`
`
`
`
`PHIGENIX
`PHIGENIX
`Exhibit 1019
`Exhibit 1 0 1 9
`
`
`
`[CANCER RESEARCH SI. 375-qu. July I. ma]
`
`Recombinant Humanized Anti-HERZ Antibody (Herceptinm) Enhances the
`Antitumor Activity of Paclitaxel and Doxorubicin against HERZ/neu
`Overexpressing Human Breast Cancer Xenograi‘tsl
`
`Jose lirrselga,2 Larry Norton, Joan Albanell, Young-Moe Kim, and John MendelsohnJ
`laboratory ofReceptor Biology and Deponnrenl of Medicine (J. 8..
`l’-M. K.. .l. M] and Breast Cancer Medicine Service [.I. 8.. L N.1. Memorial Sloan-Kettering Cancer Center.
`New l’ori New York [(172]: and Medical Oncology Service. Vall d'Hebron University Hospital. WIS Barcelona. Spain I]. 5.. .I. A.)
`
`tients with HERZ-overexpressing metastatic breast cancer. Weekly
`administration of rhuMAb HERZ 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-HERZ MAbs might be
`to administer them in combination with chemotherapy. Previous stud-
`ies with anti-HERZ antibodies have shown enhancement of the anti-
`tumor activity of cisplatin (7. 15). it has been postulated that the
`mechanism for this interaction is the interference of anti=HER2 anti-
`bodies with repair of cisplatin-induced DNA-damage (15, 16). Pacli-
`taxel and doxonrbicin 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-HERZ 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 ofthe same tyrosine kinase
`receptor family. markedly enhanced the antitumor activity of doxo-
`rubicin and paclitaxel against cancer cells overexpressing the EGFR
`(18. 19). Taking these results into consideration. we decided to con-
`duct the present studies with rhuMAb HER2 in combination with
`paclitaxel or doxorubicin. We have observed enhanced and concen-
`tration-dependent inhibition of growth in cultures of human cancer
`cell
`lines overexpressing HERZ treated with rhuMAb HERZ plus
`paclitaxel. and striking antitumor effects in breast carcinoma xe-
`nografts. resulting in the cure of well established tumors. RhuMAb
`i-[ERZ also enhanced. but to a lesser extent. the in viva antitumor
`effects of doxorubicin.
`
`,
`
`MATERIALS AND METHODS
`
`Compounds. RhuMAb HERZ and rhu lgGl were provided by Genenlech
`inc. (South San Francisco. CA). Paclitaxel was from the Bristol Myers-Squibb
`Company (Princeton. NJ). and doxorubicin was from Adria Laboratories
`(Columbus. OH).
`
`Cell “on. Human breast adenocarcinoma cell lines BT474. SK-BR-S.
`and MCFI/HERZ and the human ovarian carcinoma cell line SK-OV-3 were
`chosen for the present series of studies. BT474. SK-BR-J, and SK—OV-3 cells
`were obtained from the American Type Culture Collection (Msnassas. VA).
`The levels of HERZ expression in these cells relative to the normal mammary
`epithelial cell
`line 184 are: BT474. 75-fold increase; SK-BR-J. 33-fold
`increase; and SK-OV-J. 16.7-fold increase (11). MCF7IHER2-18 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 transfected with a full length
`HERZ cDNA coding region. and have a 45—fold increased expression of HERZ
`(20).
`
`ABSTRACT
`
`Recombinant humanhed anti-HER: antibody, rhuMAb HERZ, inhibits
`tbemwtbolbreutcancerceflsoverexpredngl'mluandbascflnlcal
`activity.Weesploredinpredi-lealmodelsitsespsdtytoenlnneetbe
`hmorlddalefledsolpodltasdanddoxombldmlnadtnresolmturauy
`llERI-ovmruinganeucdhrhuMAb HEM Inhibited growth-std
`eobanadtbecyhhfleeflnhdpacflhsdheaonentolweilmabflsbed
`Mfldbreuteancerxenop'sftaoverexprdngfllflzinatbymlemlee
`withrhuMAbHEflruldtedinadose-dependentanflhonoraeflrltyda
`combinationWtsealmentwltbpadltaxelandrbuMAbflEnor
`doxorubicinsndrhnMAbHERZrenrltedlngreaterlnhlbldonofgr-ontb
`tbsntbatobaenedwitbanyagentaloneTbecombinatlondpadltaxel
`sndrbuMAbHERzresultedlnthehlglseattumorgrowthinhlbltlonand
`badaslgnlflcantlysuperlorcompletetumorregruslonntewbencom-
`pandwlthdflrupadlhsdurbuMAbllERZahnflniealtrlslsthst
`stebulltontbesemitssreunderway.
