cell-cycle arrest in the G1 phase at the expense of S- or G2

phase cells (Supplementary Fig. S3). Trastuzumab was used as apositive control, which conferred equivalent extent of G1 arrestas KTN3379. Pertuzumab, on the other hand, failed to mediatesimilar G1 arrest (Supplementary Fig. S3). This is consistentwith the notion that trastuzumab specifically disrupts theconstitutively formed HER2-HER3 dimer in cells with ampli-fied HER2, whereas pertuzumab only suppresses ligand-induced HER2-HER3 dimer (24).

A B

−6 −5 −4 −3 −2 −1 10 2−20

−10

0

10

20

30

40

50Control IgGKTN3379

Control IgGKTN3379

Antibody concentration log (µg/mL) Antibody concentration log (µg/mL)

pHE

R3

bind

ing

(% in

hibi

tion

vs. c

ontro

l)

−6 −5 −4 −3 −2 −1 10 2

−20

0

20

40

60

80

100

pAK

T bi

ndin

g(%

inhi

bitio

n vs

. con

trol)

C D

−5 −4 −3 −2 −1 210

0

20

40

60

Antibody concentration log (µg/mL) Antibody concentration log (µg/mL)

Cel

l sur

viva

l(%

inhi

bitio

n vs

. con

trol I

gG)

Cel

l sur

viva

l(%

inhi

bitio

n vs

. con

trol I

gG)

−5 −4 −3 −2 −1 210

0

20

40

60

80

100

Figure 2.KTN3379 inhibits HER3 signaling and tumor growth in HER2-amplified cancer cells. A, BT474 cells were treated with the indicated doses of KTN3379 or control IgGfor 4 hours and then processed for ELISA analysis of pHER3 as indicated in Materials and Methods. Control represents untreated cells. B, BT474 cells weretreated with the indicated doses of KTN3379 or control IgG for 4 hours and then processed for ELISA analysis of pAKT. Control represents untreated cells. C,BT474 proliferation assay was performed by treating cells with control IgG or KTN3379 at the indicated concentrations for 6-day as detailed in Materials andMethods. Cells were processed by CellTiter-Glo. Control IgG was used as 100% reference to calculate the growth inhibition effect of KTN3379. D, BT474colony formation study was performed by seeding cells at 1,500 cells per well in a 6-well plate and treated with antibodies for 3 weeks as detailed in Materialsand Methods. Cells were then processed by CellTiter-Glo. Control IgG was used as 100% reference to calculate the inhibition effect of KTN3379.

Xiao et al.





Taken together, these data show that KTN3379 blocks HER3signaling and HER3-dependent tumor growth stimulated byligand or spontaneous heterodimerization with high levels ofHER2.

KTN3379 inhibits both ligand-induced and constitutiveHER2-HER3 dimerization

To explain the intriguing bifunctionality of KTN3379 insuppressing both ligand-dependent and -independent HER3activities, we carried out a biochemical study to measureHER2-HER3 dimerization in the presence or absence of ligand.For ligand-induced dimer formation, we pretreated the breastcancer cell line T47D cells with either control IgG or KTN3379for 1 hour, then stimulated the cells with HRG to induce HER2-HER3 complex formation (Fig. 3A). Cells were lysed and HER3was immunoprecipitated with a commercial anti-HER3 anti-body which binds to the intracellular domain of HER3 to avoidpotential competition with KTN3379. Then we probed theimmunoprecipitate for the presence of HER2 as evidence ofHER2-HER3 dimerization (Fig. 3A). In the absence of exoge-nous HRG, no HER2 was found in the HER3 precipitate (lane4). In contrast, HRG treatment efficiently induced HER2-HER3dimerization (lane 3). More importantly, pretreatment of thecells with KTN3379 but not the control IgG abrogated HRG-induced HER2-HER3 dimerization as indicated by the disap-pearance of HER2 (lane 2).

To determine whether KTN3379 could similarly block con-stitutive HER2-HER3 dimerization which occurs in the absenceof ligand, we measured the HER2-HER3 dimer formation in theHER2-amplified BT474 cells which have been shown to displayconstitutive HER3 activation without any endogenous HRGexpression, presumably through basal HER2–HER3 complexformation. To stabilize the constitutive HER2 and HER3 dimer,we pretreated the cells with the cross-linker DTSSP prior toimmunoprecipitation (24). Constitutive HER2–HER3 interac-tion was observed when cells were treated with the cross-linker,suggesting the presence of a constitutive HER2-HER3 dimer(Fig. 3B, lanes 1 and 2). Pretreatment with KTN3379, however,disrupted the dimer formation as evidenced by a sharp decrease

of HER2 in the HER3 immunoprecipitate (lane 3). These resultsare fully consistent with a structural analysis indicating thatKTN3379 has a unique binding epitope on HER3 that potentlyinhibits ligand-dependent and ligand-independent signalingby locking the receptor in an autoinhibited state regardless ofthe ligand presence (25).

