[CANCER RESEARCH 51. 4575-4580. September 1. 1991]
`
`A Monoclonal Antibody against
`c/5-Diamminedichloroplatinum
`Lines
`M. C. Hancock,1 B. C. Langton, T. Chan,' P. Toy,1 J. J. Monahan,1 R. P. Mischak, and L. K. Shawver2
`
`the Cytotoxicity of
`the c-erbB-2 Protein Enhances
`against Human Breast and Ovarian Tumor Cell
`
`Department of Cell Biology and Immunology, Berlex Biosciences, Inc., Alameda, California 94501
`
`reduced the rate at which the cells formed tumors in nude mice.
`Both inhibition of colony formation in soft agar and inhibition
`of tumor growth in nude mice required the continuous presence
`of antibody,
`suggesting that
`its effects were cytostatic
`rather
`than cytotoxic. Another cytostatic monoclonal
`antibody reac
`tive with gplSS has been described by Hudziak et al. (15). This
`antibody (4D5) recognizes a carbohydrate
`epitope on the extra
`cellular domain of gpl85,
`and it reversibly inhibits
`in vitro
`proliferation
`of several human breast
`tumor
`cell
`lines
`that
`overexpress the c-erbB-2 protein.
`To be of significant
`therapeutic value, monoclonal antibodies
`specific for gpl85,
`such as those described, must effectively
`mediate cytotoxicity either
`through activation of complement
`or cytotoxic spleen cells. An alternate approach is to mediate
`the efficacy of chemotherapeutic
`drugs.
`In the present
`study,
`we have investigated the effects of an anti-c-erbB-2 monoclonal
`antibody, alone and in combination with CDDP, an alkylating
`agent commonly used in the treatment
`of human neoplasms
`(for review, see Refs. 16 and 17). We discuss the effects of
`combined treatment on the proliferation
`of human tumor cell
`lines that overexpress
`the c-erbB-2 protein.
`
`MATERIALS AND METHODS
`
`ABSTRACT
`A monoclonal antibody ("IAli 250) specific to an extracellular epitope
`of the c-erbB-2 protein (gplSS)
`inhibited the in vitro proliferation of
`human breast
`tumor cell
`lines that overexpress c-erbB-2 in a dose-
`dependent manner. Treatment of cells with combinations of CM-diamme-
`dichloroplatinum (CDDP) and l'Ali 250 resulted in a significantly en
`
`hanced cytotoxic effect. This synergistic cytotoxicity was apparent over
`
`a wide range of antibody concentrations (200 pg/ml-100 «¿g/ml)including
`concentrations that showed no inhibitory effect alone. TAb 250 did not
`increase the cytotoxic effect of CDDP in a cell line exhibiting no detect
`able level of gpl85. Athymic mice bearing s.c. xenografts of human tumor
`cells expressing high levels of gpl85 showed a greatly enhanced inhibition
`of tumor growth when treated with TAb 250 and CDDP compared to
`treatment with the antibody or CDDP alone. This effect was specific
`inasmuch as TAb 250 did not enhance the growth-inhibitory effect of
`CDDP on tumor xenografts which were not expressing gplSS.
`
`INTRODUCTION
`
`a M,
`encodes
`protooncogene
`c-erbB-2 (Her-2/neu)
`The
`185,000 transmembrane
`glycoprotein with extensive homology
`to the EGF3 receptor. Studies with NIH3T3 cells have sug
`gested a direct role for overexpression ofc-erbB-2 in neoplastic
`transformation
`(1, 2). Amplification
`of the c-erbB-2 gene has
`been described in a number of cancers including human mam
`mary and ovarian carcinomas
`(3-7), as well as salivary gland
`adenocarcinomas
`(8), gastric tumors, and colon adenocarcino-
`mas (9). A survey of 189 primary breast adenocarcinomas
`by
`Slamon et al. (10) found that
`the c-erbB-2 gene was amplified
`in about 30% of the tumors and amplification was correlated
`with a poor disease prognosis.
`Immunohistochemical
`studies of
`gpl85 abundance
`in normal human tissues show reactivity in
`proximal kidney tubules, mucosal epithelium in the gastroin
`testinal
`tract, and squamous
`epithelium in skin (6, 11-13).
`Most other adult
`tissues show little or no reactivity with anti
`bodies against gpl85 including normal breast, ovary, spleen,
`liver, bone marrow, prostate, adrenal, and lung (6), suggesting
`that
`this protein may be a useful
`therapeutic
`target
`in tumors
`derived from tissues where the protein is overexpressed.
