`
`
`
`
`
`
`PHIGENIX
`PHIGENIX
`Exhibit 1020
`Exhibit 1020
`
`

`

`Oncogene (1999) 18. 2241—2251
`
`© 1999 Stockton Press All rights reserved 0950-9232/99 $12.00
`http://vmw.stocktanpress.co.u1t/onc
`
`Inhibitory effects of combinations of HER-Z/neu antibody and
`chemotherapeutic agents used for treatment of human breast cancers
`
`Mark Pegram‘, Sheree Hsu‘, Gail Lewisz, Richard Pietras‘, Malgorzata Beryt‘, Mark Sliwkowskiz,
`Daniel Coombsz, Deborah Balyz, Fairooz Kabbinavarl and Dennis Slamon*’1
`'
`
`’Division of Hematology-Oncology. UCLA School‘of Medicine, Los Angeles, California 90095, USA; ’Genentech, Inc. One DNA
`Way, South San Francisco, California, USA
`
`Previous studies have demonstrated a synergistic interac-
`tion between rhuMAb HERZ and the cytotoxic drug.
`cisplatin in human breast and ovarian cancer cells. To
`define the nature of the interaction between rhuMAb
`HERZ and other classes of cytotoxic drugs, we applied
`multiple drug effect/combination index (CI) isobologram
`analysis to a variety of chemotherapeutic drug/rhuMAb
`I-[ERZ 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 efi‘ects
`were observed with rhuMAb HERZ 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, S-fiuorouracil, wasfound
`to be
`antagonistjg___ with rhuMAb 'HERZ in
`vitro
`(CI=2.87, P=0.0001).
`_In vivo druglrbuMAb I-[ERZ
`studies were conducted with HER-Zlneu-transfected,
`MCF7 human breast cancer xenografts in athymic mice.
`Combinations of rhuMAb HERZ plus cyclophosphamide,
`doxorubicin, paclitaxel, methotrexate, etoposide, and -
`vinblastiue in vivo resulted in a significant reduction in
`xenograft 'volume compared to' chemotherapy alone
`(P< 0.05). Xenografts treated with rhuMAb HERZ plus
`S-fluorouracil were not significantly different from 5-
`fluorouracil alone controls consistent with the subadditive
`effects observed with this combination in vitro. The
`synergistic interaction of rhuMAb HERZ with alkylating
`agents, platinum analogs and topoisomerase II inhibitors,
`as well}. as
`the
`additive
`interaction with taxanes,
`anthracyclines and some antimetabolites in I-IER-Z/neu-
`overexpressing breast cancer cells demonstrates that these
`are rational combinations to test in human clinical trials.
`
`Keywords: HER--2/n_eu (c-erbB-Z); chemotherapy; breast
`cancer; multiple drug effects analysis, synergy .
`
`Introduction
`
`Overexpression of p185““‘”"“, resulting from amplifi-h
`cation of the HER-Z/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;
`Berchuek et al., 1991; Yonemura et al., 1991; Hetzel et
`al., 1992; Lukes et al., 1994; Press et al., 1994; Safiafi
`et al.,1995) The murine monoclonal antibody 4D5 has
`specificity
`for
`a
`juxtamembrane
`epitope
`in
`the
`extracellular domain (ECD) of the p185HER"m protein
`(Fendly et al., 1990) and is capable of eliciting an
`antiproliferative efi‘ect'against murme cells transformed
`by HER-Z/neu as well as human malignant cell
`lines
`and xenografts overexpressing this oncogene (Chazin et
`al., 1992). Importantly, this growth inhibitory efiect is
`specific for cells with HER-Z/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
`HERZ) has been generated by inserting the comple-
`mentary-determining regions (CDRs) of 4D5 into the
`framework of a consensus human lgG,,_(Qarter et al.,
`1992). When compared to murine 4D5,
`rhuMAb
`HERZ exhibits a, stronger binding afiinity
`for
`p185““'”""‘ but has similar specific antiproliferative
`activity against HER-2,’neu-overexpres_sing 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 effects 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 HERZ, 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 effect,
`termed
`receptor enhanced chemosensitivity (REC), specifically
`targets HER-2/neu-overexpressing cells and has no
`effect on cells or tissues expressing physiologic levels of
`the gene. The interaction between 4D5 and CDDP in
`inhibiting HER-Z/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-Z/neu-overexpressing human breast cancer xeno-
`. grafts (Pietras_eLaL,-1994)._._..._m._..-..
`Synergy, as it applies to drug-dmg 1nteract10ns is
`defined as a combination of two or more drugs which
`achieves a therapeutic effect greater than that expected
`by the simple addition of the effects of the component
`drugs Such synergistic interactions between drugs may
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`PHIGENIX
`
`Exhibit 1020-01
`
`