KTN3379 inhibits the in vivo growth of tumorsexpressing HRG

To investigate whether the in vitro antiproliferative effectof KTN3379 could translate into in vivo efficacy, we tested itin multiple tumor xenograft models. To understand thelandscape of HER3 pathway activity in vivo, we first deter-mined HER3 and HRG protein profiles in a variety of solidtumor models with diverse tissue origins through immuno-blot (Fig. 4A). While HER3 was broadly expressed, HRGhad a more restricted expression pattern and was only presentin a fraction of the models such as lung adenocarcinomamodel A549 and head and neck squamous cell carcinoma(HNSCC) model FADU (Fig. 4A). In xenograft efficacy stud-ies, KTN3379 efficiently suppressed tumor growth in bothA549 and FADU models (Fig. 4B and C: tumor growthinhibition: 75%–100%, P < 0.01). In contrast, very modest(but statistically significant) antitumor activity was seen in thecolorectal cancer model HT29 (Fig. 4D: tumor growth inhi-bition < 25%, P < 0.05); minimal activity was seen in thegastric cancer model Snu16 (Supplementary Fig. S4; tumorgrowth inhibition < 10%, P > 0.05). Both models express verylow level of HRG but high levels of HER3 (Fig. 4A). Thisinteresting dichotomy suggested that HRG may be a betterpredictive biomarker for response to HER3 antagonists thanHER3 expression itself.

As anti-EGFR mAb cetuximab has been approved for treat-ment of HNSCC, we additionally compared its efficacy againstKTN3379 in the FADU model (Fig. 4C). KTN3379 was morepotent than a similar dose of cetuximab (P < 0.001). Interest-ingly, the combination of the two appeared to be superior toeither antibody alone (P < 0.0001, Fig. 4C). These conclusionsare supported by statistical analysis (figure legends).

A

No

IP m

Ab

KTN

3379

Con

trol I

gG

Con

trol I

gG

HRG + + + − HER2

HER3

B

Con

trol I

gG

Con

trol I

gG

KTN

3379

DTSSP − + +

HER2

HER31

1 2 3 4

2 3

Figure 3.KTN3379 disrupts HER2-HER3 dimerization in both ligand-dependent and -independent models. A, T47D cells were pretreated with control IgG or KTN3379 andthen treated with HRG to induce HER2/HER3 dimer formation as detailed in Materials and Methods. Cells were lysed and HER3 immunoprecipitation wasperformed with anti-HER3 antibody that targets the cytoplasmic domain. The precipitates were run on Western blots and probed for HER2 and HER3proteins. The various lanes were cropped from the same Western blot analysis. B, BT474 cells were pretreated with control IgG or KTN3379 and then treated withthe cross-linker DTSSP to stabilize HER2-HER3 dimer prior to cell lysis as detailed in Materials and Methods. Cells were lysed and HER3 immunoprecipitationwas performed with anti-HER3 antibody that targets the cytoplasmic domain. The precipitates were run on Western blots and probed for HER2 and HER3 proteinlevels. The various lanes were cropped from the same Western blot analysis.






KTN3379 inhibits the in vivo growth of HER2-positive breastcancer models

Besides being effective against HRG-driven tumors, our in vitrodata above demonstrated that KTN3379 also inhibits HER3activity and tumor proliferation in HER2-amplified breast cancercells (Figs. 2 and 3). To ascertain whether this activity couldtranslate to the in vivo setting, we assessed the efficacy of KTN3379in theHER2-amplifiedBT474 xenograftmodel (Fig. 5A). KTN3379achieved moderate tumor growth inhibition (55%, P < 0.01)but was less efficacious than trastuzumab (P < 0.001). In con-trast,weobserved similar growth suppressioneffect in vitrobetweenthe two mAbs (Fig. 6A). This could be due to the fact thattrastuzumab derives its in vivo efficacy not only from inhibitionof HER2/HER3 signaling pathway, but also antibody dependentcell-mediated cytotoxicity (ADCC) effects due to the tremendousHER2 overexpression on tumor cells (26), which may not beavailable for HER3 antibody. The combination of the two mAbsfailed to achieve superior tumor inhibition compared with tras-tuzumab monotherapy (Fig. 5A, P > 0.05).

Trastuzumab has been shown to be less active in breastcancers with moderate levels of HER2 overexpression. Breastcancer model MDA-MB-361 fits this category. We compared thesingle-agent activity of KTN3379 with trastuzumab in thismodel. As expected, trastuzumab alone was substantially lesseffective in this model compared with BT474 and only achievedmodest tumor growth delay (Fig. 5B). KTN3379 achieved asomewhat better response than trastuzumab in delaying tumorgrowth (Fig. 5B). Interestingly, the combination of the tworegressed tumors significantly better than either antibody alone(P < 0.01).

We also assessed KTN3379 in JIMT1, a HER2-amplified breastcancer model that is known to be refractory to trastuzumab (27).As expected, trastuzumab monotherapy was inactive (Fig. 5C).Interestingly, while KTN3379 achieved modestly better tumorgrowth inhibition than trastuzumab, the combination of thetwo agents was significantly better than either antibody aloneto essentially stabilize tumor growth, in line with the MDA-MB-361 findings. Pertuzumab was recently approved for treating

B

CA

A549

DA

549

H19

93

H46

0

HC

T116

SNU

16

MK

N45

KAT

OIII

HT2

9

Det

roit

562

FAD

U

A43

1

SSC

4

HRG

HER3

GAPDH

20 25 30 35 40

100

500

1,000

1,500

05 10 15 20 25

Untreated Control IgG (10 mg/kg)Cetuximab (10 mg/kg)

KTN3379 (10 mg/kg)

KTN3379 (10 mg/kg) +

Untreated Control IgG (30 mg/kg)

KTN3379 (10 mg/kg)

200

400

600

800

1,000

1,200

1,400

1,600

20 30 40 50

Days after cell implantation


200

300

400

500

600

700

800


Treatments:Antibodies: 2×/wk, i.p. Treatments:

Antibodies: 2×/wk, i.p.

Treatments:Antibodies: 2×/wk, i.p.

