Therapy of an Animal Model of Human Gastric Cancer Using a Combination of Anti-Erbb-2 Monoclonal Antibodies1

[CANCER RESEARCH 52, 2771-2776, May 15, 1992] Therapy of an Animal Model of Human Gastric Cancer Using a Combination of Anti-erbB-2 Monoclonal Antibodies1

Philip G. Kasprzyk, Sun Uk Song, Pier Paolo Di Fiore, and C. Richter King2

Molecular Oncology, Inc., Gaithersburg, Maryland 20878 [P. G. K., S. V. S., C. R. K.J. and Laboratory of Cellular and Molecular Biology, National Cancer Institute, N1H, Bethesda, Maryland 20892 [P. P. DJ

ABSTRACT Our studies focused on the synergistic effect of a combination of two anti-erAB-2 antibodies. We chose to evaluate combina Amplification and/or overexpression of the erhti-2 gene have been tions of monoclonal antibodies because binding to gpl85"*B~2 demonstrated in 20-30% of adenocarcinomas of the breast, ovary, lung, should induce complex lattices of highly constrained protein and stomach and are associated with aggressive clinical course and poor prognosis. Interference with erbB-2 function by the use of monoclonal on the cell surface. We reasoned that these lattices might alter antibodies is a promising approach to the treatment of these diseases. In receptor function in a manner different from that of the binding this study we demonstrate that a combination of two anti-erÃ´B-2-specific of single monoclonal antibodies that can be expected to induce antibodies inhibited the growth of human gastric tumor cells in vitro. receptor dimerization. Our results show profound growth in This combination antibody therapy also inhibited the growth of human hibition with the combination of two antibodies when compared tumor cell lines growing as xenografts in nude mice and was able to to single-antibody treatment. dramatically reduce established tumors. This is the first reported obser vation of tumor regression induced by anti-erAB-2 monoclonal antibodies. Treatment was not curative in that tumors regrew after 6 weeks. Treat MATERIALS AND METHODS ment with either single antibody alone did not inhibit cell growth or Monoclonal Antibodies. Mice were immunized using a membrane tumor formation. Pulse chase and t>rosine kinase activity experiments preparation of N/erbB-2 cells (NIH/3T3 cells engineered to overexpress were used to investigate the activity of the erhH-2 gene product im>liS5"''" '). The formation of complexes by two antibodies was found to the human erbB-2 protein). Following tests of polyclonal antibody response using immunoprecipitation, three fusions were conducted interfere with receptor function and mimic some properties of a typical using the myeloma cell line Ag8.653 and standard techniques. These receptor ligand. Selective interference of the erbB-2 receptor by combi fusions produced approximately 1500 hybridoma clones which were nation antibody therapy may be advantageous for the treatment of human each screened using enzyme-linked immunosorbent assay. Membranes cancers. isolated from N/erbB-2 cells were bound to polystyrene plates, and culture medium was added to allow antibody-antigen interaction. 1m INTRODUCTION munoglobulin binding was detected using a biotinylated goat anti- mouse antibody, streptavidin horseradish peroxidase, and o-phenyl- Amplification and/or overexpression of the erbB-2 gene have enediamine hydrochloride. Positive reacting hybridomas were picked been shown in 20-30% of adenocarcinomas of the breast (1- and counter-screened using membranes from wild-type NIH/3T3 mem 5), ovary (3), lung (6), and stomach (7). Two lines of evidence branes. The molecular specificity was confirmed by immunoprecipita implicate erbB-2 overexpression in the pathogenesis of human tion analysis. Five hybridomas