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`(CANCER llESEAROl 51. ZUS-2831 . Jilly I. 19981
`
`Recombinant Humanized Anti-HER2. Antibody (Herceptin ™) Enhances the
`Antitumor Activity of Paclitaxel and Doxorubicin against HE~neu
`Overexpressing Human Breast Cancer Xenografts1
`Jose Baselga,2 Larry Norton, Joan AlbaneU, Young-Mee Kim, and John Mendelsohn3
`Laboralory of Rtctptor Biowiy and Depar11Mnl of MtdiciM [J. B .. Y-M. K .. J. M.J and BrtaSt Cortetr MtdiciM Strvice {J. B .. L N./. Mtmorial Sloan-Kenering Cancer Ctnrer,
`New Yo'*. New Yo1* 10021; and Medical OncoloKJ S.rvict, Vall d'Hebron Uni~nil)' Hospital, OllOJS Barcelona. Spain {J.B .. J. A./
`
`tients with HER2-overexpressing metastatic breast cancer. Weekly
`administration of rhuMAb HER2 induced tumor responses and the
`combined rate of clinical response and disease stabilization was half
`of the evaluable patients (14 ).
`One way to optimize the clinical role of anti-HER2 MAbs might be
`to administer them in combination with chemotherapy. Previous stud(cid:173)
`ies with anti-HER2 antibodies have shown enhancement of the anti(cid:173)
`tumor activity of cisplatin (7, I 5). It has been postulated that the
`mechanism for this interaction is the interference of anti-HER2 anti(cid:173)
`bodies with repair of cisplatin-induced DNA-damage (IS, 16). Pacli·
`taxel and doxorubicin are two of the most active chemotherapeutic
`agents for the treatment of patients with breast cancer ( 17). Thus,
`finding enhanced antitumor activity of these drugs when combined
`with anti-HER2 MAbs would have distinct clinical implications for
`breast cancer therapy. We had previously observed that MAbs C225
`and 528 directed at the EGFR. a member of the same tyrosine kinase
`receptor family, markedly enhanced the antitumor activity of doxo(cid:173)
`rubicin and paclitaxel against cancer cells overexpressing the EGFR
`(18, 19). Taking these results into consideration, we decided to con(cid:173)
`duct the present studie~ with rhuMAb HER2 in combination with
`paclitaxel or doxorubicin. We have observed enhanced and concen(cid:173)
`tration·dependent inhibition of growth in cultures of human cancer
`cell lines overexpressing HER2 treated with rhuMAb HER2 plus
`paclitaxel, and striking antitumor effcc.ts in breast carcinoma xe(cid:173)
`nografts .. resulting in the cure of well established tumors. RhuMAb
`HER2 also enhanced, but to a lesser extent. the in vivo antitumor
`effects of doxorubicin.
`
`MATERIALS AND MEmODS
`
`ABSTRACT
`
`Recombinant llwaanlud llllti-HERl antibody, rbuMAb HERl, lnh!blta
`the pvwtll of brast caacer cdll onrupraslac HERl and bu dlnkal
`acdrity. We explored In pndinlall models Its capmdty to enhance tlle
`tumoriddal e«eca of pedltaxel and doxonabidn. In c:ultara of natarally
`HEJU.overaprealna caDCa' cells, rhuMAb HERl Inhibited pvwtll and
`enhanced die eytotollk effects of paclltuel. Trealmeat of well establllhed
`BT47• brast cancer xenocrafts onrexprasi.ac HERl la athymlc mice
`wltb rlluMA~ HERZ raalted ia • ~t utltumor lldlrity. ID
`~ atudies, treatment with paditaxel wl rbuMAb HERl or
`cloxonablciD and rbuMAb HERl resulted ia creater Inhibition of pvwtll
`than that observed with any agent alone. The combination of padltuel
`and rbuMAb HERl resulted In tbe blpest tumor growtll l.ahlbltioa and
`bad • lliplftauldy superior complete tumor regression rate wbea com(cid:173)
`pend with either pedltaxel cir rbaMAb HERZ alone. Cllnlc:al trials that
`ue btlllt oa thae results ue llllder way.
