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`Cancer Research
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`
`Targeting HER2-Positive Breast Cancer with
`Trastuzumab-DM1, an Antibody—Cytotoxic Drug Conjugate
`
`Gail D. Lewis Phillips, Guangmin Li, Debra L. Dugger, et al.
`
`Cancer Res 2008;68:9280—9290.
`
`Updated version
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`PH IGEN IX
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`Targeting HER2-Positive Breast Cancer with Trastuzumab-DM1,
`
`an Antibody—Cytotoxic Drug Conjugate
`
`Gail D. Lewis Phillips,1 Guangmin Li,1 Debra L. Dugger,1 Lisa M. Crocker,1 Kathryn L. Parsons,1
`Elaine Mai,1 Walter A. Blattler,2 John M. Lambert,2 Ravi VJ. Chari,2 Robert J. Lutz,2
`Wai Lee T. Wong,1 Frederic S. Jacobson,1 Hartmut Koeppen,1 Ralph H. Schwall,1
`Sara R. Kenkare—Mitra,1 Susan D. Spencer,1 and Mark X. Sliwkowskil
`‘Genentech, Inc., South San Francisco, California and ‘ImmunoGen, Inc., Waltham, Massachusetts
`
`Abstract
`
`HER2 is a validated target in breast cancer therapy. Two drugs
`are currently approved for HER2-positive breast cancer:
`trastuzumab (Herceptin), introduced in 1998, and lapatinib
`(Tykerb),
`in 2007. Despite these advances, some patients
`progress through therapy and succumb to their disease. A
`variation on antibody-targeted therapy is utilization of anti-
`bodies to deliver cytotoxic agents specifically to antigen-
`expressing tumors. We determined in vitro and in vivo efficacy,
`pharmacokinetics, and toxicity of trastuzumab-maytansinoid
`(microtubule-depolymerizing agents) conjugates using disul-
`fide and thioether linkers. Antiproliferative effects of trastu-
`zumab-maytansinoid conjugates were evaluated on cultured
`normal and tumor cells. In vivo activity was determined in
`mouse breast cancer models, and toxicity was assessed in rats
`as measured by body weight loss. Surprisingly, trastuzumab
`linked to DMI
`through a nonreducible thioether linkage
`(SMCC), displayed superior activity compared with unconju-
`gated trastuzumab or trastuzumab linked to other maytansi-
`noids through disulfide linkers. Serum concentrations of
`trastuzumab-MCC-DMI remained elevated compared with
`other conjugates, and toxicity in rats was negligible compared
`with free DMI or trastuzumab linked to DMI through a
`reducible linker. Potent activity was observed on all HER2-
`overexpressing tumor cells, whereas nontransformed cells
`and tumor cell
`lines with normal HER2 expression were
`unaffected. In addition, trastuzumab-DMI was active on HER2-
`overexpressing, trastuzumab-refractory tumors. In summary,
`trastuzumab-DMI shows greater activity compared with
`nonconjugated trastuzumab while maintaining selectivity for
`HER2-overexpressing tumor cells. Because trastuzumab linked
`to DMI through a nonreducible linker offers improved efficacy
`and pharmacokinetics and reduced toxicity over the reducible
`disulfide linkers evaluated, trastuzumab-MCC-DMI was select-
`ed for clinical development. [Cancer Res 2008;68(22):9280—90]
`
`Introduction
`
`The HER2 (ErbB2) receptor tyrosine kinase is a member of the
`epidermal growth factor receptor
`family of transmembrane
`
`Note: Current address for H. Koeppen: M. D. Anderson Cancer Center, Houston,
`TX 77030.
`Requests for reprints: Gail D. Lewis Phillips, Genentech, Inc., 1 DNA Way, South
`San Francisco, CA 94080. Phone: 650-225-2201; Fax: 650-225-1411; E-mail: gdl@
`gene.com or Mark K Sliwkowski, Genentech, Inc., 1 DNA Way, South San Francisco,
`CA 94080. Phone: 650-225-1247; Fax: 650-225-5770; E-mail: ma.rks@gene.com.
`©2008 American Association for Cancer Research.
