Oncogene (1999) 18, 2241 ± 2251
`ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00
`http://www.stockton-press.co.uk/onc
`
`Inhibitory e€ects of combinations of HER-2/neu antibody and
`chemotherapeutic agents used for treatment of human breast cancers
`
`Mark Pegram1, Sheree Hsu1, Gail Lewis2, Richard Pietras1, Malgorzata Beryt1, Mark Sliwkowski2,
`Daniel Coombs2, Deborah Baly2, Fairooz Kabbinavar1 and Dennis Slamon*,1
`
`1Division of Hematology-Oncology, UCLA School of Medicine, Los Angeles, California 90095, USA; 2Genentech, Inc. One DNA
`Way, South San Francisco, California, USA
`
`Previous studies have demonstrated a synergistic interac-
`tion between rhuMAb HER2 and the cytotoxic drug
`cisplatin in human breast and ovarian cancer cells. To
`de®ne the nature of the interaction between rhuMAb
`HER2 and other classes of cytotoxic drugs, we applied
`multiple drug e€ect/combination index (CI) isobologram
`analysis to a variety of chemotherapeutic drug/rhuMAb
`HER2 combinations in vitro. Synergistic interactions at
`clinically relevant drug concentrations were observed for
`rhuMAb HER2 in combination with cisplatin (CI=0.48,
`P=0.003), thiotepa (CI=0.67, P=0.0008), and etopo-
`side (CI=0.54, P=0.0003). Additive cytotoxic e€ects
`were observed with rhuMAb HER2 plus doxorubicin
`(CI=1.16, P=0.13), paclitaxel
`(CI=0.91, P=0.21),
`methotrexate
`(CI=1.15, P=0.28),
`and
`vinblastine
`(CI=1.09, P=0.26). One drug, 5-¯uorouracil, was found
`to be antagonistic with rhuMAb HER2 in vitro
`(CI=2.87, P=0.0001). In vivo drug/rhuMAb HER2
`studies were conducted with HER-2/neu-transfected,
`MCF7 human breast cancer xenografts in athymic mice.
`Combinations of rhuMAb HER2 plus cyclophosphamide,
`doxorubicin, paclitaxel, methotrexate, etoposide, and
`vinblastine in vivo resulted in a signi®cant reduction in
`xenograft volume compared to chemotherapy alone
`(P50.05). Xenografts treated with rhuMAb HER2 plus
`5-¯uorouracil were not signi®cantly di€erent from 5-
`¯uorouracil alone controls consistent with the subadditive
`e€ects observed with this combination in vitro. The
`synergistic interaction of rhuMAb HER2 with alkylating
`agents, platinum analogs and topoisomerase II inhibitors,
`as well as
`the additive
`interaction with taxanes,
`anthracyclines and some antimetabolites in HER-2/neu-
`overexpressing breast cancer cells demonstrates that these
`are rational combinations to test in human clinical trials.
`
`Keywords: HER-2/neu (c-erbB-2); chemotherapy; breast
`cancer; multiple drug e€ects analysis; synergy
`
`Introduction
`
`Overexpression of p185HER-2/neu, resulting from amplifi-
`cation of the HER-2/neu gene, is associated with poor
`clinical outcome in 25 ± 30% of carcinomas of
`the
`breast (Slamon et al., 1987), as well as in other human
`
`*Correspondence: DJ Slamon, UCLA School of Medicine,
`Department of Medicine, Division of Hematology-Oncology, 11-934
`Factor Building, Los Angeles, CA 90095, USA
`Received 13 May 1998; revised 27 October 1998; accepted 27 October
`1998
`
`malignancies (Semba et al., 1985; Slamon et al., 1989;
`Berchuck et al., 1991; Yonemura et al., 1991; Hetzel et
`al., 1992; Lukes et al., 1994; Press et al., 1994; Sa€ari
`et al., 1995). The murine monoclonal antibody 4D5 has
`speci®city
`for
`a
`juxtamembrane
`epitope
`in the
`extracellular domain (ECD) of the p185HER-2/neu protein
`(Fendly et al., 1990) and is capable of eliciting an
`antiproliferative e€ect against murine cells transformed
`by HER-2/neu as well as human malignant cell lines
`and xenografts overexpressing this oncogene (Chazin et
`al., 1992). Importantly, this growth inhibitory e€ect is
`speci®c for cells with HER-2/neu overexpression and
`does not occur with cells expressing normal amounts of
`the protein (Hudziak et al., 1989; Chazin et al., 1992).
`A recombinant, humanized form of 4D5 (rhuMAb
`HER2) has been generated by inserting the comple-
`mentary-determining regions (CDRs) of 4D5 into the
`framework of a consensus human IgG1 (Carter et al.,
`1992). When compared to murine 4D5,
`rhuMAb
`HER2
`exhibits
`a
`stronger binding
`anity
`for
`p185HER-2/neu but has similar speci®c antiproliferative
`activity against HER-2/neu-overexpressing cell
`lines
`and xenografts.
