T-DM1: a giant step forwards in HER2 therapeutics

Cancer Therapy Vol. 9, page 45 Cancer Therapy Vol. 9, 45-54, 2013 T-DM1: a giant step forwards in HER2 therapeutics Research Article Dey Nandini 1, 2, Carlson Jennifer 1, Leyland-Jones Brian 1, 2, and De Pradip 1 Edith Sanford Breast Cancer, Sanford Research/USD 60th Street N, Sioux Falls, SD, 57104 2 Department of Internal Medicine, University of South Dakota, SD *Correspondence: Pradip De, MS, PhD Associate Scientist, Edith Sanford Breast Cancer, Sanford Research/USD 60th Street N, Sioux Falls, SD, 57104 Tel: 0030-25510-74622, Fax: 605-312-6071, Email: Pradip.De@SanfordHealth.org Keywords: Breast cancer, HER2, T-DM1, trastuzumab and pertuzumab Received: 15 August 2013; Revised: 17 September 2013 Accepted: 18 September 2013; electronically published: 19 September 2013 1, 2* Summary Despite treatment advances, including the humanized anti-her2 antibody trastuzumab and the dual EGFR/HER2 tyrosine kinase small molecule inhibitor lapatinib, HER2+ breast cancer will eventually progress in most patients, highlighting the need for novel, alternative therapies. Moreover, HER2-targeted therapies are rarely given as a mono-therapy but are generally given in combination with other chemotherapy. It has been known that systemic toxicities are often associated with chemotherapies for cancer patients, therefore antibody drug conjugates with potential cytotoxic agent(s) are a promising therapeutic approach for HER2+ breast cancer patients. Trastuzumab emtansine (T-DM1) is an antibody drug conjugate that optimizes delivery of chemotherapy with an anti-her2 monoclonal antibody, trastuzumab. In HER2+ patients, T- DM1 delivers DM1 to the tumor and uses HER2 as the address. The mechanism of action does not rely on functional HER2 signaling and only requires high levels of HER2 on the cell surface. In addition, the presence of downstream alterations, such as PIK3CA mutation or the absence of PTEN, should not matter. T-DM1 has successfully passed through several clinical trials for the targeted delivery of chemotherapy and anti-her2 monoclonal antibody therapy for patients with metastatic, HER2+ breast cancer. Recently, FDA gave approval to T-DM1, which will be marketed as Kadcyla, for patients with HER2+, late stage (metastatic) breast cancer. This is a novel approach to treating HER2+ breast cancer, the antibody drug conjugate (ADC) is seen as a significant advance for patients. I. Introduction The current view of breast cancer is that it is segmented into molecular subtypes defined by genomic alterations involved in tumor progression which then identify patient populations best treated with targeted inhibition. One such population is patients with HER2 gene amplification and/or overexpression (approximately 20-30% of human breast cancers) (O'Brien, Browne et al. 2010). Before the development of HER2-targeted therapy, HER2-positivity was predictive of poor clinical outcomes (Slamon, Clark et al. 1987; Ross and Fletcher 1998). This group of proteins is comprised of EGFR (HER1), HER2, HER3 and HER4 (Yarden 2001) that are involved in cell growth and survival through activation of the PI3K-AKTmTOR and the RAS-RAF-MEK-ERK pathways (Arteaga, Sliwkowski et al. 2012). Non-targeted breast cancer treatment options may include one or more of chemotherapy, radiation, and surgery, while HER2 overexpressing breast cancers will typically involve trastuzumab-based therapy, with newer agents such as lapatinib providing a second line of treatment (Geyer, Forster et al. 2006; O'Brien, Browne et al. 2010). Single agent use of trastuzumab, a humanized anti-her2 monoclonal antibody, has shown objective responses in the range of 15 20% in phase II trials when used in firstline or refractory HER2-positive advanced breast cancer settings (Baselga, Tripathy et al. 1996; Cobleigh, Vogel et al. 1999; Vogel, Cobleigh et al. 2002). This benefit is magnified by the concomitant use of trastuzumab with traditional chemotherapy (anthracycline or epirubicin plus cyclophosphamide), leading to an improvement in objective response rates that range between 50 84% and median survivals that range between 25 37 months (Burstein, Kuter et al. 2001; Slamon, Leyland-Jones et al. 2001; Esteva, Valero et al. 2002; Montemurro, Choa et al. 2004; Andersson, Lidbrink et al. 2011; Valero, Forbes et al. 2011). Based on these and other data, the FDA and other regulatory agencies around the world approved the use of trastuzumab in the metastatic setting in 1998; 45

Pradip De et al: T-DM1: a giant step forwards in HER2 therapeutics an expansion of its use to the adjuvant setting was approved in 2006. Subsequently, two other HER2-targeted agents have been approved for the treatment of HER2-positive metastatic breast cancer, lapatinib and pertuzumab. Lapatinib is an oral small molecule dual receptor tyrosine kinase inhibitor that binds and inhibits both HER1 and HER2. Preclinical data demonstrated that lapatinib plus trastuzumab enhanced apoptosis (Konecny, Pegram et al. 2006), improved tumor control in HER2-positive xenograft (Scaltriti, Verma et al. 2009) and resulted in a marketed accumulation of inactive HER2 on the cell surface in conjunction with enhanced cytotoxicity (Scaltriti, Verma et al. 2009). It was approved in 2007 for use in combination with capecitabine in patients whose disease progressed on or after an anthracycline, taxane and trastuzumab-based therapy. Data from a phase III trial showed an improved time to progression and response rate associated with lapatinib plus