`
`INTRODUCTION
`
`The HERZ gene (also known as neu and as c‘erbB-Z) encodes a
`185-kDa
`transmembrane
`tyrosinelkinase
`receptor,
`designated
`plBSHE'u. that has partial homology with the other members of the
`EGFR‘ family (1-3). HERZ 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 p185”: can inhibit the growth of tumor xenografts and trans-
`formed cells that express high levels of this receptor (7—10). The
`murine MAb 4D5. directed against
`the extracellular domain of
`plSSm’. is a potent inhibitor of growth of human breast cancer cells
`that overexpress HERZ (l 1). However. murine antibodies are limited
`clinically because they are inununogenic. To facilitate ciitrical inves-
`tigation. MAb 4D5 was humanized by inserting the complementary
`determining regions of MAb 4D5 into the framework of a consensus
`human immunoglobuiin GI (12). The resulting recombinant human-
`ized anti-p185”: monoclonal antibody. rhuMAb HER2 (Hercep-
`tin). has a higher affinity for p185” (KD==0.1 me) than the murine
`MAb 4D5. and has a cytostatic growth inhibitory effect against breast
`cancer cells overexpressing HERZ (12. 13). RhuMAb HERZ was
`found to be safe and to have dose-dependent pharmacokinetics in
`clinical phase 1 studies. The proof-of-principle of HERZ as a thera-
`peutic target for anticancer therapy was recently established in pa-
`
`.
`Reeeived 2/9/93: accepted Slim.
`Themofpublicadurofthhudeleweredehayedlnpsnbydrepaymentofpsge
`chugamsufickmunderefaebehnebynwkedadverfiumtinaceudmcewiur
`l8 USS. Section 1734 solely to indicate this fact.
`4
`'SuppuudinputbymAmeticurSocietyofGinicalChrcologyC-eaDevehpnun
`Award (to J. 3.). NIH Grant CA65746. and Specialized Programs of Research Excellence
`Grant p50-CA58207 from The National Cancer instinrte.
`'Towhomrequesuforrepdnushouldbeaddressed.uMedichncobgyService.
`Vsll d'l-iebrur University Hospital. Psseo Vsll d'Hebron 119—129. Barcelona 08035.
`Spin. Flame: 01 Hat-934746077; Fax: 011-34-93-2746059: E-msil: baselgsOhg.
`vhebtones.
`’ Present sdckess: U.T. M. D. Anderson Cancer Center. ISIS Holcombe Boulevard.
`Houston. Texas 77030.
`‘Thesbhrevistions used are: EGFR.epidermslgr-owtlrfaetorreceptor:MAb.n-rono-
`clonal antibody; rhuMAb HERZ. recombinant humaniud MAb HEM.
`
`Cell Culture and Mondnyer Growth Anny. BT474 cells were main-
`tained in 1:1 DMEM/Ham's (vlv) supplemented with 10% FCS. 300 mgll
`L-glutatnine. and 10 mglml hunran insulin. SK-BR-J and SK—OV-3 cells were
`cultured in DMEM/Hatns's (v/v) with 10% FCS. MCI-WHERZ cells were
`cultured in DMEMIH16 medium (1 gll glucose). with 10% FCS. 1m units/ml
`penicillin. 100 units/ml streptomycin. and 400 ug/ml G418. All cells were
`grown at 37'C and 5% 00,. For monolayer growth assays. cells were distrib-
`uted into 6-well plates (Falcon 3046. Lincoln Park. NJ) at 10.000 cells/Well.
`On the next day. cells were changed to medium containing 0.5% FCS for 18 h.
`and then treatment was added. Paclitaxel was dded to appropriate wells. with
`2825
`
`Downloaded from cancerres.aacrjournaiscrg on gebruaryh1 0, 2014. © 1998 American Association for Cancer
`esearc .