were picked with anti-erÃ©B-2-specifÃc reactivity and cloned by limiting dilution; two of these were designated neoplasia. First, overexpression has been linked with poor as e21 and e23 and were used in this study. Ascites was prepared by prognosis in breast (2-3, 8-11), ovarian (3, 12), stomach (13), administering injections of IO1'hybridoma cells to pristane-primed and lung cancer (14), indicating that overexpression profoundly mice. Antibodies were isolated in large amounts from ascites fluid and effects the cancer cell. Second, artificial overexpression of erbB- purified by high-performance liquid chromatography with a Gamma- 2 induces a transformed phenotype in NIH/3T3 fibroblasts (15, bind Ultra column (Genex, Gaithersburg, MD). SDS-PAGE3 was run 16) as well as in mammary epithelial cells (17), suggesting that under nonreducing conditions using Coomassie blue staining with a overexpression can contribute directly to the development of single band at M, 180,000 observed, indicating a >98% purified prep the malignant phenotype. aration. From 1 ml of ascites approximately 8-15 mg of antibody were Because of extensive homology between gpl85"*B"2 and the routinely purified. epidermal growth factor receptor, it is widely assumed that the Cell Lines and Tissue Culture. The human gastric tumor cell line used in these studies, N87, has been previously described (22) and was activation of growth signal transduction might proceed through routinely subcultura! in RPMI 1640 supplemented with 10% fetal similar mechanisms. One proposed mechanism involves recep bovine serum. The cell lines SK-Br-3, MDA-MB-468, and MDA-MB- tor dimerization or oligomerization, which is thought to be an 231 (breast) and SK-OV-3 (ovarian) were routinely subcultured in important step in the activation of the epidermal growth factor improved minimal essential medium (IMEM) supplemented with 5% receptor intrinsic tyrosine kinase function (18, 19). In this study fetal bovine serum. Cultures were maintained in humidified incubators interference with receptor-receptor interactions was evaluated at 37Â°Cin an atmosphere of 5% CO2 and 95% air. Cells were tested as a potential therapeutic approach to the treatment of cancers for Mycoplasma using a ribosomal RNA hybridization method (Gen- with erbB-2 overexpression. Previous studies have evaluated Probe, San Diego, CA). the use of single monoclonal antibodies directed against erbB- Growth Inhibition Assays. A single cell suspension of 10,000 cells/ well was plated in a serum-free defined media of RPMI 1640 containing 2 (20) and the related epidermal growth factor receptor protein bovine serum insulin (5 Mg/mO, human transferrin (10 Â¿tg/ml),17-/3- (21) as potential therapeutic agents for the treatment of cancer. estradiol (10 nM), sodium selenite (5 nM), and 10 mM 4-(2-hydroxy- ethyl)-l-piperazineethanesulfonic acid or in RPMI 1640 containing 2% Received 8/29/91; accepted 3/11/92. The costs of publication of this article were defrayed in part by the payment fetal bovine serum. PBS, e21, e23, or a combination of e21 and e23 of page charges. This article must therefore be hereby marked advertisement in (0.1, 1, 10 Mg/ml final concentration) was then added. The plates were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by Grant 2 R44 CA50077-02 from the Department of Health and 5The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate-polyacryl- Human Services. amide gel electrophoresis; PBS, phosphate-buffered saline; MTT, 3-(4-5-dime- 2To whom requests for reprints should be addressed, at Molecular Oncology, thylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; cDNA, complementary Inc., 19 Firstfield Road, Gaithersburg, MD 20878. DNA. 2771