capecitabine compared to capecitabine alone (Geyer, Forster et al. 2006). In 2010, lapatinib also received FDA approval in combination with an aromatase inhibitor (letrozole) for post menopausal ER+/HER2+ metastatic breast cancer patients (Johnston, Pippen et al. 2009). The response rate associated with lapatinib monotherapy was 24% in trastuzumab-sensitive patients but the response rate was less than 10% in trastuzumab-resistant settings (Burstein, Storniolo et al. 2008; Gomez, Doval et al. 2008; Blackwell, Burstein et al. 2010). Since each modality (trastuzumab and lapatinib) offers clinical benefit, combining trastuzumab plus lapatinib to target extracellular and intracellular domains of HER2 offers an attractive strategy. A phase III EGF 104900 study demonstrated a 4.5 month median overall survival advantage with the lapatinib and trastuzumab combination (Blackwell, Burstein et al. 2012). The phase III NeoALTTO study randomly assigned 455 patients (HER2+ primary breast cancer with tumors greater than 2 cm in diameter) to receive paclitaxel (T) with trastuzumab (TH), with lapatinib (TL), or with both HER2- targeting agents (THL). Pathological complete response (pcr) rate was significantly higher in the group given lapatinib plus trastuzumab plus paclitaxel (THL) (51%) than in the group given trastuzumab plus paclitaxel (TH) (29.5%) or lapatinib plus paclitaxel (TL) (24.7%) (Baselga, Bradbury et al. 2012). Similar results were observed from a randomized phase II CHER-LOB study. This is a noncomparative, randomized, phase II trial of preoperative taxane-anthracycline in combination with trastuzumab, lapatinib or combined trastuzumab plus lapatinib in patients with HER2-positive, stage II to IIIA operable breast cancer. The pcr rates were 25% for chemotherapy plus trastuzumab group, 26% for chemotherapy plus lapatinib group and 46% for chemotherapy plus trastuzumab plus lapatinib treated group (Guarneri, Frassoldati et al. 2012). In 2012, the third HER2-targeted therapy to receive regulatory approval was pertuzumab. Like trastuzumab, pertuzumab is a humanized monoclonal antibody that binds to the extra- cellular domain of HER2 but binds to a different domain than that of trastuzumab (domain II instead of domain IV) (Franklin, Carey et al. 2004; Sun Yuliang, Dey Nandini et al. 2013). In the NeoSphere study, 46% of the women who were administrated trastuzumab, pertuzumab, plus docetaxel (neoadjuvant) achieved a pcr (49 of 107 patients; 45.8% [95% CI 36.1 55.7]) 4 (Gianni, Pienkowski et al. 2012). The median progression free survival (PFS) in patients receiving trastuzumab, pertuzumab and docetaxel in the double-blind CLEOPATRA trial (808 patients with HER2-positive, locally recurrent, unresectable or metastatic breast cancer) was observed to be 18.5 months, as compared to 12.4 months in patients who received placebo, trastuzumab and docetaxel (Baselga, Bradbury et al. 2012). The results of these trials show that (1) in 54% of the patients enrolled in the NeoSphere trail, trastuzumab plus pertuzumab plus docetaxel combination fell short of achieving pcr, and (2) the median PFS in metastatic breast cancer patients receiving treatment with trastuzumab plus pertuzumab plus docetaxel in the CLEOPATRA trial was increased by not more than 6 months. Given the limited benefits of these latter treatment regimens, there is clearly a room for additional improvement. The above findings suggest that currently available HER2-targeted therapies are rarely given as mono-therapy but are generally given in combination with other chemotherapy or chemotherapies. Since toxicities associated with chemotherapy can be a substantial source of comorbidity for patients with cancer, tumor cellspecific delivery of cytotoxic agents (via antibody drug conjugate, ADC) is a promising therapeutic approach for HER2-overexpressed patient populations. It is clear that HER2 remains an active/key target, even after multiple lines of treatment. It has been demonstrated from different neoadjuvant studies including GeparQuinto (Untch, Loibl et al. 2012), NeoALTTO (Baselga, Bradbury et al. 2012), CHER-LOB (Guarneri, Frassoldati et al. 2012) and NeoSphere (Gianni, Pienkowski et al. 2012) that continued HER2 targeting therapy works even after disease progression. II. Antibody Drug Conjugates (ADC) Antibody-drug conjugates (ADCs) represent a promising therapeutic modality for the clinical management of cancer. ADCs are composed of recombinant chimeric, humanized or human antibodies covalently bound by synthetic linkers to highly cytotoxic drugs. The primary objective is to combine the pharmacological potency of small (300 to 1000 Dalton) cytotoxic drugs with the high specificity of monoclonal antibodies that target tumor-associated antigens (Teicher and Chari 2011). Inside the cell, the cytotoxic agent is released from the antibody and kills the tumor cells. ADCs are designed to minimize the systemic toxicities of free drug and to augment the anti-tumor activity of the monoclonal antibody targeting tumor. ADCs offer significant advantages over conventional chemotherapy they attach specifically to tumor cells with their target receptors while not affecting healthy cells that do not have sufficient number of those receptors. Patients would be expected to experience fewer and less severe adverse events than they would with systemic chemotherapy. The 46

