Barok et al. Breast Cancer Research 2014, 16:209
`http://breast-cancer-research.com/content/16/2/209
`
`R E V I E W
`Trastuzumab emtansine: mechanisms of action
`and drug resistance
`Mark Barok1*, Heikki Joensuu2 and Jorma Isola3
`
`Abstract
`
`Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that is effective and generally well tolerated when
`administered as a single agent to treat advanced breast cancer. Efficacy has now been demonstrated in randomized
`trials as first line, second line, and later than the second line treatment of advanced breast cancer. T-DM1 is
`currently being evaluated as adjuvant treatment for early breast cancer. It has several mechanisms of action consisting
`of the anti-tumor effects of trastuzumab and those of DM1, a cytotoxic anti-microtubule agent released within the
`target cells upon degradation of the human epidermal growth factor receptor-2 (HER2)-T-DM1 complex in
`lysosomes. The cytotoxic effect of T-DM1 likely varies depending on the intracellular concentration of DM1
`accumulated in cancer cells, high intracellular levels resulting in rapid apoptosis, somewhat lower levels in
`impaired cellular trafficking and mitotic catastrophe, while the lowest levels lead to poor response to T-DM1.
`Primary resistance of HER2-positive metastatic breast cancer to T-DM1 appears to be relatively infrequent, but most
`patients treated with T-DM1 develop acquired drug resistance. The mechanisms of resistance are incompletely
`understood, but mechanisms limiting the binding of trastuzumab to cancer cells may be involved. The cytotoxic
`effect of T-DM1 may be impaired by inefficient internalization or enhanced recycling of the HER2-T-DM1 complex
`in cancer cells, or impaired lysosomal degradation of trastuzumab or intracellular trafficking of HER2. The effect of
`T-DM1 may also be compromised by multidrug resistance proteins that pump DM1 out of cancer cells. In this
`review we discuss the mechanism of action of T-DM1 and the key clinical results obtained with it, the combinations of
`T-DM1 with other cytotoxic agents and anti-HER drugs, and the potential resistance mechanisms and the strategies to
`overcome resistance to T-DM1.
`
`Introduction
`Overexpression and amplification of human epidermal
`growth factor receptor-2 (HER2, ErbB2) is present in 15
`to 20% of primary human breast cancers [1]. In the past,
`patients with HER2-positive breast cancer generally had
`unfavorable outcome [2], but this changed radically after
`discovery of
`trastuzumab, a recombinant humanized
`monoclonal antibody that binds to the extracellular sub-
`domain IV of HER2. Trastuzumab showed substantial
`anti-tumor efficacy in both preclinical and clinical trials
`[3,4], and introduction of trastuzumab for the treatment
`of HER2-positive breast cancer can be considered a mile-
`stone in medical oncology [4,5]. However, resistance to
`trastuzumab eventually emerges in the great majority of
`patients treated [6].
`
`* Correspondence: barok.mark@gmail.com
`1Laboratory of Molecular Oncology, University of Helsinki, Biomedicum,
`Haartmaninkatu 8, Helsinki FIN-00290, Finland
`Full list of author information is available at the end of the article
`
`Several other HER2-targeted agents have been evaluated
`in clinical trials since the introduction of trastuzumab in
`1998. Lapatinib, an orally administered small molecule
`inhibitor of the HER1 and HER2 tyrosine kinases, was
`found to be superior in combination with capecitabine
`compared with capecitabine alone in the treatment of
`metastatic breast cancer (MBC) that had progressed after
`trastuzumab-based therapy [7]. As to trastuzumab, resis-
`tance to lapatinib develops frequently among patients who
`initially respond [8]. Recently, pertuzumab, a recombinant
`humanized monoclonal antibody that binds to subdomain
`II of the extracellular portion of HER2 and inhibits recep-
`tor dimerization, was found to be more effective in com-
`bination with trastuzumab and docetaxel compared with
`placebo, trastuzumab and docetaxel as first-line treatment
`of HER2-positive MBC [9].
`Despite these new therapeutic options, HER2-positive
`MBC still remains an incurable disease. In this review we
`discuss
`the mechanisms of action of
`trastuzumab
`
`© 2014 Barok et al.; licensee BioMed Central Ltd. The licensee has exclusive rights to distribute this article, in any medium, for
`6 months following its publication. After this time, the article is available under the terms of the Creative Commons
`Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
`reproduction in any medium, provided the original work is properly cited.
