Cancer Immunol [mmunother (1993] 37: 255 —-263
`
`_ nununo
`
`l
`Vlaneer
`Ilmmunotfigeiiapy
`
`© Springer—Verlag 1993
`
`Differential responses of human tumor cell lines
`to anti-p185HERZ monoclonal antibodies
`
`Gail D. Lewis, Irene Figari, Brian Fendly, Wai Lee Wong, Paul Carter, Cori German, H. Michael Shepard*
`
`Genentech luc., 460 Point San Bruno Boulevard, South San Francisco. CA 94080, USA
`
`Received 21 October l992]Acccpted 30 March 1993
`
`Abstract. The HER2 protooncogcne encodes a receptor
`tyrosine
`kinase,
`p185HER3. The overexpression of
`p135HER2 has been associated with a worsened prognosis
`in certain human cancers. In the present work we have
`screened a variety of different
`tumor cell
`lines
`for
`plSSHER2 expression using both enzyme-linked immuno-
`sorbent and fluorescence—activated cell sorting assays em-
`ploying murine monoclonal antibodies directed against the
`extracellular domain of the receptor. Increased levels of
`p185HER2 were found in breast (5:9), ovarian ( 1.16), stom—
`ach (2’3) and colorectal (5116) carcinomas, whereas all
`kidney and submaxillary adenoearcinoma cell lines tested
`were negative. Some monoclonal antibodies directed
`against
`the extracellular domain of plSSHER2 inhibited
`growth in monolayer culture of breast and ovarian tumor
`cell lines overexpressing plSfiHF-RZ, but had no effect on
`the growth of colon or gastric adenocarcinomas expressing
`increased levels of this receptor. The most potent growth-
`inhibitory anti-p185HER2 monoclonal antibody in mono—
`layer culture, designated mumAb 4D5 (a murine IgGlic
`antibody), was also tested in soft—agar growth assays for
`activity against pISSHERZ-overexpressing tumor cell lines
`of each type, with similar results. In order to increase the
`spectrum of tumor types potentially susceptible to mono
`clonal antibody—mediated anti—plSiSmiR2 therapies,
`to
`decrease potential immunogenicity issues with the use of
`murine monoclonal antibodies for human therapy, and to
`provide the potential for antibody—mediated cytotoxic ac—
`tivity, a mousei‘human chimeric 4D5 (chmAb 4D5) and a
`“humanized” 405 (rhu)mAb 4D5 HER2 antibody were
`constructed. Both engineered antibodies, in combination
`with human peripheral blood mononuclear cells, elicited
`antibody—dependent cytotoxic responses in accordance
`
`* Present address: Canji Loo, San Diego, CA 92121, USA
`
`Correspondence to: G. D. Lewis, Department of Cell Analysis,
`Genentech Inc., 460 Point San Bruno Blvd, South San Francisco,
`CA 94080.USA
`
`with the level ofplBSmiR2 expression. Since this cytotoxic
`activity is independent of sensitivity to mumAb 4D5, the
`engineered monoclonal antibodies expand the potential tar—
`get population for antibody-mediated therapy of human
`cancers characterized by the overexpression of pISSHEm.
`
`Key words: pl 85HER2 — Monoclonal antibodies : Growth
`inhibition — Cytotoxicity
`
`
`
`Introduction
`
`The pathogenesis of human cancer often involves altera—
`tion in the structure and expression of various oncogene
`products. Although direct causal relationships between
`oncogene amplification andfor overexpression and certain
`types of cancer remain ill—defined, there are examples of
`correlations between the occurrence of particular cancers
`and oncogene activation. For instance, amplification of the
`N—myc oncogene has been found in neuroblastomas and
`retinoblastomas [9, 33, 48], while c-myc amplification was
`reported in small-cell lung cancer, as well as in breast and
`kidney cancer [7, 26, 36, 69]. Activated ras oncogenes are
`known to Occur in a variety of tumors and tumor cell lines
`[54, 64]. Among receptor tyrosine kinascs, amplification
`and overexpression of the epidermal growth factor receptor
`(EGFR) gene has been observed most consistently in
`squamous cell carcinomas and glioblastomas [34, 38, 60,
`67, 68], although other tumors of epithelial origin, such as
`breast and kidney tumors, are reported to have elevated
`levels of this growth factor receptor [18, 41, 43, 69]. The
`product of the HER2 protooncogene (also known as new
`and c-erbB-Z) is a growth factor receptor with extensive
`homology to the EGFR [l2] and to c-erbB-3, a third mem-
`ber of the EGFR family [32]. p185HER2 can be distin—
`guished from EGFR by differences in chromosomal loca—
`tion [12, 19, 46], transcript size [l2, 60], molecular mass
`[2, 45, 57], ligand activation of the associated tyrosine
`Genentech 2065
`
`Hospira v. Genentech
`IPR2017-00737
`
`Genentech 2065
`Hospira v. Genentech
`IPR2017-00737
`
`

`

`256
`
