Proc. Nati. Acad. Sci. USA
`Vol. 83, pp. 9129-9133, December 1986
`Immunology
`
`Inhibition of tumor growth by a monoclonal antibody reactive with
`an oncogene-encoded tumor antigen
`(immunotherapy/neu oncogene)
`
`JEFFREY A. DREBIN*, VICTORIA C. LINK*, ROBERT A. WEINBERGtI, AND MARK I. GREENE§11
`*Department of Pathology, Harvard Medical School, Boston, MA 02115; tWhitehead Institute for Biomedical Research, Cambridge, MA 02142; tDepartment
`of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and §Division of Immunology, Department of Pathology and Laboratory
`Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
`Contributed by Robert A. Weinberg, August 8, 1986
`
`ABSTRACT
`The neu oncogene encodes a 185-kDa trans-
`membrane glycoprotein tumor antigen, termed p185. We have
`recently described a monoclonal antibody reactive with a cell
`surface domain of the p185 molecule. In vivo treatment with
`this anti-p185 monoclonal antibody was able to significantly
`inhibit the tumorigenic growth of neu-transformed NIH 3T3
`cells implanted into nude mice. Such treatment had no effect on
`the tumorigenic growth of Ha-ras-transformed NIH 3T3 cells.
`Furthermore, anti-p185 antibody treatment was able to inhibit
`the growth of the rat neuroblastoma cells from which the neu
`oncogene was initially isolated. These results demonstrate that
`a monoclonal antibody reactive with the extracellular domain
`of an oncogene-encoded protein can exert a significant anti-
`tumor effect; such antibodies may prove useful in the therapy
`of certain malignancies.
`
`The advent of monoclonal antibody technology (1) has
`allowed investigators to produce large amounts of homoge-
`neous immunoglobulin specifically reactive with tumor cell
`membrane antigens (2, 3). Therapeutic trials of such mono-
`clonal antibodies are now underway in both animal models
`and cancer patients, with some encouraging preliminary
`results (4-10). However, many of the antitumor antibodies
`identified thus far react with antigens whose expression bears
`no obvious relationship to the neoplastic state of the tumor
`cell. Continued expression of such antigens may be unnec-
`essary for maintenance of the neoplastic state, permitting the
`appearance of nonantigenic variants in the tumor cell popu-
`lation. These nonantigenic variants may then be responsible
`for tumor relapse. More effective treatment might be
`achieved by the administration of monoclonal antibodies
`directed against antigens whose continued expression is
`required for neoplastic cell growth. For example, monoclonal
`antibodies reactive with certain growth factors or their
`receptors have been shown to mediate antitumor effects in
`vitro and in vivo (11-13).
`Among the antigens necessary for neoplastic cell growth
`are those encoded by activated oncogenes. These genes and
`their products are thought to play a critical role in neoplastic
`transformation (14-16). Furthermore, some of these onco-
`genes appear to encode proteins expressed on the cell
`surface. The products of the erbB, fins, neu, and ros
`oncogenes are transmembrane glycoproteins possessing
`extracellular domains (17-21); the sis oncogene product may
`also be found in a membrane-associated form on the surface
`of tumor cells (22). The proteins encoded by activated
`oncogenes may represent an important class oftumor antigen
`for the targeting of monoclonal antibody-mediated tumor
`therapy.
`
`The neu oncogene, initially identified by transfection of
`DNA from several ethylnitrosourea-induced rat neuroblas-
`tomas, has been shown to encode a transmembrane glyco-
`protein of 185 kDa (p185) (17). Amplification and rearrange-
`ment of the neu oncogene have been detected in several
`human tumors, suggesting that this gene may also play a role
`in the development of these malignancies (refs. 23-25 and A.
`Schechter and R.A.W., unpublished data). We have de-
`scribed (18) a hybridoma that secretes an IgG2a monoclonal
`antibody reactive with a cell surface domain of the rat p185
`molecule. This anti-p185 monoclonal antibody exerts a direct
`cytostatic effect on neu-transformed cells growing in soft agar
`and can also mediate in vitro cytotoxic effects on neu-
`transformed cells in the presence of complement or spleen
`cells (ref. 26 and J.A.D., V.C.L., and M.I.G., unpublished
`data). We therefore examined the effects of anti-p185 anti-
`body administration on the in vivo growth of neu-transformed
`cells.
