`
`
`
`
`
`
`PHIGENIX
`PHIGENIX
`Exhibit 1005
`Exhibit 1005
`
`

`

`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
`
`p18SHER2 Monoclonal Antibody Has Antiproliferative Efiects lln
`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 ULLRICH‘H
`Departments ofDevelopmental 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
`
`The HERZ/c-erbB-Z 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
`extracellular domain of pISSHE'” specifically inhibits the growth of breast tumor-derived cell lines overex-
`pressing the HERZ/c-erbB-Z gene product and prevents HERZ/c-erbB-2-transformed NIH '3T3 cells from
`forming colonies in soft agar. Furthermore, resistance to the cytotoxic efl'ect of tumor necrosis factor alpha,
`is significantly reduced in the
`which has been shown to be a consequence of HERZ/c-erbB-Z overexpression,
`presence of this antibody.
`
`
`the human homolog of the rat proto—
`HERZ/c-erbB-Z,
`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
`HERZ/c-erbB—Z gene product,'p185”ER2, 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 HERZ/c-erbB-Z 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 HERZ/c-erbB-Z gene
`was amplified in about 30% of the cases. Most importantly,
`HER2/c-erbB-2 amplification was correlated with a negative
`prognosis and high probability of relapse. Similar although
`less frequent amplification of the HERZ/c-erbB-Z 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 HERZ/
`c-erbB-Z gene product p185”ER2 in neoplastic transforma—
`tion. Highlevels of HERZ/c-erbB-Z gene expression attained
`by coamplifieation 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 p185"ER2 are transformed, i.e.,
`have an altered morphology, are anchorage independent,
`and will form tumors in athymic mice.
`Overexpression of p185”ER2 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 fiir 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 HERZ/c—erbB-Z
`cDNA show increased resistance to the cytotoxic effects of
`activated macrophages or TNF-a in direct correlation with
`increased levels of p185"ER2 expression. Furthermore,
`breast tumor cell lines with high levels of pISSHERZ 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
`HERZ/c—erbB-Z gene expression in mammary gland neopla—
`sia and to facilitate investigation of the normal biological role
`of the HERZ/c-erbB-Z gene product, we have prepared
`monoclonal antibodies against the extracellular domain of
`p185”ER2. One monoclonal antibody (4D5) was character—
`ized in more detail and was shown to inhibit
`in vitro
`proliferation of human breast tumor cells overexpressing
`p185”ER2 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 HERZ/c-erbB—Z 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 pg 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
`
`1165
`
`iii"):
`ii
`iii
`1:;l
`
`PHIGENIX
`
`Exhibit 1005-01
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`

