MAIN SeY CISTI/ICIST NRC/CNRC
`MAIN Ser
`RC261
`0008-5472
`C21
`Received on : 07-23-98
`v. 58
`no. 13 Cancer research : the
`Jlll 1, L official organ of the
`American Association for
`1998
`Cancer Research . Inc .
`
`Capcer resea~ch <Ch-icag~, 11~1 Research
`
`~Faxon Stacks M-55
`
`E AMERICAN ASSOCIATION FOR CANCER RESEARCH
`
`July l, 1998
`Volume 58 • Number 13
`PP. 2693-2918
`ISSN 0008-5472 • CNREA 8
`
`(
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`1 of 10
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`Celltrion, Inc., Exhibit 1016
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`

`

`~ncer Research
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`AN OFFICIAL JOURNAL OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH
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`2 of 10
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`Celltrion, Inc., Exhibit 1016
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`3 of 10
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`Celltrion, Inc., Exhibit 1016
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`

`

`!CANCER RE.SEARCH 58. 2825-2831. July I. 19981
`
`Recombinant Humanized Anti-HER2 Antibody (Herceptin ™) Enhances the
`Antitumor Activity of Paclitaxel and Doxorubicin against HER2/neu
`Overexpressing Human Breast Cancer Xenografts1
`Jose Baselgat2 Larry Nortont Joan Albanellt Young-Mee Kimt and John Mendelsohn3
`Laboratory of Receptor Biology and Deportment of Medicine (J. B .. Y· M. K .. J. M.} and Breast Cancer Medicine Service (J. B .. L N.J. Memorial Sloon-Kettering Cancer Centu,
`New York. New York 10021; and Medico/ Oncology Service. Voll d'Htbron University Hospital, ()8()JS Borcelono, Spain (J. B .. J. A./
`
`ABSTRACT
`
`Recombinant humanized anti-HE Rl a ntibody, rhuMAb HERl, inhibits
`the growth of breast cancer cells overexpressing H ER2 and has clinical
`activity. We explored in prec.linical models Its capacity to enha nce the
`tumorlcldal effects of paclitaxel and doxorublcln. In cultures of naturally
`HE R2-overexpressing cancer cells, rhuMAb HER2 Inhibited growth and
`enhanced the cytotoxic effects of paclltaxel. Treatment of well established
`BT-474 breast cancer xenografts overexpresslng H ERl In athymlc mice
`with rhuMAb HER2 resulted in a dose-dependent antltumor activity. In
`combination studies, treatment with paclitaxel and rhuMAb HERl or
`doxorublcln a nd rhuMAb HERl resulted in gr eater Inhibition of gr owth
`tha n that observed with any agent alone. The combination of paclltaxel
`and rhuMA b HERl resulted in the highest tumor growth inhibition a nd
`had a signlftcantly superior complete tumor regression rate when com·
`pared with either paclitaxel or rhuMAb HERl alone. Clinical trials that
`are built on these results a re under way.
`
`INTRODUCTION
`
`tients with HER2-overcxpressing 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 e nhancement 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(cid:173)
`taxel and doxorubicin arc 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·
`duct the present studies 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 effects 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 METHODS
`
`The HER2 gene (also known as neu and as c·erbB-2) encodes a
`185-kDa
`transmembrane
`tyrosine/kinase
`receptor, designated
`p 185 HER2, that has partial homology with the o ther 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 p I 85HER2 can inhibit the growth of tumor xenografts and trans·
`fonned cells that express high levels of this receptor (7-10). The
`murine MAb 40 5, directed against the extracellular domain of
`p185HER2, is a potent inhibitor of growth of human breast cancer cells
`that overexpress HER2 (11). However, murine antibodies are limited
`clinically because they are immunogcnic. To facilitate clinical inves(cid:173)
`tigation, MAb 40 5 was humanized by inserting the complementary