`
`MATERIALS AND METHODS
`Cell Lines. Cell lines used in these studies have been
`described (17, 18). B104 is a rat neuroblastoma that contains
`an activated neu oncogene capable of transforming NIH 3T3
`cells in DNA transfection assays. B104-1-1 is a neu-trans-
`formed NIH 3T3 cell line generated by passaging B104-
`derived transforming DNA sequences through two cycles of
`DNA transfection. XHT-1-la is a Ha-ras-transformed NIH
`3T3 cell line generated by transfection of proviral DNA from
`Harvey sarcoma virus-infected cells. Cell lines were cultured
`in 100-mm tissue culture dishes (Costar, Cambridge, MA)
`containing 10 ml of Dulbecco's modified Eagle's medium
`(DMEM, KC Biological, Lenexa, KS) supplemented with
`10% heat-inactivated fetal bovine serum, 1% penicillin/strep-
`tomycin/Fungizone mixture (M. A. Bioproducts, Walkers-
`ville, MD), and 100 ,g of gentamicin sulfate (M. A.
`Bioproducts) per ml. Cell lines were passaged twice weekly
`at 1:20 dilution following release from the culture dish surface
`with trypsin/Versene (M. A. Bioproducts). Cell lines were
`maintained in a humidified 5% CO2. incubator at 37°C and
`were replaced from frozen stocks every 2-3 months.
`Experimental Animals. Inbred, congenitally athymic
`BALB/c nude (nu/nu) mice were obtained from the National
`Cancer Institute animal colony (Frederick, MD) and from the
`University of California Cancer Center animal colony (San
`Diego, CA). Male and female mice were used in different
`experiments with comparable results; within each experi-
`ment only animals of a single sex were used. Female BDIX
`rats were obtained from the National Cancer Institute animal
`colony (Frederick, MD).
`
`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.
`
`Abbreviation: HBSS, Hanks' balanced salts solution.
`ITo whom reprint requests should be addressed.
`
`9129
`
`IMMUNOGEN 2071, pg. 1
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`IPR2014-00676
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`9130
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`Immunology: Drebin et al.
`
`Proc. Natl. Acad. Sci. USA 83 (1986)
`
`Monoclonal Antibodies. Monoclonal antibody 7.16.4, a
`mouse IgG2a antibody reactive with the rat neu oncogene-
`encoded p185 molecule, has been described (18). Hybridoma
`cells secreting antibody 7.16.4 were washed free of serum and
`injected intraperitoneally into pristane-primed, x-irradiated
`(400 rads; 1 rad = 0.01 Gy) C3D2 F1 mice to induce ascites.
`After the development of ascites (in 7-14 days), ascites fluid
`was tapped with a 19-gauge needle, cellular elements and
`debris in the fluid were removed by centrifugation at 1000 x
`g for 10 min, and the fluid was stored at -70'C. Immuno-
`globulin was purified from ascites fluid by protein A-Seph-
`arose affinity chromatography as described (26). Control
`monoclonal antibody 9BG5, a mouse IgG2a antibody reactive
`with the hemagglutinin of reovirus type 3, was generously
`provided by G. Gaulton (University of Pennsylvania).
`Tumor Cell Implantation and Measurement of Tumor
`Growth. Tumor cells were released from tissue culture dishes
`with trypsin/Versene (EDTA) and were washed three times
`in Hanks' balanced salts solution (HBSS). Viable tumor cells
`(106) were then injected subcutaneously in the mid-dorsum of
`the experimental animals. Growing tumors were measured
`with vernier calipers on the days indicated, and tumor area
`was calculated as the product of tumor length and width, a
`method that has been shown to correlate with tumor weight
`(27). Data are presented as mean ± SEM. Significance was
`determined by comparing the means of different groups at the
`days shown, using Student's t test.
`Immunofluorescent Staining. Cells were removed from
`tissue culture dishes with buffered EDTA (Versene, M. A.
