Dual Targeting of HER2-Positive Cancer with Trastuzumab Emtansine and Pertuzumab: Critical Role for Neuregulin Blockade in Antitumor Response to Combination Therapy

Home , Gefitinib, Pertuzumab, Trastuzumab emtansine

Published OnlineFirst October 4, 2013; DOI: 10.1158/1078-0432.CCR-13-0358

Clinical Cancer Cancer Therapy: Clinical Research See related article by Gwin and Spector, p. 278

Dual Targeting of HER2-Positive Cancer with Trastuzumab Emtansine and Pertuzumab: Critical Role for Neuregulin Blockade 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

Abstract Purpose: 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 conjugate 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 advanced or metastatic breast cancer (mBC), T-DM1 was dose-escalated with a fixed standard pertuzumab dose in a 3þ3 phase Ib/II study design. Results: Treatment of HER2-overexpressing tumor cells in vitro with 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 from mouse 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 and mouse xenograft models 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.

Introduction ErbB2, HER3/ErbB3, and HER4/ErbB4. Numerous ligands The ErbB/HER family of receptor tyrosine kinases (RTK) have been identiﬁed that interact with all HER family plays critical roles in both development and cancer (1). receptors, with the exception of HER2. Ligand binding These receptors include EGF receptor (EGFR)/ErbB1, HER2/ activates multiple downstream signal transduction pathways. HER2 is the common coreceptor for the other HER family members. Association of HER2 with HER3 is the most potent signaling complex formed in this receptor 1 2 Authors' Afﬁliations: Research Oncology, Departments of Biostatistics, family (2, 3). HER3 is considered a pseudo-kinase (4, 5), 3Product Development Oncology, and 4Translational Oncology, Genen- tech, Inc., South San Francisco, California; 5Indiana University Simon but can potently activate phosphoinositide 3-kinase (PI3K) Cancer Center, Indianapolis, Indiana; 6Institute Gustave Roussy, Villejuif; signaling upon dimerization with other HER family mem- 7 8 Department of Medical Oncology, Institut Curie, Paris, France; Sarah bers (6). In addition to growth factor regulation of cell Cannon Research Institute, Nashville, Tennessee; 9Loyola University Med- ical Center, Maywood, Illinois; 10University of Munich, Munich, Germany; proliferation and differentiation, recent reports show an and 11Division of Medical Oncology, Centro di Riferimento Oncologico, important role for multiple RTK ligands in resistance to Instituto Nazionale Tumori Aviano, Italy kinase inhibitors (7–9). The HER3 ligand, neuregulin 1 Note: Supplementary data for this article are available at Clinical Cancer (NRG-1), was recently shown to mediate resistance to Research Online (http://clincancerres.aacrjournals.org/). chemotherapeutic agents as well (10). Moreover, HER3 was G.D.L. Phillips and C.T. Fields contributed equally to this work. reported to mediate resistance to EGFR, HER2, and PI3K Corresponding Author: Gail D. Lewis Phillips, Genentech, Inc., 1 DNA inhibitors. Treatment resulted in HER3 activation, leading Way, South San Francisco, CA 94080. Phone 650-225-2201; Fax: 650- to attenuation of the response to kinase inhibition (11, 12). 225-1411; E-mail: [email protected] Trastuzumab (Herceptin), a humanized HER2 antibody doi: 10.1158/1078-0432.CCR-13-0358 that binds domain IV of the HER2 extracellular domain 2013 American Association for Cancer Research. (ECD), is used in combination with chemotherapy for

