Monoclonal Antibodies against the Extracellular Domain of the erbB-2 Receptor Function as Partial Ligand Agonists*

In this paper we describe the isolation and charac- terization of four monoclonal antibodies (FRP5, FSPl6, FWP51, and FSP77) which specifically recognize the human erbB-2 protein. All of the antibodies recognize epitopes on the extracellular domain of the receptor protein. FRP5 and FSPl6 compete with one another for binding while FWP51 and FSP77 each recognize a different epitope. The effects of the antibodies on the erbB-2 receptor protein have been ana- lyzed. Two different erbB-2-expressing cell lines, SKBR3 breast tumor cells and HC 11 R1 1 1 cells, were examined. The SKBR3 cells express approximately 1 X 10‘ molecules of the erbB-2 protein/cell; HC 11 R1 11 cells, a clone of mouse mammary epithelial cells de- rived by transfection of a human erbB-2 expression plasmid, contain 10-fold less erbB-2 protein than the SKBR3 cells. Treatment of the two cell lines with FRPS, FSP16, and FWP51 led to a rapid increase in the phosphotyrosine content of the erbB-2 protein. Three of the antibodies, FRP5, FSPl6, and FSP77, stimulated the turnover of the erbB-2 protein. Binding of the antibodies did not stimulate DNA synthesis in HCll R l l l cells. Thus, the erbB-2-specific mono- clonal antibodies behave as partial ligand agonists. The antibodies were examined domain of the erbB-2 protein. mAbs SKBR3 tumor cells

Multiple genetic alterations have been detected in the DNA of tumor cells (1). Technological advances have made it possible to analyze these mutations at the molecular level. It has become clear that in particular tumor types such as colorectal (2) or breast cancers (3) there are consistent patterns of DNA alterations. This suggests that the mutated genes are involved in the development of the tumor. The erbB-2 is a gene which appears to be important in breast and ovarian tumor development. Amplification of the erbB-2 gene, leading to over-expression of the protein, has been observed in a high percentage of human breast and ovarian tumor cells (4)(5)(6)(7)(8). The erbB-2 protein is a member of the receptor tyrosine kinase family and is most closely related to the epidermal growth factor (EGF)' receptor (9). The erbB-2 protein has been the object of intensive study following the discovery of its involvement in cancer. Clinical studies have determined its role as a prognostic indicator in breast and ovarian cancers (8,10). Molecular biological studies aimed at understanding its function in normal and transformed cells have also been undertaken (11,12). Since the erbB-2 protein has an extracellular domain and is expressed at high levels in some tumors it is also a potential target for anti-tumor agents. As a first step in producing such reagents, we have isolated monoclonal antibodies (mAbs) which recognize the human erbB-2 protein.
In this paper we describe the characterization of four mAbs which specifically bind to the extracellular domain of the erbB-2 protein. These antibodies act as partial ligand agonists. Following binding of the antibodies to the erbB-2 protein on intact cells, there is an increase in the phosphotyrosine content of the receptor and of other cellular proteins. Three of the antibodies cause an increase in the degradation of the erbB-2 protein. In addition, one antibody has strong growth inhibitory effects upon the SKBR3 breast tumor cell line which expresses high levels of the erbB-2 protein.

MATERIALS AND METHODS
Cell Culture-The SKBR3 human breast tumor cell line and the A431 human epidermoid carcinoma cell line were maintained in Dulbecco's modified Eagle's medium containing 8% Nu-serum (Collaborative Research). H C l l mouse mammary epithelial cells (13) and clone R111, a transfected H C l l cell clone expressing the human erbB-2 protein (14) were grown in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum (FCS), 5 pg/ml bovine insulin (Sigma), 10 ng/ml murine EGF (Fluka), and 2 mM glutamine. Hybridoma cells were maintained in RPMI-1640 containing 20% heatinactivated FCS, 4 mM glutamine, 1 mM sodium pyruvate, and 50 p M P-mercaptoethanol. In one experiment the SKBR3 cells were treated 8 h with the antibiotic tunicamycin (Boehringer) at a concentration of 5 pg/ml of growth medium.