`Inhibition of the transformed
`phenotype as well as prolifer
`ation of tumor cells in vitro and in vivo by monoclonal antibod
`ies reactive with gpl85 has been reported previously. Drebin et
`al. (14) described a murine monoclonal
`IgG2a antibody reactive
`with domains of gpl85 expressed on the surface of NIH3T3
`cells transformed with the neu gene. This antibody inhibited
`anchorage-independent
`growth of these cells and significantly
`
`Cell Culture. Human tumor cell lines, HBL100 and MDA-MB-468,
`were obtained from the American Type Culture Collection (Rockville,
`MD). SKBR-3 cells were kindly provided by Dr. S. Aaronson (NIH,
`Bethesda, MD), and SKOV-3 cells were a gift from Dr. D. Slamon
`(University of California, Los Angeles, CA).
`in
`HBLIOO, MDA-MB-468,
`and SKBR-3 cells were maintained
`minimal essential medium with Earle's salts (Gibco) supplemented with
`10% heat-inactivated fetal bovine serum (Gibco), and 2 mM L-gluta-
`mine. MDA-MB-468 were also supplemented with nonessential amino
`acids and sodium pyruvate. SKOV-3 cells were maintained in Iscove's
`modified Dulbecco's medium (Gibco), 10% fetal bovine serum, and 2
`miviL-glutamine.
`Monoclonal Antibody Preparation and Characterization. A murine
`monoclonal antibody, TAb 250, was prepared as described previously
`(18) using intact NIH3T3 cells transformed with the c-erbB-2 oncogene
`(NIH3T3,, kindly provided by Dr. S. Aaronson). The antibody was
`screened for positive reactivity by enzyme-linked immunosorbent
`assay
`against
`fixed NIH3T3, and lack of reactivity against
`fixed nontrans-
`formed control NIH3T3 cells. Furthermore,
`it was also screened using
`a fluorescence-activated
`cell sorter
`for specific reactivity with live
`NIH3T3, cells. After several rounds of cloning,
`the hybridoma was
`injected into mice for ascites production. Monoclonal
`antibody was
`purified from ascites fluid by high performance liquid chromatography,
`dialyzed against PBS, and stored at —20°C.
`Radiolabeling and Immunoprecipitation of gpl85. Human tumor cell
`lines were cultured in T150 flasks and labeled with 400 /aCi of [35S]
`cysteine in 15 ml of cysteine-free medium (Dulbecco's modified Eagle's
`medium with 4.5 g/liter of glucose). Cells were labeled overnight at
`37°C.Labeling medium was removed and the cells washed twice with
`PBS. Cells were lysed in 100 mM Tris-HCI
`(pH 7.5), 100 mM NaCl,
`0.5% Triton X-IOO, 0.5% sodium deoxycholate,
`10 mg/ml bovine
`serum albumin, and 0.2 mM phenylmethylsulfonyl
`fluoride buffer and
`centrifuged at 100,000 x g for 30 min to remove insoluble material.
`4575
`
`Received 2/28/91; accepted 6/20/91.
`The costs of publication of this article were defrayed in part by the payment
`of page charges. This article must
`therefore be hereby marked advertisement
`in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`1Present address: Somatix Therapy,
`Inc., 850 Marina Village Parkway, Ala-
`meda, CA 94501.
`J To whom requests for reprints should be addressed.
`cis-
`3The abbreviations
`used are: EGF, epidermal growth factor; CDDP,
`diamminedichloroplatinum:
`PBS, phosphate-buffered saline: MTT.
`.V(4.5-dime-
`thylthiazolyl-2-yl)-2.5-diphenyltetrazolium
`bromide.
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on October 27, 2014. © 1991 American Association for Cancer
`Research.
`
`IMMUNOGEN 2068, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`INTERACTION OF A c-erbB-2 ANTIBODY AND CDDP
`
`supernatants were stripped of non
`to immunoprecipitation,
`Prior
`specific protein A binding by incubation at 4°Cfor 4 h with 100 p\ of
`a 50% slurry of protein A-Sepharose beads. The beads and nonspecifi-
`cally bound material were removed by a 30-s spin in a microfuge, and
`supernatants were removed to new tubes. TAb 250 (20 p\ containing
`approximately
`10 Mg) or anti-EGF receptor
`(Amersham) was then
`added, and the mixtures were incubated for 24 h at 4°Con a rotator.
`The following day, 50 n\ of the protein A slurry were added to the
`sample which was incubated for 4 h at 4°Con a rotator. The beads
`were then pelleted for 30 s in a microfuge and washed five times with
`ice cold 100 HIMTris-HCl
`(pH 7.5),100 mM NaCl, 0.5% Triton X-100,
`0.5% sodium deoxycholate, 10 mg/ml bovine serum albumin, and 0.2
`mM phenylmethylsulfonyl
`fluoride buffer. Between the 3rd and 4th
`wash, tubes were changed. The final pellet was suspended in 50 ¿ilof
`Laemmli
`sample buffer containing
`1% ff-mercaptoethanol.
`Samples
`were heated to 75°Cfor 5 min and spun for 30 s in a microfuge, and
`the supernatants were electrophoresed on a 7% sodium dodecyl sulfate-
`polyacrylamide gel.
