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`MOLECULAR AND CELLULAR BIOLOGY, Mar. 1989, p. 1165-1172
`0270-7306/89/031165-08$02.00/0
`Copyright C) 1989, American Society for Microbiology
`
`Vol. 9, No. 3
`
`p185HER2 Monoclonal Antibody Has An(iproliferative Effects In
`Vitro and Sensitizes Human Breast Tumor Cells to
`Tumor Necrosis Factor
`ROBERT M. HUDZIAK,1 GAIL D. LEWIS,2 MARCY WINGET,3 BRIAN M. FENDLY,3 H. MICHAEL SHEPARD,2
`AND AXEL ULLRICHlt*
`Departments of Developmental Biology,' Pharmacological Sciences,2 and Medicinal and Analytical Chemistry,3
`Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080
`Received 3 October 1988/Accepted 8 December 1988
`
`The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu
`oncogene. Amplification of this gene in primary breast carcinomas has been shown to correlate with poor
`clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the
`extraceUlular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overex-
`pressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from
`forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha,
`which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the
`presence of this antibody.
`
`HER21c-erbB-2, the human homolog of the rat proto-
`oncogene neu (4, 34), encodes a 1,255-amino-acid glycopro-
`tein with extensive homology to the human epidermal
`growth factor (EGF) receptor (4, 21, 33, 34, 42). The
`HER21c-erbB-2 gene product, p185HER2, has all of the struc-
`tural features and many of the functional properties of
`subclass I growth factor receptors (reviewed in references 43
`and 44), including cell surface location and an intrinsic
`tyrosine kinase activity. However, the ligand for this puta-
`tive growth factor receptor has not yet been identified.
`Amplification of the HER21c-erbB-2 gene has been found
`in human salivary gland and gastric tumor-derived cell lines
`(13, 34), as well as in mammary gland carcinomas (21, 22, 40,
`42). Slamon et al. (35) surveyed 189 primary breast adeno-
`carcinomas and determined that the HER21c-erbB-2 gene
`was amplified in about 30% of the cases. Most importantly,
`HER21c-erbB-2 amplification was correlated with a negative
`prognosis and high probability of relapse. Similar although
`less frequent amplification of the HER21c-erbB-2 gene has
`been reported for gastric and colon adenocarcinomas (45,
`46). Experiments with NIH 3T3 cells also suggest a direct
`role for the overexpressed, structurally unaltered HER21
`c-erbB-2 gene product p185HER2 in neoplastic transforma-
`tion. High levels of HER21c-erbB-2 gene expression attained
`by coamplification of the introduced gene with dihydrofolate
`reductase by methotrexate selection (18) or by using a strong
`promoter (6) was shown to transform NIH 3T3 fibroblasts.
`Only cells with high levels of pl85HER2 are transformed, i.e.,
`have an altered morphology, are anchorage independent,
`and will form tumors in athymic mice.
`Overexpression of p185HER2 may, furthermore, contribute
`to malignant tumor development by allowing tumor cells to
`evade one component of the antitumor defenses of the body,
`the activated macrophage (17). Macrophages play an impor-
`tant role in immune surveillance against neoplastic growth in
`vivo (1, 2, 38), and Urban et al. (39) have shown that tumor
`
`* Corresponding author.
`t Present address: Max-Planck-Institut fur Biochemie, 8033 Mar-
`tinsried, Federal Republic of Germany.
`
`cells made resistant to macrophages display enhanced tu-
`morigenicity. Tumor necrosis factor alpha (TNF-a) has been
`shown to play a role in activated macrophage-mediated
`tumor cell killing in vitro (3, 11, 23, 29, 39). NIH 3T3 cells
`transformed by a transfected and amplified HER21c-erbB-2
`cDNA show increased resistance to the cytotoxic effects of
`activated macrophages or TNF-a in direct correlation with
`increased levels of p185HER2 expression. Furthermore,
`breast tumor cell lines with high levels of p185HER2 exhibit
`resistance to TNF-a. Resistance to host antitumor defenses
`could facilitate the escape of cells from a primary tumor to
`establish metastases at distant sites.
`To further investigate the consequences of alteration in
`HER21c-erbB-2 gene expression in mammary gland neopla-
`sia and to facilitate investigation of the normal biological role
`of the HER21c-erbB-2 gene product, we have prepared
`monoclonal antibodies against the extracellular domain of
`p185HER2. One monoclonal antibody (4D5) was character-
`ized in more detail and was shown to inhibit in vitro
`proliferation of human breast tumor cells overexpressing
`p185HER2 and, furthermore, to increase the sensitivity of
`these cells to the cytotoxic effects of TNF-a.