`

`Ami-HER-Z/neu antibody and chemotherapy comblnafions
`M Pegram et al
`2242
`
`improve therapeutic results in cancer treatment if the
`synergism is specific 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
`resultin therapeutic synergy.
`In order to characterize the effects of combinations
`of rhuMAb HER2 cytotoxic chemotherapeutic drugs
`commonly used in breast cancer therapy, we utilized
`the
`median-eflect/combination~index
`isobologram
`method of multiple drug effect analysis. With this
`methodology,
`combination index
`(CI) values
`are
`calculated for different dose-effect
`levels based on
`parameters derived from median-efiect plots of the
`chemotherapeutic drugs alone, rhuMAb HER2 alone,
`and the combination of the two at fixed molar ratios.
`CI values <1 indicate
`synergy, CI=1 indicates
`addition, and CI>1 denotes antagonism (Chou and
`Talalay,
`1984). We performed this
`analysis With
`rhuMAb HER2 in combination with eight drugs
`representing
`seven
`different
`classes of
`cytotoxic
`chemotherapeutics in vitro. Assays were performed in
`vitro
`for
`drug/rhuMAb HER2
`combinations
`at
`clinically relevant drug/antibody concentrations using
`a cytotoxicityendpoint employing SK-BR-3 human
`breast cancer cells Which contain HER-Z/neu gene
`amplification/overexpression.
`In addition,
`to circum-
`vent the possibility that any observed interaction might
`be unique to an individual cell
`line or to a specific
`method of analysis, parallel studies were conducted in
`vivo with the same rhuMAb HER2/drug combinations.
`HER-ZI’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 efi’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 effect of xenograft size (i.e. tumor
`burden) on rhuMAb HER2 serum concentrations.
`
`Results
`
`Multiple drug eflect analysis of rhuMAb HER2 in
`combination with cytotoxic chemotherapy drugs on
`SK-BR-3 breast carcinoma cells in vitro
`
`‘To extend, thefobservations 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 different drug classes. Representative drugs
`included:
`the
`anthr‘acycline- antibiotic, doxorubicin
`(DOX);
`the.
`taxane
`drug,
`paclitaxel
`(TAX);
`a
`topoisornerase
`II
`inhibitor
`etoposide
`(VP-16);
`a
`platinum analog cisplatin (CDDP); a vin‘ca' alkaloid
`vinblastine (VBL);
`the alkylating agents,
`thiotepa
`(TSPA) for in vitro experiments and cyclophospha—
`mide
`(CPA)
`for
`in
`vivo
`experiments,
`and the
`antimetabolite drugs methotrexate (MTX) and 5-
`fluorouracil (5-FU).
`'
`curves were
`response
`In
`this
`analysis,
`dose
`constructed for each drug alone,
`rhuMAb HER2
`alone, and the combinations at
`fixed molar
`ratios
`defined as
`the ratio of
`the two agents at
`their
`maximally efl‘ective dose A representative example of
`the multiple drug effect 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 ahalysis‘fa and Fu are the '
`fractions of SK-BR-3
`c'ells affected or unaffected,
`respectively, by the dose (D) of either agent (drug or
`antibody). DM is the dose required to produce the
`median effect (analogous to the 1C”), and m is the
`Hill coefficient used to determine whether the dose
`efi’ect
`relationships
`follow 'sigmoidal dose-response
`curves
`(Hill,
`1913). Linear
`regression correlation
`coefl‘icients
`(r-values) of
`the median eflect plots
`(Table 1) reflect that the dose—eflect relationships for
`TSPA, rhuMAb HER2, and the Combination, con-
`
`.
`
`Lug ( Fall‘u J -
`
`
`
`w _
`
`as
`
`clul— u
`
`o.-
`
`o:
`
`v.7
`
`u
`
`n 8
`
`(a) Multiple drug efiect plot of TSPA, rhuMAb HER2 and the combination where Fa a the fraction of SK-BR-3 cells
`Figure 1
`affected by the drugs, Fu = the fraction of cells unafi‘ected, and D= drug dose. (b) Combination Index values for TSPA in
`combination with rhuMAb HER2 at multiple effect levels. CI values <1 indicate synergy
`
`PHIGENIX
`
`Exhibit 1020-02
`
`

