___ ,dH SCIENCES Ll ..
`University of Wleconeln
`
`FEB 1 0 1993
`
`1305 Linden Drive .
`Madison, WI 537
`
`IMMUNOGEN 2036, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Int. J. Cancer: 53,401-408 (1993)
`© 1993 Wiley-Liss, Inc.
`
`(~ Publication of the International Union Against Cancer
`~ Publication de I' Union Internationals Contre le Cancer
`
`ANTIBODY-INDUCED GROWTH INHIBITION IS MEDIATED THROUGH
`IMMUNOCHEMICALL Y AND FUNCTIONALLY DISTINCT EPITOPES ON
`THE EXTRACELLULAR DOMAIN OF THE c-erbB-2 (HER-2/neu) GENE
`PRODUCT p185
`Fengji XU 1, Ruth LUPU3, Gustavo C. RODRIGUEZ2, Regina S. WHITAKER2., Matthew P. BOENTE2, Andrew BERCHUCK2,
`Yinhua Yu 1, Karen A. D ESOMBRE 1, Cinda M. BOYER 1 and Robert C. BAST, JR.1•4
`Departments of 1Medicine and 20bstetrics and Gynecology and the Duke Comprehensive Cancer Center, Duke University Medical
`Center, Durham, NC 27710; and 3Department of Medicine and The Vincent T. Lombardi Cancer Center, Georgetown University
`Medical Center, Washington, DC 20007 USA .
`
`Over-expression of the c-erbB-2 (HER-2/neu) gene product
`p 185 occurs in 30% of breast and ovarian cancers. The p 185
`protein might serve as a target for serotherapy in that antibod(cid:173)
`ies against different epitopes on the extracellular domain of
`p 185 can inhibit growth of tumor cells in the absence of cellular
`or humoral effector mechanisms. To define epitopes of func(cid:173)
`tional relevance, II monoclonal antibodies (MAbs) were evalu(cid:173)
`ated for their ability to bind to the extracellular domain of p 185.
`Results of competition studies with 1251-labeled and non-labeled
`antibodies indicated that I 0 of I I epitopes were grouped in a
`linear array. Antibodies against 7 epitopes inhibited anchorage(cid:173)
`indep.Etndent growth and antibodies against 2 of these epitopes
`also inhibited anchorage-dependent growth of SKBrJ breast(cid:173)
`cancer cells that over-expressed p 185. Treatment with antibod(cid:173)
`ies exerted cytotoxic rather than cytostatic effects. When
`antibodies were used in combination, additive or supra-additive
`inhibition of anchorage-independent and anchorage-dependent
`growth was observed between pairs of antibodies. Growth
`inhibition did not relate to the affinity of the antibody or its
`isotype. Two antibodies that inhibited both anchorage-depen(cid:173)
`dent and anchorage-independent growth also blocked binding
`of the HER-2/neu ligand, whereas 5 antibodies that inhibited
`only anchorage-independent growth had no effect on ligand
`binding. Inhibition of cell growth did not correlate with internal(cid:173)
`ization of p 185 or down-regulation of p 185 on the cell surface.
`Fab fragments of active antibodies could also inhibit anchorage(cid:173)
`independent growth of SKBrJ. Thus, murine MAbs and their
`fragments recognized both immunochemically distinct and func(cid:173)
`tionally distinct epitopes on the p 185 molecule. Whereas inhibi(cid:173)
`tion of anchorage-dependent growth correlated with the ability
`of antibodies to block ligand binding, inhibition of anchorage(cid:173)
`independent growth did not correlate with effects on ligand
`binding, internalization, cell-surface expression or cross-linking
`ofpl85.
`© 1993 Wiley-Liss, Inc.
`
`Antibodies that bind to certain cell-surface receptors can
`inhibit tumor-cell proliferation. The transferrin receptor (White
`et al., 1990), epidermal-growth-factor receptor (Masui et al.,
`1984) and c-erbB-2 (HER-2 /neu) gene product (Drebin et al.,
`1985, 1988; Hudziak et al., 1989) can all serve as targets for
`antibody-mediated growth inhibition. Each of these receptors
`internalizes readily and has made an effective target for
`treatment with immunotoxin (Yu et al., 1990; Maier et al.,
`1991; Masui et al., 1989).
`The gene product of c-erbB-2 may provide a particularly
`useful target for serotherapy of ovarian and breast cancer. The
`neu oncogene was first described in rat neuroblastomas in(cid:173)
`duced with ethylnitrosourea (Schechter eta!., 1984, 1985). The
`human homolog of the rat neu oncogene encodes a 185-kDa
`protein (p185) that is closely related to, but distinct from the
`170-kDa epidermal-growth-factor receptor (EGFR). Both pro(cid:173)
`teins have a cysteine-rich extracellular domain, a transmem(cid:173)
`brane domain, and an intracellular tyrosine kinase domain.