`
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`emtansine (T-DM1), a novel agent that has challenged in
`efficacy and safety all existing systemic therapies for
`HER2-positive MBC, and the resistance mechanisms to it.
`T-DM1 is an excellent example of a principle suggested
`already in the 1970s to use antibodies as carriers of drugs
`to highly specific targets [10].
`
`Trastuzumab emtansine, a HER2-targeted
`antibody-drug conjugate
`Antibody-drug conjugates (ADCs) are a means to deliver
`cytotoxic drugs specifically to cancer cells. The delivery is
`followed by internalization of the ADC and release of free,
`highly active cytotoxic agents within cancer cells, leading
`
`eventually to cell death. The components of an effective
`ADC typically consist of: (i) a humanized or human
`monoclonal antibody that
`selectively and specifically
`delivers a cytotoxic agent to cancer cells by evoking
`receptor-mediated endocytosis; (ii) a cytotoxic agent that
`will kill the cell; and (iii) a linker that binds the cytotoxic
`agent to the antibody.
`The first ADC targeting the HER2 receptor is T-DM1
`(ado-trastuzumab emtansine; T-MCC-DM1; Kadcyla®),
`which is a conjugate of trastuzumab and a cytotoxic
`moiety (DM1, derivative of maytansine). T-DM1 carries
`an average of 3.5 DM1 molecules per one molecule of
`trastuzumab. Each DM1 molecule is conjugated to
`
`Figure 1 Intracellular trafficking of trastuzumab emtansine (T-DM1). Binding of T-DM1 onto human epidermal growth factor receptor-2
`(HER2) on the plasma membrane is followed by entry of the HER2-T-DM1 complex into the cell via receptor-mediated endocytosis. Internalized
`endocytic vesicles form early endosomes. The load of early endosomes can be recycled back to the cell membrane or the early endosome can
`mature to a lysosome. Release of DM1 occurs as a result of proteolytic degradation of the antibody part of T-DM1 in the lysosomes. Intracellular
`lysine (lys)-MCC-DM1 inhibits microtubule assembly, causing mitotic arrest, apoptosis, mitotic catastrophe, and disrupted intracellular trafficking.
`MCC, non-reducible thioether linker.
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`trastuzumab via a non-reducible thioether linker (N-succi-
`nimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate;
`SMCC, MCC after conjugation) [11].
`
`Mechanisms of action of T-DM1
`Binding of T-DM1 to HER2 triggers entry of the HER2-
`T-DM1 complex into the cell via receptor-mediated
`endocytosis [12,13]. Since the non-reducible linker is
`stable in both the circulation and the tumor microenvi-
`ronment, active DM1 release occurs only as a result of
`proteolytic degradation of the antibody part of T-DM1
`in the lysosome [11,14]. Following release from the lyso-
`some, DM1-containing metabolites inhibit microtubule
`assembly, eventually causing cell death [15] (Figure 1).
`Linkage of DM1 to trastuzumab does not affect the
`binding affinity of trastuzumab to HER2 [16,17], nor does
`it reduce the inherent anti-tumor effects of trastuzumab
`[16,18]. Consequently, T-DM1 has mechanisms of action
`consisting of the anti-tumor effects related to trastuzumab
`and those associated with intracellular DM1 metabolites
`(Table 1).
`
`Trastuzumab-mediated effects
`Both trastuzumab and T-DM1 inhibit HER2 receptor
`signaling, mediate antibody-dependent cell-mediated
`cytotoxicity, and inhibit shedding of the extracellular
`domain of HER2 [16,18]. Although the anti-tumor
`effects of DM1 are more pronounced than those of tras-
`tuzumab [16], trastuzumab-mediated effects should not
`
`be underestimated and might be particularly important
`when the target cells do not undergo rapid apoptotic
`death caused by DM1. This may be common in the
`clinic, where trastuzumab therapy of MBC often lasts
`for several months or years, and continuation of trastu-
`zumab therapy beyond breast cancer progression on
`trastuzumab-containing systemic therapy may still be
`beneficial [32,33].