`the gel was fixed in 10% acetic acid-30%
`Following electrophoresis,
`methanol
`for 1 h. After a washing in distilled water, gels were soaked
`for l h in 250 ml fresh distilled water. Gels were permeated with 250
`ml EnHance (DuPont)
`for 90 min and equilibrated in 2% glycerol prior
`to drying onto filter paper. Dried gels were exposed to Kodak X-OMAT
`AR-5 X-ray film at -80°Cfor 3 days.
`MTT Assay. MTT assays were carried out according to a modifica
`tion of Mosmann (19). Cells were removed from tissue culture flasks
`with Versene 1:5000 (Gibco), centrifuged in tissue culture medium at
`500 x g for 5 min, and resuspended in medium at a concentration of 1
`x 10s cells/ml. Cells were plated (100 ^I/well) into 96-well microtiter
`plates (Falcon) and incubated in a humidified CO2 incubator at 37°C
`for 24 h.
`On the next day, antibody and/or CDDP (Platinol; Bristol Myers)
`were added. Immediately after deposition of the highest antibody con
`centration into the first column of wells, 1:2 dilutions of TAb 250, or
`an IgGl
`isotype control
`(Chemicon), were performed directly in the
`microtiter plates using a multichannel pipet. CDDP was initially diluted
`in normal saline at room temperature
`and was added to appropriate
`wells at concentrations
`indicated in the figure legends. Plates were then
`incubated for 3 days, followed by the addition of 10 ¿il/wellof MTT
`(Sigma). MTT was prepared as a 5-mg/ml
`solution in PBS,
`filter
`sterilized, and stored at 4°Cin the dark. Plates were kept dark and
`incubated for an additional
`4 h at 37°C.The MTT crystals were
`dissolved by mixing the contents of the wells vigorously with 100 ^1 of
`isopropanol
`containing 0.04 N HC1 and 3% sodium dodecyl sulfate.
`Absorbance at 570 nm was determined using an enzyme-linked immu-
`nosorbent assay reader.
`In Vivo Subcutaneous Xenografts. Female BAlb/c-nu/n« mice (6-8
`weeks old) were implanted s.c. with 5 x 106-1 x IO7SKOV-3 or MDA-
`MB-468 cells. Tumors were measured every 3-4 days with vernier
`calipers and tumor volume was calculated as the product of length x
`width x height. Animals were treated (6-8 animals per group) via tail
`vein or i.p. injection every 7 days for 3 treatment cycles. Mice received
`either an isotype-matched
`IgGl
`antibody, TAb 250, CDDP, or a
`combination treatment. Mice receiving combination treatments were
`given injections of CDDP 45 min after injection of antibody. Statistical
`significance was determined by an analysis of the natural
`logarithms of
`the tumor volumes. Zero tumor volumes were set equal to 1 to permit
`use of the log transformation.
`For each mouse,
`the method of least
`squares was used to fit a straight
`line to the log tumor volumes as a
`function of time. Analysis of variance models were fit to the estimated
`slopes, and the Tukey multiple comparison approach was used to test
`for between-treatment
`group differences (20). An overall experiment-
`wise significance
`level of a = 0.05 was used for
`the pairwise
`comparisons.
`
`RESULTS
`
`1
`
`2
`
`4567
`
`8
`
`9 10
`
`200,000
`gp185
`c-erbB-2
`
`200,000
`
`^EGFR
`
`150,000
`
`97,400
`
`69,000
`
`46,000
`
`250 EGFR 250 EGFR
`
`250 EGFR MW
`
`SKBR3
`
`SKOV3
`
`MDA468
`
`Fig. 1. Specificity of TAb 250. 3T3, (Lanes 2-3), SKBR-3 (Lanes 4-5), SKOV-
`3 (Lanes 6-7).
`and MDA-MB-468
`(Lanes 8-9) cells were labeled with [35S]
`cysteine and cell lysates were immunoprecipitated with TAb 250 (Lanes 2, 4, 6,
`8) or an anti-EGF receptor antibody (EGFR; Lanes 3, 5, 7, 9) as described in
`"Materials and Methods."
`Immunoprecipitates were analyzed by sodium dodecyl
`sulfate-polyacrylamide
`gel electrophoresis
`and autoradiography. Lanes I and 10,
`molecular weight (MW) markers.
`
`were incubated and precipitated with TAb 250, a nonspecific
`IgGl
`isotype control antibody, and murine monoclonal
`anti
`body reactive with the EGF receptor. As shown in Fig. 1, TAb
`250 precipitates
`a protein with a molecular weight of 185,000
`from NIH3T3,, SKBR-3, and SKOV-3 cells, whereas the iso
`type control antibody shows no reactivity (data not shown). Fig.
`1 also shows that an EGF receptor
`antibody precipitates
`a
`distinct and separate M, 170,000 protein that
`is particularly
`abundant
`in MDA-MB-468
`cells, which are known to overex-
`press the EGF receptor
`(21).