`
`PHIGENIX
`
`Exhibit 1019-01
`
`
`
`mCEPflN ENHANCE? ACTIVITY OF PACIJTAXE. AND DOXORUBICIN
`
`or without rhuMAb HERZ. 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 HERZ. The medium and MM: were replenished
`every 2-3 days. After 5 days. cells were harvested by trypsiniution and
`counted with a Coulter counter.
`,
`Son Agar Colony Forming Any. For soft agar assays. a bottom layer of
`1 ml of the conesponding culture media containing 0.7% agar (DIFCO
`Laboratories. Detroit, MI) and l0% FCS was prepared in 35-mm 6-well plates
`(Falcon 3046). After the bottom layer was solidified. 20.000 cells/well were
`added in LS ml culture media containing the sample. 0.35% agar. and l0%
`FCS. RhuMAb l-IERZ and paclitaxel were added at the concentrations speci-
`fied in "Results" and the figures. Triplicates were performed for every condi-
`tion. Cells were incubated ll-l4 days at 37'C in 5% CO, annosmere.
`Colonies with more than 25 cells were then counted manually.
`Assay of Tumor Growth In Athylnlc Nude Mice. Female BALE/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 (l8). BT474 cells were selected because they express high levels
`of HERZ. have a high level of basal phosphorylation of the receptor. and are
`growth-inhibited by snti-HERZ MAbs (l l. 21). BALD/c nude mice received
`implants of slow release estrogen pellets (0.72 mg l7B-esuadlol; Innovative
`Research of America. Toledo. OH) and. on the following day. with l X 107
`BT474 cells s.c. In our initial studies. we observed significant discrepancies
`between the rate of tumor take and the tumor size among animals. To optimize
`the model and enhance tumorigeniclty. 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 HERZ expression and their response to
`rhuMAb HERZ 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-
`ume was calculated by the formula: «I6 X larger diameter x (smaller diam-
`eter)’. When tumors reached a mean size of 0.2-0.3 cm’. the aninrals were
`divided into groups with comparable tumor size and treated as described in the
`text and figures. Briefly. for rhuMAb HERZ treatment. mice received the
`antibody in PBS. at a dose range of 0. l—30 mg/ltg i.p. twice a week. Paclitaxel
`was given by slow retro-orbital iv. injection in a solution of normal saline with
`8% Cremophor EL and 8% ethanol at a dose range of 5—10 mg/ltg on days I
`and 4 (two doses total). This dose schedule was suggested by Dr. Jackie
`Plowman (National Cancer Institute. Bethesda. MD) and confirmed in our
`experimental model. Doxorubicin was given i.p.
`in distilled water at
`the
`indicated dose schedules as described previously by us ([8). The mice were
`followed for the observation of xenograft growth rate. body weight changes.
`and life span.
`Statistical Analysis. Rates of complete tumor regression among different
`treatment groups were compared using the Pearson x3 test. and statistical
`significance of differences in tumor growth among the different treatment
`groups was determined by the Mann-Whitney U test using SPSS 6.l sofiware.
`Two-sided P: are given at a 95% significance level.
`
`__
`
`Additive Inhibition of Anchorage-independent Growth by
`rhuMAb IIERZ and Paelltaxel. A series of assays were conducted
`to characterize the combined effects of rhuMAb HERZ and paclitaxel
`in soft agar. a more stringent test of rnitogenic 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 HERZ receptors to validate the data obtained
`with BT474 cells. RhuMAb I-IERZ produced a concentration-depen-
`dent inhibition of the clonogenic growth of breast cancer cells BT474
`(rhuMAb HERZ dose range. 0.5—2.5 mm) and SK-BR-3 (rhuMAb
`HERZ dose range. 0.1-10 nM) and also inhibited. but to a lesser
`degree. the growth of ovarian cancer cells SK-OV-3 (rhuMAb HERZ
`dose range. lO—lOO nM: data not shown). Clonogenic assays of these
`cell lines after l-h exposure to increasing concentrations of paclitaxel
`(dose range. 025-900 M) also showed growth inhibition in a con-
`centration-dependent manner. On the basis of the response data from
`these experiments. combined treatment assays with increasing con-
`centrations of rhuMAb HERZ and paclitaxel were performed. As
`shown in Fig. l. the cotreatment with rhuMAb HERZ and paclitaxel
`resulted in an additive inhibition of the growth of these three cell litres
`with endogenous HERZ overexpression. The magnitude of the
`rhuMAb HERZ-ttwdiated enhancement of the antiturnor effects of
`paclitaxel was up to 67% in BT474 cells. 50% in SK—BR-3 cells. and
`32% in SK-OV-3 cells (Fig. l. A—C; for each cell line. only data for
`the rhuMAb IERZ dose that produced the highest increase in pacli-
`taxel cytotoxicity are shown). In contrast. the growth of the MCFI cell
`line transfected with l-IERZ. which has been reported to be resistant in
`vitro to the antiproliferative effects of MAbs directed against the
`HERZ receptor (20). was minimally affected by rhuMAb HERZ
`(2.5—ltX) nM). Coneatment with this antibody and paclitaxel did not
`increase the growth suppressive effects of paclitaxel in these cells
`(Fig. 10).