`kinase [2, 57], and antigenicity, as determined by interac-
`tion with specific monoclonal antibodies [16, 24]. HER2
`protooncogene amplification has been demonstrated
`sporadically in adenocarcinomas of the stomach [19, 28,
`40, 70], salivary gland [49], thyroid [1], colon [10,58, 72],
`lung [29, 47], pancreas [65], and ovary [4, 56]. Amplifica—
`tion and overexpression of HER2 are found frequently in
`breast—tumor—derived cell lines [25, 30, 31] and in human
`mammary carcinomas [6, 61 —63]. Investigations of large
`numbers of tumors from breast cancer patients have re-
`vealed that approximately 20%~30% have amplified the
`HER2 protooncogene [6, 20, 21, 50, 55, 61]. Moreover,
`analyses of clinical parameters show a correlation between
`both amplification and overexpression of HERZ and a
`worsened prognosis in that HER2—0vereXpressing patients
`have a shorter disease—free and Overall survival period [20,
`21, 50, 55, 59, 66]. Similar findings were reported with
`smaller numbers of tumor samples for ovarian cancer [4,
`56], and more recently for lung [29], gastric [71], bladder
`[39] and endomctrial carcinomas [5, 8] as well. Although
`mechanistic explanations
`for
`the
`aggressiveness of
`p185HER2-overespressing tumors remain elusive,
`it has
`been shown that overexpression correlates with lower
`levels of estrogen receptor (resistance to tamoxifen ther-
`apy) [3] and with tumor cell resistance to immune cell
`cytotoxicity [23, 24, 35].
`In order to begin deciphering the biological functions of
`plSSHERZ, several groups have developed monoclonal an-
`tibodies raised in mice immunized with NIH—3T3 fibro—
`
`blasts that express large amounts of either p185HER2 or
`plSSM“. These antibodies were able to inhibit anchorage-
`independent growth of the Lransfected 3T3 cells in soft
`agar, but had no effect on their proliferation in monolayer
`culture [l3,
`.14, 24]. The anti-near antibodies were also
`shown to mediate tumor cell cytolysis in vitro in the pres—
`ence of Complement [14], and to inhibit tumor formation
`by neu-transfonned NIH 3T3 fibroblasts in nude mice [14,
`15]. In addition, we recently reported that anti~p185HER2
`monoclonal antibodies had antiproliferative effects on
`breast tumor cells overexpressing pISSHER2 [24]. In the
`present report, we have expanded these initial studies in
`order to determine whether the antiproliferative effects We
`observed on breast tumor cells overexpressing 13185HERZ
`could also be observed on non-breast tumor cell lines that
`
`overexpress this growth factor receptor. In addition, we
`constructed chimeric and humanized monoclonal antibod-
`
`ies derived from marine mAb 4D5, our most potent
`growth—inhibitory antibody, in order to provide the en—
`gineered monoclonal antibody with the ability to direct
`cytotoxic activity against the overexpressing tumor cells
`via antibody-dependent cellular cytotoxicity (ADCC). The
`results presented here demonstrate that the sensitivity of
`breast tumor cell lines to antibody-mediated growth inhibi-
`tion correlates well with their level of pISSHERZ, but that
`this relationship may not apply to other types of tumor cells
`with elevated levels of this receptor. The chimeric and
`humanized 4D5 antibodies, however, could mediate
`ADCC against different types of tumor cells overexpress-
`ing pISSHERZ, regardless of their sensitivity to the parent
`4D5 antibody.
`
`Materials and methods
`
`Cell lines and materials. The majority of our cell lines were obtained
`from the American Type Culture Collection (Rockvillc, Md.) Two ovar—
`ian carcinoma cell lines, SK—OV—G and HEY, were a gift from Dr. J an
`Vaage (RosWell Park Cancer Institute, Buffalo. N.Y.). The MICN? gas—
`tric carcinoma cell
`line was obtained from Mitsubishi Corporation
`(Tokyo, Japan). The mammary epithelial lines 184, 184Al and 184B5
`were kindly provided by Dr. Martha Stampfcr, Lawrence Berkeley Lah—
`oratory (Berkeley, Calif). Tumor cells were cultured in Ham’s F-l2
`medium plus Dulbecco’s modified Eagle medium (1 :1, w’v) supple—
`mented with 100 unilslml penicillin G, 100 ugiml streptomycin, 2 mM
`L—glutamine (all from Grand Island Biochemical Company, Grand Island,
`N .Y.) and 10% heat-inactivated (56° C, 45 min) fetal bovtne serum (FBS;
`Armour Pharmaceutical Company, Kankakee, 111.}. Normal human mam-
`mary epithelial cell lines were maintained in mammary epithelial growth
`medium plus 0.4% bovine pituitary extract (Clonetics Inc., San Diego,
`Calif), supplemented with 5 uglrnl transferrin. Anti—plSSHI“:R2 and anti—
`EGFR monoclonal antibodies Were prepared and characterized as in [ I 6].