`
`MATERIALS AND METHODS
`Cells and cell culture. Human tumor cell lines were ob-
`tained from the American Type Culture Collection. The
`mouse fibroblast line NIH 3T3/HER2-3400, expressing an
`amplified HER21c-erbB-2 cDNA under simian virus 40 early
`promoter control, and the vector-transfected control cell line
`NIH 3T3/CVN have been described previously (18).
`Cells were cultured in a 1:1 mixture of Dulbecco modified
`Eagle medium and Ham nutrient mixture F-12 supplemented
`with 2 mM glutamine, 100 u of penicillin per ml, 100 ,ug of
`streptomycin per ml, and 10% serum. Human tumor cell
`lines were cultured with fetal bovine serum (GIBCO Labo-
`ratories, Grand Island, N.Y.); NIH 3T3 derivatives were
`cultured with calf serum (Hyclone Laboratories, Inc., Lo-
`gan, Utah.).
`Immunization. Female BALB/c mice were immunized
`with NIH 3T3/HER2-3400 cells expressing high levels of
`
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`PETITIONER'S EXHIBITS
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`1166
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`HUDZIAK ET AL.
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`MOL. CELL. BIOL.
`
`or a control antibody (40.1.H1) recognizing the hepatitis B
`surface antigen. The cells were washed twice and suspended
`on ice for 30 min in 1 ml of PBS-1% fetal bovine serum
`containing 10 pug of goat anti-mouse immunoglobulin G
`F(ab')2 fragments conjugated with fluorescein isothiocyanate
`dye (Boehringer Mannheim Biochemicals, Indianapolis,
`Ind.). Unbound fluorescein dye was removed by two further
`washes. The cells were suspended at 2 x 106 per ml in
`PBS-1% fetal bovine serum and analyzed with an EPICS 753
`(Coulter) fluorescence-activated cell sorter. Fluorescein was
`excited by 300 mW of 488-nm argon laser light, and the
`emitted light was collected with a 525-nm band-pass filter
`with a 10-nm band width.
`Down-regulation assay. SK-BR-3 cells were plated at 1.5 x
`105 cells per 35-mm culture dish in normal medium. After a
`6-h period to allow attachment, the medium was replaced by
`1.5 ml of methionine-free labeling medium containing 150
`puCi of [35S]methionine per ml and 2% dialyzed fetal bovine
`serum. The cells were metabolically labeled for 14 h and then
`chased with medium containing 2% dialyzed serum and
`unlabeled methionine. Either a control monoclonal antibody
`(40.1.H1) or anti-p185HER2 (4D5) was added to a final con-
`centration of 2.5 jxg/ml. At 0, 5, and 11 h, extracts were
`prepared with 0.3 ml of lysis solution and 0.6 ml of dilution
`buffer. The p185HER2 was immunoprecipitated with 2.5 pul of
`polyclonal antibody G-H2CT17. The washed immune com-
`plexes were dissolved in sample buffer, electrophoresed on a
`SDS-7.5% polyacrylamide gel, and analyzed by autoradiog-
`raphy. Each time point determination was performed in
`duplicate. Autoradiograph band intensities were quantitated
`by using a scanner (Ambis Systems).
`Cell proliferation assays. The anti-p185HER2 monoclonal
`antibodies were characterized by using the breast tumor cell
`line SK-BR-3. Cells were detached by using 0.25% (vol/vol)
`trypsin and suspended in complete medium at a density of 4
`
`CVN
`
`HER1
`
`HER2
`
`I
`
`LO aco
`7
`8 a
`760o
`
`I e
`
`t
`It
`4 5 6
`
`I,
`
`T-eC:)
`
`t
`
`2
`
`3
`
`* t
`
`85
`*-170
`
`FIG. 1. Specificity of monoclonal antibody 4D5. Three cell lines,
`NIH 3T3/CVN, NIH 3T3/HER1-EGF receptor, and NIH 3T3/
`HER2-34m, were plated out at 2.0 x 106 in 100-mm culture dishes.
