Oncogene (1988), 2, 387-394
`
`©The Macmillan Press Ltd, 1988
`
`Monoclonal antibodies specific for the neu oncogene product directly
`n1ediate anti-tumor effects in vivo
`
`Jeffrey A. Drebin 1 , Victoria C. Link & Mark I. Greene
`
`Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115; Division of lnmnmology, Department of Labor(cid:173)
`atory Medicine, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania, USA
`
`We have produced a panel of monoclonal antibodies
`which bind cell surface domains of the 185 Kd tumor
`antigen (p185) encoded by the neu oncogene. All of these
`antibodies stain neu-transformed cells in immunofluores(cid:173)
`cence assays and immunoprecipitate p185 from metaboli(cid:173)
`cally labeled cell lysates. All of the anti-pl85 monoclonal
`antibodies, regardless of isotype, exert a selective cyto(cid:173)
`static effect on the growth of neu-transformed cells sus(cid:173)
`pended
`in soft agar, demonstrating their ability to
`directly inhibit the transformed phenotype. Anti-p185
`antibodies of the IgM, IgG2a, and IgG2b isotypes exert
`a cytolytic effect on 1zeu-transformed cells in the presence
`of complement. Only one IgG2a monoclonal antibody is
`also able
`to mediate minimal
`levels of antibody(cid:173)
`dependent cellular cytotoxicity (ADCC) (Roussel et al.,
`1984) in the presence of non-immune spleen cells. b1 vivo
`administration of anti-p185 antibodies of the IgGl,
`lgG2a, and IgG2b isotypes exerts a profound inhibitory
`effect on the tumorigenic growth of neu-transformed
`cells. This tumor inhibitory effect is unaffected by deplet(cid:173)
`ing tumor bearing animals of complement, and is only
`minimally affected by depleting tumor bearing animals of
`macrophages. This suggests that neither complement(cid:173)
`mediated killing nor ADCC are necessary for the anti(cid:173)
`tumor effects of pl85-specific monoclonal antibodies. The
`results presented here demonstrate that monoclonal anti(cid:173)
`bodies reactive with cell surface domains of an oncogene(cid:173)
`encoded protein can directly inhibit tumor growth in vitl'o
`and in vivo. Such antibodies may prove useful in the
`therapy of certain malignancies.
`
`Introduction
`
`Recent studies of the molecular genetics of cancer have
`implicated a group of genes, termed oncogenes, in the
`neoplastic process (Bishop, 1987). Genes closely related
`to these genes, proto-oncogenes, are found in somatic
`cells of all eukaryotic species examined and have been
`highly conserved in evolution; it is thought that proto(cid:173)
`oncogenes normally play critical roles in cellular growth
`and development. Oncogene amplification and chromo(cid:173)
`somal rearrangements involving oncogenes have been
`detected, in a large number of tumors. Furthermore,
`some tumors have been shown to contain activated
`oncogenes which
`in DNA transfection assays , are
`capable of conferring neoplastic properties upon non-
`
`Correspondence: Dr MJ. Greene, Division of Immunology, Depart(cid:173)
`ment of Pathology and Laboratory Medicine, University of Pennsyl(cid:173)
`vania School of Medicine, Philadelphia, PA 19104, USA
`1 Present address: Department of Surgery, John Hopkins Hospital,
`600 N. Wolfe St., Baltimore, MD 21205, USA
`Received 24 September 1987; accepted 30 October 1987
`
`neoplastic rodent fibroblast cell lines (Weinberg, 1985).
`Collectively these studies suggest that alterations in
`proto-oncogene structure and function play a critical
`role in the development of neoplasia.
`Although most oncogene-encoded proteins reside in
`the nucleus or the cytoplasm (Bishop, 1987; Weinberg,
`1985), some oncogenes encode proteins that express
`antigenic sites on the cell surface. For example, the
`erbB, fins, and ros oncogene products are
`trans(cid:173)
`membrane glycoproteins
`that possess extracellular
`domains (Berg & Hayman, 1984; Roussel et al., 1984;
`Neckameyer et al., 1986). The sis oncogene product may
`also exist in a membrane associated form on the surface
`of transformed cells (Robbins et al., 1985). Another
`oncogene which encodes a protein that exposes anti(cid:173)
`genic sites on the surface of transformed cells has been
`identified by
`transfection of DNA
`from
`ethyl
`.nitrosourea-induced rat neuroblastomas into NIH3T3
`cells; we have termed this oncogene neu (Schechter et
`al., 1984). The neu gene has been found to be amplified
`in some human tumors, particularly those of the breast,
`suggesting that this gene may play a role in the etiology
`of human cancer (Semba et al., 1985; King et al., 1985;
`Yokota et al., 1986; Slamon et al., 1987; Kraus et al.,
`1987).