`To determine how best to use this antibody both as
`a single agent and in combination with established
`cancer therapeutics, we undertook a series of studies to
`evaluate its inhibitory e€ects in preclinical models in
`vitro and in vivo. These studies were based on a
`previous
`report of enhanced activity of cisplatin
`(CDDP) when used in combination with antibodies
`directed against the epidermal growth factor receptor
`(EGFR)
`(Aboud-Pirak et al., 1988). Initial studies
`showed that when used in combination with the drug
`CDDP, 4D5, rhuMAb HER2, as well as other anti-
`HER-2/neu antibodies, potentiate cytotoxicity of the
`chemotherapeutic by decreasing DNA repair activity
`following CDDP-induced DNA damage (Hancock et
`al., 1991; Pietras et al., 1994). This e€ect,
`termed
`receptor enhanced chemosensitivity (REC), speci®cally
`targets HER-2/neu-overexpressing cells and has no
`e€ect on cells or tissues expressing physiologic levels of
`the gene. The interaction between 4D5 and CDDP in
`inhibiting HER-2/neu-overexpressing cell lines has been
`shown to be synergistic resulting in a two-log increase
`in CDDP-induced cytotoxicity as well as pathologic
`complete remissions in experimental animals bearing
`HER-2/neu-overexpressing human breast cancer xeno-
`grafts (Pietras et al., 1994).
`Synergy, as it applies to drug-drug interactions, is
`de®ned as a combination of two or more drugs which
`achieves a therapeutic e€ect greater than that expected
`by the simple addition of the e€ects of the component
`drugs. Such synergistic interactions between drugs may
`
`1 of 11
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`

`2242
`
`Anti-HER-2/neu antibody and chemotherapy combinations
`M Pegram et al
`
`improve therapeutic results in cancer treatment if the
`synergism is speci®c for tumor cells (Chou and Talalay,
`1984). Moreover, analysis of
`the nature of
`the
`interaction between two drugs (synergism, addition,
`or antagonism) may yield insight into the biochemical
`mechanisms of interaction of the drugs. For example,
`two drugs targeting the same enzyme or biochemical
`pathway may compete with one another resulting in an
`antagonistic interaction, whereas two drugs targeting
`completely independent pathways may be additive, and
`one drug which potentiates the action of another may
`result in therapeutic synergy.
`In order to characterize the e€ects of combinations
`of rhuMAb HER2 cytotoxic chemotherapeutic drugs
`commonly used in breast cancer therapy, we utilized
`the median-e€ect/combination-index
`isobologram
`method of multiple drug e€ect analysis. With this
`methodology,
`combination index (CI) values are
`calculated for di€erent dose-e€ect
`levels based on
`parameters derived from median-e€ect plots of the
`chemotherapeutic drugs alone, rhuMAb HER2 alone,
`and the combination of the two at ®xed molar ratios.
`CI values 51 indicate
`synergy, CI=1 indicates
`addition, and CI41 denotes antagonism (Chou and
`Talalay, 1984). We performed this analysis with
`rhuMAb HER2 in combination with eight drugs
`representing
`seven di€erent
`classes of
`cytotoxic
`chemotherapeutics in vitro. Assays were performed in
`for drug/rhuMAb HER2
`combinations
`at
`vitro
`clinically relevant drug/antibody concentrations using
`a cytotoxicity endpoint employing SK-BR-3 human
`breast cancer cells which contain HER-2/neu gene
`ampli®cation/overexpression. In addition, to circum-
`vent the possibility that any observed interaction might
`be unique to an individual cell
`line or to a speci®c
`method of analysis, parallel studies were conducted in
`vivo with the same rhuMAb HER2/drug combinations.
`HER-2/neu-transfected MCF7 human breast carcino-
`ma xenografts which, in contrast to SK-BR-3 cells are
`tumorigenic in athymic mice, served as the tumor
`target for the in vivo studies. Using this model we also
`investigated the e€ect of various chemotherapeutic
`drugs on the pharmacokinetics of rhuMAb HER2 in
`a subset of mice receiving either rhuMAb HER2 alone
`or rhuMAb HER-2 plus cytotoxic drug. Finally, we
`
`sought to assess the e€ect of xenograft size (i.e. tumor
`burden) on rhuMAb HER2 serum concentrations.