`‘
`Effects of rhuMAb I-IERZ upon Well Established Tumor Xe-
`nografts. We conducted animal experiments to determine the effi~
`cacy of rhuMAb HFJIZ in nude mice bearing BT474 xenografts. In
`a first set of 39 animals. rhuMAb HERZ was given at doses ranging
`from l-30 mglkg twice a week for 4 weeks. At least nine animals
`were treated in each group. The control group was treated with a
`nonspecific rhu IgG MAb at a dose of 30 mg/kg i.p. twice a week.
`which was the same as the highest dose level of rhuMAb m2.
`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 [0 mice treated
`with rhuMAbHERZat30 mykg.in50f lO micetreatedat l0 mg/kg.
`and in 3 of 8 mice treated at 1 mg/kg (Fig 2A). Antibody administra-
`tion was nontoxic. as assayed by animal survival and weight loss.
`To better define whether there was a dose-response relationship
`with rhuMAb HERZ treatment. a second animal experiment was ,
`conducted using lOWer doses of antibody. In this experiment rhuMAb
`HERZ was administered at doses of 0.]. 0.3. and to l mglkg. 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. 2B).
`The control group was treated with the nonspecific rhu lgG at a dose
`of 1 Mg i.p. Treatment was started when tumors reached a mean size
`of 0.2 cm3 (day 10). In this experiment. a dose-dependent antitumor
`activity was observed (Fig. 28), Doses of 0.]. 0.3. and l mg/kg
`resulted in an average inhibition of tumor growth at 5 weeks of 25. 40.
`and 80%. respectively. as compm with those mice treated with ,
`control antibody. No animal,
`toxicity was observed. A dose of
`rhuMAb HERZ of 0.3 mg/kg, that modestly inhibited the growth of
`the BT474 xenografts. was then chosen for the subsequent combina-
`tion treatment studies.
`2826
`
`RESULTS
`
`Additive Inhibition of Growth by rhuMAb HERZ and Paell-
`taxel ln Monolayer Cultures. To characterize the antiproliferative
`effects of rhuMAb HER2 plus paclitaxel in monolayer cultures. BT-
`474 cells were treated with increasing concentrations of these com-
`pounds. Treatment with rhuMAb HER2 (3-30 m) continuously for 5
`days produced a concentration-dependent inhibition of BT474 pro-
`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 HERZ showed an additive and concentration-dependent
`effect on the growth inhibition induced by paclitaxel. The enhance-
`ment of the growth inhibition seen with paclitaxel plus rhuMAb
`HERZ. versus paclitaxel alone. ranged from “—82% at the doses
`tested (data not shown).
`
`Downloaded from cancerres.aacrjournalsorg on Eebruaryhio. 2014. © 1998 American Association for Cancer
`esearc .
`
`PHIGENIX
`
`Exhibit 1019-02
`
`
`
`mmm ACTIVITY OF PACLITAXE. AND DOXDRUBICI'N
`
`Fig. l. Cymaxicity of paclilaxel in combination with rhuMAb
`HER: (HEM) in Iofl agar cultures of BT4'M (A). SK~BR~3 (5).
`SK-OV-3 (C). and MCFIIHERZ (D) cells. Paclitasel was added for
`lhinthecontimroupreaenceorabaeneeofrhuMAbHERZ.
`Cytotoxicity was enhanced in rhuMAb HERZoaensitive cells (ET-
`474. SK-BR-J. and SK-OV-J cells. 4-0. but not in the rhuMAb
`HERz-resiatuu MCFT/HERZ cells (D).