Cancer Therapy Vol. 9, page 47 level of acceptable target expression may depend on the target (e.g., HER2 needs high overexpression to serve as a good ADC target (Burris, Rugo et al. 2011) while an ADC targeting CD19 can be active on cell lines bearing only 30,000 or so receptors (Blanc, Bousseau et al. 2011). However, delivery of a lethal quantity of a tubulin-acting payload into cancer cell via antibody-mediated uptake of an ADC may be difficult to achieve below ~10,000 receptors/cell (Lapusan, Vidriales et al. 2012). Hence, HER2 protein is an ideal target for ADC as breast cancers with the amplification of HER2 gene have up to 1-2 million receptors expressed per cell (Venter, Tuzi et al. 1987). The leading ADC in the clinical arena is T-DM1, which is composed of Genentech/Roche s HER2 targeting, humanized IgG1 trastuzumab conjugated with DM1 via an non-cleavable thioether link formed using the nonreducable thioether crosslinker using ImmunoGen s platform technology (Lewis Phillips, Li et al. 2008). This non-cleavable linker means the linker does not separate from the cytotoxin (maytansine) but uses a different mechanism to activate the cytotoxin inside the cancer cells. The rapid development of T-DM1 was greatly aided by the fact that trastuzumab was already marketed and tests were already in use for patient selection of likely better responders, such as those overexpressing HER2. In 2000, gemtuzumab ozogamicin (Mylotarg) became the first ADC to be approved by the U.S. Food and Drug Administration (FDA) for the treatment of acute myelogenous leukemia (AML). However, this ADC was withdrawn from the market in 2010 because, in postmarketing follow-up clinical trials, it failed to meet prospective efficacy targets that were required as a condition of its accelerated approval by the FDA. T-DM1 is not the first effective ADC, that distinction belongs to brentuximab vedotin (SGN-35, Adcetris), developed by Seattle Genetics, which was granted accelerated approval in 2011. Indicated for the treatment of Hodgkin s lymphoma and systemic anaplastic large cell lymphoma, Adcetris is a CD30 antibody linked to the cytotoxin auristatin, which blocks cell division. 2.1 T-DM1 Chemistry Preclinical studies demonstrated synergistic or additive interactions of trastuzumab with a variety of antimicrotubulin agents, including maytansines (Baselga, Norton et al. 1998; Pegram, Hsu et al. 1999; Burstein, Keshaviah et al. 2007). Because the initial studies with maytansine proved to be associated with intolerable toxicities such as nausea and peripheral neuropathy, laboratory-based translational cancer research was geared to the development of various trastuzumab-maytansinoid conjugates (Lewis Phillips, Li et al. 2008). This strategy culminated in the development of an antibody drug conjugate (ADC), trastuzumab linked to DM1 (derivative of maytansine) (Figure.1). Trastuzumab emtansine (T- DM1) is an ADC that incorporates the HER2-targeted anti-tumor properties of trastuzumab with the cytotoxic activity of the microtubule-inhibitory agent DM1; the antibody and the cytotoxic agent are conjugated by means of a stable linker (Lewis Phillips, Li et al. 2008; Junttila, Li et al. 2011). T-DM1 allows HER2-overexpressing tumor cell-specific drug delivery, thereby improving therapeutic index and minimizing exposure of normal tissue to the toxicities of the DM component. The in vitro activity of maytansine ((DM part of the T-DM1) was highly substantial 100 fold more potent than vinca alkaloids (an anti-mitotic and anti-microtubule agent) and 24 to 270 fold more potent than paclitaxel (Remillard, Rebhun et al. 1975; Cassady, Chan et al. 2004; Junttila, Li et al. 2011). It has been previously described by others that maytansine may bind to tubulin leading to a disassembly of microtubules which prevents tubulin spiralization (E H 2008). Furthermore, microtubule polymerization /depolymerization plays an important role in tumor-induced angiogenesis through the HIF1alpha- VEGF signaling axis (Mabjeesh, Escuin et al. 2003). Figure 1: Schematic illustrations of antibody drug conjugate (ADC): ADCs are unique combination of a precise and targeted monoclonal antibody, a stable linker and a potent cytotoxic agent. 2.2 Pre-clinical and clinical efficacy Extensive analysis of T-DM1 in preclinical studies has shown that T-DM1 combines the distinct mechanisms of action of both DM1 and trastuzumab, and has antitumor activity in trastuzumab- and lapatinib-refractory experimental models (Barok, Tanner et al. 2011; Junttila, Li et al. 2011; Sun Yuliang, Dey Nandini et al. 2013). This is extremely important because trastuzumab and lapatinib are established for the treatment of HER2- postitive metastatic breast cancer (MBC). The mechanisms of action for trastuzumab include inhibition of the PI3K/AKT/mTOR signaling pathway, inhibition of HER2 shedding, and Fcγ receptor mediated engagement of immune cells, which may result in antibody-dependent cellular cytotoxicity (Spector and Blackwell 2009; De, Hasmann et al. 2013). 47