`d0i:l0.l158/0008-5472.CAN-08-1776
`
`receptors. These receptors, which also include HER3 (ErbB3) and
`HER4 (ErbB4), are known to play critical roles in both development
`and cancer (1, 2). Importantly, amplification and overexpression of
`HER2 occur in 20% to 25% of human breast cancer and are
`
`predictive of poor clinical outcome (3, 4). Because of the role of
`HER2 in breast cancer pathogenesis and the accessibility of the
`extracellular portion of the receptor, HER2 was recognized as a
`potential candidate for targeted antibody therapy. The humanized
`HER2 antibody,
`trastuzumab (Herceptin), was approved by the
`Food and Drug Administration in 1998 for use in metastatic breast
`cancer and has subsequently shown clinical benefit when used, in
`combination with cytotoxic chemotherapy, as first-line or adjuvant
`therapy (5, 6). Importantly, trastuzumab improves overall survival
`in early breast cancer after chemotherapy compared with
`observation alone (6). Increased survival after only 2 years of
`follow-up is impressive in breast cancer. Tamoxifen is the only
`other breast cancer treatment that is reported to offer a survival
`benefit in this short-time period (6).
`Although the mechanisms for response to trastuzumab are not
`completely understood, clinical benefit is attributed to interference
`with signal transduction pathways,
`impairment of extracellular
`domain (ECD) cleavage,
`inhibition of DNA repair, decreased
`angiogenesis; as well as induction of cell cycle arrest, and
`antibody-mediated cellular cytotoxicity (7, 8). Despite these diverse
`mechanisms of action, a significant proportion of patients treated
`with trastuzumab either do not respond initially or relapse after
`experiencing a period of clinical response (5, 9). Progression
`through trastuzumab-containing therapy is attributed to aberrant
`activation of signaling pathways, such as the phosphatidylinositol
`3-kinase pathway (10-12), activation of compensatory signaling
`either through up-regulation of the insulin-like growth factor-I
`receptor (13, 14) or ErbB/HER ligands (15, 16) or generation of a
`constitutively active truncated form of HER2, designated p95HER2
`(17, 18).
`Direct covalent coupling of cytotoxic agents to monoclonal
`antibodies is an alternative to naked antibody-targeted therapy.
`To date, antitumor antibodies have been linked to cytotoxic agents,
`such as the calicheamicins, auristatins, maytansinoids and
`derivatives of CC1065 (19-22). Currently, only one such conjugate,
`anti-CD33 conjugated to calicheamicin (gemtuzumab ozogamicin
`or Mylotarg), has been granted marketing approval
`for
`the
`treatment of relapsed acute myeloid leukemia (23).
`Maytansinoids are derivatives of the antimitotic drug maytan-
`sine. These agents bind directly to microtubules in a manner
`similar to the Vinca alkaloids (24, 25). Antibody-maytansinoid
`conjugates directed toward cancer antigens,
`such as CanAg
`(cantuzumab mertansine and IMGN242), prostate-specific mem-
`brane antigen (MLN2704), CD56 (IMGN901), CD33 (AVE9633), and
`
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`CD44v6 (bivatuzumab mertansine) are in early stages of clinical
`testing (20, 26-28). Because HER2 is highly differentially expressed
`on breast tumor cells (1-2 million copies per cell) compared with
`normal epithelial cells, HER2 represents an ideal
`target
`for
`antibody—drug conjugate (ADC)
`therapy. Numerous preclinical
`and clinical studies indicate that trastuzumab combines extremely
`well with microtubule-directed agents (29-32). Given the mecha-
`nism of action and potency of maytansine, it was deemed to be a
`particularly attractive cytotoxic agent to conjugate to trastuzumab.
`Herein, we describe the efficacy, pharmacokinetic properties, and
`safety of several
`trastuzumab—maytansinoid conjugates, with
`particular emphasis on the chemical nature of the linker.
`
`Materials and Methods
`
`Cell lines and reagents. Tumor cell lines (breast carcinoma BT—474, SK-
`BR—3, MCF7, MDA—MB—468, MDA—MB—361, HCC1954, lung carcinoma Calu 3,
`and ovarian carcinoma line SK—OV—3) and MCF 10A breast epithelial cells
`were obtained from American Type Culture Collection. The breast tumor
`line KPL—4 was obtained from Dr. J. Kurebayashi (33), and MKN7 gastric
`carcinoma cells were from Mitsubishi Corp. Cells were maintained in Ham’s
`F-12: high glucose DNEM (50:50) supplemented with 10% heat—inactivated
`fetal bovine serum and 2 mmol/L L—glutamine (all from Invitrogen Corp.).
`Normal human cell lines [human mammary epithelial cells (HMEC) and
`normal human epidermal keratinocytes (NHEK)] and the corresponding
`culture medium (MEGM and KGM,
`respectively) were obtained from
`Cambrex. The BT474—EEI cell line was derived by subculturing BT—474
`tumors grown in viva in the absence of estrogen pellet supplementation
`(exogenous estrogen independent) and is resistant both in Vitro and in Vii/0
`to trastuzumab treatment.