UntreatedControl IgG (30 mg/kg)KTN3379 (10 mg/kg)

Tum

or v

olum

e (m

m3 )

Tum

or v

olum

e (m

m3 )

Tum

or v

olum

e (m

m3 )

HT29

FADU

**P < 0.01

****

*P < 0.05

****

***

Cetuximab (10 mg/kg)

Figure 4.KTN3379 preferentially suppresses tumor growth in HRG-dependent models in vivo. A, HRG and HER3 expression profile across various tumor xenografts. Tumorsamples of the indicated models were harvested when they reached 500–600 mm3 volume. They were lysed and processed as in Materials and Methods andsubjected to Western blot analysis for HRG, HER3, and GAPDH. B–D, KTN3379 was tested in vivo for its antitumor effects in lung adenocarcinoma modelA549 (B), HNSCCmodel FADU (C), and colorectal cancermodel HT29 (D). Unless otherwise indicated, KTN3379was dosed at 10mg/kgwhile control IgGwas dosedat similar level or higher levels (30 mg/kg). The anti-EGFR mAb cetuximab was dosed at 10 mg/kg either as monotherapy or in combination with KTN3379in the FADU model. All mAbs treatment (twice per week) was started when tumor size reached �250 mm3. P values for KTN3379 vs. control IgG (t test):�� , P <0.01 (B); �� , P <0.0001 (C); � , P < 0.05 (D). P value for KTN3379 vs. cetuximab: �� , P <0.001, t test (C). P value for KTN3379 vs. KTN3379 þ cetuximab:�� , P <0.0001, t test (C).

Xiao et al.





HER2-positive patients in combination with trastuzumab whohave relapsed on trastuzumab. Interestingly, combination of thetwo HER2 mAbs was essentially inactive in this model and vastlyinferior compared with KTN3379 alone. However, KTN3379combination with trastuzumab conferred a better response thanKTN3379 alone (P < 0.0001, Fig. 5C).

Examination of the pharmacodynamics biomarker effects ofKTN3379 and trastuzumab on HER2 and HER3 in the JIMT1model revealed that while each mAb efficiently suppressed itsintended target (KTN3379 on HER3 and trastuzumab on HER2),they fail to suppress the other target. In contrast, combination ofthe two antibodies efficiently neutralized both pHER2 andpHER3, potentially accounting for the observed combinationeffect (Fig. 5D).

Differential impacts of PTEN loss on tumor response toKTN3379

Lesion in the critical tumor suppressor PTEN is amongthe most frequently occurring genetic abnormalities in cancer

and the resulting hyperactivation of PI3K/AKT pathway great-ly contributes to the tumorigenic process. To investigate thepotential impact of PTEN loss on tumor response toKTN3379, we knocked down PTEN expression by RNAi inboth the ligand-independent HER2-amplified BT474 cellsand the autocrine HRG-expressing HMCB cells (both modelsharbor wild-type PTEN expression, Supplementary Table S2)and measured cellular response to KTN3379 in both cellproliferation and HER3 signaling assays (Fig. 6). For controlpurposes, we first measured the cell growth rates of HMCBafter PTEN knockout and compared them with the respectivecontrol cells. No significant differences were seen, indicatingPTEN loss does not impact the baseline cell growth rate(Supplementary Fig. S5).

PTEN knockdown attenuated the antiproliferation effect ofKTN3379 in BT474 cells (extent of growth inhibition decreasedfrom �60% to �20%: ��, P < 0.001, Fig. 6A). Similarly, trastu-zumab showed the same trendbeing less active in PTEN-knockoutcells (��, P < 0.001, Fig. 6A), which is consistent with earlier

BT474

MDA-MD-361

JIMT1A

B

C

D

HER2

pHER2

HER3

pHER3

GAPDH

Control IgG KTN3379TrastuzumabKTN3379 +

Trastuzumab

Tum

or v

olum

e (m

m3 )

Tum

or v

olum

e (m

m3 )

Tum

or v

olum

e (m

m3 )

5 10 15 20 25 5 10 15 20 25 30 35100

200

300

400

500

600

700 Control IgG (30 mg/kg)

Trastuzumab (30 mg/kg)

Days after cell implantation Days after cell implantation

100

200

300

400

500

600

700

800

900

1,000

1,100


KTN3379 (10 mg/kg)

KTN3379 (10 mg/kg) +Trastuzumab (30 mg/kg)

Control IgG (30 mg/kg)Trastuzumab (10 mg/kg)Pertuzumab (10 mg/kg)

Pertuzumab (10 mg/kg)


KTN3379 (10 mg/kg)

KTN3379 (10 mg/kg) +

Trastuzumab (10 mg/kg) +

Trastuzumab (10 mg/kg)