Preparation of Cells for Immunization-Female BALB/c mice were immunized with intact SKBR3 human breast tumor cells. These cells have amplified copies of the erbB-2 gene (15) and express approximately 1 X IO6 molecules of the erbB-2 protein/cell. The cells were washed once with phosphate-buffered saline (PBS) and detached from the tissue culture plate at 37 "C in PBS containing 25 mM EDTA and 0.15 M NaC1. After low speed centrifugation, the cells were suspended in cold PBS and mixed 1:l (v/v) with Freund's complete adjuvant. 2 X lo7 cells were injected either intraperitoneally or subcutaneously into BALB/c mice. The injections were repeated intraperitoneally several times over a period of 3-4 months. Three days before fusion a final injection of SKBR3 cells was given intraperitoneally.
Fusion and Screening Procedure-Mice were sacrificed and their spleen cells were fused according to standard procedures (16) with the mouse myeloma cell line PA1 at a 5-1O:l ratio. 41% polyethylene glycol 4000 was used as the fusing agent, and the cells were plated on peritoneal macrophages at a density of 1 X lo6 cells/well of a 24-well tissue culture dish in hypoxanthine/aminopterin/thymidine selection medium.
The hybridoma cells were screened for the production of erbB-2specific antibodies in two steps. Hybridoma supernatants were tested for their immunofluorescent staining of a clone of transfected H C l l mouse mammary epithelial cells expressing the human erbB-2 protein (clone R111) (14). Nontransfected H C l l cells served as a control. Cells were grown 1-2 days on fibronectin-coated coverslips, rinsed in PBS, and fixed by 10-min treatment with 3.7% formaldehyde made in PBS. Hybridoma supernatants were added to the cells, and the coverslips were incubated 1-2 h a t room temperature in a humidified chamber. Antibody binding was detected using fluorescein-linked sheep anti-mouse Ig (Amersham). The positive supernatants were tested for specific immunoprecipitation of the erbB-2 protein from cell lysates of SKBR3 cells as described below.
Stable antibody-producing clones were obtained by limiting dilution and injected into pristane-primed mice for ascites production. Antibodies were purified from ascites fluid by ammonium sulfate precipitation and ion-exchange chromatography on DEAE-cellulose. Four anti-erbB-2 antibodies were isolated mAbs FRP5, FSP16, FWP51, and FSP77. The purified mAbs were dialyzed against PBS and stored at -70 "C.
Antibody Isotype and Subclass Determination-ELISA assays to determine the isotype and light chain class of the mAbs were done using a Bio-Rad Mouse Type TM Sub Isotyping Kit, as suggested by the manufacturer's protocol. mAbs FRP5, FWP51, and FSP77 have the IgGl isotype, and MAb FSP16 has the IgG2b isotype. All four of the antibodies have a K light chain.
Immunoprecipitation and Immunoblotting-SKBR3 cell lysates were prepared by adding 1 ml of lysis buffer (50 mM Tris-HC1, pH 7.5, 5 mM EGTA, 1% Triton-X 100, 150 mM NaCl, 1 mM paramethylsulfonyl fluoride (PMSF) to a 10-cm plate of cells for 10 min a t 4 "C. Extracts were clarified by centrifugation a t 10,000 X g for 10 min. erbB-2-Specific mAbs were added to the cell extract and allowed t o bind for 2 h at 4 "C, then rabbit anti-mouse IgG (ICN Immunobiologicals) was added for 1 h. The immune complexes were collected by the addition of protein A-Sepharose (Pharmacia). In one experiment a phospholipase C-71-specific MAb (UBI) was incubated with SKBR3 cell extract, and the immune complexes were collected by the addition of protein G-Sepharose (Pharmacia). The proteins were released by boiling in sample buffer and were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidenedifluoride membranes (Millipore Corp.) (17). The erbB-2 protein was detected using the 21N antiserum which was raised in rabbits against a peptide representing residues 1243-1255 of the human erbB-2 protein (18) followed by treatment of the filter with "'1-labeled protein A (Amersham).