`Bioproducts) and washed twice in FACS medium [HBSS
`(GIBCO) supplemented with 2% fetal bovine serum, 0.2%
`sodium azide, and 10 mM Hepes]. Cells (106) in 0.1 ml of
`FACS medium were incubated with antibody or control
`supernatant, in an additional 0.1 ml, for 1 hr at 4°C. Cells were
`diluted in 2.5 ml of FACS medium, pelleted by centrifugation
`at 1000 x g, and washed twice more with 2.5 ml of FACS
`medium per wash. After the final wash, the cell pellet was
`gently resuspended and cells were incubated with 0.1 ml of
`fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG
`(reactive with heavy and light chains, Miles) diluted 1:50 in
`FACS medium, for 1 hr at 4°C. Cells were then diluted and
`washed as after the first incubation. The cell pellet was
`resuspended, and the cells were fixed in 0.5 ml of phosphate-
`buffered saline containing 2% paraformaldehyde. Samples
`were run on a Coulter Epics V flow cytometer (85 channels
`per logarithmic unit of fluorescence). Each analysis was
`conducted on 10,000 cells.
`
`RESULTS
`Treatment with an Anti-p185 Monoclonal Antibody Inhibits
`the Tumorigenic Growth of neu-Transformed NIHI 3T3 Cells.
`NIH 3T3 cells transformed by the neu oncogene (cell line
`B104-1-1) are highly tumorigenic. As shown in Fig. 1 (trian-
`gles), injection of 106 B104-1-1 cells into nude mice produced
`rapidly growing fibrosarcomas that killed their hosts after
`15-20 days. When nude mice injected subcutaneously with
`106 B104-1-1 cells were injected intraperitoneally with ascites
`fluid containing the anti-p185 antibody 7.16.4 on days 0 and
`1 following subcutaneous tumor implantation, the tumori-
`genic growth of the B104-1-1 cells was inhibited significantly
`(Fig. 1, filled circles, P < 0.05 at all days measured). In
`contrast, injection of a control ascites fluid containing an
`IgG2a monoclonal antibody of an irrelevant specificity (anti-
`reovirus) had no effect on the tumorigenic growth of B104-1-1
`cells (Fig. 1, open circles). Not only was tumor growth
`significantly inhibited in animals treated with antibody
`7.16.4, but the median survival of tumor-bearing mice was
`almost doubled, from 18 days to 34 days, by treatment with
`
`400
`
`.~200-
`0
`
`100
`
`7
`
`Day
`
`14
`
`Inhibition of the tumorigenic growth of neu-transformed
`FIG. 1.
`NIH 3T3 cells by monoclonal antibody 7.16.4. BALB/c nude mice
`were injected subcutaneously in the mid-dorsum with 106 B104-1-1
`tumor cells on day 0. Groups of five mice received 1-ml intraper-
`itoneal injections of HBSS (A), control ascites fluid containing
`anti-reovirus antibody 9BG5 (o), or ascites fluid containing anti-p185
`antibody 7.16.4 (e) on days 0 and 1. Growing tumors were measured
`and statistical calculations were performed as described in Materials
`and Methods.
`
`the anti-p185 antibody; treatment with the control ascites
`fluid did not extend the survival of treated animals.
`Fig. 2 demonstrates that purified IgG2a immunoglobulin
`from 7.16.4 ascites fluid is able to inhibit the tumorigenic
`growth of neu-transformed cells in a dose-dependent manner.
`Intravenous injection of 1 mg of purified antibody on the day
`of tumor cell implantation markedly inhibited tumor growth
`(Fig. 2, filled circles, P < 0.05 at all days measured).
`Treatment with 100 ,ug (open triangles) or 10 u.g (filled
`triangles) of antibody had a lesser but still significant (P <
`0.05 at all days measured) effect on tumor growth when
`compared to that ofthe saline-treated control group. We have
`also found that infusion of purified 7.16.4 immunoglobulin
`can have a significant antitumor effect in mice inoculated
`with B104-1-1 tumor cells 7 days prior to antibody treatment
`(data not shown). Thus, antibody 7.16.4 treatment can inhibit
`the growth of both fresh tumor inocula and established
`tumors composed of neu-transformed cells.
`Specificity of Anti-p185 Antibody Treatment. To determine
`whether the antitumor activity of monoclonal antibody 7.16.4
`injection was specific for neu-transformed cells, we exam-
`ined the effect of antibody 7.16.4 on growth of an NIH 3T3
`cell line transformed by an oncogene unrelated to the neu
`gene. For this experiment we utilized the Ha-ras-transformed
`NIH 3T3 cell line XHT-1-la. We have previously established
`that this cell line does not display p185 reactive with antibody
`7.16.4 in either quantitative immunofluorescence or immu-
`noprecipitation assays (18). Treatment with 7.16.4 ascites
`fluid on days 0 and 7 following tumor cell inoculation had no
`effect on the tumorigenic growth of Ha-ras-transformed NIH
`3T3 cells (Fig. 3 Right). In contrast, the tumorigenic growth
`of neu-transformed NIH 3T3 cells was significantly inhibited
`by identical doses of 7.16.4 ascites fluid in an experiment
`conducted in parallel (Fig. 3 Left, P < 0.05 at all days
`
`IMMUNOGEN 2071, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Immunology: Drebin et al.