Pradip De et al: T-DM1: a giant step forwards in HER2 therapeutics Of importance, T-DM1 retains these same mechanisms of action of unconjugated trastuzumab (Junttila, Li et al. 2011). The cytotoxic antitubulin agent DM1 remains attached to trastuzumab outside the cell because of the non- cleavable linker (nonreduciable thioether) molecule until the entire ADC is transported into the cytoplasm through endocytosis. It is assumed that T-DM1 then undergoes intra-lysosomal proteolytic degradation resulting in the release of nonreduciable thioether linked-dm1 and consequent antitubulinassociated cell death (Lewis Phillips, Li et al. 2008) (Figure. 2). Figure 2: Schematic representation of mechanism of action of T-DM1: T-DM1 locks onto HER2 receptor in HER2+ breast tumor cells with drug in tow. Once internalization, the antibody and receptor begins to break each other. Then antibody breaks into pieces, thereby release the drug. The cytotoxic drug begins to disrupt the cell, killing the cell and eventually augments antitumor activity. It has been reported that T-DM1 has been shown to induce apoptotic cell death in a trastuzumab-resistant xenograft tumor model (Barok, Tanner et al. 2011). In our cell based model systems, we clearly demonstrated that T- DM1 treatment associated cell death is significantly higher (by annexinv staining) in HER2+/trastuzumabsensitive, trastuzumab-resistant and HER2+/PIK3CA mutated cells when compared to trastuzumab alone treated cells (data not shown). Initially T-DM1 was evaluated as a single agent in a dose escalation phase I trial in patients with HER2+ metastatic breast cancer (MBC) who previously received a trastuzumab- containing chemotherapy. T-DM1 was administered at various doses on a weekly (23 weeks) or every 3 weeks schedule. The maximum tolerated dose (MTD) was 3.6 mg/kg every 3 weeks, based on the doselimiting toxicity (DLT) of grade 4 thrombocytopenia at 4.8 mg/kg every 3 weeks (Krop, Beeram et al. 2010). In a group of 15 patients receiving 3.6 mg/kg every 3 weeks, the median progression free survival (PFS) was 10.4 months and the clinical benefit rate (CBR; objective response rate [ORR] plus stable disease at 6 months) was 73% (Krop, Beeram et al. 2010). Most commonly reported grade 1 or 2 adverse events at MTD were thrombocytopenia (54.2%), elevated transaminases (41.7%), fatigue (37.5%), anemia (29.2%), and nausea (25%). Cardiac toxicity was not reported. This was the first phase I breast cancer trial with T-DM1 and established MTD of 3.6 mg/kg every 3 weeks. The original phase I study also evaluated a weekly schedule of T-DM1 with 1.2 mg/kg, 1.6 mg/kg, and 2 mg/kg. In this weekly schedule, partial responses were noted in four patients (57%). Grade 3 or higher adverse events were limited to rapidly reversible thrombocytopenia in one patient. No dose limiting toxicity was observed (S. N. Holden, M. Beeram et al. 2008). A proof-of-concept phase II study by Burris et al showed that single agent T-DM1 (3.6 mg/kg given every three weeks) in 112 patients with HER2-positive MBC, who had progressed with previous chemotherapy and 48

Cancer Therapy Vol. 9, page 49 HER2-directed therapy, had anti-tumor activity (Burris, Rugo et al. 2011). In this study, the overall response rate by independent review was 25.9% and 37.5% by investigator assessment, including 4 complete responses. The median PFS was 4.6 months. In the following study, Krop and colleagues gave T-DM1 (3.6 mg/kg intravenously every three weeks) to patients (n=110) with HER2-positive MBC who had prior treatment with trastuzumab, lapatinib, an antracycline, a taxane and capecitabine. The overall response rate was 34.5%, CBR was 48.2%, median PFS was 6.9 months and median duration of response was 7.2 months. In patients with confirmed HER2-positivity (n=80) by retrospective central testing, the response rate was 41.3% and median PFS was 7.3 months (Krop, LoRusso et al. 2012). Most adverse events were grades 1 or 2; the most frequent grade 3 events were thrombocytopenia (9%), fatigue (4.5%) and cellulitis (4.6%). Sara Hurvitz and group recently published (Hurvitz, Dirix et al. 2013) their phase II randomized study of T-DM1 (n= 67) versus trastuzumab plus docetaxel (n= 70) in patients with HER2-positive MBC. The objective response rate was 64% on T-DM1 arm versus 58% for patients received trastuzumab plus docetaxel. Furthermore, the progression free survival in the T-DM1 arm was 14.2 months versus 9.2 months for patients receiving standard- of-care, an improvement in favor of T-DM1 that was significant despite the relatively small number of patients. T-DM1 had a favorable safety profile, with fewer grade 3 adverse events (AEs), and AEs leading to treatment discontinuations was 7.2% (for T-DM1) versus 40.9% (tarstuzumab plus docetaxel). The above phase I and II studies led to the landmark phase III EMILIA trial, which was published recently in New England Journal of Medicine by Verma, Blackwell and the EMILIA study group (Verma, Miles et al. 2012) and subsequently published associated biomarker analysis by Baselga and group at the AACR 2013 annual meeting (Baselga J, Verma S et al. 2013). In this trial, 991 patients with advanced HER2-positive breast cancer whose disease had progressed through treatment with trastuzumab plus taxane were assigned to T-DM1 or lapatinib plus capecitabine, an FDA approved and standard treatment option in this setting. The median PFS as assessed by independent review was 9.6 months with T-DM1 versus 6.4 months with lapatinib plus capecitabine (p<0.001, HR 0.65) and median overall survival at the second interim analysis was significantly improved in the T-DM1 arm (30.9 months versus 25.1 months, HR 90.68). The objective response rate was also significantly higher with T-DM1 (43.6%) than lapatinib plus capecitabine (30.8%). Rates of grade 3 or 4 AEs were higher with lapatinib plus capecitabine than within the T- DM1 arm (57% versus 41%). The incidences of thrombocytopenia were higher in the T-DM1 arm; although the majority of these patients were able to continue treatment (2% discontinued T-DM1 due to thrombocytopenia). The overall incidence of bleeding events was higher with T-DM1 (29.8% versus 15.8%); rates of