Effect of the Anti-erbB-2 Antibodies on the Phosphotyrosine Content ofthe Protein"SKBR3 or H C l l clone R l l l cells were grown 24 h in medium containing 1% FCS, then treated 15 min at 37 "C with the four antibodies at a concentration of 5-50 pg/ml of culture medium which contained no additional FCS or growth factors. Cell lysates were prepared using lysis buffer containing 200 p M sodium-orthovanadate and total lysates were separated by 7.5% SDS-PAGE. An imunoblotting analysis was performed as above, using a specific mAb (19) to detect phosphotyrosine. The radioactivity in the specific bands was quantitated with a phosphoimager (Molecular Dynamics).
Metabolic Labeling of Cells-SKBR3, A431, HC11, and R l l l cells were metabolically labeled overnight in methionine-free Dulbecco's modified Eagle's medium containing 2% dialyzed FCS and 50 pCi/ml of [:"S]methionine (Trans"Slabe1, ICN Radiochemicals). The cells were extracted in lysis buffer, and the erbB-2 protein was immunoprecipitated using the 21N antiserum or the four erbB-2-specific mAbs. EGF receptor was immunoprecipitated using the 15E antiserum, raised against amino acid residues 716-727 of the protein (20).
The immunocomplexes were collected and separated by 9% SDS-PAGE, and the gel was fluorographed after salicylic acid treatment (21).
Fluorescence-actiuated Cell Sorting-SKBR3 cells were trypsinized, washed in fluorescence-activated cell sorting (FACS) medium (balanced salt solution containing 10 p~ sodium azide, 4% FCS, and 25 mM EDTA), and 1 X lo6 cells were resuspended in 100 p1 of FACS medium. Nonspecific binding sites were blocked by incubation with goat serum, the cells were collected by centrifugation, and resuspended in 50 pl of hybridoma supernatant diluted 1 to 3 with FACS medium. Following incubation for 45 min on ice, the cells were washed with FACS medium, and the bound antibody was detected with fluorescein-linked sheep anti-mouse Ig. Finally, the cells were washed with, and resuspended in, 100 pl of FACS medium and analyzed without fixation for their fluorescence in a Becton-Dickson FACScan. As a control the cells were treated with a nonreactive IgGl mAb.
Iodination of erbB-2-specific mAbs-mAbs FRP5 and FSP71 were covalently linked with (as carrier-free sodium lZ5-iodide, Amer-sham) to a specific activity of 1 pCi/pg using the iodogen method (22). Purified mAbs (50 pg in PBS) and ' "I were mixed in a glass tube coated with iodogen (Pierce) and shaken for 10 min at room temperature. Radiolabeled mAbs were separated from free iodine by chromatography through PD-10-Sephadex (Pharmacia). Cross-competition Binding Assay-In order to determine if the mAbs competed with each other for erbB-2, binding cross-competition experiments were carried out. 0.5-1.0 X lo5 SKBR3 cells were seeded/ 15-mm well of a multiwell tissue culture dish. After 24 h the cells were incubated with labeled antibody at 50% of the saturating concentration and varying amounts of unlabeled antibody. The incubation was carried out in 250 p1 of RIA buffer (120 mM NaC1, 50 mM HEPES, pH 7.8, 1 mM EDTA, 2% bovine serum albumin) for 2 h at 4 "C. The cells were washed five times with RIA buffer, solubilized in 0.5 ml of 1% Triton X-100, 10% glycerol, 20 mM HEPES, pH 7.4, for 30 min a t room temperature, and the specifically bound radioactivity was quantitated in a gamma counter. The results are an average from duplicate samples.
