Proc. Natl. Acad. Sci. USA
`Vol. 88, pp. 8691-8695, October 1991
`Cell Biology
`
`Mechanistic aspects of the opposing effects of monoclonal
`antibodies to the ERBB2 receptor on tumor growth
`(growth factors/tyrine kinase/adenocarcinoma/oncogene)
`
`ILANA STANCOVSKI*, ESTHER HURWITZ*, ORIT LEITNER*, AXEL ULLRICHt, YOSEF YARDEN*,
`AND MICHAEL SELA*
`*Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel; and tInstitut fur Biochemie, Am Klopferspitz 18A, 8033
`Martinsried, Federal Republic of Germany
`
`Contributed by Michael Sela, July 11, 1991
`
`The ERBB2 (also called HER2, neu, and
`ABSTRACT
`c-erbB-2) gene product, which encodes a growth factor recep-
`tor, was implicated in the malignancy of human adenocarci-
`nomas. An antibody directed to the rat oncogenic receptor has
`been previously shown to have an antitumor effect in model
`systems. In an attempt to extend this observation to the
`protooncogenic human receptor and also to understand the
`underlying mechanism, we generated a panel of monoclonal
`antibodies specific to the extracellular portion of the ERBB2
`protein. The effects of the antibodies on tumor growth were
`compared with their cellular and biochemical actions in vitro.
`Surprisingly, opposing in vivo effects were observed: although
`some antibodies almost completely inhibited the growth in
`athymic mice of transfected murine fibroblasts that overex-
`press Erbb-2, other antibodies either accelerated tumor growth
`or resulted in intermediate responses. When tested on cultured
`human breast carcinoma cells or ERBB2 transfectants, the
`tumor-stimulatory antibody was found to induce significant
`elevation of tyrosine phosphorylation of the ERBB2 protein. In
`contrast, only partial correlation was observed between the
`capacity to restrict tumor growth and the effects of the anti-
`bodies on receptor degradation and cellular proliferation in
`vitro. This suggests that the antitumor antibodies affect both
`receptor function and host-tumor interactions. Our results
`may help establish experimental criteria for the selection of
`specific antibodies for use either alone or in cogiunction with
`other molecules as pharmacological antitumor agents.
`
`Evidence has been accumulated in recent years for the
`involvement of growth factors and their receptors in the
`process of malignant transformation. The ERBB2 protein is
`a receptor tyrosine kinase (1), homologous to the epidermal
`growth factor (EGF) receptor (2, 3). The rat homologue ofthe
`gene undergoes oncogenic activation through a single point
`mutation (4). The ERBB2 protein was found to be overex-
`pressed in several types of human adenocarcinomas, espe-
`cially in tumors of the breast and the ovary (5-7), and the
`overexpression was correlated with short time to relapse and
`poor survival of breast cancer patients (5).
`The potential use of monoclonal antibodies (mAbs) in
`diagnosis and treatment of cancer has been studied exten-
`sively (8). Receptors for growth factors constitute an ideal
`target for this approach because their location on the cell
`membrane makes them accessible to antibody molecules.
`Moreover, antibodies directed to growth factor receptors can
`potentially block biological functions essential for cell pro-
`liferation. Previous studies have demonstrated, in model
`systems, the potential therapeutic effect of mAbs against the
`epidermal growth factor receptor (9, 10). Likewise, different
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`8691
`
`mAbs to the ERBB2 receptor inhibited the proliferation of a
`human breast carcinoma cell line in culture (11), and an
`antibody directed to the rat ERBB2 protein inhibited the
`tumorigenicity of fibroblasts transformed by the mutated rat
`ERBB2 oncogene (12, 13). mAbs that recognize the protein
`product of the human ERBB2 protooncogene have been
`raised and used to study the biological function of the
`presumed receptor (14-16).
`Our studies were aimed at the generation ofantibodies with
`potential use in immunotherapy of human cancer, either
`alone or conjugated with drugs or toxins. To this end we
`raised a panel of mAbs to the extracellular portion of the
`ERBB2 receptor. These antibodies induced opposing effects
`on tumor growth in athymic mice. Our attempts to analyze
`the mechanism of antibody-mediated tumor enlargement
`suggest that activation of the tyrosine kinase is involved, but
`inhibition of tumor growth is not simply correlated with one
`receptor function.