`
`Proc. Natl. Acad. Sci. USA 83 (1986)
`
`9131
`
`7
`Day
`
`14
`
`0
`
`7
`
`Day
`
`14
`
`E J
`
`; 300-
`
`E 200-
`
`100
`
`0
`
`Antibody 7.16.4 inhibits the tumorigenic growth of
`FIG. 3.
`neu-transformed NIH 3T3 cells but not Ha-ras-transformed NIH 3T3
`cells. (Left) Groups of five BALB/c nude mice were injected
`subcutaneously with 106 neu-transformed B104-1-1 tumor cells and
`received intraperitoneal injections with 1 ml of either HBSS (o) or
`7.16.4 ascites fluid (e) on days 0 and 7 after tumor implantation.
`(Right) In an experiment conducted in parallel, groups of five mice
`were injected subcutaneously with 106 Ha-ras-transformed XHT-1-
`la tumor cells and received intraperitoneal injections with 1 ml of
`either HBSS (o) or 7.16.4 ascites fluid (n).
`
`noteworthy that B104 tumors were metastatic to regional
`lymph nodes in 3/6 and 2/6 of the B104-bearing nude mice
`treated with control antibody or saline, respectively. In
`contrast, lymph node metastases were not observed in
`B104-bearing nude mice treated with the anti-p185 antibody.
`Anti-p185 Antibody Treatment Inhibits the Growth of Rat
`Neuroblastoma Cells in Syngeneic Rats. Antibody 7.16.4 is not
`reactive with the product of the mouse neu gene, but it does
`react with the products of the activated and normal rat neu
`genes (17, 18). The experiments presented above used mono-
`clonal antibodies to inhibit the growth of tumor cells that
`express a tumor antigen (rat p185) that is antigenically
`distinct from any related proteins expressed in other tissues
`of the tumor-bearing nude mice. Although antibody 7.16.4
`was able to significantly inhibit the tumorigenic growth of
`neu-transformed cells implanted into nude mice, it was
`
`Cell-surface p185 levels on tumors progressively
`Table 1.
`growing despite anti-pl85 antibody treatment
`Immunofluorescence*
`Cell line
`with antibody 7.16.4
`38
`B104-1-1
`B104-1-1/Nul
`39
`B104-1-1/Nu2
`34
`NIH 3T3
`<1
`XHT-1-1a
`<1
`B104-1-1 cell tumors (B104-1-1/Nul and -/Nu2) that were pro-
`gressively growing in nude mice despite treatment with 1 ml of 7.16.4
`ascites fluid on days 0, 3, 7, 10, and 14 after implantation were
`excised, minced, and placed in tissue culture dishes containing
`culture medium; the medium was changed every 3 days until the cells
`had formed a dense monolayer. The cells were then processed for
`immunofluorescence staining of cell-surface p185 as described in
`Materials and Methods.
`*Values represent specific immunofluorescence, calculated by sub-
`tracting the median fluorescence channel number of populations
`stained with fluorescein isothiocyanate-conjugated rabbit anti-
`mouse IgG alone (negative control) from the median fluorescence
`channel of populations stained with saturating amounts of antibody
`7.16.4 followed by fluorescein isothiocyanate-conjugated rabbit
`anti-mouse IgG (positive staining).
`
`200
`
`200
`
`/
`H/
`0~~~~~~~~~~
`
`0
`
`7
`Day
`
`14
`
`Purified immunoglobulin from the 7.16.4 hybridoma
`FIG. 2.
`inhibits the tumorigenic growth of neu-transformed NIH 3T3 cells in
`a dose-dependent manner. Groups of five BALB/c nude mice
`received subcutaneous injections of 106 B104-1-1 cells and 0.4-ml
`intravenous injections of either HBSS (o) or HBSS containing 1 mg
`(e), 100 ,ug (A), or 10 Iug (v) of protein A-purified antibody 7.16.4 on
`day 0.
