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Cancer Therapy: ClinicalSee related article by Gwin and Spector, p. 278

Dual Targeting of HER2-Positive Cancer with TrastuzumabEmtansine and Pertuzumab: Critical Role for NeuregulinBlockade in Antitumor Response to Combination Therapy

Gail D. Lewis Phillips1, Carter T. Fields1, Guangmin Li1, Donald Dowbenko1, Gabriele Schaefer1, Kathy Miller5,Fabrice Andre6, Howard A. Burris III8, Kathy S. Albain9, Nadia Harbeck10, Veronique Dieras7, Diana Crivellari11,Liang Fang2, Ellie Guardino3, Steven R. Olsen3, Lisa M. Crocker4, and Mark X. Sliwkowski1

AbstractPurpose: Targeting HER2 with multiple HER2-directed therapies represents a promising area of

treatment for HER2-positive cancers. We investigated combining the HER2-directed antibody–drug con-

jugate trastuzumab emtansine (T-DM1) with the HER2 dimerization inhibitor pertuzumab (Perjeta).

Experimental Design: Drug combination studies with T-DM1 and pertuzumab were performed on

cultured tumor cells and in mouse xenograft models of HER2-amplified cancer. In patients with HER2-

positive locally advancedormetastatic breast cancer (mBC), T-DM1wasdose-escalatedwith afixed standard

pertuzumab dose in a 3þ3 phase Ib/II study design.

Results: Treatment of HER2-overexpressing tumor cells in vitrowith T-DM1 plus pertuzumab resulted in

synergistic inhibition of cell proliferation and induction of apoptotic cell death. The presence of the HER3

ligand, heregulin (NRG-1b), reduced the cytotoxic activity of T-DM1 in a subset of breast cancer lines; this

effect was reversed by the addition of pertuzumab. Results frommouse xenograft models showed enhanced

antitumor efficacy with T-DM1 and pertuzumab resulting from the unique antitumor activities of each

agent. In patients with mBC previously treated with trastuzumab, lapatinib, and chemotherapy, T-DM1

could be dosed at the maximum tolerated dose (MTD; 3.6 mg/kg every 3 weeks) with standard dose

pertuzumab. Adverse events were mostly grade 1 and 2, with indications of clinical activity.

Conclusions: Dual targeting of HER2 with the combination of T-DM1 and pertuzumab in cell culture

andmouse xenograftmodels resulted in enhanced antitumor activity. In patients, this combination showed

an encouraging safety and tolerability profile with preliminary evidence of efficacy. Clin Cancer Res; 20(2);

456–68. �2013 AACR.

IntroductionThe ErbB/HER family of receptor tyrosine kinases (RTK)

plays critical roles in both development and cancer (1).These receptors includeEGF receptor (EGFR)/ErbB1,HER2/

ErbB2, HER3/ErbB3, and HER4/ErbB4. Numerous ligandshave been identified that interact with all HER familyreceptors, with the exception of HER2. Ligand bindingactivates multiple downstream signal transduction path-ways. HER2 is the common coreceptor for the other HERfamily members. Association of HER2 with HER3 is themost potent signaling complex formed in this receptorfamily (2, 3). HER3 is considered a pseudo-kinase (4, 5),but can potently activate phosphoinositide 3-kinase (PI3K)signaling upon dimerization with other HER family mem-bers (6). In addition to growth factor regulation of cellproliferation and differentiation, recent reports show animportant role for multiple RTK ligands in resistance tokinase inhibitors (7–9). The HER3 ligand, neuregulin 1(NRG-1), was recently shown to mediate resistance tochemotherapeutic agents as well (10). Moreover, HER3wasreported to mediate resistance to EGFR, HER2, and PI3Kinhibitors. Treatment resulted in HER3 activation, leadingto attenuation of the response to kinase inhibition (11, 12).

Trastuzumab (Herceptin), a humanized HER2 antibodythat binds domain IV of the HER2 extracellular domain(ECD), is used in combination with chemotherapy for

Authors' Affiliations: 1ResearchOncology, Departments of 2Biostatistics,3Product Development Oncology, and 4Translational Oncology, Genen-tech, Inc., South San Francisco, California; 5Indiana University SimonCancer Center, Indianapolis, Indiana; 6Institute Gustave Roussy, Villejuif;7Department of Medical Oncology, Institut Curie, Paris, France; 8SarahCannon Research Institute, Nashville, Tennessee; 9Loyola University Med-ical Center, Maywood, Illinois; 10University of Munich, Munich, Germany;and 11Division of Medical Oncology, Centro di Riferimento Oncologico,Instituto Nazionale Tumori Aviano, Italy

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

G.D.L. Phillips and C.T. Fields contributed equally to this work.

Corresponding Author: Gail D. Lewis Phillips, Genentech, Inc., 1 DNAWay, South San Francisco, CA 94080. Phone 650-225-2201; Fax: 650-225-1411; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-0358

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(2) January 15, 2014456

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treatment of HER2-positive breast cancer. Proposedmechanisms of trastuzumab action include inhibition ofECD shedding, disruption of downstream signal-transduc-tion pathways, induction of cell-cycle arrest, inhibition ofDNA repair, decreased angiogenesis, and mediation ofantibody-dependent cell-mediated cytotoxicity (ADCC;ref. 13). Clinical benefit from trastuzumab-containing ther-apy, however, can be limited, especially in the metastaticsetting, necessitating development of alternate forms oftreatment (14, 15). We developed a cytotoxic drug-conju-gate of trastuzumab by covalently linking the antimitoticagent DM1 to trastuzumab through a stable MCC linker.Trastuzumab emtansine (T-DM1) potently inhibits growthof trastuzumab-sensitive and -insensitive HER2-amplifiedcancer cells and shows a favorable safety profile in preclin-ical toxicity studies (16). T-DM1 activity has been evaluatedin phase II and III clinical trials in patients with HER2-positive locally advanced or mBC (17, 18). T-DM1 (ado-trastuzumab emtansine [Kadcyla]) was recently approvedfor treatment of patientswithHER2-positivemBCwhohavereceived prior treatment with Herceptin and a taxane che-motherapy, based on data showing significantly prolongedprogression-free and overall survival and less toxicity com-pared with standard-of-care treatment, lapatinib (Tykerb)plus capecitabine (Xeloda; ref. 19).In addition to trastuzumab, pertuzumab is a HER2-spe-

cific humanized antibody with efficacy demonstrated inmouse xenograft models of breast, lung, prostate, andovarian cancer (20–23). Pertuzumab binds domain II oftheHER2 ECD, preventing ligand-dependent association of