`At 24 h, Triton X-100 lysates were prepared and divided into three
`portions. Either an irrelevant monoclonal antibody (6 j±g of anti-
`hepatitis B virus surface antigen, 40.1.H1; lanes 1, 4, and 7),
`anti-p185HER monoclonal antibody 4D5 (6 ,ug; lanes 2, 5, and 8), or
`anti-EGF receptor monoclonal antibody 108 (6 ,ug; lanes 3, 6, and 9)
`was added and allowed to bind at 4°C for 4 h. The immune
`complexes were collected with 30 ,u1 of protein A-Sepharose. Rabbit
`anti-mouse immunoglobulin (7 ,ug) was added to each 4D5 immuno-
`precipitation to improve the binding of this monoclonal antibody to
`the protein A-coated beads. Proteins were labeled by autophosphor-
`ylation and separated on an SDS-7.5% polyacrylamide gel. The gel
`was exposed to film at -70°C for 4 h with an intensifying screen. The
`arrows show the positions of proteins of Mr 185,000 and 170,000.
`
`p185HER2. The cells were washed once with phosphate-
`buffered saline (PBS) and detached from the plate with PBS
`containing 25 mM EDTA. After low-speed centrifugation,
`the cells were suspended in cold PBS (2 x 107 cells per ml).
`Each mouse was injected intraperitoneally with 0.5 ml of this
`cell suspension on weeks 0, 2, 5, and 7.
`On weeks 9 and 13, 100 pI of a Triton X-100 membrane
`preparation of p185HER2, partially purified by wheat germ
`agglutinin chromatography (700 ,ug of protein per ml) (25),
`was administered intraperitoneally. Three days before fu-
`sion, 100 RI of the enriched p185 E12 protein was adminis-
`tered intravenously.
`Fusion and screening. Mice with high antibody titers as
`determined by immunoprecipitation of p185HER2 were sac-
`rificed, and their splenocytes were fused as described previ-
`ously (26). Spleen cells were mixed at a 4:1 ratio with the
`fusion partner, mouse myeloma cell line X63-Ag8.653 (20),
`in the presence of 50% polyethylene glycol 4000. Fused cells
`were plated at a density of 2 x 105 cells per well in 96-well
`microdilution plates. The hypoxanthine-azaserine (12) selec-
`tion for hybridomas was begun 24 h later. Beginning at day
`10 postfusion,
`supernatants from hybridoma-containing
`wells were tested for the presence of antibodies specific for
`p185HER2 by an enzyme-linked immunosorbent assay with
`the wheat germ agglutinin chromatography-purified p185HER2
`preparation (28). Enzyme-linked immunosorbent assay-pos-
`itive supernatants were confirmed by immunoprecipitation
`and cloned twice by limiting dilution.
`Large quantities of specific monoclonal antibodies were
`produced by preparation of ascites fluid; antibodies were
`then purified on protein A-Sepharose columns (Fermentech,
`Inc., Edinburgh, Scotland) and stored sterile in PBS at 4°C.
`Immunoprecipitations and antibodies. Cells were har-
`vested by trypsinization, counted in a Coulter counter
`(Coulter Electronics, Inc., Hialeah, Fla.), and plated 24 h
`before being harvested for analysis of p185HER2 expression.
`Cells were lysed at 4°C with 0.8 ml of HNEG lysis buffer (18)
`per 100-mm plate. After 10 min, 1.6 ml of lysis dilution buffer
`(HNEG buffer with 1% bovine serum albumin and 0.1%
`Triton X-100) was added to each plate, and the extracts were
`clarified by centrifugation at 12,000 x g for 5 min.
`Antibodies were added to the cell extracts and allowed to
`bind at 4°C for 2 to 4 h. Immune complexes were collected
`by adsorption to protein A-Sepharose beads for 20 min and
`washed three times with 1 ml of HNEG buffer-0.1% Triton
`X-100. Autophosphorylation reactions were carried out for
`20 min at 4°C in 50 ,ul of HNEG wash buffer containing 5 mM
`MnCl2 and 3 puCi of [.y-32P]ATP (5,000 Ci/mmol, Amersham
`Corp., Arlington Heights, Ill.). The autophosphorylation
`reaction conditions have been described previously (18).
`Proteins were separated on sodium dodecyl sulfate (SDS)-
`7.5% polyacrylamide gels and analyzed by autoradiography.
`The polyclonal antibody, G-H2CT17, recognizing the car-
`boxy-terminal 17 amino acids of p185HER2, has been de-
`scribed previously (18). The anti-EGF receptor monoclonal
`antibody 108 (16) was provided by Joseph Schlessinger,
`Rorer Biotechnology, Inc.