`

`
`
`MOL. CELL. BIOL.
`
`"1166
`
`HUDZIAK ET AL.
`
`p185”ER2. The cells were washed once with phosphate-
`bufiered 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.
`0n weeks 9 and 13, 100 pl of a Triton X-100 membrane
`preparation of p185”ER2, partially purified by wheat germ
`agglutinin chromatography (700 pg of protein per ml) (25),
`was administered intraperitoneally. Three days before fu-
`sion, 100 pl of the enriched p185"ER2 protein was adminis-
`tered intravenously.
`Fusion and screening. Mice with high antibody titers as
`determined by immunoprecipitation of p185”ER2 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, . supematants
`from hybridoma-containing
`wells were tested for the presence of antibodies specific for
`pl85"ER2 by an enzyme—linked immunosorbent assay with
`the wheat germ agglutinin chromatography-purified pISSHERZ
`preparation (28). Enzyme-linked immunosorbent assay—pos-
`itive supematants 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.
`llmmunoprecipitations 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 p185”ER2 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 bufi‘er 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 butter-0.1% Triton
`X-100. Autophosphorylation reactions were carried out for
`20 min at 4°C in 50 pl of HNEG wash buffer containing 5 mM
`MnCl2 and 3 pCi of [y-3ZP]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-HZCT17, recognizing the car-
`bo'xy-terminal 17 amino acids of p185”ER2, has been de-
`scribed previously (18). The anti-EGF receptor monoclonal
`antibody 108 (16) was provided by Joseph Schlessinger,
`Rorer Biotechnology, Inc.
`Fluorescence-activated cell sorting. SK-BR-3 human breast
`tumor cells overexpressing the HERZ/c-erbB-Z 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
`pg of either anti-HERZ/c-crbB-2 monoclonal antibody (4D5)
`
`an.MMm,t...a
`
`
`a...“Mk’ayt‘3
`
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`
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`
`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 pg 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
`pCi of [35$]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-pISSHERz (4D5) was added to a final con—
`centration of 2.5 pg/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 p185”ER2 was immunoprecipitated with 2.5 pl 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-p185“"m2 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
`
`
`HER’i
`HER2
`._
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`
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`08;
`V's—Vt
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`_.40.10H1
`
`
`FIG. 1. Specificity of monoclonal antibody 4D5. Three cell lines,
`NIH 3T3/CVN, NIH 3T3/HER1-EGF receptor, and NIH 3T3/
`HEM-3400, were plated out at 2.0 x 10" in loo-mm culture dishes.
`At 24 h, Triton X-100 lysates were prepared and divided into three
`portions. Either an irrelevant monoclonal antibody (6 pg of anti-
`hepatitis B virus surface antigen. 40.1.1-11;
`lanes 1, 4, and 7).
`anti-pl85”ER monoclonal antibody 4135 (6 pg; lanes 2, 5, and 8), or
`anti-EGF receptor monoclonal antibody 108 (6 pg; lanes 3, 6, and 9)
`was added and allowed to bind at 4°C for 4 h. The immune
`complexes were collected with 30 pl of protein A-Sepharose. Rabbit
`anti-mouse immunoglobulin (7 pg) 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 M, 185,000 and 170.000.
`
`l
`
`PHIGENIX
`
`Exhibit 1005-02
`
`

`

`VOL. 9. 1989
`
`MONOCLONAL ANTIBODIES AND plSSHERZ
`
`1167
`
`wG-H2CT17
`
`NJ405
`
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`RelativeNumberofCells
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`104
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`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-HERZ/c-
`crbB-Z 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 HERZ/c-erbB—Z gene and expresses high levels of the HERZ/
`c-erbB-Z gene product p185’mR2. (B) Binding of the same antibodies
`to the human squamous epithelial cell
`line A431. This cell
`line
`expresses low levels of mRNA for HERZ/c—erbB-Z and high levels (2
`x 10“ receptors per cell) of the EGF receptor.
`
`labeledprotein of Mr 185,000 from NIH 3T3 cells expressing
`p185”sz (Fig. 1, lane 8). This antibody did not cross-react
`with the human EGF receptor (HERI; 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 epitdpe 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
`p185’”iR" (Fig. 2,
`lane 1). The polypeptide of 170,000 Mr
`represents unglycosylated p185’"3R2. The upper band of ca.
`185,000 Mr comigrated with glycosylated p185”ER2 from
`untreated cells (Fig. 2, lane 3). Monoclonal antibody 4D5
`efficiently immunoprecipitated only the glycosylated form of
`p185’”‘R2 (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.
`
`PHIGENIX
`
`Exhibit 1005-03
`
`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 pg/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 pl of a
`polyclonal antibody directed against the C terminus of p185”ER2: 2.
`tunicamycin-treated cell lysate (one-third of a plate) immunoprecip-
`itated with 6 pg of 4D5; 3. untreated control lysate (one-third of a
`plate) immunoprecipitated with the polyclonal antibody. The arrows
`show the locations of proteins of Mr 185,000 and 170,000.
`
`X 105 cells per ml. Aliquots of 100 pl (4 X 104 cells) were
`plated into 96—well microdilution plates, the cells were al-
`lowed to adhere, and 100 pl of media alone or media
`containing monoclonal antibody (final concentration, 5 pg/
`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 pg/ml. Cultures were
`incubated for another 4 h, and then increasing concentra-
`tions of TNF—a were added to a final volume of 200 pl.
`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’"5R2
`were prepared by immunizing mice with NIH 3T3 cells
`transfected with a HERZ/c-erbB-Z cDNA (HERZ-Lm) (17,
`18) and overexpressing the corresponding gene product,
`p185”ER2. One antibody exhibited several interesting biolog-
`ical properties and was chosen for further characterization.
`Antibody 4D5 specifically'immunoprecipitated a single 32P-
`
`l , l
`
`1 l
`
`