`
`MATERIALS AND METHODS
`Chemicals and Reagents. Affinity-purified goat anti-mouse
`F(ab')2 was from Jackson ImmunoResearch. It was radiola-
`beled with Na125I (Amersham) by the chloramine T proce-
`dure (17). [32P]Orthophosphate was from the Nuclear Re-
`search Center (Negev, Israel); [35S]methionine and
`[y-32PIATP were from Amersham. Sepharose-protein A was
`purchased from Pharmacia. The anti-phosphotyrosine mAb
`1G2 (18) was purified from ascites fluid.
`Cell Lines. The HER2 cell line has been described (19). The
`SKBR3 human breast carcinoma cell line was obtained from
`the American Type Culture Collection.
`Experimental Animals. BALB/c mice, CB6/F1 mice, and
`CD1/nude mice were obtained from the Experimental Ani-
`mals Center of the Weizmann Institute of Science.
`Generation of mAbs to the ERBB2 Receptor. BALB/c mice
`(2 mo old) were injected i.p. three times with 3-5 x 106 living
`SKBR3 human breast carcinoma cells, at intervals of 2
`weeks. Antisera were tested by an immunoprecipitation
`assay using HER2 cells (NIH 3T3 cells transfected with
`human ERBB2 gene, ref. 19), labeled metabolically with
`[35S]methionine. The spleens of mice that developed a strong
`immune response were selected for fusion. The spleen cells
`were fused with NSO myeloma cells by using polyethylene
`glycol (20), and the hybridomas were selected with hypoxan-
`thine/aminopterin/thymidine medium. Supernatants of the
`growing cells were screened by using an indirect binding
`assay. CHO cells transfected with the ERBB2 gene (HCC cell
`line) were plated on a flexible 94-well plate, previously coated
`with polylysine (1 mg/ml). The cells were fixed with 3%
`(wt/vol) paraformaldehyde, and supernatants of hybridomas
`were incubated for 1 hr at 22°C with the fixed cells. The bound
`
`Abbreviation: mAb, monoclonal antibody.
`
`IMMUNOGEN 2033, pg. 1
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`

`8692
`
`Cell Biology: Stancovski et al.
`
`Proc. Natl. Acad Sci. USA 88 (1991)
`
`antibodies were detected with 125I-labeled goat anti-mouse
`F(ab')2 antibody. As a negative control we used the parental,
`untransfected CHO cell line.
`The antibodies that specifically bound to the HCC cells
`were selected for further analysis by using either an immu-
`noprecipitation assay with [35S]methionine-labeled cells or
`immunoprecipitation followed by autophosphorylation in the
`presence of MnCl2 and [y-32P]ATP (21). Positive hybridomas
`were cloned twice by limiting dilution. Determination of
`antibody class was done with class-specific second antibod-
`ies. Large quantities of specific mAbs were produced by
`preparation of ascites fluid in CB6/F1 mice. The IgM anti-
`body was separated on a Sephacryl G300 column, and the
`IgG1 and IgG2a antibodies were purified by affinity chroma-
`tography on Sepharose-protein A, using elution conditions
`specific for each subclass.
`Indirect Binding Assay on Living Cells. SKBR3 cells or
`HER2 cells were plated in 24-well plates and assayed at
`confluence. The cells were incubated for an hour at 22TC with
`various concentrations of antibodies in phosphate-buffered
`saline (PBS)/1% bovine serum albumin. After being washed
`with the same buffer, the cells were incubated for 90 min with
`125I-labeled goat anti-mouse F(ab')2 (105 cpm per well). The
`cells were then washed and solubilized with 0.1 M NaOH; the
`radioactivity was then determined in a y counter.
`Determination of the in Vivo Effect of the mAbs. HER2 cells
`(3 x 106) were injected s.c. into nude mice, followed by three
`i.p. injections of the mAbs on days 3, 7, and 10. Tumor
`parameters were measured twice a week with callipers, and
`tumor volume was calculated according to the formula: tumor
`volume equals length x width x height. To validate volume
`measurements the correlation between the tumor volume and
`tumor weight was determined on the day of animal killing.
