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REVIEW
Trastuzumab emtansine: mechanisms of action
and drug resistance
Mark Barok
1*
, Heikki Joensuu
2
and Jorma Isola
3
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
patientstreatedwithT-DM1developacquireddrugresistance. 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].
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
* Correspondence: barok.mark@gmail.com
1
Laboratory 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
© 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.
Barok et al. Breast Cancer Research 2014, 16:209
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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 G
2
-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] Overexpression of cyclin E [27]
Inhibition of angiogenesis [28] 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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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
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.
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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 mm
3
)xeno-
grafts were resistant to T-DM1, small ones (approximately
70 mm
3
) 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
hadcompleteresponse)and55(49%)hadstabledisease
[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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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
threshold
Mitotic arrest
Rapid apoptosis
Resistance
Mitotic catastrophe
Disrupted intracellular
trafficking
Intracellular concentration of DM1
•Availability of T-DM1 to cancer cells
(e.g. pharmacokinetics, tumor vascularization)
Potential resistance mechanisms
to T-DM1
•Low HER2 expression in cancer
•Masking of the HER2 epitope
•High p95HER2 expression
•Poor HER2-T-DM1 complex
internalization
•Defective intracellular and
endosomal trafficking of the
HER2-T-DM1 complex
•Defective lysosomal degradation
of T-DM1
•A high rate of HER2-T-DM1
recycling
•Drug efflux proteins
-
Cancer cell with HER2
overexpression
+
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.
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
HER2-T-DM1 recycling to plasma membrane
Failure of HER2 intracellular trafficking
Inefficient lysosomal degradation of T-DM1
Drug efflux MDR1 expression
Other factors
Altered DM1 target Beta1-tubulin mutation
Autocrine or stromal growth factors Overexpression of a beta3-tubulin isoform
Modulators of the apoptotic pathway Microtubule-associated proteins
Activation of cell survival pathways
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
MDR1 (also known as P-glycoprotein) is an ATP-
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-1βdid 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
suppresses the phosphatidylinositol 3′-kinase (PI3K)
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.
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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 T-
DM1 is being investigated in combination with endocrine
therapy (with tamoxifen in premenopausal women and
aromatase inhibitor in postmenopausal women) as neoad-
juvant treatment of HER2-positive and hormone receptor-
positive operable breast cancer (NCT01745965).
GDC-0941, a selective and potent PI3K inhibitor, was
effective in preclinical models of trastuzumab-resistant
breast cancer, where administration of GDC-0941 in
combination with HER2-directed treatment (trastuzumab,
pertuzumab, or lapatinib) inhibited in a synergistic fashion
growth of breast cancer cells [74,75]. In an ongoing dose
escalation phase Ib study (NCT00928330), the safety,
tolerability, pharmacokinetics, and efficacy of T-DM1 and
GDC-0941 are being investigated in patients with HER2-
positive MBC who have progressed on prior trastuzumab
therapy.
Circumventing MDR1-mediated resistance by a modified
linker
Since the maytansinoids are substrates for the MDR1
transporters [53], drug efflux by MDR1 may decrease
the intracellular DM1 concentration, resulting in a
decline in efficacy. Kovtun and colleagues [53] developed
a potential strategy to circumvent MDR1-mediated re-
sistance to T-DM1 by attaching DM1 to an antibody
using a hydrophilic linker, PEG
4
Mal. The degradation of
such conjugates in cancer cells resulted in the release of
lysine-PEG
4
Mal-DM1 instead of lysine-MCC-DM1 (the
active metabolite of T-DM1). Lysine-PEG
4
Mal-DM1 is a
poor substrate of MDR1, and the conjugates with the
PEG
4
Mal linker evaded MDR1-mediated resistance both
in MDR1-expressing cells in vitro and in MDR1-
expressing xenografts in vivo [53]. Therefore, MDR1
drug transporter-mediated resistance to T-DM1 might
be overcome by replacing the SMCC linker with the
PEG
4
Mal linker.
Modulation of HER2 recycling
When intracellular HER2 is recycled to the plasma mem-
brane, trastuzumab recycles as a part of the HER2-
trastuzumab complex [50]. Heat shock protein (Hsp)90 is
a molecular chaperone that participates in the regulation
of HER2 recycling. Geldanamycin, an inhibitor of Hsp90,
reduces HER2 recycling and results in an over three-fold
increase in the concentration of the HER2-trastuzumab
complex being retained in tumor cells [50]. Geldanamycin
redistributes cell surface HER2 into the internal vesicles of
Barok et al. Breast Cancer Research 2014, 16:209 Page 8 of 12
http://breast-cancer-research.com/content/16/2/209
the endosomes, enhancing proteolytic degradation of
HER2 [50,76].
It has currently not been established whether intracellular
T-DM1 is also recycled, but inhibition of recycling is of
potential interest from the therapeutic point of view. Hypo-
thetically, sequential administration of T-DM1 followed by
geldanamycin (or one of its derivatives [77]) might inhibit
recycling of T-DM1 and direct the HER2-T-DM1
complexes for lysosomal degradation, thus increasing the
intracellular levels of DM1 and cytotoxicity. Sequential
administration of the two drugs in this order could be
important, since Hsp90 inhibitors decrease cell surface
HER2 and might reduce T-DM1 binding to cells [50].