15 20 25 30 350

200

400

600

800 Control IgG (30 mg/kg)KTN3379 (10 mg/kg)Trastuzumab (30 mg/kg)KTN3379 (10 mg/kg) +Trastuzumab (30 mg/kg)


Treatments:Antibodies: 2×/wk, i.p.****

P < 0.0001

**P < 0.01 ***

P < 0.001

****P < 0.0001

**P < 0.01

***P < 0.001

Figure 5.KTN3379 inhibits in vivo tumor growth of orthotopic HER2-positive breast cancer models with divergent sensitivity to trastuzumab. A, KTN3379 or trastuzumabwas dosed at 10 or 30 mg/kg, respectively, alone or in combination, in the HER2-amplified (3þ) BT474 xenograft model as described in Materials and Methods.P value between KTN3379 and control IgG: �� , P < 0.01, t test. P value between KTN3379 and trastuzumab: ��, P < 0.001, t test. B, KTN3379 or trastuzumabwas dosed at 10 mg/kg or 30 mg/kg, respectively, alone or in combination, in the Her2 2þ MDA-MB-361 xenograft model. P value between KTN3379 andcontrol IgG: �� , P < 0.0001, t test. P value between KTN3379 vs. trastuzumab: �, P < 0.05. P value between KTN3379 vs. KTN3379 þ trastuzumab: ��, P < 0.01. C,KTN3379, trastuzumab, or pertuzumab were dosed at 10 mg/kg, alone or in combination, in the HER2-positive but trastuzumab-resistant JIMT-1 xenograftmodel. All mAbs were dosed twice per week when tumor size reached �250 mm3. P value between KTN3379 and control IgG: �� , P < 0.001, t test; P valuebetween KTN3379 vs. KTN3379 þ trastuzumab: �� , P < 0.0001). D, biomarker analysis in JIMT-1 models undergoing HER2 and HER3 therapies. Tumorsamples from the indicated treatment groups were processed and run on immunoblots for HER2/pHER2, HER3/pHER3, and GAPDH analysis, as described inMaterials and Methods.






findings demonstrating trastuzumab resistance by PTEN deficien-cy in both clinical and preclinical settings (28–30). As expected,PTEN knockdown increased the basal pAKT activity in BT474cells, consistent with PTEN's role as a suppressor of the PI3K/AKTpathway (Fig. 6B). KTN3379wasunable to efficiently suppress theelevated pAKT signal in the BT474 following knockdown of PTEN

despite successful blockade of the pHER3 signal (Fig. 6B, lane 4 vs.lane 2).

In sharp contrast, however, KTN3379 conferred equally effi-cient growth repression in HMCB cells regardless of the PTENstatus, indicating that PTEN loss has no impact on the activity ofanti-HER3 mAb in ligand-driven models (Fig. 6C, lanes 1 and 2;

Figure 6.PTEN depletion impedes tumorresponse to KTN3379 in HER2-amplified and HRG-independent butnot in HRG-driven cancers. A, BT474cells with nontargeting (NT) or PTEN-targeted knockout were generated byRNAi and treated with KTN3379 ortrastuzumab at 10 mg/mL in the 6-dayproliferation assay to characterize theirantigrowth effects. P value for PTENRNAi versus control RNAi (KTN3379treatment): �� , P < 0.001; P value forPTEN RNAi versus control RNAi(trastuzumab treatment): �� ,P<0.001,t test. B, control or PTEN knockdownBT474 cells were treated with controlIgG or KTN3379 to characterize theirimpacts on HER3/AKT signalingpathway. PTEN, pHER3, pAKT, andGAPDH were immunoblotted asdescribed in Materials and Methods.C and D, stable PTEN-knockout cloneswere generated in the HRG-autocrineHMCB cells by transductionwith shRNAvectors. Control and PTEN-knockoutcells were treated with KTN3379 orpertuzumab at 10 mg/mL for 6 days. Theantiproliferative effects of thesetreatments were assessed by Cell TiterGlo (C). Cells were also lysed andHER3/AKT signaling pathway wasanalyzed by immunoblotting (D). In Aand C, data were plotted as percentageof growth inhibition as compared withcontrol IgG. E and F, CHL1 cells withPTEN knockdown were generatedand treated with KTN3379 at 10 and0.3 mg/mL levels for 6 days in cellproliferation assay (E). Control orPTEN-knockout cells were treated withcontrol or KTN3379 at 10 mg/mL for48 hours and immunoblotted forHER3 pathway markers: HRG, pHER3,AKT/pAKT, PTEN, and GAPDH (F).G and H, BxPC3 cells with or withoutPTEN-knockout were processed forimmunoblot for confirmation of PTEN-knockout (H) or spheroid growth assayas described in Materials and Methods(G). The spheroids were treated withKTN3379, trastuzumab, or pertuzumabto analyze their antiproliferative effects.

Xiao et al.





Supplementary Fig. S6). We additionally demonstrated that per-tuzumab, a HER2 inhibitory antibody that specifically suppressesligand-induced HER2–HER3 pathway activity, was also highlyactive in this model regardless of the PTEN status (Fig. 6C, lanes 3and 4; Supplementary Fig. S6). On the other hand, cetuximab, theanti-EGFR mAb, failed to suppress cell growth (SupplementaryFig. S6), suggesting that this model is driven by a ligand-inducedHER2-HER3 complex but not EGFR–HER3 complex. To ourknowledge, this represents the first study illustrating the potentialimpact of PTEN status on the efficacy of an anti-HER3 mAb orpertuzumab.

As was observed in the BT474 cells, PTEN knockdown in theHMCB cells resulted in an increase in the basal activation state ofAKT (Fig. 6D). However, despite this baseline increase, the anti-HER3mAb still efficiently suppressed the pAKT signal, potentiallyexplaining the continued ability of KTN3379 to inhibit the growthof the PTEN-depleted cells.

The failure of PTEN loss to blunt tumor response to HER3mAb in the ligand-dependent HMCB model is a novel andsomewhat counterintuitive finding. To confirm and furtherexpand on this important result, we conducted additionalstudies of PTEN knockdown in different HRG-dependent can-cer models in both 2D and 3D culture settings. On the basis ofa recent publication (31), CHL-1 melanoma cell-line is a HRG-HER3 autocrine loop-driven model and sensitive to HER3inhibitors. We validated this result and proceeded to demon-strate that PTEN knockdown does not confer resistance toKTN3379, consistent with what we observed in HMCB cells:KTN3379 conferred similar extents of growth suppression inboth control (NT RNAi) and PTEN knockdown (PTEN RNAi)cells at both 10 and 0.3 mg/mL concentrations (Fig. 6E).Successful PTEN knockdown was confirmed in Fig. 6F. Similarto HMCB, KTN3379 abrogated both pHER3 and pAKT signalsin PTEN-depleted CHL-1 cells just as efficiently as in controlCHL-1 cells (Fig. 6F). Interestingly, we found that PTEN dele-tion not only increased the basal pAKT activity in this model,but also concomitantly increased HRG expression, suggestingHRG expression may be partially driven by PI3K/AKT pathwayin this model (Fig. 6F).