`We have identified a 185 000 dalton transmembrane
`protein (pl85) encoded by the neu oncogene (Schechter
`et al., 1984; Drebin et al., 1984). Recently, we described
`an IgG2a monoclonal antibody reactive with a domain
`of p185 expressed on the surface of neu-transformed
`cells, which was able to significantly inhibit the tumori(cid:173)
`genic growth of neu transformed cells (Drebin et al.,
`1986). In an effort to elucidate the mechanism(s) by
`which anti-p185 antibodies inhibit tumor growth, we
`have produced a number of additional p185 specific
`monoclonal antibodies of several different isotypes.
`Here, we describe these p185-specific monoclonal anti(cid:173)
`bodies, examine their cytostatic and cytotoxic effects on
`neu-transformed cells in vitro, and compare the in vitro
`anti-tumor effects of pl85-specific monoclonal anti(cid:173)
`bodies with their abilities to inhibit tumor growth in
`vivo.
`
`Results
`
`Production of monoclonal antibodies reactive with
`cell surface antigens on neu-transformed cells
`
`Hybridomas secreting monoclonal antibodies reactive
`with \;ell surface antigens on neu-transformed cells were
`produced and screened by' immunoftuorescence as
`described (Drebin et al., 1984). To date, five such hybri(cid:173)
`domas have been identified. The names of these hybri(cid:173)
`domas, their immunoglobulin heavy chain isotypes, and
`
`IMMUNOGEN 2032, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`..... p185
`
`388
`
`J.A. DREBIN et al.
`
`representative immunoftuorescence flow cytometry pro(cid:173)
`files of binding to the neu-transformed NIH3T3 cell line
`B104-1-1 and the Ha-ras-transformed NIH3T3 cell line
`XHT-1-la are shown in Figure 1. It is clear that all five
`monoclonal antibodies show significant
`levels of
`binding to B104-1-1 cells but not XHT-1-la cells
`(Figure 1, b-f). In contrast, a control monoclonal anti(cid:173)
`body (UPC 10, reactive with phosphorylcholine) fails to
`bind either B104-1-1 cells or XHT-1-la cells (Figure 1,
`g). Thus the monoclonal antibodies described here iden(cid:173)
`tify a cell surface antigen associated with the presence of
`the neu oncogene in transfected NIH3T3 cells.
`
`I mmunoprecipitation of p I 85 by monoclonal
`antibodies reactive with cell surf ace antigens
`on neu-transformed cells.
`
`Initial immunoprecipitation studies to identify antigens
`reactive with the neu specific monoclonal antibodies,
`conducting anti-mouse immunoglobulin and protein A(cid:173)
`bearing Staph. aureus, demonstrated that monoclonal
`antibodies 7.16.4 and 7.21.2 could specifically immuno(cid:173)
`precipitate the p185 product of the neu oncogene
`(Figure 2, lanes 2 and 3). Antibodies 7.5.5, 7.9.5, and
`7.16.5 were unable to precipitate p185 (or any other
`antigens) using these experimental conditions (data not
`shown). However when we purified these monoclonal
`antibodies and covalently coupled them to sepharose
`beads using cyanogen bromide, we were able to demon(cid:173)
`strate that they also specifically immunopreciptated
`
`Antibody
`
`lsotype
`
`8104-1-1 Cells XHT-1-1a Cells
`
`~
`
`~
`
`-
`
`-
`
`-
`
`lgG2b L
`lgG1 L
`03 L
`lgM L
`lgG1 L
`lgG2a L.
`
`a.
`
`None
`
`b.
`
`7.5.5
`
`c.
`
`7.9.5
`
`d.
`
`7.16.4
`
`e.
`
`7.16.5
`
`f.
`
`7.21.2
`
`g.
`
`UPC10
`
`lgG2a
`
`Q)
`
`....