grade 3 or 4 bleeding events were low in both groups (1.4% and 0.8% respectively). In the majority of patients, left ventricular ejection fraction of 45% or more was maintained during the study treatment (in 97% of patients in the T-DM1 group and 93% of patients in the lapatinib plus capcetabine group). The incidence of diarrhea, nausea, vomiting and palmar-planter erythrodysesthesia were much higher with lapatinib plus capecitabine group. Tumor biomarkers data from the EMILIA trial also revealed that PIK3CA mutations in HER2-positive tumors did not compromise the effectiveness of T-DM1, but led to poorer responses to conventional HER2- targeted therapies such as trastuzumab and lapatinib. T-DM1 patients with mutated PIK3CA had 10.9 months progression-free survival, and 9.8 months for those with wild-type PIK3CA. However, PIK3CA status did make a difference for the capecitabinelapatinib treated patients: progression-free survival was 4.3 months for patients with mutated PIK3CA and 6.4 months for those with the wild type PIK3CA (Baselga J, Verma S et al. 2013). The two major lessons we learned from the above mentioned trials with T-DM1 are: a) there is a relative lack of side-effects following the treatment with T-DM1 and b) T-DM1 obviates the need for chemotherapy. Trastuzumab-induced cardio-toxicity (symptomatic or asymptomatic) is a major concern. Although, the cardio-toxicity varies according to the definition used in different studies, it has been reported to be as high as 30% when associated with anthracyclines (Telli, Hunt et al. 2007; Guglin, Hartlage et al. 2009). T-DM1 had a better safety profile compared to trastuzumab in this context and no significant doselimiting cardio-toxicity was observed with T-DM1 in heavily pre-treated patients. T-DM1 has exhibited a unique capacity to discriminate between cancer cells and normal cells in terms of cytotoxic drug delivery. EMILIA study also showed that patients receiving T-DM1 whose tumors had above-median levels of HER2 had a median overall survival of 34.1 months, vs. 26.5 months for patients with lower levels. Progression-free survival with T-DM1 was 10.6 months for patients with higher HER2 levels vs. 8.2 months for lower levels (Baselga J, Verma S et al. 2013). Data suggest that the greater the overexpression of HER2 in a breast tumor, the more sensitive the tumor is to anti-her2 therapy. Overall these results take targeted therapy for HER2-positive breast cancer to a newer level. MARIANNE, is a phase III randomized study of T-DM1 with or without pertuzumab (T- DM1 and pertuzumab bind to different epitopes of HER2) compared with trastuzuamb plus taxane for the first line treatment of HER2-positive, progressive or recurrent locally advanced or metastatic breast cancer patients. With 1,092 enrolled patients, MARIANNE will compare the efficacy of T- DM1 plus pertuzumab, T-DM1 plus placebo, and the combination of trastuzumab plus a taxane. The independent Data Monitoring Committee which assesses safety data has recently recommended continuation of the study without any modification. Brisk completion of 49

Pradip De et al: T-DM1: a giant step forwards in HER2 therapeutics accrual and data analyses are eagerly awaited (Ellis EA, CH et al. 2011). Roche/Genentech also is actively recruiting metastatic HER2+ breast cancer patients into the TH3RESA trial (NCT01419197) at 210 sites, including 90 within the US. Women entering this trial will be randomized at a 2 to 1 ratio to receive T-DM1 (3.6 mg/kg intravenously for 3 weeks) or treatment of the physician's choice. Anticipated time on study treatment is until disease progression or unacceptable toxicity occurs. A randomized, multicenter, open-label phase III KATHERINE study (NCT01772472) of T-DM1 vs. trastuzumab as adjuvant therapy for patients with HER2+ primary breast cancer who have residual tumor present pathologically in the breast or axillary lymph nodes following preoperative therapy, which was initiated in April 2013. Other ongoing phase II trials are currently exploring the feasibility and/or antitumor potency of T- DM1 in the adjuvant (NCT01196052) and neoadjuvant setting (NCT01745965) of HER2-positive BC (see Table 1). Table 1: Ongoing trials with T-DM1 in HER2 + breast cancer MBC: Metastatic breast cancer HT: Hormone treatment AEs: Adverse events DLTs: Dose limiting toxicities PFS: Progression free survival pcr: Pathological complete response IDFS: Invasive disease free survival 2.3 Pharmacokinetic and metabolic profile of T-DM1 The pharmacokinetics of T-DM1 has been assessed in preclinical and clinical studies. The results showed that T-DM1 exhibits dose-proportional pharmacokinetics in non trastuzumab-binding species (i.e., mice and rats) (LoRusso, Weiss et al. 2011) and a dose- dependent decrease in clearance associated with increasing dose in trastuzumab binding species (i.e., cynomolgus monkeys and humans) (Girish S, Gupta M et al. 2011). Results from a preclinical absorption, distribution, metabolism and excretion study of T-DM1 in rats suggest that T-DM1 nonspecifically distributes to tissues without accumulation. The major elimination routes of DM1- containing metabolites are through fecal/biliary (~80%) and urine (<10%) (Shen, Bumbaca et al. 2012). The clinical PK of T-DM1 shows that while the conjugate is quite stable in circulation, it nevertheless appears to show a slow rate of maytansiniod loss over time (Krop, Beeram et al. 2010; Burris, Rugo et al. 2011) and very low levels of free DM1 were reported to be present in plasma samples from patients treated with T- DM1 (Burris, Rugo et al. 2011). The median half-life of T-DM1 is 4.5 days and steady state is achieved in cycle 2 (Gupta, Lorusso et al. 2012). Pharmacokinetics-based drug interactions between T-DM1 and the HER2-targeted monoclonal antibody pertuzumab, or T-DM1 and paclitaxel showed that the combination had no effect on the pharmacokinetics of the individual agents and had a low risk for drug interactions (Lu D, Krop I et al. 2010; Lu, Burris et al. 2012). 50