`The cloning and expression of chimeric mAb 4D5 and humanized rrLAb
`l-[ER2 are described in [l l].
`
`FACS analysis tlfp1’85”"4“2 and EGFR expression. The procedure used to
`measure cell-surface levels of plSSHER? by FACS (fluorescence—acti~
`vated cell sorter) analysis is as follows. Cells were detached from T-75
`flasks with 25 mM EDTA in 150 mM NaCl, centrifuged at 1000 rpm for
`10 min, and resuspended in 1% (vlv) PBS in phOSphate—buffered saline
`(PBS). Cell suspensions were then counted, adjusted to 106 cellslml, and
`incubated for 60 min on ice with 10 pg either anti-p185“Jam mumAb
`4D5 or anti—EGFR monoclonal antibody 6C5, or with diluent (PBS). All
`samples were washed twice, resuspended in 0.1 ml 1% FBSlP‘BS, and
`incubated with 12.5 pg fluorescein-isothiocyanate—conjugated F(ab’)2
`fragment of goat anti—{mouse IgG) (Organon Teknika—Cappel, Malvern,
`Pen.) for 45 min on ice. Following this incubation period, the cell suspen—
`sions were washed twice with 1% FBSlPBS to remove any unbound
`fluorochrome, resuspended in 1 ml assay buffer and analyzed using a
`FACScan cell sorter (Beeton Dickinson, Mountain View, Calif). These
`measurements were repeated three or four times per cell line, giving
`identical results each time.
`
`Measurement by ELISA. Cell lines were plated in 20 x lOU—mm dishes
`and allowed to grow to ”10% confluence. The monolayers were then
`washed once with PBS. lyscd with 0.5% INF—40 in PBS, and allowed to
`sit on ice for 60 min. Cell lysates were clarified by centrifugation, and the
`supernatants were aSsayed for pl BSHER2 and EGFR levels by correspond—
`ing specific ELISA and for protein by the Pierce Micro BCA* assay
`(Pierce, Rockford, 111.). The pltiSHE122 ELISA procedure utilized micro—
`n'ter plates coated with anti-1318571“? mumAB TF3 to which samples or
`standards and horseradish—peroxidase—labeled mumAb 4135 were added
`for color development [53]. The EGFR ELISA was performed in a
`similar manner using anti-EGFR mumAb 13A9 for coating the plates,
`and two horseradish—peroxidase—conjugated mAbs (362 and 503) for
`color detection [16]. Results are expressed as concentration of recep—
`torlconcentration of total cellular protein.
`
`Cell proliferation assays. Tumor cell lines were plated in 96—well mien}
`titer plates at the following densities: 104 cellslwell for HBL—ltlfl and
`SK—OV-3; 2 X 104cellslwell for 184, 184Al, 184B5. SK—BR—3, BT-4'r'4,
`COLD 201, KATO Ill, MKNi', SW14”, ZR-TS-l, MCFT, MBA-MB-
`231, MBA—M3436, MDA—MB—453; 4 x 104 cellslwcll for MBA-MB—
`l'iS—Vll; B x 1114 cellslwell for NEDA—M13661. After the cells had been
`allotted to adhere for 2 h, medium alone or medium containing anti—
`p185”ERZ monoclonal antibodies (final concentration of 10 uglml) was
`added to give a total volume of 0.2 ml. After incubation for 5 days, the
`monolayers were carefully washed twice with PBS and stained with
`crystal Violet dye {0.5% in methanol) for determination of relative cell
`proliferation as described previously [24]. 'l‘reatment groups consisted
`ofS—lo replicates, and the coefficient of variation was always less
`than 12%.
`
`

`

`25?
`
`specific lysis (‘56) :2 (A—BlC—B) x 100, where A represents 51Cr (cpm)
`from test supematants, 3 represents Spontaneous release (“Q from
`untreated target cells), and C represents maximum release (5'Cr from
`target cells lysed with 0.4% NP—4t)). Each treatment was performed in
`triplicate. Spontaneous release from target cells alone was less than 20%
`of the maximum for all experiments.
`
`Results
`
`Tumor cell expression ofpl 8511332 as measured by FA CS
`
`Cell-surface expression of pl SSHERZ and EGFR was deter-
`mined for a number of breast tumor cell lines by measuring
`binding of fluoreseein-eonjugated anti—(mouse antibody)
`to cells that had been pretreated with murine monoclonal
`antibodies specific for the extracellular domains of either
`pl 855113RE (mumAb 4D5) or EGFR (mumAb 6C5).