`Fluorescence-activated ceHl sorting. SK-BR-3 human breast
`tumor cells overexpressing the HER21c-erbB-2 gene (17, 22)
`or A431 human squamous carcinoma cells overexpressing
`the EGF receptor gene (14) were grown in T175 flasks. They
`were detached from the flasks by treatment with 25 mM
`EDTA-0.15 M NaCl, collected by low-speed centrifugation,
`and suspended at 1 x 106 cells per ml in PBS-1% fetal bovine
`serum. One milliliter of each cell line was incubated with 10
`,ug of either anti-HER21c-erbB-2 monoclonal antibody (4D5)
`
`PETITIONER'S EXHIBITS
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`VOL. 9, 1989
`
`MONOCLONAL ANTIBODIES AND p185HER2
`
`1167
`
`C)
`
`0
`
`0E zC
`
`)
`
`CD
`
`N-
`
`0
`
`Nr-
`0
`
`12
`
`185-.- dI
`17QO- o
`
`FIG. 2. Binding of monoclonal antibody 4D5 to unglycosylated
`receptor. NIH 3T3/HER2-34. cells were plated into two 100-mm
`plates at 2 x 106 cells per plate. After 14 h, the antibiotic tunicamy-
`cin was added to one plate at 3 jig/ml. After a further 5.5 h of
`incubation, Triton X-100 lysates were then prepared from each
`plate. Immunoprecipitations, the autophosphorylation reaction, and
`SDS-polyacrylamide gel electrophoresis were performed as de-
`scribed in the legend to Fig. 1. Lanes: 1, tunicamycin-treated cell
`lysate (one-third of a plate) immunoprecipitated with 2.5 ,ul of a
`polyclonal antibody directed against the C terminus of p185HER2; 2,
`tunicamycin-treated cell lysate (one-third of a plate) immunoprecip-
`itated with 6 ,ug of 4D5; 3, untreated control lysate (one-third of a
`plate) immunoprecipitated with the polyclonal antibody. The arrows
`show the locations of proteins of M, 185,000 and 170,000.
`
`x 105 cells per ml. Aliquots of 100 ,u1 (4 x 104 cells) were
`plated into 96-well microdilution plates, the cells were al-
`lowed to adhere, and 100 ,u1 of media alone or media
`containing monoclonal antibody (final concentration, 5 ,ug/
`ml) was then added. After 72 h, plates were washed twice
`with PBS (pH 7.5), stained with crystal violet (0.5% in
`methanol), and analyzed for relative cell proliferation as
`described previously (36).
`For assays in which monoclonal antibodies were com-
`bined with recombinant human TNF-a (5.0 x 107 U/mg;
`Genentech, Inc.), cells were plated and allowed to adhere as
`described above. Following cell adherence, control medium
`alone or medium containing monoclonal antibodies was
`added to a final concentration of 5 ,ug/ml. Cultures were
`incubated for another 4 h, and then increasing concentra-
`tions of TNF-a were added to a final volume of 200 ,Il.
`Following 72 h of incubation, the relative cell number was
`determined by crystal violet staining. Some samples were
`analyzed by crystal violet staining following cell adherence
`for determination of the initial cell number.
`
`RESULTS
`Specificity of monoclonal antibody 4D5. Monoclonal anti-
`bodies directed against the extracellular domain of p185"ER2
`were prepared by immunizing mice with NIH 3T3 cells
`transfected with a HER21c-erbB-2 cDNA (HER2-3400) (17,
`18) and overexpressing the corresponding gene product,
`p185HER2. One antibody exhibited several interesting biolog-
`ical properties and was chosen for further characterization.
`Antibody 4D5 specifically immunoprecipitated a single 32P-
`
`103
`104
`0.1
`1
`102
`10
`Relative Fluorescence Intensity
`FIG. 3. Fluorescence-activated cell sorter histograms of human
`tumor cells binding anti-p185 monoclonal antibody 4D5. ,
`Binding by the control antibody, 40.1.H1, directed against the
`hepatitis B surface antigen; . , binding by the anti-HER2/c-
`erbB-2 antibody, 4D5. The antibodies were first allowed to react
`with the cell surface. After a wash step, bound antibody was labeled
`by addition of fluorescein-conjugated F(ab')2 fragment of goat
`anti-mouse immunoglobulin G. (A) Binding of the antibodies to the
`human breast tumor line SK-BR-3, which contains an amplification
`of the HER21c-erbB-2 gene and expresses high levels of the HER21
`c-erbB-2 gene product p185HER2. (B) Binding of the same antibodies
`to the human squamous epithelial cell line A431. This cell line
`expresses low levels of mRNA for HER21c-erbB-2 and high levels (2
`x 106 receptors per cell) of the EGF receptor.
`
`labeled protein of Mr 185,000 from NIH 3T3 cells expressing
`p18SIIER2 (Fig. 1, lane 8). This antibody did not cross-react
`with the human EGF receptor (HER1; Fig. 1, lane 5), even
`when overexpressed in a mouse NIH 3T3 background (Fig.