HER2 with other HER family members (24) and demon-strated clinical activity as a single agent and in combinationwith chemotherapy in ovarian, non–small cell lung andprostate cancer (25–29). Pertuzumab combined with tras-tuzumab has antitumor activity in cell culture (30) andanimal models of HER2-amplified cancer (23, 31) and inpatients with HER2-positive mBC (32, 33). Combiningpertuzumab, trastuzumab, and docetaxel significantlyimproves the pathologic complete response rate in theneoadjuvant setting compared with treatment without per-tuzumab (33). In June 2012, the U.S. Food and DrugAdministration (FDA) approved Pertuzumab (Perjeta) incombination with Herceptin and chemotherapy for treat-ment of patients with HER2-positive mBC who have notreceived prior anti-HER2 therapy or chemotherapy formetastatic disease (34).We therefore explored the potentialof combining T-DM1with pertuzumab inmodels of HER2-amplified breast and lung cancer. Our data underscore thefeasibility of using multiple strategies to target HER2-pos-itive cancer.

Materials and MethodsCell lines and reagents

Human breast tumor lines BT-474, SK-BR-3, UACC-812,ZR-75-30, HCC1954, and MDA-MB-175-VII (MDA-175),and the non–small cell lung carcinoma line Calu-3 wereobtained from the American Type Culture Collection.KPL-4 breast cancer cells were provided by Prof. JunichiKurebayashi, Kawasaki Medical Hospital, Kurashiki,Okayama, Japan (35). Cells were maintained as previouslydescribed (16). T-DM1, pertuzumab, N297A-pertuzumab(36), and E. coli-derived NRG-1b (EGF domain) wereproduced at Genentech, Inc. Unconjugated DM1 wasobtained from ImmunoGen, Inc. and chemotherapeuticdrugs from Sigma-Aldrich. Western blot antibodies werefrom R&D Systems (cleaved PARP), Cell Signaling Tech-nology (phospho-HER3, phospho-Akt, Akt), and SantaCruz Biotechnology, Inc. (HER3).

Cell viability and apoptosis assaysCell viability and apoptosis assays were performed as

described in ref. (16). T-DM1, pertuzumab, or the combi-nationwas added to cells; treatments were in duplicate to fitall groups onto one plate and experiments were repeated 5times. Studies performed without NRG-1bwere inmediumsupplemented with 10% FBS and studies with NRG-1b (2nmol/L) were in 1% FBS-containing medium. Cell viabilitywas measured at 3 (SK-BR-3, KPL-4) or 5 (BT-474, Calu-3)days after drug addition. Assays to test NRG-1b effects on T-DM1 or chemotherapeutic agent activity were 3 or 5 days(n ¼ 3 per group) and studies were performed three times.Combination Index (CI) values, as described by Chou (37),were generated using CalcuSyn software (Biosoft, Inc).ADCC assays were performed as described in ref. (38) usingCytoTox-ONE Homogeneous Membrane Integrity Assay(Promega Corp.). Treatments were T-DM1 alone or com-bined with pertuzumab or the Fc-mutant N297A-

Translational RelevanceThe antibody–drug conjugate ado-trastuzumab

emtansine (T-DM1; Kadcyla) increases progression-freeand overall survival versus lapatinib plus capecitabine inpatients with HER2-positive metastatic breast cancer(mBC) who have received prior treatment with trastu-zumab (Herceptin) and a taxane chemotherapy, with abetter safety profile than the comparator treatments.Dual antibody blockade of HER2 with pertuzumab,trastuzumab plus docetaxel showed increased progres-sion-free and overall survival with the addition of per-tuzumab to trastuzumab plus docetaxel. Neoadjuvanttreatment with trastuzumab plus pertuzumab (Neo-Sphere trial) or trastuzumab plus lapatinib (NeoALTTOtrial) in conjunction with taxane-based therapy resultedin better clinical activity than with single-agent HER2-directed therapy. The studies described here showenhanced antitumor activity with T-DM1 plus pertuzu-mab in preclinical models, with an encouraging safetyprofile and preliminary evidence of efficacy in a phase Ibstudy. Moreover, our data show suppression of T-DM1activity upon ligand activation of HER2/HER3, support-ing the rationale for combining pertuzumab withT-DM1.

Enhanced Antitumor Activity with Trastuzumab Emtansine plus Pertuzumab

www.aacrjournals.org Clin Cancer Res; 20(2) January 15, 2014 457




pertuzumab; trastuzumab and anti-gD isotype-matchedantibody were positive and negative controls, respectively.

Western blot analysisFor measuring PARP cleavage, MDA-175 cells were

seeded and allowed to adhere for 48 hours. T-DM1 (10mg/mL), pertuzumab (100 mg/mL), or the combinationwas added and cells were incubated for 48 or 72 hours.For experiments with NRG-1b, SK-BR-3, Calu-3, KPL-4,and MDA-175 were plated and allowed to adhere over-night. Cells were then starved in medium with 0.1% FBSfor 24 hours. NRG-1b (1 nmol/L) was added to all cells,except MDA-175, after a 1-hour pretreatment with pertu-zumab or N297A-pertuzumab, T-DM1, or the combina-tion. After 15 minutes or 24 hours, cells were lysed inRIPA buffer and Western blotting procedures were per-formed as described previously (16).