`Overall, p185 has approximately 40% sequence homology with
`EGFR. Recently, several ligands of 30, 44 and 75 kDa have
`been described that bind to p185 (Lupu et al., 1990, 1992; Peles
`
`et al., 1992). In contrast to the neu gene in rats, where a
`mutation in the intramembranous domain is associated with
`oncogenic activity, only the proto-oncogene c-erbB-2 has been
`associated with human cancers. Over-expression of p185 gene
`product occurs in 15 to 40% of breast and ovarian cancers and
`is associated with a poor prognosis (Slamon et al., 1987, 1989;
`Berchuck et al., 1990). Antibodies that recognize different
`epitopes on cell-surface molecules can exert different func(cid:173)
`tional activities (Stancovski et al., 1991). A series of murine
`MAbs have been produced that are reactive with the extracel(cid:173)
`lular domain of p185. In this study we have evaluated the
`epitope specificities and growth-regulatory activities of 11
`anti-p185 antibodies. Antibodies that bind to different epi(cid:173)
`topes with similar affinity can either inhibit or fail to inhibit
`tumor growth. Inhibition of anchorage-dependent tumor growth
`correlated with blocking of ligand binding, whereas inhibition
`of anchorage-independent growth did not correlate with ef(cid:173)
`fects on ligand binding, internalization of p185 or down(cid:173)
`regulation of p185. Moreover, inhibition of anchorage(cid:173)
`independent growth did not require cross-linking of the
`extracellular domains of p185 by bivalent antibody.
`
`MATERIAL AND METHODS
`
`MAbs
`Murine MAbs that react with the extracellular domain of
`p185 were obtained from Applied BioTechnology, Cambridge,
`MA (TAl, BDS, IDS, NB3, RCl, RC6, PB3, OD3) (McKenzie
`et al., 1989) and from Cetus, Inc., Emeryville, CA (520C9,
`741F8 and 454Cll) (Bjorn et al., 1985; Ring et al., 1991). Aside
`from PB3 (IgG 23), 454Cll (IgG2a) and OD3 (IgM), all other
`antibodies were of the lgG 1 isotype. MOPC21 (IgGl) and 9C6
`(IgM), obtained from Cetus (Norwalk, CT), were used as
`isotype-matched controls that did not bind to p185.
`To prepare antibodies, hybridoma cells were washed free of
`serum and injected i.p. into pristane-primed Balb /c mice.
`When tense ascites had formed, fluid was harvested asepti(cid:173)
`cally. IgG antibodies were purified from hybridoma-induced
`ascites fluid using protein A-sepharose chromatography (Phar(cid:173)
`macia-LKB, Uppsala, Sweden). Fractions containing protein
`measured by absorbance at 280 nm were dialyzed for 24 hr
`against 50 mM phosphate buffer, pH 7.2, and concentrated
`using an Am icon filter and compressed nitrogen gas. Immuno(cid:173)
`globulin concentration was calculated by dividing absorbance
`at 280 nm by the extinction coefficient for IgG. Immunoglobu-
`
`4To whom requests for reprints should be addressed, at Box 3843,
`Duke University Medical Center, Durham, NC 27710 USA.
`
`Abbreviations: EGF, epidermal growth fac tor; EGFR, epidermal(cid:173)
`growth-factor receptor; TCM, tissue-culture medium.
`
`Received: July 6, 1992 and in revised form October 2, 1992.
`
`IMMUNOGEN 2036, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`402
`
`XU ETAL.
`
`lin purity was confirmed by SDS-PAGE. Purified immunoglob(cid:173)
`ulin was aliquoted and stored at -7ooc. The IgM antibody
`OD3 was precipitated in 50% ammonium sulfate, dissolved in
`PBS and purified on a Sephacryl S-300 column (Pharmacia
`LKB). The initial peak was dialyzed and concentrated as
`described above.
`
`Fab fragments
`The Avidchrom Fab kit (Bioprobe, Tustin, CA) was used to
`papain-digest the IDS IgG MAb to Fe and Fab fragments. A
`protein-A Avidchrom cartridge was used to separate Fab
`fragments from Fe fragments and intact IgG. Fab and Fe
`fragments were evaluated for size and purity with SDS-PAGE.
`
`Cell lines
`SKBr3 (Fogh and Trempe, 1975), a human breast-cancer
`cell line, was maintained in RPMI 1640 medium supplemented
`with 15% FBS and 2 mM L-glutamine, 100 units/ml penicillin
`and 100 fLg/ml streptomycin. The MCF-7 breast-cancer cell
`line was maintained in Dulbecco's modified Eagle's medium
`(DMEM) supplemented with 5% FBS and sub-cultured every
`2 weeks. The OVCA 429 and OVCA 432 human epithelial
`ovarian-cancer cell lines were maintained in minimum essen(cid:173)
`tial medium supplemented with 10% FBS, 2 mM L-glutamine,
`non-essential amino acids, 1 mM sodium pyruvate, and antibi(cid:173)
`otics as above. Breast-cancer cell lines BT-474, MDA-MB-453
`and MDA-MB-468 were propagated as described by Lupu et
`al. (1990, 1992). A human foreskin-fibroblast cell line (Huff)
`was obtained from Dr. Kay Singer (Duke University Medical
`Center) and was grown in DMEM supplemented with 10%
`FBS. SKBr3, BT-474 and MDA-MB-453 had strong expression
`of pl85, whereas OVCA 429 and OVCA 432 had weak pl85
`expression. No p185 could be detected in MDA-MB-468,
`MCF-7 and HUFF. MDA-MB-468 and SKBr3 expressed
`EGFR, BT-474 had weak EGFR expression, and MDA-MB-
`453 was EGFR-negative.