Tritiated Thymidine Incorporation Assay-1 X lo5 R l l l cells were seeded/3.5-cm culture dish. When the cells reached 80% confluence the medium was replaced with one containing 1% FCS. One day later the cells were stimulated for 22 h with 10 ng/ml EGF or with 10 pg/ ml of each mAb. [3H]Thymidine, a t a concentration of 1 pCi/ml, was included for the final 4 h. The incorporation of ["]thymidine into DNA was measured by washing the cultures with cold PBS, followed by a 5-min treatment with cold 5% trichloroacetic acid, then cold ethanol/ether (2:l). The plates were allowed to dry, then the cells were solubilized in 0.5 N NaOH, and the radioactivity was quantitated by liquid scintillation counting. The experimental points were done in duplicate.
Cell Proliferation Assay-1 X lo4 SKBR3 cells were seeded/3.5-cm well of a multiwell tissue culture dish in growth medium containing 1-10 pg/ml of each antibody. The medium was changed every 2 or 3 days, and at day 12 the cells were counted. The results are an average from duplicate wells.

RESULTS
Isolation of Monoclonal Antibodies specific for the Human erbB-2 Protein"SKBR3 human breast tumor cells, which express high levels of the erbB-2 protein ( E ) , were used as the immunogen for the production of erbB-2-specific mAbs. Intact cells were injected into BALB/c mice in order to optimize the chance of producing antibodies specific for the extracellular domain of the protein. Hybridomas were initially screened by an immunofluorescence assay using human erbB-!&transfected H C l l mouse epithelial cells, clone R l l l (14), and control-untransfected H C l l cells. Those hybridomas whose supernatants stained the former cells positively and the latter negatively were cloned by limiting dilution, and their supernatants were screened by immunoprecipitating the erbB-2 protein from lysates of SKBR3 cells.
In order to test for specificity of the four mAbs, the following experiments were performed. SKBR3 human breast tumor cells and the human erbB-2-transfected mouse H C l l cells, clone R111, were labeled with [35S]methionine, and the erbB-2 protein was immunoprecipitated from lysates with the four mAbs. Fig. 1, A and C, shows that the four mAbs, FRP5, FSP16, FWP51, and FSP77 specifically immunoprecipitate the p185 erbB-2 protein from lysates of, respectively, SKBR3 cells a n d R l l l cells. Fig. 1D shows that the mAbs do not recognize any proteins in the untransfected H C l l cells. The endogenous mouse erbB-2 protein can be immunoprecipitated from the HCll cells by the 21N antiserum which recognizes the p185 protein from rodent cells ( D , lane 1 ) . These results show that the mAbs specifically recognize the human erbB-2 protein.
The A431 human epidermoid carcinoma cell line expresses approximately 2 x lo6 EGF receptors/cell (23) and only low levels of the erbB-2 protein (15). These cells were examined in order to show that the mAbs do not recognize the structurally related EGF receptor. The A431 cells were metabolically labeled with [35S]methionine, and the erbB-2 protein was immunoprecipitated with each of the mAbs. Lanes 1 and 2 of Fig. 1B show the results of precipitating, respectively, the EGF receptor and the erbB-2 protein with specific sera. Lanes 3-6 show that each of the mAbs precipitates the p185 erbB-2 protein and does not recognize the p170 EGF receptor.

erbB-2-specific Monoclonal Antibodies
The mAbs Bind to the Extracellular Domain of the erbB-2 Receptor-In order to ascertain whether the mAbs recognize the extracellular domain of the erbB-2 protein, their binding was investigated by fluorescence-activated cell sorting. mAbs FRP5 and FSP16 were bound to intact SKBR3 cells.  In order to characterize the nature of the epitopes recognized by the mAbs, SKBR3 cells were treated with the antibiotic tunicamycin, which prevents the addition of N-linked oligosaccharides to proteins (24) . Fig. 3, lane 2, shows that the polyclonal21N antiserum, which recognizes the carboxylterminal peptide of erbB-2, immunoprecipitates two proteins from tunicamycin-treated SKBRB cells. The upper band comigrates with the mature erbB-2 protein present in untreated cells, while the 170-kDa band represents unglycosylated protein. All four of the mAbs immunoprecipitate both forms of the erbB-2 protein from lysates of tunicamycin-treated cells (Fig. 3 lanes 4, 6, 8, and 10).