`..a
`E
`::J c
`
`u
`
`Log fluorescence intensity
`Identification of monoclonal antibodies which specifi(cid:173)
`Figure 1
`cally bind cell surface determinants on neu-transformed NIH3T3
`cells. NIH3T3 cells transformed by transfection with an activated
`neu oncogene (cell line B104-l-1) or an activated Ha-ras oncogene
`(cell line XHT-1-la) were processed for indirect immunofiuores(cid:173)
`cence using saturating amounts of the indicated antibodies, as
`described in Materials and methods. Antibody isotypes were deter(cid:173)
`mined by indirect immunodilfusion according to the method of
`Ouchterlony (Hudson & Hay, 1980)
`
`Immunoprecipitation of p185 by monoclonal antibodies
`Figure 2
`reactive with cell surface determinants on neu-transformed
`NIH3T3 cells. Immunoprecipitation and SDS-polyacrylamide· gel
`electrophoresis of [3 5S]-cysteine labeled Bl04-1-1 cell lysates were
`performed as described in Materials and methods. Lanes 1-3:
`immunoprecipitation with free monoclonal antibodies, anti-mouse
`immunoglobulin, and protein A-bearing Staph. aureus. Lane 1 -
`normal mouse serum. Lane 2 - antibody 7.16.4. Lane 3 - antibody
`7.21.2. Lanes 4--8: immunoprecipitation with antibodies covalently
`coupled to sepharose beads. Lane 4 - protein A purified normal
`mouse immunoglobulin. Lane 5 - antibody 7.16.4. Lane 6 - anti(cid:173)
`body 7.9.5. Lane 7 - antibody 7.5.5. Lane 8 - antibody 7.16.5
`
`lysates of neu(cid:173)
`labeled
`from metabolically
`pl85
`transformed cells (Figure 2, lanes 6, 7, and 8). Thus all
`of the monoclonal antibodies which specifically bound
`the surface of neu-transformed cells were reactive with
`the p185 molecule encoded by the neu oncogene.
`
`Effects of anti-p185 monoclonal antibodies on the
`anchorage-dependent growth of neu-transformed cells
`
`One of the most stringent characteristics distinguishing
`malignant from non-malignant cells is the capacity for
`anch6rage-independent growth. We have
`recently
`shown that exposure of neu-tra'nsformed cells to the
`pl85 specific monoclonal antibody 7.16.4 caused the
`down-modulation of p185 from the cell surface and
`
`IMMUNOGEN 2032, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MONOCLONAL ANTIBODIES SPECIFIC FOR NEU ONCOGENE
`
`389
`
`Table 1 Anti-p 185 monoclonal antibodies inhibit the anchorage-independent growth of 11e11-
`transformed cells
`
`Antibody (specificity)
`
`0
`
`JOO·ng
`
`1 Jig
`
`JO Jig
`
`JOO 110
`
`Anclwrage-indepe11de11t colonies* (percent inhibition)
`
`25 ± 2.2
`
`None
`7.5.5 (anti-pl85)
`
`7.9.5 (anti-p185)
`
`7.16.4 (anti-p185)
`
`7.16.4 (anti-p185)
`
`7.21.2 (anti-p185)
`
`9BG5 (IgG2a, anti-reovirus)
`
`87.92.6 (IgM, anti-beta
`adrenergic receptor)
`
`15 ± 0.3
`(40)
`14 ± l.2
`(44)
`3 ± 3.3
`(88)
`16 ± l.9
`(36)
`25 ± 2.0
`(0)
`21 ± 1.5
`(16)
`22 ± 1.2
`(12)
`
`IO± 2.5
`(60)
`9 ± 0.9
`(64)
`1±0.6
`(96)
`3 ± 0.9
`(88)
`11±0.9
`(56)
`23 ± 2.0
`(8)
`26 ± 2.6
`(<0)
`
`6 ± 1.0
`(76)
`7 ± 0.6
`(72)
`J ± 0.6
`(96)
`1±0.3
`(96)
`12 ± l.9
`(52)
`21±3.2
`(16)
`29 ± 2.9
`(<0)
`
`4 ± 0.9
`(84)
`0.3 ± 0.3
`(99)
`0.7 ± 0.3
`(97)
`0.3 ± 0.3
`(99)
`12 ± 1.2
`(52)
`22 ± 1.3
`(12)
`23 ± 1.2
`(8)
`
`*colonies> 0.5mm were counted using a dissecting microscope after 14 days, described in
`Materials and methods
`
`p185 antibodies. As shown in Figure 3, antibody 7.16.4
`has no effect on the adherent growth of neu-transformed
`cells in liquid medium, even at concentrations that
`the anchorage-independent growth of neu(cid:173)
`inhibit
`transformed cells by > 95%, as shown above. We have
`examined the density of p 185 on cells grown under a
`variety of conditions and have not discerned any major
`changes in p185 expression (data not shown). These
`studies exclude differential expression patterns of p 185
`as a significant element in the failure to observe effects
`
`fr--/';. 104 8104-1-1
`o--0 104 8104-1-1 + 1 µ,g 7.16.4
`e--e 104 8104-1-1+10 µ,g 7.16.4
`
`';-
`2 ro
`0.
`.!!!