`
`Results
`
`Multiple drug e€ect analysis of rhuMAb HER2 in
`combination with cytotoxic chemotherapy drugs on
`SK-BR-3 breast carcinoma cells in vitro
`
`To extend the observations on anti-HER2 monoclonal
`antibodies
`in combination with CDDP, and to
`conduct a comprehensive survey of rhuMAb HER2
`in combination with other
`classes of
`cytotoxic
`chemotherapeutic drugs available for clinical use,
`rhuMAb HER2 was analysed in combination with
`seven di€erent drug classes. Representative drugs
`included:
`the anthracycline antibiotic, doxorubicin
`(DOX);
`the
`taxane drug, paclitaxel
`(TAX);
`a
`topoisomerase
`II
`inhibitor
`etoposide
`(VP-16);
`a
`platinum analog cisplatin (CDDP); a vinca alkaloid
`vinblastine (VBL);
`the alkylating agents,
`thiotepa
`(TSPA) for in vitro experiments and cyclophospha-
`mide
`(CPA)
`for
`experiments;
`and the
`in
`vivo
`antimetabolite drugs methotrexate (MTX) and 5-
`¯uorouracil (5-FU).
`curves were
`response
`In this
`analysis, dose
`constructed for each drug alone,
`rhuMAb HER2
`alone, and the combinations at ®xed molar ratios
`de®ned as
`the ratio of
`the two agents at
`their
`maximally e€ective dose. A representative example of
`the multiple drug e€ect analyses performed for all of
`the chemotherapeutic agent/rhuMAb HER2 combina-
`tions is shown for the alkylating agent TSPA (Figure
`1 and Table 1). In this analysis Fa and Fu are the
`fractions of SK-BR-3 cells a€ected or una€ected,
`respectively, by the dose (D) of either agent (drug or
`antibody). DM is the dose required to produce the
`median e€ect (analogous to the IC50), and m is the
`Hill coecient used to determine whether the dose
`e€ect
`relationships
`follow sigmoidal dose-response
`curves
`(Hill, 1913). Linear
`regression correlation
`coecients
`(r-values) of
`the median e€ect plots
`(Table 1) re¯ect that the dose-e€ect relationships for
`TSPA, rhuMAb HER2, and the combination, con-
`
`(a) Multiple drug e€ect plot of TSPA, rhuMAb HER2 and the combination where Fa = the fraction of SK-BR-3 cells
`Figure 1
`a€ected by the drugs, Fu = the fraction of cells una€ected, and D = drug dose. (b) Combination Index values for TSPA in
`combination with rhuMAb HER2 at multiple e€ect levels. CI values 51 indicate synergy
`
`2 of 11
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`

`Anti-HER-2/neu antibody and chemotherapy combinations
`M Pegram et al
`
`2243
`
`form to the principle of mass action (in general, r-
`values 40.9 con®rm the validity of this methodology)
`(Chou and Talalay,
`1984). CI
`values
`for
`the
`combination of TSPA and rhuMAb HER2 were
`signi®cantly less
`than 1.0 across all combination
`doses
`tested (P=0.0008)
`indicating a synergistic
`interaction (Figure 1b). A summary of the data from
`the same analysis applied to each of
`the eight
`cytotoxic drug/rhuMAb HER2 combinations tested
`(Table 2) demonstrates that CDDP, TSPA, and VP-16
`(CI51;
`exhibit
`synergistic
`therapeutic
`interactions
`P50.001) with rhuMAb HER2 across a wide range
`(*0.2 ± 0.8) of Fa
`values. Additive
`interactions
`(CI=1) were observed for TAX, DOX, MTX, and
`VBL in combination with rhuMAb HER2, while only
`one drug, 5-FU, was found to exhibit an antagonistic
`(CI41; P=0.0001) interaction (Table 2).
`
`P185HER-2/neu expression and tyrosine phosphorylation
`following exposure to cytotoxic agents
`
`Previous work has demonstrated that exposure of
`several cancer cell
`lines to the anthracycline DOX
`results in an increase in expression of the EGFR and/
`ligand TGF-a (Zuckier and Tritton, 1983;
`or
`its
`Hanauske et al., 1987; Baselga et al., 1992, 1993).
`This phenomenon has been proposed to explain the
`synergistic cytotoxic e€ects of DOX used in combina-
`tion with anti-EGFR monoclonal antibodies (Baselga
`et al., 1992). To test whether p185HER-2/neu expression is
`similarly altered by DOX, protein expression levels
`were measured at various
`times
`following DOX
`exposure (Figure 2a). These studies demonstrate that
`following exposure to DOX, p185HER-2/neu expression
`levels in SK-BR-3 breast carcinoma cells are unaltered,
`unlike
`the
`reported e€ects of DOX on EGFR
`expression in A431 cells (Baselga et al., 1992). We
`next considered the possibility that cytotoxic drugs
`may impact p185HER-2/neu functional activity rather than
`expression levels. We therefore determined the e€ect of
`the various cytotoxic drugs on heregulin B-1 and 4D5-
`induced
`tyrosine
`phosphorylation
`of
`p185HER-2/neu
`
`Figure 2 (a) Expression of p185HER-2/neu
`in SK-BR-3 cells
`following exposure to DOX at the IC30 (30 nM) concentration
`for
`the
`times
`indicated.