`
`INTRODUCTION
`
`The HER2 gene (also known as neu and as c-erbB-2) encodes a
`receptor, designated
`185-kDa 1ransmembrane
`tyrosinelkinase
`p18S"ER2, that has partial homology with the other members of the
`EGFR4 family (1-3). HER2 is overexpressed in 25-30% of breast
`cancers and predicts for a worse prognosis as measured by lower
`overall survival and disease free survival (4-6). Antibodies directed
`at pl8SHER2 can inhibit the growth of tumor xenografts and trans(cid:173)
`formed cells that express high levels of this receptor (7-10). The
`murine MAb 405, diiected against the extracellular domain of
`pl 8SHER2 , is a potent inhibitor of growth of human breast cancer cells
`that overexpress HER2 (11). However, murine antibodies are limited
`clinically because they are immunogenic. To facilitate clinical inves(cid:173)
`tigation, MAb 405 was humanized by insening the complementary
`determining regions of MAb 405 into the framework of a consensus
`human immunoglobulin G 1 (12). The resulting recombinant human(cid:173)
`ized anti-pl8SHER2 monoclonal antibody, rhuMAb HER2 (Hercep(cid:173)
`tin), has a higher affinity for pl8S"ER2 (K0 =0.l nM) than the murine
`MAb 405. and has a cytostatic growth inhibitory effect against breast
`cancer cells overexpressing HER2 (12, 13). RhuMAb HER2 was
`found to be safe and to have dose-dependent pharmacokinetics in
`clinical phase I studies. The proof-of-principle of HER2 as a thera(cid:173)
`peutic target for anticancer therapy was recently established in pa-
`
`Compounds. RhuMAb HER2 and mu lgGI were provided by Gcnen1eeh
`Inc. (South San Francisco, CA). Paclitaxel was from the Bristol Myers-Squibb
`Company (Princeton, NJ). and doxorubicin was from Adria Laboratories
`(Columbus. OH).
`Cell LI-. Human breast adcnocarcinoma cell lines BT-474, SK-BR-3.
`and MCF7/HER2 and the human ovarian can:inoma cell line SK-OV-3 wm:
`chosen for the present series of studies. BT-474. SK-BR-3. and SK-OV-3 cells
`were obl&incd from the American Type Culture Collection (Manassas, VA).
`The levels of HER2 expression in these cells relative to the nonnal mammary
`epithelial cell line 184 are: BT-474, 25-fold increase; SK-BR-3, 33-fo'ld
`incrcasc; and SK-OV-3. 16.7-fold increase (11). MCF7/HER2-18 cells were a
`gift of Dr. C. C. Benz (University of California. San Francisco, CA). These
`cells are a subclone of MCF7 cells that have been transfcctcd with a full leng;th
`HER2 cDNA coding region. and have a 45-fold increased expression of HER2
`(20).
`Cell Culture aad Moaolayer Growth AMay. BT-474 cells were main(cid:173)
`tained in 1:1 DMEM/Ham's (v/v) supplemented with 10% FCS, 300 mg/I
`L-glutamine. and 10 mg/ml human insulin. SK-BR-3 and SK-OV-3 cells wc:rc
`cultured in DMEM/Hams's (v/v) with 10% FCS. MCF7/HER2 cells wc:rc
`cultured in DMEM/Hl6 medium (I g/I glucose), with 10% FCS. 100 units/ml
`· penicillin, 100 units/ml stttptomycin. and 400 µ.g/ml G-418. All cells were
`grown at 37"C and 5% C02 . For monolayer growth assays. cells were distrib(cid:173)
`uted inlo 6-well.plates (Falcon 3046. Lincoln Putt. NJ) at 10.000 cells/well.
`On the next day. cells wen: changed to medium containing 0.5% FCS for 18 'h,
`and then treatment was added. Paclitaxel was added to appropriate wells. wit!h
`2825
`
`Received 219198; ICCepied S/1198.
`The costs o( publication of lhis article were defnyed in pan by the payment of pe.ge
`chlr&es. This article musl lhetcfore be hereby nwtt:d advtrtiHmtnt in accordance with
`18 U.S.C. Section 1734 oolely 10 iDdicale this flCL
`1 SUpponcd ill pan by u American Society of Clinical Oncology C-- Developmenl
`Awud (10 J. 8 .). NIH Gnni CA6S746, #Id Specialiud Programs of Research Excellence
`Gnni p50-CAS8207 from The Nllion-1 Cancer lnstilUte.
`2 To whom requesta for reprints should be llddiased, at Medical Oncology Sctvice.
`Vall d'Hcbroa Universily Hospilal. Pueo Vall d' HebnSo 119-129. Barcelona 0803S.