`

`Anti-HER-Z/neu antibody and chemotherapy comblnafions
`M Pegram et aI
`2243
`
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`_ T=0
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`2h
`
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`
`24h
`
`in SK-BR-3 cells
`(9) Expression of p185HER'1/"6"
`Figure .2
`following exposure to ‘DOX at the ICy) (30 rm) concentration
`for
`the
`times
`indicated.‘
`(1) MAb 4D5-induced tyrosine
`phosphorylation of p185mm'2 "e"
`in SK-BR-3 cells following.
`exposure to chemotherapeutic agents at the IC30 concentration at
`the indicated time points. 4D5-aséociated tyrosine phosphoryla-
`Lion (lane 2) was observed under all of the chemotherapy
`conditions tested (lanes 3—11 compared to-.control (lane 1). (c)
`Heregulin-induced pIBSKER‘ F‘“
`tyrosine phosphorylation in
`MCF7 cells following exposure to chemotherapeutic drugs at
`the ICgo concentration. These data demonstrate that p185HE ‘2’”
`expression and phosphorylation state are unaltered by prior
`exposure to the chemotherapeutic agents tested
`,
`
`form to the principle of mass action (in general, r-
`values >0.9 confirm the validity of this methodology)
`(Chou and Talalay,
`1984).
`-CI
`values
`for
`the
`combination of TSPA and rhuMAb HER2 were
`significantly 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-l6
`exhibit
`synergistic therapeutic interactions
`(CI<1;
`P<0.001) with rhuMAb HERZ across a wide range
`interactions '
`(~0.2-0.8)
`of Fa
`values. Additive
`(CI=1) Were observed for TAX, DOX, MTX, and
`VBL in combination with rhuMAb HER2, while only
`one drug, S-FU, was found to exhibit an antagonistic
`(CI> 1; P=0.0001)‘ interaction (Table 2).
`
`P185””"’“‘“ 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/
`or
`its
`ligand TGF-a (Zuckier and Tritton,
`198‘3;
`Hanauske et al., 1987; Baselga et al., 1992, 1993):
`This phenomenon has been proposed to explain the
`synergistic cytotoxic effects of DOX used in combina-
`tion with anti-EGFR monoclonal antibodies (Baselga
`et al., 1992). To test whether p185“5"71”'n 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, p185““""“" expression
`lev'elsin SK-BR—3' breast carcinoma cells are unaltered, -
`unlike
`the
`reported efl‘ects of DOX on EGFR
`expression in A431 cells (Baselga et al., 1992). We
`next considered the possibility that cytotoxic drugs
`may impact p18SHER'1/M" functional activity rather than
`expression levels. We therefore determined the efiect of
`the various cytotoxic drugs on heregulin B-‘l and 4D5-
`~ induced
`tyrosine
`phosphorylation
`of
`pISSHEle’m
`
`
`
`Table .1 ' éalculated'values for the Combination Index as a function of fractional inhibition of SK-liR-Zi cell proliferation by a mixture of TSPA . , . and rhuMAb HERZ
`
`Combblation Index Valuer
`Parameters
`£050
`5 gpoo
`m
`0.81
`0.15
`0.59
`
`Drug
`TSPA
`rhuMAb HERZ
`TSPA+rhuMAb HJERZ
`Diagnosis 'of combined efi‘ect
`
`.
`
`.
`
`....£1930_
`
`_
`
`5040
`
`£1970
`
`0.52
`Synergy
`
`0.37 ‘
`Synergy
`
`.
`
`0.41
`Synergy
`
`0.49
`Synergy
`
`0.60
`Synergy
`
`Dm
`66.2 11M
`675.0 nM
`27.1 on
`
`r
`0.99
`0.96
`0.99
`
`Table 2 Mean combination index values for chemotherapeutic drug/rhuMAb HERZ combinations in vitro
`rhuMAb HEM/drug
`‘ Drug Dose Range
`Combination Index
`
`Synergy
`0.0008
`0.671012
`8.25- 1.06 x 103
`6.4 x10"
`TSPA
`Synergy .
`0.001
`0.561015
`6.5 x 10"- 1.7 x102
`4.0 x10"
`CDDP
`Synergy
`0.0003
`0.54i0.15
`2.6 x10“—6.8 ><10l
`9.9 x 10"
`vP-16
`Addition--
`0.13
`~1.-16i0.-1-3—-—
`~
`2.7 x10'2-6.9 .
`9.8x 10-3
`DOX
`Addition
`0.21
`0.91:0,23
`1.8 ><10_3-5.0><10'l
`1.4x 10-'
`TAX
`Addition
`0.21
`1.361017
`8.0x 10“—2.0x 10'1
`3.3x 10-l
`MTX
`Addition
`0.26
`1.09:0.19
`1.6 x 10"—3.9 x 10'2
`1.7
`VBL
`S-FU Antagonismw—8.8 x 107’ 3.0-7.65 x 102 . 2.87:0.5l 0.0001
`
`
`
`
`
`P values indicate level of significance compared to CI- 1.0
`
`.1
`
`PHIGENIX
`
`Exhibit 1020-03
`
`

