MOLECULAR AND CELLULAR BIOLOGY, Mar. 1989, p. 1165-1172
`0270-7306/89/031165-08$02.00/0
`Copyright © 1989, American Society for Microbiology
`
`Vol. 9, No. 3
`
`p185HER2 Monoclonal Antibody Has Antiproliferative
`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 ULLRICH1t*
`Departments of Developmental Biology, 1 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
`
`gene encodes the epidermal growth factor receptorlike human homolog of the rat neu
`
`The HER2/c-erbB-2
`
`
`
`oncogene. Amplification of this gene in primary breast carcinomas has been shown to correlate with poor
`
`
`
`clinical prognosis for certain qlncer patients. We show here that a monoclonal antibody �rected against the
`domain of pl8SHER2 specifically
`
`
`inhibits the growth of breast tumor-derived cell lines overex­
`extracellular
`pressing the HER2 /c-erbB-2 gene product and prevents HERllc-erbB-2-traqsformed
`NIH 3T3 cells from
`
`
`forming colonies in son 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.
`
`* Corresponding author.
`t Present address: Max-Planck-lnstitut filr Biochemie, 8033 Mar­
`tinsried, Federal Republic of Germany.
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`HER2/c-erbB-2, the human homolog of the rat proto­
`
`cells made resistant to macrophages display enhanced tl!·
`
`
`oncogene neu (4, 34), encodes a 1,255-amino-acid glycopro­
`
`
`
`morigenicity. Tumor necrosis factor alpha (TNF-a) has been
`
`tein with extensive homology to the human epidermal
`
`shown to play a role in activated macrophage-mediated
`growth factor (EGF) receptor (4, 21, 33, 34, 42). The
`
`tumor cell killing in vitro (3, 11, 23, 29, 39). NIH 3T3 cells
`
`
`HER2/c-erbB-2 gene product, pl8511t:Rz, has all of the struc­
`
`transformed by a transfected and amplified HER21c-erbB-2
`
`
`tural features and many of the functional properties of
`
`
`
`
`cDNA show increased resistance to the cytotoxic effects of
`
`
`
`subclass I growth factor receptors (reviewed in references 43
`
`
`
`activated macrophages or TNF-a in direct correlation with
`
`and 44), including cell surface location and an intrinsic
`
`increased levels of pl85Ht:Ri expression. Furthermore,
`breast tumor cell lines with high levels of pl8511ERz exhibit
`
`
`tyrosine kinase activity. However, the ligand for this puta­
`tive growth factor receptor has not yet been identified.
`
`
`
`resistance to TNF-a. Resistance to host antitumor defenses
`
`
`Amplification of the HER2/c-erbB-2 gene has been found
`
`could facilitate the escape of cells from a primary tumor to
`
`
`in human salivary gland and gastric tumor-derived cell lines
`
`
`establish metastases at distant sites.
`
`(13, 34), as well as in mammary gland carcinomas (21, 22, 40,
`To further investigate the consequences of alteration in
`
`
`
`42). Slamon et al. (35) surveyed 189 primary breast adeno­
`
`
`HER2/c-erbB-2 gene expression in mammary gland neopla­
`gene
`
`carcinomas and determined that the HER2/c-erbB-2
`
`
`
`sia and to facilitate investigation of the normal biological role
`was amplified in about 30% of the cases. Most importantly,
`
`of the HER2/c-erbB-2 gene product, we have prepared
`
`
`
`HER2/c-erbB-2 amplification was correlated with a negative
`
`
`monoclonal antibodies against the extracellular domain of
`pl85HERz. One monoclonal antibody (4D5)
`
`
`
`prognosis and high probability of relapse. Similar although
`was character­
`
`
`less frequent amplification of the HER2/c-erbB-2 gene has
`ized in more detail and was shown to inhibit in vitro
`
`
`been reported for gastric and colon adenocarcinomas (45,
`
`
`proliferation of human breast tumor cells overexpressing
`
`46). Experiments with NIH 3T3 cells also suggest a direct
`
`
`
`pl8511£R.? and, furthermore, to increase the sensitivity of
`
`role for the overexpressed, structurally unaltered
`
`
`
`these cells to the cytotoxic effects of TNF-a.