`1, lane 6). Furthermore, it did not immunoprecipitate any
`proteins from NIH 3T3 cells transfected with a control
`plasmid (pCVN) which expresses the neomycin resistance
`and dihydrofolate reductase genes only (Fig. 1, lane 2).
`To determine the nature of the epitope recognized by 4D5,
`NIH 3T3/HER2-3400 cells were treated with tunicamycin,
`which prevents addition of N-linked oligosaccharides to
`proteins (15, 41). Cells treated with this antibiotic for 5.5 h
`contained two proteins which were immunoprecipitated by a
`polyclonal antibody against the carboxy-terminal peptide of
`p185LIER2 (Fig. 2, lane 1). The polypeptide of 170,000 Mr
`represents unglycosylated p185HER2. The upper band of ca.
`185,000 Mr comigrated with glycosylated p1851HER2 from
`untreated cells (Fig. 2, lane 3). Monoclonal antibody 4D5
`efficiently immunoprecipitated only the glycosylated form of
`p185LIER2 (Fig. 2, lane 2). This experiment suggests either
`that the epitope recognized by 4D5 consists partly of carbo-
`hydrate, or, alternatively, that the antibody recognizes a
`conformation of the protein achieved only when it is glyco-
`sylated.
`
`PETITIONER'S EXHIBITS
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`1168
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`HUDZIAK ET AL.
`
`MOL. CELL. BIOL.
`
`X 2.8
`
`2.4-
`
`2.0 -
`
`o1.6-
`
`ow
`CD
`
`LO
`
`I 1.2-
`
`4D5
`
`0.8-
`0.0
`
`3.0
`
`2.0
`1.0
`Antibody gg/ml)
`FIG. 5. Growth of SK-BR-3 cells in different concentrations of
`monoclonal antibody 4D5. The human breast tumor line SK-BR-3
`was plated into 35-mm culture dishes at 20,000 cells per dish. Either
`0.1, 0.5, 1.0, or 3.0 ,ug of a control monoclonal antibody (40.1.H1,
`anti-hepatitis B surface antigen) or monoclonal 4D5 antibody per ml
`was added at the time of plating. After 8 days of growth, the plates
`were trypsinized and the cells were counted in a Coulter counter.
`Each concentration of antibody was plated and counted in duplicate,
`and the cell numbers were averaged.
`
`Effects on cell proliferation. We used the human mammary
`gland adenocarcinoma cell line, SK-BR-3, to determine
`whether monoclonal antibodies directed against the extra-
`cellular domain of p185HER2 had any effect on the prolifera-
`tion of cell lines overexpressing this receptorlike protein.
`SK-BR-3 cells were coincubated with several HER21c-erbB-
`2-specific monoclonal antibodies or with either of two dif-
`ferent control monoclonal antibodies (40.1.H1, directed
`against the hepatitis B surface antigen; 4F4, directed against
`recombinant human gamma interferon). Most anti-HER2I
`c-erbB-2 monoclonal antibodies which recognize the extra-
`cellular domain inhibited the growth of SK-BR-3 cells (Table
`
`co
`
`o
`
`1.2-_
`
`1.0
`
`0 S) 0.8
`
`LL
`
`CD0.6-
`
`a)
`
`0.4 -
`
`TABLE 1. Inhibition of SK-BR-3 proliferation by anti-p185HER2
`monoclonal antibodies'
`
`Relative cell
`Monoclonal
`proliferationb
`antibody
`79.3 ± 2.2
`7C2 ........................................
`79.5 ± 4.4
`2C4 ........................................
`83.8 ± 5.9
`7D3 .......................................
`4D5 .......................................44.2 ± 4.4
`66.2 ± 2.4
`3E8 ........................................
`98.9 ± 3.6
`6E9 .......................................
`62.1 ± 1.4
`7F3 .......................................
`66.5 ± 3.9
`3H4 .......................................
`92.9 ± 4.8
`2H11 .......................................
`40.1.H1 .......................................
`105.8 ± 3.8
`94.7 ± 2.8
`4F4 .......................................
`a SK-BR-3 breast tumor cells were plated as described in Materials and
`Methods. Following adherence, medium containing 5 ,ug of either anti-
`p185HER2 or control monoclonal antibodies (40.1.H1 and 4F4) per ml were
`added.
`b Relative cell proliferation was determined by crystal violet staining of the
`monolayers after 72 h. Values are expressed as a percentage of results with
`untreated control cultures (100%).