`

`AmHlER-Z/neu antibody and chemotherapy combinations
`”
`M Pegram er al
`2244
`
`(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 rig/ml). Protein lysates were then analysed
`'by ”anti-phosphotyrosine‘ iimnunoblotr’firese ‘studies
`demonstrate an. increase in
`pISSHER‘I’m tyrosine
`phosphorylation following incubation with 4D5 com-
`pared to a non-specific 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-Z/neu antibody (CDDP, TSPA, and ‘
`VP-l6) had no effect 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 effects on4D5-induced
`p185 tyrosine phosphorylation (Figure 2b, lanes 8 and
`11). In addition, when heregulin B-l is used to activate
`plSSHER'Z’M" kinase, preincubation' of~ MCF7-breast
`carcinoma cells with CDDP or DOX had no effect
`on heregulin-induced p18SHER'1’m tyrosine phosphoryla-
`tion (Figure 20). Preincubation of MCF7 cells with
`TSPA, VP-16,,TAX, MTX, VBL, or S-FU likewise had
`no eflect on heregulin-induced plSSHER‘Z/m tyrosine
`phosphorylation (data not shown). Taken together
`
`.these data demonstrate that none of the synergistic,
`additive, or antagonistic effects of chemotherapeutic
`drugs with anti-HER-Z/neu antibody can be explained
`on the basis of either chemotherapy-induced alteration
`of p185m'74m protein
`expression
`levels
`or
`its
`phosphorylation.
`
`Anti-HER—Z/neu antibodies alter cell cycle distribution
`of HER—Z/neu-overexpressing human breast cancer cells
`
`The cytotoxic effects of antimetabolite drugs are cell
`cycle dependent
`(Tannock,
`1978). To identify a
`possible mechanism for the antagonism of S-FU with
`rhuMAb HERZ we investigated the eflects of murine
`4D5 and rhuMAb HERZ .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 HERZ antibodies reduce the percentage-of
`cells undergoing S phase as well
`as
`increase the
`percentage of cells' in GO/Gl, and these efiects are
`dose-dependent with the ‘maximal antiproliferative
`activity occurring at antibody concentrations between
`1 and 10 pg/ml (Figure 4). There was no significant
`difference in the magnitude of decrease inS phase
`
` MCF7
`
`
`
`Control MAb
`
`%S=23.88
`96601636117
`
`muMAb 4D5
`%s=23._57
`70001056753
`
`
`
`rhuMAb HERZ
`%S=23.76
`%GJG,=67.75
`
`
`
`°——.Hem-wfia—“xu\-v_..
`
`“9":-
`
`Number
`
`
`
`Number
`
`.
`
`
`rhuMAb mam
`muMAb 4D5
`Control MAb
`
`
`%S=20.88
`%S=l2.4l
`%S=l3.87
`
`
`%GOIG,=76.99
`%G°/G,=67.l_3
`%G°/G,=78.0l
`
`
`
`
`-
`
`
`
`
` 150 100 1
` Giannels
`Channels -
`Channels
`
`
`
`Figure 3 DNA fluorescence flow cytcmetry'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-pISSHER‘M‘“ antibody 4D5, or
`humanized anti-piss’ER‘W" antibody (rhuMAb HERZ) at a dose of 1 ug/ml for 72 h. These data demonstrate a significant
`reduction in the fraction of breast cardnoma cells undergoing S phase following treatment with anti-HER-Z antibodies 4D5 and
`rhuMAb HER2. This effect is specific for cells with HER-Zlneuwverexpression (SK-BR-3 cells)
`‘
`
`PHIGENIX
`
`Exhibit 1020-04
`
`