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. erbB-2 THERAPY FOR GASTRIC CANCER incubated for 5-7 days in a CO2 incubator with humidity at 37Â°C.Cell RESULTS viability was monitored by one of two different methods. The first, the MTT assay (23), is based on the ability of live cells to reduce a Monoclonal antibodies directed against the extracellular do tetrazolium-based compound, MTT, to a purplish colored formazan main of gpl85'r*B2 were prepared by immunizing mice using a product that can be measured spectrophotometrically. After 7 days, 50 membrane preparation from NIH/3T3 cells engineered to ov- /il of MTT reagent (0.1 mg) were added and allowed to incubate for 4 erexpress the human erbB-2 protein (N/erbB-2 cells). Two of h at 37Â°C.Ninety % of the media was then removed, and the crystals these, designated e21 and e23, were used in this study. Both were solubilized in 0.175 ml dimethyl sulfoxide with absorbance meas antibodies specifically immunoprecipitated a single 35S-labeled ured at 540 nm in a Molecular Devices max kinetic microplate reader. protein of molecular weight 185,000 from SK-Br-3 cells (a The second method involves the cell number measurement in mono- breast cancer cell line which overexpresses gpl85'r*B'2 protein) layer cultures by crystal violet staining (24). Cells were plated as above and after 7 days cells were fixed by the addition of 20 ^1 of a 11% (26) as shown in Fig. 1. No immunoprecipitation was detected in cells which do not overexpress the gpl85"*B2 protein (e.g., glutaraldehyde solution. After being shaken on a BÃ©licoOrbitalShaker for 15 min the plates were washed three times with deionized water. MDA-MB-468; data not shown). Plates were then air-dried and stained by the addition of 100 /proteins were immunoprecipitated with 10 n\ of the indicated antibodies. The immune complexes were recovered by protein G-Aga- levels of erbB-2 gene amplification in N87, as shown in Fig. 3, rose (Genex) and analyzed by SDS-PAGE on an 8-16% Tris-glycine surpassed those found in the well-characterized SK-Br-3 and gel. The gel was exposed to film at â€”¿70Â°Covernightwith an intensifying SK-OV-3 cell lines (26). screen. The effect of these antibodies on growth was first studied in Western Blots. Cells or tumors were lysed in sample buffer which vitro using a semiautomated colorimetrie MTT assay. A dose- contained 0.125 M Tris-HCl, 4% SDS, 0.002% bromophenol blue, and 15% glycerol. Five % /3-mercaptoethanol was added after the protein concentration was determined. Samples (10 Mg total protein) were boiled for 3 min, fractionated by SDS-PAGE on 8-16% Tris-Glycine gel (Novex, Encinitas, CA), and transferred to nitrocellulose. Detection of gpl85"*B"2 was performed with a monoclonal antibody (E2-4001; Molecular Oncology, Inc.) to the COOH-terminal portion of the protein. Southern Blots. DNA was extracted from cell lines and human placenta tissue using guanidine thiocyanate and cesium gradient centrifugation. DNA (15 ^g) was cleaved with restriction enzyme Hindlll, separated by electrophoresis on a 1% agarose gel, transferred to nitro cellulose, and probed with a radioactive erbB-2 cDNA probe as previ ously described (25). The cDNA probe corresponds to the entire erbB- 2 protein coding region. CO In Vivo Antitumor Assay. Tumor cells (5 x 106/mouse) were injected O CM CM s.c. into the flanks of BNX (beige, nude, xid) mice. The day after cell C 0) 0) inoculation treatment was begun which consisted of four trial groups (3 mice/group), each given 0.2-ml i.p. treatment injections twice a 200- â€”¿ week. Tumor growth was monitored at least once a week and reported -gp185 as an average relative tumor volume. The effect of treatment after the formation of small tumors was also carried out. Cells were injected using the same treatment protocol as above except that the treatment was begun 4 days after cell injection instead of 1 day after. Animal care 97.4- â€”¿ was in accordance with institutional guidelines. Statistical analysis was carried out using a SAS Computer Package (SAS Institute, Cary, NC). gpl85"4i" Stability Assay. Subconfluent N87 cell monolayers were 69- pulse-labeled l h with 20 /