`
`measured). Thus, the ability of antibody 7.16.4 to inhibit
`tumor growth is specific for tumor cells that display p185 on
`their surface.
`Analysis of Cell-Surface p185 on neu-Transformed Cells
`from Tumors That Have Escaped Antibody Inhibition. Al-
`though treatment with antibody 7.16.4 was able to signifi-
`cantly inhibit the tumorigenic growth of neu-transformed
`cells, tumors eventually progressed in all treated animals. To
`determine whether tumor progression was due to selection
`for tumor cells that no longer expressed cell surface p185, we
`explanted fragments from tumors progressing despite anti-
`body treatment and examined cell surface p185 expression
`after a single passage in vitro. As shown in Table 1, cells from
`two independent B104-1-1 tumors that had progressed in the
`face of continued administration of antibody 7.16.4 express
`levels ofp185 comparable to that expressed by B104-1-1 cells
`maintained in vitro. Thus, the eventual progression of tumor
`growth despite anti-p185 antibody treatment is not due to the
`selection of variants that have ceased to express p185 on their
`surface.
`Anti-p185 Antibody Treatment Inhibits the Growth of Rat
`Neuroblastoma Cells Implanted in Nude Mice. The rat neu-
`roblastoma cells in which the activated neu oncogene arose
`express much less cell surface p185 than do NIH 3T3
`transfectants (17, 18). Studies by Herlyn et al. (28) have
`suggested that high antigen density is strongly correlated with
`the success of monoclonal antibody-mediated tumor therapy.
`It was therefore of interest to determine whether anti-p185
`antibody therapy could interfere with the tumorigenic growth
`of the rat neuroblastoma cells. Fig. 4 shows that the
`tumorigenic growth ofrat neuroblastoma cells (cell line B104)
`was indeed significantly inhibited in nude mice that received
`injections of purified antibody 7.16.4 (open circles) when
`compared to mice that received injections of control mono-
`clonal antibody 9BG5 (filled circles, P < 0.05 at all days
`measured) or normal saline (unpublished data). It is also
`
`IMMUNOGEN 2071, pg. 3
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`
`

`

`9132
`
`Immunology: Drebin et A
`
`Proc. Natl. Acad. Sci. USA 83 (1986)
`
`600
`
`E400-
`
`200-
`
`E
`
`N
`
`0E
`
`°
`
`7
`
`14
`Day
`
`21
`
`Antibody 7.16.4 inhibits the growth of B104 rat neuro-
`FIG. 5.
`blastoma cells in syngeneic BDIX rats. Groups of four BDIX rats
`received subcutaneous injections of 106 B104 tumor cells on day 0
`and received intravenous injections of 100 jug of antibody 7.16.4 in
`0.4 ml of HBSS (o) or 0.4 ml of HBSS alone (o) on days 0, 1, 3, 7,
`10, and 14 following tumor cell implantation. Tumor growth was
`measured and statistical calculations were performed as described in
`Materials and Methods except that rats were anesthetized with ether
`prior to tumor measurement.
`
`and some adenocarcinomas express high levels of the neu
`oncogene product (23-25). Such oncogene-encoded cell-
`surface proteins represent particularly attractive targets
`towards which to direct antitumor immunotherapy, since
`they are expressed at high levels and may play an important
`role in the neoplastic transformation of the cells that display
`them. In this report we have shown that a monoclonal
`antibody reactive with the rat neu oncogene product, pl85,
`can mediate antitumor effects in vivo.
`Systemic injection of a p185-specific monoclonal antibody,
`of the. IgG2a isotype, profoundly inhibits the tumorigenic
`growth in nude mice of NIH 3T3 cells transformed by an
`activated neu oncogene. Injection of this monoclonal anti-
`p185 antibody has no effect on the growth of NIH 3T3 cells
`transformed by a Ha-ras oncogene. The inhibition of tumor
`growth which occurs following the administration of anti-
`p185 monoclonal antibodies is dose-dependent, with as little
`as 10 ,ug of antibody causing a significant effect. Both fresh
`tumor inocula and established tumors are inhibited by the
`administration of the appropriate monoclonal antibodies.