`

`
`
`MOL. CELL. BIOL.
`
`1168
`
`HUDZIAK ET AL.
`
`TABLE 1. Inhibition of SK-BR-3 proliferation by.anti-p185”ER2
`monoclonal antibodies"
`___________.____.——.——-————
`Monoclonal
`Relative cell
`antibody
`proliferation"
`7C2 ................................................................ 79.3 t 2.2
`
`......
`2C4 .....
`79.5 t 4.4
`
`7D3 ....................................
`83.8 i 5 9
`
`4D5 ......................................
`44.2
`4 4
`
`3E8 .....
`66.2
`7
`6E9 .....
`
`
`
`” SK-BR-3 breast tumor cells were plated as described in Materials and
`Methods. Following adherence, medium containing 5 pg of either anti-
`pfiS’m’" or control monoclonal antibodies (40.1.Hl and AH) per ml were
`a
`ed.
`” 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%).
`1
`
`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 p185"ER2. 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 HERZ/c—erbB-Z gene and expresses high levels of
`p185"ER2 (17, 22). In contrast, the squamous carcinoma cell
`line A431, which expresses about 2 X 10" EGF receptors per
`cell (14) but only low levels of p185”ER2 (4), exhibited only
`a twofold increase in fluorescence with 4D5 (Fig. 38) when
`compared with a control monoclonal antibody.
`The binding of 4D5 correlated with the levels of p185”ER2
`expressed by these two cell lines. SK-BR-3 cells, expressing
`high levels of p185"ER2. 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 p185”ER2.
`
`100
`
`
`
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`
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`
`8
`
`12
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`Days of Culture
`FIG. 4. Growth curve of SK-BR-3 cells treated with anti-HERZ/
`c-erbB-Z monoclonal antibody 4D5. Cells were plated into 35-mm
`culture dishes at 20,000 cells per plate in medium containing 2.5 pg
`of either control antibody (40.1.H1, anti-hepatitis B surface antigen)
`(E1) or anti-p185""R" antibody 4D5 (
`) per ml. 0n 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.
`
`
`
`«sigma».‘22.xi;~“
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`
`0.8
`
`0.0
`
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`
`2.0
`
`3.0
`
`Antibody (pg/ml)
`FIG. 5. Growth of SK-BR-3 cells in difierent 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 pg 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 p18SHER2 had any effect on the prolifera-
`tion of cell
`lines overexpressing this receptorlike protein.
`SK-BR-3 cells were coincubated with several HERZ/c-erbB—
`2-specific monoclonal antibodies or with either of two dif—
`ferent control monoclonal antibodies (40.1.1—11, directed
`against the hepatitis B surface antigen; 4F4, directed against
`recombinant human gamma interferon). Most anti-HERZ/
`c-erbB-Z monoclonal antibodies which recognize the extra-
`cellular domain inhibited the growth of SK—BR-3 cells (Table
`
`0....won)
`
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`
`
`
`.0N
`RelativeNumberofCells(4D5/9F6) O 0)
`
`0.0
`
`157
`
`351
`
`175
`
`3T3
`HER2
`
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`
`-
`231 MCF-7 SK-
`BR-a
`Cell Line
`
` 11 i l
`
`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 gp120) or the
`anti-pISSHER" monoclonal antibody 4D5 was added on day 0 to 2.5
`pig/ml. Because the diflerent cell lines grow at different rates, the cell
`lines NIH 3T3/HER2-3.00 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—175Vll and MDA-MB-361
`were counted after 14 days. The diflerence in growth between cells
`treated with 4D5 and 40.1.Hl
`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.
`
`PHIGENIX
`
`Exhibit 1005-04
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`VOL. 9. 1989