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HER2 with other HER family members (24) and demon- Translational Relevance strated clinical activity as a single agent and in combination The antibody–drug conjugate ado-trastuzumab with chemotherapy in ovarian, non–small cell lung and emtansine (T-DM1; Kadcyla) increases progression-free prostate cancer (25–29). Pertuzumab combined with tras- and overall survival versus lapatinib plus capecitabine in tuzumab has antitumor activity in cell culture (30) and patients with HER2-positive metastatic breast cancer animal models of HER2-amplified cancer (23, 31) and in (mBC) who have received prior treatment with trastu- patients with HER2-positive mBC (32, 33). Combining zumab (Herceptin) and a taxane chemotherapy, with a pertuzumab, trastuzumab, and docetaxel significantly better safety profile than the comparator treatments. improves the pathologic complete response rate in the Dual antibody blockade of HER2 with pertuzumab, neoadjuvant setting compared with treatment without per- trastuzumab plus docetaxel showed increased progres- tuzumab (33). In June 2012, the U.S. Food and Drug sion-free and overall survival with the addition of per- Administration (FDA) approved Pertuzumab (Perjeta) in tuzumab to trastuzumab plus docetaxel. Neoadjuvant combination with Herceptin and chemotherapy for treat- treatment with trastuzumab plus pertuzumab (Neo- ment of patients with HER2-positive mBC who have not Sphere trial) or trastuzumab plus lapatinib (NeoALTTO received prior anti-HER2 therapy or chemotherapy for trial) in conjunction with taxane-based therapy resulted metastatic disease (34). We therefore explored the potential in better clinical activity than with single-agent HER2- of combining T-DM1 with pertuzumab in models of HER2- directed therapy. The studies described here show amplified breast and lung cancer. Our data underscore the enhanced antitumor activity with T-DM1 plus pertuzu- feasibility of using multiple strategies to target HER2-pos- mab in preclinical models, with an encouraging safety itive cancer. profile and preliminary evidence of efficacy in a phase Ib study. Moreover, our data show suppression of T-DM1 activity upon ligand activation of HER2/HER3, support- Materials and Methods ing the rationale for combining pertuzumab with Cell lines and reagents T-DM1. 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 were obtained from the American Type Culture Collection. treatment of HER2-positive breast cancer. Proposed KPL-4 breast cancer cells were provided by Prof. Junichi mechanisms of trastuzumab action include inhibition of Kurebayashi, Kawasaki Medical Hospital, Kurashiki, ECD shedding, disruption of downstream signal-transduc- Okayama, Japan (35). Cells were maintained as previously tion pathways, induction of cell-cycle arrest, inhibition of described (16). T-DM1, pertuzumab, N297A-pertuzumab DNA repair, decreased angiogenesis, and mediation of (36), and E. coli-derived NRG-1b (EGF domain) were antibody-dependent cell-mediated cytotoxicity (ADCC; produced at Genentech, Inc. Unconjugated DM1 was ref. 13). Clinical benefit from trastuzumab-containing ther- obtained from ImmunoGen, Inc. and chemotherapeutic apy, however, can be limited, especially in the metastatic drugs from Sigma-Aldrich. Western blot antibodies were setting, necessitating development of alternate forms of from R&D Systems (cleaved PARP), Cell Signaling Tech- treatment (14, 15). We developed a cytotoxic drug-conju- nology (phospho-HER3, phospho-Akt, Akt), and Santa gate of trastuzumab by covalently linking the antimitotic Cruz Biotechnology, Inc. (HER3). agent DM1 to trastuzumab through a stable MCC linker. Trastuzumab emtansine (T-DM1) potently inhibits growth Cell viability and apoptosis assays of trastuzumab-sensitive and -insensitive HER2-amplified Cell viability and apoptosis assays were performed as cancer cells and shows a favorable safety profile in preclin- described in ref. (16). T-DM1, pertuzumab, or the combi- ical toxicity studies (16). T-DM1 activity has been evaluated nation was added to cells; treatments were in duplicate to fit in phase II and III clinical trials in patients with HER2- all groups onto one plate and experiments were repeated 5 positive locally advanced or mBC (17, 18). T-DM1 (ado- times. Studies performed without NRG-1b were in medium trastuzumab emtansine [Kadcyla]) was recently approved supplemented with 10% FBS and studies with NRG-1b (2 for treatment of patients with HER2-positive mBC who have nmol/L) were in 1% FBS-containing medium. Cell viability received prior treatment with Herceptin and a taxane che- was measured at 3 (SK-BR-3, KPL-4) or 5 (BT-474, Calu-3) motherapy, based on data showing significantly prolonged days after drug addition. Assays to test NRG-1b effects on T- progression-free and overall survival and less toxicity com- DM1 or chemotherapeutic agent activity were 3 or 5 days pared with standard-of-care treatment, lapatinib (Tykerb) (n ¼ 3 per group) and studies were performed three times. plus capecitabine (Xeloda; ref. 19). Combination Index (CI) values, as described by Chou (37), In addition to trastuzumab, pertuzumab is a HER2-spe- were generated using CalcuSyn software (Biosoft, Inc). cific humanized antibody with efficacy demonstrated in ADCC assays were performed as described in ref. (38) using mouse xenograft models of breast, lung, prostate, and CytoTox-ONE Homogeneous Membrane Integrity Assay ovarian cancer (20–23). Pertuzumab binds domain II of (Promega Corp.). Treatments were T-DM1 alone or com- the HER2 ECD, preventing ligand-dependent association of bined with pertuzumab or the Fc-mutant N297A-