`The antiproliferative
`effects of TAb 250 in vitro were tested
`on cell lines using a 72-h colorimetrie MTT assay. Growth of
`SKBR-3 cells, a human metastatic breast
`tumor
`line that ex
`presses high levels of gpl85, was inhibited 40-50% of either
`untreated cells or cells exposed to a nonspecific isotype control
`antibody (Fig. 2). This effect decreased with dilution of the
`antibody such that
`there was no significant difference in prolif
`eration between cells treated with 0.8 Mg/ml TAb 250 or the
`IgGl
`isotype control. MDA-MB-468
`cells, which express high
`numbers of EGF receptors
`(21), but an undetectable
`level of c-
`erbB-2 protein (as determined by 125I-TAb250 binding4), or the
`immortalized
`breast cell
`line, HBL100, were not affected by
`treatment with TAB 250. These data suggest
`that TAb 250
`could specifically inhibit
`in vitro proliferation of cells expressing
`high levels of c-erbB-2, and that cells lacking this protein or
`expressing high levels of EGF receptor were unaffected.
`The nature of the growth inhibition
`of SKBR-3 cells was
`further
`investigated and the antibody effects were found to be
`cytostatic. Cells resumed proliferation
`following a 72-h incu
`bation with antibody if the cells were washed, refed with culture
`medium lacking TAb 250, and incubated for an additional
`5
`days (data not shown).
`Because a cytostatic antibody would not be likely to provide
`significant
`antitumor
`efficacy, we combined TAb 250 with
`
`the reactivity and specificity of TAb 250 for
`To determine
`gp 185 in human tumor cell lines, radiolabeled whole cell lysates
`4576
`
`4 L. K. Shawver et al., unpublished observation.
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on October 27, 2014. © 1991 American Association for Cancer
`Research.
`
`IMMUNOGEN 2068, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`INTERACTION OF A c-erbB-2 ANTIBODY AND CDDP
`
`inhibited tumor growth (P < 0.05). Treatment with 500 ßgTAb
`250 alone did not significantly inhibit tumor growth (P> 0.05).
`However,
`the inhibitory effect on tumor growth was greatly
`enhanced by injecting 500 Mgof TAb 250 followed by treatment
`with CDDP as observed in Fig. 5A. The combined treatment
`was significantly better than either of the treatments
`alone (P
`< 0.05) suggesting that
`these agents are acting synergistically.
`The weight of the animals did not change during the course of
`treatment
`and no other toxicities were observed. Tumors from
`animals treated with CDDP and TAb 250 resumed growth after
`day 42 although at a much reduced rate (data not shown). The
`in vivo effect of CDDP combined with TAb 250 appears to be
`specific for cells expressing c-erbB-2 because the combination
`effect on animals bearing tumors from MDA-MB-468 cells was
`not
`significantly
`greater
`than the inhibition
`observed with
`CDDP alone (Fig. 5Ä).
`The combination effects of TAb 250 and CDDP were signif
`icantly challenged by examining their effect on a tumor burden
`that was 6-fold greater
`at
`the beginning of treatment
`than
`described for Fig. 5. Fig. 6 shows that treatment of tumors with
`established growth are significantly inhibited with CDDP or
`with TAb 250 (P < 0.05). However, while the combined treat
`ment resulted in a >65% reduction of tumor growth,
`this was
`not significantly greater than treatment with TAb 250 or CDDP
`alone.
`
`90-
`
`80-
`
`70-
`
`60-
`
`40-
`
`30-20-
`
`10-
`
`0r^3—0-.—»—»--Ã(cid:141)-*.1--3-•*.•a-•-Ã(cid:173)v--3,i"f'ta
`1.00
`10.00
`0.00
`
`100.00
`
`B
`
`110-
`100-
`
`Avg. IgG, control
`- •
`. D.... MDA-MB-468
`-O
`HBL100
`..9
`SKBR-3
`
`1—
`0.10
`
`1.00
`
`10.00
`
`100.00
`
`Ab (ug/ml)
`
`120-,
`
`110-
`
`100-
`
`90-
`
`80-
`
`70-
`; 6o-
`Ii 50-
`. 40-
`
`I'
`
`30-
`
`20-
`
`10-
`
`0
`
`0.00
`
`lines. SKBR-3.
`Fig. 2. Inhibitory effects of TAb 250 on human breast cell
`MDA-MB-468,
`and HBL100 were obtained from the American Type Culture
`Collection and grown to confluence in minimum essential medium containing
`10% fetal bovine serum and L-glutamine. Growth inhibition was determined using
`a MTT assay as described in "Materials and Methods." Ab, antibody.