`leach:
`represent
`the
`mean + SE of triplicle rulings.
`
`
`
`Eflects of rhuMAb HERZ Combined with Paclltaxel or Dolo-
`rublcin upon Well Established Tumor Xenogratts. We explored
`next the effects of paclitaxel or doxorubicin plus rhuMAb HERZ in a
`series of experiments with well established BT474 xenografts in nude
`nuce.
`
`First. we studied whether rhuMAb HERZ could enhance the anti-
`tumor activity of equipotent doses of paclitaxel or doxorubicin (Fig
`3.4). We chose a doxorubicin dose of 10 mg/kg body weight. because
`we had previously determined it to be a dose killing 10% of the
`animals (18). The dose of paclitaxel was 10 mglkg i.v. on day l 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 HERZ of 0.3 mglkg i.p. twice
`a week for 5 weeks was chosen to prevent tumor regressions attrib-
`utable to antibody alone. Control animals were treated with the
`nonspecific rhu MAb lgG at 0.3 rug/kg i.p. twice a week for 5 weeks.
`
`Fifty-one animals were allocated into the different treatment groups
`afier tumors reached an avenge volume of 0.2 cmJ (day ll). At least
`seven animals were treated in each group. Treatment groups consisted
`of: control MAb; rhuMAb HERZ; paclitaxel plus control MAb; pac-
`litaxel plus rhuMAb HERZ; doxorubicin plus control MM); and
`.doxorubicin plus rhuMAb HERZ (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 HERZ (P = 0.2). 27% with doxorubicin (P = 0.38). and
`35% with paclitaxel (P = 0.3). Combined therapy with rhuMAb
`HERZ plus doxorubicin inhibited growth by 70% versus control
`treated mice (P = 0.04). but it was not statistically superior than
`doxorubicin alone (P = 0.l6) or rhuMAb HERZ alone (P = 0.59).
`The enhancement of antitumor activity was more profound with the
`combination of rhuMAb HERZ plus paclitaxel, resulting in growth
`inhibition of 93% (P = 0.006). In addition, growth inhibition at 5
`
`A
`
`
`
`.
`-'
`n
`
`U
`
`I
`
`day-
`2827
`
`a
`
`Fig. 2. Activity of MI: HER! (HERZ)
`Slain“ well established N474 nirmrxcnografis
`iaatbymicmieeinlwoleparateexperiments.4.
`rhuMAbl-lEllZwasgiveui.p.lwieeaweekfor-4
`weeksatdoaesofl,l0.lrd30mm.1'heconuol
`mwummm-Mmum "
`atadoaeolBOmmRhaMAbl-Eluatdoaea
`equaltoagreatadlanlmmmarkadlylap- g
`presseddsepowtholBT-H‘xeooyanaminlhis v
`usedtodeflnevhetiu'lhuMABHERZhadadoae-
`experimentlowerdoaeaofrhuMABl-IERZwere
`g
`responaerelationahithhMAbI-lflluwaagivea
`i.p.uviceaweekforSweehatdaaeaofOJJj.
`andlmdng‘heeontrolgroupwutnatedwith
`noospeeificrhuMAblgOatado-eoflmylrg.m
`theaedusehvehr‘huMAhl'lElzindueedadoae-
`dependent iahibitionofgroanhoftl: BT-4'M se-
`nognltaJleauIs-egiveaasmeanmvol-
`ume+SEAnowsahowdlyswwhichtreanuent
`withiniltfltd.
`
`‘0
`
`.
`
`
`
`manna
`
`HH HH
`
`O
`
`I.
`
`I
`
`,
`
`Downloaded from cancerres.aacrjournalsorg on Februaryh10, 2014. © 1998 American Association for Cancer
`Researc .
