(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74) (cid:82)(cid:81)(cid:3)(cid:36)(cid:88)(cid:74)(cid:88)(cid:86)(cid:87)(cid:3)(cid:26)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:26)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:27)(cid:3)(cid:3)
`
`Genentech 2138
`Hospira v. Genentech
`IPR2017-00737
`
`

`

`HERCEPTIN ENHANCES ACTIVITY OF PACLITAXEL AND DOXORUBICIN
`
`or without rhuMAb HERZ, at concentrations indicated in “Results." Paclitaxel
`was removed after 1 h by washing the cells, followed by the addition of cell
`culture medium and rhuMAb HERZ. The medium and MAb were replenished
`every 2—3 days. After 5 days, cells were harvested by trypsinization and
`counted with a Coulter counter.
`.
`Soft Agar Colony Forming Assay. For soft agar assays. a bottom layer of
`1 ml of the corresponding culture media containing 0.7% agar (DIFCO
`Laboratories, Detroit, MI) 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 HERZ and paclitaxel were added at the concentrations speci-
`fied in “Results” and the figures. Triplicates were performed for every condi-
`tion. Cells were incubated 11—14 days at 37°C in 5% C02 atmosphere.
`Colonies with more than 25 cells were then counted manually.
`Assay of Tumor Growth in Athymic 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 (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-HERZ MAbs (l l, 21). BALB/c nude mice received
`implants of slow release estrogen pellets (0.72 mg l7B—estradiol; Innovative
`Research of America, Toledo, OH) and, on the following day, with l X 107
`BT-474 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 tumorigenicity. 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 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 vemier calipers. Tumor vol-
`ume was calculated by the formula: 1r/6 X larger diameter X (smaller diam-
`eter)? When tumors reached a mean size of 0.2—0.3 cm’, the animals 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 01-30 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 5—10 mg/kg on days 1
`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 (18). 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 x2 test, and statistical
`significance of differences in tumor growth among the different treatment
`groups was determined by the Mann-Whitney U test using SPSS 6.1 software.
`Two-sided P5 are given at a 95% significance level.
`
`Additive Inhibition of Anchorage-independent Growth by
`rhuMAb HERZ and Paclitaxel. A series of assays were conducted
`to characterize the combined effects of rhuMAb HERZ 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 HERZ receptors to validate the data obtained
`with BT—474 cells. RhuMAb l-IERZ produced a concentration-depen-
`dent inhibition of the Clonogenic growth of breast cancer cells BT-474
`(rhuMAb HERZ dose range, 0.5—2.5 nM) and SK-BR-3 (rhuMAb
`1-IER2 dose range, 01—10 mm) and also inhibited, but to a lesser
`degree, the growth of ovarian cancer cells SK-OV—3 (rhuMAb HERZ
`dose range, 10-100 nM; data not shown). Clonogenic assays of these
`cell lines after 1-h exposure to increasing concentrations of paclitaxel
`(dose range, 025-900 MM) 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 I-IER2 and paclitaxel were performed. As
`shown in Fig. l, the cotreatrnent with rhuMAb HER2 and paclitaxel
`resulted in an additive inhibition of the growth of these three cell lines
`with endogenous HERZ overexpression. The magnitude of the
`rhuMAb HERZ-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-OV-3 cells (Fig. 1, A—C; for each cell line, only data for
`the rhuMAb HERZ dose that produced the highest increase in pacli-
`taxel cytotoxicity are shown). In contrast, the growth of the MCF7 cell
`line transfected with HER2, which has been reported to be resistant in
`vitro to the antiproliferative effects of MAbs directed against the
`I-IER2 receptor (20), was minimally affected by rhuMAb I-IER2
`(2.5—100 nM). Cotreatment with this antibody and paclitaxel did not
`increase the growth suppressive effects of paclitaxel in these cells
`(Fig. ID).
`Effects of rhuMAb HERZ upon Well Established Tumor Xe-
`nografts. We conducted animal experiments to determine the effi-
`cacy of rhuMAb l-IERZ in nude mice bearing BT—474 xenografts. In
`a first set of 39 animals, rhuMAb HER2 was given at doses ranging
`from 1—30 mg/kg 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 I-IERZ.
`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 rhuMAb I-IER2 at 30 mglkg, in 5 of 10 mice treated at 10 mglkg,
`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 I-IER2 treatment, a second animal experiment was
`conducted using lower doses of antibody. In this experiment rhuMAb
`HERZ was administered at doses of 0.1, 0.3, and to 1 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 IgG at a dose
`of 1 m/kg 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 0.1, 0.3, and 1 mg/kg
`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
`rhuMAb I-IER2 of 0.3 mglkg, that modestly inhibited the growth of
`the BT—474 xenografts, was then chosen for the subsequent combina-
`tion treatment studies.
`2826
`
`RESULTS
`
`Additive Inhibition of Growth by rhuMAb HERZ and Pacli-
`taxel in Monolayer Cultures. To characterize the antiproliferative
`effects of rhuMAb HERZ plus paclitaxel in monolayer cultures, BT—
`474 cells were treated with increasing concentrations of these com-
`pounds. Treatment with rhuMAb HER2 (3—30 nM) continuously for 5
`days produced a concentration-dependent inhibition of BT—474 pro—
`liferation. Exposure of cells to paclitaxel for 1 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
`HER2, versus paclitaxel alone, ranged from 41—82% at the doses
`tested (data not shown).
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:88)(cid:74)(cid:88)(cid:86)(cid:87)(cid:3)(cid:26)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:26)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:27)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`Downloaded from cancerres.aacrjournals.org on August 7, 2017. © 1998 American Association for Cancer Research.
`
`