`Spein. Pholie: 011 -34-93-2746077; Fax: Oll-34-93-27460.59; E-mail: baselga@ha.
`vhebron.es.
`1 Present ..idress: U. T. M. D. Anclmon Cancer Cenler, ISl.5 Holcombe Boulevud,
`HOIUIDll. Tc.us 77030.
`
`4 The abbrevillions 11sed -= EGFR. epidermal growth flClor recepcor. MAb. mono(cid:173)
`
`clonal onlibody; rbuMAb HER2. n:combinant h11manit.ed MAii HER2.
`
`Downloaded from cancerres.aacrjournals.org on March 6, 2018. © 1998 American Association for Cancer Research.
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`HERCEl"l1N ENHANCES ACTlVlTY OF PACUTAXEL AND DOXORUBICIN
`
`or without rhuMAb HER2, at concentrations indicated in "Results." Paclitaxel
`was removed after I h by washing the cells, followed by the addition of cell
`culture medium and rhuMAb HER2. The medium and MAb were replenished
`every 2-3 days. After 5 days, cells were harvested by ttypsinization and
`counted with a Coulter counter.
`Soft Apr Coloay Formlllg Assay. For soft agar assays. a bottom layer of
`I ml of the corresponding culture media conlaining 0.7% agar (DIFCO
`Laboratories. Detroit, Ml) and 10% FCS was prepared in 35-mm 6-well plates
`(Falcon 3046). After the bottom layer was solidified, 20.000 cells/well were
`added in 1.5 ml culture media containing the sample, 0.35% agar, and 10%
`FCS. RhuMAb HER2 and paclilaXel were added at the concentrations speci(cid:173)
`fied in "Results" and the figures. Triplicates were performed for every condi(cid:173)
`tion. Cells were incubated 11-14 days at 37°C in 5% C02 aimospbere.
`Colonies with more than 25 cells were then counted manually.
`Assay of Tumor Growth in Athymlc Nude Mice. Female BALB/c nude
`mice. 6-8 weeks of age, were used. These mice were bred and maintained in
`the animal facility at Memorial Sloan-Kettering Cancer Center as described
`previously (18). BT-474 cells were selected because they express high levels
`of HER2, have a high level of basal phosphorylation of the receptor, and are
`growth-inhibited by anti-HER2 MAbs (11, 21). BALB/c nude mice received
`implants of slow release estrogen pellets (0.72 mg 17jkstradiol; Innovative
`Research of America, Toledo, OH) anci. on the following day, with I X 107
`BT -:474 cells s.c. In our initial studies, we observed significant discrepancies
`between the rate of rumor take and the tumor size among animals. To optimize
`the model and enhance rumorigenicity. a large and rapidly growing tumor was
`removed from one of the mice and these cells were subcultured and expanded.
`These cells retained both the level of HER2 expression and their response to
`rhuMAb HER2 when compared with control cells (data not shown), and they
`were used in all of the experiments described in this study.
`Tumors were measured every 3-4 days with vernier calipers. Tumor vol(cid:173)
`ume was calculated by the formula: 1rl6 X larger diameter X (smaller diam(cid:173)
`eter)2. When tumors reached a mean size of 0.2-0.3 cm1• the animals were
`divided into groups with comparable tumor size and treated as described in the
`tell! and ti~$. Briefly, for rhuMAb HER2 treatment, mice received the
`antibody in PBS. at a dose rangeof0.1 -30 mg/kg i.p. twice a week. Paclitaxel
`was given by slow retro-orbital i.v. injection in a solution of normal saline with
`8% Cremophor. EL and 8% ethanol at a dose range of S-10 mg/kg on days I
`and 4 '(two doses total). This dose schedule was suggested by Dr. Jackie
`Plowman (National Cancer Institute, Bethesda, MD) and confinned in our
`expe.rirnental moocl. Doxorubicin was given i.p. in distilled water at the
`
`indicated dose schedules as described previously by us ( 18). The mice were
`followed for the observation of xenograft growth rate; body weight changes,
`and life span.
`Statistical Analysis. Rates of complete rumor regression among different
`treatment groups ·were compared using the Pearson x2 test, and statistical
`significance of differences in rumor growth among the diff~t treatment
`groups was determined by the Mann-Whittley U test using SPSS 6.1 software.
`Two-sided Ps are given at a 95% significance level.