`
`MONOCLONAL ANTIBODIES AND p185’"’""'z
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`1169
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`11hr
`5hr
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`123456789
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`FIG. 8. Effect of antibody binding on pISSH’”u turnover. SK-
`BR-3 cells were labeled for 14 h with [35$]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 pg/ml. The cells on the plates were lysed at (1, 5, and 11"
`h, and 3SS-Iabeled p185"E"" 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 floor-treated gel was exposed to film
`for 4 h at room temperature. The arrow indicates the position of a
`protein of MI. 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).
`
`”3::
`
`elfects were achieved by using a concentration of between
`0.5 and 1 pg/ml.
`The effect of 4D5 on the proliferation of six additional
`breast tumor cell lines, as well as mouse NIH 3T3 fibroblasts
`transformed by p185”ER2 overexpression (NIH 3T3/I-IER2-
`3400), was tested in monolayer growth assays. Cells were
`plated at low density in medium containing 2.5 ug of either a
`control antibody or 4D5 per ml. When the cultures ap-
`proached confiuency, 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/HER2-3m, 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-Vll (52% of control),
`which express high levels of p185"ER2 (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 HERZ/c-erbB-Z 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
`1). Maximum inhibition was obtained with monoclonal anti-
`yield any colonies.
`body 4D5, which inhibited cellularproliferation by 56%. The
`Monoclonal antibody 4D5 down-regulates p185’m’". To
`3,:
`control antibodies had no significant effect on cell growth.
`determine whether the antiproliferative effect of 4D5 was
`1a]
`Figure 4 compares the growth of SK—BR—3 cells in the
`due to enhanced degradation of p185"”2, we measured its
`he
`presence of either a control antibody, 40.1.H1, or the
`rate of turnover in the presence or absence of antibody.
`2.5
`anti-p185"ER2 antibody. Proliferation of the cells was inhib-
`p185’"‘"’2 was metabolically labeled by culturing SK-BR-3
`=6“
`ited when antibody 4D5 was present. The generation time
`cells for 14 h in the presence of[”S]methionine. Cells were
`ys.
`increased from 3.2 to 12.2 days. To determine whether 4D5
`then chased for various times, and either a control antibody
`55d '
`treatment was cytostatic or cytotoxic, antibody was re-
`or 4D5 was added at the beginning ofthe chase period. At 0,
`‘61
`moved by medium change 11 days after treatment. The cells
`5. and 11 h, cells were lysed and p185"sz levels were
`2;
`resumed growth at a nearly normal rate. suggestingthat the
`assayed by immunopreclilpi’tzation and SDS-polyacrylamide
`ich
`antibody affected cell growth rather than cell Viability. The
`gelelectrophoresrs. p185
`‘ ‘ ts degraded more rapidlyafter
`ms
`dose-response curve (Fig. 5) shOWed that aconcentration of
`exposure of SK-BR-3 cells to 4D5 (Fig. 8). pensitometric
`200 ng/ml
`inhibited growth by 50%. whereas maximum
`evaluation of the data showed that the p185”sz half-life of
`
`
`
`
`FIG. 7. Inhibitionofanchorage-independent growth of'NlH m/
`HERZ-Smcells by 4D5. Cells (20,000 per60-mm plate) were plated
`in 0.2% soft agar over a 0.4% agar base. After 3 weeks. the plates
`were photographed at X100 magnification by using a Nikon micro-
`scope with phase-contrast optics: (a) HER2'34(X)CCIIS Plaléd in agar
`comaining290 “3OfaconIr‘.“ anttbody (TF'CS) permI' (b) ”'8 same
`““5 Plated ‘" agar ”mammg 200 ng 0f 4D5 per m"
`
`PHIGENIX
`
`Exhibit 1005-05
`
`