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pertuzumab; trastuzumab and anti-gD isotype-matched escalation was complete, additional patients were enrolled antibody were positive and negative controls, respectively. into an expansion phase (phase II). Patient inclusion criteria and study assessment guidelines are in Supplementary Western blot analysis Methods. For measuring PARP cleavage, MDA-175 cells were seededandallowedtoadherefor48hours.T-DM1(10 Results mg/mL), pertuzumab (100 mg/mL), or the combination Combination effects of T-DM1 and pertuzumab in cell was added and cells were incubated for 48 or 72 hours. culture and xenograft models For experiments with NRG-1b,SK-BR-3,Calu-3,KPL-4, T-DM1 combined with pertuzumab in MDA-175, a model andMDA-175wereplatedandallowedtoadhereover- of autocrine heregulin growth. Growth of MDA-175 breast night. Cells were then starved in medium with 0.1% FBS cancer cells is dependent on autocrine production of g-here- for 24 hours. NRG-1b (1 nmol/L) was added to all cells, gulin (36) and provides a unique model for studying except MDA-175, after a 1-hour pretreatment with pertu- pertuzumab activity. In addition, MDA-175 cells slightly zumab or N297A-pertuzumab, T-DM1, or the combina- overexpress HER2 (1þ expression) and thus are moderately tion. After 15 minutes or 24 hours, cells were lysed in sensitive to trastuzumab (39) and T-DM1. The combination RIPA buffer and Western blotting procedures were per- of pertuzumab with T-DM1 showed enhanced antiproli- formed as described previously (16). ferative activity compared with either agent alone (Fig. 1A, top left). CI values were determined from these studies. CI In vivo efficacy studies values less than 1 denote a synergistic drug interaction, CI MDA-175 breast cancer xenograft studies were performed values more than 1 denote antagonism, and CI values as described previously (21). KPL-4 breast tumor cells were approximately 1 denote additivity. In MDA-175 cells, T- inoculated (3 106 per mouse) into mammary fat pads and DM1 combined with pertuzumab resulted in synergy at all Calu-3 lung tumor cells were inoculated subcutaneously drug concentrations tested (CI range, 0.03–0.75). The aver- (5 106 per mouse) into flanks of female CB-17 severe age CI value for the entire drug-effect range was 0.19 0.05, combined immunodeficient (SCID) Beige mice (Charles with the CI values at or near the IC50 markedly less than 1, as Rivers Laboratories). Animals were randomly assigned into depicted in Supplementary Fig. S1A (CI vs. drug effect). The groups with mean tumor volumes of 300 mm3. Animals average CI values for five different experiments ranged from received a single intravenous injection of T-DM1 (10 mg/kg 0.10 to 0.50, indicating synergy across multiple studies. No for MDA-175; 0.3, 1, or 3 mg/kg for KPL-4; and 1, 3, or 7 mg/ difference was observed between pertuzumab and N297A- kg for Calu-3) on study day 0. Mice bearing MDA-175 pertuzumab on MDA-175 cell proliferation, alone or com- tumors were administered a single intraperitoneal dose of bined with T-DM1. pertuzumab (3 mg/kg); for studies with KPL-4 or Calu-3 Additional studies were performed to determine if T- tumors, mice were first injected intraperitoneally with a 2 DM1 plus pertuzumab induced enhanced apoptosis in loading dose of pertuzumab, and received two additional MDA-175 breast cancer cells. Treatment with pertuzumab doses once per week. Pertuzumab doses were 30 mg/kg resulted in enhanced caspase-3/7 activity as early as load/15 mg/kg weekly for KPL-4 and 50 mg/kg load/25 mg/ 24 hours after treatment (Fig. 1A, top right), whereas kg weekly for Calu-3. Treatment groups consisted of 8 to 10 T-DM1–induced apoptosis was observed only after pro- animals per group. ANOVA and Tukey–Kramer HSD (hon- longed (72 hour) treatment (Fig. 1A, bottom). Maximal estly significant difference) analysis were used for calcula- caspase activation was observed with the combination of tion of means, SEs and statistical comparisons of time-to- T-DM1 and pertuzumab. Similar to the results shown for tumor volume doubling (time-to-progression, TTP). Anal- caspase activation, maximal PARP cleavage, as indicated yses were performed using JMP Software, version 6.0 (SAS by the appearance of the 23 kDa cleavage fragment, was Institute). demonstrated in the T-DM1–pertuzumab combination group, compared with single-agent treatment, and was Clinical trial design observed at both 48 and 72 hours of treatment (Fig. 1A, A single-arm, phase Ib/II study (TDM4373g) was bottom right). designed to investigate the safety and efficacy of T-DM1 The combination of T-DM1 with pertuzumab was tested in combined with pertuzumab in patients with HER2-positive vivo in the MDA-175 xenograft model. Treatments were single locally advanced or mBC who had been previously treated intravenous injections of 3 mg/kg pertuzumab, 10 mg/kg T- with a HER2-directed therapy. The study was reviewed and DM1, or the combination (Fig. 1B). Log-rank P values were approved by the institutional review board at each site, obtained for differences in time-to-tumor doubling (Supple- according to local clinical guidelines. All patients provided mentary Fig. S2). Single-agent treatment with T-DM1 or informed consent. The phase Ib results are reported herein. pertuzumab resulted in moderate tumor growth inhibition Study design. In the 3þ3 design, patients received per- that was statistically different from vehicle-treated controls tuzumab (840 mg cycle 1; 420 mg cycle 2, and beyond) with (P ¼ 0.0247 for pertuzumab vs. control; P ¼ 0.0017 for T-DM1 [3.0 mg/kg in Cohort 1 and, in the absence of dose- T-DM1 vs. control). In contrast, tumors in mice treated with limiting toxicity (DLT), 3.6 mg/kg in Cohort 2]. Both agents T-DM1 combined with pertuzumab regressed for 3 to 4 were administered by intravenous infusion. Once dose weeks, with only modest tumor regrowth occurring after