`HER2/
`
`
`c-erbB-2 gene product pl8511ERz in neoplastic transforma­
`
`
`tion. High levels of HER2/c-erbB-2 gene expression attained
`MATERIALS AND METHODS
`
`
`by coamplification of the introduced gene with dihydrofolate
`Cells and cell culture. Human tumor cell lines were ob­
`reductase
`
`
`by methotrexate selection (18) or by using a strong
`
`tained from the American Type Culture Collection. The
`
`promoter (6) was shown to transform NIH 3T3 fibroblasts.
`
`
`
`mouse fibroblast line NIH 3T3/HER2-3400, expressing an
`
`
`Only cells with high levels of pl8511ERz are transformed, i.e.,
`
`amplified HER2/c-erbB-2 cDNA under simian virus 40 early
`
`have an altered morphology, are anchorage independent,
`
`
`promoter control, and the vector-transfected control cell line
`and will form tumors in athymic mice.
`
`
`NIH 3T3/CVN have been described previously (18).
`
`
`Overexpression of pl85HERZ may, furthermore, contribute
`
`Cells were cultured in a 1:1 mixture of Dulbecco modified
`
`
`to malignant tumor development by allowing tumor cells to
`
`Eagle medium and Ham nutrient mixture F-12 supplemented
`
`evade one component of the antitumor defenses of the body,
`
`100 u of penicillin per ml, 100 µ.g of
`with 2 mM glutamine,
`
`
`the activated macrophage (17). Macrophages play an impor­
`
`streptomycin per ml, and 10% serum. Human tumor cell
`
`
`tant role in immune surveillance against neoplastic growth in
`lines were cultured with fetal bovine serum (GIBCO Labo­
`vivo (1, 2, 38), and Urban et al. (39) have shown that tumor
`
`were ratories, Grand Island, N. Y .); NIH 3T3 derivatives
`
`
`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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`HUDZIAK ET AL.
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`MOL. CELL. BIOL.
`
`CVN
`HER2
`HER1
`,....
`,....
`I
`
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`d 0 0 ci 0 0 ci 0 0
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`
`1
`
`2
`
`p185HER2• The cells were washed once with phosphate­
`or a control antibody (40.1.Hl) recognizing the hepatitis B
`
`
`
`buffered saline (PBS) and detached from the plate with PBS
`
`surface antigen. The cells were washed twice and suspended
`
`containing 25 mM EDT A. After low-speed centrifugation,
`on ice for 30 min in 1 ml of PBS-1% fetal bovine serum
`
`the cells were suspended in cold PBS (2 x 107 ce!Js per ml).
`
`
`
`containing 10 µg of goat anti-mouse immunoglobulin G
`
`Each mouse was injected intraperitoneally with 0.5 ml of this
`
`
`F(ab'h fragments conjugated with ftuorescein isothiocyanate
`
`cell suspension on weeks 0, 2, 5, and 7.
`
`
`dye (Boehringer Mannheim Biochemicals, Indianapolis,
`On weeks 9 and 13, 100 µI of a Triton X-100 membrane
`
`Ind.). Unbound fluorescein dye was removed by two further
`of pl85HER2, partially
`washes. The cells were suspended at 2 x 106 per ml in
`purified by wheat germ
`preparation
`
`
`agglutinin chromatography (700 µg of protein per ml) (25),
`
`PBS-1% fetal bovine serum and analyzed with an EPICS 753
`
`
`was administered intraperitoneally. Three days before fu­
`
`
`
`
`(Coulter) fluorescence-activated cell sorter. Fluorescein was
`sion, 100 µI of the enriched p185HER2 protein was adminis­
`excited by 300 mW of 488-nm argon laser light, and the
`tered intravenously.
`
`emitted light was collected with a 525-nm band-pass filter
`Fusion and screening. Mice with high antibody titers as
`with a 10-nm band width.