`
`Additive Inhibition or Anchorage-independent Growth by
`rbuMAb HERl and Paclltaxel. A series of assays were conducted
`to characterize the combined effects of rhuMAb HER2 and paclitaxel
`in soft agar, a more stringent test of mitogenic capacity because
`several. cycles of cell division are required to form a detectable colony.
`The experiments were conducted in a series of cancer cell lines
`expressing high levels of HER2 receptors to validate the data obtained
`with BT-474 cells. RhuMAb HER2 produced a concentration-depen(cid:173)
`dent inhibition of the clonogenic growth of breast cancer cells BT-474
`(rbuMAb HER2 dose range, 0.5-2.5 OM) and SK-BR-3 (rbuMAb
`HER2 dose range, 0.1- 10 OM) and also inhibited, but to a lesser
`degree, the growth of ovarian cancer cells SK-OV-3 (rbuMAb HER2
`dose range, 10-100 nM; data not shown). Clonogenic assays of these
`cell lines after l ·h exposure to increasing concentrations of paclitaxel
`(dose range, 0.25-900 µM) also showed growth inhibition in a con(cid:173)
`centration-dependent manner. On the basis of the response data from
`these experiments, combined treatment assays with increasing con(cid:173)
`centrations of rbuMAb HER2 and paclitaxel were performed. As
`shown in Fig. 1, the cotreatment with muMAb HER2 and paclitaxel
`resulted in an additive inhibition of the growth of these three cell lines
`with endogenous HER2 overexpression. 1be magnitude of the
`rltuMAb HER2-mediated enhancement of the antitumor effects of
`paclitaxel was up to 67% in BT-474 cells, 50% in SK-BR-3 cells, and
`32% in SK-OY-3 cells (Fig. I, A-C; for each cell line, only data for
`the muMAb HER2 dose that produced the highest increase in pacli(cid:173)
`taxcl cytotoxicity are shown). In contrast, the growth of the MCF7 cell
`line transfected with HER2, which has been reponed to be resistant in
`vitro to the antiproliferative effects of MAbs directed against the
`HER2 receptor (20), was minimally affected by rbuMAb HER2
`(2.5-100 nM). Cotreatmcnt with this antibody and paclitaxel did not
`increase the growth suppressive effects of paclitaxel in these cells
`(Fig. lD).
`Effects or rhnMAb HERl upon Well Established Tumor Xe(cid:173)
`aografts. We conducted animal experiments to determine the effi(cid:173)
`cacy of rbuMAb HER2 in nude mice bearing BT-474 xenografts. In
`a first set of 39 animals, muMAb HER2 was given at doses ranging
`from 1-30 mgfkg twice a week for 4 weeks. At least nine animals
`were treated in each group. 1be control group was treated with a
`nonspecific rbu IgG MAb at a dose of 30 mgfkg i.p. twice a week,
`which was the same as the highest dose level of muMAb HER2.
`Treatment was started when xenografts reached a mean size of 0 .3
`cm3 (day 7). Marked antitumor activity was observed at all dose
`levels. Complete tumor eradication was seen in 3 of 10 mice treated
`with rbuMAb HER2 at 30 mgfkg, in 5 of 10 mice treated at 10 mg/kg,
`and in 3 of 8 mice treated at 1 mg/kg (Fig 2A). Antibody administra(cid:173)
`tion was nontoxic, as assayed by animal survival and weight loss.
`To better define whether there was a dose-response relationship
`with rbuMAb HER2 treatment, a second animal experiment was
`conducted using lower doses of antibody. In this experiment rltuMAb
`HER2 was administered at doses of 0.1, 0.3, and to 1 mgfkg, given i.p.
`twice a week for 5 weeks. The total number of mice was 24, allocated
`into different treatment groups of at least 5 animals/group (Fig. 28).
`The control group was treated with the nonspecific rbu IgG at a dose
`of 1 mlkg i.p. Treatment was staned when tumors reached a mean size
`of 0.2 cm3 (day 10). In this experiment, a close-dependent antitumor
`activity was observed (Fig. 28). Doses of 0.1, 0.3, and 1 mglkg
`resulted in an average inhibition of tumor growth at 5 weeks of 25, 40,
`and 80%, respectively, as compared with those mice treated with
`control antibody. No animal toxicity was observed. A 'dose of
`rbuMAb HER2 of 0.3 mgfkg, that modestly inhibited the growth. of
`the BT-474 xenografts, was then chpsen for the subsequent combina(cid:173)
`tion treatment studies.