Cancer Therapy Vol. 9, page 51 2.4 T-DM1 and resistance mechanism The design of cancer therapy has become increasingly sophisticated, yet there is no cancer treatment that is 100% effective against disseminated cancer. Resistance to treatment with anticancer drugs results from a variety of factors including individual variations in patients, somatic cell genetic differences in tumors, and signaling pathway alteration/upregulation. Frequently, resistance is intrinsic to the cancer but as therapy becomes more and more effective, acquired resistance has also become common. Trastuzumab-containing therapy is now an established standard of care across all HER2-positive breast cancer disease stages. However, despite the robust clinical efficacy of trastuzumab in HER2-positive MBC, primary and secondary resistance remains a clinical challenge. Alterations of signal transducers lying downstream of HER2, which facilitate signaling independently of the HER2 kinase, have been extensively studied as potential mechanisms of trastuzumab resistance. T-DM1 is a relatively new drug in the horizon and the EMILIA trial revealed that PI3K pathway upregulation (via activating mutation of PIK3CA) did not hinder T-DM1 s efficacy. Pegram and group recently reported that despite inactivation of the PI3K-AKT signaling pathway, HER2 overexpressed lapatinibresistant cells exhibited activation of mtorc1 and its downstream signaling pathway (Jegg, Ward et al. 2012). Recently, Elkabets and group also reported that breast cancer cells (containing PIK3CA mutation) that were resistant to p110α- isoform-specific inhibitor (BYL719) had persistently active ps6 expression, although AKT phosphorylation was inhibited (Elkabets, Vora et al. 2013). Along the same line, our cell-based preclinical data showed that T-DM1 blocked AKT activation in HER2- positive breast cancer cells (BT474) but failed to block downstream of mtorc1 signaling molecule (phosphorylation of P70S6K) (Sun, Dey et al. 2013). This data suggest that T-DM1 alone may not be sufficient to completely inactivate the PI3K-AKT-mTOR signaling pathway. Literature references suggest that activation of the PI3K-AKT-mTOR pathway is one of the major causes for drug resistance including chemotherapy and trastuzumab therapy in different cancer models (LoPiccolo, Blumenthal et al. 2008; De, Hasmann et al. 2013). These findings suggest that simultaneous administration of mtorc1 inhibitors may enhance the clinical activity of T-DM1 and delay the appearance of resistance. III. Conclusion T-DM1 is a novel antibody-drug conjugate with a mechanism of action that differs from that of classical anti-her2 therapies. T-DM1 allows intracellular chemotherapy drug delivery specifically to HER2 enriching cells, thereby improving the therapeutic index and minimizing toxicity exposure to normal tissue. T- DM1 (trade name Kadcyla ) is one of the most successful ADCs representing a completely new way to treat HER2-positive metastatic breast cancer and it has helped participants in the EMILA study live nearly 6 months longer. Fewer people who received Kadcyla experienced severe side effects compared to those receiving standard therapy (40.8% versus 57%). T-DM1 has recently been approved by the FDA. Acknowledgement: Authors thank Dr. Brian Smith for his thoughtful comments towards the improvement of the MS. Authors also acknowledged Edith Sanford Breast Cancer Research in Sanford Research/USD. Conflict of Interest: None References: Andersson, M., E. Lidbrink, et al. (2011). "Phase III randomized study comparing docetaxel plustrastuzumab with vinorelbine plus trastuzumab as first-line therapy of metastatic or13locally advanced human epidermal growth factor receptor 2-positive breast cancer: the HERNATA study." J Clin Oncol 29(3): 264-271. Arteaga, C. L., M. X. Sliwkowski, et al. (2012). "Treatment of HER2-positive breast cancer: current status and future perspectives." Nat Rev Clin Oncol 9(1): 16-32. Barok, M., M. Tanner, et al. (2011). "Trastuzumab- DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo." Breast Cancer Res 13(2): R46. Barok, M., M. Tanner, et al. (2011). "Trastuzumab- DM1 is highly effective in preclinical models of HER2-positive gastric cancer." Cancer Lett 306(2): 171-179. Baselga J, Verma S, et al. (2013). Relationship between tumor biomarkers and efficacy in EMILIA, a phase III study of trastuzumab emtansine (T- DM1) in HER2-positive metastatic breast cancer. AACR Annual Meeting, Washington DC. Baselga, J., I. Bradbury, et al. (2012). "Lapatinib with trastuzumab for HER2-positive early breast cancer (NeoALTTO): a randomised, open-label, multicentre, phase 3 trial." Lancet 379(9816): 633-640. Baselga, J., L. Norton, et al. (1998). "Recombinant humanized anti-her2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing 51