`Rat-1A and 1174 cell lines were obtained from Applied
`BioTechnology (Cambridge, MA). Rat-1A is a rat fibroblast
`cell line. The cell line 1174 was derived from rat 1A cells by
`infection with a defective retrovirus that contained the
`pMX1112 plasmid with the full-length wild-type human c(cid:173)
`erbB-2. Immunoprecipitation (McKenzie et al., 1989) of 1174
`cells with anti-p185 antibodies yielded a protein of 185 kDa.
`The rat cell lines were maintained in DMEM supplemented
`with 10% FCS and 2 mM L-glutamine. In the case of 1174, 400
`fLg/ml G418 sulfate (GIBCO, Grand Island, NY) was added to
`the medium. Cells were cultured at 37°C in 5% C02 and 95%
`humidified air.
`For experiments, cells were detached with 0.25 % trypsin-
`0.02% EDTA. Cells were washed once in complete medium
`before use.
`
`Radioiodination of MAbs
`MAbs were labeled with Na 125I using the iodogen method
`(Fraker and Speck, 1978). In brief, 50 1-1! of phosphate buffer
`(0.5 M, pH 7.4) was added to a 15 x 75 mm borosilicate tube
`coated with 10 1-1g of iodogen (Pierce, Rockford, IL). MAb (50
`1-1g) was added in a volume of 95 1-1! PBS (50 mM phosphate
`buffer, 0.15 M NaCI). Radioiodination was initiated by the
`addition of 0.5 mCi of Na 125I (5 1-1!) and the mixtures were
`incubated for 30 min on ice. The protein-bound iodine was
`separated from free 125I by gel filtration on a PD-10 column
`(Pharmacia, Pleasant Hill, CA) which had been equilibrated
`with PBS. A sample of 3 1-1! from each fraction was counted in a
`Packard gamma counter (Packard, Downers Grove, IL) to
`measure protein-bound radioactivity. Iodination efficiency was
`calculated using the following formula:
`
`iodination efficiency =
`
`protein bound cpm
`
`1 tota cpm
`
`x 100%
`
`The efficiency of iodination ranged between 75 and 90%.
`Competitive binding assays
`For competitive binding assays, SKBr3 cells (104 / well),
`Rat-1174 cells (2 x 104/well), or OVCA429 cells (105/well)
`were seeded in RemovaCell 96-well plates (Dynatech, Chan(cid:173)
`tilly, VA) and grown overnight at 37oc in 5% C02 and 95%
`humidified air. After washing once with 1% FBS in TCM,
`different amounts of non-labeled MAb (10 fLg/ml, 1.0 fLg/ml,
`0.1 fLg/ml, 0.01
`fLg / ml and diluent alone) were added to
`replicate wells in 50-~-LI aliquots of TCM supplemented with
`0.1 % sodium azide. After 1 hr incubation at 37°C, 125I-labeled
`MAb (40 ng/ ml) was added in an additional 50 1-11 TCM with
`0.1% sodium azide. After another 2 hr incubation at 37°C,
`each well was washed 4 times with 1% FBS in TCM, and
`binding of 125I-Iabeled antibody was determined in a Packard
`gamma counter. Per cent inhibition of 1251-labeled antibody
`binding by non-labeled antibody was calculated.
`percent inhibition = 1
`1251-Ab bound in the presence of non-labeled Ab] (cpm)
`[ 125I -Ab bound in the absence of non-labeled Ab J ( cpm)
`X 100%
`
`(
`
`Affinity of binding to p185
`A live-cell radioimmunoassay was used to determine the
`apparent affinity for binding of different MAbs to p185. Cells
`were trypsinized and seeded at a density of 104 /well in 96-well
`RemovaCell plates. After overnight incubation, monolayers
`were washed with 1% FBS in TCM supplemented with 0.1%
`sodium azide. Different amounts of 1251-labeled MAb were
`added in volumes of 50 1-11 to cell monolayers. Non-specific
`binding was determined by adding different amounts of 1251-
`Iabeled MAb (50 1-11) to empty wells. After incubation on ice
`for 4 hr, unbound antibodies were removed by washing the
`wells 4 times with ice-cold TCM containing 5% FBS with 0.1 %
`sodium azide. Individual wells were then detached, and
`radioactivity was determined in a Packard gamma counter.
`The EBDA program was used to calculate the number of
`binding sites (McPherson, 1983, 1985).