`2g26
`
`RESULTS
`
`Additive Inhibition or Growth by rbuMAb HERl and Padi(cid:173)
`taxel in Mooolayer Cultures. To characlerize the antiproliferative
`effects of muMAb HER2 plus paclitaxel in monolayer cultures, BT-
`474 cells were treated with increasing concentrations of these com(cid:173)
`pounds. Treatment with rhuMAb HER2 (3-30 nM) continuously for 5
`days produced a concentration-dependent inhibition of BT-474 pro(cid:173)
`liferation. Exposure of cells to paclitaxel for l h (2-50 nM) also
`resulted in a concentration-dependent inhibition of cell proliferation
`(data not shown). We then proceeded to combination experiments.
`RhuMAb HER2 showed an additive and concentration-dependent
`effect on the growth inhibition induced by paclitaxel. The enhance(cid:173)
`ment of the growth inhibition seen with paclitaxel plus rbuMAb
`HER2, versus paclitaxel alone, ranged from 41-82% at the doses
`tested (data not shown).
`
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`HERCEP11N ENHANCES AcnvrTY OF PACUTAXEL AND DOXORUBICIN
`
`Fla. I. Cytoc.o.Uci1y or J*litaxel in combiution wilh rhuMAb
`HER2 (HEIUJ in soft apr culllln:s of BT-474 (AJ, SK-BR-3 (BJ,
`SK-OV-3 (CJ, and MCF7/HER2 (DJ cells. Paclitaxel was oddcd for
`I b in lbe continllOUS presence or absence of rhuMAb HER2.
`Cytoc.ollicily wu enbulccd in rhuMAb HER2·sensitive cells (BT-
`474, SK-BR-3, and SK-OV-3 cells, A-C), but noc in tbe rlwMAb
`HER2-resistut MCF7/HEIU cells (DJ. Results rqnseot the
`mean + SE oC lriplicale readings.
`
`A
`BT-474
`
`I.I
`
`...
`c
`
`f"' ....
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`...............
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`~a:=::= • HD2(l0 ...
`
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`
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`
`10
`
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`~f,if 11+f&t(t00•
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`
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`
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`
`FJrects of rbuMAb BER2 Combined with Padituel or Do10-
`nablcin upon Well En.blisbed Tumor Xenografts. We explored
`next the effects of paclitaxel or doxorubicin plus rhuMAb HER2 in a
`series of experiments with well established BT-474 xcnografts in nude
`mice.
`First, we studied whether rhuMAb HER2 could enhance the anti(cid:173)
`tumor activity of equipotent doses of paclitaxel or doxorubicin (Fig
`3A). We chose a doxorubicin dose of 10 mg/kg body weight, because
`we had previously determi.ned it to be a dose killing 10% of the
`animals (18). The dose of paclitaxel was 10 mg/kg i.v. on day 1 and
`day 4. This dose was nontoxic, but had antitumor activity similar to
`the dose of doxorubicin used in preliminary experiments (data not
`shown). A modest schedule of rhuMAb HER2 of 0.3 mg/kg i.p. twice
`a week for S weeks was chosen to prevent tumor regressions attrib(cid:173)
`utable to antibody alone. Control animals were treated with the
`nonspecific rhu MAb lgG at 0.3 mg/kg i.p. twice a week for 5 weeks.
`
`Fifty--0ne animals were allocated into the different treatment groups
`after tumors reached an average volume of 0.2 cm3 (day 11). At least
`seven animals were treated in each group. Treatment groups consisted
`of: control MAb; rhuMAb HER2; pacli!allcl plus control MAb; pac(cid:173)
`litaxel plus rhuMAb HER2; doxorubicin plus control MAb; and
`doxorubicin plus rhuMAb HER2 (Fig. 3A). In this experiment, the
`growth inhibition resulting from the single modality therapies was
`similar, average tumor volume at 5 weeks was reduced by 36% with
`rhuMAb HER2 (P = 0.2), 27% with doxorubicin (P = 0.38). and
`35% with paclitaxel (P = 0.3). Combined therapy with rhuMAb
`HER2 plus doxorubicin inhibited growth by 70% versus control
`treated mice (P = 0.04), but it was not statistically superior than
`doxorubicin alone (P = 0.16) or rhuMAb HER2 alone (P = 0.59).