In order to determine whether the antibodies recognize the same or different determinants on the extracellular domain of the erbB-2 protein, cross-competition experiments using '251-labeled mAbs were performed. The concentration of labeled mAb which saturated approximately 50% of the erbB-2-binding sites on the SKBR3 cells was determined, and the assays were performed in this range. SKBRB cells were incubated with either 12sI-labeled FRP5 or FSP77 in the presence of increasing concentrations of the four unlabeled antibodies. Fig. 4A shows that unlabeled mAb FRP5 and FSP16 were able to compete with labeled FRP5 for binding to the SKBR3 cells. Fig. 4B shows that only unlabeled mAb FSP77 was able to compete with labeled FSP77 in the binding assay. These results suggest that the four mAbs recognize at least three different antigenic determinants on the erbB-2 protein. mAbs FRP5 and FSP16 bind to similar regions on the extracellular erbB-2-specific Monoclonal Antibodies  in lanes 4,6,8, and 10, lysates from tunicamycin-treated cells were immunoprecipitated with, respectively, mAbs FRP5, FSP16, FWP51, and FSP77. The precipitated proteins were electrophoresed, electroblotted, and the erbB-2 protein detected with 21N serum followed by "'1-protein A treatment of the filter. The positions of the glycosylated p185 erbB-2 protein and the unglycosylated p170 erbB-2 protein are indicated.
domain of the erbB-2 protein, while FWP51 and FSP77 bind to separate domains.
Binding of erbB-2-specific mAbs to Cells Leads to an Increase in the Phsphotyrosine Content of the Receptor and Other Cellular Proteins-Ligand binding to a receptor tyrosine kinase leads to its activation which can be measured by an increase in the phosphotyrosine content of the protein (reviewed in Ref. 25). Antibodies which mimic this characteristic of a ligand have been described for the EGF receptor (reviewed in Ref. 26)) and they have been helpful in studying the biology of this receptor tyrosine kinase. Therefore, we have examined the effects which the erbB-2-specific mAbs have upon the phosphotyrosine content of the erbB-2 protein. SKBR3 and H C l l clone R l l l cells were treated for 15 min at 37 "C with 10 pg/ml of each mAb, and cell lysates were prepared in the presence of phosphatase inhibitors. Equal amounts of protein were examined by SDS-PAGE, and Western analyses were performed using a mAb specific for phosphotyrosine (19) and the 21N erbB-2-specific serum. The results obtained with SKBR3 cells and with R l l l cells are presented in, respectively, Fig. 5, A and B, and C. Treatment of cells with the mAbs leads to an increase in the phosphotyrosine content of the receptor. The increase was quantitated with a phosphoimager. In SKBR3 cells, treatment with mAbs FRP5, FSP16, FWP51, and FSP77 led to an increase in the erbB-2 phosphotyrosine content of, respectively, 3.7-, 2.3-, 5.6-, and 1.8-fold (Fig. 5A, lanes 2-5). As a control, Fig, 5B shows that equal amounts of erbB-2 receptor were present in each of the extracts. Treatment of R l l l cells with mAbs FRP5, FSP16, and FWP51 led to an increase in the erbB-2 phosphotyrosine content of, respectively, 3.0-, 3.3-, and 1.7-fold (Fig. 5C, lanes  3-5). The phosphotyrosine content of the erbB-2 receptor was not affected by treatment of R l l l cells with mAb FSP77 (Fig. 5C, lane 6). Equal amounts of erbB-2 protein were present in all lanes3 The results obtained with the two cell lines generally agree in that the mAbs FRP5, FSP16, and FWP51 have the strongest effect upon the erbB-2 phosphotyrosine content. mAb FSP77 had a weak effect upon the receptor in SKBR3 cells and no apparent effect in R l l l cells. This may reflect the fact that R l l l cells have approximately I. ".