We additionally used an elaborate 3D spheroid modeling ofthe pancreatic cancer cell line BxPC3 to further validate ournovel finding that PTEN status fails to impact tumor responseto HER3 inhibitors. Spheroid models are potentially morerepresentative of tumor growth in vivo. In BxPC3 spheroids,HRG behaves as a growth factor and induced substantialspheroid growth (3- to 4-fold growth induction over the assaytime-frame), which remains the same in BxPC3 spheroid withPTEN knockdown (unpublished observation; Z. Xiao). Conse-quently, KTN3379 inhibited the ligand-induced growth to asimilar extent (50%–60%) in both control and PTEN knock-down spheroids (Fig. 6G, columns 1 and 2). Pertuzumabdisplayed a similar profile in terms of suppressing ligand-induced growth in both wild-type and PTEN-knockdownspheroids (Fig. 6G, columns 5 and 6). In contrast, trastuzumabfails to suppress the ligand-induced spheroid growth (Fig. 6G,columns 3 and 4). This is consistent with the notion that onlypertuzumab but not trastuzumab was active in suppressingligand-induced HER2 signaling. Successful PTEN knockout inthe spheroids was confirmed (Fig. 6H).

We summarized the gene expression profiles and mutationstatus of keyHER3pathway components, HER3,HER2,HRG, and

PTEN, of the many cancer models used in our study in Supple-mentary Table S2.

In summary, our conclusion that PTEN knockdown failed toblunt the antiproliferative effect of KTN3379 may broadly holdtrue for most ligand-dependent tumors.

DiscussionWe discovered an HER3 antagonistic mAb, KTN3379, and

demonstrated its efficacy in both ligand-dependent as wellas ligand-independent/HER2-amplified tumors. This intriguingdual functionality, not possessed by a previously published anti-HER3 mAb (23) but reportedly shared by a more recently dis-closed anti-HER3mAb(32), is likely attributable to the capacity ofKTN3379 to block HER2-HER3 heterodimers induced either byligand, or constitutively formed in HER2-amplified cells (Fig. 3).This is further supported by a structure mapping study showingthat KTN3379 has a unique binding epitope that locks HER3 intoan autoinhibited state incompatible with HER2 dimerizationregardless of ligand presence (25).

In non-HER2–amplified cancer models, we observed a strik-ing correlation between HRG expression and tumor response,and a lack of correlation between HER3 expression and efficacy.This indicates that the presence of ligand is a much betterpredictor of tumor reliance on HER3 activity than the mereexpression of HER3 itself. This is to be expected due to thekinase-deficient nature of HER3: unlike other RTKs, it cannotundergo autoactivation through autophosphorylation, hence itcritically depends on the ligand to trigger its activation throughinducing heterodimerization with kinase-proficient partnerRTKs. HER3 has been shown to be capable of formingligand-induced heterodimers with divergent RTKs includingEGFR, HER2, HER4, MET, IGF1R, and FGFR2. However, ourstudies showed that HER2-HER3 dimer is likely the majoractive species because we could replicate the antitumor activityof our HER3 mAb with the anti-HER2 mAb pertuzumab inmany ligand-dependent models we have explored (Fig. 6).Because HRG expression seems to be more restricted than thebroad HER3 expression profile across different tumors, a HRGdetection assay may be pivotal to select the patients more likelyto benefit from HER3 therapy. Earlier studies have suggestedthat HNSCC may have higher HRG expression than othertumor types (33, 34). In addition, acquired HRG expressionmay drive HER2-positive breast cancer resistance to trastuzu-mab-DM1 (T-DM1/Kadcyla; ref. 35), recently approved anti-body–drug conjugate for the treatment of breast cancer patientswho have relapsed on trastuzumab therapy. This indicates thatHER3 antagonist may also be useful to help abrogate tumorresistance to T-DM1, the new generation of HER2-targetedbiologic therapy.

To characterize the activity of KTN3379 in HER2-positivetumors in-depth, we explored three distinct models reflectingdifferent levels of HER2 overexpression and sensitivities to tras-tuzumab: (i) BT474 as the canonical HER2-amplifiedmodel with3þ IHC staining that is highly sensitive to trastuzumab; (ii)MDA-MB-361 as an intermediate HER2-overexpressing model with 2þIHC staining and only partial sensitivity to trastuzumab; (iii)JIMT1 as a HER2-positive but trastuzumab-refractory model.Consistent with literature reports, we found trastuzumab to bemore effective in BT474 than MDA-MB-361, and inactive inJIMT1. LowerHER2 stainingmaynot be theonly reason to explain






the decreased activity of trastuzumab in MDA-MB-361, whichalso harbors a well-established E545K-activating mutation inPIK3CA, the catalytic component of PI3K. Oncogenic activatingmutations of PIK3CA may lead to less tumor dependence onHER2 and have been shown to lower tumor response totrastuzumab (24). Interestingly, KTN3379 was less active thantrastuzumab in BT474 (tumor growth delay vs. partial regres-sion), but achieved similar tumor growth suppression in MDA-MB-361, and more potent than trastuzumab in JIMT1. Moreimportantly, combination of the two produced a clear superioreffect inducing tumor regression or stabilization in trastuzu-mab-insensitive models such as MDA-MB-361 and JIMT1,suggesting that a HER2/HER3 combination therapy mayachieve a superior outcome than trastuzumab alone in thissetting with high unmet medical needs.