`Determination of Tyrosine Phosphorylation in Living Cells.
`The SKBR3 or HER2 cells were grown in a 24-well plate and
`labeled for 4 hr in Dulbecco's modified Eagle medium
`(DMEM) without phosphate but in the presence of 1%
`dialyzed fetal calf serum and [32P]orthophosphate (0.5 mCi/
`ml; 1 Ci = 37 GBq). The cells were washed with PBS and
`incubated for 15 min at 22°C with fresh medium containing
`antibodies at a concentration of 10 ,ug/ml. After being
`washed, the cells were lysed in solubilization buffer (21), and
`the tyrosine-phosphorylated ERBB2 protein was immuno-
`precipitated with an agarose-immobilized antibody to phos-
`photyrosine (18). The immune complexes were eluted with
`solubilization buffer containing 50 mM p-nitrophenylphos-
`phate and subjected to immunoprecipitation with a rabbit
`polyclonal anti-ERBB2 antibody, directed to the carboxyl
`terminus of the receptor (21).
`Determination of the Effect of the mAbs on Receptor Turn-
`over. SKBR3 or HER2 cells were grown in 24-well plates to
`80% confluence and then labeled for 16 hr at 37°C with
`[35S]methionine (50 ,Ci/ml). After being washed with PBS,
`the cells were incubated with fresh medium in the absence or
`presence of the antibodies (at a concentration of 10 ,ug/ml) for
`various periods of time. The cells were then washed, and cell
`lysates were subjected to immunoprecipitation with a rabbit
`polyclonal antibody to the ERBB2 protein (21).
`Complement-Dependent Cytotoxicity (CDC) Assay. The
`SKBR3 tumor cells were incubated at 37°C for 2 hr in a
`volume of 0.1 ml of fetal calf serum, with 300 ,uCi of
`Na[51Cr]04 (DuPont/NEN). At the end of the labeling period
`the cells were washed three times in PBS, and 1.5 x 104 cells
`(in 25 ,ul) were plated in each well ofa 96-well microtiter plate.
`Various concentrations of the mAbs (25 ul) were added and
`incubated with the cells for 1 hr followed by the addition of
`human or rabbit complement and incubation for further 3 hr.
`Appropriate control wells containing cells alone, cells with no
`antibody or no complement, and cells lysed in 10% SDS were
`
`set up in parallel. 51Cr release was determined in a 'y counter.
`The means of triplicate determinations are given.
`Antibody-Mediated Cell-Dependent Cytotoxicity (ADCC)
`Assay. The SKBR3 tumor cells were labeled with Na[5'Cr]04
`as described above. Cells (5 x 103) in 25 4ul were incubated
`for 1 hr with various concentrations of the mAbs and then for
`5 hr with effector cells, human peripheral blood lymphocytes
`(0.1 ml, lymphocytes/tumor cells = 140:1), or with mouse
`splenocytes (120:1). 51Cr release was determined as de-
`scribed above.
`
`RESULTS
`Generation of mAbs Directed to the ERBB2 Receptor. Five
`hybridomas were selected after the fusion of NSO myeloma
`cells with splenocytes obtained from mice immunized with
`intact cells of the human breast carcinoma SKBR3 cell line.
`This immunization procedure elicited specific antibodies to
`the extracellular portion of the human ERB1D2 antigen. The
`isotypes and subclasses of the resulting mAbs are given in
`Table 1. Three of these antibodies were found to be of the
`IgG1 subclass (N12, N24, N28), one was an IgM (N10), and
`one an IgG2a antibody (N29). As depicted in Fig. 1, all the
`mAbs specifically bound to cultured cells that express the
`ERBB2 receptor, yet they bound with different apparent
`affinities. Antibodies N28 and N24 displayed the highest
`apparent affinity, whereas N10 mAb exhibited the lowest
`apparent affinity. All five mAbs immunoprecipitated a single
`protein of 185 kDa from metabolically labeled HER2 cells, as
`shown in Fig. 2A. This result was also reflected in an in vitro
`kinase assay performed on the immunoprecipitates (Fig. 2B).
`None ofthese mAbs reacted with the epidermal growth factor
`receptor or with the rat p1850cu (data not shown). Immuno-
`blot analysis of the ERBB2 protein showed that only the N12
`and N29 antibodies could react with the denatured form ofthe
`receptor (Table 1).