`(b) MAb 4D5-induced tyrosine
`phosphorylation of p185HER-2/neu in SK-BR-3 cells following
`exposure to chemotherapeutic agents at the IC30 concentration at
`the indicated time points. 4D5-associated tyrosine phosphoryla-
`tion (lane 2) was observed under all of
`the chemotherapy
`conditions tested (lanes 3 ± 11) compared to control (lane 1). (c)
`Heregulin-induced p185HER-2/neu
`tyrosine phosphorylation in
`MCF7 cells following exposure to chemotherapeutic drugs at
`the IC30 concentration. These data demonstrate that p185HER-2/neu
`expression and phosphorylation state are unaltered by prior
`exposure to the chemotherapeutic agents tested
`
`Table 1 Calculated values for the Combination Index as a function of fractional inhibition of SK-BR-3 cell proliferation by a mixture of TSPA
`and rhuMAb HER2
`
`Drug
`
`TSPA
`rhuMAb HER2
`TSPA+rhuMAb HER2
`Diagnosis of combined e€ect
`
`ED30
`
`Combination Index Values
`ED50
`
`ED40
`
`ED60
`
`ED70
`
`0.52
`Synergy
`
`0.37
`Synergy
`
`0.41
`Synergy
`
`0.49
`Synergy
`
`0.60
`Synergy
`
`Parameters
`m
`
`Dm
`
`66.2 mM
`675.0 nM
`27.1 mM
`
`0.81
`0.15
`0.59
`
`r
`
`0.99
`0.96
`0.99
`
`Table 2 Mean combination index values for chemotherapeutic drug/rhuMAb HER2 combinations in vitro
`
`Drug
`
`TSPA
`CDDP
`VP-16
`DOX
`TAX
`MTX
`VBL
`5-FU
`
`rhuMAb HER2/drug
`molar ratio
`6.461075
`4.061074
`9.961074
`9.861073
`1.461071
`3.361071
`1.7
`8.861075
`P values indicate level of signi®cance compared to CI=1.0
`
`Drug Dose Range
`(mM)
`
`8.25 ± 1.066103
`6.561071 ± 1.76102
`2.661071 ± 6.86101
`2.761072 ± 6.9
`1.861073 ± 5.061071
`8.061074 ± 2.061071
`1.661074 ± 3.961072
`3.0 ± 7.656102
`
`Combination Index
`(Mean+s.e.m.)
`
`0.67+0.12
`0.56+0.15
`0.54+0.15
`1.16+0.18
`0.91+0.23
`1.36+0.17
`1.09+0.19
`2.87+0.51
`
`P value
`
`0.0008
`0.001
`0.0003
`0.13
`0.21
`0.21
`0.26
`0.0001
`
`Interaction
`
`Synergy
`Synergy
`Synergy
`Addition
`Addition
`Addition
`Addition
`Antagonism
`
`3 of 11
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`

`2244
`
`Anti-HER-2/neu antibody and chemotherapy combinations
`M Pegram et al
`
`(Yarden, 1990; Holmes et al., 1992). MCF7 or SK-BR-
`3 breast carcinoma cells were treated with cytotoxic
`drugs, then allowed to incubate with heregulin (10 nM),
`or 4D5 (12.5 mg/ml). Protein lysates were then analysed
`by anti-phosphotyrosine immunoblot. These studies
`demonstrate an increase in
`p185HER-2/neu
`tyrosine
`phosphorylation following incubation with 4D5 com-
`pared to a non-speci®c isotype control antibody
`(Figure 2b, lanes 1 and 2). Prior exposure of the cells
`to the three drugs which were found to be synergistic
`with anti-HER-2/neu antibody (CDDP, TSPA, and
`VP-16) had no e€ect on 4D5-induced p185 tyrosine
`phosphorylation (Figure 2b, lanes 3 ± 7 and lanes 9 and
`10). Similarly, neither DOX which is additive, nor 5-
`FU which is antagonistic, had e€ects on 4D5-induced
`p185 tyrosine phosphorylation (Figure 2b, lanes 8 and
`11). In addition, when heregulin B-1 is used to activate
`p185HER-2/neu kinase, preincubation of MCF7 breast
`carcinoma cells with CDDP or DOX had no e€ect
`on heregulin-induced p185HER-2/neu tyrosine phosphoryla-
`tion (Figure 2c). Preincubation of MCF7 cells with
`TSPA, VP-16, TAX, MTX, VBL, or 5-FU likewise had
`no e€ect on heregulin-induced p185HER-2/neu
`tyrosine
`phosphorylation (data not shown). Taken together
`
`these data demonstrate that none of the synergistic,
`additive, or antagonistic e€ects of chemotherapeutic
`drugs with anti-HER-2/neu antibody can be explained
`on the basis of either chemotherapy-induced alteration
`of p185HER-2/neu protein expression levels or
`its
`phosphorylation.