Pradip De et al: T-DM1: a giant step forwards in HER2 therapeutics human breast cancer xenografts." Cancer Res 58(13): 2825-2831. Baselga, J., D. Tripathy, et al. (1996). "Phase II study of weekly intravenous recombinant humanized anti-p185her2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer." J Clin Oncol 14(3): 737-744. Blackwell, K. L., H. J. Burstein, et al. (2010). "Randomized study of Lapatinib alone or in combination with trastuzumab in women with ErbB2-positive, trastuzumab-refractory metastatic breast cancer." J Clin Oncol 28(7): 1124-1130. Blackwell, K. L., H. J. Burstein, et al. (2012). "Overall survival benefit with lapatinib in combination with trastuzumab for patients with human epidermal growth factor receptor 2-positive metastatic breast cancer: final results from the EGF104900 Study." J Clin Oncol 30(21): 2585-2592. Blanc, V., A. Bousseau, et al. (2011). "SAR3419: an anti-cd19-maytansinoid Immunoconjugate for the treatment of B-cell malignancies." Clin Cancer Res 17(20): 6448-6458. Burris, H. A., 3rd, H. S. Rugo, et al. (2011). "Phase II study of the antibody drug conjugate trastuzumab- DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy." J Clin Oncol 29(4): 398-405. Burstein, H. J., A. Keshaviah, et al. (2007). "Trastuzumab plus vinorelbine or taxane chemotherapy for HER2-overexpressing metastatic breast cancer: the trastuzumab and vinorelbine or taxane study." Cancer 110(5): 965-972. Burstein, H. J., I. Kuter, et al. (2001). "Clinical activity of trastuzumab and vinorelbine in women with HER2-overexpressing metastatic breast cancer." J Clin Oncol 19(10): 2722-2730. Burstein, H. J., A. M. Storniolo, et al. (2008). "A phase II study of lapatinib monotherapy in chemotherapy-refractory HER2-positive and HER2-negative advanced or metastatic breast cancer." Ann Oncol 19(6): 1068-1074. Cassady, J. M., K. K. Chan, et al. (2004). "Recent developments in the maytansinoid antitumor agents." Chem Pharm Bull (Tokyo) 52(1): 1-26. Cobleigh, M. A., C. L. Vogel, et al. (1999). "Multinational study of the efficacy and safety of humanized anti-her2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease." J Clin Oncol 17(9): 2639-2648. De, P., M. Hasmann, et al. (2013). "Molecular determinants of trastuzumab efficacy: What is their clinical relevance?" Cancer Treat Rev. E, H. (2008). An overview of compounds that interact with tubulin and their effects on microtubule assembly. Cancer Drug Discovery and Development: The role of microtubules in cell biology, neurobiology and oncology., Humana press, Totowa, NJ. Elkabets, M., S. Vora, et al. (2013). "mtorc1 Inhibition Is Required for Sensitivity to PI3K p110alpha Inhibitors in PIK3CA-Mutant Breast Cancer." Sci Transl Med 5(196): 196ra199. Ellis EA, B. CH, et al. (2011). MARIANNE: A phase III, randomized study of trastuzumab-dm1 (T- DM1) with or without pertuzumab (P) compared with trastuzumab (H) plus taxane for first-line treatment of HER2-positive, progressive, or recurrent locally advanced or metastatic breast cancer (MBC). ASCO Annual Meeting Chicago, IL. Esteva, F. J., V. Valero, et al. (2002). "Phase II study of weekly docetaxel and trastuzumab for patients with HER-2-overexpressing metastatic breast cancer." J Clin Oncol 20(7): 1800-1808. Franklin, M. C., K. D. Carey, et al. (2004). "Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex." Cancer Cell 5(4): 317-328. Geyer, C. E., J. Forster, et al. (2006). "Lapatinib plus capecitabine for HER2-positive advanced breast cancer." N Engl J Med 355(26): 2733-2743. Gianni, L., T. Pienkowski, et al. (2012). "Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multicentre, open-label, phase 2 trial." Lancet Oncol 13(1): 25-32. Girish S, Gupta M, et al. (2011). Clinical pharmacology of trastuzumab emtansine (T- DM1): a unique antibodydrug conjugate in development for the treatment of HER2-positive cancer Clin Pharmacol Ther Gomez, H. L., D. C. Doval, et al. (2008). "Efficacy and safety of lapatinib as first-line therapy for ErbB2- amplified locally advanced or metastatic breast cancer." J Clin Oncol 26(18): 2999-3005. Guarneri, V., A. Frassoldati, et al. (2012). "Preoperative chemotherapy plus trastuzumab, lapatinib, or both in human epidermal growth factor receptor 2-positive operable breast cancer: results of the randomized phase II CHER-LOB study." J Clin Oncol 30(16): 1989-1995. Guglin, M., G. Hartlage, et al. (2009). "Trastuzumabinduced cardiomyopathy: not as benign as it looks? A retrospective study." J Card Fail 15(8): 651-657. Gupta, M., P. M. Lorusso, et al. (2012). "Clinical implications of pathophysiological and demographic covariates on the population pharmacokinetics of trastuzumab emtansine, a HER2-targeted antibody-drug conjugate, in patients with HER2-positive metastatic breast cancer." J Clin Pharmacol 52(5): 691-703. Hurvitz, S. A., L. Dirix, et al. (2013). "Phase II randomized study of trastuzumab emtansine versus trastuzumab plus docetaxel in patients with 52

Cancer Therapy Vol. 9, page 53 human epidermal growth factor receptor 2- positive metastatic breast cancer." J Clin Oncol 31(9): 1157-1163. Jegg, A. M., T. M. Ward, et al. (2012). "PI3K independent activation of mtorc1 as a target in lapatinib-resistant ERBB2+ breast cancer cells." Breast Cancer Res Treat 136(3): 683-692. Johnston, S., J. Pippen, Jr., et al. (2009). "Lapatinib combined with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hormone receptor-positive metastatic breast cancer." J Clin Oncol 27(33): 5538-5546. Junttila, T. T., G. Li, et al. (2011). "Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer." Breast Cancer Res Treat 128(2): 347-356. Konecny, G. E., M. D. Pegram, et al. (2006). "Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumabtreated breast cancer cells." Cancer Res 66(3): 1630-1639. Krop, I. E., M. Beeram, et