`
`DM1-mediated effects
`At least four molecular mechanisms have been suggested
`for DM1 anti-tumor activity. First, active DM1 metabo-
`lites disrupt the microtubule networks of the target cells,
`which causes cell cycle arrest at the G2-M phase and
`apoptotic cell death [11,18]. Second, prolonged treatment
`of breast cancer xenografts with T-DM1 caused both
`apoptosis and mitotic catastrophe, the latter being identi-
`fied as presence of cells with aberrant mitotic figures and
`a giant multinucleated structure (Figure 2) [18]. Third,
`disruption of microtubule network-mediated intracellular
`trafficking may occur. Microtubule targeting agents often
`disrupt intracellular trafficking via microtubules [34,35],
`and prolonged treatment with T-DM1, but not with tras-
`tuzumab, caused defective intracellular
`trafficking of
`HER2 in a preclinical breast cancer model [18]. Impaired
`intracellular trafficking may be an important mechanism
`of action of T-DM1, particularly in non-dividing cells.
`Finally, as we discuss below, free intracellular DM1 may
`lead to cell death in a concentration-dependent manner.
`
`Table 1 Mechanisms of action of trastuzumab and trastuzumab emtansine
`Mechanism of action
`
`Mechanism causing trastuzumab resistance
`
`Trastuzumab
`
`Fab-mediated
`
`Down-regulation of HER2 on the plasma membrane [19]
`
`Masking of trastuzumab binding epitope of HER2 [20,21]
`
`Inhibition of HER2 ectodomain shedding [22]
`
`Expression of p95HER2 [23]
`
`HLA-I-restricted antigen presentation of HER2 [24]
`
`Activation of the IGF-IR pathway [25]
`
`Inactivation of the PTEN-PI3K/AKT pathway [26]
`
`Defects in the PTEN-PI3K/AKT pathway [26]
`
`Induction of apoptosis [19]
`
`Inhibition of angiogenesis [28]
`
`Overexpression of cyclin E [27]
`
`Autocrine production of EGF-related ligands [29]
`
`Fc-mediated
`
`ADCC [30]
`
`Impaired ADCC [31]
`
`T-DM1
`
`Trastuzumab part
`
`Fab-mediated
`
`Inhibition of HER2 ectodomain shedding [16]
`
`Inhibition of PI3K/AKT signaling pathway [16]
`
`Fc-mediated
`
`ADCC [16,18]
`
`DM1 part
`
`Mitotic arrest [11]
`
`Apoptosis [11,17,18]
`
`Mitotic catastrophe [18]
`
`Disruption of intracellular trafficking [18]
`
`ADCC, antibody-dependent cell-mediated cytotoxicity; AKT, protein kinase B; EGF, epidermal growth factor; HER2, human epidermal growth factor receptor-2; HLA,
`human leukocyte antigen; IGF-IR, insulin-like growth factor-I receptor; PI3K, phosphatidylinositol 3′-kinase; PTEN, phosphatase and tensin homolog; T-DM1,
`trastuzumab emtansine.
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`Figure 2 Histological findings in a human epidermal growth factor receptor-2-positive, trastuzumab and lapatinib-resistant breast can-
`cer (JIMT-1) xenograft following trastuzumab emtansine treatment. Numerous apoptotic cells are present (stained brown with CytoDeath
`staining). Hematoxylin counterstain reveals multinucleated giant cells and pathological mitoses (arrows), which are hallmarks of mitotic
`catastrophe. Mitotic catastrophes were absent in trastuzumab-treated tumors.
`
`Activity of T-DM1 in preclinical models and
`clinical trials
`A comprehensive review of the efficacy and safety results
`obtained with T-DM1 is beyond the scope of the current
`review but, in brief, T-DM1 has shown substantial anti-
`tumor efficacy in preclinical studies and clinical trials.
`T-DM1 has superior activity compared with trastuzumab
`on trastuzumab-sensitive breast cancer cell cultures and
`tumor xenografts (Additional file 1) [11,18]. Importantly,
`T-DM1 is effective in in vitro and in vivo models of
`trastuzumab-resistant breast cancer, and in trastuzumab
`and lapatinib cross-resistant breast
`cancer models
`(Additional file 2) [11,18].