Modification of the cytotoxic drug moiety
Since the intracellular DM1 concentration is crucial for
the cell-killing potency of T-DM1, delivery of greater
quantities of DM1 into the tumor cells would likely im-
prove efficacy. This could be achieved using more heavily
loaded T-DM1 that delivers more cytotoxic drug to the
target cells. However, increasing the drug-antibody ratio
(DAR) of an ADC usually results in a faster clearance of
the ADC. ADCs with a DAR of 2 to 4 have a more favor-
able pharmacokinetic profile than those with a higher
DAR [78,79]. Therefore, increasing the number of DM1
molecules from the average of 3.5 per one trastuzumab
might result in a shorter half-life and destabilization of the
complex, and decreased efficacy. Alternative strategies in-
clude binding of another cytotoxic drug in addition to
DM1 to trastuzumab, or administering another ADC in
combination with T-DM1, such as a cytotoxic drug linked
to pertuzumab. The second cytotoxic drug could have an
alternative (non-tubulin) mechanism of action [39].
Improving the Fc part of trastuzumab
Trastuzumab retains its anti-cancer activity when conju-
gated to DM1, and improving the antibody component of
the conjugate could thus result in a more efficient ADC.
Afucosylated trastuzumab has superior efficacy compared
with trastuzumab in some preclinical breast cancer
models [80], and amino acid modifications of the Fc part
of trastuzumab may also improve efficacy [81]. Yet, DM1
remains a key component regarding the overall anti-
tumor activity of T-DM1.
Radioimmunotherapy conjugates
Auger electron emitting
111
In-NLS-trastuzumab is effective
in the treatment of trastuzumab-resistant breast cancer cells
[82]. Radioimmunotherapy conjugatesmightfindarolein
the treatment of patients who have failed T-DM1 therapy.
Conclusion
T-DM1 is a valuable new agent for the treatment of
HER2-positive breast cancer. T-DM1 has several
mechanisms of action consisting of the anti-tumor effects
associated with its key components, trastuzumab and the
cytotoxic drug DM1. Clinical research carried out suggests
superior efficacy of T-DM1 compared with trastuzumab
or trastuzumab plus chemotherapy in the treatment of
MBC. However, both primary and secondary resistance to
T-DM1 occurs. In addition to the identified resistance
mechanisms related to trastuzumab, several factors that
influence the intracellular DM1 concentration may confer
resistance to T-DM1. Understanding of these factors may
lead to the development of strategies that improve efficacy
of T-DM1 and may circumvent drug resistance.
Additional files
Additional file 1: Time-lapse microscopy of SKBR-3 breast cancer
cells grown with 1 μg/mL trastuzumab or T-DM1. SKBR-3 is a HER2-
positive, trastuzumab-sensitive breast cancer cell line. SKBR-3 is much
more sensitive to T-DM1 than to trastuzumab. Images were taken by
Cell-IQ
W
system (Chip-Man Technologies Ltd, Tampere, Finland).
Additional file 2: Time-lapse microscopy of JIMT-1 breast cancer
cells grown with 1 μg/mL trastuzumab or T-DM1. JIMT-1 is a HER2-
positive, trastuzumab and lapatinib cross-resistant breast cancer cell line.
JIMT-1 is sensitive to T-DM1. Images were taken by Cell-IQ
W
system
(Chip-Man Technologies Ltd, Tampere, Finland).
Additional file 3: Time-lapse microscopy of SNU-216 gastric cancer
cells grown with 1 μg/mL trastuzumab or T-DM1. SNU-216 is a HER2-
positive, trastuzumab-resistant gastric cancer cell line. SNU-216 is resistant
to trastuzumab and T-DM1. Images were taken by Cell-IQ
W
system (Chip-
Man Technologies Ltd, Tampere, Finland).
Abbreviations
ADC: Antibody-drug conjugate; DAR: Drug-antibody ratio; DM1: Derivative of
maytansine 1; FDA: Food and drug administration; HER2: Human epidermal
growth factor receptor-2; Hsp: Heat shock protein;
IHC: Immunohistochemistry; MBC: Metastatic breast cancer; PFS: Progression-
free survival; PI3K: Phosphatidylinositol 3′-kinase; SMCC: N-succinimidyl-4-
(N-maleimidomethyl)cyclohexane-1-carboxylate; T-DM1: Trastuzumab-emtansine;
V-ATPase: Vacuolar H
+
-ATPase.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Laboratory of Molecular Oncology, University of Helsinki, Biomedicum,
Haartmaninkatu 8, Helsinki FIN-00290, Finland.
2
Department of Oncology,
Helsinki University Central Hospital Haartmaninkatu 4, Helsinki FIN-00029,
Finland.
3
MioMediTech, University of Tampere, Biokatu 6, Tampere 33014,
Finland.
Published: 5 March 2014
Note: This article is part of a series on ‘Recent advances in
breast cancer treatment’,edited by Jenny Chang. Other
articles in this series can be found at http://breast-cancer-
research.com/series/treatment.
Barok et al. Breast Cancer Research 2014, 16:209 Page 9 of 12
http://breast-cancer-research.com/content/16/2/209
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Cite this article as: Barok et al.:Trastuzumab emtansine: mechanisms of
action and drug resistance. Breast Cancer Research 2014 16:209.
Barok et al. Breast Cancer Research 2014, 16:209 Page 12 of 12
http://breast-cancer-research.com/content/16/2/209