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40 to 50days. The difference in time-to-tumordoublingfor T- zumab of T-DM1 activity in the presence of NRG-1b was DM1 plus pertuzumab compared with single-agent treat- independent of NRG-1b mitogenic activity. In contrast, no ment groups or vehicle-treated controls was significant (P combination effect was observed in cells treated in the < 0.0001 for combination vs. vehicle or pertuzumab; P ¼ absence of NRG-1b; i.e., pertuzumab did not enhance the 0.0003 for combination vs. T-DM1; Supplementary Fig. S2). growth-inhibitory effect of T-DM1 under ligand-indepen- Thus, T-DM1 combined with pertuzumab induced apoptotic dent conditions (Fig. 2A–C, right). In KPL-4 breast cancer cell death in the MDA-175 breast cancer line and resulted in cells, T-DM1 cytotoxicity was not increased by pertuzumab significant tumor growth inhibition/regression compared in ligand-dependent (Fig. 2D, left) or ligand-independent with T-DM1 or pertuzumab alone. (Fig. 2D, right) conditions. Antitumor mechanisms for combination treatment efficacy, in addition to DM1-mediated cell death, include Inhibition of T-DM1 cytotoxicity by NRG-1b ADCC and different antisignaling properties of the two In the earlier experiments, it was noted that the cytotoxic antibodies. We therefore investigated the contribution of response to T-DM1 was reduced in the presence of NRG-1b. these different mechanisms to combination activity. The We therefore investigated NRG-1b inhibition of T-DM1– N297A Fc mutation, which prevents binding between the induced cytotoxicity on a larger panel of HER2-amplified antibody Fc portion and Fc receptors on immune effector breast cancer lines using two experimental formats, com- cells, was introduced into pertuzumab for ADCC and signal paring a fixed concentration of NRG-1b (2 nmol/L) versus a transduction studies. ADCC assays performed in vitro with dose range of T-DM1 and the converse, NRG-1b dose pertuzumab, N297A-pertuzumab, T-DM1, or the combina- response with a fixed concentration of T-DM1. In BT- tion of T-DM1 with either wild-type or mutant pertuzumab 474, SK-BR-3, ZR-75-30 (Fig. 3A, left), and UACC812 (data showed minimal tumor cell lysis (Fig. 1C): 12.8% lysis with not shown) breast cancer lines, 2 nmol/L NRG-1b sup- control antibody versus 14.7% for pertuzumab, 15.4% for pressed T-DM1 activity. Concentrations as low as 0.01 to T-DM1, and 18.6% for T-DM1 þ pertuzumab. Percentage 0.2 nmol/L NRG-1b reduced the antiproliferative activity of lysis with the N297A-mutant pertuzumab (10.6%) was T-DM1 in all four cell lines (Fig. 3A, right). In contrast, NRG- similar to the negative control, whereas cell lysis after 1b did not block T-DM1 activity in KPL-4 and HCC1954 treatment with T-DM1 þ N297A-pertuzumab (17.0%) was breast cancer cells (Fig. 3B). Interestingly, these two breast similar to T-DM1 þ wild-type, ADCC-competent pertuzu- cancer lines harbor activating PIK3CA mutations (H1047R mab (18.9%). for both; ref. 40), whereas the four cell lines that displayed Constitutive pathway activation from autocrine heregu- reduced T-DM1 activity in the presence of NRG-1b do not lin production in MDA-175 cells was demonstrated by high (40). Finally, the addition of NRG-1b also inhibited T- basal levels of phospho-HER3 (p-HER3) and phospho-AKT DM1–induced apoptosis in BT-474 and ZR-75-30 cells, as (p-AKT). Both pertuzumab and N297A-pertuzumab sup- indicated by reduced caspase activity. Blockade of NRG-1b pressed p-HER3 and p-AKT to a similar degree, indicating signaling by the addition of pertuzumab fully restored the that the N297A Fc mutation did not interfere with HER2 apoptotic response to T-DM1 (Fig. 3C and Supplementary binding and subsequent inhibition of downstream signal- Fig. S3, left). Similar to the observations in proliferation ing pathways. T-DM1 treatment resulted in modest inhibi- assays, T-DM1–induced apoptosis in KPL-4 and HCC1954 tion of p-HER3 and p-AKT. Importantly, T-DM1 combined cells was not reduced by NRG-1b (Fig. 3C and Supplemen- with either wild-type or N297A-pertuzumab markedly sup- tary Fig. S3, right). These results provide additional evidence pressed p-HER3 and p-AKT (Fig. 1D). From these studies, for the rationale of blocking ligand-induced receptor acti- we conclude that antitumor activity of combination treat- vation with pertuzumab for optimal T-DM1 response in a ment in vivo is likely due to enhanced antisignaling activ- subset of HER2-amplified cancer cells. ities, not increased ADCC. T-DM1 combined with pertuzumab in HER2-amplified lung and breast cancer cells. Additional studies were per- NRG-1b inhibits activity of antimitotic agents formed in tumor cells with 3þ HER2 expression. NRG-1b The DM1 component of T-DM1 is a potent antimitotic was included in these experiments as these cells do not agent (41). Additional studies were performed to determine express endogenous ligand. In Calu-3, BT-474, and SK-BR-3 if the protective effect of NRG-1b applied to free DM1 and to cells, T-DM1 was more active than pertuzumab, whereas the antimitotic agents used for breast cancer treatment. Similar combination was more potent than single-agent treatment to the findings with T-DM1, NRG-1b reduced the cytotoxic (Fig. 2A–C, left). The average CI value for the entire drug effects of unconjugated DM1 as well as docetaxel and effect range was 0.24 0.06 for Calu-3 and 0.37 0.17 for vinorelbine in BT-474 cells (Supplementary Fig. S4, left). BT-474, indicating synergy. (CI values were not determined Similar results were obtained with SK-BR-3 (data not in SK-BR-3 cells because of minimal pertuzumab activity shown). In contrast, NRG-1b did not reduce the cytotoxic under these assay conditions). Graphical representations of effects of these agents in KPL-4 cells (Supplementary Fig. S4, CI versus fractional effect are in Supplementary Fig. S1B and right). Overall, these data show NRG-1b inhibition of the C. Calu-3 and BT-474 cell growth was stimulated by NRG- cytotoxic activity of T-DM1, docetaxel, and vinorelbine, 1b, whereas SK-BR-3 growth was unaffected under these further validating the importance of blocking NRG-1b experimental conditions. Thus, the enhancement by pertu- activity with pertuzumab.