`Figure 1 shows histograms representative of the range
`of plBSHER2 levels expressed on different breast tumor
`lines. p l SSHERZ expression is low (arbitrarily designated as
`1.0, Table 2) on HBL—100, an immortalized mammary ep—
`ithelial cell line (Fig. l b) and slightly higher on the MCF’lr
`breast tumor cell line (12-fold increase, Fig. 1c). MDA—
`MB- UPS—VII breast tumor cells display intermediate levels
`of p185HERZ (45—fold higher than HBL~100, Fig. 1d),
`whereas both MDA—MB—361 and SK—BR—3 breast tumor
`
`cell lines are high overexpressors (16.?—fold and 33.0-fold
`greater than I-[BL—lOU, Fig. 1e and f respectively). The
`A431 epidermoid carcinoma line, which OVBrCXpTCSSBS
`EGFR (approximately 2 x 106 teceptorsleell
`[17]), was
`used as a positive control for measuring EGFR (Fig. la).
`The data obtained for each of the cell lines indicate distinct
`
`profiles for the anti —EGFR and anti-plilSHER2 monoclonal
`antibodies, and support our earlier conclusion {24] that
`mumAb 4D5 specifically recognizes p185HER2.
`Figure 2 summarizes all the FACS data obtained from
`cell
`lines with mumAb 4D5. These data are arranged
`according to tumor type from which the cell line was
`derived. One submaxillary (A253), one gastric (H5746T),
`two ovarian (Caov-4 and NIH20VCAR—3), and all renal
`carcinoma cell lines tested were negative for plSSHER2
`expression. The majority of the colon and ovarian tumor
`cell lines tested, as well as the two rectal adenccarcinoma
`lines, displayed low levels of p185HER2. Moderate amounts
`of pl 85141332 were exhibited on two other breast tumor cell
`lines (MDA—MB—436 and ZR-75-1), on several colon lines
`(e. g. SW948), and on the gastric carcinoma cell
`line,
`KATO Hi. In addition, the colon lines designated COLO
`201 and SW14] 7" demonstrated higher levels of p] SSHERZ.
`However, striking overexpression of p185HERZ was mea~
`sored on SK~OV—3 ovarian carcinoma cells, MKN-7 ga-
`stric carcinoma cells, and four breast tumor cell
`lines
`(MDA—MBAS3, MDA—MB—Bol, BT474, and SK‘BR-S).
`The proportion of cell lines overexpressing p185H'I3R2 for
`each tumor type coincides well with the percentages of
`tumors with HER2 amplification and overexpression re—
`ported in a number of different patient populations [4, 28,
`40, 56, 59, 63].
`
`100
`
`
`-
`—
`
`- b
`
`
`
`
`
`
`
`10“
`
`
`
`.
`
`l
`
`
`
`too
`
`I
`
`l
`-l
`
`—
`_
`
`to
`
`u.
`:5
`
`"0E;
`:s
`o
`U
`
`I
`
`.
`
`{)1
`‘1
`,
`A.
`
`-
`
`:l
`
`“R
`
`,
`
`\l
`
`—
`
`d
`
`t
`
`:11!
`5.5.
`
`\l
`“l
`103
`Fluorescence
`
`I
`
`Fluorescence
`
`Fig. la~f. Fluorescence-activated cell sorting (FACS) histograms of
`anti—pl85HERE mAb 4D5 (stippled line) or anti-(epidermal grth factor
`receptor) mAb 6C5 (dotted line) binding to cell lines, Solid line repre
`sents background fluorescence [binding of fluoresccin—isothioeyanate—la—
`beled goat anti—(mouse 1g) in the absence of murine mAb 4D5 or 6C5].
`Cell
`lines analyzed are: 3 A43! epidermoid carcinoma; b HBL—100
`mammary epithelial cells; C MCF? breast adenocarcinoma; d MBA-MB-
`l75—Vl'l breast ductal carcinoma; a MDA—M‘B—361 breast adenocarci—
`noma; f SK—BR—3 breast adenocarcinoma
`
`Sufi agar assays. Assays to determine colony formation in soft agar by
`tumor cell lines were performed as follows: using 15 x60-mm tissae-
`culture dishes, at bottom layer consisting of 4 ml culture medium contain—
`ing 0.5% purified agar (Difco Laboratories, Detroit, Mich.) was first
`allowed to solidify. Aliquots of cells (IDs/dish) were added, and 3 ml
`medium containing 0.25% agar was then layered on top. Experiments
`were performed in triplicate, with three dishes of each cell type receiving
`1.0 pg 4D5, and three dishes receiving 10 pg irrelevant, isotype—matched
`antibody, 40.1.Hl, directed against the hepatitis B surface antigen [24].
`Colonies were counted after a 3— to 6-week incubation using the Omni—
`eon 3600 tumor colony analysis system (Imaging Products International
`Inc., Chantilly, Va.)