`
`Binding sites
`maximum binding (mol) x 6.23 x 1023 x volume (L)
`cell number
`Assay of anchorage-independent growth
`Anchorage-independent cell growth was measured in 35-mm
`tissue-culture dishes (Nunc, Naperville, IL). A 1-ml layer of
`0.6% agar (Difco, Detroit, MI) in TCM was solidified in the
`bottom of each dish. Cells to be assayed were suspended in 1
`ml 0.3% agar in TCM supplemented with one or more
`antibodies or Fab fragments. As clonogenic efficiency differed
`between cell lines, different numbers of cells were seeded:
`Rat-1A(2 x 103/dish),1174(2 x 103/dish),SKBr3(104/dish),
`OVCA432 (IQS/dish), MCF-7 (104 /dish), Huff (2 x 104/dish)
`MDA-M8-468 (2 x 104/dish), BT-474 (2 x 104/dish), MDA(cid:173)
`MB-453 (2 x 104/ dish). MOPC21, a MAb that did not bind to
`p185 or other cell-surface determinants, was used as a control.
`To determine cytotoxic activity of the antibodies, intact immu(cid:173)
`noglobulin or Fab fragments were mixed with SKBr3 cells and
`incubated with shaking for 3 hr at 37°C. After incubation cells
`were washed once with medium and anchorage-independent
`growth was measured as described above.
`For studies of competition with ligand, the gp30 and p75
`HER-2/neu ligands (Lupu et al., 1990, 1992) were added to
`different cell lines suspended in 0.3 % agar. Within a given
`experiment, all assays were performed in triplicate. Cells were
`incubated 10 to 14 days at 37°C in 5% C02 and 95% humidified
`air. Colonies containing more than 50 cells were counted using
`inverted-phase microscopy.
`
`IMMUNOGEN 2036, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`•
`
`t •
`
`GROWTH INHIBITION BY ANTI-p 185 ANTIBODIES
`
`403
`
`Assays of anchorage-dependent growth
`Short-term assays of anchorage-dependent tumor-cell prolif(cid:173)
`eration were performed in 96-well flat-bottom plates (Costar,
`Cambridge, MA). Different wells were seeded with 2.5 x 104
`tumor cells in 200 fLl RPMI 1640 medium with 2% FBS and
`different concentrations of antibodies. After 42 hr, 1 fLCi
`3H-thymidine was added to each well. After 6 hr additional
`incubation, cells were washed 3 times with 50 mM PBS, pH 7.4,
`and harvested by adding 50 fLl 2N sodium hydroxide to each
`well. Solubilized cells were transferred to counting vials and
`radioactivity measured using a Packard Beta Counter.
`
`Internalization of antibodies
`125I-labeled TAl (0.25
`fLg/ ml) or 125I-labeled IDS (0.25 ·
`j.Lg/ ml) was added to aliquots of 106 SKBr3 cells. After
`incubation for 1 hr at 4°C to allow binding, cells were washed 3
`times with RPMI 1640 medium with 1% BSA and then either
`counted immediately to determine the total amount of anti(cid:173)
`body bound or incubated at 4°C or 37°C for 1 hr to permit
`internalization of immunoglobulin . To remove antibody still
`bound to the cell surface, 2.5 mg/ ml of protease K (Sigma, St
`Louis, MO) was added to the cells for 1 hr at 37oc. The cells
`were washed 3 times in RPMI 1640 medium with 1% BSA and
`0.1 % sodium azide. Radioactivity associated with cell pellets
`was counted in a Packard gamma counter. The amount of
`antibody internalized was determined by subtracting the cpm
`obtained after incubation at 4°C followed by protease stripping
`from the cpm obtained after incubation at 37°C for the same
`time interva l, followed by protease stripping. The percentage
`internalized was then calculated by dividing the cpm of the
`antibody internalized by the total cpm initially bound to the
`cell surface.
`
`Down-regulation ofpl85
`SKBr3 cells (5 x 104) were plated in 96-well flat-bottom
`plates (Costar). TAl, IDS or MOPC21 (1 fLg / ml) were added
`and incubated at 37oC for 1 hr to allow internalization of
`antibody. The plate was washed 3 times with medium and
`125I-labeled TAl or 125I-labeled IDS with 0.1% sodium azide
`(200,000 cpm/well) was added. After incubation at 37oc fo.r an
`additional 1 hr, cells were washed 3 times with med ium.
`
`Individual wells were then detached, and radioactivity was
`measured in a Packard gamma counter.
`
`Statistical analysis
`Statistical analysis was performed using Student's t-test.
`
`RESULTS
`Distinct epitopes on the extracellular domain of pl85 defined by
`monoclonal antibodies
`Eleven murine MAbs were used to analyze epitopes on the
`extracellular domain of pl85. After labeling with 1251, each
`antibody still bound to SKBr3 breast-cancer cells that over(cid:173)
`expressed pl85. Non-labeled antibodies were tested for their
`ability to block binding of the same or different MAbs.