(monoclonal; Upstate Biotechnology, Inc.) and immunodetected using Fig. 1. Specificity of monoclonal antibodies e21 and e23. Subconfluent SK- 12!I-protein A. The gel was exposed to film at â€”¿70"Covernightwith an Br-3 monolayers were metabolically labeled with ["SJCys and immunoprecipi intensifying screen. tated with the indicated antibodies. 2772

ies or PBS and grew substantially over the period measured. In contrast to in vitro experiments, each monoclonal antibody alone may have limited activity, thus partially restricting the rate of tumor growth. To determine if the combined therapy with e21 and e23 was able to eradicate established tumors, an experiment was performed in which tumors were allowed to grow to measurable sizes prior to antibody treatment. The results are illustrated in Fig. 6. In animal groups randomized so that the starting size of the tumors was approximately the same volume, the tumors continued to grow when the animals were given single-antibody treatment of e21 or e23. In contrast, in the animals given combination treatment with e21 and e23, tumors completely regressed after 11 days (4 treatments of 200 fig of total antibody) and continued to be absent for another 2 weeks. After another month, the tumors reappeared, demon strating that some of the cells had escaped the treatment. To investigate the molecular basis for the antiproliferative QQ 185 effects of e21 and e23, we measured the rate of gplSS"*82 turnover in the presence or absence of antibodies. N87 cells were pulse-labeled with p'SjCys and then chased for various times in the presence of single antibody or the e21/e23 combi-

O DÃ› coco 23.1 9.4

Fig. 2. gpl85"*B 2overexpression in the gastric cell line N87 and a tumor from 6.5 N87 mouse xenografts compared to high (SK-Br-3) and low (MDA-MB-231) Probe: gpl85"*t:-expressing cell lines. Cells or tumors were lysed in sample buffer, boiled for 3 min, fractionated by SDS-PAGE on 8-16% Tris-glycine gel, and erb ÃŸ-2 transferred to nitrocellulose. Detection of gplSS""" was performed with a monoclonal antibody to the COOH-terminal portion of the protein. response analysis of the effects of the antibodies on N87 cell proliferation is shown in Fig. 4. Either e21 or e23 alone had no effect on the monolayer growth of cells up to a concentration of 10 Mg/ml (6 fiM). A 1:1 combination of the two, however, markedly affected cell proliferation at doses as low as 1 ng/m\. Fab fragments prepared from both antibodies also had no effect on cell growth alone or in combination (data not shown). The Fab fragments were found to inhibit >80% of the binding of 125I-e23at the concentrations used. This was comparable to the Probe: complete antibody. In analogous experiments with three other ÃŸ-actin gastric cell lines displaying little or no overexpression by im munoblot analysis, no activity was observed with the antibody combination or the antibodies alone (data not shown). The same results were obtained using crystal violet staining. The efficacy of combination antibody therapy was subse quently tested on the growth of N87 cells growing as tumor xenografts. N87 cells injected s.c. into nude mice produce Fig. 3. Southern blot analysis of the erbB-2 gene in N87 (gastric), SK-Br-3 rapidly growing tumors with a short latency. Tumor growth at (breast), and SK-OV-3 (ovarian) cell lines and human placenta. DNA was ex tracted from cell lines and human placenta tissue using guanidine thiocyanate the injection site was easily quantitated. As shown in Fig. 5, and cesium gradient centrifugation. DNA (15 *ig) was cleaved with restriction the N87 cells did not form tumors in the animals treated with enzyme Hind\\\, separated by electrophoresis on a 1% agarose gel, transferred to nitrocellulose, and probed with a radioactive erbB-2 cDNA probe which corre the combination of e21 and e23. In sharp contrast they were sponds to the entire erbB-2 protein coding region and a 0-actin cDNA probe as a potently tumorigenic in animals treated with the single antibod- control. 2773

50000 r e23 combination mimics the ligand, then treatment should result in increased gpl85cr*B2 autophosphorylation upon e21/ e23 combined treatment. We used antiphosphotyrosine immu- noblots to test this hypothesis. As shown in Fig. 8, increases in the tyrosine phosphors hi ion of gpl85"*B 2from N87 cells were CD 30000 â€¢¿ observed 15 min and l h after the addition of the single antibodies or the antibodies in combination. (The same results -J3L^r-.p1PBS were observed at 30 min and 2 h; data not shown.) This suggests that activation of tyrosine kinase activity may be necessary but O is probably not sufficient for growth inhibition.