`
`Active agents used for cell culture and animal studies were the antibody
`trastuzumab (Genentech, Inc.), trastuzumab—maytansinoid ADC (Immuno-
`Gen, Inc.), and the control ADC, anti—H—8—MCC—DM1. The maytansinoid,
`DM1, was conjugated to trastuzumab through SPP, SMCC, or SPDP linkers
`(Fig. 1; refs. 24, 34); the thiol—containing maytansinoids, DM3 and DM4,
`
`Trastuzumab-DM1 Antibody-Drug Conjugate
`
`which have methyl groups adjacent to their sulfhydryl group were linked to
`trastuzumab with the SSNPP linker (ImmunoGen, Inc.). All trastuzumab
`ADCs had an average molar ratio of 3 to 3.6 maytansinoid molecules per
`antibody. The drug—antibody molar ratio for trastuzumab—MCC—DM1 and
`trastuzumab—SPP—DM1 was 3.2 for cell culture and xenograft studies, 3.6 for
`trastuzumab—SPP—DM1 used in the rat toxicity study, and 3.8 for anti—H—8—
`MCC—DM1.
`
`Cell viability and cell death assays. The effects of trastuzumab and
`trastuzumab—maytansinoid conjugates on tumor cell viability were assessed
`using Cell Titer—Glo (Promega Corp.). Cells were plated in black—walled
`96-well plates (20,000 per well for BT—474; 10,000 cells per well for all other
`lines) and allowed to adhere overnight at 37 °C in a humidified atmosphere
`of 5% CO2. Medium was then removed and replaced by fresh culture
`medium containing different concentrations of trastuzumab, trastuzumab
`ADC, or free DM1, and the cells incubated for varying periods of time. After
`each time point, Cell Titer—Glo reagent was added to the wells for 10 min at
`room temperature and the luminescent signal was measured using a
`Packard/Perkin—Elmer TopCount. For measurement of apoptosis, BT—474
`and SK—BR—3 were exposed to trastuzumab or trastuzumab—MCC—DM1 for
`48 h. Caspase activation was assessed by adding Caspase—Glo 3/7 reagent
`(Promega Corp.) for 30 min at room temperature, and the luminescence
`was recorded using a Packard TopCount Induction of cytotoxicity was
`assessed in cells treated with trastuzumab or trastuzumab—MCC—DM1 for
`
`72 h using ToxiLight Bioassay kit (Cambrex/Lonza). This assay measures
`release of the intracellular enzyme adenylate kinase as a result of cell lysis.
`Normal HMEC and NHEK were plated in clear 96-well plates at densities
`of 10,000 and 8,000 cells per well, respectively, and allowed to adhere
`overnight. Cells were treated with trastuzumab or trastuzumab—MCC—DM1
`for 72 h. Alamar Blue reagent (Trek Diagnostics Systems) was added to all
`wells, plates were incubated for 3 h at 37 °C, and fluorescence was measured
`on a SpectraMax 190 (Molecular Devices) using 530—nm excitation and
`590—nm emission. Because the normal cell lines were not healthy when
`grown in black multiplates (which is necessary for use of Cell Titer—Glo),
`Alamar Blue was used as the proliferation read—out. For all cellular assays,
`dose—response curves were generated using Kaleidagraph 4.0 (Synergy
`Software) four—parameter curve fitting.
`
`finker
`
`T:‘s,.siLl2umab {Tm 53)
`
`E’
`
`'l'rs3s‘1E3-Si-‘D5-7-DM1
`
`Figure 1. Structure of trastuzumab
`(Tmab)—maytansinoid conjugates (stability of
`linker, least to greatest): Tmab—SPDP—DM1,
`Tmab-SPP—DM1, Tmab—SSNPP—DM3,
`Tmab—SSNPP—DM4, and Tmab—SMCC-DM1
`(nonreducible).
`
`-
`
`Me
`
`Drug Maytansinoid
`(DM)
`
`Tamil}-SP?-DM1
`
`Trst.=;‘zE3-SSNPP-DM3
`
`'i'rn.=szt3-€~Z€~1E\E{JP-DM4
`
`f T:*n.aE3-MEL”,-C-DM1
`
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`Research.
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`PHIGENIX
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`Cancer Research
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`Western immunoblot analysis. SK—BR—3 cells were seeded at a density
`of 1 million per dish in 100 X 15 mm dishes and allowed to adhere for 2 d.