`The Effect of mAbs upon Tumor Growth in Vivo. The mAbs
`were assayed for their ability to affect tumor growth of
`murine fibroblasts transformed by overexpression of the
`ERBB2 gene (HER2 cells), in nude mice. The mAbs or a
`control antibody to dinitrophenol (anti-DNP) were injected
`i.p., into groups of five mice, on days 3, 7, and 10 after tumor
`inoculation. Fig. 3A depicts tumor volumes of each group of
`mice, on day 21, postinoculation. The tumorigenic growth of
`HER2 cells was significantly inhibited (P < 0.05, as calcu-
`lated by using Duncan's multiple comparison test) in nude
`mice injected with mAbs N29 and N12, when compared with
`mice that received no antibody or the control anti-
`dinitrophenyl antibody. As depicted in Fig. 3B, the inhibitory
`
`00
`
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`
`100
`
`10
`.01
`.1
`1
`Antibody concentration (gg/ml)
`Binding of monoclonal anti-ERBB2 antibodies to HER2
`FiG. 1.
`cells. Confluent monolayers of HER2 cells were incubated for 1 hr
`at 220C with various concentrations of mAbs. Bound antibodies were
`subsequently determined with 125I-labeled goat anti-mouse F(ab')2.
`Control cells were incubated without the murine antibody, and their
`background binding was subtracted. A, N10 (IgM); *, N12 (IgGl); o,
`N24 (IgGl); *, N28 (IgGl); *, N29 (IgG2a).
`
`IMMUNOGEN 2033, pg. 2
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`
`

`

`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`8693
`
`C)E
`
`U 0E 2
`
`0 E
`
`I-.
`
`control
`
`N10
`
`N28
`
`N29
`
`Antibody
`
`treatment
`
`16
`
`00
`*8
`
`10
`
`20
`
`30
`
`Day
`Effect of mAbs on tumor growth in athymic mice. Cells
`FIG. 3.
`(3 x 106) were injected, s.c. into CD1/nude mice. Groups offive mice
`received three i.p. injections on days 3, 7, and 10 at a total mAb dose
`of 2 mg per mouse. Tumor size was measured as described. As
`control, an irrelevant antibody anti-dinitrophenol or buffer alone
`(PBS) was used. (A) Effects of antibody treatment after 21 days
`postinoculation. (B) Kinetics of tumor growth in antibody treated
`mice. o1, Control; a, N10; *, N12; o, N24; *, N28; and *, N29.
`Statistical analysis was done by using the analysis of variance and
`Duncan's multiple comparison test.
`
`receptor. For this purpose, HER2 cells were biosynthetically
`labeled with radioactive methionine and then chased for
`various periods of time with fresh medium that contained
`different mAbs. At the end of the chase period, the residual
`labeled proteins were immunoprecipitated and analyzed by
`gel electrophoresis and autoradiography. The results of this
`experiment are shown in Fig. 5: all the mAbs accelerated, to
`different extents, the rate of turnover of the receptor, with
`antibody N29 being the most effective.
`
`DISCUSSION
`Overexpression of ERBB2 protein is frequently found in
`human adenocarcinomas, and it is believed to be involved in
`the progression ofthe malignancy state (5-7). This possibility
`was supported by gene transfer experiments demonstrating
`that overexpression of the apparently normal gene, driven by
`heterologous promoters, confers tumorigenicity on murine
`fibroblasts (19, 24). These observations, together with the
`tyrosine kinase activity of the ERBB2 protein, have made
`this human receptor an excellent target candidate for anti-
`body-mediated therapy of human solid tumors. Indeed, many
`different mAbs to the human protein have been generated
`(14-16), but their anti-tumor activity was not extensively
`investigated in vivo. On the other hand, a mAb to the rat
`ERBB2 protein efficiently inhibited the growth of tumori-
`
`Cell Biology: Stancovski et al.
`
`A
`
`mAb
`
`q- OcD
`CD
`ctj
`c)>
`z z z z z z m
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`1041040
`
`B
`
`mAb
`
`oc)J t-G
`0)
`- - cj cj N ._
`z z z z z z
`
`kDa
`0-180
`
`-1 16
`
`kDa
`
`-180
`
`-116
`
`FIG. 2.