`dctcnnining regions of MAb 405 into the framework of a consensus
`human immunoglobulin 0 1 (12). The resulting recombinant human(cid:173)
`ized anti-p185HER2 monoclonal antibody, rhuMAb HER2 (Hercep(cid:173)
`tin), has a higher affinity for p185HER2 (K0 =0. l nM) than the murine
`MAb 40 5, 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·
`
`Received 219198: accepted 5/1/98.
`The costs of publication of this article were defrayed in pan by the payment of page
`charges. This article must therefore be hereby marked odvertis~ment in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`1 Supported in part by an American Society of Clinical Oncology Career Development
`Award (lo J. B.), NTH Grant CA65746, and Specialized Programs of Research Excellence
`Grant p50-CA58207 from The National Cancer Institute.
`2 To whom requests for reprints should be addressed, at Medical Oncology Service,
`Vall d'Hebron University Hospital. Paseo Vall d'Hebr6n 119-129, Barcelona 08035,
`Spain. Phone: 011-34-93-2746077: Fax: 011-34-93-2746059: E-mail: baselga@hg.
`vhebron.es.
`3 Present address: U. T. M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,
`Houston, Texas 77030.
`•The abbreviations used are: EGFR. epidennal growth factor receptor: MAb, mono(cid:173)
`clonal antibody; rhuMAb HER2. recombinant humanized MAb HER2.
`
`Compounds. RhuMAb HER2 and rhu lgG I were provided by Genentech
`Inc. (South San Francisco, CA). Paclitaxel was from the Bristol Myers-Squibb
`Company (Princeton, NJ), and doxorubicin was from Adria Labor.11ories
`(Columbus, OH).
`Cell Lines. Human breast adenocarcinoma cell lines BT-474. SK-BR-3.
`and MCF7/HER2 and the human ovarian carcinoma cell line SK-OV-3 were
`chosen for the present series of studies. BT-474, SK-BR-3, and SK-OV-3 cells
`were obtained from the American Type Culture Collection (Manassas. VA).
`The levels of HER2 eltpression in these cells relative to the normal mammary
`epithelial cell line 184 are: BT-474. 25-fold increase: SK-BR-3. 33-fold
`increase: and SK-OV-3, 16.7-fold increase ( I I). MCF7/HER2- 18 cells were a
`gift of Dr. C. C. Benz (University of California, San Francii;co. CA). These
`cells are a subclone of MCF7 cells that have been transfected with a full length
`HER2 cDNA coding region, and have a 45-fold increased expression of HER2
`(20).
`Cell Cultu re a nd Monolayer Growth Assay. BT-474 cells were main(cid:173)
`tained in I: I 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 were
`cultured in DMEM/Hams's (v/v) with 10% FCS. MCF7/HER2 cells were
`cultured in DMEM/Hl6 medium (1 g/I glucose), with 10% FCS, JOO units/ml
`penicillin, 100 units/ml streptomycin, and 400 µ.glml G-418. All cells were
`grown at 37°C and 5% C02. 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 added to appropriate wells. with
`2825
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`4 of 10
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`Celltrion, Inc., Exhibit 1016
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`HERCEPTIN ENHANCES ACTTVITY OF PACLITAXEL AND OOXORUBICIN
`
`or without riluMAb 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 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. 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 paclitaxel 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 atmosphere.
`Colonies with more than 25 cells were then counted manually.
`Assay or Tumor Growth In Athymic 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 17/3-estradiol; Innovative
`Research of America, Toledo. OH) and, 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 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 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: 'TT/6 x larger diameter x (smaller diam(cid:173)
`eter)2. When tumors reached a mean size of 0.2-0.3 cm3• the animals were
`divided into groups with comparable tumor size and treated as described in the
`text and figures. Briefly. for rhuMAb HER2 treatment. mice received the