`

`Anti-HER-Z/neu antibody and chemotherapy combinations
`M Pegram et all
`
`2245
`
`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
`finding experiments for this specific 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
`significant
`reduction (P<0.05)
`in day 21 xenograft
`volume compared to either agent alone (Figure 5a).
`The combination of the anthracycline antibiotic DOX
`plus rhuMAb HER2 also significantly 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 significant reduction in day 20 xenograft volume
`compared to treatment with TAX alone (Figure 5c).
`However,
`the difierence between rhuMAb HER2
`alone and rhuMAb HER2 plus TAX did not reach
`statistical
`significance. ‘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 LP. 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'l-IERZ 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 significant
`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
`significantly-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 significant reductionin 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.yie1d.,a,signifieantreduction in xenogratt,
`volume when compared to S-FU alone in vivo (Figure
`6d). Although the combination of rhuMAb HER2 plus
`5-FU was superior to rhuMAb HER2 alone in this
`experiment
`(P<0.05),~ the S-FU, dose used had
`sufficient anti-tumor eflicacy as a single agent 'such
`
`PHIGENIX
`
`Exhibit 1020-05
`
`4 o
`
`o
`
`MCF7 Cells
`'
`
`20
`l
`
`.
`
`
`-—-o—_ Control MAb
`
`1 o
`
`0
`
`o
`
`_
`
`.
`-
`10
`_1
`0.1
`0.01
`‘, Antibody Concentratlon (pg/ml)
`
`100.
`
`SK-BR-a Cells
`.
`
`—o—muMAb 405
`—I-muMAb HER2
`
`o
`
`1 o
`[1
`0.1‘
`' 0.01
`Antibody Concentration (pg/ml)
`
`'1’00 ,
`
`Figure 4 Efi‘ect of anti-p18SHER'2’m MAb dose on cell cycle
`distribution of breast cells without (a) ”and with (b) HER-Zlneu
`overexpression
`
`fraction of SK-BR-3 cells comparing 4D5 and rhuMAb
`HER-2 indicating the humanization of the murine
`antibody did not adversely impact its antiproliferative
`activity. The lack of any effect On cell cycle distribution
`of MCI-7 cells demonstrates the specificity of these
`antibodies for cells with HER-Z/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 beldue
`to the longer incubation period required for MTX
`(120 vs 72 h) to elicit cytotoxicity in the assay used for
`the multiple drug effect analysis, and the fact
`that
`MTX exerts cytotoxic efl‘ects in other phases of the cell,
`cycle in addition to S phase (Buick, 1994).
`
`Eflect of rhuMAb HER2 in combination with multiple
`chemotherapeutic drugs on growth of HER-Z/neu-
`transfected MCF? breast xenografts in vivo
`
`To further evaluate the potential therapeutic effects 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
`
`m
`3
`0 3
`00’
`.3
`n'.
`a)
`..
`
`.
`
`CO 2
`
`on
`
`.
`
`2
`T:o
`aIn
`N
`.C
`n.-to
`'E
`
`33
`
`(L
`
`'
`
`