`Binding of each labeled antibody could be inhibited 81 to
`100% by an excess of the identical reagent (Fig. 1). Several
`pairs of MAbs inhibited each other's binding by more than
`50%, including TAl and RC6, NB3 and 454Cll, 454Cll and
`RC6, and RC6 and NB3. Cross-blocking of more than 50% was
`also observed between BDS and IDS, PB3 and 741F8, as well
`as 741F8 and 520C9. In some cases, less than 50% cross(cid:173)
`blocking was observed between pairs of antibodies. In other
`cases blocking was not reciprocated, in that the binding of
`125I-labeled PB3 was inhibited 47% by non-labeled RC6, and
`58% by non-labeled 454Cll, whereas non-labeled PB3 failed
`to inhibit the binding of 125I-labeled RC6 or 454Cll. Unidirec(cid:173)
`tional blocking was also observed between BDS, 520C9, 741F8
`and 454Cll. Binding of OD3 was not inhibited by other MAbs,
`suggesting that the epitope recognized by OD3 was not related
`to those recognized by the other antibodies.
`Taken together, the data for the eleven MAbs are consistent
`with the model in Figure 2, where 10 of the 11 epitopes are
`grouped in a linear array. OD3 appears distinct. Eight MAbs
`were evaluated in competitive binding assays using the rat 1174
`cell line that expressed human pl85. Essentially similar result~
`were obtained (data not shown) .
`
`Inhibitor (lOug/ml)
`
`l2S 1-Iabeled Antibodies (40ng/ml)
`
`RCl
`
`TAl
`
`4S4C11 NBJ
`
`RC6
`
`PBJ
`
`S20C9 741F8 805
`
`IDS
`
`ODJ
`
`RCl
`
`TAl
`
`4S4C11
`NBJ
`
`RC6
`
`PBJ
`
`IS20C9
`741F8
`
`IBDS
`IDS
`
`~············
`
`t
`
`~7S-100%
`
`~ S0-74%
`
`b
`
`I2S-49%
`
`c:J Below 2S%
`
`FIGURE 1- Competitive binding of anti-pl85 MAbs to SKBr3 cells. Non-labeled antibodies were pre-incubated with tumor cells for 1
`hr before the add ition of 1251 -labeled antibodies for 2 hr.
`
`IMMUNOGEN 2036, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`i
`t
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`404
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`XU ET AL.
`
`RCl-T~l~r-105
`
`454Cll~N83
`
`741F8~520C9
`
`003
`
`FIGURE 2- Relative epitope map of the extracellular domain of
`p185 determined by competitive inhibition. Solid lines indicate
`> 50% blocking, dotted lines indicate <50% blocking. Arrows
`indicate the ability of non-labeled antibody to inhibit binding of
`J25I-labeled antibody.
`
`Inhibition of anchorage-independent and anchorage-dependent
`growth by individual anti-p185 antibodies
`Growth of SK.Br3 breast-cancer cells could be inhibited by
`some, but not all, antibodies reactive with pl85 when colonies
`were enumerated after 14 days incubation in soft agar (Fig.
`3a ). In 4 replicate experiments, anchorage-independent growth
`of SKBr3 was significantly inhibited 45 to 85% by RCl, NB3,
`RC6, PB3, 741F8, BD5 and IDS (p < 0.01), but not by TAl,
`454C11, 520C9, or OD3. Over a range of 1.0 to 10 ~J-g/ml , a
`dose-response effect on anchorage-independent growth was
`obtained with inhibitory antibodies (Fig. 4) . In the case of IDS,
`as little as 0.1 fLg /ml produced 41 % inhibition. The effect of
`different antibodies on anchorage-dependent growth of SKBr3
`cells was assessed by measuring incorporation of [3H ](cid:173)
`thymidine. Only IDS and BDS significantly inhibited growth
`(p < 0.01) in 48 hr (Fig. 3b ).
`Growth inhibition did not correlate with differences in
`relative affinity or apparent epitope number when determined
`on 2 cell lines (SKBr3 and rat 1174) with different levels of
`pl85 expression (Table I). Cytotoxic rather than cytostatic
`effects were observed, in that excess antibody could be washed
`from cells after 3 hr incubation, and clonogenic growth was
`inhibited at 14 days (Table II).
`Expression of pl85 was required for growth inhibition.
`Anchorage-independent growth of Huff and MCF-7 cells was
`not affected by treatment with any of the 11 anti-pl85
`antibodies. Mean percent inhibition for the 11 antibodies
`ranged from -4 to 2% for both Huff and MCF-7 cells. The
`degree of pl85 expression may be important in that anti-pl85
`antibodies produced qualitatively similar, but quantitatively
`less inhibition (54-66%) of OVCA 432 and rat 1174 cells that
`expressed fewer copies per cell of pl85 (data not shown).
`
`Inhibition of anchorage-independent and anchorage-dependent
`growth by anti-p185 antibodies in combination
`Additive or supra-additive inhibition of anchorage-indepen(cid:173)
`dent growth was observed with 17 of the 55 possible combina(cid:173)
`tions of the 11 antibodies when tested at 1 ~J-g / ml (p < 0.05;
`underlined values in Table III). Interestingly, antibodies that
`were individually ineffective produced significant inhibition
`when used in combination, e.g., TAl and 454C11, TAl and
`520C9, 520C9 and 454C11, 454C11 and OD3, OD3 and 520C9
`(Fig. 5). Several co.mbinations of individually active antibodies
`also produced additive growth inhibition that was greater than
`that produced by NB3, BDS or IDS alone. In sever(!! cases,
`antibodies that produced additive inhibition appeared to
`recognize distinct epitopes, e.g., TAl in combination with NB3,
`520C9, BD5 or IDS.