`The medium was then removed and replaced with fresh medium
`containing either trastuzumab, free DM1, or a range of concentrations of
`trastuzumab—MCC—DM1. After a 48-h incubation,
`floating cells were
`collected and combined with detached adherent cells. The total cell
`
`population was then centrifuged and resuspended in lysis buffer [50 mmol/L
`HEPES (pH 7.5), 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1.0 mmol/L EGTA,
`10% glycerol, 1% Triton X-100, 10 mmol/L Na4P2O4, 1 mmol/L Na3VO4,
`50 mmol/L NaF,
`1
`].l1’I1()l/L leupeptin, 0.3 ].l1’I1()l/L aprotinin,
`1
`].l1’I1()l/L
`pepstatin A, 10 iimol/L bestatin, and 1.4 ].l1’I10l/L E—64]. Lysates were cleared
`by centrifugation at 4°C for 15 min at 20,800 X g in a microcentrifuge, and
`protein concentrations were determined using the bicinchoninic acid (BCA)
`protein assay kit (Pierce). Proteins were resolved by SDS—PAGE, transferred
`to nitrocellulose, and immunoblotted with a polyclonal antibody against
`poly(ADP—ribose) polymerase (PARP), which recognizes intact 116—kDa PARP
`and the 23-kDa cleavage fragment (R&D Systems). Blotting was carried out
`in TBS containing 0.1% Triton X-100 and 5% nonfat dry milk, followed by
`incubation with horseradish peroxidase (HRP)—conjugated secondary anti-
`bodies (Amersham Biosciences). Proteins were visualized using enhanced
`chemiluminescence reagents (Amersham Biosciences).
`Measurement of total and phosphorylated HER2 and p95HER2 in
`transgenic tumors was performed as follows. Tumors from the founder 5
`(Fo5;
`ref. 35) and founder 2#1282 (F2#1282)
`lineages of MMTV—HER2
`transgenic mice (Genentech, Inc.) were excised from the animals, placed in
`lysis buffer containing protease inhibitors, and homogenized on ice. Tumor
`lysates were centrifuged, and total protein levels in the supernatant were
`determined using a BCA protein assay kit. HER2 was immunoprecipitated
`overnight at 4°C using the mouse monoclonal antibody Ab—15 (Lab Vision)
`complexed to protein A/G sepharose, with 1 mg total protein from at least
`three independent tumor lysates. Complexes were pelleted by centrifuga-
`tion, washed twice with lysis buffer, resuspended in SDS sample buffer, and
`boiled. Samples were separated on a 4% to 12% Tris—glycine gel and
`transferred to nitrocellulose membranes. Blots were probed with mouse
`monoclonal antibody Ab—18 (Lab Vision)
`to detect the phosphorylated
`forms of HER2 and p95HER2 or with Ab—15 to detect total HER2 and
`p95HER2.
`In viva efficacy and pharmacokinetic studies. Tumor tissue from
`Fo5 or F2#1282 HER2 transgenic mice was collected aseptically, rinsed in
`HBSS, and cut into pieces of ~2 X 2 mm in size. These pieces were
`surgically transplanted into the mammary fat pad of female nu/nu mice
`(Charles River Laboratories). For efficacy studies using BT474EEI cells,
`naive female beige nude XID mice (Harlan Sprague—Dawley) were
`inoculated in the mammary fat pad with 20 million tumor cells
`suspended in 50% phenol red—free Matrigel (Becton Dickinson Bioscience)
`mixed with culture medium. All animals were randomly assigned into
`treatment groups, such that the mean tumor volume for each group was
`100 to 200 mm°. Trastuzumab or trastuzumab—maytansinoid conjugates
`were given by either a single i.v. injection or injection once every 3 wk.
`Vehicle control was either PBS (for pharmacokinetic studies) or ADC
`formulation buffer
`[10 mmol/L sodium succinate, 0.1% polysorbate
`(Tween) 20, 20 mg/mL trehalose dihydrate (pH5.0)]. Similarly, KPL—4
`human breast tumor cells were inoculated (3 million cells per mouse, in
`Matrigel) into the mammary fat pads of SCID beige mice (Charles River
`Laboratories). Trastuzumab (15 mg/kg) was given i.p. once per week for
`4 wk; trastuzumab—MCC—DM1 (15 mg/kg) was given i.v. (single injection
`on treatment day 0). All treatment groups consisted of 6 to 10 animals per
`group, and tumor
`size was monitored twice weekly using caliper
`measurement. Mice were housed in standard rodent microisolator cages.
`Environmental controls for the animal rooms were set
`to maintain a
`
`temperature of ~70°F, a relative humidity of 40% to 60%, and an
`approximate 14-h light/10-h dark cycle.