`
`Antibody—dependent cell—mediated cytotoxiciry (ADCC). Peripheral
`blood mononuclear cells (PBMC) were isolated from heparinized whole
`blood obtained from normal donors by density gradient sedimentation
`using lymphocyte separation media (Organon Teknika Corp, Durham,
`N.C.). After three washes with PBS, the cells were resuspended at a
`density of 3 x 106m] in culture medium containing 100 unitsl’ml recom—
`binant human interleukin—2 (Boeho'nger Mannheim, Germany). Follow~
`ing overnight incubation, the PBMC were washed twice with culture
`medium and serially diluted into 96-well round—bottom microfiter plates
`to give effector: target ratios of 25 : 1, 12.5 : 1, 6.25 : l or 3.13 : 1. Various
`dilutions of the different antibodies (mumAb 4D5, chmAb 4D5, and
`(rhu)mAb HERZ) or medium were then added. Antibody concentrations
`used were 1, 10, 100 or 1000 nyml. Target cell lines were labeled with
`150 tLCi N351Cr04(Amersham Corp, Arlington Heights, Ill.) for45 min
`at 37°C, then washed three times in culture medium. A total of 0.1 ml
`target cells (10511111) was added per well for a final volume of 0.2 mllwell.
`The plates were then incubated for 4 h at 37°C, after which the super-
`natants were harvested and the radioactivity determined in an automatic
`gamma counter. Percentage specific lysis was calculated as follows:
`
`

`

`258
`
`-
`
`MDA-
`Colon
`
`Submaxillary[ '
`
`Rectal
`
`0
`
`so
`
`100
`
`150
`
`zoo
`
`Fluorescence (units)
`Fig. 2. Summary of pISSHE'u expression on different types of tumor cell
`lines as measured by FACS analysis. Bars (fluorescence units) represent
`peak fluorescence intensity in the prcsmee of mumAb 4D5 minus back-
`ground fluorescence
`
`Table 1. Measurement of p 1 35mm and epidermal growth factor receptor
`(EGFR) by enzyme-linked immunosorbent aSsay (ELISA)a
`
`Cell
`Cell line
`p1 8539-“?
`EGFR
`
`type
`(ngimg protein)
`(ngirng protein}
`
`Breast
`
`HBL-100
`MCFl
`MDA-MB-231
`MDA—MB—l75—Vli
`MBA—M3453
`BT474
`SK-BR—3
`MDA—MB—468
`
`Ovarian
`
`SK-OV-3
`
`Gastric
`
`Colon
`
`H87461
`KATO 1.11
`MKN7
`
`SW14] 7
`COLO 205
`[ff—29
`SW403
`I-lCT—IS
`WiDr
`SW948
`COLO 320DM
`COLO 201
`HCT116
`SW48
`SW480
`LoVo
`DLD—l
`
`5.89
`7.27
`5.40
`17.43
`43 .73
`547.65
`917.27
`ND"
`
`537.26
`
`3 .3 1
`10.39
`194.10
`
`10.12
`l .87
`l .89
`3.] 1
`3.81
`1.28
`43.23
`1.65
`11.25
`2.86
`12.73
`1.95
`7 .65
`10.96
`
`242.41
`1.25
`168.02
`42.07
`1.47
`1 16.02
`123.82
`65355
`
`328.03
`
`156.77
`132.3]
`340.77
`
`359.28
`68.97
`228.08
`44.49
`262.53
`143.31
`359.44
`2.93
`79.69
`227.49
`1255.] 8
`184.30
`314.34
`392.75
`
`165.95
`5.85
`SW837
`Rectal
`
`
`4.40SW1463 307 .73
`
`*1 Cells were lyscd in 0.5% NP-40 and aliquots were assayed for
`pIBSHER? or EGFR levels by specific ELISA, and for protein by the
`Micro BCA* assay
`‘3 ND, not detectable
`
`on live cells, whereas the ELISA measured total pISSHER2
`in cell lysates. It is hoped that further work will allow us to
`distinguish whether different tumor cell types may have
`varying quantities of intracellular receptor, or if some
`differences may be due to slight differences in cell growth
`conditions.
`
`Measurement of,oi851ll'5R2 by ELISA
`
`Growth inhibition of tumor ceiis by anti—p185HER2
`monoclonai antibodies
`
`Levels of 131 RSHERQ on these different tumor cell lines were
`also measured by the ELISA method. These results corre—
`5pond well with the FACS data, with a few discrepancies.
`Cell
`lines overcxpressing plSSHERZ, such as SK-OV-3,
`BT474, and SK—BR—3, show higher levels of the receptor,
`relative to HBL—IOO,
`in the ELISA compared to the
`amounts measured by FACS analysis (Table 1). In con-
`trast, relative differences in p185'IER2 levels in the moder—
`ate overexpressors, as described in the previous section,
`were less pronounced when measured by ELISA. These
`differences might be due to the types of assays used. The
`FACS analyses measured surface expression of p185H‘3R2
`
`We previously reported that arlli-p185HFR2 monoclonal an—
`tibodies have antiproliferative activity on the SK—BR-3
`breast tumor cell line [24]. From the FACS data shown in
`Fig. 2, We selected all of the breast carcinoma lines, the
`SK—OV-3 ovarian carcinoma line, the MKN7 and KATO
`11}. gastric carcinoma lines and two colon carcinoma cell
`lines, SW14]? and COLO 201., for more detailed growth
`inhibition studies using a variety of different monoclonal
`antibodies that recognize the extracellular domain of
`plSSHERQ. Cell lines were incubated with 10 ttg/ml each
`antibody for 5 days and proliferation was compared to that
`of untreated control cells.