`Immunoprecipitation of the ERBB2 protein by mAbs. (A)
`HER2 cells were metabolically labeled with [35S]methionine, and the
`cell lysates were separately subjected to an immunoprecipitation
`assay with 10 ,ug of each mAb. As a control nonimmune serum (Ni)
`was used. Proteins were separated on a SDS/7.5% polyacrylamide
`gel. (B) The immunoprecipitation assay was done as described but
`with unlabeled cells. Before electrophoresis, the proteins from the
`cell lysate were labeled by autophosphorylation with [y-32PIATP and
`10 mM MnC12. Autoradiograms are shown. NI, nonimmune serum;
`Polycl, polyclonal anti-ERBB2 antibody.
`
`effect persisted over 31 days after tumor injection. Antibod-
`ies N10 and N24 exhibited less efficient inhibition of tumor
`growth. In contrast, mAb N28 consistently stimulated tumor
`growth. Essentially identical results were obtained in three
`separate experiments. To test the possibility that the effects
`seen in vivo are reflected in vitro, we used cell proliferation
`assay in culture and cytotoxicity assays on SKBR3 human
`breast tumor cells. Partial, if any, correlation was found
`between the results obtained in these assays and the in vivo
`experiments (Table 1).
`Stimulation of Tyrosine Phosphorylation of ERBB2. It has
`been shown (22) that mAbs directed against the rat ERBB2
`protein elevated tyrosine phosphorylation of this receptor.
`Two different assays were used to test the capacity of our
`mAbs to elevate tyrosine phosphorylation of the ERBB2
`protein: HER2 cells were metabolically labeled with
`[32P]orthophosphate, incubated with the mAbs, and sub-
`jected to two consecutive immunoprecipitation steps with
`anti-phosphotyrosine and anti-ERBB2 antibodies (21). Alter-
`natively, SKBR3 cells were first incubated with the mAbs
`and then subjected to two consecutive immunoprecipitation
`steps, followed by an in vitro phosphorylation assay in the
`presence of [_y-32P]ATP and MnC12. As depicted in Fig. 4,
`similar results were obtained in both experiments: mAb N28
`significantly stimulated phosphorylation of the ERBB2 re-
`ceptor on tyrosine residues, whereas the other mAbs dis-
`played low (N12, N24, N29 mAbs) or no activity (N10
`antibody) in living cells.
`The Effects of the mAbs on the Rate of Receptor Turnover.
`The interaction of receptor tyrosine kinases with their re-
`spective ligands is usually coupled to rapid endocytosis. It
`was further shown that antibodies could induce an analogous
`effect on the rat ERBB2 receptor (22) and that this activity
`was associated with disappearance of the transformed phe-
`notype (23). We, therefore, tested the potential of our mAbs
`to the human ERBB2 protein to accelerate the turnover of the
`
`IMMUNOGEN 2033, pg. 3
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`
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`

`8694
`
`Cell Biology: Stancovski et al.
`
`Proc. Natl. Acad Sci. USA 88 (1991)
`
`CD m
`N I'll
`c )
`mAb: z Z z z z z
`
`A
`
`B
`
`mAb: Z Z z z z z
`
`kDa
`
`-180
`
`- 116
`
`kDa
`
`-180
`
`Antibody-induced stimulation of tyrosine phosphoryla-
`FIG. 4.
`tion of the ERBB2 receptor. (A) Monolayers of HER2 cells were
`labeled with [32P]orthophosphate and then incubated for 15 min at
`22°C with each antibody at 10 ,ug/ml. Tyrosine-phosphorylated
`proteins were immunoprecipitated with an anti-phosphotyrosine
`antibody, followed by specific elution and a second immunoprecip-
`itation step with rabbit anti-ERBB2 polyclonal antibody, directed to
`the carboxyl terminus of the protein. (B) SKBR3 cells were first
`incubated with the antibodies, immunoprecipitated in two consecu-
`tive steps, as described above, and labeled by autophosphorylation
`with [y-32P]ATP and Mn2+. The autoradiograms of the SDS/gel-
`separated proteins are shown.