Especially notable is the JIMT1 study which illustrated a com-pelling rationale for the addition of HER3 therapy in HER2-positive cancers that are entirely resistant to trastuzumab despitehighHER2expression.Here,HER3monotherapy ismore effectivethan trastuzumab. More importantly, their combination dis-played a superior ability to control tumor growth, which maybe explained by the more efficient suppression of both HER2 andHER3 activities than either of the monotherapies. In contrast, thepertuzumab/trastuzumab combination, a recently approved reg-imen for HER2þ breast cancer patients, turned out to be mostlyinactive (Fig. 5C).

Because the major function of HER3 pathway is PI3K/AKTactivation, it is reasonable to speculate that loss-of-functionmutations in PTEN may drive tumor resistance to HER3 therapyby making the PI3K pathway constitutively active regardless ofHER3 activity. Through RNAi technology, we were able to silencePTEN expression in either ligand-independent ormultiple ligand-dependent cancer models in both 2D and 3D/spheroid format,which enabled the investigation of PTEN's impact on cancerresponse to HER3 mAbs, a critical unaddressed issue with highclinical relevance. In the sameprocess, we additionally studied theimpact of PTEN status on cancer response to pertuzumab, whichselectively inhibits ligand-driven HER2 activity. To our knowl-edge, this represents the first ever such study for both anti-HER3mAbs and pertuzumab.

Our finding that PTEN loss conferred resistance to KTN3379in the HER2-amplified breast cancer model is expected andhighly consistent with prior studies demonstrating that PTENdeficiency confers resistance to trastuzumab. The similarityprovides another proof that HER2-HER3 dimer, formed in aligand-independent manner under this setting, is the majoroncogenic driver in HER2-amplified cancers. It additionallyvalidates the established paradigm that PI3K/AKT constitutesthe major oncogenic effector pathway of HER2/HER3 dimer inthis tumor type, as genetic lesions that hyperactivate PI3K/AKTpathway, as exemplified by PTEN loss, led to tumor resistanceto either HER2 or HER3 mAbs.

In contrast, we were surprised to find that despite the sub-stantial induction of the PI3K/AKT pathway, PTEN loss in theligand-driven cancers fails to blunt tumor response to the HER2mAb pertuzumab or to HER3 mAb. To our knowledge, thisrepresents the first study probing the potential impact of PTENstatus on tumor responses to not only an HER3 mAb, but alsofor pertuzumab, an approved HER2 antagonist antibody inhi-biting ligand-induced HER2 activity. This somewhat counter-intuitive result may be explained by the continued efficient

suppression of pAKT even in the PTEN-knockout setting (unlikein BT474 where PTEN loss interfered with the ability ofKTN3379 to turn off pAKT).

There are two different ways PTEN loss may induce the PI3K/AKT pathway. PTEN loss leads to accumulation of PIP3 (itsnatural substrate), the major product of PI3K. Heightened PIP3levels result in higher activation of downstream effector moleculeAKT. Alternatively, as revealed by an earlier study (36), PTENdeficiency further enhances PI3K pathway through stimulatingthe RTK signaling such as IGF1R and HER3. This is partly due tosensitizing the RTKs to their respective ligands, IGF and HRG.PTEN-loss blunts tumor response to HER3mAbs more in ligand-independent cancer than ligand-dependent cancers, suggestingthat PI3K activation induced byPTEN loss ismore driven byHER3activity in the former setting than in the latter setting, possibly dueto thedifferential availability of theHER3 ligand. Therefore, PTENloss not only fails to make ligand-driven tumors less dependenton HER3, it actually may make them even more critically depen-dent on HER3.

Another possible explanation is that a distinct pathway, otherthan PI3K/AKT, may constitute the key protumorigenic effectorpathway of HRG-HER3 signaling in ligand-driven cancers, and itssuppression by anti-HER3 mAb is not perturbed by PTEN loss. Inlight of the overwhelming evidences supporting HER30s role inPI3K activation, we consider this scenario unlikely.

In summary, we have discovered a HER3 antagonistic mAbthat is capable of suppressing HER3 activity and tumor growthin both ligand-dependent and -independent settings. Its com-bination with approved HER2 inhibitors further enhanced theirefficacies in various breast cancer models with divergent extentsof HER2 expression and sensitivity to HER2 drugs. Moreimportantly, PTEN deficiency impeded the antitumor effect ofHER3 mAb in HER2-amplified cancer but not in ligand-drivencancers.

KTN3379 is currently undergoing a phase Ib clinical trial inpatients with advanced solid tumors. To date, it has demonstratedgood tolerability and early signs of clinical activity in combinationwith other targeted agents (37). Our current study will providepotential guidance to patient stratification strategies for its futureclinical trials to maximize its clinical benefit.