`A key clinical trial to investigate the efficacy and safety
`of T-DM1 in the treatment of breast cancer was the
`EMILIA study, where 991 patients previously treated for
`locally advanced or metastatic breast cancer with trastuzu-
`mab and a taxane were randomly assigned to receive
`either single-agent T-DM1 3.6 mg per kilogram of body
`weight intravenously 3-weekly or lapatinib plus capecita-
`bine. The median progression-free survival (PFS) was
`9.6 months with T-DM1 versus 6.4 months with the con-
`trol regimen, and a hazard ratio for progression or death
`was 0.65 in favor of T-DM1 (95% CI 0.55 to 0.77). Import-
`antly, patients assigned to T-DM1 lived longer (30.9
`versus 25.1 months, respectively) and had fewer serious
`adverse events recorded. T-DM1 was associated with
`higher rates of thrombocytopenia and serum aminotrans-
`ferase level elevations, whereas lapatinib and capecitabine
`were associated with more frequent diarrhea, nausea and
`palmar-plantar erythrodysesthesia [36]. These data led to
`approval of T-DM1 by the US Food and Drug Administra-
`tion (FDA) in February 2013 for the treatment of patients
`
`with HER2-positive MBC who had previously received
`trastuzumab and a taxane.
`In another randomized study (TDM4450g), where 137
`patients with HER2-positive MBC or recurrent locally
`advanced breast cancer were assigned to either T-DM1 or
`trastuzumab plus docetaxel as first-line treatment, the
`median PFS was 14.2 months with T-DM1 and 9.2 months
`with trastuzumab plus docetaxel (hazard ratio 0.59; 95%
`CI 0.36 to 0.97) [37]. T-DM1 was associated with a more
`favorable safety profile with fewer serious adverse effects.
`In the TH3RESA study, 602 patients with unresectable
`HER2-positive locally advanced breast cancer or MBC
`who had progressed on at
`least
`two prior HER2-
`directed regimens were randomly assigned to receive
`either T-DM1 or therapy chosen by the physician. Patients
`treated with T-DM1 achieved longer PFS (6.2 versus
`3.3 months, respectively; hazard ratio 0.53, 95% CI 0.42 to
`0.66) and longer survival (not reached versus 14.9 months),
`and had fewer severe (grade 3 or higher) adverse effects
`compared with a regimen chosen by the physician [38].
`
`Resistance to T-DM1
`Despite these favorable efficacy results, most patients
`treated with T-DM1 eventually progress [36-38], and
`some HER2-positive breast cancers are primarily non-
`responsive or are only minimally responsive to T-DM1.
`Understanding of the resistance mechanisms is important
`for further development of T-DM1-directed therapies.
`
`T-DM1 resistance in preclinical models
`Both primary and acquired resistance to T-DM1 have
`been observed in in vitro models of HER2-positive breast
`cancer and gastric cancer (Additional file 3) [17,39,40]. In
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`in vivo preclinical models, efficacy of T-DM1 varied
`depending on the tumor mass in a trastuzumab- and
`lapatinib-resistant human breast cancer xenograft model
`(JIMT-1). While large (approximately 350 mm3) xeno-
`grafts were resistant to T-DM1, small ones (approximately
`70 mm3) were partially sensitive. T-DM1 inhibited re-
`markably well growth of very small JIMT-1 xenografts
`with no macroscopic tumor detected until resistance to T-
`DM1 emerged after prolonged treatment (16 weeks) with
`T-DM1 [18]. In another preclinical study, large HER2-
`positive human gastric xenografts (N-87) disappeared
`macroscopically totally with T-DM1, but microscopic de-
`posits of residual tumor cells remained at the tumor
`inoculation sites. The residual cells had a low cell prolifer-
`ation rate when stained for Ki-67, and survived T-DM1
`treatment despite maintaining high HER2 protein expres-
`sion [17]. These findings suggest that cancer relapse may
`occur after a long latency period despite macroscopically
`complete response to T-DM1.