`1be enhancement of antitumor activity was more profound with the
`combination of rhuMAb HER2 plus paclitaxel, resulting in growth
`inhibition of 93% (P = 0.006). In addition, growth inhibition at 5 .
`
`Fi&· 2. Activity oC rhuMAb HER2 (HER2J
`qeinst well established BT -474 tuDMY xenoanfts
`in llbymic mice in two sepuaie experiments. A.
`diuMAb HER2 was given i.p. twice 1 week for 4
`weeb at doecs of I, 10. and 30 maJkg. The c:onlrOI
`group WU lrelted wilb I nonspecific rhuMAb lgO
`II a dose ol 30 iq/q. Rln&MAb ffER2 at doses
`equal to or pater 1hlll I mg/kg nwt.edly sup(cid:173)
`praled the poWlh ol BT -474 xeoognfts. B, in this
`experimellt lower cbes of rhuMAll HER2 were
`used to derane w~ rbuMAB HER2 hid 1 dose·
`respome relationship. RhllMAb HER2 WU givai
`i.p. twice • wedc for s weeks at doses or 0.1. 0.3.
`ud I ma/kg. The concrol poup WU lrelltcd with
`ooospeci6c rhuMAb 1,0 11 I dose or I lllj/tg. At
`lbeae dose leveb, rllllMAb HER2 iDduccd a dote(cid:173)
`dependdit itthlbition of poWlh of the BT-474 xe(cid:173)
`oopafts. Rcaulta are given u mean lllDMY vol(cid:173)
`mne + SB'. Anows sllow days on which treaanait
`-
`adminilles'ed.
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`HERC'EFTIN ENHANCES AC'llVTTY OF PACUTAXEL AND OOXORUBICIN
`
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`
`F'I- 3. A, 111titumor llC1ivity of rhuMAb HER2 (HER2) in combination with paclimel (TAX) or doxorubicin (doxo) against well established BT 474 tumor xenogratu in 4thymic
`mice. The conJrOI group wu tJcated with the control rbuMAb lgG, 0.3 mg/kg twice weekly i.p. RhllMAb HER2 wu given i.p. twice a week for S weeks at a dose of 0.3 mg/kg. Pacliwel
`wu given i.v. 81adoseof10 mg/kg on days I aod 4. Ooxorubicin wu administered i.p. at a dose of 10 mg/kg body weight on day L OoJtonibicin and paclitaxel, given each in
`combinalioo with the control antibody, rcsulced in an cquipoctlll, but modest. antitumor activity. The combined trealmCDt with rhuMAb HER2 plus either paclitaxel or doJtorubicin
`~led in a nwbd enhancement of the antitumor effects of bo4h c:kmothenpeulic agents, with grwer inhibition of rumor growth in the group of animals treated with paclitaxel and
`rhuMAb HER2. B, antitumor activity of rhuMAb HER2 (HER2) in combination with two dose levels of paclitaxel (TAX) against well established BT 474 tumor xenografts in alhymic
`mice. The control group wu lrCaled with lbe nonspecific rhuMAb lgG 0.3 mg/kg twice weekly i.p. RbuMAb HER2 was given 81 a dose of 0.3 mg/kg i.p. !Wice a week for S weeks
`aod paclitaxel was given i.v. at two dose levels: Sand 10 m&fk.& on days I and 4. TRatmcnt with rhuMAb HER2 resulted in a modest inhibition of growth. Ttellment with pacliwel
`ruulled in a dose-dependent inhibition of growth with grwer inhibition of growth at the 10 mg/kg dose level than 81 the S mg/kg dose level. RhuMAb HER2 plus paclitaxel resulted
`in a Slriking inhibition of growth regardless of 1he dose of paclitaxcl. C, antinunor llC1ivity of rhuMAb HER2 (HER2} in combinalioa with repeated doxorubicin (doxo) administtation.
`The control group wu truced with PBS. RlwMAb HER2 was given i.p. twice a week for 4 weeks 81 doses of 0.3 mg/kg. and doxorubicin wu given i.p. Ill a dose of 3.75 m&fk.& body
`weigh! (days I aod 2) and~ oo days 14 and IS. RhuMAb HER2 enhanced 1he anlitumor a.;tivity of doxorubicin althou&h the combined therapy was ll04 slalislically superior
`given u mean tumor volume + SE. ""°"'' show days oo which b'ealnlCftt wu administered.