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Effects of T-DM1, pertuzumab, or the combination on zumab treatment resulted in tumor growth delay (Fig. 5A, NRG-1b–activated HER2–HER3 signaling right). Only the highest dose of T-DM1 (7 mg/kg) resulted in NRG-1b activation of HER2–HER3 signal transduction significant tumor growth inhibition. Enhanced antitumor was investigated in SK-BR-3 and KPL-4 cells to probe the activity, compared with single-agent treatment, was observed differential effects we observed of NRG-1b on T-DM1 activ- with pertuzumab plus 3 or 7 mg/kg T-DM1. Equivalent ity. As expected, NRG-1b-stimulated HER3 and AKT phos- efficacy was observed with 7 mg/kg T-DM1 compared with phorylation was inhibited by pertuzumab, but not T-DM1, pertuzumab plus 3 mg/kg T-DM1. Pertuzumab with 7 mg/kg in SK-BR-3 cells (Fig. 4, top; ref. 42); this effect was more T-DM1 was the most active combination tested. Tumors pronounced after prolonged (24 hours) incubation. regressed during and after treatment, with no regrowth until Although treatment with T-DM1 alone for 24 hours resulted several weeks after treatment was stopped. Although more in reduced p-HER3 and p-AKT, addition of pertuzumab to sustained tumor growth inhibition was reported in Calu-3 T-DM1 was required to inhibit AKT activation in the pres- xenografts treated with pertuzumab and trastuzumab (23), ence of NRG-1b. this was likely due to weekly antibody administration for the In KPL-4 cells, NRG-1b transiently activated HER3, but study duration (>45 days). In our studies, limited dosing not AKT, (Fig. 4, bottom), likely due to mutational activa- allows for monitoring of tumor regrowth and calculation of tion of the PI3K pathway. Interestingly, T-DM1 treatment time-to-tumor volume doubling (TTP). Log-rank tests for for 24 hours suppressed both HER3 and AKT phosphory- differences in TTP showed statistically significant differences lation, with inhibition of p-AKT sustained even in the between single-agent treatment for pertuzumab or T-DM1 presence of NRG-1b. All treatment groups containing versus the corresponding combination group (log-rank P T-DM1 (T-DM1 alone, T-DM1 þ NRG-1b, T-DM1 þ pertu- value for pertuzumab vs. pertuzumab þ 3 mg/kg T-DM1 ¼ zumab, and T-DM1 þ pertuzumab þ NRG-1b) showed 0.008; log-rank P value < 0.0001 for pertuzumab vs. pertu- similar inhibition of AKT phosphorylation, indicating that zumab þ 7 mg/kg T-DM1, and for 1 or 3 mg/kg T-DM1 þ T-DM1 is sufficient to suppress AKT activation (Fig. 4, right). pertuzumab vs. T-DM1 alone; log-rank P-value ¼ 0.0004 for Because KPL-4 are insensitive to trastuzumab in vitro (42), 7 mg/kg T-DM1 vs. pertuzumab þ 7 mg/kg T-DM1; Supple- this effect is mediated by the DM1 component of T-DM1 mentary Fig. S5A). (unpublished data). Thus, in NRG-1b–sensitive cells such In KPL-4 breast tumor xenografts, T-DM1 (0.3, 1, 3 mg/ as SK-BR-3, blockade of HER2–HER3 signaling by pertu- kg) or pertuzumab (30 mg/kg loading dose; 15 mg/kg zumab is essential to achieve the full inhibitory effect of weekly) caused significant tumor growth inhibition T-DM1 under ligand-dependent conditions, whereas in compared with vehicle-treated animals (log-rank P value cells such as KPL-4, which are not stimulated downstream ¼ 0.0204 for 0.3 mg/kg T-DM1 vs. vehicle group; log-rank by NRG-1b and therefore not responsive in vitro to P value < 0.0001 for all other single-agent treatment groups pertuzumab, T-DM1 alone potently suppresses HER2– vs. vehicle control group; Fig. 5B, right; Supplementary HER3 signaling. We speculate that a contributing factor is Fig. S5B). Administration of pertuzumab with each hyperactivation of the PI3K pathway in KPL-4 cells due to an dose of T-DM1 resulted in pronounced inhibition of activating PIK3CA mutation, versus SK-BR-3 cells that are tumor growth, reaching statistical significance for each PIK3CA wild-type. The mechanisms underlying these dif- combination group compared with the corresponding sin- fering activities are currently under investigation. gle-agent treatment groups (Supplementary Fig. S5B). Sus- tained tumor regressions were observed in mice treated with T-DM1–pertuzumab combination studies in vivo 3 mg/kg T-DM1 and with the combination of pertuzumab To further support our observation that T-DM1 combined plus 1 mg/kg T-DM. After approximately 40 days, tumors in with pertuzumab leads to enhanced antitumor effects, we these two groups started to regrow. However, treatment performed in vivo combination studies in two HER2-ampli- of mice with pertuzumab plus 3 mg/kg T-DM1 resulted fied tumor xenograft models. In the Calu-3 lung cancer in lasting tumor regression for the duration of the study model, 25 mg/kg pertuzumab was administered once weekly (88 days). for 3 weeks; T-DM1 was administered (1, 3, or 7 mg/kg) as a In addition to direct antisignaling activities mediated by single intravenous injection at the start of the study. Pertu- T-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), as determined 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 doses used 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 in combination, in MDA-175 cells implanted into mammary fat pads of beige nude XID mice. Mice bearing MDA-175 tumors were treated with a single intravenous injection of 3 mg/kg pertuzumab, 10 mg/kg T-DM1, or the combination of the two agents at the same doses (n ¼ 10 per group). C, lack of ADCC activity on MDA-175 cells in vitro: cells were incubated with trastuzumab, pertuzumab, N-297A-pertuzumab, T-DM1, T-DM1 þ pertuzumab, T-DM1 þ N297A- pertuzumab, or isotype control antibody in the presence of peripheral blood mononuclear cells (PBMC), 25:1 effector:target ratio. Tumor cell lysis was assessed by lactate dehydrogenase (LDH) release. D, inhibition of HER2–HER3–PI3K signaling in MDA-175 cells (24-hour treatment with 10 mg/mL pertuzumab or N-297A-pertuzumab, 10 mg/mL T-DM1, or the respective combinations).