120

0.1 1 10 0.1 1 10 0.1 1 10

e21 & e23 e21 e23

CONCENTRATION Utg/ml]

Fig. 4. EfTects of e21 and e23 on the growth of human N87 gastric tumor cells in a monolayer MTT growth assay. A single cell suspension of 10,000 cells/well was plated in a defined medium consisting of RPMI 1640 supplemented with insulin, human transferrin, 17-/3-estradiol, sodium selenite, and 4-(2-hydroxy- ethyl)-l-piperazineethanesulfonic acid buffer. Antibodies and combinations were then added. After 7 days, MTT reagent was added and allowed to incubate for 4 h at 37'C. Ninety % of the media was then removed, and the crystals were solubilized in dimethyl sulfoxide. Absorbance was measured at 540 nm with results representing the average of eight wells, with SD noted. Under the condi tions used, the cell number is directly proportional to MTT reduction.

0 4 7 11 14 19

Days after therapy started Fig. 6. Effect of treatment after the formation of small tumors. Cells were injected using the same treatment protocol as in Fig. 5 except that the treatment was begun 4 days after cell injection instead of 1 day after.

+ CO i-

0) 0) 0) 0) 0) 0) S â€¢¿* â€¢¿Â« â€¢¿V) J2 n ra (0 to (0 (O 3 .C .C .C Â£i C. 1.0 2.0 3.0 4.0 5.0 6.0 7.0 u. O O O O O Weeks following introduction of tumor cells Fig. S. Effects of treatment with e21 (O), e23 (A), a combination of e21 and 200- e23 (â€¢),orPBS (D) on the growth of N87 tumor xenografts in BNX mice. Tumor "t â€”¿gp185 cells (5 x 10" mouse) were injected s.c. into the flanks of BNX (beige, nude, xid) mice. Treatment begun on day 1 consisted of four trial groups (3 mice/group), each given 0.2-ml i.p. injections twice a week of either PBS (D), 200 ;

CO erbB-2 antibody has been proposed as a therapeutic. Following CM kinase activation, another sterically distinct receptor-receptor interaction may then facilitate internalization and down-regu lation. This alignment may be induced when the erbB-2 protein molecule is constrained by binding a combination of anti-ereB- 2 e21 and e23. This model is supported by the finding that the CMCM CM Fab fragments do not show antigrowth activity in combination, O 0) 0 0 nor do other combinations of nnti-erbB-2 monoclonal antibod ies that we have tested. Alternatively, the combined e21/e23 nÂ« -gpi85 treatment may induce a nonphysiological pattern of receptor oligomerization that shunts gplSS"*8"2 to the down-regulation pathway. 15 min Regardless of the mechanism, our results demonstrate that a combination of anti-receptor antibodies leads to different and more potent antitumor activities than single antibodies, and therefore, combination antibody therapy may be a useful ap proach to the treatment of human malignancies overexpressing gplSS"*8 2.This approach may be particularly important in the treatment of gastric cancer, a disease which responds poorly to ~W -gp185 current systemic chemotherapies.

1 hr ACKNOWLEDGMENTS We are grateful to Adi Gazdar for providing the N87 human gastric cell line. We thank Teresa Clayton, John Henry, and Laura Porter for excellent technical assistance and Michael Herman and Nancy Turner for critical reading of the manuscript. Fig. 8. Measurement of tyrosine phosphorylalion of gplSS"*8"' after incubation with antibody combination. Cells were plated as in Fig. 7. After l h cells were REFERENCES processed as in Fig. 1. The proteins were electroblotted onto nitrocellulose paper and incubated with anti-phosphotyrosine IgG (monoclonal; Upstate Biotechnol 1. King, C. R., Kraus, M. H., and Aaronson, S. A. Amplification of a novel v- ogy, Inc.) and immunodetected using 1!*I-protein A. The gel was exposed to film iT/ill related gene in a human mammary carcinoma. Science (Washington at â€”¿70"Covernight with an intensifying screen. DC), 229: 974-976, 1985. 2. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and McGuire, W. L. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. Therapy of an Animal Model of Human Gastric Cancer Using a Combination of Anti- erbB-2 Monoclonal Antibodies

Philip G. Kasprzyk, Sun Uk Song, Pier Paolo Di Fiore, et al.

Cancer Res 1992;52:2771-2776.

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