`
`PHIGENIX
`
`Exhibit 1019-03
`
`
`
`
`
`Hm“
`
`HWWH
`
`Fig.3.A.andnrmoraetivityofrhuMAbHERzfl-IERZ)incunfinuimwifipaehuxelUAX)adumubidn(doao)agdnuwuenabw3T474mu
`Whethymie
`mice.'I'hecono'olmwaatreatedwiththeeoaoulrhaMAblgG.O.3Inglkgtwiceweeklpr.RhuMAbPlEleasglveniprieeaweekfaSweeksatadoaeofOJ
`mJ’aclitaxei
`wasglveni.v.atadoaeoll0mmm¢llylland4.Doawqunwuadmiaislesedl.p.uado¢otl0mg/kg
`
`weeks was significantly superior in the group treated with rhuMAb
`rhuMAbHERZ was superiortorhuMAbI-IERZalone(P - 0.02)and
`HERZ 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 HERZ a'lone(P = 0.4). The significance of all these
`findings was statistically confirmed 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
`weeks. which was the time when overall follow-up ended) analyzed
`time points. Combined paclitaxel 5 mg/kg x 2 plus rhuMAbl-IERZ
`time points. RhuMAb HERZ did not increase the toxicity of melitaxel
`was also significantly superior titan rhuMAb HERZ alone (P = 0.02;
`or doxorubicin in mice as determined by animal survival and weight
`versus paclitaxel 5 mg/kg x 2 alone. P = 0.08). In seven mice whose
`loss (data not shown).
`tumors were completely eradicated upon treatment with rhuMAb
`The above experiment. showing the enhancement of antitumor
`HERZ plus paclitaxel at both doses and who were followed for 90
`activity when paclitartel was given in combination with rhuMAB
`days alter cell inoculation. no evidence of tumor regrowth was ob—
`I-IERZ. was performed with only one dose level of paclitaxel. There-
`served.Inanomertwomicewithsmallturnorsaftertherapyandthat
`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 determine whether the
`minimalregrowthwasobservedintheotber.
`,
`enhanced antitumor effects against xenografis were don-dependent.
`AsseminFig.3A.mecombinafionofdoxorubicinandrhuMAb
`as was observed in in vitro experiments. a follow-up experiment was
`HERZseenredtobelessactivethanpaclitaxelandrhuMAbl-IERZM
`conducted with two dose levels of pulitaxel (Fig. 38). On day 10
`attemptwasmadetoimprovetheantitumoractivltyofthiscombina-
`when tumors reached a mean size of '0.2 cm’. 50 animals were
`tion by changing the schedule of doxorubicin administration (Fig.
`allocated into treaMent groups consisting of at
`least 8 animals.
`3C). In the prior experiment. the maximally tolerated single dose of
`RhuMAb HERZ dose was unchanged at 0.3 mg/kg twice a week i.p.
`doxorubicin (IO mg/kg body weight) had been administered. Here. we
`for 5 weeks and paclitaxel was given at 5 mg/kg and ID mg/kg i.v.
`opted for two successive administrations of doxorubicin over a
`given on days I and 4. either alone or in combination with rhuMAb
`2-weekperiodtoincreasethetotaldueofdoxorubicinwithout
`HERZ (Fig SB). 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 lgG. was reduced by 42% with
`weight given on beam days I and 2. with repeat doxorubicin
`rhuMAb HERZ alone (P = 0.4). by 51% with paclitaxel 5 mg/kg x 2
`administrationontreaunentdays l4‘and lSwhenanimalshadrecov-
`(P a 0.7). and by 77% with pmlitaxel IO mg/kg X 2 (P = 0.3). In
`ered from the first doxoruhicin administration. Fifty-nine mice hear-
`mice treated with rhuMAb HERZ plus paclitaxel (5 or 10 rug/kg X 2)
`ing well establisbd BT474 tumor xenografts were allocated into
`the growth of the xenografis was strikingly affected. Average tumor
`treannentgrnupsofatleast13animalseach(Fig.30.Treatmentwas
`volume was reduced by more than 98% (P = 0.04 for paclitaxel 5
`startedwhenturnonreachedameansizeofo.2cm’.'l‘hecombined
`mg/kg X 2 plus rhuMAb HERZ versus control; P = 0.0] for pacli-
`therapyresultedinanenhanwdantitumoreffecLasshownbyan
`taxel l0 mykg X 2 plus rhuMAb HER2 versus control) and resulted
`average reduction of tumor volume at 5 weeks by 84% (P - 0.0008)
`in the eradication of well established xenografts in five of eight mice
`ascomparedwithareductionof54%withrhuMAbHER2alone
`(paclitaxel 5 mg/kg X 2) and in seven of eight mice (paclitaxel 10
`(P - 0.0l) and 75% with doxorubicin alone (P :- 0.016) versus
`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
`HERZ was not statistically better than doxorubicin alone (P - 0.4) or
`2828
`
`Downloaded from cancerresaacrjournalsorg on Februaryh10, 2014. © 1998 American Association for Cancer
`Researc .