`Qi
`u 105
`+"' c
`~
`Q)
`.I::
`-0
`<(
`
`for anchorage(cid:173)
`the capacity
`loss of
`in
`resulted
`independent growth (Drebin et al., 1985). We have sub(cid:173)
`sequently examined the ability of each of the pl85
`specific antibodies described here
`to
`inhibit
`the
`anchorage-independent growth of neu-transformed cells.
`As shown in Table 1, all of the anti-p185 monoclonal
`antibodies are able to cause over 50% inhibition of the
`anchorage-independent growth of B 104-1-1 cells at
`doses of less than 1 µg per dish. The potency of the dif(cid:173)
`ferent anti-pl85 antibodies in inhibiting anchorage(cid:173)
`independent growth parallels their relative affinity for
`binding B 104-1-1 cells, with antibody 7.16.4 having the
`highest affinity and antibody 7.21.2 having the lowest
`affinity. The relative affinity has been deduced by the
`saturable binding curves of the various purified anti(cid:173)
`bodies for p185 expressed on 8104-1-1 cells (data not
`shown). In addition, in studies to be described else(cid:173)
`where, the antibodies have been found to identify three
`distinct domains of p185. Thus, 7.16.4, 7.9.5, and 7.21.2
`react with independent epitopes of the extracytoplasmic
`portions of p185. Hence, the effects observed in this
`study cannot be attributed to the binding of the differ(cid:173)
`ent monoclonals to the same site.
`In contrast to the effects of anti-pl 85 antibodies, two
`control monoclonal antibodies fail
`to significantly
`the anchorage-independent growth of neu(cid:173)
`inhibit
`transformed cells even at 100 µg per dish (Table 1). It is
`important to note that one of these control antibodies,
`87.92.6, is reactive with a beta-adrenergic like receptor
`on B 104-1-1 cells and shows significant binding to these
`cells by immunofluorescence (data not shown), but has
`no effect on their anchorage-independent growth. This
`demonstrates that the effects of the anti-pl 85 mono(cid:173)
`clonal antibodies on the anchorage-independent growth
`of neu-transformed cells does not simply result from
`antibody binding the cell surface, but reflects a specific
`cytostatic effect resulting from antibody binding to spe(cid:173)
`cific domains of the p 185 molecule.
`The ability of anti-p 185 monoclonal antibodies to
`inhibit the growth of neu-transformed cells occurs exclu(cid:173)
`sively under conditions that are selective for neoplastic
`behaviour, such as when the cells are suspended in soft
`agar. In contrast adherent growth in the 10% fetal calf
`serum, which is a property shared by non-neoplastic
`cells as well as neoplastic cells, is unaffected by anti-
`
`0
`
`2
`
`4
`
`5
`
`6
`
`3
`Day
`Figure 3 Antibody 7.16.4 has no effect on the adherent growth of
`Bl04-1-1 cells in 10% fetal calf serum. Bl04-l-l cells were plated
`at 1 x 104 cells per dish in 60 mm tissue culture dishes containing
`Dulb~cco's Modified Eagle's ·Medium supplemented with 10%
`fetal calf serum, antibiotics, and the· indicated amounts of purified
`antibody 7.16.4. At the days indicated, 'cells from three plates in
`each experimental group were removed from the culture dish
`surface with trypsin-versene solution and were counted using a
`hemocytometer
`
`IMMUNOGEN 2032, pg. 3
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`
`

`

`390
`
`J.A. DREBIN et al.
`
`of anti-p185 antibodies on cells grown in liquid media.
`Collectively, these studies demonstrate that anti-p185
`antibodies selectively inhibit the neoplastic behavior of
`neu-transformed cells, without in any way affecting cell
`viability.