`
`Anti-HER-2/neu antibodies alter cell cycle distribution
`of HER-2/neu-overexpressing human breast cancer cells
`
`The cytotoxic e€ects of antimetabolite drugs are cell
`cycle dependent
`(Tannock, 1978). To identify a
`possible mechanism for the antagonism of 5-FU with
`rhuMAb HER2 we investigated the e€ects of murine
`4D5 and rhuMAb HER2 on cell cycle distribution of
`exponentially growing SK-BR-3 and MCF7 cells in
`vitro (Figures 3 and 4). Both the murine 4D5 and
`rhuMAb HER2 antibodies reduce the percentage of
`cells undergoing S phase as well as
`increase the
`percentage of cells in G0/G1, and these e€ects are
`dose-dependent with the maximal antiproliferative
`activity occurring at antibody concentrations between
`1 and 10 mg/ml (Figure 4). There was no signi®cant
`di€erence in the magnitude of decrease in S phase
`
`Figure 3 DNA ¯uorescence ¯ow cytometry histograms of propidium iodide-stained nuclei obtained from MCF7 (a ± c) and SK-
`BR-3 (d ± f) breast carcinoma cells following treatment with control antibody 6E10, murine anti-p185HER-2/neu antibody 4D5, or
`humanized anti-p185HER-2/neu antibody (rhuMAb HER2) at a dose of 1 mg/ml for 72 h. These data demonstrate a signi®cant
`reduction in the fraction of breast carcinoma cells undergoing S phase following treatment with anti-HER-2 antibodies 4D5 and
`rhuMAb HER2. This e€ect is speci®c for cells with HER-2/neu-overexpression (SK-BR-3 cells)
`
`4 of 11
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`

`2245
`
`Anti-HER-2/neu antibody and chemotherapy combinations
`M Pegram et al
`
`athymic mice. All of the doses, routes of administra-
`tion, and dose intervals for the various cytotoxic drugs
`and rhuMAb HER2 were based on independent dose
`®nding experiments for this speci®c strain, age, weight,
`and sex of athymic mouse. The cytotoxic drug doses
`used were at or near the maximum tolerated doses
`previously reported in the literature (Giovanella et al.,
`1977; Boven and Winograd, 1991).
`For the alkylating agent cyclophosphamide CPA,
`combination with rhuMAb HER2 resulted in a
`signi®cant reduction (P50.05)
`in day 21 xenograft
`volume compared to either agent alone (Figure 5a).
`The combination of the anthracycline antibiotic DOX
`plus rhuMAb HER2 also signi®cantly reduced MCF7/
`HER-2 xenograft volume compared to either single
`agent alone (Figure 5b). The combination of
`the
`taxane compound TAX plus rhuMAb HER2, which
`demonstrated an additive interaction in vitro, resulted
`in a signi®cant reduction in day 20 xenograft volume
`compared to treatment with TAX alone (Figure 5c).
`However,
`the di€erence between rhuMAb HER2
`alone and rhuMAb HER2 plus TAX did not reach
`statistical
`signi®cance. This
`is
`likely due
`to the
`relatively small sample size in each group and the
`fact that the dose of rhuMAb HER2 in this particular
`analysis (10 mg/kg I.P. twice weekly) yielded a marked
`reduction in xenograft growth even when used as a
`single agent.
`The following four rhuMAb HER2/drug combina-
`tions were studied in a single in vivo experiment. For
`this experiment, a `rational dose' (RD) or rhuMAb
`HER2 was
`chosen as new information became
`available based on comparative pharmacokinetic
`studies from both humans and athymic mice. RD is
`the dose of a given drug which can reproduce a serum
`level in experimental animals similar to that observed
`in human subjects (Inaba et al., 1988). The RD for
`rhuMAb HER2 resulted in a lower
`cumulative
`rhuMAb HER2 dose (16 mg/kg vs 30 ± 50 mg/kg)
`during the 21 day observation period for
`this
`experiment compared to the three in vivo studies
`reported above. With this approach, a signi®cant
`reduction in day 21 xenograft volume was observed
`for the topoisomerase II inhibitor VP-16 when used in
`combination with rhuMAb HER2 compared to either
`agent alone (Figure 6a). The combination of
`the
`microtubule inhibitor VBL with rhuMAb HER2 also
`signi®cantly reduced MCF7/HER-2 xenograft volume
`compared to treatment with VBL alone or single agent
`rhuMAb HER2 (Figure 6b). For the antimetabolite
`class of cytotoxic chemotherapeutics, two drugs with
`clinical activity against breast cancer were chosen for
`combination studies. Treatment with MTX, which
`targets dihydrofolate reductase, plus rhuMAb HER2
`resulted in a signi®cant reduction in day 21 MCF7/
`HER-2 xenograft volume when compared to either
`MTX alone or rhuMAb HER2 alone (Figure 6c).