`
`Primary and acquired resistance to T-DM1 in clinical trials
`In a phase II study (TDM4558g) conducted in a cohort of
`112 patients with HER2-positive MBC who had received
`prior chemotherapy and who had progressed on prior
`HER2-directed therapy or within 60 days after the last dose
`of trastuzumab, 29 (26%, 95% CI 18% to 34%) patients
`achieved objective response with single-agent T-DM1 (none
`had complete response) and 55 (49%) had stable disease
`[41]. In this study only 22 (20%) patients had disease
`progression as their best response, suggesting that most
`patients with HER2-positive MBC are not primarily resistant
`to T-DM1 despite prior exposure to HER2-directed therapy.
`Primary resistance to T-DM1 may be more infrequent
`when the patients are naive to trastuzumab, although only
`indirect data are currently available to support this hy-
`pothesis. In the TDM4450g trial carried out in the first-
`line setting with most patients not previously treated with
`trastuzumab, 43 (64%, 95% CI 52% to 75%) out of the 67
`patients with MBC treated with T-DM1 achieved objective
`response, including seven (10%) complete responders, and
`the median duration of response was not reached [37],
`whereas in the EMILIA trial conducted in the second-line
`setting in a patient population who had previously been
`treated with trastuzumab and a taxane, 169 (44%, 95% CI
`39% to 49%) out of the 397 patients treated with T-DM1
`had objective response,
`including four (1%) complete
`responders, and the median duration of response was
`12.6 months [36].
`While primary resistance to T-DM1 may be relatively
`infrequent, particularly in patients who have no prior ex-
`posure to trastuzumab, most initially responding patients
`eventually cease to respond despite continued treatment
`with T-DM1 [36-38], suggesting that acquired resistance
`to T-DM1 is a common problem.
`
`Potential factors that cause resistance to T-DM1
`Except for low HER2 expression in cancer, the clinical,
`biological and pharmacological factors that are related to
`poor efficacy of T-DM1 are incompletely understood.
`Yet, factors that are strongly implicated in the biological
`mechanism of action of T-DM1 are good candidates for
`having a role in resistance to T-DM1.
`DM1 and its metabolites (lysine-MCC-DM1) need to
`accumulate in cancer cells to reach a concentration that
`exceeds the threshold to evoke cell death [12]. Here we
`summarize the factors that may influence the intracellular
`DM1 concentration and thus cause resistance to T-DM1
`(Figure 3, Table 2).
`
`Low tumor HER2 expression
`Expression of HER2 on cancer cells is essential for T-DM1
`efficacy. Not surprisingly, retrospective analyses of two
`phase II trials (TDM4258g and TDM4374g) carried out in
`advanced breast cancer revealed that patients with HER2-
`positive cancer (defined either as immunohistochemistry
`(IHC) 3+ or fluorescence in situ hybridization +) had more
`frequent responses to T-DM1 than patients who had
`HER2-normal
`cancer;
`in TDM4258g
`the objective
`response rates were 34% and 5%, respectively, and in
`TDM4374g, 41% and 20%, respectively [41-43]. When
`cancer HER2 mRNA levels were quantified by quantitative
`reverse transcriptase polymerase chain reaction in the sub-
`group of HER2 IHC 3+ disease, patients with the median
`or higher HER2 mRNA concentration responded more
`often to T-DM1 than those with a lower concentration
`(in TDM4374g, the response rates were 50% and 33%,
`and in TDM4258g, 36% and 28%, respectively) [41-43].
`Quantitative HER2 assays should probably be performed
`from the most recent cancer biopsy tissue material rather
`than the primary tumor, since the primary tumor HER2
`content may sometimes be discordant with that of most
`metastatic lesions [44,45].
`
`Poor internalization of the HER2-T-DM1complexes
`Binding of T-DM1 to the extracellular domain of HER2
`triggers entry of the HER2-T-DM1 complex into cancer
`cells via receptor-mediated endocytosis [12,13]. A high
`rate of complex internalization may result in high intracel-
`lular concentrations of DM1, and deceleration of the
`endocytosis rate might cause loss of sensitivity to T-DM1.
`However, it is unknown whether the rate of internalization
`differs between cancers, and the factors affecting the rate
`have not been identified.