In vivo efficacy studiesMDA-175breast cancer xenograft studies were performed

as described previously (21). KPL-4 breast tumor cells wereinoculated (3� 106 permouse) intomammary fat pads andCalu-3 lung tumor cells were inoculated subcutaneously(5 � 106 per mouse) into flanks of female CB-17 severecombined immunodeficient (SCID) Beige mice (CharlesRivers Laboratories). Animals were randomly assigned intogroups with mean tumor volumes of 300 mm3. Animalsreceived a single intravenous injection of T-DM1 (10mg/kgforMDA-175; 0.3, 1, or 3mg/kg for KPL-4; and1, 3, or 7mg/kg for Calu-3) on study day 0. Mice bearing MDA-175tumors were administered a single intraperitoneal dose ofpertuzumab (3 mg/kg); for studies with KPL-4 or Calu-3tumors, mice were first injected intraperitoneally with a 2�loading dose of pertuzumab, and received two additionaldoses once per week. Pertuzumab doses were 30 mg/kgload/15mg/kgweekly for KPL-4 and 50mg/kg load/25mg/kg weekly for Calu-3. Treatment groups consisted of 8 to 10animals per group. ANOVA and Tukey–Kramer HSD (hon-estly significant difference) analysis were used for calcula-tion of means, SEs and statistical comparisons of time-to-tumor volume doubling (time-to-progression, TTP). Anal-yses were performed using JMP Software, version 6.0 (SASInstitute).

Clinical trial designA single-arm, phase Ib/II study (TDM4373g) was

designed to investigate the safety and efficacy of T-DM1combined with pertuzumab in patients with HER2-positivelocally advanced or mBC who had been previously treatedwith a HER2-directed therapy. The study was reviewed andapproved by the institutional review board at each site,according to local clinical guidelines. All patients providedinformed consent. The phase Ib results are reported herein.

Study design. In the 3þ3 design, patients received per-tuzumab (840mg cycle 1; 420mg cycle 2, and beyond)withT-DM1 [3.0 mg/kg in Cohort 1 and, in the absence of dose-limiting toxicity (DLT), 3.6mg/kg in Cohort 2]. Both agentswere administered by intravenous infusion. Once dose

escalation was complete, additional patients were enrolledinto an expansion phase (phase II). Patient inclusion cri-teria and study assessment guidelines are in SupplementaryMethods.

ResultsCombination effects of T-DM1 and pertuzumab in cellculture and xenograft models

T-DM1 combined with pertuzumab in MDA-175, a modelof autocrine heregulin growth. Growth of MDA-175 breastcancer cells is dependent on autocrine production of g-here-gulin (36) and provides a unique model for studyingpertuzumab activity. In addition, MDA-175 cells slightlyoverexpress HER2 (1þ expression) and thus aremoderatelysensitive to trastuzumab (39) andT-DM1. The combinationof pertuzumab with T-DM1 showed enhanced antiproli-ferative activity compared with either agent alone (Fig. 1A,top left). CI values were determined from these studies. CIvalues less than 1 denote a synergistic drug interaction, CIvalues more than 1 denote antagonism, and CI valuesapproximately 1 denote additivity. In MDA-175 cells, T-DM1 combined with pertuzumab resulted in synergy at alldrug concentrations tested (CI range, 0.03–0.75). The aver-age CI value for the entire drug-effect rangewas 0.19� 0.05,with theCI values at or near the IC50markedly less than 1, asdepicted in Supplementary Fig. S1A (CI vs. drug effect). Theaverage CI values for five different experiments ranged from0.10 to 0.50, indicating synergy across multiple studies. Nodifference was observed between pertuzumab and N297A-pertuzumab on MDA-175 cell proliferation, alone or com-bined with T-DM1.

Additional studies were performed to determine if T-DM1 plus pertuzumab induced enhanced apoptosis inMDA-175 breast cancer cells. Treatment with pertuzumabresulted in enhanced caspase-3/7 activity as early as24 hours after treatment (Fig. 1A, top right), whereasT-DM1–induced apoptosis was observed only after pro-longed (72 hour) treatment (Fig. 1A, bottom). Maximalcaspase activation was observed with the combination ofT-DM1 and pertuzumab. Similar to the results shown forcaspase activation, maximal PARP cleavage, as indicatedby the appearance of the 23 kDa cleavage fragment, wasdemonstrated in the T-DM1–pertuzumab combinationgroup, compared with single-agent treatment, and wasobserved at both 48 and 72 hours of treatment (Fig. 1A,bottom right).

The combinationof T-DM1withpertuzumabwas tested invivo in theMDA-175xenograftmodel. Treatmentswere singleintravenous injections of 3 mg/kg pertuzumab, 10 mg/kg T-DM1, or the combination (Fig. 1B). Log-rank P values wereobtained for differences in time-to-tumor doubling (Supple-mentary Fig. S2). Single-agent treatment with T-DM1 orpertuzumab resulted in moderate tumor growth inhibitionthat was statistically different from vehicle-treated controls(P ¼ 0.0247 for pertuzumab vs. control; P ¼ 0.0017 forT-DM1 vs. control). In contrast, tumors in mice treated withT-DM1 combined with pertuzumab regressed for 3 to 4weeks, with only modest tumor regrowth occurring after

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40 to50days. Thedifference in time-to-tumordoubling forT-DM1 plus pertuzumab compared with single-agent treat-ment groups or vehicle-treated controls was significant (P< 0.0001 for combination vs. vehicle or pertuzumab; P ¼0.0003 for combination vs. T-DM1; Supplementary Fig. S2).Thus, T-DM1 combinedwithpertuzumab induced apoptoticcell death in the MDA-175 breast cancer line and resulted insignificant tumor growth inhibition/regression comparedwith T-DM1 or pertuzumab alone.Antitumor mechanisms for combination treatment effi-