`
`of tumor
`drugs and evaluated proliferation
`chemotherapeutic
`cells in vitro and in vivo. Fig. 3A shows that cells in culture
`exposed simultaneously
`to TAb 250 and CDDP were dramati
`cally inhibited. During the 72-h incubation period, cells exposed
`to 1.0 or 2 Mg/ml CDDP reduced proliferation to 70 and 55%
`of control. However, cells treated with 1.0 /¿g/mlCDDP plus
`TAb 250 were inhibited to 25-30% of control, and those treated
`with 2.0 /¿g/mlCDDP and TAb 250 were inhibited to 10-15%
`of control. Treatment
`of cells with CDDP and an isotype
`control antibody did not inhibit proliferation greater than treat
`ment with CDDP alone (data not shown).
`The combined effect of TAb 250 and CDDP is specific for
`cells expressing c-erbB-2 as shown in Fig. 35. The growth of
`MD-MB-468 cells was not affected by TAb 250 alone, even at
`high concentrations.
`In addition,
`the inhibitory effect of CDDP,
`when combined with TAb 250, was not greater
`than CDDP
`alone. While MDA-MB-468
`cells appear
`to be more sensitive
`to CDDP,
`this may reflect variation between cell lines rather
`than expression ofc-erbB-2 or the EGF receptor. Regardless of
`the difference in CDDP effect, a greater
`sensitivity was not
`observed by treatment
`of the MDA-MB-468
`cells with TAb
`250.
`When SKBR-3 cells were treated with antibody and CDDP,
`followed by incubation with fresh growth medium for an addi
`tional 5 days, no evidence of cell growth was observed suggest
`ing the combination was cytotoxic
`(Fig. 4). This synergistic
`cytotoxicity was apparent over a wide range of antibody con
`centrations
`(200 pg/ml-100
`fig/ml; data not shown) and could
`be observed even at antibody concentrations
`that did not appear
`to have an effect when used alone (see Figs. 3A and 4). In
`addition,
`the synergistic effect was most readily observed when
`the dose of CDDP used alone resulted in a 30-50% inhibition.
`Under
`these conditions,
`the combined treatment
`resulted in
`80-100% cytotoxicity. Time course
`experiments
`(data not
`shown) suggested that
`the effects of the combined treatment
`occurred within the first 24 h of antibody and drug exposure.
`Because of the marked effect observed in vitro, the combina
`tion of CDDP and TAb 250 was assayed for
`inhibition of
`growth of s.c. xenografts
`in athymic mice (Fig. 5). Seven days
`following tumor
`inoculation,
`animals were treated with TAb
`250, IgGl, or the combination of TAb 250 and CDDP. Treat
`ment of tumor bearing animals with CDDP alone significantly
`
`70-î«-
`
`_
`
`1
`
`90
`80 -
`
`50 -
`40 -
`30-
`
`Q....
`
`20
`
`o.oo
`
`1.00
`
`10.00
`
`100.00
`
`Ab (ug/ml)
`
`Fig. 3. Synergistic effect of TAb 250 and CDDP. SKBR-3 cells (A) or MDA-
`MB-468 cells (B) were cultured and growth inhibition by TAb 250 and CDDP is
`carried out using a MTT assay as described in "Materials
`and Methods." For
`combination treatment,
`antibody was added to the wells at the concentrations
`indicated followed by addition of CDDP. A, —*—. TAb 250; A, CDDP (0.5 Mg/
`ml); *. CDDP.
`(l Mg/ml). Q. CDDP,
`(2 Mg/ml); --A--, TAb 250 plus CDDP
`(0.5 Mg/ml): — * —, TAb 250 plus CDDP (1 Mg/ml);
`Q
`, TAb 250 plus
`CDDP (2 Mg/ml). B. --T--,
`IgG,; —*—, TAb 250; * , CDDP (0.1
`Q, CDDP (0.2 Mg/ml);
`*
`, TAb 250 plus CDDP (0.1
`—Q—, TAb 250 plus CDDP (0.2 Mg/ml).
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on October 27, 2014. © 1991 American Association for Cancer
`Research.
`
`4577
`
`IMMUNOGEN 2068, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`INTERACTION OF A c-frftfi-2 ANTIBODY AND CDDP
`
`A
`
`800-
`
`700-
`
`"i 60°"
`
`-
`
`-•- -
`
`IgG, (500 W)
`
`TAb 250 (500 Hg)
`»
`»— CDDP (50 H«)
`
`O
`
`CDDP(3Hê/ml)
`..TAb250tCDDP(lU2/ml)
`
`0.00
`
`0.10
`
`I.(XI
`Ah (ug/ml)
`
`1IKIIHI
`
`—$-••TAh250*CDDP(lW/nil)
`
`20-
`
`I MIIIIM
`
`Ah (ng/ml
`
`In A, SKBR-3 cells were
`Fig. 4. Cytotoxic effects of TAb 250 and CDDP.
`cultured, and growth inhibition by TAb 250 and CDDP was carried out using a
`MTT assay as described in "Materials
`and Methods."
`In B, following .1 days
`exposure to TAb 250 and CDDP,
`the cells were gently washed,
`refed growth
`medium, and incubated for ¡inadditional 5 days prior to addition of MTT.