`The TAl antibody was of particular interest in that it could
`produce directly opposing effects on growth of SKBr3 in
`combination with other antibodies. TAl, 454Cll and RCl
`appeared to react with closely related epitopes in that they
`could partially inhibit each other's binding to pl85. When used
`individually, TAl did not affect anchorage-independent growth.
`TAl in combination with 454Cll produced additive inhibition
`
`90
`80
`r::::
`0 70
`:;:::
`:0 60
`E
`..5 50
`c 40
`Q)
`0 30
`Q;
`a. 20
`10
`
`0
`
`50
`
`A
`
`B
`
`r::::
`40
`0
`·.;:::
`:0 30
`E
`..5
`c 20
`Q)
`0
`Q;
`a.
`
`10
`
`0
`
`-10
`
`FIGURE 3- Inhibition of anchorage-independent growth (a) and
`anchorage-dependent growth (b) of SK.Br3 by individual MAbs.
`For anchorage-independent growth, each value represents the
`mean percent inhibition ± SD for 3 or 4 replicate experiments
`performed on different days. For anchorage-dependent growth,
`each value represents the mean ± SE for 7 or 8 replicate
`experiments performed on different days.
`
`100
`
`r:::: 80
`0
`
`:;::: :c 60
`E
`..5
`c 40
`Q)
`0
`Q;
`a. 20
`
`0
`
`TA 1
`
`NB3
`
`RC6
`
`PB3
`
`7 41 FS
`
`IDS MOPC21
`
`FIGURE 4- Dose response of antibody-mediated inhibition of
`anchorage-independent growth of SK.Br3.
`
`of anchorage-independent growth, whereas TAl decreased
`the growth inhibition observed with RCl in a dose-dependent
`manner (Fig. 6).
`Antibodies were also tested in combination at 1.25 fLg / ml
`for inhibition of anchorage-dependent growth. Additive growth
`inhibition was observed only with IDS and PB3 (p < 0.01;
`underlined value in Table IV) .
`
`Modulation of cell-swface p185 by anti-p185 antibodies
`125I-labeled anti-pl85 antibodies were internalized after
`binding. After 1 hr of incubation, 30% of 125I-TAl and 27% of
`125I-ID5 remained associated with cells after protease treat(cid:173)
`ment (data not shown). Thus, similar levels of cell-associated
`radioactivity were observed with both antibodies, despite the
`fact that IDS inhibited both anchorage-independent and
`anchorage-dependent growth, whereas TAl did not.
`To determine whether antibody internalization produced an
`apparent down-regulation of the receptor, cells were incu(cid:173)
`bated at 37°C for 1 hr with individual unlabeled antibodies to
`
`IMMUNOGEN 2036, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`• I
`
`GROWTH INHIBITION BY ANTI-p185 ANTIBODIES
`
`405
`
`TABLE 1 - BINDING CHARACTERISTICS OF ANTI-pi85 ANTIBODIES
`
`Antibody
`
`TAl
`BDS
`IDS
`RCl
`RC6
`NB3
`OD3
`S20C9
`741F8
`PB3
`4S4Cll
`
`SKBr3
`Relative affinity (M - 1)
`2.3 X lQtO
`1.1 X 109
`1.9 X 109
`2.3 X 108
`1.2 X 109
`8.0 X 108
`3.9 X 107
`2.S X 109
`2.0 X 108
`1.1 X 109
`2.7 X 108
`
`Cell line
`
`Sites ( x 105)
`
`1174
`Relative affi nity (M - 1)
`
`Sites ( x i04)
`
`3.7
`S.S
`S.8
`S.6
`l.S
`1.6
`1.3
`4.1
`S.3
`S.3
`7.8
`
`l.S X 109
`4.1 X 108
`2.1 X 108
`1.3 X 108
`S.l X 1Q8
`6.4 X 108
`3.S X 1Q8
`3.S X 109
`4.6 X 109
`1.6 X 1Q9
`4.4 X 1Q9
`
`6.2
`3.8
`4.8
`6.3
`3.3
`2.2
`2.2
`4.1
`2.0
`S.3
`6.1
`
`TABLE II - CYTOTOXIC EFFECT OF INTACT IMMUNOGLOBULIN AND
`ANTIBODY FRAGMENTS REACTIVE WITH pl85 ON THE
`ANCHORAGE-INDEPENDENT GROWTH OF SKBr3 BREAST-CANCER
`CELLS
`
`100
`
`Ant ibody
`
`TAl
`
`PB3
`
`IDS
`
`Perce nt inhibition 1
`
`Concentration
`
`Intact IgG
`
`10 J..Lg/ ml
`S J..Lg/ ml
`10 J..Lg/ ml
`S J..Lg/ ml
`10 J..Lg/ ml
`S J..Lg/ ml
`
`3
`1
`73
`6S
`87
`79
`
`Fab
`
`ND
`ND
`ND
`ND
`89
`77
`
`1Anchorage-independent growth at 14 days after 3-hr incuba(cid:173)
`tion with intact IgG or Fab fragments.-ND, not determined.