`than doxorubicin alone or rhllMAb HER2 alone (see text). Results -
`
`rhuMAb HER2 was superior to rhuMAb HER2 alone (P = 0.02) and
`weeks was significantly superior in the group treated with rhuMAb
`HER2 plus paclitaxel versus paclitaxel alone (P = 0.016), but not
`paclitaxel 10 mg/kg X 2 alone (0.02). The significance of these
`versus rhuMAb HER2 alone (P = 0.4). The significance of all these
`findings was statistically confinned at earlier (3 weeks) and latter (7
`findings was statistically confirmed at earlier (3 weeks) and latter (8 weeks, which was the time when overall follow-up ended) analyzed
`time points. Combined paclitaxel S mg/kg X 2 plus rhuMAbHER2
`weeks, which was the time when overall follow-up ended) analyzed
`time points. RhuMAb HER2 did not increase the toxicity of paclitaxel was also significantly superior than rhuMAb HER2 alone (P = 0.02;
`versus paclitaXel S mg/kg X 2 alone, P = 0.08). In seven mice whose
`or doxorubicin in mice as determined by animal survival and weight
`loss (data not shown).
`tumors were completely eradicated upon treatment with rhuMAb
`The above experiment, showing the enhancement of antitumor HER2 plus paclitaxel at both doses and who were followed for 90
`days after cell inoculation, no evidence of tumor regrowth was ob(cid:173)
`activity when paclitaxel was given in combination with rhuMAB
`served. In another two mice with small tumors after therapy and that
`HER2, was performed with only one dose level of paclitaxel. There-
`fore, we could not exclude the possibility that the results were re-
`·were followed for 90 days, tumor size was stabilized in one and
`stricted to the paclitaxel dose level used. To detennine whether the minimal regrowth was observed in the other.
`As seen in Fig. 3A, the combination of doxorubicin and rhuMAb
`enhanced antitumor effects against xenografts were dose-dependent,
`as was observed in in vitro experiments, a follow-up experiment was HER2 seemed to be less active than paclitaxel and rhuMAb HER2. An
`conducted with two dose levels of paclitaxel (Fig. 38). On day 10
`attempt was made to improve the antitumor activity of this combina(cid:173)
`when tumors reached a mean size of'0.2 cm3
`, SO animals were.
`tion by changing the schedule of doxorubicin administration (Fig.
`3C). In the prior experiment, the maximally tolerated single dose of
`allocated into treatment groups consisting of at least 8 animals.
`RhuMAb HER2 dose was unchanged at 0.3 mg/kg twice a week i.p.
`doxorubicin (10 mg/kg body weight) had been administered. Hen:, we
`for 5 weeks and paclitaxel was given at 5 mg/kg and 10 mg/kg i.v.
`opted for two successive administrations of doxorubicin over a
`2-week period to increase the total dose of doxorubicin without
`given on days I and 4, either alone or in combination with rhuMAb
`HER2 (Fig 38). The average tumor volume at 5 weeks, as compared
`causing prohibitive toxicity. The schedule used was 3.75 mg/kg body
`with the control mice treated with rhu IgG, was reduced by 42% with weight given on treatment days I and 2, with repeat doxorubicin
`rhuMAb HER2 alone (P = 0.4), by 51 % with paclitaxel 5 mg/kg x 2
`administration on treatment days 14 and 15 when animals had recov(cid:173)
`(P = 0.7), and by 77% with paclitaxel 10 mg/kg x 2 (P = 0.3). In
`ered from the first doxorubicin administration. Fifty-nine mice bear(cid:173)
`mice treated with rhuMAb HER2 plus pacJitaxel (Sor 10 mg/kg X 2)
`ing well established BT-474 tumor xenografts were allocated into
`the growth of the xenografts was strildngly affected. Average tumor
`treatment groups of at least 13 animals each (Fig. 3C). Treatment was
`started when tumors reached a mean size of 0.2 cm3• The combined
`volume was reduced by more than 98% (P = 0.04 for paclitaxel 5
`mg/kg x 2 plus rhuMAb HER2 versus control; P = O.Ql for pacli-
`therapy resulted in an enhanced antitumor effect, as shown by an
`average reduction of tumor volume at 5 weeks by 84% (P = 0.()()()8)
`taxel 10 mg/kg X 2 plus rhuMAb HER2 versus control) and resulted
`in the eradication of well established xenografts in five of eight mice
`as compared with a reduction of 54% with rhuMAb HER2 alone
`(paclitaxel 5 rn~g x 2) and in seven of eight mice (paclitaxel 10
`(P = 0.01) and 75% with doxorubicin alone (P = 0.016) ver:su.s
`mg/kg X 2). In this experiment, growth inhibition effects at 5 weeks
`control-treated mice. However, combined doxorubicin plus rhuMAb
`resulting from combined treatment with paclitaxel 10 mg/kg X 2 plus HER2 was not statistically better than doxorubicin alone (P = 0.4) or
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`

`HERCEPTlN ENHANCES AcnYITY Of PACUTAXEI. AND OOXORUBICIN
`
`DISCUSSION
`
`Table I Pookd data for rompldt fllmDr regrmiDM i11 IN difftrelll trealmttll groups
`of BT -47' xtMtraJls
`
`rhuMAb HER2 alone (P = 0.7). Although no direct comparisons were
`made in the two experiments shown in Fig. 38 and C, the augmen(cid:173)
`tation of doxorubicin activity by rhuMAb HER2 again seemed to be
`less than the augmentation of paclitaxel activily by rhuMAb HER2.