`
`DISCUSSION
`
`'s
`
`500-
`
`£ 400-
`
`§ 300-
`
`200-
`
`100-
`
`o-
`
`B
`
`•C
`
`700-1
`
`600-
`
`400-
`
`300-
`
`200-
`
`t
`
`
`
`
`
`IgQ + CDDP--*}--Q— TAb 250 +CDDP
`
`14
`t
`
`Day
`
`--
`
`IgG| (STOMS)
`
`TAb 250(500(1«)
`— CDDP (50 (ig)
`
`--
`
`IgG|+CDDP
`
`— TAb 250 + CDDP
`
`Day
`
`In A,
`activity of TAb 250 in combination with CDDP.
`Fig. 5. Antitumor
`SKOV-3 cells (1 x IO7) were implanted s.c. into athymic mice and allowed to
`grow until reaching a volume of 25-40 mm'. Three injections of TAb 250, IgG I,
`CDDP, or TAb 250 followed by CDDP 45 min later were administered once a
`week for 3 weeks (arrows). Tumor parameters were measured twice a week with
`a caliper and tumor volume calculated as
`
`Tumor volume (mm3) = length x width x height
`
`In B, MDA-MB-468 cells were implanted into athymic mice as described in I.
`Following inoculation,
`tumors were allowed to grow to a volume of 50-100 mm3
`prior to treatment. Animals were treated as described in A.
`
`the c-erbB-2 protein have been
`against
`Several antibodies
`shown to inhibit
`the growth of cell lines overexpressing c-erbB-
`2. Hudziak et al. (15) demonstrated
`antiproliferative
`effects of
`a c-erbB-2 monoclonal antibody against human tumor cell lines
`in vitro, and Drebin et al. (14) showed that growth of 3T3 cells
`transformed with neu could be inhibited in soft agar and in
`nude mice with a monoclonal
`antibody made against
`the rat
`neu protein. The effects demonstrated
`in these studies, however,
`were reversible or required the continuous presence of antibody.
`Thus,
`to be of significant
`therapeutic value, monoclonal
`anti
`bodies to gp 185 are likely to require conjugation or combination
`with other cytotoxic agents. Recently, an antibody against
`the
`EGF receptor was reported to have enhanced antitumor activity
`in vivo when combined with CDDP (22). In the present study,
`we show that combining CDDP with a monoclonal
`antibody
`specific for the extracellular
`domain of c-erbB-2 markedly en
`hances the inhibitory effect of CDDP both in vitro and in vivo.
`SKBR-3 cells exposed to TAb 250 and CDDP were dramat
`ically inhibited compared to cells exposed to either TAb 250 or
`CDDP alone. While the inhibitory effect of cells exposed to
`TAb 250 alone was cytostatic,
`the inhibitory effect appeared to
`be cytotoxic for cells exposed to both antibody and drug. The
`synergistic inhibition was apparent
`for antibody concentrations
`which did not appear
`to have an effect when used alone.
`TAb 250 also markedly enhanced
`the inhibitory effect of
`Fig. 6. Inhibitory effects of TAb 250 and CDDP on established tumor growth.
`CDDP in vivo using a s.c. xenograft model with SKOV-3 cells.
`SKOV-3 cells were implanted into athymic mice as described for Fig. 5. However,
`following inoculation,
`tumors were allowed to grow to a volume of 150-200 mm'
`This increased inhibitory effect was most readily observed when
`prior to treatment. Animals were then treated once a week for 3 weeks with TAb
`treatment began early after tumor cell inoculation. The effect
`250, IgG I, CDDP, or combination as described for Fig. 5.
`4578
`
`301X1-1
`
`2500 -
`
`5
`
`2000 -
`
`-» - IgOi <SOO(lg>
`
`-4-
`
`TAb 250 (SOOng)
`
`_$.-
`
`_Q-
`
`CDDP(50(lg)
`
`TAb 250 + CDDP
`
`28
`
`T1
`
`4
`Day f
`
`t
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on October 27, 2014. © 1991 American Association for Cancer
`Research.
`
`IMMUNOGEN 2068, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`INTERACTION OF A c-erbB-2 ANTIBODY AND CDDP
`
`was less significant when treating animals with established
`tumor growth. However,
`increasing the frequency of dosing
`with antibody or increasing the amount of antibody injected for
`each dose may result
`in increased efficacy and we are currently
`examining these parameters.
`The mechanism for the synergistic effect of TAb 250 and
`CDDP remains unclear at this time. While CDDP has signifi
`cant effects on DNA alkylation,
`its therapeutic effects on human
`cancers may occur due to several mechanisms. K562 and L1210
`cells treated with CDDP have been shown to have decreased
`methionine uptake and altered endogenous
`folate and methio-
`nine metabolism (23, 24). Several components
`of the growth
`factor-induced
`signal
`transduction
`pathway are affected by
`CDDP.