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Figure 3. The effect of NRG-1b on T-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 of T-DM1 activity. The right panels show a dose-dependent reduction of T-DM1 activity by NRG-1b;the fixed dose of T-DM1 selected for each cell line was the lowest dose giving maximum growth inhibition (1 mg/mL for BT-474 and ZR-75-30; 0.1 mg/mL for SK-BR-3). B, two HER2-amplified breast cancer lines, KPL-4 and HCC1954, are equally sensitive to T-DM1 in the absence or presence of NRG-1b. All treatments were for 3 days. C, the effect of NRG-1b on T-DM1–induced apoptosis, measured by caspase-3/7 activation, was assessed after a 2-day treatment. NRG-1b completely blocked T-DM1 apoptotic activity in BT-474 cells; this effect was reversed by the addition of pertuzumab. T-DM1– induced apoptosis in KPL-4 cells was unaffected by the addition of NRG-1b. pmab, pertuzumab.

contribute to combination antitumor efﬁcacy. We therefore mediated lysis in a dose-dependent manner in Calu-3 and performed in vitro ADCC assays comparing T-DM1, pertu- KPL-4 cells similar to trastuzumab (Fig. 5A and B, left). zumab, or N-297A-pertuzumab with the corresponding However, T-DM1 combined with pertuzumab or N297A- combinations. Trastuzumab was included as a positive con- pertuzumab did not result in greater ADCC activity compared trol and an isotype-matched antibody as the negative control. with T-DM1 or wild-type pertuzumab alone. As expected, Both T-DM1 and pertuzumab induced immune effector cell- N297A-pertuzumab and isotype-matched control antibody

Figure 2. Inﬂuence of NRG-1b on combination activity of T-DM1 plus pertuzumab. Enhanced antiproliferative activity with combination treatment was observed 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 pertuzumab inhibition 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 and BT-474, 3 days for SK-BR-3 and KPL-4 cells.

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Figure 4. Effects of T-DM1 and pertuzumab, alone or in combination, on HER2–HER3- mediated signal transduction in SK-BR-3 and KPL-4 breast cancer cells. T-DM1 (0.1 mg/mL) or pertuzumab (15 mg/mL) were added 1 hour before stimulation with 1 nmol/L NRG-1b for 15 minutes or 24 hours. Cell lysates were analyzed for total and p-HER3 and AKT. The presence of pertuzumab was required for suppression of signaling in NRG- 1b–activated SK-BR-3 cells.

showed no activity. These findings indicate that combination mg/kg dose, for a total of 9 patients. Patient demographics activity in vivo is likely due more to the antisignaling activities and baseline characteristics are shown in Supplementary of both T-DM1 and pertuzumab and warrant further inves- Table S1. The majority (8/9) of patients were White and had tigation, in models such as KPL-4, of the role of stromal Eastern Cooperative Oncology Group (ECOG) perfor- ligands in the response to pertuzumab in vivo. mance status of 1 (6/9). Patients had tumors that were either 3þ for HER2 expression by immunohistochemistry Phase Ib study of T-DM1 combined with pertuzumab (6/9) or positive by FISH (8/9). The median number of A global, single-arm, phase Ib/II study (TDM4373g) was previous systemic therapies was eight. All patients had conducted to investigate the safety and efficacy of T-DM1 received prior trastuzumab and lapatinib therapy, as well plus pertuzumab in patients with HER2-positive locally as a taxane and capecitabine (Supplementary Table S2). advanced or mBC who had received prior systemic therapy Most patients (7/9) had also received prior anthracycline for recurrent locally advanced or metastatic disease and in therapy. patients with newly diagnosed or previously untreated mBC During dose escalation, no patient treated with 3.0 mg/ (first-line). The primary objectives of this study included kg T-DM1 combined with pertuzumab developed a DLT. characterization of the safety and tolerability of this drug However, among the first 3 patients treated with 3.6 mg/ combination and to assess objective response rate (ORR) kg T-DM1, 1 patient developed a DLT (grade-4 throm- based on investigator assessment. In the 3þ3 study design bocytopenia), and the cohort was expanded to include 3 (Supplementary. Fig. S6), patients received the standard more patients, none of whom developed a DLT. There- pertuzumab dose every 3 weeks (840 mg, cycle 1; 420 mg, fore, 3.6 mg/kg T-DM1 in combination with standard cycle 2, and beyond) and T-DM1 at a dose of 3.0 mg/kg dose pertuzumab, administered every 3 weeks, was every 3 week (Cohort 1). In the absence of DLT, patients declared the recommended phase II schedule. Table 1 then received pertuzumab with 3.6 mg/kg T-DM1 (Cohort shows adverse events grade 3 for the 9 patients. The 2). In the event of DLTs, the T-DM1 dose was reduced to 2.4 most common grade 3 adverse effect was fatigue (3/9), mg/kg (from 3.0 mg/kg, Cohort 1) or to 3.0 mg/kg (from with thrombocytopenia and lung complications each 3.6 mg/kg, Cohort 2). Once dose escalation was completed, occurring in 1 out of 9 patients. All 9 patients experienced patients could then be added to the expansion phase (the at least one adverse event of any grade; these are sum- phase II part of this study, results to be published in a marized in Supplementary Table S3 and included gastro- separate article). Only the phase Ib results are reported here. intestinal, respiratory, musculoskeletal, and skin disor- In the phase Ib portion of the study, 3 patients received ders, as well as more general disorders such as fatigue, 3.0 mg/kg T-DM1 with the rest receiving the standard 3.6 fever, and chills.