`
`
`determined by immunoprecipitation of p185HER2 were sac­
`assay. SK-BR-3 cells were plated at 1.5 x
`Down-regulation
`105 cells per 35-mm culture dish in normal medium. After a
`
`rificed, and their splenocytes were fused as described previ­
`ously (26). Spleen cells were mixed at a 4:1 ratio with the
`
`
`6-h period to allow attachment, the medium was replaced by
`
`
`
`1.5 ml of methionine-free labeling medium containing 150
`
`
`fusion partner, mouse myeloma cell line X63-Ag8.653 (20),
`
`
`µCi of [35S)methionine per ml and 2% dialyzed fetal bovine
`
`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
`
`serum. The cells were metabolically labeled for 14 h and then
`
`
`microdilution plates. The hypoxanthine-azaserine (12) selec­
`
`chased with medium containing 2% dialyzed serum and
`
`tion for hybridomas was begun 24 h later. Beginning at day
`
`methionine. Either a control monoclonal antibody
`unlabeled
`
`10 postfusion, supematants from hybridoma-containing
`
`
`(40.1.Hl) or anti-p185HERz (4D5) was added to a final con­
`
`wells were tested for the presence of antibodies specific for
`
`centration of 2.5 µg/ml. At 0, 5, and 11 h, extracts were
`
`
`p185HER2 by an enzyme-linked immunosorbent assay with
`
`prepared with 0.3 ml of lysis solution and 0.6 mJ of dilution
`the wheat germ agglutinin chromatography-purified p185HER2
`
`
`
`buffer. The p185HERZ was immunoprecipitated with 2.5 µI of
`
`
`
`
`preparation (28). Enzyme-linked immunosorbent assay-pos­
`
`polyclonal antibody G-H2CT17. The washed immune com­
`
`itive supematants were confirmed by immunoprecipitation
`
`
`plexes were dissolved in sample buffer, electrophoresed on a
`
`and cloned twice by limiting dilution.
`
`SDS-7.5% polyacrylamide gel, and analyzed by autoradiog­
`Large quantities of specific monoclonal antibodies were
`
`
`
`raphy. Each time point determination was performed in
`
`
`produced by preparation of ascites fluid; antibodies were
`
`
`
`duplicate. Autoradiograph band intensities were quantitated
`
`then purified on protein A-Sepharose columns (Fermentech,
`by using a scanner (Ambis Systems).
`
`
`
`Inc., Edinburgh, Scotland) and stored sterile in PBS at 4°C.
`assays. The anti-pl85HERz monoclonal
`Cell proliferation
`and antibodies. Cells were har­
`
`
`antibodies were characterized by using the breast tumor cell
`Immunoprecipitations
`
`vested by trypsinization, counted in a Coulter counter
`
`line SK-BR-3. Cells were detached by using 0.25% (vol/vol)
`
`(Coulter Electronics, Inc., Hialeah, Fla.), and plated 24 h
`trypsin and suspended in complete medium at a density of 4
`before being harvested for analysis of p185HERZ 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 oflysis 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
`beads for 20 min and
`
`by adsorption to protein A-Sepharose
`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 µI of HNEG wash buffer containing 5 mM
`MnCl2 and 3 µCi 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.
`FIG. 1. Specificity of monoclonal antibody 405. Three cell lines,
`
`
`
`The polyclonal antibody, G-H2CT17, recognizing the car­
`17 amino acids of pl85HER2, has been de­
`NIH 3T3/CVN, NIH 3T3/HER1-EGF receptor. and NIH 3T3/
`boxy-terminal
`HER2-3400, were plated out at 2.0 x 106 in 100-mm culture dishes.
`
`
`scribed previously (18). The anti-EGF receptor monoclonal
`At 24 h, Triton X-100 lysates were prepared and divided into three
`antibody 108 (16) was provided by Joseph Schlessinger,
`portions. Either an irrelevant monoclonal antibody (6 µg of anti­
`hepatitis B virus surface antigen, 40.1.Hl; lanes 1, 4, and 7),
`
`Rorer Biotechnology, Inc.