`For pharmacokinetic analysis of trastuzumab—maytansinoid conjugates,
`female beige nude mice (age 15-20 wk; Harlan Sprague—Dawley) were
`injected i.v. with 2 mg/kg of different trastuzumab ADCs (four mice per
`group). To assess circulating levels of total and conjugated antibody, blood
`was collected via cardiac puncture from three animals at 5 min and 1, 6, 24,
`
`72, and 168 h postinjection. The samples were left at room temperature for
`30 min until the blood coagulated. Subsequently, serum was obtained by
`centrifuging the samples at 10,000 X g for 5 min at 4°C, after which serum
`samples were stored at —70°C. Total trastuzumab concentration in the
`serum samples was measured as follows: 96-well ELISA plates were coated
`with HER2 ECD in 0.05 mol/L carbonate/bicarbonate buffer (pH 9.6) at 4°C
`overnight. After removal of the coat solution, nonspecific binding sites were
`blocked by incubating with blocking solution [0.5% bovine serum albumin
`(BSA) in PBS] for 1 to 2 h. The plates were then washed with wash buffer
`(0.05% Tween in PBS), and standards or samples diluted in ELISA assay
`buffer [PBS containing 0.5% BSA, 0.05% Tween, 10 ppm proclin 300, 0.2%
`bovine 'y—globulin, 0.25% CHAPS, 0.35 mol/L NaCl, 5 mmol/L EDTA (pH 7.4)]
`were added. After a 2—h
`incubation, plates were washed and HRP-
`conjugated goat anti—human Fc (The Jackson Laboratory) was added for
`an additional 2 h. Plates were then washed again, followed by the addition
`of tetramethyl benzidine substrate for color development. The reaction was
`stopped after 10 to 15 min by the addition of 1 mol/L phosphoric acid.
`Plates were read on a Molecular Devices microplate reader at a wavelength
`of 450 to 630 nm. The concentration of trastuzumab in the samples was
`extrapolated from a four—variable fit of the standard curve. For
`measurement of trastuzumab—maytansinoid concentration, wells were
`coated with anti—DM antibody (GNE DM1—3586) and serum samples
`added as above. After the 2—h sample incubation, the plates were washed,
`60 ng/mL biotin—conjugated HER2 ECD was added to each well, and the
`plates were incubated for
`1 h. Plates were then washed, and HRP-
`conjugated streptavidin (GE Healthcare) was added for an additional
`30-min incubation. Color detection and measurement were performed as
`described above. Previous analyses of different preparations of trastuzu-
`mab—DM1 conjugates with drug—antibody ratios ranging from 1.9 to 3.8
`showed that the conjugated antibody ELISA is not sensitive to drug load.
`Circulating HER2 ECD levels from mice harboring Fo5 or F2#1282
`xenograft tumors was measured, as previously described (35). Briefly, serum
`was diluted 1:50 with ELISA assay buffer (above) followed by 1:2 serial
`dilutions. HER2 ECD was captured using goat anti—HER2 polyclonal
`antibody (Genentech,
`Inc.) and detected with biotin—conjugated rabbit
`anti—Her2 polyclonal (Genentech, Inc.), followed by addition of streptavidin-
`HRP.
`
`Rat toxicity studies. Female Sprague—Dawley rats weighing 75 to 80 g
`were obtained from Charles River Laboratories and allowed to acclimate
`
`trastuzumab—MCC—DM1,
`for 5 d before study. Trastuzumab—SPP—DM1,
`and free DM1 were diluted in PBS (Invitrogen Corp.) as vehicle and given
`as a single i.v. bolus tail—vein injection on day 1 at a dose volume of
`10 mL/kg. Body weights were measured predose on day 1 and daily
`thereafter for 5 d.
`
`Results
`
`Linker optimization. Antibody—DM1 conjugates were originally
`designed with a disulfide-based linker for release of active drug by
`intracellular reduction (24). Recently, it was discovered that the
`endocytic pathway is oxidizing and that cleavage of the disulfide
`linker, SPP,
`is very inefficient
`(36). Thus, different trastuzumab
`ADCS were constructed to investigate the effect of disulfide linker
`hindrance on the biological activity of these conjugates (Fig. 1). A
`trastuzurnab—DM1 conjugate made with the SPDP linker contains
`no methyl substitutions adjacent to the disulfide bond and is
`therefore the least hindered disulfide—containing design. Trastuzu-
`mab ADCS composed of SPP—DM1, SSNPP—DM3, or SSNPP-DM4
`contain one, two, or three methyl groups, respectively, around the
`disulfide bridge and show increasing resistance to cleavage via
`thiol-disulfide exchange reactions?’ DMS and DM4 nomenclature
`
`3 ImmunoGen, Inc., unpublished data.