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Figure 5. Comparison of in vitro ADCC activity (left) to in vivo antitumor efﬁcacy (right) in Calu-3 lung cancer cells (A) and KPL-4 breast cancer cells (B). ADCC assays were performed as described in Figure 1C. No enhanced ADCC activity was observed with combination treatment. Mice bearing Calu-3 subcutaneous tumors were administered 25 mg/kg pertuzumab once per week for 3 weeks; T-DM1 was administered as a single intravenous injection at doses of 1, 3, or 7 mg/kg (n ¼ 9/group). T-DM1 combined with pertuzumab was more efﬁcacious than treatment with single agents. Similarly, in vivo tumor growth of KPL-4 breast cancer cells is greatly reduced with T-DM1 and pertuzumab in combination. In this study, mice were treated weekly for 3 weeks with 15 mg/kg pertuzumab or once at treatment initiation with 0.3, 1, or 3 mg/kg T-DM1; n ¼ 8 per group.

About objective response in Cohort 1 (n ¼ 3patients trastuzumab in combination with cisplatin and a ﬂuoro- whoreceived3.0mg/kgT-DM1),2patientsshoweda pyrimidine for treatment of HER2-postive gastric/gastro- partial response and 1 patient had stable disease. In esophageal cancer was shown to extend survival in these Cohort 2 (n ¼ 6 patients receiving 3.6 mg/kg T-DM1), patients (44). Despite advances in the treatment of HER2- 2 patients had a partial response, 3 patients had stable positive cancers, a subset of patients will show disease disease, and 1 patient had disease progression as best progression through treatment. The antibody–drug conju- response. Therefore, for the full phase Ib cohort, the ORR gate T-DM1 has been tested in preclinical models and in was 44.4% (4/9; Table 2). Disease stability was main- multiple clinical trials as an additional method for targeting tainedforatleast6monthsfor2ofthe3patientswho HER2-positive cancer. T-DM1 provides intracellular deliv- had stable disease as best response. ery of the cytotoxic agent DM1, resulting in tumor cell death (16), along with the important therapeutic properties of Discussion unconjugated trastuzumab (38). Recently, T-DM1 (Kad- With the approval by the FDA in 1998, trastuzumab cyla) was approved for treatment of HER2-positive mBC became widely integrated into standard treatment regimens in patients who had received prior Herceptin and a taxane. for HER2-positive mBC (14). Less than a decade later, Effective targeting of HER2 is also achieved with pertuzu- favorable results were obtained with trastuzumab plus mab, a HER2-speciﬁc antibody that recognizes an epitope chemotherapy in early breast cancer (43). More recently, on the HER2 extracellular domain distinct from

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Table 1. National Cancer Institute (NCI) Importantly, these findings add to the emerging body of data describing autocrine or stromal-derived growth factor- Common Terminology Criteria for Adverse mediated resistance to targeted therapies. Several recent Events (CTCAE) grade 3 adverse events reports show that the c-Met receptor ligand HGF (hepato- in the phase Ib portion of TDM4373g cyte growth factor), as well as fibroblast growth factors (FGF), attenuate the anticancer activity of inhibitors target- Number of patients ing EGFR, B-Raf, and FGF receptors (7–9, 46). NRG-1b was Adverse event (n ¼ 9) also reported to rescue cells from the inhibitory effects of Fatigue 3 HER2 and EGFR kinase inhibitors (9, 47). Moreover, the Thrombocytopenia 1 NRG-1b receptor, HER3, mediates inhibitor resistance; Pneumonia 1 upregulation and activation of HER3 was demonstrated in in vitro Pleural effusion 1 diverse tumor cell types in response to EGFR, HER2, Deafnessa 1 and PI3K kinase inhibitors (11, 12, 48, 49). Our data showing NRG-1b–mediated resistance to the microtu- NOTE: Pertuzumab dose: 840 mg first cycle, then 420 mg bule-targeted cytotoxic agents T-DM1, docetaxel, and vinor- thereafter; all agents administered once every 3 weeks. elbine thus expand these observations beyond kinase inhi- a Patient had baseline grade 3 hearing loss that worsened but bitors to cytotoxic agents. did not increase in grade. Clinical activity of T-DM1 is being assessed in numerous phase II and III trials. Phase II single-arm trials of T-DM1 in patients with HER2-positive mBC who had received prior trastuzumab. Pertuzumab was approved by the FDA in trastuzumab and/or lapatinib in addition to chemotherapy 2012 on the basis of the findings of CLEOPATRA, a phase showed that, in these heavily pretreated patients, the ORR III trial that demonstrated significantly improved progres- ranged from 25% to 35% (17, 18). Given the small sample sion-free and overall survival with the addition of pertuzu- size and the specifics of the patient population in the study mab to trastuzumab plus docetaxel in previously untreated reported here, it is difficult to compare the response rates for HER2-positive mBC patients (34, 45). T-DM1 with pertuzumab with response rates for T-DM1 We therefore extended these studies by investigating the alone. An ongoing phase III, randomized, 3-arm study, efficacy of pertuzumab combined with T-DM1 in preclinical MARIANNE (TDM4788g/BO22589), will directly address models of HER2-amplified cancers and in an early-phase this question by evaluating the safety and efficacy of T-DM1 clinical trial. The combination of pertuzumab and T-DM1 with pertuzumab or T-DM1 plus pertuzumab placebo ver- resulted in a synergistic drug effect in multiple tumor cell sus trastuzumab plus taxane (docetaxel or paclitaxel) in lines in vitro and was more efficacious than treatment with patients with HER2-positive progressive or recurrent locally individual agents in tumor xenograft models. In vitro, NRG- advanced or previously untreated mBC. The phase Ib data 1b suppressed the cytotoxic effects of T-DM1 in a subset of reported here demonstrate that the combination of T-DM1 cell lines; the addition of pertuzumab was necessary to with pertuzumab is feasible at full doses with no unexpected overcome this inhibitory effect. As ADCC activity was safety signals; the majority of adverse events were grade 1 not increased in vitro compared to treatment with T-DM1 and 2 and were similar to those reported for single-agent T- or pertuzumab alone, enhanced antitumor activity is likely DM1 (fatigue, thrombocytopenia, and increased serum due to the multiple and diverse antisignaling properties of transaminases; refs. 17–19). Data from the phase II expan- pertuzumab and T-DM1, as well as the cytotoxic activity of sion portion of this study will be reported separately. the DM1 component of T-DM1. Because NRG-1b–mediat- Results from the EMILIA phase III trial showed that ed resistance was not observed in all cell lines tested, further patients treated with T-DM1 had significantly longer pro- studies are in progress to investigate the effect of additional gression-free and overall survival with a lower frequency of growth factors/cytokines on the response not only to T- severe adverse events compared with the control arm (lapa- DM1 but other chemotherapeutic agents as well. 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 0 Partial 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