`
`The binding of monoclonal antibody 4D5 to human tumor
`cell lines was investigated by fluorescence-activated cell
`sorting (Fig. 3). This antibody was bound to the surface of
`cells expressing p185HER2. Figure 3A shows the 160-fold
`increase in cellular fluorescence observed when 4D5 was
`added to SK-BR-3 breast adenocarcinoma cells relative to a
`control monoclonal antibody. This cell line contains an
`amplified HER21c-erbB-2 gene and expresses high levels of
`p185HER2 (17, 22). In contrast, the squamous carcinoma cell
`line A431, which expresses about 2 x 106 EGF receptors per
`cell (14) but only low levels of p185HER2 (4), exhibited only
`a twofold increase in fluorescence with 4D5 (Fig. 3B) when
`compared with a control monoclonal antibody.
`The binding of 4D5 correlated with the levels of p185HER2
`expressed by these two cell lines. SK-BR-3 cells, expressing
`high levels of p185HER2, showed an 80-fold increase in
`relative fluorescence intensity compared with A431 cells.
`This experiment demonstrates that 4D5 specifically recog-
`nizes the extracellular domain of p185HER2.
`
`100
`
`ci)
`0~
`Ci)Q.
`
`Days of Culture
`FIG. 4. Growth curve of SK-BR-3 cells treated with anti-HER2I
`c-erbB-2 monoclonal antibody 4D5. Cells were plated into 35-mm
`culture dishes at 20,000 cells per plate in medium containing 2.5 ,ug
`of either control antibody (40.1.H1, anti-hepatitis B surface antigen)
`(C1) or anti-p185HER2 antibody 4D5 (0) per ml. On the indicated
`days, cells were trypsinized and counted in a Coulter counter. The
`determination for each time point and each antibody was done in
`duplicate, and the counts were averaged. The arrow indicates the
`day the cells were refed with medium without antibodies.
`
`-*-
`
`a)s
`cc
`
`0.2
`
`0.0
`
`157
`
`361
`
`231
`
`175
`
`3T3
`HER2
`
`MCF-7 SK-
`BR-3
`Cell Line
`FIG. 6. Screening of breast tumor cell lines for growth inhibition
`by monoclonal antibody 4D5. Each cell line was plated in 35-mm
`culture dishes at 20,000 cells per dish. Either a control monoclonal
`antibody (9F6, anti-human immunodeficiency virus gpl20) or the
`anti-p185HER2 monoclonal antibody 4D5 was added on day 0 to 2.5
`,ug/ml. Because the different cell lines grow at different rates, the cell
`lines NIH 3T3/HER2-34w, and SK-BR-3 were counted after 6 days,
`cell lines MDA-MB-157, MDA-MB-231, and MCF-7 were counted
`after 9 days, and cell lines MDA-MB-175VII and MDA-MB-361
`were counted after 14 days. The difference in growth between cells
`treated with 4D5 and 40.1.H1 is expressed as the ratio of cell
`numbers with 4D5 versus a control monoclonal antibody, 9F6. Each
`cell line was assayed in duplicate for each antibody, and the counts
`were averaged.
`
`PETITIONER'S EXHIBITS
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`VOL. 9, 1989
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`MONOCLONAL ANTIBODIES AND p185HER2
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`FIG. 8. Effect of antibody binding on p185HER2 turnover. SK-
`BR-3 cells were labeled for 14 h with [35S]methionine. The label was
`then chased with cold methionine and either an irrelevant monoclo-
`nal antibody (40.1.H1, anti-hepatitis B surface antigen) or 4D5 was
`added to 2.5 ,ug/ml. The cells on the plates were lysed at 0, 5, and 11
`h, and 35S-labeled p185HER2 was quantitated by immunoprecipita-
`tion with the C-terminal specific polyclonal antibody. The 5- and
`11-h time point determinations were performed in duplicate for each
`of the two antibodies. Proteins were separated by SDS-polyacryl-
`amide gel electrophoresis. The fluor-treated gel was exposed to film
`for 4 h at room temperature. The arrow indicates the position of a
`protein Of Mr 185,000. Band intensities were quantitated by using an
`Ambis Systems scanner. Lanes; 1, 0 h; lanes 2 and 3, 40.1.H1 (5 h);
`lanes 4 and 5, 4D5 (5 h); lanes 6 and 7, 40.1.H1 (11 h); lanes 8 and
`9, 4D5 (11 h).
`
`effects were achieved by using a concentration of between
`0.5 and 1 p,g/ml.