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`PHIGENIX
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`reflects addition of methyl groups to the DM1 (drug) moiety
`(addition of one or two methyls, respectively). An additional ADC
`containing a thioether linker
`(SMCC, designated MCC after
`conjugation) was also constructed. In Vitro potency assays were
`conducted for 3 days with the HER2-amplified breast cancer lines
`BT-474 and SK-BR-3 treated with various trastuzumab ADCs. This
`
`short treatment period was selected to minimize the effects of
`unconjugated trastuzumab on these trastuzumab-sensitive lines.
`The graphs in Fig. 2A show enhanced potency of trastuzumab-
`maytansinoid conjugates compared with trastuzumab in both cell
`lines, with no significant difference in activity among the various
`ADCs tested (IC50, 0.085—0.148 ug/mL for BT-474 and 0.007-
`0.018 ug/mL for SK-BR-3). Because the effect of trastuzumab is
`cytostatic in nature, the enhanced potency of the ADC is, thus, due
`to exposure of the cells to the cytotoxic maytansinoid. In addition,
`brief exposure of SK-BR-3 cells for 10, 30, or 60 minutes to
`trastuzumab-MCC-DM1, followed by a 3-day incubation in culture
`medium, also resulted in substantial growth inhibition (data not
`shown). When comparing potency as molar DM1 equivalents, DM1
`conjugated to trastuzumab is 5-fold more potent than free L-DM1
`on SK-BR-3 and shows equal potency to free L-DM1 on BT-474 cells
`(Fig. 2B). Nontargeted effects of trastuzumab-maytansinoid con-
`jugates were assessed on breast cancer lines expressing normal
`levels (MCF7) or lacking expression (MDA-MB-468) of HER2 (37).
`All ADCs tested showed minimal antiproliferative activity in both
`cell lines (Fig. 2A), whereas free DM1 showed potency equal to that
`observed on the HER2-amplified lines (Fig. 2B). Treatment of the
`four cell lines with an isotype-matched control ADC, anti—IL-8-
`MCC-DM1,
`resulted in negligible growth inhibition (data not
`shown). Thus, like trastuzumab, maytansinoid-conjugated trastu-
`zumab ADCs are specific for HER2-overexpressing cells. Further-
`more, the different linkers behave similarly with respect to in Vitro
`antiproliferative activity.
`Pharmacokinetic analyses in nude mice were performed to
`determine the effect of different
`trastuzumab ADC linkers on
`serum concentration.
`In contrast
`to the cell culture results,
`we observed a clear correlation between ADC exposure and
`linker hindrance. The ADC with the least hindered disulfide bond
`
`(no adjacent methyl groups), trastuzumab-SPDP-DM1, showed the
`fastest clearance and was undetectable by day 3. The addition of
`methyl groups (CH3) on either side of the S-S bond (trastuzumab-
`SPP-DM1 with one CH3 on the antibody side of the S-S;
`trastuzumab-SSNPP-DM3 with two CH3, one on either side of
`the S-S;
`trastuzumab-SSNPP-DM4 with three CH3, one on the
`antibody side, and two on the drug side of the S-S) resulted in
`increasingly sustained ADC serum concentrations (Fig. 3A,
`left).
`Trastuzumab-MCC-DM1 (nonreducible) and trastuzumab-SSNPP-
`DM4 (the most hindered disulfide-containing design) showed
`similar pharmacokinetics with the ADC concentration at 70% of
`the total antibody at day 7. Studies were also performed to
`compare clearance of DM1-conjugated trastuzumab to total
`trastuzumab levels (Fig. 3A,
`right). Serum concentrations of
`trastuzumab-MCC-DM1 measured for 1 week were not different
`
`from total serum trastuzumab concentration, indicating that, with
`the nonreducible MCC linker,
`the amount of maytansinoid
`released from the antibody was negligible over the 7 days. In
`contrast, only 11% of the trastuzumab-SPP-DM1, compared with
`total
`trastuzumab antibody concentration,
`remained in the
`circulation after 7 days.