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Enhanced Antitumor Activity with Trastuzumab Emtansine plus Pertuzumab

treatment of HER2-positive mBC; ref. 19). Similarly, data Authors' Contributions from a randomized phase II study, TDM4450g, demon- Conception and design: G.D. Lewis Phillips, C.T. Fields, G. Li, G. Schaefer, S.R. Olsen, M.X. Sliwkowski strated that T-DM1 significantly prolonged progression-free Development of methodology: G.D. Lewis Phillips, C.T. Fields, G. Li, D. survival with a more favorable safety profile compared to Dowbenko, M.X. Sliwkowski patients treated with trastuzumab plus docetaxel in the first- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.T. Fields, G. Li, D. Dowbenko, K. Miller, F. line setting (50). Overall, these results, together with the Andre, H.A. Burris III, K.S. Albain, N. Harbeck, V. Dieras, D. Crivellari, E. CLEOPATRA data, show significantly improved clinical Guardino, L.M. Crocker, M.X. Sliwkowski Analysis and interpretation of data (e.g., statistical analysis, biosta- benefit of T-DM1 or trastuzumab/pertuzumab/docetaxel tistics, computational analysis): C.T. Fields, G. Li, K. Miller, H.A. Burris III, compared with the trastuzumab/docetaxel regimen. From N. Harbeck, V. Dieras, L. Fang, E. Guardino, S.R. Olsen, L.M. Crocker, M.X. these data, we anticipate the MARIANNE (TDM4788g/ Sliwkowski Writing, review, and/or revision of the manuscript: G.D. Lewis Phillips, BO22589) trial to demonstrate better clinical outcome in C.T. Fields, G. Li, K. Miller, F. Andre, H.A. Burris III, K.S. Albain, N. Harbeck, the T-DM1 plus pertuzumab arm, further supporting the V. Dieras, D. Crivellari, E. Guardino, S.R. Olsen, M.X. Sliwkowski rationale for using multiple HER2-targeted agents in the Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): C.T. Fields, G. Li, S.R. Olsen, L.M. treatment of HER2-positive breast cancer. Crocker, M.X. Sliwkowski Study supervision: G.D. Lewis Phillips, G. Li, G. Schaefer, K. Miller, H.A. Burris III, E. Guardino, S.R. Olsen, M.X. Sliwkowski Disclosure of Potential Conflicts of Interest K. Miller received commercial research grant from Genentech/Roche and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Merrimack. K.S. Albain is consultant/advisory board member of Genentech advertisement and Roche. N. Harbeck and V. Dieras have honoraria and are consultant/ in accordance with 18 U.S.C. Section 1734 solely to indicate advisory board members of Roche. L. Fang has ownership interest (including this fact. patents) in stock options. S.R. Olsen and M.X. Sliwkowski have ownership interest (including patents) in Roche. No potential conflicts of interest were Received February 11, 2013; revised August 22, 2013; accepted September disclosed by the other authors. 15, 2013; published OnlineFirst October 4, 2013.

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Dual Targeting of HER2-Positive Cancer with Trastuzumab Emtansine and Pertuzumab: Critical Role for Neuregulin Blockade in Antitumor Response to Combination Therapy

Gail D. Lewis Phillips, Carter T. Fields, Guangmin Li, et al.

Clin Cancer Res 2014;20:456-468. Published OnlineFirst October 4, 2013.

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