`
`Defective intracellular and endosomal trafficking of the
`HER2-T-DM1 complex
`The internalized endocytotic vesicles containing HER2-
`T-DM1 complexes fuse and form early endosomes. The
`contents of early endosomes can be recycled back to the
`
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`(cid:129)Availability of T-DM1 to cancer cells
`(e.g. pharmacokinetics, tumor vascularization)
`
`Cancer cell with HER2
`overexpression
`
`Mitotic arrest
`Rapid apoptosis
`
`Mitotic catastrophe
`Disrupted intracellular
`trafficking
`
`Resistance
`
`Intracellular concentration of DM1
`
`+
`
`-
`
`t h r e s h o l d
`
`Potential resistance mechanisms
`to T-DM1
`(cid:129)Low HER2 expression in cancer
`(cid:129)Masking of the HER2 epitope
`(cid:129)High p95HER2 expression
`(cid:129)Poor HER2-T-DM1 complex
`internalization
`(cid:129)Defective intracellular and
`endosomal trafficking of the
`HER2-T-DM1 complex
`(cid:129)Defective lysosomal degradation
`of T-DM1
`(cid:129)A high rate of HER2-T-DM1
`recycling
`(cid:129)Drug efflux proteins
`
`Figure 3 Factors influencing the intracellular DM1 level. DM1 may evoke cell death in a concentration-dependent manner, where a threshold
`concentration of intracellular DM1 and its metabolites needs to be exceeded for cell kill. At high DM1 concentrations mitotic arrest and rapid
`apoptotic death follow, whereas at lower levels mitotic catastrophe and disrupted intracellular trafficking occur, and at the lowest levels of DM1
`cells show resistance. HER2, human epidermal growth factor receptor-2; T-DM1, trastuzumab emtansine.
`
`cell membrane, or the early endosome can mature into a
`lysosome [13] where proteolytic degradation of the anti-
`body part of T-DM1 occurs (Figure 1). The dynamics of
`loading of the lysosomes with the HER2-T-DM1 cargo
`may influence the intracellular DM1 levels. T-DM1
`treatment results in defective intracellular trafficking of
`the HER2 protein [18], which is not in disagreement
`with a hypothesis that mitosis is not the only target of
`anti-microtubule agents, but rather trafficking on the
`microtubules [34].
`
`Defective lysosomal degradation of T-DM1
`Since DM1 release in the cytosol occurs only following
`proteolytic degradation of the trastuzumab part of the T-
`DM1 complex in the lysosomes, efficient lysosomal deg-
`radation is essential. Expression and activity of lysosomal
`enzymes may vary between tumors and even cancer cells,
`and is influenced by several factors such as tumor necrosis
`factor-α, lysosomal vacuolar H+-ATPase (V-ATPase), and
`Bax inhibitor-1 [46-48]. All of these factors may thus affect
`cancer sensitivity to T-DM1. For example, V-ATPase
`
`Table 2 Potential factors that may cause resistance to trastuzumab emtansine
`Factors decreasing intracellular DM1 level
`T-DM1 binding to HER2
`
`Low cancer HER2 expression
`
`HER2 down-regulation
`
`Shedding of HER2 ectodomain
`
`Masking of the trastuzumab binding epitope on HER2 p95HER2 expression
`
`Intracellular trafficking and lysosomal degradation
`
`Poor HER2-T-DM1complex internalization
`
`Drug efflux
`
`Other factors
`
`Altered DM1 target
`
`Autocrine or stromal growth factors
`
`Modulators of the apoptotic pathway
`
`Activation of cell survival pathways
`
`HER2-T-DM1 recycling to plasma membrane
`
`Failure of HER2 intracellular trafficking
`
`Inefficient lysosomal degradation of T-DM1
`
`MDR1 expression
`
`Beta1-tubulin mutation
`
`Overexpression of a beta3-tubulin isoform
`
`Microtubule-associated proteins
`
`HER2, human epidermal growth factor receptor-2; T-DM1, trastuzumab emtansine.
`
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`inhibition using archazolid, an inhibitor of myxobacterial
`origin, results in apoptosis, growth inhibition, and im-
`paired HER2 signaling in the trastuzumab-resistant cell
`line JIMT-1 [49].