`antibody in PBS. at a dose range of 0.1-30 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 5-10 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 confirmed in our
`experimental model. Doxorubicin was given i.p. in distilled water at the
`indicated dose schedules a~ 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 x1 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 Ps are given at a 95% significance level.
`
`Additive Inhibition of Anchorage-Independent Growth by
`rbuMAb HER2 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
`{rhuMAb HER2 dose range, 0.5-2.5 nM) and SK-BR-3 (rhuMAb
`HER2 dose range, 0. 1-IO nM) and also inhibited. but to a lesser
`degree, the growth of ovarian cancer cells SK-OV-3 (rhuMAb HER2
`dose range, 10- IOO nM; data not shown). Clonogenic assays of these
`cell lines after 1-h exposure to increasing concentrations of paclitaxel
`(dose range, 0.25-900 µ.M) 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 rhuMAb HER2 and paclitaxel were performed. As
`shown in Fig. I, the cotreatment with rhuMAb HER2 and paclitaxel
`resulted in an additive inhibition of the growth of these three cell lines
`with endogenous HER2 overexpression. The magnitude of the
`rhuMAb 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-OV-3 cells (Fig. I, A-C; for each cell line. only data for
`the rhuMAb HER2 dose that produced the highest increase in pacli(cid:173)
`taxel cytotoxicity are shown). In contrast, the growth of the MCF7 eel I
`line transfected with HER2, which has been reported to be resistant in
`vitro to the antiproliferative effects of MAbs directed against the
`HER2 receptor (20), was minimally affected by rhuMAb HER2
`(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 rbuMAb HER2 upon Well Established Tumor Xe(cid:173)
`nograrts. We conducted animal experiments to determine the effi(cid:173)
`cacy of rhuMAb HER2 in nude mice bearing BT-474 xenografis. 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 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 tre.ited
`with rhuMAb HER2 at 30 mg/kg, in 5 of IO 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 rhuMAb HER2 treatment, a second animal experiment was
`conducted using lower doses of antibody. In this experiment rhuMAb
`HER2 was administered at doses ofO.I, 0.3, and to I mg/kg, 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 rhu IgG at a dose
`of I mlkg i.p. Treatment was started when tumors reached a mean size
`of 0.2 cm3 (day IO). In this experiment, a dose-dependent antitumor
`activity was observed (Fig. 28). Doses of 0.1, 0.3, and I 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 HER2 of 0.3 mg/kg, that modestly inhibited the growth of
`the BT-474 xenografts, was then chosen for the subsequent combina(cid:173)
`tion treatment studies.
`2826
`
`RESULTS
`
`Additive Inhibition of Growth by rhuMAb HER2 and Pacll(cid:173)
`taxel in 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(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 I 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 rhuMAb
`HER2, versus paclitaxel alone, ranged from 41-82% at the doses
`tested (data not shown).
`
`5 of 10
`
`Celltrion, Inc., Exhibit 1016
`
`

`

`HERCEPTIN ENHANCES ACTIVITY OF PACLITAXEL AND OOXORUBICIN
`
`A
`BT-474
`
`B
`
`SK·BR-3 : = . HIER2 (10 nM)
`
`2-6
`
`2-6
`
`10
`
`Fig. I. Cy101oxici1y of pacli1axel in combination wi1h rhuMAb
`HER2 (HER2) in sof1 agar cuhures of BT-474 (A), SK-BR-3 (8),
`SK-OV-3 (CJ, and MCF7/HER2 (D) cells. P:iclilaxel was added for
`I h in the continuous presence or absence of rhuMAb HER2 .
`Cy101oxici1y wa> enhanced in rhuMAb HER2-sensitive cells (BT-
`474, SK-BR-3, and SK-OV-3 cells, A-C). but 001 in the rhuMAb
`HER2-resistant MCF7/HER2 cells (D). Results represent the
`mean + SE of triplicate readings.
`
`c
`
`SK-OV-3
`~==+ ltER2 (10nM)
`
`D
`MCF71HER2
`
`·==
`
`o
`
`+ HEA2 (100 nll)
`
`100
`
`2-6
`
`100
`
`Effects of rhuMAb HER2 Combin