`
`

`

`259
`
`Table 2. Effect of anti—p185HER2 ntAb on the growth of human tumor cell linesa
`
`Relative cell proliferation (% of control}
`
`
`Relative
`Cell
`line
`p185HEM
`expressionh
`4D5
`31-14
`201
`7F3
`7C2
`6E9
`
`184
`1.0
`116
`114
`109
`116
`117
`103
`184A].
`0.3
`100
`110
`103
`106
`104
`110
`13435
`0.8
`108
`107
`105
`108
`108
`106
`BBL-100
`1.0
`104
`102
`103
`96
`104
`105
`MCF?
`1.2
`101
`113
`100
`111
`112
`105
`M'DA—MB~231
`1.2
`91
`100
`93
`98
`104
`013
`ZR-75-1
`3.3
`102
`105
`99
`97
`108
`9'1
`MDA—MB-436
`3.3
`97
`9]
`98
`93
`92
`101
`MDA-MB— l 75
`4.5
`62
`T1
`29
`48
`87
`96
`MDA—MB—4S3
`16.?
`61
`65
`88
`80
`’10
`101
`MDA—MB—361
`16.7
`63
`67
`64
`76
`105
`99
`
`91
`78
`21
`60
`29
`.
`25.0
`BT474
`89
`82
`51
`73
`40
`33
`33.0
`SK-BR—3
`99
`97
`91
`87
`85
`77
`16.7
`SK-OV —3
`108
`106
`111
`103
`102
`99
`16.7
`MKN?
`99
`107
`98
`101
`102
`91
`5.0
`KATO 111
`1 10
`122
`125
`123
`132
`107
`8.3
`COL0201
`96
`98
`100
`99
`97
`98
`6.7
`SW1417
`
`
`1 Cells were seeded in 96-well microtiter plates and allowed to adhere
`before the addition of different antzi-plfii'flfl-TR2 mAb at a final concentra-
`tion of 10 gym]. Monolayers were stained with crystal violet dye after
`5 days for determination of relative cell proliferation. Each group con-
`
`sisted of 8— 16 replicates, with the coefficient of variation for each group
`always less than 12%
`1‘ Levels of p185“RE expression, measured by fluorescence—activated
`cell sorting, relative to the normal mammary epithelial cell line 184
`
`Table 2 compares the antiproliferatiVe activity of the
`different antibodies with the relative levels of plttiSHER2
`expression as determined by FACS assay. Growth of the
`normal human mammary epithelial line, 184, was slightly
`stimulated by the different anti-p185HERZ antibodies,
`whereas the growth of the immortalized mammary epithe—
`lial cell lines, 184A], 184135, and HBL-lOO, was unaf—
`fected by treatment with these antibodies. For the breast
`tumor cell lines, there is a clear relationship between the
`level of HER2 protooncogene expression and sensitivity to
`the growth—inhibitory effects of the antibodies. It appears
`that cell lines with more than 4-fold overexpression (rela-
`tive to 184 or HBL-IOO cells) are growth—inhibited. For
`example, growth of cells showing little expression (e.g.,
`MCF'i, MDA-MB-231, ZR-"r'S-l and MDA-MB-436) is
`not inhibited by the monoclonal antibodies. Accordingly,
`cell lines with higher levels of p1 8511131?2 (MDA-MB-175-
`VII, MDA—MB—453, MDA—MB—361) are increasingly
`more sensitive to antibody—mediated growth inhibition.
`Hence, SK—BR-3 and BT474 cells, which express abundant
`amounts of pISSHERZ, are the most sensitive to the anti—
`proliferative effects (showing approximately 70% growth
`inhibition).
`This work also indicates that coexpression of EGFR and
`p185HER2 may not be required for expression of the
`growth-inhibitory activity of plBSHER2 since the MDA-
`MB—4S3 and MDA-MB-361 cell lines express very little
`EGFR (Table 1, Fig. 1). The data in Table 2 also demon-
`strate that antibodies that share a common epitope have a
`similar Spectrum of activity. Antibodies mumAb 4D5 and
`mumAb 3H4 compete for binding to the extracellular
`domain of p185HER2 [16], and elicit almost identical re—
`SpOIlSCS on each tumor cell
`line. Similarly, antibodies
`mumAb 2C4, mumAb 7F3, and mumAb 7C2 show over—
`lapping activities corresponding to their shared epitopes
`
`[16]. Furthermore, these results suggest that mumAb 4D5
`has the most consistent growth—inhibitory activity toward
`various breast tumor cell lines.