`Finally, the antimetabolite drug 5-FU, which targets
`thymidylate synthetase, and which was found to be
`antagonistic when combined with rhuMAb HER2 in
`vitro, did not yield a signi®cant reduction in xenograft
`volume when compared to 5-FU alone in vivo (Figure
`6d). Although the combination of rhuMAb HER2 plus
`5-FU was superior to rhuMAb HER2 alone in this
`(P50.05),
`experiment
`the 5-FU dose used had
`sucient anti-tumor ecacy as a single agent such
`
`Figure 4 E€ect of anti-p185HER-2/neu MAb dose on cell cycle
`distribution of breast cells without (a) and with (b) HER-2/neu
`overexpression
`
`fraction of SK-BR-3 cells comparing 4D5 and rhuMAb
`HER2 indicating the humanization of
`the murine
`antibody did not adversely impact its antiproliferative
`activity. The lack of any e€ect on cell cycle distribution
`of MCF7 cells demonstrates the speci®city of these
`antibodies for cells with HER-2/neu overexpression.
`These data suggest that a decrease in the percentage of
`SK-BR-3 cells in S phase may result in a decreased
`sensitivity to 5-FU. An antagonistic interaction for the
`combination of rhuMAb HER2 with the antimetabo-
`lite MTX was not observed. The lack of antagonism
`between MTX and rhuMAb HER2 in vitro may be due
`to the longer incubation period required for MTX
`(120 vs 72 h) to elicit cytotoxicity in the assay used for
`the multiple drug e€ect analysis, and the fact that
`MTX exerts cytotoxic e€ects in other phases of the cell
`cycle in addition to S phase (Buick, 1994).
`
`E€ect of rhuMAb HER2 in combination with multiple
`chemotherapeutic drugs on growth of HER-2/neu-
`transfected MCF7 breast xenografts in vivo
`
`To further evaluate the potential therapeutic e€ects of
`rhuMAb HER2/chemotherapy combinations and to
`extend our observations beyond a single cell line and
`preclinical model, a series of
`in vivo studies were
`performed using human breast cancer xenografts in
`
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`

`

`Anti-HER-2/neu antibody and chemotherapy combinations
`M Pegram et al
`
`2246
`
`Figure 5 Combination treatment of MCF7/HER-2 breast
`carcinoma xenografts in athymic mice with rhuMAb HER2 plus
`chemotherapeutic agents CPA (a), DOX (b), and TAX (c). For
`each drug, signi®cant reduction in xenograft volume was observed
`for rhuMAb HER2/drug combinations compared to drug alone
`controls (P50.05)
`
`that it was not possible to resolve potential di€erences
`between 5-FU alone and the combination with the
`sample sizes chosen (10 mice/group).
`
`Correlation between rhuMAb HER-2 serum
`concentration and MCF7/HER-2 xenograft volume
`
`To investigate the relationship between rhuMAb HER2
`concentration and xenograft
`size,
`trough rhuMAb
`HER2 serum concentration was measured in a subset
`of mice on day 64 following extended rhuMAb HER2
`treatment at the RD (8 mg/kg loading dose and eight
`weekly i.p.
`injections of 4 mg/kg)
`(Figure 7). A
`signi®cant inverse correlation (Spearman Rank Corre-
`
`lation rho=70.543; P=0.0067) between trough
`rhuMAb HER2 concentration and xenograft volume
`was observed,
`suggesting that
`the MCF7/HER-2
`xenograft
`size signi®cantly a€ects
`rhuMAb HER2
`pharmacology. Furthermore, this e€ect is independent
`of serum shed HER-2/neu ECD concentration as this
`molecule was undetectable in any of the murine serum
`samples analysed (data not shown).
`To determine if chemotherapeutic drugs have an
`e€ect on rhuMAb HER2 pharmacology, day 64 trough
`serum rhuMAb HER2 concentrations were analysed by
`treatment group in a subset of mice used for the in vivo
`studies. Controlling for xenograft size, there was no
`signi®cant di€erence
`in rhuMAb HER2
`trough
`concentration between any of the treatment groups in
`Figure 7 (data not shown).