cacy, in addition to DM1-mediated cell death, includeADCC and different antisignaling properties of the twoantibodies. We therefore investigated the contribution ofthese different mechanisms to combination activity. TheN297A Fc mutation, which prevents binding between theantibody Fc portion and Fc receptors on immune effectorcells, was introduced into pertuzumab for ADCC and signaltransduction studies. ADCC assays performed in vitro withpertuzumab, N297A-pertuzumab, T-DM1, or the combina-tion of T-DM1with either wild-type or mutant pertuzumabshowedminimal tumor cell lysis (Fig. 1C): 12.8% lysis withcontrol antibody versus 14.7% for pertuzumab, 15.4% forT-DM1, and 18.6% for T-DM1 þ pertuzumab. Percentagelysis with the N297A-mutant pertuzumab (10.6%) wassimilar to the negative control, whereas cell lysis aftertreatment with T-DM1þN297A-pertuzumab (17.0%) wassimilar to T-DM1 þ wild-type, ADCC-competent pertuzu-mab (18.9%).Constitutive pathway activation from autocrine heregu-

lin production inMDA-175 cells was demonstrated by highbasal levels of phospho-HER3 (p-HER3) and phospho-AKT(p-AKT). Both pertuzumab and N297A-pertuzumab sup-pressed p-HER3 and p-AKT to a similar degree, indicatingthat the N297A Fc mutation did not interfere with HER2binding and subsequent inhibition of downstream signal-ing pathways. T-DM1 treatment resulted in modest inhibi-tion of p-HER3 and p-AKT. Importantly, T-DM1 combinedwith either wild-type or N297A-pertuzumabmarkedly sup-pressed p-HER3 and p-AKT (Fig. 1D). From these studies,we conclude that antitumor activity of combination treat-ment in vivo is likely due to enhanced antisignaling activ-ities, not increased ADCC.T-DM1 combined with pertuzumab in HER2-amplified

lung and breast cancer cells. Additional studies were per-formed in tumor cells with 3þ HER2 expression. NRG-1bwas included in these experiments as these cells do notexpress endogenous ligand. InCalu-3, BT-474, and SK-BR-3cells, T-DM1wasmore active than pertuzumab,whereas thecombination was more potent than single-agent treatment(Fig. 2A–C, left). The average CI value for the entire drugeffect range was 0.24� 0.06 for Calu-3 and 0.37� 0.17 forBT-474, indicating synergy. (CI values were not determinedin SK-BR-3 cells because of minimal pertuzumab activityunder these assay conditions). Graphical representations ofCI versus fractional effect are in Supplementary Fig. S1B andC. Calu-3 and BT-474 cell growth was stimulated by NRG-1b, whereas SK-BR-3 growth was unaffected under theseexperimental conditions. Thus, the enhancement by pertu-

zumab of T-DM1 activity in the presence of NRG-1b wasindependent of NRG-1b mitogenic activity. In contrast, nocombination effect was observed in cells treated in theabsence of NRG-1b; i.e., pertuzumab did not enhance thegrowth-inhibitory effect of T-DM1 under ligand-indepen-dent conditions (Fig. 2A–C, right). In KPL-4 breast cancercells, T-DM1 cytotoxicity was not increased by pertuzumabin ligand-dependent (Fig. 2D, left) or ligand-independent(Fig. 2D, right) conditions.

Inhibition of T-DM1 cytotoxicity by NRG-1bIn the earlier experiments, it was noted that the cytotoxic

response to T-DM1was reduced in the presence of NRG-1b.We therefore investigated NRG-1b inhibition of T-DM1–induced cytotoxicity on a larger panel of HER2-amplifiedbreast cancer lines using two experimental formats, com-paring a fixed concentration of NRG-1b (2 nmol/L) versus adose range of T-DM1 and the converse, NRG-1b doseresponse with a fixed concentration of T-DM1. In BT-474, SK-BR-3, ZR-75-30 (Fig. 3A, left), and UACC812 (datanot shown) breast cancer lines, 2 nmol/L NRG-1b sup-pressed T-DM1 activity. Concentrations as low as 0.01 to0.2 nmol/L NRG-1b reduced the antiproliferative activity ofT-DM1 in all four cell lines (Fig. 3A, right). In contrast,NRG-1b did not block T-DM1 activity in KPL-4 and HCC1954breast cancer cells (Fig. 3B). Interestingly, these two breastcancer lines harbor activating PIK3CA mutations (H1047Rfor both; ref. 40), whereas the four cell lines that displayedreduced T-DM1 activity in the presence of NRG-1b do not(40). Finally, the addition of NRG-1b also inhibited T-DM1–induced apoptosis in BT-474 and ZR-75-30 cells, asindicated by reduced caspase activity. Blockade of NRG-1bsignaling by the addition of pertuzumab fully restored theapoptotic response to T-DM1 (Fig. 3C and SupplementaryFig. S3, left). Similar to the observations in proliferationassays, T-DM1–induced apoptosis in KPL-4 and HCC1954cells was not reduced by NRG-1b (Fig. 3C and Supplemen-tary Fig. S3, right). These results provide additional evidencefor the rationale of blocking ligand-induced receptor acti-vation with pertuzumab for optimal T-DM1 response in asubset of HER2-amplified cancer cells.

NRG-1b inhibits activity of antimitotic agentsThe DM1 component of T-DM1 is a potent antimitotic

agent (41). Additional studies were performed to determineif the protective effect ofNRG-1b applied to freeDM1and toantimitotic agents used for breast cancer treatment. Similarto the findings with T-DM1, NRG-1b reduced the cytotoxiceffects of unconjugated DM1 as well as docetaxel andvinorelbine in BT-474 cells (Supplementary Fig. S4, left).Similar results were obtained with SK-BR-3 (data notshown). In contrast, NRG-1b did not reduce the cytotoxiceffects of these agents in KPL-4 cells (Supplementary Fig. S4,right). Overall, these data show NRG-1b inhibition of thecytotoxic activity of T-DM1, docetaxel, and vinorelbine,further validating the importance of blocking NRG-1bactivity with pertuzumab.