`Inhibitors
`of protein kinase C have been shown to
`enhance
`the antiproliferative
`activity of CDDP (25, 26). In
`addition, gene expression of c-fos has been shown to increase
`in Chinese hamster ovary cells treated with CDDP (27). Both
`c-fos and c-ras have been demonstrated
`to be amplified
`in
`patients
`failing treatment with CDDP (28). Since TAb 250 is
`directed against
`the extracellular domain of the c-erbB-2 protein
`and may possibly interfere with ligand binding,
`the synergistic
`effect with CDDP may be due to an interaction
`along this
`common pathway.
`An alternative explanation for a synergistic enhancement may
`be due to an inhibition of DNA repair. Treatment of cells with
`inhibitors
`of poly(ADP-ribose)
`polymerase
`has been docu
`mented to depress the excision repair of alkylated DNA (29-
`31). A similar phenomenon may occur after
`treatment with
`CDDP and TAb 250. The effects of TAb 250 on the repair of
`DNA-interstrand
`cross-links is being investigated.
`The c-erbB-2 oncogene is amplified and overexpressed in a
`large number of cell lines derived from human adenocarcino-
`mas. Of significance is the percentage of primary adenocarci-
`nomas of the breast which overexpress
`the c-erbB-2 protein
`(10) and the correlation with a poor disease prognosis. The
`extracellular portion of the c-erbB-2 protein provides an attrac
`tive target
`for immunotherapeutic modalities. However,
`treat
`ment using antibodies alone may be reversible or may require
`the continued
`presence of antibody. The use of monoclonal
`antibodies as adjunctive therapy with CDDP provides an alter
`native means of therapy for human tumors which overexpress
`c-erbB-2.
`
`ACKNOWLEDGMENTS
`
`The authors wish to thank Dr. Dennis Slamon for helpful discus
`sions; Janette Lenzi and Wendy Schraufnagel
`for technical assistance;
`Mary Crenshaw, Doris Hollander, KimVan Tran, and Lynn Webster
`for antibody preparation; Fai Pang and Carl Yoshizawa for assistance
`with statistical
`analyses; and Jo Ann Dornenburg
`for
`typing the
`manuscript.
`
`REFERENCES
`
`1. DiFiore, P. P.. Pierce, J. H., Kraus, M. H., Segallo, O., King, R., and
`Aaronson, S. A. erbB-2 is a potent oncogene when overexpressed in NIH/
`3T3 cells. Science (Washington DC), 237: 178-181, 1987.
`2. Hudziak, R. M., Schlessinger, J., and Ullrich, A. Increased expression of the
`putative growth factor receptor pl85HER2
`causes transformation
`and tu-
`morigenesis of NIH 3T3 cells. Proc. Nati. Acad. Sci. USA, 84: 7159-7163,
`1987.
`3. King, C. R., Kraus. M. H., and Aaronson, S. A. Amplification of a novel v-
`eréS-relatedgene in a human mammary carcinoma. Science (Washington
`DC), 229: 974-976, 1985.
`4. Kraus. M. H., Popescu, N. C., Amsbaugh, S. C., and King, C. R. Overexpres-
`
`4579
`
`sion of the EGF receptor-related proto-oncogene erbB-2 in human mammary
`tumor cell lines by different molecular mechanisms. EMBO J., 6: 605-610,
`1987.
`5. van de Vijver, M., van de Bersselaar, R., Devilee, P., Cornelisse, C., Peterse,
`J., and Nusse, R. Amplification of the neu (c-erbB-2) oncogene in human
`mammary tumors is relatively frequent and is often accompanied by ampli
`fication of the linked c-erbA oncogene. Mol. Cell Biol., 7: 2019-2023,
`1987.
`6. Natali, P. G.. Nicotra, M. R., Bigotti, A., Venturo,
`I., Slamon, D. J., Fendly,
`B. M., and Ullrich, A. Expression of the pl85 encoded by HER2 oncogene
`in normal and transformed human tissues. Int. J. Cancer, 45:457-461,
`1990.
`7. Berchuck, A., Kamel, A., Whitaker, R., Kerns, B., Olt, G., Kinney, R., Soper,
`J. T., Dodge, R., Clarke-Pearson, D. L., and Marks, P. Overexpression of
`HER-2/neu
`is associated with poor survival
`in advanced epithelial ovarian
`cancer. Cancer Res., 50:4087-4091,
`1990.
`8. Semba, K., Kamata, N., Toyoshima, K., and Yamamoto, T. A v-ereß-related
`protooncogene,
`c-erbB-2,
`is distinct
`from the c-erbB-1 /epidermal
`growth
`factor-receptor gene and is amplified in a human salivary gland adenocarci-
`noma. Proc. Nati. Acad. Sci. USA, 82: 6497-6501,
`1985.