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`,
`
`PHIGENIX
`
`Exhibit 1019-04
`
`
`
`HERCEPI'IN ENHANCE? AL‘I‘IVI'IY 0F PACIJ'I'AXEL AND DOXORUBlClN
`
`rhuMAb HERZ alone (P = 0.7). Although no direct comparisons were
`made in the two experinrents shown in Fig. 3B and C. the augmen-
`tation of doxonrbicin activity by rhuMAb l-IERZ again seemed to be
`less than the augmentation of paclitaxel activity by rhuMAb l-IERZ.
`Afier. 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 separately 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 HERZ
`plus chemotherapy were compiled. and data from the various doses of
`paclitaxel and doxorubicin administered were pooled (Table l). An-
`imals treated with either rhuMAb HERZ 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 HERZ 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 aninrals in two of the experiments for which
`additional follow-up is available. In 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. 3B), the follow-up of the various treatment groups was
`temtinated at 7 weeks. However, the animals in complete remission at
`5 weeks continued to be followed for I20 days with no evidence of
`tumor IBCIIITCI'ICC.
`
`DISCUSSION
`
`These studies demonstrate that rhuMAb HERZ results in an addi-
`tive and concentration-dependent effect on the cytotoxicity of pacli-
`taxel in cultures of human carcinoma cell lines overexpressing H1312.
`The data also indicate that rhuMAb HERZ has significant and dose-
`dependent antitumor activity against human breast cancer xenografts
`established from cells that express high levels of plasma”. Further-
`more. doses of rhuMAb l-IER2 that modestly inhibit growth can
`effectively enhance the armoricidal effects of the anti-tubulin agent
`paclitaxel. resulting in a striking rate of tumor eradication in otrr nude
`mouse model. RhuMAb HERZ enhanced the antitumor effects of
`doxorubicin as well. albeit to a lesser degree than it was observed with
`
`paclitaxel. The increased antitumor activity with the studied combi-
`nations occurred without additional animal toxicity.
`The observed effects were seen in cultures of all three cancer cell
`lines endogenously overexpressing HERZ (two breast and one ovar-
`ian), thus excluding the possibility that the findings could be unique
`to a single cell line or tumor type. In contrast. rhuMAb HER2 failed
`to potentiate the effects of paclitaxel in MCI-7/HER2 transfectants.
`Although the antibody induces pl85"Em phosphorylation in these
`cells (Ref. 20 and data not shown).
`it does not result in growth
`inhibition (Ref. 20 and Fig. ID). This observation may indicate that
`growth inhibition by the antibody is required to potentiate the antim-
`mor effects of paclitaxel or that these transfected cells are either not
`dependent on HERZ or lack critical downstream elements in their
`signal transduction pathway.
`There is only one published report that has used die recombinant
`humanized anti-HERZ antibody against human tumor xenografts (22).
`The authors administered one single i.v. dose of 36 mg/kg rhuMAb
`l-IERZ in severe combined immunodeficient mice bearing well estab-
`lished human gastric cancers overexprcssing HERZ. 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 viva antitrunor
`activity of rhuMAb HERZ against human cancer xenografts overex-
`pressing HERZ. In addition. we showed that repeated administrations
`of the humanized antibody given at doses equal to or greater than 1
`mg/kg resulted in strong growth. suppression and eradication of tu-
`mors in a significant proportion of animals. This suggests that in our
`animal model. doses of.1 mg/kg given twice a week result in maximal
`inhibition of growth. This dose level
`is similar to the one being
`administered in the current series of clinical trials in patients with
`advanced breast cancer (2 mykg X week). We also showed that at
`lower doses the growth inhibition by rhuMAb HERZ was less marked.
`thus allowing us to test the concept of combined treatment with
`rhuMAb HER2 plus chemotherapy.