`
`cell sorting. SK-BR-3 human breast
`anti-p185HER monoclonal antibody 405 (6 µg; lanes 2, 5, and 8), or
`Fluorescence-activated
`anti-EGF receptor monoclonal antibody 108 (6 µg; lanes 3, 6, and 9)
`
`tumor cells overexpressing the HER2/c-erbB-2
`gene (17, 22)
`or A431 human squamous carcinoma cells overexpressing
`was added and allowed to bind at 4°C for 4 h. The immune
`complexes were collected with 30 µI of protein A-Sepbarose. Rabbit
`
`the EGF receptor gene (14) were grown in Tl 75 flasks. They
`anti-mouse immunoglobulin (7 µg) was added to each 405 immuno­
`were detached from the flasks by treatment with 25 mM
`precipitation to improve the binding of this monoclonal antibody to
`
`
`EDTA--0.15 M NaCl, collected by low-speed centrifugation,
`the protein A-coated beads. Proteins were labeled by autophosphor­
`and suspended at 1 x 106 cells per ml in PBS-1 % fetal bovine
`ylation and separated on an SDS-7.5% polyacrylamide gel. The gel
`
`
`serum. One milliliter of each cell line was incubated with 10
`was exposed to film at -70°C for 4 h with an intensifying screen. The
`µg of either anti-HER2/c-erbB-2
`
`monoclonal antibody (405)
`arrows show the positions of proteins of M, 185,000 and 170,000.
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` on April 24, 2015 by guest
`
`-185
`-170
`
`9
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`VOL. 9. 1989
`
`MONOCLONAL ANTIBODIES AND pl85Ht'Rl
`
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`A .
`
`8.
`
`\
`J
`l '
`I
`I
`\
`
`(/)
`Q)
`(,)
`
`.... Q) .c
`E
`:::::>
`z
`Q)
`-�
`n;
`Q)
`er
`
`-0
`
`102 103 104
`10
`0.1
`Relative Fluorescence Intensity
`FIG. 3. Fluorescence-activated cell sorter histograms of human
`tumor cells binding anti-pl85 monoclonal antibody 405. -.
`Binding by the control antibody. 40.1.Hl. directed against the
`hepatitis B surface antigen: ·······. binding by the anti-HER21c­
`erb8-2 antibody. 405. 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'h 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 pl85H'"'Rl. (8)
`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
`p18511ERi (Fig. 1, lane 8). This antibody did not cross-react
`with the human EGF receptor (HER!; Fig. 1, lane 5), even
`when overexpressed in a mouse NIH 3T3 background (Fig.
`l, 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
`pl8511ERi (Fig. 2, lane 1). The polypeptide of 170,000 M,
`represents unglycosylated pl85HER2• The upper band of ca.
`185,000 M, comigrated with glycosylated p185HER2 from
`untreated cells (Fig. 2, lane 3). Monoclonal antibody 405
`efficiently immunoprecipitated only the glycosylated form of
`pl85"ERZ (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.
`
`,.....
` C\J
`t0 I
`� 6
`2 3
`
`..-1-{.)
`
`185-
`170-
`
`FIG. 2. Binding of monoclonal antibody 405 to unglycosylated
`receptor. NIH 3T3/HER2-3400 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 µ.g/ml. After a further 5.5 h of
`incubation. Triton X-100 lysates were then prepared from each
`plate. lmmunoprecipitations. 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 µ.I of a
`polyclona.I antibody directed against the C terminus of pl85H'"'Rl: 2.
`tunicamycin-treated cell lysate (one-third of a plate) immunoprecip­
`itated with 6 µ.g of 405; 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 IOS cells per ml. Aliquots of 100 µ.l (4 x 104 cells) were
`plated into 96-well microdilution plates, the cells were al­
`lowed to adhere, and 100 µ.l of media alone or media
`containing monoclonal antibody (final concentration. 5 µ.g/
`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 µ.g/ml. Cultures were
`incubated for another 4 h, and then increasing concentra­
`tions of TNF-a were added to a final volume of 200 µ.I.