`
`Discussion
`
`The protein products of transforming oncogenes have
`been a target for anti-cancer drug development since
`the initial discovery of these genes, however there is
`only one currently approved drug speci®cally targeting
`these proteins in clinical use. Identi®cation of
`the
`HER-2/neu gene alteration and its association with
`aggressive forms of human breast cancer has resulted
`in its successful therapeutic targeting (Slamon et al.,
`1987, 1989; Baselga et al., 1996; Pegram et al., 1998).
`The interaction of anti-HER-2/neu antibodies with
`p185HER-2/neu results in receptor tyrosine phosphoryla-
`tion.
`(Yarden, 1990), downregulation of
`receptor
`expression (Park et al., 1992),
`internalization of the
`antibody-receptor complex (Maier et al., 1991), and a
`decrease in the association of p185HER-2/neu with its
`heterodimeric partners HER-3 and/or HER-4 (Reese et
`al., 1996; Klapper et al., 1997). These events are
`accompanied by a number of biological
`e€ects
`including most importantly a decrease in cell prolifera-
`tion (Rodriguez et al., 1993), alteration of cell cycle
`distribution, and a marked decrease in the ability of
`the cell to excise and repair DNA damage induced by
`platinum analogs (Pietras et al., 1994; Arteaga et al.,
`1994). This enhanced cytotoxic activity is speci®c for
`malignant cell
`lines or xenografts with HER-2/neu
`receptor overexpression since anti-HER-2/neu antibo-
`dies have no such e€ect on cell lines with physiologic
`HER-2/neu expression levels (Hancock et al., 1991;
`Pietras
`al.,
`1994).
`Interaction
`between
`the
`et
`p185HER-2/neu
`signaling pathway
`and CDDP-DNA
`repair mechanisms has been con®rmed using tyrosine
`kinase
`inhibitors
`to
`block
`p185HER-2/neu
`receptor
`phosphorylation which inhibits
`antibody
`induced
`attenuation of
`repair of platinum-DNA adducts
`(Arteaga et al., 1994). Moreover, reversal of CDDP
`resistance is possible through transfection and over-
`expression of HER-2/neu cDNA followed by incuba-
`tion with anti-HER-2/neu antibody (Pietras et al.,
`1994). As a result of this work, studies demonstrating
`the clinical ecacy of the combination of an anti-
`HER-2/neu antibody plus CDDP were conducted in
`breast
`cancer patients with HER-2-overexpressing
`breast cancers who previously exhibited clinical drug
`resistance to cytotoxic therapy (Pegram et al., 1998).
`To test whether this receptor enhanced chemosensi-
`tivity mechanism could be observed with other classes
`
`6 of 11
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`Celltrion, Inc., Exhibit 1017
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`

`

`Anti-HER-2/neu antibody and chemotherapy combinations
`M Pegram et al
`
`2247
`
`Figure 6 Treatment of MCF7/HER2 xenografts with rhuMAb HER2 in combination with VP-16 (a), VBL (b), MTX (c), and 5-
`FU (d). Combination drug/rhuMAb HER2 treatment resulted in a signi®cant reduction in xenograft volume compared to drug
`alone, or rhuMAb HER2 alone, controls (P50.05) for each of the drugs indicated with the exception of 5-FU
`
`concentrations achieved in humans (Pegram et al.,
`1997, 1998). Data from the multiple drug e€ect
`analysis methodology are useful, not only in establish-
`ing hypotheses as to the mechanism of action of multi-
`drug combinations, but can also provide insight as to
`how two drugs should be administered temporally to
`gain the maximum therapeutic e€ect. For example, two
`drugs which are synergistic might best be administered
`together whereas two antagonistic drugs would be most
`e€ective if given sequentially. Data from the current
`study demonstrate
`that
`the platinum compound
`CDDP, the alkylating agent TSPA, and the topoi-
`somerase
`II
`inhibitor VP-16
`are
`synergistic
`in
`combination with rhuMAb HER2 in treating HER-2/
`neu-overexpressing SK-BR-3 breast carcinoma cells in
`vitro. These results
`suggest
`the possibility of an
`interaction between the HER-2/neu signaling pathway
`and intracellular DNA repair mechanisms involved
`with repair of DNA damage resulting from these
`speci®c DNA damaging
`agents. Other potential
`mechanisms might also explain the synergy observed
`between rhuMAb HER2 and these agents,
`including
`the possibility that rhuMAb HER2 could impact the
`cellular pharmacology of the drugs resulting in an
`increase in their cytotoxic activity. An argument
`against this hypothesis is the fact that the anti-HER-
`2/neu antibody has no e€ect on the net cellular
`incorporation of
`14C-labeled carboplatin (Pietras et
`al., 1994) or [14C]-doxorubicin in target cells (Pegram et
`al., 1992). Another possible mechanism for
`the
`observed synergy with rhuMAb HER2 is an e€ect of
`cytotoxic drugs on the expression level and/or kinase
`
`Figure 7 Inverse relationship between MCF7/HER-2 xenograft
`volume and trough rhuMAb HER2 concentration in murine
`serum (Spearman Rank Correlation rho=70.543; P=0.0067).