Harwerth, unpublished results.  10-fold less erbB-2 molecules/cell than do SKBR3 cells? EGF addition to a variety of cells leads to an increase in the erbB-2 phosphotyrosine level (27)(28)(29)(30). This phosphorylation most likely occurs via the formation of heterodimers between these two receptors (31) . Fig. 5C, lane I, shows the results obtained following EGF addition to R l l l cells. Both the EGF receptor (p170) and the erbB-2 receptor (p185) display increased phosphotyrosine compared to the level seen in untreated cells (C, lane 2).
In order to test if the effect of the mAbs upon the erbB-2 phosphotyrosine content is dosage dependent, SKBR3 cells were treated 15 min at 37 "C with 5 or 50 pg/ml of mAb FSP16, FWP51, or FSP77. Only treatment of the cells with mAb FWP51 results in a dosage-dependent increase in erbB-2 phosphotyrosine content (Fig. 6). Quantitation of the filter revealed a 2.8-and 4.2-fold increase in phosphotyrosine in cells treated with, respectively, 5 and 50 pg/ml FWP51. Treatment of the cells with both concentrations of FSP16 and FSP77 led to increases of, respectively, 2.0 and 1.7 in the erbB-2 phosphotyrosine content. These results agree well with those presented in Fig. 5A.
Activation of receptor tyrosine kinases leads to elevated levels of phosphotyrosine on cellular proteins which are putative substrates of the receptors. Fig. 7 shows that, in addi- tion to the elevated phosphotyrosine content found in the erbB-2 receptor, increased tyrosine phosphorylation of other cellular proteins can be detected in SKBR3 cells treated with erbB-2-specific mAbs. In particular, cells treated with FRP5 and FWP51, (Fig. 7, lanes 3 and 5), the two mAbs which had the strongest effect upon the erbB-2 phosphotyrosine content, contain 145-, 115-, and 86-kDa proteins with elevated tyrosine content. The former protein comigrates with the p145 PLCy l which was immunoprecipitated from SKBR3 extracts (Fig.  7, lane 7). Lane 1 shows that the pattern of phosphotyrosinecontaining proteins in EGF-treated SKBR3 cells is different from the pattern seen in the mAb-treated cells.
Binding of Antibodies Accelerates the Turnover of the erbB-2 Protein in Intact Cells-Ligand-activated receptors are rap-  FRP5, FSP16, FWP51, FSP77 (lanes 3-6 ) or with 100 ng/ml EGF (lane I ) . Equal amounts of cell lysates were separated by SDS-PAGE, and a protein blotting analysis using the phosphotyrosine specific mAb was performed as described in Fig.  5. In lane 7, PLC-1 was immunoprecipitated from SKBR3 extracts with a specific mAb, and the precipitated material was run on the same gel. The PLC--yl was detected in a blotting analysis using the specific mAb. The positions of the p185 erbB-2 protein and the p145 PLC"y1 protein are indicated on the right. The arrours on the left show the positions of the prestained protein markers of 180, 116, and 84 kDa.
idly cleared from the cell surface via endocytosis through the coated pits (reviewed in Ref. 25). For the EGF receptor this can be measured by an increase in the turnover of the protein.
Since binding of the antibodies enhances the in vivo phosphorylation of the erbB-2 protein, this suggests that they act as ligand agonists. We studied the next step in the activation pathway, ie. the turnover of the erbB-2 protein following mAb binding. SKBR3 and H C l l clone R l l l cells were metabolically labeled with [35S]methionine, then chased for 24 h with medium containing unlabeled methionine plus 10 pg/ml of each of the mAbs. The erbB-2 protein was immunoprecipitated, and the content of [35S]methionine-labeled p185 was analyzed by SDS-PAGE followed by autoradiography. The results of the experiment are shown in Fig. 8. The binding of mAbs FRP5, FSP16, and FSP77 accelerates the turnover of the erbB-2 protein in SKBR3 cells (panel A) and in clone R l l l cells (panel B). In both cell lines the binding of mAb FWP51 has no effect upon the turnover of the erbB-2 protein.