`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 405. Monoclonal anti­
`bodies directed against the extracellular domain of pl8511ERi
`were prepared by immunizing mice with NIH 3T3 cells
`transfected with a HER2/c-erbB-2 cDNA (HER2-34o<» (17.
`18) and overexpressing the corresponding gene product.
`pl8511ER2• One antibody exhibited several interesting biolog­
`ical properties and was chosen for further characterization.
`Antibody 4D5 specifically immunoprecipitated a single 32P-
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`1168
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`HUOZIAK ET AL.
`
`MOL. CELL. BIOL.
`
`TABLE l. Inhibition of SK-BR-3 proliferation by anti-pl85H£Ri
`monoclonal antibodies"
`
`Relative cell
`Monoclonal
`proliferation•
`antibody
`7C2 ... .. ......... .. .. ... .. .. . .... .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. ... . 79.3 :!: 2.2
`2C4 ... ..... ... ... .. .. ... .. . . . .. . . . . .. ... . .... .. ... ... ... . . ... . . ... . 79.5 :!: 4.4
`703 . ... . ... . . .. .. .. . .. .. ... .. . . .. .. .. ... . ...... ... ...... .. ... . .. .. . 83.8 :!: 5.9
`405 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . 44.2 :!: 4.4
`3E8 .. .. . . .. .. .. ..... .. ... .. .. .. .. ..... .. .. .... .. .. .. .. .. . . .. .. .. .. . 66.2 :!: 2.4
`6E9 .. .. ........... .... ... .. ....... .. .. . . .. .. . . .. .. .. .. .. .. .. ....... 98.9 :!: 3.6
`7P3 ............................................... ................. 62.1 :!: 1.4
`3H4 ................................................................ 66.5 :!: 3.9
`2Hll ... ...... .. .. ... .. . . ....... .. .... ...... ...... ... . . .... .... .. .. 92.9 :!: 4.8
`40.l.Hl ........................................................... 105.8 :!: 3.8
`4F4 ................................................................ 94.7 :!: 2.8
`
`3.2
`
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`
`1.2
`
`0.8
`0.0
`
`1.0
`
`2.0
`
`3.0
`
`"SK·BR-3 breast tumor cells were plated as described in Materials and
`
`medium containing 5 µg of either anti­Methods. Following adherence,
`
`
`pl8511"°R2 or control monoclonal antibodies (40.1.Hl and 4F4) per ml were
`added.
`• 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 pl8511ERz. Figure 3A shows the 160-fold
`increase in celluJar fluorescence observed when 405 was
`added to SK-BR-3 breast adenocarcinoma cells relative to a
`control monoclonal antibody. This cell line contains an
`amplified HER2/c-erbB-2 gene and expresses high levels of
`pl8511ERz (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 p18511ERz (4), exhibited only
`a twofold increase in fluorescence with 405 (Fig. 3B) when
`compared with a control monoclonal antibody.
`The binding of 4D5 correlated with the levels of p18511ERz
`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 405 srecifically recog­
`nizes the extracellular domain of pl8511ER •
`
`100
`
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`a.
`� Q) a.
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`
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`
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`8
`
`12
`
`16
`
`Days of Culture
`FIG. 4. Growth curve of SK-BR-3 cells treated with anti-HER2/
`c-erbB-2 monoclonal antibody 405. Cells were plated into 35-mm
`culture dishes at 20,000 cells per plate in medium containing 2.5 µ.g
`of either control antibody (40.1.Hl, anti-hepatitis B surface antigen)
`(0) or anti-pl85H£Ri antibody 405 <•> 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.
`
`Antibody (µg/ml)
`FIG. 5. Growth of SK-BR-3 cells in different concentrations of
`monoclonal antibody 405. 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 µ.g of a control monoclonal antibody (40.1.Hl,
`anti-hepatitis B surface antigen) or monoclonal 405 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.
`
`Eft'ects 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 p18511ERz had any effect on the prolifera­
`tion of cell lines overexpressing this receptorlike protein.