`
`The apparent correlation between HER2 expression and
`susceptibility to antibody—induced growth inhibition in
`monolayer culture does not persist for the MKN7, KATO
`[I], COLO 20] or SW14” cell lines. SK-OV—3 cells, al-
`though somewhat sensitive to the growth-inhibitory effects
`of mumAb 4D5, appear to be less sensitive than would be
`expected from their
`level of p185H5R2
`expression
`(Table 2). Moreover, the KATO 111 gastric line and the
`SW1417 and COLD 201 colon lines, with levels of recep-
`tor expression equal to or greater than the MDA-MB— l 75—
`VII breast tumor line, are completely resistant to the anti-
`proliferative effects of mumAb 4D5 or mumAb 3H4. In
`fact, growth of the COLO 201 cells appears to be stimu-
`lated by several of the antibodies (e. g., mumAb 3H4 and
`mumAb 7F3, Table 2). The MKN? gastric carcinoma line,
`which has a 17—fold overexpression of surface receptor
`(similar to the breast tumor cell lines MDA—MB-361, or
`MDA-MB-453), is also resistant to growth inhibition by
`the monoclonal antibodies. The reason for this discrepancy
`is still not understood. Labeling of these tumor cells with
`[35S]rnethionine followed by immunoprecipitation showed
`the receptors to be of the expected size and amount (data
`not shown). Figure 3 graphically displays the results from
`Table 2, clearly depicting the relationship between
`p185HER2 expression and growth inhibition by the mono—
`clonal antibodies on the breast tumor cell lines, as well as
`the resistance of the other tumor cell lines to the different
`
`antibodies. In this figure, the cell lines with less than the
`expected extent of growth inhibition by mumAb 4D5 ap-
`pear above the “best-fit” line derived from the data obtain—
`ed using breast tumor cells expressing inereasing levels of
`p185HER3.
`
`

`

`260
`
`120
`
`
`
`100
`
`00
`
`Proliferation 8
`RelativeCell
`
`4o
`
`20
`
`1
`
`10
`
`100
`
`1000
`
`ptt‘lS'F'EF‘2 Expression
`Fig. 3. Relationship between p] 85HEm expression and growth inhibition
`mediated by mumAb 4D5 on human breast cell lines, normal and tumor
`(0), and other types of tumor cell lines overexpressing plfiSHER2 (0)
`
`Several investigators have previously described the ina—
`bility of anti—nan or anti-p185HERZ antibodies to inhibit the
`monolayer growth of NIH 3T3 cells transformed with ei-
`ther the activated c-neu or overexpressed HER2 pro-
`tooncogene [13, 24]. However, anchorage-independent
`growth in soft agar was abrogated in cultures treated with
`the appropriate monoclonal antibodies [13, 24]. Therefore,
`we performed soft-agar growth assays on the same tumor
`cell lines studied in monolayer assays (Table 3). The mam—
`mary epithelial line 18435 did not form colonies in soft
`agar. Although the BBL-100 line is also an immortalized
`marmnary epithelial line, anchorage—independent growth
`was observed (also documented by the American Type
`Culture Collection). Treatment with murnAb 4D5, how-
`
`Table 3. Colony formation in soft agar“
`
`Cell line
`
`Percentage
`ColonyIr Count after
`of
`treatment with:
`———-— control
`
`0
`4D5
`{%)
`
`
`
`—
`0
`0
`18435
`1.27
`694195
`546i112
`REL—100
`99
`290 :— 25
`293 i 25
`MCF'?
`108
`997 : 32
`923 i 20
`MDA —MB—23l
`100
`805 2‘. 32
`805 i30
`ZR-7 5—1
`99
`457 :59
`461 i30
`MDA-MB—436
`67
`518 $47
`778 i 39
`MDA~MB-453
`30
`101:1b
`35 1‘5
`MDA~MB-3f)l
`0
`'3"
`321 i 25
`BT474
`0
`0"
`462 1-17
`SK—BR—3
`45
`107 4:713
`239i?
`SK~OV-3
`75
`1089 i 144
`1449 i 198
`KATE) HI
`128
`1913i465
`1489:150
`COLD 201
`
`SW1417 65 1078-:31 705i32b
`
`
`
`*1 Cells were seeded onto a bottom layer of 0.5% agar and covered with
`a top layer containing 0.25% agar with or without 10 pg mumAb 4135.
`Colony formation was determined after 3—6 weeks using the Omnicon
`3600 tumor colony analysis system. Numbers represent mean colony
`counts 1 SE
`
`‘3 Values significantly different from control group (for P values, see
`text)
`
`ever, had no effect on HBL— 100 colony formation. Tumor
`cell
`lines expressing low levels of p185HER2 (MCI-17,
`MDA—MB-23 l, ZR-T’S-l, MDA-MB-436) formed equiva—
`lent numbers of colonies in soft agar in the presence or
`absence of mumAb 4D5, as expected. Anchorage-indepen-
`dent growth of breast tumor lines expressing high levels of
`13185HERZ (16.7-fold or greater, relative to the 184 line)
`was inhibited upon treatment with mumAb 4D5 (P = 0.06
`for MDA—MB-453, P = 0.008 for MDA—MB—361,
`P : 0.002 for BT4'14, and P = 0.0001 for SK—BR—3 com-
`pared to the respective controls). Complete suppression of
`colony formation was observed in the SK~BR—3 and
`BT474 lines. As with the monolayer growth assays, there is
`an apparent correlation between p185HER2 expression and
`extent of growth inhibition by the anti—p185HER2 antibody.