`
`Masking of the HER2 epitope
`The trastuzumab binding epitope of HER2 can be masked
`at least partly by MUC4 or hyaluronan inhibiting the
`binding of trastuzumab to HER2 [20,21]. Although no
`similar data are available regarding T-DM1, masking of
`the epitope may also decrease the binding of T-DM1 to
`HER2.
`
`High p95HER2 expression
`p95HER2 is an amino-terminally truncated form of
`HER2 that lacks most of the extracellular domain of the
`protein, including subdomain IV recognized by trastuzu-
`mab. Therefore, trastuzumab or T-DM1 cannot bind to
`p95HER2 [23]. No studies have thus far correlated breast
`cancer p95HER2 expression with sensitivity to T-DM1.
`
`A high rate of HER2-T-DM1 recycling
`After internalization, trastuzumab-HER2 complexes can
`evade degradation and undergo rapid and efficient recyc-
`ling to the cell membrane. About 50% of internalized
`HER2-bound trastuzumab is recycled back to the cell
`membrane within 5 minutes and 85% within 30 minutes
`in in vitro breast cancer cell culture [50]. It is currently
`unknown whether cytoplasmic recycling of T-DM1 differs
`from that of trastuzumab. Extensive recycling of T-DM1
`could yet lead to decreased efficacy, since in the absence
`of proteolytic degradation of trastuzumab no release of
`intracellular DM1 can occur.
`
`Drug efflux pumps
`is an ATP-
`MDR1 (also known as P-glycoprotein)
`dependent transporter that mediates efflux of drugs and
`toxins from the cell. Tumor MDR1 expression is associ-
`ated with poor response to chemotherapy in many types
`of cancer [51,52]. DM1 and other maytansinoids are sub-
`strates of MDR1, and MDR1 expression is linked with a
`maytansine-resistant cancer phenotype [53]. In one study,
`one out of three T-DM1-resistant breast cancer cell
`lines showed upregulation of multi-drug resistance
`transporters [40], but the role of drug efflux proteins in
`resistance to T-DM1 may be complex and requires
`further study [39].
`
`Neuregulin-HER3 signaling
`Presence of the HER3 ligand neuregulin-1β (NRG-1β,
`heregulin) suppressed the cytotoxic activity of T-DM1 in
`four out of the six breast cancer cell lines tested, this effect
`being reversed by pertuzumab [54]. Activating PIK3CA
`mutations were present in the two breast cancer cell lines
`
`where NRG-1β did not inhibit T-DM1 activity, while the
`four cell lines where T-DM1 activity was reduced did not
`harbor PIK3CA mutations [54]. As trastuzumab, T-DM1
`the phosphatidylinositol 3′-kinase (PI3K)
`suppresses
`signaling pathway [40]. The potential association between
`PIK3CA mutational status and T-DM1 efficacy remains
`unknown, but the results from clinical breast cancer series
`suggest that trastuzumab benefit does not depend on the
`mutational status of PIK3CA [55,56] or tumor PTEN
`expression [57].
`
`Altered tubulins
`Since DM1 binds to tubulin, altered or mutant tubulins
`[58,59] or altered modulators of the microtubule dynamics
`might also impact on the response to T-DM1 [39,47].
`
`Concentration-dependent mechanism of action of free
`intracellular DM1
`A high intracellular concentration of DNA damaging
`agents often leads to terminal mitotic arrest and apop-
`tosis [60,61]. Besides apoptosis, aberrant cytokinesis
`(pathological mitoses) and multinucleation may take
`place at low concentrations of DNA damaging agents
`[60-62], which is called mitotic catastrophe [60,63].
`T-DM1 caused rapid tumor shrinkage of human gastric
`cancer xenografts with high HER2 expression (IHC 3+),
`the type of cell death being predominantly apoptosis [17],
`whereas T-DM1 was less effective on human breast cancer
`xenografts expressing moderate HER2 levels (IHC 2+),
`but prolonged treatment times eventually evoked apop-
`tosis and mitotic catastrophe in these xenografts [18]. T-
`DM1 may thus cause cell death through two molecular
`mechanisms depending on the intracellular DM1 concen-
`tration, high concentrations of DM1 causing mitotic arrest
`with no or few mitotic catastrophes followed by apoptosis,
`whereas cell exposure to low DM1 concentrations of long
`duration may lead to mitotic catastrophes and cell death.