`In Vivo efficacy studies examining the different trastuzumab-
`maytansinoid conjugates given as a single i.v. dose were carried
`
`Trastuzumab-DM1 Antibody-Drug Conjugate
`
`out using the MMTV-HER2 Fo5 mammary tumor transplant
`(trastuzumab-resistant) model. This model was developed by
`serial
`transplantation of tumors derived from the HER2
`transgenic mouse lineage founder 5 (35). These mouse tumors
`overexpress human HER2 (3+ expression) as measured by
`immunohistochemistry and quantitative reverse transcription-
`PCR. Interestingly, established tumors from this model do not
`respond to single-agent trastuzumab therapy. Consistent with the
`pharmacokinetic analysis,
`increased linker
`stability in viva
`correlated with increased antitumor activity for
`trastuzumab
`ADCs (Fig. 3B). Tumor volume in both trastuzumab-MCC-DM1
`and trastuzumab-SSNPP-DM3 treatment groups were statistically
`different from trastuzumab-SPP-DM1 (log-rank P values of 0.0165
`and 0.0414, respectively). Further characterization of the HER2
`transgenic models revealed the presence of high circulating levels
`of shed HER2 ECD in the serum of Fo5 tumor-bearing mice.
`Moreover, analysis of these tumors revealed increased expression
`of the transmembrane-containing fragment p95HER2, which is
`highly tyrosine-phosphorylated, indicating constitutive activation.
`In comparison, the trastuzumab-sensitive F2#1282 model showed
`1,000-fold lower levels of circulating HER2 ECD, no detectable
`p95HER2, and significant tumor growth reduction after a single
`injection of different doses of trastuzumab-DM1 (data not
`shown). Thus, the trastuzumab-DM1 conjugate is efficacious in
`models,
`such as Fo5, which express
`reported markers of
`resistance to trastuzumab,
`i.e., high circulating HER2 ECD and
`activated p95HER2.
`Short-term,
`single-dose toxicity studies were performed in
`female rats comparing the effects on body weight of free or
`conjugated (SMCC or SPP) DM1. ADCs were given as DM1
`equivalents (Hg/m2). As
`trastuzumab does not
`recognize rat
`erbB2, this model measures antigen-independent ADC effects. Of
`the agents tested, trastuzumab-MCC-DM1 showed the best safety
`profile. Body weight gain in rats given 1,632 ug/m2 DM1 (25 mg/
`kg antibody) was comparable with vehicle control animals (15.9%
`and 16.3%, respectively; Fig. 3C). In contrast, administration of
`trastuzumab-SPP-DM1 at an equivalent DM1 dose resulted in
`considerable body weight
`loss (—10%) by end of study. Rats
`treated with 3,264 Hg/m2 (50 mg/kg) trastuzumab-MCC-DM1 did
`not exhibit body weight
`loss; however,
`the amount of weight
`gained during the course of study (+6.7%) was less than in the
`vehicle control and the 1,632 ug/m2 trastuzumab-MCC-DM1
`groups. The change in body weight with 3,264 ug/m2 trastuzu-
`mab-MCC-DM1 was similar to that observed with free DM1 at a
`
`dose of 653 Hg/m2 (+6.5%). In a separate study, clinical chemistry
`analysis of rats treated with trastuzumab-MCC-DM1 showed
`transient elevation of liver enzymes (aspartate aminotransferase,
`alanine aminotransferase, and ~/-glutamyl
`transpeptidase) and
`mild, reversible thrombocytopenia at a dose of 20 mg/kg, and no
`clinical signs of toxicity at 6 mg/kg (data not shown). Evidently,
`substituting a reducible linker (SPP) with a nonreducible linker
`(SMCC) results in a less toxic but still efficacious trastuzumab-
`DM1 conjugate. Consequently, fiirther studies focused on defining
`the activity of trastuzumab-MCC-DM1.
`tumor cells
`HER2-overexpressing,
`trastuzumab-resistant
`respond to trastuzumab-MCC-DM1, whereas normal cell lines
`are unaffected. The effects of trastuzumab-MCC-DM1 on in Vitro
`
`proliferation were fiirther examined in several tumor lines shown
`to be resistant to trastuzumab. Additional breast cancer lines,
`HCC1954, KPL-4, and BT-474 EEI, all with 3+ HER2 expression,
`were first
`tested. The BT-474 EEI cell
`line was developed by
`
`www.aacrjourna|s.org
`
`9283
`
`Cancer Res 2008; 68: (22). November 15, 2008
`
`Downloaded from cancerres.aacrjourna|s.org on February 9, 2014. © 2008 American Association for Cancer
`Research.
`
`PHIGENIX
`
`Exhibit 1004-05
`
`

`
`Cancer Research
`
`
`Relativecell
`
`
`
`viability(cellularATPcontent,percentofcontrol)
`
`>3:
`".5
`E5>
`T;L)
`cu
`.2..