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

Authors' ContributionsConception and design: Z. Xiao, K. Kinneer, P. Chowdhury, P. Steiner,R.E. Hollingsworth, D.A. TiceDevelopment of methodology: Z. Xiao, K. Kinneer, C. Yang, P. Chowdhury,P. Tsui, P. Steiner, B. Jallal, R.E. HollingsworthAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.A. Carrasco, K. Schifferli, K. Kinneer, R. Tammali,H. Chen, R. Rothstein, L. Wetzel, C. Yang, P. Chowdhury, P. TsuiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Xiao, R.A. Carrasco, K. Kinneer, H. Chen,R. Rothstein, C. Yang, P. Chowdhury, P. Tsui, P. Steiner, B. Jallal, D.A. TiceWriting, review, and/or revision of themanuscript:R.A. Carrasco, K. Schifferli,K. Kinneer, R. Tammali, L. Wetzel, C. Yang, P. Chowdhury, R. Herbst,R.E. Hollingsworth, D.A. TiceAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Kinneer, L. Wetzel, D.A. TiceStudy supervision: Z. Xiao, K. Kinneer, P. Chowdhury, P. Tsui, P. Steiner,D.A. Tice

Xiao et al.





AcknowledgmentsThe authors thank Rob Woods for the SPR-based affinity measurement

of KTN3379 to human, cynomolgus monkey, and murine HER3. Theauthors also thank Sandrina Phipps for generating the stable knockoutclones of PTEN in various tumor models. We also like to thank Dr. TheresaLaVallee of Kolltan Pharmaceutical Inc. for review and comments on themanuscript.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 15, 2015; revised February 4, 2016; accepted February 9, 2016;published OnlineFirst February 15, 2016.

References1. Yarden Y, Pines G. The ERBB network: at last, cancer therapymeets systems

biology. Nat Rev Cancer 2012;12:553–63.2. ArteagaCL, Engelman JA. ERBB receptors: fromoncogenediscovery tobasic

science to mechanism-based cancer therapeutics. Cancer Cell 2014;25:282–303.

3. Guy PM, Platko JV, Cantley LC, Cerione RA, Carraway KL III. Insectcell-expressed p180erbB3 possesses an impaired tyrosine kinase activity.Proc Natl Acad Sci U S A 1994;91:8132–6.

4. Klapper LN,Glathe S, VaismanN,HynesNE, AndrewsGC, SelaM, et al. TheErbB-2/HER2 oncoprotein of human carcinomas may function solely as ashared coreceptor for multiple stroma-derived growth factors. Proc NatlAcad Sci U S A 1999;96:4995–5000.

5. Sierke SL, Cheng K, Kim HH, Koland JG. Biochemical characterization ofthe protein tyrosine kinase homology domain of the ErbB3 (HER3)receptor protein. Biochem J 1997;322:757–63.

6. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al.MET amplification leads to gefitinib resistance in lung cancer by activatingERBB3 signaling. Science 2007;316:1039–43.

7. Kunii K,Davis L,Gorenstein J,HatchH, YashiroM,DiBaccoA, et al. FGFR2-amplified gastric cancer cell lines require FGFR2 and Erbb3 signaling forgrowth and survival. Cancer Res 2008;68:2340–8.

8. Lee-Hoeflich ST, Crocker L, Yao E, Pham T, Munroe X, Hoeflich KP, et al. Acentral role for HER3 in HER2-amplified breast cancer: implications fortargeted therapy. Cancer Res 2008;68:5878–87.

9. Prigent SA, Gullick WJ. Identification of c-erbB-3 binding sites for phos-phatidylinositol 30-kinase and SHC using an EGF receptor/c-erbB-3 chi-mera. EMBO J 1994;13:2831–41.

10. Soltoff SP, Carraway KL III, Prigent SA, Gullick WG, Cantley LC. ErbB3 isinvolved in activation of phosphatidylinositol 3-kinase by epidermalgrowth factor. Mol Cell Biol 1994;14:3550–8.

11. Wheeler DL, Huang S, Kruser TJ, Nechrebecki MM, Armstrong EA, Bena-vente S, et al. Mechanisms of acquired resistance to cetuximab: role of HER(ErbB) family members. Oncogene 2008;27:3944–56.

12. Ritter CA, Perez-Torres M, Rinehart C, Guix M, Dugger T, Engelman JA,et al. Human breast cancer cells selected for resistance to trastuzumab invivo overexpress epidermal growth factor receptor and ErbB ligands andremain dependent on the ErbB receptor network. Clin Cancer Res2007;13:4909–19.

13. Wang SE, Xiang B, Guix M, Olivares MG, Parker J, Chung CH, et al.Transforming growth factor beta engages TACE and ErbB3 to activatephosphatidylinositol-3 kinase/Akt in ErbB2-overexpressing breast can-cer and desensitizes cells to trastuzumab. Mol Cell Biol 2008;28:5605–20.

14. Sergina NV, Rausch M, Wang D, Blair J, Hann B, Shokat KM, et al. Escapefrom HER-family tyrosine kinase inhibitor therapy by the kinase-inactiveHER3. Nature 2007;445:437–41.

15. Garrett JT, Olivares MG, Rinehart C, Granja-Ingram ND, S�anchez V,Chakrabarty A, et al. Transcriptional and posttranslational up-regulationof HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase.Proc Natl Acad Sci U S A 2011;108:5021–6.

16. Chakrabarty A, S�anchez V, Kuba MG, Rinehart C, Arteaga CL. Feedbackupregulation of HER3 (ErbB3) expression and activity attenuates anti-tumor effect of PI3K inhibitors. Proc Natl Acad Sci U S A 2012;109:2718–23.

17. Chandarlapaty S, Sawai A, Scaltriti M, Rodrik-Outmezguine V, Grbovic-Huezo O, Serra V, et al. AKT inhibition relieves feedback suppression ofreceptor tyrosine kinase expression and activity. Cancer Cell 2011;19:58–71.

18. Turke AB, Song Y, Costa C, Cook R, Arteaga CL, Asara JM, et al. MEKinhibition leads to PI3K/AKT activation by relieving a negative feedback onERBB receptors. Cancer Res 2012;72:3228–37.