`The effect of 4D5 on the proliferation of six additional
`breast tumor cell lines, as well as mouse NIH 3T3 fibroblasts
`transformed by pl85HF42 overexpression (NIH 3T3/HER2-
`3400), was tested in monolayer growth assays. Cells were
`plated at low density in medium containing 2.5 ,uag of either a
`control antibody or 4D5 per ml. When the cultures ap-
`proached confluency, cells were removed with trypsin and
`counted. 4D5 did not have any significant effect on the
`growth of the MCF-7, MDA-MB-157, MDA-MB-231, or
`NIH 3T3/HER2-3400 cell lines (Fig. 6). It did, however,
`significantly affect the growth of the cell lines MDA-MB-361
`(58% of control) and MDA-MB-175-VII (52% of control),
`which express high levels of p185HER2 (17).
`Interestingly, monoclonal antibody 4D5 had no effect on
`the monolayer growth of the NIH 3T3/HER2-3400 cell line.
`However, it completely prevented colony formation by
`these cells in soft agar (Fig. 7), a property which had been
`induced by HER21c-erbB-2 amplification (18). In the pres-
`ence of 200 ng of a control monoclonal antibody (antitissue
`factor, TC-C8) per ml, 116 (average of two plates) soft-agar
`colonies were counted, while the same cells plated simulta-
`neously into soft agar containing 200 ng of 4D5 per ml did not
`yield any colonies.
`Monoclonal antibody 4D5 down-regulates p185HER2. To
`determine whether the antiproliferative effect of 4D5 was
`due to enhanced degradation of p185HER2, we measured its
`rate of turnover in the presence or absence of antibody.
`p185HER2 was metabolically labeled by culturing SK-BR-3
`cells for 14 h in the presence of [35S]methionine. Cells were
`then chased for various times, and either a control antibody
`or 4D5 was added at the beginning of the chase period. At 0,
`5, and 11 h, cells were lysed and p185HER2 levels were
`assayed by immunoprecipitation and SDS-polyacrylamide
`gel electrophoresis. p185HER2 is degraded more rapidly after
`exposure of SK-BR-3 cells to 4D5 (Fig. 8). Densitometric
`evaluation of the data showed that the p185HER2 half-life of
`
`FIG. 7. Inhibition of anchorage-independent growth of NIH 3T3/
`HER2-34m cells by 4D5. Cells (20,000 per 60-mm plate) were plated
`in 0.2% soft agar over a 0.4% agar base. After 3 weeks, the plates
`were photographed at x 100 magnification by using a Nikon micro-
`scope with phase-contrast optics. (a) HER2-34m cells plated in agar
`containing 200 ng of a control antibody (TF-C8) per ml. (b) The same
`cells plated in agar containing 200 ng of 4D5 per ml.
`
`1). Maximum inhibition was obtained with monoclonal anti-
`body 4D5, which inhibited cellular proliferation by 56%. The
`control antibodies had no significant effect on cell growth.
`Figure 4 compares the growth of SK-BR-3 cells in the
`presence of either a control antibody, 40.1.H1, or the
`anti-p185HER2 antibody. Proliferation of the cells was inhib-
`ited when antibody 4D5 was present. The generation time
`increased from 3.2 to 12.2 days. To determine whether 4D5
`treatment was cytostatic or cytotoxic, antibody was re-
`moved by medium change 11 days after treatment. The cells
`resumed growth at a nearly normal rate, suggesting that the
`antibody affected cell growth rather than cell viability. The
`dose-response curve (Fig. 5) showed that a concentration of
`200 ng/ml inhibited growth by 50%, whereas maximum
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`1170
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`HUDZIAK ET AL.
`
`MOL. CELL. BIOL.
`
`A. SK-BR-3
`
`B. SK-BR-3
`
`C. MDA-MB-175-VII
`
`1L
`
`D. MDA-MB-231
`
`E. HBL-100
`
`F. T24
`
`1.5
`
`1.0 -
`
`0.5 -
`
`_.
`
`0.0 -
`1.5
`
`0 -
`
`5
`0~
`
`1.0
`
`CZ
`
`0.5
`
`FIG. 9. Monoclonal antibody 4D5 sensitizes breast tumor cells to the cytotoxic effects of TNF-a. Cells were plated in 96-well
`microdilution plates (4 x 104 cells per well for SK-BR-3, MDA-MB-175-VII, and MDA-MB-231; 104 cells per well for HBL-100 and T24) and
`allowed to adhere for 2 h. Anti-HER21c-erbB-2 monoclonal antibody 4D5 (5 p.g/ml) or anti-hepatitis B surface antigen monoclonal antibody
`40.1.H1 (5 p.g/ml) was then added for a 4-h incubation prior to the addition of TNF-a to a final concentration of 104 units/ml. After 72 h, the
`monolayers were washed twice with PBS and stained with crystal violet dye for determination of relative cell proliferation. In addition, some
`cell monolayers were stained with crystal violet following adherence in order to determine the initial cell density for comparison with cell
`densities measured after 72 h. The symbols denote initial cell density (E), untreated (control) cells (1), cells treated with TNF-a (E), 4D5
`(n), TNF-a plus 4D5 (O), 40.1.H1(1 ); or TNF-ot plus 40.1.H1(l'o!.).