`Prolonged T-DM1 treatment led to disruption of intracel-
`lular trafficking of HER2 in the breast cancer xenografts
`with moderate HER2 expression (IHC 2+) [18].
`Based on these findings, we hypothesize that the anti-
`cancer effects of T-DM1 depend on the intracellular
`concentration of DM1 and the duration of exposure.
`When the intracellular concentration of DM1 exceeds a
`critical threshold level, mitotic arrest and rapid apoptotic
`death follows, whereas mitotic catastrophe and disrupted
`intracellular trafficking occur at lower DM1 levels pro-
`vided that the exposure time is long enough (Figure 3).
`This hypothesis requires further research in preclinical
`models, but it could support carrying out clinical trials
`evaluating prolonged administration of T-DM1 in cancer
`patient populations with low to moderate tumor HER2
`expression levels.
`
`IMMUNOGEN 2111, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Barok et al. Breast Cancer Research 2014, 16:209
`http://breast-cancer-research.com/content/16/2/209
`
`Page 8 of 12
`
`Strategies to improve T-DM1 efficacy and
`circumvent resistance
`Here we summarize the potential strategies to improve
`efficacy of T-DM1 and to prevent drug resistance. Some
`of these strategies are already being tested in clinical trials.
`
`T-DM1 in the adjuvant and neoadjuvant setting
`At present T-DM1 has been approved by the FDA for
`second-line treatment of HER2-positive MBC. Ongoing
`clinical trials are evaluating the potential role of T-DM1
`as first-line treatment of MBC and in the adjuvant and
`neoadjuvant settings [64]. The trials to be carried out in
`patient populations with a small or minimal tumor bulk
`are clearly of great importance, since T-DM1 has substan-
`tial efficacy and a favorable safety profile as a single agent
`in advanced breast cancer, and T-DM1 may be particularly
`effective in eradication of cancer when the tumor mass is
`small [65].
`
`Combination therapies with T-DM1
`There is substantial interest in investigating the efficacy and
`safety of T-DM1 in combination with other anti-cancer
`agents, particularly with those that have proved effective in
`combination with trastuzumab. Both paclitaxel and doce-
`taxel are approved for the treatment of HER2-positive
`MBC in combination with trastuzumab [4,66]. Since DM1
`and taxanes bind to tubulins at different sites [12,67], a
`combination of taxanes and T-DM1 could have synergistic
`effects. Two ongoing clinical trials are evaluating such
`combinations (NCT00951665 and NCT00934856).
`An ongoing clinical trial (NCT01702558) evaluates effi-
`cacy and safety of capecitabine plus T-DM1 in MBC. This
`trial is built on the clinical activity observed in a phase II
`single cohort study that evaluated the combination of cap-
`ecitabine and trastuzumab in HER2-positive MBC [68],
`and a randomized phase II trial that compared a combin-
`ation of capecitabine, trastuzumab and docetaxel to tras-
`tuzumab plus docetaxel, the triple combination resulting
`in significantly improved PFS [69].
`Patients with HER2-positive MBC treated with pertuzu-
`mab in combination with trastuzumab and docetaxel had
`longer PFS and overall survival compared with patients
`who received placebo, trastuzumab and docetaxel
`in a
`large randomized trial (CLEOPATRA) [70]. The on-
`going trials evaluating the combinations of pertuzumab
`plus T-DM1 and the triple combination of pertuzumab
`plus T-DM1 plus a taxane are thus well founded [64].
`MARIANNE (NCT01120184) is an ongoing trial with a
`planned target population of over 1,000 patients with
`HER2-positive MBC. In this study, patients who have
`not received prior chemotherapy for MBC are randomly
`assigned to receive T-DM1 plus placebo, T-DM1 plus
`pertuzumab, or trastuzumab plus paclitaxel or doce-
`taxel. The combination of T-DM1 and lapatinib also
`
`deserves clinical evaluation considering the superior
`efficacy of lapatinib and trastuzumab in HER2-positive
`MBC over lapatinib alone [71].
`Trastuzumab has been approved for the treatment of
`patients with HER2-positive and hormone receptor-
`positive postmenopausal MBC in combination with an
`aromatase inhibitor [72,73]. The efficacy and safety of