al. (2010). "Phase I study of trastuzumab-dm1, an HER2 antibody- drug conjugate, given every 3 weeks to patients with HER2-positive metastatic breast cancer." J Clin Oncol 28(16): 2698-2704. Krop, I. E., P. LoRusso, et al. (2012). "A phase II study of trastuzumab emtansine in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer who were previously treated with trastuzumab, lapatinib, an anthracycline, a taxane, and capecitabine." J Clin Oncol 30(26): 3234-3241. Lapusan, S., M. B. Vidriales, et al. (2012). "Phase I studies of AVE9633, an anti-cd33 antibodymaytansinoid conjugate, in adult patients with relapsed/refractory acute myeloid leukemia." Invest New Drugs 30(3): 1121-1131. Lewis Phillips, G. D., G. Li, et al. (2008). "Targeting HER2-positive breast cancer with trastuzumab- DM1, an antibody-cytotoxic drug conjugate." Cancer Res 68(22): 9280-9290. LoPiccolo, J., G. M. Blumenthal, et al. (2008). "Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations." Drug Resist Updat 11(1-2): 32-50. LoRusso, P. M., D. Weiss, et al. (2011). "Trastuzumab emtansine: a unique antibody-drug conjugate in development for human epidermal growth factor receptor 2-positive cancer." Clin Cancer Res 17(20): 6437-6447. Lu D, Krop I, et al. (2010). Pharmacokinetics (PK) of trastuzumab-dm1 (T-DM1) and paclitaxel (T) in patients with HER2-positive locally advanced or metastatic breast cancer (MBC) previously treated with a trastuzumab-containing regimen.. SABCS San Antonio. Lu, D., H. A. Burris, 3rd, et al. (2012). "Drug interaction potential of trastuzumab emtansine (T- DM1) combined with pertuzumab in patients with HER2-positive metastatic breast cancer." Curr Drug Metab 13(7): 911-922. Mabjeesh, N. J., D. Escuin, et al. (2003). "2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF." Cancer Cell 3(4): 363-375. Montemurro, F., G. Choa, et al. (2004). "A phase II study of three-weekly docetaxel and weekly trastuzumab in HER2-overexpressing advanced breast cancer." Oncology 66(1): 38-45. O'Brien, N. A., B. C. Browne, et al. (2010). "Activated phosphoinositide 3-kinase/AKT signaling confers resistance to trastuzumab but not lapatinib." Mol Cancer Ther 9(6): 1489-1502. Pegram, M., S. Hsu, et al. (1999). "Inhibitory effects of combinations of HER-2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers." Oncogene 18(13): 2241-2251. Remillard, S., L. I. Rebhun, et al. (1975). "Antimitotic activity of the potent tumor inhibitor maytansine." Science 189(4207): 1002-1005. Ross, J. S. and J. A. Fletcher (1998). "The HER-2/neu Oncogene in Breast Cancer: Prognostic Factor, Predictive Factor, and Target for Therapy." Oncologist 3(4): 237-252. S. N. Holden, M. Beeram, et al. (2008). A phase I study of weekly dosing of trastuzumab-dm1 (T-DM1) in patients (pts) with advanced HER2+ breast cancer (BC). ASCO, Journal of Clinical Oncology. Scaltriti, M., C. Verma, et al. (2009). "Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity." Oncogene 28(6): 803-814. Shen, B. Q., D. Bumbaca, et al. (2012). "Catabolic fate and pharmacokinetic characterization of trastuzumab emtansine (T-DM1): an emphasis on preclinical and clinical catabolism." Curr Drug Metab 13(7): 901-910. Slamon, D. J., G. M. Clark, et al. (1987). "Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene." Science 235(4785): 177-182. Slamon, D. J., B. Leyland-Jones, et al. (2001). "Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2." N Engl J Med 344(11): 783-792. Spector, N. L. and K. L. Blackwell (2009). "Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2-positive breast cancer." J Clin Oncol 27(34): 5838-5847. 53

Pradip De et al: T-DM1: a giant step forwards in HER2 therapeutics Sun, Y., N. Dey, et al. (2013). "Bevacizumab confers additional advantage to the combination of trastuzumab plus pertuzumab in trastuzumabrefractory breast cancer model." Cancer Chemother Pharmacol. Sun Yuliang, Dey Nandini, et al. (2013). "bevacizumab confers additional advantage to the combination of trastuzumab plus pertuzumab in trastuzumab-refractory breast cancer model." cancer chemotherapy and pharmacology Teicher, B. A. and R. V. Chari (2011). "Antibody conjugate therapeutics: challenges and potential." Clin Cancer Res 17(20): 6389-6397. Telli, M. L., S. A. Hunt, et al. (2007). "Trastuzumabrelated cardiotoxicity: calling into question the concept of reversibility." J Clin Oncol 25(23): 3525-3533. Untch, M., S. Loibl, et al. (2012). "Lapatinib versus trastuzumab in combination with neoadjuvant anthracycline-taxane-based chemotherapy (GeparQuinto, GBG 44): a randomised phase 3 trial." Lancet Oncol 13(2): 135-144. Valero, V., J. Forbes, et al. (2011). "Multicenter phase III randomized trial comparing docetaxel and trastuzumab with docetaxel, carboplatin, and trastuzumab as first-line chemotherapy for patients with HER2-gene-amplified metastatic breast cancer (BCIRG 007 study): two highly active therapeutic regimens." J Clin Oncol 29(2): 149-156. Venter, D. J., N. L. Tuzi, et al. (1987). "Overexpression of the c-erbb-2 oncoprotein in human breast carcinomas: immunohistological assessment correlates with gene amplification." Lancet 2(8550): 69-72. Verma, S., D. Miles, et al. (2012). "Trastuzumab emtansine for HER2-positive advanced breast cancer." N Engl J Med 367(19): 1783-1791.17 Vogel, C. L., M. A. Cobleigh, et al. (2002). "Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer." J Clin Oncol 20(3): 719-726. Yarden, Y. (2001). "The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities." Eur J Cancer 37 Suppl 4: S3-8. 54