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Effects of T-DM1, pertuzumab, or the combination onNRG-1b–activated HER2–HER3 signalingNRG-1b activation of HER2–HER3 signal transduction

was investigated in SK-BR-3 and KPL-4 cells to probe thedifferential effects we observed of NRG-1b on T-DM1 activ-ity. As expected, NRG-1b�-stimulatedHER3 andAKT phos-phorylation was inhibited by pertuzumab, but not T-DM1,in SK-BR-3 cells (Fig. 4, top; ref. 42); this effect was morepronounced after prolonged (24 hours) incubation.Although treatmentwith T-DM1alone for 24hours resultedin reduced p-HER3 and p-AKT, addition of pertuzumab toT-DM1 was required to inhibit AKT activation in the pres-ence of NRG-1b.In KPL-4 cells, NRG-1b transiently activated HER3, but

not AKT, (Fig. 4, bottom), likely due to mutational activa-tion of the PI3K pathway. Interestingly, T-DM1 treatmentfor 24 hours suppressed both HER3 and AKT phosphory-lation, with inhibition of p-AKT sustained even in thepresence of NRG-1b. All treatment groups containingT-DM1 (T-DM1 alone, T-DM1þ NRG-1b, T-DM1þ pertu-zumab, and T-DM1 þ pertuzumab þ NRG-1b) showedsimilar inhibition of AKT phosphorylation, indicating thatT-DM1 is sufficient to suppress AKT activation (Fig. 4, right).Because KPL-4 are insensitive to trastuzumab in vitro (42),this effect is mediated by the DM1 component of T-DM1(unpublished data). Thus, in NRG-1b–sensitive cells suchas SK-BR-3, blockade of HER2–HER3 signaling by pertu-zumab is essential to achieve the full inhibitory effect ofT-DM1 under ligand-dependent conditions, whereas incells such as KPL-4, which are not stimulated downstreamby NRG-1b and therefore not responsive in vitro topertuzumab, T-DM1 alone potently suppresses HER2–HER3 signaling. We speculate that a contributing factor ishyperactivationof the PI3Kpathway inKPL-4 cells due to anactivating PIK3CA mutation, versus SK-BR-3 cells that arePIK3CA wild-type. The mechanisms underlying these dif-fering activities are currently under investigation.

T-DM1–pertuzumab combination studies in vivoTo further support our observation that T-DM1 combined

with pertuzumab leads to enhanced antitumor effects, weperformed in vivo combination studies in two HER2-ampli-fied tumor xenograft models. In the Calu-3 lung cancermodel, 25mg/kgpertuzumabwas administeredonceweeklyfor 3 weeks; T-DM1 was administered (1, 3, or 7 mg/kg) as asingle intravenous injection at the start of the study. Pertu-

zumab treatment resulted in tumor growth delay (Fig. 5A,right). Only the highest dose of T-DM1 (7mg/kg) resulted insignificant tumor growth inhibition. Enhanced antitumoractivity, comparedwith single-agent treatment,was observedwith pertuzumab plus 3 or 7 mg/kg T-DM1. Equivalentefficacy was observed with 7 mg/kg T-DM1 compared withpertuzumabplus 3mg/kg T-DM1. Pertuzumabwith 7mg/kgT-DM1 was the most active combination tested. Tumorsregressed during and after treatment, with no regrowth untilseveral weeks after treatment was stopped. Although moresustained tumor growth inhibition was reported in Calu-3xenografts treated with pertuzumab and trastuzumab (23),this was likely due toweekly antibody administration for thestudy duration (>45 days). In our studies, limited dosingallows for monitoring of tumor regrowth and calculation oftime-to-tumor volume doubling (TTP). Log-rank tests fordifferences in TTP showed statistically significant differencesbetween single-agent treatment for pertuzumab or T-DM1versus the corresponding combination group (log-rank Pvalue for pertuzumab vs. pertuzumab þ 3 mg/kg T-DM1 ¼0.008; log-rank P value < 0.0001 for pertuzumab vs. pertu-zumab þ 7 mg/kg T-DM1, and for 1 or 3 mg/kg T-DM1 þpertuzumab vs. T-DM1 alone; log-rank P-value¼ 0.0004 for7 mg/kg T-DM1 vs. pertuzumabþ 7 mg/kg T-DM1; Supple-mentary Fig. S5A).

In KPL-4 breast tumor xenografts, T-DM1 (0.3, 1, 3 mg/kg) or pertuzumab (30 mg/kg loading dose; 15 mg/kgweekly) caused significant tumor growth inhibitioncompared with vehicle-treated animals (log-rank P value¼ 0.0204 for 0.3 mg/kg T-DM1 vs. vehicle group; log-rankP value < 0.0001 for all other single-agent treatment groupsvs. vehicle control group; Fig. 5B, right; SupplementaryFig. S5B). Administration of pertuzumab with eachdose of T-DM1 resulted in pronounced inhibition oftumor growth, reaching statistical significance for eachcombination group compared with the corresponding sin-gle-agent treatment groups (Supplementary Fig. S5B). Sus-tained tumor regressionswere observed inmice treatedwith3 mg/kg T-DM1 and with the combination of pertuzumabplus 1mg/kg T-DM. After approximately 40 days, tumors inthese two groups started to regrow. However, treatmentof mice with pertuzumab plus 3 mg/kg T-DM1 resultedin lasting tumor regression for the duration of the study(88 days).

In addition to direct antisignaling activities mediated byT-DM1 and pertuzumab, enhanced ADCC may also

Figure 1. Effects of T-DM1 and pertuzumab, alone and in combination, on MDA-175 cells in vitro and in vivo. A, growth inhibition and apoptotic effects:treatment of MDA-175 cells for 5 days with T-DM1 and pertuzumab (wild-type or N297A mutant) results in synergistic reduction of cell viability (top left), asdetermined by the Chou and Talalay method. Time-course of apoptosis induction by T-DM1 and pertuzumab, as measured by caspase-3/7 activation(24 hours, top right; 72 hours, bottom left) and PARP cleavage (bottom right). Cell viability and caspase assays were run at full-dose ranges; single dosesused for PARP cleavage were 10 mg/mL T-DM1 and 100 mg/mL pertuzumab. B, antitumor activity of T-DM1 and pertuzumab, as single agents or incombination, in MDA-175 cells implanted into mammary fat pads of beige nude XID mice. Mice bearing MDA-175 tumors were treated with a singleintravenous injection of 3 mg/kg pertuzumab, 10 mg/kg T-DM1, or the combination of the two agents at the same doses (n¼ 10 per group). C, lack of ADCCactivity onMDA-175 cells in vitro: cellswere incubatedwith trastuzumab, pertuzumab,N-297A-pertuzumab, T-DM1, T-DM1þpertuzumab, T-DM1þN297A-pertuzumab, or isotype control antibody in the presence of peripheral blood mononuclear cells (PBMC), 25:1 effector:target ratio. Tumor cell lysis wasassessed by lactate dehydrogenase (LDH) release. D, inhibition of HER2–HER3–PI3K signaling in MDA-175 cells (24-hour treatment with 10 mg/mLpertuzumab or N-297A-pertuzumab, 10 mg/mL T-DM1, or the respective combinations).