`SK-BR-3 cells were coincubated with several HER2/c-erbB-
`2-specific monoclonal antibodies or with either of two dif­
`ferent control monoclonal antibodies (40.1.Hl, directed
`against the hepatitis B surface antigen; 4F4, directed against
`recombinant human gamma interferon). Most anti-HER2/
`c-erbB-2 monoclonal antibodies which recognize the extra­
`cellular domain inhibited the growth of SK-BR-3 cells (Table
`
`1.2 "T"""--------------.
`
`1.0
`
`�<O 0.8
`Q) u. .0 O> E it; 0.6
`�Q
`2-.:t 04
`a>- .
`>
`�
`Q;
`a:
`
`0.2
`
`0.0 157 361 231 MCF-7 SK- 175 3T3
`BR-3
`HER2
`Cell Line
`FIG. 6. Screening of breast tumor cell lines for growth inhibition
`by monoclonal antibody 405. 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-pl85HERi monoclonal antibody 405 was added on day 0 to 2.5
`µ.g/ml. Because the different cell lines grow at different rates, the cell
`lines NIH 3T3/HER2-3.oo and SK-BR-3 were counted after 6 days,
`cell lines MOA-MB-157, MOA-MB-231, and MCF-7 were counted
`after 9 days, and cell lines MOA-MB-175VII and MOA-MB-361
`were counted after 14 days. The difference in growth between cells
`treated with 405 and 40.1.Hl is expressed as the ratio of cell
`numbers with 405 versus a control monoclonal antibody, 9F6. Each
`cell line was assayed in duplicate for each antibody, and the counts
`were averaged.
`
`4 of 8
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`BI Exhibit 1021
`
`

`

`VOL. 9, 1989
`
`MONOCLONAL ANTIBODIES AND pl85HeRz
`
`1169
`
`Ohr - ---'-s __ hr __ _
`
`11 hr
`
`1
`
`2 3 4 5
`
`6
`
`7 8 9
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`
`FIG. 8. Effect of antibody binding on pl85H£RZ 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.l.Hl. anti-hepatitis B surface antigen) or 4D5 was
`added to 2.5 µg/ml. The cells on the plates were lysed at 0, 5, and 11
`h, and 35S-labeled pl85H£Rz 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 ftuor-treated gel was exposed to film
`for 4 h at room temperature. The arrow indicates the position of a
`protein of M, 185,000. Band intensities were quantitated by using an
`Ambis Systems scanner. Lanes; 1, 0 h; lanes 2 and 3, 40.1.Hl (5 h);
`lanes 4 and 5, 4D5 (5 h); lanes 6 and 7, 40.1.Hl (11 h); lanes 8 and
`9, 4D5 (11 h).
`
`effects were achieved by using a concentration of between
`0.5 and 1 µg/ml.
`The effect of 4D5 on the proliferation of six additional
`breast tumor cell lines, as well as mouse NIH 3T3 fibroblasts
`transformed by pl85HE�z overexpression (NIH 3T3/HER2-
`3400), was tested in monolayer growth assays. Cells were
`plated at low density in medium containing 2.5 µg of either a
`control antibody or 4D5 per ml. When the cultures ap­
`proached con fluency, 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 pl85HERz (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 HER2/c-erbB-2 amplification (18). In the pres­
`ence of 200 ng of a control monoclonal antibody (antitissue
`factor, TC-CS) 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 4DS down-regulates pl8SHER2. To
`determine whether the antiproliferative effect of 4D5 was
`due to enhanced degradation of pl85H£Rz, we measured its
`rate of turnover in the presence or absence of antibody.
`pl85HERz 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 pl85H£R2 levels were
`assayed by immunoprecipitation and SDS-polyacrylamide
`gel electrophoresis. p185HERZ is degraded more rapidly after
`exposure of SK-BR-3 cells to 4D5 (Fig. 8). Densitometric
`evaluation of the data showed that the p185HERz half-life of
`
`FIG. 7. Inhibition of anchorage-independent growth of NIH 3T3/
`HER2-3400 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 XlOO magnification by using a Nikon micro­
`scope with phase-contrast optics. (a) HER2-3400 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.Hl, or the
`anti-pl85H£Rz 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
`
`5 of 8
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`BI Exhibit 1021
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`

`

`1170 HUOZIAK ET AL.