`Injections of p185-specific monoclonal antibodies also inhibit
`the tumorigenic growth of the neu oncogene donor rat
`neuroblastoma cell line B104 in both nude mice and
`syngeneic immunocompetent BDIX rats. Collectively, these
`findings demonstrate that treatment with an anti-oncogene
`product monoclonal antibody can result in significant
`antitumor effects in vivo.
`Our studies have not defined the mechanism(s) by which
`antibody 7.16.4 inhibits the tumorigenic growth of neu-
`transformed cells. This antibody mediates multiple antitumor
`effects in vitro, including direct reversion of the transformed
`phenotype, complement-dependent lysis of antibody-coated
`
`C1E
`
`E
`
`~100
`
`H
`
`50-
`
`14
`
`0
`
`7
`Day
`Antibody 7.16.4 inhibits the growth of Bf104 rat neuro-
`FIG. 4.
`blastoma cells in nude mice. Groups of six BALB/c nude mice
`received subcutaneous injections on day 0 with 106 B104 neuroblas-
`toma cells and daily intravenous injections for 10 days with 20 ,ug (per
`day) of protein A-purified anti-p185 antibody 7.16.4 (o) or control
`antibody 9BG5 (e) in 0.4 ml of HBSS.
`
`important to address the question of whether such therapy
`would also affect the growth of B104 neuroblastoma tumors
`in syngeneic BDIX rats. Since the neu protooncogene is
`expressed at low levels in a variety of normal tissues (29),
`there was a possibility that injecting antibody 7.16.4 into rats
`might prove toxic. However, preliminary studies showed that
`intravenous injection of up to 4 mg ofpurified antibody 7.16.4
`per rat had no discernible toxic effects (unpublished data).
`We therefore injected B104 neuroblastoma cells subcuta-
`neously into syngeneic BDIX rats and examined the effect of
`intravenous antibody 7.16.4 infusion on subsequent tumor
`growth. As shown in Fig. 5, treatment with small doses of
`anti-p185 antibody was able to significantly inhibit the growth
`of B104 neuroblastoma cells in syngeneic immunocompetent
`rats compared with saline-treated controls (P < 0.05 at all
`days measured). Injections of a control IgG2a mouse mono-
`clonal antibody into BDIX rats had no significant effect on
`the growth of B104 tumors (unpublished data). These results
`demonstrate that it is possible to inhibit the growth of a tumor
`by the administration of a monoclonal antibody specific for
`an oncogene-encoded tumor antigen that is also displayed by
`the normal tissues of the tumor-bearing host.
`
`DISCUSSION
`Alterations in cellular oncogenes, resulting from gene ampli-
`fication, chromosomal translocation, or point mutations,
`have been identified in a significant fraction of tumors
`(14-16). While the proteins encoded by most of the known
`oncogenes normally reside within the nucleus or cytoplasm,
`some oncogene-encoded proteins are found on the external
`surface of the plasma membrane. Among human tumors that
`have been examined, tumors of several histologic types
`express high levels of the erbB oncogene product (30-32),
`
`IMMUNOGEN 2071, pg. 4
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`

`Immunology: Drebin et al.
`
`Proc. Natl. Acad. Sci. USA 83 (1986)
`
`9133
`
`tumor cells, and targeting of murine lymphoid cells to
`mediate modest levels of antibody-dependent cellular cyto-
`toxicity (ref. 26 and unpublished data). It is possible that the
`antitumor effects of antibody 7.16.4 reflect both the direct
`cytostatic effects of p185 crosslinking by anti-p185 antibodies
`and the cytolytic effects resulting from the recognition of
`antibody-coated tumor cells by immunologic mediators in the
`tumor-bearing host.
`Although profound inhibition of tumor growth and pro-
`longed survival resulted from treatment with anti-p185 mono-
`clonal antibodies, all of the treated animals eventually died
`from tumor progression. Examination of cells from explanted
`tumors that were progressing despite continued antibody
`treatment revealed normal levels of p185. This suggests that
`selection of variants expressing reduced levels of p185 was
`not the mechanism responsible for treatment failure.
`
`We thank Dr. Glen Gaulton (University of Pennsylvania) for
`providing control ascites fluid from the 9BG5 hybridoma and Dr.
`David Stern (Massachusetts Institute of Technology) for reviewing
`this manuscript. This work was supported by grants and fellowships
`from the National Institutes of Health, the Cancer Research Insti-
`tute, and the American Cancer Society. R.A.W. is an American
`Cancer Society Research Professor.
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