`
`Table 2 Complement-mediated lysis of 11eu-transformed cells
`by anti-pl85 monoclonal antibodies
`
`Specific lysis of tumor targets·~
`
`Antibody*
`
`Isotype
`
`Bl04-1-1 cells
`
`XHT-1-la cells
`
`Complement mediated lysis of neu-transformed cells
`by pl85-specific monoclonal antibodies
`
`All of the anti-p185 antibodies described in this report
`exert a cytostatic effect on
`the growth of neu(cid:173)
`transformed cells suspended in soft agar. In order to
`identify additional mechanisms by which anti-p185
`monoclonal antibodies might exert anti-tumor effects,
`we examined their abilities to kill tumor cells in vitro in
`the presence of rabbit complement. Figure 4 presents
`data demonstrating that purified immunoglobulin from
`the 6.16.4 hybridoma is able to lyse neu-transformed
`cells in the presence of complement in one hour [ 51Cr]
`release assay at immunoglobulin concentrations as low
`as 5ngm1- 1 (Figure 4a). The ability of antibody 7.16.4
`to kill neu-transfonned cells is completely dependent on
`
`a
`
`80
`
`60
`
`40
`
`20
`
`80
`
`60
`
`40
`
`20
`
`0.0001
`
`0.1
`0.01
`0.001
`Antibody well (µg)- 1
`
`10
`
`7.5.5
`7.9.5
`7.16.4
`7.16.5
`7.21.2
`
`IgG2b
`IgGl
`IgG2a
`IgM
`IgGI
`
`43%
`2%
`66%
`28%
`8%
`
`0%
`0%
`0%
`0%
`0%
`
`*Antibodies were used at 5µgml- 1 in a total volume of0.2ml
`tDetermined by [ 51Cr]0 4 release assay with 5% rabbit serum
`complement source, as described in Materials and methods
`
`the addition of complement since purified immuno(cid:173)
`globulin does not exert cytotoxic effects in the absence
`of complement in either short term in vitro [ 51Cr]
`release assays (Figure 4b) or longer term cell cultures, as
`shown previously (Figure 3).
`Several other anti-p185 antibodies, in addition to
`antibody 7.16.4, are able to cause significant lysis or the
`neu-transformed NIH3T3 cell line B104-1-1 in the pre(cid:173)
`sence of complement (Table 2). There is no killing of the
`control cell line XHT-1-la by any of the monoclonal
`anti-p185 antibodies. In agreement with observations
`from other laboratories, IgM and lgG2 monoclonal
`antibodies are effective and IgGl monoclonal antibodies
`are ineffective in mediating complement-dependent lysis
`(Oi et al., 1984).
`
`Antibody dependent cell mediated lysis of
`neu-transformed cells by pl85-specific monoclonal
`antibodies
`In contrast to the effective complement-mediated lysis
`observed with several of the anti-p185 monoclonal anti(cid:173)
`bodies, only the IgG2a anti-p185 antibody 7.16.4 was
`able
`to mediate even modest
`levels of antibody(cid:173)
`dependent cell-mediated lysis of neu-transformed cells
`(Table 3). Anti-p185 antibodies of other isotypes had no
`activity in antibody-dependent cellular toxicity (ADCC)
`assays (data not shown). The level of killing obtained
`with antibody 7.16.4 was relatively low regardless of
`whether spleen cells, complete Freund's adjuvant elicted
`macrophages or
`thioglycollate elicited microphages
`were used as cellular effectors (data not shown). Even
`enhancing the adherence of anti-p185 antibodies to the
`surface of effector cells with polyethylene glycol, a pro(cid:173)
`cedure that increases ADCC activity (Jones & Segal,
`1980), failed to increase the cell-mediated lysis of neu(cid:173)
`transformed cells (data not shown). We have examined
`
`Table 3 Antibody dependent cell-mediated lysis of neu-transformed
`cells by antibody 7.16.4
`
`Antibody*
`
`Effectort:
`Target
`Ratio
`
`Specific lysis of tumor target4
`
`B104-l-1 cells
`
`XHT-1-la cells
`
`7.16.4
`
`4%
`5%
`3%
`
`100: 1
`12%
`33: 1
`6%
`7%
`11: 1
`Control IgG2att
`100: 1
`l %
`* A~tibodies were used at 5 ,ig mr:- 1 in a total volume of 0.2 ml
`t ~ucleated spleen cells from Balb/c nude mice were used as
`Antibody-Dependent Cellular Toxicity (ADCC) effectors
`:j: Determined by [ 51Cr JO 4 release assay as described in Materials
`and methods
`tt Monoclonal antibody UPClO, specific for phosphorylcholine
`
`1:10
`
`0
`
`1:80
`
`1 :40
`1 :20
`Complement dilution
`Figure 4 Lysis of neu-transformed cells by antibody 7.16.4 and com(cid:173)
`plement. (a) [ 51Cr]-labeled B104-1-l cells (open circles) or XHT-1-la
`cells (solid circles) were incubated in microwells in a total volume of
`200 µl with the indicated amounts of purified antibody 7.16.4 and
`rabbit compliment diluted to a final concentration of 1 : 20. After
`incubation at 37°C for 1 hour, specific lysis was calculated as
`described in Materials and methods. (b) [ 51Cr]-labeled B104-1-1 cells
`(open circles) or XHT-1-la cells (solid circles) were incubated in
`rilicrowells in a total volume of 200 µ1 with 1 µg/well of purified anti(cid:173)
`body 7.16.4 and rabbit complement diluted to the concentrations indi(cid:173)
`cated. After incubation at 37°C for 1 hour, specific lysis was calculated
`as described in Materials and methods
`
`IMMUNOGEN 2032, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MONOCLONAL ANTIBODIES SPECIFIC FOR NEU ONCOGENE
`
`391
`
`several neu-transformed cell lines with the same result.