`

`HERCEPI'IN ENHANCES ACTIVITY OF PACLITAXEL AND DOXORUBICIN
`
`A
`
`B
`
`swan-3
`
`3Mdmflufiorccmlt S3
`
`O
`
`£100
`3 so
`2‘. so
`5 an
`
`an
`
`3 g
`
`Fig. l. Cytotoxicity of paclitaxel in combination with rhuMAb
`HFJIZ (HERZ) in soft agar cultures of [ST-474 (A). SK-BR-3 (B).
`SK—OV-3 (C). and MCHIHERZ (D) cells. Paclitaxel was added for WM °
`l h in the continuous presence or absence of rhuMAb HERZ.
`Cytotoxicity was enhanwd in rhuMAb HEM-sensitive cells (BT-
`474. SK-BR-S. and SK-OV-3 cells. 21-0. but not in the rhuMAb
`HERZ-resistam MCF'I/HERZ cells (D). Results represent
`the
`mean + SE of triplicate readings.
`
`t
`
`an
`
`i...
`
`3
`
`"
`
`o
`as WW o
`
`on u
`
`C
`
`
`
`
`
`3mw+lcmnnom
`
`
`
`Effects of rhuMAb HERZ Combined with Paclitaxel or Doxo-
`
`rubicin upon Well Established Tumor Xeuogr'afts. We explored
`next the effects of paclitaxel or doxorubicin plus rhuMAb HERZ in a
`series of experiments with well established BT—474 xenografts in nude
`mice.
`
`First, we studied whether rhuMAb HERZ could enhance the anti-
`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 determined it to be a dose killing 10% of the
`animals (18). The dose of paclitaxel was 10 mg/kg 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 mg/kg 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 mg/kg i.p. twice a week for 5 weeks.
`
`Fifty-one animals were allocated into the different treatment groups
`after tumors reached an average volume of 0.2 cm3 (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 MAb; 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
`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).
`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
`
`«3.3..
`g...
`
`s 2
`
`20
`
`.. HHiHHi
`
`A
`
`Himmfil
`
`
`
`
`
`
`
`tumorvolume(ems) ti8
`
`a9
`
`Fig. 2. Activity of rhuMAb HER2 (HERZ)
`against well established BT474 tumor xenografts
`in athymic mice in two separate experiments. A.
`rhuMAb HERZ was given i.p. twice a week for 4
`weeks at doses of l. IO. and 30 mg/kg. The control
`group was treated with a nonspecific rhuMAb lgG
`atadoseof30mg/kg.RhuMAbl-lER2atdoses
`equaltoorgreaterthan I mg/kgmarkedlysup—
`pressed the growth of BT474 xenografts. B. in this
`experiment lower doses of rhuMAB HERZ were
`used to define whether rhuMAB HERZ had a dose-
`response relationship. RhuMAb HERZ was given
`i.p. twice a week for 5 weeks at doses of DJ. 0.3.
`and l mykg. The control group was treated with
`nonspecific rhuMAb lgG at a dose of l trig/kg. At
`these dose levels. rhuMAb HERZ induced a dose-
`dependent inhibition of growth of the BT474 xe-
`nografts. Results are given as mean tumor vol-
`ume + SE. Armws show days on which treatment
`was administered.
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:88)(cid:74)(cid:88)(cid:86)(cid:87)(cid:3)(cid:26)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:26)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:27)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`Downloaded from cancerres.aacrjournals.org on August 7, 2017. © 1998 American Association for Cancer Research.
`
` nw+mrum 8"
`
`
`:Wlllol-Eflztwnl)
`
`.
`
`.
`
`T
`
`..
`
`__
`
`to
`
`as
`
`as
`
`D
`MCF'M-IEM
`
`