`
`MOL. CELL. BIOL.
`
`A. SK-BR-3
`
`B. SK-BR-3
`
`C. MDA-MB-175-Vll
`
`1.5
`
`1.0
`
`D. MDA-MB-231
`
`E. HBL-100
`
`F. T24
`
`c::
`0
`1U 0.5
`
`�
`e
`a..
`
`'"-CD
`
`0.0
`1.5
`
`CD
`()
`CD
`>
`+;::;
`co
`CD
`a:
`
`1.0
`
`0.5
`
`0.0
`FIG. 9. Monoclonal antibody 405 sens1t1zes 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, MOA-MB-175-Vll, and MOA-MB-231; 104 cells per well for HBL-100 and T24) and
`allowed to adhere for 2 h. Anti-HER2/c-erbB-2
`monoclonal antibody 405 (5 µg/ml) or anti-hepatitis B surface antigen monoclonal antibody
`(5 µg/ml) was then added for a 4-h incubation prior to the addition ofTNF-a to a final concentration of 104 units/ml. After 72 h, the
`40.1.Hl
`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<•>. untreated (control) cells (lil!I), cells treated with TNF-a <•>. 405
`(rd), TNF-a plus 405 (0), 40.1.Hl
`( ::::;): or TNF-a plus 40.1.Hl ('�'�'.).
`
`Downloaded from
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`http://mcb.asm.org/
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` on April 24, 2015 by guest
`
`(1 x 104 units/ml;
`50% and 60% inhibition, respectively).
`
`
`7 h decreased lo 5 h in the presence of antibody (data not
`
`
`However, the combination of TNF-a and monoclonal anti­
`shown).
`Monoclonal antibody 4DS enhances TNF-a cytotoxidty.
`body 405 reduced the SK-BR-3 and MOA-MB-175-VII
`
`tumor cell number to a level below that initially plated,
`The addition of certain growth factors to tumor cells has
`
`
`
`
`indicating the induction of a cytotoxic response. In a sepa­
`
`been shown to increase their resistance to the cytotoxic
`rate experiment,
`
`SK-BR-3 cell viability was determined
`
`effects of TNF-a (37). A prediction based on these findings
`
`
`
`directly by using trypan blue dye exclusion, yielding identi­
`
`would be that expression of oncogenes that mimic or replace
`
`cal results to those described above that were obtained by
`
`
`
`growth factor receptor function may also increase the resis­
`
`using crystal violet staining (data not shown). A control
`
`tance of cells to this cytokine. Recently,
`it was shown that
`monoclonal antibody, 40.1.Hl, did not inhibit SK-BR-3
`
`
`overexpression of the putative growth factor receptor
`p185H ERi in NIH 3T3 cells caused an increase
`
`breast tumor cell proliferation, nor did it induce an enhanced
`in the resis­
`
`of this cell line to the cytotoxic effects of TNF-a
`sensitivity
`tance of these cells to TNF-a (17). Furthermore, breast
`
`tumor cell lines with high levels of p18511ERi also exhibited
`
`(Fig. 98). In addition, the growth of the breast tumor cell line
`
`MOA-MB-231, which does not express detectable levels of
`TNF-a resistance.
`p18511ERi (17), was unaffected by monoclonal
`antibody 405,
`To further investigate the mechanism by which the 405
`
`
`
`
`and the growth inhibition seen with the combination of 405
`
`
`antibody inhibited cell growth, we investigated the response
`and TNF-a was similar to that observed with TNF-a alone
`of three breast tumor cell lines to TNF-a in the presence or
`
`(Fig. 90). Furthermore, neither HBL-100 (30), a nontrans­
`
`
`absence of this antibody. If the anti-p18511ER2 monoclonal
`
`
`formed but immortalized human breast epithelial cell line
`
`
`antibody 405 inhibited proliferation of breast tumor cells by
`of pl85"£Ri, addi­