`

`trough
`between
`lation rho= —0.543; P=0.0067)
`rhuMAb HERZ concentration and xenograft volume
`was observed,
`suggesting that
`the MCF7/HER—2
`xenograft
`size
`significantly affects
`rhuMAb HERZ
`pharmaéology. Furthermore, this effect is independent
`of serum shed HER-Z/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
`effect on rhuMAb HERZ pharmacology, day 64 trough
`serum rhuMAb HERZ concentrations were analysed by
`treatment group in a subset of mice used for the in vivo
`studies Controlling for xenograft size,
`there was no
`significant
`difference
`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 specifically targeting
`these proteins in clinical use.
`Identification 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-Z/neu antibodies with
`p18SHER'1/"W 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 plSSHERW" with its
`heterodimeric partners HER-3 and/or—HER-4 (Reese et
`al.,1996gKlapper et al.,1997) These events are
`accompanied by a number of biological
`effects
`including most importantly a decreasein 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 cytdtoxic activity is specific for
`malignant cell
`lines or xenografts with HER-2471a;
`receptor overexpression since anti-HER--2/neu antibo—
`dies have no such eflect on cell lines with physiologic
`HER-2-/neu expression levels (Hancock et al., 1991,
`Pietras
`et
`al.,
`1994).
`Interaction
`between
`the
`p18SHF-Rjz’m signaling
`pathWay
`and. CDDP—DNA
`repair mechanisms has been confirmed using tyrosine
`kinase
`inhibitors
`to
`block
`plSSHE‘F'W"
`receptor
`phosphorylation which
`inhibits
`antibody induced
`attenuation of
`repair _of platinum-DNA adducts
`(Arteaga et al.,
`l994). Moreover, reversal of CDDP
`resistance is possible through transfection and over-
`expression of HER-Z/neu cDNA followed by incuba—
`tion with anti--HER—2/n'eu antibody (Pietras et al.,
`1994). As a result of this work, studies demonstrating
`the clinical efficacy of the combination of an anti-
`HER-Z/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
`
`PHIGENIX
`
`Exhibit 1020-06
`
`Anti-HER-Z/neu antibody and chemotherapy combinations
`M Pegram et at
`
`+ Control
`
`+ rhuMAb HERZ
`
`
` ' —O- CPA+rhuMAb HERZ
`
`0
`
`2
`
`4
`
`6 810121416182022
`
`TreatmentDay
`
`I
`
`
`
`
` + Control hulgG1
`-I— rhuMAb HER2
`
`>
`+ DOX
`+ DOX+rhuMAb HERZ
`
`
`
`0 '-2
`
`4
`
`6 81012141618202224
`Treatment Day
`
`
` ' , + Co'ntrol hulgG1
`
`+ rhuMAb HERZI
`+ TAX
`
`
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`
`+ITAX+rhuMAbHER2
`
`
`
`0
`
`2
`
`4
`
`6 810121416182022
`TreatmentDay
`Figure 5 Combination treatment of MCF7/HER-2 breast
`carcinoma xenografts in athymic mice with rhuMAb HER; plus
`chemotherapeutic agents CPA (a), DOX (b), andvTAX (c). For
`each drug, significant reductionin xenograft volume was observed
`for rhuMAb HER2/drug combinations compared to drug alone
`controls (P<0.05)
`
`that it was not possible to resolve potential differences
`between S—FU alone and the combination with the
`sample sizes chosen (10 mice/group).
`
`Correlation between rhuMAb HER-2 serum ‘
`concentration and MCH/HER-Z xenograft volume
`
`To investigate the relationship between rhuMAb HERZ
`concentration and xenograft
`size,
`trough rhuMAb
`HERZ serum concentration was measured in a subset
`of mice on day 64 following extended rhuMAb HERZ
`treatment at the RD (8 mg/kg loading dose and eight
`weekly ip.
`injections of 4 mg/kg)
`(Figure 7) A
`significantinverse correlation (Spearman Rank Corre-
`
`