`

`HERCEPI'IN ENHANCES ACTIVITY OF PACLITAXEL AND DOXORUBICIN
`
`A
`
`B
`
`it"i'i‘i'i‘i'i
`
`pelt-III
`
`i
`
`
`
`
`
`tumorvolume(em3)
`
`8
`
`.aO
`
`1 i771”? H
`
`time-«u
`
`.a.
`
`...
`.aO
`
`.oO
`
`
`
`
`
`tumorvolume(em3)
`
` OMOOO
`
`
`mm
`
`HHHHH
`
`.5O
`.aO
`.
`
`.A-a.‘on
`
`
`
`
`
`tumorvolume(em3)
`
`8
`
`45
`
`5 d
`
`ays
`
`5
`
`15
`
`Fig. 3. A. antitumor activity of rhuMAb HERZ (l-IERZ) in combination with paclitaxel (TAX) or doxorubicin (doxo) against well established BT—474 tumor xenoyafts in athyrnic
`mice. The control group was treated with the control rhuMAb IgG. 0.3 mg/kg twice weekly i.p. RhuMAb HERZ was given i.p. twice a week for 5 weeks at a dose of 0.3 mg/kg. Paclitaxel
`was given i.v. at a dose of 10 mg/kg on days 1 and 4. Doxorubicin was administered i.p. at a dose of 10 mg/kg body weight on day 1. Doxorubicin and paclitaxel, given each in
`combination with the control antibody, resulted in an equipotent, but modest, antitumor activity. The combined treatment with rhuMAb HERZ plus either paclitaxel or doxorubicin
`resulted in a marked enhancement of the antitumor effects of both chemotherapeutic agents. with greater inhibition of tumor growth in the group of animals treated with paclitaxel and
`rhuMAb HER2. B, antitumor activity of rhuMAb HERZ (HERZ) in combination with two dose levels of paclitaxel (TAX) against well established BT474 tumor xenografts in athyrnic
`mice. The control group was treated with the nonspecific rhuMAb lgG 0.3 mglkg twice weekly i.p. RhuMAb l-IER2 was given at a dose of 0.3 mg/kg i.p. twice a week for 5 weeks
`and paclitaxel was given i.v. at two dose levels: 5 and 10 mg/kg on days 1 and 4. Treatment with rhuMAb HERZ resulted in a modest inhibition of growth. Treatment with paclitaxel
`resulted in a dose—dependent inhibition of growth with greater inhibition of growth at the 10 mg/kg dose level than at the 5 mg/kg dose level. RhuMAb HERZ plus paclitaxel resulted
`in a striking inhibition of growth regardless of the dose of paclitaxel. C, antitumor activity of rhuMAb HERZ (HERZ) in combination with repeated doxorubicin (doxo) administration.
`The control group was treated with PBS. RhuMAb HERZ was given i.p. twice a week for 4 weeks at doses of 0.3 mg/kg. and doxonrbicin was given i.p. at a dose of 3.75 rug/kg body
`weight (days 1 and 2) and repeated on days 14 and 15. RhuMAb HERZ enhanced the antitumor activity of doxorubicin although the combined therapy was not statistically superior
`than doxorubicin alone or rhuMAb HERZ alone (see text). Results are given as mean tumor volume + SE. Arrows show days on which treatment was administered.
`
`rhuMAb HERZ was superior to rhuMAb l-IER2 alone (P = 0.02) and
`weeks was significantly superior in the group treated with rhuMAb
`paclitaxel 10 mg/kg X 2 alone (0.02). The significance of these
`l-IERZ plus paclitaxel versus paclitaxel alone (P = 0.016), but not
`versus rhuMAb HERZ alone (P = 0.4). The significance of all these
`findings was statistically confirmed at earlier (3 weeks) and latter (7
`weeks, which was the time when overall follow-up ended) analyzed
`findings was statistically confirmed at earlier (3 weeks) and latter (8
`time points. Combined paclitaxel 5 mglkg X 2 plus rhuMAbl-IERZ
`weeks, which was the time when overall follow-up ended) analyzed
`was also significantly superior than rhuMAb HER2 alone (P = 0.02;
`time points. RhuMAb HERZ did not increase the toxicity of paclitaxel