`
`7 h decreased to 5 h in the presence of antibody (data not
`shown).
`Monoclonal antibody 4D5 enhances TNF-a cytotoxicity.
`The addition of certain growth factors to tumor cells has
`been shown to increase their resistance to the cytotoxic
`effects of TNF-ot (37). A prediction based on these findings
`would be that expression of oncogenes that mimic or replace
`growth factor receptor function may also increase the resis-
`tance of cells to this cytokine. Recently, it was shown that
`overexpression of the putative growth factor receptor
`p185HER2 in NIH 3T3 cells caused an increase in the resis-
`tance of these cells to TNF-a (17). Furthermore, breast
`tumor cell lines with high levels of p185HER2 also exhibited
`TNF-a resistance.
`To further investigate the mechanism by which the 4D5
`antibody inhibited cell growth, we investigated the response
`of three breast tumor cell lines to TNF-a in the presence or
`absence of this antibody. If the anti-p185HER2 monoclonal
`antibody 4D5 inhibited proliferation of breast tumor cells by
`interfering with the signalling functions of p185HER2, addi-
`tion of this antibody would be expected to enhance the
`sensitivity of tumor cells to TNF-c. Both SK-BR-3 (Fig. 9A)
`and MDA-MB-175-VII (Fig. 9C) were growth inhibited by
`both the monoclonal antibody 4D5 (5 ,ug/ml; 50% and 25%
`inhibition, respectively) and high concentrations of TNF-cx
`
`(1 x 104 units/ml; 50% and 60% inhibition, respectively).
`However, the combination of TNF-a and monoclonal anti-
`body 4D5 reduced the SK-BR-3 and MDA-MB-175-VII
`tumor cell number to a level below that initially plated,
`indicating the induction of a cytotoxic response. In a sepa-
`rate experiment, SK-BR-3 cell viability was determined
`directly by using trypan blue dye exclusion, yielding identi-
`cal results to those described above that were obtained by
`using crystal violet staining (data not shown). A control
`monoclonal antibody, 40.1.H1, did not inhibit SK-BR-3
`breast tumor cell proliferation, nor did it induce an enhanced
`sensitivity of this cell line to the cytotoxic effects of TNF-aL
`(Fig. 9B). In addition, the growth of the breast tumor cell line
`MDA-MB-231, which does not express detectable levels of
`p185HER2 (17), was unaffected by monoclonal antibody 4D5,
`and the growth inhibition seen with the combination of 4D5
`and TNF-ot was similar to that observed with TNF-ot alone
`(Fig. 9D). Furthermore, neither HBL-100 (30), a nontrans-
`formed but immortalized human breast epithelial cell line
`(Fig. 9E), nor T24 (27), a human bladder carcinoma cell line
`(Fig. 9F), expressed high levels of p185HER2 (data not
`shown), and neither demonstrated growth inhibition by 4D5
`or an enhanced growth-inhibitory or cytotoxic response to
`the combination of TNF-ot and monoclonal antibody 4D5.
`These results demonstrate that only tumor cells which
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`VOL. 9, 1989
`
`MONOCLONAL ANTIBODIES AND p185HER2
`
`1171
`
`overexpress p185HER2 will become sensitized to the cyto-
`toxic effects of TNF-a by antibody 4D5.
`DISCUSSION
`We have prepared monoclonal antibodies against the
`extracellular domain of the HER21c-erbB-2 gene product,
`pl85HER2, and have found that one of these, 4D5, strongly
`inhibits the growth of several breast tumor cell lines and
`furthermore sensitizes pl85HER2-overexpressing breast car-
`cinoma cell lines SK-BR-3 and MDA-MB-175-VII to the
`cytotoxic effects of TNF-a. Monoclonal antibody 4D5 is
`specific for pl85HER2 and shows no cross-reactivity with the
`closely related human EGF receptor expressed in mouse
`fibroblasts. Of six mammary carcinoma cell lines tested,
`only the three lines which express high levels of p185HER2
`(