`

`a: O O
`
`+ control hulth
`
`60° - + control hulgG‘l
`
`Anti-HER--2/neu antibody and chemotherapy combinations
`M Pegram et al
`2247
`
`3 h
`
`0' O O
`
`& OO
`
`4— rhuMAb HER2
`
`+VP-16
`
`D o
`
`+v15-1s + rhuMAb HER2
`
`to O
`
`'(a
`
`to
`100 '
`
`
`
`
`
`TumorVolume(mm3)
`
`o
`
`2
`
`4‘
`
`6 810121416182022
`Treatment Day
`
`
`
`_TumorVolume(mm3) w o o
`
`
`
`
`
`+ control hul'gG1
`+ rhuMAb HER2
`
`
`
`o
`
`2
`
`4
`
`6 8101214161820221
`Treatment- Day
`
`500 “
`
`+rhuMAbHER2
`"’E
`.
`E 1
`:4°° —A—MTX
`.
`E
`% 30° —O—Ml'X+rhuMABl-lER2.
`..
`> 200
`2a 100
`p—
`0
`
`.
`6 810121416182022
`Treatment Day
`
`o
`
`2
`
`4
`
`
`500 +controlhulth
`D
`+rhuMAbHER2
`5‘ 500
`
`400 —A—5-FU
`
`.
`
`E E
`
`Q
`
`g 300 —O—5-FU+rhuMAbHER2
`3 200
`.6.
`E 100
`l
`
`
`
`O
`
`2
`
`4. 6
`
`8
`'1012 141618 20 22
`Treatment Day
`
`Figure 6 Treatment of MCF7/HER2 x'enografts with rhuMAb HER2in combination with VP-16 (a), VBL (l3), MTX (c), and 5-
`FU (d). Combination drug/rhuMAb HER2 treatment resulted in a significant reductionin xenograft volume compared to drug
`alone;- or rhuMAb HER2 alone, controls (P<0.05) for each of the drugs indicated with the exception of. 5-FU_
`
`.(mm3)\.
`
`Xeuogral‘tVolume
`
`'20-
`
`30
`
`40
`
`s 0
`
`[rhuMAb HER21n-ough tug/all)
`Inverse relationship between MCF7/HER-2 xenograft
`Figure 7
`volume and trough rhuMAb HER2 concentration in murine
`serum (Spearman Rank Correlation rho=—0.543; 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
`
`concentrations. achieved in humans '(chram er al.,
`1997,
`1998). Data, from the multiple drug effect
`analysis methodology are useful,.not.on1y 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 effect For example, two
`drugs which are synergistic might best be administered
`together whereas two antagonistic drugs would be most
`effective 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-Z/neu signaling pathway
`and intracellular DNA repair mechanisms involved
`with repair of DNA damage resulting from these
`specific 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—
`Z/Heu . .amib.ody_has. “not effect. .0L1..._t1_1§ net. .geflular
`incorporation of “C-labeled carboplatin (Pietras gr
`al., 1994) or ["C]-doxorubicin.in target cells (Pegram et
`a1.,
`1992). Another possible mechanism for
`the
`observed synergy with rhuMAb HER2 is an effect of
`cytotoxic drugs on the expression level and/or kinase
`
`PHIGENIX
`
`Exhibit 1020-07
`
`

`

`‘”
`
`'
`
`3-92.12a.
`
`M Pegram et al
`”
`Ami-HER-Z/neu antibody and chemotherapy combinations
`2248
`.
`
`activity of p185HER'2’m. 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 at al., 1987;
`Baselga
`et
`al.,
`1992,
`1993). The
`current data
`demonstrate no changein plSSHE“2"“ 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-Z/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 effects 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 a