`Because paclitaxel and doxorubicin are two of the most active
`chemotherapeutic agents against breast cancer. studies were con-
`ducted to analyze the capacity of rhuMAb HERZ to enhance their
`activity. In culture of cells overexpressing l-IER2. an additive and
`concentration-dependent cytotoxic effect was observed with cotreat-
`ment with rhuMAb l-IERZ plus paclitaxel. Furthermore. a strikingly
`enhanced in viva antitumor activity of these two compounds given
`together was seen. resulting in a statistically significant increase in the
`rate of tumor eradication when compared with either rhuMAb HERZ
`or paclitaxel given alone. The combined treatment with rhuMAb
`HERZ and doxorubicin also showed increased antitumor effects in
`two separate animal experiments when compared with doxorubicin or
`rhuMAb HERZ alone. The lack of significance with the doxorubicin
`and rhuMAb HERZ combination could be a reflection of insufficient
`sample size to achieve enough statistical power.
`These results are complemented by studies with antibodies against
`the closely related EGFR. ln xenograft studies with human breast
`carcinoma cell lines overexpressing the BGFR. the combined therapy
`with anti-EGFR MAbs and doxorubicin. and anti-EGFR MAbs and
`paclitaxel resulted in similar results to the ones reported here with
`rhuMAb HERZ (18. I9). Hence. the concept of combining antirecep-
`tor strategies with either paclitaxel or doxorubicin may apply to other
`members of the type I tyrosine kinase receptor family. Currently.
`clinical trials with the hurnanzmurine chimeric anti-EGFR MAb C225
`are under way (23).
`The mechanisms responsible for the observed interaction between
`paclitaxel and rhuMAb HER2 are unknown. The simplest explanation
`for the enhanced activity of paclitaxel and rhuMAb HERZ is that it is
`the result of the summation of effects of two anticancer drugs that act
`2829
`
`Treatment
`
`,
`
`'
`
`Table l Pooled data for complete tumor regression: in the dr'fl'erenr Imtnteru groups
`«JET-4742mm
`-—-—————_____._________________
`Initial
`No.0fmiee
`No.0frru’ce
`no. of
`alive at
`motor-flee
`P
`mice
`5 weeks
`n 5 weeks'
`—
`3|
`3|
`2 (6.4%)
`Control
`0.013
`30
`29
`9 (3m)
`RhuMAb max:
`0.3!
`Ls
`23
`3 (us)
`Doxorubicin
`am
`23
`2t
`7 (31.35)
`Doxmbicirr + rhuMAb HERZ‘
`0.2
`25
`23
`4 (was)
`Paclitasel
`MIN
`25
`22
`I! (59%)
`Paclitaxel + rhuMAb HEM”
`'Addrtronslcornpleteturnorregressrons wereobservedaflerSweekstnonemousera
`theconuolgroup.memouuladlerbuMAbHER2almegroup.memoureinme
`rhuMAbHERZ + doxmrbiein group. and two mice in the rhuMAbHEltZ + paclitaxel
`group.
`.
`’Two-sidedPsforPearsonxzcomparisonofcornplete tumorregressionratesofeach
`treated group versus contr'ol animals.
`.
`‘ Dosorubicin + rhuMAb HERZ versus rhuMAb HERZ alone. P - 0.8; versus
`doxorubicin alone. P I 0.09.
`dl’aclltasel + rhuMAb HERI versus rhuMAb HBIZ slate. P = 0.04: versus pacli-
`taxel slot-e. P - 0.004.
`
`Downloaded from cancerres.aacrjournalsorg on Eebruaryh10, 2014. © 1998 American Association for Cancer
`esearc .
`
`PHIGENIX
`
`Exhibit 1019-05
`
`
`
`
`
`HERCEPHN ENHANCE ACTIVITY OF PACUTAXE. AND DOXORUBICIN
`
`HERZ markedly enhances the antitumor activity of paclitaxel and.
`modestly. of doxorubicin. A phase III clinical trial in patients with
`advanced. HERZ-overexpressing breast cancer. to test the efficacy of
`this combined therapy. is currently underway.
`
`ACKNOWLEDGMENTS
`
`We thank Drs. Dan Maneval and Refaat Shalaby for expert assistance in the
`development of our BT474 human tumor senognt’t model. We also acknowl-
`edge Mark Sliwkowski (Genentech Inc.) for careful review of the manuscript
`and useful suggestions.
`
`REFERENCES
`
`l. Hynes. N. E.. and Stern. D. F. The biology ofch-Z heu/HER-Z and in role in cancer.