`
`genic cells carrying the oncogenic mutated ERBB2 protein
`(12, 13).
`In the present study we used a murine model system to
`address the potential and diversity of mAbs to ERBB2 as
`anti-tumor agents. We further attempted to understand the
`mechanisms of action of the antibodies in the hope that this
`may constitute an experimental basis for selection of an
`optimal mAb. Of the five mAbs surveyed in this study, two
`almost completely inhibited tumor growth, two displayed
`moderate inhibitory effects, and the last one significantly
`accelerated the rate of tumor growth (Fig. 3). These differ-
`ential biological activities can be attributed to different
`
`120
`
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`
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`
`c
`
`80
`
`60
`
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`CM
`
`w 20,
`
`0
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`Time, hr
`
`Effect of mAbs on the rate of turnover of the ERBB2
`FIG. 5.
`receptor. HER2 cells were labeled with [35S]methionine in a 24-well
`plate and then chased for the indicated periods of time with fresh
`medium that contained the indicated mAbs. Residual 35S-labeled
`ERBB2 protein was subjected to immunoprecipitation with a rabbit
`polyclonal antibody directed to the carboxyl terminus of the protein
`and separated on SDS gel. Quantitative analysis of receptor turnover
`is shown, as determined by measuring densitometry of the autora-
`diogram. o, Control cells without antibody treatment; A, N10; e,
`N12; o, N24; A, N28; and m, N29-antibody-treated cells.
`
`epitopes on the exoplasmic portion of the receptor. The
`mechanism by which different receptor regions may mediate
`opposing effects on tumor growth is apparently important for
`both receptor structure-function relationships and also for
`the elucidation of the biochemical mechanism underlying
`tumor inhibition. One simple explanation may be that the
`ligand-binding site ofthe putative receptor (25, 26) is involved
`in the action of the biologically active antibodies. However,
`in the absence of a well-characterized ligand for the ERBB2
`protein this possibility cannot be experimentally tested.
`Aware of its limitations, we tried to find a correlation
`between the in vivo effects of the mAbs and their actions on
`cultured cells. The results of this analysis are summarized in
`Table 1; in contrast with our inability to correlate the binding
`
`Table 1.
`
`Comparison of the biological properties of anti-ERBB2 mAbs
`Tyrosine
`Cell
`Receptor
`Tumor
`proliferationj
`ADCC,1
`CDC,§
`growth,t
`phosphorylation,
`degradation,**
`%
`t1/2 in hr
`-fold
`Immunoblot*
`Antibody
`%
`%
`%
`6.5
`-
`Anti-DNP
`ND
`ND
`1.0
`100
`100
`N1O IgM
`0.9
`68
`54
`247
`-
`3.1
`6
`7 ± 1
`N12 IgG1
`6
`1.8
`10 ± 0.01
`0.9
`9
`2
`63
`+
`3.5
`1%
`N24 IgG1
`2.5
`9 ± 2.2
`1.1
`60
`16
`-
`10 ± 1.7
`-
`18 ± 0.01
`N28 IgG1
`3
`141
`107
`14.0
`9 ± 2.2
`2.5
`1.2
`12 ± 0.33
`N29IgG2a
`0.3
`72
`+
`DNP, dinitrophenol; CDC, complement-dependent cytotoxicity; ADCC, antibody-mediated cell-dependent cytotoxicity, ND, not determined.
`*HER2 cell lysates were separated by SDS/gel electrophoresis, transferred to nitrocellulose, and blotted with the mAbs, followed by detection
`with horseradish peroxidase-conjugated goat anti-mouse F(ab')2.
`tAverage tumor volume (as percentage of control; n = 5) measured 21 days after tumor inoculation.
`*SKBR3 breast tumor cells were plated in 24-well plates (103 cells per well) and incubated for 48 hr in medium supplemented with 10%o fetal
`calf serum. The amount of serum was then decreased to 5%, and the indicated antibodies were added at 10 ,ug/ml. Five days later, the numbers
`of viable cells were determined.
`§Complement-dependent cytotoxicity assay of SKBR3 tumor cells was done as described. Values represent [51Cr]04 release from cells treated
`with the indicated mAbs (50 ,ug/ml) and human complement, as percentages of total cellular content of 51Cr. Corrections were made for
`spontaneous release, in the absence of antibody and complement. Similar results were obtained by using rabbit complement.