`In addition, colony formation by the SK-0V«3 ovarian
`carcinoma line and the SW 1417 colon carcinoma line was
`
`significantly inhibited by murnAb 4D5 compared to con—
`trols (P 2 0.0001 and P = 0.0011 respectively). However,
`the gastric carcinoma line KATO III and the colon carcino-
`ma COLO 201 were not inhibited by antibody treatment.
`The MDA—MB—li'S-VII and MKN7 lines did not form
`
`colonies in soft agar under our standard assay conditions.
`
`Antibodydependent cellular cytotoxiciry mediated by the
`chimeric and humanized versions ofmttrmlb 4D5
`
`The issue of mechanism of resistance to mumAb 4D5 in
`non—breast and ovarian tumor cells remains unresolved.
`
`However, We hypothesized that antibody-mediated anti—
`tumor effects could also be directed by overexpression of
`plSSHER2 based upon grafting of the murine hypervariable
`domains onto the human immunoglobulin framework vari-
`able and constant regions. In order to accomplish this, a
`chimeric mumAb 4D5 (ehmAb 4D5) was prepared and
`employed as a substrate to generate a family of humanized
`variants, all of which contained the human IgGl Fc region
`[17], which should allow for ADCC.
`To test this idea we performed ADCC assays on several
`different
`tumor cell
`lines expressing various levels of
`p185HER2 utilizing each of the monoclonal antibodies,
`mumAb 4DS, chmAb 4D5 and (rhu)mAb HERZ. We se—
`lected as targets the SK-BR-3 breast tumor cells (sensitive
`to mumAb 4D5, with high levels of plSSHERZ), MKN7
`gastric tumor cells (resistant to mumAb 4D5, also with
`high levels of p185flER2), COLD 201 colon tumor cells
`(resistant to mumAb 4D5, with moderate p 1 SSHERZ levels),
`MCF’? breast tumor cells (resistant to mumAb 4D5, with
`only slightly elevated membrane expression of pISSHERZ),
`and the mammary epithelial cell line 184A] (resistant to
`mumAb 4D5, with low receptor expression). The growth
`response to mumAb 4D5 and levels of p185HER2 expres-
`sion on each cell type are sununarized in Table 2.
`The chimeric and humanized 4D5 molecules have sim—
`ilar antiproliferative activities on SK-BR-3 cells (57% and
`60% inhibition of growth respectively), as compared to
`mumAb 4135 (G. D. Lewis, unpublished data). However,
`the combination of human PBMC and (rhu)mAb HER2
`caused lysis of the tumor cells as a function of their level of
`p1851'IBR2 surface expression (Fig. 4). The normal mam-
`
`

`

`:e 4°
`s
`..
`5 3°
`
`E
`'56 20
`
`
`
`
`
`
`
`
`
`¢
`2 10
`¢%
`
`
`
`
`
`
`
`.
`
`50
`
`
`7
`f
`a
`g
`
`e.
`f3}
`
`i'
`éy/
`
`1 84m
`
`MCF? COLO201 MKN
`
`SK—BH'3
`
`Cell Line
`
`Fig. 4. Antibody—dependcm cellular cytotoxiclty directed against select—
`ed tumor cell lines by recombinant human (rhu)mAb I-[ER2 in the pres—
`ence of human peripheral blood mononuclear cells. 5lCr—labeled targct
`cells (lO‘Uwell) were incubated for 4 h with different dilutions of effector
`cells to give effector:target ratios of 25:] (open bars). 12.521 (wide
`hatched bars), 6.25 :1 (smll hatched bars) and 3.13:1 (solid bars).
`Incubations were carricd out in the presence or absence of 0.1 ttgfml
`(rhu)mAb I-IER2. Net percentage cytotoxicity represents cytotoxicity in
`the presence of humAb I-IERZ minus nonspecific cytotoxicity (no antiA
`body). Values shown are means i SE
`
`mary epithelial line 1.84Al and MCF? breast tumor cells
`are relatively resistant to cytolytic attack, while the tumor
`cells with increasing levels of plSSHER2 demonstrate in-
`creasing sensitivity to ADCC mediated by (rhu)mAb
`HERZ as measured by release of chromium-51. A statisti~
`cally significant relationship was observed between the net
`percentage cytotoxicity and level of HER2 expression at all
`four effector: target ratios. The slope of the fitted line was
`significantly different (P <0.001) from zero in all cases (r2
`values of 0.93, 0.92, 0.87, and 0.66 for effectorztarget
`ratios