Effect of the erbB-2-specific Antibodies upon the Growth of Cells-In fibroblasts ligand binding to a member of the receptor tyrosine kinase family ultimately leads to a stimulation of DNA synthesis (reviewed in Ref. 32). Since treatment of intact cells with the erbB-2-specific antibodies partially mimics ligand activation of a receptor protein, we tested whether binding of the mAbs would increase cellular DNA synthesis. The R l l l clone was examined since the HCll cells can be brought to quiescence in medium-containing low serum (33). The cells were 'starved for 1 day and then stimulated with EGF or with each mAb for 22 h. [3H]Thymidine was included in the medium for the final 4 h. Table I shows that the incorporation of [3H]thymidine into DNA was stimulated 67fold over background in cells which were treated with EGF, whereas none of the cultures treated with the erbB-2-specific antibodies showed an increase in DNA synthesis. From these results we conclude that the increased level of phosphotyrosine present in the erbB-2 protein following binding of the shown that the mAbs specifically recognize the p185 erbB-2 protein and do not react with the closely related EGF receptor. Other monoclonal antibodies specific for the human erbB-2 protein (34-40) and for the rat neu protein (41,42) have been described. These antibodies display a variety of characteristics, some of which are similar to those which we have described in this paper.
We have examined the biological effects which the four erbB-2-specific antibodies have upon the receptor protein in intact living cells. Two different cell lines were analyzed SKBR3, a human breast tumor cell line, and HCll clone R111, a mouse mammary epithelial cell line transfected with a plasmid encoding the human erbB-2 protein. The results obtained with both cell lines were comparable with respect to the erbB-2 protein down-regulation. The turnover of the erbB-2 protein in intact cells is slightly stimulated following binding of mAbs FRP5, FSP16, and FSP77. In an experiment not shown, we determined that in untreated SKBR3 cells and in cells treated with the mAbs FRP5 and FSP16 the half-life of the erbB-2 protein was, respectively, 8 and 6 h.3 It is interesting that the binding of mAbs FRP5 and FSP16, which recognize similar domains on the erbB-2 protein, has the strongest effect upon the erbB-2 protein turnover.
With respect to the effects which binding of the mAbs had upon the phosphotyrosine content of the erbB-2 receptor, there were some differences observed between SKBR3 and R l l l cells. Binding of all four of the antibodies to SKBR3 cells led to an increase in the phosphotyrosine content of the erbB-2 protein, although the effect of mAb FSP77 was weak. In R l l l cells FSP77 did not effect the phosphotyrosine content of the receptor, while the other three mAbs had stimulatory effects. This difference could be due to the fact that the SKBR3 cells express approximately 10-fold more erbB-2 molecules than the R l l l cells. The increased level of phosphotyrosine present in the erbB-2 receptor following mAb binding may reflect receptor activation via binding to the domain normally used by the ligand, or may reflect dimerization and ensuing activation via the bivalent binding of the antibody. This problem will be addressed using monovalent Fab fragments. A ligand for the erbB-2 protein has been described (41, 42) but is not yet readily available. Antibody 9,371 f 403 specific antibodies does not lead to an increase in the cellular DNA synthesis. We next examined the long term effect of the antibodies on anchorage-dependent cell proliferation. SKBRS cells were plated at low densities together with three concentrations of each antibody. The antibody-containing medium was replenished every 2 or 3 days, and after 12 days in culture the cells were counted. The results are shown in Fig. 9. The cell proliferation in cultures treated with mAbs FRP5, FSP16, and FWP51 did not vary significantly from the control culture. In contrast, SKBR3 cells treated with three concentrations of mAb FSP77 were inhibited in their growth by 90%. None of the antibodies had an effect .upon the anchoragedependent growth of R l l l cells?

DISCUSSION
We have described the isolation and characterization of four mAbs which recognize at least three distinct epitopes on the extracellular domain of the erbB-2 protein. The mAbs were isolated using intact SKBR3 tumor cells which express high levels of the erbB-2 protein as the immunogen. We have activation of the erbB-2 protein may help to circumvent this problem and prove useful, e.g. in studying short term effects of receptor activation or in assays to screen for agents which inhibit erbB-2 kinase activity.