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contribute to combination antitumor efficacy. We thereforeperformed in vitro ADCC assays comparing T-DM1, pertu-zumab, or N-297A-pertuzumab with the correspondingcombinations. Trastuzumab was included as a positive con-trol andan isotype-matchedantibody as thenegative control.Both T-DM1 and pertuzumab induced immune effector cell-

mediated lysis in a dose-dependent manner in Calu-3 andKPL-4 cells similar to trastuzumab (Fig. 5A and B, left).However, T-DM1 combined with pertuzumab or N297A-pertuzumabdidnot result in greaterADCCactivity comparedwith T-DM1 or wild-type pertuzumab alone. As expected,N297A-pertuzumab and isotype-matched control antibody

Figure 3. The effect of NRG-1b onT-DM1 activity in five HER2-amplified breast cancer lines. A,BT-474, SK-BR-3, and ZR-75-30:inhibitory effect of NRG-1b (2 nmol/L) on a dose titration of T-DM1 (left)with almost complete inhibition ofT-DM1 activity. The right panelsshow a dose-dependent reductionof T-DM1 activity by NRG-1b; thefixed dose of T-DM1 selected foreach cell line was the lowest dosegiving maximum growth inhibition(1mg/mL for BT-474 andZR-75-30;0.1 mg/mL for SK-BR-3). B, twoHER2-amplified breast cancerlines, KPL-4 and HCC1954, areequally sensitive to T-DM1 in theabsence or presence of NRG-1b.All treatments were for 3 days.C, the effect of NRG-1b onT-DM1–induced apoptosis,measured by caspase-3/7activation, was assessed aftera 2-day treatment. NRG-1bcompletely blocked T-DM1apoptotic activity in BT-474 cells;this effect was reversed by theaddition of pertuzumab. T-DM1–induced apoptosis in KPL-4 cellswas unaffected by the addition ofNRG-1b. pmab, pertuzumab.

Figure 2. Influence of NRG-1b on combination activity of T-DM1 plus pertuzumab. Enhanced antiproliferative activity with combination treatment wasobserved in Calu-3 lung (A) and in BT-474 (B) and SK-BR-3 (C) breast cancer cells only in the presence of NRG-1b, indicating the importance of pertuzumabinhibition of NRG-1b signaling. There was no T-DM1–pertuzumab combination effect in KPL-4 cells (D) despite NRG-1b addition to the culture medium.Cell proliferation in the absence of NRG-1b is denoted by the closed black triangle on the y-axis. Assays were conducted for 5 days for Calu-3 andBT-474, 3 days for SK-BR-3 and KPL-4 cells.






showednoactivity. These findings indicate that combinationactivity in vivo is likely duemore to the antisignaling activitiesof both T-DM1 and pertuzumab and warrant further inves-tigation, in models such as KPL-4, of the role of stromalligands in the response to pertuzumab in vivo.

Phase Ib study of T-DM1 combined with pertuzumabA global, single-arm, phase Ib/II study (TDM4373g) was

conducted to investigate the safety and efficacy of T-DM1plus pertuzumab in patients with HER2-positive locallyadvanced or mBC who had received prior systemic therapyfor recurrent locally advanced or metastatic disease and inpatients with newly diagnosed or previously untreatedmBC(first-line). The primary objectives of this study includedcharacterization of the safety and tolerability of this drugcombination and to assess objective response rate (ORR)based on investigator assessment. In the 3þ3 study design(Supplementary. Fig. S6), patients received the standardpertuzumab dose every 3 weeks (840 mg, cycle 1; 420 mg,cycle 2, and beyond) and T-DM1 at a dose of 3.0 mg/kgevery 3 week (Cohort 1). In the absence of DLT, patientsthen received pertuzumab with 3.6 mg/kg T-DM1 (Cohort2). In the event of DLTs, the T-DM1 dose was reduced to 2.4mg/kg (from 3.0 mg/kg, Cohort 1) or to 3.0 mg/kg (from3.6mg/kg, Cohort 2). Once dose escalationwas completed,patients could then be added to the expansion phase (thephase II part of this study, results to be published in aseparate article). Only the phase Ib results are reported here.

In the phase Ib portion of the study, 3 patients received3.0 mg/kg T-DM1 with the rest receiving the standard 3.6

mg/kg dose, for a total of 9 patients. Patient demographicsand baseline characteristics are shown in SupplementaryTable S1. Themajority (8/9) of patients wereWhite and hadEastern Cooperative Oncology Group (ECOG) perfor-mance status of 1 (6/9). Patients had tumors that wereeither 3þ for HER2 expression by immunohistochemistry(6/9) or positive by FISH (8/9). The median number ofprevious systemic therapies was eight. All patients hadreceived prior trastuzumab and lapatinib therapy, as wellas a taxane and capecitabine (Supplementary Table S2).Most patients (7/9) had also received prior anthracyclinetherapy.