`These data suggest that binding of rhuMAb HER2 to HER-2/
`neu-overexpressing xenografts reduces serum rhuMAb HER2
`concentrations
`
`of cytotoxic chemotherapeutic agents, we performed a
`series of studies evaluating combinations of cytotoxic
`agents with rhuMAb HER2 testing seven classes of
`chemotherapeutics in common clinical use. All con-
`centration ranges of cytotoxic drugs and rhuMAb
`HER2 tested in these studies were conducted at serum
`
`7 of 11
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`

`

`2248
`
`Anti-HER-2/neu antibody and chemotherapy combinations
`M Pegram et al
`
`activity of p185HER-2/neu. An analogous mechanism has
`been postulated for the EGFR where low doses of
`DOX appear to increase receptor expression enhancing
`the antiproliferative activity of anti-EGFR antibody
`(Zuckier and Tritton, 1983; Hanauske et al., 1987;
`Baselga
`al.,
`1992,
`1993). The
`current data
`et
`demonstrate no change in p185HER-2/neu expression levels
`or in HER-2/neu receptor tyrosine phosphorylation
`following exposure to cytotoxic drugs, suggesting that
`unlike the EGFR, this mechanism is not operative for
`the HER-2/neu receptor.
`Most of
`the rhuMAb HER2/drug combinations
`evaluated in this study demonstrate additive rather
`than synergistic
`interactions
`suggesting
`that
`the
`majority of observed antiproliferative
`e€ects of
`rhuMAb HER2 plus cytotoxic drugs are due to a
`mechanism of action involving each agent acting
`independently.
`It
`is
`interesting to note that
`the
`mechanisms of
`action of many of
`the drugs
`demonstrating additivity do not involve direct DNA
`damage, but rather disruption of microtubule poly-
`merization/depolymerization (taxanes and vinca alka-
`loids) or inhibition of DNA synthesis (antimetabolites).
`This observation is consistent with the hypothesis that
`the synergy between cytotoxic drugs and rhuMAb
`HER2 involves an interaction between the HER-2/neu
`signaling and DNA repair pathways. Subsequent to
`our initial demonstration of the additive e€ects of
`rhuMAb HER2 with TAX (Hsu et al., 1997), studies
`con®rming this additive interaction were published
`(Baselga et al., 1998). The antimetabolite drug 5-FU is
`the only drug which demonstrated antagonism when
`used in combination with rhuMAb HER2 in vitro. We
`have not yet de®ned the mechanism of this interaction,
`but it may be the result of alterations in cell cycle
`distribution caused by rhuMAb HER2 as seen in the
`current data. It could also be the result of intracellular
`pharmacological e€ects, alteration of
`the enzymatic
`activity responsible for conversion of 5-FU to 5-
`¯uorodeoxyuridine monophosphate, or an impact on
`the level of the target enzyme thymidylate synthetase.
`Further work is needed to explore these possibilities.
`The multiple drug e€ect model is not easily applied
`to analysis of in vivo studies since such analyses, with
`the number of drugs reported in this study, would
`require at least 600 athymic mice (assuming ®ve mice
`per group, ®ve data points for each dose response
`curve, and three dose response curves ± for each drug
`alone, and in combination with rhuMAb HER2).
`Consequently we used a more conventional approach
`for analysis of
`the in vivo data (i.e. single factor
`ANOVA at ®xed time points following treatment of
`mice with optimal drug or rhuMAb HER2 doses). The
`cytotoxic drug doses chosen for these experiments are
`at or near the MTD reported in the literature for each
`of the cytotoxic drugs. The rhuMAb HER2 doses and
`schedules were designed to achieve target
`serum
`concentrations of 510 ± 20 mg/ml
`in mice bearing
`HER-2/neu-overexpressing xenografts of 50 ± 500 mm3
`in size. This antibody concentration is associated with
`our previously published maximal antiproliferative
`e€ect in vitro (De Santes et al., 1992). With this in
`vivo approach, we demonstrated signi®cantly superior
`anti-tumor ecacy of rhuMAb HER2 in combination
`with CPA, DOX, MTX, TAX, VP-16, and VBL when
`compared to e€ects of each chemotherapeutic drug
`
`alone. These results are consistent with the in vitro data
`which demonstrate that
`rhuMAb HER2 is either
`additive or synergistic with each of these drugs. For
`the drug 5-FU, which was antagonistic w