`
`100
`90
`c:
`80
`0
`:;::; 70
`:0
`£ 60
`E 50
`c 40
`Ql
`0 30
`Q;
`a.. 20
`10
`0
`
`FIGURE 5- Inhibition of anchorage-independent growth of SK(cid:173)
`Br3 by combinations of MAbs (1 J..Lg / ml).
`
`2
`
`8
`6
`4
`TA1 Concentration (llg/ml)
`
`10
`
`12
`
`FIGURE 6- Dose-dependent neutralization by TAl antibody of
`RCl-mediated inhibition of anchorage-independent growth of
`SKBr3.
`
`in a competitive radioimmunoassay did not block ligand(cid:173)
`mediated stimulation of anchorage-independent growth.
`
`Growth inhibition by intact immunoglobulin, F(ab'hfragments
`and Fab fragments
`In earlier studies with antibodies against rat neu, Fab
`fragments did not inhibit tumor growth (Drebin et al., 1985;
`Yarden, 1990). By contrast, both F(ab')z and Fab fragments of
`ID5 RCl and PB3 could inhibit anchorage-independent growth
`of SKBr3 that over-expressed human pl85 (Table VII). Both
`F(ab')2 and Fab fragments were approximately as effective as
`intact immunoglobulin.
`
`DISCUSSION
`
`prompt internalization of pl8S. After internalization, labeled
`antibody to a distinct epitope on pl85 was used to detect
`receptors remaining on the cell surface. Pre-incubation with
`TAl did not affect subsequent binding of IDS and pre(cid:173)
`incubation with IDS did not affect binding of TAl. Internal(cid:173)
`ized receptor was apparently replaced within 1 hr, since
`down-regulation of the receptor was not detected after incuba(cid:173)
`tion with either TAl or IDS (Table V).
`
`Antibody-mediated inhibition of ligand stimulated
`anchorage-independent growth
`Stimulation of anchorage-independent growth of 3 cell lines
`expressing pl8S by gp30 ligand at 0.2 ng/ ml or p7S ligand at 0.4
`ng/ ml could be partially blocked by the BD5 and ID5 antibod(cid:173)
`ies (Table VI). BD5 and IDS had no effect on the pl8S(cid:173)
`negative cell line MDA-MB-468. BD5 and IDS also blocked
`binding of the gp30 ligand in a competitive radioimmunoassay
`(data not shown). TAl antibody as well as the other anti-p18S
`antibodies (data not shown) that did not block the gp30 ligand
`
`Antibodies against the extracellu lar domain of the c-erbB-2
`gene product can inhibit growth of tumor cells that over(cid:173)
`express pl85, in the absence of cellul ar or humoral effectors of
`antibody-dependent cytotoxicity. Drebin et at. (1985) reported
`down-regulation of rat tumor cell growth with intact IgG
`antibodies directed against the extracellu lar domain of the rat
`neu gene product. Our data indicate that murine MAb against
`the human c-erbB-2 (HER-2/neu) gene product p18S can
`inhibit the growth of human tumor cells. Of the 11 antibodies
`stud ied, 7 reagents inhibited anchorage-independent growth
`and 2 of these 7 inhibited anchorage-dependent growth of
`SKBr3 breast-cancer cells. Since fewer antibodies inhibited
`anchorage-dependent growth than
`inhibited anchorage(cid:173)
`independent growth, it is possible that mechanisms for inhibi(cid:173)
`tion in these 2 assays differ. Antibodies to rat neu inhibited
`anchorage-independent growth but not anchorage-dependent
`growth (Drebin et at., 198S).
`Antibodies directed against different p185 epitopes exerted
`different effects on tumor cell growth. When tested individu-
`
`t I
`
`IMMUNOGEN 2036, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`406
`
`XU ET A L.