`9. Yokota, T., Yamamoto, T., Miyajima, N., Toyoshima, K., Nomura, N.,
`Sakamoto, H., Yoshida, T., Terada, M., and Sugimura, T. Genetic alterations
`of the c-erbB-2 oncogene occur frequently in tubular adenocarcinoma of the
`stomach and are often accompanied by amplification of the v-erbA homo
`logue. Oncogene, 2: 283-287, 1988.
`10. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and
`McGuire, W. L. Human breast cancer: correlation of relapse and survival
`with amplification of the HER-2/neu oncogene. Science (Washington DC),
`235: 177-182, 1987.
`J., Maertens, G., Van Daele, S., Pauwels, C.,
`11. DePotter, C. R., Quatacker,
`Verhofstede, C., Eechaute, W., and Roels, H. The subcellular localization of
`the neu protein in human normal and neoplastic cells. Int. J. Cancer, 44:
`969-974, 1989.
`12. Maguire, H. C., Jr., Jaworsky, C., Cohen, J. A., IMiman. M., Weiner, D.
`B., and Greene, M. I. Distribution of neu (c-erbB-2) protein in human skin.
`J. Invest. Dermatol., 92: 786-790. 1989.
`13. Cohen, J. A., Weiner, D. B., More, K. F., Kokai. Y., Williams, W. V.,
`Maguire, H. C., Jr., LiVolsi, V. A., and Greene, M. I. Expression pattern of
`the neu (NGL) gene-encoded growth factor receptor protein (pl85neu)
`in
`normal and transformed epithelial
`tissues of the digestive tract. Oncogene,
`4:81-88,
`1989.
`14. Drebin, J. A., Link, V. C., Stern, D. F., Weinberg, R. A., and Greene, M. I.
`Down-modulation
`of an oncogene protein product and reversion of the
`transformed phenotype by monoclonal antibodies. Cell, 41: 695-706, 1985.
`15. Hudziak, R. M., Lewis, G. D., Winget, M., Fendly, B. M., Shepard, H. M.,
`and Ullrich, A. Monoclonal antibody has antiproliferative effects in vitro and
`sensitizes human breast
`tumor cells to tumor necrosis factor. Mol. Cell.
`Biol., 9: 1165-1172, 1989.
`16. Chabner. B. A., and Myers, C. E. Clinical Pharmacology of Cancer Chemo
`therapy.
`In: V. T. DeVita, Jr., S. Mellman,
`and S. A. Rosenberg (eds.).
`Cancer, Principles and Practice of Oncology, Ed. 3, Vol. 1. pp. 349-395.
`Philadelphia: J. B. Lippincott Co., 1989.
`17. McClay, E. F.. and Howell, S. B. A review: ¡ntraperitoneal cisplatin in the
`management of patients with ovarian cancer. Gynecol. Oncol., 36:1-6,1990.
`18. Langton, B. C., Crenshaw, M. C, Chao, L. A., Stuart, S. G., Akita, R. W.,
`and Jackson, J. E. An antigen immunologically related to the external domain
`of gp 185 is shed from nude mouse tumors overexpressing the c-erbB-2 (HER-
`21neu) oncogene. Cancer Res., 51: 2593-2598,
`1991.
`19. Mosmann, T. Rapid colorimetrie
`assay for cellular growth and survival:
`application to proliferation and cytotoxicity assays. J. Immunol. Methods,
`65:55-63,
`1983.
`20. SAS Institute Inc. SAS/STAT User's Guide, Version 6, Ed. 4, Vol. 2. Cary,
`
`NC: SAS Institute, 1989.
`21. Filmus. J., Pollak, M. N., Cailleau, R., and Buick, R. N. MDA-468, a human
`breast cancer cell line with a high number of epidermal growth factor (EGF)
`reeptors, has an amplified EGF receptor gene and is growth inhibited by
`EGF. Biochem. Biophys. Res. Commun., 128: 898-905, 1985.
`22. Aboud-Pirak, E., Hurwitz, E., Pirak, M. E., Beilot, F., Schlessinger, J., and
`Sela, M. Efficacy of antibodies to epidermal growth factor receptor against
`KB carcinoma in vitro and in nude mice. J. Nati. CáncerInst., 21: 1605-
`1611, 1988.
`23. Shionoya, S., Lu, Y., and Scanlon, K. J. Properties of amino acid transport
`systems in K562 cells sensitive and resistant
`to fis- diamminedichloropla-
`iimimil I). Cancer Res., 46: 3445-3448, 1986.
`24. Gross, R. B., and Scanlon, K. J. Amino acid membrane transport properties
`of L1210 cells resistant
`to cisplatin. Chemioterapia, 5: 37-43, 1986.
`25. Hofmann, J., Doppler, W., Jakob, A., Maly, K., Posch, L., Ãœberall,F., and
`Grunicke, H. H. Enhancement of the antiproliferative
`effect ofcis- diammi-
`nedichloroplatinum(II)
`and nitrogen mustard by inhibitors of protein kinase
`C. Int. J. Cancer, 42: 382-388, 1988.
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on October 27, 2014. © 1991 American Association f