`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
`l-IER2 plus paclitaxel at both doses and who were followed for 90
`The above experiment, showing the enhancement of antitumor
`days after cell inoculation, no evidence of tumor regrowth was ob-
`activity when paclitaxel was given in combination with rhuMAB
`served. In another two mice with small tumors after therapy and that
`HERZ, was performed with only one dose level of paclitaxel. There-
`were followed for 90 days, tumor size was stabilized in one and
`fore, we could not exclude the possibility that the results were re-
`minimal regrowth was observed in the other.
`stricted to the paclitaxel dose level used. To detemiine whether the
`As seen in Fig. 3A, the combination of doxorubicin and rhuMAb
`enhanced antitumor effects against xenografts were dose-dependent,
`l-IER2 seemed to be less active than paclitaxel and rhuMAb HERZ. An
`as was observed in in vitro experiments, a follow-up experiment was
`attempt was made to improve the antitumor activity of this combina-
`conducted with two dose levels of paclitaxel (Fig. 33). On day 10
`when tumors reached a mean size of'0.2 cm3, 50 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.
`doxorubicin (10 mg/kg body weight) had been administered. Here, we
`RhuMAb HERZ dose was unchanged at 0.3 mg/kg twice a week i.p.
`opted for two successive administrations of doxorubicin over a
`for 5 weeks and paclitaxel was given at 5 mg/kg and 10 mg/kg iv.
`2-week period to increase the total dose of doxorubicin without
`given on days 1 and 4, either alone or in combination with rhuMAb
`HERZ (Fig 3B). 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 1 and 2, with repeat doxorubicin
`administration on treatment days 14 and 15 when animals had recov-
`rhuMAb HER2 alone (P = 0.4), by 51% with paclitaxel 5 mg/kg x 2
`(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-
`ing well established BT-474 tumor xenografts were allocated into
`mice treated with rhuMAb I-{ER2 plus paclitaxel (5 or 10 mg/kg X 2)
`treatment groups of at least 13 animals each (Fig. 3C). Treatment was
`the growth of the xenografts was strikingly affected. Average tumor
`started when tumors reached a mean size of 0.2 cm’. The combined
`volume was reduced by more than 98% (P = 0.04 for paclitaxel 5
`mg/kg X 2 plus rhuMAb HERZ versus control; P = 0.01 for pacli-
`therapy resulted in an enhanced antitumor effect, as shown by an
`taxel 10 mg/kg X 2 plus rhuMAb HER2 versus control) and resulted
`average reduction of tumor volume at 5 weeks by 84% (P = 0.0008)
`as compared with a reduction of 54% with rhuMAb l-IER2 alone
`in the eradication of well established xenografts in five of eight mice
`(P = 0.01) and 75% with doxorubicin alone (P = 0.016) versus
`(paclitaxel 5 mg/kg X 2) and in seven of eight mice (paclitaxel 10
`mg/kg X 2). In this experiment, growth inhibition effects at 5 weeks
`control-treated mice. However, combined doxorubicin plus rhuMAb
`HERZ was not statistically better than doxorubicin alone (P = 0.4) or
`resulting from combined treatment with paclitaxel 10 mg/kg X 2 plus
`2828
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:88)(cid:74)(cid:88)(cid:86)(cid:87)(cid:3)(cid:26)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:26)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:27)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`Downloaded from cancerres.aacrjournals.org on August 7, 2017. © 1998 American Association for Cancer Research.
`
`