During dose escalation, no patient treated with 3.0 mg/kg T-DM1 combined with pertuzumab developed a DLT.However, among the first 3 patients treated with 3.6 mg/kg T-DM1, 1 patient developed a DLT (grade-4 throm-bocytopenia), and the cohort was expanded to include 3more patients, none of whom developed a DLT. There-fore, 3.6 mg/kg T-DM1 in combination with standarddose pertuzumab, administered every 3 weeks, wasdeclared the recommended phase II schedule. Table 1shows adverse events � grade 3 for the 9 patients. Themost common � grade 3 adverse effect was fatigue (3/9),with thrombocytopenia and lung complications eachoccurring in 1 out of 9 patients. All 9 patients experiencedat least one adverse event of any grade; these are sum-marized in Supplementary Table S3 and included gastro-intestinal, respiratory, musculoskeletal, and skin disor-ders, as well as more general disorders such as fatigue,fever, and chills.

Figure 4. Effects of T-DM1 andpertuzumab, alone or incombination, on HER2–HER3-mediated signal transduction inSK-BR-3 and KPL-4 breast cancercells. T-DM1 (0.1 mg/mL) orpertuzumab (15 mg/mL) wereadded 1 hour before stimulationwith 1 nmol/L NRG-1b for 15minutes or 24 hours. Cell lysateswere analyzed for total andp-HER3and AKT. The presence ofpertuzumab was required forsuppression of signaling in NRG-1b–activated SK-BR-3 cells.

Phillips et al.





About objective response in Cohort 1 (n ¼ 3 patientswho received 3.0 mg/kg T-DM1), 2 patients showed apartial response and 1 patient had stable disease. InCohort 2 (n ¼ 6 patients receiving 3.6 mg/kg T-DM1),2 patients had a partial response, 3 patients had stabledisease, and 1 patient had disease progression as bestresponse. Therefore, for the full phase Ib cohort, the ORRwas 44.4% (4/9; Table 2). Disease stability was main-tained for at least 6 months for 2 of the 3 patients whohad stable disease as best response.

DiscussionWith the approval by the FDA in 1998, trastuzumab

becamewidely integrated into standard treatment regimensfor HER2-positive mBC (14). Less than a decade later,favorable results were obtained with trastuzumab pluschemotherapy in early breast cancer (43). More recently,

trastuzumab in combination with cisplatin and a fluoro-pyrimidine for treatment of HER2-postive gastric/gastro-esophageal cancer was shown to extend survival in thesepatients (44). Despite advances in the treatment of HER2-positive cancers, a subset of patients will show diseaseprogression through treatment. The antibody–drug conju-gate T-DM1 has been tested in preclinical models and inmultiple clinical trials as an additionalmethod for targetingHER2-positive cancer. T-DM1 provides intracellular deliv-ery of the cytotoxic agentDM1, resulting in tumor cell death(16), along with the important therapeutic properties ofunconjugated trastuzumab (38). Recently, T-DM1 (Kad-cyla) was approved for treatment of HER2-positive mBCin patients who had received prior Herceptin and a taxane.Effective targeting of HER2 is also achieved with pertuzu-mab, a HER2-specific antibody that recognizes an epitopeon the HER2 extracellular domain distinct from

Figure 5. Comparison of in vitroADCC activity (left) to in vivoantitumor efficacy (right) in Calu-3lung cancer cells (A) and KPL-4breast cancer cells (B). ADCCassays were performed asdescribed in Figure 1C. Noenhanced ADCC activity wasobserved with combinationtreatment. Mice bearing Calu-3subcutaneous tumors wereadministered 25 mg/kgpertuzumab once per week for 3weeks; T-DM1 was administeredas a single intravenous injectionat doses of 1, 3, or 7 mg/kg(n ¼ 9/group). T-DM1 combinedwith pertuzumab was moreefficacious than treatment withsingle agents. Similarly, in vivotumor growth of KPL-4 breastcancer cells is greatly reduced withT-DM1 and pertuzumab incombination. In this study, micewere treated weekly for 3 weekswith 15 mg/kg pertuzumab or onceat treatment initiation with 0.3, 1, or3 mg/kg T-DM1; n ¼ 8 per group.






trastuzumab. Pertuzumab was approved by the FDA in2012 on the basis of the findings of CLEOPATRA, a phaseIII trial that demonstrated significantly improved progres-sion-free and overall survival with the addition of pertuzu-mab to trastuzumab plus docetaxel in previously untreatedHER2-positive mBC patients (34, 45).

We therefore extended these studies by investigating theefficacy of pertuzumab combinedwith T-DM1 inpreclinicalmodels of HER2-amplified cancers and in an early-phaseclinical trial. The combination of pertuzumab and T-DM1resulted in a synergistic drug effect in multiple tumor celllines in vitro and was more efficacious than treatment withindividual agents in tumor xenograft models. In vitro, NRG-1b suppressed the cytotoxic effects of T-DM1 in a subset ofcell lines; the addition of pertuzumab was necessary toovercome this inhibitory effect. As ADCC activity wasnot increased in vitro compared to treatment with T-DM1or pertuzumab alone, enhanced antitumor activity is likelydue to the multiple and diverse antisignaling properties ofpertuzumab and T-DM1, as well as the cytotoxic activity ofthe DM1 component of T-DM1. Because NRG-1b–mediat-ed resistance was not observed in all cell lines tested, furtherstudies are in progress to investigate the effect of additionalgrowth factors/cytokines on the response not only to T-DM1 but other chemotherapeutic agents as well.

Importantly, these findings add to the emerging body ofdata describing autocrine or stromal-derived growth factor-mediated resistance to targeted therapies. Several recentreports show that the c-Met receptor ligand HGF (hepato-cyte growth factor), as well as fibroblast growth factors(FGF), attenuate the anticancer activity of inhibitors target-ing EGFR, B-Raf, and FGF receptors (7–9, 46). NRG-1bwasalso reported to rescue cells from the inhibitory effects ofHER2 and EGFR kinase inhibitors (9, 47). Moreover, theNRG-1b receptor, HER3, mediates inhibitor resistance;upregulation and activation of HER3 was demonstrated indiverse tumor cell types in vitro in response to EGFR, HER2,and PI3K kinase inhibitors (11, 12, 48, 49). Our datashowing NRG-1b–mediated resistance to the microtu-bule-targeted cytotoxic agents T-DM1, docetaxel, and vinor-elbine thus expand these observations beyond kinase inhi-bitors to cytotoxic agents.