The pattern of phosphotyrosine-containing proteins present in SKBR3 cells following treatment with the mAbs displayed differences when compared to unstimulated cells. This was most obvious in cells treated with FRP5 and FWP51, the two antibodies which stimulate the erbB-2 phosphorylation to the greatest extent. The elevated level of phosphotyrosine on intracellular proteins most likely reflects the activation of the receptor following antibody treatment of the cells. In fibroblasts it has been reported that the stimulated erbB-2 receptor is coupled to p145 PLC-71 phosphorylation and activation (45,46). A protein which comigrates with PLC-yl displayed enhanced phosphorylation in cells treated with both mAbs, and we are currently examining the phosphotyrosine content of the isolated PLC-71. It is interesting that EGF treatment of the SKBR3 cells leads to a different pattern of phosphotyrosine-containing proteins, suggesting that these two receptors couple to different intracellular signaling pathways. This is in agreement with our previous publications describing these two receptors in mammary epithelial cells (14, 33) and from others who examined the receptors in fibroblasts and in transfected hematopoietic cells (46,47).
The increased phosphotyrosine found in the erbB-2 and other cellular proteins following antibody binding, while suggesting receptor activation, did not result in a measurable increase in DNA synthesis in H C l l R l l l cells. Similar results have been described for anti-EGF receptor antibodies. In one study it was found that the binding of three EGF receptorspecific mAbs to intact cells led to an increase in the phosphotyrosine content of the receptor, but none of the antibodies stimulated DNA synthesis (48). In contrast, there are two reports of anti-EGF receptor antibodies which behave as full ligand agonists (49,50). One mAb competes with EGF for receptor binding (49) and the other does not (50), but both were able to stimulate kinase activity, receptor internalization, and DNA synthesis. None of the erbB-2-specific antibodies described in this paper appear to have full ligand agonistic activity.
The most striking result which we obtained was the growth inhibitory effect which mAb FSP77 had upon the SKBR3 cells. This antibody inhibits the anchorage-dependent growth of SKBR3 cells up to 90%. We have analyzed the effect of mAb FSP77 on the growth of H C l l clone R l l l cells and other human tumor cell lines expressing various levels of the erbB-2 protein. None of these cells were as sensitive to the growth inhibitory effects of FSP77 as were SKBR3 cells.3 There are a number of possible explanations for the results. One straightforward interpretation would be that mAb FSP77 disrupts an autocrine loop between the erbB-2 receptor and its ligand. SKBR3 cells might be particularly dependent upon this autocrine loop while the other cell lines which we tested are not. It is highly unlikely that the growth of all in uitro cultured breast tumor cell lines would be dependent upon such an autocrine loop. Despite the fact that TGF-a can be found in the medium conditioned by many tumor cell lines (51) there is only one example of a breast tumor cell line, MDA-468, in which an autocrine loop between TGF-a and EGF receptor is important for growth. In this case it was shown that all antibodies which competed with EGF for binding to its receptor also inhibited the growth of the MDA-468 cells (52). We are presently unable to test whether FSP77 competes with the erbB-2 ligand, but it is interesting that another antibody, mAb 4D5 (34), which also preferentially inhibits the anchorage-dependent growth of SKBR3 cells, does compete for binding with a protein which appears to be an erbB-2 ligand (43). Other erbB-2-specific mAbs have been reported to inhibit the proliferation of SKBR3 cells (40) or Calu-3 tumor cells which also express high levels of the receptor (39). It will be interesting to see if these antibodies bind to similar epitopes on the extracellular domain.
In summary, the four anti-erbB-2 antibodies behave as partial ligand agonists and one of them, FSP77, strongly inhibits the in uitro growth of SKBR3 breast tumor cells.
Preliminary results show that FSP77 also inhibits nude mouse tumor growth of erbB-2-transformed cells.3 In a modified form these erbB-2-specific monoclonal antibodies may prove useful as therapeutic agents in the treatment of human malignancies.