`
`TABLE Ill - PERCENT INHIBITION O F ANCHORAG E-INDEPENDENT GROWTH OF SKBr3 WITH ANTI-piSS ANTIBOD IES USED IND IVIDUALLY AND IN
`COMB INATION'
`
`RC I
`
`TA l
`
`4S4Cll
`
`NB3
`
`RC6
`
`PB3
`
`S20C9
`
`741F8
`
`BDS
`
`IDS
`
`OD3
`
`80 ± 1
`RCl
`5 ± 0
`38 ± 18
`TAl
`454C11
`0.3 ± 0.5
`84 ± 4
`61 ±52
`63 ± 74
`NB3
`78 ± 6
`95 ± 33
`83 ± 5
`RC6
`73 ± 9
`64 ± 2
`62 ± 3
`80 ± 1
`67 ± 6
`PB3
`83 ± 4
`81 ± 1
`80 ± 3
`80 ± 1
`88 ± 8
`80 ± 2
`520C9
`4±4
`81 ± 2
`69 ± 6
`79 ± 7
`66 ± 22
`86 ± 12
`80 ± 1
`741F8
`79 ± 4
`74 ± 2
`81 ± 2
`81 ± 3
`78 ± 7
`79 ± 4
`79 ± 2
`81 ± 1
`80 ± F
`BDS
`40 ± 2
`84 ± 6
`44 ± 12
`90 ± 8
`91 ± 12
`79 ± 8
`86 ± 22
`83 ± 4
`IDS
`99 ± p
`99 ± 33
`79 ± 10
`87 ± 2
`97 ± 64
`99 ± 23
`70 ± 14
`92 ± 94
`99 ± 14
`90 ± 34
`27 ± 7
`81 ± 4
`24 ± 63
`81 ± 4
`59± 1
`75 ± 5
`72 ± 43
`3 ± 3
`79 ± 2
`OD3
`55± 7
`1Each value represents the mean ± SD for 3 experiments performed on different days.-2p < 0.001.-3p < O.Ol.-4p < 0.05.
`
`0.8 ± 1.5
`
`TABLE IV - PERCENT INH IBIT ION OF ANCHORAGE-DEPENDENT GROWTH OF SKBr3 WITH ANTI-pl8S ANTIBOD IES USED IND IVIDUALLY AND IN
`COMB INATION'
`
`RCI
`
`TA l
`
`4S4C II
`
`NB3
`
`RC6
`
`PB3
`
`S20C9
`
`74 IF8
`
`BDS
`
`IDS
`
`OD3
`
`-.6 ± .2
`RCl
`-4 ± 3
`TAl
`10 ± 4
`-9 ± 5
`15 ± 6
`8±4
`454C11
`7±4
`8 ± 4
`14 ± 8
`4±2
`NB3
`-1 ± .5
`-.7±.4
`0.3 ± 0.1
`9±5
`10 ± 6
`RC6
`-12 ± 7
`18 ± 7
`12 ± 7
`4±2
`-6 ± 3
`7±4
`PB3
`18 ± 7
`13 ± 7
`5 ± 2
`21 ± 12
`22 ± 10
`13 ± 6
`520C9
`15 ± 8
`12 ± 4
`16 ± 8
`12 ± 7
`16 ± 9
`741F8
`15 ± 8
`15 ± 7
`10 ± 6
`13 ± 8
`BDS
`30 ± 11
`15 ± 8
`31 ± 18
`13 ± 16
`32 ± 16
`18 ± 9
`19 ± 10
`19 ± 8
`13 ± 7
`IDS
`35 ± 17 32 ± 16 36 ± 7
`61 ± 182
`22 ± 11
`32 ± 16
`24 ± 12
`57± 26
`20 ± 10
`-7 ± 4
`-.7 ± .4
`-4 ± 2
`6 ± -4
`20 ± 10
`25 ± 13
`9 ± 5
`2 ± 1
`7 ± 4
`OD3
`1Each value represents the mean ± SD for 3 experiments performed on different days.-2p < 0.01.
`
`35 ± 10
`22 ± 11 3±3
`
`TABLEV - LAC K O F DOWN-REGULATION OF pi SS WIT H TA l AND IDS ON
`SKBr3 CELLS
`
`A ntibodies for
`pre-incubation 1
`
`MOPC21
`TAl
`IDS
`11 fi.g /ml.
`
`Binding of 125 1-labeled ant ibodies (cpm ± SD)
`TAl
`ID5
`
`36650 ± 2338
`1280 ± 216
`35129 ± 2093
`
`36760 ± 5458
`38430 ± 1490
`1677 ± 297
`
`ally, antibodies with similar affinity and isotype could ei th er
`inhibit or fail to inhibit growth of SKBr3. Antibodies that
`lacked activity individually could, however, inhibit anchorage(cid:173)
`independent growth when used in combination. The TAl
`antibody could either augment or reduce growth inhibition
`produced by other antibodies such as 4S4Cll or RCl. Additive
`effects upon anchorage-dependent growth were much more
`restricted, and were observed on ly between IDS and PB3.
`Growth inhibition might be due to several mechanisms.
`Antibodies that bind within or near the site(s) for ligand
`binding might block or mimic the activity of ligand . In the case
`of tumor cells that over-express p18S, treatment with ligand at
`low concentration c_an stimulate and at high concentration can
`inhibit tumor cell growth (Lupu et al., 1990, 1992). Interest(cid:173)
`ingly, the 2 antibodies (BDS and IDS) that can inhibit both
`anchorage-dependent and anchorage-independent growth of
`SKBr3 also inhibited binding of the gp30 and p7S ligands
`described by (Lupu eta /. (1990, 1992). Antibodies reactive with
`S other epitopes cou ld inhibit anchorage- independent growth ,
`but did not appear to interact with ligand. Consequently,
`epitopes at a distance from the ligand binding site can provide
`a target for antibody-mediated g