`Therefore, we conclude, ADCC is not an important
`effector mechanism in this system.
`
`400
`
`Mechanisms of in vivo tumor growth inhibition by
`pJ85-specijic monoclonal antibodies
`We have recently shown that antibody 7.16.4 is able to
`exert a profound inhibitory effect on the tumorigenic
`growth of neu-traiisformed cells implanted into nude
`mice (Drebin et al., 1986). The present study has identi(cid:173)
`fied a number of in vitro anti-tumor effects mediated by
`antibody 7.16.4,
`including a direct effect on
`the
`anchorage-independent growth of neu-transformed cells,
`as well as the targeting of immunologic effectors to
`mediate complement-dependent cytotoxicity but only
`negligible levels of ADCC. Evidence that host immu(cid:173)
`nologic factors play only a minimal role in the in vivo
`anti-tumor effects of pl85 specific immunotherapy with
`antibody 7.16.4 comes from studies examining the
`effects of depleting complement or macrophage func(cid:173)
`tions in the tumor bearing host. As shown in Figure 5,
`depleting complement with cobra-venom factor does
`not inhibit the anti-tumor effects of antibody 7.16.4.
`Independently, we verified that mice treated with cobra
`venom factor had no discernible complement activity
`(data not shown). Similarly, depleting macrophage func(cid:173)
`tions with carrageenan, only modestly (although sta(cid:173)
`tistically significant on the day of the assay in this
`experiment) inhibited the antitumor effects of antibody
`7.16.4 (Figure 5). Both of these studies have been per(cid:173)
`formed with long periods of observation of greater than
`21 days. The absence of any significant effect during this
`time indicates that complement components and ADCC
`are not critical to the mechanism of action. These
`results suggest that a direct effect of antibody 7.16.4 on
`the neoplastic growth of neu-transformed cells is prin-
`
`Antibody Cobra Venom
`7.16.14
`Factor
`
`Complement depletion
`
`+
`+
`
`+
`+
`
`+
`+
`Carrageenan
`
`+
`+
`
`Macrophage depletion
`+--<
`I
`;:
`
`J p < 0.01
`J NS
`]P < 0.01
`
`p < 0.01
`p < 0.01
`p < 0.01
`
`0
`
`1 00 1 20
`80
`60
`40
`20
`Tumor size (mm 2 ) ± s.e.m.
`Figure 5 Effects of complement-depletion and macrophage deple(cid:173)
`tion on the anti-tumor activity of antibody 7.16.4. Balb/c nude
`mice received subcutaneous injections of 1 x 106 B104-1-1 tumor
`cells on day 0. Some groups of mice received an intravenous injec(cid:173)
`tion of 50 µg of purified antibody 7.16.4 within 2 hours of tumor
`cell implantation. Some groups of mice were treated with cobra
`venom factor (to deplete serum complement activity) or lambda
`carrageenan (to deplete macrophage activity) as described in
`Materials and methods. Tumor size was measured on day 9 fol(cid:173)
`lowing tumor implantation in the complement depletion experi(cid:173)
`ment (upper panel) and on day I 0 following tumor implantation in
`the macrophage depletion (lower panel). NS = not significantly
`different
`
`300
`
`E
`ai
`ui
`+I
`N'
`E 200
`E
`Ql
`N
`·u;
`,_
`0
`E
`:J
`f-
`
`100
`
`0
`
`7
`Day
`
`14
`
`Inhibition of tumor growth by pl85-specific mono(cid:173)
`Figure 6
`clonal antibodies of several different isotypes. Balb/c nude mice
`received subcutaneous injections of I x 106 B 104-1-1 tumor cells
`bn day 0. Groups of 5 mice received 1 ml intraperitoneal injections
`of Hank's Balanced Salt Solution (open circles), antibody 7.5.5
`ascites fluid (open triangles), antibody 7.9.5 ascites fluid (solid
`circles), or antibody 7.16.4 ascites fluid (solid triangles) on days 0
`and 7. Tumor size was measured as described in Materials and
`methods
`
`cipally responsible for its in vivo anti-tumor effects. ln
`recent studies, a role for direct oligomerization followed
`by internalization of p185 has been shown to affect the
`malignant phenotype (Drebin et al., 1985). We believe
`that this might also occur in vivo.