`‘.5an
`
`
`
`
`
`LC(cellularATPcontent,percentofcontrol)
`
`Relativecell
`
`
`
`
`
`viability(cellularATPcontent,percentofcontrol)
`
`0.001
`
`0.01
`
`0.1
`
`1
`
`Trastuzumab-DM concentration (ug/mL)
`
`;mS.-”~‘F‘mD:'\/it
`
`ii‘.*a2ii:i~5.§§.‘>N§-‘t-‘~L:
`
`
`Relativecell
`
`
`
`viability(cellularATPcontent,percentofcontrol)
`
`00O
`
`-l>O
`
`|\JCD0O
`
`00O
`
`0.001
`
`0.01
`
`0.1
`
`1
`
`Trastuzumab-DM concentration (ug/mL)
`
`MDA-MB-468
`
`0.001
`
`0.01
`
`0.1
`
`1
`
`0.001
`
`0.01
`
`0.1
`
`1
`
`Trastuzumab-DM concentration (pg/mL)
`
`Trastuzumab-DM concentration (pg/mL)
`
`In vitro activity of trastuzumab-DM1 conjugates versus free DM1
`
`(K250, nmol/L DM1 equivalents)
`
`trastuzumab-SPF’-DM1
`
`trastuzumab-MCC-DM1
`
`free L-DM1
`
`SK-BR-3
`
`BT-474
`
`MCF7
`
`MDA-MB-468
`
`0.24
`
`2.71
`
`>100
`
`18.37
`
`0.22
`
`4.26
`
`>100
`
`41.37
`
`1.00
`
`4.52
`
`2.92
`
`0.59
`
`Cells were treated with trastuzumab-DM1 conjugates or L-DM1 as free drug for72 h;
`|C50 values were determined from four-parameter curve fitting.
`
`Figure 2. Trastuzumab—maytansinoid conjugates are selective for HER2—amp|ified breast cancer lines, whereas free DM1 shows equivalent potency on all cell lines
`tested regardless of HER2 expression. A, trastuzumab—maytansinoid conjugates show enhanced antiproliferative activity on HER2—overexpressing breast tumor cell
`lines compared with trastuzumab. The nature of the linker, however, does not affect in vitro activity. B, in vitro potency of Tmab—SPP—DM1 and Tmab—MCC—DM1
`compared with the free drug L-DM1 afler 3 d of treatment (lC50 values, nmol/L DM1 equivalents). HER2 expression: SK—BR—3 (HER2 3+), BT-474 (HER2 3+), MCF7
`(normal HER2 expression), MDA-MB-468 (HER2—negative).
`
`Cancer Res 2008; 68: (22). November 15, 2008
`
`9284
`
`www.aacrjourna|s.org
`
`Downloaded from cancerres.aacrjournals.org on February 9, 2014. © 2008 American Association for Cancer
`Research.
`
`PHIGENIX
`
`Exhibit 1004-06
`
`

`
`Trastuzumab-DM1 Antibody-Drug Conjugate
`
`If
`E\
`cs:
`3C
`.9..
`cu
`-.5C
`0:C.)C
`
`oDE3.
`
`_
`as
`CD
`
`DM1-conjugatedantibody(%oftotal
`
`antibody)
`
`‘M.-.'-.
`
`-.5.”
`
`’3‘i':':£.‘ii::f-J5“.-‘('";?€§"‘§*"*3'.'?i“‘fi3
`
`00O0
`
`ETAO0
`
`
`
`Percentweightchange
`
`
`
`(meaniSE)
`
`
`
`Meantumorvolume(mm
`
`
`
`
`
`Figure 3. Trastuzumab conjugated to DM1 through the thioether SMCC linker displays better efficacy and pharmacokinetics and lower toxicity than conjugates
`with disulfide linkers. A, pharmacokinetic analysis of trastuzumab—maytansinoid conjugates shows increased serum concentrations of conjugates with more hindered
`disulfide linkers. Female beige nude mice were given a single i.v. injection (2 mg/kg) of different trastuzumab—maytansinoid conjugates (n = 4 mice per group).
`Serum samples were collected at various time points afler injection for measurement of total and conjugated trastuzumab as described. B, increased in vivo linker
`stability results in improved efficacy in vivo. Mice bearing mammary tumor transplants from the MMTV—HER2 Fo5 line were given a single i.v. injection (10 mg/kg)
`of Tmab—SPP—DM1, Tmab—SSNPP—DM3, Tmab—SSNPP—DM4, Tmab—MCC—DM1, or vehicle (n = 7 mice per group), and tumor growth was monitored for 25 d.
`C, Tmab—MCC—DM1 displays the best safety profile, as assessed by changes in body weight, of the conjugates tested. Female Sprague—Dawley rats were given a
`single i.v. injection of Tmab—MCC—DM1 (1,632 or 3,264 pg/m2, 25 or 50 mg/kg, respectively), Tmab—SPP—DM1 (1,6