`

`HERCEPTIN ENHANCES ACTIVITY OF PACLITAXEL AND DOXORUBICIN
`
`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 HERZ failed
`to potentiate the effects of paclitaxel in MCF7/HER2 transfectants.
`Although the antibody induces p185"E"‘2 phosphorylation in these
`cells (Ref. 20 and data not shown),
`it does not result in growth
`inhibition (Ref. 20 and Fig. 1D). This observation may indicate that
`growth inhibition by the antibody is required to potentiate the antitu-
`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 the recombinant
`humanized anti-HER2 antibody against human tumor xenografts (22).
`The authors administered one single i.v. dose of 36 mg/kg rhuMAb
`HERZ in severe combined immunodeficient mice bearing well estab-
`lished human gastric cancers overexpressing HERZ. The single dose
`of rhuMAb HERZ 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
`activity of rhuMAb HERZ against human cancer xenografts overex-
`pressing HER2. 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 mg/kg 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 HERZ 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 HER2 to enhance their
`activity. In culture of cells overexpressing HER2. an additive and
`concentration-dependent cytotoxic effect was observed with cotreat-
`ment with rhuMAb HERZ plus paclitaxel. Furthermore. a strikingly
`enhanced in vivo 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 HER2
`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. In xenograft studies with human breast
`carcinoma cell lines overexpressing the EGFR. 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, 19). 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 human:murine chimeric anti-EGFR MAb C225
`
`rhuMAb HERZ alone (P = 0.7). Although no direct comparisons were
`made in the two experiments shown in Fig. 3B and C, the augmen-
`tation of doxorubicin activity by rhuMAb l-IERZ again seemed to be
`less than the augmentation of paclitaxel activity by rhuMAb HERZ.
`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 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 1). 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 I-IERZ plus
`paclitaxel, which was significantly higher than in those animals
`treated with paclitaxel
`(P = 0.004) or
`rhuMAb HERZ 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. 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
`terminated at 7 weeks. However, the animals in complete remission at
`5 weeks continued to be followed for 120 days with no evidence of
`tumor recurrence.
`
`DISCUSSION
`
`These studies demonstrate that rhuMAb HER2 results in an addi-
`
`tive and concentration—dependent effect on the cytotoxicity of pacli-
`taxel in cultures of human carcinoma cell lines overexpressing I-IERZ.
`The data also indicate that rhuMAb I-IER2 has significant and dose-
`dependent antitumor activity against human breast cancer xenografts
`established from cells that express high levels of plSSHEm. Further~
`more. doses of rhuMAb I-IER2 that modestly inhibit growth can
`effectively enhance the tumoricidal effects of the anti-tubulin agent
`paclitaxel, resulting in a striking rate of tumor eradication in our nude
`mouse model. RhuMAb I-IER2 enhanced the antitumor effects of
`
`doxorubicin as well, albeit to a lesser degree than it was observed with
`
`Treatment
`
`Table l Pooled data for complete nanor regressions in the difi'erent treatment groups
`of BT474 xenografts
`No. of mice
`Initial
`No. of mice
`tumor-free
`no. of
`alive at
`P"
`at 5 weeks”
`mice
`5 weeks
`—
`2 (6.4%)
`31
`31
`Control
`0.013
`9 (31%)
`30
`29
`RhuMAb HERZ
`0.38
`3 (13%)
`25
`23
`Doxorubicin
`0.01
`7 (33.3%)
`23
`21
`Doxorubicin + rhuMAb HERZ‘
`0.2
`4 (17.3%)
`26
`23
`Paclitaxel
`0.004
`13 (59%)
`25
`22
`Paclitaxel + rhuMAb HERZ"
`" Additional complete tumor regressions were observed after 5 weeks in one mouse in
`the control group. one mouse in the rhuMAb HERZ alone group, one mouse in the
`rhuMAbHERZ + doxorubicin group. and two mice in the rhuMAbHERZ + paclitaxel
`”BF
`.
`Two-sided [’5 for Pearson A} comparison of complete tumor regressron rates of each
`treated group versus control animals.
`cDoxorubicin + rhuMAb HERZ versus rhuMAb HERZ alone. P = 0.8; versus
`doxorubicin alone. P = 0.09.
`dPaclitaxel + rhuMAb HERZ versus rhuMAb HERZ alone. P = 0.04: versus pacli—
`taxel alone. P = 0.004.
`
`are under way (23).
`The mechanisms responsible for the observed interaction between
`paclitaxel and rhuMAb HERZ are unknown. The simplest explanation
`for the enhanced activity of paclitaxel and rhuMAb HER2 is that it is
`the result of the summation of effects of two anticancer drugs that act
`2829
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:88)(cid:74)(cid:88)(cid:86)(cid:87)(cid:3)(cid:26)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:26)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:27)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`Downloaded from cancerres.aacrjournals.org on August 7, 2017. © 1998 American Association for Cancer Research.
`
`

`

`HERCEP‘I’IN ENHANCES ACTIVITY OF PACLITAXEL AND DOXORUBICIN
`
`HER2 markedly enhances the antitumor activity of paclitaxel and,
`modestly, of doxorubicin. A phase III clinical trial in patients with
`advanced, HER2—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 BT-474 human tumor xenograft model. We also acknowl—
`edge Mark Sliwkowski (Genentech Inc.) for careful review of the manuscript
`and useful suggestions.
`
`on different targets; rhuMAb HER2 acts on the HER2 receptor sig-
`naling pathway and paclitaxel acts on tubulin. In fact, responses to
`rhuMAb HER2 were seen in patients previously treated with taxanes
`or anthracyclines in our phase II study (14). However, the magnitude
`of the enhanced antitumor activity with the combination may be well
`beyond a simple summation of effects.
`A number of studies have shown that therapies leading to HER2
`receptor down—regulation or inhibition of its phosphorylation may
`enhance sensitivity to paclitaxel. Transfection of mouse fibroblasts
`and human breast cancer cell lines overexpressing HER2 with the
`adenovirus type 5 EM, which results in repression of HER2
`expression at the transcriptional level, sensitiz