`The effects of other pl85-specific monoclonal anti(cid:173)
`bodies, which possess a more limited repertoire of in
`vitro anti-tumor activities, on the tumorigenic growth of
`B104-1-1 cells implanted into nude mice have also been
`examined. These studies provide additional evidence
`that a direct effect on the tumorigenic growth of neu(cid:173)
`transformed cells is responsible for the anti-tumor activ(cid:173)
`ity of anti-p185 antibodies. As shown in Figure 6,
`antibodies 7.5.5 (open triangles), 7.9.5 (solid circles), and
`7.16.4 (solid triangles) are all able to profoundly inhibit
`B 104-1-1 tumor growth. Antibody 7.21.2 has a some(cid:173)
`what less striking effect, whereas antibody 7.16.5 has no
`significant effect on Bl04-1-1 tumor growth (data not
`shown). Antibodies 7.9.5 (IgGl) and 7.21.2 (IgGl) are
`unable to effect complement-mediated cytolysis, and
`antibodies 7.5.5 (IgG2b) and 7.9.5 are unable to effect
`any ADCC, as shown above. The potent in vivo anti(cid:173)
`tumor activity of these antibodies suggest that neither
`complement-mediated lysis nor ADCC play a critical
`role in ~he inhibition of tumor growth by p 185-specific
`monoclonal antibodies. In independent studies not
`shown, we have also demonstrated that some of these
`antibodies can be shown to localize directly to the
`tumor in vivo in labeling and homing experiments. It
`appears
`therefore
`that
`the principle mechanism
`
`IMMUNOGEN 2032, pg. 5
`Phigenix v. Immunogen
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`
`

`

`392
`
`J.A. DREBIN et al.
`
`responsible for the in vivo anti-tumor effects on p185-
`specific monoclonal antibodies is the ability to directly
`inhibit the neoplastic properties of neu-transformed cells
`upon interaction with the oncogene encoded cell surface
`protein.
`
`Discussion
`
`The neu oncogene encodes a transmembrane glycopro(cid:173)
`tein tumor antigen, p185, which plays a critical role in
`the neoplastic behavior of neu-transformed cells
`(Schechter et al., 1984; Drebin et al., 1984; Drebin et al.,
`1985). We have produced a panel of monoclonal anti(cid:173)
`bodies which bind cell surface antigenic domains on
`neu-transformed NIH3T3 cells and which immuno(cid:173)
`precipitate p185 from metabolically labeled lysates of
`neu-transformed NIH3T3 cells. All of the p185-specific
`monoclonal antibodies including antibodies of the IgGl,
`IgG2a, IgG2b, and IgM isotypes, are able to exert a
`cytostatic effect on the growth of neu-transformed cells
`suspended in agar. Anti-p185 monoclonal antibodies of
`the lgM, IgG2a, and IgG2b isotypes are also able to
`mediate significant in vitro lysis of neu-transformed cells
`in the presence of complement.
`In contrast to the high levels of cell killing effected by
`these monoclonal antibodies in the presence of com(cid:173)
`plement, none of the anti-p185 monoclonal antibodies
`were able to target cellular effectors to mediate high
`levels of ADCC, and only the lgG2a antibody 7.16.4'
`was able to target even a detectable level of ADCC.
`This most likely reflect the relatively low cell surface
`density of p185 on B104-1-1 cells. Direct binding
`studies, using [ 1251]-labeled monoclonal antibodies,
`have shown that B104-1-1 cells express less
`than
`1 x 105 p185 molecules per cell (data not shown).
`Studies by Herlyn et al. have shown that target cells
`must express about 1 x 106 antigenic sites per tumor
`cell for effective ADCC mediated tumor killing to occur
`(Herlyn et al., 1985). Thus the B104-1-1 cells probably
`fail to bind enough antibody to allow high levels of
`tumor cell killing by cellular effectors.
`We have recently shown that the IgG2a p185-specific
`monoclonal antibody, 7.16.4 is able to profoundly
`inhibit the tumorigenic growth of neu-transformed cells
`implanted into nude mice or syngeneic rats (Drebin et
`al., 1986). The present study has demonstrated that anti(cid:173)
`body 7.16.4 mediates multiple anti-tumor activities in
`vitro including a cytostatic effect on the growth of neu(cid:173)
`transformed cells suspended in soft agar, as well as a
`cytotoxic effect on neu-transformed cells in the presence
`of complement. We have examined the relationship of
`the in vitro anti-tumor effects mediated by pl85-specific
`monoclonal antibodies to the anti-tumor effects seen in
`vivo. We have shown that depletion of complement or
`macrophage activity in the tumor-bearing host has little
`or no effect on the anti-tumor activity of antibody
`7.16.4. Furthermore, additional anti-p185 monoclonal
`antibodies which are unable to effect significant levels of
`complement-dependent lysis or ADCC in vitro are able
`to inhibit the tumorigenic growth of neu-transformed
`cells in vivo. These results suggest that the ability to
`directly
`inhibit
`the neoplastic growth of neu(cid:173)
`transformed cells is primarily responsible for the anti(cid:173)
`tumor activity of pl85-specific monoclonal antibodies.