`lAntibody-mediated cell dependent lysis of SKBR3 cells was assayed as described, using each antibody at 50 ,ug/ml, and human effector cells,
`and expressed as percentages (see §). Similar results were obtained with mouse splenocytes.
`Extent of induction of tyrosine phosphorylation of ERBB2 protein by mAbs was determined by densitometry of autoradiograms, according
`to the assay of Fig. 4A.
`**Down-regulation of ERBB2 protein was determined with [35S]methionine-labeled HER2 cells, as described in text and Fig. 5, and expressed
`as half-life of the labeled protein (t1/2).
`
`IMMUNOGEN 2033, pg. 4
`Phigenix v. Immunogen
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`
`

`

`Cell Biology: Stancovski et al.
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`8695
`
`and the National Institutes of Health (Grant CA 51712). Y.Y. is an
`incumbent of The Armour Family Chair for Cancer Research.
`
`1.
`
`2.
`
`4.
`
`5.
`
`6.
`
`7.
`
`affinities of the various antibodies (Fig. 1) or their effects on
`cell proliferation in culture with their actions on tumors, an
`interesting correlation was found with direct responses ex-
`hibited by the receptor protein. Thus, our single tumor
`stimulatory mAb, N28, dramatically stimulated the tyrosine
`kinase activity of the receptor (Fig. 4). On the other hand,
`both tumor-inhibitory antibodies were the only mAbs capable
`of recognizing the fully denatured protein. This may reflect
`similar characteristics of the epitopes recognized by these
`antibodies, but the- correlation to tumor effect is not readily
`apparent: Although the most efficient tumor inhibitory anti-
`body, N29, led to the shortest receptor half-life (Fig. 5), this
`correlation is difficult due to the effects seen with the other
`antibodies.
`The simplest interpretation ofthese observations is that the
`positive effect on tumor growth involves stimulation of the
`tyrosine kinase function of the ERBB2 receptor, whereas
`tumor inhibition may involve other effects including reduc-
`tion in the cellular level of intact receptor-kinase molecules.
`The in vivo and in vitro effects of mAbs N28 and N29 are in
`line with the oncogenic role of the overexpressed ERBB2
`protein and are also consistent with the tumor-inhibitory
`function of a mAb directed to the rat ERBB2-transforming
`protein (12). Nevertheless, tumor inhibition may occur even
`without a significant effect on receptor down-regulation, as
`reflected by the action of the N12 mAb. It is, therefore,
`conceivable that several independent mechanisms may lead
`to inhibition of tumor growth. Cellular proliferation of either
`SKBR3 cells (Table 1) or HER2 cells (data not shown) in the
`presence of the mAbs turned out to be a limited predictor of
`the anti-tumor potential of each rilAb (Table 1). What is the
`significance then ofthe lack ofreflection in vitro ofthe effects
`of the various antibodies on tumors in vivo? One possible
`explanation is that the antibodies interfere with a process that
`occurs only in the living animal. This process may involve
`changes in tumor invasiveness, attraction of blood vessels
`(angiogenesis), or a paracrine loop. Interestingly, the differ-
`ential tumor inhibitory potential of the mAbs also did not
`correlate with cell lysis in vitro (Table 1), suggesting that
`neither complement- nor antibody-mediated cell lysis signif-
`icantly contributes to the inhibitory function.
`In summary, our results stress the caution with which
`antibody therapy should be considered, as different mAbs to
`the same protooncogenic receptor may have opposing effects
`on tumor growth. Nevertheless, the presented study provides
`further support to the notion that overexpression of a growth
`factor receptor leads to oncogenic transformation. It also
`demonstrates that a carefully selected mAb may be an
`efficient antitumor agent, at least in an experimental animal
`system.
`
`We gratefully thank R. Frackelton for the 1G2 antibody, E. Peles
`for the HCC cell line, and Y. Yaniv for technical assistance. This
`work was supported by grants from the Ministry of Health, Israel
`(Grant 1760), The Mario Negri-Weizmann Joint Research Program,
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`IMMUNOGEN 2033, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
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