`After having shown that rhuMAb HER2 enhances the inhibition of
`growth of both paclitaxel and doxorubicin in our xenograft tumor
`model (Fig. 3), we decided to i;eparately analyze the rate of complete
`tumor erradication induced in the different treatment groups. In this
`analysis, only animals with tumors that could not be detected at the
`time of tumor measurement were considered to have achieved a
`complete tumor regression. Complete tumor regression rates obtained
`in the different animals experiments with combination rhuMAb HER2
`plus chemotherapy were compiled, and data from the various doses of
`paclitaxel and doxorubicin administered were pooled (Table I). An(cid:173)
`imals treated with either rhuMAb HER2 alone or with rhuMAb plus
`chemotherapy had a significantly higher complete tumor regression
`rate than control animals. The highest complete tumor eradication rate
`was observed in those animals treated with rhuMAb HER2 plus
`paclitaxel, which was significantly higher than in those animals
`treated with paclitaxel (P = 0.004) or rhuMAb HER2 alone
`(P = 0.04).
`The observed complete tumor regressions were maintained beyond
`5 weeks in all of the animals in two of the experiments for which
`additional follow-up is available. ln the experiment with antibody and
`equipotent doses of paclitaxel or doxorubicin (Fig. 3A), the remissions
`lasted until the experiment was terminated at 8 weeks of follow-up. In
`the subsequent experiment with antibody plus two dose levels of
`paclitaxel (Fig. 38), the follow-up of the various treatment groups was
`terminated at 7 weeks. However, the animals in complete remission at
`S weeks continued to be followed for 120 days with no evidence of
`tumor recurrence.
`
`paclitaxcl. The increased antitumor activily with the studied combi(cid:173)
`nations occurred without additional animal toxicily.
`The observed effects were seen in cultures of all three cancer ccU
`lines endogenously ovcrexpressing HER2 (two breast and one ovar(cid:173)
`ian), thus excluding the possibilily that the findings could be unique
`to a single cell line or tumor lypC. In contrast, rhuMAb HER2 failed
`to potcntiatc the effects of paclitaxel in MCF7/HER2 transfcctants.
`Although the antibody induces pl85mR2 phosphorylation in these
`cells (Ref. 20 and data not shown), it docs not result in growth
`inhibition (Ref. 20 and Fig. ID). This observation may indicate that
`growth inhibition by the antibody is required to potentiatc the antitu(cid:173)
`mor effects of paclitaxcl or that these transfccted cells arc either not
`dependent on HER2 or lack critical downstream clements in their
`signal transduction pathway.
`There is only one published rcpon that has used the recombinant
`humaniud anti-HER2 antibody against human tumor xcnografts (22).
`The authors administered one single i.v. dose of 36 mg/kg rhuMAb
`HER2 in severe combined immunodeficient mice bearing well estab(cid:173)
`lished human gasttic cancers ovcrexpressing HER2. The single dose
`of rhuMAb HER2 inhibited tumor growth by 50%. This effect was
`maintained during animal follow-up but did not result in tumor
`eradication. In the present study, we confirmed the in vivo antitumor
`activily of rhuMAb HER2 against human cancer xcnografts ovcrex(cid