19. Hoet RM, Cohen EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, et al.Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity.Nat Biotechnol 2005;23:344–8.

20. Wu H, An LL. Tailoring kinetics of antibodies using focused combinatoriallibraries. Methods Mol Biol 2003;207:213–33.

21. Dall'Acqua WF, Kiener PA, Wu H. Properties of human IgG1s engineeredfor enhanced binding to the neonatal Fc receptor (FcRn). J Biol Chem2006;281:23514–24.

22. Stove C, Boterberg T, Van Marck V, Mareel M, Bracke M. Bowes melanomacells secrete heregulin, which can promote aggregation and counteractinvasion of human mammary cancer cells. Int J Cancer 2005;114:572–8.

23. Schoeberl B, Faber AC, Li D, LiangMC,Crosby K,OnsumM, et al. An ErbB3antibody, MM-121, is active in cancers with ligand-dependent activation.Cancer Res 2010;70:2485–94.

24. Junttila TT, Akita RW, Parsons K, Fields C, Lewis Phillips GD, Friedman LS,et al. Ligand-independent HER2/HER3/PI3K complex is disrupted bytrastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 2009;15:429–40.

25. Lee S, Greenlee EB, Amick JR, Ligon GF, Lillquist JS, Natoli EJ Jr, et al.Inhibition of ErbB3 by a monoclonal antibody that locks the extracellulardomain in an inactive configuration. Proc Natl Acad Sci U S A. 2015;112:13225–30.

26. Junttila TT, Parsons K, Olsson C, Lu Y, Xin Y, Theriault J, et al. Superior invivo efficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancer. Cancer Res 2010;70:4481–9

27. Tanner M, Kapanen AI, Junttila T, Raheem O, Grenman S, Elo J, et al.Characterization of a novel cell line established from a patient withHerceptin-resistant breast cancer. Mol Cancer Ther 2004;3:1585–92.

28. Nagata Y, LanKH,ZhouX, TanM, Esteva FJ, SahinAA, et al. PTENactivationcontributes to tumor inhibition by trastuzumab, and loss of PTEN predictstrastuzumab resistance in patients. Cancer Cell 2004;6:117–27.

29. Chandarlapaty S, Sakr RA, Giri D, Patil S, Heguy A, Morrow M, et al.Frequent mutational activation of the PI3K-AKT pathway in trastuzumab-resistant breast cancer. Clin Cancer Res 2012;18:6784–91.

30. Rexer BN, Shyr Y, Arteaga CL. Phosphatase and tensin homolog deficiencyand resistance to trastuzumab and chemotherapy. J Clin Oncol 2013;31:2073–5.

31. Capparelli C, Rosenbaum S, Berman-Booty LD, Salhi A, Gaborit N, Zhan T,et al. ErbB3-ErbB2 Complexes as a Therapeutic Target in a Subset of Wild-type BRAF/NRAS cutaneous melanomas. Cancer Res 2015;75:3554–67.

32. Garner AP, Bialucha CU, Sprague ER, Garrett JT, Sheng Q, Li S, et al. Anantibody that locks HER3 in the inactive conformation inhibits tumorgrowth driven by HER2 or neuregulin. Cancer Res 2013;73:6024–35.

33. Wilson TR, Lee DY, Berry L, Shames DS, Settleman J. Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibi-tors in a subset of human cancers. Cancer Cell 2011;20:158–72.

34. Shames DS, Carbon J, Walter K, Jubb AM, Kozlowski C, Januario T, et al.High heregulin expression is associated with activated HER3 and maydefine an actionable biomarker in patients with squamous cell carcinomasof the head and neck. PLoS One 2013;8:e56765.

35. Gwin WR, Spector NL. Pertuzumab protects the achilles' heel of trastuzu-mab–emtansine. Clin Cancer Res 2014;20:278–80.

36. Miller TW, P�erez-Torres M, Narasanna A, Guix M, Sta�l O, P�erez-Tenorio G,

et al. LossofPhosphataseandTensinhomologuedeletedon chromosome10engages ErbB3 and insulin-like growth factor-I receptor signaling to promoteantiestrogen resistance in breast cancer. Cancer Res 2009;69:4192–201.

37. Koltan press release. Available from: http://www.businesswire.com/news/home/20150530005013/en/Kolltan-Pharmaceuticals-Announces-Presen-tation-KTN3379-Interim-Phase.






2016;15:689-701. Published OnlineFirst February 15, 2016.Mol Cancer Ther Zhan Xiao, Rosa A. Carrasco, Kevin Schifferli, et al.

Status on Tumor ResponsePTENof ImpactsLigand-Dependent and -Independent Activities: Differential

A Potent HER3 Monoclonal Antibody That Blocks Both

Updated version

10.1158/1535-7163.MCT-15-0555doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2016/02/13/1535-7163.MCT-15-0555.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/15/4/689.full#ref-list-1

This article cites 36 articles, 22 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/15/4/689.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

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

Subscriptions

Reprints and

[email protected]

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

Permissions

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

.http://mct.aacrjournals.org/content/15/4/689To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-15-0555

http://mct.aacrjournals.org/content/suppl/2016/02/13/1535-7163.MCT-15-0555.DC1

http://mct.aacrjournals.org/content/15/4/689.full#ref-list-1

http://mct.aacrjournals.org/content/15/4/689.full#related-urls

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

mailto:[email protected]

http://mct.aacrjournals.org/content/15/4/689


a potent her3 monoclonal antibody that blocks …...large molecule therapeutics a potent her3...

Documents