`

`
`
`1170
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`HUDZIAK ET AL.
`
`1.5
`
`1.0
`
`0.5
`
`0.5
`
`1.0
`
`
`
`RelativeCellProliferation
`
`c efl‘ects of TNF-a. Cells were plated in 96—well
`0.0
`L—100 and T24) and
`tumor cells to the cytotoxi
`FIG. 9. Monoclonal antibody 4D5 sensitizes breast
`MB-231; 10‘ cells per well for HB
`en monoclonal antibody
`microdilution plates (4 x 10‘ cells per well for SK—BR-3, MDA-MB-l75-Vll. and MDA-
`-hepatttis B surface anttg
`“ units/ml. After 72 h, the
`'
`allowed to adhere for 2 h. Anti—HERZ/c-erbB-Z monoclonal antibody 4DS (5 pg/ml) or ant
`ration. In addition. some
`40.1.H1 (5 pg/ml) was then added for a 4—h incubation prior to the addition of TN F-a to a .
`for comparison with cell
`monolayers were washed tWIce with PBS and stained with crystal violet dye for
`ated with TNF-u (E). 4D5
`
`
`
`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
`p185"ER2 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 p185"'5’"2 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-p185"ER2 monoclonal
`antibody 4D5 inhibited proliferation of breast tumor cells by
`interfering with the signalling functions of p185”"5R2. addi-
`tion of this antibody would be expected to enhance the
`sensitivity of tumor cells to TNF-u. Both SK-BR—3 (Fig. 9A)
`and MDA-MB-175—Vll (Fig. 9C) were growth inhibited by
`both the monoclonal antibody 4D5 (5 (Lg/ml: 50% and 25%
`inhibition, respectively) and high concentrations of TN F-u
`
`and 60% inhibition, respectively).
`(1 X 10" units/ml; 50%
`d monoclonal anti-
`f TNF-a an
`However, the combination 0
`BR-3 and MDA-MB—175-Vll
`uced the SK-
`body 4D5 red
`low that
`initially plated,
`r to a level be
`e. In a sepa-
`tumor cell numbe
`n of a cytotoxic respons
`indicating the inductio
`determined
`3 cell viability wa
`rate experiment, SK-BR-
`‘elding identi-
`ye exclusion. yt
`pan blue d
`btained by
`directly by using try
`ove that were 0
`described ab
`cal results to those
`0t shown). A control
`olet staining (data n
`using crystal vi
`t
`inhibit SK—BR—3
`body, 40.1.H1, did no
`uce an enhanced
`oliferation, nor did it ind
`breast tumor cell pr
`s of TN F—a
`cell line
`
`and the growth inhibition seen w
`similar to that o
`and TNF-u was
`ore. neither HBL-100 (30). a nontrans-
`(Fig. 9D). Furtherm
`d human breast epithelial cell line
`immortalize
`formed but
`man bladder carcinoma cell line
`(Fig. 9E). nor T24 (27). a hu
`evels of p185”":R2 (data not
`(Fig. 9F). expressed high 1
`growth inhibition by 4D5
`shown). and neither demonstrated
`ytotoxic response to
`an enhanced growth-inhibitory or c
`nal antibody 4D5.
`ation of TN F-a and monoclo
`the combin
`cells which
`at only tumor
`These results demonstrate th
`
`PHIGENIX
`
`Exhibit 1005-06
`
`

`

`
`
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`
`MONOCLONAL ANTIBODIES AND pISSHERZ
`
`1171
`
`mammary gland neoplasia. We have shown here that HER2/
`c-erbB-2 gene overexpression in NIH 3T3 cells is associated
`with increased resistance to the monokine TNF-ot and that
`breast
`tumor cell
`lines which overexpress p185”ER2 are
`resistant to the cytotoxic efl‘ects of TNF-ct. The mechanism
`by which 4D5 inhibits breast tumor cell proliferation and
`reverses phenotypes associated with high levels of p185”ER2
`expression, such as resistance to TNF—a,
`is not clear.
`However, these results suggest that in addition to its ability
`to transform cells by virtue of overexpression (6, 18),
`HERZ/c-erbB-Z could play a role in tumor progression by
`allowing tumor cells overexpressing p18SHER2 to evade one
`component of the antitumor immunosurveillance of the host,
`the activated macrophage (17). These properties of the
`HERZ/c-erbB-Z gene product may in part explain the aggres-
`sive, single-step induction of mammary adenocarcinoma in
`transgenic mice bearing the neu oncogene (24), which en-
`codes the mutated rat homolog of pISSHERZ.
`The experiments presented here demonstrate that a mono-
`clonal antibody which recognizes the extracellular domain of
`p185”ER2 inhibits the proliferation of breast
`tumor cells
`which overexpress this receptorlike protein. Moreover,
`treatment with this antibody also sensitizes these tumor cells
`to the cytotoxic efi‘ects of TNF-a. Monoclonal antibodies
`specific for p185”ER2 may therefore be useful therapeutic
`agents for the treatment of human neoplasias,
`including
`certain mammary carcinomas, which are characterized by
`the overexpressing of p18SHER2
`ACKNOWLEDGMENTS
`
`VOL. 9