`The immunotherapy of neoplastic disease with mono-
`
`clonal antibodies has shown significant anti-tumor
`effects in laboratory animals and man (reviewed in Reis(cid:173)
`feld, 1986; Houghton & Scheinberg, 1986). The effec.
`tiveness of antibodies raised against random structures
`present on the malignant cell surface depends on immu(cid:173)
`nologic mechanisms in the tumor-bearing host to elimi(cid:173)
`nate antibody-coated
`tumor cells
`(Reisfeld, 1986·
`Houghton & Scheinberg, 1986). Such studies hav~
`shown that IgG2a and lgG3 antibodies have the most
`potent in vivo anti-tumor activity as a result of their
`abilities to target complement-dependent cytotoxicity
`and/or ADCC. In an effort to enhance the anti-tumor
`effects of monoclonal antibodies, a number of groups
`have produced antibodies specifically reactive with
`structures necessary for neoplastic cell growth or which
`directly inhibit the neoplastic properties of tumor cells
`in vitro (Trowbridge & Domingo, 1981; Masui et al.,
`1984; Cuttitta et al., 1985; Vollmers et al., 1986; Roth et
`al., 1986; Hollingsworth et al., 1986; Hamada &
`Tsuruo, 1986). The results presented here demonstrate
`that monoclonal antibodies specific for cell-surface
`domains of the neu oncogene product are able to exert
`in vitro anti-tumor effects via multiple mechanisms, and
`are also able to inhibit tumor growth in vivo.
`Although the neu oncogene was initially identified by
`transfection of DNA from rat neuroblastomas into
`NIH3T3 cells (Schechter et al. 1984), enhanced expres(cid:173)
`sion of the human homologue of the neu gene may play
`a role in the development of a variety of human adeno(cid:173)
`carcinomas, particularly those of the breast (Semba et
`al., 1985; King et al., 1985; Yokota et al., 1986; Slamon
`et al., 1987; Kraus et al., 1987). Since the neu oncogene
`appears to be selectively expressed in embryogenesis
`with low levels of expression in non-dysplastic adult
`tissues (Coussens et al., 1985), antibodies directed
`against the neu oncogene encoded p185 molecule may
`be relatively specific in their
`targeting for
`tumors
`expressing high levels of p185. It will clearly be of inter(cid:173)
`est to determine whether antibodies specific for human
`p185 can exert direct or immunologically-mediated
`anti-tumor effects on human cancer cells expressing ele(cid:173)
`vated levels of the neu oncogene-encoded p185 mol(cid:173)
`ecule.
`
`Materials and methods
`
`Transformed cell lines
`The cell lines used here have been described previously
`(Drebin et al., 1984). Bl04-1-1 is a neu oncogene transformed
`NIH3T3 cell line derived by passing rat neuroblastoma trans-
`forming DNA sequences through two cycles of transfection in
`NIH3T3 cells. XHT-1-la is an Ha-ras-transformed NIH3T3
`cell line. Cells were cultured in 100 mm tissue culture dishes
`(Corstar), in 10 ml of Dulbecco's Modified Eagle's Medium
`(DMEM, K.C. Biologicals) supplemented with 10% fetal calf
`serum, 1 % pen-strep-fungizone mixture (M.A. Bioproducts)
`and 100 µg ml- 1 gentamycin sulfate (M.A. Bioproducts). Cell
`cultures were maintained in .a humidified 5% C0 2 incubator
`at: 37°C and replaced from frozen stocks every 2-3 months.
`
`.
`.
`Elperimental animals
`qH and [C 3H x DBA/2] Fi (C3D2 F 1) mice were obtained
`from the Jackson Laboratory,· Bar Harbor, ME. Inbred
`congenitally athymic Balb/c nude (nu/nu) mice were obtai~ed
`from the National Cancer Institute animal colony (Fredenck,
`MD) and from the University of California Cancer Center
`
`IMMUNOGEN 2032, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MONOCLONAL ANTIBODIES SPECIFIC FOR NEU ONCOGENE
`
`393
`
`animal colony (San Diego, CA). Animals used in this study
`were maintained in accordance with the guidelines or the
`Committee on Care a