Clinical activity of T-DM1 is being assessed in numerousphase II and III trials. Phase II single-arm trials of T-DM1 inpatients with HER2-positive mBC who had received priortrastuzumab and/or lapatinib in addition to chemotherapyshowed that, in these heavily pretreated patients, the ORRranged from 25% to 35% (17, 18). Given the small samplesize and the specifics of the patient population in the studyreported here, it is difficult to compare the response rates forT-DM1 with pertuzumab with response rates for T-DM1alone. An ongoing phase III, randomized, 3-arm study,MARIANNE (TDM4788g/BO22589), will directly addressthis question by evaluating the safety and efficacy of T-DM1with pertuzumab or T-DM1 plus pertuzumab placebo ver-sus trastuzumab plus taxane (docetaxel or paclitaxel) inpatients with HER2-positive progressive or recurrent locallyadvanced or previously untreated mBC. The phase Ib datareported here demonstrate that the combination of T-DM1withpertuzumab is feasible at full doseswithnounexpectedsafety signals; the majority of adverse events were grade 1and 2 and were similar to those reported for single-agent T-DM1 (fatigue, thrombocytopenia, and increased serumtransaminases; refs. 17–19). Data from the phase II expan-sion portion of this study will be reported separately.

Results from the EMILIA phase III trial showed thatpatients treated with T-DM1 had significantly longer pro-gression-free and overall survival with a lower frequency ofsevere adverse events compared with the control arm (lapa-tinib plus capecitabine, a standard of care for second-line

Table 2. Best clinical response, by cohort, in the phase Ib portion of TDM 4373g

Cohort 1 (n ¼ 3) Cohort 2 (n ¼ 6) Total (n ¼ 9)

Complete response 0 0 0Partial response 2 (66.7%) 2 (33.3%) 4 (44.4%)Stable disease 1 (33.3%) 3 (50.0%) 4 (44.4%)Progressive disease 0 1 (16.7%) 1 (11.1%)

NOTE: Pertuzumab dose: 840 mg first cycle, then 420 mg thereafter; all agents administered once every 3 weeks.Cohort 1, 3.0 mg/kg T-DM1 plus pertuzumab; Cohort 2, 3.6 mg/kg T-DM1 plus pertuzumab

Table 1. National Cancer Institute (NCI)Common Terminology Criteria for AdverseEvents (CTCAE) grade � 3 adverse eventsin the phase Ib portion of TDM4373g

Adverse eventNumber of patients(n ¼ 9)

Fatigue 3Thrombocytopenia 1Pneumonia 1Pleural effusion 1Deafnessa 1

NOTE: Pertuzumab dose: 840 mg first cycle, then 420 mgthereafter; all agents administered once every 3 weeks.aPatient had baseline grade 3 hearing loss thatworsenedbutdid not increase in grade.

Phillips et al.





treatment of HER2-positive mBC; ref. 19). Similarly, datafrom a randomized phase II study, TDM4450g, demon-strated that T-DM1 significantly prolonged progression-freesurvival with a more favorable safety profile compared topatients treatedwith trastuzumab plus docetaxel in the first-line setting (50). Overall, these results, together with theCLEOPATRA data, show significantly improved clinicalbenefit of T-DM1 or trastuzumab/pertuzumab/docetaxelcompared with the trastuzumab/docetaxel regimen. Fromthese data, we anticipate the MARIANNE (TDM4788g/BO22589) trial to demonstrate better clinical outcome inthe T-DM1 plus pertuzumab arm, further supporting therationale for using multiple HER2-targeted agents in thetreatment of HER2-positive breast cancer.

Disclosure of Potential Conflicts of InterestK. Miller received commercial research grant from Genentech/Roche and

Merrimack. K.S. Albain is consultant/advisory board member of Genentechand Roche. N. Harbeck and V. Dieras have honoraria and are consultant/advisory boardmembers of Roche. L. Fang has ownership interest (includingpatents) in stock options. S.R. Olsen and M.X. Sliwkowski have ownershipinterest (including patents) in Roche. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design:G.D. Lewis Phillips, C.T. Fields, G. Li, G. Schaefer,S.R. Olsen, M.X. SliwkowskiDevelopment of methodology: G.D. Lewis Phillips, C.T. Fields, G. Li, D.Dowbenko, M.X. SliwkowskiAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): C.T. Fields, G. Li, D. Dowbenko, K. Miller, F.Andre, H.A. Burris III, K.S. Albain, N. Harbeck, V. Dieras, D. Crivellari, E.Guardino, L.M. Crocker, M.X. SliwkowskiAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):C.T. Fields, G. Li, K.Miller, H.A. Burris III,N. Harbeck, V. Dieras, L. Fang, E. Guardino, S.R. Olsen, L.M. Crocker, M.X.SliwkowskiWriting, review, and/or revision of the manuscript: G.D. Lewis Phillips,C.T. Fields, G. Li, K. Miller, F. Andre, H.A. Burris III, K.S. Albain, N. Harbeck,V. Dieras, D. Crivellari, E. Guardino, S.R. Olsen, M.X. SliwkowskiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.T. Fields, G. Li, S.R. Olsen, L.M.Crocker, M.X. SliwkowskiStudy supervision: G.D. Lewis Phillips, G. Li, G. Schaefer, K. Miller, H.A.Burris III, E. Guardino, S.R. Olsen, M.X. Sliwkowski

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 11, 2013; revised August 22, 2013; accepted September15, 2013; published OnlineFirst October 4, 2013.

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2014;20:456-468. Published OnlineFirst October 4, 2013.Clin Cancer Res Gail D. Lewis Phillips, Carter T. Fields, Guangmin Li, et al. in Antitumor Response to Combination TherapyEmtansine and Pertuzumab: Critical Role for Neuregulin Blockade Dual Targeting of HER2-Positive Cancer with Trastuzumab

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